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Summarize: TOKYO (Reuters) - The plight of a lonely dolphin and dozens of penguins that have been abandoned in a derelict aquarium in Japan since the start of the year sparked protests this week, with activists and ordinary Japanese alike calling for the animals to be saved. The female bottlenose dolphin, nicknamed Honey, was captured in 2005 near Taiji, a western port town that has become notorious for its annual dolphin hunt that was featured in the Oscar-winning 2009 documentary “The Cove”, media reports say. The practice of Japanese aquariums buying dolphins from Taiji came under heavy criticism following the release of the film. The hunt involves driving hundreds of dolphins into a cove, where some are taken alive for sale to marine parks, while others are killed for meat. The Japan Association of Zoos and Aquariums has since agreed to stop buying dolphins from Taiji. Honey, a bottle-nose dolphin, is seen at abandoned Inubosaki Marine Park Aquarium in Choshi, Japan August 15, 2018. Picture taken August 15, 2018. PEACE/Handout via REUTERS The operator of the Inubosaki Marine Park Aquarium in the city of Choshi in Chiba prefecture, just east of Tokyo, shuttered the facility in January citing a decline in visitors after the 2011 earthquake and nuclear crisis. Honey and 46 penguins, along with hundreds of fish and reptiles, remain at the aquarium, an official with the Chiba prefectural Health and Welfare department said. Employees have been regularly feeding the animals, he added, but photos and video taken by activists in March and August from outside the park show Honey floating in a tiny pool in an eerily empty facility. In another picture, dust-covered penguins can be seen perched on a crumbling structure near a pile of debris. “Honey is a symbol of both the problem of marine parks and Taiji’s hunting practices,” said Akiko Mitsunobu, chief of aquarium issues for Animal Rights Center, a local group. Slideshow (3 Images) “When we went to check on the facility, she was showing signs of stress, putting her head weakly in and out of the water.” Repeated calls to Inubosaki Marine Park and its parent company went unanswered. A Choshi city official said they have also been unable to reach park representatives. “I get feelings of danger and doubt from the fact that they are so silent about this,” said Sachiko Azuma, a representative of local activist group PEACE (Put an End to Animal Cruelty and Exploitation). “As a group that handles animals, they have a responsibility to explain what they intend to do with Honey and the other animals.” News of the abandoned animals spread quickly over social media, with Twitter users posting photos captioned “Save Honey”. A resort hotel’s offer to give them a new home sparked a flood of retweets. “I beg the authorities to get in close contact with each other and push ahead with this,” wrote one Twitter user. Anger after hundreds of fish and reptiles have been left in tiny pools amid crumbling concrete since January Anger is mounting in Japan after a dolphin, 46 penguins and hundreds of fish were found to have been abandoned for months in a derelict aquarium. Animal rights campaigners have warned that the marine animals could die if they are not rescued from the Inubosaki marine park aquarium in the Pacific coastal town of Choshi north-east of Tokyo. The plight of Honey, a female bottlenose dolphin, as well as scores of Humboldt penguins and hundreds of fish and reptiles, has triggered outrage following reports that they were abandoned when the facility closed seven months ago. Images taken from outside the marine park in March this year show the solitary dolphin languishing in a tiny pool. In another photograph, dishevelled-looking penguins can be seen perched on a structure near what appear to be piles of loose concrete. 'Not ashamed': dolphin hunters of Taiji break silence over film The Cove Read more The marine park closed at the end of January following a dramatic drop in visitor numbers blamed on the earthquake and tsunami that struck Japan’s north-east in March 2011. Reports said employees of the marine park were feeding the animals, although it is unclear how they are sourcing food and how much they have left. It is possible that the park still has large stocks of frozen food or that employees are purchasing fresh fish in Choshi, a fishing port. Animal rights campaigners have been refused entry to the facility, while local authorities have been unable to contact its private owner, Inubosaki Marine Park. Calls to the park’s owner went unanswered. Facebook Twitter Pinterest Penguins at the Inubosaki Marine Park Aquarium Photograph: Animal Rights Center Japan/Peace “I am worried that Honey will die if this situation continues,” Akiko Mitsunobu, head of aquarium issues at the Animal Rights Centre Japan, told the Guardian. “Lately she has just been repeating the same movements – dipping her head in and out of the water – and is showing definite signs of stress. “ Despite claims by the prefecture’s public health centre that the dolphin and penguins are being properly cared for, Mitsunobu said they needed to be seen by animal welfare experts who can offer a second opinion. Sachiko Azuma, a representative of Japanese animal rights group Peace (Put an End to Animal Cruelty and Exploitation), said the former operator’s silence left her fearing the worst. “As a group that handles animals, they have a responsibility to explain what they intend to do with Honey and the other animals,” she said. “Compared to a year ago you can see that her condition has deteriorated. It’s impossible to say that she’s healthy.” Peace has launched a postcard campaign calling for Honey to be rescued, while the animals’ plight prompted a wave of criticism on social media. “This makes me so sick to my stomach,” one Twitter user wrote in a post with the hashtag #SaveHoney. “Animals deserve much better than this.” Another wrote: “I beg the authorities to get in close contact with each other and push ahead with this.” By last week, the marine park had received more than 800 emails and letters demanding that the animals be moved to a new home. The Mainichi Shimbun reported that the operator had been in talks with another aquarium about transferring Honey and the penguins but had abruptly ended negotiations and refused to respond to enquiries from the Choshi town government. Local officials are not legally entitled to enter the facility without permission and cannot compel the owner to relocate the animals, the newspaper said. Honey was taken to the marine park in 2005 after being captured in Taiji, a town on the Pacific coast that drew international criticism after its hunts were featured in the 2009 Oscar-winning documentary The Cove. Facebook Twitter Pinterest The abandoned aquarium Photograph: Reuters/Peace In 2015, the Japan Association of Zoos and Aquariums agreed to stop buying dolphins from Taiji after it was threatened with expulsion from the World Association of Zoos and Aquariums (Waza). The move came after the Guardian revealed that Waza had been targeted in a court action launched by the conservation group Australia for Dolphins, which accused it of being complicit in the hunts by failing to take decisive action against Japanese aquariums. Aquariums in Japan voted to stop buying live specimens from Taiji to avoid expulsion, but a facility in the town quit Jaza in protest, and local fishermen have vowed to continue the hunts. In a typical hunt the fishermen pursue pods of dolphins across open seas, banging the top of metal poles that extend beneath the water to confuse their hypersensitive sonar and driving them into a narrow cove. Some are killed for their meat, which is sold in local supermarkets and restaurants, while the most attractive specimens are selected for sale to aquariums and sea parks. The animal rights centre said it feared that Honey could be recaptured if she was returned to waters off Taiji. Instead, it is demanding that she be sent to a sanctuary that closely resembles her natural habitat. Mitsunobu said Honey’s plight “reflects the lack of awareness people in Japan generally have about animal welfare and keeping animals in captivity. Aquariums and marine parks are still seen as places for families to have a fun day out. “The issue has been complicated by the fact that it is connected to the Taiji dolphin hunts, but for now this is about the need to do something to help Honey before it is too late.” (CNN) Animal rights groups in Japan have raised the alarm over a dolphin, 46 penguins and hundreds of fish and reptiles that are locked in an aquarium that has been closed for months. The bottle-nosed dolphin, known as Honey, is being kept on her own in a small pool and is showing signs of mental stress, activists say. The Inubosaki Marine Park, in the city of Choshi, just east of Tokyo, shut down in January because of a lack of visitors. Park staff continue to feed the marine life at the aquarium. But with no companions, Honey -- who was captured from the wild in 2005 -- is condemned to a lonely existence. Read More | Summary: A solitary bottlenose dolphin swims in a small pool enclosed by a rusty railing. Not far away, dust-covered penguins wander over chunks of concrete and other debris. This has been life at Japan's Inubosaki Marine Park Aquarium since January when its owner abandoned it, citing a decline in visitors. Employees have reportedly been feeding the remaining animals-Honey the dolphin, 46 penguins, and hundreds of fish and reptiles, per Reuters-but animal rights activists and regular citizens who've penned 1,400 letters to the town of Choshi are furious. "I am worried that Honey will die if this situation continues," the head of aquarium issues at the Animal Rights Centre Japan tells the Guardian, noting the dolphin has "been repeating the same movements... and is showing definite signs of stress." Officials with the Chiba Prefecture's hygiene control division visited the aquarium this week and found the animals to be healthy, with Honey recovering well from a sunburn, per CNN. A prefecture rep says the private owner, Inubosaki Marine Park, is working to find them a new home. Animal Rights Centre Japan hopes Honey, who's been at the aquarium since 2005, can be moved to a sanctuary, fearing recapture if she's returned to her original home off Taiji, the Pacific coast town known for its annual dolphin slaughter. But local media previously reported that transfer negotiations had abruptly ended, per the Guardian. "They have a responsibility to explain what they intend to do with Honey and the other animals," one activist says. Others are elevating the case on social media using #SaveHoney. | 2,113 | 378 | multi_news | en |
Write a title and summarize: The osteoblast-lineage consists of cells at various stages of maturation that are essential for skeletal development, growth, and maintenance. Over the past decade, many of the signaling cascades that regulate this lineage have been elucidated; however, little is known of the networks that coordinate, modulate, and transmit these signals. Here, we identify a gene network specific to the osteoblast-lineage through the reconstruction of a bone co-expression network using microarray profiles collected on 96 Hybrid Mouse Diversity Panel (HMDP) inbred strains. Of the 21 modules that comprised the bone network, module 9 (M9) contained genes that were highly correlated with prototypical osteoblast maker genes and were more highly expressed in osteoblasts relative to other bone cells. In addition, the M9 contained many of the key genes that define the osteoblast-lineage, which together suggested that it was specific to this lineage. To use the M9 to identify novel osteoblast genes and highlight its biological relevance, we knocked-down the expression of its two most connected “hub” genes, Maged1 and Pard6g. Their perturbation altered both osteoblast proliferation and differentiation. Furthermore, we demonstrated the mice deficient in Maged1 had decreased bone mineral density (BMD). It was also discovered that a local expression quantitative trait locus (eQTL) regulating the Wnt signaling antagonist Sfrp1 was a key driver of the M9. We also show that the M9 is associated with BMD in the HMDP and is enriched for genes implicated in the regulation of human BMD through genome-wide association studies. In conclusion, we have identified a physiologically relevant gene network and used it to discover novel genes and regulatory mechanisms involved in the function of osteoblast-lineage cells. Our results highlight the power of harnessing natural genetic variation to generate co-expression networks that can be used to gain insight into the function of specific cell-types. The osteoblast-lineage consists of a spectrum of cells beginning with osteoprogenitors derived from mesenchymal stem cells that then differentiate to form mature bone-forming osteoblasts and bone-lining cells. The final stage in the life-cycle of the lineage occurs when a subset of mature osteoblasts become entombed in bone as mechanosensitive osteocytes [1]. As the only known bone-forming cell, osteoblasts are essential for skeletal development, growth and maintenance [1]. In addition to their critical role in the skeleton, osteoblast-lineage cell have been shown to be important for other physiological systems. Osteoprogenitors can support and modulate erythropoiesis [2] and mature osteoblasts are responsible for many of the endocrine functions of bone, including the regulation of energy expenditure [3]–[5] and male fertility [6]. Furthermore, osteocytes play important roles in mineral metabolism [7] and bone resorption [8], [9]. Therefore, the development of a more comprehensive understanding of the molecular networks operative in osteoblast-lineage cells will have important implications not only for osteoporosis, but many other common complex diseases. Genetic, molecular and biochemical approaches have been used over the last decade to identify many of the key genes that are required for osteoblast progenitor commitment, proliferation, differentiation and apoptosis as well as mature osteoblast and osteocyte activity [1]. An example of this has been the discovery that the Wnt signaling pathway plays a central role in many functional aspects of the osteoblast lineage [10]. However, these investigations have been reductionist in nature and therefore have not provided information on how key signaling genes interact in complex cellular networks, which is critical to fully understand the molecular mechanisms that underlie cellular processes and disease. In many cases, the insight gained regarding how genes interact in networks goes well beyond what can be learned about a process using only traditional approaches [11]. Systems genetics is an emerging approach that provides a systems-level perspective of the role of genetic variation in cell function and disease [12]. Systems genetics relies on the principles and methods of systems biology, but focuses on determining how naturally occurring genetic variation perturbs cellular phenotypes [13]. The foundation of systems genetics is a suite of analytical approaches that include genome-wide association, expression quantitative trait locus (eQTL) discovery, causality modeling and network analysis [14]. One of the most powerful systems genetics tools is network analysis. Biological networks can be based on many different types of interactions [15], such as genetic, protein-protein and transcription factor binding. However, the most common networks used in systems genetics research are based on co-expression. In a systems genetics context, co-expression networks are generated using global expression data collected across many genetically unique individuals [16]. The patterns of gene expression that result from each unique set of genetic perturbations are used to quantify correlational relationships among genes on a genome-wide scale. Co-expression networks typically display two important behaviors, i) they are modular, with distinct modules representing dense clusters of genes that are highly co-expressed and ii) the co-expression modules are often enriched for genes that share similar functions [17]. Because modules contain functionally similar gene sets, they can be used to extract many pieces of information about a system. For instance, a number of studies have shown that summarized measures of module behavior often correlate with complex processes or disease phenotypes [18]–[20]. The identification of such modules provides a list of genes and pathways that likely play a role in the process or disease. In addition, genes within a module can be organized by connectivity. Highly connected genes are called “hubs” and in a co-expression network hub genes are those that are the most strongly correlated with the largest number of other module genes [21]. Importantly, a number of studies have found that connectivity correlates with biologically relevant properties. For example, hubs in yeast networks have been found to be more likely essential for growth than non-hub genes [22] and connectivity was found to be predictive of survival in a human brain co-expression module associated with glioblastoma [23]. Recently, we demonstrated that in a co-expression module associated with Bone Mineral Density (BMD) in humans, hub genes were more likely to be genetically associated with BMD than non-hub genes [24]. We have also shown that hubs within a chrondrocyte co-expression network play key roles in chrondrocyte differentiation [20]. In this study, we reconstructed a bone co-expression network using Weighted Gene Co-expression Network Analysis (WGCNA) and gene expression microarray profiles from femur samples collected from 96 Hybrid Mouse Diversity Panel (HMDP) strains. The resulting network was used to identify a core module of genes (module 9; M9) specific to cells of the osteoblast-lineage. We then demonstrated that the top two M9 hub genes were regulators of osteoblast function and one was a regulator of BMD in vivo. In addition, we showed that a local eQTL regulating the expression of secreted frizzled-related protein 1 (Sfrp1), orchestrated the transcriptional behavior of the M9. Notably, Sfrp1 is an antagonist of Wnt signaling, which is a major pro-osteoblastic signal, and Sfrp1 transgenic and knockout mice display alterations in BMD and osteoblast functions [25], [26]. We further demonstrated the physiologically relevant nature of the M9 by identifying a strong non-linear relationship between the M9 and BMD in the HMDP and that the M9 is enriched for genes implicated in the regulation of human BMD through genome-wide association studies. In summary, our results begin to clarify the composition and role of cellular networks in the osteoblast cell lineage. A genome-wide co-expression network for bone was constructed by applying WGCNA to microarray gene expression profiles from femur samples collected on 96 HMDP strains [27], [28]. The network was generated using all 45,719 expression probes (representing 30,264 unique genes) present on the Illumina Mouse WG6 microarrays. Of the total, 13,759 probes (10,968 unique genes) were assigned to one of 21 co-expression modules (Table 1). All other probes were not assigned to a module. Of the 21 modules, all but module 15 was enriched for genes belonging to similar genome ontology (GO) or Kyoto Encyclopedia of Genes and Genomes (KEGG) functional groupings (Table 1). Lists of module assignments for all genes and all significant (FDR≤0. 05) functional enrichments are provided in File S1 and File S2. Given that bone is a heterogeneous tissue, we expected that a subset of modules from the network would represent cell-type specific networks. Therefore, we next set out to identify the co-expression module that was the most relevant for the function of osteoblast-lineage cells. We calculated two metrics, the Gene Significance (GS) and Module Significance (MS) scores. The GS for each network gene was defined as the absolute value of the correlation between its expression and the eigengene summarizing the expression of a group of nine (Col1a1, Col1a2, Akp2, Bglap1, Sp7, Ibsp, Sost, Mmp13, Tnfrsf11b, Dmp1 and Phex) osteoblast/osteocyte markers. These genes were selected prior to the network analysis based on mining the literature for widely used markers of osteoblasts and osteocytes. The MS was defined as the mean GS for each module. In bone, the above marker genes are preferentially (or exclusively) expressed in cells of the osteoblast-lineage; therefore, a module with a high MS would be expected to represent a sub-network of genes that function specifically in these cells. Of the 21 modules, module 9 (M9) had the highest MS (MS = 0. 62±0. 02; P<0. 002). The next highest MS scores were observed for module 6 (MS = 0. 32±0. 02; P<0. 002) and module 16 (MS = 0. 27±0. 01; P<0. 002) (Figure 1A). All other modules had MS scores at or below 0. 20 (P>0. 002). We also expected that the most relevant module would contain genes more highly expressed in osteoblasts than other bone cells. To evaluate the expression patterns of all network genes we utilized an independent set of microarray data which surveyed global gene expression in primary osteoblasts (at 5,14 and 21 days of in vitro differentiation), primary osteoclasts, bone marrow and whole bone [29]. M9 genes were over 4-fold (P<0. 05) more highly expressed in osteoblasts than whole bone or bone marrow and 2. 8-fold (P<0. 05) more highly expressed in osteoblasts than osteoclasts (Figure 1B–1D). The M9 was significantly (FDR≤0. 05) enriched for 53 GO and KEGG pathway terms (File S1). These included terms such as “extracellular matrix” (FDR = 1. 9×10−16), “collagen” (FDR = 4. 9×10−7), “ossification” (FDR = 3. 0×10−5), “bone development” (FDR = 8. 6×10−5), “skeletal system development” (FDR = 4. 9×10−4), “Wnt receptor signaling pathway” (FDR = 1. 2×10−3) and “regulation of bone mineralization” (FDR = 3. 7×10−2), which are relevant to osteoblasts. Additionally, we found that the M9 was enriched (Fisher' s exact test P = 2. 8×10−8) in genes belonging to a list of 254 that were members of 11 GO terms containing the term “osteoblast” (such as “regulation of osteoblast differentiation” (GO: 0045667) and “osteoblast proliferation” (GO: 0033687) ) or their perturbation in a mouse model has been observed to affect osteoblast function. Lastly, the M9 contained many known genes that, in bone, are specific to or define the osteoblast-lineage. Examples include Akp2 (alkaline phosphatase), Bglap1 (bone gamma carboxyglutamate protein; osteoclacin), Cd276 (CD276 molecule), Col1a1 (collagen, type I, alpha 1), Col1a2 (collagen, type I, alpha 2), Lrp5 (low density lipoprotein receptor-related protein 5), Mmp2 (matrix metallopeptidase 2), Pthr1 (parathyroid hormone receptor 1), Sp7 (Sp7 transcription factor 7), Tnfrsf11b (tumor necrosis factor receptor superfamily, member 11b) and Wnt5a (wingless-related MMTV integration site 5A). Together, these data indicate that M9 represents a core gene network specific to cells of osteoblast-lineage. To characterize the M9 network we first determined if M9 topology was important for the function of osteoblast lineage cells. We began by evaluating connectivity, a parameter of network topology. Recently, a number of studies have shown that highly connected “hub” genes tend to play critical roles in module organization [19], [24]. We defined connectivity (kme) for each gene as the correlation between its expression and its module eigengene [21]. Importantly, kme is a property inherent to each gene and would not be expected to correlate with other individual gene metrics (such as GS), unless the organization of the module was an important property of the system. A strong positive correlation was observed between M9 gene kme and GS (r = 0. 89, P = 7. 7×10−141) (Figure 2), indicating that the more highly connected an M9 gene, the more closely its expression resembles that of a prototypical gene of the osteoblast lineage. The immediate implication of this finding is that we could use kme to determine if M9 hubs play a role in osteoblast function. The top 10 M9 hub genes are listed in Table 2. We focused on the two genes with the highest kme (Table 2), melanoma antigen, family D, 1 (Maged1) and par-6 partitioning defective 6 homolog gamma (C. elegans) (Pard6g). Maged1 is a transcriptional co-activator that has been implicated in the regulation of myogenic differentiation [30], sexual behavior [31], obesity [31] and the transcriptional function of Dlx5 [32], a positive regulator of osteoblast differentiation and bone mass [33], [34]. Pard6g is a homologue of the Par (partitioning defective) family of proteins that are involved in the regulation of cell polarity. We characterized the broad expression profiles of Maged1 and Pard6g using microarray data from 96 mouse tissues and cell-types [29]. Maged1 and Pard6g were expressed in multiple samples including primary calvarial osteoblasts (pcOBs) (Figure 3A). To confirm these data, the expression of both genes was measured during differentiation in an independent set of pcOBs. Maged1 and Pard6g were differentially expressed (P = 0. 03 for both genes) as a function of osteoblast differentiation (Figure 3B). Maged1 expression increased rapidly after the induction of differentiation, peaked at day 6 and then decreased through day 20, possibly indicating a more important role for Maged1 in early osteoblastogenesis. In contrast, Pard6g expression increased more slowly, peaking at day 14 and then decreasing by day 20. The expression of Pard6g was highly similar to established markers of osteoblast maturation (Figure 3C–3F), especially Sp7, Akp2 and Ibsp. We next determined the effects of Maged1 and Pard6g knockdown on osteoblast proliferation and differentiation. Two independent siRNAs (M1 and M2 for Maged1 and P1 and P2 for Pard6g) were used to target each gene. At 48 hours post-transfection in undifferentiated pcOBs, Maged1 transcript levels were reduced to 18% and 14% of control in M1 and M2 transfected cells, respectively (P<0. 05) (Figure 4A). At 96 hours post-transfection in these cells, Maged1 knockdown was lower at 42% and 33% of control in M1 and M2 transfected cells, respectively (P<0. 05) (Figure 4A). Knockdown at the transcript level resulted in similar reductions in MAGED1 protein levels 48 hours after transfection (Figure 4B). Maged1 knockdown resulted in a 12% (P<0. 10) and 31% (P<0. 05) increase in proliferation rate in M1 and M2 transfected undifferentiated pcOBs, respectively (Figure 4C). In differentiating pcOBs (4 days), M1 and M2 treatment led to 40% (P<0. 10) and 118% (P<0. 05) increases in alkaline phosphatase activity (a marker of maturing osteoblasts), respectively (Figure 4D). We also observed 69% and 74% increases in the transcript levels of the osteoblastic genes Sp7 and Akp2, respectively, in M2 treated cells (P<0. 05) (Figure 4E). No differences were observed for the other osteoblast markers assayed (Figure 4E). Surprisingly, we observed a significant (P<0. 05) decrease in the ability of M1 and M2 transfected osteoblasts to form mineralized nodules (a marker of mature osteoblast function) at 14 days post-differentiation (Figure 4F–4H). All the differences demonstrated a dose-dependent relationship with the extent of Maged1 knockdown a the transcript level (the effect of M2>M1), suggesting that the effects were specific for the reduction in Maged1 levels. At 48 hours post-transfection in undifferentiated pcOBs, Pard6g transcript levels were reduced to 50% and 33% of control in P1 and P2 transfected cells, respectively (P<0. 05) (Figure 4I). At 96 hours post-transfection, Pard6g knockdown was lower at 74% and 63% of control in P1 and P2 transfected cells, respectively (P<0. 05) (Figure 4I). Knockdown at the transcript level resulted in similar reductions in PARD6G protein levels 48 hours after transfection (Figure 4J). Pard6g knockdown resulted in a 12% (P = 0. 10) and 62% (P<0. 05) decrease in proliferation rate in P1 and P2 transfected cells, respectively (Figure 4K). In differentiating pcOBs (4 days), P1 and P2 treatment led to 66% and 71% decreases (P<0. 05) in alkaline phosphatase activity, respectively (Figure 4L). Additionally, 40% to 95% decreases (P<0. 05) were seen in the levels of the osteoblast markers Sp7, Runx2, Akp2, Col1a1, Bglap1 and Ibsp (Figure 4M). Consistent with these observations, there was a significant (P<0. 05) impairment in the formation of mineralized nodules in P1 and P2 transfected cells (Figure 4N–4P). All the differences demonstrated a dose-dependent relationship with the extent of Pard6g knockdown at the transcript level (the effect of P2>P1), suggesting that the effects were specific for the reduction in Pard6g levels. These data indicate that the M9 hub genes, Maged1 and Pard6g, are novel regulators of osteoblast activity. To further assess the physiological function of Maged1, we measured total body BMD in Maged1 deficient mice (Maged1−). In 18 week-old male Maged1− mice, we observed significantly (P<0. 05) decreased BMD, relative to wild-type littermates (Maged1+) (Figure 5A). This difference was primarily due to a decrease in bone mineral content (BMC) and not skeletal size (Figure 5B and 5C). Additionally, the difference was not a reflection of alterations in lean body mass that would alter BMD (Figure 5D). We next sought to identify genetic loci responsible for the coordinate expression of M9 genes. Two strategies were employed for this analysis, the identification of expression QTL (eQTL) hotspots and genome-wide association (GWA). Based on previous studies [35], we anticipated that the identification of eQTL hotspots would have more statistical power than genome-wide association, but less mapping resolution. In contrast, genome-wide association would be less powerful, but would allow us to more precisely define the location of potential regulators. We choose a priori to focus only on regions that were implicated by both analyses, as these were the most likely to harbor true regulators. We used the Efficient Mixed Model Algorithm (EMMA) [36] to perform GWA for all network genes. The number of M9 genes with eQTLs (−logP>4) in 5 Mbp bins across the genome was counted and compared to the frequency of eQTL for all other network genes. Several significant (Bonferroni corrected P<9. 4×10−5) bins were identified, suggesting that the regulation of M9 gene expression was polygenic. The most prominent hotspots were located on Chrs. 8 (P = 6. 1×10−32) and 3 (P = 5. 1×10−19) (Figure 6A). We next used EMMA to directly identify associations for the M9 eigengene. In both cases, the top two hotspots/associations were concordant. The SNP (rs33030926, P = 9. 0×10−6) that was the most strongly associated with the M9 eigengene was located on Chr. 8 at 24. 587852 Mbp. (Figure 6B). Both analyses provided strong evidence for the presence of a regulator of the M9 on Chr. 8 at ∼24. 5 Mbp. It is possible that such a regulator influences M9 gene expression through a genetically regulated difference in its own expression and this would be detectable as a local eQTL. To determine if this was the case we identified all microarray probes mapping between 20 and 30 Mbp on Chr. 8. A total of 237 probes corresponding to 137 unique genes were located within the region. EMMA was used to perform genome-wide association for each probe [36]. We then selected the probe for each gene with the most significant local eQTL. A total of 15 genes were found to be regulated by significant (P≤3. 6×10−4) local eQTL after correcting for multiple comparisons (Table 3). We would expect that the causal genes expression should be correlated with M9 gene expression. Thus, we calculated the proportion of M9 genes whose expression was correlated (r>|0. 25|, nominal P<0. 01) with the expression of each candidate regulator. Most of the 15 candidates showed little overlap (between 0 and 24%). However, the expression of Sfrp1 correlated with 88. 8% of M9 genes at a threshold of r>|0. 25| (Table 3). Sfrp1 was correlated with all 400 M9 probes if the threshold was reduced to r>|0. 15|. The most significant SNP regulating Sfrp1 expression was rs33030926 (P = 5. 0×10−11) located at 24. 587852 Mbp. This SNP was also the most significantly associated with the M9 eigengene (P = 9. 0×10−6). Rs33030926 is located 27. 7 Kbp downstream of the 3′ end of Sfrp1. We hypothesized that rs33030926 (or the causal variant linked to rs33030926) regulates Sfrp1 expression, which in turn influences the co-expression of M9 genes. To test this hypothesis, we used the Network Edge Orienting (NEO) causality modeling R package [37]. NEO is statistical approach used to determine the relationship between genetic variation and two traits. In our case we wanted to determine if rs33030926 affected Sfrp1 expression and if this in turn perturb the M9. NEO was used to orient the relationships between rs33030926, each of the 15 candidate regulators and the M9 eigengene by determining if the data best fit a causal model (rs33030926→candidate eQTL→M9 eigengene) or one of four competing models that could be used to explain the data. The causal model was the best fit for the Sfrp1 expression data with a causal score (LEO. nb. AtoB) of 2. 80 (Table 3). The LEO. nb. AtoB scores for the other 14 candidate eQTLs were negative (Table 3). To further characterize the effect of Sfrp1 on the bone network we stratified mice by rs33030926 genotype and calculated the percent difference in transcript level in Sfrp1 and all M9 probes. For Sfrp1, there was a 10. 1% increase in transcript levels in strains (N = 52) homozygous for the rs33030926 “C” allele, relative to strains (N = 44) homozygous for the rs33030926 “T” allele. Similarly, its effect on M9 gene expression was subtle. The max percent difference in expression between rs33030926 genotypes for M9 probes was 27. 2% with a mean of 7. 4% (Table 4). We conclude that Sfrp1 induces strong correlations between M9 genes through the subtle coordinate regulation of their expression. To add additional support for the causal role of Sfrp1, RNAi was used to knockdown Sfrp1 expression in pcOBs. At four days post differentiation, the expression of Sfrp1 was measured using qPCR and network-wide gene expression using microarrays. In cells transfected with a siRNA targeting Sfrp1, its expression was reduced to 44% (P = 0. 001) of the level seen in cells transfected with the scrambled control. Similar to the in vivo data in the HMDP, the knockdown of Sfrp1 early in differentiation (four days post-differentiation) exerted only minor perturbations in network gene expression. Only a small number of the network genes were classified as differentially expressed (FDR<0. 05). However, significant (P<0. 002) mean differences in expression (difference in expression between pcOBs transfected with Sfrp1 siRNA and the scrambled siRNA) were observed for modules 9 and 21. In addition, the M9 was the only module with a significant mean percent difference in module gene expression in which there was a significant correlation (r = 0. 32, P = 4. 8×10−8) between the mean percent difference in the HMDP and in response to Sfrp1 knockdown, indicating that the same M9 genes that were perturbed in the HMDP were also altered due to Sfrp1 knockdown (Table 4). Our in vitro experiments are consistent with Sfrp1 regulating the coordinate expression of the M9. Together, with the systems genetics analysis from the HMDP, these data identify Sfrp1 as a regulator of the M9. If M9 behavior in the HMDP is reflective of osteoblast/osteocyte activity then we would expect that it would be associated with changes in bone mass in the HMDP strains. We previously measured BMD in all HMDP strains [27]. To assess its relationship with BMD, we determined the correlation between the M9 eigengene and BMD. The M9 eigengene was not linearly correlated (r = −0. 03; P = 0. 71) with femoral BMD, however, as shown in Figure 7 there was a U-shaped relationship between the two. Based on this observation we fit a quadratic model (M9 eigengene = BMD+BMD∧2) to the data (Figure 7). The quadratic model was a highly significant fit (P = 1. 1×10−6). A shown in Figure 7, strains with the highest M9 had either low or high BMD. As would be expected based on the observation that rs33030926 regulates Sfrp1 and the M9 eigengene, all the strains with high expression of the M9 eigengene and the lowest and highest BMD were ‘TT’ homozygotes (Figure 7). Similar patterns were observed for total body and spinal BMD (data not shown). Importantly, these data provide additional evidence of the biological relevance of the M9. It also suggests that the M9 reflects the complex, and often contradictory, role of osteoblast-lineage cells in bone homeostasis. To evaluate the potential relevance of the M9 to BMD in humans we determined if it contained genes that have been implicated in the regulation of BMD through human GWA studies. We used information from the largest GWA analysis for BMD performed to date. This study meta-analyzed data from 17 BMD GWA studies (N = ∼32 K in the discovery phase and N = ∼50 K in the replication phase) [38]. In this meta-analysis, a total of 64 independent SNPs reached genome-wide significance implicating 56 regions and 61 unique genes (these genes were the closest to the most significant independent GWA SNPs). We were able to identify a mouse homolog for 57 of the 61 genes (93%) and 39 were located within one of the 21 network modules (Table 5). Of these, five (8. 7% of the total) (Lrp5, Tnfrsf11b, Wnt4, Gpr177 and Sp7) were members of the M9. The probability of identifying five M9 genes among 57 randomly chosen genes from the network was P = 5. 0×10−4. After a Bonferroni correction for the 21 modules, the M9 was the only module to demonstrate this enrichment. These data indicate the M9 is enriched for genes that have been implicated in the regulation of BMD in humans. In this study, we generated a co-expression network for bone that consisted of 21 “modules”, each of which contained genes that shared similar expression patterns and were enriched for functionally similar genes. We then focused on one module, the M9, which was predicted to be specific for cells of the osteoblast-lineage. We demonstrated that the perturbation of M9 hub genes altered osteoblast proliferation and differentiation and for one hub, Maged1, BMD in vivo. Additionally, we discovered that an Sfrp1 local eQTL was the key driver of M9 gene expression, that the M9 was associated with BMD in the HMDP and was enriched for the homologs of genes implicated in the regulation of BMD through human GWA studies. Traditional genetic and molecular approaches are powerful tools for dissecting cellular function, however, reductionist techniques may not be able to capture the overall organization of cellular interactions. Systems genetics is an approach that can provide an unbiased and more comprehensive view of not only the genes involved in cell function, but also key gene-gene interactions. By using the hundreds of thousands of genetic perturbations that exist in the HMDP to identify correlational patterns between genes on a genome-wide scale we were able to discover a core group of 354 genes that are highly co-expressed and function together in a network. We believe that this network acts to propagate or modulate major osteoblastic stimuli, such as Wnt signaling. Importantly, the M9 represents a wealth of information that can be mined in future experiments to increase our understanding of the genes and interactions that are critical for proper osteoblast-lineage function. The use of network analysis provided a number of unique advantages. First, WGCNA gave us the opportunity to group genes into modules based on their in vivo patterns of expression in whole bone and then determine which module was the most relevant to cells of the osteoblast-lineage. Second, in a traditional differential expression analysis across strains, only a small percentage of M9 genes would have been identified as differentially expressed and thus, potentially important in bone. Third, the discovery that M9 connectivity was highly correlated with GS could have only been made via network analysis. Lastly, integrating network analysis and GWA identified Sfrp1 as a regulator of the M9. Although Sfrp1 is known to play an important role in the osteoblast lineage, our results have identified an entire network of genes that are novel downstream targets of Sfrp1. A number of recent works have identified “module quantitative trait loci (mQTL) ” (as examples [35], [39], [40]). Here, we identified Sfrp1 as the gene and its local eQTL as the mechanism underlying the mQTL regulating the M9 eigengene. This represents one of the first successful attempts at identifying the molecular basis of an mQTL. This was possible due to the ability to perform high-resolution genome-wide association in the HMDP and the tools of systems genetics. This study highlights the advantages of disentangling the genetics of co-expression module regulation using a high-resolution genetic reference population such as the HMDP. We observed that the M9 eigengene was inversely correlated with BMD in low bone mass mice and positively correlated with BMD in high bone mass mice. This nonlinear association is likely due the complex roles of osteoblast-lineage cells in bone [1]. Osteoblasts directly control bone formation and secrete Osteoprotegerin, a strong inhibitor of bone-resorbing osteoclasts [1]. Moreover, pre-osteoblasts and recently osteocytes, have been shown to secrete RANKL, which promotes osteoclastogenesis and bone resorption [8], [9], [41]. Therefore, M9 “activity” likely represents a balance between osteoblast-mediated bone formation and osteoblast/osteocyte-directed bone resorption. It is possible that the differential effect of high M9 activity in the HMDP is due to differences cell composition (e. g. differences in the relative numbers of osteoprogenitors, mature osteoblasts and osteocytes) or other factors that are determined by genetic background. Consistent with the role of genetic background, many of the low BMD HMDP strains with high M9 eigengene expression belonged to the AXB recombinant inbred set. More detailed phenotyping of strains with high M9 expression and low or high BMD will be needed to clarify the difference. At any rate, the association between M9 and BMD indicates that it reflects physiologically relevant differences in the activities of osteoblast-lineage cells. We identified a strong correlation between M9 connectivity (kme) and GS. This finding is important since it suggests that not only are M9 genes important, but the topology of the M9 network is also important for the function of osteoblast-lineage cells. This finding allowed us to use kme information to prioritize genes for validation. Because the most highly connected genes were the most correlated with GS, we choose the top two hubs for further investigation. Many of the top ten hubs are known to function in osteoblasts (Table 2); however, the top two, Maged1 and Pard6g, have not been shown to directly participate in osteoblast proliferation, differentiation or mineralization. Using RNA interference we demonstrated that both genes play a role in osteoblast activity. The siRNA knockdown of Maged1 increased the proliferation of primary calvarial osteoblasts. It also increased the early expression of alkaline phosphatase, a marker of maturing osteoblasts. Surprisingly though, we found that it decreased mineralized nodule formation. Maged1 is a transcriptional co-activator involved in a wide-array of cellular processes such as the regulation of myogenic differentiation, circadian rhythms, sexual behavior and obesity, to name a few [30], [31], [42]. Maged1 has been shown to bind to the homeodomain protein DLX5 and is required for its transcriptional function [32]. Consistent with Maged1 affecting osteoblast function through DLX5, mineralized nodule formation in osteoblasts from Dlx5−/− knockout mice is also lower. However, proliferation in Dlx5−/− osteoblasts is also decreased in contrast to the increase in proliferation we observed when Maged1 is knocked-down. The effect of Maged1 on proliferation in osteoblasts, however, is consistent with the observations that it inhibits proliferation in other cell-types [43], [44]. This suggests the Maged1 may have effects on osteoblast function independent of DLX5 activity. The increase in alkaline phosphatase and Sp7 expression at 4 days post-differentiation (both markers of osteoblast differentiation) is hard to reconcile with the decreased mineralized nodule formation at 14 days post-differentiation. It is worth noting that Sp7 and Akp2 were the only osteoblast markers that were increased, which suggests that Maged1 knockdown may selectively result in increased Sp7 and Akp2 expression without inducing the complete differentiation cascade. However, as suggested above it could also reflect diverse roles for Maged1 in the osteoblast. An alternative explanation is that the conflicting early increase and late decrease in osteoblast differentiation is a result of the transient nature of Maged1 knockdown with siRNA. Most importantly, however, we demonstrate that Maged1 deficiency in vivo results in decreased BMD. This is consistent with decreased mineralized nodule formation in vitro. It is also consistent with Maged1 mediating its effects on osteoblast function through DLX5, as Dlx5 deficient mice also have decreased bone mass [33]. Further work is needed to define the precise role of Maged1 in osteoblasts and how this translates into lower bone mass. The Par6 (partition defective) family of proteins was first identified in C. elegans and Drosophila as proteins required to establish cell polarity [45]. There are three homologues of Par6 in mammals, PARD6A, 6B and 6G [46]. The siRNA knockdown of Pard6g decreased both osteoblast proliferation and differentiation. It is possible that these effects of Pard6g are due strictly to the fact that cell polarity is an essential cellular process and are not necessarily reflective of Pard6g function in osteoblasts. However, we feel this is unlikely given its membership in the M9 and the fact that its expression is high in osteoblasts and its expression differs as a function of osteoblast differentiation. In addition, Par6 has recently been shown to be involved in skeletogenesis and biomineralization in the sea urchin [47]. Although more work is needed it is tempting to speculate that Pard6g is involved in Wnt signaling. Non-canonical Wnt signaling is a major regulator of cell polarity and the M9 contains non-canonical Wnts such as Wnt4 (also a gene associated with BMD in humans) [48]. The Wnt signaling pathway is a major pro-osteoblast stimulus [10]. In osteoblasts, Wnts bind frizzled (Fzd) receptors and their co-receptors (LRP5 and LRP6) and induce the stabilization and translocation of ß-catenin to the nucleus [10]. Sfrp1 antagonizes Wnt signaling by interfering with the interaction between Wnts and Fzd receptors [49]. Sfrp1 knockout mice are resistant to age-related decreases in trabecular bone mass and display reduced osteoblast/osteocyte apoptosis and increased osteoblast proliferation and differentiation [50]. Conversely, Sfrp1 transgenic mice have decreased trabecular and cortical BMD and decreased osteoblast proliferation and differentiation [25]. Using genome-wide association we identified Sfrp1 as a regulator of M9. Based on its known role in cells of the osteoblast-lineage, its discovery as a major regulator of the M9 is consistent with M9 representing a core network operative in osteoblasts/osteocytes. There has been interest in developing therapeutics targeting Wnt signaling in general and Sfrp1 specifically. In fact, Bodine et al. demonstrated that piperidinyl diphenylsulfone sulfonamide could bind and inhibit the activity of SFRP1 [51], [52]. However, there is concern that Sfrp1 is a poor drug target based on its broad tissue expression and the link between Wnt signaling and various cancers [26]. Given that Sfrp1 regulates M9 gene expression it is likely that many M9 genes are downstream targets of Sfrp1 and more generally Wnt signaling. This notion is supported by the observation that the perturbation of Maged1 and Pard6g expression resulted in similar effects on pcOBs as did Sfrp1 alteration [25]. Targeting M9 genes could promote increased bone formation in a more bone-specific manner. Recent studies have demonstrated that co-expression modules can be conserved across species; therefore, it is of significant interest to know if the human homologs of M9 genes function in a similar network. We will directly investigate this in future studies, however, the fact that conserved pathways (primarily Wnt signaling) are represented in the M9 suggests that the majority of M9 genes will also play a role in the function of osteoblast-lineage cells in humans. Additionally, the fact that nearly 10% of genes implicated in the most comprehensive BMD GWA meta-analysis performed to date are members of the M9 further support the notion that the M9 is relevant to the regulation of human BMD. It is worth noting that 4 of the 5 M9 human BMD genes are involved directly in Wnt signaling, which again suggests that this signaling pathway is a major component of the M9. Our approach is not without limitations. One possible limitation, which also may have been an advantage, was the generation of expression data from bone tissue. An alternative approach would have been to profile isolated cell populations. This may have been more informative since it is clear that the different cells in the osteoblast-lineage perform distinct and often contradictory functions. On the other hand, the profiling of bone cells in their native complex cellular milieu may have resulted in expression profiles that were more representative of their true in vivo state. At any rate, we do believe that it will be informative in future studies to repeat this analysis using isolated cell populations as this may remove some of the noise associated with averaging expression across multiple cell-types. A second limitation was our use of siRNA in primary calvarial osteoblasts to validate the role of Sfrp1, Maged1 and Pard6g. In terms of Sfrp1, we perturbed its expression in vitro at one time point in osteoblasts isolated from neonatal mice. Although this system allowed us to test the hypothesis that Sfrp1 preferentially modulated the expression of M9 genes, it did not fully recapitulate the effects of Sfrp1 expression differences in the HMDP. It is known that Sfrp1 has many effects on osteoprogenitors, mature osteoblasts and osteocytes [50]. Thus, it would have been more ideal to test the effect of Sfrp1 perturbation in vivo, which will be the focus of future investigations. The same is true for Maged1 and Pard6g. Although we clearly demonstrate their involvement in osteoblast proliferation and differentiation, it is possible that we missed many aspects of their function by focusing on just two stages in the complicated life-cycle of an osteoblast-lineage cell. This is especially true in the case of Maged1 were we observed that its reduction increased certain aspects of early osteoblast differentiation, but later decreased mineralized nodule formation. In summary, we have used a systems genetics approach consisting of co-expression network analysis, eQTL analysis, genome-wide association and causality modeling in a powerful mouse genetic reference population to identify a module (M9) of co-expressed genes that play an important role in the function of osteoblast-lineage cells. These data improve our understanding of the gene networks important for osteoblast function and demonstrates the ability of systems genetics to unravel gene networks involved in complex cellular processes. The Institutional Care and Use Committee (IACUC) at the University of California, Los Angeles approved the animal protocol for the HMDP. The animal protocol for the isolation of primary calvarial osteoblasts was approved by the University of Virginia IACUC. The manipulations of Maged1-deficient mice has been approved by the local ethics committee of the University of Namur and follow the European legislation. Data from Hybrid Mouse Diversity Panel (HMDP) were generated from 16-week old male mice from 96 inbred strains. More details regarding the population can be found in [27], [28]. RNA isolation and Illumina microarray processing for bone tissue samples from the HMDP are described in [26]. The expression data are available from the NCBI Gene Expression Omnibus (GEO) database (GSE27483). The quantification of femoral, spinal and total BMD in the HMDP has been described in [27]. The Maged1-deficient mice and control littermates used in this study have already been described [53]. Experiments were done with male mice aged 18 weeks backcrossed for >10 generations in the C57Bl/6J genetic background. Body composition and BMD was measured using a dual-energy X-ray absorptiometry (DEXA) scanner (Lunar PIXImus2; GE Healthcare). All scans were analyzed using the PIXImus2 software (version 2. 10). For the calculation of total body BMD the skull was excluded from the analysis. Network analysis was performed using the WGCNA R package [54]. All 45,759 array probes were used to construct the bone network. We did not collapse multiple probes per genes down to a single probe representing each gene since many of the seemingly redundant probes actually recognize alternatively spliced isoforms. We also did not have to worry that the inclusion of probes that were not expressed would add noise, since the vast majority of such probes would not be expected to exhibit biologically meaningful correlations with a large number of other transcripts. The approach also allowed for the inclusion of probes that are truly expressed, but at a level that may not have exceeded a particular “expressed/not expressed” threshold. To generate the co-expression network, we first calculated Pearson correlation coefficients for all gene-gene comparisons across all microarray samples. The matrix of correlations was then converted to an adjacency matrix of connection strengths. The adjacencies were defined as where and are the and gene expression traits. The power was selected using the scale-free topology criterion previously outlined by Zhang and Horvath [55]. In this study a = 8 was used. Modules were defined as sets of genes with high topological overlap [21]. The topological overlap measure (TOM) between the and gene expression traits was taken as, where denotes the number of nodes to which both and are connected, and indexes the nodes of the network. A TOM-based dissimilarity measure was used for hierarchical clustering. Gene modules corresponded to the branches of the resulting dendrogram and were precisely defined using the “Dynamic Hybrid” branch cutting algorithm [56]. A principal component analysis was used to generate a vector of values (first principal component) that summarized or were the most representative of each modules expression. Intramodular connectivity (kme) was defined as the correlation between a gene' s expression and its module eigengene. Highly similar modules were identified by clustering and merged together. Network depictions were constructed using Cytoscape [57]. The gene content of each module was characterized using the DAVID gene enrichment analysis tool [58], [59]. Gene Significance (GS) for each network gene was defined as the absolute value of its correlation with the eigengene of a set of nine osteoblast/osteocyte marker genes identified from the literature. Module Significance (MS) was calculated as the mean GS for each module. Significance of the MS was determined for each module by randomly selecting x GS scores from the set of 13,579 network GS scores; where x is equal to the number of genes in that module. The mean for each set was then calculated and this was repeated 10,000 times. The true MS score was then compared to the distribution of random mean GS scores and P-values were calculated by counting the number of random mean GS scores that were greater than the true MS score divided by 10,000. An MS score with a P<0. 002 (Bonferroni adjusted for the 21 modules tested) was deemed significant. To determine the expression patterns of network genes in bone and bone cells microarray data on primary osteoclasts, primary osteoblasts, whole bone and bone marrow were downloaded from GEO (GSE11339 and GSE10246). The samples were derived from C57BL6/J mice in triplicate. The osteoblast samples were comprised of three different time-points (5,14 and 21 days of differentiation) assayed in triplicate. For each module the log2 fold expression of its genes in osteoblasts (highest of the three time-points) were compared to the other samples. Statistical significance of the increase in expression in osteoblasts was determined as described above for GS and MS. EMMA and its application to HMDP data has been described in [27], [28], [36], [60]. EQTL hotspots for the M9 were identified by performing genome-wide association for the expression of all 13,759 network probes using EMMA. SNPs were clustered into 531,5-Mbp bins across the genome and for each network probe, the minimum association p-value was recorded for each bin and the number of probes with p-values that exceeded −logP≥4 were counted. Enrichment P-values were then assigned to each bin using a Fisher' s exact test to compare the frequency of significant associations for M9 probes relative to all other network probes. Bins with P<0. 05/531 = 9. 4×10−5 were deemed significant. Causality modeling was performed as described in [61], [62]. Briefly, NEO is an R function designed to orient the relationships between genetic markers, gene expression traits and clinical traits [37]. NEO utilizes the fact that all cellular information begins with DNA and therefore, the many possible relationships that can exist between DNA variation, gene expression and clinical traits can be distilled to three. The three relationships (or models) are: 1) causal – flow of information goes from DNA to gene to BMD (gene' s expression is causing the change in the trait); 2) reactive – flow of information goes from DNA to BMD to gene (gene' s expression is reacting to the change in the trait) and 3) independent – DNA variation affects both traits independently. NEO uses structural equation modeling to estimate the probabilities for each of the three relationships. The log10 ratio of the causal model probability relative to the next best model probability (of the two remaining) is then calculated. This ratio (referred to as the LEO next best or LEO. NB score) quantifies the relative likelihood that a gene' s expression is causal for a trait such as BMD. Simulation studies have demonstrated that single marker LEO. NB scores above 1. 0 are highly suggestive of causal relationships [37]. The mouse homologs for human genes nearest the most significant SNP for all genome-wide significant associations identified in [38] were identified. A clear homolog was identified for 57 associations. The module membership for each of the 57 genes was then determined. Enrichment p-values for each module were calucated by randomly selecting×genes, where x is the number of genes in a given module, out of the 30,264 unique genes used to generate the network. This was repeated 10,000 times. The number of randomly selected genes that overlapped the GWA set in each random selection was then recorded. The enrichment P-value was calculated as the number of times (out of 10,000) the overlap equaled or exceeded the actual number observed for each module. An enrichment P-value corrected for the 21 modules (0. 05/21 = 0. 0023) was deemed significant. Three to nine day old neonates from C57BL/6J breeding pairs (obtained from Jackson Laboratory, Bar Harbor, ME) were euthanized by CO2 inhalation followed by decapitation. The heads were sprayed with 70% ethanol and placed in sterile cold DPBS (Gibco). Using sterile instruments, the skin was removed from the skull, calvariae were removed and placed into sterile cold DPBS (Gibco). Harvested calvariae were then placed in sterile digestion solution (0. 05% Trypsin-EDTA, 1. 5 U/ml Collagenase P, MEM Alpha) and incubated at 37°C, with 120 rpm shaking for 15 minutes. Four digests were performed, the first being discarded. For the remaining three digests an equal volume of sterile plating media (DMEM, 10% heat-inactivated FBS, 100 U/ml penicillin, 100 ug/ml streptomycin) was added to each immediately following its collection and stored on ice. The fractions were combined, filtered through a 100 uM sterile vacuum filtration tube and counted. Cells isolated from 4–6 independent groups of 3–9 day old neonates were plated into 6 well plates at 300,000 cells/2 ml sterile plating media (DMEM, 10% heat-inactivated FBS, 100 U/ml penicillin, 100 ug/ml streptomycin) per well. After 24 hours, confluent cells (Day 0) were washed 1× with DPBS (Gibco) and placed in sterile differentiation media (DMEM, 10% FBS, 100 U/ml penicillin, 100 ug/ml streptomycin, 0. 1 M ascorbic acid, 1 M B-glycerophosphate). Every 48 hours thereafter cells were washed one time with DPBS (Gibco) and differentiation media replaced. PcOBs were plated at 150,000 cells/2 ml plating media per well. Transfections were performed using Lipofectamine 2000 Reagent (Invitrogen) according to manufacturer' s directions. A total of three Stealth Select RNAi siRNAs (Invitrogen) per gene were first tested using three different concentrations (0. 2 nM, 2. 0 nM or 10 nM). Knockdown of the target gene was tested at 48 hours using qPCR in undifferentiated pcOBs (see below). All siRNAs demonstrated the most effective knockdown at 10 nM. The two most effective siRNAs for each gene were used for all downstream experiments with the exception of Sfrp1 and only one of the three provided more than 50% knockdown. The sense strand of the duplex siRNA sequences were as follows: Sfrp1 - MSS277026; CCGAGAUGCUCAAAUGUGACAAGUU; Maged1 – MSS294723; GCAAGGUUAAUAACUUGAAUGUGGA and MSS235163; UCAGAACGUGGAGUCCCGGACUAUA; Pard6g MSS234948; GCAACGGCAGCAUCCACAGAUUUCU and MSS234949; CAUAAGUCUCAGACCCUACGCUUCU. The Stealth RNAi Negative Control Duplex (Invitrogen) was used as a scrambled control. As a control, we also demonstrate in File S3 that knockdown of Kdelr3 (a gene expressed in primary calvarial osteoblasts) has no effect on mineralized nodule formation, providing additional support that the effects of target gene siRNA are due specific to the knockdown of Maged1 and Pard6g (File S3). At 24 hours post-transfection, cells were trypsinized (0. 25% Trypsin-EDTA) and re-plated in a 12 well plate. The following day cells reached 100% confluency (Day 0) and were washed 2× with sterile DPBS (Gibco) and placed in sterile differentiation media (DMEM, 10% FBS, 100 U/ml penicillin, 100 ug/ml streptomycin, 0. 1 M ascorbic acid, 1 M B-glycerophosphate). Every 48 hours thereafter cells were washed 1× with DPBS (Gibco) and differentiation media replaced. Protein was extracted from pcOBs in 10% NP40 detergent containing protease inhibitors (Thermo Scientific). The extracts were separated on 12% NativePAGE™ Novex Bis-Tris Gels (Life Technologies) and transferred to PVDF membranes. Antibodies were obtained from Santa Cruz Biotechnology (PARD6G) and Milipore (MAGED1). Bound antibodies were visualized using the Western Lightning Plus-ECL (Perkin-Elmer). ImageJ (NIH) was used to quantify individual bands by normalizing the density of the target band (MAGED1 or PARD6G) by the density of the ß-ACTIN band for each sample. Total RNA was isolated using RNeasy Mini-Kit (Qiagen) according to manufacturer' s instructions followed by genomic DNA decontamination using DNA-free kit (Applied Biosystems) according to manufacturer' s directions. cDNA was synthesized using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems) according to manufacturer' s instructions using C1000 Thermal Cycler (Bio-Rad). Quantitative real-time PCR (qPCR) was performed on 50 ng cDNA template, 10 uM each forward and reverse primer, and SensiMix Plus SYBR kit (Quantace) according to manufacturer' s directions in 20 ul total volume using an ABI 7900 thermocycler. The following primer sets were used (all sequences 5′-3′): Maged1–F, AGATGGCTCCCAGACTCAGA; Maged1–R, CCTTTGATCCCCACTGTTGT; Pard6g–F, TGACGACAACTTCTGCAAGG; Pard6g–R, GCTCCGAAGCTGTAATGGTC; Sfrp1-F, TACCACGGAAGCCTCTAAGC; Runx2-F, ACAGTCCCAACTTCCTGTGC; Runx2-R, CACAGTCCCATCTGGTACCTC; Sfrp1-R, TCGCTTGCACAGAGATGTTC; Sp7–F, TGCCCCAACTGTCAGGAG; Sp7–R, GATGTGGCGGCTGTGAAT; Akp2–F, CCTTGAAAAATGCCCTGAAA; Akp2–R, TTACTGTGGAGACGCCCATA; Ibsp-F, GAGGAGACTTCAAACGAAGAGG; Ibsp-R, ACACCCGAGAGTGTGGAAAG; Col1a1-F, CCCAAGGAAAAGAAGCACGTC; Col1a1-R, AGGTCAGCTGGATAGCGACATC; Bglap2–F, GAACAGACAAGTCCCACACAGC; Bglap2–R, AGAGACAGAGCGCAGCCAG; 36B4-F, ACTGAGATTCGGGATATGCTGT; 36B4-R, TCCTAGACCAGTGTTCTGAGCTG. Relative quantification was determined by the 2 (−Delta Delta CT) ) method using the 36B4 gene as reference gene [63]. The results were obtained from N = 4 independent experiments. Microarray expression profiles were generated using the MouseWG-8v2 BeadChips (Illumina, San Diego, CA). Briefly, biotin-16-UTP labeled cRNA was synthesized using the Illumina TotalPrep RNA Amplification Kit (Ambion, Austin, TX). A total of 850 ng of cRNA was then hybridized to the Illumina BeadChips. Microarrays were scanned using the Illumina iScan system and background corrected signal intensities were extracted using the GenomeStudio software (Illumina). The lumi R package was used to transform the data using a Variance Stabilizing Transformation (VST) and normalized using quantile normalization [64]. Proliferation was measured in pcOBs by plating cells at a density of 2000 cells/dish in 96 wells, treating as indicated and proliferation rate was determined using the BrdU ELISA assay (Roche). The results were obtained from N = 6 independent experiments. Quantitative analysis of soluble alkaline phosphatase activity in cell extracts was performed using a colorimetric kit (AnaSpec) that measures the conversion of p-nitrophenyl phosphate to p-nitrophenol according to the manufacturer' s instructions. Alkaline phosphatase activity was normalized to protein concentration. Protein levels were determined using using the bicinchoninic acid (BCA) assay according to the manufacturer' s instructions (Pierce). Mineralized nodule formation was measured by staining cultures at 12 days post-differentiation with Alizarin Red (40 mM) (pH 5. 6). The stained cells were imaged and nodule number was measured using ImageJ (NIH). Alizarin Red was quantified by destaining cultures with 10% Acetic Acid and determining the optical density (405 nM) of the resulting solution. The results were obtained from N = 4 independent experiments. | Title: Systems Genetic Analysis of Osteoblast-Lineage Cells Summary: The osteoblast-lineage consists of a range of cells from osteogenic precursors that mature into bone-forming osteoblasts to osteocytes that are entombed in bone. Each cell in the lineage serves a number of distinct and critical roles in the growth and maintenance of the skeleton, as well as many extra-skeletal functions. Over the last decade, many of the major regulatory pathways governing the differentiation and activity of these cells have been discovered. In contrast, little is known regarding the composition or function of gene networks within the lineage. The goal of this study was to increase our understanding of how genes are organized into networks in osteoblasts. Towards this goal, we used microarray gene expression profiles from bone to identify a group of genes that formed a network specific to the osteoblast-lineage. We used the knowledge of this network to identify novel genes that are important for regulating various aspects of osteoblast function. These data improve our understanding of the gene networks operative in cells of the osteoblast-lineage. | 14,528 | 232 | lay_plos | en |
Summarize: AUGUST 28--After their friend apparently died of a drug overdose, two Missouri residents posed for a “selfie” with the corpse, an image that one of them later uploaded to Facebook after dumping the body on a rural road, according to police. As detailed in a probable cause affidavit, Chelsie Berry, 24, told cops that she was driving around with Dennis “Nathan” Meyer last week when he began acting “crazy” after injecting himself with the pain killer Dilaudid. Claiming that she was “nervous” around the unstable Meyer, Berry (seen at right) said that she called one of his friends, Jared Prier, whom she had met the prior evening. Prier, 28, picked up Berry at a McDonald’s and they “hung out in the parking lot for a little bit.” Meyer, Berry said, was “passed out.” After driving to a convenience store, Prier told Berry that “he believed that Nathan had quit breathing.” Berry told cops that she “checked Nathan and did not think he was breathing either.” Berry claimed that she and Prier were “scared to call for an ambulance and did not want to take Nathan to a hospital because she and Jared were high on meth and Xanax and thought they would get in trouble.” So the duo drove to a back road and “dragged Nathan from the front seat to the back seat because they did not want to look at him any longer and also the fact that he began to smell bad.” However, before they moved the body, Berry and Prier posed for a “selfie” with the 30-year-old Meyer, said Newton County Sheriff Ken Copeland. A tipster subsequently alerted investigators to the photo after Berry uploaded it to Facebook, said Copeland, who added that the image showed Berry, Prier, and the “passed out” Meyer in the front seat of Meyer’s Nissan Pathfinder. Berry told police that she and Prier (pictured above) drove around “looking for a place to dump the body.” They eventually settled on a rural driveway and “pushed the body out of the vehicle onto the ground.” An autopsy determined that Meyer’s “breathing would have been faint and it would have taken a while for Nathan to die.” Berry and Prier were charged yesterday with abandonment of a corpse. According to investigators, Meyer and Prier have been linked to the Joplin Honkies street gang. Earlier this month, Meyer posted to Facebook a series of photos showing him posing with Prier. The images were captioned, “Me and my homie prier... I'd ride with him with or without a patch....” (3 pages) Close Get email notifications on Emily Younker daily! Your notification has been saved. There was a problem saving your notification. Whenever Emily Younker posts new content, you'll get an email delivered to your inbox with a link. Email notifications are only sent once a day, and only if there are new matching items. | Summary: When Dennis "Nathan" Meyer apparently overdosed on Dilaudid, a painkiller, while hanging out with Chelsie Berry, 24, and Jared Prier, 28, last week, police say the Missouri pair reacted in perhaps the worst possible way: They allegedly took a selfie with his corpse, dumped the body, and then posted the selfie to Facebook. A tipster told police about the picture, which allegedly shows Berry and Prier with a "passed out" Meyer in the front seat of his car. When police questioned Berry, she said she was hanging out with Meyer when he started acting "crazy" after injecting himself with the painkiller, so she called Prier, one of Meyer's friends, who came to pick her up at a McDonald's. But after Meyer lost consciousness, the pair eventually realized he was no longer breathing. They were too scared to call an ambulance or go to a hospital because they were also high on meth and Xanax, Berry told police, so they took the selfie and then moved Meyer from the front seat to the back seat because he started to smell bad, police say. Then, Berry told cops, she and Prier drove around "looking for a place to dump the body" and, upon finding a rural driveway that apparently seemed suitable, "pushed the body out of the vehicle onto the ground." Police were tipped off about the selfie while investigating Meyer's death, the Joplin Globe reports. Berry and Prier have been charged with abandonment of a corpse, the Smoking Gun reports. | 658 | 341 | multi_news | en |
Summarize: Background Job Corps was established as a national employment and training program in 1964 to mitigate employment barriers faced by severely disadvantaged youths. Job Corps enrolls youths aged 16 to 24 who are economically disadvantaged, in need of additional education or training, and living in disorienting conditions such as a disruptive homelife. Students may enroll in training programs throughout the year and progress at their own pace. Job Corps provides participants with a wide range of services, including basic education, vocational skills training, social skill instruction, counseling, health care, room and board, and recreation. The program offers vocational skills training in areas such as business occupations, automotive repair, construction trades, and health occupations. Participation in Job Corps can lead to placement in a job or enrollment in further training or education. It can also lead to educational achievements such as attaining a high school diploma and skills in reading or mathematics. Job Corps is unique in that, for the most part, it is residential. About 90 percent of the youths enrolled each year live at Job Corps centers and are provided services 24 hours a day, 7 days a week. The premise for boarding participants is that most come from a disruptive environment and, therefore, can benefit from receiving education and training in a different setting in which a variety of support services are available around the clock. Job Corps operates in a very structured and disciplined environment. For example, established daily routines must be followed, as must specific rules and regulations governing such areas as acceptable dress and behavior. Furthermore, Job Corps participants must have permission to leave the Job Corps center grounds, and participants “earn” home leave, which must be approved before being taken and can be denied for a number of reasons such as failure to follow a center’s rules of conduct. Job Corps typically employs residential staff to oversee dormitory living and security staff for the safety and well-being of its participants. The program recently implemented a “zero tolerance” policy for violence and drugs. This policy includes a “one-strike-and-you’re-out” provision for the most serious violent or criminal offenses as well as for drug violations. Job Corps currently operates 109 centers throughout mainland United States, Alaska and Hawaii, the District of Columbia, and Puerto Rico. Most states have at least one center, and several states have four or more centers. Job Corps’ nine regional directors are responsible for the day-to-day administration of the Job Corps program at the centers within their geographic boundaries. Private corporations and nonprofit organizations, selected through competitive procurement, operate the majority of the centers. However, the departments of Agriculture and Interior directly operate 28 centers, called civilian conservation centers, under interagency agreements. The regional directors are also responsible for overseeing the recruitment of youths for program participation and the placement of participants after they leave Job Corps. Recruitment, referred to as outreach and admissions by program managers, and placement services are provided by private contractors, the centers, or state employment service agencies under contract with the regional offices. During program year 1995, Job Corps spent about $60 million on outreach and admissions as well as placement contracts. This included amounts paid contractors solely for outreach and admissions and placement services. In addition, a portion of the funding for some Job Corps center operation contracts was specifically designated for outreach and admissions and placement services. Job Corps contractors are expected to meet certain levels of achievement in order to continue to participate in the program and receive program funding. A performance standard has been established for outreach and admissions contractors with respect to “quotas” of male and female youths to be enrolled (as specified in the contract), and a second standard relates to the proportion of participants who are to remain in the program for more than 30 days (90 percent). A third standard relates to the percentage of participants who are eventually placed following termination from the program (70 percent). Similarly, placement contractors are required to meet established standards related to the percentage of participants placed in jobs, the military, schools, or other training programs (70 percent). Additional standards are applied to participants who are placed in jobs. These standards relate to the percentage obtaining full-time jobs (70 percent) and jobs directly related to the vocational training received (42 percent). A fourth placement standard relates to the average wage received at placement. Individuals enroll in Job Corps by submitting applications through outreach and admissions contractors. The length of time students stay in Job Corps can vary substantially—from 1 day to 2 years. In program year 1995, about 15 percent of the enrollees left Job Corps within 30 days of entering the program and more than one-fourth left within 60 days. On the average, however, students spend about 7 months in the program. Students leave Job Corps for a variety of reasons, including successful completion of the program objectives, voluntary resignation, disciplinary termination, and being absent without leave (AWOL) for 10 consecutive training days. With a few exceptions, participants terminating from Job Corps are assigned to a placement contractor for assistance in finding a job or enrolling in other education or training programs. Placement contractors are to give priority to finding full-time, training-related jobs for participants. Job Corps Eligibility Guidance Is Inadequate We found that Job Corps’ policy guidance on two of its eligibility criteria was ambiguous and incomplete. As a result, the program’s eligibility process was not following all the requirements of the law or program regulations. The law specifies program eligibility requirements, including age, economic status, educational needs, medical condition, and behavioral condition—all defined in the legislation, implementing regulations, or Labor policy guidance. Another legislative requirement—living in an environment characterized by disorienting conditions—has not been clearly defined in the statute, regulations, or Labor’s guidance. Further, Labor has not provided adequate guidance regarding the requirement that participants have the capability and aspirations to complete and secure the full benefits of Job Corps. Contractors are required to follow Labor’s Policy and Requirements Handbook, which sets out 11 eligibility criteria for the program that all participants must satisfy: age, economically disadvantaged, requires additional education or training, environment, health history, behavioral adjustment history, capability and aspirations to participate, legal U.S. resident, child care, parental consent, and Selective Service registration (see app. II). The first seven are specified in the law. The policy handbook generally provides guidance on what is needed to meet most of these criteria. For example, to be eligible under the education or training criterion, an applicant must be a dropout or in need of additional education, training, or related support services in order to hold meaningful employment, participate in regular school work, qualify for other training, or satisfy armed forces requirements. However, guidance on two of the criteria (environment and capability and aspirations) is vague. Environmental Criterion Is Open to Interpretation One of Job Corps’ eligibility criteria specified in the law for participation in the program relates to environment: A participant must come from “an environment so characterized by cultural deprivation, a disruptive homelife, or other disorienting conditions as to substantially impair prospects for successful participation in other programs providing needed training, education, or assistance.” Program regulations go on to explain that the disorienting condition must be one that would impair the applicant’s chance of success in a nonresidential program rather than a residential Job Corps program. Job Corps legislation, Labor’s program regulations, and Job Corps’ policy handbook list environmental factors to be considered when assessing eligibility, but these sources of program guidance are not entirely consistent nor do they contain adequate definitions (see table 1). With the exception of the regulatory definition of disruptive homelife, program guidance does not define the factors that make up the environmental criterion. In the absence of specific definitions of the environmental criterion, admissions counselors applied their own interpretations. As shown in table 1, Labor includes “limited job opportunities” in its policy handbook as a disorienting condition that fulfills the environmental eligibility requirement. However, none of the sources of program guidance specifically defines this factor or gives any direction to assessment counselors to help them interpret it, nor do they explain how limited job opportunities affect the chance of success in a residential program compared to a nonresidential one. In prior Job Corps regulations, Labor included among “disruptive conditions” that could impair an applicant’s prospect to participate fully in nonresidential training “a neighborhood or community characterized by high crime rates, high unemployment rates, high school dropout rates, and similar handicaps.” Unlike the present regulations, the prior version made clear that applicants might be subject to more than one disruptive factor and that several factors in combination might satisfy this impairment criterion. Labor’s present guidance does not explain how “limited job opportunities” by themselves can satisfy this criterion. Nonetheless, limited job opportunities was the factor cited as fulfilling the environmental eligibility requirement for 92 percent of the 68,000 Job Corps enrollees in program year 1995. Because admissions counselors generally indicate only one environmental factor on the Job Corps application form, we have no way of knowing how many of these participants would have met the environmental criterion had limited job opportunities not been used to fulfill the requirement. Further, the admissions counselors we interviewed had varying interpretations of limited job opportunity. Some thought that it referred to the applicants’ lack of job skills or lack of education, whereas others thought that it referred to the economic condition of the geographic areas in which applicants resided or their being too young or lacking transportation. Cultural deprivation, another eligibility factor that could fulfill the environmental criterion, was not clearly defined—in fact, it is not even listed in Labor’s policy handbook—and was also interpreted differently by various admissions counselors. One contractor referred to persons who had never gone to a museum or the beach; another thought it applied to a situation such as raising a minority child in a nonminority family; a third referred to living in a housing project. Most admissions counselors we interviewed admitted that they had no idea what this term meant. Finally, Labor’s policy handbook restricts what can be considered under the environmental criterion, stating that to be eligible an applicant must be living in an environment characterized by disruptive homelife; unsafe, overcrowded dwelling; limited job opportunities; or disruptive community; high crime rates. However, the handbook excludes cultural deprivation—specified in the statute and Labor’s own regulations—from permitted environmental factors. Inadequate Guidance on Capability and Aspirations Criterion The Job Corps law states that to enroll in Job Corps, an applicant must, after careful screening, have the present capability and aspirations to complete and secure the full benefit of the program. However, in determining whether applicants meet this requirement, Labor relied primarily on an evaluation form that assesses behavior that would be expected of any and all applicants. Without more detailed guidance on the use of this criterion, the program may not always be serving those who are most likely to benefit from it. In previous work, we found that ensuring that project participants are committed to training and getting a job is a key feature of successful employment training projects. The law does not define “capabilities and aspirations” but leaves to Labor the tasks of defining this term and providing guidance on how it is to be implemented. Labor has developed the “Capability and Aspirations Assessment Tool,” which admissions counselors must complete for each applicant (see app. III). This “tool” formulates four categories of factors—commitment, attitude, capability, and compatibility of applicant and program goals—that are used to assess capability and aspirations and to demonstrate suitability for the program. Factors under commitment include meeting scheduled appointments on time, providing requested documents such as birth certificates, and reacting favorably to program requirements such as following center rules and living away from home. Attitude includes willingly responding to questions and behaving respectfully during the interview. Capability involves obtaining documentation that supports an applicant’s ability to benefit from the program such as school, court, or medical records or a letter from a former employer. Compatibility of applicant and program goals relates to the admissions counselor’s opinion that an applicant’s expressed goals—for example, for job placement or vocational training—can be realistically achieved through Job Corps. The factors specified in Labor’s assessment tool include characteristics that if not displayed would be an appropriate basis for rejecting an application. However, the possession of these characteristics does not necessarily demonstrate that an applicant has the ability and motivation to benefit from Job Corps. Job Corps outreach and admissions contractors and regional staff whom we spoke with pointed out shortcomings in the current approach to assessing applicants’ capability and aspirations. Staff in one of Labor’s regional offices stated that admissions counselors have asked for additional guidance in making better decisions on capability and aspirations. An admissions contractor with statewide recruiting responsibility in one state said that there is a need for a valid assessment tool for this criterion because the current tool is inadequate. Another contractor stated that it filled out Labor’s assessment tool because it is a program requirement but did not use it in assessing the suitability of applicants. One of Labor’s regional offices has started to develop a more meaningful tool. Recruitment and Selection of Job Corps Participants Could Be Improved A substantial number of Job Corps participants leave the program within a short time after enrollment—about one-fourth of program year 1995 participants left within 2 months. Therefore, we believed that it would be useful to identify ways contractors could target recruitment efforts and the selection of applicants to the eligible youths who are more likely to stay in the program and, thus, more likely to benefit from it. To determine the factors that might be related to program retention, we visited a number of outreach and admissions contractors to examine their practices in assessing and screening applicants for the program. We also analyzed the characteristics of the more than 68,000 program year 1995 participants to determine the characteristics that were associated with remaining in Job Corps for at least 60 days. In our visits, we identified several procedures that distinguished outreach and admissions contractors with higher retention rates from other outreach and admissions contractors. In general, these procedures were aimed at identifying applicants with the commitment and motivation to remain in and benefit from the program. Our statistical analysis provides some information about characteristics significantly related to the likelihood of remaining in the program for at least 60 days that Labor could use to design outreach efforts, establish priorities among applicants, or improve the retention rate for those who might otherwise leave the program early. Contractors With Higher Retention Rates Have Better Assessment Procedures Of the 11 outreach and admissions contractors that we visited, those with higher retention rates (10 percent or fewer of their enrollees dropping out within the first 30 days) tended to have better procedures for identifying applicants with the commitment and motivation to remain in and benefit from the program. That is, these contractors emphasized making sure that applicants met the programs’ statutory eligibility criterion of having the capability and aspirations to complete and secure the full benefit of the program. These more-successful contractors’ procedures included “commitment checks” and preenrollment tours and briefings, which gave applicants a more realistic basis for deciding whether they wanted to enroll. The emphasis in these programs was consistent with the finding we reported in a May 1996 report on successful training programs—that a key job-training strategy shared by successful programs was a focus on ensuring that participants are committed to training and getting a job. It was also consistent with the opinions expressed by several regional directors we interviewed. The “commitment checks” contractors’ used were designed to test Job Corps applicants’ initiative. For example, several contractors required individuals interested in Job Corps to set up application appointments. Admissions counselors at four contractors also mentioned that they required applicants to arrive for their meetings dressed in proper attire; otherwise, they had to schedule another appointment. In addition, three admissions counselors required applicants to submit written statements of why they wanted to participate in the program and what they hoped to accomplish. Several admissions counselors required applicants to call weekly between the date of application and the enrollment date to determine the status of their application and to demonstrate their continued interest in the program. Finally, one contractor also used a nine-point checklist of documents that all interested persons had to acquire before they set up their application appointment. Some outreach and admissions contractors considered preenrollment tours and briefings to be extremely useful, although they were not practical in every situation. They provided applicants with a firsthand opportunity to obtain a thorough understanding of Job Corps rules and requirements, observe the living conditions, erase false expectations, and determine whether they were suited for regimented life. In some instances, these preenrollment briefings were given prior to application while others took place afterward. For example, one contractor required that all interested individuals attend a prearranged tour and briefing. After taking the tour, attending the briefing, and participating in a question and answer session, those still interested had to set up an appointment to complete an application. Another contractor required potential enrollees to take a tour after the application process. Following the tour, applicants attended a briefing and question and answer session, followed by one-on-one interviews with center staff. The value of preenrollment tours and briefings was also confirmed by Job Corps participants at two of the centers we visited who thought the tours and briefings were definitely worthwhile and by two regional directors who agreed that the preenrollment tours and briefings were very effective in preparing applicants for Job Corps and in improving program retention. These tours and briefings would help meet the law’s requirements that applicants be given a full understanding of Job Corps as well as what is expected of them after enrollment. Several regional directors commented on the importance of identifying applicants who are ready for Job Corps and can benefit from its training. For example, one regional director stated that because the program cannot afford to squander its resources on applicants who do not really want to be in the program, admissions counselors should ensure that applicants are ready and can benefit from the investment. Another regional director noted that because so many people are eligible for Job Corps (over 6 million) it was important to provide this opportunity to those most likely to benefit and that commitment should be “first and foremost” when assessing applicants. Another regional director agreed that commitment was important but considered the program’s Capability and Aspirations Assessment Tool to be ineffective in measuring it. Characteristics Associated With Program Retention In our analysis, we identified several characteristics associated with program retention that Labor might consider in designing outreach efforts, establishing priorities among applicants, or improving participant retention rates. Some of these characteristics would be of limited value nationwide, however, because so few participants nationwide had those characteristics. In addition, when considering how to use the results from our analysis, Labor also needs to consider other factors. Two of the characteristics most strongly related to the likelihood of remaining in the program were need for bilingual education and years of education. Of the characteristics we examined, the need for bilingual education had the strongest relationship with the likelihood of remaining in the program. Participants needing bilingual training—Spanish as well as other languages—were much more likely than others to remain in the program for at least 60 days. Education was also an important factor—participants with 12 or more years of education were more likely to remain than participants with 8 or fewer years of schooling. Another characteristic with a strong relationship to retention was age. Our analysis indicated that older participants had a greater likelihood than younger participants of remaining in the program. Specifically, when compared to 15-17-year-old participants, those aged 18 to 20 and 21 to 25 were more likely to remain in the program for at least 60 days. This analysis supported the concern expressed by many of the admissions counselors we interviewed regarding enrollment, retention, and placement of 16- and 17-year-old youths, who make up nearly 40 percent of the program year 1995 enrollees. The concerns they expressed were that these younger youths are often victimized by older participants at the center, have a harder time adjusting to center life, are more likely to drop out, and are difficult to place. Labor program year 1995 outcome data showed that 16- and 17-year-old terminees were less likely to be placed once they left the program (see fig. 1). Because of the difficulty in placing 16- and 17-year-old participants, one regional Labor official believed that the minimum age for enrollment should be increased, while another thought that there should be separate standards for these participants. In contrast, a third regional Labor official thought that maturity, and not age, should be the deciding factor for enrollment. He acknowledged, however, that the program should probably have different expectations and performance standards for 16-year-old participants. Another Labor official told us that a work group has been established to look into the problem of serving 16- and 17-year-old participants. Appendix IV discusses our statistical analysis of characteristics related to remaining in the program at least 60 days, including limitations associated with the analysis. Table IV.3 in that appendix contains the final model and significance levels. For example, it shows that other factors that had a significant relationship to the likelihood of remaining in the program for at least 60 days included residing less than 50 miles from the assigned Job Corps center, being a nonresidential student, having no dependents, and having served in the military. Additionally, some of the factors that proved to be useful predictors of remaining in the program were characteristics of only small subsets of participants. For example, because relatively few participants had a need for bilingual education (less than 3 percent of the Job Corps population), that characteristic was limited in its value as a feature for nationwide use in screening. Because we found no large subgroups with great differences, the ability of the model we used in our analysis to predict 60-day retention for the program’s full population is limited. In deciding how to use the results of this analysis, Labor would need to consider more than the statistical results. For example, it would clearly be inappropriate to use these findings to exclude applicants who met the statutory eligibility requirements because they had characteristics associated with a low likelihood of completing the program. If Labor chose to consider these characteristics in designing outreach efforts or establishing priorities for eligible applicants, it would be faced with the complexity of integrating these results with existing eligibility requirements and program policy. For example, our results showed that participants with at least 12 years of education were more likely to remain for 60 days than those with less education. Many youths with that many years in school, however, might not meet the eligibility requirement of needing additional education or training to secure and hold meaningful employment, participate successfully in regular school work, qualify for other suitable training programs, or satisfy armed forces requirements. The most clear-cut use of this information on participant characteristics may be in designing efforts to improve the retention rate of participants with characteristics associated with leaving the program early. Performance Measures Are Inadequate for Assessing Placement Contractor Performance Labor uses performance measures in deciding whether contractors are to continue to participate in the program. However, Labor does not have the information it needs to accurately assess the performance of its placement contractors. We found that two of the four measures Labor used in assessing placement contractor performance were not meaningful. One of the measures held contractors accountable for placing participants who were realistically unemployable. A second measure, relating to the placement of terminees in training-related occupations, included terminees who received little vocational training and also gave placement contractors wide latitude in deciding whether placements were related to training. Job Corps requires placement contractors to assist all terminees with placement regardless of how long they were in the program or the reason they left, and it has established the following standards to measure contractor performance: 70 percent of all terminees assigned to a contractor are to be placed, 70 percent of all placements are to be in full-time jobs, the average wage paid to participants placed in jobs is to be equal to or greater than a specified level, and 42 percent of all job placements are to be in occupations related to the training received. Measurement of Job Placements Includes Unemployable Terminees In calculating a contractor’s placement performance, Labor includes participants who remained in the program for as little as 1 day, those who were AWOL, and those who were expelled from Job Corps after 30 days for using drugs or committing violent acts—all individuals a placement contractor would have difficulty recommending for employment. During program year 1995, about one-third of the participants leaving Job Corps were in these categories. If Labor’s methodology were modified to include only participants who were in the program for sufficient time to obtain at least minimal benefits (that is, stayed for at least 30 days) and were employable (that is, were not terminated for drug violations and violence and were not AWOL), the average placement rate for the 12 placement contractors we visited would be about 8 points higher—ranging from an increase of 2.6 points for one contractor to 13.6 points for another contractor—and the rank order among the 12 contractors would change somewhat. (See fig. 2.) About half of the placement contractors we visited suggested that Labor should exclude certain individuals when calculating placement rates. For example, one contractor noted that it is unreasonable to expect contractors to recommend to an employer someone who was expelled for taking drugs or committing a violent act. Another contractor believed that it was a waste of resources to try to place participants who were AWOL because they were not only difficult to locate but also undependable to an employer. A third contractor suggested that Labor’s methodology include only participants who are truly employable. Similarly, a regional director stated that it is ridiculous to require placement specialists to be responsible for placing participants who stayed in the program a very short time, were expelled for drug use or violence, or were AWOL. He said that this responsibility asks the placement specialist to lie to employers by recommending they hire these people. Another regional director agreed that placement contractors should not be responsible for participants who received no benefit from the program or who were kicked out for violating the program’s drug and violence policies. Training-Related Placement Measure Is Flawed The job-training match measure is used to evaluate the effectiveness of vocational training programs and placement contractors by determining the percentage of jobs terminees obtain that matches the training they received while in Job Corps. Labor allows placement contractors wide discretion in deciding whether a job placement they obtain for a terminee is related to the training received—another measure of performance. At the same time, Labor requires that terminees who receive little vocational training be included in the calculation of this measure. As a result, the value of the current job-training match performance measure is questionable. Labor is developing a new system to determine job-training matches that, it believes, will be more accurate. Labor’s guidance gives placement contractors wide latitude in deciding whether a job placement was a job-training match. According to Labor guidance, a job-training match results when a participant is placed in a job requiring skills similar to those included in the participant’s training. Placement contractors are responsible for recording this information. Labor’s guidance for these decisions consists of 16 broad categories of training programs, and within each category are a varying number of detailed occupations in which Job Corps participants may be trained. In addition, each of the 16 broad categories contains a list of jobs that would be considered a match with the training received. To illustrate, the broad training category of construction trades includes 47 detailed training occupations and 357 placement occupations. An individual who was trained in any one of the 47 training occupations and then was placed into any one of the 357 placement occupations would be counted as having made a job-training match. Overall, Labor’s system includes nearly 300 detailed training occupations and more than 5,700 job placement occupations. In addition to the wide range of jobs that are considered to be training matches under each of the broad training categories, Labor’s guidance includes jobs that appear to bear little, if any, relationship to the training received. For example, a position as a key cutter would be considered to be a training match for any of the 51 training categories under the broad category of mechanics and repairers, which includes auto mechanic, electronics assembler, and parts clerk. A position as a general laborer would be considered to be a job-training match for any of the 30 training occupations under the precision production category, which includes mechanical drafter, sheet metal worker, and welder. Table 2 lists examples of some possible matches under Labor’s guidance. Many of the positions that are considered to be related to Job Corps training require relatively little training to perform. The job placement occupational categories contained in Labor’s guidance for job-training match come from its Dictionary of Occupational Titles. The dictionary includes, for each occupation, the average time required to learn the techniques, acquire information, and develop the facility for average performance in a specific job situation. For more than 700 of the jobs included in Labor’s guidance, the average training time is indicated as either only a short demonstration or training up to and including 1 month. Thus, Labor is allowing job-training match credit for occupations requiring relatively short training time even though participants spend an average of about 7 months in the program at an average cost of about $15,300 each.While we recognize that some of these positions provide entry into an occupational area that may lead to a better job, in our view it is questionable to consider such positions to be a job-training match until the participant advances into a job commensurate with the training received. Further, Labor guidance encourages placement contractors to search among the allowable jobs for a job-training match. Its policy handbook states that, if a job-training match is not generated when a job placement code is entered in its automated system, the placement contractor is allowed to enter a different code that may generate a job-training match, “so long as integrity of data is maintained.” We found that placement contractors’ practice of recording job-training matches does indeed raise questions about the integrity of the data. One contractor told us that if a placement specialist obtained a job for a terminee that was not a job-training match under Labor’s guidance, then the manager and placement specialist would meet to determine how to make it a match. This same contractor claimed that it is possible to get a job-training match in fast-food restaurants for participants trained as bank tellers, secretaries, and welders. For the most part, the placement contractors we visited similarly indicated that creativity is used when entering the code for the placement job in order to obtain a job-training match and raised concerns about the validity of reported job-training match statistics. The job-training match performance measure may also unfairly hold placement contractors accountable for placing certain terminees in training-related jobs. All participants placed in a job or the military are included in the calculation of job-training match, regardless of how long they received vocational training. Thus, participants for a few days or weeks who had little chance to participate to any extent in vocational skill training would be included in the calculation of the job-training match measure. Most of the placement contractors and regional staff we spoke with agreed that when calculating this measure it would be more meaningful to include only participants who completed their vocational skills training. Labor officials told us that they are revising the methodology for determining job-training matches. The proposed methodology will use an existing system used by the Bureau of Labor Statistics to collect occupational employment data by various industry classifications. This system uses about 830 five-digit codes rather than the 5,700 nine-digit codes used in the current methodology based on the Dictionary of Occupational Titles. In its comments on a draft of our report, Labor acknowledged that we made a legitimate point about the need to strengthen the job-training match process. According to Labor, the proposed system will be more accurate and easier to maintain and monitor in terms of egregious job-training matches. Labor hopes to have implemented the new methodology by July 1, 1998. In addition, Labor stated that the job-training match issue is one of the primary projects being addressed by a Job Corps committee to improve the quality of vocational outcomes. Characteristics of Contractors With Higher Placement Rates We found that a characteristic common to the contractors we visited that had higher placement rates was having staff solely responsible for providing placement services to Job Corps participants. In addition, most of these placement contractors were either Job Corps centers or had staff located at the centers they served. In contrast, Labor regional officials have been concerned with the performance of state employment service agencies and have not renewed many of their contracts during the past 2 years. We also noted that Labor and several of the Job Corps centers we visited were starting to improve links to the business community in an effort to increase placements. The placement contractors we visited had had varying success in placing Job Corps participants in program year 1995. Placement included getting a job, entering the military, or returning full-time to school. The seven contractors that had relatively high placement rates (over 73 percent) included four Job Corps centers and three private organizations. A common characteristic among these contractors was having staff who had only one responsibility—placing Job Corps participants. Other contractors that were not as successful used the same staff to perform outreach and admissions as well as placement. One contractor whose staff performed these functions noted that with the program’s emphasis on maintaining centers at full capacity, placement is often secondary to admissions. We also noted that most of the contractors with higher placement rates were either Job Corps centers or had staff at the center. Placement specialists at the Job Corps center contended that being at the center allowed them easy access to instructors, counselors, and participants. One Labor regional director also mentioned the importance of having a continuity of services from the time enrollees arrive at the center until they are placed, noting that it was no accident that every center in his region also has a placement contract. In contrast, the placement contractor we visited with the highest placement rate was not a Job Corps center and did not have staff at a center. The program manager of this private company viewed Job Corps placement as a business and ran the organization accordingly—either placement specialists produced jobs for Job Corps participants or else the program manager found someone who could. Thus, having a focus on the ultimate goal—placement in a job—is a strategy associated with a high placement rate. One type of contractor that generally has not had high placement rates is state employment service agencies. Between program years 1994 and 1996, Labor regional offices did not renew two-thirds (12 of 18) of the placement contracts they had with state employment service agencies (see table 3). Labor officials in three regional offices informed us that they cancelled the placement contracts with state employment service agencies because of poor performance. A Labor official in a fourth region stated that the agency had sent a letter of concern to the state employment service agency because it was the worst-performing placement contractor in the region. Five of the six remaining state employment service placement contractors had placement rates in program year 1995 below the national Job Corps standard of 70 percent. Officials from two of the three state employment service agencies we visited expressed reservations about continuing to contract with Job Corps for placement services. For example, one employment service official said that the agency might not seek contract renewal because of its strained relations with Labor’s regional office. An official from another employment service commented that its Job Corps contract was really “small potatoes” and insufficient to provide for adequate staffing and that the only reason it was still involved was that the employment service commissioner believed that Job Corps was worthwhile and wanted to assist disadvantaged youths. An official from the third employment service agency we visited noted that the Labor regional office threatened to cancel its placement contract 2 years ago for poor performance and gave the agency another 6 months to improve. The official noted that, under new management, performance did improve and Labor renewed the agency’s contract for another 2 years. Placement specialists at the three employment service offices we visited stated that they have no contact with Job Corps participants before their termination. It also appeared that the major placement emphasis was to register Job Corps participants in the employment service databank. While this did provide access to a major source of potential jobs, it was the same service provided to regular job seekers using the employment service and was not any kind of specialized assistance. As pointed out by the Chairman of the Senate Subcommittee on Employment and Training, Committee on Labor and Human Resources, during hearings on Job Corps in April 1997, a key to program success is the development of links to the business community. However, concerns were raised about whether Job Corps had developed such links. We noted that several of the centers we visited that had higher placement rates also had good relationships with local businesses. For example, one center had established a physical therapy program to meet the needs of local health facilities, and another center used temporary agencies as a springboard for their computer services trainees to gain access to the area’s computer industry. A third center was working on improving its work experience component to better match participants’ skills and abilities to the needs of local businesses so that more permanent hires would result. Labor regional offices are also exploring ways to improve links to the business sector. For example, one office has recently started a business roundtable of 18 employers in the region who discuss placement issues. Another regional office has begun a project to get local employers involved with training and placement. The idea is to have employers identify what they need in terms of training curriculum, equipment, and skills and then determine how the program can meet these needs. Recognizing the importance of employer links, Labor has launched a new school-to-work initiative within Job Corps to involve more employers in placing program terminees and to establish the basic framework for a school-to-work program. It started as a pilot program at three Job Corps centers and will be expanded to 30 centers this year. Further expansion will depend on the availability of funding. Conclusions Labor’s program guidance to admissions counselors on two eligibility requirements was ambiguous and incomplete. One of the program’s eligibility criteria—living in an environment characterized by disorienting conditions—has not been clearly defined in the statute, regulations, or Labor’s guidance. In addition, Labor has not provided adequate guidance regarding the requirement that participants have the capability and aspirations needed to complete and secure the full benefits of Job Corps. As a result, outreach and admissions contractors may not be enrolling the applicants who are most appropriate for the program. In the absence of specific Labor guidance, we noted that outreach and admissions practices varied among contractors. Those with higher participant retention rates tended to have better procedures to identify applicants who have the capability and aspirations to remain in and benefit from the program. A particularly effective tool in preparing applicants for Job Corps appeared to be preenrollment tours and briefings. Most admissions counselors expressed concern about the enrollment of 16- and 17-year-old applicants. Labor data confirm that these youths are more likely to drop out early for disciplinary reasons and less likely to be placed once they leave the program. Although Job Corps is a performance-driven program, the measures used to assess placement performance may not be meaningful and thus may not provide Labor with the information it needs to accurately assess placement contractor performance. Labor’s system for calculating a contractor’s placement performance included program terminees who were realistically unemployable. Determining what happens to every program participant is an important indicator of how well Job Corps is performing but not necessarily an appropriate measure of a contractor’s placement performance. Guidance related to another placement measure—the extent to which terminees were placed in training-related occupations—gave contractors such wide latitude when deciding whether a job was related to the training received that the validity of the measurement was questionable. In addition, the performance measure included terminees who received little vocational skills training and, therefore, were unlikely to be placed in jobs requiring an acquired skill. Labor is redesigning the methodology for determining job-training matches, which may help address some of these problems. However, any system would still be susceptible to manipulation by placement contractors without proper oversight and monitoring. Recommendations to the Secretary of Labor To help ensure that Job Corps’ resources serve the most appropriate participants, we recommend that the Secretary of Labor provide clear and complete guidance on program eligibility criteria, ensuring that the guidance is consistent with the law, and provide better guidance to ensure that outreach and admissions contractors assess each applicant’s capability and aspirations to complete training and attain a positive outcome. Improvements are also needed to make the measures used to assess placement contractor performance more meaningful. Therefore, we recommend that the Secretary of Labor modify certain measures for placement contractors, including eliminate from the placement pool participants whom contractors realistically could not or should not be expected to place, such as participants who were expelled for criminal or violent behavior; replace the current job-training match system with one that captures realistic information and provide guidance to regional offices to ensure that the data are accurately recorded; establish separate placement performance standards for participants with different levels of program accomplishment—for example, those who completed program requirements and those who dropped out early. Agency Comments In Labor’s comments on a draft of this report, the agency disagreed with our recommendation that it clarify and expand its program eligibility criteria in order to ensure that they are consistent with the law. Labor stated that our report lacked acknowledgment of the detailed specifications for eligibility requirements developed over the years in conjunction with the Office of Inspector General and that the eligibility, verification, and documentation requirements contained in its policy handbook are detailed and specifically related to guidance for Job Corps admissions counselors. Labor gave no indication of any formal action it planned to take on this recommendation. Although Labor expressed some concern with our remaining recommendations, it acknowledged that they have merit, warrant consideration, and identify actions that the agency would take in response to them. Labor’s specific concerns with our report are in three broad areas—adequacy of program eligibility guidance, potential effect of additional assessment procedures, and recommended changes to placement performance measures, including training-related placements. Labor also pointed out a number of items in the draft report that it believed should be modified or clarified, and we acted on these, where appropriate. For example, we clarified that our discussion of the ambiguity of program eligibility guidance related to only 2 of the 11 criteria. We also made a number of other technical changes to our report to respond to Labor’s comments. Following is a summary of Labor’s concerns and our responses. Labor’s full comments are printed in appendix VI. Eligibility Guidance Labor stated that our report lacked acknowledgment of the detailed specifications for eligibility requirements developed over the years in conjunction with the Office of Inspector General and that the eligibility, verification, and documentation requirements contained in its policy handbook are detailed and specifically related to guidance for Job Corps admissions counselors. Labor expressed concern with our characterization of the program eligibility guidance as inadequate. For example, regarding the lack of definition in Labor’s policy handbook for “limited job opportunities,” Labor commented that training conducted in program year 1995 for all admissions counselors included technical assistance material that defined this term as follows: “scarcity of jobs, commensurate with the skill levels of Job Corps-eligible youth and which has been designated as an area of substantial unemployment.” Labor added that “In essence, any applicant who lacks the specific skills required by the local labor market to obtain meaningful employment is a legitimate candidate for Job Corps.” Labor acknowledged that another eligibility factor—cultural deprivation—is not included in the policy handbook because more-specific factors—including (1) disruptive homelife, (2) unsafe or overcrowded dwelling, (3) disruptive community with high crime rates, and (4) limited job opportunities—were more useful to admissions counselors than the general term itself. Finally, Labor expressed concern with our discussion of the tool used in assessing another eligibility requirement—capability and aspirations. According to Labor, this assessment by its very nature must rely on the judgment of admissions counselors and determining aspirations is very difficult and challenging; Labor stated that the current assessment tool will be revisited and modified according to suggestions from regional offices and admissions counselors. We disagree that sufficient policy guidance defining “limited job opportunities” was provided to admissions counselors at a training seminar. Even if all admissions counselors at that time received such guidance, contractors and staff have since turned over. And, as mentioned in our report, admissions counselors we interviewed had different interpretations of “limited job opportunities,” indicating that something more is needed to ensure the consistent interpretation of limited job opportunities. Because Labor’s policy handbook was created to be “the single document containing all policy and requirements which would be: clear and concise, and up-to-date, and consistent with legislative provisions,” any definition of “limited job opportunities” that Labor develops should be incorporated into this policy handbook. In addition, the law states that environmental factors substantially impair an individual’s ability to succeed in training, not his or her ability to find employment. But Labor fails to explain the connection between its definition and the impairment of ability to succeed in training. And there is a separate eligibility requirement in the law that the applicant must “require additional education, training, or intensive counseling and related assistance in order to secure and hold meaningful employment....” Labor’s interpretation of limited job opportunities appears to duplicate or at least overlap that separate requirement. Finally, Labor fails to explain how its definition satisfies the program regulations that stipulate that the environmental criteria are to be used in the context of residential versus nonresidential programs. Nowhere in its guidance does Labor mention this distinction. We also disagree that Labor provided adequate guidance regarding the term “cultural deprivation.” On the Job Corps application form, Labor not only lists each of the four factors it says define “cultural deprivation” as separate and distinct eligibility factors (any one of which would satisfy the eligibility requirement) but also adds the term “cultural deprivation” as a fifth factor that can be used to meet program eligibility. Guidance for completing the application form does not define this term and, as noted in our report, most of the admissions counselors we spoke with admitted that they did not know what the term meant. Furthermore, cultural deprivation cannot include disruptive homelife, as Labor says it does, because the law lists these as two separate environmental conditions. Regarding the eligibility requirement that participants have the capability and aspirations to complete and benefit from Job Corps, we agree with Labor that making such a determination is very difficult and challenging and, therefore, we believe that it is important that admissions counselors have guidance adequate to assist them in making these judgments. Furthermore, we agree with one regional official’s portrayal of the current assessment tool as a beginning step in providing guidance on this criterion. Accordingly, we support Labor’s decision to revisit this assessment tool and to obtain regional office and admissions contractors’ suggestions for improving it. Assessment Procedures With respect to assessment procedures, Labor agreed that Job Corps should not enroll youths who obviously have no desire to be in the program or capability to succeed and that assessing the interest and ability to benefit are important parts of the intake procedure. Labor also noted that participants’ leaving the program within the first 2 months is a cost that Job Corps must do whatever it can to minimize. However, Labor points out the need for a balance between this goal and the goal of serving youths who truly need the program, noting that overly strict assessment procedures could be a barrier to many severely disadvantaged youths. Furthermore, Labor states that the Congress clearly intended that Job Corps serve a severely at-risk population. Labor acknowledged that our report contained a number of positive suggestions (that is, “best practices”) that will be made available to outreach and admissions as well as placement contractors. Labor cautioned that the results of our analysis of characteristics associated with program retention could be misinterpreted because the report lacks the proper context. Labor further suggested that the detailed appendix related to this discussion be removed. Finally, Labor stated that the age data relating to participants who were 15 and 25 years old was inaccurate because Job Corps serves individuals aged 16 to 24. While we do not disagree that the program is to target persons most in need, the law states that the purpose is to assist youths who both need and can benefit from an intensive program. And the law requires that enrollees have the capabilities and aspirations to complete and secure the full benefits of the program. Several Labor regional directors commented on the importance of identifying applicants who are ready for Job Corps and can benefit from its training. For example, one director stated that with more than 6 million people eligible for Job Corps, admissions counselors have to identify those most likely to benefit from the program and that commitment should be first and foremost when they assess applicants. We also note that, in a previous report, we found that a key element of successful job-training projects was ensuring that participants are committed to training and to getting a job. Accordingly, we endorse Labor’s decision to make available to admissions contractors the procedures noted in our report that help identify the applicants who have the commitment and motivation to remain in and benefit from the program. We modified the report to provide our reasons for performing our analysis of characteristics associated with program retention and to highlight the limitations associated with our approach as well as the results. However, we do not believe the detailed appendix should be eliminated. In addition to describing our analysis and results in detail, it describes the related limitations. Regarding our mention of 15- and 25-year-old program participants being inaccurate, we obtained our data from Labor’s national database, which showed that less than 1 percent of program year 1995 enrollees were either 15 or 25 years old. We have added a relevant footnote. Placement Performance Measures Labor expressed concern with our recommendation with respect to placement performance measures that Job Corps eliminate from the contractors’ placement pool individuals who realistically could not or should not be expected to be placed, such as those expelled from the program for using drugs or engaging in violent behavior. Labor believes that the program has the responsibility to provide placement services to all participants and that it is not asking placement contractors to mislead or lie to employers during placement. Labor further commented that the current placement measure resulted from a recommendation by its Office of Inspector General that all participants who leave the program should be included in the placement pool, thus creating incentives to keep students as long as possible. Labor acknowledged that the points we made in this portion of the report merit serious consideration and, therefore, it will convene a workgroup to discuss our recommendations and examine the incentives and disincentives resulting from any proposed changes to the performance management system. Labor also acknowledged that our report contained “some good points” with respect to training-related placements but expressed concern about our use of hypothetical examples of questionable job-training matches and the lack of data to indicate the degree to which these occur. Labor also commented that the claim by a contractor about obtaining a job-training match for participants trained as bank tellers, secretaries, and welders and placed in fast-food restaurants is inaccurate, noting that the system does not permit such matches. Although Labor may not be asking its placement contractors to lie to or mislead employers when attempting to place individuals who realistically could not be placed, by holding contractors responsible for placing individuals expelled for criminal or violent behavior, the program may be encouraging such practices. We agree with Labor that determining what happens to every participant is an important indicator of program performance, but we do not believe that it is necessarily an appropriate measure of a contractor’s placement performance. We also acknowledge that establishing an effective performance management system is complex and agree with Labor that, before any changes are made to this system, the incentives and disincentives should be thoroughly examined, and we commend Labor for its proposed action. We used “hypothetical” examples of job-training matches to illustrate the wide latitude Job Corps permits. Labor data were not available to identify the extent of abuse, but as we mentioned in the report, most placement contractors we interviewed indicated that creativity is used when entering codes for placement jobs, and they expressed their concern about the validity of reported job-training match statistics. In response to Labor’s contention that the system does not permit job-training matches for participants trained as bank tellers, secretaries, and welders who obtain jobs in fast-food restaurants, we agree that if such jobs were reported as “fast-food workers,” the system would not permit a job-training match. But, as a contractor we spoke with pointed out, reporting such jobs in fast-food restaurants as “cashier” would be an allowable match for participants trained as bank tellers and secretaries, and reporting such placements as “machine cleaners” would be an allowable match for participants trained as welders. As arranged with your office, unless you publicly announce the contents of this report earlier, we plan no further distribution of it until 15 days after its issue date. We will then send copies to the Secretary of Labor, the Director of the Office of Management and Budget, relevant congressional committees, and others who are interested. Copies will be made available to others on request. If you or your staff have any questions concerning this report, please call me at (202) 512-7014 or Sigurd R. Nilsen at (202) 512-7003. GAO contacts and staff acknowledgments are listed in appendix VII. Scope and Methodology We designed our study to identify whether Labor’s policy guidance on eligibility was consistent with legislation and regulations and to collect information on contractors’ practices in enrolling individuals for the program and in placing them in jobs after they leave Job Corps. We reviewed Job Corps legislation as well as Labor’s program regulations and policy guidance on program eligibility, outreach and assessment of individuals for participation in the program, and placement of participants after termination. We also interviewed national and regional Job Corps officials and conducted site visits to 14 outreach, admissions, and placement contractors. We augmented the information we collected during the site visits with data from Labor’s Student Pay, Allotment, and Management Information System (SPAMIS), a database containing nationwide Job Corps data on all Job Corps participants as well as information on the outreach, admissions, and placement contractors for each participant. We analyzed program year 1995 enrollee data, the most recent full program year for which SPAMIS data were available. While we did not verify the accuracy of Labor’s SPAMIS data, we performed internal validity checks to ensure the consistency of the database. We performed our work between October 1996 and July 1997 in accordance with generally accepted government auditing standards. Site Visits We visited 14 Job Corps outreach and assessment and placement contractors. We selected the sites judgmentally to provide a mixture of contractors that were private contractors, Job Corps centers, and state agencies. We also selected contractors that provided both outreach and assessment services and placement services or that provided only one of these services. In addition, we considered past contractor performance in making our selections. We selected contractors located in 5 of Labor’s 10 regions to provide some regional management diversity and geographic dispersion and to allow us to visit multiple contractors during individual trips. In making our site selections, we identified contractors that had outreach and admissions or placement contracts with Labor during program years 1994 and 1995 and that were still under contract in program year 1996. This provided us with contractors that had multiyear program experience and were currently under contract with Job Corps. In order to select among the larger contractors, we included only contractors who enrolled or were responsible for placing at least 150 participants each year. We then ranked outreach and admissions contractors according to the percentage of program year 1995 enrollees who stayed in the program for more than 30 days and placement contractors according to the percentage of program year 1995 terminees placed in jobs, school, the military, or other training. We then selected contractors from among the top, middle, and bottom third of these rankings. Table I.1 lists the contractors we visited and their characteristics. Table I.1: Outreach, Admissions, and Placement Contractors We Visited North Carolina Department of Human Resources Dynamic Educational Systems, Inc./Hubert H. Humphrey Job Corps CenterDynamic Educational Systems, Inc. Oklahoma City, Okla. Oklahoma Sacramento, Calif. Sacramento, Calif. Did not meet selection criteria (continued) Carson City, Nev. San Francisco, Calif. Women In Del Jen, Inc. We visited 11 outreach and admissions contractors from which varying percentages of program year 1995 enrollees left the program within the first 30 days. As shown in figure I.1, the percentages ranged from about 1 percent for one contractor’s enrollees to nearly 20 percent for another’s. We also selected 12 placement contractors to visit that had varying success in placing Job Corps participants in program year 1995. As shown in figure I.2, placement rates ranged from about 54 percent to about 85 percent. To obtain information on how contractors enroll individuals in Job Corps and place them after their termination, we interviewed contractor personnel using a semistructured interview protocol. We asked outreach and admissions contractors questions related to their practices and procedures in attracting youths to Job Corps and in screening applicants. We also asked about their understanding and implementation of program eligibility criteria as specified by Labor and about their views on what affects program retention. We questioned placement contractors on their procedures in placing terminees in jobs, the military, or other training; the types of services they provided to terminees; and their practices when deciding whether a placement is a job-training match. We asked both groups of contractors about their views on current Labor performance standards related to recruitment and placements and their opinions on improvements needed in the Job Corps program. At three centers (David L. Carrasco, Kittrell, and Sacramento), we also interviewed Job Corps participants (approximately six from each center) to learn about their experiences when they were recruited for Job Corps and to obtain their views about the enrollment process. National and Regional Job Corps Offices We interviewed Labor officials at national and selected regional offices to obtain an overview of Job Corps enrollment, placement, and contracting. We also obtained information on Labor’s policy guidance on eligibility and how it relates to the Job Corps legislation; outreach, admissions, and placement contractors’ performance; and the program’s performance management system. In addition, we reviewed Labor’s Policy and Requirements Handbook, which was designed to include all program policy and requirements concerning eligibility criteria and policies and standards related to program enrollment and participant placement. Data Analysis We analyzed Job Corps participant retention data, reasons for termination, and placement information for program year 1995. We used 30-day retention data, part of Labor’s standard for evaluating outreach and admissions contractor performance, as a basis for selecting outreach and admissions contractors to visit. We expanded our analysis of retention beyond the 30-day standard and determined how many terminees left Job Corps within 60 days of enrollment in order to look at retention beyond the realm of outreach and admissions contractor performance. We also used one of Labor’s placement standards—the extent to which terminees are placed in jobs, the military, school, or other training—as a basis for selecting placement contractors to visit. Furthermore, we used the data from our analysis to supplement information obtained in discussions with admissions counselors and placement specialists. Labor’s Job Corps Eligibility Criteria Labor’s Capability and Aspirations Assessment Tool Analysis of the Relationship Between Participant Characteristics and the Likelihood of Remaining in Job Corps for at Least 60 Days In our analysis, we examined the relationship between the characteristics of Job Corps participants and the likelihood of their remaining in the program for at least 60 days. We used the data that were available from Labor’s Student Pay, Allotment, and Management Information System (SPAMIS) on the characteristics of the more than 68,000 participants enrolled in Job Corps during program year 1995. We performed a three-stage analysis resulting in a logistic regression model that used these characteristics to predict the odds of a participant’s remaining in the program for at least 60 days. While the information from our analysis provides some indication of whether participants with specific characteristics will remain in Job Corps for at least 60 days, we do not intend to imply that only individuals with these characteristics should be enrolled in the program or that outreach and assessment efforts should be focused on them. Rather, this information is a source of insight into early program attrition for Labor’s use in Job Corps management. We also recognize that being in the program for at least 60 days indicates only longevity, not necessarily success. For our initial exploration of the data, we selected the participant characteristics from SPAMIS that appeared to be conceptually relevant to the likelihood of remaining in the program for at least 60 days. These included age at enrollment, distance between a participant’s home and the assigned Job Corps center, and educational status. We first used crosstabulations to examine the relationship of these variables to whether the participant remained in the program for 60 days. The chi-square statistics from these analyses showed the variables that seemed to exhibit no relationship to 60-day retention and helped us eliminate certain characteristics and select a set of variables for further analysis. The set of variables we selected is shown in table IV.1. No need for bilingual education Distance from home to center (continued) With these variables, we then performed a bivariate logistic regression to estimate the effects of each individual factor on remaining in Job Corps for at least 60 days. The results from the regression models are stated as odds ratios, which tell us how much more likely participants with certain characteristics are to remain in Job Corps for at least 60 days than participants without those characteristics. We give a chi-square test of significance for each of these odds ratios. To calculate the odds of a specific group remaining in Job Corps for at least 60 days, the percentage remaining and not remaining must be determined. For example, 26,687 participants aged 15-17 enrolled in Job Corps during program year 1995. As shown in table IV.1, 19,148 of these participants remained in the program for at least 60 days, while 7,539 did not. The odds of 15-17-year-old participants remaining in the program for at least 60 days were calculated by dividing the number remaining (19,148) by the number not remaining (7,539). Therefore, the odds for this group’s remaining were 2.54, meaning that 2.54 individuals remained for every 1 who did not. Similar calculations for participants aged 18 to 20 and 21 to 25 yield higher odds of 3.04 and 3.47, respectively. The logistic regression model provides us with odds ratios that tell us how different the odds are for each group and whether the differences are statistically significant. For example, when the 15-17-year-old group is used as a benchmark for comparing the two other groups, the resultant odds ratios are 3.04/2.54 = 1.20 and 3.47/2.54 = 1.37 for participants aged 18 to 20 and 21 to 25, respectively. Thus, the odds of 18-20-year-old participants remaining in Job Corps at least 60 days are 1.20 times the odds of 15-17-year-old participants, and the odds of 21-25-year-old participants remaining are 1.37 times the odds of 15-17-year-old participants. Odds ratios that deviate from 1.0 the most, in either direction, represent the most sizable effects (for example, odds ratios of 0.5 and 2.0 represent effects that are similar in size, since 0.5 indicates that one group is half as likely as the other to remain in the program for at least 60 days, while 2.0 indicates that one group is twice as likely as the other to remain). We performed this type of bivariate analysis for each characteristic we selected. The resulting odds ratios are shown under the “bivariate results” column of table IV.2. Distance from home to center Less than 50 miles vs. 300 miles or more 50-149 miles vs. 300 miles or more 150-299 miles vs. 300 miles or more (continued) Size of participant’s home city or town 250,000 or over vs. under 2,500 * Statistical significance =.05. After performing the bivariate analysis, we used the same set of variables in a multivariate logistic regression analysis, identical to the bivariate analysis except that it provides estimates of the effects of each characteristic on the likelihood of remaining in the program for at least 60 days while holding constant, or controlling for, the effects of the other characteristics. We included all factors (and levels of factors), even if their effects were not statistically significant in the bivariate analysis because, in some cases, effects that are suppressed in bivariate analysis emerge as significant in multivariate analysis. Similarly, effects that were significant in the bivariate analysis may be insignificant in the multivariate analysis. The results of the multivariate logistic regression are shown in column 2 of table IV.2 (“multivariate result”). As this column shows, when we entered all variables into the model, some variables and levels of variables had odds ratios that were not significantly different from the reference category. We dropped these variables or, in cases in which levels of variables were not significantly different from other levels within the same variable, we combined levels. For instance, in the multivariate model, the odds of remaining in Job Corps for at least 60 days for participants having a prior conviction were not significantly different from the odds of remaining for participants not having had a conviction. As shown in table IV.2, the odds ratio of.94 is not statistically significant. Therefore, we dropped this variable from subsequent analysis. Similarly, the odds of remaining for two levels of the variable “distance from home to center” (50-149 miles and 150-300 miles) were not significantly different from the odds of the reference category (over 300 miles). Therefore, we combined these two levels with the reference category to create a two-level variable for subsequent analysis. Thus, we included in the final model only the variables, and levels of variables, that were shown to be significant in the previous multivariate analysis. The results of this final model, as well as statistics related to how well the model performs, are shown in table IV.3. Model performance can be measured by the likelihood ratio method, which evaluates the probability of the observed results, given the parameter estimates. These results are shown under the –2 Log Likelihood (–2LL) entries in the note to table IV.3. As shown, the model containing the predictor variables shows an improved (smaller) –2LL compared with the model containing only the constant (that is, the model that assumes no differential effects resulting from individual variables). The model chi-square, which tests that the coefficients for all the terms in the model (except the constant) are 0 (that is, the null hypothesis), was significant at the.0000 level. Distance from home to center Less than 50 miles vs. 50 miles or more Size of participant’s home city or town Over 250,000 vs. under 250,000 * Statistical significance =.05. Results of Multivariate Analysis The results of our multivariate analysis revealed that older participants have greater odds of remaining in the program 60 days or more. When compared to 15-17-year-old participants, those aged 18 to 20 and 21 to 25 had odds of remaining that were 15-percent and 27-percent greater, respectively. In addition, we found that participants with 12 or more years of school had about 80-percent greater odds of remaining in Job Corps for at least 60 days than participants with 8 years or less of school. (See table IV.3.) We also found a relationship between the need for bilingual education and the likelihood of remaining in the program for at least 60 days. Of the variables we examined, the need for bilingual education yielded the highest odds ratio. Spanish-speaking participants needing bilingual training had odds of remaining that were almost twice the odds of those not needing bilingual education. Other non-English-speaking participants needing bilingual assistance had odds that were more than 3 times the odds of those not needing bilingual education. Limitations of the Analysis Our attempt to construct a model for predicting the characteristics of participants who are more likely to remain in the program for at least 60 days was limited by the variables available to us in Labor’s SPAMIS extracts. Most of these variables were demographic characteristics. We were unable to include in the analysis measures of such things as student ability, attitude, and motivation, as well as other characteristics that could potentially affect the likelihood of participants remaining in the program for at least 60 days. Additionally, the factors that proved to be the most useful predictors of remaining in the program for at least 60 days were characteristics of small subsets of participants. For example, there is evidence that participants in need of bilingual education are more likely to remain, but this group made up less than 3 percent of the Job Corps population. Similarly, participants who had completed 12 years or more of school had odds of remaining that were more than 80-percent greater than those of participants who completed 8 or fewer grades, but almost two-thirds of the participants were in neither of these groups. Consequently, while the model is very useful in predicting whether participants with specific characteristics will remain in Job Corps for at least 60 days, the model’s ability to predict 60-day retention for the program’s full population is limited because we found no large subgroups with great differences. Finally, in this analysis, we examined only main effects for the variables we investigated. An examination of the interactions among the variables might produce useful information and improve the predictive ability of the model. Data Supporting Report Figures Comments From the Department of Labor GAO Contacts and Staff Acknowledgments GAO Contacts Acknowledgments In addition to the contacts named above, the following persons made important contributions to this report: Thomas N. Medvetz, Wayne Dow, Deborah Edwards, Jeremiah Donoghue, Robert Crystal, and Sylvia Shanks. Related GAO Products Job Corps: Where Participants Are Recruited, Trained, and Placed in Jobs (GAO/HEHS-96-140, July 17, 1996). Employment Training: Successful Projects Share Common Strategy (GAO/HEHS-96-108, May 7, 1996). Job Corps: Comparison of Federal Program With State Youth Training Initiatives (GAO/HEHS-96-92, Mar. 28, 1996). Job Corps: High Costs and Mixed Results Raise Questions About Program’s Effectiveness (GAO/HEHS-95-180, June 30, 1995). The first copy of each GAO report and testimony is free. Additional copies are $2 each. 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A recorded menu will provide information on how to obtain these lists. | Summary: Pursuant to a congressional request, GAO provided information on the Job Corps program's recruitment and placement contractors, focusing on: (1) whether Job Corps' policy guidance regarding eligibility criteria is consistent with the legislation and regulations; (2) how the use of recruiting contractors could be improved to increase participant retention in the program; and (3) how the use of placement contractors could be improved to enhance positive outcomes. GAO noted that: (1) Job Corps' policy guidance for 2 of the 11 eligibility criteria was ambiguous and incomplete, which has led to an eligibility determination process that fails to follow the requirements of the law and program regulations; (2) in GAO's visits to several outreach and admissions contractors, GAO found that those with higher retention rates follow procedures aimed at identifying applicants with the commitment and motivation to remain in and benefit from the program; (3) in GAO's analysis of participant characteristics, GAO identified certain characteristics significantly related to the likelihood of remaining in the program for at least 60 days; (4) the Department of Labor (DOL) could use some of these characteristics to design outreach efforts or to establish priorities among eligible applicants; (5) although Job Corps is a performance-driven program and DOL uses performance measures to make decisions on placement contractor renewal, two of the measures DOL used were not meaningful and, thus, DOL did not have the information it needed to accurately assess the performance of placement contractors; (6) placement measures held contractors responsible for placing individuals who may have received little or no benefit from the program or who demonstrated behavior that normally would be unacceptable to most employers; (7) the job-training match measure did not accurately portray the extent to which participants obtained jobs related to their vocational training because of the wide latitude placement contractors have in deciding whether a job is related to the training received and the creativity contractors used in recording the occupational titles of the jobs obtained; (8) one aspect of placement contractors' operations associated with better performance was having staff solely responsible for placing Job Corps participants; (9) seven contractors visited by GAO with high placement rates had staff solely responsible for placing Job Corps participants; (10) in contrast, four of the five contractors having lower placement rates had the same staff responsible for performing outreach and assessment as well as placement; and (11) as a result of its concern about performance, DOL has not renewed 12 of the 18 contracts with state agencies. | 16,074 | 536 | gov_report | en |
Summarize: FIELD [0001] The present invention relates generally to drug delivery. More particularly, the present invention relates to fluid driving systems for portable drug delivery devices. BACKGROUND [0002] Drug therapies are a primary component of an overall patient health plan. Oral tablets and patches are an available means for many drugs, but some drug treatments, such as protein-containing drugs like insulin, cannot be administered in this fashion. Since insulin is a protein which is readily degraded in the gastrointestinal tract, those in need of the administration of insulin administer the drug by subcutaneous injection. In addition, there are many other occasions where liquids, such as blood, saline solution, or water, must be injected into the body. [0003] Drug delivery using current drug delivery devices can be problematic in that certain issues, such as control of the fluid flow rate of the drug being administered, need to be addressed. For example, in applications such as delivery of the drug insulin into a diabetic patient's body, it is desirable to mimic the function of a normally operating pancreas; the ability to more precisely control the flow rate of insulin would enable that objective. A number of flow-regulators have been proposed for the purpose of controlling the fluid flow rate, but the known regulators have not proved satisfactory with respect to the precision and the compactness of the drug delivery device. Conventional drug delivery devices attempt to control flow by using so-called volume controlled flow. Volume-controlled flow is an open-loop system that generates precision pressures by driving precision pumps such as syringe pumps with stepper motors. In these open-loop systems, there is no measurement of the rate of fluid flow and no subsequent use of that measurement as feedback to change the actual flow rate to conform to a desired flow rate. [0004] The ability to measure fluid flow and change the flow rate based on the feedback of the measurement would not only allow for more effective dosing to patients, it would increase the safety of drug delivery as well. Given the fact that drug chamber pressure is above body pressure, there remains a remote possibility for an overdose of drug due to component failure, allowing for excess fluid flow into the body. Although adding backup mechanisms could decrease the risk of excess fluid flow due to component failure, there remains some risk of multiple component failure which could result in overdosing. Depending on the type of drug being administered, such overdosing could potentially be fatal. If a drug delivery device could more precisely measure the fluid flow rate, a large excess of fluid flow could quickly be detected. [0005] There is a need for a drug delivery system for administering drug therapies that can measure the actual flow rate of a fluid, and use that measurement to change the flow rate, obtaining precise control over the fluid flow of the drug being administered. SUMMARY [0006] The present invention overcomes many of the disadvantages of the prior art by providing a drug delivery device that remains compact and wearable, yet maintains a more precise control over the flow rate than conventional systems. This is preferably achieved using two control loops, a pressure control loop and a variable impedance control loop, in a closed loop system. Such a drug delivery device may help improve healthcare of patients by providing a fluid flow that is able to conform more accurately to the desired flow rate. In the case of the delivery of insulin to diabetes patients, for example, a more precise fluid flow rate and the ability to measure and adapt the rate accordingly may allow for the drug delivery device to more accurately mimic a normally functioning pancreas. [0007] For purposes of this disclosure, the term “drug” means any type of molecules or compounds deliverable to a patient to include being deliverable as a fluid, slurry, or fluid-like manner. The term “drug” is also defined as meaning any type of therapeutic agent/diagnostic agent which can include any type of medicament, pharmaceutical, chemical compounds, dyes, biological molecules to include tissue, cells, proteins, peptides, hormones, signaling molecules or nucleic acids such as DNA or RNA. [0008] As previously stated, the present invention uses a pressure control loop and a variable impedance control loop; both are controlled by a closed loop feedback path. In one illustrative example, the pressure control loop and variable impedance control loop are electronically powered. The pressure control loop is powered by a stepper motor, which is coupled to a removable cartridge that contains a reservoir of fluid. The stepper motor uses a piston to apply pressure to a removable cartridge that contains a reservoir of fluid, increasing the pressure in the removable cartridge. Once the pressure has built, the fluid exits the reservoir, flowing through a chamber and a tube to an outlet. To further control the rate of the fluid before the fluid exits through the outlet, the variable impedance control loop, also powered by a stepper motor, inserts a wire into the tube to provide an impedance to fluid flow. The fluid then exits the tube and flows through a flow sensor and into the body of a patient. [0009] The flow sensor measures the fluid flow rate (“measured flow rate”), and sends output signals regarding the measured flow rate to the control electronics, which receives the signals. The control electronics compares the measured flow rate to a pre-programmed or user-input desired flow rate, and adjusts the appropriate stepper motor to conform the measured flow rate to the desired flow rate. [0010] The range of control requested by a drug delivery, such as insulin, is very large, the maximum/minimum flow ratio being approximately 1000. This large range can be controlled using the two control loops, each in charge with the control of a flow ratio of approximately 30. [0011] The miniaturized portable drug delivery system may be provided in a housing sufficiently small to be appropriately and comfortably “wearable” on a person. In one illustrative example of the invention, the housing is sized similar to a personal digital assistant. The wearable housing may include, for example, a base, cover, and hinge that secures the base to the cover. BRIEF DESCRIPTION OF THE DRAWINGS [0012] Various embodiments are described herein with reference to the following drawings. Certain aspects of the drawings are depicted in a simplified way for reason of clarity. Not all alternatives and options are shown in the drawings and, therefore, the invention is not limited in scope to the content of the drawings. In the drawings: [0013] FIG. 1 is a perspective view of the preferred portable drug delivery device; [0014] FIG. 2 is a schematic view of the drug delivery device of FIG. 1, at the initial start-up phase; [0015] FIG. 3 is a schematic view of the drug delivery device operating at maximum flow; [0016] FIG. 4 is a schematic view of the drug delivery device operating with an impedance control restricting the flow of fluid; and [0017] FIG. 5 is a schematic view of the drug delivery device in the re-charging phase, with a valve open for air intake. DETAILED DESCRIPTION [0018] FIG. 1 is a perspective view of an illustrative portable drug delivery device in accordance with the present invention. The drug delivery device is generally shown at 10, and includes a display screen 11, a housing 12, and user input buttons 13. Preferably, drug delivery device 10 is approximately 35 mm by 40 mm; however, the device is not limited to these dimensions. The display screen 11 and user input buttons 13 comprise a configuration interface, wherein a user may navigate through a menu shown on display screen 11, and introduce the profile rate of the flow needed for the particular drug delivery application. Although three user input buttons 13 are shown in FIG. 1, the device is not limited to three user input buttons, and other numbers of user input buttons 13 may be used. [0019] FIG. 2 is a schematic view of the drug delivery device 10 of FIG. 1, and includes control electronics 14, a first stepper motor 16, a second stepper motor 18, a removable or replaceable cartridge 20, a container 22, and a flow sensor 24. [0020] The first stepper motor 16 includes a first piston 26. The removable cartridge 20 includes a rigid wall 28, a flexible wall 30, a reservoir 32, an aperture 34, and a valve 36. The reservoir 32 is preferably filled with a fluid before removable cartridge 20 is shipped for use in drug delivery device 10. Second stepper motor 18 includes a second piston 38, a block 40, and a wire 42. Container 22 includes a chamber 44, a tube 46, and an outlet 48. [0021] Drug delivery device 10 may also include a battery 50 for powering the device. Preferably, battery 50 is a single AA battery; alternatively, battery 50 may be one or a plurality of batteries of varying types. [0022] Control electronics 14 includes a controller or processor that is able to receive and process output signals, as well as control and power motors in accordance with the signals received. [0023] Preferably, both first stepper motor 16 and second stepper motor 18 are light weight, low power, compact, high precision motors. It is desirable to have very small motors for this application. First stepper motor 16 may be the same motor as second stepper motor 18. Alternatively, first stepper motor 16 may be a different motor from second stepper motor 18. [0024] Flow sensor 24 is preferably a high-performance, liquid nano-flow sensor. The primary features of this sensor are high accuracy, high sensitivity, wide dynamic range, automatic temperature and viscosity compensation, small package size and analog signal output. The Honeywell X119177 flow sensor is a suitable flow sensor for this drug delivery device; the flow sensor can measure very small flow rates, from 5 nL/min to 5 uL/min. [0025] For ease of manufacture, rigid wall 28 may be made from another rigid material used on drug delivery device 10. As an example material, rigid wall 28 may be made from a polycarbonate. Flexible wall 30 may be made from an elastomeric material. In the alternative, flexible wall 30 may be the same material as removable cartridge 20. As an example, removable cartridge 20 may be made from a deformable polycarbonate, allowing for any wall to be flexible wall 30. [0026] Valve 36 may be a passive valve; that is, the valve opens due to the removal of pressure from removable cartridge 20, and closes when the pressure increases. Alternatively, valve 36 may be an active valve, such as an electrostatic valve, that is controlled by control electronics 14, and can be opened or closed electronically. An active valve may be desired when more power is required to open valve 36. Additionally, if valve 36 is an active valve, control electronics 14 may be programmed to open valve 36 when the rate of the fluid drops below a predetermined value. When the pressure drops too low to initiate fluid flow, valve 36 is opened to replenish air supply into removable cartridge 20. Control electronics 14 is then re-started. The re-start process, however, would only take a matter of seconds. [0027] In the portable drug delivery device 10, the removable cartridge 20 is removably affixed to first piston 26. Rigid wall 28 of the replaceable cartridge 20 may be affixed to first piston 26 with a screw. Although rigid wall 28 and second stepper motor 18 are shown on the side of the cartridge opposite control electronics 14, rigid wall 28 and second stepper motor 18 may be located on the same side as control electronics 14. In fact, rigid wall 28 and second stepper motor 18 may be located on any side of removable cartridge 20, as long as first piston 26 is able to push rigid wall 28, and increase the pressure. Control electronics 14 is connected to first stepper motor 16. Control electronics 14 may be connected to the first stepper motor 16 with a wire, so that they are in electronic communication. Control electronics 14 is also connected to second stepper motor 18. Control electronics 14 may be connected to the second stepper motor 18 with a wire, so that they are in electronic communication. Aperture 34 connects reservoir 32 to chamber 44. Tube 46 connects chamber 44 to flow sensor 24. Flow sensor 24 is connected to and is in electronic communication with control electronics 14. [0028] To initiate drug delivery, removable cartridge 20 is inserted into drug delivery device 10. Rigid wall 28 is then affixed to first piston 26 with a screw. To pressurize the system as shown in FIG. 2, control electronics 14 powers second stepper motor 18 to push block 40 and wire 42 into chamber 44, so that block 40 completely blocks aperture 34 and there is zero fluid flow into chamber 44. Control electronics 14 then powers first stepper motor 16, pushing first piston 26 toward rigid wall 28. First piston 26 makes contact with rigid wall 28, and then continues to push against rigid wall 28. As rigid wall 28 is pushed toward reservoir 32, flexible wall 30 depresses, applying pressure to reservoir 32. As the pressure increases, valve 36 closes to prevent air seepage out of removable cartridge 20. Once reservoir 32 is properly pressurized, control electronics powers second stepper motor 18 to remove block 40 from aperture 34, allowing fluid to flow into chamber 44. [0029] FIG. 3 is a schematic view of drug delivery device 10 operating at maximum flow. Once sufficient pressure has been built in the removable cartridge, control electronics 14 causes second stepper motor 18 to pull block 40 back, uncovering aperture 34, as shown in FIG. 3. Once aperture 34 is uncovered, it is in fluid communication with chamber 44, and the fluid exits reservoir 32 via aperture 34, flowing into chamber 44. The fluid then continues to flow through tube 46, through outlet 48, and into the body of the patient. Flow sensor 24 is provided in-line with the fluid prior to delivery into the body. Flow sensor 24 measures the rate of fluid flow. An output signal from flow sensor 24 is provided to control electronics 14. Control electronics 14 receives the output signals from flow sensor 24. [0030] FIG. 4 is a schematic view of an illustrative drug delivery device during operation, using a variable impedance loop 52 to control the fluid flow rate. After control electronics 14 receives the output signals from flow sensor 24, control electronics 14 may compare the measured rate of the flow with a desired rate. The desired rate may be a pre-programmed rate. In the alternative, the desired rate may be manually entered by a user. As an example, for use as a drug delivery device to deliver insulin to a diabetes patient, it is desirable to mimic the function of the pancreas, and thus control electronics 14 may be pre-programmed to increase or decrease the flow rate of insulin into a patient at specific, pre-determined times of the day, to mimic a normally functioning pancreas. However, if an emergency arises, in which the patient requires an immediate dosage of insulin that was not part of the pre-programmed fluid flow, a separate input access may be available on control electronics 14 for a user to manually input a desired rate. An LCD display may be connected to control electronics 14 to display the fluid flow rate and include a user input. [0031] If the fluid's measured rate does not match the desired rate, control electronics 14 may adjust either first stepper motor 16 or second stepper motor 18, or both, to attain the desired rate. [0032] Control electronics 14, first stepper motor 16, first piston 26, and flow sensor 24 comprise pressure control loop 52. Control electronics 14 controls pressure control loop 54 by powering first stepper motor 16 to increase or decrease the pressure applied to removable cartridge 20 by either pushing first piston 26 against rigid wall 28, or not pushing piston 26 against rigid wall 28. As the pressure is increased, the flow rate of the fluid through aperture 34 is increased. [0033] Control electronics 14, second stepper motor 18, second piston 38, block 40, wire 44, tube 46, and flow sensor 24 comprise variable impedance loop 52. Variable impedance loop 52 is able to control fluid flow very precisely, due to the impedance determined by tube 46 and wire 42. FIG. 4 illustrates variable impedance loop 52 in operation. In FIG. 4, as fluid flows through chamber 44 and into tube 46, second stepper motor 18 pushes wire 42 into tube 46 by a distance, thus impeding the flow of fluid through tube 46. The impedance section of wire 42 inside tube 46 is determined by the equation: [0000] Impedance˜ *[(Π(radius tube) 2 )−(Π(radius wire) 2 )] [0034] The total impedance of the flow in the tube is defined by the summation of the impedance of the section of tube 46 with wire 42 inserted and the impedance of the section of tube 46 without the insertion of wire 42. [0035] The maximum flow rate occurs when wire 42 is completely removed from tube 46 and the pressure applied to reservoir 32 is at a maximum. [0036] Tube 46 preferably has a diameter in the range of 6-8 mils (a mil being a unit of length equal to 0.0254 millimeters), and wire 42 is preferably in the range of 4-6 mils; however, other values outside of those ranges may be possible. The preferred embodiment uses an approximate 0.5 mil to 1 mil difference between the diameter of tube 46 and wire 42. Wire 42 is preferably made from a material strong enough so that it will not be damaged from the force of fluid flow. [0037] By increasing or decreasing length, second stepper motor 18 is able to precisely control the flow rate. After exiting tube 46, flow sensor 24 measures the flow rate, sends the flow rate as an output signal to control electronics 14, which may then fine-tune the rate of the flow by further adjusting first stepper motor 16 and second stepper motor 18. This closed loop system provides feedback to control electronics 14 and uses that feedback to adjust the flow rate using both a pressure control loop and a variable impedance control loop. [0038] If a large enough quantity of air seeps out of removable cartridge 20, there will not be enough air inside removable cartridge 20 for sufficient pressure to maintain the desired flow of fluid through the system. In this case, as shown in FIG. 4, control electronics 14 will stop first stepper motor 16 from pushing first piston 26 into rigid wall 28, allowing valve 36 to open. Valve 36 opens to allow for sufficient air intake to re-pressurize fluid reservoir, so that the drug delivery process may begin anew. Control electronics 14 then re-starts, and the drug delivery device 10 returns to the initiation phase as described in FIG. 1. [0039] Although the invention has been described in detail with particular reference to a preferred embodiment, other embodiments can achieve the same results. Variations and modifications of the present invention will be obvious to those skilled in the art and it is intended to cover in the appended claims all such modifications and equivalents. The entire disclosures of all references, applications, patents, and publications cited above, are hereby incorporated by reference. | Summary: A drug delivery system for delivering a fluid to a desired location within a body that uses two control loops to control fluid flow: a first control loop in which a pressure source moves the fluid at an approximate rate, and a second control loop where a variable impedance mechanism more precisely controls the flow rate. After the fluid has moved through the chambers of these two control loops, a flow sensor measures the flow rate, sends the flow rate information to the control electronics, which then adjusts the pressure and impedance in a closed-loop manner to maintain a constant, desired flow rate. The drug delivery device may be used in portable or wearable mechanisms. | 4,794 | 138 | big_patent | en |
Summarize: A man indicted for the murder of his second wife and under investigation for the suspicious death of his first in 'glaringly similar' circumstances may have plotted to kill a third woman – his sister-in-law – according to newly submitted court documents. Harold Henthorn was indicted for the murder of his second wife, Toni, 50, last November. The indictment was the result of a two-year investigation during which the FBI eavesdropped on the Colorado widower's telephone conversations, combed through his finances, scrutinized his employment history and concluded that the 'freak' hiking accident that claimed the mother-of-one's life was murder. Dr Toni Jill Bertolet Henthorn plunged 140 feet to her death while on a remote hike with Henthorn who was the only witness to her death. Accused: Harold Henthorn, left, has been accused of murdering his wife, Dr Toni Henthorn, right, who fell to her death while the couple took a hike at Rocky Mountain National Park in September 2012. Twenty years earlier Henthorn was the only witness to the 'freak accident' that killed first wife, Lynn Rishell. She was crushed under the front of his Jeep when, he told first responders, the jack gave way as she reached under the vehicle for a lug night while changing a flat tire in the dark. In both cases Henthorn stood to benefit from substantial life insurance policies taken out on the women in his name. Now new court documents have revealed that the prosecution will submit 'evidence that Harold Henthorn killed his first wife and collected life insurance on her to establish intent, motive, planning, preparation and lack of accident' in the death of his second. And, shockingly, the court papers have revealed that, in 2009, Henthorn took out a life insurance policy on his sister-in-law, Grace Rishell, who was married to his first wife's brother. Grace Rishell was going through a divorce and 'concerned for her financial stability,' when she initially agreed to Henthorn taking out a policy that would pay $50,000 to Grace's brother and each of her four daughters for a total of $250,000. According to the prosecution, 'The evidence will show that Henthorn had a romantic interest in Rishell.' The documents reveal that Rishell had a change of heart in spring 2010 and told the insurance agent to stop the policy from going through. Instead she took out a policy of her own with another insurer. But unbeknownst to Grace Rishell, 'her signature was forged to procure the original policy as well.' Henthorn made payments on the policy until December 2012 and instead of listing her daughter as brothers as beneficiaries, the policy listed only Henthorn. The policy was cancelled in 2013 when the insurer concluded that Henthorn had no insurable interest in Rishell. Family: Dr Henthorn, a skilled ophthalmologist, left behind a seven-year-old daughter when she died in 2012. Mystery: Harold Henthorn told authorities his wife slipped on a steep mountainside while taking a photo. Henthorn, who was arrested on November 6 when FBI swooped on the 59-year-old returning home after dropping his daughter, Hayley, 9, at school, was was denied bail by US District Judge Kathleen Latoyfa. Ms Latoyfa described him as a 'danger' and noted, 'there are rather glaring similarities between the loss of his first wife and the loss of his second,' and pointed to large sums of money recently transferred by Henthorn to his brother as suggestive that he was 'hiding funds.' Loss: His first wife, Sandra Lynn, was killed when their car crushed her as she changed a flat tire. Though Henthorn claimed to work as a fundraiser for a charity the economic crime auditor for the US Attorney found no evidence that the geology graduate from Virginia had any earnings other than some in 1993, 1999 and 2000. The only job he appears to have had was one briefly held in the petroleum industry – the job that saw him and his then new bride, Lynn Rishell, move from their hometown of Harrington, Virginia, across the country to Colorado. Now in documents that provide further damning detail of the deaths of both Toni Bertolet Henthorn and Lynn Rishell Henthorn the prosecution has highlighted the similarities between both women's passing. Both deaths, the documents state, 'involved bizarre 'accidents.' Both left Henthorn with significant insurance benefits. He is beneficiary or trustee on three policies on Toni, totaling around $4.5million and received a $500,000 pay out on the death of his first wife. Both occurred in remote locations. Both times Henthorn was the lone witness. Both deaths occurred after about 12 years of marriage. Henthorn told wildly varying stories about both deaths. According to the documents the man who met his second wife on a Christian dating website, even told a Sunday school class that his first wife died of cancer and lost a baby she was carrying to chemotherapy. In the case of his second wife, an incident which happened just two years after she almost died when she was struck by a wooden beam Harold had thrown off their deck is disclosed in the documents. After that incident, Harold joked to friends: 'I was trying to kill my wife.' In both situations, the documents continue, the women were doing atypical things when they died. Lynn Henthorn was 'fastidious in dress and a cautious women who would not ordinarily crawl under a jacked-up car to search for lug nuts while wearing a nice sweater, especially after exploratory surgery only two weeks earlier.' Scene: The couple were on an anniversary hike - they had been married 12 years - on Deer Mountain in Rocky Mountains National Park on September 29, 2012 when Dr Henthorn fell to her death. Toni Henthorn 'was not likely to scramble down a rocky face on bad knees to look over a cliff in the midst of a tough hike at elevation.' Investigators have also established that Henthorn 'extensively scouted' the fatal hike. Contrary to what he told law enforcement he made nine different trips to the area around Rocky Mountain Park that summer. And the fortnight before their anniversary – the date of her death – Henthorn had hiked six trails to 'find the perfect one for the trip.' Damningly a map found in Henthorn's car suggested that the detours Henthorn claimed to have preceded his wife's fall were not, as he stated spontaneous: it marked the spot near where Toni fell with a pink X. In both situations Harold was eager to have the bodies quickly cremated despite family requests against it. And, disturbingly, Harold spread both women's ashes on the same mountain near Ouray, Colorado. Henthorn has not been indicted for the murder of his first wife and has entered a not guilty plea on the murder of his second. The trial date is set for May 4 – 15 | Summary: Harold Henthorn is being charged with the murder of his wife Toni who he claims fell to her death in Rocky Mountain Park. Toni had life insurance policies worth $4.5m and had almost died two years earlier when she was hit with a wooden beam while alone with her husband. Harold told different stories as to what happened at the time, and later joked 'I was trying to kill my wife' Court documents show he had a $250,000 life insurance on his sister-in-law and a 'romantic interest' in her. Court case will hear his first wife's death was in similar circumstances - although he has not been charged with her murder. | 1,585 | 142 | cnn_dailymail | en |
Summarize: By. Nick Pisa. Last updated at 4:03 PM on 20th January 2012. Dramatic footage of a Costa Concordia crew member telling passengers to 'Go back to your cabins' has emerged. The plea came despite the luxury liner being fatally holed and underlines the chaotic situation in the moments after the ship struck rocks. Holidaymakers wearing red life jackets can be seen milling on the deck of the Concordia in the mobile phone footage which was posted on Italian newspaper Corriere Della Sera's website. Emergence of the footage came as a cook from the ship said Captain Francesco Schettino had ordered dinner at 10.30pm - nearly an hour after the liner struck rocks at 9.41pm. Scroll down to see the videos... Calling for calm: This crew member may have asked passengers not to panic, but it came after the ship had struck rocks. Chaos: A crew member tried to calm passengers down who stood in the ship's corridors wearing lifejackets. Disaster: An incredible aerial shot of the doomed Costa Concordia shows just how far it has listed onto its side. Questions have already been raised as to the slow response from captain Francesco Schettino in waiting more than an hour to abandon the stricken 290m Concordia. And the footage confirms passenger accounts of how they were told to return to their cabins sending some of the victims to certain death. The film also again underlines how Schettino and his crew were still insisting that the situation was all down to a 'electrical black out'. But by then he knew that the ship had suffered a 70m gash in its hull after hitting a reef off the coast of the Italian island of Giglio as he sailed past too close in a mad moment of showing off. Debris: A bench engraved with the name of the grounded Costa Concordia has washed up on the shore. Rescue effort: Firefighters approach the stricken cruise liner as stormy weather sees the search being called off over fears the ship could slip from its rocky resting place. Busy: Italian firefighters have scoured the doomed Costa Concordia as they try to find the 21 missing people. Service: Lorenzo Pasquotti, Giglio Island parish priest (centre) prays with Orbetello bishop Girolamo Borghetti during a Mass on the harbour next to the Costa Concordia. Further light has been shed on what happened to the youngest person still missing from the doomed Costa Concordia. Dyana Arlotti, five, and her father William have not been accounted for since the ship ran aground last Friday. But a children's entertainer has revealed the Rimini girl was part of a group of eight youngsters he took charge of after they were separated from their parents in the chaos. John Lazzarini, who was at the time dressed as Spiderman, shepherded them to a quiet corner, organised them into a chain and led them hand in hand to the deck. He told the Telegraph: 'We heard the signal to abandon ship. We went down to Deck 4, the pickup area, where I divided them up according to which muster station they belonged to and helped them find their parents.' But some of the children, including Dyana, whose mother (top right) was pictured on Giglio yesterday, became lost in the chaos. The female crew member is seen telling. passengers in Italian: 'The situation is under control. Go back to your. cabins. We ask you that you all return to your cabins. 'Once the electrical problem is sorted out everything will be back to normal shortly. Everything is under control. We are resolving the problem.' However, just 30 minutes after the video was shot cowardly captain Schettino eventually gave the order to abandon ship. But by then it was too late as the ship had begun to list too far over for the lifeboats to be launched safely and passengers had to make their way down the superstructure of the hull using rope ladders. This morning the search was called. off after movement was detected by lasers pointed onto the ship and it. was deemed too dangerous for the dive teams to carry on the hunt. There. are still 21 people still missing, who are believed to be lying in. muster stations on decks submerged below the water. A total of 11 people. have been confirmed dead. The. main concern for teams working on the ship is the weather, as although. they have been lucky in the week following the tragedy the forecast is. for strong winds. Relentless: Scuba divers have searched large swathes of the stricken ship over the last five days as hopes fade of finding any more survivors. Looking: Divers have had to postpone their efforts several times amid fears they could become trapped. Shipwreck: The main concern for teams working on the ship is the weather, as although they have been lucky in the week following the tragedy the forecast is for strong winds. The ceremony of the'salute' (inchino) - where captains sail dangerously close to shore - appears to be widespread among captains of Italian cruiseships. TradeWinds publication reported 'it has become frequent in Italy to be dazzled by the sight of one of Costa's giant ships blaring its horns a few hundred metres from shore'. Italian environment minister Corrado Clini spoke out about the manoeuvre, and made it clear it will no longer be tolerated. The fear. is that it could whip up waves around the Concordia and shift it further. - even possibly dislodging it from the rock shelf it is resting on. Fire brigade spokesman Luca Cari said: 'The search was suspended at around midnight because movement was detected. 'It was thought best to pull the dive teams out and we are now deciding the best course of action.' The. revelation that Schettino had ordered dinner at 10.30pm - less than an. hour after the liner struck rocks at 9.41pm - was made by ship's cook. Rogelio Barista. Claims: Moldovan ballerina Domnica Cemortan (left) was seen 'cavorting' with captain Francesco Schettino on the bridge. Defended: Captain Francesco Schettino (left) has been defended by Domnica Cemortan (right) Illuminated: The Costa Concordia lights up the Tuscan coast last night as rescue efforts were suspended once again. He told Filipino TV station GMA: 'We wondered what was going on. 'At that time, we really felt something was wrong. 'The. stuff in the kitchen was falling off shelves and we realised how grave. the situation was. I have had 12 years of experience as a cook on a. cruise ship. 'I have even witnessed fires, so I wasn't that scared. But I did wonder, though, what the captain was doing. Why was he still there?' Investigators. are looking into the role played by the owner of the Costa Concordia as. the order to abandon ship was delayed by 68 minutes. They. are looking in particular at the requirement that, once the order is. given, each passenger is entitled to €10,000 euros in compulsory. compensation. It would mean a €30million bill for the 3,000 tourists. Costa Cruises declined to comment on the compensation allegations. But. it has labelled itself as an 'aggrieved party' in what could be one of. the biggest maritime claims in history. It has also suspended Captain. Francesco Schettino. Prosecutor Francesco Verusio has also said that the only suspects were Captain Schettino and the Second Officer Ciro Ambrosio. Suspension of the search comes after it was revealed Moldovan ballerina Domnica Cemortan, 25, was seen cavorting with Schettino on the bridge of the ship in the minutes before his cruise liner foundered on rocks. Italian authorities believe Schettino - dubbed Captain Coward - may have been distracted from his command by trying to impress the dancer who they now consider to be a 'key witness'. Miss Cemortan, who has a two-year-old daughter, had worked as a translator for the Concordia but was on holiday on the ship at the time of Friday night’s disaster. She has now defended the captain, who she told Moldovan TV had'saved over 3,000 lives'. She also said she was summoned to the bridge to help translate for the evacuation of Russian passengers. Prosecutor Francesco Verusio declined to comment on Italian media reports that Ms Cemortan was being sought as a witness. Schettino is under house arrest and faces. possible charges of manslaughter, abandoning ship and causing a. shipwreck. He admitted he made an unauthorised detour from the programmed route. that caused the vessel to slam into a reef and capsize off the Tuscan. island of Giglio. Hard at work: The ship is at risk of completely sinking in the next few hours. Yesterday an audiotape of the doomed vessel's first communications with maritime authorities showed the ship's officers continued to report only an electrical problem for more than 30 minutes after hitting the reef. Seven of the 11 dead were also identified yesterday by authorities - four French passengers, one Spanish and one Italian passenger and one Peruvian crew member. Italian passenger Giovanni Masia, who would have been 86 next week, was buried in Sardinia. Italian authorities have identified 32 people who have either died or are missing: two Americans, 12 Germans, seven Italians, six French, two Peruvians and one person each from Hungary, India and Spain | Summary: Crew member's plea came as luxury liner was fatally holed. Ship's cook claims captain ordered food nearly an hour after striking rocks. Moldovan ballerina 'cavorting' with Schettino on bridge defends captain. Investigators probe owner's role in 68-minute delay of abandoning ship. Search suspended as ship slipped slightly from its rocky resting place. Fears rose again that it could soon plummet 100m to bottom of sea. 11 confirmed dead, 21 still missing. | 2,186 | 120 | cnn_dailymail | en |
Write a title and summarize: Autochthonous cutaneous and visceral leishmaniasis (VL) caused by Leishmania martiniquensis and Leishmania siamensis have been considered emerging infectious diseases in Thailand. The disease burden is significantly underestimated, especially the prevalence of Leishmania infection among HIV-positive patients. A cross-sectional study was conducted to determine the prevalence and risk factors associated with Leishmania infection among patients with HIV/AIDS living in Trang province, southern Thailand, between 2015 and 2016. Antibodies against Leishmania infection were assayed using the direct agglutination test (DAT). DNA of Leishmania was detected by ITS1-PCR using the buffy coat. Species of Leishmania were also identified. Of 724 participants, the prevalence of Leishmania infection was 25. 1% (182/724) using either DAT or PCR assays. Seroprevalence of Leishmania infection was 18. 5% (134/724), while Leishmania DNA detected by the PCR method was 8. 4% (61/724). Of these, 24. 9% (180/724) were asymptomatic, whereas 0. 3% (2/724) were symptomatic VL and VL/CL (cutaneous leishmaniasis). At least five species were identified: L. siamensis, L. martiniquensis, L. donovani complex, L. lainsoni, and L. major. Multivariate analysis showed that CD4+ levels <500 cells/μL and living in stilt houses were independently associated with Leishmania infection. Those who were PCR positive for Leishmania DNA were significantly associated with a detectable viral load, whereas non-injection drug use (NIDU) and CD4+ levels <500 cells/μL were potential risk factors of Leishmania seropositivity. A magnitude of the prevalence of underreporting Leishmania infection among Thai patients with HIV was revealed in this study. Effective public health policy to prevent and control disease transmission is urgently needed. Co-infection of leishmaniasis and human immunodeficiency virus (HIV) is a major public health problem globally. Leishmania and HIV each promote the activation of the other, causing host immune impairment. The co-infection results in treatment failure, high relapse, and high mortality rate [1]. Meta-analysis has revealed that the direct agglutination test (DAT) gave high sensitivity and specificity for serodiagnosis of VL when compared to other serological tests [2]. However, low sensitivity of serological tests for VL diagnosis has been shown among these patients due to defective host immunity [3]. To increase the sensitivity of Leishmania DNA detection, the polymerase chain reaction (PCR) using blood samples has been suggested [4]. The internal transcribed spacer 1 (ITS1) -PCR method has been recommended to detect Leishmania DNA [5]. In Thailand, the first autochthonous VL case was documented in a 3-year-old girl living in a southern province in 1999 [6]. Until 2012, L. siamensis was firstly reported in a patient with HIV in Trang province. Since then, CL and/or VL have been sporadically reported in immunocompetent and immunocompromised patients predominantly in the south and north of Thailand and about 40% were patients with HIV/AIDS. L. martiniquensis was the predominant causative agent while L. siamensis was indigenously reported in only one Thai patient [7]. Information of the true prevalence of Leishmania infection among Thai patients with HIV, a high risk group, is still lacking. Thus, the objectives of this study were to determine the prevalence and the risk factors associated with Leishmania infection among patients with HIV/AIDS in Trang province, southern Thailand. A cross-sectional study of Leishmania infection was conducted between February 2015 and February 2016. Eligible participants were >18 years old and attending an HIV clinic, Trang Hospital, Trang province. They visited the clinic every 6 months for follow-up testing and to receive antiretroviral therapy (ART). They lived in ten districts of Trang province, other nine provinces located in the south, and three provinces in other regions of Thailand. Clinical information of participants was collected from patients’ medical records. Written informed consent was obtained from all participants. All participants were >18 years old. All analyzed data were anonymized. The research protocol was approved by the Ethics Committee of the Royal Thai Army Medical Department and the Ethics Committee of Mahidol University, Thailand. Eight milliliters of EDTA anti-coagulated blood samples were collected. The whole blood was centrifuged at 900 × g for 10 minutes to separate the plasma and buffy coat and was then kept at −20°C until further use. Seropositivity of Leishmania infection was defined as detection of antibodies in individuals who were exposed to Leishmania infection and being either symptomatic or asymptomatic. Asymptomatic Leishmania infection was defined as individuals who experienced no symptoms of VL but presented a positive test by DAT or PCR assays. Symptomatic VL was defined as individuals having a history of fever lasting at least 2 weeks with splenomegaly. One or more of the following clinical characteristics may be observed: hepatomegaly, weight loss, anemia, leucopenia, thrombocytopenia, and hypergammaglobulinemia. Detection of the parasites must be confirmed under microscopic examination or by PCR assay using any clinical samples (e. g., bone marrow aspirates, lymph node, blood, and/or other biopsy samples). Leishmania antibodies were assayed using the commercial DAT kit (Biomedical Research) according to the manufacturer’s instruction. The positive plasma control was obtained from confirmed VL cases using the PCR method. For the negative control, plasma from healthy individuals was used. The cutoff value of positive DAT titers was ≥1: 100 following manufacturer recommendation. DNA was extracted from 200 μL of buffy coat sample using Gen UP gDNA Kit (Biotech). Nested PCR was used to amplify the ITS1 region of the ribosomal DNA (rDNA) gene of Leishmania. In the primary PCR, primers LITSR and L5. 8S were used to amplify the 319–348 amplicons [8]. The newly designed secondary primers LITSR2 (CTG-GAT-CAT-TTT-CCG-ATG-ATT) and L5. 8S inner (GTT-ATG-TGA-GCC-GTT-ATC-C) generated 230–280 amplicons depending on Leishmania species. PCR reactions were performed using the MJ Mini thermal cycler (BioRad) in volumes of 25 μL, containing 12. 5 pmol of each primer, 0. 2 mM dNTP, 1. 5 mM MgCl2,1× PCR buffer, 1 U of Taq DNA polymerase, and 4 μL of DNA template. DNA of L. martiniquensis promastigotes (MHOM/MQ/92/MAR1) was used as the positive control. The condition was started by pre-denaturation at 94°C for 3 minutes followed by 35 cycles: denaturation at 94°C for 1 minute, annealing temperature at 54°C for 30 seconds, and extension at 72°C for 30 seconds. Final extension was at 72°C for 5 minutes. PCR products were separated by electrophoresis in 1. 5% agarose gel stained with SYBR Safe (Invitrogen). The results were visualized and documented by Molecular Imager Gel Doc XR+ System with Imager Lab 3. 0 (BioRad). Positive PCR products were sent to U2Bio Co. Ltd., South Korea for sequencing. Chromatograms were validated using BioEdit version 7. 0. 1. The sequences were multiple-aligned with reference Leishmania strains retrieved from GenBank. The phylogenetic tree was constructed by using the neighbor-joining (NJ) method using the MEGA program, version 7. 0. The reliability was tested by 1,000 bootstrap replications and Tajima-Nei was selected for the DNA substitution model of phylogenetic analysis. To determine the risk factors and outcomes of Leishmania infection, standardized questionnaires were used. Enrolled subjects with HIV were interviewed face-to-face covering demographic data, socioeconomic status, clinical symptoms, and associated risk behaviors. The association between potential risk factors and Leishmania infection was assessed by univariate and multivariate logistic regression analysis. Odds ratios and 95% confidence intervals (CI) were calculated and p values <0. 05 were considered statistically significant. All analyses were performed using STATA, version SE14 (Stata Corporation, College Station, TX, USA). (http: //dx. doi. org/10. 17504/protocols. io. j2dcqa6) A total of 724 participants with HIV were enrolled in this study. Of these, 643 (88. 8%) filled out questionnaires. Living areas of participants were as follows: 570 (88. 6%) lived in Trang province and 67 (10. 4%) lived in nine other provinces located in the south. Only five (0. 8%) were from other regions of Thailand. The mean age was 43. 6 ± 8. 5 years. The characteristics of the enrolled subjects are shown in Table 1. Their clinical characteristics and risk behaviors during the past one year (S1 and S2 Tables) included history of injection drug users (IDUs) at 16. 6% while 13. 7% were non-injection drug users (NIDUs). A total of 512 (79. 6%) subjects lived in non-stilt houses while 131 (20. 4%) lived in stilt houses. Most of the individuals (68. 6%) had CD4+ levels more than 500 cells/μL and only 9. 6% were less than 200 cells/μL. Three categories of data analysis of the prevalence of Leishmania infection were performed (Table 1). The first group comprised patients who were either seropositive by DAT analysis with titers of ≥100 or positive by PCR assay. The second category involved patients who were seropositive by DAT analysis with titers of ≥100, and the last group comprised patients who were positive only by PCR assay. The prevalence of Leishmania infection using positive results either by DAT or PCR assays was 25. 1% (182/724). Seropositive cases comprised 18. 5% (134/724) while Leishmania DNA detection by PCR was 8. 4% (61/724). Only 1. 8% (13/724) were positive using both methods (Table 2). Table 3 shows numbers of positive Leishmania infection using DAT, the titer of DAT, and PCR. Thus, the overall prevalence of asymptomatic Leishmania infection was 24. 9 (180/724). Tables 4 and 5 show affected areas of Leishmania infections. Regarding the analysis of those who were either seropositive by DAT or positive by PCR assay, the prevalence of Leishmania infection significantly differed among participants who lived in stilt houses compared to those living in non-stilt houses (p = 0. 03), those who developed jaundice (p = 0. 02), having opportunistic infection (p = 0. 002) especially tuberculosis (p = 0. 001), and those having low CD4+ levels <500 cells/μL (p = 0. 003) (Fig 1). No significant difference was found among age group, gender, educational level, occupation, working outdoors at night, average income, years of HIV diagnosis, viral load, history of going abroad, drug use (IDUs/NIDUs), pet/animal owner, animal shed nearby the house, plantation nearby the house, and bed net use (S1 and S2 Tables). Two patients showed symptomatic VL. The first case was a 39-year-old herdsman living in Phuket province. He had a history of NIDU. He presented with a fever for more than two weeks. Many nodules were observed on the trunk. Laboratory findings revealed CD4+ levels at 173 cells/μL with an undetectable viral load. The DAT titer was 1: 6400. The causative agent was L. martiniquensis, which was identified by the nested ITS1-PCR using the buffy coat and skin biopsy. The patient was treated with amphotericin B. However, he died of disease progression one year after initial VL diagnosis. The second case involved a 41-year-old male who originally lived in Trang province. He worked on a rubber plantation. He was both an IDU and NIDU. He developed epistaxis and bleeding gums. Laboratory findings revealed pancytopenia and CD4+ levels of 622 cells/μL with an undetectable viral load. Intracellular amastigotes were observed from the lymph node biopsy with Giemsa stain. The DAT titer was 1: 6400 and the causative agent was L. martiniquensis, which was identified using the nested ITS1-PCR of the buffy coat. The patient was treated with amphotericin B but he died within one week after treatment. Univariate and multivariate analysis of risk factors for acquiring Leishmania infections by DAT titers of ≥100 or positive by PCR assays are shown in Table 6. Univariate analysis showed that participants who lived in stilt houses had higher risk (OR = 1. 58,95% CI = 1. 04–2. 39) of contracting Leishmania when compared with those living in non-stilt houses. CD4+ levels between 200 and 500 cells/μL were at higher risk than those who had CD4+ levels >500 cells/μL (OR = 1. 70,95% CI = 1. 29–2. 97). After adjusting for age, gender, NIDUs, history of traveling abroad, pet owners and raising animals in housing areas, bed net use, animal shed and plantation nearby the house, underlying diseases, viral load, and duration of HIV diagnosis, multivariate logistic regression analysis revealed that those living in stilt houses had greater risk (OR = 1. 60,95% CI = 1. 04–2. 47) of acquiring the infection when compared with those living in non-stilt houses. In addition, those who had CD4+ levels 200–500 cells/μL (OR = 2. 13,95% CI = 1. 36–3. 32) and <200 cells/μL (OR = 1. 98,95% CI = 1. 06–3. 73) had higher risk of contracting Leishmania than those who had CD4+ levels >500 cells/μL. In addition, multivariate analysis of the associated risk factors of Leishmania infection using only seropositivity showed that participants who were NIDU had higher risk of presenting Leishmania seropositivity than those who were not (OR = 2. 23,95% CI = 1. 27–3. 92). In addition, those who had CD4+ levels 200–500 cells/μL were also at higher risk of being seropositive than those who had CD4+ levels >500 cells/μL (OR = 2. 09,95% CI = 1. 27–3. 44) (S3 Table). Multivariate analysis of the associated risk factors of Leishmania infection using only positive PCR results showed that those who had a detectable viral load >50 copies/mL were at higher risk of acquiring detectable Leishmania DNA in the blood (OR = 2. 31,95% CI = 1. 01–5. 29) than those who had an undetectable viral load after adjusting for those variables as mentioned above (S4 Table). Of 61 samples, nucleotide sequencing was successful for 49. These sequences, together with 16 reference sequences of different Leishmania species, were included to construct the phylogenetic tree using the NJ method (Fig 2). The phylogenetic analyses grouped the sequences into five separated clades. The majority of the samples (20 (40. 8%) and 13 (26. 5%) ) were closely related to L. siamensis and L. martiniquensis, respectively, whereas ten (20. 4%) were closely related to L. donovani complex, five (10. 2%) were related to L. lainsoni, and one (2. 1%) was related to L. major. This was the first study providing important information of the prevalence of co-infection of Leishmania among Thai patients with HIV who had been regularly attending the HIV clinic in Trang province. The prevalence of Leishmania infection was approximately one-fourth of the 724 participants determined by either DAT or PCR assays. In the past, Leishmania infections were previously reported in five provinces in the south (Surat Thani [6], Phang-Nga [9], Trang [10], Songkhla [11], and Satun [12]) where most people mainly earn their living in agricultural sectors. The climate and humidity in the south are suitable for the sand fly’s habitat where potential sand fly vectors have been reported [13,14]. Our study revealed four new affected areas of Leishmania infections in the south (Phuket, Krabi, Nakhon Si Thammarat, and Phatthalung provinces). Thus, at present, overall affected areas cover nine southern provinces. For VL/HIV co-infection, the sensitivities of DAT to detect antibodies against Leishmania infection were 50–84% [15]. Related studies of VL in immunocompetent subjects in endemic areas in India and Iran used DAT titers at different cutoff values ranging from 1: 800 to 1: 3200 [16–18]. A cutoff value at a titer of 1: 100 was previously used to screen VL among HIV-positive patients who developed clinical symptoms in northeast Iran [19]. A low DAT titer of 1: 200 was also detected in an immunocompetent VL Thai patient caused by L. martiniquensis [9]. In this study, one patient having a DAT titer of 1: 100 also produced a positive PCR result. Therefore, a positive serological test at low titers could have diagnostic value to detect the infection. Thus, the cutoff values of DAT varied when conducted in different study populations as well as areas of study where cross-reactivity of DAT against other blood parasite infections could have occurred. A systematic review revealed that DAT titers detected in symptomatic patients were higher than those of asymptomatic patients [20]. Patients presenting very high DAT titer would have significantly greater disease progression than those presenting low titers [21]. In this study, clinical characteristics of VL were observed in two symptomatic cases that had DAT titers of 1: 6400 together with positive PCR results. Thus, a close follow-up for those asymptomatic infections showing positive results of DAT or PCR is needed. In this study, serological and molecular diagnosis among individuals with HIV was not in concordance with other studies [22]. Our results showed that PCR positivity was low when compared with numbers of DAT positivity. The potential reasons among DAT positive individuals who might become PCR negative could include degradation and clearance of Leishmania DNA after infection, which corresponds to development of protective immunity due to the use of antiviral drugs for HIV. A positive PCR test among DAT negative individuals could occur when the individual was bitten by a Leishmania infected sand fly, but either immunity has not yet developed or antibody levels are too low to be detected by the methods employed, especially in HIV-positive populations [23]. Before using HAART, asymptomatic infection in HIV-Leishmania co-infection in Europe was 4–33% [24]. However, the incidence has been reduced to 20% after ART drugs had been given to all individuals with HIV [3]. In this study, all patients with HIV regularly received HAART treatment that could restore TH1 cytokine and antibody production [25]. HAART-treated HIV patients demonstrated a better ability to control Leishmania infection [26]. Many patients with subclinical VL did not develop clinical symptoms after taking HAART medications while some developed the disease [3]. Other risk factors (e. g., stage of HIV infection, parasite virulence, drug resistance, nutritional status, age, and gender) may be involved in disease progression [1,27]. Prospective studies are needed to determine disease progression in this population. In this study, most enrolled participants (88. 6%) were not randomly selected and originally lived in Trang province. Thus, our results do not represent the prevalence of co-infection of Leishmania and patients with HIV of the country, and they do not represent the prevalence in each district of Trang province. However, this study showed the magnitude of the seroprevalence and significant numbers of subclinical results of Leishmania DNA detection circulating in the blood in Thai individuals with HIV. In southern Europe, IDUs were the most important risk factor accounting for more than 90% of all cases [28]. Our results showed no significant difference in prevalence among IDUs, while seroprevalence was associated with NIDUs. Leishmaniasis was also associated with socioeconomic status. In India, housing materials such as mud, plants, and earthen floors were risk factors for VL [20,29]. Our study showed that living in stilt houses was an independent associated risk factor for Leishmania infection. The presence of stilts provided an open area under the house that might have increased chances of sand fly bites to humans as well as providing resting sites for sand flies. Sand flies frequently bite at dusk [30] when most people spend their time at the open area of the house. In addition, CD4+ levels play an important role to protect the host from opportunistic infections. Related studies showed that the first episode of symptomatic VL diagnosis involved more than 80% of patients with HIV who had low CD4+ levels [26,31]. Our results also confirmed that low CD4+ levels (<200 cells/μL) as well as 200 to 500 cells/μL significantly increased the risk of Leishmania infection. In this study, Leishmania DNA detection by PCR assay was associated with detectable viral load. No correlation has been reported between PCR positivity and CD4+ levels, whereas the correlation between HIV viral load and parasitemia was observed among asymptomatic patients [22]. Clinical progression of HIV/AIDS was simultaneously promoted by VL. HIV infection enhances parasite growth by modulating significant cytokine response to Leishmania while the parasite upregulates viral expression [3]. Public health awareness of Leishmania infection in Thailand started when one autochthonous VL was reported in 1996. However, at that time, the disease was uncommon as well as unfamiliar to most physicians, which could have led to a lot of underreporting of leishmaniasis cases in the past 20 years. Using the molecular method, this cross-sectional study was the first to systematically estimate the prevalence of Leishmania infection among patients with HIV, a high risk group, revealing not only L. siamensis and L. martiniquensis infection but also infections of other species (e. g., L. donovani complex, L. lainsoni, and L. major) that already existed in the affected area. From the phylogenetic analysis, the ITS1 region is one of the more powerful targets used to discriminate the Leishmania species [32]. Our previous study showed that the ITS1 region had the highest sensitivity to detect L. martiniquensis and L. siamensis compared with the other genes: hsp70, cyt b, and SSU-rRNA [5]. Additionally, we used hsp70-PCR and kDNA-PCR to amplify DNA samples of L. major, L. donovani, L. lainsoni, L. siamensis, and L. martiniquenensis. Unfortunately, negative PCR results were obtained due to lower sensitivities of PCR amplification using these genes. L. martiniquensis and L. siamensis were the predominant species detected in this study. Additionally, L. infantum (which causes VL) was previously reported in an HIV negative individual living in Bangkok [33]. The L. donovani complex species are the major causative agents of VL worldwide. The distribution of the complex species might have been introduced by travelers or workers from VL-endemic areas to Thailand [34]. L. major, a causative agent for CL in the Old World, causes zoonotic transmission, especially Afghanistan and India [31]. VL, caused by L. major, has occasionally been reported among patients with HIV [3]. L. donovani complex species and L. major have been reported in China, Bangladesh, India, and Nepal [15]. Thus, distribution of these two species in Thailand could be possible. L. lainsoni infection causes localized CL and was reported in South America in Bolivia, Peru, Suriname, French Guiana, and Brazil [15,35]. This is the first report of L. major and L. lainsoni infection among individuals with HIV in Thailand. In addition, no report of L. lainsoni has been documented in the Old World, especially among patients with HIV. To prevent and control VL, understanding disease epidemiology is extremely important. A cohort study conducted in this population as well as studies of potential vectors and animal reservoirs are needed. Moreover, large scale molecular epidemiological studies in other high morbidity areas are required for this emerging disease. | Title: Prevalence and risk factors associated with Leishmania infection in Trang Province, southern Thailand Summary: Visceral leishmaniasis (VL) in Thailand is caused by two causative agents, Leishmania martiniquensis and Leishmania siamensis. A public health concern brought us to investigate the magnitude of Leishmania infection among individuals with HIV living in an affected area, Trang province, southern Thailand. The results showed a high seroprevalence of Leishmania infection. Using PCR-based technique, DNA detection in the buffy coat was 8. 4%, and 1. 8% were symptomatic VL. Asymptomatic Leishmania infection could play an important role in disease transmission. Risk factors associated with Leishmania infection among Thai patients with HIV were firstly described. Those who were NIDU and lived in stilt houses were associated with Leishmania infection. Individuals who had lower immunity with detectable viral load were more likely to contract the infection. Interestingly, not only L. martiniquensis and L. siamensis were identified but also L. donovani complex, L. major, and L. lainsoni, were firstly reported among indigenous Thai people. These findings could lead to effective intervention and prevention methods to control leishmaniasis in Thailand. Further studies are needed to investigate the disease development in asymptomatic infections as well as evaluate the prevalence in other regions of Thailand. | 6,000 | 301 | lay_plos | en |
Summarize: BACKGROUND This invention relates to training devices for sports involving balls which are thrown or projected in some other manner, such as baseball, tennis, lacrosse, and golf, among other things. The strong, patriotic tradition of baseball in the United States has led to the creation of various training devices for the particular benefit of baseball players. Baseball throwing, fielding and pitching training devices are desirable for a number of applications. Many coaches have a number of individuals to train simultaneously, and a training device can allow for more practice for the trainees without requiring constant one on one interaction with the coach. This not only increases efficiency, but prevents injury to the coach that can result from overworking certain muscle groups, such as a throwing arm. By incorporating the use of a training device into their routine, coaches can enable or provide the same amount of practice to their trainees without requiring undue physical exertion on the coach's part. Even a coach who has teammates practice between each other still has use for these devices. Some players will have substantially more or less skill level than other players, and to meet their individual needs, the coach can incorporate a training device into the training to either help the trainee continue to excel beyond the level of the others, or to allow this individual to work at his or her own pace to reach the level of the other players. These training devices are also desirable for players who do not have access to another individual with whom they can practice. Whether for financial reasons, or as a result of social or family circumstances, some individuals cannot utilize another individual in practice. Instead, they are required to use a training device to hone their skills. There are many reasons for using pitching or throwing training devices. Two are particularly relevant to the present invention. First, players need to refine their accuracy, especially pitchers with regards to pitching. Pitchers can make or break a game for the entire team depending on a margin of error that could be less than an inch. Other players also need accuracy training, because the infielder who throws just a little too high over the head of the first baseman, or cannot field the ball properly, will not receive jovial cheers in the post game celebration. Second, players need to build up the strength and endurance of their throwing arm, in order to throw farther and faster in the game. A pitcher with razor-sharp accuracy is virtually worthless if his arm gets tired after a few pitches. Likewise, the infielder who cannot throw the ball to the first baseman fast enough will never get the runner out, no matter how accurate his throw. Thus, in baseball training, players need to utilize not only those exercises that generate accuracy, but also exercises that increase strength and endurance and improve reflexes. With regards to exercises that utilize training devices, some exercises require a device which will absorb the kinetic energy of a thrown ball, and simply let it fall to the ground without any rebound. For instance, a pitcher is vulnerable to a ricocheting ball in the moments after releasing the pitch, and it is desirable that, for the purposes of practice, the pitcher not be subject to the risk of getting knocked in the head with every returning pitch. Other exercises require a device to rebound the ball with substantially the same amount of force with which it was thrown. Ball stopping training devices are known in the art. For example, see U.S. Pat. No. 5,573,240, issued to Geoffrey Humboldt on Nov. 12, 1996, for a baseball backstop for pitching training; U.S. Pat. No. 5,002,274, issued to Mark D. Bidema on Mar. 26, 1991, for a baseball batting practice device; U.S. Pat. No. 5,088,740, issued to Leroy L. Peterson on Feb. 18, 1992, for a practice backstop for ball playing sports; U.S. Pat. No. 5,333,8561, issued to Jonathan S. Gery on Aug. 2, 1994, for a pitching practice apparatus. None of the existing ball stopping devices include any mechanism whereby a ball will rebound to the person throwing it. Ball rebounding training devices are also known in the art. For example, see U.S. Design Pat. No. D462, 733, issued to Christopher Smith on Sep. 10, 2002, for a baseball rebounder; U.S. Patent Application No. 2004/0178585 A1, by Anthony Consenza, on Mar. 14, 2003, for a strike zone for return throw pitching assemblies. The existing ball rebounding devices do not include any mechanism whereby the kinetic energy of a ball will be absorbed, allowing the ball to simply drop to the ground. It is inconvenient to maintain two different devices for these various exercises. Not only will they require substantial space to use, even when placed immediately next to each other, they are also difficult to transport to such places as a practice field, or a game for warm-ups. Furthermore, the expense of maintaining two devices for all of the players on a team becomes too costly for a coach or team to bear. The cost may also be too high for an individual whose family is far enough below median income levels, thus preventing them from practicing necessary exercises at home on their own time. SUMMARY OF THE INVENTION The present invention provides a training device for ball sports that has both a rebounding mat and a stopping mat mounted to the same frame, which allows those using the invention to practice strength, endurance and/or reflex training, or to work on accuracy training, without having to use more than one device. An optional feature of this invention provides a combination ball rebounding and stopping training device that is transportable for use in places other than a fixed location. Another optional feature of one embodiment provides a single device which may be used for both ball rebounding exercises and ball stopping exercises, which has a smaller footprint than the two separate devices that would otherwise be required to practice these exercises. The prior art does not include a combination device which has both a ball stopping mat and a ball rebounding mat. All of the prior art includes frames that have some kind of brace or other device that creates a substantial footprint that, in order to maintain the stability of the device, cannot be impinged upon by the frame of another device. None of the prior art devices have a mechanism whereby a ball stopping apparatus may be easily combined with a ball rebounding apparatus in the same footprint. Thus, if an individual using a device from the prior art wishes to alternate between exercises that involve a ball stopping mat and exercises that involve a ball rebounding mat, they must use two separate devices that have sizeable, independent footprints. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a front perspective view of a preferred embodiment of the pitching and throwing training device. FIG. 2 is a side view of the pitching and throwing training device shown in FIG. 1. FIG. 3 is a rear perspective view of the pitching and throwing training device shown in FIG. 1. FIG. 4 is a front fragmentary view of the pitching and throwing training device shown in FIG. 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT While it is possible to incorporate the teachings of the present invention into various devices in a number of ways, FIGS. 1-4 show a preferred embodiment of a training device for ball sports constructed according to the teachings of the present invention, with the understanding that the present disclosure is not intended to be limited to the specific constructions illustrated in the drawings. The preferred embodiment shown in the figures has a rectangular frame 100 which is made of any suitable material, i.e. metal, wood or plastic. One preferred material for the frame is metal tubing, such as aluminum, which is both lightweight and strong. The frame 100 includes a horizontal top cross bar 102, made of metal tubing, having a left end 102 a and a right end 102 b. The frame 100 also includes left and right vertical supports 106 and 108 having upper ends 106 a, 108 a and lower ends 106 b, 108 b. The left and right vertical supports 106, 108, may consist of a series of separate sections of tubing that can be disassembled into shorter lengths for shipping. The upper ends 106 a, 108 a of the left and right vertical supports 106, 108 are connected to the left and right ends 102 a, 102 b of the horizontal top cross bar 102, respectively, at rounded L-shaped joints 120, 121. The L-shaped joints 120, 121 are also made of metal tubing. The frame 100 also has horizontal bottom crossbar 104, made of metal tubing, having a left end 104 a and a right end 104 b. The horizontal bottom crossbar 104 is connected to the lower ends 106 b, 108 b of the left and right vertical supports 106, 108, respectively. The horizontal bottom crossbar 104 is connected to the vertical supports 106, 108 in a manner parallel to the top cross bar 102. The frame 100 also has horizontal midsection crossbar 110, made of metal tubing, having a left end 110 a and a right end 110 b. The horizontal midsection crossbar 110 is connected to a midsection of the left and right vertical supports 106, 108, respectively, between the lower ends 106 b, 108 b of the left and right vertical supports 106, 108, respectively, and the upper ends 106 a, 108 a of the left and right vertical supports 106, 108, respectively. It is preferable that the horizontal midsection crossbar 110 be attached to the left and right vertical supports 106, 108, approximately halfway between the upper ends 106 a, 108 a and the lower ends 106 b, 108 b, of the left and right vertical supports 106, 108, respectively. The horizontal midsection crossbar 110 serves as the base of an upper frame 130 that supports a rebounding mat 200, and receives and secures the same by springs 210, a plurality of which are spaced at substantially similar intervals 209 apart from each other, and are attached between a bottom edge 200 a of the rebounding mat 200 and the horizontal midsection crossbar 110. More specifically, the springs 210 are made from metal, similar to the springs in a trampoline, and include a first hook end 211 which is connected into to the rebounding mat 200 through a metal loop 212 which is sewn into the rebounding mat. They also include a second hook end 213, which is connected into the frame 100 through a hole 214 in the frame 100. The springs 210 also continue at intervals between a left edge 200 b of the rebounding mat 200 and the portion of the left vertical support 106 above the horizontal midsection crossbar 110, between a top edge 200 c of the rebounding mat 200 and the horizontal top crossbar 102, and between a right edge 200 d of the rebounding mat 200 and the portion of the right vertical support 108 above the horizontal midsection crossbar 110, in a manner that the rebounding mat 200 is, in general, evenly suspended across the portion of the frame 100 above the horizontal midsection crossbar 110, and is pulled or tensioned into the position shown in FIG. 1. The rebounding mat 200 will thus have the attribute of causing a thrown ball that contacts it to bounce back in the direction from which the ball was thrown with substantially the same force and velocity with which the ball was thrown. In other words, rebounding mat 200 has the attribute of translating the kinetic energy of a thrown ball at the initial impact of the ball with the rebounding mat 200 into a reflection of such. The rebounding mat 200 may be made of various durable materials, but it is preferred that it be constructed of plastic similar to that used in the construction of conventional trampoline mats. The metal loops 212 are preferably made of the same metal as the springs 210. A different embodiment could include a plurality of ring and grommet fasteners to support the rebounding mat 200 at a tension that will cause the same elastic reflection of the kinetic energy of a thrown ball. The rebounding mat 200 is generally about thirty six inches wide and forty eight inches tall, and may also have some kind of decal or target zone painted or marked on its face. The rebounding mat 200 also has an apron 244 sewn around its periphery which may be pulled across the springs 210, to provide a cover for them. A series of straps 245 extend out from the apron, which may be used to firmly place the apron 244 over the springs 210. This apron 244 protects the springs from wear caused by rust from rain, snow or other moisture caused by weather. The apron further has the effect of diminishing the number of dangerous ricochets that may occur from an aberrantly thrown ball. The preferred embodiment further includes a ball stopping mat 300 that is made of a heavy material, preferably thick natural rubber that is of high quality and durability, that has relatively high friction to prevent slippage of a ball on contact. The ball stopping mat 300 hangs below the horizontal midsection crossbar 110 in a lower frame 140 section of the frame 100. The horizontal midsection crossbar 110 acts as the top of the lower frame 140. The ball stopping mat 300 is attached to the horizontal midsection crossbar 110 by a series of fasteners 310, which may consist of a series of straps, clips, or bolts, for example plastic cable ties, or nylon or dyneema webbing. It is preferred that the fasteners 310 be straps made of nylon webbing, which will tend not to damage the ball stopping mat 300 upon the repeated impacts of balls over time. In this preferred embodiment, the ball stopping mat 300 is not attached to the left and right vertical supports 106, 108, and is also not attached to the horizontal bottom crossbar 104. The ball stopping mat 300 is allowed to hang flaccidly from the horizontal midsection horizontal crossbar 110, which facilitates the absorption of the kinetic energy of the thrown ball. The ball stopping mat 300 may alternatively be attached to all four sides of the lower frame 140, including the left and right vertical supports 106, 108, and the horizontal bottom crossbar 104. This is not preferred, however, because the ball stopping mat 300 will be subject to excess wear, and will likely experience tearing around the fasteners 310. In the preferred embodiment, the ball stopping mat 300 has the approximate dimensions of thirty six inches in height and thirty six inches in width, and about one half of an inch thick. Moreover, the ball stopping mat 300 may be perforated with holes, preferably one inch in diameter, evenly spaced in a pattern, as long as the perforation does not substantially weaken the ability of the ball stopping mat 300 to withstand the repeated impacts of thrown balls. The preferred embodiment also includes a strike zone 399 painted on the ball stopping mat 300, substantially in the center of the ball stopping mat 300 using a color which contrasts with the color of the ball stopping mat 300. The strike zone 399 is approximately twelve inches high and twelve inches in width. The preferred embodiment has a frame brace member 150 which extends behind the frame 100 from a point above the horizontal midsection crossbar 110 to the ground at such an angle as to provide sufficient support to the ball training device. This may be an angle from fifteen degrees to approximately seventy degrees away from either the left and right vertical supports 106, 108. The frame brace member 150 is also preferably made from metal tubing, and consists of a left brace support 126 and a right brace support 128, with a horizontal brace bottom crossbar 124 extending between them. The horizontal brace bottom crossbar 124 makes contact with the ground simultaneously with the horizontal bottom crossbar 104, which in the preferred embodiment creates a compact footprint for the training device, allowing it to be utilized in a smaller space than would be required for two separate devices. The frame 100 may alternatively have a unique pivot point along the axis of the horizontal midsection crossbar 110, which enables it to be folded substantially in half. This may be accomplished simply by attaching the ball stopping mat 300 pivotally to the horizontal midsection crossbar 110, and at the same time by creating points on the left and right vertical supports 106, 108 that will allow them to be disassembled at some point along their length which is near the horizontal midsection crossbar 110. This allows the training device for ball sports to be shipped more easily, and to be more transportable by the coach or player who may be using it in different locations at various times. For example, a coach may take it from home to a practice field where the trainees will use it. The foldability of the device will be desirable in this circumstance. The frame 100, may also include a tilt adjustment mechanism 199 which allows the frame to be tilted at a less or more vertical angle, depending on the rebound angle that is desired for exercises utilizing the rebounding mat 200. The tilt adjustment mechanism 199, utilizes the same technology that is available in the prior art. It may incorporate a cotter pin with different possible placements upon which the frame brace member 150 may rest in a stable position, or may involve a clamp that may be tightened to fix the frame at the desired angle. Alternatively, it may incorporate any of the tilt adjustment mechanisms that are adaptable to the present invention. Detailed descriptions of the preferred embodiment are provided herein. It is to be understood, however, that the present invention may be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in virtually any appropriately detailed system, structure or manner. While the invention has been described in connection with a preferred embodiment, it is not intended to limit the scope of the invention to the particular form set forth, but on the contrary, it is intended to cover such alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims. | Summary: A training device for ball sports includes a frame member which supports both an elastic surface for rebounding a ball, along with an inelastic surface which absorbs the kinetic energy of the ball and allows it to drop to the ground, which is designed as a combination apparatus that includes both a ball rebounding surface and a ball stopping surface, in order to provide a variety of training activities and which may be transported easily by a coach or an individual using it for practice. | 4,531 | 98 | big_patent | en |
Write a title and summarize: GWAS of prostate cancer have been remarkably successful in revealing common genetic variants and novel biological pathways that are linked with its etiology. A more complete understanding of inherited susceptibility to prostate cancer in the general population will come from continuing such discovery efforts and from testing known risk alleles in diverse racial and ethnic groups. In this large study of prostate cancer in African American men (3,425 prostate cancer cases and 3,290 controls), we tested 49 risk variants located in 28 genomic regions identified through GWAS in men of European and Asian descent, and we replicated associations (at p≤0. 05) with roughly half of these markers. Through fine-mapping, we identified nearby markers in many regions that better define associations in African Americans. At 8q24, we found 9 variants (p≤6×10−4) that best capture risk of prostate cancer in African Americans, many of which are more common in men of African than European descent. The markers found to be associated with risk at each locus improved risk modeling in African Americans (per allele OR = 1. 17) over the alleles reported in the original GWAS (OR = 1. 08). In summary, in this detailed analysis of the prostate cancer risk loci reported from GWAS, we have validated and improved upon markers of risk in some regions that better define the association with prostate cancer in African Americans. Our findings with variants at 8q24 also reinforce the importance of this region as a major risk locus for prostate cancer in men of African ancestry. Genome-wide association studies (GWAS) have revealed more than 30 variants that contribute susceptibility to prostate cancer, with most of the discoveries having been made in populations of European ancestry [1]–[14]. However, as so far observed for most common diseases, variants identified through GWAS are of low risk both individually and in aggregate, and therefore provide only limited information about disease prediction [15], [16]. Most risk variants for prostate cancer are located outside of annotated genes, with some positioned in gene poor regions and some regions harboring more than one independent signal [1], [10], [14], [17], [18]. Thus, for the vast majority of risk loci, the identity, frequency and risk associated with the underlying biologically relevant allele (s) are unknown. The risk variants revealed through GWAS have also been found to vary in frequency across racial/ethnic populations [19]. Even in the absence of functional data, the associated risk variants may highlight a genetic basis for differences in disease risk between populations, such as at 8q24 where genetic variation is suggested to contribute to population differences in risk of prostate cancer [10]. Testing of the risk variants and fine-mapping in diverse populations will help to identify and localize the subset of markers that best define risk of the functional allele (s) within known risk loci, as well as to determine their contribution to racial and ethnic differences in prostate cancer risk. In the present study, we tested common genetic variation at the prostate cancer risk loci identified in men of European and Asian descent in a large sample comprised of 3,425 African American prostate cancer cases and 3,290 controls, to identify markers of risk that are relevant to this population. More specifically, we conducted GWAS and imputation-based fine-mapping of each risk locus to both improve the current set of risk markers in African Americans as well as to identify new risk variants for prostate cancer. We then applied this information to model the genetic risk of prostate cancer in African American men. The African American prostate cancer cases (n = 3,621) and controls (n = 3,502) in this study are part of a collaborative genome-wide scan of prostate cancer that includes 11 individual studies (Table S1, Methods). Samples were genotyped using the Illumina Infinium 1M-Duo bead array, and following quality control exclusions (see Methods), the analysis of variants at the known risk loci was performed on 3,425 cases and 3,290 controls. The ages of cases and controls ranged from 23 to 95, with cases and controls having similar ages (mean 65 and 64 years, respectively). We tested 49 known prostate cancer risk variants located in 28 risk regions (Table S2, Table 1, and Table 2); 43 SNPs were directly genotyped (with call rates >95%), while 6 were imputed with high accuracy (see Methods) [1], [3], [4], [6]–[14], [17], [18], [20]–[23]. The minor allele frequencies (MAF) of all 49 variants were common (≥0. 05) in African Americans, except for rs721048 at 2p15 (MAF, 0. 04) and rs12621278 at 2q21 (MAF, 0. 02; Table 1, Figure 1). On average, across all variants tested, the risk allele frequencies (RAFs, i. e. alleles associated with an increased risk of prostate cancer in previous GWAS) were 0. 05 greater in African Americans than in Europeans. However, when removing the 12 risk variants at 8q24 (Table 2) the average difference in RAF over the remaining risk loci was only 0. 03. We examined the association of local ancestry with prostate cancer risk at each of the 28 risk regions (Table S3). In addition to 8q24, which we had previously found to be strongly associated with African ancestry [5] (OR per European chromosome = 0. 81, p = 4. 7×10−5), we observed significant associations at 22q13 (OR = 0. 88, p = 0. 01), 7p15 (OR = 1. 16, p = 1. 6×10−3) and 10q26 (OR = 1. 14, p = 6. 2×10−3). To address the potential for confounding by genetic ancestry, we adjusted for both global and local ancestry in all analyses (see Methods). In previous GWAS, the index signals outside of 8q24 had very modest odds ratios (1. 05–1. 30 per copy of the risk allele) and our sample size provided ≥80% power to detect the reported effects for 24 of the 37 variants (at p<0. 05; Table S2). We observed positive associations with 28 of the 37 variants (odds ratios (OR) >1) in African Americans and 18 reached nominal statistical significance (p≤0. 05; Table 1). Results were similar without adjustment for local ancestry in each region (Table S4). Of the 19 variants that were not replicated at p<0. 05, power was <80% for 9 of the variants. While power was limited to detect associations at some loci, the lack of replication at loci where power was acceptable (>80%) suggests that the particular risk variant revealed in GWAS in European and Asian populations may not be adequately correlated with the biologically relevant allele in African Americans. In an attempt to identify a better genetic marker of the biologically relevant allele in African Americans we conducted fine-mapping across all risk regions using genotyped SNPs on the 1 M array and imputed SNPs to Phase 2 HapMap (Table S5, see Methods). If a marker associated with risk in African Americans represents the same signal as that reported in the initial GWAS, then it should be correlated to some degree with the index signal in the initial GWAS population. Using HapMap data (CEU or JPT+CHB depending upon the initial GWAS population) we catalogued and tested all SNPs that were correlated (r2≥0. 2) with the index signal (within 250 kb), applying a significance criteria αa, of 0. 004 given the large number of correlated tests. This level of significance was based on the number tag SNPs in the HapMap YRI population that capture (r2≥0. 8) all SNPs that were correlated with the index signal in the HapMap CEU (r2≥0. 2; see Methods). We also looked for novel independent associations, focusing on the genotyped and imputed SNPs that were uncorrelated with the index signal in the initial GWAS populations. Here, we applied a Bonferroni correction for defining novel associations as significant in each region, with αb estimated as 0. 05/the total number of tags needed to capture (r2≥0. 8) all common risk alleles across all risk region in the YRI population (αb = 5. 6×10−6). This is similar to the genome-wide-type correction of 5×10−8, which accounts for the number of tags needed to capture all common alleles in the genome. For each region, stepwise regression was used with SNPs kept in the final model based on αa or αb (results for each model are provided in Table S6). Among the SNPs correlated with the index signal in the GWAS population, a more significantly associated marker was identified at 12 regions. For 5 of these regions, the new marker showed only a slightly more significant association than the index signal (<1 order of magnitude change in the p-value; Table 1). However, for 7 regions (2p24,2p15,3q21,6q22,8q21,11q13, and 19q13), the new marker appeared to capture risk more strongly than the index signal in African Americans. The risk region at 3q21 is provided in Figure 2 as an example. Here the index signal was not significantly associated with risk in African Americans (rs10934853, OR = 1. 03,95% CI, 0. 95–1. 03, p = 0. 43), with the most significantly associated marker in African Americans located ∼200 kb centromeric from the index signal (rs7641133, OR = 1. 16,95% CI 1. 08–1. 25, p = 1. 0×10−4). These two markers are strongly correlated in Europeans (HapMap CEU, r2 = 0. 91) but not in Africans (HapMap YRI, r2 = 0. 11; Table 1), which suggests that in African Americans rs7641133 is a better proxy of the biologically important allele and may better localize the true association. For some of these regions, the size of the LD blocks differ in populations of African ancestry compared with the GWAS population and thus, may assist in localizing the functional allele (Figure S1). Using a strict αb of 5. 6×10−6 for discovery of novel risk variants we observed no evidence of a second independent signal at any risk region. For variants identified as significantly associated with risk (Table 1), odds ratios for homozygous carriers were generally greater than for heterozygous carriers, which provides support for their associations (Table S7). We examined 12 risk variants at 8q24 that had been reported previously to be associated with prostate cancer risk [1], [7], [10], [13], [14], [20] with 7 being statistically significant and positively associated with risk (p<0. 05). The risk SNP BD11934905 [10] is not on the Illumina 1 M array and was not genotyped in this study. In contrast with what has been reported in Europeans, the risk allele for rs12543663 was observed to be significantly inversely associated with risk in African Americans (OR = 0. 89, p = 0. 028; Table 2). The RAFs for 8 of the 12 alleles are more common in African Americans than Europeans, with the average RAF being 0. 46 in African Americans and 0. 32 in Europeans. The largest difference in RAFs between populations are noted with variants rs13252298, rs13254738, rs6983561, rs6983267 and rs7000448, which have RAFs that are >0. 20 greater in African Americans than in Europeans. When all 12 variants were included in a multivariate model, only 5 remained nominally associated with risk (Table 2). In African Americans, many of these index signals were weakly correlated (Figure S2) and demonstrated stronger multi-allelic correlations (Table 2), which suggests that some variants may define similar haplotypes marking the same biologically relevant variants in this population. No significant association was observed with rs7008482 (OR = 0. 96, p = 0. 52, computed using data included in the initial report [24]) or markers of risk at 8q24 for cancers of the breast, bladder, ovary, or leukemia (rs13281615: OR = 1. 03, p = 0. 48; rs9642880: OR = 1. 07, p = 0. 13; rs10088218: OR = 0. 91, p = 0. 06; rs2456449: OR = 1. 06, p = 0. 24) [25]–[28]. To identify markers at 8q24 that best capture risk in African Americans we performed a stepwise analysis of 1,549 genotyped and imputed SNPs spanning the established risk locus (127. 8–129. 0 Mb). This region contained 132 SNPs with nominal p-values<0. 001 (Figure 3), and 9 common alleles with per allele ORs of 1. 16–1. 42 (Table 2) defined the most parsimonious model. Similarly to the previously reported risk variants at 8q24 four of these markers are substantially more common in African Americans than Europeans (average RAF difference = 0. 07). Eight of these markers show some degree of correlation with the known risk variants and thus are likely to be tagging the same functional allele, albeit for 4 SNPs the correlations are quite weak in the CEU and YRI populations (r2<0. 2; Table S8) suggesting that they may be marking independent risk variants. For example, SNP rs6987409 (RAF = 0. 15), which is monomorphic in Europeans, remains significantly associated with risk conditional on the 12 known risk alleles at 8q24 (OR = 1. 31,95% CI, 1. 16–1. 47, p = 7. 1×10−6), which suggests that this SNP may be marking a novel variant that is relevant in African Americans; rs6987409 was the most significant marker in the region (Figure 3). We next estimated the cumulative effect of all prostate cancer risk alleles, and compared a summary risk score comprised of unweighted counts of all GWAS reported risk alleles to a risk score that included variants we identified as being associated with risk in African Americans (Table 3). Using index signals from GWAS (see Methods), the risk per allele was 1. 08 (95% CI, 1. 06–1. 09; p = 6. 0×10−26) and individuals in the top quartile of the risk allele distribution were at 2-fold greater risk of prostate cancer compared to those in the lowest quartile (Table 3). As expected, the risk score was improved when utilizing the markers that we identified at the known risk loci as being more relevant to African Americans (OR = 1. 17 95% CI, 1. 15–1. 19; p = 5. 1×10−74), with risk for those in the top quartile being 3. 5-times those in the lowest quartile. When stratifying by first-degree family history of prostate cancer, risk was 4. 7-fold greater for those with a family history and in the top quartile of the risk score distribution (3. 5% of the population) compared to those without a family history and in the first quartile (Table 3). The risk score was associated equally with risk for advanced (n = 1,087) and non-advanced (n = 1,968) prostate cancer (case-only test: OR = 1. 02,95% CI, 1. 00–1. 05 phet = 0. 082). Using this risk score, we estimate (see Methods) that in the aggregate, all risk alleles tested explain approximately 11% of risk in first-degree relatives of cases. In this large study of prostate cancer risk in African American men we tested 49 variants that had been reported primarily in populations of European and Asian ancestry, and we were able to replicate associations (at p≤0. 05) with roughly half of these markers. We had adequate power (>80%) to detect relative risks of the magnitude reported previously for the majority of risk variants (although we realize that power was overestimated as the effect estimates from the initial report may be inflated due to the winner' s curse phenomenon [29].) Through fine-mapping, we identified markers in many regions that were more strongly associated with risk in African Americans than the index variant, and thus, are likely to be better proxies of the biologically relevant allele in this population. Our ability to detect associations in African Americans with either the index signal or correlated variants suggests that most loci contain a biologically relevant allele that is not unique to the initial GWAS population. These findings improve upon previous studies to replicate associations in African Americans [30], efforts which included some of these same studies, but in substantially smaller sample sizes for most variants examined [19], [31]. Within 12 regions, fine-mapping in African Americans revealed a more significantly associated marker (with evidence over the index signal being clearly greater at 7 loci). For some of the regions, the signal in African Americans was located in a smaller region of LD than that observed in the GWAS population which should aid in localizing the functional variant (s). Confirmation of these associations in the initial GWAS populations will be required before they can be declared as proxies of the underlying functional alleles; however in many cases, given their modest to strong correlation, based on HapMap data, with the index signal in the GWAS population, most markers are expected to be strongly associated with risk. At each locus, fine-mapping was based on the Illumina 1 M-Duo content supplemented with SNPs imputed from Phase 2 HapMap (CEU/YRI), which is expected to provide good coverage of the vast majority of common alleles in the admixed African American population. Of the ∼1. 5 million common SNPs (MAF≥0. 05) in the HapMap YRI population that we did not genotype, we were able to impute ∼1. 4 million with Rsq≥0. 8. Our inability to detect associations at 10 regions (p>0. 05 for an index signal and p>0. 004 for a proxy) could be due to low power, the functional allele being rare or non-existent in African Americans and/or inadequate tagging in these specific regions. Because of limited LD, fine-mapping in African Americans is thought to be an effective approach for localizing functional risk alleles for common phenotypes as populations of African ancestry are expected to have, on average, fewer alleles that are correlated with a functional variant. Fine-mapping in multiple racial/ethnic populations should prove to be even more powerful for isolating these variants as only a subset SNPs that are correlated with the functional allele in different populations will be similar. Thus, conducting association testing across multiple populations should narrow the subset of potentially functional alleles in a region. A complete resource of genome-wide variation data from multiple populations provided by the 1000 Genomes Project will assist in further interrogating these risk loci and together with large-scale association testing in diverse samples, will guide researchers in defining the subset of alleles that are correlated with risk across populations and hence are the most logical candidates for functional characterization. A number of prostate cancer risk regions have been found to harbor more than one risk variant (e. g. 8q24,17q12 and 11q13) [1], [10], [17], [18]. Aside from 8q24, the search for independent markers at known risk loci has been limited to populations of European ancestry. Using a relatively strict threshold for declaring significance (average α<5. 6×10−6), we observed no evidence of association that is independent of the index signal. While suggestive associations were observed at many loci, testing of these variants in additional African American samples will be needed to confirm these associations, followed by testing in other populations to assess whether the associations may be limited to African Americans. The risk region at 8q24 is the strongest susceptibility locus for prostate cancer that has been identified to date, with a number of different risk variants having been reported in different populations [1], [6], [7], [10], [13], [14]. We identified nine SNPs at 8q24 that best captured the genetic risk in African Americans, including SNP rs6987409 [1] which is not observed in Europeans (or is present at an extremely low frequency). Like the reported index signals at 8q24 (Table 2), many of these markers are more common in African Americans than in Europeans (average RAF difference = 0. 07). This is in contrast to the index signals in regions outside of 8q24 where the RAF average difference was only 0. 03. If the frequency of these 8q24 variants is a good correlate of the frequency of the underlying biologically relevant alleles then some of the variants in this region may to contribute to the excess risk of prostate cancer in African Americans, as suggested previously [10]. A precise estimate of its contribution will only come once the functional alleles have been found and we understand their associations in the context of other genetic and environmental factors (or host factors such as age). The cumulative effects of GWAS-identified variants for common cancers are not yet clinically informative for risk prediction [15], [16]. Until the functional alleles are identified and their effects are accurately estimated, modeling of the genetic risk will rely on markers that best capture risk at an established susceptibility locus for a given population. Many of the markers we identified at these risk loci in African Americans appear to provide substantial improvement over the GWAS-identified variants in defining those who are at greater risk of prostate cancer in this population. However, as estimated with the index signals in European populations [3], these alleles likely account for only a small fraction of the familial risk of the disease (∼10%) in African Americans. Validation of this risk model in African Americans and in other populations will be needed, as will incorporating novel risk variants identified through this GWAS in African American men. The Institutional Review Board at the University of Southern California approved the study protocol. Nine studies were genotyped as part of the GWAS of prostate cancer in African American men. Below is a brief description of each study. Genotyping of 7,123 samples from these studies (3,621 cases and 3,502 controls) was conducted using the Illumina Infinium 1 M-Duo bead array at the University of Southern California and the NCI Genotyping Core Facility (PLCO study). Following genotyping samples were removed based on the following exclusion criteria: 1) unknown replicates across studies (n = 24, none within studies); 2) call rates <95% (n = 126); 3) samples with >10% mean heterozygosity on the X chromosome and/or <10% mean intensity on the Y chromosome - we inferred 3 samples to be XX and 6 to be XXY; 4) ancestry outliers (n = 108, discussed below), and; 5) samples that were related (n = 141, discussed below). To assess genotyping reproducibility we included 158 replicate samples; the average concordance rate was 99. 99% (≥99. 3% for all pairs). Starting with 1,153,397 SNPs, we removed SNPs with <95% call rate, MAFs<1%, or >1 QC mismatch based on sample replicates (n = 105,411). The analysis included 1,047,986 SNPs among 3,425 cases and 3,290 controls. | Title: Characterizing Genetic Risk at Known Prostate Cancer Susceptibility Loci in African Americans Summary: Prostate cancer is one of the most common cancers in men and is especially frequent in men of African origin, as incidence rates in African Americans in the United States are >1. 5-fold greater than rates in European Americans. In order to gain a more complete understanding of the genetic basis of inherited susceptibility to prostate cancer in men of African origin, we examined the associations at risk loci identified in men of European and Asian descent in a large African American sample of 3,425 cases of prostate cancer and 3,290 male controls. In testing 49 known risk variants, we were able to demonstrate that at least half of these variants also contribute to risk in African American men. We were able to find additional risk variants in many of the previously reported regions that better captured the pattern of risk in African American men. In addition, we verified and improved upon the evidence we previously reported that there are multiple risk variants in a region of 8q24 that are important in men of African origin. | 5,986 | 239 | lay_plos | en |
Write a title and summarize: Evidence derived from human clinical studies and experimental animal models shows a causal relationship between adverse pregnancy and increased cardiovascular disease in the adult offspring. However, translational studies isolating mechanisms to design intervention are lacking. Sheep and humans share similar precocial developmental milestones in cardiovascular anatomy and physiology. We tested the hypothesis in sheep that maternal treatment with antioxidants protects against fetal growth restriction and programmed hypertension in adulthood in gestation complicated by chronic fetal hypoxia, the most common adverse consequence in human pregnancy. Using bespoke isobaric chambers, chronically catheterized sheep carrying singletons underwent normoxia or hypoxia (10% oxygen [O2]) ± vitamin C treatment (maternal 200 mg. kg−1 IV daily) for the last third of gestation. In one cohort, the maternal arterial blood gas status, the value at which 50% of the maternal hemoglobin is saturated with oxygen (P50), nitric oxide (NO) bioavailability, oxidative stress, and antioxidant capacity were determined. In another, naturally delivered offspring were raised under normoxia until early adulthood (9 months). Lambs were chronically instrumented and cardiovascular function tested in vivo. Following euthanasia, femoral arterial segments were isolated and endothelial function determined by wire myography. Hypoxic pregnancy induced fetal growth restriction and fetal oxidative stress. At adulthood, it programmed hypertension by enhancing vasoconstrictor reactivity and impairing NO-independent endothelial function. Maternal vitamin C in hypoxic pregnancy improved transplacental oxygenation and enhanced fetal antioxidant capacity while increasing NO bioavailability, offsetting constrictor hyper-reactivity and replenishing endothelial function in the adult offspring. These discoveries provide novel insight into mechanisms and interventions against fetal growth restriction and adult-onset programmed hypertension in an animal model of complicated pregnancy in a species of similar temporal developmental milestones to humans. Cardiovascular disease kills 1 in 3 people [1]. The annual costs for patient care and lost workforce due to heart disease are over US$130 billion in the United States and Canada [2] and over £30 billion in the United Kingdom [3]. Therefore, cardiovascular disease is a significant problem imposing a substantial burden on every nation’s health and wealth [4]. It is widely accepted that our genes interact with traditional lifestyle factors, such as smoking, obesity, and/or a sedentary lifestyle, to promote an increased risk of cardiovascular disease [5]. It is also established that the gene–environment interaction early in life may be just as, if not more, important in “programming” heart health and heart disease [6–8]. Evidence from human sibling-pair studies suggests that these relationships are causal, that they occur independently of genotype, and that they are significantly influenced by the quality of the intrauterine environment during pregnancy [9–12]. For instance, studies in Pima Indians showed a greater prevalence of type 2 diabetes in siblings born from pregnancies during which the mother had gestational diabetes compared to those whose mother did not [9]. Bariatric surgery to decrease the weight of obese women reduced the risk of obesity, insulin resistance, and raised blood pressure in children born after surgery compared to those born before surgery [10–12]. Therefore, these studies highlight a disproportionate risk of disease in offspring born from the same mother but under different in utero conditions, providing strong evidence in humans that the environment experienced during this critical period of development directly influences long-term cardiovascular health. One of the most common outcomes of complicated pregnancy in humans is chronic fetal hypoxia leading to reduced fetal growth, as can occur during placental insufficiency, preeclampsia, or inflammatory conditions during pregnancy, such as in chorioamnionitis, gestational diabetes, or maternal obesity [13,14]. In humans, low birth weight is related to poor neonatal outcome [15] and endothelial dysfunction [16] and high blood pressure at adulthood [17]. In turn, increased blood pressure is associated with an increased risk of cardiovascular disease [18], with this risk being greatest in those who were smallest at birth but with the most accelerated weight gain in childhood [19]. To date, there is no cure for pregnancy complicated by chronic fetal hypoxia to protect against fetal growth restriction or programmed cardiovascular dysfunction in the offspring. Treatment options are restricted to monitoring surrogate measures of fetal hypoxia and fetal growth, ultimately ending up in elective delivery of the offspring [20]. This highlights the need for experimental studies addressing underlying mechanisms to identify plausible intervention. Studies in animal models of adverse pregnancy have confirmed causality, reporting that oxidative stress during complicated pregnancy, including one involving chronic fetal hypoxia, may be a potential underlying mechanism [8,21–31]. Chronic fetal hypoxia is a powerful stimulus for reactive oxygen species (ROS) generation [8]. Under physiologic conditions, ROS are important mediators of a wide variety of cell functions, for instance, via signaling or by interacting with nitric oxide (NO) to provide a vascular oxidant tone [32–34]. However, excessive ROS and/or a fall in antioxidant defenses can lead to cellular oxidative stress and a fall in the bioavailability of NO, predisposing to cardiovascular dysfunction [8,31]. A small cluster of investigations including our own, mostly through studies of isolated hearts and vessels or echocardiography in rodents, has provided evidence for possible intervention with maternal treatment with antioxidants to protect against the ill effects of hypoxic pregnancy on the offspring [28–31,35,36]. However, when working with animal models of cardiovascular dysfunction before birth, the temporal profile of cardiovascular development between species is a highly important consideration for successful interventional translation to the human clinical situation. Rodents are altricial species, in which cardiovascular maturation continues past birth, becoming completed by the second week of postnatal life [28]. In contrast, sheep and humans share similar prenatal tempos of cardiovascular development [28] and some breeds of sheep, like Welsh Mountain, give birth primarily to singleton lambs of similar weight to term human babies. To date, no study has addressed maternal antioxidant intervention to protect against systemic cardiovascular dysfunction in the adult offspring in a human translational model of hypoxic pregnancy, such as in sheep, which additionally permits detailed cardiovascular analysis of in vivo mechanisms of action. In the present study, we tested the antioxidant vitamin C, as it is widely supplemented in human populations. In an elegant study, Jackson and colleagues [37] reported that the capacity of the antioxidant vitamin C to scavenge O2− ex vivo and its ability to prevent O2−-induced impairment of endothelial function in vivo occurred at very different concentrations, requiring a much higher effective concentration in vivo. Therefore, the dose regimen used in the preset study was derived from previous studies in our laboratory, which achieved elevations in circulating ascorbate within the required range for vitamin C to act effectively in vivo in ovine pregnancy [33,34]. To put into context, the dose of vitamin C used in the present study was 8 times higher than the dose employed in human clinical trials to prevent preeclampsia [38]. Here, we tested the hypothesis using Welsh Mountain sheep that maternal treatment with vitamin C protects against fetal growth restriction and programmed hypertension in adulthood in gestation complicated by chronic fetal hypoxia. While experimental models, which affect uterine blood flow or placental function, impair both fetal nutrition and fetal oxygenation, the isolated effect of chronic hypoxia on the fetus can be best studied by exposing the ovine pregnancy to an environment of reduced oxygenation. We therefore created 4 isobaric chambers [39,40] able to maintain pregnant sheep for long periods of gestation (Fig 1A). Adopting an integrative approach at the in vivo, isolated organ, and molecular levels, we show that maternal treatment with vitamin C in ovine hypoxic pregnancy protects against both fetal growth restriction and hypertension in the adult offspring. Mechanisms underlying this antioxidant protection include improved transplacental oxygenation, enhanced endogenous antioxidant capacity, increased in vivo NO bioavailability, offset in vivo vasoconstrictor hyper-reactivity, and replenished endothelial function in the peripheral vasculature of the offspring (see Summary illustration, Fig 2). By studying a species of similar developmental milestones to humans, the study therefore presents a conceptual advance to this field of research. It allows not only in vivo investigation of basal and stimulated cardiovascular physiology in the chronically instrumented adult offspring in an animal model that closely recapitulates human pregnancies involving fetal oxygen insufficiency, but it also provides a spring board toward human clinical translation, offering viable treatment options for the developmental origins of hypertension. In control ewes undergoing normoxic pregnancy, maternal arterial blood gases and pH remained unaltered from baseline until 135 days of gestation (dGA) (Fig 1B–1D and Table 1). However, in these ewes, there was a significant increase in maternal bicarbonate (HCO3−) on dGA 130 and dGA 135 and significant reductions in maternal hemoglobin concentration ([Hb]) from 110 dGA compared to baseline (Table 1). In control ewes, maternal plasma total levels of plasma concentrations of total NO3− + NO2− (NOx) also showed an increase with advancing gestation, with a significant difference from baseline at 135 dGA (Fig 1E). Exposure of pregnant sheep to a 10% inspired fraction of oxygen for a month in the last third of gestation, from 105 to 135 dGA, led to a sustained controlled reduction in the maternal PaO2 and arterial blood percentage saturation of hemoglobin with oxygen (Sat Hb) (Fig 1B–1D). Chronic hypoxia in untreated ewes led to a transient maternal respiratory alkalosis, with significant falls in maternal arterial blood partial pressure of carbon dioxide (PaCO2) throughout exposure and a significant increase in maternal pH at 106 dGA, the day after the onset of hypoxia (Table 1). This maternal respiratory alkalosis was buffered by reductions in maternal HCO3− throughout the chronic hypoxia period (Table 1). In contrast to control ewes undergoing normoxic pregnancy, ewes exposed to chronic hypoxia did not show a significant fall from baseline in maternal [Hb] and levels of maternal [Hb] were significantly higher than those in control ewes from 110 dGA (Table 1). Maternal hypoxia in untreated ewes enhanced the ontogenic increase in maternal plasma NOx (Fig 1E), and it did not affect maternal food intake or maternal weight gain (Fig 1F and 1G). Maternal vitamin C treatment in both control and hypoxic ewes produced similar increments from baseline (N: 38. 0 ± 3. 9; H: 36. 2 ± 3. 1 μmol/L) in maternal plasma vitamin C, doubling the circulating concentration (Fig 1H). Maternal vitamin C treatment in hypoxic pregnancy did not affect the alterations in maternal blood gases or pH or maternal plasma NOx seen in untreated hypoxic ewes. In addition, ewes undergoing hypoxia treated with vitamin C also did not show any changes in maternal food intake (Fig 1B–1F and Table 1). However, maternal treatment with vitamin C in hypoxic pregnancy led to a greater fall in maternal Sat Hb despite a similar fall in maternal PaO2 compared to untreated ewes (Fig 1C and 1D). This meant a rightward shift in the oxygen–hemoglobin dissociation curve and thereby a significant increase in the maternal arterial P50 from 23. 5 ± 0. 9 to 26. 6 ± 10 mmHg (P < 0. 05; Fig 1I). The total number of placentomes, the placental weight, the fetal: placental weight ratio, and the distribution of placentome type did not differ among all 4 groups (see S1A–S1C Fig). Fetuses from hypoxic pregnancy showed growth restriction with brain sparing. They had a significant reduction in body weight, body mass index (BMI), and lower limb length (LLL) and significant increases in the absolute and relative brain weight, in the relative weight of the hypothalamus, the absolute weight of the cerebral hemispheres, and in the ratio of the biparietal diameter relative to LLL when compared to fetuses from normoxic pregnancy (Table 2 and Fig 3A–3F). Fetuses from hypoxic pregnancy showed an increase in [Hb] and in plasma total NOx (Fig 3G and 3H). They also had increased hepatic nitrotyrosine concentrations and decreases in plasma homocysteine and in the activities of catalase and superoxide dismutase (SOD) in the liver (Fig 3I–3L). Fetuses from hypoxic pregnancy treated with maternal vitamin C no longer showed significant reductions in body weight and BMI, no longer showed a significant increase in relative brain weight, and the ratio of the biparietal diameter relative to LLL was restored when compared to fetuses from normoxic pregnancy. However, the reduction in LLL and the increase in the relative weight of the hypothalamus persisted (Table 2 and Fig 3A–3F). Fetuses from hypoxic pregnancy treated with maternal vitamin C still showed an increase in [Hb] and total plasma NOx and a fall in liver SOD, and the increase in hepatic nitrotyrosine was significantly greater than in fetuses from untreated hypoxic pregnancy. However, they showed restored levels of plasma homocysteine and of hepatic catalase activity when compared to hypoxic fetuses of untreated pregnancy (Table 2 and Fig 3J and 3K). Maternal treatment with vitamin C in normoxic pregnancy had no effect on any outcome variable measured in the ewes or fetuses when compared to untreated normoxic pregnancy (Tables 1 and 2, Figs 1 and 3). When blood was taken at post mortem immediately following the end of the experiment at 138 dGA, the fetal plasma concentrations of cortisol were not different among all groups (N: 17. 6 ± 3. 0; H: 16. 6 ± 2. 9; HC: 27. 7 ± 2. 9; NC: 28. 5 ± 6. 2 ng. mL−1). We have also reported that maternal plasma stress hormone mean levels are not different in ewes undergoing normoxic or chronic hypoxic pregnancy [39]. A separate cohort of animals was allowed to deliver naturally at term under normoxic conditions, and lambs were maintained with their mothers until weaning and then lived on pasture in fields surrounding the Barcroft Centre at the University of Cambridge. Lambs from hypoxic pregnancy were born earlier in gestation (N: 147. 4 ± 0. 6 versus H: 143. 6 ± 0. 6 days, P < 0. 05) with a reduced birth weight (N: 3. 5 ± 0. 1 versus H: 2. 9 ± 0. 1 Kg, P < 0. 05). Despite being born lighter, lambs from hypoxic pregnancy showed accelerated postnatal fractional growth rates (N: 25. 1 ± 1. 2 versus H: 32. 4 ± 1. 8 mg. d−1, starting weight−1, P < 0. 05), such that body weight at 9 months was no longer different (N: 26. 5 ± 1. 2 versus H: 30. 4 ± 1. 7 Kg). There were also no differences in the weight of the brain, heart, and liver nor in the weight of components of the brain, such as the hemispheres, hypothalamus, or cerebellum between lambs from normoxic or hypoxic pregnancy at 9 months (Table 2). In vivo experiments in chronically instrumented 9-month-old lambs revealed that offspring from hypoxic pregnancy were hypertensive, and they showed greater resting values for femoral blood flow and vascular conductance (Fig 4A–4F). Addressing in vivo mechanisms contributing to the hypertension, adult offspring of hypoxic pregnancy showed enhanced in vivo femoral constrictor responses to phenylephrine (PE) and to angiotensin II (AngII). They also showed enhanced in vivo femoral dilator responses to sodium nitroprusside (SNP), with no significant effect on basal femoral vascular resistance of N (ω) -nitro-L-arginine methyl ester (L-NAME) treatment (Fig 4G–4J). To further address mechanisms, third order femoral vessels isolated from adult offspring of hypoxic pregnancy showed reduced relaxation to methacholine because of endothelial dysfunction due to a decrease in NO-independent mechanisms, including prostanoid and endothelium-derived hyperpolarizing factor (EDHF) dilator reactivity (Fig 4K and 4L). Adult offspring of hypoxic pregnancy treated with maternal vitamin C were no longer hypertensive (Fig 4A–4C); they showed a significant increase in femoral vascular resistance following L-NAME treatment, indicating greater circulating NO bioavailability (Fig 4J), and had constrictor and dilator responses in vivo and ex vivo, which were more similar to adult offspring of control pregnancy (Fig 4G–4L). The weight of organs collected at 9 months was similar in adult offspring from treated and untreated hypoxic pregnancies (Table 2). Adult offspring of normoxic pregnancy treated with vitamin C showed similar responses in all outcomes relative to offspring of untreated normoxic pregnancy, with the exception of reduced endothelial function in isolated femoral vessels (Table 2 and Fig 4L). During healthy pregnancy, maternal cardiovascular adaptations are essential to maintain appropriate fetal growth and development as well as the maternal well-being. These adaptations include the expansion of the maternal blood volume, which places the mother in a state of physiological anemia. Following Poiseuille’s Law, reductions in maternal [Hb] decrease the maternal blood viscosity, thereby promoting lower systemic vascular resistance and increased blood flow in the uteroplacental circulation to match fetal growth in the last third of pregnancy [41,42]. Therefore, the fall in maternal [Hb] in control ewes in the present study is consistent with healthy maternal adaptations during late gestation. In humans and sheep, exposure to hypoxia during pregnancy, such as that associated with high altitude, leads to a fall in the maternal PaO2 and PaCO2 and an increase in maternal [Hb] [42–44]. In human high-altitude pregnancy, there is also an increase in the maternal blood pH and a decrease in HCO3− aiming to buffer the maternal alkalosis [43,44]. Therefore, the maternal circulatory responses to chronic hypoxia in the present study are consistent with these maternal adaptations to hypoxic pregnancy, and they explain the significantly higher levels of maternal [Hb] and lack of a fall in maternal [Hb] with advancing gestation in the chronically hypoxic relative to the normoxic ewe. Fetuses of hypoxic pregnancy were not only lighter but also showed a reduced BMI with an increase in relative brain weight. These are robust indices of fetal brain sparing at the expense of redistribution of blood flow away from the fetal peripheral circulation [14]. Regional differences in brain weight changes suggest that the hypothalamus and cerebellum were preserved more so than the hemispheres. Seminal studies in fetal sheep have previously reported a hierarchy in maintaining blood flow to subcortical regions at the expense of blood flow to the brain hemispheres during chronic hypoxia [45]. In a separate study, we have previously reported that a comparative level of maternal hypoxia in sheep, as used in the present study, reduced the fetal PaO2 from normal baseline to values of 12 ± 1 mmHg [40]. This is of significant clinical translation, as this is the level of reduced fetal oxygenation measured by cordocentesis in human pregnancies complicated by significant fetal growth restriction [46]. Additional data in the present study show that increased oxidative stress in the fetus contributes to the mechanisms mediating the adverse consequences of developmental hypoxia. 3-nitrotyrosine (3-NT) is a product of protein tyrosine nitration following oxidative damage to proteins by peroxynitrie [47]. An increase in oxidative stress will cause an increased demand for the synthesis of endogenous antioxidant enzymes. Therefore, a decrease in the activities of hepatic catalase and SOD in fetuses from hypoxic pregnancy provides further evidence of increased oxidative stress and impaired antioxidant defenses in the hypoxic fetus in the present study. It is widely recognized that individuals with high-plasma homocysteine (Hcy) levels have an increased risk of developing cardiovascular disease [48]. However, it must also be recognized that Hcy is an important intermediate in the conversion of methionine to cysteine, which is then used in the production of the antioxidant glutathione. During an oxidative challenge, this pathway is up-regulated rapidly [48,49]. However, in situations of low Hcy, there is a limit to how much glutathione can be produced, thereby restricting the body’s ability to respond to oxidative stress [49]. Any condition that causes an increase in oxidative stress will promote an increased demand on the liver to produce glutathione, and consequently, low availability of glutathione and homocysteine is also associated with a large range of diseases [49]. In hypoxic pregnancies, oxidative stress will drive Hcy into glutathione synthesis, leading to a fall in plasma Hcy levels, as was observed with the hypoxic fetuses in the present study. These data in the sheep fetus are also in keeping with reports of oxidative stress in the fetal cardiovascular system in rodent models of hypoxic pregnancy [8,28–31,35,36]. In the present study, treatment of hypoxic pregnancies with vitamin C maintained the increase in fetal [Hb], and in fetal total plasma NOx, it augmented the increase in nitrotyrosine in the fetal liver but prevented the fall in the fetal hepatic activities of Hcy and catalase without affecting the reduction in SOD in the fetus of hypoxic relative to normoxic pregnancy. Maintained or augmented increases in fetal [Hb], fetal total plasma NOx, and in nitrotyrosine in the fetal liver in hypoxic pregnancy suggests that maternal treatment with vitamin C is not protective in hypoxic pregnancy by affecting oxygen sensing pathways, the induction of oxidative stress, or compensatory adaptations to increase NO bioavailability in the fetus. Rather, maternal treatment with vitamin C in hypoxic pregnancy confers antioxidant protection by additional exogenous antioxidant supplementation. This normalizes the fetal hepatic activities of Hcy and catalase but not SOD, thereby reducing the demand for 2 out of 3 fetal endogenous antioxidant defenses in hypoxic pregnancy. Maternal vitamin C treatment increases umbilical blood flow in vivo in ovine pregnancy by quenching O2− production and increasing NO bioavailability [33,34]. Here, we show that maternal vitamin C treatment additionally increases the maternal P50 and restores fetal plasma Hcy levels and hepatic catalase activity. Therefore, maternal vitamin C treatment by increasing umbilical blood flow, fetal endogenous antioxidant defenses, and the transplacental PO2 gradient will buffer reductions in fetal oxygen delivery despite chronic hypoxia. These mechanisms may explain the capacity of the chronically hypoxic fetus whose mother is treated with vitamin C to be able to maintain appropriate growth, no longer requiring fetal brain sparing. Pregnancy complicated by fetal growth restriction is itself a major killer in perinatal medicine today [15]. Therefore, treatment to protect fetal growth in human high-risk pregnancy is clinically important. Systemic hypertension affects 1 billion adults worldwide [50]. Increased systolic blood pressure is an independent risk factor for coronary events, stroke, and heart failure [51]. A few studies in rodents have shown that hypoxic pregnancy can program hypertension in the adult offspring and that this was associated with constrictor hyper-reactivity in the peripheral vasculature and exacerbated cardiovascular responses to stress [52–55]. However, intervention against the programmed hypertension was not addressed in these studies. Here, we show that ovine pregnancy complicated by developmental hypoxia can also program significant hypertension in the adult offspring. The mechanisms mediating hypertension in the present study include increased in vivo vasoconstrictor reactivity to α-adrenergic agonists and to AngII in the peripheral circulation. In addition, we show endothelial dysfunction due to impaired EDHF and prostanoid-dependent reactivity in femoral arteries of adult offspring of hypoxic pregnancy. There is also in vivo evidence of programmed compensatory increases in NO function in the cardiovascular system in adult offspring of hypoxic pregnancy. We show in vivo that they have enhanced basal femoral blood flow and vascular conductance and a greater femoral dilator response to SNP, a NO donor. In the present study, maternal vitamin C treatment in hypoxic pregnancy prevented hypertension in the adult offspring. Therefore, we provide novel in vivo evidence of successful intervention with antenatal maternal antioxidant administration against programmed systemic hypertension in the adult offspring. Further, the successful intervention against programmed hypertension is in an ovine model of chronic fetal hypoxia, thereby of comparable developmental milestones to humans. In the present study, maternal treatment with vitamin C conferred protection against hypertension in the offspring by programming an increase in NO bioavailability, since in vivo treatment of adult offspring with L-NAME led to significantly greater increases in femoral vascular resistance. Further, maternal vitamin C also improved endothelial function. Combined, therefore, these protective mechanisms induced by maternal vitamin C treatment meant that NO-compensatory responses in the peripheral vasculature of adult offspring of hypoxic pregnancy, such as enhanced basal femoral blood flow and vascular conductance and a greater femoral dilator response to SNP, were no longer required. In the present study, fetal sheep were not instrumented with catheters, as the focus was on offspring of hypoxic pregnancy being allowed to be born naturally and maintained until adulthood for cardiovascular study. This study design precluded the measurement of fetal arterial blood pressure and knowledge of whether chronic fetal hypoxia led to fetal arterial hypertension in utero, which was maintained or exacerbated until adulthood. However, in separate studies, which determined changes in fetal arterial blood pressure during hypoxic pregnancy in sheep using isobaric chambers, we [40] and others [56] have reported that the chronically hypoxic fetus showed an impaired or unchanged ontogenic increase in basal arterial blood pressure. Similarly, late-gestation fetal sheep conceived, gestated, and studied at high altitude had lower resting arterial blood pressure relative to sea level controls [57]. Combined, therefore, past and present evidence suggests that chronic fetal hypoxia programs adult-onset hypertension and that this develops in association with aging, rather than it persisting from fetal origin. Several studies in rodents and in sheep have reported that pregnancy exposed to excess glucocorticoids can also program hypertension in the adult offspring [58–62]. In the present study, fetal plasma glucocorticoid levels were not elevated by the end of hypoxic pregnancy relative to normoxic pregnancy. This is again consistent with data derived from high-altitude pregnancy, which reports in sheep that the fetal hypothalamo–pituitary–adrenal axis desensitizes in response to chronic hypoxia. This is an adaptive response to prevent stress-induced preterm birth and to maintain normal circulating levels of fetal cortisol for appropriate development and maturation [63]. Therefore, in the present study, chronic fetal hypoxia programmed adult-onset hypertension independent of fetal hypercostisolemia. In the clinical setting, people are diagnosed as hypertensive when the systolic/diastolic reading exceeds 140/90, with systolic blood pressure being the greatest predictor of later disease (National Institute for Health and Care Excellence [NICE] guidelines) [64]. There is no equivalent definition for sheep, and although a direct comparison cannot be made, adult offspring of hypoxic pregnancy in the present study had a mean increase in systolic and diastolic blood pressure values of 13 and of 8 mmHg above control values, respectively. The magnitude of these effects is of high clinical significance, as reports on the prevention and treatment of high blood pressure in humans show that the risk of cardiovascular disease doubles with each increment of systolic/diastolic pressure of 20/10 mmHg and that such individuals require active health-promoting lifestyle modifications to prevent overt cardiovascular disease [18]. The Framingham study reported that modest increases in mean blood pressure milder than those observed in lambs from hypoxic pregnancy in the present study can dramatically increase the risk of a future cardiovascular event [65]. A recent randomized clinical trial also reported that targeting a systolic blood pressure to <120 mmHg compared with <140 mmHg in humans resulted in lower rates of fatal and nonfatal cardiovascular events [66]. In the present study, offspring of hypoxic pregnancy therefore already fall under the human classification of prehypertensive despite being young adult and female and thereby likely to show even greater risk of cardiovascular disease with aging. Interestingly, adult offspring born from normoxic pregnancy treated with vitamin C also showed evidence of endothelial dysfunction in the present study. While antioxidant supplementation may benefit conditions of increased oxidative stress, antioxidant excess resulting from antioxidant supplementation under normal conditions may paradoxically promote oxidative stress [32]. One mechanism may be by providing excess NO bioavailability, which also serves as a precursor for peroxynitrite generation [32]. This reveals an equally important translational message in the present study, and that is that clinically, maternal treatment with antioxidants should only be administered to pregnancies diagnosed with chronic fetal hypoxia rather than given prophylactically to all pregnancies. This will only be feasible if risk can be identified prior to maternal therapy. Expert obstetric opinion today dictates that in human pregnancy, chronic fetal hypoxia can be reliably identified according to severity by ultrasound scan between 20–26 weeks of gestation. Diagnosis encompasses early onset intrauterine growth restriction, reduced fetal heart rate variability and body movements and/or abnormal Doppler blood flow velocimetry indices in the fetal middle cerebral artery and the umbilical circulation [67,68]. Although this supports possible translation to the human clinical setting, there are several important points for additional discussion and further key basic science experiments that are required to inform double-blind, randomized, placebo-controlled human clinical trials before maternal antioxidant supplementation in human obstetric practice could be considered. A limitation of the current program of work is that although the study design controlled for sex differences in the offspring, it did not address them. To make the study viable ethically and economically, every singleton pregnancy generated was used. Therefore, studies in the fetal period used the male offspring while studies in the adult period used the female offspring, as ewe lambs are easier to group house when compared to growing rams. Longitudinal comparisons from the fetal through the adult period thus require caution. A second limitation of the current program of work is that outcome variables in the fetal and adult periods were not balanced for sex. Several recent reviews have reported important sex-dependent differences in the programming effects on cardiovascular function in the adult offspring of adverse intrauterine conditions, usually highlighting that the male offspring is more vulnerable [69–71]. It is also well accepted that aging increases cardiovascular risk [72–74] and that ovarian estrogens at adulthood confer protection against cardiovascular disease [75]. Given that the present study reported significant hypertension in young adult females born from hypoxic pregnancy, the data may underestimate the potential adverse impact of developmental hypoxia on systemic hypertension and cardiovascular dysfunction in adult male offspring in this ovine model of hypoxic pregnancy. However, the study does emphasize that hypoxic pregnancy in sheep increases the risk of cardiovascular dysfunction and that maternal antioxidant treatment is protective in both male and female offspring. A third limitation of the current study design is that pregnancies exposed to hypoxia from 105 to 135 dGA were returned to normoxia at 135 days to allow delivery under normal oxygen conditions. This study design ensured a fixed length period of fetal exposure to chronic hypoxia prior to birth. However, it also means that adult-onset hypertension could be programmed by fetal exposure to chronic hypoxia with some reoxygenation rather than by chronic fetal hypoxia alone. Since hypoxia and reoxygenation is a more potent stimulus of oxidative stress than hypoxia alone [32], the data highlight the protective impact of maternal antioxidant therapy even under these conditions. The final limitation and perhaps the most important in terms of human clinical translation is that multicenter human clinical studies have been unable to confirm benefits of maternal treatment with antioxidant vitamins in pregnancies at risk of preeclampsia [38,76]. These studies include the vitamins in preeclampsia (VIP) and international trial of antioxidants in the prevention of preeclampsia (INTAPP) trials, which found that administration of vitamin C (1 g. d−1) and E (400 IU) to mothers once clinical signs of the preeclampsia were established did not affect the incidence of preeclampsia but instead led to an increase in the rate of low-birth weight babies by 4%. While preeclampsia was not the focus of the present study and the adverse effect on birth weight of maternal vitamin C in human studies appears no longer present with meta-analysis [38,76], these clinical findings are significant enough to warn against possible adverse side effects of maternal vitamin C treatment in human pregnancy. Hence, we strongly agree that vitamin C may not be the antioxidant of choice for translation to human therapy. However, the data we show provide proof-of-principle that maternal treatment with antioxidants can protect against developmental programming of adult-onset hypertension. Future key experiments to inform human clinical trials should consider alternative maternal antioxidant therapy of improved translational value using human clinically relevant animal models of adverse pregnancy, such as sheep. Plausible alternative candidate antioxidant therapies for human translation may include melatonin, allopurinol, or the mitochondria-targeted antioxidant MitoQ. Rodent models show that maternal treatment with melatonin confers protection on fetal growth and cardiovascular function in the offspring of adverse pregnancy at doses similar to or lower than those required to avoid jet lag in humans [77,78]. An alternative antioxidant strategy may be to prevent the synthesis of free radicals through specific pathways rather than quench them once formed, as in the case of the xanthine oxidase inhibitor allopurinol. Hypoxia is a potent stimulus for xanthine oxidase-induced superoxide anion generation [32], and recently, we have reported that maternal treatment with allopurinol in hypoxic pregnancy in rats protects against cardiac dysfunction in the adult offspring [79]. Yet another strategy may be for targeted antioxidant therapy. Mitochondria are a major site of ROS production; therefore, targeting them specifically should be one of the most effective antioxidant therapeutic strategies. This is now possible with mitochondria-targeted antioxidants. MitoQ is composed of a lipophilic triphenylphosphonium cation covalently attached to an ubiquinol antioxidant [80,81]. Lipophilic cations can easily move through phospholipid bilayers without requiring a specific uptake mechanism. Therefore, the triphenylphosphonium cation concentrates MitoQ several hundred-fold within the mitochondria, driven by the large mitochondrial membrane potential [78,79]. Only within the mitochondria, MitoQ is reduced by the respiratory chain to its active ubiquinol form, which is a particularly effective antioxidant that prevents lipid peroxidation and mitochondrial damage [80,81]. The benefits of MitoQ have been revealed in a range of in vivo studies in rats and mice and in two human trials [82–85]. In contrast to vitamin C and other conventional antioxidants, MitoQ demonstrates no pro-oxidant activity at high doses and long-term administration to mice for 28 weeks [83] and to human patients in two Phase II trials, one that lasted 1 year revealed no toxicity [84,85]. Two recent studies in rats have also reported that nano-particle bound MitoQ, which prevents passage of the drug through the placenta and to the fetus, protected against programmed cardiac diastolic dysfunction, endothelial dysfunction, and neurodegeneration in the adult offspring, using an established model of hypoxic pregnancy in rats [35,86]. However, the antioxidant benefits of either melatonin or allopurinol or MitoQ in protecting against fetal growth restriction and programmed hypertension at adulthood in offspring of high-risk pregnancy in sheep have yet to be determined. In conclusion, our discoveries provide compelling evidence of clinical translational importance for chronic fetal hypoxia programming of adult-onset hypertension in a species of similar developmental milestones to humans. Further, the data provide novel insight into underlying mechanisms and thereby successful intervention against cardiovascular dysfunction in the next generation programmed by pregnancy complicated by developmental hypoxia (See Summary illustration, Fig 2). All procedures were performed under the Home Office Project Licence PC6CEFE59 under the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012, following ethical review by the University of Cambridge Animal Welfare and Ethical Review Board (AWERB). At 100 ± 1 days gestational age (term ca. 145 days), pregnant Welsh Mountain ewes carrying singleton pregnancies determined by ultrasound scan (Toshiba Medical Systems Europe, Zoetermeer, the Netherlands) underwent a laparotomy and catheterization, as previously described [33,34,39,40]. In brief, under general anesthesia (1. 5%–2. 0% isofluorane in 60: 40 O2: N2O) maintained by use of a positive pressure ventilator (Datex-Ohmeda Ltd, Hatfield, Hertfordshire, UK), a midline abdominal incision and uterotomy was used to expose the fetal hind limbs and determine fetal sex. If male, then the fetuses were assigned to the fetal studies group; female fetuses were assigned to the adult offspring studies group. The fetus was returned into the intrauterine cavity, and the uterine and maternal abdominal incisions were closed in layers. A Teflon catheter (i. d. 1. 0 mm, o. d. 1. 6 mm, Altec, UK) was placed in the maternal femoral artery and positioned in the descending aorta, in addition to a maternal femoral venous catheter into the inferior vena cava (Critchly Electrical Products, NSW, Australia). Catheters were filled with heparinized saline (80 i. u. ml−1 heparin in 0. 9% NaCl), tunnelled subcutaneously, and exteriorized via a keyhole incision made in the maternal flank to be kept inside a plastic pouch sewn onto the maternal skin. Following surgery, ewes were housed in individual floor pens with a 12: 12-hour light–dark cycle. From 103 dGA, ewes were fed daily a bespoke maintenance diet made up of concentrate and hay pellets to facilitate the monitoring of food intake (Cambridge ewe diet: 40g nuts/kg and 3g hay/kg; Manor Farm Feeds Ltd; Oakham, Leicestershire, UK) [39,40]. On day 105 of gestation, ewes were randomly assigned to 1 of 4 experimental groups: normoxia (N), chronic hypoxia (H), chronic hypoxia with vitamin C treatment (HC), and normoxia with vitamin C (NC); n = 18 for all groups. Ewes were housed in bespoke isobaric hypoxic chambers (Telstar Ace, Dewsbury, West Yorkshire, UK) supplied with controlled volumes of nitrogen and air provided by nitrogen generators and air compressors, respectively, from a specially designed nitrogen generating system (Domnick Hunter Gas Generation, Gateshead, Tyne & Wear, UK) [39,40]. Ambient PO2, PCO2, humidity, and temperature within each chamber were monitored via sensors, displayed, and values recorded continuously via the Trends Building Management System of the University of Cambridge through a secure Redcare intranet. In this way, the percentage of oxygen in the isolators could be controlled with precision over long periods of time. For experimental procedures, each chamber had a double transfer port to internalize material and a manually operated sliding panel to bring the ewe into a position in which daily sampling of blood could be achieved through glove compartments. Each chamber incorporated a drinking bowl on continuous water supply and a rotating food compartment, which could be removed for determining food intake. Therefore, all experimental and maintenance procedures could be carried out without interruption of the hypoxic exposure. Pregnancies randomly assigned to the chronic hypoxia group were placed inside the chambers at 103 dGA under normoxic conditions (11 L. sec−1 air, equating to 39. 6 m3. h−1). At 105 days, pregnancies were exposed to ca. 10% O2 by altering the inspirate mixture to 5 L. sec−1 air: 6 L. sec−1 N2. The inspirate air mixture underwent a minimum of 12 changes per hour in each chamber, and the incoming air mixture was passed via silencers able to reduce noise levels within the hypoxic chamber laboratory (76 dB[A]) and inside each chamber (63 dB[A]) to values lower than those necessary to abide by the Control of Noise at Work Regulations. This not only complied with human health and safety and animal welfare regulations but also provided a highly tranquil environment for the animal inside each chamber. Starting on day 105 of gestation, vitamin C (Ascorbate; A-5960; Sigma Chemicals, UK; 1. 14 mmol/kg/day dissolved in 0. 6 ml/kg saline and administered as a slow IV bolus injection) or saline vehicle (0. 6 ml/kg slow IV bolus injection) were administered every day to the mothers at approximately 09: 00. The dose of vitamin C used in this study was derived from previous studies in sheep pregnancy in our laboratory, which achieved elevations in circulating ascorbate concentrations within the required range for vitamin C to compete effectively in vivo with NO in ovine pregnancy [33,34,37]. Samples of descending aortic maternal blood (0. 3 ml) were taken daily for measurement of maternal blood gases and pH and hemoglobin concentration, as previously described [33,34,39,40]. Chamber oxygenation and maternal blood gases and pH were recorded at 104,105, and 106 dGA then as summary averages of the preceding 5 days for 110,115,120,125,130, and 135 days. For all data, baseline for each animal was calculated as the average values of 104 and 105 days. On days 104,105,106, and 110 of gestation, and every 5 days after that, an additional 9 ml of maternal blood was taken and divided into tubes containing the anticoagulant EDTA or heparin. Samples were spun in a centrifuge for 5 minutes at 1000 x g and 4°C, after which the plasma was aliquoted into storage tubes and immediately frozen for subsequent analysis. At 138 dGA, pregant ewes with male fetuses were transferred from the hypoxic chambers to the post mortem laboratory wearing a respiratory hood providing the same hypoxic mixture. Under hypoxic conditions, ewes and their fetuses were humanely killed by overdose of sodium pentobarbitone (0. 4 ml. kg−1 IV Pentoject; Animal Ltd, York, UK) and the fetus exteriorized by Cesarean section. An 8-ml sample of fetal blood was taken from the umbilical artery using a syringe with an attached needle, divided into tubes containing the anticoagulant EDTA or heparin, then spun in a centrifuge for 5 minutes at 1000 x g and 4°C. The plasma was aliquoted into storage tubes and immediately frozen for subsequent analysis. Fetal crown rump length, biparietal diameter, and hind limb lengths were then determined. The upper hind limb comprised the length of the femur, the middle hind limb ranged between the patella and tuber calcis, and the lower hind limb ranged between the tuber calcis and the tips of the phalanges. Remaining fetal organs were dissected, weighed, and immediately frozen for subsequent analysis. The maternal arterial blood P50, the PO2 at which 50% of the maternal hemoglobin is saturated with oxygen, was calculated using the average of the maternal values for PO2 and Sat Hb from day 115–135 of gestation, according to the Hill equation [87]. Maternal and fetal plasma concentrations of NOx species (NO2− and NO3−) were determined by a commercially available assay kit (Caymen Chemical, USA, Cat No. 780001) according to the manufacturer’s instructions. A standard curve was plotted in Excel using a straight line fit, allowing NOx concentrations to be calculated for each sample. The inter- and intra-assay coefficients of variation were 3. 4% and 2. 7%, respectively, and the lower limit of detection of the assay was 2. 5 μmol/L. Maternal plasma concentrations of ascorbic acid were measured by a fluorimetric technique using a centrifugal analyzer with a fluorescence attachment, according to the method of Vuilleumier and Keck [88], in collaboration with the Core Biochemical Assay Laboratory, Cambridge, UK. The interassay coefficients of variation were 7. 9% at 27. 1 μmol/L and 5. 0% at 89. 7 μmol/L. The lower limit of detection of the assay was 10 μmol/L. Fetal plasma concentrations of total L-homocysteine were measured using a commercially available enzyme immunoassay kit (Axis-Shield diagnostics Ltd., UK, Cat No. FHCY100), in collaboration with the Core Biochemical Assay Laboratory, Cambridge, UK. The inter- and intra-assay coefficients of variation for a 6. 1 μmol/L sample were 2% and 8%, respectively, and the lower limit of detection of the assay was 1. 0 μmol/L. Fetal plasma cortisol concentrations were measured using a commercially available ELISA kit (IBL international, Germany, Cat No. RE52061), according to the manufacturer’s instructions. The lower limit of detection of the assay was 2. 46 ng/mL. The cross-reactivity of the antiserum with other cortisol-related compounds was 4. 2% cortisone, 1. 4% corticosterone, 0. 4% progesterone, and 7. 0% deoxycortisol. The expression of 3-NT and SOD and the activity of catalase in frozen, powdered, fetal right liver lobe samples were determined by commercial assay kits (3-NT: ab116691, Abcam, Cambridge, UK, AMS biotechnology, Abington, UK, SOD: Sigma-Aldrich, Catalase: 707002), according to the manufacturer’s instructions. Placentomes were classified into 4 categories by their gross morphological appearance, according to Vatnick and colleagues [89]. Following classification, the individual types were counted and weighed. At 138 dGA, pregnant ewes carrying female fetuses were transferred from the hypoxic chambers to individual pens in a barn with a 12: 12-hour light–dark cycle, where they were returned to normoxic conditions. Ewes were allowed to deliver naturally and remained with their offspring until weaning. Newborn lambs were weighed within 12 hours of birth and then daily for the first week of life. Thereafter, body weight was recorded at 2 weeks of age, 30 days of age, and then at subsequent 30-day intervals until they were 9 months old. At 265 ± 5 days (9 months) of age, when sheep are sexually mature and classified as young adults, the female offspring were surgically instrumented under general anesthesia with vascular catheters and a femoral artery flow probe. In brief, food but not water was withdrawn 10–15 hours before surgery in order to minimize the risk of bloat and regurgitation of the reticulo-rumen contents. On the day of surgery, anesthesia was induced by injection of Alfaxan (1. 5–2. 5 ml/kg IV alfaxalone; Jurox Ltd., Worcestershire, UK) into the jugular vein and maintained by spontaneous inhalation of 1. 5% isoflurane in 60: 40 O2: N2O (2 L/min; IsoFlo; Abbott laboratories Ltd., Berkshire, UK). Surgery was performed under aseptic conditions. An incision, approximately 3. 5 cm long, was made in the medial surface of each of the hind limbs in order to expose the femoral artery and vein, which were catheterized as before. On the contra-lateral hind limb, a 4SB Transonic flow probe (Transonic Systems Inc., Ithaca, New York, USA) was placed around the main femoral artery for measurement of femoral blood flow. Catheters and the flow probe lead were then tunneled subcutaneously on their respective sides of the body and exteriorized via keyhole incisions made in the animal’s flanks. All skin incisions were then sutured closed and plastic pouches were sewn onto each flank to house the exteriorized catheters and flow probe. Following surgery, lambs were housed in individual floor pens with a 12: 12-hour light–dark cycle with free access to hay and water. Antibiotics (30 ml/kg IM procaine benzylpenicillin; Depocillin; Intervet UK Ltd., Milton Keynes, UK) were administered daily to the lamb for 5 days following surgery, and catheters were flushed daily with heparinised saline (100 i. u/ml heparin, 0. 9% NaCl). Following at least 5 days of postoperative recovery, basal and stimulated cardiovascular function was assessed in vivo. Post mortem, third-order femoral arteries (internal diameter < 300μm) were isolated and reactivity was determined via in vitro wire myography. Experimental protocols were performed following at least 5 days of postoperative recovery. On the morning of an experiment, ewe lambs were moved into a metabolic crate where their arterial catheter was connected to a sterile pressure transducer (Argon Division, Maxxim Medical, Athens, Texas, USA) and their flow probe to a Transonic flow meter (T206; Transonics Systems Inc., Ithaca, NY, USA). On each experimental day, lambs were allowed to acclimatize for 2–3 hours before commencing recordings. Basal cardiovascular variables were recorded continuously for 4–6 hours. A custom-built Data Acquisition System (Maastricht-Programmable AcQuisition system, M-PAQ and IDEEQ software, Maastricht Instruments, the Netherlands; 1000-Hz sample rate) was used to record arterial blood pressure. Blood flow signals from the Transonic flow meter were also directly recorded by the IDEEQ software, and heart rate was calculated continuously online by the program using the femoral blood flow or systolic pulse as a trigger. On a separate day, stimulated cardiovascular function in the ewe lambs was determined by measuring the change in mean arterial pressure, heart rate, and femoral blood flow in response to increasing bolus doses of the vasoconstrictors PE (0. 25,0. 5,2, 4,8, 16,32, and 64 μg/kg IA, diluted in 1 ml dH2O; L-phenylephrine; P-6126; Sigma Chemicals, UK); and Ang II (5,10,20,40,80,160, and 320 ng/kg IA, diluted in 1ml dH2O; MP Biomedicals, California, USA) or in response to increasing doses of the NO donor SNP (0. 625,1. 25,2. 5,5, and 10 μg/kg IA, diluted in 1 ml dH2O; S-0501; Sigma Chemicals, UK). Suitable dose ranges were derived from pilot experiments and previous studies in the literature [90,91]. All doses were administered in a random order. On a separate day, the change in femoral vascular resistance was also determined after intravenous treatment with the NO synthase inhibitor L-NAME (100 mg/kg; Cayman Chemicals, Cambridge, UK). At the end of all experimental procedures, ewe lambs underwent euthanasia with a lethal overdose of sodium pentobarbital administered to the indwelling venous catheter (200 ml/kg IV Pentoject; Animalcare Ltd., York, UK). The brain, heart, and liver were dissected and weighed. The brain was further dissected into the midbrain plus diencephalon, cerebellum, and right and left hemispheres for compartmental weighing and storage of each section. Third-order femoral arteries (internal diameter < 300μm) were isolated and placed in physiologic buffer solution (PBS). A segment of approximately 2 mm in length was cut and threaded with two 40-μm diameter stainless steel wires. The vessel segment was then mounted on a wire myograph (Multi Wire Myograph System 610M; DMT, Denmark), while bathed in Krebs solution (mM: NaCl 118. 5, KCl 4. 75, MgSO4 7, H2O 1. 2, KH2PO4 1. 2, NaHCO3 25. 0, CaCl2 2. 5, and glucose 5. 5; Sigma) and constantly exposed to a gas mixture of 5% CO2 and 95% O2 at 37°C in the myograph chamber. Following a 30-minute equilibration period, the vessel was stretched in a stepwise manner to a standardized tension equivalent to physiologic transmural pressure. Following a 20-minute equilibration period, all vessels were then precontracted with PE (10−5 M) before assessing endothelium-dependent vasodilator responses to different concentrations of methacholine (MetCh; 10−9 to 10−4 M; Sigma Aldrich). To determine the relative contribution of endogenous NO, EDHF, and prostanoid to endothelium-dependent relaxation, concentration-response curves to MetCh were also generated following incubation for 10 minutes with L-NAME (10−5 M; Sigma Aldrich) and L-NAME plus indomethacin (10−6 M; Sigma Aldrich), as previously reported [92]. Vessels were washed repeatedly with Krebs solution and allowed to equilibrate for at least 20 minutes between different concentration-response curves. For the in vivo cardiovascular experiments at adulthood, variables representing basal cardiovascular function represent the average of the 4–6-hour recording period, which was always during the same time of the day. Femoral vascular resistance (FVR) and femoral vascular conductance (FVC) were calculated by applying Ohm’s Law to the circulation, using the following equations, for which ABP is the arterial blood pressure and FBF is femoral blood flow: FVR = ABP/FBF and FVC = FBF/ABP. An increase in vascular resistance signifies a reduction in blood flow greater than can be accounted by a reduction in arterial blood pressure, therefore active vasoconstriction. An increase in vascular conductance signifies an increase in blood flow greater than can be accounted by an increase in arterial blood pressure, therefore active vasodilatation. For the in vivo dose-response experiments at adulthood, maximal changes from baseline in cardiovascular variables were recorded for each dose. The baseline was taken as the preceding 1–2 minutes of stable recording before each dose was given. After each dose, cardiovascular variables were allowed to return to baseline and remain stable for at least 2 minutes before preparing for the next dose. For the in vitro wire myography experiments at adulthood, femoral arterial responses were analysed using Prism (v. 5. 0, GraphPad software). Concentration-response curves were analysed using a sigmoidal fit curve. The maximal vessel relaxation (percent Rmax) was expressed as percentage of the contraction induced by PE. The contribution of NO-dependent mechanisms to the relaxation induced by MetCh was calculated by subtracting the area above the curve (AAC) for MetCh–the AAC for MetCh + L-NAME. The contribution of NO-independent mechanisms was the AAC for MetCh + L-NAME. The contribution of prostanoid to the relaxation induced by MetCh was calculated as the AAC for MetCh + L-NAME–the AAC for MetCh + L-NAME + indomethacin. The remaining AAC following MetCh + L-NAME + indomethacin was taken as EDHF [92]. For all in vivo and ex vivo experiments, data are expressed as the mean ± SEM. All variables were assessed as appropriate either using two-way ANOVA comparing the interactions between oxygenation and treatment or two-way ANOVA with repeated measures comparing the effects of group and dose or time. Where a significant effect was indicated, Tukey’s posthoc test was used to isolate the statistical differences (Sigma-Stat 3. 5; Chicago, IL, USA and GraphPad Prism 6). For all comparisons, statistical significance was accepted when P < 0. 05. | Title: Intervention against hypertension in the next generation programmed by developmental hypoxia Summary: Adverse conditions during pregnancy can increase the cardiovascular risk of the adult offspring. However, the mechanisms underlying these effects remain unclear, precluding the identification of candidate therapy. In this interventional study in sheep, a species of similar temporal developmental milestones to humans, we adopt an integrative approach, combining studies in vivo with those at the isolated organ, cellular, and molecular levels to investigate consequences of suboptimal pregnancy on the offspring at two stages of life: in the near-term fetus and in the adult. We show that developmental hypoxia, the most common outcome in human suboptimal pregnancy, slows fetal growth and programs high blood pressure in the sheep adult offspring. Maternal treatment with vitamin C in hypoxic pregnancy restored fetal growth and protected against adult-onset hypertension by improving transplacental oxygen delivery, enhancing fetal antioxidant capacity, and increasing the bioavailability of nitric oxide (NO) in the sheep adult offspring. Our discoveries highlight that when considering strategies to reduce the overall burden of heart disease, a much greater attention to prevention rather than treatment is required. Treatment should start as early as possible during the developmental trajectory, rather than waiting until adulthood when the disease process has become irreversible. | 13,353 | 285 | lay_plos | en |
Write a title and summarize: For the last three decades, evolutionary biologists have sought to understand which factors modulate the evolution of parasite virulence. Although theory has identified several of these modulators, their effect has seldom been analysed experimentally. We investigated the role of two such major factors—the mode of transmission, and host adaptation in response to parasite evolution—in the evolution of virulence of the plant virus Cucumber mosaic virus (CMV) in its natural host Arabidopsis thaliana. To do so, we serially passaged three CMV strains under strict vertical and strict horizontal transmission, alternating both modes of transmission. We quantified seed (vertical) transmission rate, virus accumulation, effect on plant growth and virulence of evolved and non-evolved viruses in the original plants and in plants derived after five passages of vertical transmission. Our results indicated that vertical passaging led to adaptation of the virus to greater vertical transmission, which was associated with reductions of virus accumulation and virulence. On the other hand, horizontal serial passages did not significantly modify virus accumulation and virulence. The observed increases in CMV seed transmission, and reductions in virus accumulation and virulence in vertically passaged viruses were due also to reciprocal host adaptation during vertical passages, which additionally reduced virulence and multiplication of vertically passaged viruses. This result is consistent with plant-virus co-evolution. Host adaptation to vertically passaged viruses was traded-off against reduced resistance to the non-evolved viruses. Thus, we provide evidence of the key role that the interplay between mode of transmission and host-parasite co-evolution has in determining the evolution of virulence. Understanding which factors determine the evolution of virulence–the negative effect of parasites on host fitness [1], [2] –and how they act, is a long-standing goal in evolutionary biology, and central for the control of infectious diseases [3], [4]. Indeed, changes in virulence have been associated with the effects of parasites on host population dynamics [1], [5], the reduction of ecosystem biodiversity [6], [7], and the emergence and re-emergence of infectious diseases [8], [9]. In the last three decades considerable effort has been devoted to developing theoretical models that predict conditions favouring the increase or decrease of parasite virulence. Most models are based on the hypothesis that the level of virulence is determined by trade-offs between the within-host and between-host components of the parasite' s fitness; this is known as the trade-off hypothesis [2], [10]–[12]. According to the trade-off hypothesis the level of virulence is generally, but not always [13], [14], a consequence of optimizing the within-host multiplication and between-host transmission components of parasite fitness [10], [13]–[16]. Two key assumptions underlie the trade-off hypothesis. First, virulence is positively correlated with parasite multiplication within the infected host; and second, greater parasite load in an infected host increases the probability of transmission to a susceptible, uninfected host. A trade-off occurs because higher virulence may also increase host mortality, reducing the infectious period and the probability of transmission. Experimental analyses have shown that multiplication rates and/or transmission rates are positively correlated with virulence for most parasites of humans, animals and plants when transmitted horizontally, i. e., between host individuals that are not parent and offspring [2], [17]–[19]; [ see 20], [ 21 for exceptions]. However, a wide range of human, animal and plant parasites that cause severe diseases, are vertically transmitted, i. e., from parent to offspring, or are transmitted both horizontally and vertically. Evolution of virulence in these parasites may challenge the trade-off hypothesis and derived models, since the presence of an alternative mode of transmission may reduce the relative importance of the trade-off between horizontal transmission and virulence. For instance, under strict vertical transmission, virulence should be negatively correlated with transmission rate [2], [13], [14], [22]: The fitness of vertically transmitted parasites is highly dependent on host reproductive potential, as hosts need to reproduce for the parasite to infect new individuals. Since virulence, by definition, reduces host fitness, vertically transmitted parasites should evolve towards lower virulence to maximize their own fitness [10], [13], [14], [23]–[25]. Accordingly, the ‘continuum hypothesis’ proposes that the optimum virulence in parasites transmitted both vertically and horizontally will vary along a continuum depending on the relative weight of each transmission mode on the parasite' s fitness: parasites mostly vertically transmitted will tend towards lower virulence and parasites mostly horizontally transmitted will tend towards higher virulence [14], [16], [22]. Despite the abundance of theory, and the numerous examples of important parasites with vertical or mixed modes of transmission, the largest fraction of experimental analyses of the effect of transmission mode on the evolution of virulence has been done under strict horizontal transmission. Studies of parasite evolution under vertical transmission have mostly reported a negative correlation between virulence and rate of vertical transmission, supporting the ‘continuum hypothesis’ [26]–[33]. However, this might not be a universal trend. For instance, in some of the few plant-parasite interactions studied, virulence was not negatively correlated with vertical transmission [34], [35]. Therefore, understanding the relationship between mode of transmission and virulence evolution requires further analysis and is in need of more experimental data from a larger variety of host-parasite systems [36]. Most theoretical and experimental analyses of virulence evolution overlook the important fact that virulence and transmissibility are not just parasite traits [8]. Rather, virulence and transmissibility are the result of the parasite' s interaction with the host, and thus are potentially subjected to host-parasite co-evolution [8], [37], [38]. For instance, reduced virulence might result from selection on the parasite to increase the efficiency of its vertical transmission, or from reciprocal selection on the host to reduce the damage caused by the parasite during vertical transmission [3], [39]. Selection on the host will thus result in higher tolerance, where tolerance is defined as the host' s ability to reduce the effect of infection on its fitness [8], [40]. For example, tolerance of Arabidopsis thaliana plants to virus infection is achieved through developmental reprogramming, so that resources are reallocated from the vegetative growth to reproduction [41]. Demonstrating co-evolution presents the daunting challenge of demonstrating reciprocal effects of both host and pathogen [37]. Thus, empirical evidence for the co-evolution of host- and parasite-related components of virulence is limited [8], [37]. Here we analyse experimentally the role of the mode of transmission, vertical or horizontal, on the evolution of virulence, while also accounting for the effect of host-parasite co-evolution in virulence-related traits. For this, we used the plant-virus system Arabidopsis thaliana L. Heynh. (Brassicaceae) - Cucumber mosaic virus (CMV, Bromoviridae). A. thaliana (from here on, Arabidopsis) has been developed as model organism for molecular and genetic analyses of a wide range of plant traits, and also is increasingly used in analyses of host-parasite co-evolution [e. g. 42–48]. The short life cycle [49], [50] of Arabidopsis facilitates performing serial passage experiments of vertical transmission, and the large numbers of seeds produced by a plant [49], [51] ensures the maintenance of parasite lineages between host generations even at low rates of vertical transmission. CMV is a generalist parasite with the broadest host range known for a plant virus, and its genomic structure, replication, gene expression and pathogenicity have been analysed extensively [reviewed in 52], [ 53]. CMV isolates are highly diverse and they have been classified into two subgroups (subgroup I and subgroup II) based on the nucleotide sequence similarity of their genomic RNAs. CMV is horizontally transmitted by more than 70 species of aphids in a non-persistent manner, and vertically through seeds, with rates that vary depending on CMV and plant genotypes [53]. In Arabidopsis, the efficiency of CMV seed transmission ranges between 2 and 8% [unpublished data]. In this work, we serially passaged three CMV strains in one Arabidopsis genotype for five generations under three transmission modes: strict vertical transmission, strict horizontal transmission, and alternation of vertical and horizontal transmission. We monitored the vertical transmission rate through seeds, virus accumulation and virulence after each passage. After the last passage, we compared virus accumulation and virulence of the evolved and the non-evolved virus lineages, both in the original plant stock, and in the progeny of plants derived from the fifth passage of vertical transmission. Results indicate that vertical passaging led to adaptation of the virus to greater vertical transmission, which was associated with reductions of virus accumulation and virulence. Increases in seed transmission and reductions in virus accumulation and virulence were determined also by reciprocal host adaptation during vertical passages, which additionally reduced virulence and multiplication of vertically evolved viruses. Host adaptation to vertically passaged viruses was traded-off against reduced resistance to the non-evolved viruses. We studied the evolution of three strains of CMV; Fny-CMV (a well-characterized strain isolated in New York State, belonging to subgroup I of CMV strains), De72-CMV (isolated from a species in the Brassicaceae in Central Spain and also belonging to subgroup I), and of LS-CMV (a well-characterized strain isolated in New York State, belonging to subgroup II) [53] in Arabidopsis under strict vertical, strict horizontal or alternated vertical and horizontal transmission (Figure 1, and see Material and Methods for experimental details). The vertical transmission rate, estimated as the percentage of CMV-infected seeds; virus accumulation, quantified as µg of viral RNA per gr of fresh plant tissue; and virulence, measured as the effect of infection on fecundity, estimated from total seed weight, 1− (SWi/SWm) (i and m denote infected and mock plants, respectively), were determined in each of five passages of vertical transmission (Figure 2, Table 1). CMV vertical transmission rate, virus accumulation and virulence in each passage were quantified in the same plants in which the virus was passaged. For each of the three virus strains, the five replicate lineages yielded similar values for seed transmission rate (F4,25≤2. 19; P≥0. 120), virus accumulation (F4,25≤1. 51; P≥0. 248), and virulence (F2,72≤1. 50; P≥0. 251). Thus, lineage was not considered as a factor for further analyses. Seed transmission rate increased as the number of vertical passages increased, either when the three CMV strains were analysed together (F4,72 = 48. 72; P<1×10−5), or independently (F4,25≥12. 79; P≤1×10−5). Indeed, a significant positive linear correlation between seed transmission rate and number of vertical passage was found either when the three strains were considered together (r = 0. 84; P<1×10−5) or individually (r≥0. 83; P<1×10−5). Slopes and intercepts of these regression lines did not differ significantly between CMV strains (F2,72≤1. 03; P≥0. 363), indicating that seed transmission increased at the same rate for the three viral strains (Figure 2a, Table 1). Virus accumulation decreased as the number of vertical transmission passages increased, either considering all strains together (F4,72 = 8. 16; P<1×10−4) or separately (F4,25≥6. 17; P≤0. 002) (Figure 2b, Table 1). A negative logarithmic correlation between virus accumulation and vertical passage was found considering all strains together (r = −0. 58; P<1×10−5) and independently (r≥−0. 69; P≤1×10−4). The slope and intercept of the De72-CMV regression were different from those of the Fny-CMV and LS-CMV regressions, so that virus accumulation decreased more slowly in the former than in the latter two strains (F2,72≥9. 41; P≤0. 010) (Figure 2b). Virulence decreased as the number of passages increased for all viral strains together and individually (F4,72≥6. 53; P≤0. 002) (Figure 2c, Table 1). A significant negative linear correlation between virulence and vertical passage was observed, either for all strains together (r = −0. 80; P<1×10−5) or separately (r≤−0. 78; P<1×10−5). Slopes and intercepts of the regression lines for each viral strain were similar (F2,72≤1. 23; P≥0. 298). The analyses above strongly suggest that increases in seed transmission rate might be accompanied by reductions in virus accumulation and virulence. To explore this relationship, we analysed the association between these traits during the serial vertical transmission passages considering all strains together and independently (Figure 3). Regression analyses indicated that the seed transmission rate was negatively (exponentially) correlated with virus accumulation for all strains together (r = −0. 32; P = 0. 005) and separately (r≤−0. 40; P≤0. 049) (Figure 3a). In addition, seed transmission rate was also negatively (linearly) correlated with virulence in all CMV strains (r≤−0. 56; P≤0. 006) (Figure 3b). Finally, virus accumulation and virulence were always positively (exponentially) correlated (r≥0. 45; P≤0. 034) (Figure 3c). Overall, these results indicate that the increase of seed transmission rate through vertical passages is associated with a reduction of virus accumulation and virulence in Arabidopsis. These changes might be due to virus evolution and/or host evolution. In the next sections we address these possibilities. We further analysed how the mode of transmission affected CMV accumulation, and the effect of infection on plant growth and fitness. To do so, virus lineages evolved under vertical, horizontal and alternated transmission (from here on, referred to as vertically evolved, horizontally evolved and alternately evolved viruses), as well as the initial non-evolved strains, were inoculated in plants from the ‘original’ seed stock. In these plants, we determined the effect of virus infection on plant vegetative and reproductive growth, through the weight of the rosettes (RW) and inflorescences (IW), respectively. We previously reported that tolerance of Arabidopsis to CMV is attained by reallocating resources from vegetative to reproductive structures [41]. For these analyses, we used the RW and IW ratios (Traiti/Traitm, where i and m denote infected and mock-inoculated plants, respectively). We also quantified the effect on SW (virulence) and virus accumulation as described above. Vertical transmission rate in plants from the ‘original’ seed stock was quantified only for vertically evolved viruses. The five virus lineages for each combination of CMV strain and mode of transmission did not differ for any of the parameters estimated (F≤2. 27; P≥0. 077) (Table S1). Thus, lineage was not considered as a factor for further analyses. Indeed, GLM analyses nesting lineage in mode of transmission did not change our results (not shown). Virus accumulation differed between evolved and non-evolved viruses, and between evolved viruses under different transmission modes for the three CMV strains (F3,135≥7. 53; P≤1×10−5). In general, evolved viruses of the three strains showed lower virus accumulation than non-evolved viruses (P≤0. 024), with the exception of horizontally evolved Fny-CMV (P = 0. 378) (Table 2). In addition, the effect of the mode of transmission (vertical, horizontal or alternated) varied depending on the CMV strain. In Fny-CMV, virus accumulation was similar for vertically and alternately evolved viruses (P = 0. 154), and in both cases was significantly lower than for horizontally evolved viruses (P≤0. 001). In LS-CMV, the virus accumulation of vertically evolved viruses was lower than that of horizontally and alternately evolved viruses (P<1×10−4), which were not significantly different (P = 0. 898) (Table 2). The mode of transmission did not affect the accumulation of De72-CMV (P≥0. 200) (Table 2). The mode of transmission did not significantly affect the effect of infection on vegetative growth (RW ratio) of any CMV strain (Table 2) (F3,135≤0. 73; P≥0. 538). The effect of virus infection on reproductive growth (IW ratio) (Table 2) varied significantly among transmission modes (F3,135≥3. 29; P≤0. 023). In Fny-CMV and LS-CMV, the IW ratio was significantly higher in vertically and alternately evolved viruses than in horizontally evolved and non-evolved viruses (P≤0. 051) (Table 1). In De72-CMV, the effect of infection on the IW ratio was significantly lower in vertically evolved viruses than in the other three treatments (P≤0. 017), which were not significantly different (P>0. 645) (Table 2). The effect of infection by Fny-CMV and LS-CMV on seed weight (i. e., virulence) significantly differed among transmission modes (F3,135≥5. 15; P≤0. 002). In both strains, virulence was lower when viruses were vertically and alternately evolved than when they were horizontally evolved or non-evolved (P≤0. 030). No differences in virulence were observed between evolved and non-evolved De72-CMV viruses regardless of transmission mode (F3,135 = 1. 36; P = 0. 260). Finally, Fny-CMV and LS-CMV viruses had significantly higher vertical transmission rates when vertically evolved than non-evolved viruses in plants from the ‘original’ stock (F1,42≥4. 42; P≤0. 043), while no differences were observed for De72-CMV (F1,33 = 1. 15; P = 0. 289) (Table 2). Thus, CMV evolution under strict vertical transmission results in decreased virus accumulation and virulence. Vertical transmission also results in a reduced effect of infection on plant growth, particularly of the plant reproductive structures. These effects of passaging are higher in Fny-CMV and LS-CMV than in De72-CMV. Serial passages under strict vertical transmission may result not only in virus evolution, but also in host adaptation; that is, we might be selecting for plant individuals that transmit the virus to seed at higher rates. To analyse this possibility, seeds from non-infected plants of the fifth vertical passage representing plant lineages evolved with each of the three virus strains were grown and inoculated with the corresponding evolved lineages and with the non-evolved CMV isolates. In these plants, virus accumulation and effects of infection on plant growth (RW and IW) and on fecundity (SW) were analysed as in ‘original’ stock plants, but percentage of CMV seed transmission was not determined. For each combination of CMV strain and mode of transmission, the different virus lineages did not differ in any of the parameters estimated (F≤2. 45; P≥0. 060) (Table S2), and therefore lineage was not considered as a factor. As above, GLM analyses using ‘lineage’ as a nested factor did not alter the results. Virus accumulation in evolved plants differed among treatments (F3,200≥3. 59; P≤0. 015). Fny-CMV and LS-CMV evolved viruses accumulated at lower levels than non-evolved viruses (P<1×10−4), while the opposite was observed in De72-CMV (P≤0. 055) (Table 3). The effect of the mode of transmission varied depending on the CMV strain. In Fny-CMV, virus accumulation of vertically evolved viruses was lower than that of alternately evolved viruses (P = 0. 001), with intermediate accumulation values of horizontally evolved viruses (P≤0. 005) (Table 3). In LS-CMV, virus accumulation was not significantly different in vertically and alternately evolved viruses (P = 0. 446), but was significantly less in horizontally evolved viruses (P<1×10−4). The mode of transmission did not affect the accumulation of De72-CMV (P≥0. 216) (Table 3). Differences in the effect of CMV infection in RW and IW were observed among the different modes of transmission (F3,200≥6. 27; P<1×10−4), and followed similar patterns in the three virus strains (Table 3). In Fny-CMV and LS-CMV, vertically evolved viruses had significantly higher RW and IW ratios than the non-evolved viruses (P<1×10−4), with intermediate values for horizontally and alternately evolved viruses (Table 3). In De72-CMV the effect of infection on RW and IW ratios was less in vertically evolved viruses than in the other three treatments (P≤0. 041), which were not significantly different among themselves (P≥0. 068) (Table 2). In all strains, virulence significantly differed among modes of transmission (F3,200≥21. 17; P<1×10−4), with vertically evolved viruses always being less virulent than viruses in the other three treatments (P<1×10−4). In addition, virulence was significantly lower in alternately evolved viruses than in the other two treatments (P≤0. 052) (Table 3). Finally, we explored if there were differences in virus accumulation and virulence of vertically evolved viruses in plants derived from the fifth vertical passage with respect to whether they were infected by vertical transmission (via seed) or horizontal transmission (mechanical inoculation). To do so, we compared the data above for plants mechanically inoculated (Table 3) with results for these traits in vertically infected plants derived from the fifth vertical passage (Table 1). Virus accumulation was higher in the horizontally inoculated than in the vertically infected plants for lineages of the three strains combined (F1,333≥450. 11; P<1×10−5), and for each strain individually (F1≥720. 45; P<1×10−5). Accordingly, virulence was always lower in vertically infected plants (F1≥12. 58; P≤0. 001). In summary, CMV evolution under vertical transmission decreases virus accumulation, virulence and effect of infection on the growth of plants derived from the fifth vertical passage. Differences among modes of transmission were larger in these plants than in those from the ‘original’ stock for most traits, which suggests that plants changed during the vertical passages by increasing their resistance to virus infection. Whether these changes co-evolved with those observed in the viruses is analysed in the next section. If vertical transmission of CMV resulted in selection for Arabidopsis plants with better performance under virus infection, the effect of infection with evolved and non-evolved strains would differ between ‘original’ stock plants and plants derived from the fifth vertical passage. To test this hypothesis, virus accumulation, RW and IW ratios and virulence in ‘original’ stock plants were compared with plants derived from the fifth vertical passage (Tables 2–3). Vertical transmission rate was compared between plants of the ‘original’ seed stock (Table 2) and plants of the fifth vertical passage (fifth passage in Figure 2). Fny-CMV and LS-CMV vertically evolved viruses accumulated to lower levels (F1,100≥3. 97; P≤0. 054), and infected plants had higher RW and IW ratios, and lower virulence (F1,100≥3. 61; P≤0. 068) in plants derived from the fifth vertical passage than in ‘original’ stock plants (Table 3). The opposite was observed in the non-evolved viruses of these two strains (F1,100≥7. 23; P≤0. 009, for virus accumulation; and F1,100≥1. 65; P≤0. 085, for RW and IW ratios, and virulence). The exception was that no differences in RW and IW ratios were observed between LS-CMV-infected plants of the ‘original’ stock and those derived from the fifth vertical passage (F1,100≤1. 68; P≥0. 198) (Table 3). For De72-CMV, an increase of RW and IW ratios, and a reduction in virulence was found in plants derived from the fifth vertical passage relative to ‘original’ stock plants infected with vertically evolved viruses (F1,80≥5. 36; P≤0. 023), but virus accumulation was similar in both types of plants (F1,80 = 0. 34; P = 0. 560). Non-evolved viruses were marginally more virulent in plants derived from the fifth vertical passage than in ‘original’ stock plants (F1,80≥2. 97; P≤0. 090) (Table 3). Vertical transmission was higher in plants of the fifth passage of vertical transmission than in plants of the ‘original’ stock (compare Figure 2 and Table 2; F≥6. 93; P≤0. 013). Fny-CMV and LS-CMV strains horizontally evolved (Table 3) caused less reduction in IW and were more virulent in plants derived from the fifth vertical passage compared with ‘original’ stock plants (F1,100≥4. 69; P≤0. 033). In De72-CMV virus accumulation was higher in plants derived from the fifth vertical passage than in the ‘original’ stock plants (F1,80 = 301. 33; P≤1×10−4). Finally, Fny-CMV and LS-CMV alternately evolved were more virulent in plants derived from the fifth vertical passage compared with ‘original’ stock plants (F1,100≥4. 40; P≤0. 040), but virus accumulation of De72-CMV was higher in ‘original’ stock plants than in plants derived from the fifth vertical passage (F1,80 = 41. 92; P≤1×10−4). No significant differences were found in the rest of comparisons (Tables 1–3). Thus, differences in the traits analysed between ‘original’ stock plants and plants derived from the fifth vertical passage indicate that the latter group of plants have mechanisms to reduce virus accumulation, the effects of infection on plant growth and on fecundity (virulence) by vertically transmitted viruses, often at the cost of increased virus accumulation and/or virulence of non-evolved viruses. These results support the hypothesis of plant-virus co-evolution during vertical transmission passages. Most experimental analyses of virulence evolution are based on the hypothesis that virulence, which is correlated with parasite multiplication, is determined by trade-offs with parasite transmission rate [2], [10]–[12]. However, the factors that modulate this trade-off and how they act are only partially understood. Although theory has identified several of these potential modulators, their effect has seldom been analysed experimentally. Here we investigated the role of two such major factors in the evolution of virulence: the mode of transmission, and host adaptation in response to parasite evolution [8], [14], [16], [22]. The paucity of information on this subject has been attributed, in part, to the lack of suitable experimental systems [2]. For our experiments, we used the plant virus CMV and its host plant Arabidopsis thaliana, a system with several traits suitable for our objectives: i) CMV is a pathogen of Arabidopsis that is found at high incidence in Arabidopsis wild populations [45], ii) CMV is transmitted both horizontally and vertically in Arabidopsis [45; unpublished data], iii) Arabidopsis has a short generation time, which allows serial passages of vertical transmission to be performed in a reasonable time frame; and iv) recovery of Arabidopsis plants to CMV infection has not been described. From the perspective of viral fitness, host recovery is equivalent to host death in which the virus can no longer replicate or be transmitted. Therefore, recovery would affect the transmission rate, potentially blurring the virulence-transmission trade-off [54]. We serially passaged three CMV strains in Arabidopsis under strict vertical or strict horizontal transmission, and quantified virulence and traits related to viral fitness in the evolved and non-evolved viruses. We used the effect of virus infection on plant fecundity as a measure of virulence. Virulence encompasses the negative effect of a parasite on host longevity and fecundity [8], [55], [56]. In vertically transmitted parasites, the effect of infection on host fecundity is the most relevant trait for transmission success [10], [13], [14], [23]–[25]. Thus, host fecundity is the best proxy for virulence to analyse the vertical transmission-virulence trade-off. However, longevity of hosts infected with horizontally transmitted parasites, which is linked to the length of the infectious period, is the most obvious determinant of parasite transmission rate. Indeed, in such parasites longevity of infected hosts has been proposed to be a good proxy for virulence [57]. Although CMV infection significantly affects host fecundity, it has little effect on the lifespan of Arabidopsis [41]. This mimics the effect of sterilizing parasites on their hosts. For sterilizing parasites horizontal transmission may be correlated with host fecundity, in which case the effect on host fecundity is the best proxy for virulence [8], [55], [58]. As predicted by theory, our results show a key role of the mode of transmission in the evolution of virulence. The comparison of non-evolved viruses with those evolved under strict vertical transmission indicated that the latter increased their rate of vertical transmission, and that adaptation to this transmission mode was associated with the reduction of virus multiplication and virulence. In contrast, evolution under strict horizontal transmission did not result in changes of virus multiplication or virulence. We did not quantify the rate of vertical transmission of horizontally evolved lineages. However, the absence of evolution in virus multiplication and in virulence of horizontally passaged viruses − two traits that are correlated with vertical transmission rate in our system − may be suggestive of a lack of change in vertical transmission rate. Thus, our results support predictions of the models of virulence evolution based on the trade-off hypothesis [14], [16], [22] in that we found a negative correlation between rate of vertical transmission and virulence. This negative correlation was also found in previous studies with bacteria and insect parasites [26]–[28], [30]–[33], [59], [60], and in the only other study of a plant virus, Barley stripe mosaic virus (BSMV) in barley [29]. Predictions of the trade-off hypothesis were not supported by results of the only other reported analyses of a plant-parasite system, in which the rate of vertical transmission was positively correlated with virulence. However, this is an unusual system as it involves a sterilizing fungus. This fungus invades the plant reproductive structures, and more virulent fungal strains have better access to the seeds. Thus, more virulent strains compensate the higher reduction of plant fecundity by infecting more seeds than less virulent strains [34], [35]. More plant-parasite systems need to be characterized to determine the generality of the trade-off predictions. Perhaps one of the most striking results of this work is the observed negative correlation between virus multiplication and transmission rate. In other plant-parasite systems higher parasite load is associated with higher percentage of infected seeds [29], [34], [35]. Limited knowledge on the mechanisms of CMV seed transmission hinders the interpretation of this result. CMV is present in the embryo, the endosperm, and the coat of infected seeds [61]; and the virus is thought to gain access to seed tissues either through the ovules or pollen [62], or through the suspensor that connects the mother plant and the developing seed [63]. Hence, vertical transmission rate would be determined by the capacity of the virus to reach the seed during gametogenesis and/or while the suspensor is still functional, and by the ability of plant defense to block virus access to the seed. If this model holds for CMV and Arabidopsis, a negative correlation between virus multiplication and transmission rate would be explained: 1) if lower virus titer would result in a less efficient triggering of plant defenses that prevent seed infection; and/or 2) if serial passages of vertical transmission selects for virus variants with mutations that facilitate direct or indirect access for CMV to the seed even at low multiplication levels. Virulence and within-host multiplication were positively correlated in viruses evolved under strict vertical transmission in this study, consistent with the central assumption of the trade-off hypothesis [2], [10]–[12]. This result is at odds with observations in BSMV and barley where neither vertical transmission nor virulence correlated with virus multiplication [29], [64]. Virulence and within-host multiplication does not correlate across CMV and Arabidopsis genotypes, however, as genotype-specific host tolerance to virus infection uncoupled both traits [65]. Interestingly, the Arabidopsis genotype used in the present work (Cen-1) was rated as a low-tolerance genotype [41], so that the relationship between virulence and within-host multiplication will not be blurred by tolerance. Note also that the relationship between virulence and within-host multiplication is not linear (Figures 2–3). Thus, it is possible that this relationship could go unnoticed in some studies [66] if data are within the range in which this relationship is saturated. The reduction of CMV virulence associated with increased rates of vertical transmission is also associated with smaller effects of infection in both the vegetative and reproductive efforts of the host (RW and IW). Vertically transmitted parasites may select for modifications of host life-history traits that enhance host reproductive success and parasite transmission. For instance, insects have increased survival when infected by bacteria such as Rickettsia or Wolbachia [67], [68], or increased reproductive time span when infected by microsporidian parasites [69]. The higher RW and IW ratios of Arabidopsis infected with vertically evolved viruses compared with non-evolved viruses might be interpreted similarly, as Arabidopsis fecundity is positively correlated with both the vegetative and the reproductive growth of the plant [41]. Thus, the observed evolution towards lower virulence may represent a selective advantage for the virus, as it increases the chances for its vertical transmission. In certain contexts, the parasite and its host may have sufficient alignment of interests to boost host-virus co-evolution: Parasite infection may be still detrimental for the host, but maximization of host fitness upon infection also maximizes the parasite fitness [70]. Such maximization might occur when parasites adapt to vertical transmission [3], [71], [72]. In this scenario, the host may also evolve to increase the parasite' s rate of vertical transmission [39], [73], a possibility that has seldom been analysed [36]. Our data provide evidence that this may be the case for the Arabidopsis-CMV interaction, as vertical transmission rate was higher, and virulence was lower, in plants selected during serial passages of vertical transmission as compared with plants from the ‘original’ stock. Although these changes in the passaged plant were not enough to completely compensate the negative effect of CMV infection in plant fitness, they significantly increased plant fecundity as compared with ‘original’ stock plants. The observed changes in the host plant during vertical passages are related to increased resistance, i. e., reduced parasite multiplication [74]. Resistance was particularly effective when infection was the result of vertical transmission compared with horizontal transmission (Tables 1–3), although we cannot rule out that this would reflect that plants infected horizontally may be weaker as they come from seeds already challenged (unsuccessfully) with CMV. In either case, a degree of plant resistance may paradoxically represent a benefit for CMV strains adapted to vertical transmission, as long as multiplication does not go below a threshold that importantly decreases seed transmission rate. These patterns of plant-virus co-evolution may be due to selection during passages. In wild Arabidopsis populations in Spain, the inbreeding coefficient, i. e., probability of autozygosity [75], ranges between 0. 87 and 0. 99 [76]. Hence, wild accessions are not homozygous at all loci, and genetic variation may be expected in an experimental plant population derived from a single individual. Therefore, selection may have acted upon genetic variation in the original stock plant. Evolution in the Arabidopsis-CMV interaction is compatible with conditions required for host-parasite co-evolution as defined by [37] in which hosts and pathogens exert reciprocal selection on each other. Additionally, we can speculate on genetic changes induced by virus infection, resulting either in genomic rearrangements or in epigenetic variation [77], [78] that may explain the observed changes in the host plant. Since all passages of horizontal transmission were done in plants from the ‘original’ stock, we cannot address whether plant adaptation to horizontally passaged viruses occurred in our experiments. The hypothesis that adaptation to vertical transmission is advantageous for both the virus and the plant is in apparent contradiction with the observation that vertical transmission of CMV occurs only at low rates across Arabidopsis genotypes [45; this study]. However, adaptation in Arabidopsis came at the cost of increased virulence and increased multiplication of the non-evolved viruses, i. e., a trade-off in host fitness when infected with non-evolved virus genotypes. In natural populations of Arabidopsis, CMV spreads both by horizontal and vertical transmission [45; unpublished data], and it is reasonable to hypothesize that both the optimal level of vertical transmission for the host plant, and of virulence for the virus would be influenced by the observed adaptation trade-off. In nature, most virus genotypes are likely to be better adapted to horizontal than to vertical transmission, and plant genotypes adapted to vertical transmission would suffer from extra fitness penalties when infected by most virus genotypes. Thus, virulence evolution could not be explained only by trade-offs between the relative rates of vertical and horizontal transmission, as stated by the ‘continuum hypothesis’: our results illustrate the key role that the complex interplay between mode of transmission and host-parasite co-evolution has in determining virulence evolution. This type of interplay should be considered in theoretical and experimental analyses of virulence evolution, and in the design of better strategies for virulence management. Three virus strains were used: Fny-CMV and De72-CMV belonging to subgroup I of CMV isolates, and LS-CMV, belonging to subgroup II. Fny-CMV and LS-CMV are well characterized and were derived from biologically active cDNA clones [79], [80] by in vitro transcription with T7 RNA polymerase (New England Biolabs, Ipswich, MA, USA). De72-CMV was obtained from a field-infected plant of Diplotaxis erucoides (Brassicaceae), a host closely related to Arabidopsis [81]. Transcripts of Fny-CMV and LS-CMV, and purified viral RNA from De72-CMV were used to infect tobacco (Nicotiana tabacum) plants for virus multiplication. CMV virions from tobacco leaves were purified as described in [82], and viral RNA was extracted by virion disruption with phenol and sodium dodecyl sulphate. Accession Cen-1 (Centenera, Spain) was selected from a panel of eighteen Arabidopsis accessions as it combined a higher efficiency of CMV vertical transmission − ranging between 2–8% depending on the virus isolate [unpublished data] −, with a relatively short life cycle [65]. For plant growth, seeds of Cen-1 were sown on filter paper soaked with water in single plastic Petri dishes, and stratified in darkness at 4°C for five days before transferring for germination to a growth chamber (22°C, 14 h light and 70% relative humidity). Five day-old seedlings were planted in soil in 10. 5-cm-diameter pots (0. 43 l volume), and grown in a greenhouse (25/20°C day/night, 16 h light). The three CMV isolates were serially passaged in Cen-1 plants five times by strict vertical transmission, strict horizontal transmission, or alternating both modes of transmission (Figure 1). To analyse virus evolution under strict vertical transmission, Cen-1 plants from a seed stock (referred to as ‘original’ stock) generously provided by Dr. Carlos Alonso-Blanco (CNB-CSIC, Spain) were mechanically inoculated with purified CMV RNA of the three CMV strains (100 ng/ml) in 0. 1 M Na2HPO4 when rosettes had 4–5 leaves (stages 1. 04–1. 05 in [50]) with five replicates per treatment. Seeds from each infected plant were harvested at complete senescence (stage 9. 0 as in [50]) generating five independent vertically transmitted CMV lineages per virus strain. One hundred Cen-1 seeds per CMV-infected plant were grown as described above, and CMV infection in twenty-day-old plants was detected by dot-blot hybridization (see below) to estimate the percentage of seed transmission. Infected plants were allowed to complete their life cycle and seeds were harvested at senescence. One infected plant per CMV lineage was randomly selected to start the next plant generation, and the process was repeated for five generations; i. e., five passages of vertical transmission (Figure 1). The percentage of seed transmission was determined in every passage as described above. Seeds from uninfected individuals of the fifth generation were also harvested. To analyse virus evolution under strict horizontal transmission, sap extracts from the fifteen Cen-1 plants inoculated to generate the vertically transmitted lineages were used to mechanically inoculate fifteen uninfected Cen-1 plants from the ‘original’ Cen-1 seed stock; these fifteen plants represented the first passage of horizontal transmission in five independent lineages for each virus strain. Sap extract from each of these fifteen infected plants was used to inoculate ten plants at stages 1. 04–1. 05 [50] again from the ‘original’ Cen-1 seed stock. CMV infection was detected fifteen days post-inoculation (dpi) by dot-blot hybridization (see below). Sap extract from one randomly chosen infected plant per lineage was used to inoculate ten new plants of the ‘original’ stock of Cen-1 seeds. This procedure was repeated for five passages (Figure 1). In parallel, five viral lineages per CMV strain were evolved alternating vertical and horizontal transmission to generate five alternately transmitted lineages. To do so, 100 seeds from each of the fifteen plants used to generate the vertically transmitted CMV lineages were grown, and CMV infection was detected, as described above. Sap extracts from one infected plant per lineage were used to inoculate ten Cen-1 plants from the ‘original’ seed stock. The procedure for this treatment to this point is identical to that used for the strict horizontal treatment. However, seeds from these horizontally infected plants were harvested at complete senescence. One hundred seeds per plant were grown, virus infection was detected, and one plant per lineage was randomly chosen to generate the new vertically transmitted generation. This process was repeated until the third horizontal passage was completed, for a total of three vertical passages and three horizontal passages (Figure 1). Multiplication and virulence of non-evolved and evolved CMV strains after vertical, horizontal and alternated passages, referred to as vertically evolved, horizontally evolved and alternately evolved viruses, were analysed in plants from the ‘original’ seed stock and in plants derived from the fifth vertical transmission passage. Sap extracts from CMV-infected plants of the fifth vertical and horizontal passages were used to inoculate ten Cen-1 plants from the ‘original’ seed stock and ten plants from seeds from the fifth passage of vertical transmission per virus lineage. Only five plants were inoculated for each of the alternately evolved viruses. In the fourth vertical transmission passage, no infected seeds were detected for one of the De72-CMV lineages; therefore, only four replicates were analysed for this treatment. Plants derived from the fifth passage of vertical transmission were chosen randomly to represent plants passaged with each of the three CMV strains, and inoculated with evolved lineages of the corresponding strain. In addition, ten plants of the ‘original’ Cen-1 stock and ten plants from one randomly chosen uninfected replicate after five vertical passages per each of the three strains were inoculated with sap from Cen-1 plants infected with non-evolved Fny-CMV, De72-CMV and LS-CMV. Mock-inoculated plants, with ten replicates per seed stock, were included as a control. Virus multiplication, and effect of virus infection on rosette and inflorescence growth, and on seed production were determined in each plant as described below. CMV multiplication was quantified as virus RNA accumulation. Total nucleic acid extracts from four leaf discs (0. 01 g fresh weight) collected from four different rosette and inflorescence leaves were obtained using TRI-reagent (Sigma-Aldrich, St. Louis, MO, USA). RNA quantification was done by dot-blot hybridization with 32P-labeled RNA probes obtained by transcription from cDNA clones representing the 3′ non-coding region of the three genomic RNAs, which is highly similar within a CMV isolate. For Fny-CMV and De72-CMV, a probe representing nucleotides 1933 to 2215 of Fny-CMV RNA3 (GeneBank Acc. No. D10538) was used, and for LS-CMV the probe represented nucleotides 1861 to 2193 of LS-CMV RNA3 (Acc. No AF127976). Internal CMV standards for subgroup I (Fny-CMV or De72-CMV), and subgroup II (LS-CMV) were included as a two-fold dilution series of purified RNA (0. 5 to 0. 001 µg) in nucleic acid extracts from mock-inoculated Arabidopsis plants. RNA extracts from infected plants were blotted at different dilutions to ensure that hybridization signal was on the linear portion of the RNA concentration-hybridization signal curve. All hybridizations were done at 65°C overnight in 6× SSC, 5× Denhardt' s mixture, 0. 1% sodium dodecyl sulphate, and yeast tRNA at 50 mg/ml [83]. RNA hybridization signal was detected using a Typhoon 9400 scanner (GE Healthcare, Chalfont St. Giles, UK) after exposure of the Eu+2 store phosphor screens to the labelled samples, and CMV multiplication was quantified by using Image-Quant 5. 2 (Molecular Dynamics, GE Healthcare) [84]. Virulence is defined as the negative effect of infection on host fitness [1], [2], a good proxy for host fitness being total fecundity. Our previous work showed that CMV infection does not affect the viability or weight of individual seeds in Cen-1 [56]; therefore we used total seed weight (SW) as a measure of host fitness. Thus, virulence was estimated as one minus the ratio of the total seed weight of infected (SWi) to total seed weight of mock-inoculated (SWm) plants, 1− (SWi/SWm). We also measured plant dry weight at complete senescence after drying at 65°C until constant weight, as a measure of tolerance (see Introduction). Rosette weight (RW) and inflorescence weight including seeds (IW) were measured separately. To quantify the effect of CMV infection on RW and IW, the value of each infected plant was divided by the mean value of the mock-inoculated plants (Traiti/Traitm, i and m denote infected and mock-inoculated plants, respectively). In each vertical passage, the rate of seed transmission was determined as the percentage of infected individuals out of the 100 germinated plants per lineage. Two leaves of twenty-day-old plants were harvested and pooled in groups of ten individuals, and total nucleic acid extracts of these pools were obtained. We pooled leaves among plants because of the low percentage of plants infected [unpublished data]. The presence of CMV in each pool was detected by dot-blot hybridization with the same probes used for quantification of virus accumulation. As negative controls, total nucleic acid extracts from pools of ten twenty-day-old non-infected plants were used. Samples with hybridization signal more than two-fold higher the negative controls were considered as positive. Two leaves were harvested from each plant within each positive pool for detection of CMV individually by dot-blot hybridization as described above. In the ‘original’ stock plants infected with the vertically evolved CMV strains, five infected plants per lineage were randomly chosen and 100 seeds per plant were grown as described above. Five-day-old seedlings were harvested in pools of ten individuals and the presence of CMV in each pool was detected essentially as described above. Because we pooled seedlings for this analysis, seed transmission rate was estimated assuming that the number of seedlings infected follows a Poisson distribution [85]. Estimates obtained assuming a binomial distribution yielded the same results. Data on virus multiplication, seed transmission rate, and rosette, inflorescence and seed weights, and their various transformations, including virulence, were homoscedastic and were analysed using full factorial General Linear Models (GLM). Virus strain/lineage, type of plant (stock vs. passaged), and mode of transmission were considered as fixed effect factors. Significance of differences among classes within each factor was determined by Least Significant Difference (LSD) analyses. Linear associations between virus multiplication, seed transmission rate and virulence during serial passages was analysed by bivariate tests and using Pearson' s correlation test [85]. To investigate whether non-linear models better explained these associations, we fitted them to logarithmic, exponential and quadratic models [86]. Regression lines were compared using ANOVA to test the equality of slopes and intercepts; non-linear curves were log transformed for this analysis. All statistical analyses were performed using the statistical software packages SPSS 21. 0 (SPSS Inc., Chicago, IL, USA). | Title: Vertical Transmission Selects for Reduced Virulence in a Plant Virus and for Increased Resistance in the Host Summary: Virulence is a key property of parasites, and is linked to the emergence of new diseases and to the reduction of ecosystem biodiversity. Consequently, scientists have devoted a great effort to build theoretical models that predict which factors may modulate virulence evolution. However, whether (and how) these factors affect virulence evolution has been seldom analysed experimentally. Using the plant virus Cucumber mosaic virus (CMV) and its natural host Arabidopsis thaliana, we studied the role of two such factors: the mode of transmission, and host adaptation in response to parasite evolution. We serially passaged CMV under strict vertical and strict horizontal transmission, and a combination of both. Subsequently, we analysed differences in CMV seed (vertical) transmission rate, accumulation and virulence between evolved and non-evolved viruses. We also compared whether these differences varied in original plants and in plants evolved during vertical passaging. Vertical passaging increased CMV seed transmission, and reduced accumulation and virulence, while horizontal passaging had no effect. Changes during vertical passaging were determined also by reciprocal host adaptation, which additionally reduced virulence and accumulation of vertically transmitted viruses. Hence, we provide evidence that the interplay between the transmission mode and host-parasite co-evolution is central in determining virulence evolution. | 12,172 | 313 | lay_plos | en |
Summarize: CROSS-REFERENCE TO RELATED APPLICATION [0001] This application is based upon co-pending U.S. provisional patent application Serial No. 60/354,467, filed Feb. 5, 2002, incorporated herein by reference. FIELD OF THE INVENTION [0002] The invention relates to extracorporeal hemodialysis. More particularly, the invention related to a method and apparatus for dialyzing a patient's blood with a single venipuncture or cannulation. BACKGROUND OF THE INVENTION [0003] Historically, kidney diseases have been of critical concern to human life. Many kinds of kidney diseases interfere with the function of the kidney such that the kidney ceases to remove waste and excess water from the blood. When the kidney is sufficiently impaired that large portions of the waste products and water are not removed from the blood, the life of the patient cannot be preserved unless a way is provided for artificially performing the function of the impaired kidney. Even today, the same general procedure is used for dialyzing patients' blood that was used very early in the treatment of kidney disease. [0004] For example, the most commonly accepted practice for dialyzing a patient's blood extracorporeally requires the surgical creation of a subcutaneous, arterio-venous fistula. Thereafter, the subcutaneous venous system dilates secondary to the increase of blood flow derived from the artery to the vein through the fistula. Sufficient blood flow for dialysis is then obtainable by venipuncture with large bore needles. Normally, two hollow needles or cannulas are used to perform two venipunctures on the patient so that two blood-communication sites exist simultaneously in the patient. Conventionally, blood is withdrawn from one of the punctured blood vessels, forced through a hemodialyzer and thereafter forced into the other. The needles have to be substantially distant from one another to prevent recirculation of blood. [0005] The aforementioned procedure has been found to have serious disadvantages both to the patient and to the attending physicians, nurses, and technicians. The problems are particularly aggravated because most patients requiring extracorporeal hemodialysis must undergo treatment as frequently as three to four times per week. This means that if every venipuncture were completely successful, a patient would need to undergo from 6 to 8 venipunctures or cannulations each week. [0006] It is well-known that the duration and well-function of a fistula created by venipuncture is inversely related to the number of venipunctures. Tissue repeatedly subjected to the trauma of venipuncture is much more susceptible to thrombophlebitis, paravascular hemorrhage, clotting and infection. In fact, it is commonly found in patients who have experienced a number of venipunctures, that the tissues surrounding the most accessible veins develop large hematomas which obscure the veins, making successful venipuncture extremely difficult because of insufficient blood flow in the damaged blood vessels. [0007] Also contributing to the problem is the fact that once one successful venipuncture is made and blood is allowed to flow from the patient's body toward a hemodialyzer, the blood volume in the patient's body is reduced, making the second venipuncture very difficult. It has historically been found that while most skilled physicians or technicians are able to perform the first venipuncture with little difficulty, frequently a plurality of attempts is necessary before a second venipuncture can be performed on the same patient. [0008] In addition, while the pain and discomfort suffered by a hemodialysis patient is understandable, the multiple attempts at venipuncture often necessary to place the second needle result in increasing apprehension, and anxiety on the part of both the patient and the physician, nurse, or technician attending the patient further reduces the likelihood of successful venipuncture. SUMMARY OF THE INVENTION [0009] The present invention, including a novel method and apparatus, reduces patient trauma and tissue damage by accommodating extracorporeal hemodialysis with a single venipuncture. Generally, once the venipuncture has been performed, blood is conducted away from the venipuncture site through one passageway in a double-lumen needle system. The blood is forced through the extracorporeal hemodialyzer and thereafter through the other passageway of the double-lumen needle system again to the venipuncture site. The current invention also reduces recirculation by preventing blood returned to a graft from being aspirated back into the hemodialyzer. A mechanical barrier, membrane, or other structure, such as a balloon, physically substantially separates blood that enters one needle from the blood exiting the other needle. [0010] In another embodiment of the invention, an access needle system comprises a cannula, a sheath around the cannula to form an annular space, and a hemostasis valve surrounding at least the proximal end of the cannula and being in fluid communication with the annular space. The sheath has one or more lateral ports or openings, and hoses in fluid communication with the proximal end of the cannula and the hemostasis valve or the annular space extend to a blood hemodialyzer. Preferably there will be a barrier to block or partially obstruct fluid flow. The barrier may comprise an inflatable balloon membrane, or other mechanical structure. Optionally a second sheath may partly surround the first sheath and uncover the one or more ports in the inner sheath when the outer sheath is moved proximally. Alternatively, the outer sheath may have one or more lateral pores that may align with one or more lateral pores of the first sheath when the second sheath is slid along the first sheath. OBJECTS OF THE INVENTION [0011] It is a primary object of the present invention to provide an improved method of extracorporeal hemodialysis using a single venipuncture for each treatment. [0012] It is another object of the present invention to provide an improved apparatus facilitating extracorporeal hemodialysis with a single venipuncture. [0013] It is yet a further object of the present invention to provide a single access dialysis needle having two lumens and a barrier wherein extracorporeal homodialysis can be performed using a single venipuncture. [0014] These and other objects and features of the present invention will become more fully apparent from the description below. BRIEF DESCRIPTION OF THE DRAWINGS [0015] [0015]FIG. 1 is a partly cross-sectional schematic view of an embodiment of the system of the invention for dialyzing a patient's blood using a single venipuncture; [0016] [0016]FIG. 1A is a partly cross-sectional schematic view of a variation of the embodiment shown in FIG. 1 where the two cannulae are coaxial; [0017] [0017]FIG. 2 is a schematic illustration of a portion of the system shown in FIG. 1 in position in a patient's dialysis conduit; [0018] [0018]FIG. 3 is a cross-sectional view of another embodiment of the system of the invention where at least one sheath is arranged circumferentially around a cannula; and [0019] [0019]FIGS. 4 and 5 are each an oblique view of the embodiment shown in FIG. 3. DETAILED DESCRIPTION OF INVENTION [0020] The invention can perhaps be better understood from the drawings. In FIG. 1 a dialysis needle system 2 comprises a first cannula 4 with an annular balloon 6 and a second cannula 8. Balloon 6 is in fluid communication with an inflation lumen 12 and an inflator 14. Balloon 6 is deployed after insertion of the cannula and positioning within a dialysis fistula. The proximal end 16 of first cannula 4 and the proximal end 18 of second cannula 8 are separated or otherwise configured so that each of proximal ends 16 and 18 can be attached to hoses 20, 22 attached to a hemodialyzer (not shown). First cannula 4 and second cannula 8 are shown in FIG. 1 to be essentially parallel. It should be noted that it is within the scope of the invention that first cannula 4 and second cannula 8 can be coaxial to one another. For example, as shown in FIG. 1A, second cannula 8 a circumferentially surrounds first cannula 4 a. Balloon 6 a is in fluid communication with an inflation lumen 12 a and an inflator 14 a. Balloon 6 a is deployed after insertion of the needle system and positioning within a dialysis fistula. The proximal end 16 a of first cannula 4 a and the proximal end 18 a of second cannula 8 a are separated or otherwise configured so that each of proximal ends 16 a and 18 a can be attached to hoses 20 a, 22 a attached to a hemodialyzer (not shown). [0021] Needle system 2 is shown in FIG. 2 in position in a patient's dialysis conduit or vessel 26. Annular balloon 6 is inflated to cause blood flow in dialysis conduit 26 to go in the direction of arrows 28 into a lumen 30 of second cannula 8. Blood flows into hose 22, to a hemodialyzer (not shown), through hose 20, and then into a lumen 32 of first cannula 4. Annular balloon 8 functions to stop the flow of blood and separate the blood streams into and out of dialysis needle system 2, thereby preventing recirculation of blood. It is within the scope of the invention that blood could also flow in the opposite direction, that is, in the directions opposite to the arrows shown in FIG. 2. [0022] In the embodiment of the invention shown in FIG. 3, a first or inner sheath 38 is circumferentially arranged around a cannular or needle 40 to form annular space 42, and optionally a second or outer sheath 44 is circumferentially and slidably arranged around inner sheath 38. The distal portion 52 of inner sheath 38 is sealingly attached to dilating member 50, which surrounds the distal portion 53 of cannula 40. [0023] Adjacent to dilating member 50 is an annular support member 54 through which cannula 40 extends. Support member 54 is affixed to or integral with dilating member 50 and provides support for sheath distal portion 52. [0024] Inner sheath 38 preferably has an expandable section 56 having longitudinal cut or score lines 57, for example, from at least about 4 to about 20, or more, such cut or score lines and optionally a latitudinal score line 58. When sheath 38 is pushed or slid distally, section 56 forms a barrier structure 59 in the radial direction, as shown in FIG. 5. If outer sheath 44 is used, it is slid proximally to expose barrier section 56 and at least one opening 60. Alternatively, an opening 60 in inner sheath 38 could become aligned with an opening 62 in outer sheath 44 when sheath 44 is moved in the proximal direction. [0025] It is within the scope of the invention that the embodiment of the invention shown in FIGS. 3 to 5 may have a barrier element other than the structure described above. For example, this embodiment may have an inflatable balloon with an inflation lumen and an inflator, as described for needle system 2. Other barrier structure known now or to be developed would be similarly operable. [0026] The proximal end 68 of cannula 40 is in fluid communication with a stationery shaft 70, which is connected to a flexible hose (not shown) which preferably has a threaded luer connector 74 at its proximal end. A hemostasis valve 76 slidably encompasses the distal end 78 of stationary shaft 70 and the proximal portion 80 of inner sheath 38. Also, hemostasis valve 76 is in fluid communication with a port 82, which is connected to a flexible hose (not shown) which preferably has a luer connector 86 at its proximal end. Preferably hemostasis valve 76 has at least one sealing ring 88, such as an O-ring or a rubber membrane, on or in the surface adjacent shaft 70. Preferably valve 76 is affixed to the proximal portion 80 of inner sheath 38. Both luer connectors 74, 86 are intended to connect to a hemodialysis machine (not shown). [0027] [0027]FIGS. 4 and 5 are each an oblique view of the embodiment of the invention depicted in FIG. 3. In FIG. 4 outer sheath 44 is in its initial position relative to inner sheath 38, where port 60 in inner sheath 38 and barrier structure section 56 are covered. [0028] If outer sheath 44 is used, it is moved longitudinally in the proximal direction, for example, by gripping the outer surface of outer sheath 44, and then port 60 in sheath 38 and barrier section 56 are uncovered. Hemostasis valve 76 and the proximal 80 of inner sheath 38 are displaced in the distal direction to cause distal section 56 to expand radially to form barrier structure 59. Blood flow is represented by arrows 108, 110, 112, and 114. [0029] The device of the invention comprises conventional physiologically acceptable materials, especially those that can be sterilized, as would be appreciated by those skilled in the art. Typically the components described herein will be made from rigid or slightly flexible polymers and/or co-polymers, several as polypropylene, polyethylene, polystyrene, polybutylene, and co-polymers thereof. Sealing ring 88 would preferably be comprised of a suitable flexible polymer, rubber or elastomer. [0030] The outer surface of the device should be configured or designed to minimize friction or interference with movement or body surfaces or tissue. The outer surfaces of the device according to the invention should be as smooth as possible. For example, the distal surface 92 of hemostasus valve 76 can be rounded as in FIG. 3 or tapered as shown in FIGS. 4 and 5. [0031] The invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive and the scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope. | Summary: A single access dialysis needle system comprises a first cannula, a second cannula or sheath, and a barrier arranged on the outer surface of the first cannula. The distal end of the first cannula extends distal to the distal end of the second cannula or outer sheath, and the barrier is positioned between the respective distal ends. When the barrier is inflated or otherwise activated, it prevents or minimizes recirculation. | 3,578 | 98 | big_patent | en |
Summarize: BACKGROUND [0001] The present invention relates to methods and apparatuses for closing punctures and apertures in human and animal tissue and to methods and apparatuses for inserting such an apparatus into such tissue to perform such closure functions. [0002] During angiography and related procedures, catheters are inserted through an incision or puncture in the skin and underlying tissues to access an artery or vein, typically in the groin, neck, or subclavian areas of a patient. The catheter can be inserted through a puncture in the blood vessel and guided to the desired site to perform interventional procedures such as angiography, angioplasty, plaque removal, and infusion of a therapeutic substance. After the procedure is completed and the catheter is removed from the patient, the access hole must be closed to prevent massive hemorrhage. This is conventionally achieved by applying pressure over the blood vessel manually and then by applying a pressure bandage, compressive weight, or clamp device. With conventional methods, the rate of post-puncture hemorrhage is high, which causes considerable complications. This complication is exacerbated by the concomitant use of anticoagulant medications such as heparin or warfarin and by antiplatelet drugs, which are commonly used to treat vascular disease. [0003] Sutures have been used to close access puncture wounds in blood vessels. U.S. Pat. No. 5,613,974 describes a device and method for applying sutures to a vascular puncture. US2004/0093027A1 describes barbed suture-like material that apposes the puncture site. US 2005/0121042 A1 describes a device and method for applying suture to a vascular puncture. Difficulties with these methods include the large number of steps necessary to deploy the needles, capture the suture, withdraw the suture, tie the knot, and cut the suture. In addition, the hole in the blood vessel is often widened by insertion of the instrument, and the suture remains intravascularly on the endothelial surface, and thus can be a nidus for thrombus or intravascular mural hyperplasia with later spontaneous and catastrophic closure of the vessel. [0004] Extravascular plugs have also been proposed for closure of vascular punctures. U.S. Pat. No. 5,254,105 and U.S. Pat. No. 5,330,445 describe an extravascular plug which is slid down the external surface of the catheter or introducer and is placed into the puncture site in this manner. U.S. Pat. No. 5,643,318 relates to a similar device that has its own vessel locator device. US22022822A1 and US2004/0158287A1 describe an extravascular plug that is delivered with a specialized system. US24215232A1 describes an extravascular plug with an intravascular anchor set with a sheath with a detection port. US2005/0085855A1 describes an extravascular collagen plug, held in place with an intravascular anchor, and a device that locks over a piece of suture. U.S. Pat. No. 5,906,631 describes a plug made of hydrophilic material. U.S. Pat. No. 6,126,675 describes an intravascular anchor and a bioabsorble extravascular plug. U.S. Pat. No. 6,623,509 describes a bioabsorbable plug. U.S. Pat. No. 6,296,657 and U.S. Pat. No. 6,743,195 describe an inflatable balloon that puts pressure on the puncture site. U.S. Pat. No. 6,569,185 describes an injectable vascular plug. U.S. Pat. No. 6,663,655 describes a plug that screws in the puncture tract. US2004/0143290 A1 describes a combination of an intraluminal balloon and injectable sealant. Disadvantages to these methods are related to the high likelihood of thrombosis associated with the intravascular plug or anchor, and the presence of collagen or other bioabsorble materials which cause inflammation, activate the clotting cascade, and increase the likelihood of thrombosis, which, in an arterial system, is catastrophic. [0005] Vascular patches have also been used for repairing blood vessels, but usually only for large areas of damage. U.S. Pat. No. 5,100,422 describes a vascular patch that is sutured to the external surface of the damaged blood vessel. U.S. Pat. No. 5,100,422 describes a vascular patch achieved by instilled adhesives and the device for doing such. These are generally impractical for catheter-based methods. U.S. Pat. No. 6,248,124 and U.S. Pat. No. 5,507,744 describe devices and methods that use electrocautery for sealing vascular punctures. This also requires a complicated device, and perforation and thrombosis are very real possibilities. [0006] Vascular clips or staples delivered through a catheter device have also been proposed. These devices have penetrating members that bring the edges of the tissue together. U.S. Pat. No. 6,695,867 describes a clip or staple that is delivered by a specialized device. U.S. Pat. No. 6,749,622 describes a number of different clips with sharpened barbs or ends that include both intra- and extravascular portions, made of metal with memory characteristics. U.S. Pat. No. 5,861,005 describes an arterial staple that is delivered with a specialized device. U.S. Pat. No. 5,919,207 describes a stapling system based on long hooked wires that appose the surfaces, with a small staple gun to close the lesion. U.S. Pat. No. 6,022,372 describes a similar staple gun. U.S. Pat. No. 6,296,657, U.S. Pat. No. 6,663,655, and U.S. Pat. No. 6,749,621 describe a clip that is external to the vessel, but clips the two sides of the puncture together, and a device for achieving such. U.S. Pat. No. 5,782,861 and U.S. Pat. No. 5,964,782 describe clip devices composed of two or more prongs or hooks that, depending on the direction of the prongs, can clip together the puncture site from the intra- or extravascular position, through the use of a collar which forces the prongs together or other mechanisms. These clip devices are composed of thick semi-rigid material, and can be placed only with a specialized instruments, and because of the rigidity have great potential to injure or cut the blood vessel. Disadvantages of these clip devices in general include difficulty in retrieving the device if misplaced, excessive manipulation required, the thickness of the clip material which tends to cut or shear the blood vessel, the large forces that must be used to curve the staples and fix the clips, the increased possibility of tearing the blood vessel, and the general lack of control of the forces being applied to the blood vessel. [0007] Accordingly, there is a need for methods and apparatuses that are suitable for closure of vascular punctures or other openings, and that do not suffer from the drawbacks of conventional approaches. SUMMARY OF THE INVENTION [0008] The present invention provides methods and apparatuses that are suitable for closure of vascular punctures or other openings, and that do not suffer from the drawbacks of conventional approaches. [0009] The present invention comprises a tissue closure device, comprising a plurality of tissue engagement elements, mounted with each other such that in a first compressed configuration the tissue engagement elements can pass through a tissue opening to be closed, and such that in a second compressed configuration the tissue engagement elements bring the edges of the opening into apposition, and such that in an expanded configuration the tissue engagement elements span the opening, wherein the tissue engagement elements engage the tissue in the compressed configuration and in the expanded configuration. [0010] The present invention also comprises methods for closing tissue openings, comprising passing a device while in the first compressed condition through a sheath that penetrates the proximal surface of the tissue; expelling the device from the sheath into space beyond the proximal surface of the tissue such that the device assumes the expanded configuration; c) manipulating the device such that the tissue engagement elements engage the tissue; d) causing the device to assume the second compressed configuration, bringing the edges of the opening into apposition. [0011] The present invention can provide a catheter-delivered umbrella-like device comprising fine, strong, flexible material that after delivery expands in a blood vessel so that the individual members extend beyond the catheter edges and/or puncture dimensions. The device can be viewed as analagous in structure and design as contemporary expandable vascular filters and closure umbrellas, although its purpose and function is completely different. As the catheter is withdrawn, the device is pulled against the interior of the blood vessel and the hooks or grasping devices on the ends of the members seize the interior of the vessel wall. Because of the very fine and flexible nature of the members and their multiplicity, there is minimal shear force applied to the blood vessel. While pulling on the retaining suture to keep the device against the blood vessel, a retaining lock is then advanced distally starting at the proximal portion of the members, which causes the members to first angle the device into a conical shape and then force the individual members together in a linear parallel direction, which because the members are engaged with the vessel wall, brings the edges of the punctured tissue together into apposition. The retainer lock is then locked onto the parallel members and can keep tension on the wound externally, and can prevent intravascular migration of the device. If there is no blood leakage through the closure and the device is properly positioned and stable, then the guidewire can be removed and the retaining suture or string loop cut, resulting is complete and rapid closure, which can then heal. [0012] Since this device brings the puncture edges together, there is true blood vessel healing with little endothelial disruption, reducing the chances of thrombosis or intimal hyperplasia. The device can be supplied in different diameters (e.g., french) to accommodate different sizes of catheters and different sizes of puncture holes. [0013] The present invention can comprise a device with umbrella-like structure, which can be viewed as analogous to the various designs of intravascular filters and aperture seals, which are delivered in a folded or compressed form, and then expanded to their filter shapes. U.S. Pat. No. 4,969,891 describes a self-expanding removable filter device that is placed with a sheath. U.S. Pat. No. 5,634,942 and U.S. Pat. No. 5,634,942 describe a similar device but with two sets of arms which protrude in opposite directions. U.S. Pat. No. 6,241,746 B1 is a similar version that can be converted to a vascular stent. U.S. Pat. No. 6, 361, 546 is a version with a central guidewire lumen. U.S. Pat. No. 6,428,559 describes a variable-diameter vascular filter system. U.S. Pat. No. 6,485,501 B1 also describes a filter with a guidewire. US 2003/0208227 describes different construction configurations of a filter. US 2004/0087999 A1 reveals various types of structures to retain the filter in the vessel. U.S. Pat. No. 5,709,707 describes a typical umbrella-type closure device used to close apertures. Each of the preceding patents and applications are incorporated herein by reference. The present device, analogous to many of the vascular filters and umbrella closure devices noted above, is both expandable like an umbrella and retrievable if it had to be retrieved because of misplacement. Unlike previous umbrella closure devices, the present device engages the tissue in both the expanded and compressed configurations, and functions by bringing tissue edges into apposition rather than by providing a patch that covers the opening. In addition, although the embodiments shown here generally have linear members, these members, like the structure of the intravascular filters and umbrella-type devices, need not be strictly linear, but can assume a number of complex geometrical shapes and structural patterns. [0014] The present device, like some contemporary vascular filters, can utilize an expanding material, preferably with memory characteristics, that opens up spontaneously within the blood vessel. The device also, like some contemporary filters, can have tissue hooks or penetrators, in order to seize the vessel wall and stabilize the device. However, unlike an umbrella-style vascular filter, the device uses this opening-closing quality to seize the edges of the puncture site, and close them, resulting in a complete vascular closure. Although the device can be viewed as analogous to some contemporary self-expanding and retractable vascular filters, it is unlike them in that in certain embodiments it has a retaining lock to force the umbrella to reassume its folded state. DESCRIPTION OF THE FIGURES [0015] The invention is explained by using embodiment examples and corresponding drawings, which are incorporated into and form part of the specification. [0016] FIG. 1 ( a,b ) is a schematic depiction of a vascular closure apposition device according to the present invention. [0017] FIG. 2 ( a,b ) is a schematic depiction of a vascular closure apposition device according to the present invention. [0018] FIG. 3 ( a,b ) is a schematic depiction of a vascular closure apposition device with a guidewire lumen, according to the present invention. [0019] FIG. 4 is a schematic depiction of a vascular closure apposition device according to the present invention. [0020] FIG. 5 is a schematic depiction of a vascular closure apposition device according to the present invention. [0021] FIG. 6 ( a,b,c ) are schematic depictions of example embodiments of vascular closure apposition devices according to the present invention. [0022] FIG. 7 ( a,b,c,d,e,f ) is a schematic illustration of a method of closing a vascular opening according to the present invention. [0023] FIG. 8 ( a,b,c,d ) is a schematic depiction of a double-action vascular closure apposition device according to the present invention. [0024] FIG. 9 ( a,b,c ) is a schematic depiction of a double-action vascular closure apposition device according to the present invention. DETAILED DESCRIPTION [0025] The present invention provides apparatuses and methods for closing a vascular puncture wound or any tissue aperture, for example those resulting from the insertion of a vascular catheter or surgical instrument, trauma or disease. The present invention embraces both apparatus and method aspects of devices for closing a vascular puncture, and the methods for delivering such a device. The present device can be closed in the delivery system (catheter or sheath) and when discharged, be open in the blood vessel. In some embodiments, at least a portion of the device can be formed of a memory metal or similar material, as is currently done in vascular filters. The stress free state corresponds to the state at which the apparatus has opened in a blood vessel, and the stressed state in the catheter and when a retaining lock is put into position. Example embodiments of tissue closure apposition devices according to the present invention are shown in FIGS. 1, 2, 3, 4, 5, 6, and 8. The descriptions refer to “vessels” for convenience; the present invention is applicable to facilitate closure of various types of tissue openings. [0026] FIG. 1 ( a,b ) is a lateral view of a vascular closure apposition umbrella. A plurality of members 101 are disposed substantially radially about a central junction 103. Each of the members 101 comprise a tissue hook 102, in the figure a double hook. The central junction 103 is adapted to engage a closed loop of string or suture 104. The hooks 102 engage the tissue, and are brought and maintained together by operation of a retaining lock 105. In FIG. 1 b, the device is shown in a closed position, where the central junction 103 has passed through the retaining lock 105. [0027] The vascular closure apposition umbrella of FIG. 1 ( a,b ) comprises 2 or more members 101 placed in apposition to each other, shown in the figure as disposed substantially radially. The members 101 in the figure are shown as straight wires, but can be curved or have a wave structure or other design, for example a design to engage a retaining lock. The members are flexible for manipulation in tissue and delivery, yet rigid enough when extended to push the tissue engagement structures against the vessel wall. The tissue engagement structures 102 in this example comprise double hooks, allowing engagement of the tissue in 102 different directions simultaneously. The tissue engagement structures can also comprise multiple hooks, a single hook, or straight engaging devices. The tissue engagement structures can be sharp in order to penetrate in one direction, but not to cut, thus, would generally not have a cutting surface other than the point. The members join in a central junction 103 which can be continuous with each of the members or can be joined to the members by any suitable method. The central junction 103 comprises an eyelet or recovery loop in which initially a closed loop of string or suture 104 engages. The eyelet or recovery loop can be used to recover the device into a catheter in the event of misplacement. A retaining lock 105 can encourage closure of the device, and can also prevent unintended intravascular migration of the device. The retaining lock 105 is shown in the figure as a washer-like device, but can take a number of different shapes and can comprise a number of different materials. For example, the retaining lock 105 can comprise plastic, metal, or composite. [0028] In operation, the tissue closure apposition umbrella is closed within the catheter or sheath, corresponding to the illustration of FIG. 1 ( b ). Once placed within the blood vessel, the umbrella can be opened within the blood vessel, corresponding to the illustration of FIG. 1 ( a ), so that the hooks on the members engage the vessel wall. The umbrella can then be closed with the retaining lock. As the umbrella closes with the retaining lock, the hooks hold the edges of the puncture wound and, as they align with each other, bring the puncture wound edges in apposition. Undulations or excresences on the members or central junction can engage corresponding locking surfaces on the retaining lock. More specific locking devices such as angled dentates, peg and hole, and male-female locking surfaces can also be suitable. A guidewire can go between the members in this particular embodiment without a specific lumen for the guidewire. [0029] FIG. 2 ( a,b ) is a lateral view of a vascular closure apposition double umbrella. A plurality of members 201 are disposed substantially radially about a central junction 203. Each member comprises a tissue engagement structure 202, shown in the figure as a hook on the end of the member. The central junction can comprise a structure compatible with a string or suture 204 to facilitate deployment and removal. A retaining lock 205, in the figure an umbrella oriented opposite the umbrella formed by members 201, closes the device. FIG. 2 ( b ) shows the device with the umbrella formed by members 201 closed, bringing tissue sides in apposition, and the retaining lock 205 open, maintaining the closed position of the umbrella and providing tissue stability on the opposite side of the vessel wall. [0030] The radial members 201 in FIG. 2 ( a,b ) are shown as straight, but can have dentates or other devices compatible with engagement of the retaining lock. The string or suture 204 can be engaged with the central junction 203 to urge the central junction 203 through the retaining lock 205, encouraging the members 201 into apposition. The retaining lock of the device of FIG. 2 ( a,b ) comprises another expanding umbrella, but facing the opposite direction. In the figure, the retaining lock umbrella has straight members with hooks on the ends. The retaining lock umbrella can also comprise a variety of configurations, including bent or curved members, members with various hooks or no hooks, web-like structures, and film-like members. This retaining lock comprise more complicated structure members, as examples like many constructions of intravascular stents and filters. The members 201 close and bring the tissue together as in FIG. 2 ( b ); the retaining lock can provide for tissue stability in the extravascular tissues. A guidewire in this embodiment can go between the members without a specific lumen for the guidewire. [0031] FIG. 3 ( a,b ) is a lateral view of a vascular closure apposition umbrella with a guidewire lumen. The device comprises members 301 disposed substantially radially about a central junction 303. Each arm 301 comprises a tissue engagement structure 302, in the figure shown as a double hook on the end of the arm 301. The central junction 303 comprises a columnar guidewire lumen with a recovery loop or device for engaging a closed loop of string or suture 304. A retaining lock 305 that closes the device. FIG. 3 ( b ) shows the device in a closed position with the retaining lock 305 engaged. [0032] The embodiment of FIG. 3 ( a,b ) comprises a closure apposition umbrella with a guidewire lumen. Inclusion of a guidewire lumen can reduce interference of the guidewire with placement of the umbrella, and allows the guidewire to remain in place in case the seating of the device is not optimal and then the device must be retrieved. The device can be delivered and placed with a guidewire in place. The apposed tissue might close the lumen once the guidewire is withdrawn. If desired, a soft one-way flap valve (not shown) or other structure can be placed in the lumen to occlude any blood flow that might occur when the wire is withdrawn. [0033] FIG. 4 is a lateral view of a vascular closure apposition umbrella comprising members 401 disposed substantially radially about a central junction 403, forming an overall conical shape. The members have tissue engagement structures 402, shown in the figure as hooks at the ends of the members 401. The members also have reversed barbs or feathers 406 to prevent intravascular migration of the device and to maintain the members in a closed state by engaging the tissue, a retaining lock, or both. In operation, the embodiment of FIG. 4 is similar to those discussed previously. [0034] FIG. 5 is a lateral view of a vascular closure apposition umbrella. A plurality of members 501 are disposed substantially radially about a central junction 503. The members have tissue engagement structures 502, shown in the figure as a hook at the end of the member 502. The central junction 503 can have a recovery loop or device, and can engage a string or suture 504 for delivery, placement, and recovery. A retaining lock 505 can engage the members 501, the central junction 503, or both, to encourage the device to and maintain the device in a closed position. A backing or coating 507 can be mounted with the members as a fabric, web, or film. The backing can carry, or be made of, a material that can elute drug to prevent coagulation or to prevent endothelial hyperplasia or can be hemostatic initially, and fill the puncture track later. [0035] FIG. 6 ( a,b,c ) are lateral views of example embodiments of vascular closure apposition devices according to the present invention. In FIG. 6 ( a ), members 601 comprise elongated diamond shapes (rather than the straight wires depicted previously), which shape can have advantages in manufacture and in operation. The members 601 have tissue engagement structures 602, in the figure shown as hooks mounted with members at various locations. In FIG. 6 ( b ), a device having a reduced number of members 611 is shown. A reduced number of members can provide for a simpler device, which can have manufacturing advantages and can be suitable for certain applications. In FIG. 6 ( c ), members 621 comprise a non-linear geometry, and are connected by intermember struts 628. The non-linear geometry and intermember struts can allow specific opening and closing trajectories, and can allow optimization of forces when opening and closing. A wide variety of specific geometries and structures can be suitable with the present invention, as examples including geometries and structures currently used in vascular filters and stents. [0036] FIG. 7 ( a,b,c,d,e,f ) is a schematic illustration of a method of closing a vascular opening according to the present invention. A blood vessel 701 is penetrated by a sheath 702 and a guidewire 703. In FIG. 7 ( a ), an apposition device 704, for example as described previously, is in a closed configuration within the sheath, with the loop of string or suture 705 engaged. In FIG. 7 ( b ) the device 704 has been extruded from the sheath 702 and is in the expanded configuration within the vessel 701. In FIG. 7 ( c ), the device 704 has been positioned against the wall of the vessel 701, seating the tissue engagement structures 706 in the tissue 701. The sheath 702 has been partially removed to facilitate seating of the device. The lock can be preseated on the device (not shown) or can be placed on the device after intravascular placement of the device by threading the lock down the guidewire or suture onto a central junction of the device after the device has been positioned in the blood vessel, or positioned by a separate sheath). In FIG. 7 ( d ), a retaining lock 707 has been advanced over the members of the apposition device, forcing them into the closed configuration, and bringing the edges of the opening 709 together. If no bleeding occurs, then the guidewire 703 can be removed as shown in FIG. 7 ( e ). The loop of string or suture 705 can be cut and removed, leaving the device 704 safely seated and locked with the opening closed, as shown in FIG. 7 ( f ). [0037] Delivery of the device can be done sequentially, or can be done with a dedicated device. For sequential delivery of the device, the following sequence of steps are suitable: 1) the guidewire and sheath are in place, 2) the device is pushed down the sheath, either next to the guidewire or with the guidewire in the lumen of the umbrella; 3) the umbrella is extruded, and then using the thread or suture, pulled tight against the lumen of the vessel; 4) the retaining lock is pushed down the thread and/or guidewire, and is pushed onto the umbrella while applying traction (the sheath can be removed partially at this stage); 5) after the retaining lock is seated, the sheath is observed for bleeding; 6) if there is no bleeding, then the sheath and guidewire are removed. For a dedicated device, there can be a sheath with the umbrella, a pushing device to push the umbrella out (another sheath), a sheath to push the retaining lock, and a thread/suture to oppose the other sheaths and to retrieve the umbrella if it is misplaced. [0038] FIG. 8 ( a,b,c,d ) is a lateral view of a double-action vascular closure apposition device. In FIG. 8 a, the unassembled device is in the closed position. The device comprises a plurality of umbrella members 801 disposed substantially radially about a central junction 803. Each umbrella member 801 can comprise a tissue hook 802, in the figure a double hook, spaced from the junction of the member and the central junction 803. The central junction 803 can include a central lumen for a guidewire, and is adapted to engage a plurality of opposite facing members 804, which opposite facing members can optionally have hooks, tissue penetrators, or feet. The opposite facing members can comprise memory material, and be configured such that they force a retaining lock 805 over the open umbrella members 1 (shown open in FIG. 8 c ) forcing them to close (as shown in FIG. 8 d ). [0039] A closed loop of string or suture (not shown) can be joined to the device by ways of a lumen or loop. FIG. 8 a shows the device preassembly. In FIG. 8 b, the device is in assembled form and in a closed position, where the central junction 3 has passed through the retaining lock 805. In this form the double action vascular closure apposition device can reside within a delivery catheter before being placed in the puncture wound of a blood vessel. FIG. 8 c shows the device partially expelled from the sheath (not shown), where the umbrella members 801 have opened and engaged the vessel wall, analogous to the embodiments previously described. In the arrangement of FIG. 8 c, the opposite facing members 804 are retained in the sheath so that they are prevented from forcing the lock 805 over the umbrella members 801. FIG. 8 d shows the device completely expelled from the delivery sheath, where the opposite facing members 804 are now forcing the retaining collar or lock 805 down the umbrella members 801, causing the portions of the umbrella members 801 with tissue engagement features (hooks in the figure) together (i.e., closing of the umbrella). The opposite facing members 804 are shown for ease of illustration as wire-like; they can be configured as coiled or semi-coiled structures, strut-like, multiple angles, spring-like, curled in an opposite direction, single or multiple members, elbow-like, or other geometrical or curvolinear shapes that when extended are neutral to the retaining lock, but when expelled, force the lock over the umbrella members, initiating closure. [0040] Accordingly, the double action vascular closure apposition device of FIG. 8 is first closed, then opens, and then closes again, the second closure occurring spontaneously by contraction of the opposite facing members against the retaining lock. After the device has been delivered and vascular closure is complete, then the guidewire can be removed. [0041] FIG. 9 ( a,b,c,) is a lateral view of a collarless double-action vascular closure apposition device. FIG. 9 a shows the device in the closed position. A plurality of umbrella members 901 are disposed substantially radially about a central junction 905, which can have a lumen for a guidewire. The central junction can comprise a retaining ring which permits the members to flex along their length. Each of the umbrella members 901 has a tissue engagement feature spaced apart from the central junction 905, in the figure a double hook 902. The central junction is adapted to engage a plurality of opposite facing members 903, which optionally can have hooks, tissue penetrators, or feet 904. The opposite facing members 904 can be composed of memory material, and can be directly joined to a corresponding umbrella member, with the memory forces in the opposite facing members dominant over the memory forces in the umbrella members. FIG. 9 a depicts the form that the collarless double action vascular closure apposition device would have while within a delivery catheter before being placed in the puncture wound of the blood vessel. [0042] FIG. 9 b shows the device partially expelled from the sheath (not shown). The umbrella members 901 have opened and engaged the vessel wall, analogous to embodiments described previously. The opposite members are still in the closed form, restrained there by the sheath. FIG. 9 c shows the device completely expelled from the delivery sheath, where the opposite facing members 903 have curled or contracted. Since the opposite facing members are directly joined to the umbrella members and have dominant memory characteristics, they force the umbrella members 901 to close. The opposite facing members are shown in figure as wire-like for ease of illustration; they can be configured as coiled or semi-coiled structures, strut-like, multiple angles, spring-like, curled in an opposite direction, more than two members, elbow-like, or other geometrical or curvolinear shapes that when extended are neutral to the umbrella members, but when expelled, dominate over the umbrella members, and force closure of the umbrella members, initiating puncture wound closure. [0043] Accordingly, the collarless double action vascular closure apposition device is first closed, then opens, and then closes again, with the second closure occurring spontaneously, by contraction of the opposite facing members which have dominant memory characteristics over the umbrella members. The central junction can be loose enough (e.g., made of a flexible polymer) to permit the forces from the contraction of the opposite facing members to be exerted on the umbrella members. In some embodiments each opposite facing member can be continuous with a corresponding umbrella member (i.e., a first portion of a single wire comprises an umbrella member, a second portion of the same wire comprising an opposite facing member). For simplicity, only two opposing members are shown in the figure; in embodiments where an umbrella member and a opposite facing member are formed from a single wire, the number of umbrella members can equal the number of opposite facing members, and each opposing member-umbrella member can be an integrated piece of memory material. After the device has been delivered and vascular closure is complete, then the guidewire can be removed. [0044] In any of the embodiments described, the umbrella-like structure, members of this structure or components of the umbrella can be made from any number of suitable materials, including radiopaque materials and materials coated to be made radiopaque, including bioabsorbable polymers or compounds, non-absorbable alloys and compounds including stainless steel, MP35, Nitinol, Nickel-Titanium ally, Kevlar, nylon polyester acrylic, gold, platinum, tantalum, niobium, molybdenum, rhodium, palladium silver, hafnium, tungsten, iridium. Materials with memory would also be preferable to allow the umbrella to spontaneously open after placement by the sheath. These can be made in the form of wires, fibers, filaments, small beams, and other extruded, woven, or formed shapes. Piano wire, super elastic memory wire, chromium allows, alloys of titanium and nickel, and other elastic memory materials previously mentioned as well as others can be used as well The umbrella fabric can be made from a number of suitable materials, including flexible polymeric materials with elastomeric properties including polyurethane, polyethylene, polyestenurethane, polyimide, olyethreimide, polycarbonate, polysiloxane, polyvinyls, hydroxyethylmethacrylate, related polymers, co-polymers of these or other polymers, or drug-embedded or drug-eluting polymers to prevent coagulation or intimal hyperplasia (such as Taxol), also which can be made radiopaque by markers and addition of appropriate radiopaque materials. EXAMPLE EMBODIMENTS [0045] The present invention can comprise a device to close puncture wounds caused by catheter procedures and especially angiography comprised of an expandable umbrella-like device that in the compressed state resides in a sheath, and after being expelled from the sheath assumes a planar or conical or other shape, engages vessel wall by means of tissue hooks or penetrators, is collapsed, analogous to umbrella tines, and brings the edges of the vessel wound or puncture into apposition. The device can have a retaining locking device that prevents the umbrella-like structure from reopening. This locking can be achieved by mechanical means including deformable enlargements on the members, dentates, male-female connectors, peg and hole, or other directional mating/locking devices on the members and retaining locking device. This locking device can have a washer like appearance, but can also take a number of different forms, including an inverted umbrella device made of metal, plastic, composites, or biodegradable material. [0046] The device can have single or multiple hooks and penetrating devices to engage and seize the vessel wall. Each hook can be a single or multiple hook. The device can have members (or tines) of the umbrella-like structure that are linear, curvilinear, spiral, leaf-like, diamond shaped, woven, or other complex shapes, but still function as an opening-closing structure that can accommodate a retaining lock. The device when expanded can have a planar or conical or reverse conical geometry, or other complex shape that can collapse into near-linear form with traction and locking of the retaining lock. The device can have a retrieval fitting, usually a loop, fitted with a closed loop thread, string, or suture in order to apply traction to the device. The device can have a lumen for a guidewire. [0047] The device can have members that are coated or backed with a fabric or membrane, either completely or partially. The device can elute therapeutic material to prevent thrombogenesis, hemorrhage, inflammation, and intimal hyperplasia with vascular closure. The device can be used in angiography, angioplasty, vascular puncture, tissue biopsy, or trauma that cause a puncture wound that should be closed. The device can comprise materials with memory, so that the device spontaneously assumes it therapeutic shape when expelled from the sheath. The device can comprise at least 2 or more members; 3 or more members can be preferable in some applications. The device can have members with angled dentates or tissue penetrators to prevent movement or migration of the device into the lumen of the blood vessel. These can also be used to retain the retaining lock. [0048] A tissue opening can be closed according to the present invention by a) introducing a guidewire and sheath, b) penetrating the proximal surface of the blood vessel by the sheath and the guidewire, c) placing a device in the closed form in the sheath, with the loop of string or suture, d) extruding the device from the sheath and expanding while in the tissue (e.g., while inside a blood vessel), e) pulling the device against the tissue wall (e.g., the blood vessel wall), seating the hooks in the tissue, f) partially removing the sheath is to seat the device while a retaining lock is advanced, g) advancing the retaining lock over the members of the device while applying traction with the string, forcing them in the closed position, h) bringing the edges of the puncture wound together; if no bleeding occurs, i) then removing the guidewire, j) cutting the loop of string, leaving the device safely seated and locked with the puncture repaired. [0049] A tissue opening can be closed according to the present invention with sequential delivery of a device. For example, the following steps can be taken 1) first the guidewire and sheath are in place, 2) next the umbrella is pushed down the sheath, either next to the guidewire or with the guidewire in the lumen of the umbrella; 3) the umbrella is extruded, and then using the thread or suture, pulled tight against the lumen of the vessel; 4) next the retaining lock is pushed down the thread and/or guidewire, and is pushed onto the umbrella while applying traction (the sheath can be removed partially at this stage, 5) after the retaining lock is seated, the sheath is observed for bleeding, 6) if there is no bleeding, then the sheath and guidewire are removed. [0050] A tissue opening can be closed according to the present invention employing a dedicated device consisting of a sheath containing a device, a pushing device to push the device out (e.g., another sheath), a sheath to push the retaining lock, and a thread/suture to oppose the movement of the other sheaths and to retrieve the umbrella if it is misplaced. [0051] The particular sizes and equipment discussed above are cited merely to illustrate particular embodiments of the invention. It is contemplated that the use of the invention may involve components having different sizes and characteristics. It is intended that the scope of the invention be defined by the claims appended hereto. | Summary: A vascular closure device comprised of a sheath-delivered expandable, umbrella-like device with structural radial members with terminal and non-terminal hooks that engage the vessel wall. Unlike other vascular closure umbrella-type devices that effect closure by opening of the umbrella to cover an opening, the present invention effects closure of the aperture with closure of the umbrella. The closure can be maintained by a retainer lock that slides down the members, causing contraction, bringing the members into a compressed configuration (e.g., a parallel orientation of linear members) and the wound edges together, permitting immediate vascular closure and healing of the blood vessel. The device can be delivered and recovered by an intravascular sheath. | 9,745 | 156 | big_patent | en |
Summarize: BACKGROUND OF THE INVENTION FIELD OF THE INVENTION This invention relates to machines for harvesting cotton and, in particular, to a novel structure for pulling the mature cotton plant from the stalk or burr. More particularly, this invention relates to a machine for harvesting cotton which includes a novel paired disk picking mechanism mounted for rotation about a central shaft in which the individual disks of the disk pair are separated to allow a portion of the cotton plant to pass therebetween and which are brought to a closed position to squeeze or pull the mature cotton from the stalk of the plant. The most widely used mechanical harvesting machine for cotton is the spindle picker in which the mature cotton is collected on a plurality of pins positioned around the rotating spindle arms. Although other machines, characterized as strippers, are used to harvest cotton, such machines gather too much extraneous portions of the cotton plant to obtain the high quality harvest the cotton mills demand. The spindle picker has undergone only minor improvements in design since its widespread introduction in the late 1940's, and although the picking efficiency is very satisfactory, the large mass of individual components in every row unit or header of this type of cotton harvesting machine is disadvantageous for several reasons. The large amount of components consequently demands a large amount of time for daily maintenance, such as for lubrication, and seasonal attention to replace worn parts which comprise a large portion of the operation cost of the spindle picker. The weight of the individual headers which contain all the picking machinery is another serious disadvantage since the number of headers that can be mounted on a harvesting machine is severely limited. Only by removing some of the component parts to reduce weight and thereby sacrifice picking efficiency can this disadvantage be somewhat overcome. Accordingly, a need exists for a cotton harvesting machine which eliminates the problems associated with the conventionally used spindle picking machines. Thus, in accordance with the present invention, such problems are eliminated by utilizing a novel method of picking the mature cotton from the stalk in which less machinery is required, thereby reducing daily and seasonal servicing of the cotton harvesting machine and reducing the respective labor and machinery cost. Another advantage of the novel picking device of the present invention is the drastically reduced weight per header, allowing more headers to be mounted on each harvesting machine and thereby allowing a single machine to cover more acreage at greater speed than present machines, cutting both labor and fuel costs. DISCLOSURE STATEMENT Cotton harvesting machines, such as the spindle picker, have existed as far back as the turn of the 20th century. As described above, such machines include an endless carrier with rapidly rotating spindles. As the machine travels along the rows of cotton, the spindles enter the plants and gather the cotton, the spindles then being doffed and the cotton collected in suitable receivers. Examples of patented spindle picker cotton harvesting machines include U.S. Pat. No. 1,208,591, issued Dec. 12, 1916, to Lovejoy, and U.S. Pat. No. 2,143,901, issued Jan. 17, 1939, to Rust et al. U.S. Pat. No. 1,213,529, issued Jan. 23, 1917, to Neil, also discloses a cotton picker in which a rotating roller picks the mature cotton from the plant. U.S. Pat. No. 3,164,942, issued Jan. 12, 1965, to Middlesworth et al, discloses a fruit harvester having gathering fingers or spindles in which the fingers are adapted to be advanced into a tree and pursuant to rotation of the spindles to auger into the tree and then be withdrawn to strip the fruit off the plant. The fingers are shaped in the form of helical convolutions and each group of four spindles are arranged so that the crest of adjacent helically shaped spindles always oppose each other. No mention is made in the patent to Middlesworth et al of using the harvesting machine to pick cotton. The novel paired disk picking mechanism of the present invention is not taught by any of the above references and is considered to be an improved substitute for the spindle picking cotton harvesters. SUMMARY OF THE INVENTION Briefly, the cotton harvesting machine of the present invention utilizes a novel mechanism for separating the mature cotton from the cotton stalk or burr. The novel separating mechanism comprises a plurality of paired disks mounted for rotation about a central shaft, the harvesting machine including two or more columns of paired disks per header. Each opposed column of paired disks rotate in opposite directions so that the perimeters of each disk move in the same direction as the cotton plants pass through the picking area of the harvesting machine. Each disk of a disk pair is provided with a resilient opposing pad and is mounted so that opposed disks of a disk pair separate as the cotton plants are passing through the picking area and close to pinch the mature cotton between the opposed resilient pads, pulling the mature cotton from the stalk as the opposed disks rotate in the closed position. The opening and closing movement of the opposed paired disks is effected by a pair of opposed cams mounted in a stationary position around each central shaft. A separate cam follower attached for rotation about the central shaft and to one of the paired disks follows a specifically structured cam track placed on the cams to move each disk in position during rotation of the shaft. During the picking operation, the cotton plant enters the picking area of the header as the harvesting machine travels through the rows of cotton. At this point, the opposed disks of each of the paired disks are in the open position so that the plant limbs are funneled between the pads of the opposed open disks. As the harvesting machine moves forward along the rows of cotton plants, the cotton plant in the picking area moves toward the back of the header at which point rotation of the central shaft causes the cam follower to close the opposed disks, squeezing the mature cotton with sufficient pressure to pull the cotton from the burr as movement of the cotton harvester and rotation of the central shaft continues. The pressure that the opposed pads exert on the cotton plant can be varied to meet changing field conditions by an adjustment device which various the relative position between opposed cams. After the cotton has been pulled from the burr, the disks continue to rotate to an appropriate place adjacent a vacuum area in the header whereupon the cam tracks for the opposed paired disks are such that the cam followers cause the opposed disks to open and through centrifugal force throw the cotton from between the opposed disks to a vacuum area which pulls the cotton into a storage area situated on the cotton harvesting machine. Each row of paired disks are in the form of a circle about the central shaft, each row containing a plurality of upper and lower disks in which each disk is attached to the central shaft and is associated with its own cam follower positioned within the cam track. Each rotating column of paired disks in the header hold several rows of paired disks vertically spaced along the central shaft, the number of rows varying depending upon height requirements. An object of the present invention is to provide a cotton harvesting machine which will eliminate the problems associated with conventional spindle pickers. Another object of the invention is to provide a cotton harvesting machine which includes a novel mechanism to pull the mature cotton from the stalk or burr. Another object of the present invention is to provide a novel picking mechanism which will require less daily and seasonal maintenance than cotton spindle pickers. Still another object of the invention is to provide a novel picking mechanism which will drastically reduce the weight of a header placed on a cotton harvesting machine over conventional spindle pickers, allowing more headers to be mounted on each harvesting machine. Still yet another object of the invention is to provide a cotton harvesting machine which includes a picking mechanism which is adjustable to meet varying field conditions. 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 side elevational view illustrating a cotton harvesting machine including the novel rotating paired disk picking mechanism of the present invention. FIG. 2 is a top plan view illustrating the arrangement of components within the header of the cotton harvesting machine of FIG. 1. FIG. 3 is an enlarged fragmentary elevational view illustrating two of the novel disk pairs of the present invention in which one pair is in the fully open position and the other in the fully closed position. FIGS. 4A and 4B are fragmentary elevational views of the adjusting mechanism for altering the relative position between opposed cams, the dashed line illustrating phantom positions of the adjustment arms, the adjustment being indicated by the arrows. FIG. 5 is an elevational view of the cam adjustment mechanism without the hand adjustable nut threaded onto the adjustment shaft. FIG. 6 is a transverse sectional view of the cam adjustment device taken generally along the line 6--6 of FIG. 4B. FIG. 7 is a transverse sectional view of the cam adjustment device taken generally along the line 7--7 of FIG. 5. FIG. 8 is an exploded perspective view illustrating the disk support collar which supports the paired disks for rotation about the central shaft. FIG. 9 is a transverse sectional view illustrating the cam follower and spring attachment to the individual disks. DETAILED DESCRIPTION OF THE INVENTION Referring now to FIGS. 1 and 2, cotton harvesting machine 10 includes an operator's station 12 including seat 14, storage area 16 for storing the picked cotton harvest, header 18, vacuum conduit 20 for directing the picked cotton into storage area 16, guards 21 and 22 which assist in directing the cotton plants into the picking area of header 18 and the novel picking mechanism 24 placed inside header 18 and operated by gear drive train 26 which is rotated from a power source (not shown) in cotton harvesting machine 10. Picking mechanism 24 comprises three rows of paired disks, rows 28, 29 and 30 mounted and spaced vertically along a central rotating shaft 32 which is driven by gear train 26. The rows of paired disks form a single column about each rotating shaft 32 and 42. Each row of paired disks forms a circle surrounding the central shaft in which the individual disks forming the row are separately mounted to respective central shafts 32 and 42. In FIG. 2, it can be seen that each row is formed from eight disk pairs in which each disk is pie-shaped or triangular, such as disk 34. The side edges of each disk being contiguous with the adjacent disk, such as shown by disks 36, 38 and 40. The number of rows of paired disks in each column can be varied depending upon height requirements. Likewise, the number of each picking disk column per header can be varied and can include one, two columns, such as the picking disk columns disposed about central shafts 32 and 42 in FIG. 2, and even three or more disk columns per header. As opposed to the spindle picking mechanism of conventionally used cotton harvesting machines, in which rotating spindles gather mature cotton from the plant, picking mechanism 24 of the present invention utilizes the opening and closing of each paired disk to funnel the cotton plant between the open paired disks and for gripping the mature cotton from the cotton plant as the paired disks close during movement towards the rear of the header. FIG. 3 illustrates the structure utilized to move the paired disks into the open and closed position. Two rows of paired disks are shown, 44 and 46, only two of the disk pairs being shown per row, though it is to be understood that as many as eight disk pairs can exist per row to form a complete circle about the central shaft. Row 44 includes paired disks 48 and 50 in the closed and open positions, respectively. Paired disk 48 is formed from disks 52 and 54 while paired disk 50 is formed from disks 56 and 58. Each disk is provided with a rubber pad 60 which is fastened to the opposed faces of each disk pair. The use of pliable rubber allows pads 60 to grip and conform to a certain extent to the irregularities of the cotton plant, thus providing for increased picking efficiency. A pair of cams, 62 and 64, having contained therein cam tracks 66 and 68, respectively, are associated with the upper or lower disks of the disk pairs which form the paired disk row. Accordingly, cam 62 and cam track 66 guide upper disks 52 and 56 during rotation of shaft 70 by means of individual cam followers 72 and 74, respectively, while lower disks 54 and 58 are moved into the open and closed positions by respective cam followers 76 and 78 travelling in cam track 68 of cam 64. The movement of each disk pair will be explained with respect to disk pair 48. The movement of cam follower 72 in respective cam track 66, is transferred to disk 52 through spring loaded cam support 80, pivotally mounted to the upper face of disk 52 opposite the face containing rubber pad 60. Cam support 80 can be mounted to disk 52 by any conventional pivot means, such as a pivot pin 82, passing through a lug 84 secured to the upper face of disk 52. In a like manner, disk 54 is associated with cam follower 76. Cams 62 and 64 are mounted to central shaft 70 in a nonrotatable position, while disks 52 and 54 are mounted for rotation to shaft 70 via support collars 86 and 88, respectively, so that as central shaft 70 rotates, each individual cam follower moves in the respective cam track about the perimeter of the cam and through the respective cam supports moves each disk in a circle about the axis of rotation of central shaft 70. The cam tracks are structured in the respective cams so that during each rotation of the cam follower within the cam track, each paired disk is in the fully opened and fully closed positions only once. As can be seen in FIG. 3, paired disks 52 and 54 are in the fully closed positions, since cam followers 72 and 76 are positioned in respective cam tracks 66 and 68 at a location where the distance between the cam tracks is the smallest with respect to the remaining distances between the cam tracks along the perimeter thereof. Similarly, disks 56 and 58 are in the open position as their respective cam followers 74 and 78 are at a position along the perimeter of the respective cam tracks which comprises the greatest distance between the respective cam tracks. This position represents the fully opened position of the disk row. Each upper and lower disk in the paired disk row is supported for rotation with central shaft 70 by respective upper and lower support collars, such as upper support collar 86 which supports disk segments 52 and 56 and lower support collar 88 which supports disks 54 and 58. Each individual disk is pivotally mounted to the respective support collar in any conventional manner, such as by a pivot pin 90 passing through lug 92 attached to the respective disk. To prevent the individual cam followers from binding in the cam track during rotation, each cam support 80 is maintained in a vertical position by means of a parallel linkage 94 pivotally mounted to each support collar. In FIG. 3, linkage 94 can be seen pivotally mounted to respective support collars 86 and 88 by means of pivots 96 and 98, respectively, and to the cam supports by means of pivots 100, each of which are preferably pins movable within a lug attached to the support collars. FIG. 8 illustrates a support collar 102 equivalent to support collars 86 and 88 shown in FIG. 3. Support collar 102 is in the form of a hollow cylinder containing a hollow space 104 which enables the support collar to slip over the central rotating shaft. A roll pin placed through aperture 106 formed through the body of the support collar maintains the support collar firmly secured to the central shaft. Placed around the perimeter of disk support collar 102 are a plurality of elongated vertical grooves 108 placed along the full length of the support collar cylinder. Placed within each groove 108 is a hinge bar 110 fastened to support collar 102 by means of screw 112 through aperture 114 contained in hinge bar 110. Apertures 116 and 118 placed through hinge bar 110 are utilized for pivotally supporting either linkages 94 or one of the disks depending upon whether the support collar is used for mounting the upper or lower disk of the disk pair. FIG. 9 illustrates cam support 80 for converting the movement of the individual cam followers in the cam tracks into the open and close movements of the attached disks. Cam support 80 includes a pair of interfitting cylinders 120 and 122 interconnected by means of a rigid spring 124 attached to cylinder 122 by means of hook 126 and to cylinder 120 by means of hook 128. Cylinder 122 contains a lug 130 which supports the cam follower indicated by reference numeral 132 for movement within cam track 134 of cam 136. Lug 138 attached to the bottom surface of cylinder 120 holds the pivot pin for pivotally mounting cam support 80 to the individual disk. Spring 124 allows some relative movement between interfitting cylinders 120 and 122 so that the individual picking disks can conform to the different types of materials passing between the paired disks. Pivot mechanism 100 for pivotally mounting cam support 80 to linkage 94 is fastened to the exterior of cylinder 120 by any conventional means, such as by welding, a strong adhesive, etc. Referring back to FIG. 3, it is seen that attached to cam 64 of paired disk row 44 and cam 140 of paired disk row 46 is a yoke assembly 141 comprising a pair of pivotal linkages 142 and 144, attached to cams 64 and 140, respectively, the linkages being mounted to a connecting rod 146 at pivot point 143. By movement of connecting rod 146 to the right, cams 64 and 140 move closer together and consequently move the individual cams in respective paired disk rows a farther distance apart. Likewise, moving connecting rod 146 to the left moves the cam members of individual paired disk rows closer together. This vertical adjustment of the cams is an accessory which is used to position the cams at varying distances from the disks and which varies the amounts of spring pressure applied to the disk segments via spring 124 in cam support 80. Thus, the amount of squeezing or pulling action by the closed paired disks can be adjusted to meet varying field conditions. Connecting rod 146 and attached pivot arms 142 and 144 are part of a cam adjustment 148 illustrated in FIGS. 4 through 7. Cam adjustment 148 comprises a tube 150 enclosing a threaded shaft 152 containing threads 154. A hand adjustable nut 156 moves threaded shaft 152 relative to the surrounding tube 150. Yoke assembly 158 is pivotally fastened to connecting rod 146 at pivot point 147 and includes arm 160 fastened to shaft 152 and arm 162 fastened to outer tube 150. Arms 160 and 162 are pivotally mounted to support arms 164 and 166, respectively, each arm 164 and 166 being mounted at pivot point 147 to connecting rod 146. Referring to FIG. 4A, as threaded shaft 152 is moved further into tube 150 by means of nut 156, arms 160 and 164 are moved closer to arms 162 and 166 causing a scissoring action whereby connecting arm 146 is moved toward the cams causing attached linkages 142 and 144 to scissor outwardly from pivot point 143 bringing the pair of cams in each row of paired disks closer together, FIG. 3. Likewise, pulling shaft 152 out of 150 causes yoke assembly 158 to spread further apart as shown in FIG. 4B causing connecting rod 146 to pull away from the attached cams causing the individual cams in each paired disk row to separate. FIGS. 5-7 show that arms 160 and 162 can be attached to shaft 152 and tube 150, respectively, by means of lugs 167 screwed or welded thereto. Referring again to FIGS. 1 and 2, header 18 is shown containing two columns of paired disk rows. Each column comprises three rows of paired disks equivalent to the structure set forth in FIG. 3 and includes a cam adjustment 168 equivalent to that shown in FIGS. 4A and 4B. Header 18 also includes vacuum areas 170 and 172 which communicate with vacuum conduit 20 to direct the pulled cotton into storage area 16. Plant dividers 174 and 176 funnel the cotton plant limbs between the paired disks rotating about respective shafts 32 and 42. Vacuum areas 170 and 172 are bounded by shields 178 and 180, respectively, the ends of each shield protruding between the open disks to prevent the picked cotton from being carried back into the picking area. Shields 182 and 184 positioned at the back of the header are used as buffers to ease the cotton limbs back into the picking area. Operation of a cotton harvesting machine manufactured in accordance with the teachings of the present invention will be described with respect to FIGS. 1 and 2 which illustrate cotton harvesting machine 10 containing picking mechanism 24. Picking mechanism 24 comprises a pair of columns picking disks, each column containing three rows of paired disks, rows 28, 29 and 30 each constructed in an equivalent manner to the picking mechanism illustrated in FIG. 3. As cotton harvesting machine 10 moves forward, the cotton plants are funneled into the space between the columns with the aid of guards 21 and 22 and plant dividers 174 and 176. Rotation of central shafts 32 and 42 by a drive mechanism (not shown) in cotton harvesting machine 10 also rotates the individual paired disks which are opened and closed by movement of the cam followers in the cam tracks as described above. Area A, as shown in FIG. 2, is the picking area where the rubber pads on the paired disks are closed completely applying pressure to the plant limbs to pull the mature cotton from the burrs as shafts 32 and 42 continue rotation. Area B is a transitional area in which the cotton is released from between the closed pads as the paired disks start to separate due to the diverging direction of the respective cam tracks. The paired disk segments go from a fully closed to a fully opened position at the end of area B adjacent vacuum areas 170 and 172, the cotton being thrown by centrifugal force into the vacuum area. In area C, the paired disk segments are fully open. Area D is the other transitional area whereby the disks go from the fully open position to the closed position as the plant limbs come between the pads to complete the cycle. During movement of harvesting machine 10 through the rows of cotton plants, a constant vacuum is being applied to pull the pulled cotton from the vacuum area into storage area 16. 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 cotton harvesting machine which utilizes a novel structure for pulling the mature cotton plant from the burr includes a plurality of paired rotating disks mounted on a drive shaft for rotation in the same relative direction as the movement of the cotton plant through the picking area of the machine, each disk pair being moved into an open position whereby the cotton plant is funnelled between the open disk pair and a closed position whereby the disk pair grips the mature cotton and pulls it from the burr. The opening and closing movement of each disk pair is provided by a pair of cams and associated cam followers, one for each disk in the pair, each cam being mounted in a stationary position around the rotating drive shaft, the cam followers being fastened to each disk and following a path of movement along a cam track during the rotation of the shaft. The cam tracks associated with each pair of cams converge and diverge around the shaft causing the closing and opening position of the disk pair as the cam followers move in the track. An adjusting mechanism can be provided to change the relative positions between opposing disks by relative movement of the associated pair of cams. | 5,675 | 239 | big_patent | en |
Write a title and summarize: Diabetes impacts approximately 200 million people worldwide, of whom approximately 10% are affected by type 1 diabetes (T1D). The application of genome-wide association studies (GWAS) has robustly revealed dozens of genetic contributors to the pathogenesis of T1D, with the most recent meta-analysis identifying in excess of 40 loci. To identify additional genetic loci for T1D susceptibility, we examined associations in the largest meta-analysis to date between the disease and ∼2. 54 million SNPs in a combined cohort of 9,934 cases and 16,956 controls. Targeted follow-up of 53 SNPs in 1,120 affected trios uncovered three new loci associated with T1D that reached genome-wide significance. The most significantly associated SNP (rs539514, P = 5. 66×10−11) resides in an intronic region of the LMO7 (LIM domain only 7) gene on 13q22. The second most significantly associated SNP (rs478222, P = 3. 50×10−9) resides in an intronic region of the EFR3B (protein EFR3 homolog B) gene on 2p23; however, the region of linkage disequilibrium is approximately 800 kb and harbors additional multiple genes, including NCOA1, C2orf79, CENPO, ADCY3, DNAJC27, POMC, and DNMT3A. The third most significantly associated SNP (rs924043, P = 8. 06×10−9) lies in an intergenic region on 6q27, where the region of association is approximately 900 kb and harbors multiple genes including WDR27, C6orf120, PHF10, TCTE3, C6orf208, LOC154449, DLL1, FAM120B, PSMB1, TBP, and PCD2. These latest associated regions add to the growing repertoire of gene networks predisposing to T1D. Diabetes impacts approximately 200 million people worldwide [1], with microvascular and cardiovascular disease being the primary complications. Approximately 10% of cases are type 1 diabetes (T1D) sufferers, with ∼3% increase in the incidence of T1D globally per year [2]. It is expected that the incidence is 40% higher in 2010 than in 1998 [3]. T1D is a clear example of a complex trait that results from the interplay between environmental and genetic factors. There are many lines of evidence that there is a strong genetic component to T1D, primarily due to the fact that T1D has high concordance among monozygotic twins [4] and runs strongly in families, together with a high sibling risk [5]. Prior to the era of GWAS, only five loci had been fully established to be associated with T1D. However, the majority of the other reported associations in the pre-GWAS era [6]–[8] remain highly doubtful, where an initial report of association does not hold up in subsequent replication attempts by other investigative groups. This previous hazy picture of the genetics of T1D can be put down to the use of the only methodologies that were available at the time and which were much more limited than GWAS i. e. the candidate gene approach (where genomic regions were studied based on biological reasoning) and family-based linkage methodologies. Inconsistent findings can also be attributed to small sample sizes i. e. when power is low the false discovery rate tends to be high; GWAS per se has not improved consistency, rather it has leveraged large, well powered sample sizes combined with sound statistical analyses. It has been long established that approximately half of the genetic risk for T1D is conferred by the genomic region harboring the HLA class II genes (primarily HLA-DRB1, -DQA1 and -DQB1 genes), which encode the highly polymorphic antigen-presenting proteins. Other established loci prior to the application of GWAS are the genes encoding insulin (INS) [9]-[12], cytotoxic T-lymphocyte-associated protein 4 (CTLA4) [13]–[16], protein tyrosine phosphatase, non-receptor type 22 (PTPN22) gene [17], [18], interleukin 2 receptor alpha (IL2RA) [19]–[21] and ubiquitin-associated and SH3 domain-containing protein A (UBASH3A) [22]. The application of genome wide association studies (GWAS) has robustly revealed dozens of genetic contributors to T1D [23]–[29], the results of which have largely been independently replicated [30]–[36]. The most recently reported meta-analysis of this trait identified in excess of forty loci [29], including 18 novel regions plus confirmation of a number of loci uncovered through cross-disease comparisons [34]–[36]. As such, the risks conferred by these additional loci are relatively modest compared to the ‘low-hanging fruit’ described in the first studies and could only be ultimately uncovered when larger sample sizes were utilized. We sought to expand further on this mode of analysis by combining our cohort with all publically released genome wide SNP datasets to identify additional loci contributing to the etiology of T1D. Unfortunately, there is a relative paucity of control genotype data in these publically available sources. To circumvent this problem, we combined individual level data from each available cohort and we then compared the cases with controls from two sources. We next separated all the individual level data into two groups, characterized by the type of genotyping platform that was used to genotype the samples, which would later be recombined using inverse-variance meta-analysis. The 6,523 cases genotyped on an Illumina BeadChip included subjects from McGill University, The Children' s Hospital of Philadelphia (CHOP), The Diabetes Control and Complications Trial – Epidemiology of Diabetes Interventions and Complications (DCCT-EDIC) cohort, and the Type 1 Diabetes Genetics Consortium (T1DGC), which in turn were compared with 6,648 similarly genotyped controls recruited at CHOP. The 3,411 cases genotyped on Affymetrix arrays included subjects from the Genetics of Kidneys in Diabetes Study (GoKinD) and the Wellcome Trust Cases Control Consortium (WTCCC) that were then compared with 10,308 similarly genotyped controls, including being derived from non-autoimmune disease related cases from the WTCCC, as well as from the British 1958 Birth Cohort and the UK National Blood Service [24]. We compared the power of our meta-analysis to that of the previous largest meta-analysis to date. We have more than double the power of the Barrett et al. meta-analysis to find variants with a relative risk of 1. 2 and approximately three times the power to detect variants with a relative risk of 1. 1 [29] (Figure S1). We used principal components analysis (PCA) [37] in order to minimize the potential impact of population stratification in our case/control sample sets. Eigenstrat 3. 0 was employed to remove outliers and to subsequently calculate the principal components in the Illumina and Affymetrix assigned groups separately. The principal components were then used as covariates in a logistic regression, using the software PLINK [38], to compute the P-values, betas and standard errors which were combined in our fixed effects inverse variance meta-analysis. After controlling for population stratification, the λ in the Affymetrix and Illumina cohorts was 1. 11 and 1. 17, respectively (see Figure 1 for Q-Q plot). A full description of the correlation of each eigenvector with known continental ancestry appears in Text S1. Mach was used to impute ∼2. 54 million SNPs, including HapMap Phase 2 SNPs in the Illumina and Affymetrix datasets in order for the statistics to be uniform and amenable to being combined [39]. Results from the meta-analysis of this resulting ‘discovery’ cohort are shown Table 1 and graphically in Figure 2. 53 SNPs were brought forward to the replication stage based on satisfying the following criteria; however one of these SNPs consistently failed genotyping in the replication effort. The most significantly associated SNP at a given locus if the meta-analysis P-value was less than 1×10−5 (an independent locus was defined as a region for a given focal SNP, where we extended the region in both directions until either 250 kb had been traversed or until reaching another SNP with P<10−5), the Cochran' s Q statistic P-value was greater than 0. 05 and the locus had not been already reported from a previous GWAS of T1D. A table outlining the results for all previously described T1D associated SNPs plus our strongest associations for known regions associated with the disease are shown in Table 2 and Table S1, respectively. The replication cohort consisted of additional T1D affected trios from the T1DGC and McGill which had not been part of the original discovery cohort. The replication cohort was genotyped using the Sequenom iPLEX system and the results were analyzed using the transmission disequilibrium test in PLINK. Results for both the discovery and replication cohorts for the six SNPs that replicated with P≤0. 05 are shown in Table 1 (the full outcomes for all SNPs tested are in Table S2). We combined the ‘discovery’ and ‘replication’ meta-analysis P-values using Fisher' s combined P-value method implemented in Haploview, comparable to what has been previously employed by others [40]. Three of the SNPs, namely rs539514, rs478222 and rs924043, had combined P-values <5×10−8, the statistical threshold for genome wide significance, while the remaining three, namely rs550448, rs12679857 and rs6547853, failed to reach this bar but were suggestive of association as the alleles yielded both a consistent direction of effect and P-values <0. 05 in the replication cohort. These two categories of outcome are summarized in Table 1; in addition, these six SNPs were further investigated with respect to adjustments of the discovery and met-analysis P-values based on the lambdas of each respective cohort (Table S3). We have carried out the largest meta-analysis of genome wide genotyped datasets for T1D to date. The replication of three loci using the stratification-free TDT with minimal Mendelian error clearly indicates that they are not false positives due to artifacts such as uncorrected systematic error from stratification or genotyping bias. The most significantly associated SNP (rs539514, P = 5. 66×10−11) resides in an intronic region of the LMO7 (LIM domain only 7) gene on 13q22. We investigated the associated region using LocusZoom [41] and determined that it is the only gene residing within the block of linkage disequilibrium harboring the signal (Figure S3). Regional plots showing P-values, linkage disequilibrium, and recombination rate for all SNPs in Table 1 are outlined in the Figures S2, S3, S4, S5, S6, S7. LMO7 encodes a protein that contains multiple domains, including a calponin homology domain, a PDZ domain and a LIM domain. There are multiple LMO7 isoforms already known but their full nature and the actual extent of different isoforms remains unclear [42]. Mice with homozygous deletions of LMO7 display retinal, muscular, and growth retardation [43]. Although the function of LMO7 doesn' t clearly relate to the etiology of T1D, LMO7 is expressed in pancreatic islets and thus is a possible biological candidate at this locus [44]; however it should be noted that the retinal, muscular development and islet patterns are a key element in Emery-Dreifuss Muscular Dystrophy, caused by mutations in LMO7 [45], but bears very little similarity to T1D. The second most significantly associated SNP (rs478222, P = 3. 50×10−9) resides in an intronic region of the EFR3B (protein EFR3 homolog B) gene on 2p23; however the region of linkage disequilibrium is approximately 800 kb and harbors additional multiple genes, including 3NCOA1, C2orf79, CENPO, ADCY3, DNAJC27, POMC, and DNMT3A. (Figure S2). A previous meta-analysis of a subset of the data used in this current study found suggestive association with T1D in the same LD block with the independent SNP, rs2165738 (r2 = 0. 115), but did not achieve genome wide significance at that time (P = 3. 65×10−6) [27]; however, we only found modest evidence of association with rs2165738 (P = 4. 78×10−3) in our discovery cohort. There has also been association to inflammatory bowel disease [46] height [47], [48] and BMI [49] reported at this locus, where in both cases the risk allele for increased height or BMI was protective for T1D risk. The third most significantly associated SNP (rs924043, P = 8. 06×10−9) lies in an intergenic region on 6q27, where the region of association is approximately 900 kb and harbors multiple genes including WDR27, C6orf120, PHF10, TCTE3, C6orf208, LOC154449, DLL1, FAM120B, PSMB1, TBP and PCD2 (Figure S5). In addition, despite not reaching the bar for genome wide significance, we did observe evidence for association at three additional loci (Table 1) containing the candidate genes LOC100128081, TNFRSF11B and FOSL2. Of these, it is notable that TNFRSF11B is a strongly associated locus with bone mineral density, also as a consequence of GWAS [50], [51]. In addition, the locus harboring LOC100128081 has also been reported in the context of a GWAS of SLE [52]. Further work will be required to fully validate the role of these particular loci in the pathogenesis of T1D. The Barrett et al. meta-analysis was able to use British controls with British cases and American controls with American cases [29]. We did not have the same control data to be able to make the same comparisons. In the case of the Affymetrix analysis, some American cases were analyzed with purely British controls and, in the case of the Illumina analysis, some British cases with purely American controls. As such, we were forced to make our corrections using eigenvectors as covariates in our analysis; this will have the effect of modestly weakening the level of significance for associations that vary in allele frequency between the cases and controls, as now the case and controls will both vary with the eigenvectors to some degree. This in effect will make our analysis overly conservative with estimating the true effect of a SNP, and in fact every SNP that had a P-value less than 0. 05 in the replication set did indeed have a greater effect than that which was estimated from the discovery set. In summary, we provide convincing evidence for the existence of three additional loci associated with the T1D, adding to the repertoire of over 50 loci already demonstrated to be associated with the disease. The study was approved by the institutional review board and the ethics committee of each institution. Written informed consent was obtained from each participant in accordance with institutional requirements and the Declaration of Helsinki Principles. Cases in the discovery set were obtained from four publically available resources and combined with those from a previous publication for the meta-analysis. Samples descriptions are available on dbgap (http: //www. ncbi. nlm. nih. gov/sites/entrez? db=gap) for the T1DGC (http: //www. ncbi. nlm. nih. gov/projects/gap/cgi-bin/study. cgi? study_id=phs000180. v1. p1), GoKinD (http: //www. ncbi. nlm. nih. gov/projects/gap/cgi-bin/study. cgi? study_id=phs000088. v1. p1), and DCCT-EDIC (http: //www. ncbi. nlm. nih. gov/projects/gap/cgi-bin/study. cgi? study_id=phs000086. v2. p1) patients. The WTCCC sample information is available from [24]. Samples from the T1D segment of the WTCCC were used as cases, while controls were derived from the 1958 Birth Cohort, UK Blood Service, Bipolar disorder, Coronary heart disease, Hypertension, and Type 2 Diabetes segments. The remaining cases used in the meta-analysis were previously described [23]. The total number of individuals used in the meta-analysis discovery set was 26,890 (9,934 cases/16,956 controls). The replication set consisted of 1120 case-parent trios from the T1DGC and those identified through pediatric diabetes clinics in Canada. The replication set was identical to that used in Hakonarson et al. with an extension of patients identified through pediatric diabetes clinics in Montreal, Toronto, Ottawa, and Winnipeg. All individuals were of Caucasian ancestry. A breakdown of the number of samples in each cohort in the discovery phase and a comparison with the numbers used in the Barrett et al. meta-analysis are shown in Table 3 [29]. The minor variation in the number of cases reflects that, despite using slight differences in both quality control and methods for dealing with population stratification, we have comparable numbers of cases from this cohort remaining in our analysis. Primarily, this small difference is due to the fact that we strictly accounted for relatedness and duplicates within and across cohorts in this current setting. Power analysis was performed with the genetic analysis calculator which can be found at (http: //pngu. mgh. harvard. edu/~purcell/gpc/) [53]. Various assumption were made included perfect LD between the causative variant and the markers that were genotyped, an additive genetic model, a disease prevalence of 0. 0033 and an alpha of 1×10−5. Discovery samples from Philadelphia, Canada, T1DGC, and DCCT-EDIC were genotyped on a mixture of the Illumina HumanHap 550v1,2, and 3, whereas samples from GoKinD and WTCCC were genotyped on the Affymetrix 500 K Chip. Sequenom iPlex was used to replicate the findings of the meta-analysis in 1,120 affected offspring trios from the T1DGC and from Canada. All individuals needed an individual genotyping call rate greater than 0. 98 to be included in the analysis pre-imputation and individuals were removed that showed evidence of cryptic relatedness and duplication within and across cohorts using identity-by-state. SNP quality control was performed on all samples pre-imputation. SNPs were excluded from the analysis if the minor allele was below 1%, the genotyping call rate was less than 95%, or the Hardy Weinberg equilibrium P-value was less than 0. 00001. To control for population stratification, Eigenstrat 3. 0 was used to compute the top 10 principal components of the individuals genotyped on the Illumina SNP chips and the Affymetrix SNP chips separately [37]. Individuals were removed from the analysis if they were 6 standard deviations away from the mean of one of the top 10 principal components. After controlling for population stratification, the estimated lambda in the Affymetrix data was 1. 11 and 1. 17 in the Illumina data. Mach 1. 0 was used to impute ∼2. 54 millions SNPs from the HapMap CEU panel for all individuals [39]. SNPs were excluded after imputation if they had a minor allele frequency less than 0. 01 and an r2 value less than 0. 3. PLINK [38] was used to perform a logistic regression using the 10 principal components as covariates, T1D status as the outcome, and in the case of the Affymetrix cohort, an extra dummy covariate specifying WTCCC or GoKinD cohort membership. Results from the logistic regression of 2,436,110 SNPs from the Affymetrix samples and 2,062,307 SNPs from the Illumina samples separately were combined using inverse-variance meta-analysis in PLINK. A fixed effects meta-analysis was performed and 53 SNPs were chosen for replication who had a fixed effects P-value <0. 00001, a Cochran' s Q statistic P-value greater than 0. 05 and were not previously known to be associated with type 1 diabetes. However one of the SNPs consistently failed during the replication effort. | Title: A Genome-Wide Meta-Analysis of Six Type 1 Diabetes Cohorts Identifies Multiple Associated Loci Summary: Despite the fact that there is clearly a large genetic component to type 1 diabetes (T1D), uncovering the genes contributing to this disease has proven challenging. However, in the past three years there has been relatively major progress in this regard, with advances in genetic screening technologies allowing investigators to scan the genome for variants conferring risk for disease without prior hypotheses. Such genome-wide association studies have revealed multiple regions of the genome to be robustly and consistently associated with T1D. More recent findings have been a consequence of combining of multiple datasets from independent investigators in meta-analyses, which have more power to pick up additional variants contributing to the trait. In the current study, we describe the largest meta-analysis of T1D genome-wide genotyped datasets to date, which combines six large studies. As a consequence, we have uncovered three new signals residing at the chromosomal locations 13q22,2p23, and 6q27, which went on to be replicated in independent sample sets. These latest associated regions add to the growing repertoire of gene networks predisposing to T1D. | 5,160 | 277 | lay_plos | en |
Summarize: CROSS-REFERENCES TO RELATED APPLICATIONS This non-provisional application claims priority under 35 U.S.C. §119(a) on Patent Application No. 103142261 filed in Taiwan, R.O.C. on Apr. 12, 2014, the entire contents of which are hereby incorporated by reference. BACKGROUND Technical Field The instant disclosure relates to a robot, in particular, to a robot with state-detection ability, a mobile device, and a state detecting method. Related Art With the advancement in technology, robots are being widely used in today's modern day life. Some examples include robotic arms, security robots, broom-sweeping robots, etc. Robots can perform precise operations, repeat routine tasks, and help humans with basic chores such as broom-sweeping robots. One type of broom-sweeping robots is self-propelled robot vacuum. This robot vacuum is applicable for home cleaning. When people are sleeping at night or out for work during the day, the robot vacuum can move around the house and clean up dusts, particles, etc. Hence, the residents are saved from cleaning the house room-by-room tediously. The self-guided robots are often deployed with obstacle detectors. For instance, an infrared emitter emits infrared in a forward direction. When the infrared is reflected by a forwardly standing obstacle and received by the infrared receiver equipped on the robot, the robot can determine whether an obstacle exists in the path. However, aside from detecting obstacles to prevent from hitting the robots, the robots may stick or flip over due to uneven ground geometry or steep slopes, thus causing work interruption. Another issue is if a child is present, the child may be curious enough to lift up the robot. If the robot is not stopped from its operation in time, the situation could cause injury to the child. SUMMARY In light of above, the instant disclosure provides a state detection method applicable for mobile devices, such as robots, cellular phones, electric charging stations, and other movable devices. First, the mobile device is furnished with a depth sensor on an inner side thereof. After a depth sensing signal is obtained, based on the numerical value of the signal, the mobile device is determined to be in a lifted state, a tilted state, or an edge-bordering state. The lifted state is defined as without touching the support surface. The tilted state is defined as one end touching the support surface, while another end is without touching the support surface. The edge-bordering state is defined as being at the edge of the support surface. Therefore, when the mobile device is in any of the above-mentioned states, a response procedure can be implemented. In one embodiment, the response procedure implements immediate or gradual change of the motion for the mobile device (such as non-linear or linear stop, turnaround, linear or non-linear back up, etc). The response procedure prevents the mobile device from continuing its original motion or remaining in the original state. In another embodiment, the response procedure issues a warning. The warning could be sent out continuously until it is lifted or lifted automatically after a time duration. The warning reminds the user to make the mobile device exiting any of the above-mentioned states. In yet another embodiment, the response procedure makes the mobile device to return to the original position or starting location. Alternatively, the mobile device is made to return to a previous state. After the mobile device has reached the previous state, the mobile device can change its current state proactively or passively. In some embodiments, based on at least one depth sensing signal, the moving direction of the mobile device and its travelled distance can be obtained via the state detection method. The obtained data is further used to retrieve the travelling path of the mobile device. Therefore, based on the travelling path, the mobile device can be returned to its original position or starting location. In further yet another embodiment, a multiplicity of depth sensors is employed. When the signal changes of all sensors surpass a first threshold value, the mobile device is determined to be in the lifted state. If the signal changes of some sensors surpass a second threshold value, while the sensor signals of all other sensors do not change, the mobile device is determined to be in the tilted state. Meanwhile, if the signal changes of some sensors surpass a third threshold value, while the sensor signals of all other sensors do not change, the mobile device is determined to be in the edge-bordering state. In one embodiment, the state detection method further includes disposing a shield in front of the depth sensor, along with detecting the sensor signal. When the numerical value of the sensor signal is zero, the mobile device is determined to be in a collision state. The instant disclosure also provides a robot. The robot comprises a main body, a moving unit, at least one depth sensor, and a control module. The moving unit and the depth sensor are arranged on one side of the main body. The control module is electrically connected to the moving unit and the depth sensor. Based on the numerical value of the sensor signal, the control module determines whether the robot is in the lifted state, the tilted state, or the edge-bordering state. The lifted state is defined as the robot not touching a support surface. The tilted state is defined as one end of the robot touching the support surface, while another end thereof is not touching the support surface. The edge-bordering state is defined as the robot being adjacent to the edge of the support surface. Therefore, when the robot is in any of the above-mentioned states, the afore-mentioned response procedure can be implemented. The instant disclosure further provides a mobile device. The mobile device comprises a main body, at least one depth sensor, and a control module. The depth sensor is arranged on one side of the main body, and the control module is electrically connected to the depth sensor. Based on the numerical value of the sensor signal, the control module determines whether the mobile device is in the lifted state, the tilted state, or the edge-bordering state. The lifted state is defined as the mobile device without touching a support surface. The tilted state is defined as one end of the mobile device touching the support surface, while another end thereof does not touch the support surface. The edge-bordering state is defined as the mobile device being adjacent to the edge of the support surface. Based on the above, the state detection method, the robot, and the mobile device disclosed by the instant disclosure utilize the depth sensor to identify the state (lifted, tilted, collision, or edge-bearing) of the robot or the mobile device. None of the other instruments are needed. When any of the four above-mentioned states is identified, the appropriate response procedure is triggered to restrict the motion of the robot/mobile device, or to change the internal state or procedure of the system. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a perspective view of a robot for a first embodiment of the instant disclosure. FIG. 2 is a schematic view of a depth sensor for the first embodiment of the instant disclosure. FIG. 3 is a block diagram of the robot in FIG. 1. FIG. 4 is a schematic view showing the lifted state for the first embodiment of the instant disclosure. FIG. 5 is a schematic view showing the tilted state for the first embodiment of the instant disclosure. FIG. 6 is a schematic view showing the edge-bordering state for the first embodiment of the instant disclosure. FIG. 7 is a flow chart of a state detection method for the first embodiment of the instant disclosure. FIG. 8 is another schematic view of the depth sensor in FIG. 2. FIG. 9 is a schematic view of a mobile device for the first embodiment of the instant disclosure. FIG. 10 is a top view of the robot for a second embodiment of the instant disclosure. FIG. 11 is a flow chart for the state detection method for the second embodiment of the instant disclosure. FIG. 12 is another flow chart for the state detection method for the second embodiment of the instant disclosure. DETAILED DESCRIPTION Please refer to FIG. 1, which shows a perspective view of a robot 100 for a first embodiment of the instant disclosure. The robot 100 comprises a main body 110, a moving unit 120, and at least one depth sensor 130. For the present embodiment, the robot 100 is for broom-sweeping purpose. The main body 110 includes a casing 111, a vacuum opening 112 formed on the casing 111, a brush 113, and a vacuum unit 114 (as shown in FIG. 3 ). In other embodiments, the robot 100 may serve other purposes with the main body 110 having the casing 111, but including other accessories (e.g. video camera, robotic arm, etc.). The selection of accessories is based on desired capabilities of the robot, which does not necessarily need to have aforementioned vacuum opening 112, brush 113, and the vacuum unit 114. As shown in FIG. 1, the vacuum opening 112 and the brush 113 are formed and disposed, respectively, on the bottom portion of the casing 111. The vacuum unit 114 is disposed internally of the casing 111. The vacuum unit 114 may include a motor, a dust bag, a filter, etc. The moving unit 120 is disposed on the bottom portion of the main body 110. The depth sensor 130 is arranged on one side of the casing 111 (the depth sensor 130 is disposed internally of the casing 111 but adjacent to the bottom portion thereof and partially exposed from the bottom portion thereof). Detection away from the robot 100 is made by the depth sensor 130. The moving unit 120 includes a swivel wheel 121, a pair of fixed wheels 122, and a drive motor (not shown). The depth sensor 130 can be an infrared sensor, an ultrasonic sensor, a static sensor, or other non-contact type distance sensor. Please refer to FIG. 2, which shows the depth sensor 130. For the present embodiment, the depth sensor 130 is an infrared sensor, which includes a cover 131, an emitter 132, and a receiver 133. The cover 131 is formed with a pair of light-permitting openings 134 corresponding to the emitter 132 and the receiver 133. One of the light-permitting openings 134 allows the emitter 132 to output infrared externally of the cover 131. The other light-permitting opening 134 allows the reflected infrared to be received by the receiver 133 internally of the cover 131. As shown in FIG. 2, one end of the receiver 133 adjacent to one of the light-permitting holes 134 is bent toward the emitter 132. Such configuration maximizes the receiving area and the angle for the receiver 133 to receive reflected infrared. FIG. 2 further illustrates a protrusion 135 formed between the light-permitting openings 134 on the cover 131. In particular, the protrusion 135 extends from a surface flushed with the light-permitting openings 134. The protrusion 135 serves to isolate the emitter 132 and the receiver 133 from each other, so as to prevent the receiver 133 from receiving emitted infrared directly without reflection. The isolation allows the light-permitting openings 134 to be radially maximized, in order to increase the luminous flux off the emitter 132 and reflected influx to the receiver 133. The above configuration enhances detection precision and flexibility. Now please refer to FIG. 3, which shows a block diagram for the robot 100 further having a control module 140. The control module 140 can be a processor of embedded type. The control module 140 electrically connects the moving unit 120 and the depth sensor 130. Based on the numerical value of the detection signal of the receiver 133 (i.e., electrical signal generated by the optical-electrical conversion of the reflected infrared), the control module 140 determines whether the robot 100 is in the lifted state, the tilted state, or the edge-bordering state. Particularly, the numerical value and the distance between the depth sensor 130 and a support surface 200 ( FIGS. 4-6 ) are negatively correlated for determining the state of the robot 100. FIGS. 4-6 are discussed in details below to describe the three states of the robot 100. FIG. 4 shows the lifted state for the robot 100, in which the robot 100 is lifted from the support surface 200. The support surface 200 may be a ground surface, a table surface, etc. The lifted state means not one part of the robot 100 is in touch with the support surface 200. For example, when a child lifts up the robot 100, the robot 100 is suspended off the support surface 200. Please refer to FIG. 5, which shows the tilted state of the robot 100 with one end thereof suspended off the support surface 200. However, another end of the robot 100 is in contact with the support surface 200. In particular, the right side of the robot 100 is suspended off the support surface 200, while the left side thereof touches the support surface 200. The edge-bordering state is shown in FIG. 6, as can be seen when the robot 100 is at the edge of the support surface 200. For instance, the robot 100 moves to the edge of a step. Next, FIG. 7 shows a flow chart for a state detection method for the first embodiment of the instant disclosure. This method is performed by the aforementioned control module 140. First, at least one depth sensor 130 is disposed on one side of the mobile device (step S 710 ). The mobile device may be a self-propelled device like a robot. For the present embodiment, the mobile device is the robot 100. In other cases, the mobile devices may be mobile phones or other portable devices. In step S 720, the control module 140 obtains a detection signal of the depth sensor 130. In step S 730, based on the numerical value of the detection signal, the control module 140 determines whether the mobile device is in the lifted state, the tilted state, or the edge-bordering state. If the mobile device is in any of the above-mentioned states, the method will proceed to step S 740, which will implement a response procedure. If not, the method will return to step S 720 for continuing detection by the depth sensor 130. The response procedure referred herein may include any of the following features. First, the mobile device is put into a different motion, such as powering off or switch to standby mode, to stop the current motion of the mobile device, so the child would not be injured by the continuous motion of the mobile device. Other attribute is reducing power consumption by the mobile device. The mobile device can also be instructed to turn around from its original direction or trek backward, in order to change its current motion or exit from its current state. Secondly, the response procedure can issue a warning, to alert the user to get the mobile device out of its current state. The third option is to make the mobile device return to its original location or previous state, so the mobile device can exit from any of the abovementioned states. The response procedure is executed by the control module 140 based on switching between different software operations like interrupting, polling, threading, etc. In FIG. 8, another schematic view of the depth sensor 130 is shown. This depth sensor 130 differs from the one in FIG. 2 by having a shield 136. The shield 136 is connected to the cover 131 by a flexible member (e.g., spring). The shield 136 is disposed in front of the receiver 133. When the robot 100 is hit on the bottom portion thereof, the shield 136 would be displaced toward the receiver 133, so as to block the light from entering the light-permitting opening 134 corresponding to the receiver 133. As suggested by the description, the shield 136 provides covering and blocking functions. Thus, when a collision occurs, the numerical value of the detection signal of the depth sensor 130 would be zero or close to zero. Once the collision state has been detected, any of the aforementioned response procedures can be carried out accordingly. For the present scenario, the protrusion 135 also serves to block and limit the movement of the shield 136. In other embodiment, the shield 136 can be arranged in front of the emitter 132. When a collision occurs, the shield 136 would block the infrared output by the emitter 132. For another embodiment, the depth sensor 130 further includes an on/off switch (not shown), which is disposed between the cover 131 and the shield 136. The purpose is that when the shield 136 is displaced toward the cover 131, the shield 136 would actuate the switch. By connecting electrically to the switch, when the control module 140 receives the triggering signal of the switch, a collision state is determined to have occurred. FIG. 9 shows a mobile device 300 for the first embodiment of the instant disclosure. For the present scenario, the mobile device 300 is a cellular phone, with the depth sensor 130 shown in FIG. 8 being disposed on the back surface thereof. Under the collision state (i.e., the back surface of the cellular phone is facing toward and disposed on the support surface), the aforementioned first response procedure is implemented. In other words, the cellular phone is powered off or put in standby mode, so as to save power consumption. Please refer back to FIG. 3. The robot 100 can further include a warning module 150 electrically connected to the control module 140. The warning module 150 can issue a warning to execute the aforementioned second response procedure. Also, for different types of warning, the warning module 150 can be made up by different parts. For example, if the warning is provided in audio mode, the warning module 150 can be a speaker or a buzzer. When the warning is provided in lighting mode, the warning module 150 can be an indicator light or a display. Alternatively, if the warning is in a message form, the warning module 150 may be a mobile communication module or a wireless internet module. Thus, a warning message can be sent to a designated device (e.g., cell phone or computer) of the user. The warning may last for a period of time before terminating automatically. Another option may be the warning would be on continuously until being terminated by the user. For example, the robot 100 has a disarm button (not shown) for pressing by the user to terminate the warning. In some embodiments, if the third type response procedure is employed, and based on the detection signal provided by the depth sensor 130, the state detection method can covert the detection signal into an image. The before and after images are then compared to identify the moving direction of and distance traveled by the mobile device. By using the obtained data, the travelling path of the mobile device can be stored. Accordingly, the robot 100 can further have a storage module 160, such as a memory unit, memory card, hard disk, etc. As illustrated in FIG. 3, the storage module 160 is electrically connected to the control module 140. When the mobile device intends to return to its original location, the control module 140 can read the travelling path saved in the storage module 160. Based on the recorded travelling path, the control module 140 can guide the moving unit 120 in trekking back to the original location. For the present scenario, the original location is an electric charging station or a starting location. In another embodiment, after the robot 100 has returned to its previous state, the robot 100 can change its state again proactively or passively, such as moving toward another direction. For a second embodiment of the robot 100 for the instant disclosure, please refer to FIG. 10, which shows a top view of the robot 100. The present embodiment differs from the first embodiment in that the robot 100 has three depth sensors 130 substantially evenly distributed on the bottom portion thereof. More detailed description of the embodiment having a multiplicity of depth sensors 130 is provided below. Please refer to FIG. 11, which shows a first flow chart of the state detection method for the second embodiment. The detection process is carried out while the robot 100 is in motion. In step S 910, the robot 100 starts to move. Then, the control module 140 determines if any of the three depth sensors 130 does not indicate the presence of the support surface 200 (step S 920 ). If at least one depth sensor 130 does not detect the support surface 200, the method proceeds to step S 940. In step S 940, the control module 140 determines whether a predetermined time period has passed with the support surface 200 going undetected. If yes, the robot 100 is determined to be in lifted or tilted state and its current motion must be stopped (step S 950 ). If no, the state detection process returns to step S 910. Please note, step S 930 is between step S 920 and step S 940. Step S 930 is for the robot 100 to avoid any obstacle if encountered upon. FIG. 12 shows a second flow chart for the state detection method for the second embodiment of the instant disclosure. Unlike the first flow chart, the present detection process is implemented while the robot 100 is still. For step S 960, the control module 140 determines if any of the three depth sensors 130 does not detect the presence of the support surface 200. If at least one of the depth sensors 130 does not detect the support surface 200, the robot 100 is determined to be in the lifted state or the tilted state, and the detection process must proceed to step S 970. In step S 970, the robot 100 is stopped from its current operation. The determination of whether any depth sensor 130 has detected the presence of the support surface 200 is based on if the changes of numerical value for the detection signals surpass a threshold value. For example, if the robot 100 is in the lifted or the tilted state, at least one depth sensor 130 would not be able to receive reflected infrared. A resulting change in the numerical value of the detection signal would be over the threshold value. Thus, when the changes in magnitude of detection signals for all depth sensors 130 surpass a first threshold value, the robot 100 is determined to be in the lifted state. When the changes in magnitude of detection signals for some depth sensors 130 surpass a second threshold value, with the detection signals for the rest of the depth sensors 130 do not change, the robot 100 is determined to be in the tilted state. When the changes in magnitude of detection signals for some depth sensors 130 surpass a third threshold value greater than the second threshold value, with the detection signals for the rest of the depth sensors 130 remain constant, the robot 100 is determined to be in the edge-bordering state. Based on the above, the instant disclosure provides a state detection method, a robot 100, and a mobile device 300. At least one depth sensor 130 is employed to identify if the robot 100 or the mobile device 300 is in the lifted state, the tilted state, the collision state, or the edge-bordering state. No complex sensing instruments are needed to achieve the above purpose. After any of the four abovementioned states has been identified, the appropriate response procedure is implemented, in order to limit the motion or adjust the internal state or procedure of the system for the robot 100 or the mobile device 300. While the present invention has been described by the way of example and in terms of the preferred embodiments, it is to be understood that the invention need not be limited to the disclosed embodiments. On the contrary, it is intended to cover various modifications and similar arrangements included within the spirit and scope of the appended claims, the scope of which should be accorded the broadest interpretation so as to encompass all such modifications and similar structures. | Summary: A state detecting method applied to a mobile device includes: arranging a depth sensor at the bottom of the mobile device, obtaining a detection signal of the depth sensor, and determining if the mobile device is in a lifted state, a tilted state, or an edge-bordering state, based on the numerical value of the detection signal of the depth sensor. The lifted state is associated with the mobile device without contacting with a support surface. The tilted status is associated with one end of the mobile device contacting the support surface and the other end of the mobile device without contacting the support surface. The edge-bordering state is associated with the mobile device located at the edge of the support surface. Accordingly, when the mobile device is in any of the aforementioned states, an appropriate response can be implemented. | 5,857 | 175 | big_patent | en |
Summarize: A filmmaker has gone into hiding after his movie attacking Islam's Prophet Muhammad sparked assaults on U.S. missions in Egypt and Libya, where an American State Department officer was killed. Writer and director Sam Bacile spoke on the phone Tuesday from an unidentified location. He remained defiant, saying Islam is "a cancer" and he wanted his film to make a political statement. The 56-year-old identifies himself as an Israeli Jew and says he believes his video will help his native land by exposing Islam's flaws to the world. Excerpts dubbed into Arabic were posted on YouTube. Among other claims that have caused outrage, the film claims Muhammad was a philanderer who approved of child sexual abuse. Bacile says he's sorry for the person who died, but blames lax embassy security. Read this story in Arabic. Cairo (CNN) -- The United States said it was taking measures to protect its citizens worldwide after protesters angry about an online film considered offensive to Islam attacked U.S. diplomatic compounds in Libya and Egypt on Tuesday, killing an American. In Cairo, several men scaled the walls of the U.S. Embassy and tore down its American flag, according to CNN producer Mohamed Fadel Fahmy, who was on the scene. In Libya, witnesses say members of a radical Islamist group called Ansar al-Sharia protested near the U.S. Consulate in Benghazi, where NATO jets established no-fly zones last year to blunt ground attacks from then Libyan strongman Moammar Gadhafi. The group then clashed with security forces in the city, blocking roads leading to the consulate, witnesses said. A U.S. State Department officer was killed in the violence in Benghazi, Secretary of State Hillary Clinton said in a statement late Tuesday. "We are heartbroken by this terrible loss," Clinton said. "Our thoughts and prayers are with his family and those who have suffered in this attack." Clinton said that she condemned the attack on the U.S. facilities "in the strongest terms" and that following Tuesday's events, the U.S. government was "working with partner countries around the world to protect our personnel, our missions and American citizens worldwide." Libya's General National Congress also condemned the attack, saying it "led to the regrettable injury and death of a number of individuals." Lawmakers said in a statement Tuesday night that they were investigating. It was unclear whether the two attacks were coordinated, CNN national security contributor Fran Townsend said Tuesday night. "One such breach of an embassy or consulate's walls or security on any given day would be tremendous news.... The fact that two of them happened on the same day that is the 9/11 anniversary where Americans are remembering those that we lost, you have to ask yourself, what are American officials trying to understand about this and whether or not these two are related?" she asked. In Egypt, police and army personnel formed defensive lines around the U.S. Embassy in an effort to prevent demonstrators from advancing, but not before the protesters affixed a black flag atop a ladder in the American compound. The black flag, which hangs in full view from inside the complex, is adorned with white characters that read, "There is no God but Allah and Mohammed is his messenger," an emblem often used by Islamic radicals. A volley of warning shots were fired, as a large crowd gathered around the compound, although it is not clear who fired the shots. Are you there? Share your images and videos. Egyptian groups point to U.S. websites, including YouTube, that have scenes from the film. Some anti-Muslim blogs also have flagged the movie. In a series of disjointed scenes, the film depicts Prophet Mohammed as a child molester, womanizer and ruthless killer. The movie was made by Sam Bacile, an Israeli-American real-estate developer, according to the Wall Street Journal. Bacile -- who wrote, directed and produced the film -- said he wanted to showcase his view of Islam as a hateful religion, the Journal reported, citing a telephone interview with him. Bacile, 52, told the newspaper that to make the film, he had raised $5 million from about 100 Jewish donors, who he declined to identify. He said he made the two-hour movie over a three-month period last year in California, using about 60 actors and 45 crew members, the Journal reported. Most of the Muslim world considers depictions of Mohammed to be blasphemous and deeply offensive. "Some have sought to justify this vicious behavior as a response to inflammatory material posted on the Internet," Clinton said. "The United States deplores any intentional effort to denigrate the religious beliefs of others. Our commitment to religious tolerance goes back to the very beginning of our nation." But she stressed that "there is never any justification for violent acts of this kind." Embassy officials issued a warning to Americans in Egypt, telling them to avoid the demonstrations which "may gather in front of the U.S. Embassy, or Egyptian government buildings such as the People's Assembly and Ministry of Interior." "It is unclear if large numbers will take to the streets, but clashes may occur should two opposing groups come into contact with one another," the U.S. Embassy said in a statement. "Large gatherings and non-essential travel in and around downtown and Garden City should be avoided this afternoon." Frenzied protesters could been seen Tuesday afternoon holding up bits of a shredded American flag to television camera crews while chanting anti-U.S. slogans. An embassy phone operator told CNN that the compound had been cleared of diplomatic personnel earlier in the day ahead of the apparent threat, while Egyptian riot police and the army were called in. "This is an expression of a feeling that is thought to be an insult," said Nizih El Naggary, a spokesman for the Egyptian Foreign Ministry. "But events like this are extremely deplorable. And we have to work to get things under control." The Foreign Ministry issued a statement Tuesday, pledging to protect embassies and warning of the protests' potentially debilitating effects on the Egyptian economy. "There are police forces at the demonstrations," El Naggary said. "They should be protecting the embassy and asking people to leave." Several individuals claimed responsibility for organizing the demonstrations Tuesday, including Salafist leader Wesam Abdel-Wareth, who is president of Egypt's conservative Hekma television channel. Mohamed al-Zawahiri -- the brother of al Qaeda leader Ayman al-Zawahiri -- added, "We called for the peaceful protest joined by different Islamic factions including the Islamicc Jihad (and the) Hazem Abu Ismael movement." "We were surprised to see the big numbers show up, including the soccer Ultra fans," he said. "I just want to say, how would the Americans feel if films insulting leading Christian figures like the pope or historical figures like Abraham Lincoln were produced?" He added that "the film portrays the prophet in a very ugly manner, alluding to topics like sex, which is not acceptable." The U.S. Embassy in Cairo announced that it had canceled visa services for Wednesday. It also said in a statement that it "condemns the continuing efforts by misguided individuals to hurt the religious feelings of Muslims -- as we condemn efforts to offend believers of all religions." "Respect for religious beliefs is a cornerstone of American democracy," the statement said. "We firmly reject the actions by those who abuse the universal right of free speech to hurt the religious beliefs of others." The embassy statement set off a political spat back in the United States after the Republican presidential nominee, Mitt Romney, criticized its message and linked it to his opponent for the White House. "It's disgraceful that the Obama Administration's first response was not to condemn attacks on our diplomatic missions, but to sympathize with those who waged the attacks," Romney said in a statement released late Tuesday. He said he was "outraged" by the attacks in Libya and Egypt. The Obama campaign quickly responded to Romney's comments. "We are shocked that, at a time when the United States of America is confronting the tragic death of one of our diplomatic officers in Libya, Governor Romney would choose to launch a political attack," Ben LaBolt, an Obama campaign spokesman said in an email. Demonstrations elicited a mixture of reactions from the Egyptian street, where last year tens of thousands turned out in opposition to former Egyptian President Hosni Mubarak. This summer, Egypt's first Islamist president, Mohamed Morsy, was sworn into power at Tahrir Square, the scene of the nation's revolution in 2011. Though Tuesday's embassy protests are the first that Morsy has dealt with, Egypt recently produced similar scenarios when protesters attacked the Israeli and Syrian embassies in unrelated episodes. "These protests are a bad image for Egypt," said a Cairo street vendor named Ahmed. "Of course I'm against insulting Islam, but it's the undereducated, poor people who are out here causing problems." "All I want for Egypt is security and stability," he said. "And as you can see this isn't it." The incident occurred on the 11th anniversary of the 9/11 attacks as crowds gathered across the United States in somber remembrance of a day that left nearly 3,000 people dead. Tuesday's focus on the controversial film also drew comparisons to outcry generated from a 2008 movie produced by an anti-Muslim Dutch lawmaker to portray Islam as a violent religion. Geert Wilders' film "Fitna," which he released online, featured images of terrorist acts superimposed over verses from the Quran. Report: Egypt's ex-PM Ahmed Shafik faces arrest, extradition order Egypt kills militants, seizes weapons in Sinai offensive U.S. companies eye Egypt for investment CNN's Ian Lee in Cairo, Jomana Karadsheh, Matt Smith, Brian Walker, Elise Labott, Paul Cruickshank and Tracy Doueiry contributed to this report | Summary: Moving quickly to make a political point following yesterday's attacks on the US diplomatic missions in Egypt and Libya, Mitt Romney called an early US statement "disgraceful" for "sympathizing" with protesters. A statement released in Cairo shortly after angry demonstrators began to gather at the embassies referred to an anti-Muslim film posted online that triggered the protests, and said that the US condemns "the continuing efforts by misguided individuals to hurt the religious feelings of Muslims." The statement was posted hours before an American State Department officer was shot to death as suspected members of the radical Islamist group Ansar al-Sharia torched and looted the US consulate in Benghazi. "It's disgraceful that the Obama administration's first response was not to condemn attacks on our diplomatic missions, but to sympathize with those who waged the attacks," said Romney. Secretary of State Hillary Clinton, in a statement released about the same time as Romney's, condemned the attack in Libya "in the strongest terms." Some have "sought to justify this vicious behavior as a response to inflammatory material posted on the Internet," Clinton said. "The US deplores any intentional effort to denigrate the religious beliefs of others. But let me be clear: There is never any justification for violent acts of this kind." The White House condemned Romney for launching a political attack in the wake of tragedy. Americans worldwide are being warned by the State Department to take extra precautions in the wake of the violence, reports CNN. The filmmaker of the controversial anti-Islam movie, 56-year-old Israeli Sam Bacile, is in hiding, AP reports. The film reportedly depicts the Prophet Mohammed as a child molester, womanizer, and killer. | 2,254 | 395 | multi_news | en |
Summarize: Yesterday morning, two maids — still unnamed at this time — walked into what they believed to be an empty cabin in California's Big Bear mountains. Instead, they stumbled upon fugitive ex-cop Christopher Dorner, who tied them up and fled in a purple Nissan. Eventually, one of the maids managed to break loose and call the police. About a half-hour later, according to the Los Angeles Times, "officers with the California Department of Fish and Wildlife spotted the stolen vehicle and called for backup," and the chase was on. Following a shoot-out, Dorner took refuge in another cabin, where he died in a fire. Normally, the maid's actions, which clearly led to Dorner's demise, would merely earn her the gratitude of the police force and the community. But this was hardly just any old criminal investigation. Three days ago, a group of local businesses, organizations, and private citizens, led by the Los Angeles mayor's office, cobbled together a record-breaking $1 million reward for information leading to Dorner's "arrest, apprehension, and conviction," in the words of Riverside County District Attorney Paul Zellerbach. Does the maid, in a fairy-tale twist, get that million dollars? It's not clear. The maid's phone call didn't technically lead to the "arrest, apprehension, and conviction" of Dorner, only to his death, so if the terms of the reward are taken literally, it would seem she's out of luck. However, the city did get exactly what it wanted — the chance to bring Dorner to justice and end his reign of terror. Being stingy with the money, in such a high-profile case, could arguably dissuade citizens from making an effort to help the LAPD when rewards are offered in the future. The ultimate decision for how to dole out that million-dollar bounty rests with the mayor's office, LAPD spokesperson Richard French tells Daily Intelligencer. "When there are rewards like this, they have to sit down with investigators and others, including the people who are offering the reward, the organizations who were offering the reward, and they have to kind of figure out how, or if, the reward is going to be distributed." There are no standard guidelines for rewards of this nature; they're handled on a "case by case basis," French says. A message left with the mayor's office has not been returned. Update, 5:34 p.m.: TMZ points out that there are two other rewards for $100,000 each, but each of them require the capture or conviction of Dorner. Chris Dorner $1 Million Reward... Big Loophole EXCLUSIVE Several private citizens were instrumental in cornering cop killer... but there may be a gigantic loophole preventing any of them from collecting the loot.There are 3 rewards. The Mayor of L.A. announced a $1 million reward -- funded by private groups -- for information leading to the "capture and conviction" of Dorner. Big problem -- technically speaking, Dorner must be both capturedconvicted to trigger the reward.The L.A. City Council offered a $100,000 reward for information leading to "the identification, apprehension, and conviction" of Dorner. Again.... City Council sources tell us there's already a disagreement between the Legislative Analyst and the City Attorney over how to interpret the reward language.And finally... the L.A. County Board of Supervisors offered a $100,000 reward for information " leading to the capture of Christopher Dorner." One source at the Board of Supervisors tells TMZ,"Dorner was cornered but not captured."Translation... technically speaking, no one may be entitled to the reward. It's unclear if the Mayor, the City Council and the Board of Supervisors will bend the rules. | Summary: Christopher Dorner's all-but-certain demise came thanks to the help of a few civilians, notably the two people who entered what they thought was an empty cabin only to be accosted by the fugitive and tied up. (Update: Turns out, they weren't maids.) One of them managed to call for help after he left, and police were in hot pursuit of Dorner in no time. So at least one of the pair has a claim to the mayor's $1 million reward, right? Maybe not, reports TMZ. The reward stipulates that it's for information leading to the "capture and conviction" of Dorner, who is believed to have died in yesterday's cabin showdown before he could be convicted of anything. Similar wording applies to two lesser awards of $100,000 each. In short, it's a judgment call, and all depends on how generous the mayor's office and the organizations that donated the money are feeling. Rewards this big are handled on a "case-by-case basis," an LAPD spokesman tells Daily Intel, which has more details. | 856 | 249 | multi_news | en |
Summarize: Introduction and Overview More than half the states require a voter to provide a specified identification document (ID) to cast a ballot at the polling place, and a few extend the requirement to absentee or mail-in voting. Many states require an ID with a photograph of the voter (photo ID). Some require a document that does not need to include such a photograph (nonphoto ID). Others do not require any type of ID to vote, but a voter may be asked to provide certain information to verify what is contained in the registration record or otherwise confirm his or her identity, such as stating an address or birth date or providing a signature. Voter identification requirements across the states vary in flexibility, in the types of documents allowed, in exceptions made to the requirements, and in the recourse available to a voter who cannot comply with the ID requirement at the polls. Photo ID requirements in particular have been a major issue of policy debate in recent years, but for both photo and nonphoto ID, the range of IDs accepted and how strictly the state enforces the requirement have also been sources of controversy. Debates over such requirements are typically complex and can be contentious. This report provides an updated overview of state requirements for voters to present some form of ID before casting a ballot in a federal election. The report also discusses the origins of voter ID, relevant federal legislative action in the 114 th Congress, and selected legal and policy issues related to state voter ID laws. The scope is limited to identification requirements for voting; the report does not address voter registration requirements. Status of Voter ID Requirements in the States Thirty-two states require voters to show an ID to cast a ballot at a polling place. Nine of those permit no alternatives to photo IDs. Another 9 states require a voter to show a photo ID, if available, but also permit means of identification other than an ID. Examples of such alternatives include signing an affidavit and permitting the voter to cast a provisional ballot, with the election office confirming identity subsequently by matching information or a signature provided by the voter to what the office has on file (see Table A-1 ). Fourteen states require a voter to present an ID but accept documents that do not include a photo, such as a voter registration card, current utility bill, hunting or fishing license, bank statement, paycheck, tribal ID, Social Security card, or other approved document (see Table A-2 ). See " Differences in Voter Identification Requirements among the States " for further discussion of requirements among the states. The Help America Vote Act Identification Requirement and the Origins of Voter Photo ID A number of notable developments are relevant to the increased attention to voter identification in policy debates during the past 15 years. Requirements for voters to present an ID have reportedly been in force in some states since at least the 1950s. By the year 2000, about a dozen states had voter ID requirements. After the 2000 election, numerous studies and reports assessed the nation's voting process, or aspects of it, and made policy recommendations. Perhaps the best known study was issued in August 2001 by the National Commission on Federal Election Reform, often referred to as the "Carter-Ford Commission." The study was sponsored by the Miller Center of Public Affairs at the University of Virginia and The Century Foundation, and it was co-chaired by former Presidents Gerald R. Ford and Jimmy Carter. The report noted that states should work to improve "verification of voter identification at the polling place" and recommended that they require those who are registering to vote and those who are casting their ballot to provide some form of official identification, such as a photo ID issued by a government agency (e.g., a driver's license). A photo ID is already required in many other transactions, such as check-cashing and using airline tickets. These Commissioners point out that those who register and vote should expect to identify themselves. If they do not have photo identification then they should be issued such cards from the government or have available alternative forms of official ID. They believe this burden is reasonable, that voters will understand it, and that most democratic nations recognize this act as a valid means of protecting the sanctity of the franchise. Many of the report's recommendations were incorporated in the Help America Vote Act (HAVA, P.L. 107-252 ) which was enacted in October 2002. Title III of HAVA includes requirements for states on voting systems, voter information, provisional voting, and voter registration. Since 2006, states have been required to maintain a single, computerized list of all registered voters that every election official in the state can access. Title III also includes a limited voter identification requirement. An individual who registers to vote by mail and has not previously voted in a federal election in the jurisdiction must provide a current, valid photo ID or a copy of a current utility bill, bank statement, government check, paycheck, or other government document with the voter's name and address, whether voting in person or by mail. The requirement does not apply to a voter who registers under the National Voter Registration Act of 1993 (NVRA, P.L. 103-31, also known as the "motor-voter" law), and submits with the registration application one of the required identifications, or who provides a driver's license number or the last four digits of the voter's Social Security number that matches an existing state record with the same number, name, and date of birth as provided in the registration. A voter who does not provide required documentation may submit a provisional ballot that is counted in accordance with state law if the appropriate election official determines that the voter is eligible. Another provision in HAVA made clear that states are free to adopt more stringent election administration requirements than those imposed by the act: The requirements established by this title are minimum requirements and nothing in this title shall be construed to prevent a State from establishing election technology and administration requirements that are more strict than the requirements established under this title so long as such State requirements are not inconsistent with the Federal requirements under this title or any law described in section 906. Following passage of HAVA, states enacted laws to implement the act's identification requirement, and in some cases, more stringent requirements, leading to a doubling over the next few years in the number of states with voter ID requirements. After the 2004 election, another study was issued in September 2005 by the Commission on Federal Election Reform. Often referred to as the "Carter-Baker Commission," it was organized by the Center for Democracy and Election Management at American University and co-chaired by former President Carter and former Secretary of State James A. Baker, III. Its report repeated the recommendation of the Carter-Ford Commission for states to adopt an ID requirement for voters. Further, it recommended that states require a photo ID that meets requirements specified in Title II of the REAL ID Act of 2005 ( P.L. 109-13 ), and that states provide IDs with no charge to voters without them. However, some members of the commission considered that recommendation "troublesome," for reasons similar to the concerns of some observers about stringent photo ID requirements discussed in the section on " Implementation Issues and Policy Considerations." Some attempts have been made to enact federal photo ID requirements. A notable bill from the 109 th Congress, H.R. 4844, passed the House of Representatives in September 2006. It would have required photo ID and proof of citizenship to vote in federal elections. It would also have required that voters who cast a provisional ballot because they did not have the required ID present an approved ID within 48 hours for the ballot to be counted. The bill included an exception for military and overseas voters. It would have required states to provide photo IDs to qualified voters who did not have them, and to provide them to indigent voters at no cost. It would have authorized appropriations to cover the costs of providing such IDs to indigent voters. The bill was not taken up by the Senate before the 109 th Congress adjourned, but several states have adopted similar requirements (see Table A-1 ). Public Opinion on Voter ID Requirements Much of the public discussion about voter ID focuses on how to balance the goals of preventing voter fraud and protecting voter rights (see " Impacts on Turnout and Voter Fraud "). Public opinion surveys over the past decade have consistently found significant majority support for requiring a photo ID to vote. The wording of the questions has varied, with some surveys providing more context about the issues than others. Although all of the surveys described broad categories of ID, several specified that they be "valid," "official," or "government" documents. Some points from the surveys relevant to this report are summarized below: According to one study that compared results from 19 polls between 2006 and 2014, overall support for voter ID has decreased somewhat, from more than 80% in 2006 to about 75% in 2014. In a 2012 survey by the Pew Research Center, respondents were asked if they possessed the needed identification, and 98% said they did. Other studies, however, have found a broader range, from 80% to 95%. A Washington Post poll from 2012 asked whether voter fraud was a problem in presidential elections. About half of respondents said they believed it to be a major problem, one-third considered it a minor problem, and about one in six said it was not a problem. The same poll asked whether voter suppression—described as eligible voters taken off registration lists or denied the right to vote—was a problem in presidential elections. About 40% of respondents believed it to be a major problem, one-third believed it a minor problem, and 20% believed it not to be a problem. One question from that poll joined those two concepts by asking which was more of a concern to the respondent, the potential for vote fraud or the potential that eligible voters could be denied the right to vote. The response was about evenly split, with a few percent more stating they thought vote fraud was more of a concern. It is not clear to what extent respondents in any of the polls were aware of evidence on the degree to which voter fraud occurs; nor is it possible to know how such information would have affected their opinions. No information was found in the surveys about how voters respond to the different specific kinds of photo ID that different states permit (e.g., a driver's license, an employee ID card, a passport). These and similar policy concerns remain factors in the ongoing debate about whether obtaining required ID presents an undue hardship for some who wish to vote, or whether voter IDs are essential, despite any such hardships, to prevent voter fraud (see " Implementation Issues and Policy Considerations "). Voter ID Legislation in the 114th Congress Several bills introduced in the 114 th Congress contain provisions pertaining to voter ID. Some of those bills contain provisions would promote or protect voter ID requirements, while provisions in others would modify, restrict, or eliminate the use of such requirements. H.R. 885 would shield voter ID requirements from some challenges. It would amend Section 2 of the Voting Rights Act (VRA) relating to the requirement for federal courts to block procedures that deny or abridge voting rights. It would expand the kinds of violations that would trigger such actions by the courts, but it would exclude voter photo ID requirements from the list of violations under the section. H.R. 2867 and S. 1659 would restrict the use of voter ID. It would amend the VRA to require preclearance under Section 4 of state changes to voter ID requirements that would make them more stringent than either those required of first-time voters under HAVA or, for voter registration, those in effect in the state at the time of enactment of the bill. H.R. 3277 would eliminate voter ID requirements. It would amend HAVA to prohibit election officials from requiring photo ID for registering to vote or for voting. H.R. 3364 would not directly prohibit voter ID requirements but would require states to provide an alternative. It would amend HAVA to permit a voter—other than a first-time voter who registered by mail—to meet an identification requirement for voting by signing a sworn statement attesting to his or her identity. The bill would require states to make preprinted statements available to absentee voters and at the polls, and it would prohibit a state from requiring a voter who presents the form to be required to vote a provisional ballot. Similar proposals for first-time voters were debated in Congress when HAVA was being considered. Several states that do not require voter ID currently require an affidavit or signature by a voter before the voter can cast a ballot. H.R. 5557 would restrict voter ID requirements. It would amend HAVA and NVRA to prohibit requiring a voter ID that has an associated cost. S. 1912 would modify state voter ID requirements. The bill stipulates that states or jurisdictions with such requirements shall accept a tribal identification card as valid for that purpose. As of June 2016, none of those bills had received further consideration by the committees to which they were referred. A discharge petition was filed that month for H.R. 2867. Differences in Voter Identification Requirements among the States As with many aspects of election administration, states vary widely with respect to verifying voter identity. Some require photo ID, others nonphoto ID, and yet others nondocument identification. Some states with ID requirements accept a broad range of documents, while others permit only a narrow range. Some states permit voters without ID to confirm their identity through another means, while others do not. As shown in Figure 1 and the two tables in the Appendix, 32 states require a voter to show ID before voting at a polling place. The other 18 states and the District of Columbia do not require a voter to provide any ID to vote—they have a range of nondocument requirements instead. Figure 1 depicts state ID requirements as organized into five categories, based on whether the state requires a document ID, whether an ID must have the voter's photograph, and whether the ID requirement is strictly enforced. Table A-1 describes the specific requirements for photo ID states, and Table A-2 for states requiring an ID that need not be photographic. The differences among requirements in the states are sufficiently nuanced that observers may reasonably differ in characterizing a state's requirements as strictly enforced or not. For purposes of this report, whether an ID requirement is considered strictly enforced is based on interpretation of the requirement as described in state law or available guidance. In a state with a strictly enforced requirement, a voter who does not present the required ID at the polling place—with certain exceptions that vary among the states—either cannot cast a ballot at all or must cast a provisional ballot and take action after leaving the polling place to verify his or her identity in order for the ballot to be counted. By those criteria, 9 states have a strictly enforced photo ID requirement (Alabama, Georgia, Indiana, Kansas, Mississippi, North Dakota, Tennessee, Virginia, and Wisconsin), and 4 states have a strictly enforced nonphoto ID requirement (Arizona, Missouri, Texas, and Utah). A voter without an ID is permitted other means of identification in 9 other states requiring photo ID (Florida, Idaho, Louisiana, Michigan, New Hampshire, Rhode Island, South Carolina, South Dakota, and Washington), and the 10 others requiring nonphoto ID (Alaska, Arkansas, Colorado, Connecticut, Delaware, Hawaii, Kentucky, Montana, Ohio, and Oklahoma). Examples of such alternative means of identification include signing an affidavit or providing a nonphoto ID. In some states voters may cast a provisional ballot, and the election office will attempt to confirm identity subsequently by matching information or a signature that is provided by the voter against the information that the office has on file. Georgia and Indiana were the first states to enact strictly enforced photo ID requirements, in 2003 and 2005, respectively. The most recent such requirement is that of North Dakota, enacted in 2015. Oregon and Washington conduct elections entirely by mail. In these two states, election officials mail ballots to all registered voters, who are not required to provide IDs when submitting those ballots. Both states also permit voters to cast a ballot in person during a designated voting period that ends on Election Day. Washington requires either a photo ID or signature declaration for in-person voting. Oregon uses signature verification for both mail-in and in-person ballots. Some states in the "strictly enforced" category also provide exceptions or other recourse to a restricted group of voters. For example, Kansas permits voters with religious objection against being photographed to sign a form declaring the objection either in advance or at the polling place. Alabama and Missouri permit voters without IDs to cast regular ballots if two election officials at the polling place sign an affidavit attesting to the voter's identity and eligibility. The 19 states with ID requirements not categorized as strictly enforced permit ballots to be counted for most or all voters who do not have ID without the need for them to take action after leaving the polling place. For example, in Florida, a voter without an ID must cast a provisional ballot, but it will be counted if the signature on the ballot matches that in the registration record. In South Carolina, a voter without a photo ID must cast a provisional ballot along with a declaration of a "reasonable impediment" to obtaining such an ID. The ballot will be counted unless someone presents proof to the election commission that the voter is lying about his or her identity or the impediment. Sixteen states with photo ID requirements had them in effect for the federal election in November 2014. Among the other states that had enacted such requirements before the 2014 election, Wisconsin's went into effect in 2015. Because North Dakota enacted its requirement in 2015, the 2016 federal election will be the first for which it will be in effect. The requirements enacted in Arkansas and Pennsylvania were rejected by state courts. See also the section on " Legal and Constitutional Issues Regarding Voter Photo ID Laws." In the Appendix, Table A-1 describes the photo ID requirements for the 19 states with such a requirement. Table A-2 describes requirements for the 14 states in which a voter is to provide some form of identification document that may be a photo or a nonphoto ID. About 60% of voters live in states with voter ID requirements—30% in photo ID states and 20% in nonphoto ID states—and about 30% live in the 13 states with strictly enforced voter ID requirements. Some patterns can be discerned from the tables, such as on the kinds of IDs accepted, application to mail-in and absentee voting, and the recourse voters have if they do not have an accepted ID. The discussion below illustrates both commonalities and the complexity of variation among state requirements. Commonly Accepted IDs There is a common set of IDs accepted by most or all states. All states accept driver's licenses or nondriver IDs issued by that state. All but one state (North Dakota) will accept a U.S. passport or other federal photo ID, although some states stipulate that a voter produce an ID showing the voter's address (e.g., Arizona, Ohio), in which case a second ID might be necessary. Other IDs are specified as acceptable by several states. For example, tribal IDs are explicitly mentioned by 16 states. Overall, while the requirements in some states, such as Alabama and Mississippi, appear to be similar overall, no two states have clearly identical requirements. Range of Accepted IDs The range of other authorized IDs listed in the tables for different states is broad. In addition to those discussed above, photo IDs specified as accepted by one or more states in Table A-1 include employee, neighborhood association, public assistance, retirement center, and student IDs, credit and debit cards, weapon licenses, election identification certificates, certificates of naturalization, government-issued medical cards, military, veterans, and other government IDs, and others as determined by election officials. Additional IDs specified by one or more states in Table A-2 include birth certificates, Bureau of Indian Affairs cards, certified court records with adoption or name change, hunting or fishing licenses, Indian census cards, leases or mortgages, local election authority IDs, naturalization documents, official election material mailed to voters, pilot's licenses, property tax statements, recorder's certificates, Social Security cards, tribal treaty cards, vehicle registration or insurance cards, verification of residency in group facility or medical confinement, voter confirmation notices, and voter registration cards, as well as documents required for some voters by Section 303(b) of HAVA—bank statement, government check, paycheck, utility bill, and other government document with the name and address of the voter (see " The Help America Vote Act Identification Requirement and the Origins of Voter Photo ID "). Some states in Table A-1 accept only a narrow range of photo IDs. The narrowest requirement is that of North Dakota, which accepts only a driver's license or photo ID issued by the North Dakota Department of Transportation or a photo ID issued by a tribal government. Twelve states permit state-issued IDs only from that state but also permit other classes of ID such as those issued by the federal government. Some states permit IDs to be expired, at least in some cases, while others require that they be current. All of the states with strictly enforced photo ID requirements issue free IDs to voters who qualify. Several of the other photo ID states do as well. However, there may be other costs associated with obtaining a free voter ID (see " Obtaining an ID "). Application to Absentee and Mail-in Voting The ID requirements for polling-place voters apply to those voting by absentee or mail-in ballots in eight states (Alabama, Alaska, Kansas, North Dakota, Ohio, South Dakota, Virginia, and Wisconsin). Three of those (Alaska, Ohio, and South Dakota) do not have strictly enforced voter ID requirements. Eight states with strictly enforced requirements do not extend them to absentee voters (Arizona, Georgia, Indiana, Mississippi, Missouri, Tennessee, Texas, and Utah). Three of those (Arizona, Georgia, and Utah) do not require voters to provide a reason when applying to vote absentee. Among the states with voter ID requirements for absentee voting, notable exceptions occur in two: North Dakota permits absentee voters who do not have approved IDs to have their identities attested to by other qualified voters. In Virginia, this ID requirement pertains only to those applying in person for an absentee ballot. See also " Impacts on Turnout and Voter Fraud." Recourse for Voters without ID In 8 of the 9 states with strictly enforced photo ID requirements, (Alabama, Georgia, Indiana, Kansas, Mississippi, Tennessee, Virginia, and Wisconsin), a voter who does not have the required photo ID may cast a provisional ballot that will be counted if the voter presents an accepted ID to election officials within a specified period after the election. North Dakota does not provide such an option. Two states (Kansas and Virginia) specify that a voter can provide the ID via mail or other designated means rather than in person. Five of the photo ID states where the requirement is not strictly enforced permit a voter without ID to cast a regular ballot after signing an affidavit (Idaho, Louisiana, Michigan, New Hampshire, and South Dakota). The other four permit the voter to cast a provisional ballot, which will be counted after confirmation of a signature match (Florida and Washington) or information provided in an affidavit (Rhode Island and South Carolina). The voter's eligibility may be challengeable or subject to subsequent investigation to verify eligibility. Among the 14 nonphoto ID states in Table A-2, a voter who does not have an acceptable ID can establish his or her identity by some other means at the polling place in 10 (Alaska, Arkansas, Colorado, Connecticut, Delaware, Hawaii, Kentucky, Montana, Ohio, and Oklahoma). Arizona, Missouri, Texas, and Utah are the four states where the requirement is strictly enforced. In Arizona, Texas, and Utah, a voter who cannot produce a nonphoto ID from the list of approved documents may vote provisionally and provide the ID to county election officials within a specified time after the election. Missouri's requirement does not contain such an option. A voter without ID can cast a ballot only if two supervising election judges at the polling place who are from different major political parties sign an affidavit attesting to the voter's identity. No data were available on the frequency of such attestations, but they would seem unlikely except for polling places in small, tight-knit communities where most voters would be known to the pollworkers. That suggests that among the nonphoto ID states, Missouri's requirements are most similar to those of North Dakota, which has a strictly enforced photo ID requirement. Among the 11 states with strictly enforced ID requirements permitting subsequent verification of identity, the time period within which verification must occur varies. It might be a specified day or number of days after the election—ranging from 2 to 10 days—or before a meeting of election officials or county certification of the election, or by a deadline to be provided to the voter at the polling place. Ohio permits a voter who cannot present identifying information at the polling place to present it within 7 days to the election office. To further illustrate differences in state laws, in Louisiana, a voter without a "generally recognized" photo ID with name and signature must sign an affidavit and present other identifying information to an election official, and may be subject to challenge. In Indiana, a voter without a current or recently expired photo ID issued by the Indiana or federal government can cast a provisional ballot that will be counted if the voter appears at the county elections office by noon on the Monday after the election and either brings the required ID or signs an affidavit affirming indigence or religious objection to being photographed. In Ohio, an in-person or absentee voter must present an Ohio or federal government ID which is unexpired or other specified document dated within the last year, with ID or document containing the voter's name and address (except for a military ID); a voter without an accepted ID may cast a provisional ballot and provide an Ohio driver's license or nondriver ID number or the last four digits of the social security number, either as part of the provisional ballot information or within seven days after the election. For additional details and other examples, see the Appendix. For further discussion, see " Voters Who Do Not Have an Accepted ID." Legal and Constitutional Issues Regarding Voter Photo ID Laws41 State voter photo ID laws have been the subject of litigation. (While nonphoto ID laws have also been challenged, this section focuses on legal issues relating to the generally stricter voter photo ID laws.) These laws have been challenged under the Fourteenth Amendment to the U.S. Constitution, Section 2 of the Voting Rights Act (VRA), and state constitutional provisions. Such challenges have drawn attention in view of a 2008 U.S. Supreme Court ruling, discussed below, that upheld the constitutionality of an Indiana voter photo ID law. Challenges under Section 2 of the VRA are also notable because in the past, Section 2 has generally been invoked in the context of redistricting. Section 2 of the VRA provides a right of action for private citizens or the government to challenge discriminatory voting practices or procedures. The law prohibits any voting qualification or practice by any state or political subdivision that results in the denial or abridgement of the right to vote based on race, color, or membership in a language minority. The statute further provides that a violation is established if, based on the totality of circumstances, electoral processes are not equally open to participation by members of a racial or language minority group in that its members have less opportunity than other members of the electorate to elect representatives of their choice. Until the final weeks and months preceding the November 2014 election, due to ongoing appeals, the question of whether some state voter ID laws would be in effect was unknown. Likewise, leading up to the November 8, 2016, presidential election, there has been ongoing litigation challenging certain state laws. This section of the report analyzes challenges to voter photo ID laws under the Fourteenth Amendment and Section 2 of the VRA, specifically addressing, by way of example, recent court rulings in North Carolina and Texas; provides an overview of a challenge to a voter photo ID law under a state constitutional provision; and finally, discusses some potential implications of these challenges. Fourteenth Amendment and Voting Rights Act Supreme Court Ruling In a 2008 ruling, Crawford v. Marion County Election Board, the Supreme Court upheld an Indiana voter photo ID law against a facial challenge under the equal protection clause of the Fourteenth Amendment. The Indiana law requires voters to present a photo identification card issued by the government. A majority of the Court in Crawford did not agree on a rationale for upholding the voter photo ID law. The lead opinion found that although the law imposes a "somewhat heavier burden" on a "limited number" of people, the severity of that burden is mitigated by the fact that eligible voters may cast provisional ballots that will ultimately be counted. Moreover, the opinion reasoned, even if the burden cannot be justified as to a few voters, that fact would be insufficient to require the relief sought by the petitioners, which was to invalidate the voter photo ID law in all its applications. In conclusion, the lead opinion determined that Indiana's voter photo ID law imposed only a "limited burden" on voting rights that is justified by the state interest in protecting election integrity. Notably, the opinion announced that if a law is nondiscriminatory, and supported by valid, neutral justifications, then such justifications are still relevant to consider even if one of the legislature's motivations in enacting the law was to pursue partisan political interests. Importantly, although the lead opinion in Crawford rejected a facial challenge, i.e., a case seeking to invalidate the statute in all its applications, to a voter photo ID law, it appears to have left open the possibility of "as applied" challenges to such laws if greater evidence of the burdens imposed on voters' rights could be shown. Lower Court Rulings Litigation challenging a number of state voter photo ID laws has occurred, or is currently ongoing, in the lower courts. By way of example, this report discusses recent court rulings in long running litigation evaluating two state laws. In the first ruling, a partially divided U.S. Court of Appeals for the Fourth Circuit invalidated North Carolina's voter photo ID law, holding that it was enacted with a racially discriminatory intent in violation of the Equal Protection Clause of the Fourteenth Amendment and Section 2 of the VRA. In the second ruling, issuing a plurality opinion, a divided en banc panel of the U.S. Court of Appeals for the Fifth Circuit ruled that a Texas voter photo ID law has a discriminatory effect on minorities' voting rights in violation of Section 2 of the VRA. While not invalidating the Texas law, the court required that it be administered on November 8 with modifications. On the issue of whether the law was enacted with a discriminatory intent, however, in contrast to the Fourth Circuit ruling, the court remanded. North Carolina In July 2016, a partially divided U.S. Court of Appeals for the Fourth Circuit (Fourth Circuit) invalidated North Carolina's voter photo ID requirement, along with other provisions of its election law. In North Carolina State Conference of the NAACP v. McCrory, the court held that the 2013 law was enacted with a racially discriminatory intent in violation of the Equal Protection Clause of the Fourteenth Amendment and Section 2 of the VRA. Reversing and remanding a lower court ruling, the court enjoined implementation of the law. On August 31, by a 4-4 vote with regard to the voter photo ID provision, the U.S. Supreme Court denied North Carolina's request for a stay of the appellate court ruling. As a result, the law will not be in effect for the November 8 election. As a threshold matter, the Fourth Circuit observed that, similar to laws that expressly discriminate on the basis of race, if a law is discriminatorily motivated, it is unconstitutional. In determining whether discriminatory intent motivates a facially neutral law, the court interpreted Supreme Court precedent as requiring consideration of several factors, including first, the historical background in a case. Here, the appellate court determined that the lower court clearly erred in finding minimal evidence of official discrimination in North Carolina since the 1980s, and instead identified evidence of attempts by the legislature "to suppress and dilute" African American voting rights. Second, a court is required to consider the sequence of events leading to the challenge. According to the court, the record of the case showed that immediately after a 2013 Supreme Court ruling that rendered the preclearance requirements of the VRA inoperable, the North Carolina legislature substantially expanded an earlier photo ID bill and "rushed through... the most restrictive voting legislation seen in North Carolina since enactment of the Voting Rights Act of 1965." The court held that the lower court erred by not drawing "the obvious inference" of discriminatory intent from this sequence of events. Third, Supreme Court precedent recognizes the relevance of legislative history. In this case, the court determined it relevant that the legislature requested and utilized racial data, including a breakdown by race of DMV-issued ID ownership, absentee and early voting, and same-day registration, which showed that African Americans disproportionately use such procedures. When compared to the "unpersuasive non-racial explanations the State proffered for the specific choices it made," the court considered this element of the legislative history probative. Last, a court is instructed to consider whether the law impacts one race more than another. The court found error in the lower court's conclusion that the voting procedures eliminated by the law were simply "more convenient," and "preferred" by African Americans. African-American voters disproportionately use the voting procedures eliminated or reduced by the challenged law as a result of socioeconomic disparities, the court held, and such use is not borne from a simple "preference." The court concluded: "Registration and voting tools may be a simple 'preference' for many white North Carolinians, but for many African Americans, they are a necessity." The court was also careful to note that its holding was not meant to suggest that any member of the North Carolina legislature "harbored racial hatred or animosity toward any minority group." The court concluded that the totality of the circumstances evidenced that the law was enacted to "entrench" the majority party's control of the legislature, and even if enacted for such "partisan ends," the court held, "targeting voters who, based on race, were unlikely to vote for the majority party... constituted racial discrimination." Finally, the court held that the challenged provisions of the North Carolina law, including the voter photo ID requirement, were not tailored to achieve the stated justifications, and in several ways, were "solutions in search of a problem." Texas Also in July 2016, issuing a plurality opinion, a divided en banc panel of the U.S. Court of Appeals for the Fifth Circuit (Fifth Circuit) ruled that a Texas voter photo ID law must be administered in such a manner to rectify a discriminatory effect on voters who do not have the required ID or are unable to obtain such ID reasonably. Affirming a lower court, in Veasey v. Abbott, the plurality found that the law has a discriminatory effect on minorities' voting rights and therefore violates Section 2 of the VRA. While the court did not invalidate the law, it remanded for consideration by the district court of an appropriate remedy. The court also held that the indirect cost on voters who were not born in Texas to obtain an ID was not the equivalent of a poll tax. On the issue of whether the law was enacted with a discriminatory intent, however, in contrast to the Fourth Circuit ruling discussed above, the court reversed the lower court's judgment and remanded for the district court to consider in light of guidance that the appellate court provided. In August, the district court entered an order approving a plan that, among other things, for the November 8 election, allows certain Texas voters without the required voter photo ID, and who cannot obtain such ID due to a reasonable impediment, to cast a ballot after completing a "reasonable impediment declaration." Furthermore, in September, the district court ordered the State of Texas to insure that voter education materials accurately reflect the court's August order setting forth how the voter photo ID law is to be administered for the November 8 election. In Veasey, regarding the finding that the voter photo ID law has a discriminatory effect in violation of Section 2 of the VRA, the plurality opinion invoked Supreme Court precedent. As required under such precedent, the opinion determined that the challengers showed not only that the law imposes a burden on minorities, but also that it interacts with social and historical conditions to cause an inequality in the opportunities of minority voters to elect preferred representatives. The opinion approved of the lower court's analysis and resulting determination that (1) the law burdens Texans living in poverty, who are less likely to have, or to be able to procure, the requisite ID; (2) a disproportionate number of Texans living in poverty are African Americans and Hispanics; and (3) such minority voters are more likely to be living in poverty because they bear the socioeconomic effects of historical racial discrimination. Further, the opinion determined that the district court thoroughly evaluated the totality of the circumstances, with each finding well supported, and that the State of Texas had failed to contest many of the factual findings. On September 23, the State of Texas filed a petition for writ of certiorari in the U.S. Supreme Court, arguing for a reversal of the Fifth Circuit's holding that the law has a discriminatory effect. If the Court agrees to hear this case, a decision would be expected after November 8, and therefore would not affect the 2016 election. State Constitutional Qualifications Beyond challenges under the U.S. Constitution or federal law, challenges to voter photo ID laws may also arise under state constitutional provisions. For example, similar to certain other state constitutions, the Arkansas Constitution sets forth qualifications for voters. Based on that constitutional provision, less than three weeks prior to the November 2014 election, the Arkansas Supreme Court invalidated a voter photo ID law. According to the court, the framers of the constitutional provision intended that only the four listed voter qualifications be required, and nothing further. The court found that upholding the voter ID law would disenfranchise Arkansas voters, and negate the intent of the framers of the state constitution. Implications The question of whether voter photo ID laws comply with the U.S. Constitution, the VRA, and state constitutional provisions continues to unfold. Although the Supreme Court upheld the constitutionality of an Indiana voter photo ID law in 2008 against a facial challenge, as discussed above, some courts have found other state laws distinguishable or have evaluated such laws under the VRA or state constitutional provisions. Litigation in this area is ongoing, and it is unclear how various courts will rule. Most notably, case law addressing the question of whether voter photo ID laws violate Section 2 of the VRA is just beginning to develop. In the past, litigation under Section 2 was generally invoked in the context of redistricting. Therefore, case law applying Section 2 to voter photo ID laws is evolving and ultimately, may be considered by the U.S. Supreme Court. Implementation Issues and Policy Considerations Several issues may arise in the application of voter ID requirements. Among them are these: implementation problems, especially for new or modified requirements voter difficulties in obtaining IDs issues with recourse for voters with no ID effects on turnout and risk of fraud Implementation Election administration changes have the potential to introduce a degree of uncertainty in the voting process simply because they involve new procedures. That is especially true in the first election for which they are implemented. Some states, such as Florida, Georgia, Indiana, and Michigan, have had photo ID requirements in effect for two or more presidential elections. In 8 others (Alabama, Mississippi, New Hampshire, North Dakota, Rhode Island, South Carolina, Virginia, and Wisconsin), the November 2016 election will mark the first time the current requirements will be in effect in a presidential election. In addition, lawsuits that could affect voter ID requirements for that election have been filed in some states. The administration of federal elections by state and local jurisdictions is a complex, interconnected process, in which changes to any part may have both expected and unexpected effects, not only on what those changes affect directly, but on other parts of the process, and on individual voters, as well. Implementing such changes may reduce the resources available for other tasks before the election, or may have unforeseen effects that would require correction. It may therefore be advantageous for policymakers to provide as much time as possible for implementation, so that election officials, pollworkers, and voters have time to adjust. Some changes, such as moving the location of an individual polling place, affect a limited number of voters. Others, such as changing voting systems or identification procedures, may affect all the voters in the state. Election officials may be required to educate the voting public about the changes and make the necessary adjustments to pollworker training and procedures before the election to ensure a smooth implementation on Election Day. Voters need to understand the changes and may need to undertake actions, such as obtaining an ID, to insure that they do not jeopardize their ability to cast a ballot. Other issues that could arise because of new photo ID laws include the potential for long lines and the possibility that pollworkers could misapply the rules. Long lines may develop in high-turnout elections, such as presidential ones, if new check-in procedures require each voter to present an ID. Voters who are unaware of such new requirements and those who do not have an acceptable ID may cause delays and complications if they need to execute affidavit votes or cast provisional ballots. Finally, there is the possibility that some pollworkers will not be sufficiently trained to know which IDs are acceptable (particularly in states that accept a range of federal, state, and other IDs), which voters, if any, are exempt from the requirement, the procedures to be followed if a voter lacks the proper ID, and how to interpret an ID photograph, especially if the voter has changed in appearance in some way, such as hair color or facial hair. Pollworker training is one of several kinds of costs that state and local governments may incur in implementing voter ID requirements. Such considerations suggest that implementation of new or newly modified voter ID requirements could increase the risk of polling-place problems, but that such risks can be mitigated through administrative preparation, training of pollworkers, and timely education of voters. According to survey data, most local election officials believe that pollworker training and voter education can be important factors in preventing problems at the polling place, and many have said there is a need for improvement. While no systematic studies were available for this report on the effects of changes in election procedures, examples can be found where problems are attributed to such changes. Even after implementation issues have been addressed, the wide variation in voter ID requirements among the states may create difficulties for voters who move to a different state. Census data suggest that 8-10% of the U.S. population relocates to another state approximately every five years. In 2014, about 2.3% (7.3 million people) moved to a new state, with about 1.4% (4.4 million people) moving to a state with a different class of voter ID requirement (e.g., photo ID to nonphoto ID, no ID to photo ID). Obtaining an ID There is no universal voter ID that is used in the United States (including the voter registration card, which is mostly used to provide information for the voter rather than for identification). Acceptable forms of identification differ by state and may be obtained from agencies or other entities that vary among the states. Voters who possess one of the acceptable IDs need not take any action except to bring it with them to the polling place. However, it is also possible that a voter may possess an approved ID that does not match the information in the voter's registration record, for example because of a recent name change due to marriage or divorce, which would require the voter to rectify the discrepancy. Or the ID might be of a type that is accepted, such as a tribal ID, but does not have information that may be required under state law, such as an address and date of birth. In several studies published between 2007 and 2013 for five states, and one nationwide study from 2013, the percentage of registered voters with valid ID ranged from 80% to 95%. Some of the studies found that the percentage of voters with IDs was lower for some minority groups or for voters without regular access to vehicular transportation. In the 12 states with strictly enforced ID requirements, voters who do not have an acceptable ID must secure one in order to cast a ballot. All 9 states with strictly enforced photo ID requirements as well as some others provide free IDs to voters who qualify. It is not only the kinds of IDs accepted that may affect a voter's ability to cast a ballot, but also the kinds of information required to obtain an accepted ID. The voter may need to obtain one or more other documents first—such as a birth certificate, government-issued ID, or proof of residence such as a current utility bill—to apply successfully for a voter ID. The documents required vary among the states, may have associated costs, and may be difficult to obtain for some eligible voters. Whether such costs and other factors, such as difficulty in obtaining supporting documents, place an inappropriate burden on some voters, especially those who are poor, elderly, or members of minority groups, has been a subject of debate for several years. Voters Who Do Not Have an Accepted ID In states with strictly enforced voter ID requirements, except Missouri and North Dakota, voters who do not bring an accepted ID to the polling place may cast a provisional ballot. Such voters need to present required documentation at the county election office within a specified time period for the provisional ballot to be counted. One study found that in two states, Kansas and Tennessee, in the 2012 election, fewer than one in a thousand voters cast a provisional ballot because of ID problems, with fewer than half of those being counted. No information was available for this report on the application and impacts of that requirement in other states, however. Impacts on Turnout and Voter Fraud The term turnout refers in this report to the number of voters or the proportion of the electorate that votes in a given election. Definitions of the term voter fraud vary. Herein, the term comprises voter impersonation and illicit voter registration—namely, illegal activities that voter ID might potentially help to reduce. It does not include other electoral crimes such as vote buying. Whatever their individual views on voter ID or other voting requirements, most observers would probably agree on these two goals: 1. All eligible voters should have equal opportunity to cast a ballot. 2. All necessary steps should be taken to protect the election process from fraud, abuse, and error at any stage. Both of those goals are arguably essential to ensuring the integrity of elections, but they are sometimes thought of as conflicting. On the one hand, it may be reasonable to suppose that the more focus is placed on providing access to the ballot box for all eligible voters, the greater the risk that people who do not meet the criteria for eligibility—for example by reason of noncitizenship, nonresidence, or criminal history—will be improperly included on the voter rolls, or succeed in voting despite not being on the rolls. Also, once a voter is registered, it may be reasonable to suppose that the less stringent the identification requirements at the polling place, the greater the risk that someone might successfully impersonate a legitimately registered voter either at the polling place or on an absentee or mail-in ballot. Voter ID laws would appear to be best suited to preventing voter impersonation rather than illicit registration. Some observers might argue, however, that voter ID requirements can serve as an additional line of defense against illicit or erroneous voter registration. On the other hand, it may also be reasonable to suppose that the more focus is placed on preventing fraud and abuse, the greater the risk that people who do meet the fundamental criteria for eligibility will be improperly excluded from the voter rolls, or not succeed in voting despite being on the rolls. Such concerns have been raised especially for specific demographic groups such as elderly, poor, and minority voters. Some observers have proposed solutions that might reduce the risk of such a conflict, for example, by placing digital photographs of registered voters in electronic pollbooks, thereby eliminating the need for most voters to present separate identification documents. It could be that the apparent conflict is in fact a false one—for example, changes to the election process aimed at increasing access or at decreasing fraud might not have significant effects on actual access and fraud—or that the impact of any particular measure that seems likely to be effective is in fact minimal. Some observers argue that examples of voter fraud at the polling place are rare. Some also claim that voter fraud is much more of a risk with absentee and mail-in voting than at the polling place. Among the 33 states with voter ID requirements, 8 apply those requirements to voters casting absentee or mail-in ballots. However, there does not appear to be sufficient information available to determine the degree to which more broadly applied ID requirements for absentee and mail-in voting would reduce the risk of fraud or how they would affect turnout. In general, no broad consensus has emerged on how to interpret the data on voter fraud that exist. That uncertainty is not surprising, given the complexities of the election process, the difficulties of collecting data about it that are amenable to scientific analysis, the difficulty of controlling for the effects of various factors other than voter ID requirements, the relative recency of ID requirements in several states, variation among states in the stringency of the requirements, and other factors. In addition, as with many aspects of election administration, the impact of any effect of voter ID requirements on the results of the election will depend on factors such as the closeness of the contest. For example, if the implementation of an ID requirement caused a change in turnout, either a reduction or an increase, of 2%, but the margin of victory for a contest was 5%, there would be no effect on the outcome of the election for that contest. In contrast, if the margin of victory were 1%, a 2% reduction in turnout might change the outcome. The observed tendency for some demographic groups to vote more frequently for one major political party than another has raised questions for some commentators about the impacts of voter ID requirements that might affect turnout more for some groups than others. However, given the range of results found in various studies, no compelling consensus has yet emerged about the strength or direction of such impacts. The lack of conclusive data may also help explain seemingly paradoxical views of election officials on voter ID. Two scientific surveys of local election officials in 2006 and 2008 found that on average the officials supported a photo ID requirement, but they believed it would have a negative effect on turnout. They also believed it would increase election security, even though they found voter fraud uncommon and not a serious problem in their jurisdictions. A systematic approach to achieving the two goals discussed in this section would presumably include a risk analysis of all steps in the election process with respect to each goal. In recent elections, attention has shifted among different points in the process, although voter ID has been subject to significant legislative attention for several election cycles. But in the absence of systematic risk analyses, it is difficult to determine what points in the election process—voter registration, voting systems, polling place location and hours, pollworker training, voter identification, vote tabulation, or other steps—actually involve the greatest potential risks to election integrity with respect to fraud, access, and other factors, and therefore what priorities would be most effective for reducing those risks. Concluding Observations Given recent state legislative activity on photo ID, and identification requirements generally, it is likely that legislators in the states will continue to consider similar legislation in the future. According to the National Conference of State Legislatures (NCSL), more than 200 voter ID bills were considered in the states during legislative sessions in both the current (2015-2016), and previous (2013-2014) federal election cycles, although that is a significant decrease from the more than 300 bills considered in the preceding cycle (2011-2012). Further action in the courts and the Department of Justice should be expected as well on voter ID in response to the several new laws that have recently gone into effect. The 2016 election may provide useful data on the implementation and performance of voter ID laws, data that Congress may choose to examine. As more experience is obtained with the impacts of the range of voter ID requirements in different states on both individual voters and elections, a consensus may emerge about the benefits and disadvantages of those requirements, including answers to questions such as the following: Does attempted voter fraud occur frequently enough that it poses significant risk to the validity of the outcome of elections? Is there a significant difference in that risk for polling place versus absentee or mail-in voting? Do voter ID requirements significantly reduce or eliminate the chances of voter fraud? Do voter ID requirements in any states prevent a large enough number of legitimate voters from casting ballots to pose significant risk to the validity of the outcome of elections? Do those risks vary significantly depending on the type and stringency of the requirements? In any case, voter ID is likely to continue to be a topic of significant interest well beyond the November 2016 election. Appendix. State Voter ID Requirements This appendix contains two tables describing ID requirements for in-person voting in the states that have voter ID requirements. Table A-1 covers states that require a voter to present a photo ID; Table A-2 covers states that require an ID that need not include a photograph. The tables briefly summarize the major requirements for each state, describing the types of IDs accepted, whether the requirements apply to absentee and mail-in voting as well as in-person balloting, exceptions to the ID requirements for specified classes of voters, the recourse available to voters who do not present an accepted ID, year of enactment of the requirement (for photo ID states) and additional information such as whether an expired ID will be accepted and whether the state makes an ID available without charge to qualified voters. Specific types of accepted IDs (e.g., passport) are listed in the tables if they are specified in state law or guidance. A glossary of summary terms and abbreviations in the table (in addition to postal abbreviations, used for all states except Idaho) is below: In both tables, states with names in italics (e.g., Alabama ) are categorized in this report as having a strictly enforced voter ID requirement. The states with names not in italics (e.g., Florida) are categorized as not having a strictly enforced requirement. | Summary: About 60% of U.S. voters live in the 32 states that require a voter at a polling place to produce an identification document (ID) before casting a ballot. Among those states, 19 permit voters without ID to cast a ballot through alternative means, such as signing an affidavit; 13 strictly enforce the ID requirement. The other 18 states and the District of Columbia have a range of nondocument requirements instead. Over the last two decades, the number of states requiring voter IDs has tripled. The stringency of those requirements is controversial. States vary substantially in the range of IDs accepted, the information they must contain, and the ease with which a voter can procure an ID. Although all states requiring voter ID accept a local driver's license, no two states have the same overall requirements. Among states with voter ID laws, 18 require photographic identification (photo ID), while 14 permit a nonphoto ID. In addition, eight states require ID for voters casting absentee or mail-in ballots. Several states enacted voter ID laws that have been struck down by courts or are not yet in effect. Recent congresses have seen a number of bills with voter ID provisions, including H.R. 885, H.R. 2867, H.R. 3277, H.R. 3364, H.R. 5557, S. 1659, and S. 1912 in the 114th Congress. State legislatures also continue to consider the issue. Supporters of the more stringent requirements often emphasize the need to prevent voter fraud, while opponents emphasize the need to avoid disenfranchising legitimate voters who do not have ready access to an accepted ID. Polling data suggest that most voters and most local election officials support a voter ID requirement but that many are also concerned about the risk of disenfranchisement. Both voter fraud and disenfranchisement pose potential risks to the integrity of the electoral process, but the policy debate is being conducted in the absence of a consensus about the evidence pertaining to those risks, with available studies producing a broad range of results. As with the 2014 election, leading up to the November 8, 2016, presidential election, state voter photo ID laws have been challenged under the Fourteenth Amendment to the U.S. Constitution, Section 2 of the Voting Rights Act (VRA), and state constitutional provisions. Such challenges have drawn attention in view of a 2008 U.S. Supreme Court ruling that upheld the constitutionality under the Fourteenth Amendment of a voter photo ID law, and because some suits have been brought under Section 2 of the VRA, which in the past, has generally been invoked in the context of redistricting. As the case law challenging voter photo ID laws under Section 2 of the VRA is just beginning to develop, it ultimately may be considered by the U.S. Supreme Court. Election administration is complex, and changes in voter ID requirements may affect elections in unanticipated ways, such as a need for more provisional ballots, increased waiting times at polling places, and misapplication of the rules by pollworkers. The longer that election officials have to implement changes to voting procedures, the lower the risk of unintended and potentially harmful consequences may be. The impact of state voter ID laws is likely to continue to be a topic of high interest beyond November 2016. It seems likely that state legislators will continue to consider such legislation in the future. The 2016 election may provide useful data on the implementation and performance of voter ID laws, which Congress may choose to examine, and which may lead to greater consensus about the benefits and disadvantages of voter identification requirements. | 12,520 | 833 | gov_report | en |
Summarize: A man who lost half of his face to a vicious cancer can now look forward to getting his life back on track after having a implants for a new face as part of the Embarrassing Bodies show. Eric, 60, tragically lost the left side of his face four years ago after being diagnosed with squamous cell carcinoma, which occurs when small growths called polyps grow into tumours. Unfortunately Eric didn’t discover the lump until it was too late and underwent dramatic surgery to have his left eye removed in order to save his life. Scroll down for video. It's back: Channel 4's Embarrassing Bodies is back and in a new episode we meet Karen and Eric, who lost half of his face to a vicious cancer. Speaking on Channel 4 show Embarrassing Bodies, Eric’s partner Karen said: ‘I had no idea it was going to end up like this, it's just something I have to live with. 'It doesn't bother me, who I fell in love with is in here, this bit is just the outside. ‘Eric is now completely cured of the cancer so you have to take some things to gain others.’ Eric and 48-year-old Karen decided to visit Dr Christian Jessen on the TV show to see what could be done to help Eric. ‘We want to tell our story and show others that life isn’t all doom and gloom and that there are ways of curing these things; there is a light at the end of the tunnel,’ said Karen. The couple, from Waltham Abbey, Essex, can be seen on the show meeting with Dr Andrew Dawood of Dawood & Tanner, who is an expert in dental implant surgery and has been working on a bespoke procedure for Eric. Using a 3d technique, he has created a new face for the cancer survivor and the show sees him try it on for the first time. ‘It’s going to be the first time in nearly four years, that you’ve got a whole face and we will see you as you were,’ says Mr Dawood as he helps Eric fit his new face into place. Watching avidly, Karen cannot hide her overwhelming joy and exclaims: 'That's my Eric, he's back. ‘You have got your face back and it looks perfect, it's amazing.’ Tragic: The couple explain to Dr Christian how an aggressive cancer caused Eric to lose half of his face after he had to have surgery to remove his left eye. Describing the moment in an interview with MailOnline, Karen said: ‘It was unbelievable, I’ve got him back. ‘Hopefully he can now go out with his friends to the pub; it will give him a real confidence boost.’ Eric has had his first transplant but is still gradually undergoing the procedures to fit his new face fully. He will have a magnetic bar fitted to hold it into place and the couple are thrilled. Karen, who met Eric when they were both working together at a hotel, has been his absolute rock. ‘It’s not been easy. I have down days but family support helps me and my granddaughter Amelia brightens up my days. Back on track: The show will help Eric have a new face fully fitted and Karen says it will change his life and give him a real confidence boost. ‘She is great with Eric, she is only three and never frightened of him, it’s remarkable. ‘When he had his first implant she saw him and said “Granddad, you’re still broken, the hospital will still fix you won’t they?”’. The couple are still waiting to get married after Eric popped the question on Christmas Day. ‘He covered my eyes and took me into the kitchen. He’d prepared this lovely romantic meal with champagne. ‘We will wait to get through this and then we are hoping to get married this year,’ said Karen. See Eric's full story tonight on Embarrassing Bodies, 9pm on Channel 4 | Summary: New series of Channel 4 show sees Eric, who has lost half of his face to cancer, meeting with Dr Christian to see if there is anything that can be done. He and partner Karen meet dental implant expert who designs a bespoke face for Eric. Eric lost left side of face four years ago after he had operation to remove eye and cure cancer. When Karen sees him with new face she says: 'That's my Eric, he's back, it looks perfect, it's amazing' | 873 | 107 | cnn_dailymail | en |
Summarize: By. Sarah Dean. Wife killer Gerard Baden-Clay's former mistress,Toni McHugh, has revealed how terrified she now is of the man she thought she was going to marry and once loved 'unconditionally'. Ms McHugh was having an affair with real estate agent Baden-Clay when he killed his wife Allison on 19th April 2012. She appeared on Channel Nine's 60 Minutes on Sunday night to make it clear that Baden-Clay didn’t kill his wife Allison for her. ‘I loved him already he didn’t need to, he already had me – he killed for himself,’ she said. In an emotional interview with 60 Minutes reporter Tara Brown, Ms McHugh broke down as she explained: ‘I want this man to never, ever, ever enter my life ever again.’ Scroll down for video. Gerard Baden-Clay's mistress, Toni McHugh, has revealed how terrified she is of the man she once loved 'unconditionally' Ms McHugh broke down as she spoke about her relationship with the wife killer. She said the pair had planned to get married once he left his wife Allison. She added that she is ‘genuinely fearful’ that she was involved with a man who was not who he said he was. ‘That worries me and that terrifies me,’ she said. Ms McHugh appeared on the Channel Nine documentary, which detailed the tragic murder of mother-of-three Allison, to tell her side of the story after she was a key prosecution witness at Baden-Clay’s trial. 'I believed I was in a loving, caring relationship that did have a future,' she told the show. 'He had told me that he would marry me one day.' Gerard Baden-Clay took his wife Allison's body and dumped it at a nearby creek after murdering her. Ms McHugh appeared on the Channel Nine documentary, which detailed the tragic murder of mother-of-three Allison, to tell her side of the story after she was a key prosecution witness at Baden-Clay's trial. Ms McHugh said she loved the convicted murderer'very much' and wanted to be Mrs Baden-Clay. Allison Baden-Clay was the mother of three young daughters. 'I don't like to use that word "unconditional" anymore but I did love him unconditionally. 'And I was very forgiving, too forgiving.' The three-and-a-half year affair between Baden-Clay, 43, and Ms McHugh commenced in August 2008. When asked if she thought the real estate agent killed his wife, Ms McHugh said: ‘I don’t think he’s innocent’. Ms McHugh said she was devastated when she first heard Alison was missing. 'I never ever wanted harm to her… I didn’t intentionally go out to hurt Allison or her family. I honestly believed it [their affair] was going to be handled properly,' she said. The 60 Minutes programme also gave an insight into the incredible police investigation that helped secure Baden-Clay’s conviction. Detective Superintendent Mark Ainsworth, who led the investigation, said Baden-Clay is ‘a guy I wouldn’t trust to take my dog for a walk’. The morning after he killed his wife and dumped her body by Kholo Creek, about 13k from their Brisbane home, a calm Baden-Clay called 000 at 7.15am to report his wife as missing. 'My wife isn’t home, um and um, I don’t know where she is,’ he said on the phone. Allison's friends said they immediately suspected him of harming her when they heard she was missing, and claimed they never liked him. He told police that the two scratches on his face were from shaving and that the scratches on his body came from a caterpillar, while the cut on his hand came from changing a light-bulb. The program obtained 'chilling' footage of Baden-Clay laughing and saying 'everything is going to be alright', to the person behind the camera. The 60 Minutes programme gave an insight into the incredible police investigation that helped secure Baden-Clay's conviction. Baden-Clay attended corporate functions and night time events connected with his real estate business in the company of mistress Ms McHugh (left) rather than his wife Allison. Sergeant Andrew Jackson was called over to the house after Baden-Clay reported Allison missing. He told 60 Minutes: ‘I knew that they weren’t shaving scratches… they were definitely fingernail scratches.’ He added: ‘It looked like someone had cleaned the house before we arrived.’ The police were immediately suspicious of Baden-Clay and the way he was behaving, so called in 80 officers to search the local area. Acting inspector Ewan Taylor was in charge of forensics and found no blood on Baden-Clay's razor to back up his claim that he'd scratched himself shaving - but they did find blood in Allison’s car. As police hunted for Allison for 11 days, Baden-Clay continued to call his mistress Ms McHugh. Allison’s badly decomposed body was eventually found by a canoeist. Allison's best friends Helen Wilson and Nicole Morrison described how they never liked Baden-Clay and immediately suspected he had harmed Allison when they found out she was missing. Helen Wilson (left) and Nicole Morrison (right) spoke fondly about their best friend Allison and said that she would never consider leaving Baden-Clay as she still loved him despite his affairs. A female witness who did not appear before the court claims Gerard called her looking for someone to kill his wife. ‘Baden-Clay lived a life of deception and it was all about Gerard,’ Detective Superintendent Ainsworth said. He added that Allison appeared to have fought for her life as he most likely smothered her. ‘You’ve only got to look at the scratches on the side of his face,’ he said. Police found leaves knotted in her hair, and all six types of leaves were from the Baden-Clay back yard - not the creek where she was found. Police believe he dragged her body across their patio before lifting her into the car. During the 60 Minutes show, Allison's heartbroken best friends Helen Wilson and Nicole Morrison said Allison 'lived to be a mother, she adored her girls.’ Allison's mother said her family had been sentenced to a lifetime of grief. Allison's body was found dumped near a creek in Brisbane's west on April 30, 2012. Ms Morrison added: 'She had the best laugh, when I stop to think about her I can still hear her laugh.’ They said Baden-Clay was very emotionally cruel and once told her he didn’t love her. They said he picked at the dinners she made and how she cleaned the house. But despite this, they explained Allison never considered leaving him and always wanted to fix it and make it better. Allison's diaries were used as evidence in the trial and they detailed how she continued to love her husband despite his cruelty. During the murder trial, the court heard evidence that Baden-Clay attended corporate functions and night time events connected with his real estate business in the company of mistress Ms McHugh rather than his wife Allison. The 60 Minutes piece included testimony from a witness the jury never heard from, who claimed Baden-Clay called her and asked 'I'm looking for someone to kill my wife.' The program also obtained 'chilling' footage of Baden-Clay laughing and saying 'everything is going to be alright', to the person behind the camera. Justice John Byrne said Baden-Clay had shamelessly pretended to search for his wife, and had used a razor blade to disguise what were really the marks of Allison's finger nails on his face. In court, Justice John Byrne said Baden-Clay had shamelessly pretended to search for his wife, and had used a razor blade to disguise what were really the marks of Allison's finger nails on his face. He said Baden-Clay took his wife's body and dumped it at a nearby creek, and then put in place, and persisted in, a deception plan. Baden-Clay had also invented the idea of a drug overdose as a ruse about what had really happened to Allison, and he'd shown a profound absence of remorse for his crime, Justice Byrne said. He'd gone on to besmirch Allison's memory during the trial. 'You have no criminal history but you are definitely not of good character,' Justice Byrne told Baden-Clay. You took a devoted loving mother from her three girls, blighting their lives. We finally have justice for Allison. The evidence presented at this trial has proven that Gerard Baden-Clay is responsible for the murder of his wife Allison. It has been a long wait over the last two years. And this result today marks the beginning of our long journey towards healing and finally allowing us to mourn and grieve this beautiful woman. Today is not a win for our family, for it will not bring our beautiful Allison back. However it is the closure in another chapter of our journey for this family. We have lost Allison and nothing that has happened here today will bring her back. We as a family will grieve her tragic death forever. Her memory is tarnished by the fact that she was taken from us in such horrific circumstances. We would like to sincerely thank the Queensland Police Service and the officers involved in the investigation, the SES volunteers who searched night and day in all weather, the scientific experts and the Office of the Director of Public Prosecutions who have all worked tirelessly to ensure that we have justice for Allison. We would also like to thank them for their compassion and support over what has been the darkest of days. We have appreciated your efforts to protect the privacy of Allison's daughters. Our primary concern has always been and remains the physical and emotional wellbeing of Allison's three beautiful girls. We will help them to rebuild their lives and ask for your support, co-operation and privacy in order to do this. We have a long way to go to ensure that they will cope with a future without their mother. Allison was a kind-hearted, generous woman, a loving wife and devoted mother whose legacy will continue if we all remember that life is precious, and to take the time to be kind, smile at those who pass you by and live for today. We, her family and friends, didn't get a chance to say goodbye but Allison will always remain forever in our hearts. Thankyou | Summary: Gerard Baden-Clay was convicted of murdering his wife Allison in 2012. He was sentenced to life in prison with a non-parole period of 15 years. 60 Minutes screened exclusive interview with Baden-Clay's mistress. Toni McHugh thought she was going to eventually marry him. But claims Baden-Clay did not kill Allison for her. 'I loved him already he didn't need to,' she said. | 2,436 | 106 | cnn_dailymail | en |
Summarize: BACKGROUND OF THE INVENTION 1. Field of the Invention In the games of baseball and soccer, to name only two, the athlete must practice striking the ball. Pitching machines for baseball were developed, and they enabled a batter to develop his timing and swing. However, the pitching machine is expensive and cumbersome and has been the source of serious injuries. Also, it cannot place the ball precisely and repeatedly to help the batter develop a consistent swing plane. Alternatively, and again using the games of baseball or soccer as examples, two or more athletes can practice together in a game situation. However, striking a thrown or placed ball in the open or in an enclosure does not allow sufficient rhythmic and consistent repetition for maximum training effect. 2. Description of the prior art It is, of course, old to suspend a ball or toy for play. Doyle's "Baseball Batting Apparatus", U.S. Pat. No. 831,605 shows a simple suspended ball with elastic return cords. Bearn's "Batting Practice Stand", U.S. Pat. No. 4,258,916 is a swinging arm apparatus with no similarity in function to my invention. Hynes' "Batting Practice Device", U.S. Pat. No. 4,322,075 is again a swinging arm apparatus, with a cord combined with the arm. Neither of these devices allows an actual game ball to be inserted and removed, and neither allows the ball to travel away from the athlete as it would travel in game play. The means of harnessing the actual game ball in the apparatus is a further element of my invention. It is, of course, old to fasten a cord directly to a ball or to use a netting. In the field of playground tether balls, straps of fixed size are disclosed by Minchin's "Tether Ball Holder," U.S. Pat. No. 3,709,491 and Papp's "Game Ball and Tethering Means Therefore,"U.S. Pat. No. 3,351,343. Neither one is a sufficiently durable and safe harness for the functioning of my apparatus. BRIEF SUMMARY OF THE INVENTION It is the principal object of my invention to provide a training apparatus which helps the athlete to develop a consistent plane of motion while striking a ball. It is a further object of my invention to provide a training apparatus that increases the athlete's eye quickness by allowing a struck ball to travel a short, constant and limited distance, while the athlete repeats a consistent swing plane. It is further object of my invention to provide a training apparatus that trains the athlete in the placing of the ball during play. An exemplary embodiment of the invention achieves the foregoing objects in a safe and durable apparatus including a suspended ball, at rest, which is then struck towards targets upon a backstop, and which then returns to the same rest position; a strong harness to hold the suspended ball; a hook with a descending inelastic cord to which the harness appends; a transverse inelastic cord which limits and dampens ball travel; and a fork used in raising and positioning the overhead hook. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a perspective view of a batting screen and an athlete, showing a suspended ball and training targets. FIG. 2 is a perspective view of the overhead hook device made according to the invention, seated upon a fragment of chain-link mesh, with a descending cord attached to the hook device. FIG. 3 is a top plan view of the hook device. FIG. 4 is an elevation view of the hook device showing the cord which descends downward to the harnessed ball. FIG. 5 is a plan view of the empty harness device laid out upon a flat surface and made according to the invention, showing the parallelogram of strapwork which leaves the face of the ball exposed after the harness is wrapped about the ball. FIG. 6 illustrates, in perspective, the first step of the harness being wrapped about the ball. FIG. 7 illustrates, in perspective, the second step of the harness being wrapped about the ball. FIG. 8 illustrates, in perspective, the third step of the harness being wrapped about the ball. FIG. 9 illustrates, in perspective, the fourth step of the harness being wrapped about the ball. FIG. 10 illustrates, in perspective, the completely wrapped harness after the fifth step of wrapping. FIG. 11 is a side sectional view of the ball enclosed in the harness with cords attached to the two grommeted tabs which are made integral to the harness according to the invention. FIG. 12 is a top sectional view of the ball enclosed in the harness with the parallelogram of strapwork and the top grommeted tab illustrated. FIG. 13 is a top sectional view of the harness wrapped about the ball. FIG. 14 is a perspective view of a bight taken in the descending cord. FIG. 15 is a plan view of the fork which is mounted in a pole for use in conjunction with the hook. FIG. 16 is a sectional view of the fork, with an elevation view of the hook being lowered onto the fork. FIG. 17 is a sectional view of the fork, showing an elevation view of the hook positioned on the fork. FIG. 18 is an identical view as FIG. 16, but with the fork pointed upward and the cord hanging downward against the fork. FIG. 19 is an identical view as FIG. 18, but with the fork and hook being raised upwards through the interstices of the wire mesh. FIG. 20 is perspective view that illustrates the fork being lowered downward through the chain-link, and the hook being released from the fork by the action of the chain-link mesh-work against the tailpiece of the hook. FIG. 21 is a perspective view of the hook securely positioned upon a fragment of the chain-link, with the fork being fully withdrawn downwards away from the hook, and with the descending cord still within the center of the fork. DETAILED DESCRIPTION Exemplary embodiments of a sports training apparatus made according to the invention are illustrated in the drawings. As illustrated in FIG. 1, there is provided a hook (1) of cold rolled metal, from which is suspended a descending inelastic cord (3) and a ball (4) encased in a harness (5). A transverse inelastic cord (6) goes from the lower portion of the harness (5) and is hooked or tied to the backstop (8). Targets (7) of a light colored material are movably arranged on the backstop. Chain-link mesh (2) is an integral part of the backstop. Turning now to FIG. 2, the preferred embodiment of the hook device made according to the invention is shown to be in place atop the chain-link mesh (2) which is an integral part of the backstop (8). The hook device is made of cold rolled metal and is formed with a headpiece (9) which is securely captured by the elements of the chain-link mesh when the hook device is in place atop the mesh. The mesh (2) can be either horizontal or inclined upwards from the horizontal. The tailpiece (10) of the hook device also serves to rest upon the chain-link mesh (2). The tailpiece (10) generally points down the overhead slope of and toward the rear of the backstop (8), the rear of the backstop being the portion of the backstop upon which the targets (7) are fastened. The hook device also includes a bend (1) which serves a place to fasten the descending cord (3). The configuration of the headpiece (9), the tailpiece (10), and the bend (11) is further illustrated in FIG. 3 and FIG. 4. The unique structural design of the hook is such that it is a continuous piece of metal configured in such a way as to interlock with the chain-link fencing material. That portion of the hook between the headpiece and the tailpiece, which includes the bend (11) for the fastening of the descending cord, is the portion of the hook which spans the interstices of a chain-link fence material. The hook therefore provides a secure point from which to suspend the harnessed ball. It can be readily understood that the configuration of the hook enables the hook to be relocated so that the harnesses ball can be positioned closer to the targets or further from the targets which are hung upon the backstop. Turning to FIG. 5, the harness is formed of a fabric having inherent strength, durability and flexibility. The material is day-glow orange color in the preferred embodiment. The material must also be resistant to stretching or deformation, either along the direction of the weave, or along the bias of the fabric. The center of the harness comprises a parallelogram (60) fashioned of fabric strapping. Fabric strapping (51), (52), (56) and (57) extends radially from each corner of the parallelogram so as to form equal obtuse angles (61) between the extending strap and each adjacent face of the parallelogram. Two radially opposing straps (51) and (52) include grommeted tabs (53) and (54). The grommeted tab (53) is located on strap (51) immediately beyond the corner of the parallelogram. Radially outward on the embodiment illustrated in FIG. 5, is Velcro material (58) and (59) which is attached to one face of each of the projecting fabric straps. The companion portions of the Velcro material are indicated as Velcro pile (58) and Velcro loop (59). In the embodiment illustrated in FIG. 5, the projecting fabric strap (51) is the first strap to be wrapped around the ball which will ultimately be securely wrapped within the harness. For purposes of describing the positioning of the Velcro material on one of the two faces of the extending fabric strap, the outward face of the harness will be the face that appears in FIG. 5 and will be the face to which the grommeted tabs (53) and (54) are affixed. The other face will be referred to as the inner face. Thus, beginning with fabric strap (51), the pile portion of the Velcro material (as distinguished from the loop portion of the Velcro material) will be securely fastened to the outer face of the fabric. The Velcro would extend from the radially outmost portion of the fabric strap, inward to the base of the grommeted tab (53). On the radially opposite fabric strap (52) the companion loop portion (59) of the Velcro material would be securely fastened to the inner face of the fabric strap, with the Velcro material being of approximately equivalent length to the companion Velcro which is attached to fabric strap (51). As shown, there are two further fabric straps (56) and (57) extending radially outward from the central parallelogram in the center of the harness. Strap (56) has Velcro pile material (58) fixed to the outward face of the strap, extending from the extremity of the strap radially inward for a suitable distance. Strap (57) is radially opposite from strap (56), and has the companion Velcro loop (59) fastened to the inward face of said strap (57). In the central portion of the harness assembly, a parallelogram (60) is constructed of the fabric strapping. In the preferred embodiment, two opposing sides of the parallelogram are comprised of the middle section of the lengthy pieces, continuous from the radial extremes of straps (51) and (57), and from the radial extremes of straps (52) and (56). Two substantially shorter pieces of strapping form the final opposing sides of the parallelogram. By folding the longer strips on the half-bias, and attaching them where folded to the shorter strips, the preferred embodiment of the parallelogram is constructed and securely fastened at the corners thereof. The best mode of attaching the folded strappings to the shorter strappings, to construct the central parallelogram, is by hand or machine stitching using a strong and durable thread. Similarly, the best mode of attaching the Velcro material to the strapping, and of attaching the grommeted tabs to the strapping, is by means of hand or machine stitching using a strong and durable thread. The grommets (55) are affixed by hand or machine to the fabric tabs (53) and (54). FIGS. 6, 7, 8, 9 and 10 illustrate the method of sequentially wrapping the harness strappings about the ball, in order to attain the overlap of wrappings that is further illustrated in FIGS. 11 and 13. In FIG. 6, a strap (51) which has Velcro pile material upon the outward face of the strapping and which has a grommeted tab (53) on the same fabric strap face as the Velcro pile, is wrapped downward along a longitudinal circumference so that the grommeted tab is at the uppermost point of the ball. FIG. 7 illustrates the next step of wrapping, which requires the radially opposite strap (52) to be brought around the bottommost portion of the ball and then upwards along the same longitudinal circumference. The Velcro loop material on the second strap (52) is pressed to the Velcro pile on strap (51). As illustrated in FIG. 7, the second strap (52) is not brought upwards above the horizontal circumference of the ball as of yet. That second strap (52) also has a grommeted tab (55). FIG. 8 illustrates the next step of wrapping the harness, in which one of the latitudinal straps (56) is wrapped around the horizontal circumference of the ball with the Velcro pile facing outward. FIG. 9 illustrates the fourth step in wrapping the harness, in which the ascending strap (52), which was only partially wrapped in FIG. 7, is now brought upwards along the longitudinal circumference of the ball to complete the circumferential wrapping that includes both of the grommeted tabs. FIG. 10 illustrates the fifth and final step in wrapping the harness about the ball, in which the remaining latitudinal strap is brought around the horizontal circumference of the ball, said remaining strap having Velcro loop material upon the inner fabric strap face, with the Velcro loop material being used to fastened against the Velcro pile material of the prior latitudinally postitioned strap (56). FIG. 12 illustrates the appearance of the ball in perspective with the parallelogram (60) portion of the harness positioned on one face of the ball, and the grommeted tab (53) appearing at the uppermost portion of the ball. FIG. 11 illustrates, in a side sectional view, the successive wrappings applied against the face of the softball opposite from the face upon which the parallelogram is situated. The reference numerals in FIG. 11 correspond to FIG. 5. FIG. 13 illustrates, in a top sectional view, the same pattern of alternating harness wrappings about the horizontal circumference of the ball, with the point of intersection of all wrappings commencing outward from the face of the ball with the vertically descending strap being first against the face of the ball, the first horizontal strap being next positioned against the first strap, the verically ascending strap being the third strap proceeding outward from the ball at the point of intersection, and with the remaining horizontal strap being the outermost wrapping at the point of intersection. The reference numerals in FIG. 13 correspond to FIG. 5. FIG. 14 illustrates the bight (12) taken in the descending cord (3). The mode of operation of the apparatus with the harnessed ball in position can be readily understood. In the preferred embodiment, the length of descending cord is adjusted by a bight which places the harnessed ball in the proper position relative to the athlete. Keeping in mind the purpose of training for a consist swing plane, the descending cord is of sufficient overall length to establish the hand, arm, foot or leg swing plane which is desirable. Also, the harnessed ball in the preferred embodiment hangs 8 to 12 feet away from the rear of the backstop upon which the targets are fastened. The targets are fastened upon the backstop in such a fashion that the extreme left hand and the extreme right hand targets are separated by a distance which is no greater than double the distance between the harnessed ball in the rear of the backstop upon which the targets are fastened. A transverse cord is affixed to the lower grommeted tab upon the harness, with the other end of the transverse cord affixed to the backstop by knotting. Adjustment of the length of the transverse cord is made so that it dampens the oscillation of the ball after the ball is propelled towards the screen, allowing the ball to quickly return to a resting position, and enabling the athlete to repeat his motion in a rhythmic fashion, without altering his stance. It will be appreciated that the progressive steps of wrapping Velcroed strapping about the ball makes this apparatus strong, durable, safe and easy to use. The athlete is able to concentrate on his plane of swing and his stance or approach to the ball. It will also be appreciated that the placement of targets upon the backstop allows the athlete to practice placement of the ball. The functions of swing plane, together with stance or approach, are well understood as being the essential elements in any sport where the athlete must place a ball in a given location during the course of play. It will also be appreciated that the athlete will be trained in eye quickness through the use of the apparatus. It is well understood that the athlete must keep his eyes fixed on the ball in order to make contact with the ball, whether the ball is in motion or at rest prior to being struck. However, the motion of the ball towards the athlete before being struck, or away from the athlete after being struck, can disturb the focus of the athlete's eye on the ball if the athlete involuntarily moves his head, rather than allowing the eyes alone to follow the ball's actual or anticipated motion. Because this apparatus permits motion of the ball only in constant, short and limited field of travel, the athlete will be encouraged to allow his eyes to track the ball, rather than allowing his head to move in tracking the ball. Turning to FIG. 15, the preferred form of fork device made according to the invention is shown inserted in a pole (62), for use together the hook device. It will be appreciated that if the apparatus is used with an existing backstop upon a playing field, it could be unsafe for the athlete to clamber up the backstop in order to fasten the hook device to which the descending cord is attached. Also, it will appreciated that the athlete may wish to move the ball so at to increase or decrease the distance between the resting ball and the targets. In the embodiment illustrated in FIG. 15, the preferred embodiment of fork device made according to the invention is made of cold rolled metal and is formed with a bend (61) which is seated against the face of the supporting pole (62). A gate (63) and prongs (64) are furnished so as to accommodate the shape of the hook device. The distance between the prong base (65) and the gate (66) is equal to the span between the headpiece (9) and the tailpiece (10) of the companion hook device. FIG. 16 illustrates the hook device being lowered onto the fork device, after the cord has been routed through the gate and into the center (67) of the fork device. In FIG. 17, the hook has been seated on the fork device with the headpiece radius (69) seated against the prong base (65), the tailpiece (10) resting upon the gate base (66), the headpiece angle (70) descending between the prongs (64), and the tailpiece extremity (68) extending above the plane of the fork. FIG. 18 shows the combination of fork and hook pointed upward, with the fork and hook in the identical orientation as in FIG. 17. However, the descending cord is now hanging downward, preparatory to raising the fork and hook upwards through the chain mesh above. FIG. 19 shows the fork and hook assembly passing through a section of chain-link mesh (2), with the tailpiece not yet being above the chain-link mesh. The mode of operation of the fork and hook device can be readily understood, as illustrated in FIG. 20 and FIG. 21. The tailpiece (10) is raised through and above the chain-link mesh, and then the fork is lowered as in FIG. 20 so that the tailpiece extremity (68) catches on the chain-link mesh (2), and the tailpiece of the hook is carried away from the fork as the fork is further lowered through the chain-link mesh. The headpiece radius (69) remains seated upon the prong base (65). As the fork moves downward towards the chain-link mesh, the headpiece angle (70) will be brought to bear against the chain-link mesh, and the headpiece radius (69) will seat upon the mesh. FIG. 21 illustrates the fork being lowered completely away from the chain-link mesh, with the headpiece radius (69) now seated against the chain-link mesh (2). It can be readily understood that the tailpiece of the hook device will always point in the direction of the back screen, and that the headpiece radius (69) will remain seated on the chain-link mesh, and keep the headpiece angle (70) pointed toward the ground, the hook thereby remaining firmly in place. It will, of course, be understood that various details of construction, combination and assembly may be modified throughout a range of equivalence, and it is, therefore, not the purpose to limited the scope of the present invention otherwise than as necessitated by the scope of the appended claims. | Summary: A backstop supports a ball which is encircled by non-fabric straps whose ends overlap and are secured by pile and loop fasteners. A vertical elastic tether is fastened at one end to one of the straps and at its other end to a metal hook which detachably engages a horizontal overhead chain-link mesh portion of the backstop. The backstop also has vertical chain-link mesh portions which support targets. A transverse inelastic cord is secured at one end to a ball strap and at its other end to one of the vertical mesh portions near a target. A pole with a fork on its end is used to engage and disengage the metal hook relative to the overhead chain-link portion. | 5,166 | 157 | big_patent | en |
Summarize: FIELD OF THE INVENTION [0001] The present invention relates to a method and holder for supporting a vacuum hose, for example a vacuum hose of the type used in a central vacuuming system and the like. BACKGROUND [0002] In a central vacuuming system, tubing is typically installed within a building structure, connected between wall outlets dispersed throughout the building and a central fixed vacuum collector. A vacuum head is provided which is supported on a rigid section of pipe in communication therewith which acts as an upright handle for pushing the vacuum head along the floor. An elongate flexible hose is provided for connection between the rigid section of pipe of the vacuum head and one of the wall outlets of the tubing. [0003] The elongate flexible hose, in one example, can be 35 feet in length or other common lengths which may be longer, resulting in a hose which often tangles and is awkward for carrying and storage. Various reels and wall mounted units are known, but none are suitably portable for being carried with the vacuum head in a central vacuuming system. [0004] U.S. Pat. No. 5,331,714 to Essex et al and U.S. Design Pat. No. 383,882 to Medema disclose examples of carriers for a vacuum hose. In each instance, brackets are disclosed for coupling to the body of an upright vacuum and accordingly are only intended to support a small coil of hose supported thereon. The hose cannot readily be separated from the vacuum for storage nor are the brackets adaptable to the rigid section of pipe of a typical central vacuuming system. SUMMARY [0005] According to one aspect of the present invention there is provided a hose holder for a vacuum system including a vacuum head supported on an elongate rigid pipe for connection, using an elongate vacuum hose, to a central vacuum tubing system installed in a building, the hose holder comprising: a frame for supporting the vacuum hose wrapped thereabout; and clamp means for supporting the frame on the rigid pipe. [0008] According to a second aspect of the present invention there is provided a hose holder for a vacuum system including a vacuum head supported on an elongate rigid pipe for connection, using an elongate vacuum hose, to a central vacuum tubing system installed in a building, the hose holder comprising: first and second cradles for supporting the vacuum hose wrapped thereabout; and first and second clamp members for supporting the respective cradles on the rigid pipe. [0011] According to a further aspect of the present invention there is provided a method of storing an elongate vacuum hose in a vacuum system including a vacuum head supported on an elongate rigid pipe for connection, using the vacuum hose, to a central vacuum tubing system installed in a building, the method comprising: providing a frame for supporting the vacuum hose thereon; wrapping the hose about the frame; and supporting the frame on the rigid pipe. [0015] The use of clamps for connection at spaced positions along a rigid pipe as in a central vacuuming system, readily permits the flexible hose of such a vacuuming system to be supported for movement with the vacuum head. The clips can readily be separated from the pipe for separately storing the hose. [0016] The clamp means are preferably releasable for selective separation and reattachment on the rigid pipe. The clamp means may comprise at least one spring clip including a pair of opposed jaws which are biased towards one another to permit release and reattachment thereof. [0017] The frame may span first and second cradles which are at spaced positions from one another at opposing ends of the frame. In this instance, the frame preferably comprises a pair of elongate rails supported spaced apart from one another by crossbars spanning therebetween, each cradle being defined by a crossbar and a pair of end portions of the rail projecting beyond the respective crossbar. [0018] There may be provided a hose clamp suitably arranged for securement of a free end of the vacuum hose therein and which is supported on the frame. [0019] The holder may be provided in combination with a rigid pipe of a vacuum upon which the clamp means are supported. [0020] Preferably, the first and second cradles and the first and second clamp members are all integrally moulded with one another. [0021] The method may include wrapping the hose about the frame in a coil such that a pipe connecting end of the hose is supported at an inner side of the coil and a wall connecting end of the hose is supported on an outer side of the coil. [0022] A wall connecting end of the hose is preferably supported in the hose clamp on the frame. [0023] A pipe connecting end of the hose may be secured in the clamp means when the frame is removed from the rigid pipe for storage. [0024] The frame may be suitably sized to support a vacuum hose greater than 25 feet in length fully within the area defined by each cradle, and preferably would support longer hoses in the order of 35 feet or more. BRIEF DESCRIPTION OF THE DRAWINGS [0025] In the accompanying drawings, which illustrate an exemplary embodiment of the present invention: [0026] FIG. 1 is a perspective view of the hose holder supported on the rigid pipe of the vacuum head with the vacuum hose wrapped thereabout. [0027] FIG. 2 is a perspective view of the hose holder with the vacuum hose removed therefrom. [0028] FIG. 3 is a top plan view of the hose holder. [0029] FIG. 4 is a front elevational view of the hose holder. DETAILED DESCRIPTION [0030] Referring to the accompanying drawings, there is illustrated a vacuum hose holder generally indicated by reference numeral 10. The vacuum hose holder 10 is particularly suited for carrying the vacuum hose 12 of a typical central vacuuming system. [0031] The central vacuuming system includes tubing installed in a building for communication between a plurality of wall outlets and a central vacuum collector. A push type vacuum head 14 includes a motor for rotating bristles therein and an elongate rigid pipe 16 forming a handle for pushing the vacuum head 14. The rigid pipe 16 communicates between the vacuum head and a pipe connecting end 18 of the hose 12. The hose comprises an elongate flexible tubing extending in a longitudinal direction between the pipe connecting end and a wall connecting end 19 for being received in one of the wall outlets of the central vacuuming system. [0032] The hose holder 10 generally includes a frame comprising two rails 20 which are supported parallel and spaced apart from one another. A pair of cross bars 22 extend between the rails adjacent to but spaced inwardly from the ends of the rails. The crossbars 22 are oriented perpendicularly to the rails and are spaced inwardly sufficiently from the ends of the rails to define first and second cradles 24 and 26 for supporting the hose thereon. Each cradle is generally U-shaped defined by end portions of the respective rails projecting beyond the crossbar and the respective one of the cross bars 22 spanning therebetween such that open ends of the U-shaped cradles extend outwardly in a longitudinal direction of the frame opposite from one another. A central cross bar 28 spans between the rails, perpendicularly thereto at a central location along the rails. The cross bars and rails lie in a generally common plane. [0033] First and second clamp members 30 and 32 are supported at spaced positions along one of the rails 20. Each clamp member comprises a spring clip formed of resilient material extending partway about a circular periphery in a relaxed position. The material forming the clamp members is such that it is biased towards the relaxed position when the jaws 34 of each clamp member are pulled apart from one another. The pair of jaws 34 forming each clamp member confront one another and each extend through an arc of approximately 120 degrees. [0034] The jaws 34 of the clamp members can be flexed apart from one another to receive the circular cross section of the rigid pipe through the gap between the jaws to be subsequently retained within the clamp members when the jaws return to the relaxed position. The resilient material forming the clamp members ensures that the clamp members remain releasable for selective separation and re-attachment as desired. [0035] The first and second clamp members 30 and 32 are aligned with respective ones of the first and second cradles 24 and 26 which they support on the rigid pipe. The jaws 34 of each clamp member are curved about a respective axis which lies parallel to the rails 22 and which is concentric with the jaws 34 of the other clamp member so that both clamp members can be clamped about a common round pipe extending therebetween. [0036] A hose clip 36 is supported on the centre cross bar 28 of the frame. The hose clip 36 comprises a spring clip having a pair of confronting jaws 38 identical in configuration to the jaws 34 of the clamp members. In the relaxed position, the jaws extend partway about a circular periphery of the hose clip while remaining flexible to receive the wall connecting end 19 of the pipe therein which is subsequently retained therein by gripping action of the jaws being biased inwardly towards the relaxed position similarly to the clamp members 30 and 32 described above. The jaws 38 of the hose clip are curved about an axis lying perpendicular to the plane of the frame. [0037] The rails and crossbars of the frame, as well as the clamp members 30 and 32 and the hose clip 36 are all integrally moulded with one another of a suitable plastic material which provides sufficient strength and stiffness for supporting typical central vacuuming hoses thereon while being sufficiently resilient to permit the jaws of the clamp members to be sufficiently separated for receiving the rigid pipe therein. [0038] In use, the pipe connecting end 18 of the hose is connected to the free end of the rigid pipe 16 of the vacuum head. The frame of the holder 10 is then attached to the rigid pipe by means of the clamp members 30 and 32 at spaced positions along the pipe. The hose is then wrapped in a coil about the frame between the opposed first and second cradles starting with the pipe connecting end 18 on an inner side of the coil and terminating with the wall connecting end 19 on an outer side of the coil. The wall connecting end can then be secured in the hose clip 36. [0039] Releasing the hose from the rigid pipe and the clamp members 30 and 32 from the rigid pipe permits the hose holder with the vacuum hose coiled thereon to be released and stored separately as desired. When it is desired to vacuum, the clamp members 30 and 32 and the pipe connecting end of the hose are secured to the rigid pipe, while the wall connecting end 19 is released from the hose clip 36. A desired length of hose is unwrapped from the frame and connected to the appropriate wall outlet. The frame is suitably sized for supporting typical 35 foot length hoses wrapped thereon while being fully contained within the area defined by the respective cradles between the crossbar 22 and the respective end portions of the rails. [0040] While one embodiment of the present invention has been described in the foregoing, it is to be understood that other embodiments are possible within the scope of the invention. The invention is to be considered limited solely by the scope of the appended claims. | Summary: A hose holder includes a frame comprising first and second opposed cradles for supporting a vacuum hose wrapped thereabout. The frame is suitably sized for supporting long hoses, for example in the order of 35 feet or more, of the type typically associated with a central vacuuming system. The frame includes first and second clamp members for securement at spaced positions along the rigid pipe typically attached to the vacuum head in the central vacuuming system. | 2,782 | 99 | big_patent | en |
Summarize: FIELD OF THE INVENTION The present invention relates generally to refrigeration systems for refrigeration devices such as frozen beverage machines, ice cream machines, ice makers and similar devices, and more particularly, to an electronic control that regulates the flow rate of refrigerant being supplied to the mixing chamber of a frozen carbonated beverage machine. BACKGROUND OF THE INVENTION Frozen carbonated beverage machines are well known in the art. Such equipment is designed to produce a partially frozen beverage from the partial freezing of a combination of carbonated water and syrup. This product is made by continuously scraping the partially frozen beverage from the interior surface of a refrigerated mixing chamber. Frozen carbonated beverage machines were originally operated electro-mechanically. However, it was found that electro-mechanical systems produced product of inconsistent quality. In particular, electro-mechanical systems would react too slowly or would overcompensate for changes in ambient conditions, rate of beverage dispensing and the like. Such slow reaction or overcompensation had a negative effect on the viscosity of the beverage, causing a drink to become either unacceptably loose or firm and thus of inconsistent quality. Electronic controls have been recently used to provide for an improved ability to maintain the beverage viscosity within a predetermined desired range. As is known in the art, pulse modulated expansion valves (PMVs) or process control valves can provide for precise refrigeration control. These valves are typically controlled by a computer software algorithm so that the flow rate of refrigerant being supplied to the mixing chamber can be changed quickly in response to sudden changes in the refrigeration requirements in the system. When a PMV or control valve is used in a refrigeration cycle, varying control schemes are used to increase or decrease flow of the refrigerant through the system. Typically, the inlet and/or outlet temperatures to the evaporator (or other components of the refrigeration cycle) are measured and then used to adjust the flow rate of the refrigerant in response to changing conditions. Such a system is proposed in U.S. Pat. No. 5,095,710 issued to Black et al. The pressure in the refrigeration cycle may also be used with temperature in the algorithm to modify the flow rate of the refrigerant. A drawback of this approach, however, is that it requires the use of several temperature and/or pressure sensors and thus makes for a somewhat complex system for controlling the refrigeration cycle. The present invention is directed to a simple control scheme for regulating the flow of refrigerant to a refrigeration device that does not use temperature or pressure sensors. The present invention is applicable in any type of frozen beverage machine, ice cream machines, ice makers, and similar devices. SUMMARY OF THE INVENTION In one aspect of the present invention, an apparatus for controlling the flow of refrigerant to a refrigeration device is provided. The apparatus includes a mixing chamber that produces a product, an evaporator secured to the exterior of the mixing chamber that freezes the product being produced within the mixing chamber, a compressor connected to the evaporator, and a valve coupled to the evaporator that regulates the flow of refrigerant through the evaporator. The apparatus also includes a scraper within the mixing chamber that scrapes the frozen product from the interior surface of the mixing chamber, and a beater motor that operates the scraper. The apparatus further includes a control circuit connected to the beater motor, the compressor and the valve that controls the refrigeration cycle by controlling the activation of the compressor and the position of the valve. The valve may be a pulse modulated expansion valve, control valve or any other valve well suited to control refrigerant flow. If a pulse modulated expansion valve is used, it is adjusted by controlling the frequency at which the valve opens and closes. The control circuit receives signals from the beater motor indicative of the viscosity of the product being produced within the mixing chamber and transmits control signals to adjust the position of the valve in response to the signals received from the beater motor so as to control the rate of refrigerant flowing through the evaporator. The control circuit includes a voltage detection circuit and an EPROM connected to a microprocessor. The EPROM is programmed with an algorithm for determining when to turn the compressor on and off and how much the valve should be opened, if at all, in controlling the rate of refrigerant through the evaporator. The voltage detection circuit includes a current-to-voltage converter connected to the signal received from the beater motor, an isolation device connected to the current-to-voltage converter, and a zero crossing detector connected to the isolation device and the microprocessor. The voltage detection circuit further includes a second isolation device connected to a line voltage supplied to the beater motor and a second zero crossing detector connected to the second isolation device and the microprocessor. In another aspect of the present invention, a method for controlling the flow of refrigerant to a refrigeration device is provided. In the preferred method, the control circuit instructs the compressor to turn off and the; valve to close thereby stopping the flow of refrigerant through the evaporator when the viscosity of the product reaches a predetermined high value. The control circuit also instructs the compressor to turn on the compressor and open the valve thereby initiating the flow of refrigerant through the evaporator when the viscosity of the product reaches a predetermined low value. The control circuit further instructs the valve to remain partially open when the viscosity is between the predetermined low value and the predetermined high value so as to control the rate at which refrigerant flows through the evaporator and thereby control the rate at which the product in the mixing chamber freezes. The percent that the valve is open is directly proportional to the viscosity of the product being produced in the mixing chamber and is determined by the microprocessor utilizing the algorithm stored in the EPROM. BRIEF DESCRIPTION OF THE DRAWINGS Other aspects and advantages of the present invention will become apparent upon reading the following detailed description and upon reference to the drawings which: FIG. 1 is a schematic diagram of the basic components of a frozen carbonated beverage machine according to the present invention. FIG. 2 is a schematic diagram of the refrigeration system of a frozen carbonated beverage machine according to the present invention. FIG. 3 is a schematic diagram of the circuit which controls the refrigeration cycle according to the present invention. FIG. 4 is a flow diagram showing the steps performed by an algorithm according to the present invention which controls the flow of refrigerant through the refrigeration system. FIG. 5 is a graph showing the optimum freeze curve utilized by the algorithm according to the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Turning now to the drawings and referring initially to FIG. 1, a system diagram of a frozen carbonated beverage machine according to the present invention is shown generally by reference numeral 10. The system 10 includes an ingredient supply source 12, an electronic control circuit 14, a process flow block 16, a pair of mixing chambers 18 and 20 and a refrigeration system 22. The supply source 12 supplies carbon dioxide, water and syrup to the process flow block 16 via supply lines 24. The process flow block 16 mixes the ingredients and in turn delivers them to a pair of expansion chambers 26 and 28 via lines 30 and 32, respectively. The expansion chambers 26 and 28 are of the type well known in the art and therefore will not be further described herein. The ingredients are fed into the mixing chambers 18 and 20 from the expansion chambers 26 and 28 via lines 34 and 36, respectively. The mixing chambers 18 and 20 are each provided with scraper blades 38 and 40 which scrape the partially frozen product off of the interior circumferential surface of the mixing chambers 18 and 20, as the product begins to freeze. The scraper blades 38 and 40 are attached to shafts 42 and 44 which are in turn rotated by beater motors 46 and 48, respectively. The beater motors 46 and 48 provide output signals which are in turn communicated to the control circuit 14 via communication lines 50 and 52, respectively. The output signals contain the readings of the current being drawn by the beater motors 46 and 48. This information is useful in determining the output torque of the beater motors 46 and 48 and thus viscosity of the product in the mixing chambers 18 and 20, as is further explained below. The refrigeration system 22 includes a compressor 54, an accumulator 56, a condensor 58, and a pair of evaporators 60 and 62, as shown in FIG. 2. The accumulator 56 is connected by a line 64 to pulse modulated expansion valves 66 and 68, which control delivery of the refrigerant to the evaporators 60 and 62, respectively. In an alternate design, the pulse modulated expansion valves 66 and 68 may be substituted by control valves. The evaporators 60 and 62 are each defined by a sleeve having an advancing helical groove formed along its inner circumferential surface. The evaporators 60 and 62 are preferably shrink fitted onto the outer surfaces of mixing chambers 18 and 20, respectively. The helical grooves define flow paths 70 and 72 which encircle the mixing chambers 18 and 20, respectively. The refrigerant flows through the flow paths 70 and 72 so as to come into direct contact with the walls of the mixing chambers 18 and 20, respectively. This provides for an efficient heat transfer. The flow paths 70 and 72 empty into a common outlet 74 which, in turn, is connected to the accumulator 56 by line 76. The accumulator 56 delivers the refrigerant to the compressor 54 via line 77. The compressor 54 delivers the refrigerant to the condensor 58 via line 78. The condensor 58, in turn, delivers the refrigerant to the accumulator 56 via line 80. The operation of the various components of the refrigeration system is well known in the art, and therefore will not be further discussed herein. The control system for regulating the flow of refrigerant through the refrigeration system will now be discussed as the present invention is directed to this aspect of the frozen carbonated beverage machine. In particular, the present invention is concerned with the flow of the refrigerant through the evaporators 60 and 62. The regulation of this flow is controlled directly by the expansion valves 66 and 68. The expansion valves 66 and 68 are in turn controlled by the control circuit 14, based upon the viscosity of the beverage in the mixing chambers 18 and 20 as will be further explained below. The expansion valves 66 and 68 are connected to the control circuit 14 via communication lines 82 and 84, as shown in FIG. 1. The control circuit 14 includes a microprocessor 86, an EPROM (erasable programmable only memory) 88, and a voltage detection circuit 90, as shown in FIG. 3. The microprocessor 86 is preferably Motorola part no. 68 HC811. The EPROM is preferably SGS Thomson part no. M27/C512. The microprocessor 86 utilizes an algorithm stored in the EPROM 88 to control the refrigeration cycle for each of the mixing chambers 18 and 20, based upon information provided from the voltage detection circuit 90. The EPROM 88 communicates with the microprocessor 86 via communication line 92. The voltage detection circuit 90 receives various inputs including the output signals from the beater motors 46 and 48 via communication lines 50 and 52 and the line voltage 94 supplied to the beater motors, as shown in FIG. 3. A pair of current-to-voltage converters 96 and 98 convert the currents drawn by the beater motors 46 and 48, respectively, into sinusoidal voltage values having a maximum value of 1 volt. These current-to-voltage converters 96 and 98 are resistors of a type well known in the art. The signals associated with these sinusoidal voltage values are communicated to a pair of isolation devices 100 and 102 via communication lines 104 and 106. The isolation devices 100 and 102 are transformers of the type well known in the art. They function to step up the output voltage from a maximum of 1 volt to a maximum of 5 volts. Thus, the output of the isolation devices 100 and 102 is a sinusoidal voltage having a maximum value of 5 volts. The voltage outputs from the isolation devices 100 and 102 are in turn communicated via communication lines 108 and 110 to a pair of zero crossing detectors 112 and 114, respectively. The zero crossing detectors 112 and 114 are the type well known in the art. The zero crossing detectors 112 and 114 convert the sinusoidal voltage values received from the isolation devices 100 and 102 into logic values of "0" or "1" which can be understood and processed by the microprocessor 86. The output is one logic value, e.g., 0 if the voltage value is non-negative and another logic value, e.g., 1 if the voltage value is negative. These logic values are communicated to the microprocessor via communication lines 116 and 118. The line voltage 94 is communicated to an isolation device 120 which is identical to isolation devices 100 and 102. It reduces the line voltage from 240 volts to a maximum of 5 volts. The reduced sinusoidal line voltage is then communicated to a zero crossing detector 122 via communication line 124. The zero crossing detector 122 in turn converts the voltage signal received from the isolation device 120 into a logic value and in turn communicates this logic value to the microprocessor 86 via communication line 126. The microprocessor 86, employing a timer (not shown), determines the time difference between the instant that the zero crossing detector 112 begins detecting a non-negative voltage signal emanating from the beater motor 46 load circuit and the instant that the zero crossing detector 120 begins detecting a non-negative voltage signal from the line voltage 94. This time difference or phase difference between the voltage signals is known as the "beater count " and relates directly to the power consumption of the beater motor 46. Similarly, the microprocessor 86 determines the time difference between the instant that the zero crossing detector 114 begins detecting a non-negative voltage and the instant that the zero crossing circuit 120 begins detecting a non-negative voltage signal from the line voltage. This latter "beater count" relates directly to the power consumption of the beater motor 48. It is well known in the art that the power consumption of the beater motors 46 and 48 relate directly to the torque of the beater motors and that the torque of the beater motors relates directly to the viscosity of the beverage product in the mixing chambers 18 and 20. The present invention uses the power consumption/torque information from the beater motors 46 and 48 to control the refrigeration cycles for the mixing chambers 18 and 20. A flow diagram of the process steps performed by the microprocessor 86 for controlling these refrigeration cycles is shown in FIG. 4. For simplicity sake, this discussion will be directed to the flow sequence as it pertains to one of the mixing chambers, e.g., mixing chamber 18. As should be understood by those of ordinary skill in the art, the same routine applies to the mixing chamber 20. Furthermore, for simplicity sake the viscosity value will be used to describe the process steps. In the preferred embodiment, however, the "beater count" measurement which is indicative of the viscosity is actually used. As will be further appreciated by those of ordinary skill in the art, there is no need to convert the "beater count" measurement into a torque or viscosity value to carry out the process according to the present invention. After a start point (block 200), the viscosity of the product in the mixing chamber 18 is measured (block 210) based upon the phase difference in the current signal 50 and the voltage signal 94, as shown in FIG. 4. The measured viscosity value 210 is then compared to a predetermined low set point viscosity value. This comparison is made in the decision block 220. The predetermined low set point viscosity value is stored in the EPROM 88 and is selectable by the operator. It is the value at which the viscosity of the product being produced is at its lowest "acceptable" value, i.e., the product is as liquidy as is desired. This value is also known as the "thaw value" and may be empirically determined. If the measured viscosity 210 is not lower than the predetermined low set point value then the viscosity of the product in the mixing chamber 18 is again measured. The viscosity of the product in the mixing chamber 18 is repeatedly measured until its value is below the predetermined low set point value, at which point the compressor 54 is turned on (block 230). The compressor 54 receives its instruction to turn on (and off) from the control circuit 14 via communication line 130, as shown in FIGS. 1-2. Once the compressor 54 is turned on, the viscosity of the product in the mixing chamber 18 is continuously measured and compared to an optimum freeze curve (block 240) which is part of the algorithm stored in the EPROM 88 and utilized by the microprocessor 86. From the optimum freeze curve, the algorithm sets the open percent of the expansion valve 66 (block 250) based on the viscosity measurement and thereby controls the flow of refrigerant to the mixing chamber 18. So long as the measured viscosity value exceeds the predetermined low set point value, the expansion valve 66 remains open. If, however, the measured viscosity value should exceed a predetermined high set point value, then the compressor 54 is turned off (block 260) and the expansion valve 66 is closed (block 270). The comparison of the measured viscosity value to the predetermined high set point value is made in the decision block 280. Like the predetermined low set point value, the predetermined high set point value is stored in the EPROM 88 and is selectable by the operator. It is the value at which the viscosity of the product being produced is at its highest "acceptable" value, i.e., the product is as frozen as is desired. This value is also known as the "freeze value" and may be empirically determined. The optimum freeze algorithm employs the optimum freeze curve shown in FIG. 5 which plots the open percent of the expansion valve as a function of product viscosity. This optimum freeze curve is established through a process of empirical testing. The curve is constructed first by selecting maximum and minimum percent open values. The maximum percent open value indicated by the point F is the value that maximizes heat transfer when the product is a liquid. This value varies depending upon whether one or both mixing chambers are being supplied with refrigerant, the compressor being used, and other components being used. This value may vary, for example, between 60% and 100%. The minimum percent open value indicated by the point G is the value that maximizes heat transfer in the refrigeration cycle when the product is in a semi-frozen state. This value may also vary depending upon the same factors affecting the maximum percent open value, and is typically between 20% and 60%. As the refrigeration cycle continues and the product begins to freeze, the viscosity of the product increases and the optimum refrigerant flow is found to follow the path illustrated in FIG. 5. This path is generally linear, i.e., follows the equation y=mx+b. As will be appreciated by those skilled in the art, other curves could be used, e.g., a second order polynomial expression to fit the empirical test data. It has been found that optimum results are obtained by allowing the flow to remain constant once the minimum flow condition (i.e., where the open percentage is at a minimum) is reached to allow the semi-frozen liquid to freeze at a slow rate and mix in the chamber thoroughly. This reduces the gradient of temperature that occurs from the wall to the center of the mixing chamber. The point A is a baseline value. It is the point at which the product is completely liquid and, thus, the point at which the viscosity is at an absolute minimum. The point B is the thaw value. It is the point at which the frozen product begins to thaw. This value is a settable value and is empirically determined. The point C is the freeze value. It is the point at which the product has reached its desired consistency. It also is a settable value and is empirically determined. The point E is thee point at which the rate of refrigerant flow becomes constant. It is equal to (B+(C-B)/D) where D is 2. The point D is a settable value and has been derived empirically. As the product becomes frozen, the viscosity continues along the constant rate portion of the graph until the viscosity reaches the maximum viscosity setting or "freeze" value. At this point the compressor 54 is turned off and the expansion valve 66 (or 68) is fully closed. Those skilled in the art who have the benefit of the present disclosure will appreciate that the present invention may take many forms and embodiments. For example, other control routines could be used to control refrigerant flow using the measured viscosity of the product. It is intended that the embodiments described herein should be illustrative only, and not limiting of the present invention. Rather, it is intended that the invention cover all modifications, equivalents and alternatives falling within the spirit and scope of the invention as defined by the appended claims. | Summary: An apparatus and method for controlling the flow rate of refrigerant to a refrigeration device is provided. The system includes a mixing chamber that retains and produces the product, a compressor, and an evaporator secured to the exterior of the mixing chamber for providing freezing of the product being produced within it. The system further includes a expansion valve coupled to the evaporator that regulates the flow of the refrigerant through the evaporator. A scraper rotatably mounted within the mixing chamber is provided for scraping the frozen product from the interior surface of the mixing chamber. A beater motor rotates the scraper. An electronic control is provided for controlling the refrigeration cycle. The electronic control employs a microprocessor which is operated in accordance with an algorithm for controlling the operation of the expansion valve. The microprocessor periodically receives signals from the beater motor indicative of the viscosity of the product being produced within the mixing chamber. The electronic control transmits signals to turn the compressor on and off and adjust the position of the valve in response to the signals received from the beater motor so as to control the rate of refrigerant flowing through the evaporator. | 5,150 | 238 | big_patent | en |
Summarize: La rossa più nominata e premiata di Hollywood, il 3 dicembre, compie 55 anni. Protagonista di circa 62, Julianne Moore ha esordito sul grande schermo – come molte altre star – col film horror “I delitti del gatto nero”(1990), di John Harrison, ma da allora ha inanellato tantissimi successi come “Il mondo perduto – Jurassic Park”(1997), “Boogie Nights”(1997), “Il grande Lebowski”(1998), “Hannibal”(2001). La Moore ha collezionato 5 nomination agli Oscar, facendolo suo solo nel 2015 grazie allo straordinario ruolo da protagonista in “Still Alice”, diretto dalla coppia formata da Richard Glatzer e Wash Westmoreland. Ultimamente, abbiamo avuto modo di ammirarla nella saga di “Hunger Games”, nel ruolo del Presidente Alma Coin e nello straordinario e commovente “FreeHeld – Amore, Giustizia, Uguaglianza” diretto da Peter Sollett. La Moore (il cui vero nome è Julie Anne Smith) è nata il 3 dicembre del 1960 a Fayetteville, in Carolina del Nord, ma a causa del lavorod di suo padre, un avvocato militare, è cresciuta e ha studiato tra Stati Uniti ed Europa. A 23 anni si è trasferita a New York, lavorando a teatro e mantenendosi facendo la cameriera. Grazie allo spettacolo “Serious Money”, viene notata da alcuni agenti e le viene offerto un ruolo nel serial “Così gira il mondo”(1985-1988), per il quale vincerà un Emmy Award. Da quel momento, la Moore diventerà una vera e propria macchina da guerra, dividendosi tra televisione e cinema. Dal 1986 al 1995 è stata sposata con l'attore e regista John Gould Rubin. Nel 1990 esordisce sul grande schermo con l’horror “I delitti del gatto nero”, per la regia di John Harrison, basato sulla famosa serie tv “Un salto nel buio”. Il film è composto da 3 storie dell’orrore e la Moore compare nel ruolo di Susan nella seconda storia, “Lotto 249”, dove si troverà alle prese con una mummia, intenzionata ad ucciderla. Il suo carisma, il talento e il fascino naturali la portano all’attenzione dei media e dei più grandi registi. Nel 1992 è già sul set del thriller “La mano sulla culla” di Curtis Hanson e l’anno dopo gira altri 4 film: “Body of Evidence – Il corpo del reato”, di Uli Udel, “Benny & Joon”, per la regia di Jeremiah Chechick, “Il fuggitivo” di Andrew Davis e “America oggi” di Robert Altman. Gli anni ’90 sono estremamente prolifici e ricchi di soddisfazioni. Nel 1994, la Moore gira “Vanya sulla 42esima strada” di Louis Malle, seguito dal profondo e straordinario “Safe”, di Todd Haynes e dall’esilarante commedia “Nine Months – Imprevisti d’amore”, diretto da Chris Columbus, accanto a Hugh Grant, che le da grandissima visibilità presso il grande pubblico. Nel 1997 è la volta del blockbuster “Il mondo perduto – Jurassic Park”, di Steven Spielberg, nel ruolo della Dott.ssa Sarah Harding. Grazie al ruolo di Amber Waves nel film “Boogie Nights – L’altra Hollywood”, di Paul Thomas Anderson, riceve la sua prima candidatura agli Oscar, diventando anche l’attrice feticcio del regista. Nel 1998 è Maude Lebowski ne “Il grande Lebowski” dei fratelli Coen, accanto a Jeff Bridges, John Goodman, Steve Buscemi e John Turturro, seguito da “Psycho”(1998) di Gus Van Sant, “La fortuna di Cookie”(1999) di Robert Altman, “Fine di una storia”(1999) di Neil Jordan e “Magnolia”(1999), di Paul Thomas Anderson, dove recita accanto a Tom Cruise. Nel 2001 è nei panni dell’agente sepciale dell’FBI Clarice Sterling in “Hannibal”, diretto da Ridley Scott. In seguito, gira i meno fortunati “Evolution”(2001) di Ivan Reitman e “World Traveler”(2001) per la regia di Bart Freundlich. Il 2003 è un anno ricchissimo di soddisfazioni. La Moore ottiene una doppia candidatura agli Oscar come Migliore attrice protagonista, per “Lontano dal paradiso” di Todd Haynes (che le vale, comunque, la Coppa Volpi a Venezia) e come Migliore attrice non protagonista per “The Hours”(2002) di Stephen Daldry, ma purtroppo non porta nulla a casa. Dal 2003, è sposata con il regista Bart Freundlich, conosciuto nel 1996 sul set de “I segreti del cuore”. La coppia ha avuto due figli, Caleb e Liv. Negli anni a seguire, prende parte a pellicole che hanno scarso seguito come: “The Forgotten”(2004), “I figli degli uomini”(2006), “Next”(2007) e “Savage Grace”(2009). Sempre nel 2009, Tom Ford la vuole per il ruolo di Charlotte nel meraviglioso “A Single Man”, esordio alla regia del famoso stilista. La Moore recita accanto ad un fenomenale Colin Firth, vincitore della Coppa Volpi a Venezia e candidato all’Oscar come Miglior attore protagonista. Nel 2010 recita accanto ad Annette Bening nella commedia “I ragazzi stanno bene”, diretto da Lisa Cholodenko ed è sul set di “Shelter – Identità paranormali”. L’anno dopo gira la commedia “Crazy, Stupid, Love” di Glenn Ficarra e John Requa e nel 2012, grazie al ruolo di Sarah Palin nel film tv “Game Change”, porta a casa il Golden Globe, un Emmy e uno Screen Actors Guild Award. Nel 2013 è già sul set della pruriginosa commedia “Don Jon”, di Joseph Gordon-Levitt e l’anno è suggellato dalla stella sulla Hollywood Walk of Fame. Archiviata la commedia, torna all’horror con il remake “Lo sguardo di Satana – Carrie”, di Kimberly Peirce e con “Maps to the Stars”(2014), di David Cronenberg. La pellicola ha partecipato, in concorso, alla 67esima edizione del Festival di Cannes, dove la Moore ha vinto il Prix d'interprétation féminine. Sempre nel 2014, entra nel cast dell’amatissima saga di “Hunger Games”, nei panni del Presidente Alma Coin. | Summary: La rossa più talentuosa di Hollywood ha collezionato 5 nomination all'Oscar, portato a casa solo nel 2015 per "Still Alice", 62 film e, ultimamente, si sta godendo il successo di "Hunger Games: Il canto della rivolta - Parte 2", dove interpreta il ruolo del Presidente Alma Coin. Il 3 dicembre compie 55 anni e, grazie a "Freeheld - Giustizia, amore, uguaglianza", potrebbe attirare nuovamente l'attenzione dell'Academy. | 1,515 | 114 | fanpage | it |
Summarize: RELATED APPLICATIONS [0001] Not Applicable BACKGROUND OF THE INVENTION [0002] The present invention relates to swing training devices used in the field of golf. [0003] Years of exposure to the game of GOLF have lead to examination of the fundamental mechanics that must take place in a golf swing to produce a desired outcome with a high percentage of consistency. In the profession of GOLF, commonly noted documentation exists of early stage back swing mechanics relative to the proper wrist hinging position located approximately during the half way back stage and pertinent to proper swing plane golf. [0004] For example: Nick Faldo writes in Golf Today Newsletter, (http://www.golftoday.co.uk/proshop/features/how_to_plug_in_a_repeating_swing.html), “How to plug in a repeating swing.” “For the better part of 20 years, ever since the reconstruction of my swing in the mid-1980s, I have focused on this halfway-back position via what's become known as an ‘early wrist set’. It's quite simple: I look for my wrists to be fully hinged and the club ‘set’ up on a good plane by the time my left arm is at horizontal.” [0005] “From here, the swing is pretty well plugged in” (Faldo). [0006] “The beauty of working on this halfway-back position is that all the details of a technically sound swing are encapsulated within it: you have a full wrist hinge, the club is swinging up on plane, and you maintain good body angles. Completing your shoulder turn gets you to the top, whereupon unwinding the body invites the hands and arms into the perfect hitting position” (Faldo). [0007] Problems that occur while trying to duplicate the proper “halfway-back” swing position are trying to train the eyes, muscles, and brain to react to what you perceive as reproducing the proper mechanical moves. [0008] While golf teaching professionals are great for identifying swing problems verbally, most students of the game cannot carry out what is conveyed to them because they can't see the before or after in order to make reliable corrections. For example, to be told that at the top of ones swing, one did not have the club face properly positioned in relationship to ones spine and chosen target line due to the collapse of ones wrist coupled with the collapse of ones elbow during the “halfway-back” point of the back-swing is unimaginable to absorb, forbid carrying out the change next time because there's nothing to compare with. [0009] There is little replacement for repetition to establish good “muscle memory” while training in the sport of golf; however, golf swings are habit forming and once established are very difficult to correct if needed. For example: a multitude of people know who Charles Barkley is mostly for his outstanding basket ball career and others relate him to his past appearance on the TV show, “The Haney Project.” Most would agree that Hank Haney, the number 1 golf instructor in the World, had his hands full to correct the infamous hitch Charles developed over the years of repeating golf swing habits that are considered non conforming to good golf mechanics theory. [0010] The Brain; thousands of practice balls, one after another and as fast as you can empty the bucket, you find yourself frustrated because that new move is not working like it did during lesson day with the golf pro. The problem is that most students of the game try to process multiple changes all at once, why not; it worked when the instructor was present. It works to some degree because the golf instructor is constantly fixing the problems that the new move has created thus eventually, bang, it's on. An hour later when the lesson is over, multiple new myelin changes have violated your neural circuits taking up battle with old habits and it seems the only way to fix it is with more lessons, $$$. [0011] In Golf Magazine 50 th Anniversary Issue, September 2009, Dr. Robert Christina, dean emeritus of the School of Health and Human Performance at UNC-Greensboro and Golf Magazine's learning expert in residence, writes; “The Problem.” “You empty bucket after bucket on the range, but the new swing change you′re trying to ingrain just won't stick. I fact, you find yourself repeating the fault you′re trying to lose over and over.” “The Solution.” “When you practice, work on one change only, or you'll literally short-circuit your brain. Repeating a movement-like swinging a golf club-causes changes in your central nervous system that increase the efficiency of the brain circuits controlling the muscles involved.” “One of these changes is myelination, the production of a fatty tissue called myelin around your neural circuits. Each time you use a circuit, this myelin cocoon gets thicker and increases the timing and speed of the signal traveling through the circuit, making it more efficient. Here's the problem: Myelin doesn't recognize a good golf move from a bad one. This means each lousy swing you make creates myelin and just makes that bad move easier to repeat, adding to the need to practice the right things. “The key,” says Dr. Christina, “is to practice while someone qualified is watching you, or with drills or training aids that provide you with feedback to ensure that you are performing the skill correctly.” [0012] Its been observed recently that there is an enormous amount of focus on resilience training devices, a shift perhaps from the typical audio and digital training devices, which may be attributed to resistance bands having direct influence on trainable muscles with excellent efficiency. The present invention meets and exceeds Dr. Christina's' aforementioned suggestions because it uses all three: use with drills, visually, it's like having an instructor with you, and as a training aid, it provides excellent feedback through out the entire swing. [0013] An over-whelming amount of golf swing training devices are available to the public that focus on parts of the golf swing and in whole but may not be practical or encapsulate what Dr. Christina suggests. For example, a strap that ties your arms together forming a V-shape to discourage disconnect of the major muscle groups during a swing. Another training device would be an elastic strap that is attached to a body part attached to a stationary object that focuses on a particular move or muscle group exercise. Another training device would be a golf club grip formed such that when grasped it aligns your hands in the proper grip position. Yet another, digital video recording software that allows one to view what one did right or wrong during his or her swing. And yet another, and another, and another, etc..... [0014] Given the enormous amount of golf training devices on the market today, the Annual Golf Participation reports (according to National Golf Foundation (NGF)) for 2011 was 25.7 million and an estimated growth for the U.S. in 2012 at 7.4%, and that the average amateur golfer scores 100 on a regulation course for 18 holes, one can deduce that a golf swing training device that comprises multi-learning techniques while maintaining as much as possible the natural characteristics of a playing golf club transferable to the golf course is desperately needed. [0015] The uniqueness of the present invention incorporates a multitude of the aforementioned training techniques and necessities, namely visual aids, muscle memory, and confidence that, what is learned is reliable. [0016] The present invention, a golf swing training device utilizable by a golfer comprising of: an elastic band at relaxed state forming a loop with a length relative to the golfer's size and having two distal ends; the first distal end of the elastic band is connected to the playing club component grip proximal to the butt end of the grip; the second distal end of the elastic loop is attached to the component grip located longitudinally linier from the first connection point proximal to the bore end of the component grip. [0017] Connection points of the band to the golf component grip and the golfer play key role in the stability and performance of the present swing training device. The connection points also provide repeatable natural assistance to proper hand-to-grip position. By using a flat elastic resistance band, used in nearly every fitness center in the U.S., there is little noticeable friction while connection around the back neck during the swing. [0018] While swinging the device, a visual aid is displayed via the stretched elastic band loop that runs parallel to the player's arms; the player can slow or stop his/her swing at any point in the swing for instant visual relationship feedback. [0019] Furthermore, the present invention provides an elastic band loop capable of positioning it on the body in a multitude of different set ups to train while providing numerous visual and muscle memory feedback scenarios. [0020] It is these connections, around the back of neck nearest the upper spine coupled with the connection at the golf swing device, that provide outstanding performance of the present swing training device. BRIEF SUMMARY OF THE INVENTION [0021] In general, the present invention golf swing training device is a tool to improve control, power, accuracy and distance long-term while executing an unrestricted golf swing. [0022] Problems that occur while trying to execute the perfect swing are trying to train the eyes, muscles, and brain to react to what you perceive proper mechanical moves without costly inconsistent advice. [0023] The present invention uses different types of learning techniques that promote long term memory, such as the coined Mechanically Assisted Muscle Memory (MAMM) in that strategically connected elastic bands actually help set the “halfway-back” position in the golf swing producing excellent stability and consistency with little effort and instant muscle feedback. If the elastic band is loose during the swing, the feel is obvious. [0024] The present invention also uses the coined learning technique Simplified Visual Aid (SVA) in that a strategically connected elastic band is visual at any chosen point in the swing to compare the relationship with the arms, chest, and shoulders. [0025] Some advantages with the present invention consist of, low cost to make therefore low cost for the consumer, replaces instruction and cost, use to train or practice round, wide range of swing stroke, flexible to training body groups, can be used for instruction, can be placed in a golf bag next to the other clubs, durable, adjustable to fit any body type and size, not weather dependant, can be installed on any golf club, helps proper set up, aligns hands on grip, although the assembled golf component grip is intended as a unit, it can be obtained separately and installed per component grip install procedure by any qualified club-maker, assembles to any standard tubular golf grip, quick and easy to use, no belts, no slings, no set-up, and esthetically professional to name a few. Most of all, the present invention golf swing training device works. [0026] BRIEF DESCRIPTION OF THE DRAWINGS [0027] FIG. 1 of the present invention depicts prospectively a golfer in set up position with the elastic bands attached to a golf club and looped around the lower neck. [0028] FIG. 2 of the present invention depicts prospectively an exploded view of a golfer's hands, swing position “halfway-back,” and elastic band distal end locations. [0029] FIG. 3 of the present invention depicts prospectively a golfer swing “halfway-back” with the elastic band looped around the lower neck and over both shoulders. [0030] FIG. 4 of the present invention depicts prospectively a golfer swing “halfway-back” with the elastic band looped over the right shoulder and under the left arm pit. Golf club stays in “halfway-back” position with open hands in any represented band location. [0031] FIG. 5 of the present invention depicts prospectively a golfer swing “halfway-back” with the elastic band looped over the left shoulder and under the right arm pit. [0032] FIG. 6 of the present invention depicts prospectively a golfer swing ¾ follow-through stage of a golf swing. [0033] FIG. 7 of the present invention depicts prospectively a golfer in the full follow-through stage of a golf swing [0034] FIG. 8A of the present invention depicts prospectively a standard golf grip component. [0035] FIG. 8B of the present invention depicts prospectively a standard golf grip component with two longitudinal perimeter holes. [0036] FIG. 8C of the present invention depicts prospectively a standard golf grip component with a hole punch back stop inserted. [0037] FIG. 8D of the present invention depicts prospectively an assembled golf grip with an elastic band installed onto a golf club shaft, band untrimmed. [0038] FIG. 8E of the present invention depicts prospectively an assembled and installed golf grip with a double sided tape band connector and a band loop for band length adjustment. [0039] FIG. 9 of the present invention depicts prospectively a tool for assembly of an elastic band to a component grip with holes. [0040] FIG. 10 of the present invention depicts prospectively the install of an assembled golf grip with an elastic band untrimmed. DETAILED DESCRIPTION OF THE INVENTION [0041] As with FIG. 1 of the present invention a right handed golfer ( 10 ) is in set up position with an elastic band ( 3 ) composed of flat elastic resistance training exercise band attached to a golf club ( 9 ) and looped around the back lower neck nearest upper spine while resting on the right ( 1 ) and left ( 2 ) shoulders. For a right hand golfer, the intended use but not limited to, the right hand of the elastic loop ( 3 ) donned over the right shoulder ( 1 ) correlates with the right hand ( 6 ) grasp location on the golf club grip ( 8 ). The golfer extends his/her arms to take set up position thus stretching the elastic band to form parallelism view points between both right ( 4 ) and left ( 5 ) arms and the elastic band ( 3 ). [0042] The golfer is now ready to take swing, drill check, or full unrestricted swing with or without a golf ball. During the swing, the golfer ( 10 ) will diagnose swing mechanics compared to the elastic band ( 3 ) i.e. if the elastic band ( 3 ) tension decreases at critical points in the swing, then a collapse between the upper spine and the hands took place, commonly the left or right elbow. The arm muscles will feel this collapse and help diagnose where failure took place in the swing. The golfer ( 10 ) can then take intermediate drill swings to visualize the comparison of the elastic band ( 3 ) parallel to the right ( 4 ) and left ( 5 ) arms to both see and feel for the corrections of the previous swing. [0043] Flat elastic resistance bands were chosen as to provide comfort to the user, flexibility in methods of use, and flexibility in the manufacturing of the present invention. Flat elastic resistance bands are widely used in the fitness and exercise industry because of their durability, flexibility of use, and non friction surfaces. [0044] An exploded view of the present invention in use by a right hand golfer FIG. 2 shows the elastic band ( 3 ) properly positioned between the thumb and forefinger of the right hand ( 6 ) and properly positioned between the thumb and forefinger of the left hand ( 7 ) with the elastic band ( 3 ) attached to a golf club ( 9 ) and the golfers swing at “halfway-back” position or hinge point position. [0045] FIG. 3 of the present invention shows a golfer ( 10 ) in the “halfway-back” swing position with the elastic band ( 3 ) looped around the lower neck and resting on both the right and left shoulders ( 1 ) and ( 2 ) respectively. With the elastic band ( 3 ) looped around the lower neck nearest the upper spine, the present invention provides the golfer with excellent resistance feedback and unmatched golf club stability as the golf club ( 9 ) rotates on plane around the spine. [0046] FIG. 4 of the present invention shows a golfer in the “halfway-back” position or hinge position with the elastic band ( 3 ) around the lower back neck and resting on the right shoulder ( 1 ) and the elastic band ( 3 ) is positioned under the left arm ( 5 ) pit which then runs parallel with the inside left arm ( 5 ). With this elastic band ( 3 ) position, the golfer ( 10 ) can focus on the left arm ( 5 ) position through out the swing and compare his/her left arm ( 5 ) with the elastic band ( 3 ) flex, i.e., if the left elbow bends, the elastic band ( 3 ) will send resistance force loss to the left arm ( 5 ). Again, the golfer ( 10 ) can then take drill stroke swings to visualize the comparison of the elastic band ( 3 ) parallel to the right ( 4 ) and ( 5 ) left arms to both see and feel for the corrections of the previous swing. [0047] In addition, FIG. 4 displays a golfer in the “halfway-back” position or hinge position with the golfers right hand ( 6 ) and the left hand ( 7 ) wide open. The upwardly projected resistance forces of the elastic band ( 3 ) are vectored through the arms and directly toward the loop around the lower back neck/upper spine which is then translated to the distal ends connected to the golf club ( 9 ), right hand ( 6 ) between thumb and forefinger, left hand ( 7 ) between thumb and forefinger, which forms a balanced connection between the golfer and the present invention. This shows that the resistance forces are in perfect plane with the golfer ( 10 ). For example, with the present invention, a golfer ( 10 ) can swing to any intermediate check point in the swing, stop, open his/her hands and the golf club ( 9 ) will stay in position without fingers grasping. This is proof that the forces are positioned most efficiently throughout the present invention thus will provide the utmost stability and reliability. For contrast, imagine if the connection point of the elastic band ( 3 ) to the golf club ( 9 ) was at the butt end of the golf club handle, the forces would be pulling up on the handle and pushing the golf club head into the ground. The golfer ( 10 ) would then be confused by the off plane forces distracting him/her from focusing on what is needed. [0048] FIG. 5 of the present invention shows a golfer in the “halfway-back” position or hinge position with the elastic band ( 3 ) around the lower back of neck and resting on the left shoulder ( 2 ) and the elastic band ( 3 ) is positioned under the right arm ( 4 ) pit which then runs parallel with the inside right arm ( 4 ). With this elastic band ( 3 ) position, the golfer ( 10 ) can focus on the right arm ( 4 ) position through out the swing and compare his/her right arm ( 4 ) with the elastic band flex, i.e., if the right elbow bends, the elastic band ( 3 ) will send resistance force loss predominantly through the right arm ( 4 ). Again, the golfer ( 10 ) can then take intermediate stroke swings to visualize the comparison of the elastic band ( 3 ) parallel to the right arm ( 4 ) and ( 5 ) left arm to both see and feel for the corrections of the previous swing. The resistance forces are positioned most efficiently throughout the present invention with all lower neck/upper spine combinations of the elastic band ( 3 ) loop and will provide the utmost stability and reliability. [0049] FIG. 6 of the present invention shows a golfer ( 10 ) in the ¾ follow-through stage of a golf swing. Again, the elastic band ( 3 ) will remain straight and in parallel reference with both the right ( 4 ) and left ( 5 ) arms. [0050] FIG. 7 of the present invention shows a golfer ( 10 ) in the full follow-through stage of a golf swing. Again, the elastic band ( 3 ) will remain straight and in parallel reference with the right arm ( 4 ) but the left arm ( 5 ) on a right hand golfer will relax slightly as the left arm ( 5 ) elbow bends to complete the typical golf swing. A golfer can use the present invention to check the final position easily via resistance and visual elastic band ( 3 ) as the ball is already hit and the golf club ( 9 ) is at rest. [0051] FIG. 8A of the present invention depicts a standard rubber golf club grip ( 8 ) with a bore hole ( 11 ) for inserting onto a golf club shaft. [0052] FIG. 8B of the present invention depicts a standard rubber golf club grip ( 8 ) with small punched holes ( 12 ) and ( 13 ) that go only through one surface of the golf grip ( 8 ) and preferably longitudinal linier with the golf grip manufacture label facing up. Typical install of a golf grip is with the label up and in line with the golf club head leading edge of the blade. [0053] The small holes shown in FIG. 8B ( 12 ) and ( 13 ) are 3/16 inches in diameter and positioned (for a medium to large size hand) 3¾ inches from the butt end of the golf club grip ( 8 ), first hole ( 12 ), and 6¼ inches from the butt end of the golf grip ( 8 ), second hole ( 13 ). The aforementioned hole location distances are set as base and flexible to change dependant upon the hand size of the golfer ( 10 ). The hole size is also base and can change dependant upon elastic band ( 3 ) size. [0054] The small holes ( 12 ) and ( 13 ) in the golf grip ( 8 ) are designed to receive the distal ends of the elastic band ( 3 ). For assembly purpose, we will call golf grip small hole ( 12 ) the butt proximal hole and for small hole ( 13 ) the bore ( 11 ) proximal hole. [0055] FIG. 8C of the present invention depicts a standard rubber golf club grip ( 8 ) with a wooden trim piece (12″×¾″×¼″) punch back stop ( 14 ) inserted into the bore ( 11 ) end of the rubber golf grip approximately 8″ deep. The punch back stop ( 14 ) will serve as a back stop for using a 3/16″ punch to install the two small holes ( 12 ) and ( 13 ) into the rubber golf grip ( 8 ). [0056] FIG. 8D of the present invention depicts a standard rubber golf club grip ( 8 ) with an elastic band ( 3 ) installed into the rubber golf club grip ( 8 ). The two distal ends of the elastic band ( 3 ) are inserted one at a time into the small holes ( 12 ) and ( 13 ) with the first end into proximal hole ( 12 ) and extends through the bore ( 11 ) end by approximately 2″. The second distal end of the elastic band ( 3 ) is inserted into the proximal hole ( 13 ) and extends through the bore ( 11 ) end by approximately 2″. The length of the elastic band ( 3 ) loop can now be set by pulling one or both of the elastic band distal ends through the bore ( 11 ). [0057] The length of the elastic band ( 3 ) loop is adjusted per the golfers' outstretched arm and fingers held horizontal to the ground and measured from the middle of either thumb to the side of either neck at shoulder height. For example, if you choose the right arm ( 4 ), measure from the right side of the neck along the outstretched right arm horizontal to the ground to the middle of the outstretched right thumb. This measurement will correlate with distance between the extended, but not stretched, elastic band ( 3 ) loop and the golf club grip ( 8 ) at the point between the two small holes ( 12 ) and ( 13 ). [0058] By using this measurement, the width of the golfers' neck is the elastic stretch incorporated into the elastic band ( 3 ) when the golfer ( 10 ) takes address with the golf club ( 9 ). Once the elastic band ( 3 ) length is determined, the excess elastic band ( 3 ) extending past the bore ( 11 ) can be trimmed. The elastic band ( 3 ) distal ends extending out of the golf club grip ( 8 ) bore ( 11 ) should be trimmed back by ½″ so that the bore end of the golf grip ( 8 ) will cover the elastic band ( 3 ) distal ends. See FIG. 8E. [0059] FIG. 8E of the present invention depicts a standard rubber golf club grip ( 8 ) with an assembled elastic band ( 3 ). Double sided tape ( 15 ) used in the golf industry to install golf grips can be used to secure the elastic band ( 3 ) distal ends. FIG. 8E also shows the elastic band ( 3 ) looped around the golf club grip ( 8 ) at small hole position ( 13 ) as an adjustment for the elastic band ( 3 ) length if desired. [0060] Several combinations of the elastic band ( 3 ) looped around the golf club grip ( 8 ) will produce numerous different effects. For instance, if a golfer ( 10 ) wanted to practice the feel of the golf club ( 9 ) head turning over or closing at impact, he/she could wrap the elastic band as shown in FIG. 8E around the golf club grip ( 8 ). The elastic band ( 3 ) will produce a turning tension in the right hand ( 6 ) giving the golfer ( 10 ) a muscle memory feedback motion of rotating the golf club head closed at impact. [0061] FIG. 9 of the present invention shows the elastic band ( 3 ) install tool ( 16 ). The elastic band ( 3 ) install tool ( 16 ) is used to install the elastic band ( 3 ) into the small holes FIG. 8B ( 12 ) and ( 13 ) on the golf club grip with holes. First, thread one distal end of the elastic band ( 3 ) through the tool hole ( 17 ) and push it through the small hole ( 12 ) then through the bore ( 11 ) end of the golf club grip. Second, thread the remaining distal end of the elastic band ( 3 ) through the tool hole ( 17 ) and repeat the previous step. To help with installing the elastic band ( 3 ) through the small holes ( 12 ) and ( 13 ), one can use a small amount of soapy water. Clean thoroughly afterwards with a cloth. [0062] FIG. 10 of the present invention shows an exploded view a golf club makers hands ( 18 ) and ( 19 ) right and left respectively installing an assembled golf grip FIG. 8D onto a golf club shaft ( 9 ) butt end. [0063] First, prepare the golf club shaft as would a golf club maker by installing double sided golf club grip tape onto the butt end of the golf shaft. Apply a small amount of solvent into the golf club grip ( 8 ) through the bore ( 11 ) end and onto the prepared golf club shaft over the grip tape. Hold the distal ends of the elastic band ( 3 ) in the right hand ( 18 ). Position the elastic band ( 3 ) ends upward and above the golf club shaft butt end with the right hand ( 18 ). Hold the assembled golf grip ( 8 ) FIG. 8D with the left hand ( 19 ) and guide the bore ( 11 ) end of the golf club grip ( 8 ) onto the butt end of the golf shaft while holding the golf club grip ( 8 ) at approximately 20 degrees of horizontal. Pull slightly with the right hand ( 18 ) the elastic band ( 3 ) ends at the same time pushing with the left hand ( 19 ) the golf club grip ( 8 ) onto the golf club shaft while keeping the elastic band ( 3 ) on top relative the golf club shaft. Install the golf club grip ( 8 ) all the way on until the butt end of the golf grip ( 8 ) stops at the butt end of the golf club ( 9 ) shaft. Align the golf club grip ( 8 ) as needed to ensure that the elastic band ( 3 ) is aligned up with the golf club head blade leading edge, as per typical club making process of installing golf component grips. Clean thoroughly with a cloth the golf club grip ( 8 ) and elastic band ( 3 ) of excess solvent. Lean the present invention against a wall or back board with the grip end up in room temperature and let cure for approximately 8 hours before use. [0064] The present invention sets itself apart from other golf swing training devices because it addresses with simplicity the most common mistakes made in the complexity of a golf swing. “Chicken Winging” is where the left elbow collapses during the down swing follow-through leaving the club face wide open for slices, chunks, topping the ball, etc. It's visible at post ball impact where the left arm disconnects from the left side rib cage area with the left elbow pointing down the target line when it should be rotating around the body turn. The present invention will capture this visually and mechanically. Visually, the elastic band is no longer parallel with the left arm forming a gap between the two. Mechanically, the elastic band will send feedback through the left arm in the form of resistance force loss. [0065] The dreadful “flying right elbow” is where the right elbow disconnects from the right side rib cage area pointing upward and back during the backswing. Again, this causes major failure to produce good results in the sport. The present invention addresses this by visual and mechanical aid. Visually, during a “flying right elbow” the elastic band will no longer run parallel to the right arm forming a V-shape but rather form a rectangle leaving a noticeable gap between the elastic band and the right arm. Mechanically, again, resistance force will be lost during the collapse of the right elbow sending feedback through the right arm. [0066] The present invention incorporates strategically placed connection points; the golfer is joined with the golf club in the most efficient use of forces and natural feeling of proper position with out restrictive devises. These balanced forces coordinate and guide the golfer unrestricted through the desired swing plane from the beginning of the swing set up to the end of the full swing follow through. The connection is evident when a golfer uses the present invention for drill swing check points by stopping the swing and holding the position with his/her hands wide open and the golf club will stay in that position. [0067] Setting the “halfway-back” position is one of the most import parts of the golf swing because it sets the stage for the rest of the golf swing outcome. The present invention was originally design for this purpose and works well. As Nick Faldo pointed out “The beauty of working on this halfway-back position is that all the details of a technically sound swing are encapsulated within it: you have a full wrist hinge, the club is swinging up on plane, and you maintain good body angles. Completing your shoulder turn gets you to the top, whereupon unwinding the body invites the hands and arms into the perfect hitting position” (Faldo). [0068] The present invention has no over-whelming fixtures that get in the way so the golfer can focus on the muscle feedback and visual aids that can be transferred to the golf course under pressure situations. It produces the most natural realistic feeling of a golf club because the contact points are kept intact to resemble as much as possible a typical golf club. [0069] The present invention re-enforces the correct changes, all desired changes through out the entire swing as apposed to most devices that focus on one or two aspects. It allows you to repeat simultaneously good memory habits subconsciously. Changes are easily noticed to ensure what is practiced is reliable. The golfer takes ownership of his/her swing with confidence apposed to someone watching and providing feedback that may not be useful or even comprehendible. [0070] Self diagnosing good swing habits from bad ones is one of the most difficult challenges in golf. Who hasn't walked away from a bad shot and said “what am I do wrong?” Most golfers don't have the knowledge to differentiate good from bad swing mechanics. The present invention golf swing training device makes it simple; during the swing, visually ensure that the arms parallel the elastic band while keeping resistance between the connection points. If resistance is lost at any point in the swing, note the location and run drills on that point, most likely a collapse in the elbows. [0071] The doctrine herein the present invention reflect examples, drawings, advice, claims and “best mode contemplated” to facilitate comprehension by those possessing skills in the art of golf. Embodiments of the present invention are subject to change within the scope of the claims. | Summary: A golf swing training device teaches proper golf swing. The present invention comprises a golf club with attached elastic band loop. An aspect of the present invention is the elastic band/golf club connection located at the mid longitudinal section of the golf club grip. In use, the player loops the elastic band over his/her head. The elastic band left side rests on the left shoulder; elastic band right side rests on the right shoulder. The player grasps the present invention by placing the right and left hands on the golf club with forefingers and thumbs between the elastic band distal ends. This teaches proper alignment of the hands. The player takes set-up position with out-stretched arms creating resistance forces through the arms provided by the elastic band. A collapse in either elbow will result in resistance loss and noticeable elastic band sag. Re-enforce good habits through drills, intermediate, or, unrestricted full-swing ball contact at the practice range. The present invention incorporates adjustability in resistance and dimensions to accommodate golfers of different body types and sizes. | 7,948 | 229 | big_patent | en |
Summarize: North Korean dictator Kim Jong-un visited a cat fish farm as computer engineers struggled to restart the hermit state's internet connection. The nation's 1,000 internet connected computers suffered a service outage for ten hours during a suspected cyber attack. While the computers were offline, Kim visited the Pyongyang Catfish farm where he provided 'field guidance' to managers at the plant. Scroll down for video. Kim Jong-un, second from left, inspects a large tank containing catfish at the fish farm in Pyongyang. Kim Jong-un, centre, looked disinterested as he wondered through the fish farm with hands in his pockets. The North Korean leader, second from left. issued orders to his minions who scribbled furiously on a notepad. Wearing his traditional heavy woolen coat, Kim was surrounded by a group of senior officials, unusually, none of whom were wearing military uniforms, as he looked into a tank containing cat fish. The United States refused to comment whether they were responsible for the ten-hour shut down. The hermit state has been accused of being responsible for last month's highly embarrassing attack on Sony Pictures. South Korean officials said the Korean Central News Agency and the Rodong Sinmun newspaper, which are both used to disseminate propaganda. The outage was probably more inconvenient to foreigners, who can access the Internet through 3G networks, than to North Korean residents, most of whom have never gone online. Outside the plant, Kim, pictured centre, gave officials the benefit of his experience following the inspection. The Korean National News Agency website, pictured, was off-line for ten hours earlier today. The top news item on the website's return was Kim Jong-un's visit to the Pyongyang Catfish plant. There are only about 1,000 Internet Protocol addresses in North Korea for a population of 25 million, South Korean analysts say. The privileged are also allowed to view a self-contained domestic Intranet that carries state media propaganda and a limited amount of information pulled and censored from the real Internet. North Korea did not immediately release a response to the shutdown. But, a communique on the official North Korean news agency website, which had been off line for ten hours, condemned the United States, comparing it to the Roman Empire, which 'was thrown into a dumping ground of history as it collapsed while seeking prosperity through aggression and war'. Internet connections are incredibly rare within North Korea, although more than one million people are believed to have access to a mobile phone. Although the North Korean phone network does not allow subscribers to call abroad or receive foreign phone calls | Summary: North Korean dictator Kim Jong-un inspected a cat fish farm in Pyongyang. Local media claimed the leader gave staff at the facility instructions. The hermit state is believed to only have 1,000 computers with web access. North Korea's internet service was knocked off-line for almost ten hours. The U.S. State Department refused to comment on if it was responsible. Kim said the U.S. would be 'thrown into the dumping ground of history' | 584 | 107 | cnn_dailymail | en |
Summarize: BACKGROUND AND SUMMARY OF THE INVENTION [0001] The present invention relates to insulated ice chests or coolers and, more particularly, to an insulated ice chest having beverage and other accessory holders detachably secured thereto. [0002] Insulated ice chests and coolers (hereinafter generically referred to as “coolers”) are regularly used to transport food and beverages on recreational outings such as to the beach, to sporting events and on picnics. Insulated coolers are used not only to transport food and beverages to remote locations, but also to maintain the food and beverages at a reduced temperature, often with the aid of ice cubes or ice packs. [0003] In many environments it is advantageous to provide receptacles in the cooler structure so that as the beverages are consumed over time, they need not be placed on the ground where they are more likely to be knocked over or become soiled. Thus, many insulated coolers have been provided with beverage receptacles. Typically, the beverage receptacles are included in the cooler lid or as permanent receptacles on a side or front face of the cooler. A problem with the providing beverage receptacles in the lid, however, is that the beverages must be removed each time the interior of the cooler is accessed. A disadvantage of incorporating beverage receptacles on a front or side wall of the cooler is that such receptacles may not always be required, but add to the dimensions of the unit whether or not they are used. [0004] There are other articles which may from time to time be used by the consumer in conjunction with a cooler. For example, coolers are often taken along when fishing. Therefore, it would be advantageous to associate a fishing rod receptacle with the cooler to hold the fishing rod while the fisherman waits for the fish to bite, or takes a break for a snack. Insulated coolers are also taken to the beach where the customer may wish to use a beach umbrella. It is often desirable to brace the beach umbrella against the side of the cooler and, therefore, an umbrella holder accessory would be another advantageous accessory for association with an insulated cooler. [0005] The present invention provides beverage and other accessory holders which may be selectively detachably secured to an outer wall of an insulated cooler. Because the accessory holders may be removed and stored, they do not contribute to the cooler dimensions during periods of non-use. Detachably secured holders also allow the consumer to selectively add or remove accessory holders on a portion of the cooler where it is convenient and in a number and at a location suitable to the customer's immediate needs. [0006] As will be appreciated, a variety of accessory holders may be provided in accordance with the invention and selectively detachably secured to the insulated cooler. By way of example, an accessory holding ring, which may be used for example to hold beverage bottles, cans, or cups, is provided. Also provided as an exemplary embodiment is an elongated accessory holder which may be used, for example, to receive a fishing rod. [0007] Thus, the invention may be embodied in an insulated cooler comprising upstanding insulating front, rear and side walls, a bottom wall and a lid, the walls and lid together defining an insulated enclosed space; at least one accessory holder having a first coupling portion; at least one second coupling portion complimentary to said first coupling portion provided on one of said front, rear and side walls for selectively coupling said accessory holder to said respective wall; wherein said accessory holder comprises a frame defining a perimeter of an accessory receiving space having an upwardly open mouth and wherein said first coupling portion is provided on an outer periphery of said frame. BRIEF DESCRIPTION OF THE DRAWINGS [0008] These and other objects and advantages of this invention will be more completely understood and appreciated by careful study of the following more detailed description of the presently preferred exemplary embodiments of the invention taken in conjunction with the accompanying drawings, in which: [0009] FIG. 1 is a perspective view of an insulated cooler having accessory holders coupled thereto in accordance with an exemplary embodiment of the invention; [0010] FIG. 2 is a view similar to FIG. 1 with a fishing rod and cup holder exploded away from the cooler to illustrated the respective coupling portions; [0011] FIG. 3 is an enlarged perspective view showing an exemplary accessory holder and coupling assembly; [0012] FIG. 4 is a perspective view of an accessory holder embodying the invention, taken from above; [0013] FIG. 5 is a perspective view of an accessory holder embodying the invention, taken from below; and [0014] FIG. 6 is a perspective view of a fishing rod holder, from behind, in accordance with an embodiment of the invention. DETAILED DESCRIPTION OF THE INVENTION [0015] As will be appreciated from the description provided hereinbelow, a variety of accessory holders may be provided in accordance with the invention. FIG. 1 illustrates by way of example an insulated cooler 10 having various accessory holders 12, 14 detachably secured thereto to illustrate exemplary embodiments of the invention. [0016] The insulated cooler 10 is comprised of upstanding insulating front 16, rear 18, and side 20, 22 walls; a bottom wall 24 ; and a lid 26. The walls and lid together define an insulated enclosed space. Each accessory holder 12, 14 has a first coupling portion 28, 30. Second coupling portions 32, 34 complimentary to the first coupling portions 28, 30 are provided on the front 16, rear 18, and/or side 20, 22 wall(s) for selectively coupling the accessory holder 12, 14 to the cooler. [0017] In the illustrated embodiment, accessory holder 12 is comprised of a truncated holder frame 36 defining a perimeter of an accessory receiving space 38 having an upwardly open mouth. As shown, the first coupling portion 28 is provided on an outer periphery of the frame 36. The holder frame 36 preferably defines a closed perimeter to fully encircle the accessory receiving space 38. However, the holder frame may be generally c-shaped, that is circumscribing more than one half and preferably about ¾ of the accessory receiving space 38, if deemed necessary or desirable to accommodate the accessory, as described in greater detail hereinbelow. The holder frame 36 is illustrated as being generally circular in cross-section since in an exemplary embodiment it is used to receive a beverage container, which is typically also circular in cross-section. It is to be understood, however, that a holder frame provided in another, e.g., straight-sided shape, such as a square or hexagon could be used without departing from the invention. [0018] In the embodiment illustrated in FIG. 1, the holder frame 36 is used to receive a beverage container such as a bottle, can or glass (not shown). To support the beverage container from below, a bag like receptacle 40 is provided to extend downwardly from the holder frame 36 so that the holder is well suited to beverage containers of any of the variety of sizes and shapes. Such a receptacle 40 may be omitted, however, where the holder frame's shape and size generally correspond to that of the beverage container and the beverage container has a flared or enlarged top end or has a tapered outer wall that is larger adjacent its upper portion, to engage and be held by the frame on insertion. [0019] As mentioned above, the holder frame 36 is secured to the outer wall of the cooler 10 by providing complimentary coupling portions 28, 32 on the frame and on the cooler outer wall. In the illustrated embodiment, the coupling portion 32 provided on the cooler outer wall defines a generally dovetail shaped receptacle having first and second inclined walls 42, 44 and a bottom wall 46 to limit the displacement of the holder with respect to the receptacle. The holder frame 36 likewise includes a pair of outwardly flared walls 48, 50 for laterally locking the holder frame 36 with respect to the coupling portion 32. Stop walls 52, 54 are defined adjacent the upper end of the flared walls 48, 50 to limit downward displacement of the frame 36 with respect to the coupling portion 32 provided on the cooler. Thus, when the holder is attached to the cooler, the inclined/flared walls 42, 44, 48, 50 limits or precludes lateral displacement of the holder away from the cooler and the stop walls 52, 54 and bottom wall 46 respectively limit the downward displacement of the holder. However, the holder can be readily removed from the cooler by lifting as shown by the exploded view of FIGS. 2 and 3. [0020] Although radially projecting legs to define a dovetail like projection 28 and a dovetail like receptacle 32 are respectively provided to secure holder 12 and cooler 10 in the illustrated embodiment, it is to be understood that other complimentary coupling structures, such as a solid dove-tail projection or a T-shaped projection and corresponding slot or groove, may be provided without departing from the invention. Moreover, it is to be appreciated that the inverse of the connector arrangement shown could be provided so that the protrusion is defined on the cooler and the receptacle on the holder. [0021] When the holder frame is not in use, advantageously it may be seated in the lid 26 of the cooler 10 in a respective receptacle 56 as illustrated in FIG. 1. Advantageously, when provided, the container receptacle 40 can be collapsed into a lower part 58 of the two-tiered holder receptacle 56. The frame 36 of the holder 36 is then seated on the ledge 60 defined by the larger portion 62 of the two-tiered receptacle 56. A cutout 64 is provided to accommodate the flared walls 48, 50 of the holder. It is further seen in FIG. 1 that a at least one locking projection 66 is defined on an inner wall of the larger portion 62 of receptacle 56. Furthermore, at least one projecting lip 68 is provided on the holder frame 36. In the illustrated embodiment, primarily for aesthetic reasons, the projecting lip 68 of the holder 36 extends substantially about the entire periphery. However, spaced projecting lip segments may be provided at spaced locations about the holder frame. Thus, when the holder frame is not in use, it may be snap-locked into position in the larger portion 62 of the two-tiered receptacle 56, with the receptacle portion 40 of the holder 12 seated, e.g., collapsed, in the lower part 58. When it is desired to remove the holder frame from its respective receptacle, the consumer can reach under the holder frame 36 into the finger accommodating space 70 defined in the lower part 58 of receptacle 56 and snap the holder 36 out of its storage position. Because the container receptacle or bag 40 (if provided) is flexible, it does not serve as an impediment to the unlocking motion described above. [0022] While in the illustrated embodiment a bag-like container receptacle 40 is provided to receive the bottom portion of the beverage container, it is to be understood that a rigid, partially rigid, and/or telescopically collapsible container receptacle can be provided integral with or attached to the frame 36 in lieu of a flexible bag or net. [0023] Reference will now be made to the elongated frame 72 defining the accessory holder 14, illustrated in particular in FIGS. 1, 2, and 6, which may be used to receive and hold, e.g., a fishing rod handle. The frame 72 defines an accessory receiving area 74 having and an upwardly open mouth. This holder includes first connector portion 30 for engaging complimentary second connector portion 34 provided on an outer wall of the insulated cooler 10. Because holder 14 is vertically elongated, the first and second connector portions are similarly elongated to support the holder body securely with respect to the cooler. [0024] In the illustrated embodiment, the first and second connector portions 30, 34 are respectively defined by a generally dovetail projection or protrusion and a corresponding generally dovetail receptacle. The dovetail protrusion 30 in this embodiment is provided on the outer wall of the frame or housing 72 of the elongated holder 14 and the dovetail receptacle 34 is defined on the cooler wall. It is to be appreciated that the protrusion may be provided on the cooler and the receptacle on the holder, albeit inverted to support the holder. The coupling receptacle may be the same or similar to the complimentary couplings provided for cup dispensers provided, e.g., on conventional 5 and 10 gallon plastic beverage coolers manufactured by Igloo Products Corporation. Thus, the second coupling portion 34 includes first and second walls 76, 78, joined by base 77. Walls 76, 78 are inclined towards one another to define the generally dovetail receptacle. Coupling walls 80, 82 project from the elongated holder frame or housing 72. In the illustrated embodiment, the legs project generally radially and receptacle engaging walls 84, 86 having inclined edges are provided for being slidably and lockingly disposed in the dovetail receptacle. Upper walls 88, 90 are also provided for limiting the displacement the elongated holder 14 with respect to the second coupling portion 34. It is to be understood that rather than providing wedge walls 84, 86, the radial walls 80, 82 may be flared outwardly to engage the inclined walls of the receptacle. In the illustrated embodiment, the elongated frame or housing 72 includes a generally cylindrical side wall 92 and a slot 94 for accommodating the reel mounted to the fishing rod. Thus it may be said that the frame or housing 72 is substantially circular in cross-section. It is to be understood, however, that the frame may have, e.g., a straight-sided shape, such as a square or hexagon, without departing from the invention. Moreover, as illustrated, the generally cylindrical side wall 92 and the receptacle defined thereby can be flared towards the upper open end thereof, so that the fishing rod holder is narrowest adjacent its closed bottom wall 96. [0025] While the bottom of the holder 14 may be open, it is preferred that a bottom wall 96 be provided on which, e.g., the fishing rod handle can rest so that the weight of the fishing rod is not borne by the reel attachment structure engaging the slot 94. Although the holder 14 has been characterized as particularly suitable for a fishing rod, it is to be understood that an elongated holder may be provided to accommodate other accessories such as a flashlight, utensils, or other elongated tool(s) or device(s). [0026] As mentioned above, coolers are often transported onto the beach and braced against a beach umbrella to not only be shaded, but also to help hold the umbrella shaft in position. In accordance with a further advantageous characteristic of the invention, an accessory holder embodying the invention such as a holder frame 36 having no container receptacle 40 can be used to engage a beach umbrella shaft. For example, such a holder frame 36 can be slipped over the insertion end of the umbrella shaft before the umbrella is planted into the ground. Then, after the umbrella shaft is planted, the holder frame can be displaced along the shaft to align with the complementary coupling portion 32 provided on the ice chest, thereby defining a support ring to aid in the support of the umbrella. In the alternative, a holder frame embodying the invention defined as a part circumferential or c-shaped holder may be provided that can be engaged with the umbrella after planting and then coupled to the ice chest. [0027] By way of example, holder frames which may be used as a beverage holder, a fishing rod holder, and/or and umbrella holder have been illustrated and described as detachably secured to an insulated cooler. However, those skilled in the art will appreciate from the disclosure provided that these are merely examples of a variety of holders which may be detachably secured to an insulated cooler using the principles of the invention. Likewise, the holders described may be used to receive and hold a variety of devices, components, and accessories, not just beverage containers, fishing rods, and umbrellas. Therefore, the specification is not to be limited to the particular holder types and accessories described herein. Indeed, while the invention has been described in connection with what is presently considered to be the most practical and preferred embodiment, it is to be understood that the invention is not to be limited to the disclosed embodiment, but on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. | Summary: An improved insulated cooler has accessory holders detachably secured to its outer wall. An exemplary holder is provided for containerized drinks. The holder is made of a rigid plastic upper ring at the top and may have flexible fabric or mesh lower portion at the bottom such that the holders can be nested into receptacles in the top surface of the lid for convenient storage. This arrangement provides the benefit of permitting the user to access conveniently the contents of the ice chest without having to remove drinks that otherwise would be sitting on the lid of the ice chest. There also may be one or more connections on the ice chest that permit a fishing rod holder, an umbrella holder, or other accessory holder to be affixed to the cooler. | 4,105 | 159 | big_patent | en |
Summarize: RELATIONSHIP TO OTHER APPLICATION [0001] This application claims priority from provisional Application Serial No. 60/287 320, filed Apr. 30, 2001 and entitled CHAIR. FIELD OF THE INVENTION [0002] This invention relates to a chair of the type used in offices and the like, and in particular to an improved chair back having limited vertical swinging movement about an axis disposed adjacent the upper edge of the back. BACKGROUND OF THE INVENTION [0003] Chairs of the type used in offices and the like are often utilized for permitting a seated occupant to carry out work-intensive tasks adjacent a desk or worksurface, including keyboarding and other tasks which require the person to sit generally upright or even lean forwardly so as to partially overlie a worksurface. When used in this manner, the back of the chair generally loses contact with the occupant's back, and thus provides no supportive engagement therewith. [0004] It is an object of this invention to provide an improved office-type chair wherein the back of the chair has limited vertical swinging movement generally about the upper portion thereof so that when a person using the chair leans forwardly, the back of the chair will be urged forwardly, as by a spring, through at least a limited extent and hence the lower portion of the chair back, such as in the lumbar region, will continue to maintain supportive engagement with at least the lower back of the chair occupant. SUMMARY OF THE INVENTION [0005] This invention is directed to a new and useful chair including a frame having laterally spaced first and second rigid uprights. A back is attached to the frame and includes upper and lower ends. Also attached to the frame adjacent to the lower end of the back is a seat. A pivot assembly couples the first and second uprights to the back for permitting pivoting of the back about a substantially horizontal pivot axis that projects laterally of the back and is positioned in the vicinity of the upper end of the back. The pivot assembly includes a spring arrangement that exerts a force on the back a substantial distance below the pivot axis for biasing the back toward a forward position. [0006] Other objects and purposes of the invention will be apparent to persons familiar with constructions of this type upon reading the following specification and inspecting the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS [0007] [0007]FIG. 1 is a perspective view of a chair according to the present invention shown positioned adjacent a conventional desk. [0008] [0008]FIG. 2 is a side view of the chair shown in FIG. 1. [0009] [0009]FIG. 3 is a further perspective view taken generally from the rear of the chair shown in FIG. 1. [0010] [0010]FIG. 4 is a perspective view which illustrates solely the back frame for the chair back of this invention and its connection to the rear inner shell of the chair back. [0011] [0011]FIG. 5 is a back elevational view of the construction illustrated in FIG. 4. [0012] [0012]FIG. 6 is a side elevational view of the arrangement shown in FIGS. 4 and 5. [0013] [0013]FIG. 7 is a side elevational view showing in cross sectional view the chair arm and its connection to the upright associated with the back frame, and specifically showing in solid lines the chair arm in both its uppermost and lowermost height adjusted positions. [0014] [0014]FIG. 8 is a top view of the arrangement shown in FIG. 7 and showing both positions of the chair arm in solid lines. [0015] [0015]FIG. 9 is a rear elevational view of the arrangement shown in FIGS. 7 - 8 and again showing both elevational positions of the chair arm in solid lines. DETAILED DESCRIPTION [0016] Referring to FIGS. 1 - 3, there is illustrated a chair 10 according to the present invention. This chair includes a conventional base 11 having legs 12 and a central height-adjustable pedestal 13 projecting upwardly therefrom. The pedestal at its upper end connects to the underside of a generally horizontally enlarged seat arrangement 14. The seat arrangement 14, as is generally conventional, includes a generally rigid structural inner shell 15 having a cushion thereover 16, with the cushion and shell being generally enclosed by a surrounding covering such as a fabric or vinyl covering. [0017] A back frame structure 21 joins to the underside of the seat structure 14 and projects upwardly for supportive engagement with a back arrangement 22 which projects upwardly from the seat arrangement 14 in the vicinity of the rear edge thereof. This back arrangement 22, in the illustrated embodiment of the invention, has chair arms 61 associated therewith, which chair arms are cantilevered forwardly from the back frame arrangement 21 and are mounted for height adjustment with respect thereto. [0018] The back arrangement 22 includes an inner structural back member or shell 23 typically constructed of wood or rigid plastic, and this inner shell is appropriately covered on a front side thereof with a cushion 24 such as of plastic foam, and the inner shell and foam cushion are appropriately enclosed within an outer covering of fabric, vinyl or the like. The rear of the back arrangement is typically closed by a rear cover or shell 27 which overlies the inner structural shell and is secured thereto. The general construction of the back arrangement 22, like the seat arrangement, is conventional. [0019] The back frame arrangement 21 as illustrated in FIGS. 4 - 6 includes a pair of generally upright frame members 31 which are substantially identical except for being mirror images of one another so as to be disposed adjacent the right and left sides of the chair back. Each upright frame member 31 includes a main elongate center part 32 which extends generally vertical and which at a lower end joins to a curved portion 33 which projects forwardly so as to terminate at a lower free end part 34. The lower free end parts 34 of the upright frame members 31 are rigidly joined by a cross strap or plate 35, the latter in turn being fixedly secured to the underside of the structural shell 15 associated with the seat arrangement 14. The upright frame members 31, at the upper ends thereof, are also provided with curved portions 36 which form an upper leg which projects toward and terminates in a free end 37 disposed adjacent the rear surface 28 of the inner back shell 23 in the vicinity of the upper edge 45 thereof. [0020] The pair of sidewardly-spaced upright frame members 31, at their upper ends, are rigidly joined by a top cross rod or bar 38 which has the free ends thereof non-rotatably and fixedly joined to the upper free end parts 37 of the side frame members 31. This cross bar 38, extending inwardly from the free ends thereof, has generally aligned and substantially horizontally extending rod portions 41 which project inwardly from the side frame members toward the center of the back shell. These horizontal rod members 41 are bent through about 90° angles and joined to a generally U-shaped center rod portion 42. This center rod portion 42 includes side legs 43 which project generally vertically downwardly adjacent the rear surface of the back shell 23, and these side legs 43 join through generally right angle bends to a bottom cross rod 44 which extends generally horizontally. The cross bar 38 and its rigid securement between the upper ends of the spaced side frame members 31, and the bottom strap 35 and its rigid securement between the lower ends of the side frame members 31, thus define a rigid frame assembly which is of a generally closed endless configuration, and provides a connection to support the back arrangement 22 from the seat arrangement 14 as described hereinafter. [0021] To connect the back arrangement 15 to the frame arrangement, the back shell 23 fixedly mounts thereon, in the vicinity of the upper corners thereof, a pair of sidewardly spaced journals or bearings 46 which are fixed to and project outwardly from the rear surface 28 of the back shell 23. This pair of spaced journals 46 define aligned openings 47 therein in which are snugly but rotatably accommodated the horizontal rod parts 41 of the cross bar 38. This connection of the horizontal rod parts within the journals secured to the back shell thus couples the back shell 23, and hence the back arrangement 22, to the frame assembly 21 while permitting relative pivoting of the back arrangement 22 about the longitudinally extending horizontal axis 48 defined by the horizontal rod parts 44. [0022] To control and limit the amount of pivoting movement of the back arrangement 22 relative to the back frame assembly 21 about the pivot axis, the back assembly 22 has a restraining member 51 fixedly secured to and projecting rearwardly from the rear surface of the back shell 23 at an elevation which is spaced downwardly a substantial distance below the horizontal pivot axis 48. This restraining member 51 in the illustrated arrangement is formed generally as a horizontally elongate strap Am,; which is fixedly secured to the back shell 23, and the strap has a pair of control parts 52 in sidewardly spaced relationship therealong. These control parts 52 are formed generally as U-shaped parts, or yokes, and effectively extend around and provide control over the vertical rod portions 43. More specifically, each of the control yokes 52 has generally parallel side legs 53 which are spaced apart so as to permit the side rods 43 to move lengthwise of the control yoke until restricted by the closed end 54 of the yoke which is spaced from the rear surface 28 of the seat shell 23 and functions as a stop. These control yokes 51 thus permit the back shell 23 to pivot about the horizontal pivot axis 48 through a limited extent as permitted by the vertical rods 43 abutting the ends of the yokes 52 as a forward limit position, and by the shell 23 swinging rearwardly into a rearwardmost position in which it effectively abuts the U-shaped center rod part 42. The forward and rearward positions are diagrammatically indicated in FIG. 6. [0023] The back arrangement 22 is normally maintained in its forwardmost position by the urging of a spring arrangement 56 which, in the illustrated embodiment, comprises two coil-type torsion springs 57 which surround the horizontal center rod part 44 and have one leg 58 thereof anchored to the rod, with the other leg 59 of each torsion type coil spring being in abutting engagement with the rear surface of the back shell 23. The legs 59 of the torsion springs which project inwardly for contact with the back shell 23 are, in the preferred embodiment, joined together to define a generally U-shaped configuration which bears against the rear surface of the seat shell at a location disposed in the vicinity of the horizontal rod part 44 and hence vertically approximately at the middle of the back shell. The contact of the spring against the seat shell is thus spaced a substantial distance downwardly from the pivot axis 48 and hence, acting through the long lever arm defined between the pivot axis and the spring, urges the seat shell 23 forwardly into the forward position as limited by the vertical rods 43 contacting the stop parts 54 defined at the ends of the control yokes 52. [0024] When the chair of this invention is not occupied, the spring 56 will normally urge the back arrangement 22 forwardly (counter-clockwise in FIG. 6) about axis 48 into the forwardmost position for the back. When the chair is occupied, however, and the occupant leans against the back in the normal manner, the force imposed on the back 22 by the occupant will overcome the spring force and the back will swing back (clockwise) into its rearwardmost position wherein the back shell 23 abuts the U-shaped rod part 42 and thus defines a generally solid or rigid back assembly. [0025] However, if the occupant leans forwardly and relieves the force against the back 22, such as when carrying out an intensive task on a table, such as a keyboarding function, then the back of the occupant will partially move away from the back and relieve the load on the back. At the same time, however, the spring 56 acting against the back shell 23 causes the lower portion of the back 22 to pivot forwardly about the top hinge axis 48, and thus the lower portion of the back 22 will be disposed so as to continue to maintain supportive engagement with at least the lower portion of the occupant's back, particularly in the lumbar area. [0026] Since the torsion springs 57 and their reaction against the rear surface of the back shell 23 occurs at a point which is spaced downwardly a substantial distance below the hinge axis 48, the springs 57 acting through the large lever arm created by this spacing thus results in creation of a significant mechanical advantage so that a rather significant moment can be applied to the back 22 about the pivot axis 48, even though the individual torsion springs themselves are small, and thus a significant force urging the lower portion of the back 22 forwardly can be achieved so as to continue to maintain partial supportive contact with the lower region of the occupant's back. [0027] At the same time, however, the overall mechanism is small and compact, and can be easily enclosed in a small space defined between the inner structural back shell 23 and the outer rear cover 27. [0028] Considering now the construction and operation of the height-adjusting chair arms 61 as associated with the chair of this invention, each height-adjusting chair arm 61 includes an elongate support sleeve 63 which is fixed to and encircles the vertically extending portion 32 of the respective side frame member 31 over a significant extent of the length thereof. This tubular support member 63 has an opening therethrough for snugly receiving therein the elongate straight portion 32 of the side frame member 31, and the tubular support member 63 is formed in two halves which enable it to be snugly clamped around the side frame member and then secured thereto by screws or similar fasteners which extend through the two halves of the support member as well as the side frame member. [0029] The tubular support member 63 has an exterior configuration which is preferably polygonal and is defined by a plurality of flat sides, which exterior polygonal configuration in the preferred embodiment is generally rectangular and more specifically square. [0030] The exterior front side wall 64 of the support tube 63 has a toothed or racklike configuration formed thereon throughout the vertical extent thereof, whereby adjacent teeth 65 are vertically separated by a notch or recess 66 which extends transversely (i.e. generally horizontally) with the upper side of this notch merging smoothly into a ramplike surface which slopes outwardly and upwardly to define the tooth. [0031] The opposite or rear flat wall 67 of the support tube 63 is generally flat but has a series of transversely (i.e. horizontally) extending notches or recesses 68 formed therein. The series of notches 68 are disposed in vertically spaced relationship along the support tube, with the vertical spacing between adjacent notches 68 generally corresponding to the vertical spacing between adjacent recesses 66 associated with the front wall of the support tube 63. [0032] The upright back frame members 31 are disposed substantially totally exteriorly of the back arrangement 22, and the elongate vertical uprights 32 associated with the back frame members 31 are disposed so that they are positioned closely adjacent but spaced slightly rearwardly and slightly outwardly from opposite sides of the back arrangement 22. Each of the elongate vertical upright portions 32 of the back frame elements 31, specifically those portions having the support tubes 63 secured therearound, support thereon one of the cantilevered arm assemblies 61. [0033] Each cantilevered arm assembly 61 includes a generally horizontally elongate arm member 71 which is mounted on and projects forwardly from the respective support tube 63, with this arm member in turn having a top cap member 72 fixedly mounted thereon, which top cap member typically incorporates some type of resilient cushioning material enclosed within an appropriate exterior cover, such as is conventional, so that further description thereof is believed unnecessary. [0034] The arm member 71 at the rearward end thereof is provided with a sleeve part 73 which has an opening 74 extending vertically therethrough, the cross section of which is noncircular and is sized so as to nonrotatably but vertically axially accommodate therein the respective support tube 63, as illustrated in FIG. 8. [0035] The sleeve part 73 defines thereon, on the front side of the interior opening 74 adjacent the lower end thereof, a transversely extending rib 75 which projects rearwardly into the interior of the sleeve part and is sized so as to engage a selective one of the recesses 66 defined between the teeth 65 on the front or rack-bearing side of the support tube 63. [0036] The rear side of the opening 74, in the vicinity of the upper end thereof, has a further rib 76 which extends transversely and projects outwardly in a forward direction so as to terminate in a generally flat outer end. This latter projection 76 is adapted to bear against the rear surface 67 of the support tube 63 in the flat regions between the notches 68. This rear projection 76 is also disposed vertically upwardly a substantial distance above the front projection 75, as illustrated by FIG. 7. [0037] The support hub 73 on the arm member 71 also has a small platelike spring 77 which is mounted interiorly thereof and has a cantilevered portion which terminates in a free end part 78 adapted to resiliently engage one of the latching notches 68 formed on the rear wall of the support tube 63. This spring 77 has the upper end thereof secured over the rear support rib 76 associated with the support hub so that the spring is fixed to and hence carried with the support hub 73. The spring 77 as it projects downwardly is cantilevered so as to be resiliently urged forwardly for engagement with the rear wall 67 of the support tube 63. [0038] With the height-adjusting arm arrangement of the present invention, the individual arms can each be vertically adjusted in height from an uppermost position as illustrated in FIGS. 7 - 9 to the lowermost position illustrated therein. This height adjustment range is preferably between about seven inches, with the arm when at the upper limit as illustrated in FIGS. 7 - 9 typically being at the uppermost height which is conventionally provided for arms associated with office type chairs. Conversely, however, when the arm is in the lowermost position illustrated in FIGS. 7 - 9, the arm is now disposed so that it is positioned closely adjacent the outer side edges of the seat arrangement 14, and elevationally is positioned closely adjacent or just slightly above the upper surface of the seat arrangement, whereby in this latter position the arms are at an elevation whereby they are compactly stored directly adjacent the seat arrangement, and thus the chair in its entirety, except for the back arrangement, can be readily stored in a position under even low tabletops or worksurfaces. Further, even when the chair is occupied, the arms can be disposed in this lowermost position whereby they do not interfere with the occupant's movements if the occupant prefers to have the sides of the chair seat free of obstructions. [0039] The operation of the height-adjusting arms is extremely simple since, if the occupant when sitting in the chair grips the arm 71 adjacent the rear end thereof and lifts upwardly, this causes the arm to rock about the bearing rib 76, thereby causing the locking rib 75 to be withdrawn from engagement with the rack. The operator can then move the arm vertically, either upwardly or downwardly, since the spring 77 will merely function like a releasable detent and effectively “click” upwardly or downwardly along the support tube 63 and hence define the various locking positions. When the arm reaches the desired elevational position, the operator then allows the arm to tilt back downwardly causing the locking rib 75 to engage the respective recess 66 associated with the rack, thereby relocking the arm in the selected position, substantially in the manner illustrated by FIG. 7. In this locking position, the weight of the arm tending to swing it downwardly (counter-clockwise in FIG. 7) thus effectively maintains the support hub 73 of the arm in locked engagement with the support tube 63. No additional complex locking mechanisms are required, and in addition no separate levers or trigger mechanisms are required so as to release the arm for height adjustment purposes. [0040] Although a particular preferred embodiment of the invention has been disclosed in detail for illustrative purposes, it will be recognized that variations or modifications of the disclosed apparatus, including the rearrangement of parts, lie within the scope of the present invention. | Summary: A chair having a swingable chair back with a top pivot includes a frame having laterally spaced first and second uprights. A back having upper and lower ends is attached to the frame. Also attached to the frame adjacent the lower end of the back is a seat. A pivot assembly couples the first and second uprights to the back and permits pivoting of the back about a substantially horizontal pivot axis. The pivot axis projects laterally of the back and is positioned in the vicinity of the upper end of the back. A biasing device cooperates with the back and normally urges the lower portion of the back forwardly away from a rearward position. | 5,211 | 142 | big_patent | en |
Summarize: By. Ap Reporter. Kevin Spacey has hit back at accusations made by Toronto mayor Rob Ford and Ford's brother that he doesn't take photos with fans, Tweeting a photoshopped image of himself wedged between the two politicos. Spacey, who plays cold and calculating U.S. Vice President Francis Underwood on hit drama House of Cards, was reacting to a complaint made earlier in the week by the Rob and Doug Ford, who were backstage at Jimmy Kimmel Live on the same night as the actor. The embattled mayor said he wouldn't know Spacey 'if I ran over him'. Ford's brother called the actor 'an arrogant SOB', saying there were strict instructions that no photos were to be taken with Spacey by anyone. Kevin Spacey Tweeted a photoshopped image of himself between Toronto mayor Rob Ford and his brother on Saturday, in response to the siblings' criticism that he won't take photos with fans. Take that: Kevin Spacey has his say on claims made by Rob Ford. Original: Rob and Doug Ford made a video specifically about Kevin Spacey on their YouTube show, Ford Nation, following a run-in with the actor on Jimmy Kimmel Live earlier this month. Political@ Kevin Spacey plays the cold and calculated Francis Underwood on House of Cards. The pair made the comments on their YouTube show Ford Nation, which is named after the mayor's supporters. Along with the photo he tweeted, Spacey wrote: 'When did Mayor Ford start doing what people tell him to do? All you had to do was ask, guys. Here's your pic.' During Spacey's appearance on Jimmy Kimmel Live, the actor cracked several jokes at the mayor's expense when Spacey appeared after a brief cameo by Ford. 'That's the first time I've had to follow a Ford,' Spacey quipped. 'And one that was so banged up.' He started it: Kevin Spacey made a series of jokes at Toronto mayor Rob Ford's expense when he appeared on Jimmy Kimmel Live on March 2. Toronto Mayor Rob Ford appears the late night talk show Jimmy Kimmel Live in Los Angeles. When Kimmel thanked Spacey for sharing a dressing room with the mayor, Spacey responded: 'Well, he threw up all over it, but those are the chances you take.' Ford has been under great pressure to resign since he admitted smoking crack last year, and several videos have emerged that showed him seemingly drunk or high. His antics have made him the target of late-night television comedians and embarrassed Canadians. City Council stripped him of many of his powers but does not have the authority to remove him. Undaunted, Ford has vowed to run for re-election this fall | Summary: Toronto mayor Rob Ford, along with his brother and campaign manager Doug Ford, appeared on Jimmy Kimmel Live in L.A. on the same night as Kevin Spacey on March 2. Afterwards the siblings created a video for their YouTube show, Ford Nation, about Spacey. They said there strict instructions not to take photos with the actor. Mayor Ford said he wouldn't know Spacey 'if I ran him over' Spacey responded Saturday by posting a tongue-in-cheek photo on Twitter. Spacey has not denied his 'no photo' rule. | 608 | 122 | cnn_dailymail | en |
Summarize: This application is a continuation of International Application No. PCT/AT2006/000270, filed Jun. 29, 2006. BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an article of furniture comprising a furniture carcass and a first and at least one second drawer displaceable relative to the furniture carcass, wherein in the closed position of the two drawers the front panel of the first drawer substantially completely covers the front panel or the front wall of the second drawer. 2. Description of the Related Art Items of furniture of that kind describe inter alia what are referred to as internal extension portions, the front panel or the front wall of which is disposed behind a larger or upwardly extended front panel of a front extension portion. When an internal extension portion is fitted into an article of furniture in the form of a cabinet, the front extension portion always has to be opened first so that the internal extension portion becomes accessible and can also be opened. Often those internal extension portions, for aesthetic reasons or reasons of space, do not have any handle or grip whereby the operating option is restricted. If, in addition, the internal extension portion is arranged directly beneath a transverse member, an intermediate panel portion or under additional internal extension portions, there is no comfortable and convenient possible way of opening the internal extension portion. If, moreover, the internal extension portion is provided with a closure mechanism, which in its closed position acts on the internal extension portion with a retaining force for keeping it closed, actuation is in addition impractical as the opening movement against a spring force is made considerably more difficult. SUMMARY OF THE INVENTION It is therefore an object of the present invention to propose an article of furniture of the general kind set forth in the opening part of this specification, which offers a user a high level of operating comfort. In accordance with the invention in an advantageous configuration that is achieved in that the first and the at least one second drawer have an ejection device for moving the drawers from the closed position into an open position and that arranged between the two ejection devices is a switching element which couples the two ejection devices in a first switching position and uncouples them in a second switching position. If the ejection device acts, for example, on the second drawer (internal extension portion), it can be conveniently moved into an open position without any need for the user to grip behind the internal extension portion for that purpose. If the ejection device acts on the first drawer (front extension portion), it can be selectively coupled to or not coupled to the internal extension portion, by a preferably mechanical coupling means. In order to promote the ejection movement of the drawer it can also be provided that at least one ejection device has a drive device, preferably at least one spring or at least an electric motor, by which an ejection element is or can be acted upon for ejection of the drawer. In order to avoid the drawer abruptly springing open, it can also be desirable for the relative movement between the ejection device and the drawer or the guide rails thereof to be damped by means of a damping device. An embodiment of the invention provides that the first drawer and the at least one second drawer have a separate ejection device for moving the drawers from a closed position into an open position. In that way the extension movements can be controlled independently of each other. An advantageous configuration provides in that respect that the ejection device of the first and the at least one second drawers are coupled so that the ejection device of the second drawer can be triggered by way of the ejection device of the first drawer. In that case, the at least two ejection devices can be electrically or mechanically coupled together so as to afford a very wide range of triggering and switching combinations. A configuration in that respect can be such that disposed between the two ejection devices is a switching element which couples the two ejection devices in a first switching position and uncouples them in a second switching position. In that case, the switching element can be either in the form of a mechanical switch or—in accordance with a further embodiment—it can be in the form of a logic circuit for interlinking digital signals in accordance with the rules of Boolean algebra. A further embodiment of the invention provides that at least the first drawer has a first limit position which corresponds to the closed position of the first drawer and the first drawer is movable starting from that limit position by the application of pressure in the closing direction thereof into a second limit position which is further into the furniture carcass. In that case, the configuration can be such that an ejection device is of such a configuration that it moves the drawer from the second limit position into an open position. Fitments of that kind are known as touch-latch fitments which are actuated by the drawer being pushed in by a predetermined distance in order then to be moved into an open position by a force storage device (spring device, fluid damper, electric motor). Alternatively or as a supplemental consideration, it may also be advantageous if the at least first drawer has a limit position which corresponds to the closed position of the drawer and the first drawer is movable starting from that limit position by applying a pulling force into an open position, wherein the ejection device is inactive. If the first drawer (front extension portion) is activated by way of a pulling pulse, in that case preferably only the front extension portion is extended as the ejection device of the internal extension portion does not receive a switching pulse. In that situation, the internal extension portion can selectively remain in the closed position or selectively be activated by manually applying pressure or a pulling force to the front panel thereof. In accordance with a further embodiment of the invention it can be provided that the article of furniture has a control and/or a regulating device for selectively moving the drawers. In that case for example selective activation, the moment in time of actuation and/or the movements of the drawer can be controlled separately or in all possible combinations. In that connection, it may be desirable if the ejection device has at least one electric switch which is activatable by the application of a pulling force or a pushing force to the drawer, wherein the signals thereof can be passed to the control and/or regulating device. In that respect, the electric switch can be in the form of a microswitch, the switching pulses of which are passed to an address decoder for programmed actuation of the individual ejection devices. By way of example the arrangement can be such that the control and/or regulating device is so designed that it does not activate the ejection device of the at least second drawer (internal extension portion) when a pressure is preferably applied once to the first drawer (front extension portion). In other words, the internal extension portion, when pressure is applied once to the front panel of the front extension portion, is not extended while the front extension portion alone moves into the open position. A further embodiment can be such that the control and/or regulating device is so designed that it activates the ejection device of the at least second drawer (internal extension portion) when pressure is preferably applied twice in succession to the first drawer (front extension portion) within a predetermined or predeterminable period of time. In other words, in the case of a “double click” on the front extension portion, both the front extension portion and also the internal extension portion are moved into an open position. In an advantageous development it can also be provided that the control and/or regulating device is so designed that it does not activate the ejection device or devices when a pulling force is applied to the first drawer. It is to be noted in that respect that a very wide range of switching options are possible in this connection, for one of ordinary skill in the art dealing with this object. In accordance with a preferred development of the invention it can be provided that the article of furniture has a releasable coupling device for temporarily coupling the two drawers. The additional arrangement of that releasable coupling device in combination with at least one ejection device for the drawer affords additional possible combinations. In that respect, it can be advantageous if the at least second drawer is movable at least from the completely closed position into an open position by the releasable coupling device. In this connection, it is desirable if the releasable coupling device has two or more, preferably preselectable, modes of operation. The possibility of adjusting the releasable coupling device can provide that for example the first drawer (front extension portion) can be opened, in which case in accordance with the two modes of operation it is possible selectively to establish whether the at least second drawer (internal extension portion) does or does not also perform the movement of the front extension portion. In that respect, it is advantageous if the releasable coupling device is arranged or designed in such a way that the drawers can be uncoupled, preferably in a common open position thereof, so that each drawer can be actuated in itself alone. In regard to the releasable coupling device many different configurations can be implemented in a manner with which the man skilled in the art will be familiar. In an embodiment it can be provided that the releasable coupling device is preferably operative between the rear side of the front panel of the first drawer and between the front side of the front panel or front wall of the at least second drawer. In the simplest case it may be desirable if the releasable coupling device has a mechanical latching connection (for example at least one spring-loaded latching element). An alternative embodiment can also provide that at least one, preferably spring-loaded, spring buffer and/or at least one spacer portion, preferably comprising a damping material, is or are arranged between the front panel of the first drawer and the front panel or front wall of the second drawer. That arrangement means that it is possible to effectively prevent hard impact of the front panel of the first drawer against the front panel or front wall of the second drawer so that the banging noises which occur in that case are substantially reduced. Finally, in an embodiment of the invention it can be provided that at least one of the drawers has a retraction device by which the drawer is movable into the closed limit position, wherein preferably the last closing travel to the completely closed position thereof takes place in a damped fashion. In that respect, each drawer of the article of furniture can be equipped with a retraction device of that kind, which reliably move the drawers into the closed limit position. Usually, retraction devices of that kind are also provided with a damping device which damps the movement of the drawer over the last closing range so that it is not retracted into the carcass of the article of furniture with too much momentum. In that respect fluid dampers, for example linear or rotational dampers, can advantageously be used as the damping devices. BRIEF DESCRIPTION OF THE DRAWINGS Further details and advantages of the invention are described in greater detail hereinafter by means of the specific description with reference to the drawings in which: FIG. 1 shows a perspective view of an embodiment of an article of furniture according to the invention, wherein the article of furniture has a front extension portion and two internal extension portions disposed therebehind, FIG. 2 shows a further embodiment of the invention with a releasable coupling device between the lower internal extension portion and the front extension portion and with a spring buffer between the upper internal extension portion and the front extension portion, and FIGS. 3 a - 3 d show various embodiments of the triggering and coupling options between an internal extension portion and the front extension portion. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT FIG. 1 shows an embodiment of an article of furniture 1 according to the invention as a perspective view. The article of furniture 1 which is in the form of a cabinet has a carcass 2 in which a first drawer 3 (front extension portion) with an upwardly extended front panel 3 ′ is displaceable. Also, disposed in the carcass 2 are a second and a third drawer 4 and 5 (internal extension portions), the front walls 4 ′ and 5 ′ of which in the closed position of the drawers 3, 4, 5 are behind the upwardly extended front panel 3 ′ of the front extension portion 3. In order to open the two internal extension portions 4 and 5, the front extension portion 3 always has to be opened first, which makes it more difficult to operate the drawers 3, 4 and 5. For that purpose provided on the rear wall 8 (only partially shown) of the article of furniture are three separate ejection devices 9, 9 ′ and 9 ″, the ejection elements 9 a, 9 a ′ and 9 a ″ of which preferably act on the respective rear walls of the individual drawers 3, 4 and 5 so that the drawers 3, 4 and 5, starting from the closed position, are accelerated, overcoming the mass inertia thereof, so that the drawers 3, 4 and 5 can be moved into an open position. The ejection devices 9, 9 ′ and 9 ″ preferably include a spring device (not shown) or, in accordance with an alternative embodiment, at least one electrical drive unit so that a torque is applied to the respective ejection elements 9 a, 9 a ′ and 9 a ″. The rear wall of the respective drawers 3, 4 and 5 is suitable in particular for the force of the ejection elements 9 a, 9 a ′ and 9 a ″ to act thereon, in which case the free ends of the ejection elements 9 a, 9 a ′ and 9 a ″ bear in the closed limit position against the respective rear walls of the drawers 3, 4 and 5. In principle the ejection devices 9, 9 ′ and 9 ″ can also act at another location of the article of furniture, for example on the extension rails thereof which are disposed at both sides of the drawers 3, 4 and 5. The separate ejection devices 9, 9 ′ and 9 ″ which in accordance with an embodiment of the invention can be coupled by at least one switching element afford various possible combinations which are described in greater detail in the Figures hereinafter. In order still further to enlarge the possible combinations of the drawers 3, 4 and 5, there are provided releasable coupling devices for selectively or temporarily coupling an internal extension portion 4, 5 to the front extension portion 3 so that, in an outward movement of the front extension portion 3, an internal extension portion 4, 5 also moves therewith or does not move therewith. The releasable coupling device includes substantially hook-shaped entrainment elements 10 b and 11 b respectively, and coupling elements 10 a and 11 a. The hook-shaped entrainment elements 10 b and 11 b are disposed in the region of the front walls 4 ′ and 5 ′ and can be releasably coupled to the coupling elements 10 a and 11 a of the front panel 3 ′. In that respect it may desirably be provided that the entrainment elements 10 b and 11 b and/or the coupling elements 10 a and 11 a have at least two operating positions, wherein in a first operating position the entrainment elements 10 b and 11 b automatically latch to the coupling element 10 a and 11 a respectively when the front extension portion 3 is brought together with the internal extension portions 4, 5 while in a second operating position no latching occurs between the entrainment elements 10 b and 11 b and the coupling elements 10 a and 11 a. In that connection, it is desirable if the entrainment element 10 b and 11 b respectively has an adjustment option by which the two operating positions can be activated, for example by manual actuation. That can be affected by either the entrainment elements 10 b and 11 b respectively, or the coupling elements 10 a and 11 a respectively, being adapted to be rotatable through 180° so that coupling is activated in one position and is deactivated in the position of being turned through 180°. FIG. 2 shows a slight modification to the embodiment of FIG. 1. The structure of the article of furniture 1 is identical to that of FIG. 1, with the exception that no coupling option is provided for the upper internal extension portion 5 or the front wall 5 ′ thereof, to the upwardly extended front panel 3 ′ of the front extension portion 3. In the illustrated embodiment provided on the rear side of the front panel 3 ′ of the front extension portion 3 is a spring buffer 12 which in the closed position of the two drawers 3 and 5 bears against the front wall 5 ′ of the upper internal extension portion 5. In that way, the spring buffer 12 prevents unwanted panel impact in respect of the front panel 3 ′ when the drawer 3 is closed. The spring buffer 12 can be replaced by a spacer portion preferably formed from a damping material with rubber-elastic properties. FIGS. 3 a - 3 d show various embodiments of the triggering and coupling options by means of a simplified view of the front extension portion 3 and an internal extension portion 4, as a side view. FIG. 3 a shows the front extension portion 3 with its upwardly extended front panel 3 ′ which in the closed position of the drawers 3 and 4 substantially completely covers the front wall 4 ′ of the internal extension portion 4. Provided at the rear wall 8 of the article of furniture 1 are two separate ejection devices 9 and 9 ′, the ejection elements 9 a and 9 a ′ of which act on the respective drawer rear wall. In the illustrated embodiment, it is provided that the front extension portion 3 has a first limit position which corresponds to the closed position of the front extension portion 3 and that the front extension portion 3 is movable from that setting by the application of pressure in the closing direction thereof into a second limit position which is disposed inwardly of the carcass 2 so that the ejection element 9 a of the ejection device 9 can be acted upon by pressure applied to the front extension portion 3. In the illustrated embodiment the ejection devices 9 and 9 ′ are not coupled and there is not a releasable coupling option as between the front panel 3 ′ and the front wall 4 ′. The opening process for the drawers 3, 4 takes place in mutually independent fashion, that is to say the internal extension portion 4 can only be extended after the front extension portion 3 has been deliberately moved into an open position by way of the ejection device 9. Subsequently, the internal extension portion 4 can be moved into the open position after separate activation of the ejection device 9 ′. FIG. 3 b shows an alternative embodiment of the invention as a side view. In contrast to FIG. 3 a, the internal extension portion 4 or the front wall 4 ′ thereof is coupled by way of a releasable coupling device 11 to the front panel 3 ′ of the front extension portion 3. The releasable coupling device 11 comprises the entrainment element 11 b shown in FIG. 1, and the coupling element 11 a. The releasable coupling device 11 is preferably operative between the rear side of the front panel ( 3 ′) of the first drawer ( 3 ) and between the front side ( 4 ′) of the front panel or front wall of the at least second drawer ( 4 ) and preferably has two preselectable modes of operation, wherein in a first mode of operation the two drawers 3 and 4 are coupled together and in a further mode of operation they are not coupled. If the ejection device 9 of the front extension portion 3 is activated—for example by applying a manual pressure or pulling force to the front panel 3 ′—then the internal extension portion 4, in a situation involving active coupling of the releasable coupling device 11, moves together with the front extension portion 3 out of the carcass 2. If the releasable coupling device 11 is not active, separate activation of the internal extension portion 4 (also by applying pressure or a pulling force to the front panel 4 ′) can be effected for moving it from its closed limit position into an open position. FIG. 3 c shows a further embodiment of the invention. A spring buffer 12, as shown in FIG. 2, or a spacer portion is operative between the front panel 3 ′ of the front extension portion 3 and the front wall 4 ′ of the internal extension portion 4. When a pressure is applied to the front panel 3 ′ the drawer 3 —starting from the illustrated limit position corresponding to the closed position of the front extension portion 3 —will be moved into a second limit position which is further into the carcass 2 so that pressure is also applied to the ejection element 9 a. Pressure is also applied to the ejection element 9 a ′ of the ejection device 9 ′ by the spring buffer 12 or the spacer portion so that this leads to joint triggering of the two ejection devices 9 and 9 ′. In that case, it can be provided that when a pulling force is applied to the front panel 3 ′, only the ejection device 9 of the front extension portion 3 is activated. The internal extension portion 4 can be activated independently of the front extension portion 3 by way of pressure or pulling pulses applied to its front panel 4 ′. FIG. 3 d shows a further embodiment of the invention. The illustrated Figure diagrammatically shows a switching element 13 operative between the two ejection devices 9 and 9 ′. The switching element 13 has at least two switching positions, wherein the switching element 13 couples the two drawers 3 and 4 in a first switching position and uncouples them in a second switching position. When the ejection device 8 of the front extension portion 3 is activated the internal extension portion 4 also moves or does not move with the front extension portion 3, depending on the respective switching position. The switching element 13 can also be part of a program logic of a control and/or regulating device 14 by which the at least two ejection devices 9 and 9 ′ are selectively controllable and actuatable. Various triggering pulses can be implemented by preferably manually applying a pulling and/or pressing force to the front panel 3 ′ of the front extension portion 3. In that respect, it can be provided for example that the ejection device 9 ′ of the internal extension portion 4 is not activated when pressure is preferably applied once to the front panel 3 ′ of the front extension portion 3. In an advantageous development, it can also be the case that the ejection device 9 ′ is activated upon preferably two successive applications of pressure to the front panel 3 ′ of the front extension portion 3 within a predetermined or predeterminable period of time. The invention is not limited to the illustrated embodiments by way of example but embraces or extends to all technical equivalents which can fall within the scope of the appended claims. The positional references adopted in the description such as for example up, down, lateral and so forth refer to the directly described and illustrated Figure and in the event of a change in position are to be correspondingly applied to the new position. The activation options described in respect of the individual ejection devices are to be interpreted only by way of example as a large number of further possible options are to be implemented for one of ordinary skill in the art concerned with the technical object here. | Summary: A piece of furniture with a furniture body and a first and at least one second drawer, displaceable relative to the furniture body, the front panel of the first drawer essentially completely covering the front panel or front wall of the second drawer in the closed position for both drawers, characterized in that the first and the at least one second drawer comprise an opening device for displacing the drawers from a closed position to an open position and a switch element is provided between both opening devices which couples both opening devices in a first switch position and uncouples the same in a second switch position. | 5,422 | 124 | big_patent | en |
Summarize: RELATED APPLICATION This application claims priority to U.S. provisional application 60/708,124 filed Aug. 12, 2005, which is incorporated in its entirety herein by reference. BACKGROUND OF THE INVENTION 1. Field of Invention This invention relates generally to medical devices, more particularly to devices for removal of stone, foreign bodies and the like from the body. 2. Discussion of Prior Art Existing techniques for extraction of stones from the body such as in the case of stones lodged in the urinary collecting system and in the biliary tree can be cumbersome, inefficient and risky with respect to complications. For example, the extraction of urinary stones often requires cystoscopic balloon dilation of the distal ureter using a high pressure balloon to increase the capacity of the ureter in order to allow decreased resistance with passage of the ureteroscope and extraction of the stone or its fragments. This high pressure balloon requires a costly pressure gauge and can be traumatic to the ureter placing the ureter at risk for stricture formation. After dilation, an ureteroscope is inserted and lithotripsy performed if the stone is too large for extraction. The stone is then engaged under direct vision with a basket or similar device and then withdrawn into the bladder where the stone is then considered passed. This technique requires that the stone be of sufficiently diminutive size (which is usually not the case with a lodged stone) or that the stone be fragmented with a device such as a laser which in itself carries the risk of injury to the ureter. Furthermore, extraction of a stone engaged in a wire basket carries the risk of frictional damage to the ureteral mucosa and wall, the risk of a retained basket engaged with stone requiring tertiary referral or open surgery, or the risk of catastrophic avulsion injury of the ureter. The above time consuming, costly and risky standard techniques begs for the development of a device which will provide a less cumbersome, safer, more efficient and less costly technique to treat the extremely common problem of urinary stone disease. Other inventors have attempted to address this vexing task. Hardwick, U.S. Pat. No. 4,469,100, proposed a device in which the stone is drawn into the balloon by intussusception, protecting the ureteral walls from the friction with stone extraction. The deficiencies of this idea include the blind passage of the device to the stone (i.e.: not under direct visualization as with the standard technique), dependence upon suction to engage a stone which has an irregular surface not amenable to suction seal for traction, and, most significantly, the device's construction where the balloon is attached to the catheter near its proximal and distal ends. The result of the latter construction is that, while the stone achieves sanctuary within the confines of the balloon's wall during intussusception, the external surface of the balloon is withdrawn in direct opposition to the ureteral wall when extracting the stone which places the ureter at risk for injury. Another inventor who attempts to solve the existing problems with stone extraction is Drettler, U.S. Pat. No. 4,927,426. Here a catheter-like device is used which allows a laser fiber for lithotripsy but suffers the same deficiencies as Hardwick's device. U.S. Pat. Nos. 4,243,040 and 4,295,464, likewise, suffer similar problems. Current techniques for biliary stone extraction also can be cumbersome, inefficient and risky for complication. Gallstones may become lodged in the biliary tree, often at the sphincter of Oddi which may result in biliary colic and cholangitis or pancreatitis. Many surgical devices and techniques exist for treatment of these stones confined to the biliary tree and unable to pass to the duodenum. Access to and extraction of biliary stones often require balloon dilation or sphincterotomy at the duodenal papilla which carries the risk of bleeding and perforation. The stone is then engaged with a basket, such as Cook's The Web™ Extraction Basket which risks, as with a urinary stone, injury of the biliary ductal system and retention of the basket. Another technique for stone extraction uses a balloon such as that described by Karpeil, et al., U.S. Pat. No. 6,692,484 B1, where the sphincter is dilated and a second balloon is used to push the stone through into the duodenum. Similar balloons, such as the Cook Endoscopy Tri-Ex® Triple Lumen Extraction Balloon, often requires sphincterotomy. These balloons in general work well but do not directly control the stone as with a basket which can sometimes leave the stone wedged between the balloon and ductal wall. Whatever the precise merits, features, and advantages of the above cited references, none of them achieves or fulfills the purposes of the present invention. SUMMARY OF THE INVENTION The device consists of a balloon, inflated and deflated through a small catheter, which is applied to the outside of a ureteroscope (or inserted through a separate guide when used in a separate application) which inverts to safely guide the removal of a stone, surgical specimen, foreign body or organ. As the object is drawn through the balloon, there is significantly less friction and therefore less risk for injury to surrounding tissue. The present invention's toroidal balloon is activated via inflation. The unwanted object is manipulated with existing techniques and then drawn into the balloon, inverting the balloon. With extraction, the object in drawn internally through the balloon, protecting the tissue from the object. The balloon is taken down from the inside and does not require the sliding of the external balloon surface against the tissue. This allows a low friction extraction since only the two opposing inner balloon surfaces are sliding against each other. Since friction is minimized and the anatomy protected from the object, larger objects may be extracted without the need for dilation or fragmentation of the object. It is thus a highly efficient safety device for removing objects from the body. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 illustrates an exemplary embodiment of the present invention's toroidal balloon used to remove an object that is lodged, for example, between the walls of the ureter. FIG. 2 illustrates one embodiment of the present invention's toroidal balloon device for attachment to a scope. FIG. 3 illustrates such an example, wherein an attachment is configured without the inflation catheter, wherein the coupling attachment inserts directly into a channel of a scope for inflation. FIG. 4 illustrates another embodiment wherein the present invention's toroidal balloon comprises a luer lock connector, valve, an inflation/deflation port, a toroidal balloon, a coupling attachment to the catheter, and a catheter containing wire to open a basket. FIG. 5 illustrates scenarios wherein the basket is closed, open, and open with balloon being inflated. FIG. 6 illustrates a first design to apply the present invention's balloon to the end of the scope. FIGS. 7 a - c illustrate another design to apply the present invention's balloon to the end of the scope. FIGS. 8 a - c illustrate yet another design to apply the present invention's balloon to the end of the scope. FIGS. 9 a - 9 c illustrates the present invention's balloon having the scope being advanced to the stone, the stone being fragmented and engaged with a basket, and the balloon being inflated, respectively. FIG. 10 illustrates how the stone is withdrawn with minimal friction based on the teaching of the present invention as illustrated and described according to FIG. 1. FIG. 11 a - f illustrate the technique of how the present invention's device can be used as a balloon instrument. DESCRIPTION OF THE PREFERRED EMBODIMENTS While this invention is illustrated and described in a preferred embodiment, the device may be produced in many different configurations, forms and materials. There is depicted in the drawings, and will herein be described in detail, a preferred embodiment of the invention, with the understanding that the present disclosure is to be considered as an exemplification of the principles of the invention and the associated functional specifications for its construction and is not intended to limit the invention to the embodiment illustrated. Those skilled in the art will envision many other possible variations within the scope of the present invention. The present invention provides for a toroidal balloon used in the extraction of objects from the body. FIG. 1 illustrates an exemplary embodiment of the present invention's toroidal balloon used to remove an object 102 that is lodged, for example, between the walls of the ureter. The inflated toroidal balloon is taken down internally, wherein the external balloon surface does not move relative to the ureter (i.e., there is no sliding of the external balloon surface against ureter). The inflated toroidal balloon inverts with minimal friction during extraction. Object 102 is drawn internally through the balloon. Reference points 104, 106, 108, and 110 are shown to illustrate how the balloon is taken down internally. The method illustrated in FIG. 1 teaches the low friction extraction of object 102 with the sliding of the two opposing inner balloon surfaces, allowing for the safe removal of a stone, surgical specimen, foreign body or organ. Inflation of balloon is preferable as it minimizes friction between the ureteroscope/basket/stone complex with extraction. It should be noted that the only significant force with the extraction described in FIG. 1 is a radial force involved with the low friction extraction, wherein the force distribution similar to that of a wedge splitting wood. Also, since the stone itself dilates the ureter (just enough for stone passage, no more), there are no overdilation problems. Hence, the present invention's toroidal balloon minimizes ureteral dilation injury. FIG. 2 illustrates one embodiment of the present invention's toroidal balloon device for attachment to a scope. The device as per the embodiment of FIG. 2 comprises luer lock connector 201, valve 202, inflation/deflation catheter 203, toroidal balloon 204, coupling attachment 205 and, optionally, a sheath 205 along catheter to guide insertion of scope. Connector 201 is a conventional luer lock threaded to receive a conventional syringe in an air/watertight connection. It should be noted that a no pressure gauge is required for low pressure balloon for most applications. The Luer lock connection may be part of a locking valve 202 to control inflation or deflation. The valve is then connected to a catheter 203 which on the opposite end is connected with the toroidal balloon 204. The catheter 203 provides a conduit for the fluid such as saline, or a biocompatible lubricant used for inflation of the balloon. Catheter 203 is sufficient long to allow extracorporal inflation of balloon 204 (e.g., approximately 20-50 cm in length). The flexible catheter 203, constructed of polyethylene or other appropriate material, runs through the inner opening in the balloon 204, attaching at the leading end of the balloon only. Inflation/deflation catheter 203 provides a conduit for fluid such as saline or other biocompatible lubricant used for in/deflation of balloon. The proximal end of the inflation/deflation catheter 203 attaches to syringe, distal to balloon and is sufficiently long to allow extracorporal inflation (e.g., 20-50 cm in length). The inflation/deflation catheter 203 attaches at balloon's leading edge and runs internal to balloon, external to scope, and is made of polyethylene or other material. Further, the inflation/deflation catheter 203 may not be necessary with specialized endoscope (i.e., if balloon attaches directly to scope channel) In one embodiment, the inflation/deflation catheter 203 has a non-cylindrical construction for low-profile. Balloon 204 may be constructed of an expandable material such as latex rubber, silicone or other medical grade material/elastomer. Balloon 204 may have a lubricious coating to facilitate insertion into the ureter. Balloon 204 may be coated with a biologically active substance such as an alpha-blocker or smooth muscle relaxant. Balloon 204 may have reinforcements in the wall to maintain shape and pressure. Dilation of the ureter is only a minor role of the balloon so construction with a high tension substance is unnecessary. A purpose of inflation is to minimize friction between the ureteroscope/basket/stone complex with extraction. Balloon 204 achieves this low friction extraction with the sliding of the two opposing inner balloon surfaces. In one non-limiting example, the length of the balloon is 2 to about 20 cm long. The length depends upon the distance from the stone to just beyond the ureteral orifice, as determined by the Urologist. Attachment 205 of the catheter to the balloon may be of plastic or metal (alloy) and be shaped to allow simultaneous filling (or emptying) of the balloon 204 and fixation of the balloon/catheter device to the distal end of the endoscope such as a ureteroscope. Existing ureteroscopes vary in construction so the construction of the attachment will be specific to the ureteroscope. The attachment will also be formulated to allow loading of the balloon over the ureteroscope. Attachment 205 attaches inflation/deflation catheter 203 to balloon 204 and attaches the device to a telescope. Attachment 205 facilitates loading of balloon 204 onto the telescope and facilitates insertion of the scope into ureteral orifice (e.g., may be. tapered/curved at the leading edge). Attachment 205 could also helps seal the balloon closed in manufacturing process. Attachment 205, in one non-limiting example, is made using plastic or metal (alloy). Attachment 205 may be constructed for a water-tight attachment to endoscope channel for inflation. FIG. 3 illustrates such an example, wherein attachment 205 is configured without inflation catheter wherein the coupling attachment 205 inserts directly into a channel of a scope for inflation. The toroidal balloon device may also be fashioned with dimensions and materials for other applications such as removal of gall stones, foreign bodies, surgical specimens, etc. The device is of simple construction, of low cost and disposable. The balloon is attached to the extraction device at just one level at the distal aspect, so the stone can both be drawn via intussusception into the protective confines of the inner surface and also drawn out the entire length of the balloon and out of the patient via intussusception. The device in Hardwick's patent (U.S. Pat. No. 4,469,100) intussuscepts slightly to cover the stone but is not a toroidal balloon since it is attached both distally and proximally. Hardwick's double attachment may allow minimal inversion of the balloon to cover the stone before the balloon is pulled out, but intussusception of the balloon is not the mechanism by which a stone is extracted. The ability to invert our entire balloon is the important and unique benefit of our device. During stone extraction using Hardwick's doubly attached balloon, after a short length of intussusception, the outer surface of his balloon must be dragged across the surface of the ureter during extraction. It seems doubtful that the friction and other physical forces would allow extraction of an inflated balloon through a tight tube such as the ureter. There is no balloon in common use which is withdrawn in the ureter while inflated. The present invention's 204 balloon is taken down internally with minimal friction. By contrast, Hardwick's balloon is extracted by sliding the external balloon wall against the ureteral wall. In our balloon, the only walls that slide against each other are the two internal surfaces of the balloon, wetted by the fluid used to inflate the balloon. Unlike Hardwick's balloon, the outside wall of our balloon remains stationary with respect to the ureteral wall until it inverts during the extraction. Another important difference is that the present invention's balloon facilitates existing techniques for stone extraction. The present invention's device is added external to the ureteroscope without affecting existing techniques for stone manipulation and adds safety and efficiency to the process of stone extraction. Ureteral stones are addressed, fragmented if necessary and engaged per routine with existing devices (ureteroscopes, laser fibers and wire baskets, respectively). Urologists won't need to learn a new technique for stone manipulation. They'll need only to inflate the sleeve (balloon) for extraction. Hardwick device's, in contrast to our balloon, replaces the ureteroscope and depends upon suction to grasp the stone. This requires blind passage of the device, not under safe direct visualization as with ours, to the level of the stone, which would be difficult and risky. The ability to grasp a jagged stone by suction is suspect. In addition, suction would engage the ureteral wall. FIG. 4 illustrates another embodiment wherein the present invention's toroidal balloon is used as an instrument, wherein the setup comprises a luer lock connector 401, valve 402, inflation/deflation port 403, toroidal balloon 404, coupling attachment to the catheter 405, and catheter containing wire to open basket 406. The principle of functionality is similar to that of what is outlined in FIG. 1. FIG. 5 illustrates scenarios wherein the basket is closed, open, and open with balloon being inflated. FIG. 6 illustrates a first design to apply the present invention's balloon to the end of the scope. In this example, the balloon is constructed over a tube matching the external conformation of the scope 601. The length of the balloon is at least the distance from the stone to uretral orifice. FIGS. 7 a - c illustrate another design to apply the present invention's balloon to the end of the scope. As shown in FIG. 7 a, the balloon is pre-rolled and attached to the end of the scope 701 and, as shown in FIGS. 7 b - c, the pre-rolled balloon is then unrolled, line a condom, onto the telescope. As mentioned previously, the length of the balloon is at least the distance from the stone to uretral orifice. FIGS. 8 a - c illustrate yet another design to apply the present invention's balloon to the end of the scope. As shown in FIG. 8 a, the balloon is inflated before application to the scope 801. As shown in FIG. 8 b, the inflated balloon is attached to the end of the scope 801 and, then, rolled back over the scope 801. As shown in FIG. 8 c, the balloon is then deflated and is ready for insertion into the patient. FIG. 9 a illustrates the present invention's balloon having the scope being advanced to stone 901. The stone is then fragmented with a laser, if necessary, and is engaged within basket 902 as shown in FIG. 9 b. In FIG. 9 c, the toroidal balloon is then inflated. FIG. 10 illustrates how the stone 901 is withdrawn with minimal friction based on the teaching of the present invention as illustrated and described according to FIG. 1. FIG. 11 a - f illustrate the technique of how the present invention's device can be used as a balloon instrument. According to FIG. 11 a, basket 1102 is inserted into the scope. According to FIG. 11 b, the basket 1105 is advanced into the duct and opened. According to FIG. 11 c, the stone 1106 is engaged in basket 1105. Next, as shown in FIG. 11 d, the balloon is inflated. FIG. 11 e depicts an example wherein the balloon is inflated within the duct with the stone engaged in the basket. FIG. 11 f illustrates how the stone is withdrawn with minimal friction based on the teaching of the present invention as illustrated and described according to FIG. 1. The present invention can be used in the extraction of various objects, including, but not limited to: percutaneous stone extraction (PCNL), bladder stones, urethral stones, tracheal foreign bodies, rectal foreign bodies, surgical specimens, endotracheal tubes, or virtually in any endoscopic procedure. Therefore, the present invention provides a toroidal balloon of a simple construction and has a low-cost of manufacturing. Further, the present invention's toroidal balloon provides for a low risk of device failure. The present invention's device may be a disposable toroidal balloon. The present invention's device may also be fashioned with dimensions and materials for other applications such as removal of gall stones, foreign bodies, surgical specimens, etc. The present invention allows for the development of specialized ureteroscopes, such as a smaller scope with a balloon attachment for stone basketing and extraction only. Further, the present invention's toroidal balloon is applicable to multiple medical and veterinary specialties and body systems. CONCLUSION A system and method has been shown in the above embodiments for the effective implementation of a method and device for extracting objects from the body. While various preferred embodiments have been shown and described, it will be understood that there is no intent to limit the invention by such disclosure, but rather, it is intended to cover all modifications and alternate constructions falling within the spirit and scope of the invention, as defined in the appended claims. For example, the present invention should not be limited by size, materials, or specific manufacturing techniques. | Summary: A device for extracting objects from the body, such as urinary stones, using a low pressure inflatable toroidal balloon that serves to engulf the object during extraction while dilating and protecting the passageway. The balloon loads onto an ureteroscope prior to insertion, rather than through the ureteroscope as do existing balloons. The toroidal balloon is a simple and unique device that may be applied external to the extracting telescope and does not interfere with existing methods for stone manipulation such as laser lithotripsy, irrigation and basket extraction in the case of urinary stone manipulation. | 5,213 | 130 | big_patent | en |
Write a title and summarize: The fate of neural progenitor cells (NPCs) during corticogenesis is determined by a complex interplay of genetic or epigenetic components, but the underlying mechanism is incompletely understood. Here, we demonstrate that Suppressor of Mek null (Smek) interact with methyl-CpG–binding domain 3 (Mbd3) and the complex plays a critical role in self-renewal and neuronal differentiation of NPCs. We found that Smek promotes Mbd3 polyubiquitylation and degradation, blocking recruitment of the repressive Mbd3/nucleosome remodeling and deacetylase (NuRD) complex at the neurogenesis-associated gene loci, and, as a consequence, increasing acetyl histone H3 activity and cortical neurogenesis. Furthermore, overexpression of Mbd3 significantly blocked neuronal differentiation of NPCs, and Mbd3 depletion rescued neurogenesis defects seen in Smek1/2 knockout mice. These results reveal a novel molecular mechanism underlying Smek/Mbd3/NuRD axis-mediated control of NPCs’ self-renewal and neuronal differentiation during mammalian corticogenesis. Neural stem cells (NSCs) are self-renewing, multipotent cells that generate major neural cell types, including neurons and glia, in the developing central nervous system (CNS) [1,2]. During neurogenesis, NSCs are derived from neuroepithelial cells (NECs), which first divide symmetrically to expand the population and then undergo a series of asymmetric cell divisions to produce neural progenitor cells (NPCs), lineage-restricted precursor cells (RPCs), and mature neural cells [3]. NSC fate determination is tightly regulated by intrinsic and extrinsic factors [4–6]. Recent findings suggest that neurodevelopmental and neurological anomalies, such as schizophrenia, autism, and depression, can emerge from abnormal specification, growth, and differentiation of NSCs [6–8]. Suppressor of Mek null (Smek), an evolutionarily conserved protein family, consists of two isoforms, Smek1 (PP4R3A) and Smek2 (PP4R3B), first reported as playing a role in the formation of a functional phosphatase group with PP4c, PP4R1, and PP4R2 complex [9]. Smek was initially identified in Dictyostelium discoideum as a playing a role in cell polarity, chemotaxis, and gene expression [10]. Smek also has several functions in lower eukaryotes, such as Caenorhabditis elegans, including roles in longevity by modulating DAF-16/FOXO3a transcriptional activity [11], DNA repair through dephosphorylation of phosphorylated H2AX (g-H2AX) during DNA replication [12], and glucose metabolism by controlling cAMP-response element binding protein (CREB) -regulated, transcriptional coactivator 2 (CRTC2) -dependent gene expression [13]. Notably, Smek also plays a critical role in cell-fate determination in higher eukaryotes. In Drosophila neuroblasts, PP4R3/Falafel (Flfl), which is an orthologous of Smek and is conserved throughout eukaryotic evolution, regulates asymmetric cell division by controlling localization of Miranda [14–16]. In mice, which express orthologous Smek 1 and 2, both Smek proteins suppress brachyury expression in embryonic stem cells (ESCs), and Smek1, especially, promotes NSC neuronal differentiation by negatively regulating Par3 [14–16]. Although we have shown that the Smek isoform Smek1 promotes NSC neuronal differentiation, signaling pathways required for that activity remain unclear [15]. Methyl-CpG–binding domain protein 3 (Mbd3), a core component of the repressive nucleosome remodeling and deacetylase (NuRD) complex, possesses a conserved methyl-CpG–binding domain (Mbd) [17,18]. Unlike other family members, which recognize 5′-methyl-cytosine (5′-mC) -modified DNA, Mbd3 specifically recognizes 5′-hydroxymethyl-cytosine (5′-hmC), an epigenetic marker highly enriched in NSCs [19,20]. Mbd3 plays an important role in brain development. Mbd3 expression is reported to be predominant in cortical NECs of the embryonic forebrain [21]. Mice lacking Mbd3 die in utero before neurogenesis is completed [22]. Conditional knockout of Mbd3 in neural progenitor cells leads to defects of differentiation of appropriate cell types during neurogenesis [23]. Despite emerging evidence that Mbd3 has a critical function in the CNS, little is known about its regulatory mechanism in NSCs. To understand Smek protein function during mammalian CNS neurogenesis, we screened for novel Smek-binding proteins that regulate NPC neuronal differentiation and identified Mbd3, a potent epigenetic regulator, as a Smek-interacting protein. We found that Mbd3 is highly expressed in NPC populations in the ventricular zone, and it was predominantly expressed in the nucleus. Smek interacted directly with the Mbd3’s Mbd domain, destabilizing Mbd3 protein and its interaction with NuRD components, and sequentially, preventing accumulation of the Mbd3/NuRD complex on target gene loci functioning in neurogenesis. Such dissociation of Mbd3/NuRD complex promotes NPC neuronal differentiation. Moreover, overexpression of Mbd3 significantly inhibited neuronal differentiation of wild-type NPCs, while Mbd3 depletion rescued neurogenesis defects seen in Smek knockout mice. This work identifies a novel pathway of Smek and Mbd3/NuRD complex in brain development and could encourage discovery of novel epigenetic regulators governing neuronal differentiation. Recently, we reported that Smek1 promotes neurogenesis during mouse cortical development [15]. To further characterize Smek function in neurogenesis, we generated Smek1 and Smek2 double knockout (dKO) mice and set out to analyze cortical development in Smek1 knockout (KO) and Smek1 and Smek2 dKO embryo brains (S1A and S1B Fig). To do so, we undertook immunohistochemical analysis of the embryonic cortex derived from WT and Smek1/2 dKO mice and observed a decrease in the number of cells positive for Tuj1, an early neuronal marker (~15% and ~25% fewer Tuj1+ cells at E12. 5 and E14. 5, respectively) (Fig 1A). We observed similar decreases in postmitotic cortical neuron marker Tbr1-positive cells (~15% and ~20% reductions at E12. 5 and E14. 5, respectively) and Tuj1 and Tbr1 double-positive cells (near 20% reduction in each stage) (Fig 1A). The number of mature microtubule-associated protein 2 (MAP2) -positive neurons also significantly decreased by ~12% in the E12. 5 cortex (Fig 1A, right and S1C Fig). In Smek1/2 mice, the number of Pax6-positive NPCs increased significantly (by ~23% at both E12. 5 and E14. 5), as did Nestin/Ki67 double-positive cells (~24% increase at E12. 5) compared with wild-type (WT) mice (Fig 1B and S1D–S1F Fig). As expected, neurogenesis defects in Smek1/2 dKO embryonic brains were greater than those seen in Smek1 KO mice (S2A and S2B Fig, S1 Table). These results demonstrated that in the Smek1/2 dKO mice, the number of neurons is reduced while the number of neural stem cells is increased. To assess direct effects of Smek loss on NPC differentiation capacity, we cultured Smek1/2 dKO NPCs derived from E11. 5 mouse embryo brains in the presence of basic fibroblast growth factor (bFGF) and then withdrew bFGF to induce neural differentiation. Consistent with in vivo results, the number of Tuj1-positive cells decreased in Smek1/2 dKO cultures while the number of Nestin positive cells increased slightly in Smek1/2 dKO cultures over the course of differentiation (Fig 1C and S3 Fig). In addition, we assessed the role of Smek in maintenance of self-renewal activity using the single-cell clonal neural sphere formation assay. Smek1/2 dKO NPCs showed higher sphere-forming ability than those derived from WT NPCs in both primary and secondary sphere-forming assays (Fig 1D). Further analysis using quantitative PCR (qPCR) showed that Smek1/2 dKO cells exhibited decreased expression of Dlx1, Dlx2, Tuj1, Gad67, NeuN, and NeuroD1, as well as other neural differentiation genes such as Gfap and Mbp (the latter an oligodendrocyte marker), and increased expression of Nestin (Fig 1E and S3 Fig). Severe differentiation defects seen in cortical NPCs lacking both Smek 1 and 2 suggest that these factors compensate for each other during cortical development. All these experiments demonstrated that Smek plays a role in self-renewal and neural differentiation of NPCs in vivo and in vitro. In order to determine a detailed molecular mechanism modulating the differentiation of NPCs by Smek, we sought to identify proteins interacting with Smek protein. To identify how Smek mediates neuronal differentiation of NPCs, a yeast two-hybrid (Y2H) screening assay was performed and revealed that Smek interacts with full-length Mbd3 (Fig 2A and S2 Table). An immunoprecipitation (IP) assay confirmed the interaction of Smek1 and Smek2 with Mbd3 in 293T cells (Fig 2B, S4A and S4B Fig). As shown in Fig 2B and S4B Fig, Smek1 or Smek2 coimmunoprecipitate with Mbd3, while no signals were detected in cells transfected with a negative control vector. Furthermore, colocalization of endogenous Mbd3 and Smek2 protein was also observed in the nucleus of in vitro cultured NPCs (Fig 2C). Both Smek1 and Mbd3 proteins were expressed in NPCs of the ventricular zone (VZ) and subventricular zone (SVZ) in vivo (Fig 2D, S4C and S4D Fig), and merged images reveal that Smek and Mbd3 staining was nuclear (Fig 2C and 2D). Mbd3 is highly enriched in the nucleus of the VZ progenitor cells and its expression in the nucleus showed a gradually decreasing pattern in the direction of the intermediate zone (IZ) and cortical plate (CP). Smek1 is expressed in the nuclear or perinuclear region of cortical progenitor cells in the VZ, but its expression and nuclear localization is significantly increased in the differentiated neurons in the IZ and CP (Fig 2D, S4C and S4D Fig). The interaction of endogenous Smek and Mbd3 were further confirmed by co-IP of Smek and Mbd3 using NPC lysates. Smek/Mbd3 interaction, however, was apparently disrupted during neuronal differentiation of wild-type NPCs, suggesting that protein complexes may function in NPC differentiation (Fig 2E and S4C Fig). To map Smek and Mbd3 domain (s) required for interaction, we generated Smek1 and Mbd3 mutants and assessed their interaction (Fig 2F–2H). A Smek N-terminal deletion mutant (ΔRanBD) did not interact with Mbd3 protein, shown by co-IP of Smek and Mbd3 in HEK293T cells, whereas interaction of other Smek deletion mutants with Mbd3 was comparable to the full-length protein, suggesting the RanBD domain of Smek mediates Smek’s interaction with Mbd3 (Fig 2F). To map to the domains on Mbd3 required for Smek interaction, we generated glutathione-S-transferase (GST) fusion Mbd3 mutant proteins in bacteria and incubated these proteins with HEK293T cell lysates expressing Smek2. GST pull-down assays revealed that the Smek-Mbd3 interaction was disrupted in Mbd3 deletion mutants lacking the first 92 N-terminal amino acids (ΔN92), a region encompassing the Mbd domain (Fig 2G, upper panel). This finding was confirmed by IP experiments in HEK293T cells transfected with full-length or ΔN92 forms of Mbd3 and Smek (Fig 2H). Those results indicated that the Mbd domain of Mbd3 is required for Smek interaction. To characterize Smek and Mbd3 expression during neural differentiation, we examined protein and mRNA levels after withdrawal of bFGF from NPC culture media. In NPCs, Mbd3 protein levels gradually decreased by day 1 of differentiation, while Mbd3 protein levels did not decrease in Smek1 and Smek2 single KO or dKO NPCs (Fig 3A–3C and S5A, S5B Fig). However, Mbd3 mRNA levels did not change during differentiation in both WT and Smek1, Smek2, or Smek1/2 dKO NPCs, suggesting that changes in Mbd3 protein levels that occur during differentiation require Smek (Fig 3D and 3E and S5A Fig, lower panel). These observations led us to further analyze Mbd3 stability. We found that Mbd3’s half-life was ~6 h in NPCs and 293T cells in which new protein synthesis was inhibited and significantly prolonged in the presence of the proteasomal inhibitor MG132 (Fig 3F). Endogenous Mbd3 protein levels in NPCs were increased by MG132 treatment (Fig 3G). Overexpressed Mbd3 protein was polyubiquitylated, and polyubiquitylated proteins accumulated in cells treated with MG132 or MG-101, respectively (Fig 3H and 3I and S5C, S5D Fig). Furthermore, endogenous Mbd3 in NPCs was found to be polyubiquitylated as well (Fig 3J). To determine whether Smek regulates Mbd3 protein stability, we monitored endogenous Mbd3 protein levels in wild-type NPCs, in HEK293T cell lines stably overexpressing Smek1 or Smek2 (S5E and S5F Fig), or in NPCs derived from wild-type and Smek1/2 dKO embryonic mouse brains (Fig 4A and 4B). Mbd3 protein turnover rate was increased by overexpression of either Smek1 or Smek2 and decreased upon Smek loss (Fig 4A and 4B). Consistent with Mbd3 degradation, Smek1 or Smek2 overexpression in HEK293T cells significantly promoted Mbd3 polyubiquitylation (Fig 4C and S6A Fig). Furthermore, Mbd3 was ubiquitylated in wild-type NPCs but not in Smek1/2 dKO NPCs (Fig 4D). To further assess the effects of Smek expression on Mbd3 degradation, we examined Mbd3 ubiquitylation following expression of the Mbd3 ΔN92 mutant, which cannot interact with Smek. Polyubiquitylation of this mutant was not significantly changed by overexpression of either Smek1 or Smek2 (Fig 4E and S6B Fig). These results suggest that interaction with Smek destabilizes Mbd3. Smek has a nuclear localization signal (NLS) and is nuclear localized [15], suggesting a potential role in regulating transcription. To determine whether the Smek protein is associated with chromatin—and if so, whether it is enriched on chromatin loci of neurogenesis-associated genes—we performed chromatin immunoprecipitation sequencing (ChIP-seq) in NPCs using a Smek1 antibody. Genome-wide binding profiling demonstrated that Smek proteins bind to chromatin loci of genes related to organ morphogenesis, cell-fate determination, and CNS development and differentiation (Fig 5A and 5B and S7A, S7B Fig). Furthermore, Smek specifically bound proximal promoter regions and gene bodies of neuronal genes such as Dlx1, Dlx2, Tlx3, NeuroD1, Ascl1, and Lbx1, which were known to highly express in neuron or proneuronal cells (Fig 5A–5C and S7C, S7D Fig). Unlike Smek, which lacks a known DNA-binding motif, Mbd3 exhibits the Mbd domain, which reportedly binds 5′-hydroxymethyl cytosine (5′-hmC) regions [20]. We therefore asked whether Smek and Mbd3 share similar genomic regions in NPCs, initially by determining whether Mbd3 binds neuronal gene promoters that Smek binds to. To do so, we undertook ChIP-qPCR with a Mbd3 antibody in wild-type and Smek1/2 dKO NPCs cultured in undifferentiation or differentiation conditions. This analysis confirmed enrichment of Mbd3 on the Smek-bound loci of genes including Dlx1, Dlx2, Tlx3, NeuroD1, Ascl1, and Lbx1 in undifferentiated conditions (Fig 5D and S7C Fig). Moreover, Mbd3 enrichment on these gene loci significantly decreased under differentiation conditions in wild-type NPCs but was unchanged in Smek1/2 dKO NPCs under the same conditions (Fig 5D and S7C Fig). Then we asked whether enrichment of the Smek1 protein on chromatin loci of neurogenesis-associated genes is dependent on Mbd3 protein, and we performed ChIP-qPCR with Smek1 and Mbd3 antibodies in shScramble or shMbd3 knockdown (KD) NPCs cultured in undifferentiation or differentiation conditions. These results demonstrated that occupancy of Smek1 on the promoters of Dlx1, Dlx1as, Tlx3, NeuroD1, Ascl1, and Lbx1 genes, but not Gfap genes, is dependent on Mbd3 protein (Fig 5E and S7F Fig). Mbd3 has been reported to represses transcription by recruiting the NuRD complex to target gene loci [20]. Co-IP experiments showed that Mbd3 interacted with MTA1, RbAP46, HDAC1, and HDAC2, which are components of NuRD complex (S7G Fig). We then undertook ChIP-qPCR with HDAC1, HDAC2, MTA1, and acetyl histone H3 antibodies using wild-type and Smek1/2 dKO NPCs cultured in differentiation conditions. ChIP analysis revealed that enrichments of NuRD components HDAC1, HDAC2, and MTA1 to target gene loci were significantly increased in Smek1/2 dKO NPCs when compared with those of the wild type (Fig 5F). Inversely, the amount of acetyl histone H3 was decreased (Fig 5F). These findings suggest that Smek inhibits enrichment of Mbd3/NuRD complex to neurogenesis-associated gene loci and increases acetyl histone H3 activity for their gene transcription during neurogenesis. These data also suggest that NuRD activity is dependent on Mbd3 ability, which can bind to target DNA, and Smek, as an upstream regulator of Mbd3/NuRD complex, promotes Mbd3 degradation, potentially allowing transcription of neuronal differentiation–associated genes by disrupting association and enrichment of Mbd3/NuRD complex on target gene loci. Our findings suggest that Mbd3 regulates expression of neuronal target genes in NPCs, an activity modulated by Smek. To assess whether Mbd3 represses neurogenesis-associated target genes, we overexpressed full-length or mutant (ΔN92) Mbd3 in NPCs and then induced differentiation over 2 d. Full-length Mbd3 (but not mutant form) overexpression attenuated Dlx1, Tlx3, NeuroD1, Tuj1, Gad67, and NeuN gene expression, all neuronal lineage markers, but had no effect on glial cell differentiation or gene expression (Fig 6A and S8A–S8D Fig). As noted above, Mbd3 bound specifically to the Dlx1, Tlx3, NeuroD1, Ascl1, and Lbx1 gene loci, and this association decreased upon induction of differentiation conditions (Fig 5D). Thus, we asked whether decreased neuronal gene expression following Mbd3 overexpression paralleled increased occupancy of Mbd3 on target promoters (Fig 6B). As expected, the amount of overexpressed Mbd3 bound to gene promoters in NPCs in differentiation conditions over 2 d was similar to that seen in nondifferentiation conditions, leading to attenuate Dlx1, Tlx3, NeuroD1, Tuj1, Gad67, and NeuN gene expression, all markers of neuronal lineage (Fig 6A and S8B Fig). In contrast, there was little or no accumulation of Mbd3 on the Gfap promoter under the differentiation condition over 2 d for NPCs after Mbd3 overexpression, suggesting that Mbd3 blocks neuronal rather than glial cell differentiation (Fig 6B and S8B Fig). Immunocytochemistry analysis also showed that Mbd3 overexpression prevented NPC neuronal differentiation but did not affect astrocyte differentiation (Fig 6C, 6D and S8C, S8D Fig). Moreover, in both control and Mbd3-overexpressing NPCs, Nestin staining was comparable in staining intensity (S8C Fig). These data suggest that Mbd3 is a novel regulator for neuronal cell-fate determination of NPCs. To further investigate whether the Smek-Mbd3 axis regulates neurogenesis, we knocked down endogenous Mbd3 using an shMbd3 lentiviral vector in cultured Smek1/2 dKO NPCs and then induced differentiation for 2 d. Mbd3 KD significantly rescued effects of Smek loss on Dlx1, Tlx3, NeuroD1, Tuj1, Gad67, and NeuN expression but had no effect on astrocyte differentiation or gene expression (Fig 7A). Increased neuronal gene expression seen following Mbd3 KD was accompanied by decreased occupancy of target gene promoters by Mbd3 (Fig 7B). The epistatic relationship of Mbd3 and Smek in neurogenesis was analyzed by Mbd3 knockdown in Smek1/2 dKO NPCs. Mbd3 shRNA were expressed from the same vector that coexpressed enhanced green fluorescent protein (EGFP). The percentage of EGFP and Tuj1 double-positive cells among EGFP-positive cells was increased in cultures expressing Mbd3 shRNA but not in cells expressing Scramble shRNA (Fig 7C). We next assessed Mbd3 function in neurogenesis using an in utero electroporation system. Electroporated embryos were readily identifiable by EGFP expression (Fig 8A). About 74% of total EGFP-positive Mbd3 knockdown cells migrated toward the IZ or CP, while only ~39% of EGFP-positive control cells showed a similar migration pattern (Fig 8B and 8C). Quantitative analyses showed that the number of Tuj1-positive cells significantly increased in the VZ, SVZ, and IZ regions in Mbd3 KD EGFP-positive cells relative to control EGFP-positive cells (Fig 8D). These results strongly suggest that Mbd3 regulates NPC neuronal differentiation in the VZ or SVZ during cortical development. Here, we have analyzed mouse embryos lacking functional Smek1 and Smek2 genes as well as cultured NPCs derived from those animals to understand Smek function during cortical development. We discovered that Smek1/2 dKO NPCs exhibit significantly reduced capacity for neuronal differentiation and increased self-renewal activity. Furthermore, we employed a Y2H screen to search for Smek binding partners and identified Mbd3 as a novel Smek-interacting protein (Fig 2A and S2 Table). Importantly, we observed that Smek promotes Mbd3 protein degradation and reduces Mbd3 occupancy of neural differentiation–associated gene promoters, likely increasing transcription of those genes via inhibiting recruitment of the repressive NuRD complex. Interestingly, in the developing CNS, increased Mbd3 instability had an effect only on neuronal differentiation, with little or no effect on glial cell fate (Figs 6,7 and S7F and S8B–S8D Figs). We could not determine the molecular mechanism by which the Smek-Mbd3 axis specifically regulates neuronal cell-fate determination but not glial cell fate. In our previous study, we found that protein phosphatase PP4c interacts with Smek and this complex suppressed Par3 activity for differentiation of NPCs [15]. PP4c is known to regulate neuronal cell-fate determination and organization of early cortical progenitors in the ventricular zone of the embryo brain by modulating spindle orientation during mitosis [24], and we could confirm the role of PP4c in neuronal differentiation of NPC by a PP4c loss-of-function study (S9A Fig). Interestingly, knockdown of PP4c significantly abolished neuronal cell as well as glial cell differentiation of NPCs, similar to Smek loss of function, and this finding suggests that Smek/PP4c/Par3 might have a different biological function from Smek/Mbd3 in at least regulating glial cell gene expression of NPCs (Fig 1E and S9A Fig). Moreover, Par3 regulation of Smek/PP4c during neurogenesis exclusively occurs in a cytosolic fraction but not in the nucleus of NPCs [15]. However, Smek and Mbd3 expression and transcriptional repression of Mbd3/NuRD complex mainly occurs in the nucleus of NPCs (Fig 2C and 2D). Our preliminary investigation of the relationship between Smek-PP4c complex and Mbd3 protein stability also reveals that loss of PP4c could not affect Smek-mediated Mbd3 polyubiquitylation (S9B Fig). In addition, ChIP-seq and ChIP-qPCR data show that Smek and Mbd3 are not significantly enriched at Gfap gene loci (S7F Fig). Thus, overall data suggest that the Smek-Mbd3 axis likely functions independently of the Smek-PP4c-Par3 axis, at least in regulation of Gfap gene expression, and that the Smek-Mbd3 interaction plays a crucial role in neuronal cell-fate determination in NPCs. So far, five vertebrate MBD proteins have been identified as members of the MBD protein family: Mbd1, Mbd2, Mbd3, Mbd4, and MECP2 [25,26], and these members are more highly expressed in the brain than in other tissues, leading investigators to hypothesize that they may play a critical role in normal brain development and in behavior [27,28]. Our data indicate that Mbd3 represses neurogenesis and likely functions differently from other family members. For example, Mbd2 and Mbd3 are closely related and share a highly conserved methyl-CpG–binding domain, but mouse studies indicate that the two proteins are not functionally redundant [24], possibly because Mbd3 specifically recognizes methylated DNA, especially, 5′-hydroxymethylcytosine (5′-hmC) [19]. Deletion of Mbd3 gene in neural progenitor cells leads to generation of neurons expressing both deep- and upper-layer markers [23], suggesting that Mbd3 is required to maintain appropriate transcription in progenitor and neurons during neural development. A recent study suggests that Mbd3 may fine-tune expression of both active and silent genes [29]. Other studies suggest that conversion of 5′-mC to 5′-hmC coincides with increased transcriptional activity by excluding Mbd proteins from target genes [30]. Consistent with these findings, we found that Mbd3 is specifically bound to neuronal gene loci, and our findings suggest that it is likely released from these loci by Smek during NPC differentiation (Fig 5D). Mbd3 is a subunit of the NuRD complex, which has nucleosome remodeling and histone deacetylase activities [17,18] and thus regulates gene expression. The molecular function of this complex has been extensively studied in the context of tumorigenesis, stem cell pluripotency, and brain development [23,31–34]. Mbd3 mutation or abnormal expression may function in tumorigenesis by perturbing gene expression. Like Mbd3, other Mbd proteins, especially Mbd2 and Mbd4, are associated with progression of cancer such as colorectal cancer, albeit by different mechanisms [34–36]. Furthermore, Mbd3 knockdown during somatic cell reprogramming significantly increases reprogramming efficiency [31–33]. Although Mbd3 activity is likely relevant to pathologies seen in cancer, neurological disease, and developmental defects, mechanisms underlying its regulation remain unclear. We propose a novel function in which Mbd3 protein levels, depending on Smek activity, decrease during neurogenesis (Figs 2D, 3A–3C and S4C and S4D, S5A and S5B Figs). Smek1/2 dKO NPCs or the embryonic cortex show aberrantly high Mbd3 levels that may repress neuronal gene expression and underlie developmental defects seen in the latter. In accordance, we report that Smek promotes ubiquitylation and degradation of Mbd3 (Figs 3A–3C, 4A–4D and S5A–S5B, S6A Figs). Our data also indicate that Smek regulation of Mbd3 is not transcriptional, based on the lack of significant change in Mbd3 mRNA levels over NPC differentiation. Conversely, Mbd3 protein levels decreased during neuronal differentiation in the embryonic cortex starting at E12. 5 in mice. In addition, decreased Mbd3 levels seen in cultured NPCs are blocked by MG132 treatment concomitant with accumulation of polyubiquitylated Mbd3. These results overall indicate that Mbd3 activity is regulated at the level of protein stability and that Smek likely governs this process. Changes in protein stability often constitute a more rapid means of regulating protein activity than does modulation of transcription. Therefore, regulation of Mbd3 protein stability might function epigenetically to recruit the NuRD complex to 5′-hmC–modified gene promoters. To our knowledge, this is the first report of regulation of an Mbd family protein by stability changes. We also examined potential factors or complexes that might function in Smek-dependent Mbd3 degradation. To do so, we sought potential E3 ligase proteins that might catalyze Mbd3 ubiquitylation by using Biograph software and identified the E3 ligase TRIpartite Motif protein 33 (TRIM33) protein, which has an N-terminal Really Interesting New Gene (RING) -domain (S10 Fig). TRIM33 specifically targets phosphorylated nuclear proteins for degradation [37]. Interestingly, we have previously identified protein kinase C (PKC) lambda/iota (λ/ι), a serine/threonine kinase, as a binding partner of Smek1 from a mass spectrometry analysis [15]. PKC isoforms contain an NLS and contribute diverse cellular physiology [38–40]. Smek1/2 also have NLS sequences and are localized exclusively in the nucleus in interphase [15]. To further investigate the involvement of PKCλ/ι in the molecular mechanism for Mbd3 protein stability, we performed prediction of putative kinases for phosphorylation of Mbd3 protein by GPS (group-based prediction system) software 3. 0 (http: //gps. biocuckoo. org/). (S3 Table). Interestingly, we predicted PKCλ/ι as putative kinases for Mbd3 phosphorylation. Although it still remains unclear how Smek1/2 promotes ubiquitylation and stability of Mbd3, accumulating data and predictions suggest that nuclear-localized Smek1/2-PKCλ/ι complex with TRIM33 may function in Mbd3 ubiquitylation and degradation. Alternatively, Aurora-A protein, a serine/threonine kinase, reportedly physically associates with Mbd3 at centrosomes in early M phase in vivo and phosphorylates Mbd3 protein in vitro [41]. These findings suggest that Aurora-A may also be involved in the regulation of Mbd3 protein stability as a different mechanism from Smek-PKCλ/ι complex. This topic will be addressed in future studies. Smek orthologues in Drosophila play a critical role in neuroblast mitosis [16]. In Smek-deficit neuroblasts, cell-fate determinants, such as Prospero and Miranda, are no longer localized to the cell cortex; instead, they are distributed in the cytoplasm of dividing neuroblasts [16]. As a result, asymmetric cell division and neurogenesis are defective. In the Drosophila system, another class of asymmetric cell division regulators are epigenetic modulators. However, it is not clear if Smek functions through epigenetic modulators. Our studies suggest that Smek and Mbd3 have the opposite function in the NPCs’ differentiation in vertebrate systems. Although these studies do not address asymmetric cell division, our research may shed light on asymmetric cell division and neurogenesis in Drosophila and mammals. Our findings also highlight the importance of Smek/Mbd3 interaction in regulating NPC differentiation. Other studies suggest that Smek and Mbd3 may have overlapping functions or activities in brain development, stem cell activity, and regulation of transcription [15,16,20,21,42]. Our studies support a functional relationship of Smek and Mbd3 in NPCs. Our mapping analysis shows that Smek/Mbd3 interaction is mediated by the Mbd domain of Mbd3. Immunofluorescence analysis confirmed close proximity of these proteins in the NPC nucleus, and we have observed coincident expression of Smek and Mbd3 in the mouse embryonic brain [16,21]. Finally, we found that Smek and Mbd3 target the same neuronal gene loci for regulating transcription (Fig 5D). Further analysis suggests a model in which Smek regulates target gene transcription by regulating Mbd3 protein stability, interaction with NuRD components, and recruitment of Mbd3/NuRD complex to the promoters of target genes. Smek1/2 dKO exhibits reduced neuronal differentiation and decreased expression of Dlx1, Dlx2, NeuroD1, Tuj1, Gad67, NeuN, and stabilizing Mbd3 protein, while Mbd3 overexpression attenuated Smek-mediated neuronal differentiation (Figs 1E, 3A–3C, 6A, 6C and 6D). Thus, this study is significant not only for demonstrating Smek-mediated Mbd3 protein degradation but also in providing evidence that Smek/Mbd3 interaction regulates neuronal gene expression and neuronal differentiation during cortical development. In conclusion, we report functional interaction of Smek with Mbd3 in neuronal differentiation of NPCs. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) and the National Institutes of Health (IACUC protocol number: 11489). Mouse embryos and primary neural progenitor cells were obtained from a deceased pregnant mouse following CO2 asphyxiation. For in utero electroporation experiments, timed-mated pregnant mice had been anesthetized with Avertin (2. 5%) (Sigma, St. Louis, MO) following IACUC instruction. At the experimental endpoints, mice were euthanized by CO2 asphyxiation. Smek1/2 dKO mice were generated using gene trap mutant ES cells obtained from the Gene Trapping Consortium. Gene trap vectors were targeted between exons 3 and 4 of the Smek1 gene and between exons 10 and 11 of the Smek2 gene, respectively. Smek1 and Smek2 mutant ES cells (E14) were injected into mouse blastocysts and chimeric mice were backcrossed with C57BL/6 mice. Smek1/2 dKO mice were generated by crossing C57BL6J-Smek1+/- with Smek2+/- mice. After six generations, mice were used for analysis. Although Smek1/2 dKO mice can die at later stages of embryonic development, we were able to obtain dKO embryos as late as E14. 5 with a normal Mendelian distribution. Thus, we have conducted functional analysis of Smek1/2 dKO embryos at E11. 5, E12. 5, and E14. 5. Embryos and pups of wild-type and heterozygous KO mice were collected from timed-mated pregnant females. Antibodies used in this study were anti-Smek1 (rabbit polyclonal 1: 500 dilution) anti-Smek2 (rabbit polyclonal 1: 500 dilution), anti-Flag (mouse monoclonal 1: 2,000 dilution) (Sigma), anti-Mbd3 (rabbit polyclonal 1: 500 dilution), anti-CDH3 (rabbit polyclonal 1: 500 dilution), anti-RbAP46 (rabbit polyclonal 1: 2,000), anti-GFAP (rabbit polyclonal 1: 200 dilution), anti-MTA1 (rabbit polyclonal 1: 2,000 dilution), anti-RbAp46 (rabbit polyclonal 1: 2,000 dilution), anti-HDAC1/HDAC2 (mouse monoclonal 1: 2,000 dilution) (Cell Signaling Technology, Beverly, MA), anti-HA (rabbit polyclonal 1: 500 dilution), anti-GST (rabbit polyclonal 1: 500 dilution), anti–α-tubulin (mouse monoclonal 1: 5,000 dilution), anti-HA (mouse monoclonal 1: 3,000 dilution) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-MAP2ab (rabbit polyclonal 1: 200 dilution) (Chemicon, Temecula, CA), anti-NeuN (rabbit polyclonal 1: 200 dilution) (EMD Millipore, Billerica MA), anti-Nestin (mouse monoclonal 1: 350 dilution) (BD Biosciences, San Jose, CA), anti-Tuj1 (mouse polyclonal 1: 200 dilution) (Covance, Princeton, NJ), anti-Tbr1 (rabbit polyclonal 1: 200 dilution), and anti-Pax6 (rabbit polyclonal 1: 200 dilution) (Abcam Ltd, Cambridge, MA). Secondary antibodies were anti-rabbit Alexa Fluor 488-, anti-mouse Alexa Fluor 488-, anti-rabbit Alexa Fluor 555-, or anti-mouse Alexa Fluor 555-conjugated IgG (1: 200 dilution) (Molecular Probes, Eugene, OR). bFGF was purchased from PeproTech (Rocky Hill, NJ). The protease inhibitor cocktail was from Roche Applied Science (Indianapolis, IN). To HDAC inhibition, Trichostatin A (TSA) and 4- (dimethylamino) -N-[6- (hydroxyamino) -6-oxohexyl]-benzamide (DHOB) were purchased from Santa Cruz Biotechnology. TRIzol, Protein A/G agarose beads, and DAPI were from Sigma. The ECL Kit and KOD Hot Start DNA polymerase were from EMD Millipore. Glutathione magnetic beads, phenol: chloroform: isoamyl alcohol, and the First Strand cDNA Synthesis Kit were from Thermo Fisher Scientific (Rockford, IL). Cells were gently lysed with IP buffer (50 mM Tris-HCl, pH 7. 4,130 mM NaCl, 10mM NaF, 2 mM EGTA, 2 mM EDTA, 0. 5% Triton X-100,0. 5% NP-40,5% glycerol, 1 mM dithiothreitol [DTT], and a protease inhibitor cocktail) for 1 h on ice and then centrifuged at 14,000 rpm at 4°C for 15 min. The supernatant was collected and precleared with 30 μl of Protein A/G beads (Santa Cruz Biotechnology) for 2 h, and then precleared lysates were incubated with 4 μg of each specific antibody overnight at 4°C. Lysates were then incubated with 30 μl of Protein A/G beads for 4 h at 4°C. After immune complexes were washed six times with IP buffer, they were eluted by boiling for 3 min at 95°C in SDS sample buffer and separated on 10% SDS-PAGE. After blocking, membranes were incubated with primary antibody and then with a peroxidase-conjugated secondary antibody. Bound secondary antibody (anti-mouse or anti-rabbit 1: 10,000) (Santa Cruz Biotechnology) was detected using the enhanced chemiluminescence (ECL) reagent (Santa Cruz Biotechnology). For immunohistochemistry, embryonic brains were dissected and fixed in 4% parafomaldehyde (PFA) at 4°C, cryoprotected in 30% sucrose, embedded and frozen in Tissue Tek OCT compound, and sectioned at 30 μm on a cryostat. Sections were incubated with primary antibody at 4°C for 18 h. For immunocytochemistry, cells cultured on coverslips were fixed with 4% PFA/PBS for 30 min and immunostained after permeabilizing with 0. 2% Triton X-100. Tissues and cells were incubated with secondary antibodies at room temperature for 1 h and counterstained in 4' -6-diamidino-2-phenylindole (DAPI) (Boehringer Mannheim, Mannheim, Germany), and images were visualized using confocal microscopy (LSM5 PASCAL; Zeiss, Jena, Germany). Values obtained from at least three independent experiments were averaged and reported as means ± SD. The two-tailed Student’s t test was used to compare two experimental groups. DH5 bacteria were transformed with GST-tagged plasmids (Mbd3, ΔN36, ΔN92, ΔC249, ΔC221, ΔC174, and ΔC93) and protein expression was induced by addition of 0. 5 mM isopropyl 1-thio-β-D-galactopyranoside (IPTG) at 25°C at mid-log phase. Cells were lysed with B-PER Bacterial Protein Extraction Reagent (Thermo Fisher Scientific), lysates were purified, and proteins were captured using with Glutathione magnetic beads. HEK293T cells were transfected with Flag-tagged Smek2 plasmid and lysed with lysis buffer for 1 h on ice. Cell lysates were centrifuged at 14,000 rpm at 4°C for 15 min, and collected supernatants were incubated with Glutathione magnetic beads bound to GST or GST proteins. Bound proteins were eluted by boiling for 3 min at 95°C in SDS sample buffer, followed by immunoblotting. NPCs were prepared from E11. 5 cortex of Wild-type, Smek1-/-, Smek2-/-, and Smek1/2 dKO mice in Hank’s balanced salt solution (HBSS) (Invitrogen) and cultured as described [43]. To maintain stem cell characteristics, NPCs were cultured in N2 medium containing bFGF for 4 d. Stemness of cultured NPCs was confirmed by Nestin and Sox2 expression. To induce NPC differentiation, cells were seeded and further cultured in the absence of bFGF2. NPCs derived from Wild-type or Smek1/2 dKO E11. 5 forebrain were transfected with 4 μg pUltra-hot-Mbd3-flag or a Myc-tag vector for Mbd3 gain-of-function experiments and with pLKO3G-shMbd3 for Mbd3 loss-of-function experiments using Lipofectamine LTX and Plus Reagent (Invitrogen) or electroporation with an AMAXA nucleofector (Ronza AG, Basel, Switzerland). pLKO3G-shcontrol vector was used for negative control of pLKO3G-shMbd3 vector. After 12 h, transfection efficiency was confirmed to be >90% by monitoring mcherry or EGFP signaling. After two more days in differentiation conditions, cells were analyzed by qPCR or ChIP. Mbd3 expression vectors were constructed by subcloning full-length mouse Mbd3 from a lentiviral FUIGW-Mbd3-Flag vector we previously created into XbaI/EcoRI sites of pUltra-hot (Addgene plasmid # 24130). For Mbd3-myc, the reverse primer included the full Myc sequence. PCR was carried out using the KOD Hot Start Polymerase Kit (EMD Millipore) with corresponding primer pairs. PCR products were ligated into double-digested pUltra-Hot vector and inserted ligations were confirmed by PCR and DNA sequencing (Genewiz, Inc). For the shRNA vector, the Mbd3 target sequence was designed based on the RNAi Consortium library top hits for mouse Mbd3. Details for pLKO3G shMbd3 and shcontrol construction are listed in S4 Table. Cells were harvested and total RNA was isolated using TRIzol reagent (Invitrogen). The SuperScript III qRT-PCR kit (Invitrogen) was used to synthesize cDNA from total RNA. Quantitative PCR was carried out using the ABI PRISM 7900 Sequence Detection System with SYBR Green Master Mix (iTaq) with conditions of 95°C for 10 min followed by 50 cycles at 95°C for 15 sec and 60°C for 3 sec. Samples were run in triplicate and Dlx1, Dlx2, Tlx3, NeuroD1, Tuj1, Gad67, NeuN, Mbp, Gfap, Ascl1, and Id1 transcript quantitation was undertaken by comparing Cycle Threshold (Ct) values for each reaction with the Gapdh reference. Primer sets for quantitative PCR are listed in S5 Table. For the ChIP assay, NPCs derived from wild-type or Smek1/2 dKO E11. 5 forebrain or transfected with 4 μg pUltra-hot-Mbd3-flag or a pUltra-hot-empty vector for Mbd3 gain-of-function experiments and with pLKO3G-shMbd3 or pLKO3G-shScramble for Mbd3 loss-of-function were treated with 1% formaldehyde for 10 min at room temperature and quenched with 0. 125 M glycine for ten more minutes at room temperature. Cross-linked chromatin was sonicated to fragment DNA to 200–1,000 base pairs, and then immunoprecipitation was performed with rabbit anti-IgG, anti-Smek1 (Sigma), anti-Mbd3 (Cell Signaling), anti-HDAC1, anti-HDAC2, anti-MTA1, and acetyl histone H3 (Santa Cruz Biotechnology) antibodies overnight at 4°C, followed by incubation with 50 μl of magnetic Protein A/G Dynabeads (EMD Millipore). Abundance of sequences in immunoprecipitates was determined by PCR and normalized as a fold-value relative to input chromatin. Smek ChIP-seq data were analyzed with the MACS online tool, and cis-regulatory sequences were analyzed using the Genomic Regions Enrichment of Annotations Tool (GREAT) interface (http: //bejerano. stanford. edu/great/public/html/). We also utilized the Intergrative Genomics Viewer (IGV v2. 3) to visualize distribution of ChIP-seq–identified peaks in different genomic regions. Primer sets for ChIP-qPCR are listed in S6 Table. All procedures followed guidelines of the Institutional Animal Care and Use Committee (IACUC) and the National Institutes of Health. A total of 2. 5% (w/v) avertin (1 g/ml solution of 2,2, 2-Tribromoethanol, 97% in tert-amylalcohol [99%]; Aldrich, catalog numbers T4,840–2 and 24,048–6, respectively) in 0. 9% saline was injected i. p. (15 μl/g of body weight) to anesthetize pregnant mice (E13. 5). A laparotomy was performed, and the uterus with embryos was exposed. A total of ~2–5 μl of plasmid DNA (approximately 2 μg/ μl, dissolved in water) was injected into the lateral ventricle using a fine-glass microcapillary and a PV830 pneumatic PicoPump. Electroporation was performed using a Nepagene CUY21SC electroporator (amplitude, 50 V [E13. 5]; duration, 50 ms; intervals, 150 ms). To deliver electrical pulses, tweezer-type circular electrodes (7-mm diameter) were used with the positive side directed to the medial wall of the ventricle into which DNA was injected. Uterine horns were repositioned in the abdominal cavity, and the abdominal wall and skin were sewed with surgical sutures. Mice were kept on a warm plate (37°C) for recovery. Two to three days later, embryos were taken from mothers and fixed with 4% (w/v) PFA (Sigma) in PBS (pH 7. 4). After a 24 h fixation at 4°C, embryo brains were transferred to a 30% (w/v) sucrose solution in 4% PFA. Tissues were sectioned at 30 μm using a cryotome (Leica) and analyzed by immunohistochemistry. Smek2 DNA was fused in frame to the LexA bait vectors pBTM116 for use as bait in the yeast two-hybrid screen. Preys were expressed as fusions to the activation domain of GAL4 in pACT2 (BD Biosciences Clontech, Palo Alto, CA, US). Transformed bait strains with or without transformed prey strains were mated and analyzed by using β-galactosidase activity. The following preys were used: mbd3. The Saccharomyces cerevisiae strain L40 (MATa trp1 leu2 his3 LYS2: : lexA-HIS3 URA3: : lexA-lacZ) was cotransformed with bait and prey plasmids using the PEI method and selected for histidine prototrophy on minimal medium, containing 2% glucose; 6. 7% yeast nitrogen base (BD Diagnostic Systems, Sparks, MD, US); complete amino acid mixture lacking histidine, leucine, and tryptophan (Qbiogene, Carlsbad, CA, US); and 2% bacto agar (BD Biosciences, Franklin Lakes, NJ, US). Yeast transformants were grown for 3 d at 30°C. Statistical differences among groups were analyzed using Student’s t test and are indicated in each Fig as follows: *p <. 05, **p <. 005, and ***p <. 0005. *p <. 05 was considered statistically significant. | Title: Smek promotes corticogenesis through regulating Mbd3's stability and Mbd3/NuRD complex recruitment to genes associated with neurogenesis Summary: Neural progenitor cells are self-renewing, multipotent cells that generate major neural cell types, including neurons and glia. Their fate during development of the cerebral cortex is determined by a complex interplay of genetic and epigenetic components. It has been shown that Suppressor of Mek null (Smek) proteins-which are evolutionarily conserved-play a role during the asymmetric cell division of neuroblasts in invertebrates. Methyl-CpG-binding domain 3 (Mbd3) protein, a core component of the repressive nucleosome remodeling and deacetylase (NuRD) complex, is an important epigenetic regulator that plays an essential role in mammalian development. In this study, we discovered that Smek interacts with Mbd3 and promotes its degradation via a posttranslational modification called polyubiquitylation. Degradation of Mb3, in turn, blocks recruitment of Mbd3/NuRD complex on target gene promoters, leading to an increase in neuronal differentiation during cortical development. This study not only elucidates a distinct mechanism for Smek-mediated neuronal differentiation but also identifies Smek as a negative regulator of the Mbd3 protein during cortical brain development. | 12,346 | 305 | lay_plos | en |
Summarize: He and his wife already have an adorable two-year-old son whom they adopted in 2012. Now, Bubba Watson has welcomed a second child into his family - a baby girl named Dakota. The professional golfer - who is currently fourth in the world golf rankings - shared a selfie with the newborn on Instagram on Tuesday, alongside the caption: 'Daddy-daughter selfie!! #ProudDad.' In the photo, which has been 'liked' nearly 6,000 times in just three hours, the 36-year-old is pictured beaming at the camera as he holds Dakota, who is donning a pink animal-themed babygro. Scroll down for video. Adorable: Bubba Watson - who is currently fourth in the world golf rankings - shared this selfie with his new adopted daughter, Dakota, on Instagram on Tuesday. The golfer already has a two-year-old son, Caleb. Proud: Alongside the picture, the 36-year-old, from Florida, wrote: 'Daddy-daughter selfie!! #ProudDad' Athlete: Watson is seen playing a shot in round three of the Thailand Golf Championship on December 13. Last week, Watson publicly revealed the latest addition to his family for the first time, posting an image of his son, Caleb, hugging his new sister, whom he and his wife, Angie, are also adopting. Alongside the image, he wrote: 'Caleb has a brand new baby sister, Dakota. Watson Family is now 4 and we are so blessed! Look forward to releasing more information once our adoption is final.' He also shared a picture of himself and Angie, 37, on their 'first date night in 12 weeks', during which they watched King James and the Cleveland Cavaliers triumph 98-89 over Orlando Magic. Watson, who married his wife, a former pro basketball player in Europe, in 2004, has known from their first date that she is unable to have children for biological reasons. Siblings: Last week, Watson publicly revealed the latest addition to his family for the first time, posting an image of his son, Caleb, hugging his new sister (pictured), whom he and his wife, Angie, are also adopting. To be finalized: Alongside the image, he wrote: 'Caleb has a brand new baby sister, Dakota. Watson Family is now 4 and we are so blessed! Look forward to releasing more information once our adoption is final' Date night: He also shared a picture of himself and Angie on their 'first date night in 12 weeks' (pictured), during which they watched King James and the Cleveland Cavaliers triumph 98-89 over Orlando Magic. Excited: Watson captioned the iamge: 'We can't wait to hug you @kingjames at the @orlandomagic game!' In March 2012, the couple adopted Caleb, then only one month old, after their adoption of a girl fell through. The following month, Watson won his first Masters title in a playoff with Louis Oosthuizen. Earlier this year, the PGA star scooped his second Masters title, triumphing over Jordan Spieth and Jonas Blixt by three shots. With Dakota's arrival, he could be on track to win his third green jersey. Shortly after his second Masters win, Watson spoke to USA TODAY Sports about the process of adoption, adding that he was trying 'every day... to get better as a father and husband'. 'When we were going through the process of the adoption, I didn’t know what to expect being a dad. I mean, who wants to change a dirty diaper, you know what I’m saying?' he said. Success: In 2012, Watson and Angie - who cannot have children - adopted Caleb, then only one month old. Earlier this year, the golfer scooped his second masters title. Above, he is pictured with Caleb after his win. In good spirits: Also in 2012, Watson won his first Masters title. With Dakota's arrival, some have predicted he could be on track ton win his third green jersey. Above, he lifts Caleb into the air following his April 2014 win. 'But now, changing his diapers doesn’t even bother me. That’s my boy. I’ll do anything for him. Caleb has changed my personality, changed who I am, and hopefully made me a better person.' Earlier this month, Watson released a hilarious new Christmas rap video in which he assumed his festive-season alter ego named Bubba Claus. In it, alongside an elvish crew, he rapped about his golf hovercraft, delivering gifts on a John Deere tractor, his Christian faith and his son's inability to sleep on Christmas Eve. The song, titled 'The Single', was firmly tongue-in-cheek, with lyrics also detailing mundane details of Christmas in his home of Bagdad, Florida as a child. Tearful: Watson, pictured hugging his wife after his 2014 win, is among the longest drivers on the PGA Tour. From a family of three to four: The couple are pictured with Caleb in April this year before Dakota's arrival. His mother, he rapped, was cooking something on the stove, while his father was chilling in the living room as his sister played on the floor. Watson is one of the more colourful characters on the professional golf circuit. He is a member of the Golf Boys, a boy band consisting of Watson, Ben Crane, Rickie Fowler and Hunter Maham. In recent years, he has also heavily promoted his golf cart hovercraft. The vehicle, named the BW1, has been promoted by the golfer as being able to drive through water features and sand traps. It is believed that Dakota's adoption is pending paperwork. Rapping: Earlier this month, Watson released a hilarious new Christmas rap video in which he assumed his festive-season alter ego named Bubba Claus. Above, footage of the rap sees Watson posing with golf gear | Summary: Bubba Watson posted adorable selfie with baby Dakota on Instagram. Golfer, 36, captioned the image: 'Daddy-daughter selfie!! #ProudDad' Dakota is his second adopted child - he already has son, Caleb, now 2. Watson found out wife, Angie, 37, could not have children on first date. One month after Caleb's 2012 adoption, golfer won first Masters title. He won tournament again in April this year; is now world number four. | 1,379 | 114 | cnn_dailymail | en |
Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Wind Power Tax Incentives Act of 2003''. SEC. 2. OFFSET OF PASSIVE ACTIVITY LOSSES AND CREDITS OF AN ELIGIBLE TAXPAYER FROM WIND ENERGY FACILITIES. (a) In General.--Section 469 of the Internal Revenue Code of 1986 (relating to passive activity losses and credits limited) is amended by redesignating subsections (l) and (m) as subsections (m) and (n) and by inserting after subsection (k) the following new subsection: ``(l) Offset of Passive Activity Losses and Credits From Wind Energy Facilities.-- ``(1) In general.--Subsection (a) shall not apply to the portion of the passive activity loss, or the deduction equivalent (within the meaning of subsection (j)(5)) of the portion of the passive activity credit, for any taxable year which is attributable to all interests of an eligible taxpayer in qualified facilities described in section 45(c)(3)(A). ``(2) Eligible taxpayer.--For purposes of this subsection-- ``(A) In general.--The term `eligible taxpayer' means, with respect to any taxable year, a taxpayer the adjusted gross income (taxable income in the case of a corporation) of which does not exceed $1,000,000. ``(B) Rules for computing adjusted gross income.-- Adjusted gross income shall be computed in the same manner as under subsection (i)(3)(F). ``(C) Aggregation rules.--All persons treated as a single employer under subsection (a) or (b) of section 52 shall be treated as a single taxpayer for purposes of this paragraph. ``(D) Pass-thru entities.--In the case of a pass- thru entity, this paragraph shall be applied at the level of the person to which the credit is allocated by the entity.'' (b) Effective Date.--The amendments made by this section shall apply to facilities placed in service after the date of the enactment of this Act. SEC. 3. CREDIT FOR WIND ENERGY FACILITIES OF AN ELIGIBLE TAXPAYER ALLOWED AGAINST MINIMUM TAX. (a) In General.--Section 38(c) of the Internal Revenue Code of 1986 (relating to limitation based on amount of tax) is amended by redesignating paragraph (4) as paragraph (5) and by inserting after paragraph (3) the following new paragraph: ``(4) Special rules for wind energy credit.-- ``(A) In general.--In the case of the wind energy credit of an eligible taxpayer-- ``(i) this section and section 39 shall be applied separately with respect to such credit, and ``(ii) in applying paragraph (1) to the credit-- ``(I) the tentative minimum tax shall be treated as being zero, and ``(II) the limitation under paragraph (1) (as modified by subclause (I)) shall be reduced by the credit allowed under subsection (a) for the taxable year (other than the wind energy credit). ``(B) Wind energy credit.--For purposes of this subsection, the term `wind energy credit' means the portion of the renewable electric production credit under section 45 determined with respect to a facility using wind to produce electricity. ``(C) Eligible taxpayer.--For purposes of this paragraph, the term `eligible taxpayer' has the meaning given such term by section 469(l)(2).'' (b) Conforming Amendments.--Paragraphs (2)(A)(ii)(II) and (3)(A)(ii)(II) of section 38(c) of such Code are each amended by inserting ``or wind energy credit'' after ``employee credit''. (c) Effective Date.--The amendments made by this section shall apply to taxable years ending after the date of the enactment of this Act. SEC. 4. APPLICATION OF CREDIT TO COOPERATIVES. (a) In General.--Section 45(d) of the Internal Revenue Code of 1986 (relating to definitions and special rules) is amended by adding at the end the following new paragraph: ``(8) Allocation of credit to shareholders of cooperative.-- ``(A) Election to allocate.-- ``(i) In general.--In the case of a cooperative organization described in section 1381(a), any portion of the credit determined under subsection (a) for the taxable year may, at the election of the organization, be apportioned pro rata among shareholders of the organization on the basis of the capital contributions of the shareholders to the organization. ``(ii) Form and effect of election.--An election under clause (i) for any taxable year shall be made on a timely filed return for such year. Such election, once made, shall be irrevocable for such taxable year. ``(B) Treatment of organizations and patrons.--The amount of the credit apportioned to any shareholders under subparagraph (A)-- ``(i) shall not be included in the amount determined under subsection (a) with respect to the organization for the taxable year, and ``(ii) shall be included in the amount determined under subsection (a) for the taxable year of the shareholder with or within which the taxable year of the organization ends. ``(C) Special rules for decrease in credits for taxable year.--If the amount of the credit of a cooperative organization determined under subsection (a) for a taxable year is less than the amount of such credit shown on the return of the cooperative organization for such year, an amount equal to the excess of-- ``(i) such reduction, over ``(ii) the amount not apportioned to such shareholders under subparagraph (A) for the taxable year, shall be treated as an increase in tax imposed by this chapter on the organization. Such increase shall not be treated as tax imposed by this chapter for purposes of determining the amount of any credit under this subpart or subpart A, B, E, or G.''. (b) Effective Date.--The amendments made by this section shall apply to taxable years ending after the date of the enactment of this Act. | Title: A bill to amend the Internal Revenue Code of 1986 to encourage investment in facilities using wind to produce electricity, and for other purposes Summary: Wind Power Tax Incentives Act of 2003 - Amends the Internal Revenue Code to allow: (1) passive activity losses and credits attributable to qualified wind energy facilities; (2) the wind energy credit to be used against the alternative minimum tax; and (3) the pass-through of a cooperative's wind energy credit to the cooperative's members. | 1,496 | 111 | billsum | en |
Summarize: I trust Stephen Hawking's word when it comes to black holes and quantum mechanics, but I'm more dubious when the famous cosmologist says, as he does in an upcoming BBC special, that it is urgent for humans to colonize another planet in the face of catastrophes like climate change, asteroid strikes, epidemics and overpopulation. The Beeb is resurrecting its long-running science program, "Tomorrow's World" with a documentary called "Expedition New Earth," in which Hawking says the human species must set up shop beyond Earth within the next hundred years in order to ensure the survival of our species, according to the Telegraph. I really hope someone on the show's production team, or the BBC's promotions people, or maybe the outlets picking up the story are misrepresenting Hawking's level of hysteria and urgency here, because it's sorely lacking in the logic department. The most likely worlds for colonization are our moon or Mars (which is also Elon Musk's target of choice for a colony in the next century), and in case you hadn't heard, neither of these places are habitable. Even if Earth were to suffer the catastrophes that Hawking worries about -- and they all worry me, too -- it would still be more habitable than the moon or Mars. This is to say nothing of the trip to each place, which is highly risky, expensive and involves exposure to an awful lot of cosmic radiation and weightlessness that can do very real damage to the human body, especially on the long trip to Mars. I feel like I should be able to drop the mic here, but openly disagreeing with one of the smartest men on Earth demands a little extra diligence. So to be clear, I think pursuing a small human colony is a great idea. The scientific discoveries and technological innovations that will come out of such a venture are well worth the effort. But this notion that Mars or the moon is our salvation because the end of the world is nigh is really silly. Let's just run through the scenarios: rising sea levels, famine, epidemics, ecological collapse... If all those came to pass, even all at the same time, Earth would still be more habitable than anywhere else in our solar system. Really, you can't undersell the value of a working magnetic field and an atmosphere, even one with a little too much carbon dioxide in it. What about nuclear war though? Well, that depends on where you are and if it has been contaminated by as much radiation as the entire planet of Mars gets blasted by all the time. You might need to go underground, which is what we'd also have to do on Mars. That big asteroid strike could be a really big problem for us, though, right? Yes, it could, but I'm not sure that running away to Mars, which neighbors the asteroid belt, is a better option. But what if society just completely falls apart and we devolve into anarchic, violent states thanks to any combination of the above? Wouldn't we want to somewhere we could just start over? Again, just cleaning up our own mess and starting over by rising from the rubble seems more practical, but what if another planet was an option? This gets at the real urgent problem that we should aim to solve long before we start looking for a panacea beyond our own planet. I'm talking about the very ugly but real conundrum of global inequality. Many of the disasters Hawking warns of are much more likely to impact, say, the low-income residents of low-lying Bangladesh than the average Forbes reader (or contributor). Just as our terrestrial problems are more likely to affect the poor, pie-in-space solutions like a Martian colony are likely to be an option for only the wealthy and less apocalyptically challenged among us when the stuff finally hits the fan. Martian condos certainly aren't going to be cheap, after all. This is why the off-planet colonies in so many space operas like "The Expanse," "Elysium" and even "Star Wars" tend to be run by the bad guys. Colonizing a new planet would be just as much about who gets left behind as it would be about who gets to start over. Yes, there are all kinds of problems that we face on Earth, but all the solutions are here too. All the natural resources, manufacturing facilities, intellectual capital and other tools we use to, you know, live, are here. We'd have to recreate them under far more difficult conditions anywhere else. You think recycling and conservation are a pain? Just wait until you have to try to grow potatoes in toxic Martian soil and reduced gravity while dealing with your latest bout of radiation poisoning. Yet somehow, the grass is always greener for some people, even when it's on a dead Red Planet. Updated Renowned physicist Stephen Hawking has warned that humanity needs to become a multi-planetary species within the next century in order to avoid extinction. Hawking made the prediction in a new documentary called Expedition New Earth, which is set to be released this summer as part of the BBC’s Tomorrow’s World science season. Existential risks include climate change, overpopulation, epidemics and asteroid strikes, according to Hawking. In his program, Hawking will explore the latest advances in astronomy, biology and rocket technology that will make it possible to live on Mars—from plasma rockets to human hibernation. “In this landmark series, Expedition New Earth, [Hawking] enlists engineering expert professor Danielle George and his own former student, Christophe Galfard, to find out if and how humans can reach for the stars and move to different planets,” the BBC said. “The journey shows that Prof Hawking’s ambition isn’t as fantastical as it sounds—that science fact is closer to science fiction than we ever thought.” Efforts to create a human colony on Mars are already underway, with billionaire Elon Musk hoping to establish a settlement within the next few decades through his aerospace firm SpaceX. “I don’t have a doomsday prophecy,” Musk said in 2016, “but history suggests some doomsday event will happen.” Hawking has repeatedly called for humans to colonize the moon and Mars as an insurance policy against an apocalyptic catastrophe on Earth. “The moon could be a base for travel to the rest of the solar system,” Hawking said in a 2008 lecture marking NASA’s 50th anniversary, adding that Mars would be “the obvious next target.” Hawking predicted last year that the chance of a species-ending event on Earth was a “near certainty” when all possibilities were taken into consideration. “Although the chance of disaster to planet Earth in a given year may be quite low, it adds up over time, and becomes a near certainty in the next 1,000 or 10,000 years,” Hawking told the Oxford University Union in November. “By that time, we should have spread out into space and to other stars, so a disaster on Earth would not mean the end of the human race.” Despite the dire warning, Hawking did have some positive news for the assembled students. He pointed to how our fundamental understanding of the universe has advanced in his lifetime and said it is a “glorious time to be alive and doing research into theoretical physics.” He added: “Our picture of the universe has changed a great deal in the last 50 years and I am happy if I have made a small contribution. The fact that we humans, who are ourselves mere fundamental particles of nature, have been able to come this close to understanding the laws that govern us and the universe is certainly a triumph.” Update: This article has been update to include a comment from the BBC on the Expedition New Earth series. Humans will need to colonise another planet within one hundred years to ensure our survival, according to Professor Stephen Hawking. The astrophysicist has made a new documentary, Expedition New Earth, as part of the BBC’s new science season Tomorrow’s World. In it he will claim that time is running out for Earth and if humanity is to survive climate change, asteroid strikes, epidemics and overpopulation we will need to leave our planet and venture further afield. In the landmark series, Prof Hawking and his former student Christophe Galfard will travel the world to find out how humans could live in outer space. It is 14 years since the BBC cancelled its future-gazing series Tomorrow’s World after 38 years on air, but the corporation and the scientists involved promise the new season will be even better. | Summary: Stephen Hawking is giving humanity a tall order: Colonize Mars in the next century or watch as life on Earth fizzles out. After last year claiming that humans have 1,000 years left on Earth, Hawking says in a new documentary that we instead have about 100 years until we'll need to jump ship as Earth is overwhelmed by overpopulation, climate change, disease, and artificial intelligence. It might be a bit premature to start packing, but the BBC's Expedition New Earth will explore technological and scientific advances that will enable life in space or a colony on another planet, reports the Telegraph. It will show "Hawking's ambition isn't as fantastical as it sounds-that science fact is closer to science fiction than we ever thought," the BBC says, per Newsweek. Elon Musk of SpaceX is already planning to send humans to Mars in the next decade. But while a Mars colony is a good idea, bringing new scientific discoveries, columnist Eric Mack says Hawking needs to give his head a shake if he honestly believes Mars, the moon, or anywhere else in our solar system will be more hospitable than Earth even after a host of disasters. "Just cleaning up our own mess and starting over by rising from the rubble seems more practical" and more affordable than figuring out how to grow food or survive radiation poisoning on Mars, he writes at Forbes. The solution to all of our problems is here on Earth, he adds. "Yet somehow, the grass is always greener for some people, even when it's on a dead Red Planet." | 1,898 | 350 | multi_news | en |
Write a title and summarize: National mapping of soil-transmitted helminth infections (STH) was conducted for the first time in all of the 77 districts of Benin (West Africa) from 2013 to 2015. This mapping aimed to provide basic epidemiological data essential for the implementation of the national strategy against the neglected tropical diseases (NTDs) in the context of achieving the WHO target of controlling these infections by 2020. In each district, 5 schools were purposively selected in 5 villages and 50 school-children (25 girls and 25 boys) from ages 8 to 14 years were randomly enrolled in each school. In total, 19,250 stool samples of school children (9,625 girls and 9,625 boys) from 385 schools were examined by Kato-Katz technique. The three major species of STH (hookworm, Ascaris lumbricoides and Trichuris trichiura) were observed with intra- and inter-specific variations in the prevalence and the intensity of these parasites. Hookworm infection was present in all of the surveyed districts with an average prevalence of 17. 14% (95% CI 16. 6%-17. 6%). Among the infected schoolchildren, at national level, 90. 82%, 6. 73% and 2. 45% of infections were of light, moderate and heavy parasite intensities respectively. A. lumbricoides infection, with a national average prevalence of 5. 35% (95% CI 5. 00%-5. 60%), was the second most prevalent STH, and 84. 37%, 14. 27% and 1. 36% of the infections were of light, moderate and heavy parasite intensities, respectively. T. trichiura had a national average prevalence of 1. 15% (95% CI 0. 90%-1. 20%) and 80. 45%, 13. 18% and 6. 36% infections were of light, moderate and heavy parasite intensities, respectively. The national cumulative prevalence of the three STH infections was 22. 74% (95% CI 22. 15%-23. 33%), with58. 44% (45/77) of the districts requiring mass treatment according to WHO recommendations. In all of the surveyed districts, multiple infections by STH species were common, and boys seemed more at risk of hookworm and Ascaris infections. This first national mapping provided an overview of the epidemiological pattern of STH infections and was essential for the implementation of a control strategy with an effective preventive chemotherapy treatment (PCT). Results show that while preventive chemotherapy is not indicated for children in 32/77 districts, 43 require annual deworming and two require twice yearly deworming. If no environmental change occurs, and no mass treatment is delivered, prevalence is likely to remain stable for many years owing to poor hygiene and sanitation. The soil-transmitted helminthiasis (STH) are one of eighteen groups of diseases termed as Neglected Tropical Diseases by the World Health Organization (WHO) and which have caught the attention of donors following World Health Assembly resolution WHA54. 19 in 2001 [1]. Indeed, more than 1. 5 billion people, or 24% of the world population, have been estimated to be infected with STH worldwide [2]. Infections are widely distributed in tropical and subtropical areas and affects the poorest and most deprived communities, with the greatest numbers occurring in sub-Saharan Africa, the Americas, China and East Asia [3,4]. These diseases are transmitted by eggs present in human excreta, which contaminate the soil in areas with populations who have limited access to latrines and potable water. The main species of STH that infect humans are nematodes (A. lumbricoides, T. trichiura, Necator americanus and Ancylostoma duodenale) with relatively similar cycles involving the presence of adult worms in the intestine. Infection is caused by larval penetration of the skin (in the case of hookworm) or ingestion of parasite eggs (for other STH). High to moderate intensity of STH infections have been associated with increased risk of malnutrition, iron-deficiency related anemia and other adverse physical and cognitive morbidities, particularly in children and pregnant women [2,5, 6,7]. In sub-Saharan Africa (SSA), the majority of pregnant women are anemic (more than 60% in Benin) [8]and STH infections are among the most significant causes of anemia diagnosed during pregnancy in SSA [9]. In Benin, several studies have highlighted STH infections as a major public health problem and the related associations have been recently demonstrated on a small Beninese cohort [8–11]. Anemia had a negative impact on maternal health but also on newborn health due to significant decrease of the hemoglobin during the first months of life, leading to elevated risk of mortality and morbidity of the young child [8–11]. These studies have been the basis of sporadic preventive chemotherapy according to WHO guidelines. With the London Declaration on NTDs by 2020, the National Communicable Disease Control Program developed a strategy for the control of five NTDs in 2012 (Trachoma, Onchocerciasis and Lymphatic Filariasis, schistosomiasis and STH). Since 2013, the national plan has been funded by USAID through the ENVISION project led by Research Triangle Institute (RTI). This study aimed to investigate the national epidemiological situation regarding the three major STH infections (hookworm, ascariasis and trichuriasis) as baseline data was essential for implementation of the PCT strategy in Benin. This study was approved by the Comité National d’Ethique pour la Recherche en Santé (CNERS) under the authorisation reference 009/CNERS-MS from the Ministry of Health in Benin. Informed consent was obtained from the head teacher of each school and sometimes from the chief of the village, parents and teachers associations. In some districts where parents and teachers associations (PTA) exist, the head of the PTA and the head teachers received detailed explanation about the study. Individual parents were informed by the PTA and consents were secured orally. The PTA then provided a formal written approval on behalf of school children and parents. In cases where those that were to give authorisation were unable to read and write, a detailed verbal explanation of the form was given so that informed consent was obtained. Two copies of the written consent form were signed and dated. The person giving consent kept one copy and the second copy was returned to the NTD national control program. During the sampling all school children from whom no consent statement was received were replaced by other volunteers according to inclusion criteria. Participants detected with high intensity of STH or any other intestinal parasite infections were directed to healthcare centres in order to receive appropriate treatment before PCT school-based organized the following year. The republic of Benin is divided into 12 departments (political subdivisions), which are further divided into 77 districts. A total population is 10,008,749 inhabitants with 29. 7% of total population of school age children (5–14 year old). These districts are divided into 545 sub-districts and into 3755 villages. Each sub-district has at least one unit of health and each village has at least one public school. All of the 77 districts were sampled in this study. Rainfall intensifies from the south to the north of the country. In the northern departments (Atacora, Donga, Borgou and Alibori), the annual rainfall varies between 900 mm and 1200 mm with numerous lakes and rivers feeding the region. In the southern departments (Collines, Zou, Atlantic, Littoral, Mono, Couffo Plateau and Oueme), the annual rainfall varies from 800 mm to 1200 mm [12]. The geographical location of each school surveyed, including altitude andUniversal Transverse Mercator coordinates are provided in S1 Table. This study was carried out from 2013 to 2015 in all 77 districts of Benin (S1 Table). In each district, five primary schools were selected as previously described [13]. From each selected school, 50 children (25 girls and 25 boys) aged between 8 and 14 years were randomly selected. The children were given a container to provide stool samples. The containers were distributed by a team of lab technicians and the samples were collected within an hour. The stool samples were analyzed using the Kato-Katz method as previously described [13]. Although the Kato-Katz method has some limitations, especially in terms of sensitivity in settings with low infection intensities[14,15], this technique is the standard approach for highly endemic areas such as Benin [16]. In this study, the kits used were manufactured by Vestergaard Frandsen Group and 41. 7 mg of stool was filtered through a nylon mesh and covered with cellophane previously soaked in 50% green-malachite [17]. Only one smear was prepared and examined per stool sample. The slides were observed under microscope by two technicians and their results were validated by a third slide-reader. Especially, hookworm eggs were counted from 20 to 60 minutes after the slide was preparedand other soil-transmitted helminth eggs (A. lumbricoides, T. trichiura) were counted 24 hours later. The presence of infection was recorded; the number of eggs for each parasite was tallied and the intensity of infection was reported as the number of eggs per gram of feces (epg). Egg counts were used to classify the intensity of infection into light (L), moderate (M), or heavy (H) as follows: for A. duodenale / N. americanus (not distinguished hookworms) 1–1,999 epg (L), 2,000–3,999 epg (M) and ≥4,000 epg (H); for A. lumbricoides, 1–4,999 epg (L), 5,000–49,999 epg (M) and ≥50,000 epg (H); for T. trichiura, 1–999 epg (L), 1,000–9,999 epg (M) and ≥10,000 epg (H) [18]. The cumulative prevalence of STH infections reflects the number of individuals infected with any one of the three STH parasites. Cases of co-infection were counted once. For quality control purposes, 10% of the slides prepared on the previous day were examined each day by an independent team of biologists. Data were double entered into Microsoft Excel 2008 (Redmond, Washington, USA). Range and consistency checks were conducted for all non-string variables. Descriptive statistics and prevalence estimates were calculated using Epi-Info 7 (CDC, Atlanta, USA) and all results with a P value of <0. 05 were considered significant. The multiple comparison test chi square proportions were used to compare the prevalence by departments. The Fisher exact method of maximum likelihood and calculation of confidence intervals was used to calculate odds ratios by gender in each of the districts. A Z-test was used to compare prevalence values between two districts and between the gender and Chi square to compare prevalence values between districts of each department. Any district with a prevalence of infection above 50% was defined as a “hotspot”of the corresponding parasite. Hookworm infection was detected in all the surveyed districts with a prevalence ranging from 0. 4% to 60% (Table 1). The national average prevalence of hookworm infection was 17. 4% (95% CI: 16. 6%-17. 6%). The district of Djakotomey in the department of Couffo had the highest prevalence (60%) with hookworm infection detected in 150 children, whereas only one case was detected in the districts of Aguegue and Porto-Novo in the department of Oueme. In two districts (Toffo and Djakotomey), hookworm infection was detected in more than 50% of the surveyed children. The prevalence of hookworm infection was below 10% in 29/77 districts, and in 18 and 28 districts the prevalence ranged from 10% to 19. 2% and from 20% to 43. 2%, respectively. The intensity of the infection was light in most of the surveyed schools. However, moderate and heavy infections have been detected in different regions of the country with the highest prevalence of heavy infections observed in the districts of Copargo (16. 67% of the infections) and Ouake (17. 58%), both in the department of Djougou. In these two districts located in the northern part of the country, 33. 33% (Copargo) and 27. 47% (Ouake) of the hookworm infections were defined as moderate. Ascariasisis less widely distributed compared to hookworm and the overall prevalence of infection was 5. 35% (95% CI5. 00%-5. 60%). No Ascaris infection was detected in 15 districts (Table 2). In the other districts where Ascaris infection was detected, the prevalence ranged from 0. 4% to 26. 4%. The highest prevalence was observed in Toffo (26. 4%), in the department of Atlantique in southern Benin, along with the district of Allada (21. 20%) from the same department, and the districts of Bante (22. 40%) and Glazoue (23. 20%) both from the department of Collines. In these 4 districts, the prevalence of Ascaris infection was ≥20%. In 48/77 districts, the prevalence of Ascaris infection was <10%, whereas 10/77 districts had prevalence values between 10% and 18%. Most of the detected infections had light parasite load. All over the country, Ascaris infections with heavy parasite intensity were detected in only 6 districts, the highest being observed at Ouake (13% of infections) in the department of Djougou (Table 2). Ascaris infections with moderate parasite intensity were found in the 20 districts including the 6 districts with heavy parasite intensity. T. trichiura had the lowest prevalence at district and national level. The overall prevalence was 1. 15% (Table 3). Trichuris infection was absent in 40/77 districts and most of the infections were detected at prevalence <10% in the other 37 districts. In these districts, the prevalence of Trichuris infection varied between 0. 4% and 9. 6%. Districts of Dassa-Zoume and Dogbo in the departments of Collines and Couffo respectively, had the highest (9. 6%) prevalence of Trichuris infections. Along with these two districts, the districts of Materi in the department of Atacora, and Glazoue in the department of Collines were the only 4 districts with prevalence >5%. As for the other parasites screened in this study, the intensity of the Trichuris infections was light, whereas moderate and heavy infections were observed in 12 and 4districts, respectively. In the district of Dassa-zoume, heavy infection (41. 67%; 95% CI: 35. 56%-47. 78%) was more prevalent as compared to moderate (20. 83%: 95° CI: 15. 79%-25. 86%) and light (35. 50%; 95% CI: 29. 57%-41. 43%) infections. Cumulative prevalence of the surveyed STH parasites was analyzed by combining the determined prevalence of hookworm, Ascaris and Trichuris infections (Table 4). At least one of the three species of the targeted STH was found in all 77 surveyed districts). The average cumulative prevalence of STH infection was 22. 74% (95% CI 22. 15%-23. 33%) and 58. 44% (45/77) of the surveyed districts needed preventive chemotherapy (Table 4), as defined by WHO (STH prevalence ≥20%). Two rounds of PCT per year was needed in the districts of Toffo and Djakotomey where the detected STH prevalence was >50% (Table 4) using WHO guidelines [15]. Boys were significantly more likely to be infected compared to girls (S2 Table), with both hookworm and Ascaris, with average prevalence of 20. 31% v. 14. 03% (Z = 11. 53; p<0. 00001) and 5. 79% v. 4. 91% (Z = 2. 68: p = 0. 01), respectively. This difference was not observed with Trichuris infections. In all the surveyed districts, multiple infections by STH species was common in school age children as determined by the prevalence of coinfections with the highest number of cases (prevalence >10%) being found in the districts of Toffo (18. 00%), Glazoue (16. 50%), Bante and Lalo (14. 40%), Djakotomey (14. 00%) and Lokossa (10. 80%). This study was part of national schistosomiasis and soil-transmitted helminthiasis mapping performed to collect baseline epidemiological data prior to launching STH MDA in Benin using WHO guidelines [18]. Results from this study provided evidence of nationwide distribution of STH parasites with variable prevalence and intensity of infection throughout the country. Among the three STH species studied in the present work, T. trichiura had the lowest prevalence at the districts and national level. This overall low prevalence of trichuriasis confirmed data reported from previous studies in several districts of Benin [19,20]. The reported prevalence of trichuriasis in Benin, was also low as compared to other West Sub-Saharan African (WSSA) countries [7,21,22]. In contrast, ascariasis and hookworm infections are widely distributed with high prevalence in different regions of the countries. Hookworm infections were widely distributed throughout the country and have been detected in 100% of the surveyed districts. This study clearly highlighted the predominance of hookworm infections nationwide with several hotspots where the prevalence reached 50% and more. Ascariasis was detected in 62/77 districts with the highest prevalence observed in Toffo. In this district, elevated prevalence of hookworm and Ascaris infections was detected, suggesting that populations living in that area are more vulnerable to these infections. Although, moderate to heavy hookworm and Ascaris infections have been observed in several districts of the country, most of the detected infections had a light parasite load. These findings confirmed that Ascaris and hookworm infections are endemic in all the departments of Benin and that the prevalence of STH in Benin varied steadily over localities and the current results are similar to those from our previous studies [13,19,20] and reports from other SSA countries [2,4, 7,21]. Ascariasis appeared to be the predominant STH infection in the border districts between Benin and Burkina Faso (Materi and Boukoumbe), Benin and Togo (Copargo, Bante and Aplahoue), and Benin and Nigeria (Pobe, Ouinhi, Adja-Ouere and Segbana), whereas hookworm infections appeared to be predominant in peripheral districts in the northern and in the central districts in the southern part of the country. This distribution of STH infection could be explained by recently published data from Ivory Coast [22], which found a significantly positive association between STH infection rates and activities involving close contact to water and access to latrines. On the other hand, a negative association between STH infection and deworming, higher socioeconomic status, living in urban settings has been reported [22]. Our study showed that prevalence estimates in some districts are higher than those reported in previous studies from Benin [19,20], suggesting an increased exposure to STH in those districts. We believe that Benin follows the trends reported from other countries in SSA where the prevalence of the neglected tropical diseases appeared higher [7,22] than previously thought. However, this general trend of high prevalence of STH in WSSA is exceptional. In Burkina Faso for example, a recent study conducted on 3514 schoolchildren aged from 7 to 11 years randomly selected from 22 schools in 11 regions revealed low prevalence of hookworm, A. lumbricoides and T. trichiura infections [21]. In the current study the predominance of STH infections in boys compared to girls (hookworms: 21. 31% of boys vs. 14. 03% of girls, Z = 11. 53, p<0. 0001; Ascaris: 5. 79% of boys vs. 4. 91% of girls, Z = 2. 68, p<0. 01) might be explained by a difference in terms of behavior and activities [19,21]. Our study has a few limitations. First, the prevalence of STH in adult populations and particularly in pregnant women has not been investigated. Although having data on prevalence/intensity in other population groups such as pre-school-age children (PSAC) and women of child-bearing age (WCBA) is certainly useful, WHO does not strictly recommend collecting such information. However, since age prevalence curves have been well established for all three infections, information collected among school-age children can guide decision-making on treatment of this age-group, as well as of PSAC and WCBA. Data shown in this report are therefore sufficient to justify treatment in these two additional population groups as well [1]. Second, only one sample was analyzed for each participant due to the nationwide scale of this study and the limited financial and human resources. Day-to-day variation in STH detection that may influence the prevalence estimates reported in this study could not be considered [23]. Third, the impact of STH infections on the health of the Beninese population has not been addressed in the current study. Many studies conducted in Benin [8,11] and in other West Africa countries [10,24] showed that people living in the endemic areas are at risk of helminth infections, with the most vulnerable populations being pregnant women and children. Helminth and hookworm infections have been associated with poor cognitive and gross motor outcomes in infants, as well as maternal anemia in pregnant women [8,10,11,24]. The high prevalence of hookworm and its predominance in all the districts has an implication in terms of maternal child health policy, which should be strengthened. PCT for STH should be tailored to prevent sequelae and disabling consequences in these populations at risk. Those infections may cause not only maternal anemia but also affect infants' hemoglobin levels, their growth, their susceptibility to helminth infections, their cognitive development, their selective attention, their socioeconomic status, their physical fitness and their immunological responses to vaccination [25–30]. | Title: Results of the first mapping of soil-transmitted helminths in Benin: Evidence of countrywide hookworm predominance Summary: Benin, like other low or moderate-income countries in the African continent, is endemic for several neglected tropical diseases, including soil-transmitted helminthiases. The National Program for Neglected Tropical Diseases of the Ministry of Health has conducted the national STH mapping using the Kato-Katz method to assess the baseline epidemiological status in all 77 districts of Benin, in order to guide implementation of a preventive chemotherapy program using albendazole. The results of the survey showed that infection with at least one of the three targeted species (hookworm, roundworm or whipworm) affected 20% or more of school aged children in 45 out of 77 districts, and which therefore require PCT. Hookworm infection was the most prevalent followed by ascariasis and trichuriasis. Boys were significantly more likely than girls to be infected with hookworm or ascariasis. | 5,441 | 225 | lay_plos | en |
Summarize: Tweet with a location You can add location information to your Tweets, such as your city or precise location, from the web and via third-party applications. You always have the option to delete your Tweet location history. Learn more The two posts struck a more combative tone for President Donald Trump, who was decidedly more conciliatory in a statement released by the White House Wednesday night. AP Photo Trump calls appointment of special prosecutor 'the single greatest witch hunt' The president's tweets are a stark contrast from his more restrained tone on Wednesday night. President Donald Trump on Thursday blasted the appointment of Robert Mueller to be the special prosecutor overseeing the investigation into Russia’s meddling into the 2016 election, calling the probe “the single greatest witch hunt of a politician in American history.” He also accused former President Barack Obama’s administration and the campaign of Democratic presidential nominee Hillary Clinton of committing “illegal acts,” complaining that his campaign will now face the scrutiny of a special prosecutor while neither his predecessor nor 2016 opponent ever have. Story Continued Below “With all of the illegal acts that took place in the Clinton campaign & Obama Administration, there was never a special councel [sic] appointed!” the president wrote on Twitter on Thursday morning, without elaborating further on the illegal acts he accused the Clinton campaign and Obama administration of committing. (He later fixed the spelling error.) The flurry of posts comes as Trump's White House is mired in controversy, most of it self-created, stemming from the president's decision to fire FBI Director James Comey last week, a decision he said he made based in part on the bureau's Russia investigation, whichwill now be taken over by Mueller. The most reliable politics newsletter. Sign up for POLITICO Playbook and get the latest news, every morning — in your inbox. Email Sign Up By signing up you agree to receive email newsletters or alerts from POLITICO. You can unsubscribe at any time. The Trump administration was also left reeling earlier this week in the wake of a Washington Post report alleging that the president divulged highly sensitive intelligence in a meeting with top Russian diplomats last week, a story that the White House has disputed without outright denying that classified information was shared. The whiff of scandal intensified when allegations surfaced that Trump had pressured Comey to drop a probe into former national security adviser Michael Flynn — a charge the White House denies. All of it has taken the focus away from what was to have been the week's focus, Trump's first international trip as president, an eight-day, five-nation journey that will include stops in Saudi Arabia, at Israel's Western Wall and at the Vatican. The two Thursday morning posts struck a combative tone for Trump, who was more conciliatory in a statement released by the White House on Wednesday night. “As I have stated many times, a thorough investigation will confirm what we already know — there was no collusion between my campaign and any foreign entity,” said the Wednesday-night statement, credited to Trump. “I look forward to this matter concluding quickly. In the meantime, I will never stop fighting for the people and the issues that matter most to the future of our country.” The Department of Justice announced Wednesday night that former FBI Director Mueller will lead an independent investigation into Russian efforts to interfere in last year’s presidential campaign as well as into the possibility of collusion between the Kremlin and Trump associates. The White House had regularly objected to the idea of a special prosecutor, characterizing such an appointment as unnecessary. With the White House battling multiple scandals, Trump and his allies have adopted a posture of being unfairly under siege. Commerce Secretary Wilbur Ross, appearing Thursday morning on CNBC, said “we have to just get on with it, get this over. It's a sideshow. But what the media forgets in the midst of this sideshow of media frenzy, the president is running the country.” Trump’s aides, both current and former, have come to the president’s defense, as have family members working outside the White House. Corey Lewandowski, Trump’s combative first campaign manager, who maintains close ties to the White House and recently left the lobbying firm he helped found shortly after last year’s election, said Wednesday on Fox News that “I can tell you firsthand because I was on the campaign for a long time: Never, ever, ever did I ever see anybody have contact with any agent of any foreign government, Russia or anybody else.” White House social media director Dan Scavino chimed in, citing Lewandowski’s remarks in a post to Twitter, adding that he, too, “was there from the very beginning, and never, ever, ever....did I see either!” Eric Trump, the president’s son who, along with his brother, took over leadership of the family business when their father took office, tacked on his own tweet, linking back to Scavino’s and writing that “I was too! This entire thing is a witch hunt propagated by a failed political campaign.” House Oversight Committee Chairman Jason Chaffetz, who wrote Wednesday evening on Twitter that Mueller was a "great selection" with "impeccable credentials," said Thursday on ABC's "Good Morning America" that despite his approval of the former FBI director as a special prosecutor, he remained unconvinced that one was necessary in the first place. Chaffetz also said that congressional inquiries into Russian meddling should continue, even though the possibility exists that the separate probes might interfere with one another. Sen. Susan Collins (R-Maine) took a similar tack, telling MSNBC's "Morning Joe" that Comey should still testify before the Senate Intelligence Committee. Even before Mueller’s appointment, Trump was griping about his perceived unfair treatment, telling graduates of the Coast Guard Academy at their commencement ceremony on Wednesday that “no politician in history — and I say this with great surety — has been treated worse or more unfairly.” He implored the new graduates to take a lesson from his fledgling presidency. “Over the course of your life, you will find that things are not always fair. You will find that things happen to you that you do not deserve and that are not always warranted,” Trump said. “You have to put your head down and fight, fight, fight. Never ever, ever give up.” With all of the illegal acts that took place in the Clinton campaign & Obama Administration, there was never a special counsel appointed! | Summary: "This is the single greatest witch hunt of a politician in American history!" That was President Trump's Thursday morning tweet following news that a special prosecutor had been appointed to investigate whether Trump's campaign had ties to Russian tampering with the presidential election. He followed that tweet with: "With all of the illegal acts that took place in the Clinton campaign & Obama Administration, there was never a special counsel appointed!" (Per Politico, Trump originally misspelled "counsel" as "councel.") The official White House statement on the matter was less combative. | 1,433 | 123 | multi_news | en |
Summarize: [0001] This application is a divisional application of and claims priority to pending U.S. patent application Ser. No. 13/082,287, filed Apr. 7, 2011, entitled “Ankle-Foot Orthosis”. BACKGROUND OF THE INVENTION [0002] Ankle-Foot Orthoses (AFOs) are braces that structurally compensate for weak or deformed ankle joints. Typically, an AFO will limit the range of motion of an ankle to prevent the foot from dropping or rotating into a position that hinders walking Ankle braces dating back fifty years or more were commonly constructed from metal frames with leather straps. Shoes typically were characterized by prominent heels, with a heel breast that could engage a stirrup, so early ankle braces often attached to or around a heel structure. [0003] Over the decades, shoe structures and materials have evolved considerably. Typical modern footwear has become relatively more flexible and compliant compared to footwear contemporary to earlier foot braces. With few exceptions, modern shoes no longer have a heel structure to use as a point of attachment. In addition, a focus on comfort in the construct of the sole of modern footwear has led to the use and development of resilient synthetic materials, sometimes containing foams or gels, for added cushioning. This development, although preferable for shoe comfort, poses specific challenges to cutting-edge shoe orthosis companies. SUMMARY OF THE INVENTION [0004] The present invention is an Ankle Foot Orthosis, or AFO, that is fixed to a shoe. Throughout this description, the term shoe includes any footwear characterized by a sole and an upper, the shoe generally being used for walking Because there are so many different shoe styles to choose from, it is desirable to be able to modify a wearer's existing choices of shoes such that a wearer can experience exceptional stability without diminishing the fit of the shoe. Many wearers of AFOs suffer from a loss of sensation, so previous modern AFOs that incorporate hard plastic into the interior of a shoe can result in ulceration and other problems that a wearer can avoid by using the present invention. After separating at least a portion of the outsole from the rest of the sole, a horizontal portion of the preferred AFO is inserted into the sole before reattaching the outsole. When a vertical portion of an AFO is strapped to the leg of a wearer, the AFO provides assistance to those suffering from a weak or unstable ankle condition, such as foot drop or other neurogenic or congenital palsies. BRIEF DESCRIPTION OF THE DRAWINGS [0005] FIG. 1 is perspective view of a shoe incorporating a preferred embodiment of the present invention. [0006] FIG. 2 is vertical sagittal section through the shoe and AFO of FIG. 1. [0007] FIG. 3 is a side view of the AFO used in FIG. 1. [0008] FIG. 4 is a top view of the AFO used in FIG. 1. [0009] FIG. 5 is a perspective view of an alternate embodiment of a midsole and AFO of the present invention. [0010] FIG. 6 is a perspective view of an alternate embodiment of the present invention, characterized by a hinged ankle joint. [0011] FIG. 7 is a perspective view of an alternate embodiment characterized by at least two long pins secured into the midsole of a shoe. [0012] FIG. 8 is a top view of the AFO device used in FIG. 7. [0013] FIG. 9 is a perspective view of an alternate embodiment of the present invention that is secured to the quarter of a shoe. [0014] FIG. 10 is a perspective view of an alternate embodiment of an AFO, similar to the one in [0015] FIG. 9, except that press studs are used to removably fasten the AFO to the shoe such that the AFO can be reattached to the same or another shoe. [0016] FIG. 11 is a vertical cross-section through a press stud used to secure the AFO in FIG. 10 to a shoe. [0017] The following is the list of numerical callouts used in FIGS. 1-11 : 10 Shoe 12 Sole 14 Outsole 16 Midsole 18 Insole 20 Upper 22 Vamp 24 Quarter 26 Rear 28 Sides 30 Ankle-Foot Orthosis 32 Horizontal portion 34 Vertical portion 36 Fastener 38 Strap 40 Slot 44 Channel portion 46 Upwardly bent section 48 Hinge 50 Pad 52 Joint stop 54 Button 56 Pins 58 Flared portion 60 Stud 62 Rivet 64 Groove 66 Aperture 68 Ankle strap DETAILED DESCRIPTION OF THE INVENTION [0047] This detailed description will describe the present ankle-foot orthosis (AFO) substantially from the bottom up, as assembled. Throughout the remainder of this description, the term “bottom” refers to that surface or portion of a part or feature that is relatively closest to the bottom of a referenced figure. Generally, an AFO 30 has a horizontal portion 32 that is fixed to a shoe 10, and a vertical portion 34 that is strapped to a wearer's calf or shin. In the most preferred embodiment, a midsole of a shoe is cut apart along a horizontal plane such that the horizontal portion of the AFO can be adhered into the midsole 16, and the vertical portion of the AFO is fastened to the quarter 24 of the shoe. A common hook-and-loop strap 36 is used to secure the vertical portion to the leg of a wearer. Alternate embodiments, shown in FIGS. 5-11, will also be described. Where reference numbers in one figure are the same as another figure, those reference numbers carry substantially the same meaning Preferred sizes, materials and methods of attachment will be discussed, but these preferences are not intended to exclude other suitable or functionally equivalent sizes, materials or methods of attachment. [0048] A shoe 10 that incorporates the present invention is characterized by a sole 12 and an upper 20. Examples of shoes include athletic shoes, men's shoes, women's shoes, boots and other footwear commonly used for walking The sole, which is the part of a shoe below a wearer's foot, is comprised of an outsole 14 that is fixed to the bottom of a midsole 16 that is covered by an insole 18. The outsole is the part of the sole that contacts the ground, the midsole is one or more layers that adds cushion and support to the sole, and the insole is the comfortable part of the sole that contacts the bottom of a wearer's foot. The upper, which is the part of a shoe that covers a wearer's foot, includes a vamp 22 for covering the front of the foot and a quarter 24 for covering the heel of the foot. The quarter has a rear 26 and sides 28 that wrap around the heel of a wearer. [0049] The preferred ankle-foot orthosis (AFO) 30 has a horizontal portion 32 and a vertical portion 34 joined together. In the most preferred embodiment, shown in FIGS. 1-4, the entire AFO structure is made from a plastic such that the horizontal portion and vertical portion are a continuous, one-piece molded or thermo-formed construction. Other suitable materials include but are not limited to, high performance resins, thermoplastics or other synthetic materials, especially those that can be combined with tougheners to provide higher impact resistance, with pigments and UV stabilizers to provide and maintain a desired appearance, with glass fibers to provide higher stiffness, or with Teflon® or Kevlar® to provide improved wear and friction characteristics. Combinations of materials can also be used, such as a plastic vertical portion with a spring steel horizontal portion, or any other desired combination of materials to form the horizontal and vertical portions as dictated by the needs of users in a customizable fashion. When a common plastic is used, such as polypropylene, polyethylene or polyvinylchloride, it can be approximately one to four millimeters in thickness. The thickness and materials can be varied proportionately to the demands of strength, durability and flexibility for the needs of the user. For example, the proximal end of the vertical portion can be thinner and more flexible so as not to impede the mechanics of the user's gait, whereas, by way of additional example, the vertical portion surrounding a user's heel may be of a thicker and more heavy-duty construction appropriate to a stress and weight-bearing functionality. [0050] The horizontal portion 32 of the AFO 30 is essential for shoes that are lacking the rigidity historically provided by a shank, which is typically minimal or absent in modern footwear, such as a running shoe. Removal of a volume of the midsole 16 equivalent to the inserted volume of the horizontal portion of the AFO avoids addition of undesired asymmetric height relative to the contra lateral shoe. Another concern would involve those shoes having a midsole design that has a specific function, such as a honey-comb design for cushioning. In this instance, it would be sub-optimal to cut through the midsection of such a midsole, as the function will likely be compromised in the process. Attaching the horizontal portion of the AFO above or below such a midsection structure would circumvent this concern. In any case, the horizontal portion of the AFO would be inserted at an optimal location after separating the sole to create a separation between the outsole 14 and insole 18 of a user's shoe 10. [0051] The horizontal portion 32 of the AFO 30 preferably is adjoined to the vertical portion 34 of the AFO in a contiguous fashion slightly exterior to the posterior aspect of the shoe 10. Alternatively, depending on anatomic considerations, adjoinment can be at a lateral or medial aspect of the shoe. If the horizontal portion and vertical portion of the AFO adjoin posterior to the shoe, they will need to do so at an acute angle so as not to impede the natural mechanics of gait. If the point at which the horizontal portion and vertical portion of the AFO adjoin is at a medial or lateral aspect of the shoe, then the angle of adjoinment can be at or approximately ninety degrees. [0052] The vertical portion 34 of the AFO 30 is secured to the quarter 24 of a user's shoe 10, preferably at the rear 26 of the quarter, but additionally or alternatively secured to one or both sides 28 of the quarter. The vertical portion can be fastened to the quarter with any suitable fastener 36, such as a rivet, but alternatively could be affixed with an adhesive substance, or even attached via stitching. The form of the vertical portion of the AFO is anatomically structured to mimic the contour of a user's lower leg, optimally along the posterior calf. Alternatively, the vertical portion could extend proximally along the lateral or medial calf, or along the shin. However, in the absence of specific deformities, the posterior calf would likely provide the greatest tissue compressibility and padding. The material thickness comprising the vertical portion of the AFO can be varied as desired. For example, the material could be tapered superiorly to allow more flexibility where strength and durability are relatively less crucial compared to the inferior end of the vertical portion of the AFO, where thicker materials will allow the point of attachment area to withstand intensified forces, such as shear, compression and torque forces characteristic of the mechanisms of walking with an AFO. Allowing the vertical portion to flex slightly, by selecting an appropriate material and thickness relative to the weight of a user, contributes to dorsiflexion. [0053] The vertical portion 34 can be secured around the user's lower leg by many different alternate methods, depending on the anatomy of the user's lower leg and the desired proximity of the securing method. Methods can include straps secured by buckle mechanisms, hook and loop fasteners or snap fasteners, laces, or other means. A preferred strap 38, most clearly shown in FIGS. 1-4, has a hook and loop fastener fabric affixed to the outer surface of the vertical portion of the AFO, and a free end of the strap is passed through a slot 40 in the vertical portion such that the strap can overlap and fasten to itself to maximize adjustability. Foam or other comfortable materials, not shown, can be fixed to the inner surface of the vertical portion of the AFO as desired for improved comfort. [0054] In the alternate embodiment shown in FIG. 5, the horizontal portion 32 is substantially a shank that can be installed into a fitted channel portion 44 cut into the midsole 16 of a shoe 10. Because midsoles can be any number of layers, where a particular midsole is separated to install an AFO will vary. The portion of the midsole shown in FIG. 5 does not necessarily represent the entire midsole of a shoe, so additional midsole material may overlay the horizontal portion shown after the shoe is reassembled. When a strong material is used, such as spring steel, the horizontal portion can be thin such that it can be install against midsole materials that are soft enough to deform, rather than needing to cut, a channel portion into the midsole. Installing the horizontal portion near or against the bottom of a midsole should result in the best comfort to a user if the channel portion is deformed rather than cut into the midsole. Adjoinment of the horizontal portion and the vertical potion 34 of this alternate embodiment of the AFO 30 can as already described, or, where the material of the horizontal portion and vertical portion are different, insert molding or overlapping of materials can be used to join portions of the AFO. FIG. 5 shows a spring steel horizontal portion that has an upwardly bent section 46 that follows the rear 26 of the quarter 24 of a shoe 10, and a plastic vertical portion overlaps the upwardly bent section of spring steel such that the vertical and horizontal portions can be fastened together, such as with rivets. [0055] In the alternate embodiment shown in FIG. 6, the vertical portion 34 of the AFO 30 is characterized by a hinge 48 superior to where the vertical portion adjoins the horizontal portion 32, the hinge substantially approximating an ankle joint, allowing for flexion-extension of the AFO inferior to the hinge. The hinge can be spring-loaded with a resilient material to return the AFO to a dorsiflexed position after each step/extension imparted by the user. Additionally, an elastic pad 50 can be inserted along one or both opposing edges of a joint stop 52. The joint stop is composed of inferior and superior segments that prevent the horizontal portion of the AFO from excessively dropping, which in turn prevents a foot from excessively dropping during gait. The pad between the opposing edges of the joint stop will dampen impact forces and prevent audible contact of the joint stop materials during ambulatory range of motion. By way of additional example, an alternate elastic strap 38 that fastens around a user's leg using a simple button 54 is shown at the top of the vertical portion of the AFO. [0056] In the alternate embodiment shown in FIGS. 7 and 8, the horizontal portion 32 is characterized by long pins 56 that are anchored into the midsole 16 of a shoe 10. Holes can be pre-drilled or otherwise formed into the midsole such that the pins can easily be inserted. The long pins can be self tapping screws, but some midsole materials may not accept such an installation. An adhesive can be used to additionally secure the pins into the midsole. The pins can have flared heads, threads, or other fastener means for securing the horizontal portion of the AFO to the vertical portion 34. The vertical portion can additionally be secured to the quarter 24 of a shoe using one or more fasteners 36 or adhesive. [0057] In the alternate embodiment shown in FIG. 9, the inferior section of the vertical portion 34 of the AFO 30 is augmented by a flared portion 58 which forms a wrap around the quarter 24 of a shoe 10. Structurally, the flared portion is a substitute for a horizontal portion for a shoe that has a relatively stiff sole and upper. In this scenario, fasteners 36 are fastened to points that are medial, posterior and lateral points of attachment on the shoe's quarter 24. Consequently, the immobilization of the user's ankle and the anteriorly-adjusted points of circumferential attachment serve to create a functional equivalent of the vertical portion and horizontal portion combination of the preferred embodiment. This alternate embodiment, although possibly less aesthetic and hidden than the preferred embodiment, may have the advantage of being easier to install. Installation could be permanent and non-reversible, or may be by a method that would allow attachment and detachment at will by the user, such as via screw caps onto threaded pegs, metal clasps, etc. [0058] In the alternate embodiment shown in FIGS. 10 and 11, an AFO 30 similar to the one shown in FIG. 9 is removably attached to a shoe that preferably has three studs 60 fastened to the quarter 24 of a first shoe 10 using rivets 62, or another suitable fastener means. Each stud, which projects outwardly, is preferably characterized by a groove 64 that snaps into an aperture 66 formed in the flared portion 58 of the AFO. To install an AFO onto a first shoe equipped with studs, the flared portion of the AFO is pulled around the quarter of a shoe until a stud in the rear 26 of the quarter of the shoe snaps into the aperture at the posterior of the AFO. Next, apertures in the lateral and medial sides of the AFO are aligned over studs in the sides 28 of the quarter of a shoe, and the studs are snapped into the apertures. An ankle strap 68 is provided for securing the AFO around the ankle of a user. The bottom of the AFO is also preferably narrower than a shoe such that the AFO clamps around the quarter of a shoe, further securing the studs into the apertures. It should be noted that the stud at the rear of the quarter is unlikely to separate while the studs at the sides of the quarter are secured to the AFO. The AFO can easily be removed by unsnapping the studs. To equip a second shoe with studs, simply allowing the AFO to clamp around the quarter of the second shoe should allow someone to easily mark the required location of studs by passing a marker through the apertures in the AFO, and then riveting the studs into the quarter of the second shoe at the marker points. [0059] While a preferred form of the invention has been shown and described, it will be realized that alterations and modifications may be made thereto without departing from the scope of the following claims. | Summary: An Ankle-Foot Orthosis, or AFO, has a vertical portion and a horizontal portion joined together. The horizontal portion is fixed to a shoe, which is selected by a user, by separating at least part of the sole such that the horizontal portion can be adhered near or between a midsole of the sole of a shoe. The horizontal portion provides the necessary structure needed to connect a shoe to the vertical portion of the AFO such that a user suffering with a weakness or deformity can walk with a more normal gait. In an alternate embodiment, the AFO is removable from a shoe such that it can be replaced onto the same or a different shoe. | 4,523 | 139 | big_patent | en |
Summarize: This application is a continuation of application Ser. No. 163,259, filed 3/2/88, now U.S. Pat. No. 4,858,613. BACKGROUND OF THE INVENTION The present invention relates in general to the treatment of disease, tumors, etc., by the use of ultrasound. More particularly the present invention relates to a combined visualization and treatment device using ultrasound for both functions. Treatment is achieved by ablation of tissue representing the disease entity. A large number of diseases manifest themselves in whole or in part in a focal manner. These include, for example, diseases of or in the brain, breast, liver and prostate. While surgical procedures have traditionally been employed when medicinal approaches were not suitable or effective, surgery still represents a significant risk to the patient and a chance that the entirety of the disease entity will not be completely removed. There is no dispute as to the value of noninvasive treatment as such as producing volume lesions with focused ultrasound. One difficulty though with ultrasound treatment procedures is the need to visualize the disease entity and thereby determine the size, shape and location. While this concern does not normally exist with invasive techniques such as surgery, it is of critical concern in noninvasive procedures. In our pending application entitled ULTRASOUND BRAIN LESIONING SYSTEM, filed on even date herewith, a visualization technique is described for volume lesioning treatment of a brain tumor. The technique involves a use of ultrasound or CT or MRI scan transparencies whose data is digitized into a computer and the landmark references from a skull fixation apparatus are used to preprogram the drive system for the transducer. By computer control, the brain tumors are located and the transducer automatically programmed for positioning such that the focused ultrasound beam is directed at each tumor and the dosage set to produce volume lesions. An alternative to this position translation technique for brain tumors is to use ultrasound to visualize the disease entity. Since brain lesioning is somewhat unique due to the CT or MRI scans and the skull fixation apparatus, the visualization technique of our co-pending application may not be the most appropriate technique for ablation of other focal disease sites. Since some of these other disease sites may be most effectively treated by the use of ultrasound in either a transcutaneous or intraoperative mode, there is a need to insure that the transducer components which are designed and the materials selected be such so as to be suitable for steam autoclaving. The present invention provides an ultrasound localization and therapy system which is designed with both a visualization transducer and a therapy transducer. Those portions of the structure which must be sterilized are constructed from selected materials which are steam autoclavable. Another concern with the treatment of disease in a transcutaneous mode by ultrasound is the physical size and shape of the probe. Since the transducer design of the co-pending application is used external to the patient, size and packaging considerations are not substantial. However, with the modes of examination and treatment such as transrectal, transesophogeal, etc., the probe design is critical. While the specifics of our co-pending transducer design may be used in some embodiments of the present invention, it will require some scaling down in size. Further, if the transducer assembly is going to be steam autoclavable, certain material changes are advisable in order to provide a finished product which will withstand the high autoclaving temperatures. In a related embodiment the concept of utilizing a visualization transducer in combination with a treatment transducer is disclosed for treatment of the prostate. This particular configuration is adaptable for use in other body cavities. The therapy treatment from within such body cavities by ultrasound, where ultrasound is also used for imaging of the area to be treated, has not heretofore been done. SUMMARY OF THE INVENTION A visualization and treatment transducer for producing lesions in diseased tissue sites according to one embodiment of the present invention comprises a transducer housing having a main section and a detachable enclosure, movable visualization transducer means disposed within the detachable enclosure, movable treatment transducer means disposed within the detachable enclosure, first drive means providing rotary motion to the visualization transducer means in two degrees of freedom, second drive means providing rotary motion to the treatment transducer means in two degrees of freedom, the visualization transducer means and treatment transducer means having generally coaxial focal axes and the first and second drive means being operable independently of each other. A transrectal or other body cavity visualization and treatment transducer assembly for ultrasonic visualization and treatment by producing lesions in diseased tissue sites according to another embodiment of the present invention comprises a fluid-filled, flexible-walled enclosure, a movable visualization transducer disposed within the enclosure, a movable treatment transducer disposed within the enclosure, a reflective scanner disposed within the enclosure and aligned with the treatment transducer for changing the direction of the focused ultrasound beam from the treatment transducer, first drive means providing rotary motion to the treatment transducer, second drive means providing linear motion to the visualization transducer, the first and second drive means being operable independently of each other, and third drive means providing rotary motion to the visualization transducer and the treatment transducer concurrently. One object of the present invention is to provide an improved transducer assembly including both a visualization transducer and a cooperating treatment transducer. Related objects and advantages of the present invention will be apparent from the following description. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagrammatic illustration of an ultrasound treatment apparatus according to a typical embodiment of the present invention. FIG. 2 is a side elevation, diagrammatic illustration of a transducer assembly which is suitable for use in the FIG. 1 apparatus. FIG. 3 is a front elevation, diagrammatic illustration in full section of a transducer design suitable for use in the FIG. 2 transducer assembly. FIG. 4 is a perspective, diagrammatic illustration of an ultrasonic probe for prostate visualization and treatment. FIG. 5 is a side elevation, diagrammatic illustration of the FIG. 4 ultrasonic probe. FIG. 6 is a lateral section view of the FIG. 4 ultrasonic probe detailing the configuration and support of a reflective scanner. FIG. 7 is a lateral section view of the ultrasonic probe detailing the arrangement and support of the treatment transducer. FIG. 8 is a side elevation, diagrammatic illustration in full section of a control unit which is coupled to the FIG. 4 probe for imaging and treatment control. DESCRIPTION OF THE PREFERRED EMBODIMENT For the purposes of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated device, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates. Referring to FIG. 1, there is illustrated an ultrasound treatment system generally in block diagram form with the patient 20 lying on an appropriate table 21 with the transducer housing 22 diaphragm 23 in contact with the patient. A suitable coupling medium is used between the diaphragm and patient and the therapy transducer 24 is disposed in a volume of degassed water 25. In an intraoperative mode, sterile housing 22 with its diaphragm 23 is brought into contact with sterile fluid overlying on the internal organ or tissue directly. Guidance to the tissue or organ site is provided by ultrasound visualization element 28 located inside housing 22. The relative sizes and positional relationships of therapy transducer 24 and visualization element 28 which is an imaging transducer is best illustrated in FIG. 2. Housing 22 is manually placed in position by the operator while being guided by transducer 28 with the ultrasound image displayed on monitor 29. Housing 22 is supported by articulating arms 30 and 31 with rotation axes as shown by the rotary arrows. Vertical motion is shown emanating from base support 32. Once the system is appropriately located for treatment, the articulating arms and rotation axes are locked in place. From the scanning of visualization transducer 28, the treatment volume is defined and stored in computer 35. The spatial position of the treatment volume is also defined with respect to depth and orientation to surrounding tissues. By interacting with the tissue and organs displayed on monitor 36, the treatment spatial regimen is computed. Dosage parameters of sound intensity and time-on period are entered into computer 35. Once the treatment regimen is established, the system automatically progresses through the treatment volume by placing individual focal ablative lesions. Power amplifier 37 provides the drive energy to therapy transducer 24 for each focal site under control of computer 35. Degassed water system 38 provides degassed water to the interior of transducer housing 22 and temperature control system 39 keeps this degassed water at a constant temperature during the therapy procedure. The procedure can be interrupted at any time by the operator and restarted at the last stopped position, if that is desired. In the event the operating and control electronics are remote from the patient, which would be the typical case, local keyboard control 42 is provided for at-site interfacing with the computer 35. Also interfacing with computer 35 are the ultrasound guidance and site placement system 43 and the motion drive and control apparatus 44. Referring to FIG. 2, transducer assembly 27 is illustrated. Assembly 27 includes visualization transducer 28 which is a spherical ceramic piezoelectric element mounted in a metal ring. Hollow metal rod 47 attaches to this metal ring and runs through O-ring seal 48 in metal housing 49. Housing 49 is attached to metal housing 50 which runs through plate 51 and is sealed by O-ring 52. Transducer 28 is mechanically rotated (as shown by arrow) in a sector motion by rotation of rod 47 which is driven through bevel gears 55 and 56. Gear 55 is attached to rod (shaft) 47 and gear 56 is attached to drive shaft 57. Shaft 57 is driven in a rotary fashion by motor 58 which incorporates an encoder so that the angular position of transducer 28 is known. Knowing the angular position of transducer 28 provides angular information for the sector format (visualization) display. Electrical driving pulses and receiving pulses to transducer 28 go through wire lead 59 which attaches to the piezoelectric element in transducer 28 through the center hollow portion of rod 47. Transducer 28 is rotated in a plane normal to the plane of the paper from beneath transducer 22 by rotating tubular housing 50 using attached gear 62 which meshes with gear 63 driven by stepping motor 64 which has an encoder to establish the position of transducer 28 in this particular plane of rotary motion. Transducer 24 is rotated on axis elements 67. This rotation is accomplished through sprocket gear 68 driven by belt 69 which in turn is driven by sprocket 70. Sprocket 70 is driven by shaft 71 which in turn is driven in a rotary manner by meshed bevel gears 72 and 73. Bevel gear 73 is attached to and driven by shaft 74. Shaft 74 is rotatable through O-ring seal 75 in top plate 76 which is attached to tubular housing 77. Transducers 28 and 24 are positioned so that their respective ultrasound beam focal axes are substantially coaxial to each other. Tubular housing 77 is movable up and down relative to plate 51 through O-ring seal 80. Plate 51 is rotatable in ring 81 through ring gear 82 mounted to plate 51 and running entirely around the apparatus (360° circle). The parts including and below plate 51 and ring 81 are detachable from ring 83 for autoclaving. Ring 83a is illustrated as a separate piece but is in fact rigidly attached to ring 83. These components remain with the support (articulating) arms during the autoclaving procedure for the parts which are detached. Similarly, gear 85 is not removed for autoclaving. For autoclaving top plate 76 is removed with housing 77 and plate 51. After autoclaving plate 51 and ring gear 82 are inserted in ring 83 and attached by a plurality of pins 84 positioned around the periphery of ring 83a. Rotation of plate 51 is accomplished through circular ring gear 82 driven by gear 85 attached to stepping motor 86 which includes an encoder. When plate 51 rotates all attached members including transducer 24 rotate concurrently. Plate 76 meshes with tube 89 on insertion of plate 51 and ring 81. When plate 76 meshes, drive shaft 74 meshes with shaft 90 which is attached to stepper motor 91 which includes an encoder. Electrical drive power to transducer 24 is also coupled as is pressure system 92 when plate 51 and ring 81 are inserted. Vertical motion of transducer 24 is accomplished through ring 95 attached to tube 89 which links with plate 76. Ring 95 can rotate freely in element 96 which is driven up and down by gear rack 97 attached to element 96. Element 96 is constrained by slide system 98. Gear rack 97 is driven by gear 99 which is attached to stepper motor 100 and includes an encoder which is supported off the top surface 101 of ring 83 by member 102. Filling of chamber 103 with degassed water 25 is accomplished through tubing member 104 which is coupled through O-rings 105 to ring 81. Bath temperature in 103 is maintained by coils which circulate controlled-temperature fluid introduced through tubing 104. Therapy transducer 24 is provided with three degrees of freedom. The unit can be rotated about axis 106, it can be moved up and down as shown by arrow 107, and it can be rotated about axis 108. Use of these motions permits volume lesions to be made after unit 27 is locked in position. Referring to FIG. 3, internal details of therapy transducer 24 are illustrated in greater detail. It should be noted that this illustration does not include axis elements 67 and the power cable which is diagrammatically shown in FIG. 2 as a coiled wire connecting to the transducer is, in the FIG. 3 illustration a coaxial cable. While FIG. 2 discloses an air pressure system 92 for some of the interior spaces, FIG. 3 further includes a similar air pressure system 188 and an air pressure system 196 for controlling the silicone oil pressure for other interior spaces within transducer 24. Referring to FIG. 3, transducer 24 is configured with several unique features which are provided in order for a stable acoustic output to be obtained at all preselected driving levels. These driving levels are required in order to produce controlled focal lesions. In order to achieve this necessary objective, it is necessary to have a stable sound-producing source such as generally circular (disc) quartz plate 161 which is used in this particular embodiment. The quartz plate 161 is able to be maintained flat and parallel to generally circular, plano-concave lens 162 by the structure which will be described hereinafter. Lens 162 is a hard anodized aluminum lens with an elliptic concave surface for minimizing the half-intensity length of the beam at the focus. In order to maintain flatness and parallelism of plate 161 and lens 162 with a fixed spacing distance therebetween, the aluminum flat side of the lens is precisely machine flat with at least one 1/8 inch diameter rod 163 machined on the surface to extend a distance above the lens surface equal to a 1/4 wave length in the silicone oil which is disposed in space 165. In order to maintain this 1/4 wave length spacing to within plus or minus 0.0001 inches, it is required that the outer peripheral lip 162a of aluminum lens 162 provide unanodized surfaces (flat top and bottom surfaces and outer edge surface) which rest directly in contact with the flat machined surface of housing 164 and end plate 164a. Housing 164 includes an inwardly and upwardly directed lip 164b, of an annular ring configuration, whose underside abuts against the top surface of lip 162a and whose top surface supports plate 161. The height of this lip is precisely machined since it is the means to fix the 1/4 wave length separation between the plate 161 and lens 162. Rod 163 provides center stabilizing for the plate due to its span between peripheral edge supports and the pressure differential between the top and bottom surfaces of the quartz plate. The space 165 between the plate 161 and lens 162 (the 1/4 wave length spacing) is filled with silicone oil 166 which is freely exchanged through radially open channels in lip 164b. A suitable silicone oil for this application is Dow Corning 710 fluid. Gasket 164c seals the oil in space 165. One gold-plated and polished electrode, electrically connected to quartz plate 161, rests in direct contact with the top machined surface of lip 164b and provides the electrical ground contact for the quartz plate. In order to keep plate 161 in pressure contact with housing 164, a flat, flexible gasket 171 is firmly pressed against plate 161 through metal member 172. In order to provide electrical contact for power to plate 161 an electrode 173 fabricated of an approximate 0.001 thick soft metal foil (gold, brass, silver) extends part-way under compression gasket 171, while the remainder of gasket 171 acts as a seal for the silicone oil. The power and ground electrodes on plate 161 do not extend to the edge of plate 161 and the silicone oil provides insulation around the edge. The foil electrode 173 is attached to metal member 172 with a series of metal screws 174. To provide RF power to drive quartz plate 161 a coaxial cable 179, with metal sheath 180 drawn back and clamped under plate 181 to metal plate 182, is provided. The coaxial cable has an end plug 184 which side pressure contacts plate (metal member) 172 through a central hole. Space 185 is an air space so that the quartz plate 161 is not back acoustically loaded thereby directing all its acoustic output through the interspace 165 and lens 162 into the fluid which is in front of lens 162. To insure flatness of quartz plate 161 and parallelism with the flat surface of lens 162, the air space 185 and all other air spaces in the transducer housing 164 are pressurized through tube 186 into element 187. This air pressure holds quartz plate 161 against machined rod 163 to maintain the necessary parallelism. Pressure is applied from source 188. In order to maintain a positive differential pressure in space 185 relative to the pressure in interspace 165, flow communication is provided from interspace 165 via flow access channels 189 into column 190 and well 191. These areas are all silicone oil filled and in pressure equilibrium is a thin flexible diaphragm 192 which covers well 191. Above diaphragm 192, the air space 193 is exhausted through flexible tubing 194 and rigid tube 195 to the outside atmosphere. A further feature to suppress cavitation in the oil in space 165 between the quartz plate 161 and lens 162 when the system is run at the highest acoustic output power is provided by pressure system 196 providing greater-than-atmospheric pressure to space 193. Typically this pressure will be that which prevents any cavitation in space 165 (of the order of 40-50 pounds per square inch). This pressure in space 193 is readily transmitted through diaphragm 192 to the silicone oil in well 191 and hence through column 190 into space 165. The pressure provided by source 188 is in the order of 2 pounds per square inch higher than the pressure in system 196 in order to keep plate 161 flat and held against lens 162 through rod 163. Element 199 in the transducer assembly is an insulating member to which element 172 is bolted by screw(s) 200. Gasket 201 keeps the silicone oil contained in column 190 from reaching the coaxial cable 179. Metal plate 182 is bolted to housing 164 around the outer periphery of plate 182. Oil is kept in column 190 and well 191 by the use of O-ring seal 203 positioned between housing 164 and plate 182 and by gasket 205. Member 206 is bolted and sealed to plate 182. Top metal plate 207 is bolted by screws 203 to housing 164 and sealed thereto through O-rings 209. Metal tube 195 is sealed to element 187 through seal 210. The coaxial cable 179 is water-tight and sealed to top plate 207 through member 211 and O-ring 212. In order to accomplish the task of producing lesions of any complex size or shape with intense focused ultrasound it is necessary to provide for ultrasound dosage conditions which produce individual focal lesions (from which the complex volume can be generated), which do not compromise tissue outside the intended focal lesions side and permit subsequent individual focal lesions in a contiguous manner. When transducer 24 is used for the treatment of brain tumors by creating lesions in deep brain sites in both gray and white matter and abnormal brain tissue, it is necessary to inhibit the production of microbubble formation at the primary focal site so that there can be no vascular dispersion of such microbubbles away from the primary focal site which microbubbles could initiate off primary site lesion production and hemorrage due to ultrasound passage through microbubble comprised tissue. In order to accomplish this task while being able to accomplish primary site lesions, it is necessary to derive these sound intensities as a function of frequency which will not produce microbubbles at the primary lesion site. This requires that for a 1 MHz sound frequency (a frequency necessary to achieve deep penetration into the human brain), the primary site sound intensity must not exceed 300 watts per square centimeter. At this intensity and for lower intensities, gray and white matter lesions on a multiplicity of individual contiguous sites can be produced without undesirable side effects (microbubbles). As the frequency is increased above 1 MHz, the primary site sound intensity can be increased and produce no microbubbles but the penetration capability in brain tissue returns as the sound frequency is increased. At 4 MHz frequency which is the upper frequency which can be considered for more superficial brain lesion production, the intensity which will not lead to microbubble formation is at least 2100 watts per square centimeter. At these intensity limits, the time-on period of sound irradiation at each individual site can be extended to as many seconds as is needed to ablate the tissue at the focal site without microbubble formation. In order to constrict the individual lesion sites so that the boundaries of desired volume lesions can be constrained, the transducer configuration used will give a half intensity length at the lesion focal region in the order of 15 mm at 1 MHz operating frequency. This length of half intensity is consistent with the necessity of constraining lesions in the human brain so that the extending of individual lesions into white matter (white matter is more sensitive than gray matter) can also be constrained. Still referring to FIG. 3, in order to make the transducer assembly 27 capable of being steam autoclaved, gasket 171 needs to be made from fluorosilicone in order to take the high autoclave temperature and resist the uptake of the silicone oil which is used within the assembly. A suitable silicone oil for this application is Dow Corning 710 fluid which has the necessary high temperature resistance. All gaskets in contact with the Dow Corning 710 fluid must be made of fluorosilicone. All other O-rings and gaskets not in contact with the Dow Corning 710 fluid can be made of silicone. Insulator 199 must be a high-temperature plastic, such as, for example, General Electric's Ultem. Coaxial cable 179 must also include high-temperature materials such as Teflon insulation. The volume expansion chamber (well) 191 requires a fluorosilicone membrane 192 which must be capable of taking the volumetric expansion of the silicone oil during the autoclaving procedure. The system design requires that all differential expansions be accounted for when the steam autoclaving is performed. As previously pointed out, one of the primary concerns with transcutaneous and intraoperative modes of ultrasound treatment is the need to design the transducer assembly so that those portions that need to be autoclaved can be steam autoclaved. Recognizing that the entirety of the assembly will not be contaminated by use in the prior treatment procedure, only selected components need to be autoclaved and these are detachable as previously described. Another concern is the ability to visualize the area for treatment. In order to guide and manuever the therapy (treatment) transducer to the appropriate ablation sites within the body, some visualization means must be employed. In the disclosed embodiment of FIGS. 1-3, the visualization means is the visualization transducer 28. Yet another concern with a transcutaneous mode of treatment is the size and shape of the probe (transducer assembly and housing). Transcutaneous modes may include transrectal or transesophogeal, for example. Localization and treatment (tissue destruction) of the prostate by way of a transrectal route requires both the ability to localize the treatment volume and then to apply the treatment regimen in that identified volume. One configuration to accomplish this particular task is described in FIGS. 4-8. In the FIG. 4 embodiment, ultrasound probe 240 is illustrated as inserted into the rectum and positioned for visualization and treatment of the prostate 241. Also illustrated and positioned in FIG. 4 are the urinary bladder 242 and rectum 243. Diagrammatically illustrated is a cross-section area of the tapered stem of probe 240 in order to show the entry diameter 244. The probe is inserted by way of the rectal entry region 245. Referring to FIG. 5, the internal features and components of probe 240 are diagrammatically illustrated. In answer to concerns previously mentioned, probe 240 includes a focused transducer 248 for delivering the therapy (abalation) which is supported by and movable relative to arm elements 249 positioned within flexible envelope 250. Envelope 250 is filled with water so as to expand to contact the rectal wall, but by removal of some water and some rotation of transducer 248 and mirror 256, the size is reduced to make entry easier. Arm elements are curved so that when the unit is in the rectum, the diameter at the entry of the probe is smaller than the remainder. Visualization element 253 includes in its interior space transducer 254 which is operable to generate ultrasound imaging beam 255 in the direction of the prostate. Transducer 248 is movable in a rotary manner relative to elements 249 and has a focused beam directed at circular (disc) mirror 256 which is adapted to bend and redirect beam 257 toward the desired region of the prostate. The movement of transducer 248 relative to mirror 256 is used to affect the depth of the beam (focused spot) into the prostate. Since the transducer beam has a fixed focus, the less of the beam length used between the transducer and mirror, the longer the beam length reflected from the mirror. Transducer 248 is also movable linearly with mirror 256 along the longitudinal axis of probe 240. The entire probe portion is rotatable by external means as illustrated in FIG. 8. Referring to FIG. 6, the support of mirror 256 by arm elements 249 and related components is illustrated in greater detail. As previously described, elements 249 which support transducer 248 and by means of rotary and sliding extensions 260 also support mirror 256. Arm elements 249 are thin-walled, hollow, flexible tubes open at their proximal end for the exiting of elements 262 and 263 (FIG. 7) and bands 273 and 274 (FIG. 7). Extensions 260 rigidly attach to mirror 256 and extend through slot 267 so that linear movement of the mirror relative to elements 249 can be affected. Extensions 260 fit within elements 262 for rotary motion and elements 262 travel in top and bottom tracks 268 formed as part of the interior wall surface of element 249. Referring to FIG. 7, the extension of elements 249 and their coupling to transducer 248 is illustrated. Both FIGS. 6 and 7 should be regarded as lateral sections looking along the longitudinal axis of the ultrasonic probe 240 with the mirror and transducer oriented so as to reveal their full disc (circular) configuration. The structure of FIG. 7 is virtually the same as FIG. 6 with one main difference. The rotational and linear travel linkage made up of elements 263, 270, 272 and 274 for transducer 248 is outward, relative to element 249, from elements 262, 269, 271 and 273 for the mirror. This allows the linear travel of the mirror to be separately controlled as well as the rotation relative to element 263, without interference between the transducer and mirror and their linkages. Referring now additionally to FIG. 8, control unit 261 which attaches to the reduced diameter end of probe 240 is illustrated. The linear movements on both transducer 248 and mirror 256 are accomplished by the linear translation of elements 262 and 263 which are flexible strips or bands so that they are able to accommodate the configurational bend in arm elements 249. Elements 262 and 263 are coupled to linear actuators and encoders, all of which are represented by block 264 through couplers 265 and 266. This arrangement permits coordinate linear translation of the therapy transducer and reflective mirror with respect to the visualization element 253 and the beam 255 generated by transducer 254. Rotation of focused transducer 248 and reflective mirror 256 is accomplished by crank arms 269 and 270. The crank arms with pins 271 and 272 are in turn driven by bands 273 and 274. These bands are connected to the linear actuators and encoders represented by block 264 by way of couplers 275 and 276. This particular arrangement permits the coordination of linear translation and/or relative translations to rotate the focused transducer 248 and reflective mirror 256. On insertion of ultrasonic probe 240 into the rectal area, the focused transducer and reflective mirror are rotated so as to reduce as much as possible the overall outside contour of the probe upon entry into the patient. The mirror may be rotated on axis, i.e., relative to elements 249 in order to gain additional space within the probe for movement of the visualization transducer 254. Except for these two instances of mirror rotation, it remains rotationally fixed. When visualizing the prostate elements, the focused transducer and reflective mirror are translated as shown so that free visualization of the prostate can be accomplished and then the transducer 248 and reflective mirror 256 positioned in order to place beam 257 at the positions delineated by beam 255. These position determinations are made through encoder determinations arrived at by computer computations. Visualization element 253 is linearly translated and encoded by rack 279, pinion 280, shaft 281, and drive motor with encoder 282. In order to provide rotary motion in the rectum, the entire system including control unit 261 can be rotated through ring gear 285 driven by pinion or drive gear 286 and motor encoder 287. Filling (and emptying) of the unit with degassed water is done through tubes 288 and 289 so that the entire system is water-filled and means for removing trapped air bubbles provided. This arrangement avoids sliding seals at the juncture between the insertable elements and the exterior elements. Flexible envelope 250 is attached to control unit 261 by slipping band 290 over the outer surface of envelope 250. All electrical leads, some of which are shown diagrammatically, pass through water-tight seals in control unit 261. Electrical power to the focused transducer 248 is provided by an electrical lead which travels along arm element 249. While the invention has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiment has been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected. | Summary: A transducer assembly for visualization and treatment of transcutaneous and intraoperative sites includes in combination a visualization transducer and a treatment transducer, each of which are movable with both linear and rotary degrees of freedom. Movement of each transducer is by various motor and geared drive arrangements wherein certain degrees of freedom for one transducer are separate and independent from the degrees of freedom for the other transducer. At least one degree of freedom for each transducer is common and the transducers are moved concurrently. One arrangement of the transducer combination is for prostate treatment and includes a specific shape and configuration for anatomical considerations and a control unit which is operable external to the patient to control both transducers and a reflective scanner which are inserted into the patient as part of the ultrasound probe. | 8,061 | 178 | big_patent | en |
Summarize: BACKGROUND OF THE INVENTION Over a period of years synthetic sausage casings have been prepared from animal collagen which are particularly suited for the processing of pork sausages. As is known, sausage casings made from collagen are edible and they have the ability to transmit fat during the cooking of pork sausages and therefore have been found to be an acceptable substitute for natural casings. In the manufacture of collagen casings a collagen source, typically hide collagen, is converted into a slurry containing from about 2 - 8% collagen by grinding the collagen source in a meat grinder and diluting with water. The collagen is acid swollen for releasing the collagen fibrils and destroying the identity of the individual fibers. The swollen collagen is extruded through an annular die to form a collagen tube. As the casing is extruded it is passed into a coagulating bath containing a dehydrating and deswelling agent, e.g., a concentrated solution of sodium sulfate or ammonium sulfate with a minor amount of alkali, e.g., sodium hydroxide to neutralize any free acid present in the casing. After the casing is coagulated, it is tanned suitably with an aluminum tanning agent for the purpose of providing sufficient strength to the casing to permit further processing. Often a second tanning operation is conducted using a dialdehyde as the tanning agent. The tanned casing is then washed, plasticized, and dried. DESCRIPTION OF THE PRIOR ART In the past it has been common to use a collagen source which has not been fully limed. In fact, the early processes required that the collagen source be unlimed if an edible casing was to be obtained. Through later developments, it was found that a partial liming of the hide collagen could be effected without incurring undesirable results in the casing if the liming were used for dehairing the hide and was confined to a period of about 3 to 12 hours. A maximum of four days' liming treatment could be tolerated in those processes but it was very difficult to obtain a casing which could be extruded and processed. In my co-pending application having Ser. No. 347,293, and filed Apr. 2, 1973, casing is made from a collagen source which has been subjected to complete liming by preswelling the collagen source with a concentrated acid solution having a dissociation constant in water from about 1 × 10 - 6 to about 1 × 10 - 3. The swollen hide is then ground and formed into a collagen slurry and processed in conventional manner. SUMMARY OF THE INVENTION This invention relates to an improvement in a basic process for producing collagen casing from hide collagen source comprising the steps: forming a slurry containing from about 2 - 8% collagen, extruding the slurry through an annular die to form a tubular casing, coagulating, tanning, and drying the tubular casing thus formed. The improvement in this basic process comprises the steps of using hide collagen which has been subjected to substantial liming as the collagen source. In this process, hide collagen which has been subjected to substantial liming is treated with a dilute, edible acid having a pH below about 5.5 for a time sufficient to reduce the pH in the center of the hide to about 4 - 5.5 and then washing the thus formed water-soluble calcium salts from the hide collagen until the pH of the effluent is within at least 0.2 pH units of the pH of the incoming water. The basic advantage of the process is that it permits the use of a hide collagen which can be prepared substantially cheaper than any of the commercial process heretofore required for collagen casing manufacture and it permits wider flexibility in commercial collagen casing manufacturing plants because of the ability to store the collagen source for extended periods of time without bacterial degradation. DESCRIPTION OF THE PREFERRED EMBODIMENTS Collagen suitable for preparation of edible casings is obtainable from hide and tendon, although hide collagen is preferred for casing manufacture. Collagen is formed of a large number of fibers which, in turn, consists of a much greater number of fibrils of submicroscopic size. The fibrils have a diameter of the order of 10 - 50 angstroms and lengths ranging from several thousand up to several million angstroms. In the early patents, the production of edible collagen casings emphasize the necessity of using a collagen source which has not been subjected to a liming treatment. The reason which has been postulated is that the liming treatment allegedly prevents the bursting of collagen fibers to release the fibrils necessary for the formation of fibrillar films. As a result, the gel casings disintegrate in the coagulation bath or on further processing. Later patents have shown that satisfactory edible collagen casings can be prepared from hides which have been subjected to a limited liming, e.g., from 3 to 12 hours and then subjected to a deliming treatment and processed quickly. Generally, these hides are limed to effect partial dehairing of the hide and that is the extent of the operation. The hides are not shipped or stored in a saturated liming solution for an extended period of time. Thus, in the processing of partially limed hides, e.g., 3 to 12 hours and perhaps up to 4 days liming treatment, it is possible to neutralize the hide by treatment with an acid having a pH from 2.5 - 6.5 by treatment with a dilute solution of lactic acid for a period of about 10 to 12 hours, i.e., overnight. It is also easy to remove the water-soluble calcium salts from the hide by washing with water. The primary reason for the acceptability of this process was that the calcium had not penetrated the hide to any substantial degree and therefore was easy to remove. In practicing this invention, the liming period of the collagen source is at least 7 days and generally for periods up to several months. In preparing hides which have been subjected to an extended liming period in a saturated lime solution for casing manufacture, the hides are first washed thoroughly with water to remove surface lime. Washing of the hide is continued until the effleunt from the hide has a pH of about 7. Although the washing step can be eliminated and the lime neutralized, it is much more economical to wash the hide removing substantial amount of lime from the hide rather than using an expensive acid. After the hide has been washed and the surface lime removed the hide then is tumbled with a dilute aqueous acid having a pH below about 5.5 and preferably from about 4 - 5.5. The collagen source is kept in the dilute acid until the pH of the hide, at the center, is less than 5.5. The pH of the hide can be conveniently measured by phenol red. A deliming solution having a pH below about 4 is not preferred because the casing begins to swell in these solutions and it becomes extremely difficult to remove the water-soluble calcium salts or excess acid from the hide. Generally, this period of acid tumbling may extend from about 4 to 20 hours for a hide having a thickness of about 1/8 inch to about 6 to 40 hours for a hide having a thickness of about 1/4 inch. Soaking requires at least 40 hours. Acids suitable for neutralizing the lime in the hide include lactic acid, hydrochloric acid, acetic acid, ethylene diamine tetraacetic acid, ammonium chloride, propionic acid, fumaric acid, etc. Ammonium chloride or other salts of weak bases and stong acid are preferred for neutralizing the excess lime in the hide collagen because it is easier to maintain an appropriate pH range to effect this neutralization. After neutralization, the collagen is washed with water for removing substantially all of the water-soluble calcium salts from the hide. Washing is continued until the supernatant, after contact with the hide for a period of about 20 minutes, has a pH within 0.2 pH units of the pH of the incoming water. Usually the washing step is continued until the pH of the supernatant is about 6.7 - 6.8. In the past, it had been commonplace to soak the partially limed hides for about 10 - 12 hours and then wash the water-soluble calcium salts from the hide. This process was permissible for partially limed hides but when completely limed hides were used a casing could not be manufactured. It was believed that the failure was due to the fact that the collagen had been subjected to liming rather than a defect in the deliming process. I now believe the flaw in the prior art processes was that no accurate measurement of the deliming step was made and it was assumed that deliming was effected by soaking with acid for a period of from 10 to 12 hours. Deliming requires substantially longer periods of time when the hides have been completely limed than when partially limed. As a result, a time period is not the governing factor for the deliming step but rather the measurement of the pH in the hide and that pH being below about 5.5, (preferably 4 - 5.5) is the important feature. This insures that all of the calcium in the hide has been neutralized and converted to a water-soluble salt. This aspect was pointed out in my earlier filed case on the concentrated acid swelling of collagen in neutralizing collagen which had been subjected to an extended liming treatment. It was believed at that time that a concentrated acid was necessary for swelling the hide to enhance penetration of the acid into the hide and to provide a great enough concentration gradient to permit penetration of the hide by the acid and thereby effect neutralization of the hide. I have now found that it is possible to use a dilute acid solution having a pH below about 5.5 and preferably from about 4 - 5.5 for treating the completely limed collagen source for an extended period of time until the pH of the hide, at the center, is below about 5.5 and preferably anywhere from 4 - 5.5. This insures that complete neutralization of the calcium in the hide has taken place and is not left to estimate as was done in the previous prior art process. After the hide has been completely neutralized by treatment with a dilute acid, the water-soluble calcium salts are washed from the hide. Washing of the hide to remove substantially all of the calcium salts is difficult when the hide is in a swollen state. This was one of the difficulties with my earlier process where a concentrated acid solution was added to swell the hide and effect neutralization of the calcium in the hide. However, in my previous process, most of the calcium in the hide had been removed by the preliminary neutralization and washing steps. Thus, only a minor proporation of calcium remained in the hide and was neutralized by the concentrated acid treatment. But the removal of this minor proportion of calcium in the hide was extremely important in the process of the hide for casing manufacture. In practicing the process of this invention washing of the hide is continued until the pH of the supernatant, after a contact period of 20 minutes with the hide, has a pH within 0.2 pH units of the incoming water. In some areas of the country, water is slightly acidic or basic and therefore a pH of 7 is not used as the cutoff pH. This step is particularly important in the processing of limed collagen for making a collagen slurry which can be processed into a collagen casing. In applying prior art processes to hides which were subjected to a complete liming treatment, acid neutralized by a treatment somewhat similar to the one described herein and then subjected to a water wash, there was no accurate measure for determing when the hides were washed thoroughly enough for permitting manufacture of collagen casing. Washing techniques in the prior art processes were left to operator discretion as to when washing of the hides was deemed complete. Usually this was a visual procedure and washing was discontinued when the effleunt was clear. In actual practice this is not an accepted procedure as the only way to tell if enough of the water-soluble calcium salts are removed is to measure the pH of supernatant after it has been in contact with the hide for an extended period. The following examples are provided to illustrate the preferred embodiments of the invention and are not intended to restrict the scope thereof. All percentage are expressed as weight percentages. EXAMPLE 1 Selected cattle hides from carcasses certified fit for human consumption, weighing about 65 - 75 lbs. each, are washed in a large volume of circulating cool (10°C) water to remove adhering blood. After washing the hides are fleshed without curing to remove adhering fatty and muscular debris from the flaying operation. The washed and fleshed hides are treating by immersing the hides in a liming bath consisting of a saturated solution of calcium hydroxide containing about 5% solid particulate calcium hydroxide in about.5% sodium sulfhydrate for about 3 - 12 hours to effect partial dehairing of the hide. After liming, the hides are removed from the liming bath and allowed to drain for a period of about 1/2 hour. The limed hides are gently squeezed between rubber rollers to remove all excess liming liquor. The hides are then cut and split in the plane of the hide into two approximately equal portions by weight. The upper or outer hide surface contains all of the hair, hair follicles, and sebaceous and sudorfic glands. The inner or corium layer consists essentially of collagen. The outer or hair-containing layer is discarded as unsuitable for use in the preparation of casing but may be used for formation of leather laminates or other coverings. The corium splits are packed in a saturated lime solution and stored at temperatures preferably below about 5°C until processed. For convenience at the processing plant, the limed hides may be stored in this lime solution to prevent bacterial growth until the hides are ready to be used. Sometimes this period is anywhere from 1 - 12 weeks or longer. To prepare the hides for use in the manufacture of collagen casing, the corium splits are first washed with water to remove surface lime. Washing with water is continued until a liquor pH of about 7 is obtained when the liquor has been in contact with the hide for about 20 minutes. This step can be eliminated by neutralization with dilute acid as will be described in the next step except that washing of the hide is preferred for economic reasons. The hides are delimed by converting the lime to a water-soluble calcium salt. Deliming is effected by tumbling the splits with a dilute aqueous acid solution, e.g. 0.5% lactic acid having a pH from about 4 - 5.5. Deliming of the hide is continued for about 20 hours at which time the acid end point in the center of the hide is less than 5.5. The end point is conveniently measured by phenol red. After deliming, the hides are thoroughly washed with water to remove substantially all of the water-soluble calcium salts therein. Washing of the hide is continued for about 4 hours and completed only when the supernatant, when in contact with hide for about 20 minutes, has a pH of 6.7. The incoming water wash has a pH of 6.8. Then the delimed and washed hides are chopped into small pieces, e.g., 1/4 to 4 inches on a side and converted to a fine pulp by successive passes through a meat grinder. In this grinding operation, ice is mixed with the hide splits to maintain the temperature below about 20°C and preferably below about 10°C. After the hide has been passed through the meat grinder, sufficient water is added to the collagen pulp to produce a mixture consisting of about 90 - 95% water by weight. The collagen pulp is then treated with sufficient dilute lactic acid to produce a pH of about 2.5 - 3.7 and is stored overnight at a temperature of 3°C to effect swelling of the collagen. At the end of the overnight soaking period, the collagen has swollen and taken up all of the water in the slurry. The swollen collagen is mixed with additional water and acid to produce a homogenous paste containing about 4% collagen and 1.2% lactic acid (to maintain a pH of 2.5 - 3.7). The paste is further homogenized, filtered to remove solid contaminant and deaerated. The solution is pumped under pressure through an extrusion die such as the one described in Becker U.S. Pat. No. 2,046,541 into a coagulating bath consisting of 42% ammonium sulfate (sodium sulfate can also be used) in water. When the casing is extruded as a thin-walled tube into this concentration of ammonium sulfate, the collagen fibrils are dehydrated and collapsed to form a film which is sufficiently coherent for further processing. Generally the coagulation bath is circulated both inside and outside the tube to maintain the tube in an inflated condition and to insure proper coagulation of the casing both on the inside and outside. From the coagulation bath, the casing is passed into a first tanning bath comprising an aqueous solution containing about 5% aluminum sulfate expressed as Al.sub. 2 (SO 4 ).sup.. 14H 2 O, 4% sodium citrate and 4% sodium hydroxide. This tanning bath is formulated so that the sodium citrate (or citric acid) forms a complex with the aluminum sulfate and the sodium hydroxide neutralizes the portion of the aluminum-citrate complex to render the same about one-third to two-thirds basic. This results in the tanning bath having a pH of about 4. The bath is maintained at a temperature of about 15° - 30°C and the residence time of the casing in the bath is adjusted to about 5 minutes. After the casing is tanned with the aluminum complex, it is passed through one or more wash baths to wash out any unreacted tanning or hardening agent and then passed through a second tanning bath containing 200 ppm glutaraldehyde in water. Glutaraldehyde tanning bath has a pH of about 4.3 and is maintained at a temperature of about 15° - 30°C. The residence time of the casing in the glutaraldehyde bath is about 3 minutes. After the casing has been subjected to a second tanning operation, it is removed from the bath and passed through one or more wash baths to wash out any unreacted glutaraldehyde. The casing is then passed through a plasticizing bath containing about 3% glycerin, 0.1% sodium bicarbonate and sufficient sodium hydroxide is added to raise the pH to 8. The residence time of the casing in the bath is about 3 minutes. After the casing leaves the plasticizing bath, it is dried, shirred and packaged. The casing shirrs well and has excellent pan frying and deep frying characteristics. EXAMPLE 2 The procedure of Example 1 is followed except that ammonium chloride is used in place of the lactic acid for neutralizing the lime present in the hide. A 2% solution of ammonium chloride in water has a pH of 4.3 and is extremely effective and convenient to use for neutralizing the lime in the hide. The tumbling period of the hide in the ammonium chloride solution is about 8 hours and the tumbling treatment is terminated when the hide has a pH of 4.7. Casing made in accordance with the same procedures of Example 1 as acceptable process strength for commercial operation and the pan frying and deep fat frying of the resulting casing is good. EXAMPLE 3 The procedure of Example 1 is repeated except that the hides are soaked, not tumbled, in the dilute lactic acid for about 12 hours. The water-soluble calcium salts in the hide are removed by washing with water for a period of about 3 hours. A slurry is prepared in accordance with procedure of Example 1 and processed accordingly. The casing does not extrude into a continuous film and the casing breaks in the middle of the processing operation. | Summary: This invention relates to a method for preparing a collagen casing from a collagen source which had been subjected to a rigourous liming for effectively completely liming the hide collagen. The fully limed hide collagen is soaked in a dilute edible acid having a pH below about 5.5 for a time sufficient to reduce the pH in the center of the hide to about 4 - 5.5, and then washing the neutralized hide with water until the pH of the supernatant after a 20 minute exposure with the hide, is within about 0.2 pH units of that of the incoming water. The hide is then ground and formed into a collagen slurry and processed in conventional manner into edible collagen casings. | 4,610 | 158 | big_patent | en |
Write a title and summarize: Chagas disease is caused by the parasitic infection of Trypanosoma cruzi (T. cruzi). The STOP CHAGAS clinical trial was initiated in 2011 to evaluate posaconazole in treating Chagas disease, with treatment success defined as negative qualitative PCR results of detecting the parasites in blood specimens collected post-treatment. PAXgene Blood DNA tubes were utilized as a simple procedure to collect and process blood specimens. However, the PAXgene blood specimens challenged published T. cruzi PCR methods, resulting in poor sensitivity and reproducibility. To accurately evaluate the treatment efficacy of the clinical study, we developed and validated a robust PCR assay for detecting low level T. cruzi in PAXgene blood specimens. The assay combines a new DNA extraction method with a custom designed qPCR assay, resulting in limit of detection of 0. 005 and 0. 01 fg/μl for K98 and CL Brener, two representative strains of two of T. cruzi’s discrete typing units. Reliable qPCR standard curves were established for both strains to measure parasite loads, with amplification efficiency ≥ 90% and the lower limit of linearity ≥ 0. 05 fg/μl. The assay successfully analyzed the samples collected from the STOP CHAGAS study and may prove useful for future global clinical trials evaluating new therapies for asymptomatic chronic Chagas disease. Chagas disease (CD) is a tropical parasitic disease caused by the chronic infection with the protozoan T. cruzi. About 6 to 7 million people, primarily in Mexico and Central and South America, are estimated to be infected [1]. Left untreated, up to 30 to 40% of chronically infected patients will progress to have symptomatic disease, manifested as serious heart and digestive problems [2], with an estimate of more than 10,000 deaths annually [3]. Current anti-parasitic medications such as benzindazole (BNZ), although highly effective in treating acute phase infection, have limited efficacy in eliminating T. cruzi for chronically infected patients and also can induce severe side effects [4]. The STOP CHAGAS study [5] (ClinicalTrials. gov Identifier NCT01377480) was a Phase 2 proof-of-activity clinical trial comparing posaconazole (POS) with BNZ in treating patients with asymptomatic chronic CD. It was a randomized placebo-controlled study conducted in nineteen centers in five Latin American countries and Spain. The study’s primary end point was qualitative PCR results of detecting T. cruzi in whole blood samples collected post-treatment, with the treatment success response defined as the consecutive negative qualitative PCR results of blood collected on at least three post-treatment dates. The reason why persistent negative PCR results of multiple time points were utilized to define treatment success was due to low levels of circulating parasites in chronic CD patients [6]. To minimize false negative PCR results and thus reduce the false positive efficacy outcome, a PCR assay with high sensitivity and specificity was required. The PCR assay was initially performed by a contract research organization (CRO) that used a widely used commercial kit to extract DNA from a 0. 2 ml aliquot of PAXgene blood, and a published TaqMan based qPCR assay targeting minicircle DNA of T. cruzi’s kinetoplast DNA (kDNA) [7]. The study suffered from a high screen failure rate with the CRO’s assay (labeled as the Version 1 assay for the rest of the document). Among the 393 serologic positive patients screened for the study, only 123 were tested PCR positive and thus eligible for the study. Our investigation of the Version 1 assay found the persistent problems of inconsistent and low DNA extraction efficiency, poor precision among the technical replicates of reference T. cruzi DNA used to establish standard curves of kDNA qPCR assay, and highly variable parasite loads among individual 0. 2 ml aliquots of the same clinical PAXgene blood sample. We later discovered that a major cause of the substandard performance was the utilization of PAXgene Blood DNA tubes, because the Version 1 assay was designed for the commonly used method for collecting and processing CD blood, which involves the immediate mixing of freshly collected 10 ml EDTA-blood with 10 ml of 6M guanidine hydrochloride and 0. 2 M EDTA buffer (GEB), often followed by 10 minute boiling, resulting in the lysis of T. cruzi in the 10 ml blood, the release of kDNA from T. cruzi mitochondria, and the decatenation of minicircle DNA from the complex structure of kDNA. The PAXgene tubes could not lyse the cells and thus caused the variable parasite load estimates among individual 0. 2 ml aliquots. In addition, PAXgene blood prevented the Version 1 assay from efficiently and reproducibly isolating T. cruzi DNA. To accurately evaluate the treatment efficacy outcome of the STOP CHAGAS study, we developed and validated a robust, sensitive and specific assay (labeled as the Version 2 assay for the rest of the document) to detect T. cruzi DNA in PAXgene blood specimens collected from the patients randomized in the study. Reference DNA of both K98 (a representative strain of T. cruzi discrete typing unit (DTU) TcI) and CL Brener (TcVI) were obtained from Dr. Alejandro Gabriel Schijman’s laboratory at INGEBI-CONICET, Buenos Aires, Argentina. The stock concentration was 1. 5 ng/μl, equivalent to 7. 5 x 106 parasites per ml of blood (PPM). Serial titrations were made with low EDTA TE buffer (Cat. Num. 12090–015, Thermo Fisher Scientific, Waltham, MA) containing 10 ng/μl carrier RNA (Cat. Num. 4382878, Thermo Fisher Scientific) so that the DNA dilutions could be stably stored at 4°C for at least 1 month. A linearized pZErO plasmid containing a sequence of Arabidopsis thaliana was also obtained from Dr. Schijman’s laboratory. The stock concentration was 0. 6 ng/μl and serial titrations were made with the above mentioned low EDTA TE buffer containing carrier RNA. To evaluate DNA extraction efficiency and to detect potential PCR inhibition from inhibitors such as heme from whole blood, 5 μl of 0. 03 pg/μl of IAC DNA was spiked into 200 μl equivalent of clinical PAXgene blood specimens as the internal control. The amount of IAC DNA was chosen so that its corresponding PCR Ct values (around 27) in eluted DNA samples were near the midpoints of kDNA qPCR standard curves for both T. cruzi reference strains. 20 ml of whole blood specimens per donor were collected in PAXgene Blood DNA tubes (Qiagen, Hilden, Germany) from six normal healthy volunteers (NHVs) from USA-based donors that participated in the company’s Volunteer Donor Program. The routine blood collection procedures were performed by the company’s phlebotomist after the donors signed the research subject information and consent form that was reviewed and approved by Western Institutional Review Board (WIRB). The blood samples were tested negative with T. cruzi kDNA PCR assay. 5 μl of T. cruzi DNA with known concentrations spiked into 200 μl of NHVs’ PAXgene blood specimens were used to select and to optimize T. cruzi DNA isolation methods, and to establish the assay’s sensitivity. NHV PAXgene blood samples spiked with two low concentrations of T. cruzi DNA, and the ones spiked with only IAC, were utilized as the controls for batched DNA extraction of clinical samples. To lyse T. cruzi, 5 ml of PAXgene blood was mixed with 5 ml of Genomic Lysis Buffer (Zymo Research, Irvine, CA), followed by 10-minute room temperature incubation. The lysed blood specimens were stored at -80°C until DNA extraction. For DNA isolation, 5 μl of 0. 03 pg/μl of IAC DNA and 0. 6 ml of Genomic Lysis Buffer were added to 0. 4 ml of lysed blood sample (equivalent to 0. 2 ml of PAXgene blood), mixed and followed by 10 minute room temperature incubation. DNA extraction was performed with Quick-gDNA Blood MiniPrep kit (Zymo Research) per the manufacturer’s instruction except for the DNA elution step, in which 50 μl of heated low EDTA TE buffer (pre-warmed at 70°C) was added to the column and incubated at room temperature for 5 minutes before centrifugation to collect extracted DNA. The PCR reaction mix consists of 1x TaqMan Universal Master Mix II with UNG (Thermo Fisher Scientific, abbreviated as TaqMan Master Mix), 0. 5 μM of each PCR primer, with the forward primer being CCGTCATGGAACAGCACGTA and the reverse primer ACCTTCAAGAACAGATGCTCCAA, and 0. 2 μM of FAM labeled TaqMan MGB probe TTGCTGGAGAAATGACT. The PCR amplicon size is 77 base pair. The qPCR plate was prepared with Biomek NXp Laboratory Automation Workstation (Beckman Coulter, Brea, California) so that each sample would have 4 technical replicates per PCR run. We used ViiATM 7 Real-Time PCR System (Thermo Fisher Scientific), with total qPCR reaction volume at 10 μl including 2 μl of extracted DNA. PCR reaction was programed at 50°C for 2 minutes, 95°C for 10 minutes, followed by 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. For analysis settings of ViiA7 Software v1. 2. 1, we used 0. 1 as the threshold and selected automatic baseline determination. T. cruzi kDNA qPCR assay targeted the conserved 120-bp repetitive sequences of minicircle DNA. The PCR reaction mix consists of 1x TaqMan Master Mix, 0. 9 μM of each PCR primer, with the published forward primer being TTTGGGAGGGGCGTTCA and the published reverse primer ATATTACACCAACCCCAATCGAA[7], and 0. 2 μM of newly designed FAM labeled TaqMan MGB probe CCCCCGTACATTATTT. The PCR amplicon size is 117 base pair. The qPCR plate setup and the reaction conditions were the same as for the IAC qPCR assay. We used serially diluted reference T. cruzi DNA (CL-Brener or K98) spiked in NHV PAXgene blood to establish analytical sensitivity in terms of LOD. According to the CLSI EP17-A2 guideline [8], LOD is defined as the lowest concentration that at least 95% of the technical replicates have positive signals of the assay. We first searched for the lowest concentrations in which 3 of the 3 technical replicates were tested positive. We then created 21 technical replicates at these lowest concentrations for each of two references and checked if at least 20 out of the 21 replicates were tested positive. We investigated two methods of establishing T. cruzi qPCR standard curve formulas and found both methods produced highly similar results. The first method used serially diluted T. cruzi DNA spiked-in NHV PAXgene blood samples and involved DNA extraction and qPCR assays to establish an empirical standard curve for each individual run. The second method used an outlier rejection based algorithm. First, for each T. cruzi DNA dilution, we performed dozens of PCR reactions, with quad-replicates per reaction. Then for each individual reaction’s quad-replicates, we removed the replicate that was most different from the rest of the replicates and calculated the median Ct of the remaining triplicates. Finally, the median of the calculated median Cts was selected to represent the Ct at each T. cruzi DNA dilution to obtain the respective qPCR standard curve formulas for CL-Brener or K98. We selected the formulas obtained from the second method for analyzing the clinical samples because it could be implemented efficiently and was less prone to run-to-run variability and operator errors. Although the STOP CHAGAS study used the Version 1 PCR assay for patient enrollment screening, our investigation found that 75% of the clinical samples that were analyzed with the Version 1 assay failed at least one of the qPCR assay QC parameters established by the CRO (S1 Table). The QC parameters (such as PCR amplification efficiency, R2 value, and minimum number of data points required for the standard curve) were set generously for the Version 1 kDNA qPCR assay, mainly due to the challenge of establishing reproducible standard curves with reference T. cruzi DNA (see the solution to the challenge in the section called “Standard Curves of kDNA qPCR Assays”). In order for the clinical trial to accurately evaluate the treatment response, we decided to develop a more sensitive and reproducible qPCR assay accompanied with more stringent quality control and quality assurance parameters to analyze all the clinical samples collected from the enrolled patients including the ones already evaluated by the Version 1 PCR assay. Although the QIAamp Blood DNA Mini kit (Qiagen) used in Version 1 assay had consistent yields of human genomic DNA (gDNA), and had good RNase P qPCR results indicating integrity of gDNA, it had inconsistent (up to 5 fold differences) and poor (most below 50%) DNA extraction efficiency with IAC DNA, and even lower efficiency with T. cruzi DNA. We explored various experimental conditions for all major steps of the QIAamp kit and failed to improve the recovery of IAC and T. cruzi DNA. We found the solution in the Quick-gDNA Blood MiniPrep kit (abbreviated as the ZR kit) that used a simple procedure for quick isolation of total DNA, including gDNA, mitochondrial and viral DNA, without commonly used protease K or organic denaturants. DNA extraction efficiency with IAC DNA spiked in NHVs’ PAXgene blood samples was high and consistent, with an average of 93% with a CV of 7% in eleven independent DNA extraction runs with a total of 144 samples. Fig 1 shows the consistent and high DNA extraction efficiency of the ZR kit with the STOP CHAGAS study’s PAXgene blood samples, with an average of 89% and a CV of 9%. Using one-fourth the elution volume of the QIAamp kit, the ZR kit further improved assay sensitivity through concentrating DNA eluent. Based on the good recovery of T. cruzi DNA spiked in NHV PAXgene blood, we hypothesized that Genomic Lysis Buffer in the ZR kit could efficiently lyse T. cruzi and to release minicircle DNA in clinical PAXgene blood. Fig 2 shows the results that confirmed the hypothesis. Individual 0. 2 ml aliquots of a clinical PAXgene blood sample without the lysis buffer had Ct values ranging from 26. 1 to 33. 8 for the kDNA qPCR assay, while the addition of increasing amounts of lysis buffer to 5 ml PAXgene blood aliquots resulted in consistent Ct values ranging from 28. 5 to 29. 2 in 0. 2 ml equivalent of PAXgene blood aliquots. To avoid sampling error caused by small aliquots of un-lysed blood, we performed T. cruzi lysis with 5 ml of the PAXgene blood before DNA extraction. We compared Platinum Quantitative PCR SuperMix-UDG w/ROX (Thermo Fisher Scientific, abbreviated as Platinum SuperMix) used in Version 1 assay with TaqMan Master Mix used in Version 2 for both IAC and kDNA qPCR assays. S1A Fig and S2A Fig show that the Version 2 assay’s TaqMan Master Mix had better performance by lowering Ct values and increasing the exponential phases for both qPCR assays. We also re-designed the TaqMan probe, because we found two potential problems with the published probe sequence 5’-CATCTCACCCGTACATT-3′ for MGB probe-based TaqMan assays. First, there is a polymorphism at the seventh nucleotide (“A” or “C”) according to T. cruzi kinetoplast minicircle sequences in NCBI database, with “C” not “A” shown in the vast majority of T. cruzi strains. Second the original probe sequence is not optimal for MGB probe based TaqMan assay, because it was designed as a LNA probe. We removed the first five nucleotides of the published probe sequence so that the polymorphic nucleotide “C” was place at the 2nd position instead of being close to the center of the probe, thus having less influence on potential discrimination of sequences with the “A” polymorphism. We added four highly conserved nucleotides at the end for the new probe, so that the new probe could meet the design guidelines of an optimal MGB probe. The newly designed probe resulted in up to 1 Ct improvement for CL Brener (Fig 3A) and up to 7 Ct improvement for K98 (Fig 3B), further increasing the assay’s sensitivity. We used serially diluted reference T. cruzi DNA spiked in NHV PAXgene blood to establish analytical sensitivity in terms of LOD. Please note that for each technical replicate used to estimate LOD, the extracted DNA was analyzed by three separate runs of kDNA qPCR, with 4 PCR replicates per qPCR run. Only the technical replicates with at least two Ct values less than 45 among the total of twelve Ct results were called positive for the qualitative kDNA PCR assay. One major reason why at least 2 positive PCR amplifications among the 12 qPCR replicates was called positive in Version 2 assay was to be consistent with Version 1 assay in which at least one replicate had to be positive among the 6 qPCR replicates. Table 1 shows that the Version 2 assay has up to 39 fold increase in sensitivity compared to the Version 1 assay. One of the STOP CHAGAS study’s secondary objectives was to evaluate if the response to treatment could be measured by changes in T. cruzi parasite loads with quantitative PCR. To quantify T. cruzi parasite loads, the CRO of Version 1 assay established two qPCR standard curves so that K98 representing TcI could be utilized to represent the clinical samples collected from the Northern countries of South America such as Mexico and Columbia, and CL-Brener representing TcVI for the Southern Cone region of South America such as Argentina and Chile. Despite of the better performance of the Version 2 assay, we observed the same phenomenon as seen by the CRO that reference T. cruzi DNA did not behave as other DNA material, such as gDNA, plasmid DNA, or PCR DNA. S2 Fig shows an example of such atypical behavior, in which the lower concentration T. cruzi DNA dilution (10 PPM) had less variability in Ct values among the quad-replicates per sample than the higher concentration dilution (100 PPM), in which one of the four replicates tended to have much lower Ct values than the remaining three replicates that usually had consistent Ct values. We also observed another atypical behavior of reference T. cruzi DNA in which the Ct difference between the different 10 fold dilutions could be different from the expected 3. 32 value. We suspected that the atypical behaviors might be caused by the complex T. cruzi kDNA structure that is composed of thousands of interlocked minicircle DNAs and dozens of maxicircle DNAs. We hypothesize some of the reference T. cruzi DNA dilutions might still contain the interlocked minicircle DNA targeted by the qPCR assay. We explored many experimental conditions to decatenate kDNA while avoiding DNA degradation, but failed to find one that could reduce technical qPCR variability for all dilutions while maintaining the 3. 32 Ct difference expected between the 10-fold DNA dilutions. Because the observed Ct variability of T. cruzi DNA dilutions seemed to be caused by the unpredictable drop of Ct in one technical replicate (the outlier replicate), while the overall median Cts maintained the expected Cts, we decided to establish kDNA qPCR standard curves for both strains with an outlier rejection based algorithm. First, we obtained dozens of quad-replicate Ct values at each T. cruzi DNA dilution. Then for each quad-replicate, we removed the outlier replicate and calculated the median Ct of the remaining triplicates. Finally, the median of the calculated median Cts was selected to represent the Ct at each T. cruzi DNA dilution. Table 2 shows the formulas of the established qPCR standard curves for the two reference strains. To check the accuracy of the established standard curves, we measured the Ct values of T. cruzi DNA extracted from the NHV PAXgene blood samples spiked with known concentration T. cruzi DNA. Fig 4 shows the almost identical formulas and R2 between the established standard curves and the measured empirical ones for both references, demonstrating the accuracy of the established qPCR standard curves. To examine the accuracy and precision of the qPCR assay, we spiked in 100,10,1, and 0. 25 PPM equivalent T. cruzi DNA of CL Brener or K98 into 3 NHVs’ PAXgene blood specimens. The spiked-in PAXgene blood specimens were extracted on different days, and on each day at least 3 intra-run extractions were performed for each PAXgene blood sample. Table 3 summarizes the results presented in S3 Fig, which shows that the measured T. cruzi parasite loads were close to the expected values, and the measured values were consistent within the same run and among different runs for both T. cruzi strains. Table 4 lists the multiple controls for both DNA extraction and qPCR to ensure data quality. For DNA extraction, two types of controls were implemented. One was IAC DNA added to each clinical PAXgene blood samples to measure DNA extraction efficiency and PCR inhibition. The other was a set of three controls of spiked-in NHV PAXgene blood samples that were extracted along with each batch of clinical samples. For the qPCR reactions, in addition to the samples extracted from the DNA extraction controls, three individual negative controls of NTC (low EDTA TE buffer with carrier RNA) and one positive control of 10 PPM T. cruzi DNA were analyzed for each PCR run. Table 4 shows all clinical samples from patients enrolled in the STOP CHAGAS study passed the QC parameters established for the assay. Although there are a number of qPCR methods [7,9, 10] that had been published for detecting circulating T. cruzi, our method was the first to detect T. cruzi in whole blood specimens collected with PAXgene Blood DNA tubes, which had the benefits of reduced cost and the potential to reduce clinical site-specific artifacts caused by the more complicated collection and processing procedures for multicenter global clinical trials. However, due to the complex structure of T. cruzi DNA, previously published methods were not adequate for detecting T. cruzi in PAXgene blood. Our PCR assay overcame the inadequacy by finding simple methods that efficiently extract T. cruzi DNA, and by improving sensitivity of the kDNA qPCR assay. We also solved the problem of unreliable kDNA qPCR standard curves caused by kDNA structure through a statistical algorithm. The established standard curve was essentially identical to the empirical ones obtained with NHV PAXgene blood spiked with known concentrations of T. cruzi DNA, but was much easier to implement than to build empirical standard curves for each individual qPCR run. We compared the LOD of our T. cruzi qPCR assays with other published methods and find that our assay had similar or higher sensitivity compared to some published sensitive PCR methods [7,11]. The high sensitivity of Version 2 assay is likely due to both the optimized assay conditions and the stricter definition of the negative calls of the qualitative PCR assay. In one similarly designed randomized clinical trial comparing POS to BNZ in treating chronic CD [12], a sample could be called negative if it had four positive amplifications, all with Ct between 40 and 45, among the four PCR replicates. Such sample would be called positive in our definition. By lowering the chance of false negative calls, we reduced the likelihood of false positives of treatment success responses. We did not formally establish the limit of quantitation (LOQ) during assay validation. Instead, we used the following three strict criteria to evaluate the reliability of the quantitative results. First, the four technical replicates per qPCR run must pass an in-house established variability test before their mean Ct was used to calculate parasite load. If any of the three kDNA qPCR runs failed the Ct variability rules, the results would be labeled as unreliable. Second, if any of the three runs had a mean Ct above 35, the data were also called unreliable. Third, if CV% of the parasitic loads of the three kDNA qPCR assays runs was greater than 30%, the results were also labeled as unreliable. For CL-Brener DNA, we observed that when samples had at least 0. 05 fg/μl of DNA, 95% of them would pass all three criteria. However, for samples with less than 0. 05 fg/μl of DNA, 94% of them would fail at least one of the three criteria. Therefore, we estimate that the LOQ of the assay to detect CL-Brener was around 0. 05 fg/μl. Although we did not have sufficient K98 data points to estimate its empirical LOQ, because K98 has better LOD than CL-Brener, we estimated that both K98 and CL-Brener would have LOQ around 0. 05 fg/μl, equivalent to 0. 25 PPM, similar to the 0. 90 PPM LOQ of a published assay[11]. We learned a few lessons during the assay development. We found that good RNase P assay results of human gDNA [11] were not informative in selecting T. cruzi DNA extraction method due to the difference between the two DNA. NHV PAXgene blood spiked with known concentrations of T. cruzi DNA was a better approach to evaluate T. cruzi DNA extraction. To avoid false negative PCR results caused by the sampling error of small aliquots of un-lysed blood specimens, it is important to perform T. cruzi lysis with a sufficient amount of blood (5–10 ml) before DNA extraction. The commonly utilized method of mixing 10 ml of EDTA-blood with 10 ml of GEB alleviates such sampling error, while other published methods such as mixing only 0. 2 ml of EDTA-blood with 0. 2 ml of GEB or directly extracting T. cruzi DNA from 0. 2 ml of EDTA-blood would likely suffer from the sampling error. Avoiding such sample error is especially critical for samples collected from asymptomatic CD patients due to low levels of circulating T. cruzi. In the manuscript detailing the clinical results of the STOP CHAGAS study [13] that utilized our assay, one of the major conclusions is the strong support of the use of the T. cruzi qPCR assay as a marker of therapeutic response in subjects with chronic asymptomatic CD. In addition to the excellent sensitivity, specificity and precision, our assay allows the simple and cost-effective PAXgene tubes to collect blood specimens from CD patients. | Title: Development of a PCR Assay to Detect Low Level Trypanosoma cruzi in Blood Specimens Collected with PAXgene Blood DNA Tubes for Clinical Trials Treating Chagas Disease Summary: Chagas disease is caused by the infection of the protozoan parasite Trypanosoma cruzi (T. cruzi) and carries a significant tropical disease burden in the Western Hemisphere. The STOP CHAGAS study was a global clinical trial evaluating therapies for Chagas disease. PAXgene blood DNA tubes used by the study simplified and standardized the sample collection and processing procedures, but challenged published PCR assays that detect circulating T. cruzi. In this study, we report our effort of developing and validating a robust, sensitive and specific PCR assay for detecting T. cruzi in PAXgene blood specimens. The efficacy outcome results of the STOP CHAGAS study that utilized our assay further support the use of the PCR assay as a marker of therapeutic response for patients with Chagas disease. | 6,354 | 223 | lay_plos | en |
Summarize: Six million Americans who describe themselves as white have some African ancestry, according to a new study. In percentage terms, that means that roughly 3.5 percent of self-described white Americans have 1 percent or more African ancestry. To arrive at these numbers, researchers pored over the genetic records of 145,000 people who submitted a cheek swab for testing to 23andme, a private company that provides ancestry-related genetic reports. The researchers examined the genetic recordsof people of self-described European, African and Latino descent to find the genetic traces left by relatives long-since deceased. In order to hit that one percent threshold above, for instance, you'd have to have an African relative no further back than seven generations -- in other words, a great-great-great-great-great grandparent. And as you might expect, there are some fascinating differences in our genetic admixture at the state level. Southern whites are considerably more likely to have African ancestry than whites from other regions: "European Americans with African ancestry comprise as much as 12% of European Americans from Louisiana and South Carolina and about 1 in 10 individuals in other parts of the South," the authors found. That variation makes up part of the genetic inheritance of slavery. As Jenée Desmond-Harris notes over at Vox, the study finds that present-day African-Americans are far more likely to have a European male ancestor (19 percent) than a European female one (5 percent). "That, of course, reflects what historians know about white slave owners raping enslaved women who descended from Africa," she writes. Indeed, the average self-described African-American has about 24 percent European ancestry, according to the study, indicating that descriptors like "black" and "white" mean a lot less from a biological standpoint than they do from a cultural one. To dig deeper into this, the authors plotted respondents' proportion of African ancestry against their likelihood of calling themselves African American. What they found was that people who were 15 percent African or less generally didn't describe themselves as African-American, while those who were 50 percent African or greater almost universally did. But in between there was a considerable amount of variation. Those who were about one quarter African were just as likely as not to call themselves African-American. It'll be interesting to see how these proportions shift in the coming decades. In 1980, for instance, 6.7 percent of new marriages were between different-race spouses. By 2010, that share had risen to 15.1 percent. And as demographer William Frey notes, "nearly three in 10 new black marriages are multiracial, with most of them to white spouses." This is especially significant given that as recently as 1967 -- within living memory for many Americans -- interracial marriages were outlawed in 16 states. Many Americans who call themselves white might be surprised to find out that they have some African ancestry. Especially in the South. In a study published in The American Journal of Human Genetics in December 2014, researchers used the ancestry data compiled by the commercial genetic testing company 23and Me to measure the percentage of African ancestry of people who self-identified as white. It turns out that self-identified white people who live in the South have the highest concentrations of African DNA. (Read more: How a biracial woman grew up thinking she was white) In South Carolina and Louisiana — the states shaded the darkest green on the map above — researchers found that one in 20 people who called themselves white had at least 2 percent African ancestry. And in a lot of the South, about 10 percent of people who identified as white turned out to have African DNA. Racial mixture represents African female/European male couplings The researchers also used genetic information to determine the genders of the specific people who were responsible for some Americans' mixed ancestry. They found that many more (19 percent) of the ancestors of self-identified black people were European male, while only 5 percent were European females. They could even pin down the timing: the mixture generally occurred in the early 1800s, when slavery was legal. That, of course, reflects what historians know about white slave owners raping enslaved women who descended from Africa. Where black Americans have the most African ancestry Just like white people in the South had the most African ancestry, so did black people. The lowest percentage of African heritage in people who called themselves black was found in West Virginia. Next: Washington State. 28 percent: the tipping point for identifying as black? Comparing ancestry data to how people self-identified, the researchers found that Americans tended to identify as European-American, rather than African-American, when they had less than 28 percent African ancestry. h/t Quartz Further reading: 11 ways race isn't real This insanely detailed map proves race is a social construct Researchers have been thinking about race wrong WATCH: '220 years of population changes in one map' | Summary: A simple cheek swab can reveal a lot about your DNA, and for 6 million Americans who identify as white, mostly in the South, that swab has revealed African ancestry hidden in their genes, the Washington Post reports. One in 10 Southerners have at least 1% African origins, and the reason isn't hard to pinpoint: Slavery. The study, conducted by 23andMe, also found African Americans today are more likely to have a European male ancestor (19%) than a female one (5%). "That, of course, reflects what historians know about white slave owners raping enslaved women who descended from Africa," writes Jenée Desmond-Harris at Vox. The study even reveals that this blending started in the early 1800s. The study examined the DNA profiles of about 160,000 people; the data was compared to where they lived and which race they identified with. They found people with less than 15% African genes didn't identify as black and the average African American's genes are at least 24% European. "Descriptors like 'black' and 'white' mean a lot less from a biological standpoint than they do from a cultural one," writes the Post's Christopher Ingraham. Some other interesting finds, according to Discovery: 5% of African Americans have 2% Native American ancestry; the highest is 14% in Oklahoma, where Native Americans were displaced after the Trail of Tears. Iberian ancestry was common in Latinos from Florida and the Southwest. Scandinavian ancestry is highly concentrated in Minnesota and the Dakotas. Native ancestry among Latinos is concentrated in Texas and California. | 1,166 | 383 | multi_news | en |
Write a title and summarize: Sepelio de Wendy Yamileth Alfaro Mena, la primera mujer policía asesinada, en abril de 2015. Con su muerte, Adán Servellón dejó a cuatro menores en la orfandad y a su pareja embarazada. "Fue una tragedia, por cómo lo mataron, por cómo era él y por la familia que dejó", relata un agente policial en servicio, amigo cercano de Servellón, que prefiere no revelar su identidad por temor a represalias. En El Salvador, la guerra contra las pandillas ocasiona decenas de muertes de policías cada año. Asesinados a sangre fría o en enfrentamientos, en el país han muerto más de 176 agentes de forma violenta desde 2013. La mayoría han sido víctimas de alguna de las dos principales estructuras criminales que operan en el país: Mara Salvatrucha y Barrio 18, que hacen de esta pequeña nación centroamericana, una de las más violentas del mundo. Los policías asesinados dejan tras de sí a centenares de niños huérfanos que ingresan a un sistema de asistencia social que, según voces críticas, los confina al completo abandono, con apoyos económicos que no sustentan siquiera la alimentación básica y menos otros derechos fundamentales. "Ahí no matan, ahí ahorcan" BBC mundo pudo comprobar que en ese estado se encuentran los cinco hijos del agente Adán Servellón. Viven en el municipio de Huizucar, una aislada zona rural a 30 minutos de la capital, San Salvador. Para llegar hay que atravesar una complicada ruta, constantemente asediada por pandillas. Es la única forma de entrar o salir. En ese camino, cerca de un puente, mataron al agente Servellón. A las afueras del pueblo, no importa a quien se le pregunte, la advertencia es siempre la misma: no entrar en ese sendero con demasiada confianza. "Ahí no matan, ahí ahorcan", afirma, con risa nerviosa, una mujer desde la puerta de su casa. La zona muestra la pobreza en la que suelen vivir los policías y sus familias. Policías en el entierro de uno de sus compañeros. Esa condición se vuelve incuestionable al ingresar en la casa de Carolina Cornelio, la viuda de José Adán Servellón. Sus hijos ahora tienen catorce, doce, once, cinco y un año y medio de edad. Los primeros cuatro estudian, pero los mayores deben trabajar para ayudar con los ingresos del hogar. Siembran frijol y maíz, una parte de lo cultivado es para que la familia se alimente y el resto para conseguir algo de dinero. La abuela de los menores viaja, junto a una de sus nietas, al pueblo más cercano para vender el producto. Es muy poco. "A veces no hay que darle a los niños, aunque pidan, no hay qué comer", señala Carolina con resignación en su tono. Ella trabaja ocasionalmente lavando y planchando ajeno. Va solo cuando la llaman, "no es seguro salir demasiado, por la situación", declara bajando el volumen de su voz y mirando a los lados, como asegurándose de que nadie más la escuche. La mujer asegura haber entregado todos "los papeles" que la policía le solicitó: actas de matrimonio y defunción de su esposo, documentos del seguro y todo lo relacionado a ella y su familia; sin embargo, no han conseguido respuesta y afirma que tampoco se han acercado a representantes de la policía para conocer de oficio los detalles de su caso. Carolina defiende que la corporación conoce de la existencia de los cinco hijos de Adán; sin embargo, más allá de una primera ayuda para el sepelio de su pareja, no han recibido nada. "No sé a quién se lo estarán dando, pero a mi no es", añade. Asistencia En agosto de 2015, dos meses después de la muerte de José Adán, el presidente Salvador Sánchez Cerén y el entonces director de la policía, Mauricio Ramírez Landaverde, anunciaron el inicio de su plan de asistencia a hijos y familias de agentes asesinados. El programa otorgaría una cuota mensual escalada para los menores de 18 años, además de incluirlos en un sistema de becas de estudio. El proyecto, dijeron, se financiaría a través de un fideicomiso de US$787.500 impulsado por la empresa privada y la ciudadanía. Para 2017, el presupuesto general aprobado para la policía es de más de US$430 Millones, de los cuales $274.920 están dedicados al apartado de atención a víctimas de delitos, es decir un 0.063% del presupuesto total. No existe, en el presupuesto de la corporación, otro apartado referente al tema y no son públicos los detalles sobre si ese monto incluye a hijos de policías asesinados, hay quienes creen que no es así. Policías revisan la casa de un sospechoso de pertenecer a las maras. Uno de los que piensa que la policía cuenta con los fondos suficientes para acompañar a estas familias y no lo hace es Marvin Reyes, presidente del Movimiento de Trabajadores de la Policía (MTP), que vela por los derechos de los agentes policiales. Él considera que los programas de apoyo a familias de policías que murieron realizando su trabajo o a consecuencia de este, realmente no existen. Afirma que los anuncios del gobierno son una estrategia propagandística. "Realmente, la policía no le da ningún seguimiento a la familia", dice. En su opinión, lo mínimo que deberían hacer es "dejarle de por vida a la familia el salario que el policía ganaba, asegurar planes de estudio para los hijos y atención psicológica para todos los integrantes". "Parece que hubiera un desprecio hacia el policía y su familia por parte de las autoridades", asegura. Ausencia Lo cierto es que ninguno de los programas, reales o ideales, han tocado a la puerta de Carolina y sus hijos. Ellos estudian en la pequeña escuela del caserío en el que habitan, excepto el mayor, quien debe viajar hasta el pueblo más cercano para recibir clases. Lo hace caminando, por la única ruta existente, pasando todos los días por el punto exacto donde vio morir asesinado a su padre. Él no habla mucho del tema, pero su madre dice que a veces llora cuando cree que nadie lo está viendo. Aunque si hijo de doce años, que también presenció la muerte de su padre, pregunta constantemente por su él. "¿Por qué los otros niños tienen papá y yo no?", cuestiona a su madre y ella no sabe qué responder. El Salvador ha sufrido de múltiples oleadas de violencia durante su historia. En la foto, conmemoración del asesinato en 1989 de 6 padres jesuitas por parte del ejército. Carolina piensa que sus hijos, además de asistencia económica, también necesitan ayuda psicológica. "A veces solo pensando en eso pasan y tienen pesadillas. Ellos quieren que todo siga igual, pero ya no se puede". Unos días después del asesinato de José Adán, la policía lo despidió con honores, como es costumbre. A Carolina le entregaron una bandera de El Salvador, la misma que el día del funeral cubrió el ataúd. Los niños que nada más volver del cementerio comenzaron a sentir la ausencia. Su padre los acompañaba a la escuela, les compraba los uniformes, libros y zapatos. Los hacía sentir seguros. "Es duro ver cómo quedaron y el olvido en que los han dejado", señala el agente que fue amigo de Adán. Él y otros compañeros de la delegación en donde el policía estuvo destacado, apoyaron a la familia con víveres y la gestión y los trámites que hicieran falta para que los menores fueran incluidos en los programas de ayuda de la corporación policial. "Fue tan matador, viajar y viajar sin apoyo de nadie por parte de la institución", agrega. "Bienestar Policial se aprovecha de la situación y si la familia no presiona se agarran la plata", afirma el agente, en referencia al bono mensual que debería recibir cada hijo de un agente asesinado. Bienestar Policial es el nombre de la dependencia de la PNC que se encarga de organizar los sepelios. Estos suelen ser actos públicos y mediáticos a los que asisten altos dirigentes de la corporación para presentar sus respetos a la familia y despachar discursos sobre exhaustivas investigaciones para dar con los culpables. Esa misma dependencia debe dar seguimiento y asistencia a las familias de los policías asesinados Sin embargo, Carolina asegura no haber vuelto a saber de esa institución después del sepelio. Únicamente recuerda que en algún momento le mencionaron que la asistencia sería de $25 dólares por cada uno de sus hijos. "Eso en un par de zapatos se va", afirma. "¿Y para que coman todos?", lanza la pregunta al aire, mientras sostiene entre sus manos la bandera que le entregaron en el funeral de su esposo. Lo único que le queda. La historia de Wendy El asesinato de Wendy Yamileth Alfaro Mena fue el primero de una mujer policía. En similares circunstancias se encuentra la familia de la agente Wendy Yamileth Alfaro Mena, que fue asesinada también en 2015. Tenía 27 años de edad y los últimos cuatro formando parte de la policía. Al igual que el agente Servellón, fue emboscada por cinco pandilleros cuando compraba comida para su familia, a escasos metros de su hogar, en el oriente del país. En ese año, el de mayor número de muertes de policías en la historia de El Salvador, su caso fue particular por ser la primera mujer policía asesinada por pandilleros. Con su muerte dejó a un pequeño de dos años en la orfandad. Marvin Reyes conoció de cerca el caso. Recuerda que familiares de Wendy tomaron en custodia al niño para protegerlo de posibles represalias e incurrieron en gastos para cumplir con los trámites y documentos que se exigían para formar parte del programa que prometía el gobierno. Recuerda que incluso debieron demostrar que Wendy realmente había muerto en las circunstancias en que lo habían descrito Después de muchos viajes a la capital y engorrosos procesos y entrevistas, el hijo de Wendy consiguió ser incluido y aun así debió esperar cerca de medio año para recibir el primer desembolso económico: 50 dólares, que no cubrieron siquiera los gastos que tuvo la familia para incluir al menor en el programa. Cuando preguntaron por las becas que el gobierno había prometido, la respuesta fue que la corporación estaba esperando a que alguna institución se acercara a ofrecerlas, según Reyes, "la PNC no busca ni gestiona de oficio las ayudas que promete para los hijos de los policías asesinados. " "Me lo contaban a mí con una decepción tremenda", agrega el expolicía. "Para las autoridades que dirigen la corporación, la vida de un elemento policial que recibió una muerte horrible, vale 50 dólares y ningún esfuerzo más", lamenta. Ola de homicidios En lo que va de 2017, trece policías han muerto a causa de las pandillas. En el mes de julio ocurrió la mayor ola de homicidios, en pocos días fueron asesinados al menos seis agentes. Uno de los casos que causó mayor conmoción es el del agente Víctor Alcides, de 30 años, cuyo cuerpo fue encontrado desmembrado dentro de bolsas plásticas en una transitada carretera a las afueras de la ciudad. El más reciente homicidio fue el del sargento Víctor Manuel Salazar, quien, como en la mayoría de los casos, se encontraba de licencia cuando fue atacado por pandilleros dentro de su casa, a la vista de su familia. Óscar Ortiz, vicepresidente de El Salvador dijo a la prensa, que "el apoyo que le vamos a dar a las familias es como el que le estamos dando a todas las familias de policías caídos", reiteró. Militares tapan con pintura graffitis de la Mara Salvatrucha. Además dijo que "esto no va a hacer desanimar esta gran cruzada que tenemos contra el crimen y la extorsión" y agregó que "debe reforzar la moral nacional y la moral de nuestra policía" Ante el incremento del asesinato de policías, el Movimiento de Trabajadores de la Policía, liderado por Marvin Reyes llamó a los agentes a protestar por la falta de garantías para llevar a cabo su trabajo. "Hagamos temblar las calles", dice el comunicado que difundió el MTP: "vamos, a una sola voz, a denunciar la actitud de desprecio por parte de las autoridades ante la muerte de compañeros, así como la inexistencia de planes de protección para los trabajadores policiales y su familia". En repetidas ocasiones, BBC Mundo intentó conocer la opinión de las autoridades de Bienestar Policial sobre la implementación de los programas de asistencia a agentes de la corporación y sus familias; sin embargo, no hubo respuesta a pesar de la insistencia por diversas vías. Muchos casos Eso lo sabe bien Mirna Alvarez. Ella fue policía y parte del equipo de Bienestar Policial en el occidente del país. Por sus manos pasaron incontables casos de asistencia social a familias de policías asesinados. Ella confirma a BBC Mundo que, como la familia del agente Servellón o la agente Alfaro, hay decenas por todo El Salvador. Según Mirna, Bienestar Policial no se preocupa por conocer las particularidades y necesidades de cada caso y cada familia. "No lo hacen, por mi ha pasado", declara; "eso solo es teatro público", agrega. La ex policía denuncia que ella fue retirada de su cargo, en parte por explicarle a los policías y a sus familias cuáles son sus derechos. "Hay casos en donde el fallecido no tiene a nadie en los seguros y ellos se quedan con ese dinero", confirma y señala que, en efecto, la ayuda para los hijos de los policías asesinados no es suficiente como para cubrir el costo de la canasta básica de una familia y las autoridades lo saben. "No es justo que la institución a la que tanto se ha servido, trate a las familias como que son perros", agrega y advierte que el abandono de las autoridades es un factor que abona al incremento en el número de ataques a policías y a sus familias, que la impunidad en que viven quienes dañan a un agente o a sus familiares es la clave de ese aumento. La razón Los enfrentamientos entre la policía y las pandillas han aumentado en los últimos tres años. Marvin Reyes, que fue parte de la corporación policial por más de 20 años, cree que el incremento en el número de ataques a policías se debe, en parte, a la implementación de una serie de medidas extraordinarias que, entre otros puntos, refuerzan la seguridad en Centros Penales, restringen privilegios a reos y cabecillas de pandillas recluidos, además de reforzar la seguridad pública con ayuda del ejército salvadoreño. "Cuando ellos (los pandilleros) se ven acorralados en muchos aspectos, recurren a medidas que incluyen el asesinato de familiares, policías, miembros del ejército y centros penales", afirma Reyes. En su opinión, el objetivo es incidir en la moral y afectar así a los responsables de salvaguardar la seguridad pública. Reyes también cree que la muerte de policías se debe a una respuesta por parte de las pandillas a las acciones operativas que ordena la dirigencia de la corporación y que los agentes no tienen otro remedio que obedecer. "Violencia genera violencia", afirma y señala que "se está produciendo una espiral de venganza que va a ser difícil de tratar en un futuro cercano". "Cuando no lo pueden matar a uno, matan a la familia. Los padres, hijos, esposas, hermanos, tíos, sobrinos y todo aquel que tenga vínculos con alguien de la institución", señala también Mirna Álvarez. En lo que va de 2017, doce familiares de policías han sido asesinados por pandilleros, superando, por primera vez, al número de agentes muertos, que a esta fecha es de nueve. Las medidas extraordinarias que debían concluir en abril de 2017, fueron prorrogadas un año más por la Asamblea Legislativa. Desde la fundación de la Policía Nacional Civil, en 1992, más de 1.100 agentes han sido asesinados. | Title: "¿Por qué los otros niños tienen papá y yo no?": la tragedia de los huérfanos de los policías que deja la cruenta guerra contra las pandillas en El Salvador Summary: El agente José Adán Servellón Benavides fue asesinado frente a dos de sus hijos en agosto de 2015, tenía 37 años de edad y trece formando parte de la Policía Nacional Civil (PNC) Se dirigía a conseguir comida para su familia cuando fue interceptado por un grupo de pandilleros, quienes le dispararon en la cabeza. Murió en pocos minutos. | 3,435 | 126 | xlsum_es | es |
Summarize: Her husband, James Lawton, has documented some of the harassment on his Facebook page. Much is in the form of social media posts that use foul or racist language and images. Even without the harassment and threats, Ms. Morris said she found the work of state government to be rewarding but draining. “I am proud of that work, but I need the space to breathe because for that exchange it exacts a huge cost,” she said. She pointed to the low pay for legislators — $13,000 a year for five to six months of intensive work — as a barrier that keeps women, people of color and the working class out of state politics. And she said there were few positions in state government that paid enough for people to support themselves or their families. “To serve in this state requires sacrifice, literally even financial sacrifice,” she said. “It is a system set up for the wealthy and the retired.” The decision by Ms. Morris to end her re-election campaign last month sent a jolt through Vermont politics and elevated questions of racial justice in one of the most racially homogeneous states in the country. The United States Census Bureau estimated in 2017 that the population of Vermont is more than 94 percent white and slightly more than one percent African-American. “In the state of Vermont, no elected official, candidate or person should be fearful of their safety because of the color of their skin or their point of view,” Senator Bernie Sanders said in a statement to The Burlington Free Press in August. “This corrosion of political discourse is destructive to our democracy, and we cannot let it take hold." Ms. Morris and her lawyer, Robert Appel, accused the Bennington Police Department of failing to appropriately respond to her concerns since at least 2016. She said her experience illustrated that “as a state we are not familiar, we are not prepared, we are not ready at all” to deal with issues of racial justice. Kiah Morris, the only black woman in the Vermont legislature, shocked the US state when she resigned last month, citing ongoing racial harassment. Even in one of the most progressive states in America, she says white supremacy and a toxic political discourse are serious, unacknowledged problems. Kiah Morris was puzzled. Why did friends on social media keep sending her links to a Saturday Night Live comedy video? Then she watched it. In the sketch, which aired the last weekend of September, a group of Southern neo-Confederates discuss resettling in place with "no immigrants and no minorities - an agrarian community where everyone lives in harmony because every single person is white". One member, played by actor Adam Driver, raises his hand. "Yeah, I know that place," he says. "That sounds like Vermont." The studio audience laughs as the Vermont jokes continue, but for Ms Morris the lines had a bite. "It was funny," she says. "But it was sad." In 2014 Ms Morris became only the second black female member ever elected to the Vermont state legislature. Four years later, in the midst of a re-election race, she resigned from public office after repeatedly being the target of racial harassment. She received death threats. Her property was vandalised. She found swastikas painted on the trees near her house. She obtained a restraining order against a repeat online harasser, but the threats continued. For Ms Morris, racism in Vermont - long considered a bastion of liberal values - was no joke. "There were individuals in the community and throughout the state that we were finding were parts of white supremacist groups," she says. "Because we were so progressive and because we have been working so hard on so many issues of equality, we just sort of fell asleep and didn't pay attention to that." Image copyright Getty Images Image caption Vermont Governor Phil Scott and his Democratic challenger Christine Hallquist are running in the overwhelmingly white state The 42-year-old Morris, who grew up in Chicago, came to Vermont over a decade ago. She says she initially had no interest in running for public office, but friends and mentors changed her mind. She enrolled in Emerge America, an organisation that recruits and trains female Democratic candidates, and put her name on the ballot in the southern Vermont town of Bennington. "I really learned what it meant to be out there and to represent and how you can truly be this voice for the people that you are representing and bring an opportunity to bring change in a way that I couldn't do locally," she says. As she began her campaign, however, it became very clear that her ethnicity - in a state that is 95% white and 1.2% black - was going to be an issue. "We received online threats," she says. "We received hateful messages. We had things happen at our home. We had a break-in while we were asleep." She adds that she wasn't the only member of a minority in the state to experience such harassment - and she says it's only got worse since Donald Trump's victory in the highly contentious 2016 presidential race. "It brought about a complete destruction of anything that we want to call political discourse and moved into an area where the fears and the anger and the sadness and the hurt that people were feeling throughout the nation came forth and came out of their mouths and into our lives," she says. More on race in America Media playback is unsupported on your device Media caption African Americans in New Orleans on the state of race relations During her four years in office Ms Morris says she helped push legislation extending free contraceptive coverage to all Vermonters, control pollution and address racial and income disparities in the education and the criminal justice systems. It was her support for a comprehensive gun control bill, however - passed in April 2018 - that brought the harassment and threats to an intolerable level. She says opponents of the gun law, which strengthened background checks and banned large magazines and bump stocks, cited her as the "voice and face" of out-of-state efforts that they said would eventually lead to firearm confiscation. "It did not escape my attention that that type of behaviour, doing that type of a clarion call, could get me killed," she says. "It was solely directed at me, as the black woman." So in August Ms Morris, after winning the Democratic primary, decided to abandon her re-election bid. When that didn't stop the harassment, she resigned from the legislature, effective immediately. She said her son and her husband, who had just undergone heart surgery, needed her and that she had given the state all she could. She returned to her job with Tesa Collective, which makes games and tools for progressive organisations, and planned to do more writing. Ms Morris says friends and colleagues tried to convince her to change her mind. After her announcement, Vermont Governor Phil Scott, a Republican, offered to support her re-election, warning that her resignation would allow the forces of hate to win. That, according to Ms Morris, is victim-blaming. "The systems need to change to support individuals in office so that they do not have to live in fear and terror," she says. "These are incredibly violent times, and I do not feel any need to martyr myself or my family." It can't fall on her, she says - or on any one person - to try to fix a broken system. It takes a "chorus of people". "They win when you leave it up to a movement messiah to be the one to move the work forward," Ms Morris says. "They win when you place it squarely on my shoulders as somehow this emblematic person. "The change has to happen from each and every one of us because the work is that enormous. It cannot be an ask and it is wrong to ask anyone to place themselves in that type of a circumstance. "So I win because I chose to take my life back."... th no childcare) and the library doesn’t open until 1, you might as well crank up Michael Jackson and make papertoy monsters to pass the time. ❤️ We only pass this way once. At least we are enjoying it! When you can’t work because your kid has a school in service day (wi | Summary: Vermont's state legislature only had one female black lawmaker. Now, there are none-because Kiah Morris quit last month amid a rash of apparent vandalism, home invasions, and racially fueled threats. "There were individuals in the community and throughout the state that we were finding were parts of white supremacist groups," she tells the BBC. Because Vermont is considered so progressive, she says, "we just sort of fell asleep and didn't pay attention to that." Elected in 2014 to represent the town of Bennington, Morris abruptly scuttled her re-election campaign in August and quit altogether in September due to "continued harassment" and her husband's health issues, she wrote on Facebook. Now she's divulging the details. Her family suffered home invasions and found swastikas painted on nearby trees, Morris recently told the New York Times. She also got a restraining order against an online harasser, but the threats kept coming. The 42-year-old Chicago native says her support for gun control legislation-which beefed up background checks and banned bump stocks and large magazines when it passed in April-led to an intolerable level of threats and harassment. Colleagues and friends tried to stop her from quitting, and Vermont's governor, Republican Phil Scott, said he'd support her campaign because hate would win if she stepped down, but Morris wasn't buying it. "These are incredibly violent times, and I do not feel any need to martyr myself or my family," she says. "...So I win because I chose to take my life back." | 1,838 | 367 | multi_news | en |
Summarize: Just 12 months ago, a 15-year-old Martin Odegaard was in the middle of his final year at school, worrying how we would fit in his exams around training with his local football team in Norway. Now the footballing prodigy, who turned 16 last month, has signed for Real Madrid and will play alongside the likes of Cristiano Ronaldo and Gareth Bale. 2014 was a whirlwind year for Odegaard, who rose from life as a schoolboy to being the youngest player to ever represent his nation before signing a £40,000-a-week deal with the European champions this week. Scroll down for video. Martin Odegaard, 16, has signed for Real Madrid, but just months ago he was fitting his secondary school studies around playing for his local team in Norway. Odegaard holds his shirt after being unveiled as a Real Madrid player, where he will be paid £40,000 a week. Odegaard's proud family were at the signing at the Bernabeu stadium as he was announced as a Real Madrid player. Schoolboy to football star: Martin Odegaard, pictured with his brother, rose through the academy of his local team in Norway but now plays for the European champions. Odegaard became the youngest ever player to turn out for the Norwegian national side last year, just months after he made his first professional appearance in the Scandinavian nation's premier league. Odegaard was brought up in the small riverside city of Drammen in a strong Christian family, with the teenager often expressing his faith on Twitter. He played for the youth team at his local team Strømsgodset, where his father used to play, who accommodated his studies around playing football. Odegaard always stood out from children his own age and started training with the adult side, who play in the Norwegian premier league, when he was just 13. It was not long before his talent was noticed by prestigious clubs in Europe, with the teenager training with Manchester United and Bayern Munich. Aged 15 years and 117 days, the attacking midfielder made history by becoming the youngest player to take to the field in a Norwegian premier league match, in April. Just weeks later, after signing his first professional contract, he broke more records by becoming the youngest playing to score in the league. Family man: The young footballer, pictured in Sweden with his mother, was brought up in a strong Christian family. Aged 15 years and 117 days, the attacking midfielder (far left) made history by becoming the youngest player to take to the field in a Norwegian premier league match in April. Just weeks after signing his first professional contract, Odegaard broke more records by becoming the youngest player to score in the league. Here he is pictured bottom left bowling with friends. Odegaard (pictured left in both photos) has also made three appearances for the Norwegian national team. The Norwegian sensation answered only in Norwegian during his press conference in Madrid yesterday, but will be joined at the Spanish club by his father who was also signed as a coach. Odegaard celebrates scoring for Stromsgodest in Norway in May 2014 at the age of just 15. But bigger things beckoned for Odegaard, who has since made three appearances for the Norwegian national football team. Norway's manager Per-Mathias Høgmo recently told Aftenposten: 'He has developed enormously in a short time. He brought down barriers that I think neither he nor anyone else imagined would fall. 'His way of playing, his talent, his ability to make choices and think, all of this is impressive," he added.' Even his own father Hans Erik said it was a only a matter of time before a big team on the continent tried to sign the 16-year-old. There were suggestions Odegaard could be moving to England after he trained with his favourite team Liverpool, as well as Manchester City and Arsenal. Celtic, Dutch club Ajax, Bayern Munich and Real Madrid all courted the growing talent, who eventually went for Madrid - who have also signed his father as a coach as part of the deal. Aged 15 years and 117 days, the attacking midfielder made history by becoming the youngest player to take to the field in a Norwegian premier league match. Odegaard has played three times for Norway, including in a 2016 European Championship qualifier against Bulgaria. Odegaard will be paid £40,000 a week, which could rise to £80,000 when bonuses are added - small fry compared to some Real Madrid players but a huge sum of money for a teenager. Despite the money and new-found fame, the 16-year-old is keeping his feet on the ground and will play for the Spanish team's reserve side for the time being, which is managed by French World Cup winner Zinedine Zidane. Odegaard said: ‘Madrid had the best conditions for me to develop and my aim now is to become the best player I can. ‘It’s a big advantage for me that Real Madrid have a B team playing in a very competitive league and are managed by someone who was one of the best players in the world.’ Speaking of his new team mate before picking up the Ballon d'Or award, Cristiano Ronaldo said: 'A good player. I think he's a young boy, he can still grow. 'He has a good future ahead. So we must give him time to learn, to take the best decisions but I see a lot of potential in that player. A good left foot.' | Summary: A year ago Martin Odegaard was finishing his secondary school exams. This week the 16-year-old signed for European champions Real Madrid. The footballing prodigy has broken record after record in the last year. He is Norway's youngest player and youngest scorer in Norwegian league. Odegaard will be paid £40,000 a week at Madrid, as well as bonuses. He will play alongside the likes of Cristiano Ronaldo and Gareth Bale. | 1,215 | 101 | cnn_dailymail | en |
Summarize: Took the little guy out for some fresh air today... and for an interview in front of 113 million Russian viewers. 36 2 The seed for this crawl was a list of every host in the Wayback Machine This crawl was run at a level 1 (URLs including their embeds, plus the URLs of all outbound links including their embeds) The WARC files associated with this crawl are not currently available to the general public. ONCE upon a time in Adelaide, Casey Dean, 14, and his good mate Eduard Nitz, 13, stopped off at their local McDonald’s to pick up some burgers. Among them was a Quarter Pounder with cheese they’d bought for another kid. That kid never turned up, but they didn’t eat his burger. Ever. That was back in 1995. The boys have become men and the burger has turned 20 and to celebrate, Mr Dean and Mr Nitz are going to reveal it to the world for the first time with an appearance on The Project tonight. “We’re pretty sure it’s the oldest burger in the world,” Mr Dean said. “It started off as a joke, you know we told our friend we’d hold his burger for him but he never turned up and before we knew it six months had passed. The months became years and now, 20 years later, it looks the same as it did the day we bought it, perfectly preserved in its original wrapping.” To find out if Casey or Eduard take a bite of the burger after all these years, tune into The Project at 6.30pm tonight on Channel 10. She's Back In The Country And Joining Us Live At The Desk. It's Actor Extraordinaire Miriam Margolyes! From Harry Potter to the lady in the van, we chat live to the delightful Miriam Margolyes. | Summary: Wonder how long a Quarter Pounder with cheese can last? Two Australians say they bought a few McDonald's burgers for friends back in 1995, when they were teens, and one of the friends never showed up. So the kid's burger went uneaten-and stayed that way, Australia's News Network reports. "We're pretty sure it's the oldest burger in the world," says one of the men, Casey Dean. Holding onto the burger for their friend "started off as a joke," he adds, but "the months became years and now, 20 years later, it looks the same as it did the day we bought it, perfectly preserved in its original wrapping." Dean and his burger-buying mate, Eduard Nitz, even took the burger on Australian TV show The Project last night and "showed off the mold-free specimen," News 9 reports. The pair offered to take a bite of it for charity but were dissuaded by the show's hosts. They've also started a Facebook page for the burger called "Can This 20 Year Old Burger Get More Likes Than Kanye West?" that has more than 4,044 likes as of this writing. And they're selling an iTunes song, "Free the Burger," for $1.69, and giving proceeds to the charity Beyond Blue, which helps Australians battle anxiety and depression. | 416 | 305 | multi_news | en |
Summarize: Un 12enne inglese è morto vittima di una reazione allergica durante il pranzo di Natale. Cason Hallwood, di Winsford, Cheshire, è spirato in ospedale dopo essersi sentito male mentre giocava con i suoi amici in un parco il 25 dicembre dell'anno scorso. Il giovane, che soffriva di asma, ha iniziato ad avere problemi di respirazione non molto tempo dopo aver pranzato a casa dei nonni. L'inchiesta inaugurata a seguito della tragedia ha stabilito che suo nonno, Albert, si era "completamente dimenticato" della sua allergia alle noci finendo per servirgliele sottoforma di glassa. Nonostante la mamma di Cason, Louise, sia corsa al parco per somministrare il suo EpiPen (utilizzato dalle persone a rischio choc anafilattico), suo figlio è morto dopo essere andato in arresto cardiaco. Il 12enne è stato trasportato al Leighton Hospital dove i medici hanno subito fatto capire ai familiari che la situazione era compromessa. "Ero sotto choc e non sapevo cosa stesse succedendo. Mi hanno preso in disparte e li ho visti lavorare su di lui. Sapevo che se n'era andato…" ricorda la madre, Louise. Mentre erano al parco, la nonna, Helen, ha chiesto al marito, Albert "che cosa aveva fatto con il cibo". In una dichiarazione scritta, letta in tribunale dal medico legale, Alan Moore, il nonno di Cason ha affermato di essersi "completamente dimenticato" dell'allergia alle noci del ragazzo. "‘Ricordo che Cason ha leccato il suo piatto pulito e ha detto al nonno che era adorabile'" ammette la madre. | Summary: Cason Hallwood, un bambino inglese di 12 anni, è morto il giorno di Natale. Aveva appena finito di pranzare con la famiglia a casa dei nonni. Ad ucciderlo è stato uno "choc anafilattico fatale causato dall'ingestione di arachidi". Stando a quanto ricostruito dall'inchiesta sulla tragedia, il nonno si sarebbe "completamente dimenticato" della sua allergia alle noci... | 365 | 97 | fanpage | it |
Write a title and summarize: The molecular mechanisms controlling the subunit composition of glutamate receptors are crucial for the formation of neural circuits and for the long-term plasticity underlying learning and memory. Here we use the Drosophila neuromuscular junction (NMJ) to examine how specific receptor subtypes are recruited and stabilized at synaptic locations. In flies, clustering of ionotropic glutamate receptors (iGluRs) requires Neto (Neuropillin and Tolloid-like), a highly conserved auxiliary subunit that is essential for NMJ assembly and development. Drosophila neto encodes two isoforms, Neto-α and Neto-β, with common extracellular parts and distinct cytoplasmic domains. Mutations that specifically eliminate Neto-β or its intracellular domain were generated. When Neto-β is missing or is truncated, the larval NMJs show profound changes in the subtype composition of iGluRs due to reduced synaptic accumulation of the GluRIIA subunit. Furthermore, neto-β mutant NMJs fail to accumulate p21-activated kinase (PAK), a critical postsynaptic component implicated in the synaptic stabilization of GluRIIA. Muscle expression of either Neto-α or Neto-β rescued the synaptic transmission at neto null NMJs, indicating that Neto conserved domains mediate iGluRs clustering. However, only Neto-β restored PAK synaptic accumulation at neto null NMJs. Thus, Neto engages in intracellular interactions that regulate the iGluR subtype composition by preferentially recruiting and/or stabilizing selective receptor subtypes. Ionotropic glutamate receptors (iGluRs) play major roles in excitatory transmission in the vertebrate brain and at the insect neuromuscular junction (NMJ). The synapse properties are primarily shaped by the subunit composition of the receptors, which could be further modified by RNA editing and alternative splicing [1]. Changes in the subunit composition of postsynaptic iGluRs, in particular the AMPA-type receptors, have a tremendous impact on the development and plasticity of glutamatergic synapses [2,3]. Mechanisms controlling the recruitment of selective receptor subtypes exist and they integrate signals from multiple signaling pathways and regulate synaptic trafficking via posttranslational modifications within the iGluRs intracellular domains [4,5]. In addition, several auxiliary subunits, which primarily modulate the channel properties, have been implicated in the subcellular distribution of receptors [6]. Whether and how the auxiliary subunits modulate the subunit composition of iGluRs remains unclear. The Drosophila NMJ is a glutamatergic synapse similar to mammalian central synapses. In flies, the iGluRs are heterotetrameric complexes composed of three shared subunits, GluRIIC, GluRIID and GluRIIE, and either GluRIIA (type-A receptors) or GluRIIB (type-B) [7–11]. The function of the fly NMJ also requires Neto (Neuropillin and Tolloid-like), an obligatory auxiliary subunit of the iGluR complexes [12]. In the absence of Neto, or any of the shared iGluR subunits (or GluRIIA and GluRIIB together) the receptors fail to cluster at synaptic locations and the animals die as paralyzed embryos unable to develop into larval stages [12,13]. Genetic manipulation of Neto and iGluR levels indicated that Neto and the shared iGluR subunits are limiting for the synaptic localization of receptors and that GluRIIA and GluRIIB compete with each other for synaptic localization [9,14]. The type-A and type-B receptors differ in their single-channel properties, synaptic currents, regulation by second messenger, and sub-synaptic distribution [13]. The type-B channel desensitizes ten times faster than the type-A [8]. Also, the postsynaptic response to the fusion of single synaptic vesicles (the quantal size) is much reduced when only the type-B receptors are present; in fact, the dose of synaptic GluRIIA versus GluRIIB is a key determinant of quantal size. The interplay between the type-A and type-B synaptic receptors modulates synapse strength and plasticity. The synaptic accumulation of type-A and type-B receptors at the fly NMJ is differentially regulated. Two hybrid and genetics screens identified Coracle, the Drosophila homologue of the mammalian cytoskeletal protein 4. 1, as a direct binding partner for the GluRIIA cytoplasmic domain [15]. Disruptions of the actin cytoskeleton or mutations in coracle caused selective reduction in GluRIIA synaptic accumulation, suggesting that Coracle anchors the GluRIIA to the actin cytoskeleton. Mutations in the p21-activated kinase (PAK) also disrupt the localization and function of the GluRIIA. PAK co-localizes with the iGluR complexes at postsynaptic densities (PSDs) and, in conjunction with the guanine nucleotide exchange factor Pix and the adaptor protein Dreadlocks, promotes the synaptic accumulation of GluRIIA [16,17]. The synaptic accumulation of GluRIIB appears reduced in mutants of the PDZ (PSD-95/Dlg/Zona occludens-1) domain-containing scaffolding protein Discs large (Dlg) [18]. However, there is no evidence for a direct interaction between GluRIIB and Dlg, and except for GluRIIC [9], the iGluR subunits of the Drosophila NMJ lack a PDZ binding domain, suggesting that Dlg influences the synaptic abundance of GluRIIB indirectly. Also, Dlg localizes perisynaptically, at subsynaptic reticulum (SSR) [19]. In contrast, mammalian PSD-95 is a major component of the PSD, and it directly binds and modulates the synaptic targeting of iGluRs [20] and some of their interacting partners, such as the 4. 1N protein [21]. The PSD-95-mediated interactions can be further regulated by post-translational modifications such as palmitoylation and phosphorylation [5]. Here we show that Neto is key in regulating the subtype composition of iGluRs at the Drosophila NMJ. We characterized a novel Neto isoform, Neto-β, which appears to be the predominant isoform at the fly NMJ. The two Drosophila isoforms, Neto-α and Neto-β, share the highly conserved extracellular and transmembrane domains, the hallmark of the Neto family of proteins [6], but have distinct cytoplasmic domains, rich in protein interaction motifs. We generated neto-β isoform specific mutants and found that the synaptic accumulation of type-A receptors is selectively impaired at neto-β mutant NMJs. Furthermore, Neto-β-deficient synapses fail to recruit PAK, a critical PSD signaling molecule required for the synaptic stabilization of type-A receptors and for the recruitment of other postsynaptic components. Muscle overexpression of either Neto-α or Neto-β isoforms could rescue the lethality and NMJ function of neto null mutants, indicating that the shared domains of Neto function to mediate iGluRs clustering. However, only Neto-β could restore the recruitment of PAK at neto null synapses. Our data demonstrate that Neto-mediated cytoplasmic interactions control the subtype composition of iGluRs and shape postsynaptic composition. Drosophila neto codes for two transcripts, cDNA references GH11189 and RE42119. They share the first 10 exons, encoding the extracellular and transmembrane part of Neto, but have alternative exons that generate different intracellular domains (Fig 1A). Exon 11 encodes the cytoplasmic domain of Neto-α, a 206-residue acidic domain (pI 3. 88). Exons 12–14 encode the 351-residue Neto-β intracellular domain (pI 8. 90). The two domains are predicted to contain multiple phosphorylation and protein interaction motifs (Neto-β shown in S1 Fig), but they show no homology with other Neto proteins. In fact, unlike the highly conserved extracellular and transmembrane domains of Neto proteins, the intracellular parts are highly divergent or even missing, such as in the C. elegans Neto (Fig 1A) [22–24]. Previous RNA-Seq analyses indicated that neto-α and neto-β transcripts are expressed throughout development, including larval stages. Using RT-PCR and qPCR analyses we also detected both transcripts in the third instar larval carcasses and estimated that the neto-α transcript was ~4 fold less abundant than neto-β. To examine if Neto-β is present at the NMJs, we generated Neto-β isoform specific antibodies (details in Materials and Methods and S2 Fig) and compared them with previously described Neto antibodies, raised against the extracellular CUB1 domain [12]. The Neto-β positive signals co-localized with the Neto-ex puncta at control and at neto-β-rescued NMJs, but not at neto null NMJs rescued with neto-α transgenes (Fig 1B). Importantly, muscle overexpression of either isoform could rescue the embryonic lethality and paralysis of neto null mutants. This suggests that the shared part of Neto, including the CUB1, CUB2, LDLa and the transmembrane pass, contains all the components essential for iGluRs clustering and NMJ development. Indeed, muscle expression of a Neto variant in which the intracellular domains were replaced by eGFP rescued the neto null paralyzed embryos to fertile adults (Fig 1B). The caveat of these rescue experiments is that Neto overexpression highly exceeds the endogenous levels and may obscure the contribution of individual isoforms. The function of the extracellular and transmembrane domains of Drosophila Neto will be examined elsewhere. Here we will focus on characterizing the new isoform, Neto-β, and its role during NMJ development. We have previously demonstrated an essential role for Neto in the formation of iGluR clusters during synapse assembly and development. Likewise, Neto-β started to accumulate at the NMJ synapses at the onset of synaptogenesis (Fig 1C and 1D). In late embryo fillets, Neto-β antibodies labeled distinct synaptic puncta, which were also Neto-ex positive and co-localized with the iGluR synaptic signals (GluRIIA shown in Fig 1D). Neto-β signals remained at junctional locations throughout larval development and appeared to represent a significant fraction of the total Neto at the Drosophila NMJ (S2 Fig). To examine the role of Neto-β in synapse assembly and development we generated isoform specific mutants by imprecise excision of a transposable element Mi (ET1) Neto[MB07125] [25]. Several lines including precise excisions and small deletions were isolated and characterized by PCR amplification and sequencing (Fig 2A and Materials and Methods). The neto203 allele is partly missing neto-β specific exons; neto204 lacks the entire neto-β specific coding sequence and is likely a neto-β genetic null. No neto-β transcript was detectable in neto204 animals, but in neto203 larvae we found a truncated transcript, predicted to encode a short Neto-β variant (S1 Fig). For clarity we will refer to neto203 as netoβshort, neto204 as netoβnull, neto36 as netonull, and neto109 as netohypo [12]. A diagram of the neto-β alleles and predicted Neto-β proteins is shown in Fig 2A and 2B. The predicted Neto-β variants were detected by Western analysis in larval muscle extracts (Fig 2C): a full-length Neto-β was detected in control, and a band of 65 kD, corresponding to the truncated Neto-β variant, was detected in netoβshort larval muscle. Interestingly, no band corresponding to Neto-α was detected suggesting that the endogenous Neto-α levels are very low, even in the absence of Neto-β. The Neto-β variants (full length and short) appeared expressed at equivalent levels relative to Tubulin. However, the synaptic Neto levels were decreased by 34% at netoβshort NMJs, suggesting that the cytoplasmic domain influences Neto distribution (Fig 2C and S3 Fig). The Neto levels were decreased by 70% at netoβnull NMJs, consistent with the estimated 4-fold difference in the transcript levels. Further purification of Neto-β polyclonal sera against the two antigenic peptides (β1 and β2) produced two pools of antibodies (Fig 2B and S1 Fig). As expected, only Neto-β2 antibody labeled the netoβshort synapses, whereas no Neto-β1 signals were found at netoβshort or netoβnull NMJs (Fig 2D and 2E). In contrast, clear Neto-β1 synaptic signals were detected at netohypo NMJs. The netohypo allele lacks the exon containing the translation initiation codon [12]. This lesion should affect both Neto isoforms. We next examined the viability and behavior of neto-β mutants. Under optimal culturing conditions, 75% of netoβnull embryos and 90% of netoβshort developed into larval stages compared with control (n>200). In both cases, the third instar larvae were sluggish and exhibited a significant delay in rolling back when placed with dorsal side down. The netoβshort adult flies have no apparent defects, but the netoβnull homozygous have impaired climbing ability and are outcompeted by their heterozygous siblings. Further reduction to one copy of neto-α, such as in netoβnull/netonull trans allelic combinations, induced 100% lethality in more than 600 progenies examined. This lethality was rescued by a duplication covering the neto gene, Dp (1: 3) DC270 [26], indicating that the molecular lesion is confined to the neto locus. In contrast, netoβshort/netonull were viable indicating that one copy of neto-α and neto-βshort could support adult viability. To test the functionality of NMJ synapses lacking an intact Neto-β isoform we recorded spontaneous miniature potentials (minis or mEJPs) and evoked excitatory junctional potentials (EJPs) from muscle 6 of third instar larvae. The mean frequency of mEJPs was decreased in both neto-β alleles compared to control (Fig 3A–3C and S1 Table). Mini amplitude or quantal size, the postsynaptic response to the fusion of a single vesicle, was also reduced in both neto-β alleles (to 62% of control in netoβshort, and respectively 43% in netoβnull). We found no significant change in the resting potential and input resistance in the neto-β mutant larvae. The reduced mEJP frequency and amplitude at neto-β mutant NMJs were similar but less severe than at Neto- or iGluR-deprived NMJs [9,12]. But while Neto-deprived NMJs have severely reduced EJPs, the amplitude of EJPs was reduced by 12–14% compared to the control in netoβshort mutant larvae and was relatively normal at netoβnull NMJs (Fig 3D–3F and S1 Table). This suggests that neto-β mutant NMJs must compensate for the decreased postsynaptic sensitivity by a compensatory increase in quantal content, the number of vesicles released in response to each action potential. We found that quantal content, estimated as ratio of average EJP amplitude to the mEJP amplitude, was increased 1. 4 fold in netoβshort larvae and 2. 7 fold in netoβnull compared with control (Fig 3F). In contrast, the netohypo or netoRNAi larvae show no presynaptic compensatory response to reduced quantal size [12,27]. These studies show that the loss of Neto-β significantly alters the number, type and/or density of postsynaptic iGluRs and elicits a homeostatic compensatory increase of presynaptic release. These changes resemble the GluRIIA loss-of-function phenotypes, and differ from Neto- or iGluR-deprived NMJs [7–12]. Thus, the defects observed in neto-β mutants do not seem to originate from a general decrease in Neto levels and suggest isoform specific Neto activities at the NMJ. NMJ morphological analyses further emphasized these specific defects. We have previously shown that Neto-deprived NMJs have longer and fewer branches, with significantly reduced number of boutons [27]. In contrast, at similarly reduced Neto levels, the netoβnull NMJs were reduced in length and had shorter branches (Fig 3G and 3H). Compared with controls, neto-β mutant NMJs had fewer but larger type Ib boutons (Fig 3I and 3J). In particular, the distal boutons had an estimated 2. 4 fold volume increase (netoβshort: 18. 9 μm3 ± 2. 6, n = 29, netoβnull: 20. 8 μm3 ± 2. 3, n = 45, and control: 7. 8 μm3 ± 1. 3, n = 29). Together these data indicate that perturbations of Neto-β have profound effects on the NMJ morphology and function that are different from a reduction in Neto levels. At the Drosophila NMJ, Neto activities are essential for trafficking and clustering of iGluRs at synaptic sites. To assess the contribution of Neto-β to these functions we examined the iGluRs distribution at neto-β mutant NMJs. For all the quantitative analyses below control/neto-β mutant sets (5–10 larvae/genotype) were processed together and imaged under the same conditions to capture and compare the synaptic signals. At the fly NMJ synapses, the sites of neurotransmitter release are marked by presynaptic specializations called T-bars, where Bruchpilot (Brp), the fly homolog of the vertebrate active zone protein ELKS, accumulates [28]. Opposite to the T-bars, the iGluRs are concentrated and stabilized at synaptic sites by a myriad of PSD-associated proteins. We found that neto-β mutant boutons contained more synaptic contacts, as visualized by Brp/GluRIIC juxtaposing puncta, and their density was increased at proximal boutons but not at the distal ones (Fig 4A and 4B). This increase in synaptic density is consistent with the compensatory response observed at neto-β mutant NMJs (Fig 3F). The intensity of Brp signals was largely constant, but the GluRIIC levels were diminished to 78% from control at netoβshort NMJs (n = 10) and to 39% at netoβnull NMJs (n = 10) (Fig 4A and 4C). The reduction of GluRIIC synaptic signals at neto-β mutant NMJs occurred without a change in the GluRIIC net protein indicating a defect in the synaptic localization of the receptors (Fig 4D). Importantly, the GluRIIC synaptic signals paralleled the levels of synaptic Neto observed at these NMJs (Fig 2D and 2E, S3A Fig), as Neto is limiting for receptors localization. Drosophila NMJ utilizes two types of iGluRs, type-A and type-B, that differ in their subunits composition and properties [13]. The single channel current amplitude of both receptor types is identical, but the type-B channel desensitizes nearly ten times faster, thus the relative ratio of synaptic type-A/type-B receptors is a key determinant of quantal size [8]. Also, reduced levels of postsynaptic type-A receptors trigger a robust presynaptic compensatory response [7]. The physiological similarities between the GluRIIA and neto-β mutant NMJs suggest that Neto-β may primarily influence the synaptic accumulation of type-A receptors. We tested this possibility by examining the relative intensity of GluRIIA and GluRIIB signals at neto-β mutant NMJs. The netoβshort larvae had severely reduced GluRIIA synaptic levels (to 26% from control, n = 13), accompanied by no significant change in the GluRIIB levels (Fig 4C and 4E). The netoβnull NMJs showed an even stronger reduction in the GluRIIA synaptic levels (to 18. 5% from control, n = 14) and also decreased GluRIIB levels (Fig 4C and 4E). In contrast, the netohypo NMJs showed uniformly reduced levels for all iGluR subunits examined (S3 Fig). The drastic decrease of GluRIIA synaptic signals at neto-β mutant NMJs exceeded the reduction of GluRIIC and Neto synaptic levels observed at these NMJs, indicating a key role for the Neto-β isoform in regulating the GluRIIA synaptic abundance. How does Neto-β regulate the selective accumulation of type-A receptors at synaptic sites? Neto-β may preferentially recruit the type-A receptors and/or influence their synaptic stabilization. If Neto isoforms promote the synaptic accumulation of selective iGluRs, and Neto-β influences the type-A receptors, the inference is that Neto-α should preferentially impact the distribution of type-B receptors. In support for this possibility, in RNAi knockdown experiments we found that Neto-α-deprived NMJs showed a dramatic loss of GluRIIB synaptic signals (by 68%) and a modest, variable increase of synaptic GluRIIA (by 18%) (Fig 4F and 4G). The efficiency of the RNAi-mediated knockdown was verified by Western blot analysis of muscle extracts from netonull larvae rescued with a V5-tagged neto-α transgene (Fig 4H and [12]). Lack of Neto-α-specific reagents precluded us from expanding these experiments. Nonetheless, these results raise the possibility that Neto isoforms utilize their distinct cytoplasmic domains to modulate the synaptic abundance of specific receptor subtypes. In the case of Neto-β, the last 260 C-terminal cytoplasmic residues appear to be critical for the synaptic accumulation of type-A receptors. In Drosophila, several postsynaptic and perisynaptic proteins influencing the synaptic abundance of type-A and type-B receptors have been reported [15,16,18]. For example, PAK, a PSD component that co-localizes with the GluRIIA subunit, was shown to promote accumulation of type-A receptors at synaptic sites [16,17,29]. PAK also influences the size of the SSR, a stack of membrane folds that underlines the postsynaptic cell membrane of the type Ib boutons [16,17]. We found that PAK signals were severely reduced at neto-β mutant NMJs (Fig 5A). PAK synaptic levels dropped to 4% from control in netoβshort larvae (n = 11) and to 19% in netoβnull (n = 12). This drastic decrease exceeded the reduction in PAK signals observed at netohypo NMJs, suggesting that the low level of Neto-β at netohypo NMJs could partly mediate PAK stabilization at PSDs (Fig 2E and [12]). A similar, albeit less pronounced effect was observed for Dlg (Fig 5B–5D). The Dlg junctional signals were reduced to 48% from control in netoβshort third instar larvae (n = 14) and to 27% from control in netoβnull (n = 11). Importantly, the levels of PAK and Dlg net proteins were not changed in extracts from neto-β larval muscles (Fig 5E). Also, the accumulation of PAK and Dlg at junctional locations was completely restored in neto-β mutants by a duplication covering the neto locus (S4 Fig), indicating that the observed phenotypes are neto specific. In contrast, cysteine string protein (CSP), which labels clusters of synaptic vesicles in the vicinity of presynaptic membranes [30], and α-Spectrin, which labels the presynaptic axon and surrounds the bouton postsynaptically [31] appeared normal at neto-β mutant NMJs (S5 Fig). Under electron microscopy, control synapses are marked by presynaptic specializations called T-bars and electron-dense membranes, corresponding to PSDs (Fig 6A–6C). At neto-β mutant boutons, serial sections revealed that the PSDs were significantly reduced in size but increased in number (Fig 6B–6D and Fig 4). The diminished PSD length (up to 2 fold in netoβnull mutants) matched the observed reduction of postsynaptic receptor fields (Fig 4). A thick stack of SSR membranes surrounds the control type Ib boutons. In contrast, the enlarged neto-β mutant boutons had diminished or even absent SSR structures, consistent with the reduced Dlg levels observed (Fig 6E–6H). In addition, neto-β mutants appeared to have misshaped T-bar structures, including double T-bar structures (Fig 6B and 6C, details). GluRIIA deprivation induces excessive recruitment of Brp at the active zones which sometimes results in active zone profiles with two or more T-bars [32]. Likewise, the excessive T-bar structures observed primarily in netoβnull mutant boutons may correlate to a homeostatic compensatory response triggered by the GluRIIA depletion (Figs 3F and 4B and 4E). Since the SSR folds are reduced in neto-β mutants, it is possible that the reduced PAK and Dlg synaptic levels are indirectly caused by the lack of proper SSR structures. To test this possibility, we examined late embryos and first instar larvae, before the SSR develops [33]. If the SSR controls the synaptic accumulation of PAK, then PAK levels should be normal at neto-β mutant NMJs during the early stages of development. However, we found that PAK accumulated normally at the muscle attachment sites in control and neto-β mutant animals, but was severely diminished at neto-β mutant NMJs (Fig 7A). Thus, loss of synaptic PAK is not secondary to the loss of SSR and appears to be directly caused by impairments in Neto-β. If Neto-β recruits PAK at the cell membrane, then loss of synaptic PAK in neto-β mutants should be rescued by muscle expression of PAKmyr, a membrane-tethered PAK variant [34]. We found that muscle overexpression of PAKmyr in neto-β mutants induced accumulation of PAK signals at perisynaptic but not synaptic sites, and could not rescue the GluRIIA synaptic accumulation (netoβshort shown in Fig 7B). This indicates that Neto-β controls the recruitment of PAK at synaptic locations. Loss of PAK did not affect the synaptic recruitment of Neto-β: while PAK signals were lost at pak null NMJs (pak11/Df) or in mutants that fail to localize at cell peripheries (pak6/Df or pak6/11) [35], the synaptic Neto signals remained unaffected, irrespective of the pak genetic manipulations (Fig 7C). Also, the levels of GluRIIA were 4-fold reduced in neto-β mutants (Fig 4), but only 2-fold in pak mutants [16], suggesting that Neto-β has additional, PAK-independent functions in the synaptic recruitment/stabilization of GluRIIA. Together these data indicate that Neto-β is required for the synaptic accumulation of PAK. In flies and mammals, PAK membrane localization is controlled by Pix, a Rho-type guanine nucleotide exchange factor [16,36]. Mutations in dpix led to impairments in synaptic accumulation of PAK, GluRIIA, Dlg, and other postsynaptic components. Lack of Pix antibodies precluded us to test whether Neto-β recruits dPix at PSDs. Nonetheless, while the PAK synaptic signals were completely lost in dpix mutants, Neto signals accumulated at these mutant synapses albeit they appeared somewhat reduced, likely due to defects in the synapse organization (S6 Fig). Thus, dPix is required for PAK but not for Neto recruitment of at synaptic sites. We have previously demonstrated that the net levels of Neto are critical for synapse assembly and NMJ development [12,14]. In particular, Neto depletion has profound consequences for synapse assembly and function. The levels of synaptic Neto are reduced in neto-β mutants compared with controls (Fig 2 and S3 Fig) raising the possibility that the phenotypes observed in neto-β allelic series could be partly due to reduced Neto levels. To distinguish between isoform specific and suboptimal Neto phenotypes we manipulated the muscle expression of Neto-α and Neto-β isoforms and examined their effects on the development and function of NMJ. Similar to neto-α, we found that muscle expression of neto-β transgenes rescued the viability and NMJ development of netonull mutants and induced gain-of-function phenotypes in a concentration-dependent manner (Fig 8A and [27]). Low to moderate levels of Neto-β induced formation of NMJs with relatively normal morphology and clearly defined synaptic Neto clusters, resembling the neto mutants rescued with low and moderate levels of Neto-α. Excess Neto-β appeared detrimental to the NMJ development and to the overall growth and viability of rescued animals (Fig 8A). Intriguingly, Neto-β but not Neto-α enabled the stabilization of PAK at PSDs, over a wide range of concentrations tested (Fig 8A). Furthermore, increasing the levels of Neto- α in neto-β mutants could not restore the PAK synaptic accumulation, while introducing Neto-β effectively rescued this defect. This result cannot be explained by a difference between Neto- α and Neto-β cellular distribution, since both isoforms appear to concentrate at the NMJ. We confirmed that Neto-α and Neto-β synaptic levels correlate with the levels of Neto protein in the larval muscle, as detected by Western analysis (Fig 8B). Thus, both Neto isoforms can traffic and accumulate at synaptic locations where they mediate synapse assembly. However, only Neto-β isoform can enable PAK accumulation at PSDs. Since PAK contributes to synaptic stabilization of GluRIIA, Neto-α-rescued NMJs are expected to exhibit a reduction in the synaptic GluRIIA signals and thus a reduction in quantal size. Indeed, we found that netonull mutants rescued with low/moderate levels of Neto-α had significantly reduced mini amplitudes and reduced GluRIIA/GluRIIB ratio (Fig 8C and 8D, S7 Fig). In contrast, netonull NMJs rescued with neto-β transgenes had relatively normal mini amplitudes, indicating normal GluRIIA/GluRIIB ratio at these synapses. The mini frequency was largely normal, except for the neto-β transgenes which showed increased mini frequency when reared at 18°C (Fig 8E). Interestingly, the evoked potentials were in the normal range in all Neto-α and Neto-β-rescued larvae tested, likely due to compensatory presynaptic response (Fig 8F–8H). These results indicate that Neto-α-rescued NMJs have deficits in the synaptic accumulation of type-A receptors. Such defects are partly obscured by excess Neto-α, likely because the conserved domains of Neto confer high iGluRs “clustering capacity” in these rescue experiments. But under normal condition, Neto is a low abundance protein and a limiting factor for iGluRs clustering in the muscle. Furthermore, Neto-β appears to be the predominant isoform at the Drosophila NMJ. Together our findings demonstrate that Neto-β, the major Neto isoform at the Drosophila NMJ, controls the subtype composition of iGluRs partly by regulating the recruitment of the PSD-associated kinase PAK. Neto proteins have been initially characterized as auxiliary subunits that modulate the function of kainate (KA) and NMDA receptors [22,23]. In vertebrates, Neto1 and Neto2 directly interact with KAR subunits and increase channel function by modulating gating properties [23,37,38]. Since loss of KAR currents in mice lacking Neto1 and/or Neto2 exceed a reduction that could be attributed to alterations of channel gating, an additional role for Neto proteins in synaptic targeting of receptors has been proposed. The role for vertebrate Neto proteins in KAR membrane and/or synaptic targeting remains controversial and appears to be cell type-, receptor subunit-, and Neto isoform-dependent [23,39,40]. Furthermore, the C. elegans Neto has a very small intracellular domain (24 amino acids beyond the conserved domains) [24]. This implies that 1) Neto without an intracellular domain constitutes the minimal conserved functional moiety, and 2) the divergent intracellular domains of Neto proteins may fulfill tissue and/or synapse specific modulatory functions. Indeed, Neto2 bears a class II PDZ binding motif that binds to the scaffold protein GRIP and appears to mediate KARs stabilization at selective synapses [41]. In flies, Neto is an essential protein that plays active roles in synapse assembly and in the formation and maintenance of postsynaptic structures at the NMJ. The Drosophila Neto isoforms do not have PDZ binding motifs, but they use at least two different mechanisms to regulate the synaptic accumulation and subunit composition of iGluRs. First, Neto participates in extracellular interactions that enable formation of iGluR/Neto synaptic complexes; formation of stable aggregates is presumably prevented by the inhibitory prodomain of Neto [27]. Second, the two Neto isoforms appear to facilitate the selective recruitment and/or stabilization of specific iGluR subtypes. We speculate that Neto-β may selectively associate with and recruit type-A receptors, perhaps by engaging the C-terminal domain of GluRIIA, which is critical for the synaptic stabilization of these receptors [42,43]. Aside from a possible role in the selective recruitment of iGluR subtypes, Neto-β participates in intracellular interactions that facilitate the recruitment of PAK at PSDs; in turn, PAK signals through two independent, genetically separable pathways (a) to modulate the GluRIIA synaptic abundance and (b) to facilitate formation of SSR [17]. Whether Neto-β recruits PAK directly or via a larger protein complex remains to be determined. Neto-β contains an SH3 domain that may bind to the proline-rich SH3 binding domain of PAK. However, in tissue culture experiments, we failed to detect a direct interaction between PAK and Neto-β (full-length or intracellular domain). PAK synaptic accumulation is completely abolished at NMJ with mutations in dPix, although not all dpix defects are mediated through PAK [16]. Conversely, PAK together with Dreadlocks (Dock) controls the synaptic abundance of GluRIIA, while PAK and dPix regulate the Dlg distribution [17]. The reduction of GluRIIA and Dlg synaptic abundance observed at neto-β mutant NMJs suggests that Neto-β may interact with both dPix and Dock and enable both PAK activities. In addition, Neto-β may stabilize postsynaptic type-A receptors by enhancing their binding to Coracle, which anchors GluRIIA to the postsynaptic actin cytoskeleton [15]. Importantly, this study connects the complex regulatory networks that modulate the PSD composition to the Neto/iGluR clusters themselves. The Neto-β cytoplasmic domain is rich in putative protein interaction motifs (S1 Fig), and may function as a scaffold platform to mediate multiple protein interactions that act synergistically during synapse development and homeostasis. Loss of Neto-β-mediated intracellular interactions at netoβshort NMJs reduced the GluRIIA synaptic abundance, but did not affect the GluRIIB synaptic signals (Fig 4E). It is unlikely that the remaining cytoplasmic part of Neto-β facilitates the GluRIIB synaptic accumulation at these NMJs at the expense of GluRIIA and PAK. Instead, we favor a model whereby the synaptic stabilization of GluRIIA requires a Neto-β-dependent intracellular network. Disruption of this network diminishes GluRIIA and increases GluRIIB synaptic abundance, pending the availability of limiting subunits, GluRIIC-E and Neto. Conversely, the presence of this network ensures that at least some type-A receptors are stabilized at synaptic sites, even at Neto-deprived synapses, such as in netohypo larvae [12]. Assembly of this network does not require GluRIIA since both Neto-β and PAK accumulated normally at GluRIIA mutant NMJs (Fig 8I). Furthermore, in the absence of Neto-β the synaptic abundance of GluRIIA can be partly restored by excess Neto-α or a delta-intracellular Neto variant, suggesting that excess iGluRs “clustering capacity” overrides the cellular signals that shape PSD composition [27]. What intracellular domain (s) of Neto bind to and how they are modified by post-translational modifications will be critical questions to understand how postsynaptic composition is regulated during development and homeostasis. The discovery of Drosophila Neto isoforms with alternative cytoplasmic domains and isoform specific activities expands the repertoire of Neto-mediated functions at glutamatergic synapses. On one hand, all Neto proteins share the highly conserved CUB1, -2, LDLa and transmembrane domains that have been implicated in engaging and modulating the receptors, the central function of Neto proteins [22,23,44]. In flies this conserved part is both required and sufficient for iGluRs clustering and NMJ development. In C. elegans the entire Neto appears to be reduced to this minimal functional unit [24]. The only exception is a retina-specific CUB1-only Neto1 isoform with unknown function [45]. In contrast, the cytoplasmic domains are highly divergent among Neto proteins. This diversity might have evolved to facilitate intracellular, context specific function for Neto proteins, such as the need to couple the iGluR complexes to neuron or muscle specific scaffolds in various phyla. Alternatively, by engaging in different intracellular interactions, via distinct cytoplasmic domains, different Neto isoforms may undergo differential targeting and/or retention at the synapses and thus acquire isoform-specific distributions and functions within the same cell. Phylogenetic analyses indicate that the intracellular domains of Neto are rapidly evolving in insects. Blocks of high conservations could be clearly found in the genome of short band insect Tribolium castaneum (Coleoptera) or in Apis mellifera (Hymenoptera). However, most insects outside Diptera appear to have only one Neto isoform, more related to Neto-β. In fact, the only Neto-α isoform outside Drosophila was found in Musca domestica (unplaced genomic scaffold NCBI Reference Sequence: XM_005187241. 1). Other neto loci, from Hydra to vertebrates, appear to encode Neto proteins with unique and highly divergent intracellular domains. An extreme example is the C. elegans Neto/Sol-2, with a very short cytoplasmic tail, which requires additional auxiliary subunits, Sol-1 and Stargazin, to control the function of glutamate receptors [46,47]. Neto proteins appear to utilize their intracellular domains to connect to the signaling networks that regulate the distribution and subunit composition for iGluRs. Such cellular signals converge onto and are integrated by the intracellular domains of the receptors and/or by various auxiliary subunits associated with the iGluR complexes [1,6]. Neto proteins modulate the gating behavior of KAR but also play crucial roles in the synaptic recruitment of glutamate receptors in vivo [12,40,48]. At the fly NMJ, Neto enables iGluRs synaptic clustering and initiates synapse assembly. In addition, the intracellular domain of Neto-β recruits PSD components and triggers a cascade of events that organize postsynaptic structures and shape the composition of postsynaptic fields. The cytoplasmic domains of Neto proteins emerge as versatile signaling hubs and organizing platforms that directly control the iGluRs subunit composition and augment the previously known Neto functions in modulation of glutamatergic synapses. To generate neto-β alleles, the Minos transposomal element Mi (ET1) Neto[MB07125] was mobilized with Minos transposase [25]. Several lines with precise excisions and imprecise excisions/small deletions were isolated and characterized by PCR amplification over the deficiencies and DNA sequencing. The genomic fragments removed in various neto alleles were as follows: neto203 X: 13,415,115–13,418,117, including part of the exon 13 and 14 of the predicted neto gene, and neto204 X: 13,414,477–13,417,931, containing exons 12,13 and 14. The UAS-neto-β lines (B lines) were generated by insertion of the neto-β cDNA (from RE42119) in pUAST vector and germline transformation (BestGene, Inc.). Similarly, for the UAS-netoΔintra (the H4 line) the Neto coding sequence M1-R471 followed by a short linker (DVPALE) was placed in frame with the eGFP and cloned in pUAST. The neto-αRNAi lines were generated by insertion of a neto-α specific PCR fragment in pUAST-R57 followed by germline transformation. The PCR primers utilized were: RNAi-F (5′-AAGGCCTACATGGCCGGACCGGCGAACAAATGGAGGAAGACG-3′) and RNAi-Rev (5′-AATCTAGAGGTACCTGATTTTGTGCAGGAACTTGAGG-3′). Other fly stocks used in this study were as follows: neto36, neto109, and UAS-neto-α -V5 [12]; UAS-neto-α (A lines) [27]; neto (CUB1) RNAi [14]; Dp (1: 3) DC270 [26]; pak6, pak11, and pakmyr [34]; dpix1 [16]; GluRIIASP16, and Df (2L) clh4 [7] (from A. DiAntonio, Washington University). The G14-Gal4 was obtained from C. Goodman (University of California at Berkeley). The rabbit polyclonal anti-Neto-β antibodies were generated against two synthetic peptides: β1 (GRSHYGGLLVTSTAKQP) and β2 (LDDVSNRYYREAVPL) (21st Century Biochemicals) and separated by affinity purification. The rabbit polyclonal anti-GluRIIB and anti-GluRIIC were generated as previously described [8] against synthetic peptides ASSAKKKKKTRRIEK, and respectively QGSGSSSGSNNAGRGEKEARV (Pacific Immunology Corp). To analyze muscle proteins, wandering third instar larvae were dissected, and all tissues except for the body wall (muscle and cuticle) were removed. The body walls were mechanically disrupted and lysed in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100,1% deoxycholate, protease inhibitor cocktail (Roche) for 30 min on ice. The lysates were separated by SDS-PAGE on 4%–12% NuPAGE gels (Invitrogen) and transferred onto PVDF membranes (Millipore). Drosophila S2 cells were used for the production of recombinant proteins [12]. Full-length cDNA for neto-β from RE42119 was subcloned in pAcPA-based plasmids for expression in S2 cells under the actin promoter. The Neto-β truncation (neto203-like) was generated by looping out the deleted fragment using the QuickChange site-directed mutagenesis kit. All constructs were verified by DNA sequencing. Primary antibodies were used at the following dilutions: rat anti-Neto-ex [12], 1: 1000; anti-Tubulin (Sigma-Aldrich), 1: 1000; rabbit anti-Neto-β, 1: 1000; rabbit anti-GluRIIC, 1: 1000; rabbit anti-PAK, 1: 5000 (a gift from Nicholas Harden) [35]; mouse anti-Dlg (4F3), 1: 1000; mouse anti-V5 (Invitrogen), 1: 1000. Immune complexes were visualized using secondary antibodies coupled with IR-Dye 700 or IR-Dye 800 followed by scanning with the Odyssey infrared imaging system (LI-COR Biosciences). Wandering third instar larvae were dissected as described previously in ice-cooled Ca2+-free HL-3 solution [49,50]. Embryos at 18h after egg laying (AEL) were dechorinated and genotyped and, after an additional incubation of 2 h at room temperature, were dissected as described previously [12]. First instar larvae were similarly dissected within 2 h from hatching. The samples were fixed in 4% paraformaldehyde (Polysciences, Inc.) for 20 min or in Bouin’s fixative (Bio-Rad) for 3 min and washed in PBS containing 0. 5% Triton X-100. Primary antibodies from Developmental Studies Hybridoma Bank were used at the following dilutions: mouse anti-GluRIIA (8B4D2), 1: 100; mouse anti-Dlg (4F3), 1: 1000; mouse anti-Brp (Nc82), 1: 200; mouse anti-CSP (6D6), 1: 1000: mouse anti-α-Spectrin (3A9), 1: 50: mouse anti-FasII (1D4), 1: 10. Other primary antibodies were utilized as follow: rabbit anti-PAK, 1: 2000; rat anti-Neto-ex, 1: 1000 [12]; and Cy5- conjugated goat anti-HRP, 1: 1000 (Jackson ImmunoResearch Laboratories, Inc.). Alexa Fluor 488-, Alexa Fluor 568-, and Alexa Fluor 647- conjugated secondary antibodies (Molecular Probes) were used at 1: 200. All samples were mounted in ProLong Gold (Invitrogen). Samples of different genotypes were processed simultaneously and imaged under identical confocal settings in the same imaging session with a laser scanning confocal microscope (CarlZeiss LSM780). All images were collected as 0. 2m optical sections and the z-stacks were analyzed with Imaris software (Bitplane). To detect positive puncta we used the spot finding Imaris algorithm followed by manual inspection and correction. To quantify fluorescence intensities synaptic ROI areas surrounding anti-HRP immunoreactivities were selected and the signals measured individually at NMJs (muscle 4, segment A3) from ten or more different larvae for each genotype (number of samples is indicated in the graph bar). The signal intensities were calculated relative to HRP volume and subsequently normalized to control. Boutons were counted in preparations double labeled with anti-HRP and anti-Dlg. All quantifications were performed while blinded to genotype. The numbers of samples analyzed are indicated inside the bars. Statistical analyses were performed using the Student t-test with a two-tailed distribution and a two-sample unequal variance. Error bars in all graphs indicate standard deviation ±SEM. ***; p<0. 001, **; p<0. 005, *; p<0. 05, ns; p>0. 05. The standard larval body wall muscle preparation first developed by Jan and Jan (1976) [51] was used for electrophysiological recordings [52]. Wandering third instar larvae were dissected in physiological saline HL-3 saline [49], washed, and immersed in HL-3 containing 0. 8 mM Ca2+ or 0. 3 mM Ca2+ using a custom microscope stage system [53]. The nerve roots were cut near the exiting site of the ventral nerve cord so that the motor nerve could be picked up by a suction electrode. Intracellular recordings were made from muscle 6. Data were used when the input resistance of the muscle was >5 MΩ and the resting membrane potential was between -60 mV and -80 mV. The input resistance of the recording microelectrode (backfilled with 3 M KCl) ranged from 20 to 25 MΩ. Muscle synaptic potentials were recorded using an Axon Clamp 2B amplifier (Axon Instruments) and pClamp 10 software. Following motor nerve stimulation with a suction electrode (100 μsec, 5 V), evoked EJPs were recorded. Four to six EJPs evoked by low frequency of stimulation (0. 1 Hz) were averaged. For mini recordings, TTX (1 μM) was added to prevent evoked release [49]. To calculate mEJP mean amplitudes, 50–200 events from each muscle were measured and averaged using the Mini Analysis program (Synaptosoft). Minis with a slow rise and falling time arising from neighboring electrically coupled muscle cells were excluded from analysis [54,55]. Quantal content was calculated by dividing the mean EJP by the mean mEJP after correction of EJP amplitude for nonlinear summation according to previously described methods [56,57]. Corrected EJP amplitude = E[Ln[E/ (E—recorded EJP) ]], where E is the difference between reversal potential and resting potential. The reversal potential used in this correction was 0 mV [57,58]. Data are presented as mean ±SEM, unless otherwise specified; EJP amplitudes and quantal contents after the nonlinear correction are shown. Student T-test was used to assess statistically significant differences among the genotypes. Wandering third instar larvae were dissected in physiological saline HL-3 saline and fixed for 30 min on dissection plate in fixation buffer (0. 1 M Sodium Cacodylate buffer, pH7. 2; 2 mM MgCl2; 1% glutaraldehyde; 4% paraformaldehyde). The samples were trimmed to include only the abdominal segments A2 and A3, transferred in a 1. 5mL Eppendorf tube, fixed overnight at 4°C, then washed extensively with 0. 1 M Sodium Cacodylate buffer with 132 mM Sucrose, pH 7. 2. The samples were further processed and analyzed according to published protocols [59] at the Microscopy and Imaging Facility, NICHD. | Title: Neto-Mediated Intracellular Interactions Shape Postsynaptic Composition at the Drosophila Neuromuscular Junction Summary: Ionotropic receptors assembled from different subunits have strikingly different properties and uses. In mammalian brain, the molecular mechanisms controlling the subunit composition of glutamate receptors are critical for the formation of neural circuits and for the long-term plasticity underlying learning and memory. Here we investigate how subunit composition is regulated at the Drosophila neuromuscular junction (NMJ), a synapse similar in composition and physiology to mammalian AMPA/Kainate synapses. We find that an auxiliary protein, Neto, which is essential for functional receptors, has a key role in controlling which flavor of glutamate receptors will be at the synapses. In flies, synapse strength and plasticity is modulated by the interplay between two receptor subtypes, A and B. Mutations that eliminate or truncate the Neto-β isoform fail to accumulate the type-A receptors, as well as other postsynaptic proteins important for the synaptic stabilization of type-A receptors. This result indicates that Neto may use its cytoplasmic domains as signaling hubs and organizing platforms to sculpt postsynaptic composition. Neto proteins modulate the formation and function of glutamatergic synapses from worms to humans. Our findings expand the repertoire of Neto proteins and illustrate the richness in synapse modulation brought about by the growing family of auxiliary proteins. | 12,161 | 329 | lay_plos | en |
Summarize: These crawls are part of an effort to archive pages as they are created and archive the pages that they refer to. That way, as the pages that are referenced are changed or taken from the web, a link to the version that was live when the page was written will be preserved.Then the Internet Archive hopes that references to these archived pages will be put in place of a link that would be otherwise be broken, or a companion link to allow people to see what was originally intended by a page's authors.The goal is to fix all broken links on the web. Crawls of supported "No More 404" sites. Update: The Birmingham Police Department says after being reviewed by the Jefferson County District Attorney's Office, the case is ruled as justifiable homicide. No warrants will be pursued in the case. UPDATE: The Jefferson County Coroner's Office has identified the deceased shooter as Antonio Demond Sanders, 24, of Birmingham. Advertisement Birmingham Police detectives are investigating a deadly shooting at the McDonald's on Lomb Avenue Saturday night. West Precinct Officers responded to the restaurant around 10:45 p.m. on a report that multiple people had been shot. When officers arrived they found two adult men and a teenage boy had been shot. All were rushed to hospitals, but one of the adults died from his injuries. Birmingham police spokesman Sgt. Bryan Shelton said the manager of the restaurant was unlocking the door so the man and his two sons could leave the restaurant, when a masked man entered and opened fire. The father also had a gun and returned fire. The masked gunman was shot as were the father and one of his sons. The father and son have non-life threatening injuries. Shelton said investigators are not clear whether the victim was robbing the store or targeting someone in the business. Shelton said, "Things like this are difficult for both families. The gentleman who unfortunately lost his life, the teenage boy who is in the hospital recovering from his injuries and the father who is also recovering from his injuries. It's not easy being a father and watching your child get injured, get hurt like that. It's a really heartwrenching experience." Based on the preliminary information, the father won't face any charges. The investigation is ongoing. AlertMe These crawls are part of an effort to archive pages as they are created and archive the pages that they refer to. That way, as the pages that are referenced are changed or taken from the web, a link to the version that was live when the page was written will be preserved.Then the Internet Archive hopes that references to these archived pages will be put in place of a link that would be otherwise be broken, or a companion link to allow people to see what was originally intended by a page's authors.The goal is to fix all broken links on the web. Crawls of supported "No More 404" sites. | Summary: A man with a firearm may have stopped a burglary, or perhaps something worse, at an Alabama McDonald's. The man, who hasn't been named, was leaving the fast-food eatery in Birmingham with his two sons at around 10:45pm Saturday, report Fox News and AL.com. WBRC notes that a masked gunman burst into the restaurant as a worker opened the door for the family to exit, opening fire. The father whipped out his own pistol and returned fire; an employee who hid in the store's freezer with another worker says he heard more than 15 shots fired. "All we hear is like different gunfire, so... I'm imagining everybody is dead," Markus Washington tells WBRC, who said he was making hamburgers when the chaos broke out. He adds that as he and his co-worker cowered in the freezer, they were sure the gunman was "looking for us." But the gunman was felled in the shooting, later dying from his injuries. The dad and one of his sons were also hit; the son's injuries weren't life-threatening, while it's not clear on the father's condition. Sgt. Bryan Shelton, a rep for the Birmingham Police, tells WVTM they're still not sure if the gunman meant to rob the Mickey Dee's, ambush an employee, or just start shooting. "It's not easy being a father and watching your child get injured... like that," Shelton says. "It's a really heart-wrenching experience." Washington, meanwhile, calls the pistol-toting dad his "hero": "If he wasn't armed, we might not be here having this interview." | 651 | 388 | multi_news | en |
Write a title and summarize: Pathogenic microbes employ a variety of methods to overcome host defenses, including the production and dispersal of molecules that are toxic to their hosts. Pseudomonas aeruginosa, a Gram-negative bacterium, is a pathogen of a diverse variety of hosts including mammals and the nematode Caenorhabditis elegans. In this study, we identify three small molecules in the phenazine class that are produced by P. aeruginosa strain PA14 that are toxic to C. elegans. We demonstrate that 1-hydroxyphenazine, phenazine-1-carboxylic acid, and pyocyanin are capable of killing nematodes in a matter of hours. 1-hydroxyphenazine is toxic over a wide pH range, whereas the toxicities of phenazine-1-carboxylic acid and pyocyanin are pH-dependent at non-overlapping pH ranges. We found that acidification of the growth medium by PA14 activates the toxicity of phenazine-1-carboxylic acid, which is the primary toxic agent towards C. elegans in our assay. Pyocyanin is not toxic under acidic conditions and 1-hydroxyphenazine is produced at concentrations too low to kill C. elegans. These results suggest a role for phenazine-1-carboxylic acid in mammalian pathogenesis because PA14 mutants deficient in phenazine production have been shown to be defective in pathogenesis in mice. More generally, these data demonstrate how diversity within a class of metabolites could affect bacterial toxicity in different environmental niches. The Gram-negative bacterium Pseudomonas aeruginosa, a pathogen of both plants and metazoans, is a prevalent and pernicious pathogen in persons who are immunocompromised or suffer from cystic fibrosis (CF) [1], [2]. P. aeruginosa employs many mechanisms to antagonize its hosts, including the production of low molecular weight toxins [3], [4], [5]. Identifying toxins, the conditions under which they are produced, and the mechanisms by which they act, are of fundamental importance in understanding and combating the virulence of this clinically-important pathogen. The nematode Caenorhabditis elegans, which is found in decaying plants where many pathogenic microbes reside, is a useful model host for a variety of pathogens including P. aeruginosa [6], [7]. PA14 is a virulent clinical isolate of P. aeruginosa that is capable of killing C. elegans [8], [9]. Previous studies using C. elegans as a model system for PA14 pathogenesis determined that PA14 can kill C. elegans either as a consequence of intestinal infection or intoxication, depending on the media on which the bacteria are grown [8], [9]. Nematodes die in hours when PA14 is grown on a nutrient rich agar containing peptone, glucose, and sorbitol (PGS agar) [8]. This type of killing is referred to as ‘fast killing’. In contrast, when the bacteria are grown on a less rich medium, it takes several days for C. elegans to die [9]. This type of killing is referred to as ‘slow killing’. Fast killing is thought to be mediated by diffusible toxins because exudates of PA14 grown on PGS agar are sufficient to kill C. elegans; worms need not be in the presence of live PA14 in order to be killed. In contrast, slow killing requires bacterial growth in the worm gut to effect pathogenesis. Experiments in mice have demonstrated that the fast killing toxin-based model is relevant in plant and mammalian pathogenesis because mutants that are defective in fast killing are also defective in Arabidopsis thaliana and murine PA14 infection models [8]. In this study we focus on identification of the toxins responsible for fast killing. In a previously published screen for PA14 mutants that exhibited reduced levels of fast killing, several isolates displayed reduced levels of the blue-green phenazine pigment pyocyanin [8]. Phenazines are a class of tricyclic aromatic molecules produced by P. aeruginosa and several other Gram-negative and Gram-positive bacteria [10], [11], [12]. Some phenazines, especially pyocyanin, have been shown to act as toxins against bacteria, fungi, or mammals as a consequence of their redox activities [12], [13], [14], [15]. Only a subset of the previously identified C. elegans fast killing-deficient PA14 mutants were found to produce less pyocyanin than wild-type [8], suggesting that other phenazines or a different class of molecules are involved. Moreover, phenazine toxicity has not been demonstrated directly in C. elegans [8]. In order to better understand the mechanisms of P. aeruginosa PA14 toxicity, we sought to identify the toxin molecules produced by PA14 that kill C. elegans. We demonstrate that three of the phenazines produced by PA14 can rapidly kill C. elegans: phenazine-1-carboxylic acid kills C. elegans at acidic pH; pyocyanin, a product of phenazine-1-carboxylic acid, kills C. elegans at neutral or basic pH; 1-hydroxyphenazine, a second product of phenazine-1-carboxylic acid, kills C. elegans in a pH-independent manner. We also show that under the conditions of the fast killing assay phenazine-1-carboxylic acid, not pyocyanin, is the primary toxin responsible for the rapid death of C. elegans in the presence of PA14. As described above, among the previously isolated PA14 mutants that are deficient in toxin-mediated killing of C. elegans on PGS agar, those with the largest reduction in toxicity were found to produce less of the phenazine pyocyanin than wild-type PA14 [8]. Pyocyanin is one of at least four phenazines that are produced by wild-type PA14 [10], [16], [17] (Figure 1A). Phenazine-1-carboxylic acid, the precursor of all other phenazines produced by P. aeruginosa, is synthesized from chorismate by genes constituting the redundant phzA1-G1 and phzA2-G2 operons, each of which encodes a full set of functional phenazine-1-carboxylic acid biosynthetic enzymes [17]. Phenazine-1-carboxylic acid can be modified by other enzymes to make 1-hydroxyphenazine, phenazine-1-carboxamide, or pyocyanin [17]. To determine the importance of phenazines in the pathogenesis of an established C. elegans model host system, we tested the killing ability of a PA14 mutant that does not produce any phenazines (Δphz). The Δphz mutant is missing both the phzA1-G1 operon and the phzA2-G2 operon, precluding the production of phenazine-1-carboxylic acid as well as the other phenazines [16]. Lack of phenazine production by the Δphz mutant was confirmed by metabolite profiling (Table 1). Wild-type or Δphz mutant PA14 bacteria were spread on PGS agar plates and allowed to grow for 24 hours at 37°C followed by 24 hours at 23°C. L4 stage worms were then placed on the agar. Worms were scored as live or dead based on whether or not movement could be elicited by tapping their heads gently with a thin wire. We found that the Δphz mutant is severely compromised in its ability to kill C. elegans compared to wild-type PA14 (Figure 1B). Transformation of Δphz with a multicopy plasmid containing either the phzA1-G1 operon or the phzA2-G2 operon partially complemented the killing-deficient phenotype (Figure 1B). Chemical complementation of the Δphz phenotype by the addition of 100 µg/mL of synthetic phenazine-1-carboxylic acid to the agar prior to bacterial growth restored the nematode-killing phenotype (Figure 1C), suggesting that phenazine-1-carboxylic acid or another phenazine derived from phenazine-1-carboxylic acid could be a toxin involved in PA14-mediated killing of C. elegans. Alternatively, phenazine-1-carboxylic acid could function indirectly to regulate the production of a toxin that kills C. elegans. To determine if phenazines are sufficient to kill worms, we tested the killing activities of synthetic phenazines directly by adding them to PGS agar in the absence of bacteria. Under these conditions, 1-hydroxyphenazine killed worms at concentrations above 16 µg/mL with kinetics similar to a typical fast killing assay with PA14, whereas phenazine-1-carboxylic acid, pyocyanin, and phenazine-1-carboxamide did not kill worms on a relevant time scale, even at much higher concentrations (Figures 2A, S1A). To account for potential synergistic effects of phenazines with other metabolites produced by PA14, we tested the killing activities of synthetic phenazines added to PGS agar after growth of a lawn of Δphz bacteria. After growth of the lawn, the bacteria were scraped off the agar, the agar was melted in a microwave, phenazines were mixed in at several concentrations, and the agar was allowed to cool prior to introduction of the worms. We refer to the melted and cooled agar upon which the Δphz bacteria had been grown as “Δphz agar”. Similarly to the data shown in Figure 2A, when 1-hydroxyphenazine was added to Δphz agar it killed worms rapidly at concentrations above 16 µg/mL, whereas pyocyanin and phenazine-1-carboxamide killed worms poorly, even at much higher concentrations (Figures 2B, S2B). Interestingly, contrary to its activity on naive PGS agar, on Δphz agar phenazine-1-carboxylic acid also killed worms at concentrations above 16 µg/mL. These data, together with the observation that 1-hydroxyphenazine appeared to be somewhat more toxic to worms when added to Δphz agar than when added to naive PGS agar (compare Figures 2A and 2B), suggested that the Δphz strain produces a factor or factors that enhance the toxicities of 1-hydroxyphenazine and phenazine-1-carboxylic acid. The data in Figure 2B showing that both 1-hydroxyphenazine and phenazine-1-carboxylic acid kill C. elegans when added to Δphz agar indicated that either or both of these phenazines could be responsible for PA14-mediated intoxication of C. elegans in the fast killing assay. In considering the relative contributions to nematode death of phenazine-1-carboxylic acid and 1-hydroxyphenazine produced by PA14, we used metabolite profiling to determine the levels of phenazine-1-carboxylic acid and 1-hydroxyphenazine in PGS agar following growth of wild-type PA14. We found that the amount of phenazine-1-carboxylic acid (53 µg/mL) was greater than the level required for killing worms, whereas the amount of 1-hydroxyphenazine (1. 4 µg/mL) was insufficient to kill worms to a significant degree (Table 1). These observations suggested that phenazine-1-carboxylic acid is at least partially responsible for nematode killing under the conditions of our intoxication assay. To further investigate which phenazines are responsible for worm killing, we tested the killing abilities of PA14 mutants that are unable to synthesize pyocyanin, 1-hydroxyphenazine, or phenazine-1-carboxamide. A phzM mutant, which does not produce pyocyanin, killed worms more rapidly than did wild-type PA14 (Figure 2C). A phzS mutant, which does not produce pyocyanin or 1-hydroxyphenazine [17], [18], [19], did not show a significant difference in killing from wild-type. A phzH mutant, which is deficient in production of phenazine-1-carboxamide, also did not show a significant difference in killing from wild-type. The lack of production of phenazines in these mutants according to the previously described biosynthetic pathways shown in Figure 1A was confirmed by metabolite profiling (Table 1). These data, in combination with the fact that synthetic pyocyanin and phenazine-1-carboxamide do not kill worms at the concentrations they are produced under the conditions of our assay (Figure 2B, Table 1), are consistent with the conclusion that pyocyanin and phenazine-1-carboxamide do not play a significant role in fast killing. Moreover, the facts that the phzS mutant retains the ability to kill and that the level of 1-hydroxyphenazine produced by wild-type PA14 is insufficient to kill to a significant degree, are consistent with the conclusion that 1-hydroxyphenazine is either not necessary for killing or that it is one of multiple factors that cooperate to kill worms. We also observed that the phzM mutant, which kills more rapidly than wild-type PA14, produced 34% more phenazine-1-carboxylic acid than wild-type (70 µg/mL vs. 53 µg/mL; Table 1), which was the greatest amount of phenazine-1-carboxylic acid produced among the strains tested. Levels of phenazine-1-carboxylic acid were also slightly elevated with the phzH mutant (59 µg/mL). In contrast, the phzS mutant showed slightly depressed levels of phenazine-1-carboxylic acid (46 µg/mL). These data are consistent with the hypothesis that worm death requires phenazine-1-carboxylic acid. Furthermore, we observed no killing of nematodes on naive PGS agar that was supplemented with all four synthetic phenazines at concentrations comparable to those produced by wild-type PA14 (data not shown), indicating that toxicity is not induced by the combination of phenazines in the absence of other factors. These data further support the conclusion that pyocyanin and phenazine-1-carboxamide are unlikely to play major roles in worm killing under the conditions tested. The data in Figure 2 suggested that phenazine-1-carboxylic acid is most likely the primary phenazine toxin responsible for nematode killing. Because phenazine-1-carboxylic acid killed worms when mixed with Δphz agar but not when mixed with naive agar, we reasoned that at least one additional factor provided by the bacteria is necessary for the toxicity of phenazine-1-carboxylic acid. In our attempts to identify this factor, we took into consideration precedents in the literature for the pH-dependence of the activity of bacterial toxins [20], [21]. We found that the pH of PGS agar drops from approximately 6 prior to bacterial growth to between 4 and 4. 5 after growth of PA14. We tested the killing activities of phenazine-1-carboxylic acid and 1-hydroxyphenazine under different buffer conditions and found that killing by phenazine-1-carboxylic acid (at 100 µg/mL) was strongly pH dependent, with low pH supporting killing and neutral or higher pH preventing killing (Figure 3). In contrast, 1-hydroxyphenazine did not show pH-dependent toxicity at the same concentration. DMSO, the solvent for the phenazine solutions, did not kill worms under any of the buffer conditions tested, demonstrating that the worms are not dying only as a consequence of exposure to the low pH buffer. These observations explained why phenazine-1-carboxylic acid failed to kill worms when added to naive agar (pH 6), and considered together with the genetic and metabolite profiling data, suggest that phenazine-1-carboxylic acid is the primary toxin responsible for PA14-mediated killing of C. elegans under fast killing assay conditions. If phenazine-1-carboxylic acid is the primary toxic agent and if phenazine-1-carboxylic acid is only active at low pH, we reasoned that buffering the agar media to pH≥6 after growth of PA14 would block toxin-mediated killing activity of the PA14 agar. Although we observed a delay in killing when we raised the pH of the media to 7 with potassium phosphate, a substantial fraction of the worms died within seven hours (Figure 4A). When we performed the same experiment with a phzM or phzS strain, we found that killing on the relevant time scale was abrogated (Figure 4B & C). The phenotype of the phzH mutant was indistinguishable from wild-type under these conditions. Similar results were obtained when the pH was raised to 8 with Tris HCl, demonstrating that the phenomenon is not specific to potassium phosphate (Figure S2). Because both phzM and phzS are required for the synthesis of pyocyanin and phzH is not, these data suggested that pyocyanin might be toxic to worms at pH 7 or pH 8. We tested the toxicity of pyocyanin by observing worm survival on PGS agar with synthetic pyocyanin (10 µg/mL) and buffer added after growth of Δphz (Figure 4D). Addition of pyocyanin caused worm death at pH 7 and pH 8, but not at pH 6. Moreover, the kinetics of worm death at pH 7 and 8 were similar to those observed with media on which wild-type PA14 were grown and subsequently buffered at pH 7, with the toxic effects not evident until the 7-hour time point. Surprisingly, when we exposed worms to pyocyanin (10 µg/mL) at pH 8 (50 mM potassium phosphate) on PGS agar plates in the absence of bacteria, we observed no worm death within 7 hours (data not shown), suggesting that there is an unknown non-phenazine product of P. aeruginosa that sensitizes C. elegans to pyocyanin. Together, these data indicate that three of four known phenazines produced by P. aeruginosa strain PA14 are toxic to C. elegans. Toxicity of the phenazines, however, varies depending on the pH of the media as well as the presence of other factors. Pathogenesis of P. aeruginosa can occur through a variety of mechanisms including the production and excretion of toxins. In this study we identified three phenazine toxins produced by P. aeruginosa PA14 that can kill C. elegans under different environmental conditions. We showed that under the conditions of our assay phenazine-1-carboxylic acid is the predominant toxic phenazine produced by P. aeruginosa PA14 that kills C. elegans. Our work suggests that phenazine-1-carboxylic acid should be further studied as a potentially important contributor to toxin-mediated pathogenesis in other metazoan hosts besides C. elegans, including mammals. We found that the toxicity of phenazine-1-carboxylic acid to C. elegans requires an acidic environment. Acidic conditions are not uncommon, for example in wounds [22], [23], in the gut [24], [25], and intracellularly within lysosomes [23], [26] and secretory granules [27]. Phenazine-1-carboxylic acid may act as a toxin in a variety of circumstances in those locations. The pH dependence of phenazine-1-carboxylic acid toxicity may be due to the protonation state of its carboxyl group, for which the pKa is 4. 2 [28]. This hypothesis suggests that the uncharged acid species may be toxic and the negatively charged carboxylate benign, as has been observed for the antimicrobial activity of phenazine-1-carboxylic acid against Bacillus cereus and the fungus Gaeumannomyces graminis var. tritici [28]. Because the cytosol is buffered near neutral pH, we suspect that either the toxic effects of phenazine-1-carboxylic acid are occurring extracellularly or that the charge state affects the permeability of phenazine-1-carboxylic acid through the membrane. Neutral molecules typically traverse membranes more easily than charged molecules. After the uncharged phenazine-1-carboxylic acid species has diffused through the membrane, the neutral environment of the cytoplasm would result in its deprotonation. In its negatively charged carboxylate form it might be unable to diffuse out of the cell, resulting in its toxic accumulation within the cell. Phenazine-1-carboxamide, which differs from phenazine-1-carboxylic acid by only an amide group in place of the carboxyl group, is not toxic to C. elegans in our assay. The amide and the carboxyl groups should both be uncharged at acidic pH where phenazine-1-carboxylic acid is toxic and phenazine-1-carboxamide is not. Although it is possible that the chemical difference between these phenazines could affect their behavior due to other properties than their difference in pKa, the difference in toxicity of these two species suggests that charge state is not the sole determinant of phenazine toxicity. The zwitterionic species of pyocyanin is the predominant form at pH 7 and 8, and the net neutral charge may facilitate its traversal through the membrane as well [15]. The pKa of pyocyanin is 4. 9 [29], which does not explain why pyocyanin is ineffective at killing nematodes at pH 6, where the zwitterionic form should still predominate. However, as shown in Figure 4, unlike the killing activities of phenazine-1-carboxylic acid and 1-hydroxyphenazine, the killing activity of pyocyanin appears to be dependent on a non-phenazine factor produced by P. aeruginosa. It is possible that the oxidation state of pyocyanin is altered by components of P. aeruginosa exudates on PGS agar. Phenazines, including pyocyanin, exert toxic effects in a variety of mammalian tissues through the generation of reactive oxygen species [8], [12], [13], [15], [30], [31]. Oxidized and reduced phenazines also have different hydrophobicities [12], which can affect their ability to permeate membranes or remain in aqueous solution. The oxidation state of pyocyanin has been shown to change as a function of cell density in PA14 liquid culture [32], and changes in pH also affect its redox potential [33]. The difference in pyocyanin toxicity to nematodes in the presence and absence of PA14 exudates could be due to differences in the oxidation states of pyocyanin. It is also possible that the pH dependence of pyocyanin-mediated killing is due to pH-dependent activation of the accessory factor (s) and not of pyocyanin itself. Given the importance of pyocyanin as a toxin in a variety of systems, it would be valuable to identify these accessory factors and determine if they play a role in pyocyanin toxicity in other hosts. Studies have demonstrated that 5-methyl-phenazine-1-carboxylic acid (5MPCA), another phenazine produced by P. aeruginosa, is toxic to the fungus C. albicans [18], [19]. 5MPCA is an intermediate in pyocyanin synthesis that is produced by PhzM acting on phenazine-1-carboxylic acid. Given that the phzM strain is the most toxic in our assay, and that the phzS strain, which results in the accumulation of 5MPCA, is no more toxic than wild-type, we think it is unlikely that 5MPCA produced by PA14 is a toxic species to C. elegans under the conditions of our assay. CF patients are highly susceptible to lung infections by P. aeruginosa [1], [2]. CF results in abnormal hyperacidification of organelles of epithelial cells in the respiratory pathway as well as increased acidity of airway surface liquid as compared with healthy individuals [34]. Hyperacidification of the lung airway surface liquid reduces its antimicrobial effects in a porcine CF model [35] and has been speculated to have other effects on the CF lung, including tissue damage, inflammation, and thickening of the mucus [34]. Increased acidity could also lead to enhanced toxicity of phenazine-1-carboxylic acid in the P. aeruginosa infected CF lung. The concentrations of pyocyanin we determined that are active against nematodes are within the concentrations observed in human CF sputum and that have been shown to have physiological activity [36], [37], [38], [39], [40], [41]. In wild-type PA14 grown on PGS agar, we detected 2. 38 µg/mL (11. 3 µM) pyocyanin. Pyocyanin has been detected at concentrations of up to 27. 3 µg/mL (130 µM) in the sol phase of CF sputum [37]. Interestingly, a recent study showed that pyocyanin concentrations in sputum from CF patients correlated with the degree of severity of the disease, ranging from 7. 7 µM in unobstructed airways to 46. 8 µM in severely obstructed airways [36]. Phenazine-1-carboxylic acid concentrations also correlated with impairment of lung function in CF, with concentrations ranging from 5. 2 µM in unobstructed airways to 39. 9 µM in severely obstructed airways [36]. The concentrations we detected in PGS agar were considerably higher, at 52. 7 µg/mL (235 µM), but we demonstrated that phenazine-1-carboxylic acid is toxic to nematodes at concentrations as low as 16 µg/mL (71. 4 µM). Although this is still higher than concentrations observed in CF lungs, it is possible that worms are intrinsically more resistant to phenazine-1-carboxylic acid than mammalian cells. Pseudomonas infections are also common in wounds, which exhibit a variety of different pH conditions [22], [23]. During inflammation, the pH of acute wounds falls below 6, a level at which phenazine-1-carboxylic acid could exert its toxic effects. During early stages of healing, the pH increases to between 7 and 8, where phenazine-1-carboxylic acid might be ineffective as a toxin. In that pH range pyocyanin could act as a toxin. Indeed, pyocyanin has been shown to inhibit wound repair and induce cellular senescence in an in vitro model of wound healing [41]. In agreement with previous observations [8], [9], we observed that L4 worms were more susceptible to killing by phenazines than adult animals. We also noticed that older L4 worms were killed more quickly than younger L4 worms (B. Cezairliyan and R. Feinbaum, unpublished observations). We suspect that proximity to the L4 to adult molt plays a role in the susceptibility of L4 worms. Perhaps worms are most susceptible when they are first exposed to phenazines during the molt, when phenazines might be able to most easily due to the shedding of the cuticle. Earlier exposure to phenazines might allow worms time to detoxify them before reaching their most susceptible stage. It is also possible that specific gene expression patterns unrelated to toxin permeability during the L4 to adult molt are the cause of increased susceptibility. Identification of genes that reduce susceptibility to phenazines at this transition or that alter the developmental stage at which worms are susceptible to phenazines should help to elucidate the cause of the stage-dependent susceptibility of C. elegans to phenazines. An understanding of the regulation of toxin production in P. aeruginosa is critical for understanding its interactions with its hosts. Phenazine biosynthesis in P. aeruginosa is dependent on a variety of environmental factors in the media and is also largely dependent on cell density via quorum sensing signals [12], [13], [42], [43], [44], [45], [46]. In P. aeruginosa, the genes that are responsible for producing phenazine-1-carboxylic acid are present in duplicate in two differently-regulated operons in the genome. The genes that regulate production of other phenazines, however, are transcribed independently of one another and of the phzA1-G1 and phzA2-G2 operons, thus allowing for transcriptional regulation of the production of particular phenazines depending on the environmental stimuli [17], [46], [47]. Many phenazine-producing bacteria make more than one type of phenazine [11]. pH has been shown to influence the production of phenazine-1-carboxylic acid in Pseudomonas fluorescens and phenazine-1-carboxamide in Pseudomonas chlororaphis [48], [49]. Moreover, P. aeruginosa grown under different conditions can produce other low molecular weight toxins that are lethal to C. elegans, including cyanide [5], [50] and quinolone [4]. Our work suggests that one of the purposes that could be served by the diversity of phenazines is to have an armamentarium of molecules available that are active under different environmental conditions. Knowledge of the toxins that are effective in our nematode killing assay will allow for a better understanding of phenazine toxicity in C. elegans and in other hosts. Although the mutants identified in the screen for toxin-killing deficiency in PA14 were also reduced in pathogenesis in a mouse thermal injury model and an Arabidopsis leaf infiltration model [8], it is still unclear how the nematode intoxication assay serves as a proxy for the mammalian and plant models, which are based on infection and colonization by live bacteria. It will be important to determine if the same phenazine toxins that we have identified in the killing of C. elegans play a role in the murine or plant models. If the toxicity in mammals is similarly based on phenazines, C. elegans will continue to serve as a useful tool in the identification of pathogenesis mechanisms of P. aeruginosa in mammals and plants. Host pathways in C. elegans can be probed genetically to identify mechanisms by which phenazine-1-carboxylic acid acts to kill the worm and whether the three toxic phenazines exert their lethal effects in similar ways in the host. A combination of proteomics, gene expression, and mutant analysis should help to shed light on toxicity mechanisms of phenazines in the host and the ways by which their production is regulated in the pathogen. Worms were maintained on lawns of E. coli OP50 on nematode growth medium agar (NGM) plates prior to killing assays. Killing assays were performed on PGS agar (1% bacto-peptone, 1% glucose, 1% NaCl, 150 mM sorbitol, 1. 7% bacto-agar) as described [8]. 1-hydroxyphenazine was purchased from TCI America (H0289), pyocyanin from Cayman Chemical Company (10009594), phenazine-1-carboxylic acid from Apollo Scientific (OR01490), and phenazine-1-carboxamide from Princeton Biomolecular Research (PBMR030086). All phenazine stocks were dissolved in DMSO. The PA14 Δphz mutant strain lacking both the phzA1-G1 and phzA2-G2 operons was kindly provided by Lars Dietrich and Dianne Newman [16]. Other PA14 mutant strains were obtained from a non-redundant transposon insertion library [51]: phzM (mutant ID 40343), phzH (39981), phzS (44099). All transposon insertion strains were sequenced to confirm the location of the transposon. The phzM and phzS genes are on opposite sides of the phzA1-G1 operon. phzM and phzS are coregulated to some degree with phzA1-G1, but they are part of separate transcriptional units not containing other genes [17], [46]. Thus, the transposons in phzM and phzS are unlikely to exert polar effects. The phzH gene is elsewhere in the genome and is not known to be cotranscribed with other genes [17], [31]. C. elegans strain Bristol N2 [52] was used for killing assays. Killing assays were performed as previously described [8]. In order to have fourth larval stage (L4) worms for killing assays, 10 gravid worms were picked to a 60 mm plate with NGM agar and a lawn of E. coli OP50 as a food source. Gravid worms were kept on plates for 16 hours at 15°C, after which they were removed. Plates were returned to 15°C for 8 hours, after which they were transferred to 20°C. Eggs hatched and grew to L4 stage approximately 36 hours after transfer. Proper staging of L4 worms was critical to the reproducibility of the assay, as worms that were younger or older were killed less quickly than L4 stage worms. Killing agar plates were prepared by spreading 5 µL of overnight culture of PA14 in LB on a 35 mm petri plate containing 4 mL of PGS agar (1% bacto-peptone, 1% glucose, 1% NaCl, 150 mM sorbitol, 1. 7% bacto-agar). Plates were incubated for 24 hours at 37°C and then transferred to 23°C for 24 hours. L4 stage worms were put on the plates, which remained at room temperature until the completion of the assay. Worms were scored as live or dead based on movement elicited by tapping their heads gently with a thin wire. To mix the agars of plates seeded with different PA14 strains, bacteria were scraped off the surface of the agar with a cell scraper after which the agar was melted by heating in a microwave. The hot agars were mixed, repoured into plates, and allowed to cool. In experiments where phenazines or buffer were added, plate agar was melted, concentrated buffer and/or phenazine stock solution (in DMSO) was added and mixed, after which plates were repoured and allowed to cool. In order to determine absolute amounts of phenazines produced by different strains, a standard curve was constructed for each of the phenazines at concentrations of 1,4, 16,32, and 64 µg/mL. Killing agar plates seeded with Δphz were melted and doped with a range of concentrations of synthetic phenazines. Cool, dry plates were extracted with chloroform/methanol (CHCl3∶MeOH∶agar = 2∶1∶1). The organic layer was transferred to another vial and concentrated under a stream of nitrogen. Samples were dissolved in 200 µL of CHCl3 and stored at −80°C. Prior to LC-MS analysis, reconstituted samples were diluted 200 fold in CHCl3 and spiked with cytosporone B (csn-B) to the final concentration of 5 µM as a standard for normalization across samples. To obtain the most accurate concentrations of phenazines, samples from different strains were prepared in the same manner and at the same time as the standards. Three experimental replicates were used for each standard and bacterial sample. LC-MS analysis was performed using an Agilent 6410 LC-ESI-QQQ instrument in multiple reaction monitoring (MRM) mode. Samples were analyzed both in the negative and positive ion modes as previously described [53]. For the LC analysis in the negative ion mode, a Gemini (Phenomenex) C18 column (5 µm, 4. 6 mm×50 mm) was used together with a precolumn (C18,3. 5 µm, 2 mm×20 mm). Mobile phase A consisted of a 95/5 water/methanol mixture, and mobile phase B was made up of 60/35/5 isopropanol/methanol/water. Both A and B were supplemented with 0. 1% ammonium hydroxide as solvent modifiers. For the LC analysis in the positive ion mode, a Luna (Phenomenex) C5 column (5 µm, 4. 6 mm×50 mm) was used together with a precolumn (C4,3. 5 µm, 2 mm×20 mm). Mobile phases A and B, as well as the gradient, were the same as that used for the negative ion mode analysis, except that, in this case, both A and B were supplemented with 0. 1% formic acid and 5 mM ammonium formate as solvent modifiers. The gradient started at 0% B for 3 min at 0. 2 mL/min and then linearly increased to 100% B over the course of 8 min at 0. 5 mL/min, followed by an isocratic gradient of 100% B for 4 min at 0. 5 mL/min before equilibration for 5 min at 0. 5 mL/min. The total analysis time, including 3 min at 0. 2 mL/min, was 20 min. MS analysis was performed with an electrospray ionization (ESI) source. The capillary voltage was set at 4000 V. The drying gas temperature was 350°C, the drying gas flow rate was 10 L/min, and the nebulizer pressure was 45 psi. For both ionization conditions, data was collected in the profile mode. Conditions for MRM experiment were optimized using the program called Optimizer (Agilent). The resulting optimized conditions for each transition used to quantitate each phenazines are listed in Supplementary Table S1. Each run was performed using a 1 µL injection of extract. For data analysis, ions corresponding to each phenazine and csn-B were extracted from the total ion chromatograms to obtain integrated mass ion intensities (peak area; MSII) for each ion. The levels of phenazines were normalized by dividing MSII of phenazines by MSII of csn-B detected in each chromatogram to give normalized MSII (nMSII). nMSII of the standard samples was applied to construct standard curves. Finally, phenazine concentrations in the experimental samples were calculated according to the standard curves. | Title: Identification of Pseudomonas aeruginosa Phenazines that Kill Caenorhabditis elegans Summary: The bacterium Pseudomonas aeruginosa is a pathogen of a wide variety of organisms. It has been shown that P. aeruginosa factors that are critical for its toxicity to the nematode worm Caenorhabditis elegans are also important for its pathogenicity in mammals. In this report we show that phenazines, a class of small molecules produced by P. aeruginosa, act as lethal toxins against the worm. Under conditions relevant to mammalian pathogenesis, we identified one phenazine, phenazine-1-carboxylic acid, that is primarily responsible for killing worms. We found that the toxicity of this phenazine and one other phenazine, pyocyanin, are dependent on the pH of the media. We also identified a third toxic phenazine, 1-hydroxyphenazine, whose toxicity is not dependent on pH. These results show that the diversity of toxic molecules produced and released by P. aeruginosa may serve the bacterium to facilitate pathogenicity in a variety of environments. | 9,098 | 257 | lay_plos | en |
Summarize: SALVADOR, Brazil – Two sensibilities permeate the psyche of teams in the knockout stage of the World Cup. The first begs caution, conservatively defending for 90 minutes and judiciously going forward on attack – more out of necessity than a desire to score. The second is a pervasive fearlessness, which is found within the teams who have reached this point in the tournament countless times before. Those teams don’t worry about losing, because those teams don’t lose. What kind of team does the U.S. want to be? The United States has a decision to make in the 24 hours before it kicks off against Belgium. Is this a conservative, defensive U.S. team or one willing to play with the carefree swagger of a perennial favorite? Common sense would preach defense. Don’t concede and lean on the stray set piece opportunity to create scoring chances. On paper, the U.S. doesn’t have the talent to match its Belgium opponent. The numbers say bunker down. That mentality runs counter to the construct of the U.S. roster. Coach Jurgen Klinsmann has picked a team filled with players who are at their best when attacking. Klinsmann has said as much in the days leading up to this game. Yet, this team hasn’t been faithful to its identity for much of the tournament. It defended for the duration against Ghana and Germany, and for the final 20 minutes against Portugal. Perhaps it was a conscious decision to play in that conservative shell. Perhaps it was prudent in a scenario which rewarded 2-2 draws and 1-0 losses. The U.S. is going to have to throw a punch or two. There won’t be a draw in the knockout rounds. The U.S. won’t reach the quarterfinals with a 1-0 loss and a little help from Cristiano Ronaldo. Why cater to that mentality, then? Why not attack? If the U.S. is going to beat Belgium, it’s going to have to throw a punch or two. Belgium has played 270 minutes in this World Cup. It has led its opponents for 28 of those 270 minutes. Belgium is a team content with allowing its opponent to do essentially whatever they want – except score. The Belgians have managed their opponents flawlessly in the World Cup, but they have never dominated any of them. Cast in that light, the challenge for the U.S. is clear. Disrupt Belgium. Playing an attack-minded style isn’t just more entertaining; it makes tactical sense for the U.S. Belgium has proven over the first three games of the tournament that it will find a way to score at some point, and when that goal does come, it’s often too late to do anything about it. Belgium dares its opponents to beat them in the first 70 minutes of the match. Klinsmann must trust that 70 minutes of his team on offense will prevail. That means allowing Clint Dempsey and Michael Bradley to push higher up the field, sending Fabian Johnson forward from right back, and trusting Kyle Beckerman and Jermaine Jones to cover those three when possession gets lost. It’s not dissimilar from the way the U.S. match against Portugal played out. The U.S. overwhelmed an opponent who was content to absorb pressure in that situation as well. The difference between Portugal and Belgium is that Belgium doesn’t have the best player in the world on its team. In knockout stages past, the U.S. didn’t have the quality in its lineup to do anything but defend for 90 minutes. Even when the Americans advanced to the quarterfinals with a win over Mexico in 2002, that team was in a defensive shell for the duration. That’s the greatest difference between 2014’s team and every American World Cup team that came before. This team has the ability to make a semifinal with the right mentality. Klinsmann must trust his team’s ability to play at this level, and the team must have the confidence to know it can. This video will prepare you for the U.S. national team's next World Cup game, which is against Belgium. You'll sound like an expert to your friends and co-workers. (Tom LeGro/The Washington Post) This video will prepare you for the U.S. national team's next World Cup game, which is against Belgium. You'll sound like an expert to your friends and co-workers. (Tom LeGro/The Washington Post) DaMarcus Beasley speaks from experience, of which he has plenty. So no one has to explain to him what’s on the line when the U.S. national team steps on the field Tuesday. “This is what players live for,” Beasley said. “You live to play these types of games.” By now, Beasley has played many of them, and he knows if the United States can’t get past Belgium in the World Cup’s knockout round, fans could be watching the curtain close on one of the most decorated international careers any U.S. player has assembled. This is the fourth World Cup in which Beasley has appeared in a game. Fewer than 30 players — and no other Americans — have played in that many, a list that includes legends Pele and Diego Maradona. “Without a doubt,” midfielder Michael Bradley said, Beasley is “one of the best players in U.S. soccer history.” U.S. and Belgian World Cup comparison “What he has done for club teams he has been on, what he has done for the national team, the consistency, the longevity,” Bradley said, “and he continues to show how important he is for us.” Beasley’s most impressive accomplishment isn’t that he has survived this long; rather, it’s how he’s done it. He’s not the same player who slipped on a U.S. jersey 13 years ago. That guy flew up and down the side of the pitch, his legs moving faster than a hummingbird’s wings. “When he was younger, he was probably loud and brash, but he’s calmed down,” U.S. goalkeeper Tim Howard said of Beasley. “He’s quiet, he’s a dad — just all the things you’d hope from someone as they mature.” The energy is still there, enough to allow him to shuttle back and forth between the team’s veterans and its young players. Tuesday will mark his 11th appearance in a World Cup game, one behind Landon Donovan for most among Americans, and his 120th with the U.S. national team, fifth all-time. But unlike that Beasley who was fluttering his way through the 2002 World Cup, this 32-year-old version is able to appreciate the moment. He knows it could be his last chance to help his team return to the tournament’s quarterfinal round. “It seems like he gets younger and younger. He never gets tired,” U.S. Coach Jurgen Klinsmann said. “You want to go home at 3 o’clock at night. ‘Say, DaMarcus, do you want to kick the ball around?’ He’s right there.” The biggest transformation has taken place on the field. A 20-year-old Beasley was offense-minded and goal-oriented. But the Beasley in this tournament, while still a capable attacker, is a fullback tasked with significant defensive responsibilities. “Back in those days I always wanted to score goals and make the last assist or something like that. But now it’s a different mind-set and I’ve taken on a new position, a new position I like,” said Beasley, who has played for Mexico’s Puebla since 2011 after several seasons bouncing among European teams. 1 of 32 Full Screen Autoplay Close June 30, 2014 Germany vs. Algeria Skip Ad Caption France defeats Nigenia 2-0 and Germany defeats Algeria 2-1. A supporter of France waves the national flag before the round of 16 match between France and Nigeria at the Estadio Nacional in Brasilia. Robert Ghement/European Press Photo Agency Buy Photo He still gets up and down the field and seems always to be around the ball, but Beasley hasn’t scored a goal in international competition since 2008. Still, he’s as important as anyone on the field, especially with a back four that has faced plenty of scrutiny heading into the tournament. “I think that’s the thing with Beas that always excited me,” Howard said, “once he accepted playing fullback. I thought he’d be brilliant. I’m a big fan of wingers becoming fullbacks. I just think it’s a natural progression.” At times, Beasley can be a sage in cleats, a veteran who’s helping just as much off the field. The U.S. defense still feels like a work in progress, as Klinsmann tweaked the starting lineup before the Germany game, replacing veteran Geoff Cameron with 25-year-old Omar Gonzalez. The younger players say Beasley has been generous with advice but also quick to give encouragement or congratulations. Other times he sets an example without ever opening his mouth. “Nothing really freaks him out,” said Gonzalez. “He is always calm, cool, collected and it is great having a guy who’s that secure and knows what is going on in the back line.” The U.S. team’s fate could hinge on it. Belgium won its three group stage games by one goal apiece and with the U.S. offense struggling to find the net, Tuesday’s match could be a low-scoring affair. As it has for the past dozen-plus years, the American squad will look again to Beasley, who has changed himself and his game to evolve into the player his team needs most. “I still play the same,” Beasley said. “I still play with the same heart and desire to win every game, to win every tackle, to maybe score a goal — doesn’t matter. “I like big games. I like being under the lights. I like playing in front of 80,000 people.” SALVADOR, Brazil -- It seems like yesterday rather than four years ago and an ocean away. On that night in South Africa, the U.S. needed a goal to stay alive -- needed a goal just to advance out of the group stage of the 2010 World Cup -- and there was so little time left. Algeria pressed forward, pushing as many as seven men up, and the U.S. bent back, looking for any chance to counter. It's hard to describe what the stadium felt like in those moments. The Americans in the crowd had gone to an unusual place for Americans, past the far edge of their usual spectrum of expectation. At one end there's certainty, the confidence of knowing. Closer to the middle there's belief and then faith. Then comes optimism, and then comes hope. And then comes how it felt in the dying seconds of that unforgettable night. That's where the Americans were back then. Heading into Tuesday's knockout match against Belgium, the U.S. and its supporters are, for the moment, at a far different station. Before the thinning armies of American fans had even dragged themselves into sunny Salvador, many were already making plans for their next stop: Brasilia, where, depending on your own position on that spectrum, the U.S. either will play its quarterfinal, or it would play its quarterfinal, if only. On the face of things, buying plane tickets seems a little brave. The Americans did well to survive the Group of Death, but they advanced on a loss, and their best game had seen them earn only a tie. The Belgians won all three of their group-stage matches -- yes, against a far weaker draw -- allowing only a single goal. And yet it's the Belgians who now seem to be trading places with the Americans, edging toward doubt's end. That's partly because Jozy Altidore might play, and Vincent Kompany might not. It's more because in the long, slow build to this tournament, the poor Belgians were burdened with the worst mantle in sports: dark horse. The problem with dark horses is that they are saddled with all of destiny's pressure with half of the justification. They are not favorites, because they are not the best and never have been. In fact, there are several better bets. Dark horses are just good enough to make someone, somewhere, wonder. And then that wondering gets whipped by some mysterious consensus into something far closer to certainty than it has any right to be. For dark horses, that's the hole in the track. Even mighty Brazil seems doomed by the load that they're carrying. Dark horses have even harsher luck. They are forced to bear the same weight but on weaker legs. The gift and curse of games is that none of us can truly know how they will finish. This World Cup has proved once again that certainty is only an illusion. Which means that sometimes the longest shots come through, like on that magical night in South Africa. At some desperate point, past which there no longer seems even a slim chance for success, what you feel is a collective letting go. That's the release that allows miracles to happen. The Americans might soon find themselves returning to that distant edge. They might need Tim Howard to make another save and another long throw to this year's version of Landon Donovan. They more than likely will, if history and geography and their lessons hold. How much has really changed in four years? What difference is an ocean? But right now in Salvador, it doesn't feel like anyone's letting go just yet. Right now, it feels better than hope, and it feels better than optimism, and it feels better than faith. It feels like belief, and for a team that shouldn't win, there's no better feeling in the world. SALVADOR, Brazil -- Jurgen Klinsmann is never shy about offering his opinion. Say whatever you want about the U.S. coach, but the one thing you can't deny is that he usually speaks his mind. That was the case again Monday, at the end of what had been to that point a sleep-inducing prematch news conference, when Klinsmann was asked if perhaps the appointment of Algerian referee Djamel Haimoudi for Tuesday's round-of-16 match against Belgium (4 p.m. ET, ESPN/WatchESPN) was any cause for concern. Klinsmann didn't hesitate. "Well, we hope it's not a concern," Klinsmann said. "Is it a good feeling? No." Klinsmann then went on to list the reasons why having Haimoudi manning the middle might put the Americans at a disadvantage against one of Europe's most dangerous teams. Haimoudi's mother tongue is French, meaning many of the Belgian players would be able to communicate with him on the field without American players knowing what was being said. His home country faced the Belgians in the first round. Finally, he mentioned that Algeria was seconds away from advancing to the knockout round for the first time in their history (a feat they accomplished here in Brazil) at South Africa 2010 before Landon Donovan's iconic stoppage-time goal sent them home instead. Was Klinsmann implying that some measure of payback, subconscious or not, could come into play? Or maybe was it just Klinsmann being Klinsmann? Or perhaps it was just another touch of gamesmanship from a coach who has already taken every opportunity to stack the deck in his team's favor over the past few weeks. Jurgen Klinsmann could be playing mind games by implying Algerian referee Djamel Haimoudi might have a bias against the U.S. There was the time the coach trotted out backup strikers Aron Johannsson and Chris Wondolowski to speak to the media, then proceeded to start neither in place of the injured Jozy Altidore against Portugal. And on Monday, just before Klinsmann took the stage at Arena Fonte Nova, U.S. Soccer announced that Altidore suddenly was fully recovered from his hamstring injury and "ready and available" to play, which anyone who has been paying attention understands may or may not be true. That's not to say Klinsmann's mind games are a bad thing. Like U.S. players, national team coaches have long been almost naively honest in their approach. Veteran goalkeeper Tim Howard has spoken about this admirable but ultimately self-defeating trait before and even during the tournament, saying that this time around the Americans have to be smarter about taking whatever advantage is available to them. At the World Cup, the difference between winning and losing is negligible. Games are decided by the slimmest of margins. Klinsmann, a former world champion, knows this as well as anyone. As a player, he was known as a diver. But instead of denying it, he embraced the role, famously launching himself to the turf after scoring his first Premier League goal with Tottenham Hotspur in 1994 and instantly endearing himself to English fans and media in the process. So really, it was no surprise that Klinsmann jumped at the chance to put Haimoudi in the spotlight on Monday, to guarantee that the official's performance will now be scrutinized more closely than it would have been otherwise. It's a tactic iconic managers Sir Alex Ferguson and Jose Mourinho use as a matter of course, and it's not even limited to soccer. Some of the most successful American coaches, guys such as Phil Jackson and Bill Belichick, do the very same thing. Like Klinsmann, they know sports at the highest level have nothing to do with honor. What matters -- the only thing -- is giving your team every possible chance to win. Shep Messing, who was a teammate of Pele on the New York Cosmos and goalkeeper on the 1972 Olympic squad, will give Post readers his insights and opinions periodically during the World Cup. Messing also serves as MSG Network’s New York Red Bulls analyst and is calling World Cup games for ESPN Radio. As told to Brian Lewis. The great thing about this US-Belgium game is there are so many different approaches to look at it from, so many story lines. Take their roster, the dollar value, the talent, the clubs they play for: Go man-for-man, we don’t stack up. Everybody’s saying Belgium is the next great team, the dark horse to win the World Cup. I’ll take a different picture. Belgium has looked poor. I don’t take away the fact that man-for-man, we don’t match with them. But no European team has ever won a World Cup in North America or South America. England is at home. Italy is at home. Spain is at home. Holland should be home. France struggled against Nigeria. My overview is that the European teams in this World Cup are a bit arrogant, and they have an air of superiority. That’s how they act, that’s how they feel, that’s how they behave. The South and Central American teams are loving it. And here comes Belgium against the U.S., and all the pressure is on Belgium to perform. This US team, its mentality is growing. We’re not going to be beaten in terms of fight and effort over 90 minutes. My hope is we can sneak a goal. There’s a skill battle going on; just line up Belgium and they beat us. But there’s also a mental component to the game; my vote goes to the US. We’re not going to quit on the game. We’re going to fight to the end. I don’t know if Belgium doesn’t start feeling the nerves, feeling the pressure. There are a lot of expectations put on them in this tournament, and they haven’t shined in group play. The other part is tactical. The US has a really huge tactical problem — or opportunity. Jozy Altidore may be my favorite player on the team, and his importance is irreplaceable given the roster. Jurgen Klinsmann has said he is fit to go after straining his hamstring in the US opener, but I’d find it a medical miracle if he played. It’s a pure psychological play for Klinsmann, which he’s terrific at. Now he’s got Belgium preparing for what if Altidore is in the lineup, and what if he’s out? But I can’t see any way in the world that he could play. I’d find it beyond remarkable if he plays any role in the game, except psychology. The Belgians’ strength is attacking in wide positions, but I don’t think they really have an identity yet. They’re dynamite attacking from wide, but sometimes they like to feed the beast in the middle. Their attacking from wide will pin Fabian Johnson back, unless he can get forward and make them defend. Without Altidore, we’re limited offensively, so how do we tactically approach this game? I don’t know, because of Klinsmann. And I’m not saying that negatively. He could surprise everybody. Does he go with Geoff Cameron or Omar Gonzalez next to Matt Besler in the back? Does he leave the team the same or change it? The higher on the field Michael Bradley plays, the worse it is. My wish is put Kyle Beckerman on the bench, Bradley back where he’s comfortable, Clint Dempsey wide left and Graham Zusi wide right. Let’s see what midfielder Mix Diskerud can do. Do you throw in Aron Johannsson or Chris Wondolowski? I don’t think so, because Klinsmann hasn’t up until now. But I don’t think the US can afford to play against Belgium the way it did against Germany. If we’re on our heels defending the whole game, we’ll lose. If there’s a weakness, it’s that Belgium’s two centerbacks are slow, and their holding midfielders are really attacking players. The gut of Belgium’s defense is vulnerable to speed. This is a 50-50 game. What I hope for is a tight, close battle and I hope for penalty kicks, because we’re the winner with penalty kicks. I think we’re the winner with Tim Howard. We’re the winner with our mentality if it gets to that point. The longer the game goes without a goal, the better for us. I haven’t seen this young Belgium team do it yet, this expensive dark horse everybody’s talking about. All the pressure is on them, not the US. I’m so excited for today. I can’t wait. (Getty Images) SALVADOR, Brazil — Injured striker Jozy Altidore will be available for the United States’ match Tuesday against Belgium, but the team’s coach, Jurgen Klinsmann, won’t say how many minutes — if any at all — Altidore will actually play. “Just having him with us is good. How many minutes, we’ll see that in the game,” Klinsmann said at a press conference Monday. Altidore injured his hamstring in the United States’ opening World Cup match against Ghana. He missed the team’s next two matches, against Portugal and against Germany, but recently started jogging and testing the hamstring. Klinsmann was asked how much Altidore has been able to practice in the days leading up to Tuesday’s match, but he did not offer a clear answer. “This is what we want. This is what we hope for. This is what the medical staff was working (on) since the injury on him. They’ve done a tremendous job,” Klinsmann said. “It’s day and night for Jozy.” While Altidore will be in uniform against Belgium, it’s very possible he’ll be relegated to a cheerleading role. But by declaring him available, Klinsmann has forced Belgium to prepare for anything. With Altidore in the lineup, the United States used four defenders, four midfielders and two strikers against Ghana. Without Altidore, though, Klinsmann went with an extra midfielder and just one striker against Portugal and Germany, forgoing some offensive punch up-front. More on the World Cup: No Dupont watch party for U.S. match Tuesday Uruguay president calls FIFA’s ban of LuiS Suarez ‘facist’ Dark horse Belgium has the talent but is relatively untested Brazil survives massive upset, edging Chile on penalty kicks Klinsmann says U.S. has ‘absolutely no fear’ entering knockout round Video: Getting ready Belgium, the U.S.’s next opponent Suspended Suarez told FIFA that bite was unintentional Oops: Syndicated kids’ newspaper feature names Suarez a ‘Supersport’ Chiellini says suspension of Suarez was ‘excessive’ Round-of-16 bracket Scores and schedule | Summary: The US may have backed into the Round of 16, but confidence among the star-spangled faithful sure seems to be running high, observes Chris Jones at ESPN. Fans believe in this team right now. Is that belief warranted? Here's what you need to know before today's game, which kicks off at 4pm Eastern. Jozy returns? Jozy Altidore, who injured his hamstring against Ghana, is available, coach Jurgen Klinsmann said, though he wouldn't say how much the star striker would play. "Just having him with us is good," Klinsmann said, as per the Washington Post. But Shep Messing at the New York Post thinks Klinsmann is just playing mind games with Belgium. "I can't see any way in the world that he could play." Working the ref. Klinsmann also used yesterday's media appearance to sound off on referee Djamel Haimoudi, in what Doug McIntyre at ESPN sees as yet more gamesmanship. Asked about Haimoudi, Klinsmann said, "We hope it's not a concern," then listed the reasons it might be: Haimoudi speaks French, meaning the Belgian players will be better able to speak with him, and he's Algerian, meaning he might want payback for the way the US knocked Algeria out of the Cup in 2010. Belgium is good. The US might need all the help it can get. "Go man-for-man, we don't stack up. Everybody's saying Belgium is the next great team, the dark horse to win the World Cup," Messing writes. Mike Foss at USA Today agrees that "on paper, the US doesn't have the talent to match its Belgium opponent."...But the pressure is on. Messing thinks the US actually has a chance. "Belgium has looked poor," he writes, and like most European teams, arrogant. Jones adds that being the dark horse is rough; "They are saddled with all of destiny's pressure with half of the justification." Attack, attack, attack. Foss thinks the US' best chance will be to play an offense-first style, because "Belgium dares its opponents to beat them in the first 70 minutes." The US is allegedly an attack-minded club, but didn't look like one in the group stages. Beasley's last hurrah. If the US does fall, it will be the last game for one of its greats, the Washington Post points out; DaMarcus Beasley has now played in four World Cups, a feat less than 30 players in history-none of them American-have managed. "This is what players live for," Beasley tells the paper. "You live to play these types of games." For some tips on how to talk to Americans who aren't into soccer, click here. | 5,549 | 636 | multi_news | en |
Summarize: By. Richard Arrowsmith for MailOnline. CLICK HERE to follow Sportsmail's LIVE coverage of the transfer deadline day. Football legend Diego Maradona presented Pope Francis with an Argentina shirt ahead of the Interreligous Match for Peace exhibition game. The former World Cup winner, who famously credited the 'Hand of God' after scoring a goal with his fist against England at the 1986 World Cup, was among several players to meet the pontiff at the Vatican. The leader of the Roman Catholic Church, himself a dedicated fan of of Argentine side San Lorenzo, has helped arrange the celebrity exhibition match to promote a diplomatic solution to the ongoing crisis in Gaza. VIDEO Scroll down to watch Hand of God Maradona meets God's right hand man. Hand of God: Diego Maradona meets with Pope Francis at the Vatican. I come bearing gifts: The Argentina legend presents the pontiff with one of his national team shirts. Peace: Maradona is in Rome to take part in the Interreligous Match for Peace to promote a diplomatic solution to the crisis in Gaza. VIDEO Hand of God Maradona meets God's right hand man. The game will be played on Monday at the Olympic Stadium in Rome and is also expected to feature the world's current greatest player Lionel Messi alongside several 'all-star players'. Argentina manager Gerardo Tata Martino and Arsenal's Arsene Wenger will select teams from a squad of 50 players from different religous backgrounds, including Roberto Baggio, a renowned Budhist, and Zinedine Zidane who professes Islam. The two teams, named PUPI and SCHOLAS will be captained by Zanetti and Italy goalkeeper Gianluigi Buffon. All-stars: Legends past and present enjoyed an audience with football fan Pope Francis. All smiles: Maradona is embraced by Andriy Shevchenko after handing the Pope a shirt with the name 'Francisco' on the back. I'm here! Diego Maradona arrives at the Vatican after claiming it was an honour to be invited for the game. The match will be co-hosted by the Pupi Foundation, a Buenos Aires-based charity founded by the former Inter Milan midfielder Javier Zanetti, alongside the Pontifical Academy for Social Science. After agreeing to take part, Maradona posted on his Facebook page that he was excited and honored to be invited by the Pontiff to play for the cause in Rome. 'It is an honor to be invited by Pope #Francis, to participate in the Interreligious Match for Peace, on September 1st, at the Olympic Stadium of Rome,' he said. Bad boy: Diego Maradona is renowned for scoring 'Hand of God' goal against England at 1986 World Cup. It’s not too late to play MailOnline Fantasy Football… There’s £1,000 to be won EVERY WEEK by the highest scoring manager. CLICK HERE to start picking your Fantasy Football team NOW! There’s £60,000 in prizes including £1,000 up for grabs EVERY WEEK…. Andrea Ranocchia, Andrea Pirlo, Colombian midfielder Fredy Guarìn, Spaniards Mikel Arteta and Andrés Palop Cervera, Andrey Shevchenko, Argentine ex-player and current coach Antonio Mohamed, Brazilian Marcos Antonio Senna Da Silva, Chilean Arturo Vidal, Carlos Valderrama, Israelis Yossi Benayoun, Dudu Aouate and Tomer Hemed, Damiano Tommasi, David Trezeguet, Ethiopia captain Degu Debebe Gebreyes, Diego Lugano, Atletico Madrid coach Diego Simeone, Icelandic Emil Hallfreosson, Argentines Mauro Icardi, Ricky Alvarez, goalkeeper Juan Pablo Carrizo, Esteban Cambiasso, Fernando Tissone, Ezequiel Schelotto, Ezequiel Lavezzi, Juan Iturbe, Lionel Messi, Javier Mascherano, Maxi Rodríguez, Cristian Ledesma. Also featuring will be: Yuto Nagatomo, Ivan Zamorano, Ivan Cordoba, Roberto Baggio, Samuel Eto’o, Fernando Muslera, Filippo Inzaghi, Gabriel Heinze, Jose Chamot, Luca Toni, Lucas Podolski, Mesust Ozil, Nicola Legrottaglie, Radja Nainggolan, Ronaldinho, Stefano Mauri, Sulley Muntari and Belozoglu Emre | Summary: Interreligous Match of Peace organised to promote diplomatic solution to ongoing crisis in Gaza. Argentina legend Diego Maradona met with Pope Francis at the Vatican. Maradona scored 'Hand of God' goal against England at 1986 World Cup. Arsenal boss Arsene Wenger and Gerardo Martino will manage two teams. Match will played at the Olympic Stadium in Rome on Monday September 1. Teams will be captained by Javier Zanetti and Italy keeper Gianluigi Buffon. 50 player squad features players from different religous beliefs including Zinedine Zidane (Islam) and Roberto Baggio (Buddhist) Among other players taking part are Lionel Messi, Mesut Ozil and Ronaldinho. | 941 | 150 | cnn_dailymail | en |
Summarize: By. Leon Watson. Bosnia said today that more than a quarter of its four million people had been affected by the worst floods to hit the Balkans in living memory. Comparing the 'terrifying' destruction to that of the country's 1992-95 war, authorities said the extent of the devastation became apparent in neighbouring Serbia too. As waters receded in some of the worst-hit areas it was revealed that homes had been toppled or submerged in mud, trees felled and villages strewn with the rotting corpses of livestock. Scroll down for videos. A man reacts near a house tilted by floods in the village of Krupanj, west from Belgrade. Houses and agricultural lands are affected negatively due to the heavy rainfall in Obrenovac, Serbia. At least 35 people drowned after heavy rainfall since Thursday triggered floods in Bosnia-Herzegovina and Serbia, officials said. The regional death toll reached at least 39, after the heaviest rainfall since records began 120 years ago caused rivers to burst their banks and triggered hundreds of landslides. 'The consequences... are terrifying,' Bosnian Foreign Minister Zlatko Lagumdzija told a news conference. 'The physical destruction is not less than the destruction caused by the war.' Lagumdzija said more than 100,000 houses and other buildings in Bosnia were no longer fit to use and that over a million people had been cut off from clean water supplies. 'During the war, many people lost everything,' he said. 'Today, again they have nothing.' His remarks threw into sharp relief the extent of the challenge now facing the cash-strapped governments of both Bosnia and Serbia. Serbian Prime Minister Aleksandar Vucic said the cost in Serbia would run to hundreds of millions of euros, and that the death toll would certainly rise. Macedonian Red Cross workers and volunteers unload a truck containing humanitarian aid in food, hygienic products and clothing. Macedonia sends aid to flood-hit Bosnia and Herzegovina as the countries struggle to cope in the aftermath of floods which have left at least 35 people dead. Tens of thousands have been forced to evacuate their homes across the Balkan Peninsula in Sarajevo, Bosnia and Herzegovina. Even as the crisis eased in some areas, a new flood wave from the swollen River Sava threatened others, notably Serbia's largest power plant, the Nikola Tesla complex, 30 km (18 miles) southwest of the capital Belgrade. In Bosnia, one official said as many as 500,000 people had been evacuated or left their homes, the kind of human displacement not seen since more than a million were driven out by ethnic cleansing in the Bosnian war two decades ago. At least 25,000 people have been evacuated in Serbia, but many more are believed to have left of their own accord. People are transported from their homes with the help of Bosnian soldiers after flooding in the town of Bosanski Samac. A state of emergency has been declared in Bosnia and Herzegovina due to severe floods caused by rain falling for several days. Macedonia sends a special team for aid to flood-hit Bosnia and Herzegovina as the countries struggle to cope in the aftermath of floods. Many houses and agricultural lands are affected negatively due to the heavy rainfall in Obrenovac, Serbia. 'We have some indications that a half a million Bosnians have either been evacuated or have left their homes because of flooding or landslides,' said Fahrudin Solak, the acting head of the civil defence service in Bosnia's autonomous Federation. Serbian tennis star Novak Djokovic said the floods were of an 'epic, Biblical' scale. 'It has not been remembered in the last 150 years that we have had such a natural disaster,' he told Sky News. 'Our. country depends on agriculture. There are many people who lost all. their natural goods, an everything that was the mainstay of their. income.' Hundreds of volunteers in the Serbian capital filled sandbags and stacked them along the banks of the Sava. Police issued an appeal for more bags. Macedonian Red Cross workers collect boxes with food products before sending a humanitarian aid contingent to the flooded regions in Serbia and Bosnia and Herzegovina. Macedonian Red Cross workers pack bottled water at a Red Cross warehouse in Skopje, Macedonia. Serbia's Novak Djokovic celebrates at the end of his final match against Spain's Rafael Nadal, at the Italian Open. He later dedicated his win to the victims of the floods. Soldiers and energy workers toiled through the night to build barriers of sandbags to keep the water back from the Nikola Tesla energy complex and from the coal-fired Kostolac power plant, east of Belgrade. Djina Trisovic, a union spokeswoman at Serbia's EPS power utility, said some workers at the Nikola Tesla plant had worked three days with barely a break because relief teams could not reach the plant. 'The plant should be safe now,' she told Reuters. 'We've done all we could. Now it's in the hands of God.' The plant provides roughly half of Serbia's electricity. Parts of it had already been shut down as a precaution, but it would have to be powered down completely if the waters breached the defences. Flooding had already caused considerable damage, estimated by the government at over 100 million euros ($140 million), to the Kolubara coal mine that supplies the plant. Authorities in Bosnia issued a fresh warning about the danger of landmines left over from the war and now dislodged by the flooding. In the north Bosnian region of Maglaj, barely a single house was left untouched by the waters, which receded to leave a tide of mud and debris. In the village of Donja Polja, where Muslim Bosniaks returned in 1995 to homes burned or shelled during the war, Hatidza Muhic swept the mud from the hallway of her house. Dark lines on the walls indicated the water had reached some three metres high. 'I thought the war was as bad as it can get, but it can get worse,' Muhic said. 'I just pray to God that we can save our minds, because first we were hit by the war, and now this.' | Summary: A state of emergency has been declared in Bosnia and Herzegovina. Authorities compared destruction to that wreaked by 1992-95 war. Homes have been toppled or submerged in mud and trees felled. Villages were left strewn with the rotting corpses of livestock. The regional death toll has now reached at least 39. Novak Djokovic said the floods were of an 'epic, Biblical' scale. | 1,437 | 100 | cnn_dailymail | en |
Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Home School Non-Discrimination Act of 2005''. SEC. 2. FINDINGS. Congress finds as follows: (1) The right of parents to direct the education of their children is an established principle and precedent under the United States Constitution. (2) Congress, the President, and the Supreme Court, in exercising their legislative, executive, and judicial functions, respectively, have repeatedly affirmed the rights of parents. (3) Education by parents at home has proven to be an effective means for young people to achieve success on standardized tests and to learn valuable socialization skills. (4) Young people who have been educated at home are proving themselves to be competent citizens in postsecondary education and the workplace. (5) The rise of private home education has contributed positively to the education of young people in the United States. (6) Several laws, written before and during the rise of private home education, are in need of clarification as to their treatment of students who are privately educated at home pursuant to State law. (7) The United States Constitution does not allow Federal control of homeschooling. SEC. 3. SENSE OF CONGRESS. It is the sense of Congress that-- (1) private home education, pursuant to State law, is a positive contribution to the United States; and (2) parents who choose this alternative education should be encouraged within the framework provided by the United States Constitution. SEC. 4. CLARIFICATION OF PROVISIONS ON INSTITUTIONAL AND STUDENT ELIGIBILITY UNDER THE HIGHER EDUCATION ACT OF 1965. (a) Clarification of Institutional Eligibility.--Section 101(a)(1) of the Higher Education Act of 1965 (20 U.S.C. 1001(a)(1)) is amended by inserting ``meeting the requirements of section 484(d)(3) or'' after ``only persons''. (b) Clarification of Student Eligibility.--Section 484(d) of the Higher Education Act of 1965 (20 U.S.C. 1091(d)) is amended by striking the heading and inserting ``Satisfaction of Secondary Education Standards''. SEC. 5. CLARIFICATION OF ABSENCE OF CONSENT FOR INITIAL EVALUATION UNDER THE INDIVIDUALS WITH DISABILITIES EDUCATION ACT. Section 614(a)(1)(D)(ii)(I) of the Individuals with Disabilities Education Act (20 U.S.C. 1414(a)(1)(D)(ii)(I)) is amended to read as follows: ``(I) For initial evaluation.--A local educational agency may pursue the initial evaluation of a child by utilizing the procedures described in section 615, except to the extent inconsistent with State law relating to parental consent for an initial evaluation under clause (i)(I), only if the child is enrolled in public school or is seeking to be enrolled in public school.''. SEC. 6. CLARIFICATION OF THE COVERDELL EDUCATION SAVINGS ACCOUNT AS TO ITS APPLICABILITY FOR EXPENSES ASSOCIATED WITH STUDENTS PRIVATELY EDUCATED AT HOME UNDER STATE LAW. (a) In General.--Paragraph (4) of section 530(b) of the Internal Revenue Code of 1986 (relating to qualified elementary and secondary education expenses) is amended by adding at the end the following new subparagraph: ``(C) Special rule for home schools.--For purposes of clauses (i) and (iii) of subparagraph (A), the terms `public, private, or religious school' and `school' shall include any home school which provides elementary or secondary education if such school is treated as a home school or private school under State law.''. (b) Effective Date.--The amendment made by subsection (a) shall apply to taxable years beginning after the date of the enactment of this Act. SEC. 7. CLARIFICATION OF SECTION 444 OF THE GENERAL EDUCATION PROVISIONS ACT AS TO PUBLICLY HELD RECORDS OF STUDENTS PRIVATELY EDUCATED AT HOME UNDER STATE LAW. Section 444 of the General Education Provisions Act (20 U.S.C. 1232g; also referred to as the Family Educational Rights and Privacy Act of 1974) is amended-- (1) in subsection (a)(5), by adding at the end the following: ``(C) For students in non-public education (including any student educated at home or in a private school in accordance with State law), directory information may not be released without the written consent of the parents of such student.''; (2) in subsection (a)(6), by striking ``, but does not include a person who has not been in attendance at such agency or institution.'' and inserting ``, including any non-public school student (including any student educated at home or in a private school as provided under State law). This paragraph shall not be construed as requiring an educational agency or institution to maintain education records or personally identifiable information for any non-public school student.''; and (3) in subsection (b)(1), by striking subparagraph (F) and inserting the following: ``(F) organizations conducting studies for, or on behalf of, educational agencies or institutions for the purpose of developing, validating, or administering predictive tests, administering student aid programs, and improving instruction, if-- ``(i) such studies are conducted in such a manner as will not permit the personal identification of students and their parents by persons other than representatives of such organizations and such information will be destroyed when no longer needed for the purpose for which it is conducted; and ``(ii) for students in non-public education, education records or personally identifiable information may not be released without the written consent of the parents of such student.''. SEC. 8. CLARIFICATION OF ELIGIBILITY FOR STUDENTS PRIVATELY EDUCATED AT HOME UNDER STATE LAW FOR THE ROBERT C. BYRD HONORS SCHOLARSHIP PROGRAM. Section 419F(a) of the Higher Education Act of 1965 (20 U.S.C. 1070d-36(a)) is amended by inserting ``(or a home school, whether treated as a home school or a private school under State law)'' after ``public or private secondary school''. SEC. 9. CLARIFICATION OF THE FAIR LABOR STANDARDS ACT AS APPLIED TO STUDENTS PRIVATELY EDUCATED AT HOME UNDER STATE LAW. Section 3(l) of the Fair Labor Standards Act of 1938 (29 U.S.C. 203(l)) is amended by adding at the end the following: ``The Secretary shall extend the hours and periods of permissible employment applicable to employees between the ages of 14 and 16 years of age who are privately educated at a home school (whether the home school is treated as a home school or a private school under State law) beyond such hours and periods applicable to employees between the ages of 14 and 16 years of age who are educated in traditional public schools.''. SEC. 10. RECRUITMENT AND ENLISTMENT OF HOME SCHOOLED STUDENTS IN THE ARMED FORCES. (a) Policy on Recruitment and Enlistment.-- (1) In general.--The Secretary concerned shall prescribe a policy for the recruitment and enlistment of home schooled students in the Armed Force or Armed Forces under the jurisdiction of such Secretary. (2) Uniformity across the armed forces.--The Secretary of Defense shall ensure that the policies prescribed under paragraph (1) apply, to the extent practicable, uniformly across the Armed Forces. (b) Elements.--The policy under subsection (a) shall include the following: (1) An identification of a graduate of home schooling for purposes of recruitment and enlistment in the Armed Forces that is in accordance with the requirements described in subsection (c). (2) Provision for the treatment of graduates of home schooling with Tier I status with no practical limit with regard to enlistment. (3) An exemption of graduates of home schooling from the requirement for a secondary school diploma or its recognized equivalent (GED) as a precondition for enlistment in the Armed Forces. (c) Home School Graduates.--In identifying a graduate of home schooling for purposes of subsection (b), the Secretary concerned shall ensure that the graduate meets each of the following requirements: (1) The home school graduate has taken the Armed Forces Qualification Test and scored 50 or above. (2) The home school graduate has provided the Secretary concerned with-- (A) a signed home school notice of intent form that conforms with the State law of the State where the graduate resided when the graduate was in home school; or (B) a home school certificate or diploma from-- (i) the parent or guardian of the graduate; or (ii) a national curriculum provider. (3) The home school graduate has provided the Secretary concerned with a copy of the graduate's transcript for all secondary school grades completed. The transcript shall-- (A) include the enrollment date, graduation date, and type of curriculum; and (B) reflect successful completion of the last full academic year of schooling from the home school national curriculum provider, parent, or guardian issuing the home school certificate or diploma or home school notice of intent form. (4) The home school curriculum used by the home school graduate involved parental instruction and supervision and closely patterned the normal credit hours per subject as used in a traditional secondary school. (5) The home school graduate has provided the Secretary concerned with a third party verification letter of the graduate's home school status by the Home School Legal Defense Association or a State or county home school association or organization. (d) Secretary Concerned Defined.--In this section, the term ``Secretary concerned'' has the meaning given such term in section 101(a)(9) of title 10, United States Code. | Title: A bill to amend selected statutes to clarify existing Federal law as to the treatment of students privately educated at home under State law Summary: Home School Non-Discrimination Act of 2005 - Amends the Higher Education Act of 1965 (HEA) with respect to: (1) student aid eligibility of home-schooled students who have satisfied certain secondary education standards; and (2) institutional aid eligibility of the higher education institutions that such students attend. Amends the Individuals with Disabilities Education Act (IDEA) to provide that, if a parent does not consent to an initial evaluation or special education or related services for a child with a disability, the local educational agency shall not be required to convene an individualized education program (IEP) meeting or develop an IEP for such child. Amends the Internal Revenue Code with respect to qualified elementary and secondary education expenses (the Coverdell Education Savings Account) to include home schools if they are treated as a home school or private school under state law. Amends the Family Educational Rights and Privacy Act of 1974 to prohibit release of certain information on and educational records of students in nonpublic education, including any student educated at home or in a private school in accordance with state law, without written parental consent. Amends HEA to include students at home schools, whether treated as a home school or a private school under state law, among those prospective secondary school graduates eligible to apply for the Robert C. Byrd Honors Scholarship Program for higher education. Amends the Fair Labor Standards Act of 1938 to direct the Secretary of Labor to extend the hours and periods of permissible employment of employees between the ages of 14 and 16 years who are privately educated at a home school, whether the home school is treated as a home school or a private school under state law, beyond those hours and periods applicable to employees of such ages who are educated in traditional public schools. (Thus allows home-school students to be employed during the traditional school day.) Amends specified federal law with respect to policies on recruitment and enlistment of home schooled students in the Armed Forces. | 2,308 | 458 | billsum | en |
Summarize: Bloomberg News AOL Inc. (AOL:US) Chief Executive Officer Tim Armstrong, making a rare public apology, sent a memo to employees saying he was sorry for the way he fired a staff member at a meeting last week. The CEO had fired Abel Lenz, a creative director for AOL’s Patch local-news business, in front of a room full of employees, as well as a thousand others who were listening in on a conference call on Aug. 9. “I am writing you to acknowledge the mistake I made last Friday during the Patch all-hands meeting when I publicly fired Abel Lenz,” Armstrong said yesterday in the memo. “I am the CEO and leader of the organization, and I take that responsibility seriously.” Armstrong called last week’s meeting to discuss cutbacks at Patch, which he aims to turn into a profitable business by the end of the year. The division -- a collection of local websites covering community news -- has been a costly part of his strategy to transform the dial-up provider into an advertising-driven content publisher. The company has spent more than $300 million to develop the sites. Lenz’s ouster became public fodder after an audio recording of the meeting leaked out and appeared on the website of media blogger Jim Romenesko. Armstrong can be heard calmly discussing plans for the future of Patch until he notices Lenz documenting the meeting. ‘You’re Fired’ “Abel, put that camera down right now! Abel, you’re fired. Out!” Armstrong said. After a pause, he continued talking about Patch. Lenz hasn’t been rehired at the New York-based company. Earlier in the week, Armstrong replaced Patch’s top executive, Steven Kalin, with Bud Rosenthal. As part of the cuts, he’s considering shutting or finding partners for 400 of Patch’s 900 community news sites, people familiar with the matter said. AOL is still calculating the number of eliminated jobs, which could number in the hundreds, the people said. While the Patch division more than doubled its sales last year to just under $35 million, Patch’s annual costs range from $126 million to $162 million. “As you know, I am a firm believer in open meetings, open Q&A and this level of transparency requires trust across AOL,” Armstrong said in the memo. “Internal meetings of a confidential nature should not be filmed or recorded so that our employees can feel free to discuss all topics openly. Abel had been told previously not to record a confidential meeting, and he repeated that behavior on Friday, which drove my actions.” To contact the reporter on this story: Edmund Lee in New York at elee310@bloomberg.net To contact the editor responsible for this story: Nick Turner at nturner7@bloomberg.net We have some new information that may explain why one of the most bizarre sequences in AOL history happened. On Friday, AOL CEO Tim Armstrong fired Abel Lenz, the creative director of AOL's local news division Patch, during a conference call with about 1,000 coworkers on the line. An audio recording of the conference call hit the Web late Saturday night. It's pretty nuts. The point of the call was for Armstrong to rally the employees of Patch — the day after Armstrong told Wall Street analysts he planned massive cost cuts for the division. About two minutes into a speech addressed to everyone on the call, Armstrong pauses to address someone in the room. He says, "Abel, put that camera down, now." Then, without taking a breath, Armstrong says, "Abel, you're fired. Out." If you haven't heard it yet, go listen right now. One of the reasons the recording is so odd is that Armstrong hardly seems to give Lenz a chance to put down the camera before firing him. Also: We're told that Lenz, based in New York, would always take pictures of big speakers during conference calls, and later post the images on Patch's internal Web site, so the 1,000 or so remote workers could see them. He wasn't doing anything unusual at the time. It all makes you wonder: Did Armstrong have something else against Lenz? Was there another reason he was so quick to fire him? Today, we learned some new information that suggests the answer to that question is "yes." Apparently, after firing Lenz, Armstrong went on to "[crap] all over" Lenz's biggest project for the company, Patch 2.0. Says a source: The Patch 2.0 redesign was spearheaded by Abel Lenz, at least at the beginning. He was brought into Patch from another area of AOL in the first half of 2012 when it became clear that the existing Patch web and mobile design talent was not up to the task. As to whether Abel was still leading that team, I don't know. But, according to people who were on the call, Armstrong called out Patch 2.0 as being awful (paraphrase, not a direct quote). … That's why I thought it was odd that, of all the people he called out and fired on the call, it was the guy who was responsible for building and launching Patch 2.0, an update that Armstrong clearly was not happy with. Maybe Armstrong was so quick to fire Lenz over such a small offense because he was already planning on letting him go because he didn't like his work? That would certainly make Armstrong's behavior easier to explain, if not excusable. We reported yesterday that AOL’s hyper-local news service would lose hundreds of employees today, and now we have confirmation from a well-placed Patcher privy to the call that AOL CEO Tim Armstrong did indeed confirm to employees that hundreds would be laid off, with notifications of who will be let go coming throughout the coming week. (Disclosure: AOL owns TechCrunch). In a call Armstrong held with the Patch team today, he explained that “AOL is going to be running the show” at the restructured Patch along with new CEO Bud Rosenthal. Rosenthal replaces outgoing CEO Steve Kalin, who was reported to be getting the boot earlier this week. 400 Patch sites will be closed or partnered with outside sites over the coming week as part of the changes being made at Patch to try and turn things around, Armstrong explained on the call, but also reassured the Patch staff that the company is behind the initiative and told them not to “worry about what [they] read in the press,” calling it “bullshit.” Nonetheless, he encouraged any Patch non-believers still remaining at the company to get out now, emphasizing that there’s no room for equivocation in turning the effort around. In a somewhat dramatic twist, Armstrong reportedly fired an employee who took his photo (behavior he presumably didn’t approve of), while on the call with 1,000 people listening in. Our tipster said the public nature of the termination struck them as “shameful and disgusting” behavior. Our source isn’t very convinced by the bluster around the new direction put forth by Armstrong. They suggest that this new strategy is really a “correction of a bad idea” that involved scaling far too fast without proper thought and planning, and notes that original Patch sites are still among the most successful. Focusing on those regions that actually do drive good revenue and traffic is probably the only way to save Patch at this point, however, so big cuts are probably unavoidable. Armstrong put on a brave face and discussed tough choices on the call, but our source notes that the AOL CEO was also there throughout the network’s decline, and had approved the very same people that he “buried on the call today.” Overall, the source says there’s not much reason to be confident in Patch’s leadership anymore. The stakes at Patch are high because AOL has promised it will see positive revenue by the end of the year, which is a tall order given its most recent earnings results. These cuts and shifts in strategy are drastic measures, but that’s exactly what’s required if Armstrong wants to make good on a promise of Patch profitability by year’s end. Ultimately, our source says the lesson to learn is that “news works,” and that a strategy that refocuses on those editors who cover their towns well and leaving them intact is probably Patch’s best chance at still existing in some form a year from now. AOL chief executive Tim Armstrong fired Patch creative director Abel Lenz two minutes into Friday’s call with Patch employees. Lenz’s sin: Taking a picture of the CEO during his talk. (“No comment,” the fired Patcher wrote Friday from Old Town Bar.) Here’s the transcript of the first minutes of the call, which I’m told went on for an hour and 40 minutes. You can listen to part of the call (2 minutes, 33 seconds) here. Tim Armstrong: There’s a couple of things I want you guys to realize and really think about and sink in, and if it doesn’t sink in and you don’t believe what I’m about to say, I’m going to ask you to leave Patch. And I don’t mean that in a harsh way; I mean that in the way of we have to get Patch into a place where it’s going to be successful and it’s going to be successful for a long time. There’s a whole bunch of towns that are going to be successful but we need the whole enterprise to be successful. The first one is, I will take full credit and full responsibility for anything that’s not right at Patch. If the coffee machine doesn’t work, or a town doesn’t work — anything that’s going wrong at Patch you can blame me for it. I founded Patch, we brought it into AOL, we’ve been very busy turning around AOL overall. I don’t care what the press says, I don’t care if people leak information. I’ve already lived through that at AOL — when I took over AOL — so if you need somebody to blame for why we’re making changes at Patch you can blame me. I take full responsibility. … I also want to clear up the fact that leaking information or anything around Patch isn’t going to bother me, doesn’t bother me. I’m not changing direction. When you hear about what we’re doing at Patch it’s very serious and it’s very forward-thinking and anything that happens around Patch isn’t going to change that direction. Third thing is if you don’t use Patch as a product and you’re not invested in Patch, you owe it to everybody else at Patch to leave. If you think what’s going on right now is a joke, and you want to joke around about it, you should pick your stuff up and leave Patch today, and the reason is, and I’m going to be very specific about this, is Patch from an experience — Abel, put that camera down right now! Abel, you’re fired. Out! [Momentary pause.] If you guys think that AOL has not been committed to Patch, and won’t stay committed to Patch, you’re wrong. The company has spent hundreds of millions of dollars, the board of directors is committed, I’m committed. …. After a call with the AOL-owned Patch team included the public firing of Patch Creative Director Abel Lenz last week, AOL CEO Tim Armstrong has issued an internal memo apologizing for the action and providing some additional context (disclosure: AOL also owns TechCrunch). In the memo obtained from a tipster, Armstrong says that he has apologized to Lenz for the behaviour and that it was unfair to the former employee at a “human level.” The entire memo follows: AOLers – I am writing you to acknowledge the mistake I made last Friday during the Patch all-hands meeting when I publicly fired Abel Lenz. It was an emotional response at the start of a difficult discussion dealing with many people’s careers and livelihoods. I am the CEO and leader of the organization, and I take that responsibility seriously. We talk a lot about accountability and I am accountable for the way I handled the situation, and at a human level it was unfair to Abel. I’ve communicated to him directly and apologized for the way the matter was handled at the meeting. My action was driven by the desire to openly communicate with over a thousand Patch employees across the US. The meeting on Friday was the second all-hands we had run that week and people came to Friday’s meeting knowing we would be openly discussing some of the potential changes needed at Patch. As you know, I am a firm believer in open meetings, open Q&A, and this level of transparency requires trust across AOL. Internal meetings of a confidential nature should not be filmed or recorded so that our employees can feel free to discuss all topics openly. Abel had been told previously not to record a confidential meeting, and he repeated that behavior on Friday, which drove my actions. We have been through many difficult situations in turning around AOL and I have done my best to make the best decisions in the long-term interest of the employees and the company. On Friday I acted too quickly and I learned a tremendous lesson and I wanted you to hear that directly from me. We have tough decisions and work to do on Patch, but we’re doing them thoughtfully and as openly as we can. At AOL, we had strong earnings last week and we’re adding one of the best companies in the world to the team. AOL is in a great position, and we’ll keep moving forward. – TA It’s worth noting that at no point in the memo does Armstrong discuss Lenz’s current job state, so the firing likely sticks. Armstrong says the firing is a direct result of Lenz having exhibited this kind of behavior before, and being told explicitly not to, but other reports have suggested that he may have been on thin ice anyway after the Patch 2.0 redesign project failed to impress. Patch is currently in a state of considerable upheaval, as employees wait to hear more about the “hundreds” of layoffs coming down the line. TechCrunch spoke to a local editor about the original conference call discussing the changes to come for Patch, and they expressed some skepticism about its ability to turn around, though he did suggest refocusing on the core Patch sites which have proven profitable covering specific areas could effect considerable positive change. Patch was listed specifically as a significant factor negatively impacting the overall adjusted operating income of the overall AOL brand in its most recent quarterly earnings result, and AOL has promised to make Patch profitable by year’s end. | Summary: AOL's local news network, Patch, is losing money, closing hundreds of websites, and laying off hundreds of employees, TechCrunch reports. But that is not its biggest problem right now. That honor goes to the dismissal of just one employee last week. In the middle of a meeting with Patch employees, thousands of whom were listening in via conference call, CEO Tim Armstrong abruptly fired the site's creative director Abel Lenz in about 10 words, then without skipping a beat, continued on with his talk. A recording of the meeting was posted on Romenesko (and subsequently spread like wildfire online), but here's the relevant part:...I'm going to be very specific about this, is Patch from an experience - Abel, put that camera down right now! Abel, you're fired. Out! [Momentary pause.] If you guys think that AOL has not been committed to Patch... Now Armstrong has had to apologize for the "mistake," which he called an "emotional response at the start of a difficult discussion." In a company memo, he took responsibility for "acting too quickly," but defended his decision: "Internal meetings of a confidential nature should not be filmed or recorded so that our employees can feel free to discuss all topics openly," he wrote, per Businessweek. "Abel had been told previously not to record a confidential meeting, and he repeated that behavior on Friday, which drove my actions." According to Business Insider, Lenz typically took photos of speakers during meetings to post on the company's internal blog. As Tech Crunch notes, nowhere does the memo say the dismissal itself was wrong, so Lenz is probably still without a job. | 3,251 | 372 | multi_news | en |
Write a title and summarize: Laiwu District is recognized as a hyper-endemic region for scrub typhus in Shandong Province, but the seriousness of this problem has been neglected in public health circles. A disability-adjusted life years (DALYs) approach was adopted to measure the burden of scrub typhus in Laiwu, China during the period 2006 to 2012. A multiple seasonal autoregressive integrated moving average model (SARIMA) was used to identify the most suitable forecasting model for scrub typhus in Laiwu. Results showed that the disease burden of scrub typhus is increasing yearly in Laiwu, and which is higher in females than males. For both females and males, DALY rates were highest for the 60–69 age group. Of all the SARIMA models tested, the SARIMA (2,1, 0) (0,1, 0) 12 model was the best fit for scrub typhus cases in Laiwu. Human infections occurred mainly in autumn with peaks in October. Females, especially those of 60 to 69 years of age, were at highest risk of developing scrub typhus in Laiwu, China. The SARIMA (2,1, 0) (0,1, 0) 12 model was the best fit forecasting model for scrub typhus in Laiwu, China. These data are useful for developing public health education and intervention programs to reduce disease. Scrub typhus, also known as tsutsugamushi disease, is a zoonosis transmitted by chigger bites (larval trombiculid mites) and infection with Orientia tsutsugamushi (O. tsutsugamushi), a Gram-negative obligate intracellular bacterium. Globally, scrub typhus is distributed widely in the Pacific region of Asia. It is prevalent in a triangle from Northern Japan and far-eastern Russia in the north, to Northern Australia in the south, to Pakistan and Afghanistan in the west, and also involves islands of the western Pacific and Indian Oceans [1]–[3]. Scrub typhus has existed in China for thousands of years. Before 1986, the disease was found only in Southern China, with epidemics occurring mainly in summer [4]. Beginning in 1986, scrub typhus was also reported in areas of Northern China, such as Shandong Province, Tianjin City, Heilongjiang Province, Shanxi Province and Hebei Province [5]. Clinical infections in Northern China seemed to occur mainly in autumn and winter [4]. Shandong Province is most noted to the one of the most serious foci for scrub typhus in Northern China [5]. In 1986, the first outbreak of scrub typhus occurred in Linyi District, followed other outbreaks in Jinan in 1988, in Jining in 1996, in Yantai in 1997, in Weifang and in Tai′an in 2000 [6]. From 2006 to 2012, a total of 2337 scrub typhus cases were notified in the Diseases Reporting Information System of Shandong Center for Disease Control and Prevention. At present, 13 of 17 districts have reported scrub typhus cases in Shandong Province where Laiwu is the district of the highest scrub typhus incidence. In 1999, the earliest cases of scrub typhus were documented in Laiwu District [7]. Since 2006, scrub typhus cases have been included in the Notifiable Infectious Diseases System managed by Shandong Center for Disease Control and Prevention. From 2006 to 2012,308 cases were detected in Laiwu (134 males and 174 females). The average annual incidence of scrub typhus in Laiwu (3. 59/100,000) was approximately ten times of that in entire Shandong Province (0. 35/100,000). However, researchers and health authorities have more often focused their attentions to Linyi, the initial focus of scrub typhus in Shandong, where the annual incidence was 0. 89/100,000. As a new focus of scrub typhus in Northern China, Laiwu (117°19′-117°58′E, 36°02′-36°33′N) lies in the center of Shandong Province, an important geographical position in Shandong. The population of Laiwu was 1,226,393. Incidence, prevalence, duration and mortality indicators were most frequently used to estimate the burden of disease [8]. Of available indexes, we highlight disability-adjusted life years (DALYs) to measure the disease burden of scrub typhus in Laiwu, China. The DALYs metric was jointly developed by the World Bank, Harvard School of Public Health and the World Health Organization (WHO) for the Global Burden of Disease and Injury Study (GBD) [9]. The European Centre for Disease Prevention and Control (ECDC) had adopted an incidence- and pathogen-based DALYs approach to measure the Burden of Communicable Diseases in Europe Project (BCoDE) across European Member States [10]–[12]. DALY, a summary metric of population health, is often used to identify health gaps by measuring the state of a population' s health compared to a normative goal which is for individuals to live the standard life expectancy in full health. One DALY means one-year loss of ‘healthy’ life. It integrates disease-specific mortality, morbidity and severity together, and quantifies morbidity associated with different clinical outcomes by assigning disability weights on a scale between 0 and 1, where by 0 means no morbidity and 1 means death [13]. DALYs and DALY rates (DALYs per 100,000 population or DALYs per 1000 population) [8], [14], are often used to compare disease burden of the same disease among regions, areas, countries, districts [15], or disease burden of different diseases in the same place [16]. Moreover, DALYs and DALY rate could be used to identify high risk population for targeted interventions or intervention prioritization. In this research, DALYs and DALY rate were adopted to evaluate the disease burden of scrub typhus in Laiwu, China. Epidemic modeling and forecasting is more recognized as an essential tool in preventing and controlling infectious disease. Cases of scrub typhus in Laiwu, China seem to have considerable variation during the period 2006 to 2012. A seasonal time series autoregressive integrated moving average (SARIMA) modeling introduced by Box and Jenkins [17] is most useful in examining data for seasonal or periodic fluctuations that recur with about the same intensity each year. SARIMA models have been successfully applied for forecasting economic, marketing, and social problems. While this model has the advantage of accurate forecasting over short periods, it has a limitation that at least 50 observations are needed [17]. In this study, we sought to identify the most suitable SARIMA model we could use to predict scrub typhus cases in Laiwu over time. By combined the results of disease burden measured by DALYs and DALY rate with a SARIMA forecasting model of scrub typhus, we hoped to identify high-risk populations and interventions or intervention prioritization of scrub typhus. The research could help health authorities to prevent and control of scrub typhus efficiently. The dataset of the human scrub typhus cases in Laiwu, China from 2006 to 2012 was obtained from the Diseases Reporting Information System of Shandong Center for Disease Control and Prevention. The notification system recorded the detailed information for the scrub typhus cases, including gender, age, dates of symptom onset and diagnosis, and recovery or death. The symptom onset date was used in this study, and it was thought to be more useful than the date of diagnosis or the date of notification. The cases recorded were the anonymized in this study. Population data for Laiwu was obtained from the Laiwu Statistical Bureau and stratified by age group and gender [18]. In this research, we adopted the incidence- and pathogen-based DALYs approach [10]–[13] to estimate the disease burden of scrub typhus in Laiwu, China. This approach has been previously used in the BcoDE project by ECDC. Fig. 1 shows a disease outcome tree for Orientia tsutsugamushi infection. All cases of scrub typhus in this study recovered for their illness. DALYs were the sum of years of life lost due to premature death (YLLs) and years lived with disability (YLDs) [19]. According to the outcome tree, YLDs were calculated for each health outcome (l) by multiplying the number of incident cases (n) with the disability weight (w) for a specific health outcome (l), and the duration of the disabling condition (t) [see equation (1) ]. All input parameters in both YLDs and YLLs formula were chosen to be age (a) and sex (s) dependent when such information was available, where a stands for age at infection and ã for age at onset of a condition or death [11], [12]. (1) To estimate the YLLs for those health outcomes (l) that can lead to death, the number of fatal cases (d) for a specific health outcome (i) for an infection acquired at age (a) is multiplied by the remaining life expectancy (e) at age ã [see equation (2) ]. (2) In this research, the Coale and Demeny West Level 26 Life Table adopted in many disease burden studies was used in the calculation of YLLs to assure comparability to other disease burden assessments [20]. Life expectancy for males and females at birth were set at 80 and 82. 5 years, respectively. The average durations of scrub typhus in different age groups and gender were achieved by DisMod II software developed for the calculation of GBD [21]. DisMod II is powered by two basic inputs: the make-up of a region' s population by gender and age, and the overall mortality rate for each demographic group. This model stratifies the relationships between a set of indicators relevant by age and gender: incidence, prevalence, remission, mortality, duration, case fatality, and RR mortality (the relative risk on total mortality) [22]. It requires powering with a minimum of three of these variables (by age groups and gender) and these three variables allow the prediction of the others [22]. Comparing the internal epidemiological consistency of estimates of incidences, prevalence, duration and mortality, we found that when inputting the variables were incidence, remission (100%), and case fatality of scrub typhus clarified by age and gender, the outputs fitted well. Thus, the average durations for scrub typhus in gender and different age groups were obtained (Table 1). No paper has previously reported the disability weight of scrub typhus, including “Global Burden of Disease 2004 Update: Disability Weights for Diseases and Conditions, ” the widely adopted document about disability weights of disease [13], [16], [23]. Scrub typhus and dengue fever have so many similarities in duration, high-risk population, signs and symptoms, pathogenesis and prognosis [1], [24]–[26] that disability weight of dengue fever (0. 197) [13] was referred in the research of scrub typhus. According to BCoDE-project, these raw incidence data should be corrected for underestimation by pathogen-specific multiplication factors (MF), representing either correction for underestimation in one step, or separate correction for under-ascertainment and underreporting in two steps [12], [27]. The overall extent of underestimation can be explained by two major effects represented by under-ascertainment and under-reporting [27]. Since one person infected with Orientia tsutsugamushi will demonstrate acute symptoms, such as fever, rash, eschar and swollen lymph nodes, the person is likely to visit a doctor, and due to physical awareness, scrub typhus is relatively easy to diagnosis in Laiwu, China. Moreover, as a notifiable disease in Laiwu, scrub typhus must be reported to the Diseases Reporting Information System of Shandong Center for Disease Control and Prevention. So, under-ascertainment and under-reporting of scrub typhus in Laiwu was thought to be minimal and not included in the research. We used “1” as the MF value of scrub typhus in Laiwu, China in the research. Many time series data contain seasonal periodic components. To deal with seasonality, a general multiplicative SARIMA model was extended from the ARIMA model (Box et al) [28]. This building process of the SARIMA (p, d, q) (P, D, Q) s model was designed to take advantage of associations in the sequentially lagged relationships which usually exist in periodically collected data. Seven main parameters are selected when fitting a SARIMA model: p, the order of process autoregression; d, the order of difference; q, the order of process moving average; P, D and Q, the corresponding seasonal orders; s, the length of seasonal period. If d is nonzero, a general differencing can be used to remove trend. If D is not zero, seasonal differencing can be used to remove seasonality. In order to construct and validate the model, the database of scrub typhus was verified by dividing the data file into two data sets, i. e. the data between January and December 2006–2011 and data between January and December 2012. The original series of scrub typhus cases was not a stable time series, so it was necessary to make it stable by differential. After general difference of 1 order, followed by seasonal difference of 1 order and length of seasonal period was 12, the series was satisfied with stability. Thus d = 1, D = 1, S = 12. Then the order of autoregression and moving average were identified using autocorrelation function (ACF) and partial autocorrelation function (PACF) of the differenced series. The most suitable model was selected on the basis of Normalized Bayesian Information Criteria (BIC) [29] and Ljung-Box test. Lower values of Normalized BIC and Ljung-Box test (higher significant) were preferable. Furthermore, Ljung-Box test was performed to test if ACF of the residuals at different lag times were significantly different from zero, where no different from zero was expected [30]. Compare the predicted values in 2012 using the most suitable SARIMA model with the number of scrub typhus cases notified in 2012 to validate the forecasting ability of the model. The analyses were carried out with STATA version 12. 0 (Stata Corporation, College Station, USA). No cases of death were reported in Laiwu from 2006 to 2012, so DALYs of scrub typhus in Laiwu was equal to YLDs. From 2006 to 2012, DALYs of scrub typhus were 5,10,10,5, 8,10 and 13, respectively. The average annual DALYs of scrub typhus was 9, and DALYs were higher in females (5) than males (4) (Table 2). DALY rates (DALYs/100,000) of scrub typhus were 0. 3663,0. 7933,0. 8106,0. 4437,0. 6179,0. 7883 and 1. 0618 from 2006 to 2012, respectively. The average annual DALY rate was 0. 6974 DALYs/100,000. The DALY rate was higher among females (0. 8019 DALYs/1 00,000) than among males (0. 5961DALYs/100,000). Moreover, DALY rates in females and males were both highest for the 60–69 years age group (Table 3). Fig. 2 shows the breakdown of average DALY rates of scrub typhus in different age groups and gender in Laiwu. Since all cases of scrub typhus were cured in Laiwu, no sequelae were recorded. The annual incidence (/100,000) of scrub typhus was 1. 88,4. 08,4. 16,2. 28,3. 18,4. 08, and 5. 46 from 2006 to 2012, respectively. The average annual incidence was 3. 59/100,000. There was no statistical difference between incidence of scrub typhus in males and females each year (2006–2012) (P>0. 05). Related x2 and P values were showed in Table 4. But, the average annual incidence in females (4. 11/100,000) was higher than males (3. 07/100,000) (x2 = 5. 850, P = 0. 016). The average annual incidence was the highestin 60–69 age group (Table 4). The trend of DALY rates of scrub typhus was consistent with incidences from 2006 to 2012 in Laiwu, China. Since 2009, DALY rates and the incidences were both increasing year by year (Table 3,4). Fig. 2 showed that in 60–69 age group, the DALY rate in females was sharply higher than males. The series of notified cases was a non-stationary series. Therefore, by taking 1 order general difference, followed by 1 order seasonal difference and length of seasonal period was 12, the time series of scrub typhus cases was corrected into stationary series. Fig. 3 shows the autocorrelation function (ACF) and partial autocorrelation function (PACF) of scrub typhus cases in Laiwu, China after differencing. Based on the distribution characteristic, we conducted several models, SARIMA (2,1, 0) (0,1, 1) 12, SARIMA (1,1, 0) (1,1, 1) 12, SARIMA (0,1, 0) (2,1, 1) 12, SARIMA (2,1, 0) (0,1, 0) 12, SARIMA (1,1, 0) (1,1, 0) 12, SARIMA (0,1, 0) (2,1, 0) 12, SARIMA (2,1, 1) (0,1, 0) 12 and SARIMA (2,1, 1) (0,1, 0) 12. Of all the models tested, the SARIMA (2,1, 0) (0,1, 0) 12 model was the best fit for the data (Table 5). Moreover, the Ljung-Box test suggested that the ACF of residuals for the model at different lag times was not significantly different from zero, i. e. the residuals of the SARIMA (2,1, 0) (0,1, 0) 12 model was satisfied with white noise. The stationary residuals provided the evidence that the SARIMA (2,1, 0) (0,1, 0) 12 model was adequate. All the coefficients of the SARIMA (2,1, 0) (0,1, 0) 12model were significant (Table 5,6). The equation of the SARIMA was. All the number of notified cases from Jan 2012 to Dec 2012 were in the 95% confidence interval of the forecasting values by the SARIMA (2,1, 0) (0,1, 0) 12 model. The model was used to predict values from January to December 2012 for validation. The notified cases and fitted cases by the best fitted SARIMA model from 2006 to 2011, and the actual cases and predicted cases from January to December 2012, were illustrated in Fig. 4. It showed that the predicted values could follow the upturn and downturn of the observed series reasonably well. In addition, the fitted values appeared some negative values, which was a common case with the series with too many zeros as observed values in the series of scrub typhus cases. The SARIMA (2,1, 0) (0,1, 0) 12 model showed that the prevailing disease occurred mainly in autumn and peaked in October. Incidence is often used as one measure index of disease frequency, and since its calculation only concerns the cases number and basic population data, it must be standardized by some standard population when compared between different areas or different times. However, DALYs consider both survival time and life quality caused by a certain disease and its calculation requires age and sex dependent incident cases, fatal cases, severity of disease and life expectancy. Therefore, DALYs can demonstrate the threat of some disease to the whole population, and it is more objective and comprehensive than incidence [23]. In addition, standardization issues have been covered inherently in the calculation of DALYs [8], [14]–[16], [31], [32], such as different age groups and gender are assigned with different life expectancies and different diseases are assigned with different disability weights. So, DALYs and DALY rate could be compared among areas or diseases directly. From 2006 to 2012, the average annual DALYs of scrub typhus was 9, and DALY rate was 0. 6974 DALYs/100,000 in Laiwu District. The average annual DALYs and DALY rate of scrub typhus were 15 and 0. 1504 DALYs/100,000 in Linyi District, the initial foci of scrub typhus in Shandong Province. Populations of Linyi and Laiwu were 10,039,435 and 1,226,393, respectively. So, even though DALYs of scrub typhus was higher in Linyi than Laiwu, DALY rate was apparently higher in Laiwu than Linyin. Therefore, considering the limited health resources, more attention should be paid to Laiwu, a new focus of scrub typhus. No cases of death were reported in Laiwu, China from 2006 to 2012, so the YLLs were 0 and the DALYs were equal to YLDs. There were several reasons why DALYs were adopted rather than solely YLDs. Worldwide acceptance of DALYs and DALY rate enabled international comparisons or ranking of areas by disease burden [8], [15], [16], [33]–[43]. DALYs represent unified summary estimate of disease burden that takes mortality as well as morbidity into consideration, i. e., DALYs and DALY rate would be more useful in comparing the disease burden among different diseases and different areas than other indexes. In addition, the levels of diagnosis and treatment of scrub typhus in Laiwu, China were high enough to cure all the patients, but there were still death cases in some other areas in China [44], [45]. For example, there were death cases of scrub typhus reported in Guangzhou, China in 2012. When comparing the disease burden of scrub typhus between these two districts, as a single metric DALYs is better than YLDs [44]. The DALYs of scrub typhus were equal to YLDs in Laiwu demonstrated that more attention should be paid to the unhealthy conditions of scrub typhus. Since 2009, DALY rates and incidences of scrub typhus have both been increasing year by year (Table 3 and Table 4). With global warming, the predicted scenarios of increased temperature and rainfall were also causing concern for increases in vector-borne diseases, particularly, endemic arboviruses [46]. In 2010, Kim SH and Jang JY [47] reported that the incidence of scrub typhus in hyper-endemic region during the outbreak period was positively correlated with temperature and humidity during the summer. In addition, scrub typhus was transmitted to humans by the bites of chiggers which are mainly active in forest clearings, riverbanks, and grassy regions [47], [48]. With the increased urbanization and higher green coverage rate, people have more opportunity to contact chiggers. In 2012, there were several death cases of scrub typhus reported in Guangzhou, China and these patients had lied, stood, or sat on lawn in gardens before onset of the disease [44]. Green coverage rate achieved 42. 19% and park green space per capita was 18. 49 square meters in Laiwu, China in 2012 [49]. More suitable environment for chiggers' multiplication, more cases might appear in Laiwu. From 15 to 80 years old, the average annual DALY rate and incidence of scrub typhus in females were both higher than males (Table 3, Table 4). Moreover, in the 60–69 years age group, females had a sharply higher DALY rate (2. 6095 DALYs/100,000) than males (1. 5689 DALYs/100,000) (Fig. 2). Thus, females, especially those from 60 to 69 years old, were the highest risk population for scrub typhus in Laiwu, China. As a zoonosis, scrub typhus was infected with O. tsutsugamushi, and rodents were the main host of O. tsutsugamushi. With increasing touch chance with O. tsutsugamushi, people in fields were easier to suffer from the disease [50]. Being a modern industrial area, Laiwu had become one home of numerous iron and steel businesses in China. Therefore, with more and more young and males moving from rural to urban areas, more and more elderly and females now engage in agriculture activity [18]. Additionally, with sparse awareness of the disease, the elderly have a greater chance of contact with chiggers [18]. Hence, health education efforts regards scrub typhus should be focused upon high risk groups like females who are 60–69 years old, especially those who live in countryside. SARIMA modeling was useful for interpreting and applying surveillance data in disease control and prevention [51], [52]. As with many infectious diseases, the time series data of scrub typhus in Laiwu, China showed the components of trend and seasonal pattern. One of the most recognized disadvantages of this approach is the necessity of a large amount of data (i. e., a minimum of 50 observations) to build a reasonable SARIMA model [53]. In this research, monthly number of cases from 2006 to 2011 (72 observations) was used to build the SARIMA model, and monthly cases during the period of January to December in 2012 were used to validate the corresponding SARIMA model. The chosen SARIMA (2,1, 0) (0,1, 0) 12 model fit the observations well and the residual series were satisfied with white noises. Therefore, the SARIMA (2,1, 0) (0,1, 0) 12 model could be used to forecast the monthly cases of scrub typhus in Laiwu, China. The prevailing disease occurred in autumn and peaked in October, which suggests that education and other protective measures should occur just before October. In the research, we adopted the incidence- and pathogen-based DALYs approach used by BcoDE Project to estimate the disease burden of scrub typhus rather than prevalence-based DALYs method presented in the GBD 2010. In case of an infectious pathogen, and in particular for priority settings of intervention to prevent primary infection, incidence is a more appropriate input for the DALYs metric than prevalence with the reason that only with the initial start of the infection is it possible to include all the disease squeals that result from the infection [12]. In addition, the incident cases of scrub typhus in Laiwu could be obtained from surveillance systems accurately. Considering above mentioned, the incidence- and pathogen-based DALYs were adopted in this research. Limitations of this research should be acknowledged. The occurrence of scrub typhus was influenced by many factors such as pathogen prevalence, mites, rodents, human being' s activities, and social or environmental factors. While building the SARIMA model, factors mentioned above were not considered. In addition, our research regarding disease burden and time series analysis of scrub typhus only focused on Laiwu, China. The results may not generalize to the most of China' s population. The disease burden of scrub typhus has increased year-by-year in Laiwu, China. Public health authorities should make concerted efforts to control and prevent the disease. The results of disease burden can also assist authorities in identifying the high-risk population. The SARIMA (2,1, 0) (0,1, 0) 12 model developed in the research could offer prediction of scrub typhus monthly cases in Laiwu. Combined with disease burden measurement and SARIMA forecasting, these data should help official as a decision support tool in a scrub typhus risk management program and in planning various prevention efforts. | Title: Burden of Disease Measured by Disability-Adjusted Life Years and a Disease Forecasting Time Series Model of Scrub Typhus in Laiwu, China Summary: Scrub typhus, also known as tsutsugamushi disease, is a zoonosis transmitted by chigger bites (larval trombiculid mites) and the pathogen Orientia tsutsugamushi (O. tsutsugamushi), a Gram-negative obligate intracellular bacterium. It is distributed widely in the Pacific regions of Asia, and the islands of the western Pacific and Indian Oceans. People with outdoor activities that involve contact with grasses or shrubs are at highest risk. Scrub typhus has existed in Southern China for thousands of years, but it has been noted to spread from the South to the North of China in recent decades. Though this research we studied the disease burden of scrub typhus with disability-adjusted life years (DALYs), and developed a forecasting time series model for human clinical disease in Laiwu, China. Results demonstrated that the disease burden of scrub typhus was increasing year by year in Laiwu, and it was higher in females than males. Moreover, DALY rates in females and males were highest for persons in the 60-69 years age group. Of all the seasonal autoregressive integrated moving average (SARIMA) models tested, the SARIMA (2,1, 0) (0,1, 0) 12 model was the best fit for scrub typhus cases in Laiwu. The disease occurred mainly in autumn, with a peak in October. | 6,830 | 349 | lay_plos | en |
Summarize: RELATED APPLICATIONS The present invention was first described in and claims the benefit of U.S. Provisional Patent Application No. 61/003,186 filed on Nov. 16, 2007, the entire disclosures of which are incorporated herein by reference. FIELD OF THE INVENTION The present invention relates generally to an infant comfort sleeper and, more particularly, to an apparatus that simulated a mother's breathing motion with an air compressor and an air bladder regulated by a venting valve that provides a gentle motion thereto an infant bed to provide a comfortable resting and sleeping place for said infant. BACKGROUND OF THE INVENTION Babies and infants frequently have trouble falling to sleep. This forces caregivers to employ many different strategies to assist a baby to sleep. Many caregivers will place a baby in a car and drive until the child goes to sleep. Others will hold a baby and rock them until they fall asleep. Babies appear to enjoy the feeling of security of being held. The slight feeling of confinement coupled with the rise and fall of the parent's chest as they breathe, is something that is guaranteed to put any baby to sleep. Accordingly, there is a need for a means by which infants can be provided a feeling of comfort and security while parents or care providers attend to other duties. The development of the invention herein described fulfills this need. There have been attempts in the past to invent sleepers for babies. U.S. Pat. No. 7,059,000 issued to Verbovszky discloses a portable infant cushion that appears to comprise a cushion that encircles an infant. Unfortunately, this patent does not appear to disclose an infant sleeper that comprises a powered air compressor that activates a bladder contained inside of the sleeper that provides the sleeper with the ability to simulate the breathing motions of a care giver. U.S. Pat. No. 6,912,743 issued to Weil discloses a bed device that appears to comprise a mattress that is conformed to a baby. Unfortunately, this patent does not disclose an infant sleeper that possesses the ability to simulate the breathing motion of a caregiver, nor does it appear to comprise an insertable insert for use with newborns. U.S. Pat. No. 6,199,234 issued to Srour et al. discloses an infant comfort mattress that appears to comprise a surface designed to reduce surface contact with an infant. Unfortunately, this patent does not disclose an infant sleeper that comprises a base unit with the ability to simulate the breathing motion of a caregiver, nor does it appear to provide an oval shaped enclosure for placing a baby therein. U.S. Pat. No. 6,026,525 issued to Davis discloses a foldable infant mattress system with sleeping area recess. Unfortunately, this patent does not appear to disclose an infant sleeper that simulates the breathing motion of a caregiver. U.S. Pat. No. 5,937,465 issued to Carew et al. discloses an infant mattress system with sleeping recess that appears to comprise a mattress made of different materials. Unfortunately, this patent does not appear to disclose a secure and comfortable infant sleeper that possesses the ability to simulate the rise and fall of a caregiver's chest during breathing. U.S. Pat. No. 5,561,876 issued to Petruzulla discloses an infant mattress comprised of a variety of materials and possessing a bumper area around the perimeter of the bed. Unfortunately, this patent does not appear to disclose an infant sleeper with a removable insert, nor does it appear to disclose an infant sleeper that simulates the breathing motion of a caregiver. U.S. Pat. No. 3,803,646 issued to Neweroski discloses a mattress for a crib with an integral bumper. Unfortunately, this patent does not appear to disclose an infant sleeper that may be placed on any firm stable surface nor does it appear to be capable of simulating the chest motion of a caregiver during breathing. SUMMARY OF THE INVENTION In light of the disadvantages as previously described in the prior art, it is apparent that there is a need for an infant comfort sleeper which provides a “canoe” shaped infant bed comprising an internal oscillating air bladder to simulate a mother's breathing motion. An object of the infant sleeper is to provide a location to place an infant that is secure and comforts an infant by providing a motion that simulates the rise and fall of the chest of a caregiver through breathing. Another object of the infant sleeper is to simulate this motion through the use of an air compressor and an air bladder contained within a base unit. Still another object of the infant sleeper comprises components that are made of hypoallergenic substances and polyurethane foam rubber portions providing waterproof and washable outer surfaces. Still a further object of the infant sleeper comprises a foam insert that may be placed within the base enclosure to provide for newborns. Yet another object of the infant sleeper provides an insert that comprises an elevated head area which provides providing for unobstructed and clear breathing to a newborn contained therein. An aspect of the infant sleeper comprises a base unit, a base bladder, an air compressor, a pneumatic control module, batteries, and an AC power cord. Another aspect of the infant sleeper comprises a base unit further comprising an insert, a battery compartment, an air compressor, an ON/OFF switch, a rheostatic control knob, and a pneumatic control module. The base unit comprises a plastic open-topped enclosure forming a generally oval-shape enclosure suitable for cradling a baby. A further aspect of the infant sleeper comprises a base unit comprising an insert that may be removably and snuggly placed when utilizing the apparatus with a newborn infant. Still another aspect of the infant sleeper comprises a base comprising a base wall that supports a base bladder affixed on the inward facing surface of the base wall. The base bladder comprises an air compressor unit, a solenoid vent valve, and a control module and provides an oscillating breathing motion via an inflating/deflating system. Still a further aspect of the infant sleeper comprises an air compressor unit that provides an internal air filling means to the base bladder. The air compressor unit is a compact unit and further comprises an integral pneumatic control module, a rheostatic control knob, and an on/off switch being mounted to along a front surface of the base unit. Yet another aspect of the infant sleeper comprises a pneumatic control module comprises an electronic control device providing a housing means thereto all necessary electrical and pneumatic components required to provide cyclic inflating and deflating of the base bladder via a solenoid vent valve. Yet a further aspect of the infant sleeper is an external rheostatic control knob that provides manual frequency regulation of said oscillating inflating and deflating of said base bladder. Yet another aspect of the infant sleeper is a plurality of batteries and an AC adapter to provide power to the sleeper. The batteries are housed in a battery compartment which further comprises a removable plastic flush-mounted access door. A method of installing and utilizing the apparatus may be achieved by performing the following steps: installing a fresh set of rechargeable or disposable batteries into the battery compartment or, alternately, utilizing the AC adapter using an available household outlet; placing the apparatus preferably thereupon a non-traffic floor area or other safe flat surface; adding appropriate blankets and sheets required to obtain a desired sleeping temperature for an infant; pressing the ON/OFF switch to initiate the air compressor unit and solenoid vent valve, thereby producing the simulated breathing motion; regulating the frequency of said breathing motion by turning the rheostatic control knob to a desired oscillating rate; allowing continuous automatic functioning of the apparatus as needed; pressing the ON/OFF switch again to stop the breathing motion; retaining the infant in the apparatus or removing said infant from the apparatus as desired; and, benefiting from improved quality and duration of a baby or infant's sleeping experience using the present invention. An alternate method of utilizing the apparatus using the insert portion may be accomplished by performing the following additional steps: placing the insert completely down into the base unit if the apparatus is being used with a newborn baby; placing a baby thereinto said insert giving care to position said baby's head at the proper end of the padded insert floor. BRIEF DESCRIPTION OF THE DRAWINGS The advantages and features of the present invention will become better understood with reference to the following more detailed description and claims taken in conjunction with the accompanying drawings, in which like elements are identified with like symbols, and in which: FIG. 1 is a front perspective view of an infant comfort sleeper 10, according to a preferred embodiment of the present invention; FIG. 2 is a front perspective view of an insert portion 20 of an infant comfort sleeper 10, according to a preferred embodiment of the present invention; FIG. 3 a is a front perspective view of a base portion 30 of an infant comfort sleeper 10, according to a preferred embodiment of the present invention; FIG. 3 b is a section view taken along section A-A (see FIG. 3 a ) of an infant comfort sleeper 10, according to a preferred embodiment of the present invention; and, FIG. 4 is an electrical block diagram of an infant comfort sleeper 10, according to a preferred embodiment of the present invention. DESCRIPTIVE KEY 10 infant comfort sleeper 20 insert 21 insert side panel 22 insert floor 23 insert rim 30 base unit 31 base wall 32 base bladder 33 base floor 34 base rim 35 air compressor unit 36 pneumatic control module 37 ON/OFF switch 38 battery compartment 39 breathing motion 40 rheostatic control knob 41 solenoid vent valve 50 battery 52 direct current (DC) receptacle 54 alternating current (AC) adapter DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT The best mode for carrying out the invention is presented in terms of its preferred embodiment, herein depicted within FIGS. 1 through 4. However, the invention is not limited to the described embodiment and a person skilled in the art will appreciate that many other embodiments of the invention are possible without deviating from the basic concept of the invention, and that any such work around will also fall under scope of this invention. It is envisioned that other styles and configurations of the present invention can be easily incorporated into the teachings of the present invention, and only one particular configuration shall be shown and described for purposes of clarity and disclosure and not by way of limitation of scope. The terms “a” and “an” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced items. The present invention describes an infant comfort sleeper (herein described as the “apparatus”) 10, which provides a “canoe” shaped infant bed comprising an internal oscillating air bladder 32 to simulate a mother's breathing motion 39. The apparatus 10 provides an open-topped base enclosure 30 with plastic walls 31. The air bladder 32 is connected to a battery-powered air compressor 35 which pumps air in and out of the bladder 32 to simulate human breathing. The inside of the apparatus 10 is contoured to a baby's shape. Additionally, a foam insert 20 is provided for newborn babies. The use of the apparatus 10 provides a location to place an infant, which is comforting and secure. Referring now to FIG. 1, a front perspective view of the apparatus 10, according to the preferred embodiment of the present invention, is disclosed. The apparatus 10 comprises a base unit 30, an insert 20, a battery compartment 38, an air compressor 35, an ON/OFF switch 37, a rheostatic control knob 40, and a pneumatic control module 36. The base unit 30 comprises a semi-rigid plastic exterior base wall portion 31 having an open top region providing a generally oval-shaped enclosure being suitable for cradling a baby weighing approximately ten (10) to twenty (20) pounds. Affixed along a front outside surface of said base unit 30 is an air compressor unit 35 further comprising an ON/OFF switch 37, a rheostatic control knob 40, a battery compartment 38, and a pneumatic control module 36. The aforementioned components provide an automatic inflating and deflating means thereto an internal bladder 32, thereby emulating a mother's breathing motion 39 (see FIG. 3 ). Said air compressor unit 35 and associated controls are powered by a plurality of rechargeable or disposable batteries 50 contained therewithin a battery compartment 38 or receive power therefrom a removably attachable AC adapter 54 subsequently connected thereto a common household 110-volt AC circuit in an expected manner (see FIG. 4 ). The base unit 30 forms an inner oval-shaped cavity in which an insert 20 may be removably and snuggly placed therein when utilizing the apparatus 10 thereto a newborn infant. The insert 20 comprises a particular size and contour being designed to safely secure a newborn infant (see FIG. 2 ). Referring now to FIG. 2, a front perspective view of an insert portion 20 of the apparatus 10, according to the preferred embodiment of the present invention, is disclosed. The insert 20 comprises an insert side panel 21, an insert floor 22, and an insert rim 23. The insert 20 comprises an assembly of molded foam sections of particular sizes and contours designed to gently cradle and secure a newborn infant. The insert 20 and its components are envisioned to be made using hypoallergenic polyurethane foam rubber portions providing waterproof and washable outer surfaces being common in the industry and attached thereto each other using various FDA approved non-toxic adhesives. The insert side panel 21 comprises a perimeter section which forms a generally vertical and continuous surface around the insert 20 being angled outwardly along an upper edge region forming an insert rim 23. The external shape of the insert side panel 21 further comprise contours which correspond in a parallel manner thereto corresponding inner surfaces of the base unit 30, thereby fitting snuggly thereinto said base unit 30. The inside surfaces of the insert side panel 21 slope downwardly and are affixed thereto a horizontal insert floor portion 22. The insert floor 22 comprises a generally oval shape comprising ample padding, thereby providing a comfortable and safe sleeping surface for a baby. The insert floor 22 further comprises an elevated head area which provides a wide surface comprising a reduced padding area to contact an infant's head and facial areas, thereby providing for unobstructed and clear breathing. The insert floor 22 then slopes slightly downward to form a lower and narrower foot area with additional padding. Positioned along a top perimeter edge of the insert 12 is a padded insert rim 23 which provides a protective and decorative border to the insert 12. Referring now to FIGS. 3 a and 3 b, a front perspective and a section view of a base portion 30 of the apparatus 10, according to the preferred embodiment of the present invention, are disclosed. The base unit 30 comprises a base wall 31, a base rim 34, a battery compartment 38, an air compressor unit 35, an ON/OFF switch 37, a pneumatic control module 36, and a rheostatic control knob 40. The base unit 30 comprises a plastic open-topped enclosure forming a generally oval-shape enclosure approximately fourteen (14) inches wide and thirty (30) inches long with a vertical base wall 31 being approximately eight (8) inches high all around. The base wall 31 is envisioned being made of a semi-rigid plastic material such as polypropylene, acrylonitrile butadiene styrene (ABS), or the like, thereby supporting a base bladder 32 affixed all around an inward facing surface thereof. Affixed therealong a bottom edge portion of the base wall 31 is a base floor 33. The base floor 33 comprises a padded surface to comfortably support an infant without impeding normal breathing. Located along an upper edge of said base wall 31 is a base rim 34. The base rim 34 comprises a rounded edge region affixed thereto the base wall 31 using common adhesives and providing a protective and decorative padded border to the base unit 30 while supporting the insert rim portion 23 of the insert 20 when being utilized. The base wall 31 and base rim 34 are envisioned to be made using dense hypoallergenic polyurethane foam rubber sections providing waterproof and washable outer surfaces being common in the industry and assembled using various FDA approved non-toxic adhesives. The base unit 30 is envisioned being provided in a variety of decorative colors and patterns. The base bladder 32 provides an oscillating breathing motion 39 via an inflating/deflating system comprising an air compressor unit 35, a solenoid vent valve 41, and a control module 36. During an inflating and deflating cycle, the base bladder 32 provides a gentle horizontally expanding and contracting breathing motion 39 directed toward a center point of the base unit 30, thereby emulating a mother's breathing. Additionally, said breathing motion 39 is mechanically transmitted thereinto the insert portion 20 when utilized. The bladder 32 comprises an annular-shaped sealed vessel envisioned to be made of a soft and compliant grade of hypoallergenic latex rubber or equivalent expandable material. The base bladder 32 is affixed along an outer surface thereof to the base wall 31 using FDA approved non-toxic adhesives. Located therebetween the front surface of the base wall 31 and the base bladder 32, in an anticipated foot area, is a compact air compressor unit 35 comprising outwardly facing control components including an ON/OFF switch 37 and a rheostatic control knob 40. The air compressor unit 35 provides an internal air filling means to the base bladder 32. The air compressor unit 35 is envisioned to be a compact unit similar to those used to inflate popular inflatable air mattresses. The air compressor unit 35 also comprises an integral pneumatic control module 36, a rheostatic control knob 40, and an on/off switch 37 being mounted thereto along a front surface of the base unit 30. The pneumatic control module 36 comprises an electronic control device providing a housing means thereto all necessary electrical and pneumatic components required to provide cyclic inflating and deflating of the base bladder 32 via a solenoid vent valve 41 (see FIG. 4 ). The external rheostatic control knob 40 provides manual frequency regulation of said oscillating inflating and deflating of said base bladder 32, thereby selectively simulating a mother's breathing pattern. The ON/OFF switch 37 initiates a flow of DC electrical power to the air compressor unit 35, and the rheostatic control knob 40. Said DC power is provided via a plurality of batteries 50 or an AC adapter 54 being inserted thereinto a common DC receptacle 52 mounted thereto the air compressor unit 35 along a front surface (see FIG. 4 ). The battery compartment 38 comprises expected features including a plastic flush-mounted access door which may be easily removed using a small tool to access said batteries 50. Referring now to FIG. 4, an electrical block diagram of the apparatus 10, according to the preferred embodiment of the present invention, is disclosed. DC power is supplied to the apparatus 10 via a plurality of batteries 50 or therefrom the AC adapter 54 which is subsequently connected thereto a common household 110-volt AC circuit. Said DC power is then conducted thereto a pneumatic control module 36. The pneumatic control module 36 comprises a circuit board which provides input/output capability and embedded software to energize the compressor motor 35 and activate the solenoid vent valve 41 in an alternating fashion, thereby creating an oscillating pneumatic cycle. An ON/OFF switch 37 provides an input signal to said pneumatic control module 36 to initiate said continuous cycles until said ON/OFF switch 37 is pressed again. The ON/OFF switch 37 is envisioned to be a simple panel-mount momentary contact closure device common in the industry. The pneumatic control module 36 also provides a means to control a frequency of said oscillating pneumatic cycle via a rheostatic control knob 40 which provides a variable voltage input thereto said pneumatic control module 36. The pneumatic control module 36 subsequently provides an output current thereto the motor portion of the air compressor unit 35 and thereto the solenoid vent valve 41 during operation of the apparatus 10. It is envisioned that other styles and configurations of the present invention can be easily incorporated into the teachings of the present invention, and only one particular configuration shall be shown and described for purposes of clarity and disclosure and not by way of limitation of scope. The preferred embodiment of the present invention can be utilized by the common user in a simple and effortless manner with little or no training. After initial purchase or acquisition of the apparatus 10, it would be installed as indicated in FIG. 1. The method of installing and utilizing the apparatus 10 may be achieved by performing the following steps: installing a fresh set of rechargeable or disposable batteries 50 thereinto the battery compartment 38 or, alternately, utilizing the AC adapter 54 using an available household outlet; placing the apparatus 10 preferably thereupon a non-traffic floor area or other safe flat surface; adding appropriate blankets and sheets required to obtain a desired sleeping temperature for an infant; pressing the ON/OFF switch 37 to initiate the air compressor unit 35 and solenoid vent valve 41, thereby producing the simulated breathing motion 39 ; regulating the frequency of said breathing motion 39 by turning the rheostatic control knob 40 thereto a desired oscillating rate; allowing continuous automatic functioning of the apparatus 10 as needed; pressing the ON/OFF switch 37 again to stop the breathing motion 39 ; retaining the infant therein the apparatus 10 or removing said infant therefrom the apparatus 10 as desired; and, benefiting from improved quality and duration of a baby or infant's sleeping experience using the present invention 10. An alternate method of utilizing the apparatus 10 using the insert portion 20 may be accomplished by performing the following additional steps: placing the insert 20 completely down into the base unit 30 if the apparatus 10 is being used with a newborn baby; placing a baby thereinto said insert 20 giving care to position said baby's head at the proper end of the padded insert floor 22. The foregoing descriptions of specific embodiments of the present invention have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the invention and method of use to the precise forms disclosed. Obviously many modifications and variations are possible in light of the above teaching. The embodiment was chosen and described in order to best explain the principles of the invention and its practical application, and to thereby enable others skilled in the art to best utilize the invention and various embodiments with various modifications as are suited to the particular use contemplated. It is understood that various omissions or substitutions of equivalents are contemplated as circumstance may suggest or render expedient, but is intended to cover the application or implementation without departing from the spirit or scope of the claims of the present invention. | Summary: A sleeper apparatus for holding an infant designed to simulate the feeling of being held in a caregiver's arms is herein disclosed. The sleeper is similar in shape to that of a canoe and is able to accommodate most infants via a removable insert section. The insert may be added or removed, depending on the size of the infant. The sleeper is made using padded foam materials and comprises an air bladder located along an inside wall of a perimeter enclosure. The bladder is connected to a DC-powered air pump, which pumps air in and out of the bladder to simulate human breathing. The inner space of the sleeper is contoured to conform to the babies shape. The head area is larger with minimal padding to provide for unobstructed and clear breathing. The foot area is slightly declined and would be provided with additional padding. The sleeper provides a comforting and secure resting place for infants. | 5,496 | 203 | big_patent | en |
Write a title and summarize: Myelination is essential for rapid impulse conduction in the CNS, but what determines whether an individual axon becomes myelinated remains unknown. Here we show, using a myelinating coculture system, that there are two distinct modes of myelination, one that is independent of neuronal activity and glutamate release and another that depends on neuronal action potentials releasing glutamate to activate NMDA receptors on oligodendrocyte lineage cells. Neuregulin switches oligodendrocytes from the activity-independent to the activity-dependent mode of myelination by increasing NMDA receptor currents in oligodendrocyte lineage cells 6-fold. With neuregulin present myelination is accelerated and increased, and NMDA receptor block reduces myelination to far below its level without neuregulin. Thus, a neuregulin-controlled switch enhances the myelination of active axons. In vivo, we demonstrate that remyelination after white matter damage is NMDA receptor-dependent. These data resolve controversies over the signalling regulating myelination and suggest novel roles for neuregulin in schizophrenia and in remyelination after white matter damage. Myelination is essential for normal brain function as myelin sheaths provide trophic support for axons and increase the conduction speed of action potentials [1]. By speeding axonal action potentials, myelin allows rapid information transfer between different regions of the CNS. Consequently, the increased amount of white matter in human brains is thought to contribute significantly to our cognitive powers. Myelination is controlled by a complex set of factors, including growth factors, interactions between axons and their myelinating glia, and downstream signalling processes within the glia [2]. Understanding the myelination process is crucial, not only to understand in full how action potential speed can be increased to enhance the brain' s cognitive power, but also to develop therapeutic strategies for regenerating myelin in disease. Strikingly, however, we still do not know what determines whether an individual axon becomes myelinated. In particular, for the CNS, the roles of the growth factor neuregulin (NRG), neuronal activity, and neurotransmitter glutamate release from axons are highly debated. NRG on axons can regulate myelination by signalling to ErbB receptors on ensheathing Schwann cells or oligodendrocytes, but there is controversy over its actions. In the peripheral nervous system, a decrease of NRG signalling leads to less myelination [3], [4]. Decreasing NRG-ErbB signalling has also been reported to reduce myelination by oligodendrocytes in the central nervous system [5]–[8]. Contradicting this, however, knocking out NRG or ErbB was found to have no effect on myelination, although overexpressing NRG increased myelination [9]. Further uncertainty relates to how neuronal activity regulates CNS myelination. Neuronal activity can promote myelination [10], [11], yet oligodendrocytes can ensheath dead axons that lack any activity [12]. An activity dependence to myelination could reflect action potential evoked release of NRG [13], but glutamate is also released onto oligodendrocyte precursor cells by action potentials in unmyelinated axons [14]–[17] and could potentially initiate myelination. Glutamate activates both AMPA/kainate and NMDA receptors in oligodendrocyte lineage cells [14]–[18], and the presence of NMDA receptors in oligodendrocyte processes [18]–[20] is consistent with them having a role in myelination. Furthermore, glutamate signalling and NRG signalling might interact to control myelination, since, in the grey matter at least, NRG increases the expression of NMDA receptor subunits in neurons [21] and in forebrain [22]. However, contradictory data have been presented on the contribution of NMDA receptors to CNS myelination, with suggestions that they either play no role [23], [24] or that their activation by glutamate released from axons promotes myelin basic protein (MBP) expression and myelination [16], [25]. Here we show for the first time that there are two distinct modes of myelination by oligodendrocytes, one independent of neuronal activity and the other dependent on neuronal action potentials. Furthermore, we demonstrate that NRG switches oligodendrocytes between these two myelination programmes by increasing NMDA receptor-mediated currents in oligodendrocyte lineage cells, making these cells more sensitive to glutamate released from active axons. As a result, this interaction of NRG and glutamatergic signalling accelerates and increases myelination, but most importantly provides a mechanism by which myelination is focussed on the axons of active neurons. Investigations of white matter pathology based on this finding revealed that remyelination of axons in a demyelinated lesion in vivo is NMDA receptor dependent, suggesting enhancement of NRG- and NMDAR-dependent remyelination as a therapeutic approach for promoting recovery after demyelination. To assess whether NRG-ErbB signalling and activation of NMDA receptors in oligodendrocytes interact to control myelination, we studied myelination of cultured dorsal root ganglion (DRG) neurons by forebrain oligodendrocytes [8]. This allows more detailed investigation of the underlying signalling mechanisms than is possible in transgenic studies where compensation for gene knockout may occur (see Discussion). In this coculture system, compact myelin is produced (Figure S1A) [8] with a lamellar repeat distance of 10. 9±1. 8 nm (n = 6) and a g-ratio of 0. 74±0. 02 (n = 6), as expected for CNS myelination [26], [27]. Myelinating and nonmyelinating oligodendrocytes are distinguishable after labelling for MBP and neurofilament (NF) (Figure 1A, B). Myelinating oligodendrocytes enwrap NF-expressing axons with MBP-expressing processes, at the lateral ends of which the paranode labels for the axon-oligodendrocyte junction protein Caspr (Figure S1B). When two oligodendrocytes myelinate adjacent segments of the same axon, a node of Ranvier is identifiable by the presence of Caspr labelling flanked by MBP labelling on either side of the node (Figure S1C). Myelination does not occur in the absence of added OPCs, it is mediated purely by OPCs and not by Schwann cells, and only DRG neurons and not interneurons become myelinated (Figure S1D–F). ErbB signalling was activated by applying the extracellular domain of NRG (NRG1 type 1-β1,10 ng/ml, 0. 33 nM), which includes the EGF domain that binds to ErbB receptors expressed on oligodendrocytes [28] and mimics [3] the effect of membrane-bound NRG. As found previously [8], adding NRG increased the fraction of oligodendrocytes that myelinated axons (Figure 1C, E, Figure 2A). Myelination depends on the local axon density [8], so we quantified myelination (3 wk after plating OPCs on the neurons) as the slope of a plot of the fraction of oligodendrocytes that were myelinating against local axon density [8] (see Figure 1C–F, Figure 2A, and Materials and Methods; similar results were obtained with other quantification methods, see Figures S2 and S3). Measured in this way, adding NRG increased myelination by 42% (p = 7×10−4; Figure 2A). NRG also increased MBP protein expression in the cocultures (p = 0. 03; Figure S1G), without affecting MBP or MOG mRNA levels (Figure S1H), indicating a local posttranscriptional regulation of these myelin genes [16]. Varying the time at which myelination was assessed after plating the OPCs onto the neurons showed that NRG both accelerated myelination and increased its steady state level (Figure 2C). The myelination at steady-state level was 16% higher (p = 0. 024), and its onset (defined as the time to reach half the maximal value) occurred 5 d earlier (p = 0. 0037), according to the fitted curves in Figure 2C. To assess whether changes in cell proliferation, growth, or survival were responsible for the increased myelination in NRG, we counted cells of different types identified with antibodies. In the presence of NRG, there was no significant change in the density of axons (Figure 2B), the total number of DAPI-labelled cells (Figure 3A), the number of MBP-labelled oligodendrocytes (Figure 3B), or the percentage of cells that were oligodendrocyte lineage cells, OPCs, interneurons, astrocytes, or satellite cells (Figure 3C–G) in the cocultures. No microglia were detected using anti-CD11b antibody or isolectin B4. Thus, the effect of NRG is not due to an altered cell density or changes in environmental paracrine signals or trophic factors caused by altered cell numbers. To test the role of neuronal activity in regulating myelination we applied TTX (1 µM) to block action potentials. TTX had no effect on myelination in the absence of added NRG (p = 0. 65), but reduced myelination by 50% in the presence of NRG (p = 0. 006; Figure 2A) —that is, TTX abolished the increase in myelination produced by NRG (both when added at the same time as NRG and when added 3 d later; Figure S5A). Thus, whereas in the absence of NRG myelination occurs predominantly by a programme that is independent of neuronal activity, with NRG present myelination depends partly on action potentials. Because myelination can be promoted by neuronal activity [10], [11], we assessed whether NRG increased action potential activity in the DRG axons being myelinated in the cocultures, by voltage-clamping interneurons (Figure 4A) and measuring the frequency of synaptic input they received from DRG neurons (Figure 4B). NRG did not significantly affect the total frequency of (excitatory plus inhibitory) synaptic currents recorded at −64 mV with a high [Cl−]pipette (Figure 4B, C), 94% of which were blocked by TTX (Figure 4D) and so reflect action potential activity in the cocultures. At −70 mV with ECl set to −88 mV so that all inward currents are EPSCs from DRG cells (Figure 4E), there was no difference in the frequency of events in control and NRG cultures (Figure 4F; 25 µM NBQX+50 µM AP5 blocked the currents, as did TTX, while 10 µM GABAzine had essentially no effect on them; Figure 4E, G). Thus, NRG makes myelination of DRG axons become dependent on neuronal activity without increasing the level of that activity. These data suggest that NRG increases the oligodendrocyte sensitivity to events triggered by axonal action potentials. To investigate this idea, we tested the effects of glutamate receptor blockers. The NMDA receptor channel blocker MK-801 (10 µM) had no effect on myelination in the absence of added NRG (Figure 1C–D, Figure 2A; p = 0. 16) but reduced myelination by 82% when NRG was present (Figure 1E–F, Figure 2A; p = 3×10−15). In NRG+MK-801, myelination was reduced to only 25% of the level seen in control conditions in the absence of NRG (p = 4×10−9). The same results were obtained (without and with NRG) when the AMPA/kainate receptor blocker NBQX (25 µM) was added with the MK-801 (Figure 2A). Thus, NRG induces a switch in the main mode of myelination, from being completely independent of NMDA receptor activation in the absence of NRG to being mainly (82%) dependent on NMDA receptor activation when NRG is present. Similarly, the structurally unrelated NMDA receptor blocker 7-chlorokynurenate (30 µM) also had no effect in the absence of NRG (p = 0. 66) but reduced myelination in the presence of NRG by 80% (p = 4×10−6), that is, to only 28% of the control level with no NRG (p = 0. 002; Figure 2A). The blocker of the glutamate site on NMDA receptors, D-AP5 (200 µM) reduced myelination in NRG less than MK-801 or 7-chlorokynurenate (Figure 2A) for reasons discussed below. NBQX alone had no effect on myelination (p = 0. 58) in the absence of NRG, but reduced myelination by 46% in NRG (p = 0. 007; Figure 2A). Thus, the activity-dependent myelination induced by NRG depends on activation of NMDA and AMPA/kainate receptors. To assess how the effects of NRG and NMDA receptor block varied with the initial level of myelination, we took advantage of variations between cocultures in the control level of myelination. When myelination was low in control conditions the potentiation by NRG was large (reaching an extrapolated value of 2. 5±0. 2-fold at zero myelination), but the NRG effect became much smaller when the control level of myelination was large (Figure 2D). Strikingly, however, despite this variation in NRG' s potentiating action, the 5-fold reduction of myelination produced in NRG by blocking NMDA receptors with MK-801 occurred independently of the control level of myelination (Figure 2E). Thus, NRG makes myelination become highly dependent on the activation of NMDA receptors even when the increase in myelination produced by NRG is small. To assess which cells' glutamate receptors might be regulating myelination in the presence of NRG, we recorded from the cells in the cocultures at the time when myelination was measured (3 wk after adding the OPCs), using postrecording antibody labelling (Figure 4A, Figure 5A–F) and membrane I-V relations (Figure S4) to identify them. In cultures exposed to NRG, the membrane resistance of oligodendrocyte lineage cells was not altered (OPCs, control 836±369 MΩ versus NRG 894±483 MΩ, p = 0. 92; differentiated oligodendrocytes, control 130±25 MΩ versus NRG 208±65 MΩ, p = 0. 20). However, with added NRG the NMDA-evoked currents at −64 mV in oligodendrocyte precursor cells and in differentiated oligodendrocytes were ∼6-fold larger than in cells not exposed to added NRG (p = 0. 02 and p = 0. 03, respectively; Figure 5G–J). In contrast, kainate-evoked currents (mediated by AMPA/kainate receptors) were not significantly affected by NRG in either OPCs (p = 0. 48) or differentiated oligodendrocytes (p = 0. 73; Figure 5G–J). NRG did not significantly alter the size of NMDA- or kainate-evoked currents in DRG neurons (p = 0. 75 and p = 0. 93, respectively; Figure 5K), interneurons (p = 0. 30 and p = 0. 92; Figure 5L), astrocytes (p = 1 and p = 0. 78; Figure 5M), or satellite cells (p = 0. 62 and p = 0. 52; Figure 5N) that were also present in the cultures. These data indicate that NRG specifically upregulates NMDA responses in oligodendrocyte lineage cells, providing a mechanism for these cells to become more sensitive to glutamate released from active axons. NMDA receptors in oligodendrocyte lineage cells have been suggested to mainly comprise NR1, NR2C, and NR3A subunits [18]–[20], [29]. NR2B and NR2C are the main subunits known to be phosphorylated (by Fyn and Akt, respectively) downstream of NRG and other growth factor signalling [30], [31], and NR2C phosphorylation promotes subunit trafficking to the plasma membrane [31]. We therefore investigated whether the increase in NMDA responses reflected altered receptor subunit expression or phosphorylation, focussing on the possible role of NR2B, NR2C, and NR3A subunits (in fact, no NR2A or NR2D and very little NR3B were detected in the cocultures). Western blotting revealed unchanged levels of NR1, of NR2B and its phosphorylated form, of NR2C and its phosphorylated form, and of NR3B (Figure 6A, B). However, the level of NR3A protein was down-regulated by 40% in the presence of NRG (p = 0. 02; Figure 6A, B). NRG did not affect the NR3A protein level in pure DRG cultures (p = 0. 85; Figure 6C), but downregulated NR3A by 33% in pure OPC cultures provided that glutamate was also added to mimic glutamate release from DRG axons (p = 0. 02; Figure 6D and see Discussion). Removal of NR3A subunits from NMDA receptors composed of NR1, NR2, and NR3 subunits has previously been reported to increase their single channel current, their trafficking to the surface membrane, and their calcium permeability, and thus to increase NMDA-evoked currents 2. 8-fold [29], [32], [33]. Consequently, a down-regulation of NR3A synthesis or an increase of its degradation could account for the increased NMDA receptor current seen in NRG. Myelination depends on integrins [34], and integrins modulate NMDA receptor expression [35]. We therefore tested whether the NRG-evoked switch from activity-independent to NMDA receptor-dependent myelination depends on integrins, using an antibody to β1 integrin (1 µg/ml) to block its function [36]. This had no effect on myelination without added NRG (p = 0. 27), but in the presence of NRG it reduced myelination by 82% (p = 5×10−6; Figure 7A) to only 26% of the level seen without NRG (significantly lower, p = 0. 0024). Thus, unlike the activity-independent mode of myelination, the activity and NMDA receptor-dependent myelination induced by NRG is dependent on integrin function. NRG can activate the PI3K-Akt or MAPK/Erk pathways in oligodendrocytes [37]. To test which pathway mediated NRG' s effects, we measured the levels of phosphorylated Akt and Erk in the cocultures. NRG increased Akt phosphorylation by 43% (p = 0. 047; Figure 7B), with no effect on Erk phosphorylation (p = 0. 79; Figure 7C). In contrast in pure DRG cultures neither pAkt nor pErk levels were affected (Figure S5B, C). To test whether activation of Akt was downstream of the increased NMDA receptor activation produced by NRG, we measured the levels of pAkt and pErk in cocultures treated with MK-801 (Figure 7B, C). MK-801 had no effect on the actions of NRG on Akt: NRG added with MK-801 increased pAkt (p = 0. 01) with no effect on pErk (p = 0. 26), and there was no significant difference between the levels of pAkt in NRG, with or without MK-801 (p = 0. 50). Thus, activation of Akt does not occur downstream of NMDA receptor activation (it may be upstream of the NRG-evoked increase in NMDA receptor current, or on an independent NRG-activated pathway). NRG also increased by 65% (p = 0. 02) the proportion of Nkx2. 2-expressing cells (late OPCs/immature oligodendrocytes) that expressed phosphorylated CREB (pCREB), an Akt target [38], without affecting the percentage of cells lacking Nkx2. 2 that expressed pCREB (p = 0. 35; Figure 7D, E). Next we looked into CREB target genes. NRG increased more than 4-fold the message level for the early growth response gene Egr-1 (p = 0. 036; Figure 7F), a CREB target gene [39] that has been shown to be regulated by integrin β1-dependent PI3K/Akt activation [40], neuronal activity [41], activation of NMDA receptors [42], and activation of OPC glutamate receptors [43]. EGR-1 is an upstream regulator of MRF [44], an essential transcription factor needed for myelination [45]. NRG more than doubled the level of the CREB target c-Fos, but this did not reach significance (p = 0. 06) and had no effect on the level of c-Jun (p = 0. 36; Figure 7F). These data suggest that NRG promotes myelination via Akt, integrin β1, NMDA receptors, CREB, and EGR-1 signalling. This is consistent with constitutively active Akt promoting myelination [46], with β1 integrin activating Akt to promote myelination [34], and with CREB activating transcription of myelin genes [39], [47]. The role of NRG/ErbB signalling in CNS myelination is controversial, as decreasing NRG or ErbB function reduced myelination [5]–[7], while knocking NRG or ErbB out had no effect on myelination [9]. Because of the importance of myelination, there may be redundancy in the mechanisms regulating it. Like NRG, BDNF increases NMDA receptor currents in neurons by upregulating NR2C subunits [48], and via TrkB it can promote oligodendrocyte differentiation, expression of MBP [49], and CNS myelination [50]. We therefore tested the effect of adding BDNF (10 ng/ml, 0. 7 nM) to the cocultures. BDNF increased myelination (by 102%, p = 0. 006; Figure 7G) and MBP expression (p = 0. 048; Figure S1G) and, as for NRG, made myelination become dependent on activation of NMDA receptors (Figure 7G). With BDNF present, MK-801 reduced myelination by 76% (p = 6. 9×10−4), to only 47% of its value without BDNF (p = 0. 019). Like NRG, BDNF induced phosphorylation of Akt but not Erk (Figure 7H–I; p = 0. 03 and p = 0. 2, respectively), and downregulated expression of the NR3A subunit but not the NR1 subunit of NMDA receptors (Figure 7J; p = 0. 005 and p = 0. 69, respectively). Thus, by acting through similar signalling pathways, BDNF can also switch oligodendrocyte myelination to a pathway that depends on glutamate release activating NMDA receptors. Consequently, when the NRG/ErbB pathway is knocked out, myelination may still occur [9] not only via the activity-independent mode of myelination (Figure 2A) but also because of compensatory BDNF signalling (see Discussion). Our demonstration of NRG- and NMDAR-dependent myelination of axons by oligodendrocytes raises the question of whether this same mechanism operates during remyelination after white matter damage. A lack of NRG signalling has been suggested to result in poor remyelination in multiple sclerosis [51], [52]. The data above suggest this may reflect a lack of NRG-dependent upregulation of NMDA receptors in oligodendrocyte lineage cells, but NMDA receptor deletion or block has been variously reported to either have no effect on loss of myelin in the experimental autoimmune encephalomyelitis model of multiple sclerosis [24] or to delay remyelination after cuprizone demyelination [25]. We therefore tested whether successful remyelination is dependent on NMDA receptor activation, in OPCs that are recruited to remyelinate axons in a focal toxin-induced demyelinated lesion in the rat caudal cerebellar peduncle. This demyelination model provides successful spontaneous remyelination that occurs with a clear temporal separation from the acute demyelination [53]. An intracerebral implanted cannula, connected to an osmotic minipump, infused into the lesion either 0. 9% saline or 50 µM MK-801 (at a flow rate of 0. 11 µl/h) from the 3rd day postlesion (the timepoint when OPCs enter the lesion, [54]) until the animal was sacrificed at 21 d postlesion. Analysis of semithin sections stained with toluidine blue showed that there was no difference in lesion size (saline, 0. 28±0. 04 mm2; MK-801,0. 22±0. 04 mm2, p = 0. 28) nor axon density (saline, 50,800±2,100 axons/mm2; MK-801,47,500±1,900 axons/mm2, p = 0. 27) between the conditions, but a blinded analysis ranking remyelination revealed that blocking NMDA receptors with MK-801 significantly inhibited remyelination (p = 0. 036; Figure 8A–C). Moreover, at the ultrastructural level, it was clear that fewer axons were remyelinated when NMDA receptors were blocked (p = 0. 0012; Figure 8D–F), and the g-ratio (the ratio of axon diameter to outside diameter of the myelin) of remyelinated axons was higher in MK-801– compared to saline-infused lesions (p = 0. 01; Figure 8G–I), showing that the myelin was thinner. Thus, efficient remyelination depends on activation of NMDA receptors. The speeding of action potential propagation produced by myelination of axons is crucial for CNS function. We have shown that, in the absence of added NRG, myelination takes place by a mechanism that is independent of neuronal activity and glutamate release, because it is unaffected by blocking action potentials or ionotropic glutamate receptors. With NRG added, myelination is accelerated and increased, this effect is dependent on action potentials, and blocking NMDA receptors greatly reduces myelination (to well below the level seen in the absence of added NRG; Figures 1 and 2). Thus, the key result of this article is that NRG does not just speed and increase myelination; it produces a switch in the mechanism of myelination, from a default programme that is independent of neuronal activity (which allows oligodendrocytes to ensheath even fixed axons [12]) to a programme that depends on activation of NMDA receptors in oligodendrocyte lineage cells, presumably by glutamate released from active axons [14]–[18], [55] (although we cannot rule out a contribution of glutamate release, either tonic or activity-induced, from other cells). This switch implies a suppression of the default programme when the activity-dependent programme is activated by NRG. Furthermore, the influence of NMDA receptors on myelination is not limited to myelination occurring during normal development, – because remyelination of demyelinated axons in the cerebellar peduncle in vivo also depends on NMDA receptor activation (Figure 8). Adding NRG increases 6-fold the NMDA-evoked currents in oligodendrocyte precursor cells and in differentiated oligodendrocytes, but does not significantly alter NMDA-evoked currents in DRG neurons, nor NMDA- or kainate-evoked currents in any other cell type in the culture (Figure 5), nor the action potential firing of DRG axons (Figure 4). We therefore attribute the NRG-induced activity dependence of myelination to a potentiation of NMDA receptor signalling in oligodendrocyte lineage cells, making the cells more sensitive to axonal activity. This potentiation may largely reflect a decreased protein level of NR3A subunits (Figure 6). However, the 6-fold increase in NMDA receptor-mediated current that we find is larger than the 2. 8-fold increase reported in neurons when NR3A is knocked out [32]. Consequently, because of possible obscuring of changes in oligodendrocyte protein levels by neuronal NMDA receptor protein in the cocultures, we cannot rule out a contribution to the increase in NMDA-evoked current from alterations of the levels of other NMDA receptor subunits in oligodendrocyte lineage cells, of which upregulation of NR2C [21] and/or NR2B [30] subunits are the most likely candidates. In the presence of NRG, blocking action potentials reduces myelination less than does blocking NMDA receptors (Figure 2A), despite the fact that it is presumably action potentials that release the glutamate that activates the NMDA receptors. Thus, in NRG, TTX reduces myelination to a value close to that occurring without NRG present (Figures 2A and S5A). This may reflect action potential evoked glutamate release being needed for NRG to increase NMDA receptor currents, so that the switch to NMDA receptor-dependent myelination does not occur in the absence of action potentials. This is likely since NMDA receptor activation is needed both for removal of NR3A subunits from the surface membrane [56] and for NRG to upregulate NR2C subunits in neurons [21]. Consistent with this, blocking the glutamate site on NMDA receptors with D-AP5 reduced myelination in NRG less than did MK-801 or 7-chlorokynurenate, which block at other sites on NMDARs (Figure 2A), and in pure OPC cultures, NRG downregulated NR3A only if glutamate was also added (Figure 6D). The inhibitory effect of NBQX in NRG in Figure 2A may similarly reflect AMPA/kainate receptor activity maintaining neuronal firing, which evokes the glutamate release needed to upregulate NMDA currents [21], [56], or could reflect AMPA/kainate receptor-mediated depolarisation increasing current flow through NMDA receptors [18]. The exact sequence of signalling steps in oligodendrocytes that upregulates NMDA receptor currents, and thus speeds, increases, and confers activity-dependence upon myelination, remains to be defined, although we have shown that Akt, integrin β1, CREB, and EGR-1 are involved (Figure 7). Since NRG activates Akt (Figure 7B) and Fyn [30] and these kinases are known to alter NMDA receptor subunit expression and trafficking [30], [31] and to promote myelination [16], [46], [57], it is an attractive idea that NRG may initially activate Akt and/or Fyn, and that this action is potentiated by integrins (Figure 7A) [34], [35], [57]. Our results help resolve three controversies in the field. The first concerns the role of NRG in CNS myelination. Decreasing NRG or ErbB function reduces myelination [5]–[7], and in the prefrontal cortex, NRG' s control of myelination is regulated by social interactions [58]. However, knocking NRG or ErbB out had no effect on myelination, even though overexpressing NRG increased myelination [9]. Our data predict that myelination can appear to be unaffected when NRG-ErbB signalling is abolished, for three reasons. First, the activity-independent mode of myelination (Figure 2A) should still occur. Second, when myelination levels are high, as may be the case in vivo, NRG only modestly increases myelination (Figure 2D), yet it still makes myelination become very highly dependent on neuronal activity releasing glutamate to activate NMDA receptors (Figure 2E). Third, our data indicate redundancy in the growth factors that switch oligodendrocytes between the two myelination modes. Both NRG and BDNF alter NMDA receptor expression in oligodendrocyte lineage cells, induce Akt activation but not Erk activation (although promotion of myelination by BDNF can also involve Erk activation [59]), and promote myelination, and both switch oligodendrocytes to the NMDA receptor-dependent mode of myelination (Figures 2A and 7G). Since not only ErbB receptors for NRG but also TrkB receptors for BDNF (including full length receptors [49], [50], [60]–[62]) can be expressed by oligodendrocytes, our data suggest that the failure of NRG or ErbB knockout to affect CNS myelination may, in part, reflect another growth factor (BDNF) and its receptor (TrkB) acting to replace the NRG/ErbB system. The second controversy concerns the role of NMDA receptors in CNS myelination. Deleting the NMDA receptor NR1 subunit from oligodendrocyte lineage cells has been reported to have no effect on myelination [23], [24], yet activation of NMDA receptors by glutamate released from active axons is reported to promote MBP translation and myelination [16]. Interestingly, in another oligodendrocyte-specific NR1 knockout, deletion of NMDA receptors slowed myelination in the optic nerve [63], consistent with the acceleration of myelination that we observe when NMDA receptor currents are increased in the presence of NRG (Figure 2C). This effect of NMDA receptors was proposed to be caused by NMDA receptor activation upregulating glucose transporters in oligodendrocyte lineage cells (as was previously shown to occur in an Akt-dependent manner in neurons; [64]) to provide energy for myelination [63]. The third controversy concerns whether myelination is activity dependent or independent. Rearing rodents in the dark from birth, or injecting TTX into the optic nerve, can reduce or alternatively have no effect on myelination [10], [65]–[67]. Similarly, electrical activity [10], [16], or application of factors it releases [11], can promote myelination, and blocking neuronal activity with TTX in myelinating cortical cultures reduced myelination [10], whereas in spinal cord cultures TTX did not affect myelination [68]. Presumably, these differences may reflect the existence of the two alternative myelination programmes that we have characterised (whereby in the absence of added NRG, TTX has no effect on myelination, but in NRG-treated cultures, it reduces myelination by 50%; Figure 2A). Whether myelination is predominantly activity dependent or independent may depend on the amount of NRG or BDNF (or other molecules [69], [70]) being expressed or released [21], [71]. In this context it is worth noting that, although we have discussed our results as reflecting the presence or absence of NRG, in the absence of added NRG, there will be some level of endogenous NRG release, and it may be better to think of whether the NRG level is low or high as determining the main myelination mode that occurs. It is also possible that the switch from one mode to the other occurs gradually as the NRG level rises. The existence of two alternative myelination programmes regulated by NRG level, independent of, and depending on, action potentials and NMDA receptors, may reflect the evolutionary importance, but also the metabolic cost, of myelination. One can speculate that early in development it may be important to myelinate whatever axons are present, irrespective of their impulse traffic. However, once a significant density of axons is present, because myelination involves considerable investment by the oligodendrocyte in lipid production [72], it is more efficient for myelination to be focussed on axons that have a high impulse traffic, rather than on inactive axons. The NRG- and NMDA receptor-dependent mode of myelination may dominate later in development, since late myelination in the prefrontal cortex depends on NRG signalling [58] and adult remyelination depends on NMDA receptor activation (Figure 8). NRG is a susceptibility gene for schizophrenia [22], [73], and NMDA receptors are also implicated in this disease [74]. NRG affects NMDA receptor expression in the grey matter [21], [22], where the defect underlying schizophrenia is usually assumed to occur. However, correct myelination is essential for normal cognitive function, and the interaction of NRG and NMDA receptors to control myelination allows us to speculate that there could perhaps be a white matter explanation for the linkage of NRG and NMDA receptors to schizophrenia. Furthermore, NRG expression is reduced in multiple sclerosis lesions [52], and adding NRG has been suggested to promote remyelination in a mouse model of multiple sclerosis [51], suggesting that NRG- and NMDA receptor-dependent remyelination may be important after pathology. Consistent with this we have shown that, in vivo in the cerebellar peduncle, successful remyelination is reduced when NMDA receptors are blocked (Figure 8) and similar results have been found for remyelination in the corpus callosum [25]. Promoting NRG- and NMDA receptor-dependent myelination may, therefore, be a useful therapeutic strategy for increasing CNS remyelination in disease. These were made as described previously [8]. Briefly, DRG cells from E14–E16 rats were cultured for 2 wk, and then cultured oligodendrocyte precursor cells (OPCs) from P0–P2 rats were plated on top of them [8]. One coverslip was analysed for each drug condition per dissection. Three weeks later, the cocultures were fixed, labelled, and images were taken of each coverslip to assess myelination (see detailed description below) [8]. NRG (Lab Vision) or BDNF (R&D systems) and receptor blockers (Tocris) were applied with the OPCs (except when TTX was added 3 d later than the NRG and OPCs) and were included in medium changes twice per week thereafter. Culture medium contained 0. 8 mM MgCl2 and 0. 4 mM glycine, but no added glutamate. As described above, DRG cells from E14–E16 rats were cultured for 2 wk, then put in myelination medium (as for cocultures but lacking OPCs), for 3 wk, with medium changes twice per week thereafter. These were as described previously [8]. Briefly, purified oligodendrocyte precursors were obtained with minor modifications [36] of the method of McCarthy and de Vellis [75]. They were cultured in myelinating medium (the same as for the cocultures) for 6 d, either with or without 20 min 100 µM glutamate stimulation every day for the last 5 d in vitro. For each coverslip, 30 randomly located images were taken of MBP and NF staining (using either a 10× objective, with a field of view of 843 µm×636 µm, or a 20× objective, with a field of view of 709 µm×530 µm). Within each image, myelination was quantified by counting the number of myelinating MBP-positive oligodendrocytes (Figure 1B, C) as a percentage of the total number of MBP-positive oligodendrocytes [8]. The density of axons was expressed as the percentage of the same area occupied by NF (calculated from a binarised NF image using ImageJ software, version 1. 34s). There was no correlation between oligodendrocyte density and axon density. The fraction of myelinating cells was plotted against the axon density for the different images, and fitted assuming a linear dependence [8] of fraction of oligodendrocytes myelinating (F) on axon density (D), (1) where K is a free constant and the slope A is the measure of myelination plotted in Figure 2, Figure S2A, Figure S3A, and Figure 7. Fitting data in control and NRG conditions gave values for K that were not significantly different (12. 9±1. 3%, n = 32 experiments, for control, and 12. 6±1. 8%, n = 27, for NRG; p = 0. 88). This quantification of myelination gave results similar to those obtained using other assumed dependencies of fraction of oligodendrocytes myelinating (F) on axon density (D) as follows: Irrespective of the quantification used, the bar charts in different conditions (Figure S3A–C) gave the same results: blocking action potentials or NMDA receptors in control conditions had no effect on myelination, NRG significantly increased myelination, and in the presence of NRG blocking action potentials produced a significant reduction of myelination, while blocking NMDA receptors reduced myelination so strongly that it was significantly reduced below the control level with no NRG. If, instead of fitting the dependence of myelination on local axon density, we simply counted the fraction of oligodendrocytes that myelinated, by averaging over all the sampled images, then the results were broadly similar, except that NMDA receptor block in control conditions produced a reduction of myelination of borderline significance (Figure S3D). Cells were whole-cell patch-clamped [14], [18] at room temperature (21–24°C) in cocultures 3 wk after the OPCs were added. Electrodes contained solution comprising (mM) either 126 CsCl, 4 NaCl, 10 HEPES, 5 EGTA, 4 MgATP, 0. 5 Na2GTP, 12 phosphocreatine, 2 K-Lucifer yellow, pH set to 7. 3 with CsOH (ECl = 0 mV), or 130 Cs-gluconate, 4 NaCl, 0. 5 CaCl2,10 HEPES, 10 BAPTA, 4 MgATP, 0. 5 Na2GTP, 2 K-Lucifer yellow, pH set to 7. 3 with CsOH (ECl = −88 mV). Series resistance was 5–20 MΩ, and electrode junction potentials were compensated. Cultures were superfused at 24±1°C with HEPES-buffered solution containing (mM) 144 NaCl, 2. 5 KCl, 10 HEPES, 1 NaH2PO4,2. 5 CaCl2,10 glucose, 0. 1 glycine (to coactivate NMDA receptors), 0. 005 strychnine (to block glycine receptors), pH set to 7. 4 with NaOH, bubbled with 100% O2. Cells were identified (Figures 4A and 5A–F) by their postrecording dye-fill morphology [14], [18], confirmed by antibody labelling against the proteoglycan NG2 to identify oligodendrocyte precursors (17/17 tested labelled for NG2), against CNPase (11/11), MBP (10/10), MOG (4/4), or GalC (11/11) for differentiated oligodendrocytes, against GAD 65/67 for interneurons (21/21), against NF 160/200 (13/13) for DRG neurons, against GFAP for astrocytes (11/11), and against SCIP for satellite cells (10/10). In addition we checked that each recorded cell had the electrophysiological properties expected for its class: specimen responses to voltage steps from the resting potential are shown for each cell class in Figure S4. OPC morphology cells, including those confirmed as being OPCs by virtue of their labelling with antibody against NG2, fell into two classes [14] with (57% of cells) and without (43% of cells) voltage-gated Na+ current (Figure S4A–D), and had a steady-state input resistance of ∼800 MΩ. Mature myelinating oligodendrocytes had a roughly ohmic and time-independent I-V relation in the physiological range (Figure S4E), with a much lower input resistance of ∼160 MΩ. Interneurons and DRG neurons both showed a voltage-gated Na+ current on depolarization, which was too large to clamp well, so the I-V relations showed current oscillations reflecting uncontrolled action potentials occurring (Figure S4F, G). Both satellite cells and astrocytes showed roughly ohmic and time-independent I-V relations in the physiological range (Figure S4H, I) with a mean input resistance near the resting potential of ∼900 and ∼15 MΩ, respectively. A synaptic current was defined to occur if its amplitude was >3 times the standard deviation of the baseline current noise and its 10%–90% decay time was longer than its rise time. Events were detected and analysed with pClamp 10 (Axon Instruments). Cultures were fixed at 21°C for 20 min in 4% PFA and incubated for 1 h in 0. 1% Triton X-100,10% goat serum in phosphate-buffered saline at 21°C, then with primary antibody at 21°C for 2 h or for overnight at 4°C, and then for 1 h at 21°C with secondary antibody. Primary antibodies were guinea pig NG2 (a kind gift from W. B. Stallcup, 1∶100), rabbit NG2 (Chemicon, 1∶300), rabbit Olig2 (a kind gift from D. Rowitch, C. D. Stiles & J. Alberta, 1∶20,000, or Chemicon, 1∶1,000), rabbit Caspr (a kind gift from D. Colman & J. Huang, 1∶500), rabbit SCIP (a kind gift from J. R. Bermingham, 1∶100), rabbit GalC (Sigma, 1∶100), mouse NF 160/200 (Sigma, 1∶1,000), rat MBP (Serotec, 1∶100), mouse MOG (Sigma, 1∶100), mouse CNPase (Sigma, 1∶100), rabbit GAD 65/67 (Chemicon, 1∶100), rabbit GFAP (Dako, 1∶500), chicken P0 (Aves Labs, 1∶500), mouse Nkx2. 2 (DSHB, 1∶120), CD11b (Serotec, 1∶50), and rabbit pCREB (Cell Signalling, 1∶50). Alexa 488–conjugated isolectin B4 (Invitrogen, 1∶100) was used to label microglia. DAPI (Sigma, 20 µM) was used to label nuclei. Secondary antibodies (goat) were for rabbit (Molecular Probes, 1∶1,000), rat IgG (Molecular Probes, 1∶1,000), mouse IgG (Molecular Probes, 1∶1,000), chicken (Jackson Lab, 1∶1,000), and guinea pig (Jackson Lab, 1∶100). For protein analysis, cultures were scraped from 22 mm coverslips, 6 well plates, or 10 cm dishes and lysed mechanically in solution containing 0. 1 M phosphate buffered saline (PBS), 10% or 20% (w/v) sucrose and Halt protease, and phosphatase inhibitor cocktail (Thermo Scientific). Protein content was determined by Bradford assay and quantified using a standard curve obtained from Quick Start BSA protein standards (Bio-Rad). Equal amounts of protein (8 or 10 µg) from samples were resolved on 4–%12% NuPage Novex Bis-Tris mini gels (Invitrogen), with prestained molecular weight protein standards (Bio-Rad). Proteins were transferred to a nitrocellulose membrane (0. 45 µm, GE Healthcare) using a wet transfer system. Nitrocellulose membranes were blocked for 1 h at room temperature with 3% BSA in PBS with 0. 1% Tween-20 (PBS-T). Immunoblots were then incubated overnight at 4°C with goat anti-Akt (Santa Cruz, 1∶1,000), rabbit anti-phosphorylated-AktSer473 (Cell Signalling, 1∶1,000), rabbit anti-phosphorylated-ERK1/2Thr202/Tyr214 (Cell Signalling, 1∶1,000), mouse anti-ERK1/2 (Cell Signalling, 1∶2,000), rabbit anti-MBP (Sigma, 1∶3,000), mouse anti-NR1 (Millipore, 1∶1,000), rabbit anti-NR2A (Millipore, 1∶1,000), rabbit anti-NR2B (Abcam; 1∶1,000), rabbit anti-phosphorylated NR2BTyr1472 (Millipore, 1∶1,000), rabbit anti-NR2C (Millipore, 1∶1,000), rabbit anti-phosphorylated NR2CS1096 (a kind gift from Katherine W. Roche, NINDS), rabbit anti-NR3A (Millipore, 1∶500), rabbit anti-NR3B (Millipore, 1∶300), rabbit anti-NR2D (Abcam, 1∶300), or mouse anti-β-actin (Sigma, 1∶100,000) in 3% BSA in PBS-T. This was followed by incubation with the secondary horseradish peroxidise-linked anti-rabbit, anti-mouse, or anti-goat antibodies (1∶1,000, Dako) for 1 h at room temperature in 3% BSA in PBS-T. Immunoreactive proteins were visualised with enhanced chemiluminescence (GE Healthcare). When phosphorylated and total amounts of the same protein were measured, this was done by first probing with the antibody to the phosphorylated protein, then stripping the membranes to remove the antibody, and reprobing with antibody recognizing both phosphorylated and unphosphorylated forms. Stripping membranes was performed with 50 mM dithiothreitol, 50 mM Tris (pH 6. 8), and 2% SDS at 70°C for 30 min and washing three times in PBS-T before blocking and incubating with the primary antibody. Densitometric analysis was conducted using ImageJ gel analysis software (version 1. 43u). For each sample, pAkt, pERK, pNR2B, and pNR2C signals were normalised to the total levels of their respective proteins present. The levels of nonphosphorylated proteins were normalized against β-actin. RNA was extracted from DRG-OPC cocultures 21 d after addition of OPCs using an RNeasy mini kit (QIAGEN). RNA was converted to cDNA using a SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). qPCR was performed with a QuantiFast SYBR Green PCR Kit (QIAGEN) using a LightCycler 480 II. Samples were normalised to GAPDH levels using commercially available QuantiTect Primers (QIAGEN) for analysis. Female Sprague-Dawley rats aged 9–12 wk of age (200–225 g) were used for remyelination studies, which were performed in compliance with UK Home Office regulations. Focal demyelination was induced unilaterally by stereotaxically injecting 4. 0 µl of 0. 01% ethidium bromide (w/v) in saline into the caudal cerebellar peduncle (CCP) (as described previously [53]). For continuous local delivery of MK-801 (50 µM, Tocris, in 0. 9% saline) or 0. 9% saline (Vetivex) into the demyelinated lesion, an osmotic minipump with a reservoir volume of 100 µl and a flow rate of 0. 11 µl/h (Alzet Micro-Osmotic Pumps, model 1004, DURECT Corporation) was attached through a vinyl tube spacer (Plastics One Inc., Roanoke, Virginia) to a 30 gauge (6. 5 mm) cannula implanted just above the lesion. The length of the tube was cut to 2. 3 cm, to ensure that drug delivery into the lesion did not occur until the 3rd day postlesion (the start of the OPC recruitment stage [54]). The minipump was placed subcutaneously, and the cannula was fixed to the skull with cyanoacrylate gel adhesive (applied under the base of the cannula head before insertion), as well as two anchoring screws and dental acrylic cement (a 1∶1 volume mix of Paladur powder and liquid; Heraeus Kulzer). Rats were randomly assigned to treatment (50 µM MK-801) or control groups (0. 9% saline infusion). Animals were sacrificed 3 wk after lesion induction. Co-cultures were fixed at room temperature for 1 h in PBS containing 2. 5% glutaraldehyde (Agar Scientific) and 0. 72 mM CaCl2. The cocultures were scraped off the coverslips, placed into 1. 5 ml tubes with 250 µl of PBS, and centrifuged at 800 g for 3 min. For lesion samples, animals were perfusion fixed with 4% glutaraldehyde (Agar Scientific) in phosphate buffer containing 0. 72 mM CaCl2. All samples were then left to fix in 2% osmium tetroxide (Oxkem Ltd) in phosphate buffer at 4°C overnight. This was followed by dehydration in 70% ethanol for 15 min, 95% ethanol for 15 min, and 100% ethanol for 3×10 min, then the tissue was placed in propylene oxide for 2×15 min, and then left for at least 3 h in a 50%/50% mixture of propylene oxide and resin mix (containing by volume: 49% TAAB embedding resin, 33% DDSA, 16% MNA, 2% DMP-30; TAAB Laboratories Equipment Ltd.). They were then transferred to 100% resin mix overnight. New resin was made and the samples left in it for at least 6 h before being placed in embedding capsules and further incubated at 60°C for 15–24 h until the resin was solid. Embedded samples were cut in 90 nm sections on an ultramicrotome (Reichert Ultracut E) with a diamond knife (Diatome) and visualised using a Transmission Electron Microscope (Hitachi H600). Images were developed on electron microscope film 4489 (Kodak), then scanned at high resolution (12,800 dpi×12,800 dpi), and analyzed with ImageJ software, version 1. 34s. Animals were perfused with 4% glutaraldehyde (in phosphate buffer with 0. 72 mM CaCl2), and the brains were immersion fixed in 4% glutaraldehyde for 7 d. Tissue blocks encompassing the caudal cerebellar peduncle were cut as detailed previously [53]. While maintaining their correct orientation and sequence, blocks were further fixed in 2% osmium tetroxide (Oxkem Ltd.), dehydrated in increasing concentration of ethanol, and embedded in resin (TAAB Laboratories). One micron sections were cut and stained with toluidine blue. In these sections, remyelinated axons can be easily distinguished from normally myelinated axons outside the lesion by the thinness of the myelin sheath. Within the lesion, remyelinated axons can be distinguished from demyelinated axons because the former possess myelin sheaths recognizable as a dark staining rim around the axon. Data are mean ± s. e. m. The p values are from ANOVA and post hoc Student' s two-tailed t tests. For multiple comparisons p values are corrected using a procedure equivalent to the Holm–Bonferroni method (for N comparisons, the most significant p value is multiplied by N, the 2nd most significant by N–1, the 3rd most significant by N–2, etc. ; corrected p values are significant if they are less than 0. 05) or Dunnett' s post hoc test. Normality of data was assessed using Shapiro–Wilk tests, and nonparametric Kruskal–Wallis and Mann–Whitney tests, which do not assume data follow a normal distribution, gave the same conclusions for significant and nonsignificant differences in all cases. For Figure 2, when the ANOVA gave an insignificant p value (>0. 05), we used the more conservative Student' s two-tailed t test without correction for multiple comparisons to give a lower limit to the p value for post hoc comparisons (i. e., the differences among the control myelination data in Figure 2A are even less significant than is indicated by the p values). | Title: Neuregulin and BDNF Induce a Switch to NMDA Receptor-Dependent Myelination by Oligodendrocytes Summary: Myelination acts as an insulator for neurons and as such is essential for normal brain function, ensuring fast neuronal communication. Oligodendrocytes are the cells that wrap their membrane around nerve cell axons to form the myelin sheath that enables fast action potential propagation. However, what determines whether an individual axon becomes myelinated remains unknown. We show that there are two distinct modes of myelination: one that is independent of neuronal activity and the release of the neurotransmitter glutamate and another that depends on nerve cell action potentials releasing glutamate, which then activates a class of glutamate receptor (NMDA receptors) on oligodendrocyte lineage cells. We find that the protein neuregulin switches oligodendrocytes between these two modes of myelination; neuregulin increases oligodendrocyte lineage cells' sensitivity to glutamate by increasing the current flowing through their glutamate receptors. With neuregulin present, myelination is accelerated and increased. Blocking NMDA receptors reduces the amount of myelination to far below its level without neuregulin. Thus, a neuregulin-controlled switch enhances the myelination of active axons. We also demonstrate that remyelination after white matter damage (as occurs in diseases, such as spinal cord injury and multiple sclerosis) is NMDA receptor-dependent. These data help us understand the signalling that regulates myelination and suggest the possible involvement of neuregulin in schizophrenia and in remyelination after white matter damage. | 14,262 | 379 | lay_plos | en |
Summarize: ACT III (SCENE.—The editorial office of the "People's Messenger." The entrance door is on the left-hand side of the back wall; on the right-hand side is another door with glass panels through which the printing room can be seen. Another door in the right-hand wall. In the middle of the room is a large table covered with papers, newspapers and books. In the foreground on the left a window, before which stands a desk and a high stool. There are a couple of easy chairs by the table, and other chairs standing along the wall. The room is dingy and uncomfortable; the furniture is old, the chairs stained and torn. In the printing room the compositors are seen at work, and a printer is working a handpress. HOVSTAD is sitting at the desk, writing. BILLING comes in from the right with DR. STOCKMANN'S manuscript in his hand.) Billing. Well, I must say! Hovstad (still writing). Have you read it through? Billing (laying the MS. on the desk). Yes, indeed I have. Hovstad. Don't you think the Doctor hits them pretty hard? Billing. Hard? Bless my soul, he's crushing! Every word falls like—how shall I put it?—like the blow of a sledgehammer. Hovstad. Yes, but they are not the people to throw up the sponge at the first blow. Billing. That is true; and for that reason we must strike blow upon blow until the whole of this aristocracy tumbles to pieces. As I sat in there reading this, I almost seemed to see a revolution in being. Hovstad (turning round). Hush!—Speak so that Aslaksen cannot hear you. Billing (lowering his voice). Aslaksen is a chicken-hearted chap, a coward; there is nothing of the man in him. But this time you will insist on your own way, won't you? You will put the Doctor's article in? Hovstad. Yes, and if the Mayor doesn't like it— Billing. That will be the devil of a nuisance. Hovstad. Well, fortunately we can turn the situation to good account, whatever happens. If the Mayor will not fall in with the Doctor's project, he will have all the small tradesmen down on him—the whole of the Householders' Association and the rest of them. And if he does fall in with it, he will fall out with the whole crowd of large shareholders in the Baths, who up to now have been his most valuable supporters— Billing. Yes, because they will certainly have to fork out a pretty penny— Hovstad. Yes, you may be sure they will. And in this way the ring will be broken up, you see, and then in every issue of the paper we will enlighten the public on the Mayor's incapability on one point and another, and make it clear that all the positions of trust in the town, the whole control of municipal affairs, ought to be put in the hands of the Liberals. Billing. That is perfectly true! I see it coming—I see it coming; we are on the threshold of a revolution! (A knock is heard at the door.) Hovstad. Hush! (Calls out.) Come in! (DR. STOCKMANN comes in by the street door. HOVSTAD goes to meet him.) Ah, it is you, Doctor! Well? Dr. Stockmann. You may set to work and print it, Mr. Hovstad! Hovstad. Has it come to that, then? Billing. Hurrah! Dr. Stockmann. Yes, print away. Undoubtedly it has come to that. Now they must take what they get. There is going to be a fight in the town, Mr. Billing! Billing. War to the knife, I hope! We will get our knives to their throats, Doctor! Dr. Stockmann. This article is only a beginning. I have already got four or five more sketched out in my head. Where is Aslaksen? Billing (calls into the printing-room). Aslaksen, just come here for a minute! Hovstad. Four or five more articles, did you say? On the same subject? Dr. Stockmann. No—far from it, my dear fellow. No, they are about quite another matter. But they all spring from the question of the water supply and the drainage. One thing leads to another, you know. It is like beginning to pull down an old house, exactly. Billing. Upon my soul, it's true; you find you are not done till you have pulled all the old rubbish down. Aslaksen (coming in). Pulled down? You are not thinking of pulling down the Baths surely, Doctor? Hovstad. Far from it, don't be afraid. Dr. Stockmann. No, we meant something quite different. Well, what do you think of my article, Mr. Hovstad? Hovstad. I think it is simply a masterpiece. Dr. Stockmann. Do you really think so? Well, I am very pleased, very pleased. Hovstad. It is so clear and intelligible. One need have no special knowledge to understand the bearing of it. You will have every enlightened man on your side. Aslaksen. And every prudent man too, I hope? Billing. The prudent and the imprudent—almost the whole town. Aslaksen. In that case we may venture to print it. Dr. Stockmann. I should think so! Hovstad. We will put it in tomorrow morning. Dr. Stockmann. Of course—you must not lose a single day. What I wanted to ask you, Mr. Aslaksen, was if you would supervise the printing of it yourself. Aslaksen. With pleasure. Dr. Stockmann. Take care of it as if it were a treasure! No misprints—every word is important. I will look in again a little later; perhaps you will be able to let me see a proof. I can't tell you how eager I am to see it in print, and see it burst upon the public— Billing. Burst upon them—yes, like a flash of lightning! Dr. Stockmann. —and to have it submitted to the judgment of my intelligent fellow townsmen. You cannot imagine what I have gone through today. I have been threatened first with one thing and then with another; they have tried to rob me of my most elementary rights as a man— Billing. What! Your rights as a man! Dr. Stockmann. —they have tried to degrade me, to make a coward of me, to force me to put personal interests before my most sacred convictions. Billing. That is too much—I'm damned if it isn't. Hovstad. Oh, you mustn't be surprised at anything from that quarter. Dr. Stockmann. Well, they will get the worst of it with me; they may assure themselves of that. I shall consider the "People's Messenger" my sheet-anchor now, and every single day I will bombard them with one article after another, like bombshells— Aslaksen. Yes, but Billing. Hurrah!—it is war, it is war! Dr. Stockmann. I shall smite them to the ground—I shall crush them—I shall break down all their defenses, before the eyes of the honest public! That is what I shall do! Aslaksen, Yes, but in moderation, Doctor—proceed with moderation. Billing. Not a bit of it, not a bit of it! Don't spare the dynamite! Dr. Stockmann. Because it is not merely a question of water-supply and drains now, you know. No—it is the whole of our social life that we have got to purify and disinfect— Billing. Spoken like a deliverer! Dr. Stockmann. All the incapables must be turned out, you understand—and that in every walk of life! Endless vistas have opened themselves to my mind's eye today. I cannot see it all quite clearly yet, but I shall in time. Young and vigorous standard-bearers—those are what we need and must seek, my friends; we must have new men in command at all our outposts. Billing. Hear hear! Dr. Stockmann. We only need to stand by one another, and it will all be perfectly easy. The revolution will be launched like a ship that runs smoothly off the stocks. Don't you think so? Hovstad. For my part I think we have now a prospect of getting the municipal authority into the hands where it should lie. Aslaksen. And if only we proceed with moderation, I cannot imagine that there will be any risk. Dr. Stockmann. Who the devil cares whether there is any risk or not! What I am doing, I am doing in the name of truth and for the sake of my conscience. Hovstad. You are a man who deserves to be supported, Doctor. Aslaksen. Yes, there is no denying that the Doctor is a true friend to the town—a real friend to the community, that he is. Billing. Take my word for it, Aslaksen, Dr. Stockmann is a friend of the people. Aslaksen. I fancy the Householders' Association will make use of that expression before long. Dr. Stockmann (affected, grasps their hands). Thank you, thank you, my dear staunch friends. It is very refreshing to me to hear you say that; my brother called me something quite different. By Jove, he shall have it back, with interest! But now I must be off to see a poor devil—I will come back, as I said. Keep a very careful eye on the manuscript, Aslaksen, and don't for worlds leave out any of my notes of exclamation! Rather put one or two more in! Capital, capital! Well, good-bye for the present—goodbye, goodbye! (They show him to the door, and bow him out.) Hovstad. He may prove an invaluably useful man to us. Aslaksen. Yes, so long as he confines himself to this matter of the Baths. But if he goes farther afield, I don't think it would be advisable to follow him. Hovstad. Hm!—that all depends— Billing. You are so infernally timid, Aslaksen! Aslaksen. Timid? Yes, when it is a question of the local authorities, I am timid, Mr. Billing; it is a lesson I have learned in the school of experience, let me tell you. But try me in higher politics, in matters that concern the government itself, and then see if I am timid. Billing. No, you aren't, I admit. But this is simply contradicting yourself. Aslaksen. I am a man with a conscience, and that is the whole matter. If you attack the government, you don't do the community any harm, anyway; those fellows pay no attention to attacks, you see—they go on just as they are, in spite of them. But local authorities are different; they can be turned out, and then perhaps you may get an ignorant lot into office who may do irreparable harm to the householders and everybody else. Hovstad. But what of the education of citizens by self government—don't you attach any importance to that? Aslaksen. When a man has interests of his own to protect, he cannot think of everything, Mr. Hovstad. Hovstad. Then I hope I shall never have interests of my own to protect! Billing. Hear, hear! Aslaksen (with a smile). Hm! (Points to the desk.) Mr. Sheriff Stensgaard was your predecessor at that editorial desk. Billing (spitting). Bah! That turncoat. Hovstad. I am not a weathercock—and never will be. Aslaksen. A politician should never be too certain of anything, Mr. Hovstad. And as for you, Mr. Billing, I should think it is time for you to be taking in a reef or two in your sails, seeing that you are applying for the post of secretary to the Bench. Billing. I—! Hovstad. Are you, Billing? Billing. Well, yes—but you must clearly understand I am only doing it to annoy the bigwigs. Aslaksen. Anyhow, it is no business of mine. But if I am to be accused of timidity and of inconsistency in my principles, this is what I want to point out: my political past is an open book. I have never changed, except perhaps to become a little more moderate, you see. My heart is still with the people; but I don't deny that my reason has a certain bias towards the authorities—the local ones, I mean. (Goes into the printing room.) Billing. Oughtn't we to try and get rid of him, Hovstad? Hovstad. Do you know anyone else who will advance the money for our paper and printing bill? Billing. It is an infernal nuisance that we don't possess some capital to trade on. Hovstad (sitting down at his desk). Yes, if we only had that, then— Billing. Suppose you were to apply to Dr. Stockmann? Hovstad (turning over some papers). What is the use? He has got nothing. Billing. No, but he has got a warm man in the background, old Morten Kiil—"the Badger," as they call him. Hovstad (writing). Are you so sure he has got anything? Billing. Good Lord, of course he has! And some of it must come to the Stockmanns. Most probably he will do something for the children, at all events. Hovstad (turning half round). Are you counting on that? Billing. Counting on it? Of course I am not counting on anything. Hovstad. That is right. And I should not count on the secretaryship to the Bench either, if I were you; for I can assure you—you won't get it. Billing. Do you think I am not quite aware of that? My object is precisely not to get it. A slight of that kind stimulates a man's fighting power—it is like getting a supply of fresh bile—and I am sure one needs that badly enough in a hole-and-corner place like this, where it is so seldom anything happens to stir one up. Hovstad (writing). Quite so, quite so. Billing. Ah, I shall be heard of yet!—Now I shall go and write the appeal to the Householders' Association. (Goes into the room on the right.) Hovstad (sitting al his desk, biting his penholder, says slowly). Hm!—that's it, is it. (A knock is heard.) Come in! (PETRA comes in by the outer door. HOVSTAD gets up.) What, you!—here? Petra. Yes, you must forgive me— Hovstad (pulling a chair forward). Won't you sit down? Petra. No, thank you; I must go again in a moment. Hovstad. Have you come with a message from your father, by any chance? Petra. No, I have come on my own account. (Takes a book out of her coat pocket.) Here is the English story. Hovstad. Why have you brought it back? Petra. Because I am not going to translate it. Hovstad. But you promised me faithfully. Petra. Yes, but then I had not read it, I don't suppose you have read it either? Hovstad. No, you know quite well I don't understand English; but— Petra. Quite so. That is why I wanted to tell you that you must find something else. (Lays the book on the table.) You can't use this for the "People's Messenger." Hovstad. Why not? Petra. Because it conflicts with all your opinions. Hovstad. Oh, for that matter— Petra. You don't understand me. The burden of this story is that there is a supernatural power that looks after the so-called good people in this world and makes everything happen for the best in their case—while all the so-called bad people are punished. Hovstad. Well, but that is all right. That is just what our readers want. Petra. And are you going to be the one to give it to them? For myself, I do not believe a word of it. You know quite well that things do not happen so in reality. Hovstad. You are perfectly right; but an editor cannot always act as he would prefer. He is often obliged to bow to the wishes of the public in unimportant matters. Politics are the most important thing in life—for a newspaper, anyway; and if I want to carry my public with me on the path that leads to liberty and progress, I must not frighten them away. If they find a moral tale of this sort in the serial at the bottom of the page, they will be all the more ready to read what is printed above it; they feel more secure, as it were. Petra. For shame! You would never go and set a snare like that for your readers; you are not a spider! Hovstad (smiling). Thank you for having such a good opinion of me. No; as a matter of fact that is Billing's idea and not mine. Petra. Billing's! Hovstad. Yes; anyway, he propounded that theory here one day. And it is Billing who is so anxious to have that story in the paper; I don't know anything about the book. Petra. But how can Billing, with his emancipated views— Hovstad. Oh, Billing is a many-sided man. He is applying for the post of secretary to the Bench, too, I hear. Petra. I don't believe it, Mr. Hovstad. How could he possibly bring himself to do such a thing? Hovstad. Ah, you must ask him that. Petra. I should never have thought it of him. Hovstad (looking more closely at her). No? Does it really surprise you so much? Petra. Yes. Or perhaps not altogether. Really, I don't quite know Hovstad. We journalists are not much worth, Miss Stockmann. Petra. Do you really mean that? Hovstad. I think so sometimes. Petra. Yes, in the ordinary affairs of everyday life, perhaps; I can understand that. But now, when you have taken a weighty matter in hand— Hovstad. This matter of your father's, you mean? Petra. Exactly. It seems to me that now you must feel you are a man worth more than most. Hovstad. Yes, today I do feel something of that sort. Petra. Of course you do, don't you? It is a splendid vocation you have chosen—to smooth the way for the march of unappreciated truths, and new and courageous lines of thought. If it were nothing more than because you stand fearlessly in the open and take up the cause of an injured man— Hovstad. Especially when that injured man is—ahem!—I don't rightly know how to— Petra. When that man is so upright and so honest, you mean? Hovstad (more gently). Especially when he is your father I meant. Petra (suddenly checked). That? Hovstad. Yes, Petra—Miss Petra. Petra. Is it that, that is first and foremost with you? Not the matter itself? Not the truth?—not my father's big generous heart? Hovstad. Certainly—of course—that too. Petra. No, thank you; you have betrayed yourself, Mr. Hovstad, and now I shall never trust you again in anything. Hovstad. Can you really take it so amiss in me that it is mostly for your sake—? Petra. What I am angry with you for, is for not having been honest with my father. You talked to him as if the truth and the good of the community were what lay nearest to your heart. You have made fools of both my father and me. You are not the man you made yourself out to be. And that I shall never forgive you-never! Hovstad. You ought not to speak so bitterly, Miss Petra—least of all now. Petra. Why not now, especially? Hovstad. Because your father cannot do without my help. Petra (looking him up and down). Are you that sort of man too? For shame! Hovstad. No, no, I am not. This came upon me so unexpectedly—you must believe that. Petra. I know what to believe. Goodbye. Aslaksen (coming from the printing room, hurriedly and with an air of mystery). Damnation, Hovstad!—(Sees PETRA.) Oh, this is awkward— Petra. There is the book; you must give it to some one else. (Goes towards the door.) Hovstad (following her). But, Miss Stockmann— Petra. Goodbye. (Goes out.) Aslaksen. I say—Mr. Hovstad— Hovstad. Well well!—what is it? Aslaksen. The Mayor is outside in the printing room. Hovstad. The Mayor, did you say? Aslaksen. Yes he wants to speak to you. He came in by the back door—didn't want to be seen, you understand. Hovstad. What can he want? Wait a bit—I will go myself. (Goes to the door of the printing room, opens it, bows and invites PETER STOCKMANN in.) Just see, Aslaksen, that no one— Aslaksen. Quite so. (Goes into the printing-room.) Peter Stockmann. You did not expect to see me here, Mr. Hovstad? Hovstad. No, I confess I did not. Peter Stockmann (looking round). You are very snug in here—very nice indeed. Hovstad. Oh— Peter Stockmann. And here I come, without any notice, to take up your time! Hovstad. By all means, Mr. Mayor. I am at your service. But let me relieve you of your—(takes STOCKMANN's hat and stick and puts them on a chair). Won't you sit down? Peter Stockmann (sitting down by the table). Thank you. (HOVSTAD sits down.) I have had an extremely annoying experience to-day, Mr. Hovstad. Hovstad. Really? Ah well, I expect with all the various business you have to attend to— Peter Stockmann. The Medical Officer of the Baths is responsible for what happened today. Hovstad. Indeed? The Doctor? Peter Stockmann. He has addressed a kind of report to the Baths Committee on the subject of certain supposed defects in the Baths. Hovstad. Has he indeed? Peter Stockmann. Yes—has he not told you? I thought he said— Hovstad. Ah, yes—it is true he did mention something about— Aslaksen (coming from the printing-room). I ought to have that copy. Hovstad (angrily). Ahem!—there it is on the desk. Aslaksen (taking it). Right. Peter Stockmann. But look there—that is the thing I was speaking of! Aslaksen. Yes, that is the Doctor's article, Mr. Mayor. Hovstad. Oh, is THAT what you were speaking about? Peter Stockmann. Yes, that is it. What do you think of it? Hovstad. Oh, I am only a layman—and I have only taken a very cursory glance at it. Peter Stockmann. But you are going to print it? Hovstad. I cannot very well refuse a distinguished man. Aslaksen. I have nothing to do with editing the paper, Mr. Mayor— Peter Stockmann. I understand. Aslaksen. I merely print what is put into my hands. Peter Stockmann. Quite so. Aslaksen. And so I must— (moves off towards the printing-room). Peter Stockmann. No, but wait a moment, Mr. Aslaksen. You will allow me, Mr. Hovstad? Hovstad. If you please, Mr. Mayor. Peter Stockmann. You are a discreet and thoughtful man, Mr. Aslaksen. Aslaksen. I am delighted to hear you think so, sir. Peter Stockmann. And a man of very considerable influence. Aslaksen. Chiefly among the small tradesmen, sir. Peter Stockmann. The small tax-payers are the majority—here as everywhere else. Aslaksen. That is true. Peter Stockmann. And I have no doubt you know the general trend of opinion among them, don't you? Aslaksen. Yes I think I may say I do, Mr. Mayor. Peter Stockmann. Yes. Well, since there is such a praiseworthy spirit of self-sacrifice among the less wealthy citizens of our town— Aslaksen. What? Hovstad. Self-sacrifice? Peter Stockmann. It is pleasing evidence of a public-spirited feeling, extremely pleasing evidence. I might almost say I hardly expected it. But you have a closer knowledge of public opinion than I. Aslaksen. But, Mr. Mayor— Peter Stockmann. And indeed it is no small sacrifice that the town is going to make. Hovstad. The town? Aslaksen. But I don't understand. Is it the Baths—? Peter Stockmann. At a provisional estimate, the alterations that the Medical Officer asserts to be desirable will cost somewhere about twenty thousand pounds. Aslaksen. That is a lot of money, but— Peter Stockmann. Of course it will be necessary to raise a municipal loan. Hovstad (getting up). Surely you never mean that the town must pay—? Aslaksen. Do you mean that it must come out of the municipal funds?—out of the ill-filled pockets of the small tradesmen? Peter Stockmann. Well, my dear Mr. Aslaksen, where else is the money to come from? Aslaksen. The gentlemen who own the Baths ought to provide that. Peter Stockmann. The proprietors of the Baths are not in a position to incur any further expense. Aslaksen. Is that absolutely certain, Mr. Mayor? Peter Stockmann. I have satisfied myself that it is so. If the town wants these very extensive alterations, it will have to pay for them. Aslaksen. But, damn it all—I beg your pardon—this is quite another matter, Mr. Hovstad! Hovstad. It is, indeed. Peter Stockmann. The most fatal part of it is that we shall be obliged to shut the Baths for a couple of years. Hovstad. Shut them? Shut them altogether? Aslaksen. For two years? Peter Stockmann. Yes, the work will take as long as that—at least. Aslaksen. I'm damned if we will stand that, Mr. Mayor! What are we householders to live upon in the meantime? Peter Stockmann. Unfortunately, that is an extremely difficult question to answer, Mr. Aslaksen. But what would you have us do? Do you suppose we shall have a single visitor in the town, if we go about proclaiming that our water is polluted, that we are living over a plague spot, that the entire town— Aslaksen. And the whole thing is merely imagination? Peter Stockmann. With the best will in the world, I have not been able to come to any other conclusion. Aslaksen. Well then I must say it is absolutely unjustifiable of Dr. Stockmann—I beg your pardon, Mr. Mayor. Peter Stockmann. What you say is lamentably true, Mr. Aslaksen. My brother has unfortunately always been a headstrong man. Aslaksen. After this, do you mean to give him your support, Mr. Hovstad? Hovstad. Can you suppose for a moment that I—? Peter Stockmann. I have drawn up a short resume of the situation as it appears from a reasonable man's point of view. In it I have indicated how certain possible defects might suitably be remedied without outrunning the resources of the Baths Committee. Hovstad. Have you got it with you, Mr. Mayor? Peter Stockmann (fumbling in his pocket). Yes, I brought it with me in case you should— Aslaksen. Good Lord, there he is! Peter Stockmann. Who? My brother? Hovstad. Where? Where? Aslaksen. He has just gone through the printing room. Peter Stockmann. How unlucky! I don't want to meet him here, and I had still several things to speak to you about. Hovstad (pointing to the door on the right). Go in there for the present. Peter Stockmann. But—? Hovstad. You will only find Billing in there. Aslaksen. Quick, quick, Mr. Mayor—he is just coming. Peter Stockmann. Yes, very well; but see that you get rid of him quickly. (Goes out through the door on the right, which ASLAKSEN opens for him and shuts after him.) Hovstad. Pretend to be doing something, Aslaksen. (Sits down and writes. ASLAKSEN begins foraging among a heap of newspapers that are lying on a chair.) Dr. Stockmann (coming in from the printing room). Here I am again. (Puts down his hat and stick.) Hovstad (writing). Already, Doctor? Hurry up with what we were speaking about, Aslaksen. We are very pressed for time today. Dr. Stockmann (to ASLAKSEN). No proof for me to see yet, I hear. Aslaksen (without turning round). You couldn't expect it yet, Doctor. Dr. Stockmann. No, no; but I am impatient, as you can understand. I shall not know a moment's peace of mind until I see it in print. Hovstad. Hm!—It will take a good while yet, won't it, Aslaksen? Aslaksen. Yes, I am almost afraid it will. Dr. Stockmann. All right, my dear friends; I will come back. I do not mind coming back twice if necessary. A matter of such great importance—the welfare of the town at stake—it is no time to shirk trouble, (is just going, but stops and comes back.) Look here—there is one thing more I want to speak to you about. Hovstad. Excuse me, but could it not wait till some other time? Dr. Stockmann. I can tell you in half a dozen words. It is only this. When my article is read tomorrow and it is realised that I have been quietly working the whole winter for the welfare of the town— Hovstad. Yes but, Doctor— Dr. Stockmann. I know what you are going to say. You don't see how on earth it was any more than my duty—my obvious duty as a citizen. Of course it wasn't; I know that as well as you. But my fellow citizens, you know—! Good Lord, think of all the good souls who think so highly of me—! Aslaksen. Yes, our townsfolk have had a very high opinion of you so far, Doctor. Dr. Stockmann. Yes, and that is just why I am afraid they—. Well, this is the point; when this reaches them, especially the poorer classes, and sounds in their ears like a summons to take the town's affairs into their own hands for the future... Hovstad (getting up). Ahem! Doctor, I won't conceal from you the fact— Dr. Stockmann. Ah I—I knew there was something in the wind! But I won't hear a word of it. If anything of that sort is being set on foot— Hovstad. Of what sort? Dr. Stockmann. Well, whatever it is—whether it is a demonstration in my honour, or a banquet, or a subscription list for some presentation to me—whatever it is, you most promise me solemnly and faithfully to put a stop to it. You too, Mr. Aslaksen; do you understand? Hovstad. You must forgive me, Doctor, but sooner or later we must tell you the plain truth— (He is interrupted by the entrance Of MRS. STOCKMANN, who comes in from the street door.) Mrs. Stockmann (seeing her husband). Just as I thought! Hovstad (going towards her). You too, Mrs. Stockmann? Dr. Stockmann. What on earth do you want here, Katherine? Mrs. Stockmann. I should think you know very well what I want. Hovstad, Won't you sit down? Or perhaps— Mrs. Stockmann. No, thank you; don't trouble. And you must not be offended at my coming to fetch my husband; I am the mother of three children, you know. Dr. Stockmann. Nonsense!—we know all about that. Mrs. Stockmann. Well, one would not give you credit for much thought for your wife and children today; if you had had that, you would not have gone and dragged us all into misfortune. Dr. Stockmann. Are you out of your senses, Katherine! Because a man has a wife and children, is he not to be allowed to proclaim the truth-is he not to be allowed to be an actively useful citizen—is he not to be allowed to do a service to his native town! Mrs. Stockmann. Yes, Thomas—in reason. Aslaksen. Just what I say. Moderation in everything. Mrs. Stockmann. And that is why you wrong us, Mr. Hovstad, in enticing my husband away from his home and making a dupe of him in all this. Hovstad. I certainly am making a dupe of no one— Dr. Stockmann. Making a dupe of me! Do you suppose I should allow myself to be duped! Mrs. Stockmann. It is just what you do. I know quite well you have more brains than anyone in the town, but you are extremely easily duped, Thomas. (To Hovstad.) Please do realise that he loses his post at the Baths if you print what he has written. Aslaksen. What! Hovstad. Look here, Doctor! Dr. Stockmann (laughing). Ha-ha!—just let them try! No, no—they will take good care not to. I have got the compact majority behind me, let me tell you! Mrs. Stockmann. Yes, that is just the worst of it—your having any such horrid thing behind you. Dr. Stockmann. Rubbish, Katherine!—Go home and look after your house and leave me to look after the community. How can you be so afraid, when I am so confident and happy? (Walks up and down, rubbing his hands.) Truth and the People will win the fight, you may be certain! I see the whole of the broad-minded middle class marching like a victorious army—! (Stops beside a chair.) What the deuce is that lying there? Aslaksen Good Lord! Hovstad. Ahem! Dr. Stockmann. Here we have the topmost pinnacle of authority! (Takes the Mayor's official hat carefully between his finger-tips and holds it up in the air.) Mrs. Stockmann. The Mayor's hat! Dr. Stockmann. And here is the staff of office too. How in the name of all that's wonderful—? Hovstad. Well, you see— Dr. Stockmann. Oh, I understand. He has been here trying to talk you over. Ha-ha!—he made rather a mistake there! And as soon as he caught sight of me in the printing room. (Bursts out laughing.) Did he run away, Mr. Aslaksen? Aslaksen (hurriedly). Yes, he ran away, Doctor. Dr. Stockmann. Ran away without his stick or his—. Fiddlesticks! Peter doesn't run away and leave his belongings behind him. But what the deuce have you done with him? Ah!—in there, of course. Now you shall see, Katherine! Mrs. Stockmann. Thomas—please don't—! Aslaksen. Don't be rash, Doctor. (DR. STOCKMANN has put on the Mayor's hat and taken his stick in his hand. He goes up to the door, opens it, and stands with his hand to his hat at the salute. PETER STOCKMANN comes in, red with anger. BILLING follows him.) Peter Stockmann. What does this tomfoolery mean? Dr. Stockmann. Be respectful, my good Peter. I am the chief authority in the town now. (Walks up and down.) Mrs. Stockmann (almost in tears). Really, Thomas! Peter Stockmann (following him about). Give me my hat and stick. Dr. Stockmann (in the same tone as before). If you are chief constable, let me tell you that I am the Mayor—I am the master of the whole town, please understand! Peter Stockmann. Take off my hat, I tell you. Remember it is part of an official uniform. Dr. Stockmann. Pooh! Do you think the newly awakened lionhearted people are going to be frightened by an official hat? There is going to be a revolution in the town tomorrow, let me tell you. You thought you could turn me out; but now I shall turn you out—turn you out of all your various offices. Do you think I cannot? Listen to me. I have triumphant social forces behind me. Hovstad and Billing will thunder in the "People's Messenger," and Aslaksen will take the field at the head of the whole Householders' Association— Aslaksen. That I won't, Doctor. Dr. Stockmann. Of course you will— Peter Stockmann. Ah!—may I ask then if Mr. Hovstad intends to join this agitation? Hovstad. No, Mr. Mayor. Aslaksen. No, Mr. Hovstad is not such a fool as to go and ruin his paper and himself for the sake of an imaginary grievance. Dr. Stockmann (looking round him). What does this mean? Hovstad. You have represented your case in a false light, Doctor, and therefore I am unable to give you my support. Billing. And after what the Mayor was so kind as to tell me just now, I— Dr. Stockmann. A false light! Leave that part of it to me. Only print my article; I am quite capable of defending it. Hovstad. I am not going to print it. I cannot and will not and dare not print it. Dr. Stockmann. You dare not? What nonsense!—you are the editor; and an editor controls his paper, I suppose! Aslaksen. No, it is the subscribers, Doctor. Peter Stockmann. Fortunately, yes. Aslaksen. It is public opinion—the enlightened public—householders and people of that kind; they control the newspapers. Dr. Stockmann (composedly). And I have all these influences against me? Aslaksen. Yes, you have. It would mean the absolute ruin of the community if your article were to appear. Dr. Stockmann. Indeed. Peter Stockmann. My hat and stick, if you please. (DR. STOCKMANN takes off the hat and lays it on the table with the stick. PETER STOCKMANN takes them up.) Your authority as mayor has come to an untimely end. Dr. Stockmann. We have not got to the end yet. (To HOVSTAD.) Then it is quite impossible for you to print my article in the "People's Messenger"? Hovstad. Quite impossible—out of regard for your family as well. Mrs. Stockmann. You need not concern yourself about his family, thank you, Mr. Hovstad. Peter Stockmann (taking a paper from his pocket). It will be sufficient, for the guidance of the public, if this appears. It is an official statement. May I trouble you? Hovstad (taking the paper). Certainly; I will see that it is printed. Dr. Stockmann. But not mine. Do you imagine that you can silence me and stifle the truth! You will not find it so easy as you suppose. Mr. Aslaksen, kindly take my manuscript at once and print it as a pamphlet—at my expense. I will have four hundred copies—no, five or six hundred. Aslaksen. If you offered me its weight in gold, I could not lend my press for any such purpose, Doctor. It would be flying in the face of public opinion. You will not get it printed anywhere in the town. Dr. Stockmann. Then give it me back. Hovstad (giving him the MS.). Here it is. Dr. Stockmann (taking his hat and stick). It shall be made public all the same. I will read it out at a mass meeting of the townspeople. All my fellow-citizens shall hear the voice of truth! Peter Stockmann. You will not find any public body in the town that will give you the use of their hall for such a purpose. Aslaksen. Not a single one, I am certain. Billing. No, I'm damned if you will find one. Mrs. Stockmann. But this is too shameful! Why should every one turn against you like that? Dr. Stockmann (angrily). I will tell you why. It is because all the men in this town are old women—like you; they all think of nothing but their families, and never of the community. Mrs. Stockmann (putting her arm into his). Then I will show them that an old woman can be a man for once. I am going to stand by you, Thomas! Dr. Stockmann. Bravely said, Katherine! It shall be made public—as I am a living soul! If I can't hire a hall, I shall hire a drum, and parade the town with it and read it at every street-corner. Peter Stockmann. You are surely not such an errant fool as that! Dr. Stockmann. Yes, I am. Aslaksen. You won't find a single man in the whole town to go with you. Billing. No, I'm damned if you will. Mrs. Stockmann. Don't give in, Thomas. I will tell the boys to go with you. Dr. Stockmann. That is a splendid idea! Mrs. Stockmann. Morten will be delighted; and Ejlif will do whatever he does. Dr. Stockmann. Yes, and Petra!—and you too, Katherine! Mrs. Stockmann. No, I won't do that; but I will stand at the window and watch you, that's what I will do. Dr. Stockmann (puts his arms round her and kisses her). Thank you, my dear! Now you and I are going to try a fall, my fine gentlemen! I am going to see whether a pack of cowards can succeed in gagging a patriot who wants to purify society! (He and his wife go out by the street door.) Peter Stockmann (shaking his head seriously). Now he has sent her out of her senses, too. | Summary: Stockmann and his family are at home, cleaning up rocks people had thrown through their windows. It seems the glazier will not come to fix their windows, and, as Stockmann realizes when he reads a letter, their landlord evicted them. He tries to comfort Catherine that they will be happier in America but she is unconvinced. Petra comes home and announces she lost her teaching job because her supervisor received anonymous letters, one stating that she had radical ideas. Horster comes in. He is friendly, asking how the family is doing. His news, though, is not good -the ship will sail for America but he will not be on it because he was removed from his position. He says he will find another, but it seemed like the owner was a political ally of Peter. Peter himself arrives at the house, and he and Stockmann converse. Peter gives his brother a letter that fires him from the Institute. Stockmann accuses his brother of wanting to ruin him, but Peter admits he thinks it has gone too far. However, he adds that he thinks this will all go away if Stockmann will sign a statement apologizing for going overboard. Stockmann scoffs that his brother still does not understand that there are men one cannot buy. Peter retorts that he does not seem to be one of those men, and explains that Morten Kiil had gone about buying up cheap stock in the Springs last night after the meeting. Stockmann is perplexed and said he knew nothing about it, but Peter is hostile and does not believe him. He says he will draw up charges of conspiracy against Stockmann if he has to. Incredulous and irate, Stockmann starts yelling about that a maid needs to clean the room of such pestilence. Peter leaves and Kiil enters afterward. Stockmann confronts him on this and Kiil explains. His tannery, the one passed down through his family, is above the Springs and is responsible for sullying them. He used the money that he was setting aside for Catherine and the family as an inheritance to buy up stock. His hope is that Stockmann will clear his name, but he still thinks his son-in-law's theories about poison are crazy. Stockmann is aghast that his father-in-law would do this and tries to explain about the science behind the testing. Kiil nettles him, trying to get him to admit he could have even the slightest doubt about the tests' veracity. He insinuates that Stockmann was only doing this to get back at his brother. Stockmann says this is ludicrous, and that he cannot believe Kiil gambled away his family's future. After more harsh words, Kiil leaves. Hovstad and Aslaksen enter. Stockmann gruffly tells them he has a lot on his mind and they should get to the point of why they are there. In essence, they want the Doctor to help fund their paper and they will support him. However, when Stockmann mentions the tax they get annoyed and say that the paper and the town would be bankrupted. Stockmann does not budge, and mocks Hovstad for wanting to be a hero. Hovstad becomes incensed, and calls the Doctor a "madman" who is "insane with egoism". He prepares to leave, and Aslaksen nervously leaves a proposed budget for the Doctor to look at. At that moment Stockmann's sons come in. Morten was beaten up because other students called his father a traitor and he would not stand for it. Stockmann tries to comfort his son, and tells the newspapermen to leave. He officially announces that he is an enemy of the people. He says, "If the only way I can be a friend of the people is to take charge of that corruption, then I am an enemy!". Hovstad asks in amazement if he knows where this will end. Stockmann rages on -he is not a hero, he is an enemy of the people and he will do everything in his power to tell the truth even if the people must bleed for it. The other men leave, in shock, and Stockmann gathers his family close. He tells them they are besieged but will not retreat. The boys will stay out of school and he will teach them. They should find about twelve street kids too. Stockmann pauses, almost about to say that Kiil should be told, but realizes they are all alone. A rock crashes in the window. Catherine is frightened, but Stockmann says that they have the truth, which makes them strong, and they must get used to being lonely. | 9,516 | 1,013 | booksum | en |
Summarize: Afghanistan's Deputy Foreign Minister Jawed Ludin talks on the phone before an interview in Kabul March 27, 2013. KABUL (Reuters) - Afghanistan is shocked by Pakistan's "complacency" in the nascent Afghan peace process and is ready to work without Islamabad's help on reconciliation, Deputy Foreign Minister Jawed Ludin told Reuters on Wednesday. It was the first time Afghanistan has suggested the possibility of going it alone without its neighbor. Regional power Pakistan is seen as critical to stabilizing Afghanistan because of its long ties to insurgent groups. Ludin also said the government would look to senior Taliban figures recently handed over by the United States in Bagram prison to urge militants to pursue peace. He did not elaborate. Afghan officials had been pushing Pakistan hard to encourage the Taliban and other groups to join reconciliation efforts and Kabul had spoken of progress after Islamabad released some Taliban prisoners who could promote peace. But Ludin, widely believed to shape foreign policy, told Reuters in an interview that Afghanistan had noted a shift in Pakistan's position towards peace efforts that are gaining more urgency as foreign forces prepare to leave by the end of 2014. "We here in Kabul are in a bit of a state of shock at once again being confronted by the depth of Pakistan's complacency, we are just very disappointed," he said. "But what has happened in the last few months for us, (we)see that Pakistan is changing the goal post every time we reach understanding." Afghanistan also said it had canceled a military trip to Pakistan due to "unacceptable Pakistani shelling" of the country's mountainous eastern borderlands. More than two dozen Pakistani artillery shells were fired into Afghanistan's eastern province of Kunar on Monday and Tuesday. The cancellation of the trip and days of angry diplomatic exchanges have placed further strain on a fraught relationship. Afghanistan expressed its concern about what it called Pakistan's attempt to sideline President Hamid Karzai's government to U.S. Secretary of State John Kerry during his visit to Kabul this week, said Ludin. The deputy foreign minister, who is closely involved in peace efforts, said Afghanistan insisted that its High Peace Council, formed by Karzai, should spearhead any peace efforts. Ludin said Pakistan had been trying to get the Taliban to talk to other parties, like the opposition, something he said would reverse gains. "Suddenly, there is a new notion of the peace process now being introduced by Pakistan and that's 'well why should the Taliban talk to President Karzai or the High Peace Council?'" said Ludin. "They (Taliban) should in fact talk to other political parties. That's what they have told us," Ludin said. "Pakistan's concept of the peace process is one that will reverse the achievement of the last 10 years that will negate the centrality of the Afghan state." Although there have been several meetings in Western capitals over the last few months in which representatives of the Taliban met Afghan peace negotiators, there are no signs of any breakthrough. RENEWED TENSIONS Karzai is due to visit the Gulf Arab state of Qatar soon to discuss the opening of a Taliban office that could be used for peace contacts in the future. Kabul has long been suspicious of Pakistan's intentions, accusing it of harboring the Taliban leadership in the city of Quetta, and using militants as proxies to counter the influence of rival India in Afghanistan. But a few months ago, Kabul was encouraged after Pakistan released some Afghan Taliban prisoners from its jails. Now relations seem to have taken a sharp turn for the worse as the United States winds down the war, now in its 12th year. This week, Pakistani officials accused Karzai of being an impediment to the peace process. Ludin suggested Pakistan wanted Afghanistan to remain unstable so that militant groups allegedly backed by Islamabad would be in a position to capitalize on instability after 2014. "What they would like is again a fragmentation of the Afghan state and going back to the drawing board so that they can have another 10 years, at least another decade, of weak, compromised Afghan state," he said. Ludin stressed that Pakistan was a pivotal player and Afghanistan would still welcome its support. "The sad reality is though Pakistan still remains the most important missing link in this whole vision that we have," he said. Karzai had worked too hard and taken too many political risks to let Pakistan dictate how peace efforts should proceed, Ludin said. "He (Karzai) has spent his political capital on this, he has basically staked his own political capital, his own reputation, on this and he has really mobilized the whole country, the whole region in support of the process," said Ludin. "It is laughable, laughable if Pakistanis think that the whole notion of the President Karzai is impediment to peace." Karzai's government, he said, would now turn to some of the most senior, hardcore Taliban leaders behind bars in Bagram in its quest for peace. "We will try to appeal to them and say: 'Look, continued reluctance of Taliban to stay away from peace process and any buy in to this whole Pakistani design that they should not to speak to the Afghan government, is dangerous,'" said Ludin. "Now that we have them we will see who is ready to help in this process." (Reporting by Michael Georgy and Hamid Shalizi; Editing by Ron Popeski) ISLAMABAD — Pakistan's intelligence agency has accused the Afghan government of supporting Taliban splinter groups. In a report presented to Pakistan’s Supreme Court on Tuesday, the ISI agency alleged President Hamid Karzai’s administration was in league with groups linked to the main Tehrik-e-Taliban Pakistan movement, known collectively as the TTS. The report suggested the "recent nexus of TTS with Afghan government is likely to enhance the terrorist activities" in areas along the Pakistan-Afghanistan border such as Mohman, Bajaur, Dir, Swat and Chitral. Jason Reed / Reuters Secretary of State John Kerry, left, listens to Afghanistan's President Hamid Karzai during their joint news conference at the presidential palace in Kabul on Thursday. Anti-Pakistan elements, particularly from across the border in Afghanistan, had provided "strong support" in terms of money, logistics and training and this was “one of the main factors for increased militancy,” the report said. However, it added that the Taliban’s ability to act "at will and to face security forces openly has been substantially curtailed." The report said that internal rifts within the main Pakistani Taliban group had led to the creation of splinter groups. "TTS, after having been dislodged from area, has resorted to [suicide bomb and improvised explosive device] attacks" on law-enforcement agencies and other officials, the report said. The court is considering a case involving seven people who are being kept in one of several internment centers in the border area, despite being acquitted by an anti-terrorism court because of lack of evidence against them. The ISI report was submitted to justify the internment centers and military operations against militants more generally. The ISI said it was not going to release people held at the internment centers, warning that the detainees included terrorists who could go to cities like Islamabad and Lahore and launch attacks. It said that 3,871 Pakistani security personnel, more than 3,000 militants and more than 5,000 civilians had been killed in the border area in the last five years. There had been 235 suicide attacks, 9,257 rocket attacks and 4,256 bombings during the same period, the report added. Afghanistan and Pakistan have a difficult relationship. Islamabad has accused Kabul of failing to stop anti-government militants from operating from mountain havens in Afghanistan, while Kabul has blamed Pakistan’s military for cross-border shelling. U.S. Secretary of Defense Chuck Hagel responds to Afghan President Hamid Karzai's statements in which Karzai accused the U.S. and Taliban with working together. In September, Afghanistan’s foreign minister told the United Nations Security Council that diplomatic ties with Pakistan were under threat. The Afghan foreign ministry declined to comment on the ISI report. Earlier this month, Karzai claimed that the Taliban was carrying out attacks in Afghanistan "in service of America." On Monday, after a private meeting with Secretary of State John Kerry in Kabul, Karzai insisted he had not meant to suggest that the United States was colluding with the Taliban, Reuters reported. "I never used the word 'collusion' between the Taliban and the U.S. Those were not my words. Those were the [words] picked up by the media," he said. Kerry said the two men had discussed the matter but he played it down, Reuters reported. "I am confident that the president absolutely does not believe that the United States has any interest except to see the Taliban come to the table to make peace." NBC News' Akbar Shinwari and Reuters contributed to this report. Related: Taliban threat forces Pakistan's Musharraf to cancel welcome rally Karzai accuses US and Taliban of conspiring to keep troops in Afghanistan | Summary: The rift between Pakistan and Afghanistan appears to be growing. Pakistani intelligence yesterday accused Hamid Karzai's administration of working with Taliban groups allied with Pakistan's main Taliban faction, Tehrik-e-Taliban. In testimony before Pakistan's Supreme Court, the ISI said elements from Afghanistan were providing "strong support" for the Taliban, including money, logistics, and training, NBC News reports, and that the alliance with the Afghan government was "likely to enhance terrorist activities." But meanwhile, Afghan officials tell Reuters that Pakistan has been so useless in the Afghan peace process that they've decided to push forward without their neighbor's help. "We here in Kabul are in a bit of a state of shock at once again being confronted by the depth of Pakistan's complacency," a deputy foreign minister says. "Pakistan is changing the goal post every time we reach understanding." He said the government would work with Taliban prisoners in the just-handed-over Bagram prison to help negotiate a peace deal. | 2,014 | 223 | multi_news | en |
Write a title and summarize: Many viral infections, including HIV, exhibit sex-based pathogenic differences. However, few studies have examined vaccine-related sex differences. We compared immunogenicity and protective efficacy of monomeric SIV gp120 with oligomeric SIV gp140 in a pre-clinical rhesus macaque study and explored a subsequent sex bias in vaccine outcome. Each immunization group (16 females, 8 males) was primed twice mucosally with replication-competent Ad-recombinants encoding SIVsmH4env/rev, SIV239gag and SIV239nefΔ1–13 and boosted twice intramuscularly with SIVmac239 monomeric gp120 or oligomeric gp140 in MF59 adjuvant. Controls (7 females, 5 males) received empty Ad and MF59. Up to 9 weekly intrarectal challenges with low-dose SIVmac251 were administered until macaques became infected. We assessed vaccine-induced binding, neutralizing, and non-neutralizing antibodies, Env-specific memory B cells and plasmablasts/plasma cells (PB/PC) in bone marrow and rectal tissue, mucosal Env-specific antibodies, and Env-specific T-cells. Post-challenge, only one macaque (gp140-immunized) remained uninfected. However, SIV acquisition was significantly delayed in vaccinated females but not males, correlated with Env-specific IgA in rectal secretions, rectal Env-specific memory B cells, and PC in rectal tissue. These results extend previous correlations of mucosal antibodies and memory B cells with protective efficacy. The gp140 regimen was more immunogenic, stimulating elevated gp140 and cyclic V2 binding antibodies, ADCC and ADCP activities, bone marrow Env-specific PB/PC, and rectal gp140-specific IgG. However, immunization with gp120, the form of envelope immunogen used in RV144, the only vaccine trial to show some efficacy, provided more significant acquisition delay. Further over 40 weeks of follow-up, no gp120 immunized macaques met euthanasia criteria in contrast to 7 gp140-immunized and 2 control animals. Although males had higher binding antibodies than females, ADCC and ADCP activities were similar. The complex challenge outcomes may reflect differences in IgG subtypes, Fc glycosylation, Fc-R polymorphisms, and/or the microbiome, key areas for future studies. This first demonstration of a sex-difference in SIV vaccine-induced protection emphasizes the need for sex-balancing in vaccine trials. Our results highlight the importance of mucosal immunity and memory B cells at the SIV exposure site for protection. Sex differences in the pathogenesis of numerous viral diseases, including HIV, are well-known [1]. HIV-infected women exhibit lower viral loads and higher CD4 counts than men, but progress faster to AIDS [2]. Women with similar viral loads as men exhibit a 1. 6-fold higher risk of AIDS [3]. This sex bias is associated with differences in immune responses. Following viral infections, antigen recognition by pattern recognition receptors, induction of innate and adaptive immune responses, and production of inflammatory cytokines are higher in females than in males [1]. After viral clearance, immune responses in females can remain elevated, contributing to pathogenesis [1]. Less is known regarding sex differences following vaccination. Females have exhibited better immune responses to HSV-2 gD, HBV, and inactivated influenza vaccines [1], but sex-based effects following HIV/SIV vaccinations have not been reported. Using a large number of female rhesus macaques in a pre-clinical SIV vaccine study we uncovered a sex bias in vaccine-elicited immunity and protective efficacy. Our vaccine strategy is based on mucosally-delivered replicating Ad-recombinants which target myeloid dendritic cells and persist in rectal macrophages, eliciting systemic and mucosal immunity [4]. Following Ad-priming we compared the immunogenicity and protective efficacy of regimens boosted with monomeric SIV gp120 or oligomeric SIV gp140. gp120 immunogens are of interest as they were the form of antigen used as subunit boost for the RV144 clinical trial, the first to show modest protection [5]. Although that vaccine regimen failed to elicit neutralizing antibodies (nAbs) against primary HIV circulating isolates [6], non-neutralizing antibodies exhibiting binding to V1/V2 and high ADCC activity in the presence of low serum IgA levels correlated with reduced infection risk [7–8]. Nevertheless, broadly neutralizing antibodies (bnAbs) are believed important for a highly efficacious vaccine. They develop in a small proportion of HIV-1 patients over prolonged infection, and contribute to maintenance of low viremia [9]. Passive transfer of bnAbs in non-human primates has protected against SHIV infection [10]. Thus, rational design of HIV Env antigens for elicitation of bnAbs is at the forefront of HIV research [11–12]. Native gp140 trimers are thought to be more promising for this purpose compared to monomeric gp120 [13–16] due to the presence of conserved conformational and quaternary epitopes. For example, the potent bnAb 35O22 targets an epitope shared across gp120 and gp41 [17]. The SIV rhesus macaque model is extensively used in pre-clinical vaccine research as SIV transmission and disease progression in macaques resemble human HIV infection [18]. However, SIV monomeric and oligomeric Env immunogens have not been directly compared in this model. We assessed both proteins as booster immunogens, focusing on systemic and mucosal humoral immunity, and evaluating protective efficacy following repeated low-dose SIV rectal challenges. Viremia reductions were modest post-challenge, but we discovered for the first time a sex bias in SIV vaccine outcome. Female but not male macaques exhibited significantly delayed SIV acquisition. These findings are timely in view of recent NIH policy requiring balancing of males and females in animal studies [19]. The mechanisms of acquisition delay point to local mucosal B cell responses. Macaques mucosally primed with Ad5hr-SIV recombinants and boosted with monomeric gp120 or oligomeric gp140 as described in Materials and Methods and outlined in Fig 1A were challenged intrarectally with repeated low doses of SIVmac251 eight weeks following the last immunization. All macaques became infected by the 9th exposure except one gp140-immunized female. No difference in rate of infection was observed between all immunized macaques combined versus the controls (Fig 1B) or between either immunization group and the controls (Fig 1C). As the study included only 12 contemporaneous controls, to achieve greater statistical power we combined these with an additional 53 historical controls (17 females, 36 males) which had been challenged intrarectally at weekly intervals with the same low dose of the same SIV challenge stock. There was no difference in rate of SIV acquisition between the contemporaneous and historical control groups (Fig 1D). A comparison of all the immunized macaques with these combined controls (n = 65) showed a marginally significant difference in rate of SIV acquisition (Fig 1E), suggesting a vaccine effect. To explore this effect further, we evaluated the rate of SIV acquisition among the immunized male and female macaques and the combined control males and females. We observed significantly delayed acquisition in all the immunized females when compared to the combined control females (Fig 1F), whereas no acquisition delay was seen when all the immunized males were compared to the combined control males (Fig 1G). A direct comparison of all the immunized females versus all the immunized males confirmed a significant acquisition delay in the females (S1A Fig). We next explored the influence of the booster immunogen on acquisition delay. A significant difference was observed when gp120-immunized females were compared to the combined control females (Fig 1H), whereas a marginally non-significant difference was seen when comparing gp140-immunized females versus the combined control females (Fig 1I). In contrast, no delay in acquisition was seen when either gp120- or gp140-immunized males were compared with the combined control males (Fig 1J and 1K). A direct comparison of the gp120- and gp140-immunized females versus the similarly immunized males again confirmed the delayed acquisition in gp120- but not gp140-immunized females (S1B and S1C Fig). Overall the delay in SIV acquisition of the gp120 immunized females was clearly a vaccine effect and provides the first demonstration of a sex bias in SIV vaccination outcome. To understand the basis for the significantly delayed acquisition observed in gp120 immunized but not gp140 immunized females, we conducted a thorough analysis of systemic and mucosal humoral immune responses throughout the course of immunization and post-challenge. We first compared the two immunization groups. Oligomeric gp140 proved to be more immunogenic than gp120 as summarized in Tables 1 and 2 and detailed in the accompanying supplemental figures. Systemic Env-specific binding antibodies following Ad5hr-recombinant immunizations (wk 14) were boosted to titers over 106 (wk 53) in both immunization groups (S2A–S2C Fig). The gp120 group exhibited similar antibody titers against gp140 and gp120 but gp140-immunized animals developed higher titers to gp140 with an overall higher titer to gp140 compared to gp120-immunized macaques (Table 1; S2A–S2C Fig). Antibody levels were maintained between wk 53 post-vaccination and 2 weeks post-infection (2wkpi) in both groups. The gp140 immunized macaques also developed higher cyclic V2-specific binding antibody titers than the gp120 group (Table 1; S2D Fig). Serum nAb titers against tier 1 SIVmac251. 6 were comparable in both immunization groups (Table 1; S3A Fig). No neutralization of challenge-related tier 3 SIVmac251. 30 developed. Higher ADCC activity (S3B and S3C Fig) was elicited by gp140 compared to gp120 immunization, regardless of whether gp140- or gp120-coated targets were tested (Table 1). Similarly, antibody-mediated phagocytosis of gp140-coated beads pre-and post-challenge was elevated in both immunization groups compared to controls (p<0. 0001; S3D Fig). gp140-immunized macaques phagocytosed gp120-coated beads significantly above control and gp120-immunized macaque levels (wk 53, Table 1; S3E Fig), whereas phagocytosis by gp120-immunized macaques was higher than that of gp140-immunized macaques 2wkpi (p = 0. 0034; S3D Fig). Mucosal binding antibodies were also assessed during the immunization regimen. Rectal gp120- and gp140-specific IgA and IgG were elicited following mucosal Ad-recombinant priming (wk 14) in both immunization groups. After systemic Env immunization, mucosal IgG was significantly boosted (wk 53) while Env-specific IgA was maintained at post-Ad levels (S4A–S4D Fig). In most cases, IgA and IgG mucosal antibodies in both groups showed elevated reactivity to gp120 at wk 53. gp140-immunized animals developed higher levels of Env-specific rectal IgG against gp140 (wk 53) compared to gp120-immunized macaques (Table 2). Bone marrow (BM) antibody secreting cells (ASC) were next assessed by ELISpot. SIV Env-specific IgG and IgA memory B cells significantly declined after peak elicitation (wk 53), but rebounded 2wkpi (S5A and S5B Fig). IgA memory B cells developed at higher levels than IgG memory B cells at wk 53 in both groups (p = 0. 0049 and 0. 036), and also 2wkpi (p = 0. 0001 and 0. 023) (S5C and S5D Fig). Env-specific plasmablasts (PB) and plasma cells (PC) exhibited a similar response pattern as memory B cells, but displayed smaller IgG and IgA ASC declines between wks 53 and 57 as expected for long-term memory cells (S6A and S6B Fig). The gp140 group maintained higher levels of both IgG and IgA PB/PC prior to challenge (wk 53, Table 2; wk 57, S6A and S6B Fig). In both groups IgG PB/PC were elevated compared to IgA PB/PC at wk 53 (p = 0. 0072 and 0. 014, respectively), but IgA was higher than IgG in gp120-immunized macaques 2wkpi (p = 0. 023; S6C and S6D Fig). With regard to cellular immune responses, we investigated SIV specific CD4+TM and CD8+TM T-cell responses in PBMC 2wkpi. SIVsmH4 Env-specific CD4+ and CD8+ T-cell responses, representative of env encoded in the Ad-recombinant, were comparable between immunization groups, and appeared post-infection in controls (S7A and S7C Fig). In contrast, gp140- compared to gp120-immunized macaques exhibited a trend of elevated CD4+ and CD8+ T-cell responses following SIVmac239 Env stimulation, suggesting a more effective booster immunization (S7B and S7D Fig). Similar results were seen after summing responses to Env, Gag, and Nef (S7E–S7H Fig). Having shown that immune responses in general were elevated in the gp140 immunized macaques, but that SIV acquisition delay was observed in gp120 immunized female macaques, we next analyzed these data by sex. Systemic binding antibodies to the SIVmac239 Env boosting immunogens were higher in gp120-immunized males compared to females against both gp120 and gp140 targets prior to challenge (wks 53 and 57), and were maintained at higher levels against gp120 2wkpi (Fig 2A). A similar result was not seen in the gp140-immunized animals (Fig 2B). Males of both groups combined exhibited higher titers to gp120 than females at all time points (Fig 2C). Antibody responses to SIV EnvE660, representative of SIV EnvsmH4 in the Ad-recombinant, were higher in immunized males compared to females following priming (wk 14; Fig 2D). However, no significant sex differences were seen in neutralizing antibody titers or binding titers to cyclic V2 (S8A–S8C Fig); BM ASC (S9A–S9D Fig), or rectal Env-specific IgA and IgG (S10A–S10D Fig). Although no significant sex difference by group was observed in phagocytic activity, gp120-immunized females maintained higher activity against gp140-coated beads compared to the gp140 group (Fig 2E). Additionally, no sex differences were seen in ADCC activity by group; however, consistent with results of the group analysis (S3B and S3C Fig) gp140-immunized females and males maintained higher activity against both gp120 and gp140 targets (Fig 2F). Female macaques displayed significantly higher rectal Env-specific memory B cell levels than males 2wkpi (Fig 2G), regardless of immunization group. A similar trend was seen both prior to challenge (wk 53) and 8wkpi. Env-specific CD4+TM and CD8+TM T-cell responses showed no sex-based differences (S11A–S11D Fig), although females tended to exhibit higher responses following Env239 stimulation, indicative of the protein boosts derived from that strain (S11B and S11D Fig). When CD4+TM and CD8+TM responses against Env, Gag, and Nef were summed, results were similar in animals stimulated with EnvsmH4 peptides, matched to the env gene in the Ad-recombinant, (S11E and S11G Fig) whereas females showed higher CD4+TM and CD8+TM T-cell responses than males in the animals stimulated with Env239 peptides, matched to the Env booster immunogens, significantly so for the CD4 responses (p = 0. 019; S11F and S11H Fig). Analysis of all the immunogenicity data showed that neither humoral nor cellular systemic immune responses, including serum binding antibodies, serum neutralizing or non-neutralizing activities, bone marrow memory B cells and PB/PC, and CD4+ and CD8+ T cell responses, correlated with SIV acquisition delay. With regard to mucosal immune responses, Env specific IgG in rectal secretions was not associated with acquisition delay in either gp120- or gp140-immunized male or female macaques (S12 Fig). However, although present at lower levels, Env-specific IgA in rectal secretions significantly correlated with delayed acquisition (Fig 3A). All immunized animals with rectal Env-specific IgA levels above the median (0. 04ng/μg total IgA) required more SIV exposures for infection. The difference remained significant in the gp140 group alone (tested against gp140, Fig 3C) but not in the gp120 group (tested against gp120, Fig 3B). This same pattern was exhibited by immunized females. Higher Env-specific rectal IgA levels in all immunized females and in gp140-immunized females but not in gp120-immunized females were associated with an increased number of challenges (Fig 3D–3F). Env-specific rectal IgA in vaccinated males did not correlate with delayed acquisition (S13 Fig). As delayed acquisition of immunized females was most evident in gp120-immunized macaques (Fig 1H and 1I), additional factors must have been involved. To pursue the role of mucosal immunity in delayed acquisition, we next examined Env-specific memory B cells and total PB and PC in rectal tissue by flow cytometry [20–21] (S14 Fig). Consistent with the higher rectal Env-specific memory B cell levels 2wkpi in immunized females compared to males (Fig 2G), the rectal Env-specific memory B cell levels 2wkpi were significantly correlated with challenge exposures in all immunized females, but not males (Fig 3G and 3H). This correlation remained significant in gp120-immunized females and approached significance in gp140-immunized females (Fig 3I and 3J). Total rectal PC levels were significantly correlated with acquisition delay in all immunized females but not males (Fig 3K and 3L) and in gp120- and gp140-immunized females analyzed separately (Fig 3M and 3N). Overall, our data strongly implicate a local mucosal B cell contribution in delayed acquisition of vaccinated female macaques. A secondary outcome of this study was modestly reduced acute phase viremia in the immunized macaques compared to the controls. Median peak viremia for the gp120 and gp140 groups (1. 79x107 and 2. 16x107 SIV RNA copies/ml, respectively) were reduced nearly one log compared to controls (1. 71x108 SIV RNA copies/ml; p<0. 05). Viremia differences between gp120- and gp140-immunized macaques and controls were significant at 2,3 and 4wkpi, while the gp140 group also exhibited lower viremia at 6 and 8wkpi (Fig 4A). Viral loads of the individual macaques are shown in S15 Fig. In contrast to SIV acquisition, no sex bias was observed in viremia reduction. Both females and males of both immunization groups as well as the controls exhibited similar viral loads during the acute phase of infection (Fig 4B–4D). Similarly, CD4 counts over the period of follow-up were similar between the sexes (Fig 4E–4G). We did observe a decrease in viral loads of males compared to females in the gp120 group over weeks 24 to 40 post infection (Fig 4B). A similar difference was not seen in the gp140 immunized macaques, however, we cannot reach a firm conclusion regarding an immunization group difference as a number of macaques in the gp140 group had been euthanized prior to 40 weeks of follow up (see below). Viral loads during the acute phase of infection for the historical controls were available for 41 of the additional 53 macaques, however, no acute viral load difference was observed between the current and historical controls or between males and females of the combined current and historical control groups (S16 Fig). We next examined vaccine-induced immune responses associated with the modestly reduced acute phase viremia in the immunized macaques. We found that phagocytic activity prior to challenge (wk 53) against gp140 targets by the gp140-immunization group, which displayed more prolonged viremia control than the gp120-immunization group (Fig 4A), was significantly correlated with reduced viremia (Fig 5A). Phagocytosis by all macaques was inversely correlated with peak viremia 2wkpi (Fig 5B). No correlation with neutralizing antibody or ADCC activity was observed (S17 Fig). CD8+ T-cell responses contribute to viremia control in natural infection [22–23], confirmed in numerous pre-clinical vaccine studies [24–32]. SIVsmH4 Env-specific CD8+TM T-cells in all macaques significantly correlated with reduced peak and chronic viremia (Fig 5C and 5D). By immunization group, a significant inverse correlation was only observed between SIVsmH4 Env-specific cytokine-producing CD8+TM T-cells of gp140-immunized macaques and viremia levels at peak and acute-phase time points and during the chronic phase of infection (Fig 5E–5G). No correlations with SIVmac239 Env-specific CD8+TM T cells were observed. Overall, the contribution of cellular immunity to viremia reduction was most evident in gp140-immunized macaques. Although cellular responses in macaques overall and in gp140-immunized macaques were associated with better viremia control (Fig 5C–5G), this outcome was not reproduced in females. SIVsmH4 Env-specific CD8+TM responses in all males but not all females correlated significantly with reduced peak, acute-phase, and chronic viremia (p = 0. 0055,0. 0004, and 0. 0086, respectively; S18A and S18B Fig). We observed that the number of challenges necessary to infect immunized females but not males correlated inversely with peak viremia (Fig 6A and 6B). Thus we speculate that repeated exposures boosted immunity, leading to better acute viremia control. Over 40 weeks of follow-up, no group differences were seen in males or females with regard to viral loads (S19A and S19B Fig) or CD4 counts (S19C and S19D Fig), with the exception as mentioned above, that gp120 immunized females exhibited higher viral loads than similarly immunized males over weeks 24–40 of the chronic phase (Fig 4B). However, in spite of enhanced immunogenicity, significantly more gp140-immunized macaques (n = 7) met established criteria and had to be euthanized before 40wkpi compared to gp120-immunized macaques (n = 0) and the controls (n = 2) (Fig 6C). While 5 females and 2 males in the gp140 group were euthanized before 40wkpi (Fig 6D) this difference was not statistically significant. Here we report for the first time a sex bias in SIV vaccine-induced protective efficacy. Delayed SIV acquisition in females was associated with local B cell immunity, including Env-specific mucosal IgA, Env-specific rectal memory B cells, and rectal PC. Our results highlight the importance of mucosal immunity and development of memory B cells at the site of viral exposure for an effective vaccine. The correlations of anamnestic Env-specific rectal memory B cell and total rectal PC responses with acquisition delay were obtained with samples obtained 2wkpi. It is possible that these B cell responses could have been boosted by the series of repeated low dose viral exposures necessary to infect the female macaques. However, in the absence of any detectable infection over the course of these weekly challenges, these responses, initially elicited by vaccination, even if boosted, were contributing to protective efficacy. Similar responses were not observed in control macaques. Future studies should investigate more fully the possibility of antigenic boosting by repeated low-dose challenge exposures. Our previous report of vaccine-induced rectal IgA correlating with delayed SIVmac251 acquisition [33] is confirmed here and extended by demonstrating the sex bias. Other reports have also associated mucosal antibody with protection. Vaccine-induced rectal antibodies mediating transcytosis correlated with decreased chronic viremia [34]. Macaques protected against repeated vaginal SHIV challenges exhibited vaginal IgAs that blocked transcytosis and vaginal IgGs with neutralizing and/or ADCC activity [35]. Following intravenous SIVmac251 challenge, aerosol-vaccinated macaques exhibited reduced CD4+ T-cell depletion in the lung correlated with viral-specific IgA in bronchoalveolar lavage and nasal fluid [36]. Thus the rationale for continued study of mucosal antibodies in vaccine efficacy is well-substantiated. We previously reported a correlation of vaccine-elicited HIV and SIV Env-specific IgG and IgA peripheral blood memory B cells with reduced viremia [37]. Here we extend this finding, demonstrating the importance of Env-specific memory B cells and PC at the mucosal exposure site for delayed SIVmac251 acquisition. It will be important to further explore how vaccine designs can foster homing of memory B cells to the mucosa and enhance their retention. Here we believe the replicating Ad-recombinants played a major role. We have previously shown that the biodistribution of this vector following administration to the upper respiratory tract is broad, and that it exhibits persistent expression in rectal macrophages [4]. Certainly, continued exploration of vaccine-elicited mucosal immune responses in males and females is warranted, along with pursuit of vaccine regimens that target the intestinal mucosa. Females exhibited a higher percentage of SIV Env-specific memory B cells in rectal tissue, consistent with higher basal immunoglobulin levels and greater humoral responses to antigens in women compared to men [1]. While mucosal antibodies correlated with significant acquisition delay in females, male macaques exhibited higher serum antibody binding titers than females at the time of peak response, 2 weeks after the second envelope boost. Nevertheless, no sex bias was seen in neutralizing or non-neutralizing antibody activities. The proportion of IgG subtypes in males versus females should be examined, as IgG3 V1V2-specific antibodies that mediate ADCC correlated with decreased risk of HIV infection in the RV144 trial, but exhibited a short half-life [38]. Recent development of reagents for use in subtyping macaque IgG should allow this question to be addressed. Additionally, Fc-receptor differences may exist between males and females. Polymorphisms in IgG Fc-receptors modulate antibody binding affinity for IgG subtypes, and affect antibody-dependent functions [39–40]. Moreover, differences in Fc glycosylation can affect antibody function [41]. Fucosylation modulates IgG1 binding to FcγRIIIa [42]. In the absence of fucose, binding is enhanced, resulting in improved ADCC activity [43]. A non-fucoslylated variant of bNAb 2G12 exhibited greater ADCVI activity against HIV and SHIV isolates [44]. Fc glycosylation differences also modulate ADCP activity [45]. Further, Fc agalactosylation and asialyation have been associated with better HIV control [46]. Differences in Fc glycosylation patterns between males and females have been established [47] and could have impacted our results. Delayed SIV acquisition in immunized females was greatest in gp120-immunized rather than gp140-immunized macaques that exhibited enhanced humoral immunity. Moreover, gp140-immunized animals met criteria for euthanasia earlier than gp120-immunized macaques although a sex bias was not observed. Although not excluding investigations of oligomeric gp140, this result validates continued study of gp120, the form of immunogen used in the RV144 trial, as a vaccine immunogen. The basis for the different outcome in gp120 immunized macaques while gp140 immunization appeared more immunogenic, however, is not known. Differences in antibody epitope specificities elicited by the different immunogens as well as IgG subtypes and Fc/Fc-R differences as discussed above might explain these outcomes. It may also be the case that higher antibody titers are not beneficial. This has been seen in other infectious diseases. For example, high titers and avidity of vaccine-elicited non-neutralizing antibodies against influenza have been associated with development of more severe disease [48]. Moreover, some non-neutralizing antibodies may be detrimental to protective efficacy. In the RV144 trial, V1V2-specific antibodies that mediate ADCC correlated with protection against acquisition, however high serum Env-specific IgA correlated with infection risk, possibly blocking protective ADCC responses [49]. We did not examine serum Env-specific IgA levels, but they warrant evaluation. Antibody-dependent enhancement of infection can also occur via complement and Fc receptors, dependent on antibody titer and receptor affinity [50]. Both FcγRIIa and FcγRIIIa receptor genetic polymorphisms increase receptor avidity for immune complexes [40]. Notably, the FcγRIIIa genotype was associated with HIV infection rate in the VAX004 trial [51]. Thus, genotyping receptors in females and males may also help explain our complex results. Viral loads exhibited in this study not unexpectedly inversely correlated with CD8+ T-cell responses. By immunization group, a significant correlation of these cellular responses with reduced viremia was only seen in gp140 immunized animals, perhaps due to additional epitopes present in gp140. The gp140 immunized macaques also exhibited more persistent acute viremia reductions. It is possible that these cellular immune responses initially contributed to stronger acute viremia control in this immunization group while at the same time enhanced humoral immunity led to later detrimental effects as suggested above, resulting in the gp140 immunized macaques meeting euthanasia criteria earlier than the gp120 immunized animals. In this regard, significant correlations of CD8+ T cell responses with decreased viremia were exhibited in all male but not female macaques (S18 Fig), perhaps reflecting a greater waning of vaccine-induced CD8+ T-cell responses during infection delay in females. This might have abbreviated the period of time during which the CD8 T cells were able to effectively control viremia. Among humoral responses, phagocytic activity 2wkpi correlated with decreased viremia in all macaques, but a significant correlation of ADCP prior to infection (wk 53) was only present in gp140-immmunized macaques, a result possibly influenced by antibody quality as discussed above. The sex bias in immunity [52], especially mucosal immunity [53–54], is profound and can be attributed to both hormonal influences and contributions of X-linked genes. The microbiome plays a major role in shaping mucosal immune responses [55] and can impact mucosal infections. Steroid hormones can also modulate the microbiome, leading to distinct sex profiles [56]. Overall the microbiome composition is critical in HIV transmission and pathogenesis, can influence HIV acquisition [57], and is a key area for further investigation of the sex bias in SIV acquisition. Female sex hormone changes throughout the menstrual cycle impact susceptibility to vaginal HIV infection by affecting all arms of the immune system. A “window of vulnerability” in the late secretory phase of the cycle during which risk of sexually transmitted infections is highest was postulated [58], and corroborated by the demonstrations during the secretory phase of more frequent vaginal SHIV transmission to macaques [59] and better ex vivo HIV infection of human cervical explants [60]. We did not synchronize our female macaques, as rectal challenges were planned. However, fluctuations in female sex hormone levels could affect HIV/SIV acquisition by other than vaginal routes of exposure. Estrogen receptors (ER) are expressed by cells in a variety of tissues in addition to the reproductive tract. ERα is expressed in T and B lymphocytes, dendritic cells, macrophages, monocytes, natural killer cells and mast cells [61], and influences intestinal levels of proinflammatory cytokines, including TNFα [62]. An examination of gut biopsies from men and women directly demonstrated that women have higher levels of immune activation and inflammation compared to men [53]. The profound effects of ERα on DC development and function greatly influence the quality of adaptive immune responses. ERβ is expressed predominantly in the brain, cardiovascular system, and colon and is found mainly on epithelial cells [63]. It plays an important role in cellular differentiation and maintenance of cellular homeostasis in the colon [64]. In addition, by suppressing chloride ion secretion across the colonic epithelium, estrogen controls fluid retention during different stages of the menstrual cycle [65]. Estrogen also increases mucin content of the protective mucus layer in the intestine and increases mucus viscosity and elasticity [66]. ERα and ERβ play different roles in controlling B cell maturation and selection. Engagement of both by estrogen can alter B cell maturation, whereas triggering of ERα influences development of autoimmunity [67]. In rhesus macaques the frequency of ASC in not only genital mucosal but also systemic lymphoid tissues, bone marrow, and PBMC exhibited profound changes throughout the menstrual cycle [68]. Overall, little is known regarding the influence of female sex hormones on other than vaginal viral exposures, however, as illustrated above, these hormones affect innate and adaptive immune responses, intestinal homeostasis and integrity, biophysical properties of protective mucus, and immune activation and inflammation in more than just reproductive tissue. Thus, it is reasonable to take into account potential hormonal effects in future vaccine studies. Our results showing a clear sex bias in vaccine challenge outcome correlated with local mucosal humoral immunity, is timely in view of recently formulated NIH policy requiring sex balancing in animal studies [19]. Such balancing will cause increased complexity in vaccine design and may require study of the microbiome and in-depth examination of immune responses beyond mere quantitation of functional activities. This approach may provide better understanding of vaccine protective mechanisms. The knowledge gained can be applied to future sex-balanced pre-clinical studies and clinical vaccine trials, critically important as women harbor ~50% of HIV infections worldwide [69]. All animal experiments were approved by Institutional Animal Care and Use Committees prior to study initiation. During the course of this study, the study animals were housed in three facilities, each of which approved the work (Bioqual, Inc., Rockville, MD, Protocol No. 12-3507-15; Advanced BioScience Laboratories, Inc. (ABL), Rockville, MD, Protocol No. AUP526; and the NCI Animal Facility, Bethesda, MD, Protocol No. VB007). Each of these facilities is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. The standard practices closely follow recommendations made in the Guide for the Care and Use of Laboratory Animals of the United States—National Institutes of Health. The rhesus macaques (Macaca mulatta) used in this study were housed in accordance with the recommendations of the AAALAC Standards and with the recommendations in the Guide for the Care and Use of Laboratory Animals. When immobilization was necessary, the animals were anesthetized with approximately 10 mg/kg of ketamine hydrochloride injected intramuscularly. All efforts were made to minimize discomfort of all animals used in the study, including provision of peri-operative and post-operative analgesia and strict accordance to humane endpoint criteria. Details of animal welfare and steps taken to ameliorate suffering were in accordance with the Guide and the recommendations of the Weatherall report, ‘‘The use of non-human primates in research”, as approved by the relevant IACUCs. Animals were housed in temperature controlled facilities with an ambient temperature of 21–26°C, a relative humidity of 30%– 70% and a 12 h light/dark cycle. Due to the nature of the experiment the animals were housed singly in stainless steel wire-bottomed cages and provided with a commercial primate diet and fresh fruit twice daily, with water freely available at all times. All animals were monitored twice daily for activity, food and water intake, and overall health. Enrichment in the form of rotating toys, visual and auditory stimuli, and foraging opportunities were provided daily. Animals that reached IACUC defined endpoints, including pain or distress, that could not be alleviated therapeutically, were humanely euthanized with an overdose of barbiturate consistent with the recommendations of the most recent American Veterinary Medical Association Panel on Euthanasia. Sixty Indian rhesus macaques (Macaca mulatta) aged 2 to 7 years and negative for SIV, SRV, and STLV were used in this study. Males and females (see below) were assigned to immunization and control groups to achieve similar mean ages, and balanced for Mamu A*01 and B*08 haplotypes (3 Mamu A*01,2 Mamu B*08, and 1MamuA*01/B*08). Experimental and control groups (Fig 1A) were divided in two for the vaccination phase of the study, and macaques were housed and handled at either Bioqual Inc, or ABL. The challenge of all 60 macaques was conducted at the ABL facility. Post-challenge monitoring after macaques had received up to nine challenges was carried out at the NCI Animal Facility. The number of macaques used was based on a power analysis which determined that using 18 additional historical controls previously challenged with the stock to be used, and the historical infection rate, the estimated power to detect differences between the experimental groups and the controls was 84%. Twenty four macaques were included in each immunization group and primed at weeks 0 (intranasally and orally) and 12 (intratracheally) with three replication-competent Ad5hr recombinants separately encoding SIVsmH4env (gp140) /rev, SIV239gag and SIV239nefΔ1–13 (Fig 1A). The recombinants were administered in PBS at 5 X 108pfu/dose/route as previously described [24]. The SIVmac239 monomeric gp120 and oligomeric gp140 boosting immunogens were produced in CHO cells, purified and characterized as previously described [70], and administrated intramuscularly with MF59 adjuvant (Novartis Vaccines and Diagnostics, Cambridge, MA) at weeks 39 and 51. The gp120 immunization group (16 females and 8 males) received 100μg/dose of monomeric SIVmac239 gp120 in MF59 adjuvant, and the gp140 immunization group (16 females and 8 males) received 100μg/dose of oligomeric SIVmac239 gp140 in MF59. Control macaques (7 females and 5 males) received equivalent doses of Ad5hrΔE3 empty vector and MF59 adjuvant only. At week 59, all macaques were challenged intrarectally using a repeated low dose of SIVmac251 (1: 500 dilution; 120 TCID50), a challenge stock developed by Dr. Ronald Desrosiers and provided by Dr. Nancy Miller, Division of AIDS, NIAID. As SIV exposures were intrarectal, we did not synchronize females prior to initiating challenges. Challenges were continued weekly until the onset of infection determined by a plasma viral load of ≥50 SIV RNA copies/ml as assessed by the NASBA method [71–72]. Macaques were monitored for 40 weeks after infection or until euthanasia criteria were met. Samples from all macaques were included in each analysis except as specified in individual figure legends. Experiments were not blinded. By the time this study was completed, 53 additional historical control rhesus macaques, challenged intrarectally repeatedly with a 1: 500 dilution of the same SIVmac251 stock, were available to provide greater statistical power for the analyses. Twenty-three of these macaques have been reported in previous publications [73,74]. Data on the remaining 30 have not yet been published. Additionally, rectal pinch biopsies were obtained at necropsy from 6 chronically SIV infected rhesus macaques for use in validating the staining of Env-specific rectal memory B cells. Serum antibody binding titers to monomeric SIVmac239 gp120, oligomeric SIVmac239 gp140 (Novartis) and SIVsmH4 gp120 protein (ABL) were assessed by ELISA as described previously [75]. Antibody titer was defined as the reciprocal of the serum dilution at which the optical density (OD) of the test serum was two times greater than that of the negative-control serum diluted 1: 50. Binding antibody end point titers to variable region V2 of SIV gp120 Env were analyzed in serum samples collected prior to immunization and 2 weeks after the second protein boost (wk 53) by ELISA as previously described [7] using a peroxidase-labeled γ chain specific goat anti-monkey IgG (Catalog No 074-11-021, KPL, Gaithersburg, MD) and a custom-synthesized SIVmac251 cyclic V2 full-length peptide: CIAQNNCTGLEQEQMISCKFNMTGLKRDKTKEYNETWYSTDLVCEQGNSTDNESRCY (JPT Peptide Technologies, GmbH, Berlin, Germany). Serum neutralizing antibody titers against SIVmac251. 6 (tier 1) and SIVmac251. 30 (tier 3) were assayed in TZM-bl cells as described [76]. Neutralizing titers were defined as the reciprocal serum dilution at which there was a 50% reduction in relative luminescence units compared to virus control wells which contained no test sample. Serum antibody-dependent cell-mediated cytotoxicity (ADCC) was evaluated using a rapid fluorometric assay [77]. Briefly, CEM-NKR cells coated with SIVmac239 gp120 or SIVmac239 gp140 (Novartis) were used as targets along with human effector PBMC at an effector-to-target (E: T) ratio of 50: 1, and serially diluted macaque sera. Controls included unstained and single-stained target cells. The percent ADCC cell killing was determined by back-gating on the PKH-26high population of targets cells that lost the CFSE viability dye. ADCC titers are defined as the reciprocal dilution at which the percent ADCC killing was greater than the mean percent killing of the negative controls plus three standard deviations. The maximum % killing for each serum was determined. Results were expressed as the 50% maximum killing titer: the reciprocal serum dilution at which 50% maximum killing was observed, and as endpoint titers. Antibody-dependent cellular phagocytosis (ADCP) activity was measured as previously described [78], with minor modifications. Briefly, SIVmac239 gp120 or SIVmac239 gp140 was biotinylated with the Biotin-XX Microscale Protein Labeling Kit (Life Technologies, Grand Island, NY), and 3–5 μg of gp120 or gp140 was incubated with a 100-fold dilution of 1μm Yellow-Green streptavidin-fluorescent beads (Life Technologies) for 25 min at room temperature in the dark. Serial dilutions of each serum sample (1: 50 to 1: 3000) were added to 250,000–300,000 THP-1 cells in wells of a 96-well U-bottom plate. The bead-gp120/gp140 mixture was further diluted 5-fold in RPMI 1640 medium containing 10% fetal bovine serum (R10) and 50 μl was added to the cell/serum mixtures and incubated for 3 h at 37°C. Cells were then washed at low speed, fixed in 2% PFA, and assayed for fluorescent bead uptake by flow cytometry using a BD Biosciences LSRII. The phagocytic score of each sample was calculated as follows: (% phagocytosis x MFI) /106. The values were standardized to background values (cells and bead only without serum) by dividing the phagocytic score of the test sample by the phagocytic score of the background sample. Rectal secretions were collected using cotton swabs and stored in 1 ml of PBS containing 0. 1% bovine serum albumin, 0. 01% thimerosal, and 750 Kallikrein inhibitor units of aprotinin at -70°C until analyzed. Samples were tested for blood contamination using Chemstrips 5 (Boehringer Mannheim) prior to assay. To remove fecal contaminant sample was passed through a 5μm PVDF microcentrifugal filter unit (Millipore, Billerica, MA). Briefly, SIVgp120 and gp140-specific IgA and IgG antibodies were measured by ELISA as previously described [79,80]. Env-specific IgA and IgG standards derived from IgG-depleted pooled serum or purified serum IgG, respectively, obtained from SIVmac251-infected macaques and quantified as previously described [27] were used to generate standard curves. HRP-conjugated goat anti-monkey IgA and IgG (Nordic Immunology) and TMB substrate were used in sequential steps, followed by the addition of phosphoric acid prior to reading the OD at 450 nm. Total IgA and IgG antibodies were measured in each sample and used to standardize gp120 or gp140-specific IgA and IgG concentrations. Results are reported as Env-specific IgA or IgG/total IgG or IgA (ng specific/μg total). Rectal biopsies were collected at different time points and single cell suspensions were obtained from fresh samples as previously described [21]. Cells obtained were stained with a mixture of fluorescent-conjugated monoclonal antibodies. Env-specific memory B cells were identified using a biotinylated SIVmac239 gp120 or gp140 with the Biotin-XX Microscale Protein Labeling Kit (Life Technologies, Grand Island, NY) followed by APC-conjugated Streptavidin (Life Technologies) as previously described [20]. Briefly, staining was carried out at 4°C in the presence of unconjugated anti-CD4 antibodies to block reactivity to CD4. Representative gating is illustrated in S14A Fig. gp120/gp140-specific B cells were detected within the memory B cell subpopulation (CD27+/-IgD-). Rectal plasmablasts and plasma cells were similarly assessed in fresh rectal biopsies as previously described [21]. Plasmablasts were identified as CD19+CD20+/-IgD-IRF4+CD138-HLA-DR+Ki67+ and plasma cells as CD19+CD20+/-IgD- IRF4+CD138+HLA-DR-Ki67- (S14B Fig). The Env-specific memory B cell staining was validated using rectal pinch biopsies from 6 chronically SIV infected rhesus macaques (not a part of this study) in analyses by both flow cytometry and B cell ELISPOT. A significant correlation was obtained (S14C Fig). Bone marrow samples were collected at different time points, and lymphocytes were purified as previously described [75] and frozen until analysis. Lymphocytes were thawed and both total and SIVgp120 or gp140-specific IgG and IgA secreting B cells were quantified by ELISpot as previously described [37]. Briefly, plasmablasts and plasma cells were quantified on unstimulated samples while memory B cells were enumerated following 3 days of polyclonal stimulation with CpG (ODN-2006) (Operon), 0. 5 μg/ml recombinant human sCD40L (Peprotech), and 50 ng/ml recombinant human IL-21 (Peprotech). In both cases, Env-specific IgA and IgG antibody secreting cells (ASC) were standardized to the total number of IgA and IgG ASC and are reported as the percentage of SIVgp120 or gp140-specific ASC relative to the number of total ASC. Peripheral blood mononuclear cells (PBMC) were isolated from EDTA-treated blood by ficoll gradient [79] and frozen until assay. Cellular immune responses were evaluated by intracellular staining for SIV-specific IFN-γ, IL-2 and TNF-α cytokine secreting cells. After thawing, PBMC were stimulated with peptides at 1μg/ml final concentration. SIV peptide pools were made up of 15 mers overlapping by 11 amino acids and included EnvsmH4 (Advanced BioScience Laboratories, Inc), Envmac239, Gagmac239 (AIDS Research Reference and Reagents Program) and Nefmac251. Control tubes included a non-stimulated and a Leucocyte activation Cocktail (BD Pharmingen) as a positive control. Anti-CD28 PE/Texas red (clone CD28. 2; Beckman Coulter) and anti-CD49d (clone 9F10; eBioscience) were also added during stimulation along with a protein transport inhibitor (BD Pharmingen). After 6h incubation at 37°C, cells were washed with PBS, then stained as previously described [33] with the following antibodies: Anti-CD4 PerCP/Cy5. 5 (clone L220), Anti-CD8 Qdot655 (clone RPA-T8, eBioscience), and Anti-CD95 PE/Cy5 (clone DX2, eBioscience). A viability dye (Life Technologies) was added to the antibody cocktail to exclude dead cell background. Following incubation for 30 min at 4°C in the dark, intracellular staining was performed. Cells were washed twice, resuspended in 250μl fix/perm solution (BD Pharmingen) for 20 min at 4°C, washed twice with BD perm/wash buffer and resuspended in 100μl wash buffer plus the following antibodies: Anti-CD3 Pacific blue (SP34-2, BD Pharmingen), Anti- IFN-γ APC (B27, BD Pharmingen), Anti-TNF-α FITC (Mab11, BD Pharmingen) and Anti-IL-2 PE (MQ1-17H12, BD Pharmingen). After 30 min at 4°C in the dark, cells were washed twice with BD perm/wash buffer and pellets were resuspended in 2% formaldehyde solution for acquisition on an LSRII. CD3+ T cells were used as a gate for CD4+ and CD8+ T cells, and each population was further divided into CD28+CD95+ central memory (CM) and CD28-CD95+ effector memory (EM) cells. The percent of cytokine-secreting cells in each memory cell subset was determined following subtraction of the values obtained with non-stimulated samples. Both subsets were summed to give the total memory (TM) T-cell population. Flow-cytometric analysis was performed using FlowJo V9. 8. 1. (ThreeStar, Ashland, OR). All tests of quantitative data are rank-based and thus distribution-free, so the weak assumptions of the tests are met. Rank-based tests do not require similar variances. Grouped, continuous, and discrete data were analyzed using methods appropriate to each of those types. The Wilcoxon rank-sum analysis was used to test for differences between immunization groups for binding antibody titers, neutralizing and non neutralizing antibody activities, rectal SIV-Env specific B cells, mucosal Env-specific IgG and IgA, Env-specific antibody secreting B cells and cytokine responses. The Wilcoxon signed-rank test was used to test for differences in paired samples within immunization groups. The Cochran-Armitage test was used to analyze V2 peptide titers and ADCC titers. The Spearman rank correlation test was used to assess the relationships of antibody and cellular responses with number of challenges and viral loads. Acquisition and survival data were analyzed using the exact logrank test. For all comparisons a two-sided p<0. 05 was considered statistically significant. Adjustments for multiple comparisons were not made. Estimates of variation are provided as needed in individual figure legends. Analyses were conducted using SAS/STAT software version 9. 3 and GraphPadPrism V6. | Title: Mucosal B Cells Are Associated with Delayed SIV Acquisition in Vaccinated Female but Not Male Rhesus Macaques Following SIVmac251 Rectal Challenge Summary: Viral infections can have different disease courses in men and women. Following HIV infection, women generally exhibit lower viral loads and higher CD4 counts than men, but paradoxically progress faster to AIDS. Sex differences result from effects of X-linked genes and hormonal influences, and are believed to be largely based on immune response differences. Nevertheless, little is known about potential sex differences following vaccination. Here we report for the first time a sex bias in response to a SIV vaccine in rhesus macaques, showing that female animals were better protected against acquisition of SIV compared to males. The vaccine-induced immune responses that contributed to this better protection were viral-specific antibodies and immune antibody-secreting B cells, both at the local rectal site of SIV exposure. These results suggest that HIV/SIV vaccines should be better designed to target mucosal exposure sites. Additionally, they indicate that more vaccine studies should include animals of both sexes to address potential differences. Our study also illustrates that inclusion of both sexes can lead to greater complexity in vaccine trial outcomes, necessitating more in depth analyses. However, we believe sex balancing to be particularly important, as approximately 50% of HIV infections worldwide occur in women. | 13,066 | 293 | lay_plos | en |
Summarize: Background Prevalence and Consequences of Obesity among U.S. Children According to CDC, approximately 12.5 million children aged 2 to 19 years are obese. The prevalence of obesity among children and adolescents has nearly tripled since 1980. Childhood obesity can cause a number of harmful effects on health, including high blood pressure and cholesterol, breathing problems and asthma, and increased risk of type II diabetes. This rise in obesity-related health conditions also introduces added economic costs. The annual direct costs of childhood obesity in the United States are estimated at about $14.3 billion. Moreover, because studies suggest that obese children are likely to become overweight or obese adults—particularly if they are obese during adolescence—the increase in the number of obese children may also contribute to additional health care expenditures when they become adults. One study estimated the medical costs of obesity to be as high as $147 billion per year in Nearly half of all medical spending related to adult obesity is 2008.financed by the public sector, through Medicaid and Medicare. Benefits of Participating in Physical Education and Sports Research indicates that increased physical activity in general, and physical education (PE) and sports participation in particular, yields a number of important benefits for elementary and secondary students, including: Health benefits—Research has shown that regular physical activity for youth can benefit them in a number of ways, including helping build and maintain healthy bones, muscles, and joints; helping control weight and reduce fat; and preventing or delaying the development of high blood pressure. One study concluded that expanding PE programs in schools may be an effective intervention for combating obesity in the early years, especially among girls. Academic benefits—A growing body of evidence indicates a relationship between PE and sports and academic attainment and attendance. A 2010 CDC report that examined 50 existing studies found positive associations between academic performance and both PE and school-based sports. Specifically, it concluded there is substantial evidence that physical activity can help improve academic achievement, including grades and standardized test scores. Further, it suggested physical activity can affect cognitive skills, attitudes, and academic behavior, including enhanced concentration, attention, and improved classroom behavior, and concluded that increasing or maintaining time dedicated to physical education might help academic performance. Personal and social benefits—A number of studies provide some support for the premise that physical activity, and sports in particular, can positively affect aspects of personal development among young people, such as self-esteem, goal-setting, and leadership. However, evidence indicates that the quality of coaching is a key factor in maximizing positive effects. Federal, State, and Local Roles The federal government supports efforts to increase the amount of time children spend being physically active, including within the school context. For example: CDC funds 22 states and 1 tribal government to help schools develop a systematic approach to school health, including physical education, through its promotion of a coordinated school health strategy. It also publishes the Physical Education Curriculum Analysis Tool, which is designed to help school districts develop or enhance physical education curricula. The Department of Education administers the Carol M. White Physical Education Program, which awards grants to districts and community- based organizations to initiate or enhance physical education programs. In fiscal year 2011, the program had a budget of nearly $79 million, and funded 76 new awards and 152 continuation awards. The President’s Council on Fitness, Sports and Nutrition includes the President’s Challenge program, which assists schools in assessing student fitness levels, motivating and awarding student physical activity participation, and awarding model school PE programs. In 2010, the President launched the White House Task Force on Childhood Obesity, in conjunction with the First Lady’s Let’s Move! initiative aimed at increasing physical activity. The Department of Agriculture runs the HealthierUS School Challenge, an initiative to recognize schools that have created healthier school environments through the promotion of nutrition and physical activity. At the state and district levels, various parties may be involved in providing students with opportunities in PE or sports. For example, many states have requirements regarding schools’ provision of PE, according to CDC. However, policies may vary by state or district, such as the required grades in which PE should be offered, the number of minutes students should be in class, or the specific content and curriculum areas that should be taught. For extracurricular athletics, the National Federation of State High School Associations provides leadership for the administration of most high school interscholastic sports—the main form of extracurricular athletic competition in schools, which includes varsity and junior varsity level teams. In turn, each state has its own state high school athletic association that is a voluntary member of the federation. While PE Instruction Time Has Decreased, Officials Said School Sports Opportunities Have Generally Increased PE Instruction Time Has Decreased, but Schools Increasingly Require Some PE in Each Grade Opportunities The amount of PE instruction time that schools offer to students generally decreased from 2000 to 2006, according to SHPPS data, and relatively few schools offered students the opportunity to participate in daily PE or its instructional equivalent, as recommended by NASPE (see fig. 1). National data show that a higher percentage of middle schools offered daily PE than did elementary or high schools. Schools we visited differed widely in the amount of PE instruction time offered to their students. For example, only 3 out of 13 schools we visited offered daily PE or its instructional equivalent. These 3 schools had longer school days, which officials said made it easier to fit PE into the daily schedule. Among the 4 elementary schools we visited, 1 provided all students a daily 60-minute PE class, while another provided students just one 30-minute PE class per week. At the same time, the estimated percentage of schools that required students to take some PE increased at each grade level from 2000 to 2006, particularly for grades at the middle and high school levels, according to SHPPS data (see fig. 2). For example, the estimated percentage of schools that required ninth grade students to take PE increased from 13 percent in 2000 to 55 percent in 2006. Although the amount of PE instruction time has decreased, emphasis on the quality of PE programs appears to have increased, according to SHPPS data and comments from officials we interviewed. The percentage of states that required or encouraged districts or schools to follow NASPE-based PE standards—such as competency in motor skills and promotion of responsible personal and social behavior—increased from 59 percent in 2000 to 76 percent in 2006. In 2006, an estimated 65 percent of schools adopted such standards. District and school officials with whom we spoke said PE curricula now focus less on traditional sports in favor of helping students develop lifelong skills. For instance, three schools we visited offered outdoor adventure-based activities that helped students develop problem solving and teamwork skills. At one middle school we visited, PE staff members taught students how to measure their body mass index and other indicators of fitness. In addition, some school and district officials we interviewed said offering students options may increase student participation in PE. For example, officials at one high school we visited said that each semester students could select two to three different sports or fitness activities from a range of options— such as flag football, tennis, or soccer—to fulfill their PE requirement. School officials noted that students were more engaged in PE because the options were designed to motivate students across a range of athletic abilities and interests. Moreover, several school officials we interviewed said they design their PE curriculum to encourage students to move as much as possible during PE class. These YRBS estimates have a margin of error at the 95 percent confidence level of plus or minus 9.6 percentage points or less. officials told us the PE curriculum is designed to engage as many students as possible. As previously stated, national data show that many schools have PE requirements. However, some schools allow exemptions for a range of reasons. According to 2006 SHPPS data, state, district, and school policies most commonly allowed student exemptions due to long-term physical or medical disability. Our previous work has shown that students with disabilities generally attend PE class about the same amount of time as students without disabilities. In addition, according to 2006 SHPPS data, most states required schools to implement measures to meet the PE needs of students with long-term disabilities. Officials from most schools and districts we interviewed said that exemptions from PE requirements are rare, and schools generally offer students with long- term disabilities the opportunity to participate in adapted PE or general PE classes. Most Officials Reported Increased Opportunities to Participate in School Sports Opportunities School sports programs offer another opportunity for students to engage in school-based physical activity. According to 2006 SHPPS data, an estimated 77 percent of middle schools and 91 percent of high schools offered students opportunities to participate in interscholastic sports Most programs such as basketball, soccer, or softball (see app. I).national, state, district, and school officials we interviewed said that opportunities to participate in school sports have generally increased, in part because many schools have added new interscholastic sports teams over the last few years. For example, several schools we visited have added lacrosse and badminton programs as student interest in these sports has increased. Furthermore, officials from each of the four states we visited said they had added new sports programs to their statewide interscholastic competition schedules in response to increased demand. In particular, many officials we interviewed said opportunities for girls to participate in school sports have increased over time, due primarily to the addition of new interscholastic sports teams for girls. For example, one state official we interviewed noted that while only 49 high schools in the state offered girls’ soccer in 1986, about 300 high schools offered it in 2010. In addition to interscholastic sports, SHPPS data show that an estimated 50 percent of elementary schools, 49 percent of middle schools, and 45 percent of high schools offered intramural or physical activity clubs in 2006. However, several officials said that such programs, which are relatively small compared to interscholastic programs, have decreased in middle and high schools. Moreover, only a few schools we visited offered intramural programs. No-cut policies—in which schools do not limit the number of students who can participate on a sports team—have also contributed to increased opportunities for students to participate in school sports programs, according to many officials we interviewed. For example, in one district we visited, the middle school interscholastic teams adopted no-cut policies, which officials said provided interested students ample opportunities to participate in sports programs and gain exposure to new sports. Similarly, an official at another high school we visited said the school offers at least one no-cut interscholastic team per season so that students who want to participate in school sports always have at least one option. National data show that high school students’ participation in at least one school or community sports team remained about the same from 2005 to 2009. Over one-half of high school students reported participating in at least one school or community sports team in 2009, according to YRBS data. Several middle school and high school officials we interviewed reported similar student participation rates. The overall number of students who participate in school sports programs has generally increased over the years, according to most officials we interviewed. High school boys reported a higher rate of participation in school or community sports teams than high school girls in both 2005 and 2009, according to YRBS data. However, according to the officials we interviewed, the number of female athletes has increased over the years, in part due to the addition of new sports programs for girls. While participation did not vary significantly overall by race for white, black, and Hispanic high school students, white high school girls were more likely to report that they participate in at least one sports team than their black or Hispanic counterparts. Schools Cited Resource Challenges to Providing PE and Sports Opportunities but Have Mitigated Some of Them Officials Said PE Opportunities Have Been Affected by Budget Cuts and Inadequate Facilities Most officials we spoke with cited budget cuts and inadequate facilities as major challenges for schools to provide physical education opportunities for students. Specifically, officials from several of the districts and schools we visited said budget cuts have affected their ability to hire PE teachers, maintain appropriate class sizes, and purchase sufficient equipment. In one district we visited, officials told us that many PE teachers have been laid off, and some schools in the district have been forced to share a part- time PE instructor. As a result, elementary school PE instruction in the district has been reduced to as little as 30 minutes every 2-3 weeks, and a district official told us most elementary and middle schools in the district are not meeting state requirements for PE instruction. At two elementary schools we visited, PE instructors expressed a desire to conduct PE instruction on a daily basis, but cited limited funding as a barrier. Some schools have also seen increased class sizes as a result of budget cuts. In some cases, budget cuts have affected the availability and quality of equipment as well. A PE teacher at one school has stopped including several sports, such as golf, in her PE classes because the supply of equipment no longer matches the class size. In the context of limited funding, some state, district, and school officials expressed the belief that the greater emphasis on assessments for reading and math, as required has shifted priorities away under the No Child Left Behind Act of 2001,from PE. In one district we visited, an official said this focus on academic assessments had led his district to reduce the amount of PE it offers students. Officials at 9 of the 13 schools we visited told us that a lack of adequate facilities is also a major challenge in providing physical education. For example, because some schools do not have adequate indoor space, they may conduct several PE classes in the gymnasium simultaneously, or use alternative space for their activities. According to SHPPS data, an estimated 54.9 percent of elementary schools, 37.8 percent of middle schools, and 25.3 percent of high schools used a cafeteria, auditorium, or other multipurpose room for indoor physical education in 2006. During our site visits, we observed several PE classes sharing space and saw multipurpose facilities being used as gymnasiums. For example, at one school we visited, the gymnasium also served as the cafeteria. The time it takes to prepare for, serve, and clean up from lunch limits the school’s ability to schedule PE classes. Several school officials told us they have worked hard to stretch the limited funding they receive for PE instruction. Some schools partially rely on federal grant money to help maintain and augment PE opportunities. For example, two school districts we visited were able to purchase equipment, such as fitness center equipment and kick balls, for their PE programs as a result of Carol M. White grants from the Department of Education. Schools Also Face Budget and Other Challenges in Providing Sports Opportunities but Have Partially Mitigated Them Although sports opportunities have generally increased, most officials we spoke with cited budget constraints as a key challenge to providing opportunities. In particular, budget cuts have affected transportation and facilities. Some schools have also struggled to find coaches for their school sports teams. Because interscholastic sports games may involve travel and some teams practice off-site, schools often need to provide transportation for athletes. Many school and district officials we spoke with stated that, because of budget constraints, they had a difficult time providing transportation to facilitate student participation in after-school sports activities. According to SHPPS data, an estimated 29 percent of the schools that offered interscholastic sports in 2006 also provided transportation home for participating students, up from 21 percent in 2000. For schools that offered intramural activities, an estimated 31 percent of middle schools and 28 percent of high schools provided transportation home for students. Some school officials told us that transportation costs, including costs associated with maintenance and fuel, are a large part of their school’s athletic budget. To help reduce transportation costs, some schools charge students a fee for transportation, enlisting parents to provide carpools, or sharing buses with other athletic teams to transport students to and from athletic events. Several school officials we spoke with stated that budget cuts and space constraints have affected their ability to provide adequate facilities and equipment for sports opportunities. Some smaller schools do not have access to baseball or football fields or other facilities for team sports. In addition, some schools use off-site locations for practices and events due to the lack of space or adequate facilities. Furthermore, some schools, particularly schools in densely populated communities, lack the necessary space to expand their facilities. Moreover, officials at one school we visited said they were prohibited from building new athletic fields or expanding because of land use restrictions. To mitigate some of these challenges, several schools we visited have developed partnerships with local businesses, colleges, nonprofits, or community recreational centers to use their facilities for various sports programs. For example, several schools have agreements to use community athletic fields and other facilities for baseball, football, soccer, and swimming programs. In addition to space constraints, some school officials cited aging or insufficient equipment as a challenge to providing sports opportunities. For example, an official from one school told us the school had to implement a selection policy for the football program, in which some students were cut from the team, because demand exceeded the number of uniforms and helmets available for players. School officials also cited the upkeep and maintenance of fields as a challenge given budget constraints. Some state, district, and other officials cited finding quality coaches as a challenge to providing sports opportunities. Specifically, some officials told us that fewer faculty members have been coaching sports teams in recent years. Officials attribute this decline to the low pay and increased time commitments that are often required to coach a sport. In one state we visited, an official said over 60 percent of the coaches in the state were considered “walk-on coaches” who were not otherwise a part of the school community. Some officials said that non-faculty coaches may be less accessible to students. In addition, some schools may have a difficult time finding coaches for specific sports. For example, one school official reported difficulty finding cheerleading coaches given the specialized training needed to coach a cheerleading squad. Schools have mitigated some of the budgetary challenges related to providing sports opportunities by relying heavily on outside funding sources or charging fees for certain sports activities. Some school officials we interviewed said their athletics funding depended primarily on community support or the tax base of their district, both of which fluctuate with the economy. For example, one district we visited had strong community support and a high economic tax base. During our site visit, we observed that schools in this district had numerous and high-quality facilities and one official mentioned that even the district’s middle schools had swimming pools. The official added that the tax base has remained steady, and the district has not experienced some of the challenges that other school districts face in providing PE and sports opportunities for its students. However, other schools we visited reported relying heavily on booster clubs, gate receipts, private donations, and fundraising to fund their local sports programs. For example, one school official told us that the school relies heavily on ticket sales from sporting events that may total as much as $60,000 a year to maintain and fund its sports programs. Also, some schools with very strong community support benefit from community fundraising efforts. In addition, some school districts have implemented “pay-to-play” arrangements, in which students are charged a fee to participate in school sports activities. Specifically, according to SHPPS data, the percentage of schools that require students to pay an activity fee to participate in interscholastic sports was an estimated 33 percent in 2006, which did not differ significantly from the 2000 estimate of 29 percent.addition, the percentage of schools with intramural activities or physical activity clubs that required students to pay a fee for these activities increased from an estimated 23 percent in 2000 to 35 percent in 2006. However, in two states we visited, officials told us that pay-to-play arrangements are prohibited in their states. Some officials expressed concern that pay-to-play arrangements may negatively impact student participation by serving as a barrier to lower-income students. However, according to 2006 SHPPS data, an estimated 86 percent of schools that charge a fee to participate in sports activities waived the fee for students who could not afford to pay. Concluding Observations The federal government has an interest in seeing that school-aged children benefit from the positive effects regular physical activity can have on health and overall well-being. As the primary social institution where children learn and spend their time, schools can play a pivotal role in increasing students’ physical activity, in part through offering PE classes and opportunities to participate in sports programs. Although it appears schools increasingly acknowledge the benefits of PE by requiring students to take classes, they have reduced the amount of time spent on PE instruction. Opportunities to play school sports, however, appear to be on the rise. While such a trend is encouraging, school-based sports should augment, rather than replace, the experiences and skills acquired in PE, which reaches beyond student athletes to the general student population. A number of challenges inhibit further expansion of school- based PE and sports. At a time when states and districts are operating under severe fiscal constraints, acquiring resources to provide additional opportunities is difficult. Although some districts and schools have developed approaches to partially offset the challenges associated with providing sports opportunities, challenges associated with providing PE remain unresolved. Identifying practical ways to increase students’ physical activity may be difficult, but the need to address childhood obesity—and the opportunity to shore up such efforts in the school context—serves as a compelling starting point for addressing obesity- related health issues and their associated costs. Agency Comments and Our Evaluation We provided a draft of the report to the Departments of Education and Health and Human Services for review and comment. Both agencies provided technical comments, which we incorporated as appropriate. As arranged with your offices, unless you publicly announce its contents earlier, we plan no further distribution of this report until 30 days from its issue date. At that time, we will send copies of this report to relevant congressional committees, the Secretary of Education, the Secretary of Health and Human Services, and other interested parties. In addition, the report is available at no charge on the GAO website at http://www.gao.gov. If you or your staff have any questions about this report, please contact me at (206) 287-4809 or calboml@gao.gov. Contact points for our Offices of Congressional Relations and Public Affairs may be found on the last page of this report. GAO staff who made key contributions to this report are listed in appendix II. Appendix I: Estimated Percentage of All Middle and High Schools That Offered Interscholastic Sports in 2006, by School Level Appendix II: GAO Contact and Staff Acknowledgments Staff Acknowledgments Debra Prescott (Assistant Director) and Rebecca Woiwode (Analyst-in- Charge) managed this assignment. Vernette Shaw and Michelle Wong made significant contributions in all facets of the work. In addition, Dana Hopings helped with site visits; Jean McSween and Dae Park provided methodological support; Alex Galuten provided legal assistance; David Chrisinger provided writing assistance; and James Bennett provided graphics for the report. | Summary: Physical activity is a crucial part of preventing or reducing childhood obesity, and may also yield important academic and social benefits. However, many children do not attain the level of daily physical activity recommended by the Centers for Disease Control and Prevention (CDC). Schools are uniquely positioned to provide students opportunities to increase physical activity through physical education (PE) classes and involvement in sports teams. In view of the federal governments role in promoting the health and welfare of children, the Congress is currently considering a number of proposals aimed in part at increasing the physical activity of youth. To assist the Congress as it considers options for increasing physical activity among students, GAO was asked to review (1) the status of opportunities for elementary and secondary school students to participate in school-based physical education or sports activities; and (2) what challenges schools face in providing physical education and sports opportunities. To conduct this study, GAO reviewed federal survey data; interviewed state, district, and school officials in selected states, as well as federal officials and others with relevant expertise; reviewed relevant federal laws and regulations; and reviewed studies on the benefits of physical education and sports for students. GAO makes no recommendations in this report. The Departments of Education and Health and Human Services provided technical comments, which were incorporated as appropriate. While the most recent national data show instruction time for PE decreased from 2000 to 2006, officials GAO interviewed stated that school sports opportunities have generally increased in recent years. Specifically, the percentage of schools that offered PE at least 3 days a week decreased from 2000 to 2006, but the percentage of schools that required students in each grade to take some PE increased during the same period. For example, the estimated percentage of schools that required PE in ninth grade increased from 13 percent in 2000 to 55 percent in 2006. Moreover, states, districts, and schools appear to have increased emphasis on the quality of PE programs, such as helping students develop lifelong fitness skills, according to national data and GAO interviews. Data on high school students show that participation in PE varies by grade level but not by gender or across racial groups. In addition, most state, district, and school officials GAO interviewed said opportunities to participate in interscholastic sports have increased, particularly for girls, and that many schools have responded to increased demand by adding new sports teams over the last few years. Schools GAO visited cited several challenges to providing PE and sports opportunities but have found ways to alleviate some of the challenges associated with sports. In particular, school officials said that budget cuts and inadequate facilities have affected their ability to provide PE opportunities. For example, officials from one school district GAO visited reported reducing PE instruction time because of limited funding for instructors. Other schools, such as one where the gym doubled as the cafeteria, lack dedicated space to use for PE. In addition, school officials reported challenges in providing sports opportunities, as issues related to transportation, facilities, and staffing have been compounded by budgetary constraints. For example, officials from some schools said funding to transport students to outside facilities for practices or games was limited. Other school officials cited difficulty in attracting quality coaches because of low pay and the large amount of time involved. Even so, some schools have mitigated some challenges related to sports by relying heavily on outside funding sources such as booster clubs and gate receipts and leveraging community facilities. Additionally, some schools charge student fees for sports activities, which may be a barrier for lower-income students. However, many schools waive such fees. | 5,164 | 761 | gov_report | en |
Write a title and summarize: La Fiat 500 Abarth de (et par) Lapo Elkann. freakstill " Je l'ai depuis un mois et demi! Je porte un amour intangible au mouvement, aux voitures, trains, avions, bateaux. Je change d'automobile tous les ans et je ne conserve pas les anciens modèles. Je ne suis pas intéressé par la possession. On garde ses amis, ses relations, pas les choses. Ce qui m'intéresse, c'est de comprendre, d'inventer, d'apporter une valeur ajoutée. Je suis à l'écoute de l'air du temps, c'est pourquoi j'ai choisi récemment une Fiat 500. Elle symbolise l'énergie dont mon pays, l'Italie, a besoin aujourd'hui. Elle est à la fois esthétique, sensuelle, pratique et accessible en termes de prix. J'ai customisé la mienne. Elle est bleu Bugatti, car le bleu, c'est le ciel, la mer, l'océan. Je ne me sens jamais mieux qu'en contemplant l'eau, elle me permet de m'échapper pour imaginer, rêver, penser. C'est un bleu mat. L'une de mes activités préférées porte sur la recherche de nouvelles couleurs, et ce mat, j'y travaille depuis douze ans. L'intérieur est en denim, parce que le jean est une valeur démocratique. J'ai choisi des matériaux doux au toucher. Cette voiture est plus basse que la normale, ce qui la rend plus rigide et donc plus nerveuse. Je l'ai allégée de 250 kg pour rouler plus vite. J'adore conduire, c'est comme une thérapie. Parallèlement, j'ai bien sûr amélioré sa sécurité avec des pince-freins en céramique. Cette personnalisation sur mesure, tout le monde devrait y avoir droit : c'est mon credo et je travaille à cette démocratisation. Propos recueillis par Catherine Maliszewski | Title: La Fiat 500 de Lapo Elkann Summary: Il a troqué ses fonctions au sein du groupe automobile italien fondé par son arrière-grand-père contre la direction de LA Holding - qui regroupe ses agences de design et de communication. Avec ses nouvelles activités, l'héritier de la famille Agnelli compte bien se forger un nom digne de ses aïeux. A son actif, une série de courts-métrages réalisés pour le lancement de la Fiat 500 Gucci et un réfrigérateur en denim (imaginé pour Smeg) présenté au dernier Salon du meuble de Milan. Sa voiture, une Fiat 500 Abarth réinventé par ses soins, lui ressemble. | 425 | 147 | mlsum_fr | fr |
Write a title and summarize: Eukaryotic cells modulate their metabolism by organizing metabolic components in response to varying nutrient availability and energy demands. In rat axons, mitochondria respond to glucose levels by halting active transport in high glucose regions. We employ quantitative modeling to explore physical limits on spatial organization of mitochondria and localized metabolic enhancement through regulated stopping of processive motion. We delineate the role of key parameters, including cellular glucose uptake and consumption rates, that are expected to modulate mitochondrial distribution and metabolic response in spatially varying glucose conditions. Our estimates indicate that physiological brain glucose levels fall within the limited range necessary for metabolic enhancement. Hence mitochondrial localization is shown to be a plausible regulatory mechanism for neuronal metabolic flexibility in the presence of spatially heterogeneous glucose, as may occur in long processes of projection neurons. These findings provide a framework for the control of cellular bioenergetics through organelle trafficking. Cellular metabolism comprises an intricate system of reactions whose fine-tuned control is critical to cell health and function. A number of quantitative studies have focused on metabolic control through modulating reactant and enzyme concentrations and turnover rates (Grima and Schnell, 2006; Amar et al., 2008). However, these studies generally neglect the spatial organization of metabolic components within the cell. By localizing specific enzymes in regions of high metabolic demand (Laughton et al., 2007; Zecchin et al., 2015), as well as clustering together consecutively acting enzymes (O' Connell et al., 2012), cells have the potential to substantially enhance their metabolism. Spatial organization is particularly critical in highly extended cells, such as mammalian neurons, whose axons can grow to lengths on the meter scale. Metabolic demand in neurons is spatially and temporally heterogeneous, with especially rapid ATP turnover found in the presynaptic boutons (Rangaraju et al., 2014), and ATP requirements peaking during synaptic activity and neuronal firing (Shulman et al., 2004; Ferreira et al., 2011; Weisová et al., 2009). Neurons rely primarily on glucose as the energy source for meeting these metabolic demands (Peppiatt and Attwell, 2004). Due to the long lengths of neural processes, the glucose supply can vary substantially over different regions of the cell (Ferreira et al., 2011; Weisová et al., 2009; Hall et al., 2012). In myelinated neurons, for instance, it has been speculated that glucose transport into the cell is localized primarily to narrow regions around the nodes of Ranvier (Magnani et al., 1996; Harris and Attwell, 2012; Rosenbluth, 2009), which can be spaced hundreds of microns apart (Ibrahim et al., 1995; Butt et al., 1998). Glucose transporters in neurons have also been shown to dynamically mobilize to active synapses, providing a source of intracellular glucose heterogeneity (Ashrafi et al., 2017). Furthermore, varying levels of activity in the mammalian brain may lead to varying extracellular glucose levels, resulting in spatially heterogeneous nutrient access (Hawkins et al., 1979). Individual axons have been shown to span across multiple regions of the brain (Matsuda et al., 2009), enabling them to encounter regions with different glucose concentrations. Most ATP production in neurons occurs within mitochondria: motile organelles that range from interconnected networks to individual globular structures that extend throughout the cell. As energy powerhouses and metabolic signaling centers of the cell, mitochondria are critical for neuronal health (Nunnari and Suomalainen, 2012). Their spatial organization within the neuron plays a pivotal role in growth and cell physiology (Li et al., 2004). Defects in mitochondrial transport are involved in the pathologies of several neurological disorders such as peripheral neuropathy and Charcot-Marie-Tooth disease (Baloh, 2008; Baloh et al., 2007). A number of studies have shown that mitochondria are localized preferentially to regions of high metabolic demand, such as the synaptic terminals (Li et al., 2004; Chang and Reynolds, 2006). Such localization can occur via several molecular mechanisms, mediated by the Miro-Milton mitochondrial motor adaptor complex that links mitochondria to the molecular motors responsible for transport (Mishra and Chan, 2016). Increased Ca2+ levels at active synapses lead to loading of calcium binding sites on Miro, releasing mitochondria from the microtubule and thereby halting transport (Wang and Schwarz, 2009; Macaskill et al., 2009). High glucose levels can also lead to stalling, through the glycosylation of motor adaptor protein Milton by the glucose-activated enzyme O-GlcNAc transferase (OGT) (Pekkurnaz et al., 2014). This mechanism has been shown to lead to mitochondrial accumulation at glucose-rich regions in cultured neurons (Pekkurnaz et al., 2014). It is postulated to regulate mitochondrial spatial distribution, allowing efficient metabolic response to heterogeneous glucose availability. Mitochondrial positioning relies on an interplay between heterogeneously distributed diffusive signaling molecules (such as Ca2+ and glucose), their consumption through metabolic and other pathways, and their effect on motor transport kinetics. While the biochemical mechanisms and physiological consequences of mitochondrial localization have been a topic of much interest in recent years (MacAskill and Kittler, 2010; Mishra and Chan, 2016), no quantitative framework for this phenomenon has yet been developed. In this work we focus on glucose-mediated regulation of mitochondrial transport, developing quantitative models to examine the consequences of this phenomenon for metabolism under spatially varying glucose conditions. Our approach relies on a reaction-diffusion formalism, which describes the behavior of species subject to both consumption and diffusion. Reaction-diffusion systems have been applied to describe the spatial organization of a broad array of cellular processes (Kondo and Miura, 2010), ranging from protein oscillations in E. coli (Howard et al., 2001), to coordination of mitotic signalling (Chang and Ferrell, 2013), to pattern formation in developing embryos (Bunow et al., 1980; Gregor et al., 2005). The response of actively moving particles to spatially heterogeneous, diffusive regulators has also been extensively investigated in the context of chemotaxis (Van Haastert and Devreotes, 2004). In contrast to most chemotactic cells, however, mitochondria have no currently known mechanism for directly sensing glucose gradients. Instead, they are expected to accumulate in response to local glucose concentration only. Our goal is to delineate the regimes in which such a crude form of chemotaxis can lead to substantial spatial organization and enhancement of metabolism. Specifically, we model the modulation of mitochondrial density with glucose concentration in a tubular axonal region, focusing on two forms of spatial heterogeneity. In one case, we consider an axonal domain between two localized regions of glucose entry, representing the internodal region between nodes of Ranvier in myelinated neurons (Figure 1a). The second case focuses on an unmyelinated cellular region with continuous glucose permeability, embedded in an external glucose gradient (Figure 1b). In both cases, we show that mitochondrial accumulation and enhanced metabolic flux is expected to occur over a limited range of glucose concentrations, which overlaps with physiological brain glucose levels. Our simplified quantitative model allows identification of a handful of key parameters that govern the extent to which glucose-mediated mitochondrial halting can modulate metabolism. We establish the region of parameter space where this mechanism has a substantial effect, and highlight its potential importance in neuronal metabolic flexibility and ability to respond to spatially varying glucose. We begin by formulating a quantitative model to describe the spatial localization of mitochondria that halt in a glucose-dependent manner, in the presence of localized sources of glucose. This situation arises in myelinated neurons, which have glucose transporters enriched at the nodes of Ranvier, leading to highly localized sources of glucose spaced hundreds of micrometers apart within the cell (Saab et al., 2013). Neuronal glucose transporters are known to be bidirectional (Simpson et al., 2007), allowing glucose concentration within the cell to equilibrate with external glucose. For simplicity, we assume rapid transport of glucose through these transporters, so that the internal concentration of glucose at the nodes where transporters are present is assumed to be fixed. The cellular region between two glucose sources is modeled as a one-dimensional interval of length L with glucose concentration fixed to a value c0 at the interval boundaries (Figure 1a). Glucose diffuses throughout this interval with diffusivity D, while being metabolized by hexokinase enzyme in the first step of mammalian glucose utilization (Figure 1c) (Wilson, 2003). The concentration of glucose is thus governed by the reaction-diffusion equation, (1) dGdt=D∂2G∂x2−k (x) G (x) where k (x) describes the spatial distribution of the hexokinase enzyme as well as the rate of consumption. In the case of spatially uniform, linear consumption [k (x) =k, a constant] this equation can be solved directly, yielding a distribution of glucose that falls exponentially from each source boundary, with a decay length λ=D/k (Kholodenko, 2006). Hexokinase 1 (HK1), the predominant form of hexokinase expressed in neurons, is known to localize preferentially to mitochondria (John et al., 2011), which in mammalian axons can form individual organelles approximately 1 µm in length (Fawcett, 1981). We carry out numerical simulations of Equation 1 where consumption is limited to locations of individual discrete mitochondria, represented by short intervals of length Δ. Specifically, we define the mitochondria density as M (x) =n (x) / (πr2Δ), where n (x) is the number of mitochondria overlapping position x, and r is the axon radius. The phosphorylation of glucose by mitochondrial hexokinase is assumed to follow Michaelis-Menten kinetics, described byk (x) =kgM (x) G (x) +KMwhere KM is the saturation constant and kg is the turnover rate of glucose (per unit time per mitochondrion). The turnover rate kg incorporates both the catalytic rate of hexokinase and the number of hexokinase enzymes per mitochondrion. This expression reduces to the case of constant linear consumption when glucose concentration is low (G≪KM) and mitochondria are uniformly distributed throughout the region. In general, glucose consumption depends on the location of mitochondria within the domain. Mitochondrial distribution in neurons is known to be mediated through regulation of their motor-driven motility (Chang and Reynolds, 2006; Pekkurnaz et al., 2014). Individual mitochondria switch between processively moving and paused states, modulated by the interplay between kinesin and dynein motors and the adaptor proteins that link these motors to the mitochondria (Schwarz, 2013). In our model, we simulate mitochondria as stochastically switching between a processive walking state that moves in either direction with velocity v and a stationary state. The rate of initiating a walk (kw) is assumed to be constant, while the halting rate (ks (x) ) can be spatially heterogeneous. For simplicity, we assume the mitochondria are equally likely to move in the positive (+) or negative (-) direction each time they initiate a processive walk (Figure 1b). It has recently been demonstrated that the key motor adaptor protein (Milton) is sensitive to glucose levels, halting mitochondrial motility when it is modified through O-GlcNAcylation by the OGT enzyme (Pekkurnaz et al., 2014). Our model employs a highly simplified description of mitochondrial dynamics, which assumes that all pauses are associated with such an O-GlcNAcylation event. Recovery from the pause at the constant rate kw corresponds to removal of the modification through the activity of the complementary enzyme O-GlcNAcase (OGA). Although there is evidence indicating long-term glucose deprivation can reduce OGA expression (Zou et al., 2012), for simplicity we assume in our model that OGA activity is independent of glucose levels. In vivo axonal mitochondria have been observed to undergo short-lived sporadic pausing while continuing to move processively in their previous anterograde or retrograde direction (Russo et al., 2009; Wang and Schwarz, 2009). Such pauses are subsumed into an effective processive velocity v in our model. Other sources of pausing, such as Ca2+-regulated motor disengagement, PINK1/Parkin-mediated detachment of motors, and anchoring to the microtubules by syntaphilin (Schwarz, 2013), are not considered here in order to focus specifically on the effect of glucose-dependent mitochondrial spatial organization. Upon entry into the cell, the first rate-limiting step of glucose metabolism is its conversion into glucose-6-phosphate by hexokinase. Further downstream metabolic pathways split, with much of the flux going to glycolysis while a small fraction is funneled into the pentose phosphate pathway and the hexosamine biosynthetic pathway (HBP). The HBP produces UDP-GlcNAc, the sugar substrate for O-GlcNAcylation (Figure 1c) (Hart et al., 2011). In our model, we assume that the rate of UDP-GlcNAc production equals the rate of glucose conversion by hexokinase, scaled by the fraction of G6P that is channeled into the hexosamine biosynthetic pathway. This assumption is valid if, at each point of pathway branching, the Michaelis-Menten saturation constants for the two branches are similar. This in fact appears to be the case for both the branching of the pentose phosphate pathway and glycolysis from the hexosamine biosynthetic pathway which is the focus of this work (see Appendix 2). Consequently, saturation of the initial glucose conversion step will imply saturation of the entire hexosamine biosynthetic pathway. We therefore model the kinetics of Milton modification using the same Michaelis-Menten form as for hexokinase activity, with the pathway flux leading to Milton modification subsumed within a rate constant for mitochondrial stopping (ks). We note that the subcellular organization of the intermediates in the conversion from glucose into O-GlcNAcylated Milton is largely unknown. In our model, we make the extreme case assumption that all intermediates are localized to mitochondria, with only the initial glucose substrate capable of diffusing through the cytoplasm. We note that cytoplasmic diffusion of any of the pathway intermediates would attenuate the effect on mitochondrial localization. Our simplified model thus gives an upper limit on the extent to which mitochondria can localize at high glucose regions through the Milton modification mechanism. Following these simplified assumptions, we treat the kinetics of mitochondrial halting as dependent only on the local glucose concentration, according to the functional formks (x) =ksG (x) G (x) +KMwhere KM is the Michaelis-Menten constant of hexokinase. We proceed to evolve the simulation forward in time, with glucose consumption localized to regions within ±Δ/2 of each discrete mitochondrial position (details in Materials and methods). A snapshot of one simulation run is shown in Figure 2a, highlighting the accumulation of stationary mitochondria in the high glucose regions near the ends of the domain. We are interested primarily in investigating the steady-state distribution of mitochondria and glucose in this system, averaged over all possible mitochondrial trajectories. We thus proceed to coarse-grain our model by treating the distribution of mitochondria as a continuous field M (x) =W+ (x) +W- (x) +S (x), where W+ (x) is the distribution of mitochondria walking in the positive direction, W- (x) is the distribution of those walking in the negative direction, and S (x) is the distribution of stationary mitochondria. We can then write down the coupled differential equations governing the behavior of the mitochondrial distributions as: (4) dW+dt=−v∂W+∂x−ks (x) W++kwS2dW−dt=v∂W−∂x−ks (x) W−+kwS2dSdt=ks (x) [W++W−]−kwS. The glucose distribution evolves according to Equation 1 with consumption rate k (x) given by Equation 2. The boundary conditions at the ends of the domain are assumed to be reflective for the mitochondrial distributions, and to have a fixed glucose concentration c0. The stationary state for this system can be calculated numerically (see Materials and methods). The formulation with a continuous mitochondrial density faithfully represents the behavior of simulations with discrete mitochondria, as illustrated in Figure 2b. The steady-state spatial distribution of mitochondria and glucose in the continuous system depend on six parameters: ks/kw, KM, c0, D, L, kgM¯ where M¯ is the average mitochondrial density in the axon (number of mitochondria per unit volume). Estimates of physiologically relevant values are provided in Table 1. Dimensional analysis indicates that three of these parameters can be used to define units of time, length, and glucose concentration, leaving three dimensionless groups. We choose to use the following three dimensionless parameters, each of which has an intuitive physical meaning: (5) λ^=DKMkgM¯L2, c^0=c0KM, k^s=kskw Here λ^ is the length-scale of glucose decay relative to the domain length, c^0 is the boundary glucose concentration relative to the saturation constant KM, and k^s is the ratio of stopped to walking mitochondria at high glucose levels. We proceed to explore the steady-state distribution of mitochondria and glucose as a function of these three parameters. In order for mitochondria to preferentially accumulate at the source of glucose via a glucose-dependent stopping mechanism, three criteria must be met. First, the glucose concentration needs to be higher at the source than in the bulk of the cell, as occurs when the decay length due to consumption is much smaller than the size of the domain (λ^≪1). Second, if glucose levels become too high (c^0≫1) then both glucose consumption rates and stopping rates of the mitochondria become saturated, leading to a flattening of glucose and mitochondrial distributions (Figure 3). There is thus an upper limit on the possible external glucose concentrations that will yield mitochondrial localization at the edges of the domain. Finally, the mitochondria must spend a substantial amount of time in the stationary state, since walking mitochondria will be broadly distributed throughout the domain. Because the stopping rate is itself dependent on the glucose concentration, this criterion implies that very low concentrations will also not allow mitochondrial localization. Figure 3 shows the distribution of glucose and mitochondria at different values of the external glucose c^0, illustrating that accumulation of mitochondria at the edges requires intermediate glucose levels. To characterize the distribution of mitochondria along the interval, we introduce an accumulation metric A, defined byA=6σ2/L2−0. 5where σ2 is the variance in the mitochondrial distribution. This metric scales from A=0 for a uniform distribution to A=1 for two narrow peaks at the domain edges. Mitochondrial distributions with several different values of the accumulation metric are shown in Figure 3a. We use a cutoff of A=0. 2 to define distributions where the mitochondria are localized at the glucose source. We explore the dependence of the mitochondrial accumulation on the three dimensionless parameters defining the behavior of the system: the stopping rate constant k^s, the glucose decay length λ^, and the external concentration c^0. Because only the stopped mitochondria localize near the glucose sources, increasing the fraction of mitochondria in the stopped state (increased k^s) inevitably raises the overall accumulation (Figure 4a). The fraction of mitochondria in the stopped state will depend on both k^s and the overall levels of glucose, as dictated by c^0 (Figure 4b). Experimental measurements indicate that at high glucose concentrations, approximately 95% of mitochondria are in the stationary state (Pekkurnaz et al., 2014). We are thus interested primarily in the parameter regime of high stopping rates: k^s≳10. The limited range of concentrations that lead to mitochondrial accumulation at the edges of the domain can be seen in Figure 4a. For a high stopping rate (k^s=10), we then calculate the mitochondrial accumulation as a function of the remaining two parameters: λ^, c^0. Here, again, we note that only intermediate glucose concentrations result in accumulation, with the range of concentrations becoming narrower as the decay length λ^ becomes comparable to the domain size (Figure 4c). We can establish the concentration range within which substantial accumulation is expected, by setting a cutoff A=0. 2 on the accumulation metric and calculating the resulting phase diagram (Figure 4d). Below the lower concentration cutoff, insufficient mitochondria are in the stationary state and so no localization is seen. This lower cutoff disappears in the limit of infinite k^s. At intermediate concentrations, mitochondria are localized near the domain edges. Above the upper concentration cutoff, no localization is observed due to saturation of the Michaelis-Menten kinetics. Using empirically derived approximations for the rate of glucose consumption by mitochondria and the diffusivity of glucose in cytoplasm (see Table 1), we estimate the decay length parameter as λ^≈0. 03. The mitochondria are then expected to localize near the glucose source only if c^0<66. Because the saturation concentration for hexokinase is quite low (KM≈0. 03mM) (Wilson, 2003), we would expect mitochondrial accumulation for glucose concentrations below about 2 mM. We note that physiological brain glucose levels have been measured at 0. 7 − 1. 3 mM, depending on the brain region (McNay et al., 2001), implying that glucose-dependent halting of mitochondrial transport would be expected to result in localization of mitochondria at nodes of Ranvier. Localizing mitochondria to the glucose entry points is expected to increase the flux of glucose entering the cell, thereby potentially enhancing the overall metabolic rate. We calculate the overall effect of transport-based regulation on the net metabolic flux within the simplified model with localized glucose entry. Figure 5 shows the effect of increasing mitochondrial stopping rates (k^s) on the total rate of glucose consumption in the interval between nodes of glucose influx. At low k^s values, mitochondria are distributed uniformly throughout the interval. At high k^s values and at sufficiently low glucose concentrations, the mitochondria cluster in the regions of glucose entry, increasing the overall consumption rate by up to 40% at physiologically relevant glucose levels (c0 = 1 mM). We note that in hypoglycemic conditions, glucose levels can drop to 0. 1 mM (Silver and Erecińska, 1994), further increasing the magnitude of this effect. In the case of limited glucose transport into the cell, intracellular glucose levels could be significantly below the concentrations outside the cell. Measurements of intracellular glucose in a variety of cultured mammalian cell types indicate internal concentrations within the range of 0. 07 − 1mM, up to an order of magnitude lower than glucose concentrations in the medium (John et al., 2008). However, neuronal cells are known to express a particularly efficient glucose transporter (GLUT3) (Simpson et al., 2008), and these transporters have been shown to be highly concentrated near the nodes of Ranvier (Magnani et al., 1996; Rosenbluth, 2009). We therefore assume that glucose import into the nodes is not rate limiting for myelinated neurons in physiological conditions. Introducing a finite rate of glucose transport would effectively decrease the intracellular glucose concentration at the nodes c0, increasing the enhancement in metabolic flux due to mitochondrial localization. In subsequent sections, we explore the role of limited glucose import in unmyelinated axons with spatially uniform glucose permeability. Extracellular brain glucose levels exhibit substantial regional variation, particularly under hypoglycemic conditions where more than ten-fold differences in local glucose concentrations have been reported (Paschen et al., 1986). Because individual neurons can traverse multiple different brain regions (Matsuda et al., 2009), a single axon can be subjected to heterogeneous glucose levels along its length. This raises the possibility that glucose-dependent mitochondrial localization can play a role in neuronal metabolic flexibility even in the case where glucose entry into the cell is not localized to distinct nodes. We thus extend our model to quantify the distribution of mitochondria in an axon with limited but spatially uniform glucose permeability that is subjected to a gradient of external glucose. This situation is relevant, for instance, to unmyelinated neurons in infant brains, as well as to in vitro experiments with neurons cultured in a glucose gradient (Pekkurnaz et al., 2014). In this model, the extracellular environment provides a continuous source of glucose whose influx is limited by the permeability of the cell membrane. Intracellular glucose dynamics are then defined by the reaction-diffusion equationdGdt=D∂2G∂x2−k (x) G+P (x) (Gext (x) −G) where the first term corresponds to diffusive glucose spread, the second to a spatially varying metabolism of glucose, and the third to the entry of glucose into the cell. Here, Gext is the external glucose concentration, and P (x) is the membrane permeability to glucose, which we assume to depend in a Michaelis-Menten fashion on the difference between external and internal glucose concentration: P (x) = (2/r) PKMPKMP+|Gext (x) −G (x) |where P is the spatially uniform permeability constant in units of length per time. This functional form incorporates two known features of glucose transporters: (1) they are bidirectional, so that the overall flux through the transporter at low glucose levels should scale linearly with the difference between external and internal glucose (Carruthers, 1990); (2) neuronal glucose transporters saturate at high glucose levels (GLUT3 KMP≈3mM (Maher et al., 1996), with an even higher saturation constant for GLUT4 (Nishimura et al., 1993). When the difference in glucose levels is low, the overall flux of glucose entering the cell reduces to P (Gext (x) −G (x) ). Mitochondria dynamics are defined as before (Equation 4), and we again assume Michaelis-Menten kinetics for glucose metabolism by hexokinase localized to mitochondria (Equation 2). We note that the dynamics in Equation 6 are governed by three time-scales: the rate of glucose transport down the length of the axon, rate of glucose consumption, and rate of glucose entry. The first of these rates becomes negligibly small in the limit L≫D (G+KM) / (kgM¯). Because internal glucose levels can never exceed the external concentrations, in the range where Gext<10mM, the rate of diffusive transport should become negligible for L≫150μm. In the limit where intracellular glucose is much less than KM, this criterion reduces to λ^≪1, indicating that glucose diffuses over a very small fraction of the interval before being consumed. The interval length L in this model represents an axonal length which can range over many orders of magnitude. We focus on axon lengths above several hundred microns, allowing us to neglect the diffusive transport of intracellular glucose (see Appendix 3). The steady-state glucose profile can then be determined entirely by the local concentration of mitochondria and external glucose. For a given mitochondrial density M (x) and external glucose profile Gext (x), the corresponding intracellular glucose concentration can be found directly by solving the quadratic steady-state version of Equation 6 without the diffusive term. However, the steady-state mitochondrial distribution cannot be solved locally, because the limited number of mitochondria within the axon couples the mitochondrial density at different positions. We thus employ an iterative approach to numerically compute the steady-state solution for both glucose and mitochondrial density under a linear external glucose gradient Gext=Gmin+ (Gmax−Gmin) xL (see Materials and methods). For parameter combinations where intracellular glucose concentrations are above KM but well below Gext, the entry and consumption processes for glucose are both saturated. There is then a steep transition between two different regimes. In one regime, glucose entry exceeds consumption and internal glucose levels approach the external concentrations. In the other, consumption dominates and glucose levels drop below saturating concentrations. The key dimensionless parameter governing this transition can be defined as the ratio of entry to consumption rates: γ=2PKMPG¯extkgM¯r (KMP+G¯ext) This ratio can be modulated in the cell either by recruiting varying amounts of glucose transporters (adjusting P) or changing the total amount of active hexokinase (adjusting kgM¯). The remaining dimensionless parameters determining the behavior of this simplified model are the external glucose concentration relative to the hexokinase saturation constant (G^ext=G¯ext/KM), the relative magnitude of the glucose gradient, ΔG^ext= (Gmax−Gmin) /G¯ext, the ratio of stopped to walking mitochondria ks^=ks/kw, and the saturation constant for glucose transporters KMP/KM≈96. The last parameter is expected to remain approximately constant in neuronal cells. The average external glucose concentration and glucose gradient are expected to vary substantially depending on the glycemic environment to which the neuron is exposed. We note that ΔG^ext has a maximum possible value since the minimal glucose concentration cannot drop below 0zero. We proceed to analyze the limiting case where the glucose gradient is as steep as possible for any given value of average external glucose (ΔG^ext=2). We quantify the amount of mitochondrial accumulation at the high glucose side of the domain by calculating the total mitochondrial density within the distal 10% of the interval compared to a uniform distribution, in analogy to experimental measurements (Pekkurnaz et al., 2014). Substantial enrichment in the high glucose region occurs when glucose entry into the cell cannot keep up with consumption (γ≪1) and the intracellular glucose levels drop below the hexokinase saturation concentration KM, as can be seen in the glucose and mitochondrial distributions plotted in Figure 6a–c. The interplay between external glucose levels and the entry/consumption rates is illustrated in Figure 6d. For external glucose concentrations well above KM there is a sharp transition to mitochondrial enrichment at γ<1. At the lowest levels of intracellular glucose, accumulation is again reduced because a very small fraction of mitochondria are found in the stopped state. In the limit of high ks, mitochondrial accumulation would occur for arbitrarily low values of γ (Figure 6—figure supplement 1). We note that because glucose entry and turnover are much faster than diffusive spread for biologically relevant parameter regimes, the model results do not depend on the cell length L (Appendix 3). Experimental measurements of mitochondrial enrichment in cultured neurons subjected to a gradient of 0 to 5mM glucose have indicated an approximately 20% enrichment in mitchondrial counts at the axonal region exposed to high glucose. We note that using published estimates of typical glucose permeability and mitochondrial glucose turnover for mammalian cells (Table 1) yields a ratio of entrance and consumption rates of γ≈1. 9 for this experimental system. Because this ratio is above 1, we would not expect to see substantial mitochondrial enrichment. To result in the experimentally observed enrichment at high glucose, the ratio γ would need to be reduced by approximately a factor of 2, implying the existence of additional regulatory mechanisms. Modulation of γ could be achieved by either decreasing the number of glucose transporters in the cell (reducing P) or upregulating total hexokinase levels (increasing kg). Neurons are believed to regulate both the density of glucose transporters and hexokinase activity in response to external glucose concentrations and varying metabolic demand (Fujii and Beutler, 1985; Robey et al., 1999; Duelli and Kuschinsky, 2001). In particular, adaptation to glycemic levels well above physiological values, as well as possibly reduced synaptic activity in a cultured environment, may result in downregulation of glucose transporters, lowering the value of γ. The discrepancy between model prediction and observed mitochondrial accumulation highlights the existence of additional regulatory pathways not included in the current model whose role could be explored in further studies that directly quantify glucose entry and consumption rates in cultured neurons. Physiological brain glucose levels have been measured at 0. 7 mM - 1. 3 mM (McNay et al., 2001), with hypoglycemic levels dipping as low as 0. 1 mM and hyperglycemic levels rising up to 4mM (Silver and Erecińska, 1994). Axons that stretch across different brain regions with varying glucose levels can thus be subject to a glucose gradient with G¯ext on the order of 1 mM (white line on Figure 6d). We note that the physiological range overlaps substantially with the region of high mitochondrial accumulation, indicating that glucose-dependent halting can modulate mitochondrial distribution under physiologically relevant glycemic levels. By accumulating mitochondria at the cellular region subjected to higher external glucose, the metabolic flux in that region can be substantially enhanced. In (Figure 6e) we plot the enhancement in glucose consumption rates (compared to the case with uniformly distributed mitochondria) within the 10% of cellular length subjected to the highest glucose concentrations. Metabolic enhancement occurs within a narrow band of the γ parameter. The drop-off in enhancement at low values of the internal glucose concentration (low γ) is due to the coupling between glucose levels and mitochondrial localization. Specifically, mitochondrial accumulation at the region subject to high glucose concentration increases the local rate of consumption in that region, driving down local internal glucose levels. Consequently, the difference in internal glucose concentrations between the two ends of the cell is decreased when internal levels fall substantially below M (Figure 6b), reducing the enhancement of metabolic flux. Although mitochondrial accumulation decreases metabolic flux in the low glucose region, the total rate of glucose consumption integrated throughout the cell is enhanced by up to approximately 14% when γ≈1 (Figure 6f). It is interesting to note that the typical physiological range of external glucose levels spans the narrow band of parameter space where metabolic enhancement is expected (white lines on Figure 6e, f). These results implicate glucose-dependent mitochondrial stopping as a quantitatively plausible mechanism of metabolic flexibility, increasing metabolism in regions with high nutrient availability for axonal projections that span between hypoglycemic and euglycemic regions. The magnitude of this effect can be tightly controlled by the cell through modulating overall rates of glucose entry and consumption. Thus, by coupling mitochondrial transport to local glucose levels, whole-cell changes in hexokinase or glucose transporter recruitment can be harnessed to tune the cell’s response to spatially heterogeneous glucose concentrations. The minimal model described here provides a quantitative framework to explore the interdependence of glucose levels and mitochondrial motility and their combined effect on neuronal metabolic flux. Glucose-mediated halting of mitochondrial transport is shown to be a plausible regulatory mechanism for enhancing metabolism in cases with spatially heterogeneous glucose availability in the neuron. We have quantitatively delineated the regions in parameter space where such a mechanism can have a substantial effect on mitochondrial localization and metabolic flux. Specifically, mitochondrial positioning requires both sufficient spatial variation in intracellular glucose and sufficiently low absolute glucose levels compared to the saturation constant of the hexokinase enzyme. In the case of tightly localized glucose entry (as at the nodes of Ranvier), intracellular spatial heterogeneity requires a small value of the dimensionless length scale for glucose decay (λ^=DKM/kgM¯L2≪1). For physiologically estimated values, mitochondrial localization to the nodes is expected to occur for glucose levels below approximately 2 mM, comparable to physiological brain glucose concentrations (McNay et al., 2001; John et al., 2008). In the case where glucose can enter homogeneously throughout the cell surface (as with unmyelinated axons), heterogeneity can arise from an external glucose gradient. We show that metabolic enhancement through mitochondrial positioning occurs in a narrow range of the key parameter γ= (2PKMPG¯ext) / (kgM¯ (KMP+G¯ext) ), which describes the ratio of glucose entry to glucose metabolism, and that this narrow range intersects with physiological estimates. The model developed here is intentionally highly simplified, encompassing a minimal set of parameters necessary to describe glucose-dependent mitochondrial localization. Other regulatory pathways that determine mitochondrial positioning are not included in this basal model. In particular, we do not consider here calcium-based transport regulation, which is known to localize mitochondria to regions of synaptic activity (Zhang et al., 2010; Wang and Schwarz, 2009; MacAskill and Kittler, 2010; Macaskill et al., 2009). Upregulating OGT signaling in cultured cells has been shown to decrease the fraction of motile mitochondria by a factor of three, while reducing endogenous OGT nearly doubles the motile fraction, indicating that a substantial number of stationary mitochondria are stopped as a result of OGT activity (Pekkurnaz et al., 2014). Our model assumes the extreme case where all stopping events are triggered in a glucose-dependent manner, thereby isolating the effect of glucose heterogeneity. Stopping mechanisms dependent on neuronal firing activity could alter mitochondrial distribution in concert with glucose-dependent halting, increasing the density of mitochondria at presynaptic boutons or near areas of localized calcium influx as at the nodes of Ranvier (Zhang et al., 2010). We note that mitochondria have previously been shown to accumulate at spinal nodes of Ranvier in response to neuronal firing activity (Fabricius et al., 1993; Zhang et al., 2010). The mechanism described here provides an additional driving force for mitochondrial localization near the nodes even in quiescent neurons. Additional metabolic feedback loops, not included in our model, may result in a more complex dependence of mitochondrial stopping on glucose concentration. In particular, both the pentose phosphate pathway and glycolysis generate intermediates that feed back into UDP-GlcNAc production by the hexosamine biosynthetic pathway (Kruger and von Schaewen, 2003; Shirato et al., 2011). Furthermore, several of the enzymes involved in the metabolic pathways linking glucose levels to Milton O-GlcNacylation may be regulated in a glucose-dependent manner. For example, the activity of the fructose-6-phosphate metabolizing enzyme GFAT is believed to be regulated by intermediates in the hexosamine pathway (Traxinger and Marshall, 1991) and O-GlcNAc transferase (OGT) itself is directly regulated by UDP-GlcNAc levels (Hart et al., 2007). Other enzymes, such as the de-GlcNAcylating enzyme OGA exhibit long term regulation of expression in response to altered glucose levels (Zou et al., 2012). These regulatory mechanisms provide additional potential routes of metabolic control through mitochondrial positioning. Several key parameters that regulate mitochondrial localization in response to glucose heterogeneity can be dynamically regulated in neurons. Specifically, the rate of glucose consumption (kgM¯) can be tuned by modulating the concentration or activity of hexokinase within mitochondria or by altering total mitochondrial size and number. This parameter controls both the glucose decay length λ^ in the case of localized glucose influx and the ratio of glucose entry to consumption γ in the case of spatially distributed entry. We note that our model assumes hexokinase to be localized exclusively to mitochondria. The predominant form of hexokinase in the brain (HK1) is known to bind reversibly to the mitochondrial membrane, with exchange between a mitochondria-bound and a cytoplasmic state believed to contribute to the regulation of its activity (Golestani et al., 2007). Release of hexokinase into the cytoplasm would result in more spatially uniform glucose consumption, negating the metabolic enhancement achieved through mitochondrial localization. An additional parameter known to be under regulatory control is the rate of glucose entry into the neuron (P). The glucose transporters GLUT3 (Simpson et al., 2008; Duelli and Kuschinsky, 2001; Weisová et al., 2009) and GLUT4 (Ashrafi et al., 2017) have been shown to be recruited to the plasma membrane in response to neuronal firing activity. Interestingly, transporter densities are themselves spatially heterogeneous, concentrating near regions of synaptic activity (Ashrafi et al., 2017; Ashrafi and Ryan, 2017). The model described in this work quantifies the extent to which a locally increased glucose influx can enhance total metabolic flux, given the ability of mitochondria to accumulate at regions of high intracellular glucose. A number of possible feedback pathways linking glucose distribution and mitochondrial positioning are not included in our basic model. For instance, hexokinase release from mitochondria into the cytoplasm (potentially altering kg) is known to be triggered at least in part by glucose-6-phosphate, the first byproduct in glucose metabolism (Crane and Sols, 1954). Chronic hypoglycemia has been linked to an upregulation in GLUT3 in rat neurons (Uehara et al., 1997), which would in turn lead to an increased glucose uptake (P). The fraction of glucose funneled into the hexosamine biosynthetic pathway (incorporated within ks) can also be modified through feedback inhibition of GFAT by the downstream metabolic product UDP-GlcNAc (Li et al., 2007). Such feedback loops imply that several of our model parameters (P, kg, ks) are themselves glucose-dependent and may become spatially non-uniform in response to heterogeneous glucose. Incorporating these effects into a spatially resolved model of metabolism would require quantifying the dynamics of both the feedback pathways and mitochondrial positioning, and forms a promising avenue for future study. Control of glucose entry and consumption underlies cellular metabolic flexiblity, and defects in the associated regulatory pathways can have grave consequences for neuronal health. Misregulation of hexokinase has been highlighted as a contributor to several neurological disorders, ranging from depression (Regenold et al., 2012) to schizophrenia (Shan et al., 2014). Neuronal glucose transporter deficiency has been linked to autism spectrum disorders (Zhao et al., 2010) and Alzheimer’s disease (Liu et al., 2008). Furthermore, defects in mitochondrial transport, with the consequent depletion of mitochondria in distal axonal regions, contribute to peripheral neuropathy disorders (Baloh, 2008). Glucose-dependent mitochondrial localization provides an additional layer of control, beyond conventionally studied regulatory mechanisms, which allows the cell to respond to spatial heterogeneity in glucose concentration. Our analysis paves the way for quantitative understanding of how flexible regulation of metabolism can be achieved by controlling the spatial distribution of glucose entry and consumption. We simulate the internodal space of the axon, between localized nodes of glucose entry, as a one-dimensional domain for a reaction diffusion system with motile reaction sinks. The glucose concentration field is discretized over 100 equidistant points along the domain. Its dynamics are governed by the reaction diffusion equation (Equation 1), evolved forward over time-steps of δt using the forward Euler method. Because forward Euler methods have stringent conditions for stability and convergence, we use a time-step that is much smaller than both the glucose decay time-scale and the time-scale associated with diffusion over our spatially discretized grid (see below). The number of mitochondria in the domain is calculated according to N=M¯Lπr2≈38, where the mitochondrial density M¯, internodal distance L, and axonal radius r are estimated from published data (Table 1; Appendix 1). The mitochondria are treated as discrete intervals of length Δ = 1 μm, with the position of each mitochondrial center updated at each timestep. Over each time step, every motile mitochondrion moves a distance of ±vδt, (with transport velocity v = 1 μm/s) and switches to a stationary state with probability 1-exp (-ksδt), where ks (x) is a function of the center position of that mitochondrion (Equation 3). Mitochondria that reach within a distance of Δ/2 from the ends of the domain are reflected, reversing their velocity while remaining motile. Analogously, every stationary mitochondrion switches to a motile state on each time-step with probability 1-exp (-kwδt). Processive walks are initiated with equal probability in either direction. At any given time, the spatial density of mitochondria is calculated from the location of mitochondrial centers at positions x1, …xN, according to M (x) =n (x) / (πr2Δ), where n (x) =∑i=1N[θ (x−xi+Δ/2) −θ (x−xi−Δ/2) ], is the number of mitochondria overlapping spatial position x and θ is the Heaviside step function. We integrate the simulation forward in time-steps of δt=0. 2Δx2D, where Δx is the spatial discretization. This time-scale is much smaller than the relevant decay time for glucose consumption [τg= (kgM¯KM) -1]. Using these small time-steps allows for stability and robust convergence with the forward Euler method. The simulation proceeds for 107 steps. Simulations are repeated 100 times to obtain the histogram shown in Figure 2. Convergence to steady-state is established by comparing to calculations with the continuum model described in the subsequent sections. For an arbitrary spatial distribution of stopping rates ks (x) the corresponding steady-state mitochondrial distribution can be calculated directly by solving the equations for mitochondrial transport (Equation 4): (9) S=ks (x) (W−+W+) kwvdW+dx=12ks (x) (W−−W+) vdW−dx=12ks (x) (W−−W+). Because our model assumes symmetry between anterograde and retrograde mitochondrial transport, as well as equal glucose concentrations at either boundary of the domain, we take W-=W+, implying that the population of walking mitochondria must be spatially constant. Consequently, the population of stopped mitochondria is proportional to the stopping rate (S=Cks (x) /kw). The constant C can be calculated from the normalization condition, ∫0LM (x) dx=∫0L[W− (x) +W+ (x) +S (x) ]dx=M¯L The overall steady-state distribution of mitochondria is then given by, (11) M (x) =W- (x) +W+ (x) +S (x) =M¯1+1L∫0Lks (x) kwdx[ks (x) kw+1] Because the stopping rate is an explicit function of glucose concentrations [ks (x) =ksG (x) KM+G (x) ], this approach allows us to find the steady-state mitochondrial distribution for any fixed distribution of glucose. We solve for steady-state glucose and mitochondrial distributions using a numerical method that evolves the glucose concentration forward in time while explicitly setting the mitochondrial concentration to its steady-state value at each step. The glucose distribution is initialized according to the steady-state solution for uniform consumption (Equation 13). Mitochondrial density M (x) is calculated from the glucose distribution according to Equation 11 and Equation 3. The glucose distribution G (x), in turn, evolves according to the mitochondrial distribution as given by Equation 1 and Equation 2. The glucose profile is integrated forward with a timestep δt=10-5L2/D. The distributions are assumed to be converged once the root mean squared rate of glucose change drops below the minimal cutoff: 10-6kgM¯. Results of the continuous mitochondrial distribution model are shown to match the discrete mitochondria simulations (Figure 2b). All subsequent analysis is done in the continuum limit. We validate our numerical calculations by comparing to the analytically tractable solution in the limit of low glucose and nearly uniform mitochondrial distribution. In the limit of spatially uniform, linear consumption, the steady-state reaction-diffusion equation for glucose can be expressed as0=D∂2G∂x2−kG (x) where k=kgM¯/KM is the constant consumption rate. Assuming fixed glucose concentrations (c0) at the boundaries of the domain, the steady-state glucose distribution is then given byG (x) =c0cosh (xλ) cosh (L2λ) with λ=Dk defining the glucose decay length-scale. This quantity is a measure of how far glucose diffusively penetrates into the domain before being consumed by hexokinase. It is scaled by the size of the domain to give the dimensionless decay length scale λ^=DKMkgM¯L2 used as a key parameter in our model with localized glucose entry: For the model with spatially uniform glucose permeability, we solve directly for the steady state distributions of glucose and mitochondria in the limit of slow diffusivity. When diffusion along the domain is slow compared to the timescales of glucose consumption and glucose import, the steady-state equation for glucose concentration is given by a simplified form of Equation 6: −k (x) G (x) +P (x) (Gext (x) −G (x) ) =0 Substituting k (x) =kgM (x) G (x) G (x) +KM and P (x) = (2/r) PKMPKMP+|Gext (x) −G (x) |, we get a quadratic equation in G (x); (15) [1−2PKMPrkgM]G (x) 2+[2PKMPGextrkgM−2PKMPKMrkgM−Gext−KMP]G (x) +[2PKMPKMGextrkgM]=0 For a given mitochondrial profile, this quadratic equation is solved to find G (x) =G (M (x) ). The mitochondrial distribution, M (x) is then updated according to Equation 11 and Equation 3. We thus arrive at an iterative solution for G (x) and M (x). | Title: Spatial control of neuronal metabolism through glucose-mediated mitochondrial transport regulation Summary: Cells are equipped with power factories called mitochondria that turn nutrients into chemical energy to fuel processes in the cell. Hundreds of mitochondria move throughout the cell, shifting their positions in response to energy demands. This happens via molecular motors that pick the mitochondria up and carry them to new locations. Such movements enable the mitochondria to accumulate in parts of the cell with the greatest energy needs. Mitochondria of nerve cells or neurons have a particular challenging job, as neurons can be very long and different parts within the cells can have different energy needs. It has been shown that mitochondria stop in regions where nutrients such as sugar are most concentrated. So far, it has been unclear whether this regulated stopping helps control energy balance in neurons. Here, Agrawal et al. used a computational model of rat neurons to find out whether sugar levels are sufficient in guiding mitochondria. The results showed that the mitochondria only accumulated in high-nutrient regions when the sugar concentrations were moderate - not too low and not too high. A specific range of sugar levels was necessary to make this mechanism useful for increasing the efficiency of energy production. Such concentrations match the ones observed in healthy rat brains. When neurons are unable to meet their energy demands, they stop working and sometimes even die. This is the case in many diseases, including diabetes, dementia, and Alzheimer's disease. Computer models allow us to explore the complex energy regulation in detail. A better understanding of how neurons regulate their energy production and demand may help us discover how they become faulty in these diseases. | 12,254 | 363 | lay_elife | en |
Summarize: Dinosaurs are often depicted as giant, frightening beasts. But every creature is a baby once. A new examination of a rock slab containing fossils of 24 very young dinosaurs and one older individual is suggestive of a group of hatchlings overseen by a caretaker, according to a new study by University of Pennsylvania researchers. Penn’s Brandon P. Hedrick and Peter Dodson led the work, collaborating with researchers from China’s Dalian Museum of Natural History, where the specimen is held. Hedrick is a doctoral student in the School of Arts & Sciences’ Department of Earth and Environmental Science, where Dodson is a professor of paleontology. Dodson is also professor of anatomy in the School of Veterinary Medicine’s Department of Animal Biology. Gomaa I. Omar of Penn’s Earth and Environnmental Science was a coauthor of the paper, which appears in the journal Cretaceous Research. Amateur paleontologists came upon the fossils, which are about 120 million years old, in the Lujiatun beds of the Yixian Formation in northeastern China’s Liaoning Province. Though the entire specimen is only about two feet across, it contains fossils from 25 creatures, all of the species Psittacosaurus lujiatunensis. Psittacosaurs were plant eaters and are among the most abundant dinosaurs yet discovered. The specimen had previously been described only briefly, in a one-page paper in 2004. The people who found and extracted the fossils did not record their exact original location, which hampers the investigation to some degree. But Dodson and Hedrick felt there was much more to say about the specimen. “I saw a photo of it and instantly knew I wanted to explore it in more depth,” Hedrick said. To analyze the material in which the animals were preserved, the researchers examined thin slivers of rock under the microscope and samples of ground-up rock using a technique called X-ray diffraction, which relies on the fact that different kinds of minerals bend light in unique ways. Both analyses suggested the rock was composed of volcanic material, an indication that the animals were caught in flowing material from an eruption. The fossils’ orientation supported this idea. “If they were captured in a flow, the long axis — their spines — would be oriented in the same direction,” Hedrick said. “That was what we found. They were likely trapped by a flow, though we can’t say exactly what kind of flow.” Because there was no evidence of heat damage to the bones, the researchers believe the flow was likely a lahar, a slurry of water, mud, rock and other debris associated with volcanic eruptions. The 24 younger animals appeared to be quite similar in size. Though the team considered whether they might have been embryos, still in their eggs, various observations suggest they had already hatched. First, there was no evidence of eggshell material. Also, other paleontologists have identified even smaller individual psittacosaurs. And finally, Hedrick said, “the ends of their bones were well developed, which indicates they were capable of moving around.” The larger skull was firmly embedded in the same layer of rock as the 24 smaller animals. Two of the younger animals were in fact intertwined with the skull, signs that the animals were closely associated at the time of their death. The skull’s size, about 4.5 inches long, indicated that the animal was estimated to be between 4 and 5 years old. Earlier findings suggested that P. lujiatunensis did not reproduce until 8 or 9 years old, so this creature was probably not the parent of the younger dinosaurs. Given the close association of the young P. lujiatunensis with the older individual, however, Dodson, Hedrick and colleagues believe this specimen may offer evidence of post-hatchling cooperation, a behavior exhibited by some species of modern-day birds. The older juvenile may well have been a big brother or sister helping care for its younger siblings. The researchers emphasize that they can’t definitively call this assemblage of fossils a nest, as some earlier analyses have. “It certainly seems like it might be a nest, but we weren’t able to satisfy the intense criteria to say definitively that it is,” Hedrick said. “It’s just as important to point out what we don’t know for sure as it is to say what we’re certain of.” As a next step, Dodson and Hedrick are examining the microstructure of the bones of the smaller animals to establish whether they were all at the same stage of development, which would lend support to the idea of this being one clutch of animals. Additional coauthors on the paper included Gao Chunling, Zhang Fengjiao and Shen Caizhi of the Dalian Museum. The study was supported by the University of Pennsylvania Paleobiology Stipend and the National Science Foundation. This rock slab, which contains fossilized remains from 25 animals, may be evidence of a dinosaur nest, according to a study led by researchers from the University of Pennsylvania. University of Pennsylvania University of Pennsylvania researchers have uncovered evidence of a possible 120 million-year-old dinosaur nest that may have been overseen by a dinosaur “babysitter.” The finding, published in the September issue of Cretaceous Research, comes from a reexamination of a rock slab found in the Lujiatun beds of the Yixian Formation, located in northeastern China’s Liaoning province. Embedded in the slab are the fossils of 24 dinosaur hatchlings, along with the skull of one larger creature, believed to have been between 4 and 5 years old, according to the study, led by Penn environmental sciences doctoral student Brandon P. Hedrick and paleontology professor Peter Dodson. All 25 remains are from the species Psittacosaurus lujiatunensis, a type of plant-eating dinosaur that existed in the Early Cretaceous period. The older dinosaur “may well have been a big brother or sister” helping to care for its younger siblings, researchers pointed out in a news release issued by the University of Pennsylvania. The older dinosaur’s skull was found embedded in the same layer of rock as the fossils of the 24 smaller animals and was intertwined with two of them, suggesting the creatures were closely associated at the time of their death, the researchers said. Dodson and Hedrick will next examine the smaller animals’ bones in an attempt to establish whether they are all at the same developmental stage, which would support the idea the creatures were part of one clutch. Still, the researchers emphasized they can’t yet say for sure whether the fossils comprised a nest. “It certainly seems like it might be a nest, but we weren’t able to satisfy the intense criteria to say definitively that it is,” Hedrick said in the release. “It’s just as important to point out what we don’t know for sure as it is to say what we’re certain of.” Contact Alex Wigglesworth at 215-854-2305 or awigglesworth@philly.com. Follow @phila_lex on Twitter. Contact the Breaking News Desk at 215-854-2443; BreakingNewsDesk@philly.com. Follow @phillynews on Twitter. These crawls are part of an effort to archive pages as they are created and archive the pages that they refer to. That way, as the pages that are referenced are changed or taken from the web, a link to the version that was live when the page was written will be preserved.Then the Internet Archive hopes that references to these archived pages will be put in place of a link that would be otherwise be broken, or a companion link to allow people to see what was originally intended by a page's authors.The goal is to fix all broken links on the web. Crawls of supported "No More 404" sites. In a rock slab dating back some 120 million years, scientists have discovered the skeletons of 24 "baby dinosaurs" called psittacosaurs and that of an older individual, suggesting a caretaker was "babysitting" the nestlings. A nest of baby dinosaurs with what might have been a juvenile babysitter sitting atop them has been discovered in China, researchers say. These findings help shed light on how sociable these ancient reptiles might have been, scientists added. The oldest known dino nesting sites are 190 million years old, and their existence suggests that even the earliest dinosaurs may have exhibited complex family behaviors. The new findings center on a 2-foot-wide (60 centimeters) rock slab that contains skeletons of 24 very young dinosaurs and the skull of one older dinosaur. The finding suggests that a caretaker was overseeing a group of hatchlings, the researchers said. [Image Gallery: Dinosaur Daycare] "In my opinion, this fossil is one of the most beautiful dinosaur fossils known," said lead study author Brandon Hedrick, a vertebrate paleontologist at the University of Pennsylvania in Philadelphia. All of the dinosaurs were members of the species Psittacosaurus lujiatunensis. Psittacosaurs, plant-eating dinos that were about 3 to 6 feet (1 to 2 meters) long and walked on two legs. They were early relatives of horned dinosaurs such as Triceratops, though they lacked the horns and frills of these later dinosaurs. "There were a large number of small predators that probably fed on Psittacosaurus, including many different types of theropod dinosaur, ranging from maniraptoran dinosaurs similar to Velociraptor to small tyrannosaurs," Hedrick said. "Psittacosaurus is also the only dinosaur known that has been found in the stomach of a fossil mammal. It seems like pretty much everything ate it." Fossil hunters unearthed the 120-million-year-old bones in northeastern China's Liaoning province, the site of many discoveries of feathered dinosaurs over the last decade. "It is definitely one of the most important sites in dinosaur paleontology on Earth and is certainly the most productive area found in the past 20 years," Hedrick said. The 24 younger psittacosaurs are all similar in size — about 6 inches (15 cm) long. All of these appeared to have already hatched — no eggshells were found, and the ends of their bones were well developed, which suggests the animals were capable of moving around, Hedrick said. The skull of the larger psittacosaur was firmly embedded in the same layer of rock as the 24 smaller reptiles. In fact, two of the younger animals were intertwined with the skull, which suggests that the animals were near one another when they died. The larger skull was about 4.5 inches (11.6 cm) long, suggesting the dinosaur was between 4 and 5 years old, the researchers said. Previous findings suggested that P. lujiatunensis did not reproduce until age 8 or 9, so this creature was probably not the parent of the younger dinosaurs. The researchers suggest the larger dinosaur may have been a babysitter, "such as an older sibling or even a non-related animal," Hedrick told Live Science. "Some birds such as the fairy wren display helper behavior, where they stay and help their parents raise their next clutch of eggs as opposed to starting a family of their own." Even so, the "helper" behavior demonstrated by the "babysitter" is uncommon in birds, the only living members of the dinosaur family. Only 3 to 8 percent of bird species voluntarily tend another's young, Hedrick said. Microscopic analysis of the rock that encased the fossils revealed that it was volcanic material, suggesting that the dinosaurs were caught in flowing material resulting from an eruption. Since there was no evidence of heat damage to the dinosaur bones, the researchers believe the flow was likely a lahar, which refers to a slurry of water, mud, rock and other debris associated with volcanic eruptions. Hedrick cautioned that the researchers do not know for sure if the larger psittacosaur was a babysitter of the smaller psittacosaurs. It is also possible that "the juveniles and the larger individual were carried by a lahar by a river and deposited together, even though they may not have been directly interacting during life," he said. The scientists plan to analyze the microscopic structure of the bones of the smaller animals to establish whether they were all at the same stage of development. If that were true, it would lend support to the idea that these animals interacted. The scientists detailed their findings in the September issue of the journal Cretaceous Research. Follow us @livescience, Facebook & Google+. Original article on Live Science. | Summary: Even dinosaurs need a babysitter-or would that be dino-sitter? Researchers say a group of hatchlings found in a layer of rock might have been under the care of "a big brother or sister," the Philadelphia Inquirer reports. The 120-million-year-old Psittacosaurus bones were found in northeast China, the University of Pennsylvania reports. In a 2-foot-wide slab, 24 hatchlings were apparently overseen by a dinosaur about age 4 or 5, too young to be their mom or dad, LiveScience reports. Two of the young were entwined with the sitter's skull, suggesting intimacy when they all died. While the finding suggests "post-hatchling cooperation," scientists can't be certain it's a nest of related dinosaurs. And it's possible the group was flung together by a volcanic flow and "may not have been directly interacting during life," says the study's author. However, there's some support for the theory in the behavior of birds (descended from dinosaurs) who help their parents with new babies instead of having their own; up to 8% of birds do this. The scientists will further study their dino-sitter theory by testing the babies' bones. If they are at the same point in development, they may be related. LiveScience has this gallery of the discovery. | 2,833 | 308 | multi_news | en |
Summarize: Background The South Florida ecosystem encompasses a broad range of natural, urban, and agricultural areas surrounding the remnant Everglades. Before human intervention, freshwater in the ecosystem flowed south from Lake Okeechobee to Florida Bay in a broad, slow-moving sheet, creating the mix of wetlands that form the ecosystem. These wetlands, interspersed with dry areas, created habitat for abundant wildlife, fish, and birds. The South Florida ecosystem is also home to 6.5 million people and supports a large agricultural, tourist, and industrial economy. To facilitate development in the area, in 1948, Congress authorized the U.S. Army Corps of Engineers to build the Central and Southern Florida Project—a system of more than 1,700 miles of canals and levees and 16 major pump stations—to prevent flooding and intrusion of saltwater into freshwater aquifers on the Atlantic coast. The engineering changes that resulted from the project, and subsequent agricultural, industrial, and urban development, reduced the Everglades ecosystem to about half its original size, causing detrimental effects to fish, bird, and other wildlife habitats and to water quality. Figure 1 shows the historic and current flows of the Everglades ecosystem as well as the proposed restored flow. Efforts to reverse the detrimental effects of development on the ecosystem led to the formal establishment of the Task Force, authorized by the Water Resources Development Act (WRDA) of 1996. The Task Force, charged with coordinating and facilitating the restoration of the ecosystem, established three overall goals to: Get the water right: restore more natural hydrologic functions to the ecosystem while providing adequate water supplies and flood control. The goal is to deliver the right amount of water, of the right quality, to the right places at the right times. Restore, protect, and preserve the natural system: restore lost and altered habitats and change current land use patterns. Growth and development have displaced and disconnected natural habitats and the spread of invasive species has caused sharp declines in native plant and animal populations. Foster the compatibility of the built and natural systems: find development patterns that are complementary to ecosystem restoration and to a restored natural system. Figure 2 shows the relationship of the agencies participating in restoration, the Task Force, and the three restoration goals. Because of the complexity of the ecosystem and efforts underway to restore it, and the urgency to begin the long-term ecosystem restoration effort, not all of the scientific information that is needed is available to make restoration decisions. As a result, scientists will continually need to develop information and restoration decision makers will continually need to review it. According to the Task Force, scientists participating in restoration are expected to identify and determine what information is needed to fill gaps in scientific knowledge critical to meeting restoration objectives and provide managers with updated scientific information for critical restoration decisions. Generally, decisions about restoration projects and plans have been—and will continue to be—made by the agencies participating in the restoration initiative. To provide agency managers and the Task Force with updated scientific information, the Task Force has endorsed adaptive management, a process that requires key tools, such as models, continued research, and monitoring plans. Federal and State Agencies Spent $576 Million on Scientific Activities for the South Florida Ecosystem and Made Progress in Some Areas Federal and state agencies spent $576 million from fiscal years 1993 through 2002 to conduct mission-related scientific research, monitoring, and assessment in support of the restoration of the South Florida ecosystem. Eight federal departments and agencies spent $273 million for scientific activities, with the Department of the Interior spending $139 million (about half) of the funds. The level of federal expenditures, which increased by over 50 percent in 1997, has since remained relatively constant. The South Florida Water Management District—the state agency most heavily involved in scientific activities for restoration—spent $303 million from 1993 through 2002. The District’s expenditures have increased steadily since 1993, with significant increases in 2000 and 2002. Figure 3 shows the total federal and state expenditures for scientific activities related to restoration over the last decade. Eight federal agencies are involved in scientific activities for the restoration: the Department of the Interior’s U.S. Geological Survey, National Park Service, Fish and Wildlife Service, and Bureau of Indian Affairs; the Department of Commerce’s National Oceanic and Atmospheric Administration; the Department of Agriculture’s Agricultural Research Service; the U.S. Army Corps of Engineers; and the Environmental Protection Agency. Within the Department of the Interior, four agencies spent $139 million on scientific activities. The U.S. Geological Survey spent over half of the Interior funding, or $77 million, primarily on its Placed-Based Studies Program, which provides information, data, and models to other agencies to support decisions for ecosystem restoration and management. The National Park Service spent about $48 million for the Critical Ecosystem Studies Initiative (CESI), a program begun in 1997 to accelerate research to provide scientific information for the restoration initiative. The National Park Service used CESI funding to support research (1) to characterize the ecosystem’s predrainage and current conditions and (2) to identify indicators for monitoring the success of restoration in Everglades National Park, other parks, and public lands and to develop models and tools to assess the effects of water projects on these natural lands. Of the remaining Interior funding, the Fish and Wildlife Service and the Bureau of Indian Affairs spent $10 million and $3 million, respectively. Four agencies spent the other federal funds—$134 million. The Corps of Engineers and the National Oceanic and Atmospheric Administration spent approximately $37 million each, primarily on research activities. Two other federal agencies—the Agricultural Research Service and the Environmental Protection Agency—spent the remaining $60 million in federal funds. In addition to the $273 million spent by federal agencies, the State of Florida’s South Florida Water Management District provided $303 million for such activities from 1993 to 2002. The District spent much of its funding on scientific activities related to water projects in line with its major responsibility to manage and operate the Central and Southern Florida Project and water resources in the ecosystem. With these federal and state expenditures, scientists have made some progress in developing scientific information and adaptive management tools. In particular, scientists now better understand the historic and current hydrological conditions in the ecosystem and developed models that allow them to forecast the effects of water management alternatives on the ecosystem. Scientists also made significant progress in developing information on the sources, transformations, and fate of mercury—a contaminant that affects water quality and the health of birds, animals, and humans—in the South Florida ecosystem. Specifically, scientists determined that atmospheric sources account for greater than 95 percent of the mercury that is added to the ecosystem. In addition, scientists made progress in developing (1) a method that uses a natural predator to control Melaleuca, an invasive species, and (2) techniques to reduce high levels of nutrients—primarily phosphorus—in the ecosystem. Gaps Remain in the Scientific Information Needed for Restoration While scientists made progress in developing scientific information, they also identified significant gaps in scientific information and adaptive management tools that, if not addressed in the near future, will hinder the overall success of the restoration effort. We reviewed 10 critical restoration projects and plans and discussed the scientific information needs remaining for these projects with scientists and project managers. On the basis of our review, we identified three types of gaps in scientific information: (1) gaps that threaten systemwide restoration if they are not addressed; (2) gaps that threaten the success of particular restoration projects if they are not addressed; and (3) gaps in information and tools that will prevent restoration officials from using adaptive management to pursue restoration goals. An example of a gap that could hinder systemwide restoration is information on contaminants, such as fertilizers and pesticides. Scientists are concerned that the heavy use of fertilizers and pesticides—which are transported by water and soil and are deposited in sediments—near natural areas in South Florida increases the discharge of chemical compounds into these areas. Contaminants are absorbed by organisms such as aquatic insects, other invertebrates, and fish that live in the water and sediment, affecting the survival and reproduction of these organisms and those that feed on them. Scientists need information on the amount of contaminants that could be discharged into the environment, the amounts that persist in water and sediment, and the risks faced by organisms living in areas with contaminants—even low levels of contaminants on a long- term basis. If this information is not available, scientists cannot determine whether contaminants harm fish and other organisms or whether the redistribution of water will introduce potentially harmful contaminants to parts of the ecosystem that are relatively undisturbed. An example of a gap that could hinder the progress of a specific project is information needed to complete the Modified Water Delivery project, which has been ongoing for many years and has been delayed primarily because of land acquisition conflicts. The Modified Water Delivery project and a related project in the Comprehensive Everglades Restoration Plan are expected, among other purposes, to increase the amount of water running through the eastern part of Everglades National Park and restore the “ridge and slough” habitat. However, scientists identified the need for continued work to understand the role of flowing water in the creation of ridge and slough habitat. If the information is not developed, the project designs may be delayed or inadequate, forcing scientists and project managers to spend time redesigning projects or making unnecessary modifications to those already built. An example of a gap in key tools needed for adaptive management is the lack of mathematical models that would allow scientists to simulate aspects of the ecosystem and better understand how the ecosystem responds to restoration actions. Scientists identified the need for several important models including models for Florida Bay, Biscayne Bay, and systemwide vegetation. Without such tools, the process of adaptive management will be hindered because scientists and managers will be less able to monitor and assess key indicators of restoration and evaluate the effects created by particular restoration actions. The Restoration Initiative Lacks an Effective Means to Coordinate Scientific Activities The Water Resources Development Act of 1996 requires the Task Force to coordinate scientific research for South Florida restoration; however, the Task Force has not established an effective means to do so, diminishing assurance that key scientific information will be developed and available to fill gaps and support restoration decisions. The SCT’s main responsibilities are planning scientific activities for restoration, ensuring the development of a monitoring plan, synthesizing scientific information, and conducting science conferences and workshops on major issues such as invasive species and sustainable agriculture. As the restoration has proceeded, other groups have been created to manage scientific activities and information for particular programs or issues, but these groups are more narrowly focused than the SCT. These groups and a more detailed discussion of their individual purposes appear in appendix I. Although the Task Force created the SCT as a science coordination group, it established the group with several organizational limitations, contributing to the SCT’s inability to accomplish several important functions. Specifically, the Task Force did not: Provide specific planning requirements, including requirements for a science plan or comprehensive monitoring plan. A science plan would (1) facilitate coordination of the multiple agency science plans and programs, (2) identify key gaps in scientific information and tools, (3) prioritize scientific activities needed to fill such gaps, and (4) recommend agencies with expertise to fund and conduct work to fill these gaps. In addition, a comprehensive monitoring plan would support the evaluation of restoration activities. This plan would identify measures and indicators of a restored ecosystem—for all three goals of restoration—and would provide scientists with a key tool to implement adaptive management. Establish processes that (1) provide management input for science planning and (2) identify and prioritize scientific issues for the SCT to address in its synthesis reports. Scientists and managers have both noted the need for an effective process that allows the Task Force to identify significant restoration management issues or questions that scientific activities need to address. In addition, a process used to select issues for synthesis reports needs to be transparent to members of the SCT and the Task Force and needs to facilitate the provision of a credible list of issues that the SCT needs to address in its synthesis reports. One way that other scientific groups involved in restoration efforts, such as the Chesapeake Bay effort, address transparency and credibility is the use an advisory board to provide an independent review of the scientific plans, reports, and issues. Provide resources for carrying out its responsibilities. Only two agencies—the U.S. Geological Survey and the South Florida Water Management District—have allocated some staff time for SCT duties. In comparison, leaders of other large ecosystem restoration efforts—the San Francisco Bay and Chesapeake Bay area efforts—have recognized that significant resources are required to coordinate science for such efforts. These scientists and managers stated that their coordination groups have full-time leadership (an executive director or chief scientist), several full- time staff to coordinate agencies’ science efforts and develop plans and reports, and administrative staff to support functions. To improve the coordination of scientific activities for the South Florida ecosystem restoration initiative, we recommended in our report—released today—that the Secretary of the Interior, as chair of the Task Force, take several actions to strengthen the SCT. First, the plans and documents to be produced by the SCT should be specified, along with time frames for completing them. Second, a process should be established to provide Task Force input into planning for scientific activities. Third, a process—such as independent advisory board review—should be established to prioritize the issues requiring synthesis of scientific information. Finally, an assessment of the SCT’s resource needs should be made and sufficient staff resources should be allocated to SCT efforts. In commenting on a draft of our report, the Department of the Interior agreed with the premises of our report that scientific activities for restoration need to be better coordinated and the SCT’s responsibilities need to be clarified. However, Interior noted that the Task Force itself will ultimately need to agree on the actions necessary to strengthen the SCT. Although Interior agreed to coordinate the comments of the Task Force agencies, it could not do so because this would require the public disclosure of the draft report. Mr. Chairman, this concludes my formal statement. If you or other Members of the Subcommittee have any questions, I will be pleased to answer them. Contact and Acknowledgments For further information on this testimony, please contact Barry T. Hill at (202) 512-3841. Individuals making key contributions to this testimony included Susan Iott, Chet Janik, Beverly Peterson, and Shelby Stephan. Appendix I: Groups Responsible for Coordinating Scientific Activities for the South Florida Ecosystem Restoration The South Florida Ecosystem Restoration Task Force (Task Force) and participating agencies have created several groups with responsibilities for various scientific activities. One of these teams—the Science Coordination Team (SCT) created by the Task Force—is the only group responsible for coordinating restoration science activities that relate to all three of the Task Force’s restoration goals (see fig. 4). Other teams that have been created with responsibility for scientific activities include the Restoration Coordination and Verification (RECOVER) program teams, the Multi-Species Ecosystem Recovery Implementation Team, the Noxious Exotic Weed Task Team, and the Committee on Restoration of the Greater Everglades Ecosystem (CROGEE). As shown in figure 4, each of these teams is responsible for scientific activities related to specific aspects of restoration. | Summary: Restoration of the South Florida ecosystem is a complex, long-term federal and state undertaking that requires the development of extensive scientific information. GAO was asked to report on the funds spent on scientific activities for restoration, the gaps that exist in scientific information, and the extent to which scientific activities are being coordinated. From fiscal years 1993 through 2002, eight federal agencies and one state agency collectively spent $576 million to conduct mission-related scientific research, monitoring, and assessment in support of the restoration of the South Florida ecosystem. With this funding, which was almost evenly split between the federal agencies and the state agency, scientists have made progress in developing information--including information on the past, present, and future flow of water in the ecosystem--for restoration. While some scientific information has been obtained and understanding of the ecosystem improved, key gaps remain in scientific information needed for restoration. If not addressed quickly, these gaps could hinder the success of restoration. One particularly important gap is the lack of information regarding the amount and risk of contaminants, such as fertilizers and pesticides, in water and sediment throughout the ecosystem. The South Florida Ecosystem Restoration Task Force--comprised of federal, state, local, and tribal entities--is responsible for coordinating the South Florida ecosystem restoration initiative. The Task Force is also responsible for coordinating scientific activities for restoration, but has yet to establish an effective means of doing so. In 1997, it created the Science Coordination Team (SCT) to coordinate the science activities of the many agencies participating in restoration. However, the Task Force did not give the SCT clear direction to carry out its responsibilities in support of the Task Force and restoration. Furthermore, unlike the full-time science coordinating bodies created for other restoration efforts, the SCT functions as a voluntary group with no full-time and few part-time staff. Without an effective means to coordinate restoration, the Task Force cannot ensure that restoration decisions are based on sound scientific information. | 3,406 | 429 | gov_report | en |
Summarize: It was a landmark moment in her life and should have been one of her family’s most precious memories. But Harmonie-Rose Allen’s first steps are tinged with sadness for her parents. Just weeks after starting to walk, their daughter is to lose all of her limbs to meningitis. Harmonie-Rose Allen is currently in hospital facing amputation of both her arms and legs after she contracted meningitis. She is pictured here with parents Freya and Ross. The 11-month-old is on a life support machine in hospital, suffering what doctors said is one of the worst cases of the deadly condition they have seen. Despite initially being given just a 10 per cent chance of survival, Harmonie-Rose is expected to live – but her arms and legs must be amputated. The tragedy comes just weeks after she took her first tentative steps with a baby walker. Family and friends in her home city of Bath, Somerset, are now hoping to raise more than £40,000 to pay for prosthetic limbs that will enable her to live as normal a life as possible. Harmonie-Rose’s aunt Hannah Hall, 31, said doctors did not think her niece would pull through, adding: ‘The family feel like Harmonie-Rose’s whole life has been robbed.’ The baby’s parents Freya Hall, 20, and Ross Allen first noticed something was wrong on September 27, when she woke in the night coughing. The baby’s parents Freya Hall, 20, and Ross Allen first noticed something was wrong on September 27, when she woke in the night coughing. Mr Allen, a customer service worker, said: ‘She had been a little bit under the weather all week. But we just assumed it was a cold and because she was teething we put it down to that as well.’ They took her to the Royal United Hospital in Bath as she was finding it difficult to breathe, but doctors could not find anything seriously wrong and she was sent home. The next morning Harmonie-Rose turned ‘all limp and she was blue’ and the couple took her back to the same hospital. Following a thorough assessment they were told it was just a virus and were again sent away. Just weeks after starting to walk, Harmonie-Rose was diagnosed with the worst case of meningitis that doctors had seen in years. But hours later she became floppy and lethargic and her parents took her to the hospital for a third time, when a rash was spotted. Harmonie-Rose was then taken by ambulance to a specialist unit at the Bristol Royal Hospital for Children. Her devastated father said: ‘It was absolutely horrible. She was really lucky to survive. ‘Doctors told us it was the worst case they had seen in three years and they really didn’t think she would make the journey to the hospital.’ By the time she arrived at the intensive care unit, Harmonie-Rose’s arms and legs had turned black and her parents were told all four limbs would need to be amputated. The little girl needs two pairs of prosthetics to replace her arms and legs, which will cost around £40,000. She is still being closely monitored by doctors. The operation will be carried out in the coming weeks. Mr Allen said: ‘We are hoping that we can buy her two pairs of prosthetics – arms and legs that she can walk on. ‘This will cost us about £40,000 – £20,000 per set – and we’re hoping we can get her some pink ones. ‘She’s a little fighter and she is going to keep on fighting. ‘We hope that she can carry on with life like any other children her age.’ Her father Ross Allen said: She’s a little fighter and she is going to keep on fighting. We hope that she can carry on with life like any other children her age’ | Summary: Harmonie-Rose Allen, of Somereset, took first joyous steps just weeks ago. But on September 27, 11-month-old went to hospital with bad cough. Turned out to be horrendous case of meningitis and baby's limbs went black. She must now have both arms and both legs amputated in the coming weeks. Parents Freya Hall and Ross Allen desperate to pay for pink prosthetics. Father says: 'We hope she can carry on with life like other children her age' | 861 | 115 | cnn_dailymail | en |
Summarize: Published on May 6, 2016 Breaking News: OUR PLAN. CHECK IT OUT https://youtu.be/xwB5ERBjzv8 Follow Twitter for updates https://twitter.com/911REDUX FACEBOOK https://www.facebook.com/911redux/ Or at 911REDUX.com It's really heating up now and we will complete this project one way or another. Thanks for your support! Crowdfunding project: Goal: to recreate 9-11 and prove or disprove, once and for all, what happened. We need a 747 with a working blackbox, a tall building as close to the WTC as we can get, many GoPro's to record the event inside the building and inside the plane. A film crew to turn this event into a documentary. Our initial funding goal is $300,000 and we will go to any country where we can get the right ingredients to faithfully recreate 9-11. Email paul@paulsalo.com for donation information. To donate bitcoin: https://blockchain.info/address/1A7jm... A very ambitious and evidently serious American expatriate is trying to raise $1.5 million to “recreate” the September 11 attacks in an empty field in Thailand by crashing an aircraft into a building at 500 miles per hour. He’s promising front-row seats to anyone who donates $5,000 or more. Advertisement Paul Salo is a businessman of some sort whose various titles on LinkedIn include a lot of references to marketing and consulting. He says he’s lived in Shanghai for over a decade, and, in one of those swerves that happen sometimes in people’s lives, has now decided he’s the guy destined to determine whether or not September 11, 2001, was a “hoax.” In a YouTube video posted May 6, Salo announced the launch of his project, which is, in short, to buy a building that’s about to be torn down in a remote area, buy a plane with a functioning black box, and crash one into the other very hard, using autopilot. “Everything that was in 9/11, we’re putting in this one,” Salo says, joyously. “Passports, old passports, we’re going to put them around the airplane.” And no misbehavior will be tolerated, Salo explains, because the building, the airplane, and the surrounding countryside will be festooned with cameras: “Anybody who tries to mess with anything will be on camera.” Advertisement Salo says in the video he thinks, in his opinion, “that we probably will find out that it was a similar physics as what happened on 9/11, OK? I believe that actually 9/11, some guys flew airplanes in there. But at the same time, hey, I’m no dummy. Maybe it’s not true. I want to find out too. I want to fly that sucker in there. We’re going to find out exactly what happens when something goes 500 miles an hour into a building full of fuel.” Salo’s “quest” was first reported by Coconuts Bangok, which politely referred to him as “delusional.” On his crowdfunding page, which he’s titled “September 11 Redux,” Salo presents this whole thing as a way to get to the bottom of 9/11 for good and all, and insists he doesn’t mean any disrespect: Advertisement Sponsored I have total respect for the firefighters and police that gave their lives on September 11th. And for the victims of that tragedy. I have no desire to downplay the heroism on that fateful day. We all have belief in the official line somewhere between black and white on each extreme. We are not a team of tin foil hats. Of truthers. We just want to see it. And, as I said, let what happens happen. Either way, you’ll be there either online or off and can participate on some level. In his video, Salo says he needs $300,000, but the Indiegogo page is seeking $1.5 million USD. Salo acknowledges that there’s “no way to know” how much the project will cost, adding that in the event that the plane misses the building, “obviously that sucks, but we’ll do it again.” Advertisement This appears to be an entirely serious, non-trolling effort, but given how outlandish it is, there does exist the slim possibility that Salo is messing with us. At publication time, his Indiegogo page indicated that he had raised $10. On LinkedIn, he’s requesting “reputable” PR agencies to advise him on media, saying he’s been “inundated” with requests. We’ve reached out to him for comment and will update if we hear back. Nutty rich dudes with too much time on their hands and too many yes men at their beck and call have admittedly been responsible for some great innovations throughout American history. But for every Howard Hughes, you get a Paul Salo, an American expat living in Thailand who wants to recreate 9/11 by smashing a plane into a building and finding out—ONCE AND FOR ALL—whether 9/11 was a hoax, a conspiracy, a mirage, native advertising, or just some really good inspiration for high school cheer routines. Jet fuel can't melt steel beams, but can it shut down conspiracy theorists forevermore? Salo, a 51-year-old "relocation expert" and entrepreneur who has lived in Asia since 1989, seems to believe that there is no big conspiracy—he's just asking questions—but he is a true believer when it comes to attention, as seen by his newly launched social media accounts and eager promotion of the deeply misguided project on his website. Cover of Drudge Report. "Businessman to recreate 911" pic.twitter.com/c8YbhsRymL — 911 Redux (@911REDUX) May 16, 2016 "We're going to purchase a 747 or equivalent aircraft that's about to go out of service, we're going to fill it full of jet fuel, we're going to purchase a building that's about to be torn down in the countryside...and we're going to crash it at 500 miles per hour directly into that building using autopilot," he says in the video. "Everything that was in 9/11, we're gonna put into this one." "If there's just a smoking hole in the building and nothing happens, you pretty much know it was a hoax, right? Cause it's obvious, right?" he added. "Sure, some people might be upset, but we deserve to find out what happened... You deserve it. We all deserve it. We deserve to find out what happens. It's never happened before. It's never happened before. So you don't have anything to watch, anything to look at when this occurs." It's bad enough Salo has no expertise or background in scientific experiments (why is he in charge of this project?), but he seems to have made no consideration for the emotions of those actually affected by the terrible attack—he seems completely glib, even giddy, while talking about smashing planes into buildings, with no indication that he cares about the actual implications of his pet project, and the families of the people who lost their lives. And that's pushing aside the fact that the jet fuel theories have been debunked time and time again by actual experts, to the point that the whole 9/11 "truther movement" has ground to a halt in recent years and left only a few sad lonely people out there pushing the issue. Because this isn't really about some exploration for the truth, no matter how meritless that search might be—Salo is only concerned about making entertainment out of tragedy. "I really want to make something everyone in the world will want to watch," he told Coconuts Bangkok. "Don't you want to see a plane hitting a building at 500 MPH and proving or casting doubt on of the most important events of our lifetime? Sure you do." You can learn more about this dumb project at Salo's "September 11th Redux" Indiegogo page, where he has raised a whopping $10 as of 4 p.m. today (and offers you a "front row seat" to the carnage). We raise you one actual conspiracy theory worthy of consideration: in the video, he says he wants to do this project for $300K, but the Indiegogo says he is trying to raise $1.5 million for it. So where is that extra $1.2 million going? Who is it benefitting? We deserve to find out what happened to it. | Summary: Paul Salo is an American entrepreneur living in Thailand, and his latest project has earned him the descriptors of "delusional" from the website Coconuts Bangkok and "possible supervillain" from Gothamist. Why? He's trying to raise money so he can buy an old 747 and fly it into a building in a remote locale to determine "once and for all" whether 9/11 was a hoax. "I want to fly that sucker in there and see what happens," Salo says in a YouTube video. The American expat says he personally doesn't believe the conspiracy theories out there, but he's curious to see what happens to the jet. "Everything that was in 9/11, we're putting in this one," including passports and a functioning black box, he says. "Will the plane disintegrate? Will the black box disappear? Will the out-of date passports we scatter in the plane survive? You will see it all." Salo, who doesn't appear to have any background in science, says he's spoken to the Thai military about buying a 747 and a building about to be torn down, but there's no deal yet. If the stunt isn't possible in Thailand, Salo says he'll head anywhere in the world. "I have no desire to downplay the heroism on that fateful day," but "I really want to make something everyone in the world will want to watch." (It's that sentiment that has Gothamist criticizing him for trying to make "entertainment out of tragedy.") Might this be a hoax? A post at Jezebel says it "appears to be an entirely serious, non-trolling effort, but given how outlandish it is, there does exist the slim possibility that Salo is messing with us." Donate $5,000 and you'll get to see the event in person, though you'll have to pay your own way, according to his fundraising page. He's raised $105 as of this writing. | 1,993 | 452 | multi_news | en |
Write a title and summarize: Understanding the mechanisms that generate complex host-parasite interactions, and how they contribute to variation between and within hosts, is important for predicting risk of infection and transmission, and for developing more effective interventions based on parasite properties. We used the T. retortaeformis (TR) -rabbit system and developed a state-space mathematical framework to capture the variation in intensity of infection and egg shedding in hosts infected weekly, then treated with an anthelminthic and subsequently re-challenged following the same infection regime. Experimental infections indicate that parasite intensity accumulates more slowly in the post-anthelminthic phase but reaches similar maximum numbers. By contrast, parasite EPG (eggs per gram of feces) shed from rabbits in the post-treatment phase is lower and less variable through time. Inference based on EPG alone suggests a decline in parasite intensity over time. Using a state-space model and incorporating all sources of cross-sectional and longitudinal data, we show that while parasite intensity remains relatively constant in both experimental phases, shedding of eggs into the environment is increasingly limited through changes in parasite growth. We suggest that host immunity directly modulates both the accumulation and the growth of the parasite, and indirectly affects transmission by limiting parasite length and thus fecundity. This study provides a better understanding of how within-host trophic interactions influence different components of a helminth population. It also suggests that heterogeneity in parasite traits should be addressed more carefully when examining and managing helminth infections in the absence of some critical data on parasite dynamics. The large variation in disease severity and transmission often observed among individuals infected with helminths is strongly determined by the local conditions that incoming and established parasites encounter within the host, in addition to variation in the exposure of hosts to infective stages [1–3]. These conditions are mainly determined by the current and previous immuno-physiological attributes of the host, such as the local immune profile or the level of chemical homeostasis, and ecological parasite processes mostly driven by intensity-dependent competition for resources [1,4–15], where intensity represents the number of parasites in infected hosts. Host and parasite constraints have been shown to affect both the intensity and the life history of parasites with interactions that frequently lead to non-linear dynamics of infection and complex trade-offs between parasite life-history traits. The within-host regulation of gastrointestinal helminths, and the consequences for the dynamics and life history of the parasite, remains a subject of ongoing research [4,16–19]. The fundamental challenge is to provide convincing evidence for the mechanisms that affect the intensity of infection and the way parasite traits (i. e. development, fecundity and shedding) adjust to these changes. Ultimately, the understanding of these processes under ecological and immunological forces, and their relative contribution to the observed phenotype of infection, is important for explaining the often large variation in the host’s ability to control the infection. A useful approach to disentangling the contribution of these two forces is by examining how parasites adjust to external perturbations, such as anthelminthic treatments. Anthelminthics are commonly delivered over one to several days (depending on the type of treatment) and parasites are removed within hours or multiple days; however, in endemic areas reinfection is inevitably the norm. Because of this, by altering the number of parasites competing for host resources and/or the strength of intensity-dependent immune responses [20,21] treatments lead to patterns of infection and re-infection that can be fundamentally different [2,4, 22] than treatment-free settings. Indeed, the transient disruption caused by the drug treatment resets the background environment by placing the initial parasite population to 0, thus upon re-infection the new incoming parasites face a primed environment where competition for resources is minimal and/or the immunity still holds some memory from the previous history of infection. Any change in the dynamics of parasite establishment from the pre-treatment to the post-treatment phase should be due to the additional effects of memory in the immune response. In contrast, no changes in parasite dynamics between treatments should be indicative of a regulation by ecological forces driven by intensity-dependent processes in the parasite population. Irrespective of the anthelminthic treatment, following the constant exposure to infective stages and the accumulation of parasites within the host, the intensity of infection will exhibit a logistic growth with host age if regulated by intensity-dependent ecological forces, such as processes of competition for space and food [4]. A hump-shaped age-intensity profile, where older hosts carry fewer parasites, is indicative of an immune response that regulates the parasite population with a strength that increases proportionately with the force of infection [1,20]. Other processes can generate this convex profile [2,22] but here we address this shape as an immunity-generated process. Our previous observations in the Trichostrongylus retortaeformis—rabbit system have conformed to the latter pattern, indicating immune regulation [12,23–26]. The interpretation of the relative contribution of these two forces—ecological limitation through parasite competition for resources and host immune regulation—have conventionally relied on either cross-sectional measures of parasite intensity or longitudinal measures of egg shedding. However, both of these approaches have limitations. For instance, the common method of using age-related, cross-sectional measures of parasite intensity, from dead or drug-treated hosts, assumes that sequential cross-sectional observations in different animals approximate the temporal progression of parasite intensity in a single or average animal and is indicative for a time series of infection [1,10,21,22,27–31]. This necessarily averages out the within-host heterogeneity in parasite dynamics and traits, as it assumes that the unobservable or hidden dynamics of infection in individuals measured at later times behave similarly to those measured at earlier times. This problem can be circumvented by using time series of parasite shedding, which allows observations of individual host variation during the course of the infection. However, this method requires the assumption that the amount of eggs or larvae shed by a host into the environment is directly proportional to the actual parasite burden [32,33] and remains constant over time. Yet, an increasing number of studies have shown that this assumption is rarely confirmed or correct [34–43] and the view that the infection-shedding relationship remains constant over time can lead to wrong conclusions. Indeed, any feedback between parasite intensity and fecundity/shedding, or between-host immunity and parasite fecundity, would violate this assumption. The apparent limitations in the conventional measures of within-host parasite dynamics reflect two challenges in the inference for dynamical systems: the states of interest are often not directly measurable (e. g. parasite burden prior to sacrifice) and/or they are only indirectly measurable (e. g. egg shedding as an indicator of adult parasite burden). State-space or hidden Markov models provide a statistical framework that links a dynamical model of the progression of unobservable states through time to observable measures [44–47]. This class of statistical models has increasingly been used to study the dynamics of infectious diseases at the host population level, where the true incidence of infection is only partially observable through a subset of cases reported through surveillance [48–50]. To understand the relative contribution of immunity and parasite ecological constraints to patterns of helminth intensity, fecundity and shedding, we developed a within-host state-space model of parasite dynamics using a laboratory experiment and the Trichostrongylus retortaeformis (TR) -rabbit system. By linking a dynamic model to an observation model and by combining cross-sectional data on TR intensity, body length and fecundity with longitudinal data on egg shedding we are able to reconstruct the unobservable dynamics of infection within each individual and make inference on the underlying dynamic processes. Specifically, to evaluate changes in TR dynamics and traits following external perturbations and provide a mechanism of regulation we compare the model performance before and after anthelminthic treatment and under constant exposure to infective stages. The regular exposure of rabbits to a constant amount of TR infective larvae leads to a convex pattern between the intensity of infection and host age (i. e. sampling time) both in the pre- and post-treatment phase of the experiment (Fig 1). This is clearer for some rabbits, which is consistent with our previous work [22,51,55]. There was no significant difference between pre- and post- treatment in the mean and maximum intensity of infection, controlling for sampling date (ANOVA p-value = 0. 528, for additional results see S2 Table). The convex age-intensity profile in both experimental phases, slower parasite accumulation, and delayed increase in egg shedding in the post-treatment phase, is suggestive of an effect of accumulated exposure on the establishment and/or clearance of TR. However, it is difficult to make a definitive conclusion from the data as they reflect averages from rabbits sampled at a single time point and variation in TR intensity among hosts is high. The time series of eggs shed in the feces (EPG) by every animal also exhibited a tendency to a convex pattern with host age (Fig 2 (a) and 2 (b) ). The peak in EPG was reached in approximately 4 weeks in both phases of the infection. The number of eggs shed was significantly lower in the post-treatment (pre- and post-phase, mean = 1842 eggs (range 1000–3850) and 890 eggs (range 475–1325), respectively; mixed effects ANOVA p-value <0. 001 with rabbit included as a random effect, S3 Table). The ratio of the number of eggs in utero per female length was not significantly different between phases (p = 0. 35; Poisson regression, S2 Fig, S4 Table). However, we found that adult TR were significantly shorter in the post-treatment compared to the pre-treatment phase (body length post- vs pre-phase 95% CI: 6. 49 − 6. 61 mm vs 7. 12 − 7. 25 mm Fig 2), after accounting for the effect of sampling time (DPI), the section of the intestine and their interactions (p = 0. 006; mixed effects ANOVA with rabbit as a random effect; S5 Table); consistent with the overall lower rate of egg shedding in the post-treatment phase. Forward simulations from the fitted model (Fig 3) replicated the qualitative patterns described by the experimental data. The simulated intensity of infection was comparable between pre- and post-treatment, albeit more variable in the pre-treatment. The number of TR eggs shed was also lower and less variable in the post-treatment, consistent with the original data. Additionally, the fitted model also predicted smaller parasites at 60 days following initial exposure in the post-treatment phase of the experiment, consistent with the observed data (Fig 3 (c), see further discussion below). A total of nine parameters were fitted from the parasite population dynamic and the individual growth models for every rabbit, both in the pre- and post-treatment phase of the experiment. In the population dynamic model, we estimated parameters corresponding to the baseline establishment of infective larval stages (L3), γ1, adult parasite clearance, β1, and the degree of either the intensity of adult infection (γ3, β3) or the cumulative exposure (γ2, β2) on establishment and clearance rates (Table 1). Parameter estimates based on the joint likelihood for the longitudinal and cross-sectional data (Fig 3) indicate that the baseline rate of establishment was reduced in the post-, compared to the pre-treatment phase of the experiment; the posterior mean and central 95th quantiles of the fraction of L3 larvae that establish are 0. 94 (0. 84,0. 98) and 0. 88 (0. 75,0. 95), respectively (Fig 3—panel (a) ). The lower establishment rate in the post-treatment phase is consistent with the later peak in the intensity of TR infection observed. Estimates of adult parasite clearance were low in both the pre- and post- treatment phases of the experiment and the baseline clearance rate was similar in both phases. Overall, parameter estimates were highly variable among rabbits (see points below Fig 3 panels (b), (c), (e), (f) ). We note that the posterior mean for larval establishment tended to be lower (Fig 3—panel (a) ) and adult clearance tended to have higher mean (Fig 3—panel (c) ) in animals sampled later in the experiment. There is a weak trend for a stronger effect of TR intensity and cumulative exposure on establishment (Fig 3—panels (b), (c) ) in the animals sampled later in the experiment. Averaged over all rabbits in the experiment there was no consistent effect of either cumulative exposure to infective stages or current adult intensity on either larval establishment or adult clearance rate; the posterior distribution is similar to the uniform prior distribution for these four parameters when the model is fit to all rabbit simultaneously. Averaged over all rabbits, there was a strong effect of the treatment on the distribution of parasite lengths; the posterior mean and central 95th quantiles of parasite mean final length in the pre- and post-treatment phases are 9. 1 mm (8. 0,10. 4) and 7. 9 mm (7. 4,8. 8), respectively (prior mean and 95th percentiles 9. 0 mm (7. 1,10. 9) ). In both sub-models (cumulative exposure submodel and current intensity submodel (S1 Appendix) ), the general patterns remained consistent; specifically the larval establishment rate remained lower and mean final body length of parasites were shorter in the post-treatment phase. As in the full model, the posterior mean for the model fit to all rabbits simultaneously showed no strong evidence of an effect of cumulative exposure to infective stages (cumulative exposure sub-model) or TR intensity (current intensity sub-model) on establishment or clearance (S5 Fig). Compared to the full model with the cross-sectional and longitudinal data, the model fit with longitudinal data only, results in broadly similar patterns in the parameter estimates (S3 Fig). However, the estimated effect of the treatment on establishment of L3 larvae and the estimated adult parasite final length was greater than that from the fit based on the full likelihood for the longitudinal and cross-sectional data. We can directly compare the observed adult parasite length distribution to the predicted size distribution of parasites at the end of the pre- and post-treatment phases of the experiment for the models fit only to the longitudinal observations (which include no explicit length data) and to both longitudinal and cross-sectional observations. The mean (and IQR) of observed TR lengths at 60 days in the pre- and post- phases was 7. 1 mm (6. 2,7. 9) and 6. 17 mm (5. 3,6. 9) respectively; a mean difference of 0. 93 mm. For the model fit to both the longitudinal and cross sectional observations, the corresponding predicted mean (and IQR) lengths of adult parasites at 60 days in the pre- and post- phases were 8. 1 mm (7. 5,8. 7) and 7. 0 mm (6. 7,7. 5), respectively; a predicted mean difference of 1. 1 mm. For the model fit to only the longitudinal EPG observations the corresponding mean (and IQR) lengths of adult parasites at 60 days in the pre- and post- phases were 8. 8 mm (8. 0,10. 2) and 7. 3 mm (7. 0,7. 7) respectively; a predicted mean difference of 1. 5 mm. Thus, the model fit to only the EPG data, including no explicit length data, estimates a larger effect of anthelminthic treatment on adult parasite length than the full model that includes length data. Both the empirical analyses and model fits suggest a cumulative effect of time, which here reflects the cumulative exposure to both larvae and adult TR, on TR growth, namely body length. Based on the observed data, we found a significant fixed effect of both treatment and sample day on parasite length in a mixed model with a random effect for rabbit; AIC for the null model of no fixed effects, model with treatment only, and model with treatment and sample day is 9450. 1,9437. 5,9433. 7, respectively (Fig 4 (a). To investigate the role of host immunity on these patterns, we repeated the same analysis, including the additive effect of IgA and IgG. There was no significant effect of the Igs, suggesting that individual rabbit-level variation in antibody response could not explain variation over and above the cumulative effect over time. We note that when the antibody variables were included in models that did not include sampling day, all were significant; thus time was confounded with the increasing trend in antibody measures across all rabbits, but the host-to-host variation in antibody response did not provide additional explanatory power. We note that the level of both serum and mucosal IgA and IgG increased monotonically over time (Fig 4 (b) –4 (e) ) as mean TR length decreased. We note further that both serum and mucosal IgA and IgG levels at the first sampling date (day 15) of the post-treatment phase were similar to those on the last sampling day (day 60) of the pre-treatment phase (Fig 4 (c) and 4 (e) ), indicating that immunity remained relatively high during the month in which rabbits were not infected. Our modeling framework provides a novel approach to the study of helminth infections at the host level. Indeed, while offering a parsimonious explanation of the forces driving the dynamics of the parasite it also identifies the traits that are primarily constrained and the processes that generate such patterns. Moreover, the impact of external perturbations, namely anthelminthic drug treatment, is well captured in the simulated dynamics of infection. Ultimately, although this modeling approach needs information both on longitudinal and cross-sectional data it can provide quantitative predictions on changes in helminth intensity and traits under testable external disturbance. All the animal procedures were approved by the Institutional Animal Care and Use Committee of The Pennsylvania State University (USA). The intestinal helminth Trichostrongylus retortaeformis (TR) is a common parasite of the European rabbit (Oryctolagus cuniculus). Hosts become infected by ingesting herbage contaminated with infective third stage larvae (L3). Larvae colonize the small intestine where they mature into adults that reproduce and shed eggs into the environment through the host’s feces. We previously showed that rabbits develop a strong type 1 and type 2 immune response that can clear or reduce the parasite load, although there is no life-long protection and animals are constantly re—infected under endemic exposure [12,51–53]. The initial type 1 response is probably a reaction to the bacterial infiltration in the intestinal wall damaged by the movements of larvae across the tissue during maturation [25,54]. This inflammatory response does not seem to affect the long—term ability of the rabbit to control the infection, which occurs through a type 2 immune response [12,25]. We also showed that there is a negative relationship between body length, or fecundity (i. e. number of eggs in utero per body length), and both antibody IgA levels and intensity of infection [51,54]. While immunity plays an important role to the dynamics of this helminth [12,29,55], it is still unclear if ecological density-dependent parasite processes also have any impact on parasite dynamics and traits and how they adjust to these host and parasite driven forces under anti-helminthic perturbation. Here we performed a laboratory experiment where we quantified parasite variables (counts, body length and egg shedding through feces) and immune response (IgA and IgG antibodies) before and after anthelmintic treatment and over a six-month period. Out-bred, New Zealand, 2 months old, male rabbits were housed in single cages with a 12h light cycle and a daily access to 125 g of standard rabbit pellets and water ad libitum. Animals (n = 36) were orally trickle dosed every 7 days with 400 infective third stage larvae (L3) suspended in 5 ml of tap water; control animals (n = 12) only received tap water. In the current study we focus only on the infected rabbits; the feces of control animals were routinely monitored for TR eggs and were found to be zero at all time points and no parasites were found in the intestine of control animals at sacrifice; we take this as evidence of the independence of infections in rabbits. Groups of four infected animals were sacrificed at days 15,30,45 and 60 post initial infection. At day 60, animals were orally treated for five consecutive days with the broad spectrum anthelminthic Fenbendazole at dosage adjusted by animal body mass (5mg fenbendazole/kg body weight, based on a 10% suspension (100 mg/ml) ) (Panacur, Intervet Inc., USA). Infection was then suspended for 30 days, including the 5 day drug treatment, and subsequently reinstated following the same infection procedure and sampling frequency with four animals sacrificed at days 90,105,120,135 and 150 post initial infection. The removal of TR by the anthelminthic drug means that L3 larvae introduced in the post-treatment phase experience the same population dynamic conditions as in the pre-treatment, though may experience a novel immunological environment due to the history of prior exposure. We selected a one-month hiatus in the re-infection to allow the immune response enough time to decrease in the absence of parasites and mimic the natural condition of lack of exposure for a limited period, but one that was longer than the weekly dosing. This experimental design allows us to investigate the relative contribution of coupling between ecological and immunological forces within the host. Specifically, it provides a context for the effect of host immunity (which necessarily depends on parasite intensity) as well as parasite population on parasite establishment, development and reproduction of new parasites in nature. The treatment successfully removed all the parasites as no eggs were found in the feces during the one month following anthelminthic treatment also no parasites were found in the intestine of rabbits sampled prior to the start of the post-treatment phase at day 0 (or 90 days post initial infection). The sampling points were selected to be compatible with the helminth life cycle (pre-patent period about 12 days) and to provide information on the parasite variables over the course of the infection. At every sampling point, animals were processed as described previously [25]. Briefly, the small intestine was divided into four sections (from duodenum to ileum: SI–1 to SI–4) and the parasites from one half of each section were collected in a 50mL tube and then counted and sexed in 10x2. 5 ml aliquots from that tube (i. e. raw counts reflect 50% of worms in 50% of each section of the intestine). For every section, a sample of randomly selected specimens (≈ 50 parasites for each sex) was stored in 10% PBS—buffered formalin and subsequently processed for biometry data (body length (male and female) and number of eggs in female’s uterus) [51,54]. Along with these cross—sectional data, we also collected longitudinal records on the number of parasite eggs shed in feces of every host twice a week starting from the second week post initial (re–) infection, following standard parasitological procedures (eggs per gram of feces, EPG, [56]). In summary, for each host we collected four distinct parasite measurements: total count from a fraction of intestine, body length (male and female) and eggs in (female’s) uterus at the time of individual sampling, as well as eggs shed through host’s feces every week prior to rabbit sampling. Parasite-specific antibody responses (IgA and IgG) were measured in the blood serum (every week) and in the duodenum mucus (at sampling points) using indirect ELISAs. We used homogenates of adult TR as the source of antigen, as described previously [25,51]. The following dilutions were used: IgA mucus extracts 1: 10, serum samples 1: 20; IgG mucus 1: 20 and sera 1: 160. Briefly, all dilutions were prepared in 1% (w/v) non-fat milk diluted in Phosphate buffered saline (ph7. 2) supplemented with 0. 05% Tween-20 (Fisher Scientific, Hampton, NH). Antigen-antibody complexes were allowed to form overnight at 4°C prior to addition of anti-rabbit IgA or IgG detection antibody (diluted 1: 2000 in the same buffer that was used for diluting test samples). Quantification was based on spectrophotometric analysis; antibody values were then transformed and standardized into optical density (OD) indexes based on positive and negative control samples that were included in each assay plate, see full details reported in our previous work [25,51,57]. For simplicty we refer to the OD index as “antibody levels”. To examine the model for the within-host dynamics of infection and development of TR, we applied a state-space modelling framework that linked the quantified data of parasite intensity—a direct but cross-sectional measure of the state of interest, and time series of fecal egg counts—an indirect longitudinal measure of parasite intensity—to dynamic models that describe the time progression of the unobservable states (parasite counts and size) via an observation model [58]. We modeled the dynamics of parasite intensity within the host as a birth-death process (i. e. establishment of infective L3 larvae and parasite mortality/clearance), where the rates of establishment and clearance are assumed to depend on both cumulative exposure to L3 larvae and current adult parasite intensity. The population of adult parasites at time t + 1, Pt+1 is the result of the combined effect of cumulative exposure and parasite clearance P t + 1 = Λ γ 1 exp (- γ 2 ∑ t Λ - γ 3 P t) + P t - β 1 (1 - exp (- β 2 ∑ t Λ - β 3 P t) ) P t (1) where Λ is the known force of infection (i. e. weekly L3 dose: assumed to be 0 on the days when larvae are not administered and 400 on the days of infection), γ1 is the baseline probability of parasite establishment, γ2 and γ3 give the per capita reduction in L3 establishment due to either new L3 larvae or established adults, respectively. The baseline probability of clearance is given by β1, β2 and β3 give the per capita increase in clearance rate (e. g. adult mortality) due to new L3 larvae or established adults, respectively. The expected body length (in mm) of individual parasites at time t, xt is described by the discrete time logistic model [59], x t + 1 = x t + α x t (1 - x t / L) (2) where α is the mean developmental rate in length and L is the mean final length of an adult parasite. The distribution of parasite length at age t, is assumed to be normal with expectation μL and variance σ L 2. Based on our lab measurements we assumed that all L3 larvae start at an initial mean length x0 of 1. 069 mm and standard deviation of 0. 062 (a total of 53 L3 larvae were sampled to estimate the length distribution at the start of the experiment). The starting L3 size distribution is assumed to be constant at all times during the experiment. Adult male parasites are assumed to have mean final length that is 0. 75 times that of adult female parasites which is ≈ 8mm [51,54]; the mean final length μL reflects the average of all male and female parasites assuming a 1: 1 sex ratio. For every rabbit, and at each sampling point, parasite counts from one quarter of each of the 4 small intestine sections were combined and the observed number of parasites at time t, Ct, was thus assumed to be distributed as follows: C t ∼ Binomial (P t, 0. 25) where Pt is the observed parasite intensity in the whole small intestine at the time of sampling in that specific rabbit. The observed counts of parasites at each length, was binned into length classes of 0. 25 mm intervals between 2 and 20 mm. The resulting vector Z, of length 72, with elements zl is assumed to be drawn from a multinomial distribution with probability vector equal to the proportion of simulated parasites in each size class. Binning the lengths into these discrete classes allows the use of a multinomial likelihood, with expectation determined by the simulated length distribution, without assuming an explicit functional form of distribution of parasite lengths. The number of parasite eggs shed by every host (EPG, based on the average of 2 measurements a week) is a partial observation of the total number of parasite eggs, Et discharged by an animal on a given day. EPG counts are assumed to reflect approximately 3% of the average daily fecal production by a rabbit (based on 30g of dry feces from laboratory rabbits of similar age and under the same feeding regime, unpublished data) and assumed to be uniform across the different animals and constant over the day (see S4 Fig for sensitivity of fitted parameters to the assumed observation rate). The observed number of eggs shed is assumed to be distributed as: EPG t ∼ Poisson (0. 03 E t). This makes the implicit assumption that the rate of eggs shed is homogeneous through time and that the sampled feces are representative of all feces from a given animal. Parasite fecundity (i. e. the number of eggs in utero per adult female parasite at sampling day t) is known to be positively correlated with parasite length [51,54,60]. The observed relationship between eggs in utero and female parasite length (S2 Fig, S4 Table) was fit using a generalized linear mixed model (GLMM), with Poisson error distribution. We also tested whether the slope of this relationship changed from the pre-treatment to post-treatment phase of the experiment. In the simulation model, the total number of eggs produced by every female at time t was a random draw from the fitted GLMM, conditional on the predicted length of that female. Only body lengths longer than 4mm, the length at which females are assumed to be sexually mature, were considered (no eggs were observed in females smaller than 4mm throughout the experiment (S1 Fig) ). The estimated fecundity of the total helminth population at time t, Et was then quantified as the sum of eggs over all adult females, predicted by the population dynamic (Eq (1) ) and individual growth (Eq (2) ) models. We used a Bayesian particle filter method [61] to estimate the parameters of the parasite population dynamic model (Eq (1) ) and the individual parasite growth model (Eq (2) ) independently for each rabbit. The posterior distribution for all parameters was estimated by sampling from a large (50,000) set of parameter combinations with probability proportional to the product of the joint prior probability and the approximate likelihood, where the latter is calculated via a particle filter (explained below), for each parameter combination. Candidate parameters were sampled from conditionally independent uniform distributions. The upper and lower bounds on the uniform prior distributions were chosen to limit dynamics to the range of observed data. We estimated an approximate likelihood for each parameter combination using a particle filter [49]; an algorithm for estimating the likelihood for models where the exact likelihood cannot be stated analytically but realizations of the model can be generated by simulation [50]. These methods have a long history in signal processing and have been recently applied in a number of population dynamic and epidemiological scenarios with partial or imperfect observation of state variables [44–47]. The evaluation of the estimated likelihood requires two linked models: a dynamic model to generate the forward projection of the state variables—here the within-host parasite intensity and body length distribution (Eqs (1) and (2) ) —and an observation model to evaluate the likelihood of observing the data—in our case—parasite counts, EPGs and parasite lengths, conditional on the values of the unobservable state variables. The steps below outline the approximation of the likelihood for each parameter combination, ϕk for k = 1…50,000 for a single rabbit. We treat all rabbits as statistically independent and thus the likelihood of parameter combination given the observations for all rabbits is the product of the individual rabbit likelihoods; below we present estimates conditional on the observations for all rabbits jointly, and each rabbit independently. The evaluation of the particle filter for one parameter combination returns an estimated likelihood, which is used in the calculation of the posterior sampling probability. To evaluate the particle filter for the longitudinal observations of EPG for rabbit r conditional on one specific parameter combination the following approach was implemented: This process was repeated for all EPG observation points; the average of the likelihood of all N particles at each time point gives an estimate of the conditional likelihood of the observed EPG at time step t. The product of these conditional likelihoods, for all observations, 1 to Tr, (where Tr is the time of sacrifice for rabbit r in days) is then the estimated joint likelihood of the longitudinal EPG observations from each rabbit for a single parameter combination ∏ t = 1 T r L (E t | EPG t), ϕ k). On the final observation—the day of animal sacrifice, we evaluated the likelihood of the cross-sectional observations of parasite intensity and parasite length. The conditional likelihood of the observed parasite intensity (O T r) was calculated from the average of the likelihood for each N simulated parasite intensities (P T r), which is given by L (O T r | P T r, ϕ k), assuming O T r ∼ Binomial (P T r, 0. 25). The conditional likelihood of the observed parasite lengths (X T r) was calculated given the average of the likelihood for each of the N simulated lengths distributions (Z T r), is given by L (X T r | Z T r, ϕ k), assuming that the observed vector of counts in length classes is a Multinomial draw, with trials equal to the number of observed adult parasites and probability vector equal to the proportion of simulated parasites at the final time step, Tr, in each size class for particle i. The product of these two likelihoods gives the estimated likelihood of the cross-sectional observations from each individual rabbit for a single parameter combination. The product of the estimated likelihoods for the longitudinal and cross-sectional observations gives the total likelihood of all observations for a single parameter combination for each rabbit. We assume that all rabbits are independent, thus the total likelihood for parameter combination ϕk is the product of the likelihoods for all, R, rabbits L R | ϕ k = ∏ r = 1 R (∏ t = 1 T r L (E t | E P G t, ϕ k) (L (O T r | P T r, ϕ k) ) (L (X T r | Z T r, ϕ k) ) ). As many parameter combinations have very low likelihood, we implemented a two-step algorithm for computational convenience. We first estimated the likelihood for all parameter combinations using N = 100 particles and discarded all parameter combinations with estimated likelihood less than 1% of the maximum. We then re-estimated the likelihood for the remaining parameter combinations using N = 1000 particles, to obtain a more precise estimate of LR. After estimation of the likelihood for each parameter combination, we generate draws from the posterior distribution by re-sampling the parameter combinations, with replacement, using probabilities proportional to the likelihood for all rabbits. We compare this posterior distribution to that derived by re-sampling parameter combinations, with replacement, using probabilities proportional to the likelihood of the longitudinal observations only; e. g. assuming the cross-sectional data were not available. We also generated rabbit-specific parameter estimates by re-sampling parameter combinations, with replacement, using probabilities proportional to the likelihood for each rabbit independently. We fitted the same model framework to the pre- and post-treatment phase independently, assuming no explicit effects on the post-treatment dynamics from the pre-treatment conditions; as such any effect of the pre-drug exposure would be reflected in the estimated rates and strength of cumulative exposure on establishment and clearance. To examine the single effect of either cumulative exposure of infective stages or intensity of adult infection on explaining the dynamics observed, we fitted sub-models that set the effects of cumulative exposure (γ2 and β2) or the effects of parasite intensity (γ3 and β3) to 0 (S5 Fig). | Title: Changes in parasite traits, rather than intensity, affect the dynamics of infection under external perturbation Summary: Host-parasite interactions frequently lead to complex dynamics of infection that can be difficult to explain when parasite data are not accurate or complete, which is often the case in natural systems. We used the helminth-rabbit study case and developed a state-space mathematical model to capture the variation in intensity of infection and egg shedding in an experimental trial where hosts were infected weekly, then treated with an anthelminthic and subsequently re-challenged following the same infection regime. Simulations indicate that hosts control the parasite proportionally to the accumulated force of infection and intensities decrease both in the pre- and post-treatment phase. The peak and mean intensity of infection is similar before and after treatment; however, the degree of egg shedding declines proportionately with parasite body length and both traits are lower in the post- than the pre-treatment phase. The intensity of infection fails to adequately explain the variation in the degree of shedding within and between hosts. Instead, this pattern is captured by changes in parasite body length. More attention should be given to the host-parasite interactions within hosts and the impact of external perturbation on the dynamics of infection and transmission. | 8,668 | 269 | lay_plos | en |
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These people really are. They really write some entertaining, some standup stuff.’”“Robert Spencer is the Stephen King of Jihad.”“Armed with facts and fearlessness, Spencer stands up for Western civilization.”— Michelle Malkin“Widely read in conservative foreign policy circles.”— New York Times“Widely read in many quarters in Washington.”— Washington Post“A canny operative who likely has the inside track on the State Department’s Middle East affairs desk should the tea party win the White House.”— New York Magazine“A hero of the American right.”— Karen Armstrong"Spencer’s comprehensive understanding of his Christian faith and Islam along with lucidly insightful writing give the lie to his international notoriety as a bigoted 'Islamophobe.'""The leading anti-Islamic intellectual in the United States....The go-to Islam expert for the right wing."— Salon Magazine“Robert Spencer is an Edward Said turned upside down.”— Stephen Suleyman Schwartz“One of the nation's most notorious Islamophobes.”— Hamas-linked CAIR"Geller and Spencer are probably the most important propagandizing Islamophobes in the world. These people's voices speak very loudly — not just here in the United States but overseas."— Heidi Beirach, Southern Poverty Law Center“Satanic ignoramus.”— Khaleel Mohammed“The Likud anti-Christ.”— Dar al-Hayat newspaper (Saudi Arabia)“Zionist Crusader, missionary of hate, counter-Islam consultant.”— Al-Qaeda’s Adam Gadahn, “Azzam the American” Upgrade to the latest Flash Player for improved playback performance. Upgrade now or more info Nakoula Basseley Nakoula is many things: a huckster, a convenient scapegoat, a terrible filmmaker. But to the members of America’s Islamophobic fringe, the producer of “The Innocence of Muslims” is something altogether different. He’s a victim. Nakoula is a man being punished by the Muslim extremists who have infiltrated the White House, and now want to criminalize any criticism of the Prophet, according to anti-Islam crusaders like Robert Spencer. “He is a political prisoner,” Spencer says. Never mind the fact that Nakoula seems to have tricked his actors into making a viral video that depicted Muhammad as a child molester. Never mind Nakoula’s conviction for bank fraud, which earned him a 21-month sentence in federal custody and a ban on using assumed identities, after he used 14 different aliases. Never mind the fact that Nakoula not only appeared to violate that probation by using the identity “Sam Bacile” when producing his video, or the fact the he doesn’t even seem to have been convicted under his real name. [In court Friday, he said he was really Mark Basseley Youssef (.pdf). He changed it back in 2002 because "Nakoula is a girl's name and it cause me troubles," he claimed.] To his defenders, it may even be kind of appropriate for Nakoula to go by so many names. To them, he’s become less of a man and more of a symbol – a prism for projecting a thousand conspiracy theories about a Muslim president gone mad with power, ready to unleash his scimitared hordes. “He has been arrested not for the technicality of the probation violation, but for insulting Muhammad. His arrest is a symbol of America’s capitulation to the Sharia,” Spencer writes. “I am Nakoula Basseley Nakoula,” blogger Scott Johnson adds. “Hillary Clinton, I insist that you have me arrested. I am thinking of making a movie about Mohammed,” declares Roger L. Simon, who continues, “Any Jews who now vote for Obama are ‘useful idiots’ beyond anything ever conceived by Lenin.” Is there a First Amendment critique to be made of the White House’s handling of Nakoula and his video? There sure is. At first, the Obama administration tried to put the blame for the current unrest in the Middle East on Nakoula — a charge we now know to be unfair. Then the Pentagon’s top general tried in vain to talk one of the video’s promoters into abandoning “Innocence.” And the White House unsuccessfully asked YouTube to pull the video from its servers. Afterward, ACLU executive Ben Wizner said the civil liberties group was “concerned” by the federal government’s apparent attempt to “throw its weight behind a request for self-censorship.” That didn’t stop Investors’ Business Daily from fuming that “Americans might as well be living under Islamic blasphemy laws, yet the nation’s champion of free speech — the ACLU — is AWOL. That’s because it’s now largely run by Muslims.” The rhetoric only heated up with Nakoula’s arrest on Thursday. “He was hunted down like an animal,” wrote Pamela Geller, a prominent member of the anti-Islam camp. “He is being jailed for blasphemy. This is Obama sharia enforcement in America.” It’s a bit of an odd accusation, considering that Nakoula directly contradicted his terms of probation (.pdf), which explicitly forbade him from “us[ing], for any purpose or in any manner, any name other than his/her true legal name or names without the prior written approval of the Probation Officer.” But what makes the Obama comment particularly odd is that U.S. Magistrate Judge Suzanne H. Segal (.pdf), who ordered that Nakoula be taken into custody, was appointed to the federal bench in 2002 — during the administration of George W. Bush. Of course, it’s not surprising that Geller would come to the defense of Nakoula and his film. She’s a political ally of one of the men who helped make the movie. Joseph Nassralla Abdelmasih is the president of Media for Christ, a Duarte, California-based broadcasting concern. He not only obtained the permits to shoot “Innocence” and allowed Nakoula to use his soundstage. Media for Christ’s Arabic-language television channel, “The Way TV,” also hosts a regular segment by “Innocence” consultant Steve Klein, who uses the platform to let loose a series of anti-Muslim diatribes. (A sample: “We’re just telling the truth about Muhammad being a pedophile and a murderer. Why do you want to kill us? Why do you want to come to America? Why do you want to replace our Constitution? Why do you want to replace our church?”) Nassralla appeared alongside Geller, Rober Spencer, and “Innocence”-promoter Morris Sadek at a pair of rallies protesting a Muslim center in downtown Manhattan. ”I come from Egypt. Egypt was Coptic, was Christians. From one thousand four hundred years, Islamic conquer our country with their lies [sic],” Nasralla declared at the 2010 event. After portions of “Innocence” aired on Egyptian television, and rioters seized on the film as an excuse to storm the American embassy there, Nasralla published a statement on Geller’s blog saying he was “shocked” that Nakoula has turned the film into an anti-Muhammad diatribe. “The work of my ministry and my television station is to expose the brutal ideology of sharia and terrorism…. We never insult anyone.” In February of this year, Geller used her blog to promote a movie not unlike “Innocence.” The idea was to make a film so devastating to Muhammad, it would shatter the religion of his followers. “To get rid of Islam we need to reveal the truth about it,” the blog noted. ” We need to make a motion picture about Muhammad – a biopic that reveals the details of his life. The Devil is in the details.” The post was written long after Nakoula began casting “Innocence.” But it’s not hard to see why Geller would rush to the defense of the film, and its maker. She had a similar notion herself. Hillary Clinton, I insist that you have me arrested. I am thinking of making a movie about Mohammed. I don’t want to brag, but as a film professional with an Academy Award nomination in screenwriting, I may do a better job than Nakoula Basseley Nakoula, alleged creator of the Innocence of Muslims. But I have to admit one thing. Hopeless and inept as Nakoula may be as a filmmaker, I agree with the intentions of his movie. I too have a serious problem with Islam because I happen to abhor misogyny and homophobia, both mainstays of that faith. And, like most Americans, I prefer freedom of religion to jihad, Sharia law, and a global caliphate. Don’t let me criticize any of that. I also happen to agree with Nakoula that making a movie about a faith whose prophet married a six year old and deflowered her at nine is of thematic and dramatic relevance. As a father, I am seriously concerned about child abuse, as is most of our film-going public, I would imagine. Indeed, the beginnings of Islam are the very stuff of great theatre and cinema, reprehensible as the actions of the protagonist may be. In fact, it may be great because of those actions. After all, Richard III is not a classic for nothing. So I am very tempted by the subject of Mohammed. Arrest me, Hillary Clinton, before I start. Call Eric Holder! And while you are at it, tell him to round up Salman Rushdie. His novel about Mohammed is obviously blasphemous. He was lucky to escape that fatwa. We should have one of our own. And arrest those Danish cartoonists too – ink-stained wretches! Arrest everyone who dares to criticize a religion that wants to take the world back to the seventh century. After all, you’re a “progressive.” You’re on the side of human rights. And make us apologize for our work, too. We didn’t mean a word we said. I’m sure the thoughtful folks in the Arab Street will accept our apologies and return to their peaceful, meditative lives. But most of all, arrest me because I might even make things worse. My film is likely to be inspired by a fascinating lecture I heard by the very Rushdie during which the novelist, who read Islamic history at Cambridge, explained the origins of that faith. He said it began with Mohammed’s ruthless and violent battle with the mother cults that then controlled Medina over local trade routes. It was about money then, but, as I will show in my movie, that war evolved into a kind of perpetual “War on Women” that has been waged by Islam since. Interesting, huh? Good cinema. Action, adventure, sex (matriarchy vs. ultra-patriarchy), even a little meaty conversation like Lawrence of Arabia. Don’t let me do it. There’s only one “War on Women” and you know it -- the one your fellow Democrats ascribe to Mitt Romney and company. I wouldn’t want to undermine that. So stop me, Hillary, before I write. The Bill of Rights is a fusty old document anyway, obviously subject to revision by an UN-approved committee of trans-global multi-culturalists. Censor me all you want. I’m ready. I don’t want to cause any international incidents. I have enough sleepless nights as is. But you will excuse me if, in the process, I think of you as the deepest of reactionaries. I knew you were a big time liar when you blamed the “right-wing conspiracy” for your husband’s obvious serial adultery. That was nothing compared to this, however. By blaming filmmakers, even the most amateurish ones, for the murderous actions of fanatical Islamists, you have placed yourself in complete opposition to everything our country ever stood for and to the essence of the U.S. Constitution. How despicable. ADDENDUM: For an insight to the degree to which our State Department has lied to us and to themselves about the situation in the Middle East, go here. Any Jews who now vote for Obama are "useful idiots" beyond anything ever conceived by Lenin. Islamofascism: Americans might as well be living under Islamic blasphemy laws, yet the nation's champion of free speech — the ACLU — is AWOL. That's because it's now largely run by Muslims. The ACLU usually stands up strong for First Amendment rights. Not in the case of the Muhammad movie. The ACLU's executive director failed to release an official statement condemning the outrageous efforts of the White House to deep-six the film, including pressuring YouTube to remove its trailer from the Web. Amnesty International, in contrast, asserted that any Muslim hurt over the film "should not be used as a justification to curtail core freedoms or justify potential government repression." Not until The Daily Caller contacted the ACLU did it speak out, and only meekly so. It said it was "concerned" about the White House request to censor the "repellant film." The ACLU's strangely muted response contrasts sharply with its militant reaction to post-9/11 measures to crack down on Islamic terrorists. "The government has gone to extraordinary lengths to squelch dissent (in the Muslim community) — from censorship and surveillance to detention," it says on its website, complaining it was "encroaching" on the "free speech rights" of Muslims. In a 200-page report on jihad fundraising, "Blocking Faith, Freezing Charity," the ACLU argued that "federal law enforcement is engaging in practices that intimidate Muslim donors and create a climate of fear that chills American Muslims' free and full exercise of their religion." In other words, the First Amendment rights of Muslims are more precious than those of the average citizen. Where is this bias coming from? Muslims. The ACLU now counts at least eight on its national executive staff alone. In fact, a Muslim runs the ACLU's Center for Democracy, while another heads its National Security Project. The irony is not lost on Steve Emerson, director of the Investigative Project on Terrorism. "The ACLU was founded on the basis that there shouldn't be any blasphemy laws," said Emerson, who's airing a new documentary, "Jihad in America: The Grand Deception." "Yet in the last 10 years, they've appointed (to their boards) members of the Muslim Brotherhood who believe in blasphemy laws." The top Muslim lawyer in ACLU's stable is Jameel Jaffer, who successfully sued the U.S. to reveal CIA secrets for interrogating terror suspects. This national-security wrecking machine is not even American. He's Canadian. He also happens to be a Muslim activist closely tied to major Muslim Brotherhood figures and front groups. | Summary: To America's anti-Muslim bloggers, the director of Innocence of Muslims is more of a cult hero than a criminal, Wired reports. Nakoula Basseley Nakoula "is a political prisoner," writes one, who argues that recent Arab protests are designed "to intimidate the US into criminalizing criticism of Islam." Another wants to be arrested because "I am thinking of making a movie about Muhammed"; he goes on to call Jews who vote for President Obama "''useful idiots' beyond anything ever conceived by Lenin." Not all of them are typing away in basements, either. Investors' Business Daily recently seethed that "Americans might as well be living under Islamic blasphemy laws," and a California-based broadcaster called Media for Christ has welcomed guests who slam the Muslim religion (sample here). While Wired agrees that there is a good First Amendment argument against President Obama's handling of Innocence, America's anti-Islamist movement goes much further-arguing that Muhammed is "a pedophile and a murderer" and that we should "get rid of Islam." Click for the full article. | 3,840 | 251 | multi_news | en |
Write a title and summarize: Lors du bac 2016, à Strasbourg. FREDERICK FLORIN / AFP C’est la conséquence du « baby boom » de l’an 2000 : 753 148 candidats sont inscrits au bac 2018, dont les épreuves écrites débutent lundi 18 juin à 8 heures, avec l’épreuve de philosophie. Un nombre record, en hausse de 5 % par rapport à l’an dernier. Si on y ajoute les élèves de première, qui composeront lundi à partir de 14 heures lors de l’épreuve anticipée de français, les postulants sont 1,29 million, soit 40 000 de plus que l’an dernier. Cette session 2018 s’inscrit dans un contexte particulier. D’une part parce qu’a été annoncée, en janvier, une réforme de l’examen, qui sera effective en 2021 : elle supprime les séries du bac général (bac S, ES, L) et réduit le nombre d’épreuves terminales à quatre, dont un oral, au profit du contrôle continu. Article réservé à nos abonnés Lire aussi Réforme du bac : de l’ambition et des questions D’autre part, de nombreux candidats témoignent d’une préparation de l’examen perturbée par la mise en place de la nouvelle procédure d’accès aux études supérieures, Parcoursup. Sur cette plate-forme, les candidats reçoivent des propositions au fil de l’eau depuis le 22 mai : depuis, 78 % ont reçu au moins une réponse favorable à leurs voeux, tandis que l’an dernier, sur Admission post-bac, 81 % des candidats avaient reçu une réponse positive le 8 juin. Surtout, une majorité d’entre eux n’ont pas encore définitivement arrêté leur choix : seulement 41,5 % ont validé une proposition sans conserver de voeu où ils sont « en liste d’attente », tandis que l’an dernier, quand les candidats étaient obligés de classer leurs voeux, 49 % avaient directement obtenu un « oui » à leur vœu préféré, une semaine avant l’examen. « Pour que les candidats puissent se concentrer pleinement sur leurs révisions et leurs épreuves », selon les termes du ministère, les réponses sur Parcoursup ont été suspendues dimanche 17 juin pour ne reprendre que mardi 26 juin, au lendemain des dernières épreuves du bac général et du bac technologique, qui concernent respectivement 53 % et 21 % des candidats. Le délai de suspension concerne également les 26 % de candidats au bac professionnel, qui achèveront pourtant leurs épreuves ce mercredi. Le Monde Campus suivra, en direct, le premier jour des épreuves écrites du bac 2018, lundi 18 juin : l’épreuve de philosophie, prévue de 8 heures à midi, puis l’épreuve anticipée de français, que passeront les élèves de première, de 14 à 18 heures. En voici les principaux rendez-vous : 8 heures : début de l’épreuve de philosophie 9 h 15 : publication des sujets de philosophie soumis aux candidats au bac général (bac S, ES, L) et technologique (bac STMG, STI2D…) et commentez-les avec Thomas Schauder, professeur de philosophie. 10 h 30 : Tchat/Fallait-il réformer le bac? – Posez vos questions à Pierre Mathiot,professeur de science politique à Sciences Po Lille, qui a mené la mission sur la réforme du bac et du lycée. Midi : corrigés vidéo des sujets de philosophie du bac S, ES et L (en partenariat avec Les Bons profs), et tchat en direct : posez vos questions à Thomas Schauder, professeur de philosophie. 14 heures : début de l’épreuve anticipée de français. 15 h 15 : publication des sujets de français soumis aux élèves de 1re des séries S, ES, L et technologiques, puis commentaire de Sonya Laborie, professeure de lettres au lycée Martin-Luther-King de Bussy-Saint-Georges (Seine-et-Marne). 18 heures : corrigés vidéo des sujets de français du bac S, ES et L, en partenariat avec Les Bons Profs, et tchat en direct : posez vos questions sur l’épreuve à Sonya Laborie, professeure de lettres. | Title: Bac 2018: les épreuves de philo et français rassemblent un nombre record de candidats ce lundi Summary: La session 2018, qui a débuté à 8 heures avec l'épreuve de philosophie pour les lycéens des filières générales et technologiques, compte 753 000 candidats. 537 000 composeront à 14 heures pour l'épreuve anticipée de français. Retrouvez les sujets et les corrigés en suivant notre "direct". | 961 | 109 | mlsum_fr | fr |
Summarize: It turns out, the letters sounded alike because the signers were often getting their information -- and indeed, the form letters and op-eds themselves -- from lobbyists and public relations professionals with connections to the Computer & Communications Industry Association, a trade group that counts Intuit -- the maker of Turbo Tax -- among its members. (It's not the first time she's investigated Intuit's actions against proposals that would undermine its business; see her 2013 story on how the company has lobbied on federal bills on return-free filing and fought a version of pre-filled returns in the company's home state of California.) A few months back, ProPublica's director of research, Liz Day, started to notice similar language in op-eds and letters opposing return-free tax filing, a little-known proposal that would give taxpayers the option to use pre-filled tax returns to save time and money. She joined Assistant Managing Editor Eric Umansky in the Storage Closet Studio to talk about what she found when she started investigating. Strikingly, some of the letter writers Day contacted were unaware of the industry forces behind the scenes. "It's not the most natural thing to point out that someone might not have done their own independent research," Day says. Some of the op-eds and letters, she added, made claims she had trouble verifying, including one that return-free filing would end tax-prep assistance that the government gives to low-income communities. To be clear, Umanksy points out, "There's nothing necessarily wrong with the idea" of targeting community leaders and local influencers on a cause -- "grasstopping," in industry lingo -- "if you are transparent, and giving accurate information, both of which were not the case in multiple instances." Listen to the podcast for the full behind-the-scenes look at Day's article. You can hear it on SoundCloud, iTunes and Stitcher. Related Stories: Read how the maker of TurboTax, Intuit, has lobbied against free, simple tax filing, or hear how ProPublica's director of research, Liz Day, discovered Intuit's grassroots campaign. Over the last year, a rabbi, a state NAACP official, a small town mayor and other community leaders wrote op-eds and letters to Congress with remarkably similar language on a remarkably obscure topic. Each railed against a long-standing proposal that would give taxpayers the option to use pre-filled tax returns. They warned that the program would be a conflict of interest for the IRS and would especially hurt low-income people, who wouldn't have the resources to fight inaccurate returns. Rabbi Elliot Dorff wrote in a Jewish Journal op-ed that he "shudder[s] at the impact this program will have on the most vulnerable people in American society." "It's alarming and offensive" that the IRS would target the "the most vulnerable Americans," two other letters said. The concept, known as return-free filing, is a government "experiment" that would mean higher taxes for the poor, two op-eds argued. The letters and op-eds don't mention that, as ProPublica laid out last year, return-free filing might allow tens of millions of Americans to file their taxes for free and in minutes. Or that, under proposals authored by several federal lawmakers, it would be voluntary, using information the government already receives from banks and employers and that taxpayers could adjust. Or that the concept has been endorsed by Presidents Obama and Reagan and is already a reality in some parts of Europe. So, where did the letters and op-eds come from? Here's one clue: Rabbi Dorff says he was approached by a former student, Emily Pflaster, who sent him details and asked him to write an op-ed alerting the Jewish community to the threat. What Pflaster did not tell him is that she works for a PR and lobbying firm with connections to Intuit, the maker of best-selling tax software TurboTax. "I wish she would have told me that," Dorff told ProPublica. The website of Pflaster's firm, JCI Worldwide, had listed Intuit among its clients, but removed it after ProPublica contacted them. Pflaster said Intuit had been listed by mistake, but added that the firm does work for the Computer & Communications Industry Association (CCIA), a trade group of which Intuit is a member. Pflaster also said her firm has reached out to multiple groups and encouraged them to share information about the "flaws" of return-free filing. The only CCIA member that's involved with tax preparation software is Intuit, and it's also the only member of the group that has taken a public position on return-free tax filing. Intuit has long worked against return-free filing. The company has said in filings with the Securities and Exchange Commission that it views free government tax preparation as a risk to its business. Last year, the company spent more than $2.6 million on lobbying, some of it to lobby on four bills related to the issue, federal lobbying records show. Both Intuit and CCIA declined to answer questions about their connections to the letters and op-eds. An Intuit spokeswoman, Julie Miller, said in an emailed statement that Intuit works with many types of groups to support "taxpayer empowerment," and "we feel all points of view deserve to be heard." "Return Free minimizes the taxpayers' voice and instead maximizes revenue collection for government," Miller wrote. "That kind of anti-consumer policy does not advance taxpayer rights." CCIA President Ed Black said in a statement, "We think it's important to help policymakers and the public understand what many already know: ReturnFree is unfair, unworkable and unwise." The letters and op-eds — there have been at least eight of them — also appear on a new website, www.FairTaxRefunds.com, which Pflaster said her company created for CCIA. It resembles a similar previous site, www.StopIRSTakeover.org, which was also sponsored by the CCIA. Documents Op-Eds and Letters Opposing Return-Free Tax Filing Compare eight letters and op-eds against return-free tax filing have been written by community leaders over the last year. Another instance in which CCIA solicited a letter wasn't successful. Angela Martin, director of an Oregon nonprofit, said a PR professional gave her a sample anti-return-free filing letter last year for her organization to send to Sen. Ron Wyden, D-Ore. Martin knew the caller, Colin Cochran, who works for the firm Hilltop Public Solutions. Cochran used "a lot of words that advocates would be sympathetic to, like 'oh, it'll hurt people with English as a second language,'" Martin said. Martin was skeptical. So she asked Cochran who he was representing. He said he was working for the CCIA and, when asked, said yes, that Intuit is a member. Cochran confirmed the details of his discussion with Martin, including that he was working for the CCIA. His firm boasts on its website of a "winning grasstops approach" — "grasstops" is industry lingo for recruiting the support of community leaders. Two other letter-writers told ProPublica they were approached by lobbyists, though it's not clear who the lobbyists were representing. One letter-writer, Richard Smith, the president of the NAACP Delaware State Conference, was approached by a longtime acquaintance with information about how return-free filing would take dollars out of poor people's pockets. Smith felt so strongly he fired off a letter to Sen. Tom Carper, D-Del., and encouraged other local NAACP leaders to do the same. Smith said the acquaintance, Anne Farley, told him that if return-free filing was adopted, the government would stop offering free tax filing help to low-income communities. (In fact, none of the bills on return-free filing propose that.) When ProPublica told Smith that Farley is also a registered lobbyist, he said he was now questioning the information she gave him. "We may have to retract so far based on my research," Smith said. "I didn't question her." Lobbying records for Farley's firm, First State Strategies, do not list Intuit or the CCIA among its clients. The firm's website does advertise "grass tops advocacy campaigns." Farley and First State Strategies did not respond to requests for comment. Dennis Huang, executive director of the L.A.-based Asian Business Association, also told ProPublica he was solicited by a lobbyist to write about return-free filing. When the lobbyist sent him a suggested op-ed last summer and told him the proposal would hurt small businesses, Huang wrote an op-ed in the Asian Journal that claimed Asian-owned businesses would not only spend more time paying taxes, but they'd also get less of a refund each year. Huang declined to disclose the lobbyist's name, but acknowledged he didn't really do his own research. "There's some homework needed," he said. Oregon's Martin did some research on return-free filing and now supports it. She also co-published a post about the issue and the PR efforts related to it because, she says, she was alarmed that other nonprofits could easily agree to endorse a position they did not fully understand. "You get one or two prominent nonprofits to use their name, and busy advocates will extend trust and say sure, us too," Martin said. | Summary: It sounds like an uncontroversial proposal: The IRS could set up a simple, optional, online tax tool, filling out your forms automatically using info the government has on file. Yet over the past year, community leaders have been coming out of the woodwork against such a thing. Rabbi Elliot Dorff, for example, wrote an op-ed saying that he "shudder[s] at the impact this program will have." What prompted the response? Well, Dorff tells ProPublica that he heard about it from a former student-who neglected to mention that she works for a lobbying firm linked to Intuit, the makers of TurboTax. "I wish she would have told me that," Dorff says. ProPublica research director Liz Day discovered the link when she noticed that many of the op-eds and letters against the online system contained similar language-which it turned out, had been crafted by lobbyists and PR professionals. Many of the writers, like Dorff, were unaware that they were being fed by tax industry forces. Intuit has been lobbying for years against the public system, which some studies indicate could make tax prep a five-minute job for about 40% of Americans, according to Jordan Weissman at Slate. | 2,121 | 267 | multi_news | en |
Summarize: Social Security Reform Is Part of a Broader Fiscal and Economic Challenge In my role as lead partner on the audit of the U.S. government’s consolidated financial statements and the de facto Chief Accountability Officer of the United States government, I have become increasingly concerned about the state of our nation’s finances. In speeches and presentations over the past several years, I have called attention to our large and growing long-term fiscal challenge and the risks it poses to our nation’s future. Simply put, our nation’s fiscal policy is on an unsustainable course, and our long-term fiscal imbalance worsened significantly in 2004. GAO’s simulations—as well as those of the Congressional Budget Office (CBO) and others—show that over the long term we face a large and growing structural deficit due primarily to known demographic trends and rising health care costs. Continuing on this unsustainable fiscal path will gradually erode, if not suddenly damage, our economy, our standard of living, and ultimately our national security. Our current path also will increasingly constrain our ability to address emerging and unexpected budgetary needs. Regardless of the assumptions used, all simulations indicate that the problem is too big to be solved by economic growth alone or by making modest changes to existing spending and tax policies. Nothing less than a fundamental reexamination of all major spending and tax policies and priorities is needed. This reexamination should also involve a national discussion about what Americans want from their government and how much they are willing to pay for those things. This discussion will not be easy, but it must take place. In fiscal year 2004 alone, the nation’s fiscal imbalance grew dramatically, primarily due to enactment of the new Medicare prescription drug benefit, which added $8.1 trillion to the outstanding commitments and obligations of the U.S. government. The near-term deficits also reflected higher defense, homeland security, and overall discretionary spending which exceeded growth in the economy, as well as revenues which have fallen below historical averages due to policy decisions and other economic and technical factors. While the nation’s long-term fiscal imbalance grew significantly, the retirement of the baby boom generation has come closer to becoming a reality. In fact, the cost implications of the baby boom generation’s retirement have already become a factor in CBO’s baseline projections and will only intensify as the boomers age. According to CBO, total federal spending for Social Security, Medicare, and Medicaid is projected to grow by about 25 percent over the next 10 years—from 8.4 percent of gross domestic product (GDP) in 2004 to 10.4 percent in 2015. Given these and other factors, it is clear that the nation’s current fiscal path is unsustainable and that tough choices will be necessary in order to address the growing imbalance. There are different ways to describe the magnitude of Social Security’s long-term financing challenge, but they all show a need for program reform sooner rather than later. A case can be made for a range of different measures, as well as different time horizons. For instance, the shortfall can be measured in present value, as a percentage of GDP, or as a percentage of taxable payroll. The Social Security Administration (SSA) has made projections of the Social Security shortfall using different time horizons. (See table 1.) While estimates vary due to different horizons, both identify the same long- term challenge: The Social Security system is unsustainable in the long run. Taking action soon on Social Security would not only make the necessary action less dramatic than if we wait but would also promote increased budgetary flexibility in the future and stronger economic growth. Although the Trustees’ 2004 intermediate estimates project that the combined Social Security Trust Funds will be solvent until 2042, within the next few years, Social Security spending will begin to put pressure on the rest of the federal budget. (See table 2.) Under the Trustees’ 2004 intermediate estimates, Social Security’s cash surplus—the difference between program tax income and the costs of paying scheduled benefits— will begin a permanent decline in 2008. (See fig. 1.) To finance the same level of federal spending as in the previous year, additional revenues and/or increased borrowing will be needed in each subsequent year. By 2018, Social Security’s cash income (tax revenue) is projected to fall below program expenses. At that time, Social Security will join Medicare’s Hospital Insurance Trust Fund, whose outlays exceeded cash revenues in 2004, as a net claimant on the rest of the federal budget. The combined OASDI Trust Funds will begin drawing on the Treasury to cover the cash shortfall. At this point, Treasury will need to obtain cash for those redeemed securities either through increased taxes, and/or spending cuts, and/or more borrowing from the public than would have been the case had Social Security’s cash flow remained positive. Today Social Security spending exceeds federal spending for Medicare and Medicaid, but that will change. While Social Security is expected to grow about 5.6 percent per year on average over the next 10 years, Medicare and Medicaid combined are expected to grow at 8.5 percent per year. As a result, CBO’s baseline projects Medicare and Medicaid spending will be about 30 percent higher than Social Security in 2015. According to the Social Security and Medicare trustees, Social Security will grow from 4.3 percent of GDP today to 6.6 percent in 2075, and Medicare’s burden on the economy will quintuple—from 2.7 percent to 13.3 percent of the economy. GAO’s long-term simulations illustrate the magnitude of the fiscal challenges associated with an aging society and the significance of the related challenges the government will be called upon to address. Figures 2 and 3 present these simulations under two different sets of assumptions. In figure 2, we begin with CBO’s January baseline, constructed according to the statutory requirements for that baseline. Consistent with these requirements, discretionary spending is assumed to grow with inflation for the first 10 years and tax cuts scheduled to expire are assumed to expire. After 2015, discretionary spending is assumed to grow with the economy, and revenue is held constant as a share of GDP at the 2015 level. In figure 3 two assumptions are changed: discretionary spending is assumed to grow with the economy after 2005 rather than merely with inflation and the tax cuts are extended. For both simulations Social Security and Medicare spending is based on the 2004 Trustees’ intermediate projections, and we assume that benefits continue to be paid in full after the trust funds are exhausted. Medicaid spending is based on CBO’s December 2003 long- term projections under mid-range assumptions. Both these simulations illustrate that, absent policy changes, the growth in spending on federal retirement and health entitlements will encumber an escalating share of the government’s resources. Indeed, when we assume that recent tax reductions are made permanent and discretionary spending keeps pace with the economy, our long-term simulations suggest that by 2040 federal revenues may be adequate to pay little more than interest on the federal debt. Neither slowing the growth in discretionary spending nor allowing the tax provisions to expire—nor both together—would eliminate the imbalance. Although revenues will be part of the debate about our fiscal future, the failure to reform Social Security, Medicare, Medicaid, and other drivers of the long-term fiscal gap would require at least a doubling of taxes—and that seems implausible. Accordingly, substantive reform of Social Security and our major health programs remains critical to recapturing our future fiscal flexibility. Although considerable uncertainty surrounds long-term budget projections, we know two things for certain: the population is aging and the baby boom generation is approaching retirement age. The aging population and rising health care spending will have significant implications not only for the budget but also for the economy as a whole. Figure 4 shows the total future draw on the economy represented by Social Security, Medicare, and Medicaid. Under the 2004 Trustees’ intermediate estimates and CBO’s long-term Medicaid estimates, spending for these entitlement programs combined will grow to 15.6 percent of GDP in 2030 from today’s 8.5 percent. It is clear that, taken together, Social Security, Medicare, and Medicaid represent an unsustainable burden on future generations. The government can help ease future fiscal burdens through spending reductions or revenue actions that reduce debt held by the public, thereby saving for the future and enhancing the pool of economic resources available for private investment and long-term growth. Economic growth can help, but given the size of our projected fiscal gap we will not be able to simply grow our way out of the problem. Closing the current long-term fiscal gap would require sustained economic growth far beyond that experienced in U.S. economic history since World War II. Tough choices are inevitable, and the sooner we act the better. Some of the benefits of early action—and the costs of delay—can be illustrated using the 2004 Social Security Trustees’ intermediate projections. Figure 5 compares what it would take to keep Social Security solvent through 2078 by either raising payroll taxes or reducing benefits. If we did nothing until 2042—the year SSA estimates the Trust Funds will be exhausted—achieving actuarial balance would require changes in benefits of 30 percent or changes in taxes of 43 percent. As figure 5 shows, earlier action shrinks the size of the necessary adjustment. Both sustainability concerns and solvency considerations drive us to act sooner rather than later. Trust Fund exhaustion may be nearly 40 years away, but the squeeze on the federal budget will begin as the baby boom generation begins to retire. Actions taken today can ease both these pressures and the pain of future actions. Acting sooner rather than later also provides a more reasonable planning horizon for future retirees. Demographic Trends Drive Both the Long term Fiscal Outlook and Social Security’s Financing Challenge The Social Security program’s situation is but one symptom of larger demographic trends that will have broad and profound effects on our nation’s future in other ways as well. As you are aware, Social Security has always been a largely pay-as-you-go system. This means that the system’s financial condition is directly affected by the relative size of the populations of covered workers and beneficiaries. Historically, this relationship has been favorable to the system’s financial condition. Now, however, people are living longer and spending more time in retirement. As shown in figure 6, the U.S. elderly dependency ratio is expected to continue to increase.The proportion of the elderly population relative to the working-age population in the U.S. rose from 13 percent in 1950 to 19 percent in 2000. By 2050, there is projected to be almost 1 elderly dependent for every 3 people of working age—a ratio of 32 percent. Additionally, the average life expectancy of males at birth has increased from 66.6 in 1960 to 74.3 in 2000, with females at birth experiencing a rise from 73.1 to 79.7 over the same period. As general life expectancy has increased in the United States, there has also been an increase in the number of years spent in retirement. Improvements in life expectancy have extended the average amount of time spent by workers in retirement from 11.5 years in 1950 to 18 years for the average male worker as of 2003. A falling fertility rate is the other principal factor underlying the growth in the elderly’s share of the population. In the 1960s, the fertility rate, which is the average number of children that would be born to women during their childbearing years, was an average of 3 children per woman. Today it is a little over 2, and by 2030 it is expected to fall to 1.95—a rate that is below what it takes to maintain a stable population. Taken together, these trends threaten the financial solvency and sustainability of Social Security. The combination of these factors means that annual labor force growth will begin to slow after 2010 and by 2025 is expected to be less than a fifth of what it is today. (See fig. 7.) Relatively fewer workers will be available to produce the goods and services that all will consume. Without a major increase in productivity or increases in immigration, low labor force growth will lead to slower growth in the economy and to slower growth of federal revenues. This in turn will only accentuate the overall pressure on the federal budget. The aging of the labor force and the reduced growth in the number of workers will have important implications for the size and composition of the labor force, as well as the characteristics of many jobs, throughout the 21st century. The U.S. workforce of the 21st century will be facing a very different set of opportunities and challenges than that of previous generations. Increased investment could increase the productivity of workers and spur economic growth. However, increasing investment depends on national saving, which remains at historically low levels. Historically, the most direct way for the federal government to increase saving has been to reduce the deficit (or run a surplus). Although the government may try to increase personal saving, results of these efforts have been mixed. For example, even with the preferential tax treatment granted since the 1970s to encourage retirement saving, the personal saving rate has steadily declined. Even if economic growth increases, the structure of retirement programs and historical experience in health care cost growth suggest that higher economic growth results in a generally commensurate growth in spending for these programs in the long term. In recent years, personal saving by households has reached record lows while at the same time the federal budget deficit has climbed. (See fig. 8.) Accordingly, national saving has diminished but the economy has continued to grow in part because more and better investments were made. That is, each dollar saved bought more investment goods and a greater share of saving was invested in highly productive information technology. The economy has also continued to grow because the United States was able to invest more than it saved by borrowing abroad, that is, by running a current account deficit. However, a portion of the income generated by foreign-owned assets in the United States must be paid to foreign lenders. National saving is the only way a country can have its capital and own it too. Initial Social Security benefits are indexed to nominal wage growth resulting in higher benefits over time. consequences for the living standards of future generations. The financial burdens facing the smaller cohort of future workers in an aging society would most certainly be lessened if the economic pie were enlarged. This is no easy challenge, but in a very real sense, our fiscal decisions affect the longer-term economy through their effects on national saving. The persistent U.S. current account deficits of recent years have translated into a rising level of indebtedness to other countries. However, many other nations currently financing investment in the United States also will face aging populations and declining national saving, so relying on foreign savings to finance a large share of U.S. domestic investment or federal borrowing is not a viable strategy in the long run. Health Care Is a Larger and More Difficult Challenge Than Social Security As figure 4 showed, over the long term Medicare and Medicaid will dominate the federal government’s future fiscal outlook. Medicare growth rates reflect not only a burgeoning beneficiary population but also the escalation of health care costs at rates well exceeding general rates of inflation. Health care generally presents not only a much greater but a more complex challenge than Social Security. The structural changes needed to address health care cost growth will take time to develop, and the process of reforming health care is likely to be an incremental one. While the long-term fiscal challenge cannot be successfully addressed without addressing Medicare and Medicaid, federal health spending trends should not be viewed in isolation from the health care system as a whole. For example, Medicare and Medicaid cannot grow over the long term at a slower rate than cost in the rest of the health care system without resulting in a two-tier health care system. This, for example, could squeeze providers who then in turn might seek to recoup costs from other payers elsewhere in the health care system. Rather, in order to address the long term fiscal challenge, it will be necessary to find approaches that deal with health care cost growth in the overall health care system. Although health care spending is the largest driver of the long-term fiscal outlook, this does not mean that Social Security reform should be postponed until after health is addressed. On the contrary, it argues for moving ahead on Social Security now. The outlines of Social Security reform have already been articulated in many Social Security reform proposals. These approaches and the specific elements of reform are well known and have been the subject of many analyses, including GAO reports and testimonies. Reform approaches already put forward can serve as a starting point for deliberations. Considerations In Assessing Reform Options As important as financial stability may be for Social Security, it cannot be the only consideration. As a former public trustee of Social Security and Medicare, I am well aware of the central role these programs play in the lives of millions of Americans. Social Security remains the foundation of the nation’s retirement system. It is also much more than just a retirement program; it pays benefits to disabled workers and their dependents, spouses and children of retired workers, and survivors of deceased workers. In 2004, Social Security paid almost $493 billion in benefits to more than 47 million people. Since its inception, the program has successfully reduced poverty among the elderly. In 1959, 35 percent of the elderly were poor. In 2000, about 8 percent of beneficiaries aged 65 or older were poor, and 48 percent would have been poor without Social Security. It is precisely because the program is so deeply woven into the fabric of our nation that any proposed reform must consider the program in its entirety, rather than one aspect alone. To assist policymakers, GAO has developed a broad framework for evaluating reform proposals that considers not only solvency but other aspects of the program as well. Our criteria aim to balance financial and economic considerations with benefit adequacy and equity issues and the administrative challenges associated with various proposals. GAO Framework For Evaluating Reform Proposals The analytic framework GAO has developed to assess proposals comprises three basic criteria: Financing Sustainable Solvency—the extent to which a proposal achieves sustainable solvency and how it would affect the economy and the federal budget. Our sustainable solvency standard encompasses several different ways of looking at the Social Security program’s financing needs. While a 75-year actuarial balance has generally been used in evaluating the long-term financial outlook of the Social Security program and reform proposals, it is not sufficient in gauging the program’s solvency after the 75th year. For example, under the trustees’ intermediate assumptions, each year the 75-year actuarial period changes, and a year with a surplus is replaced by a new 75th year that has a significant deficit. As a result, changes made to restore trust fund solvency only for the 75-year period can result in future actuarial imbalances almost immediately. Reform plans that lead to sustainable solvency would be those that consider the broader issues of fiscal sustainability and affordability over the long term. Specifically, a standard of sustainable solvency also involves looking at (1) the balance between program income and costs beyond the 75th year and (2) the share of the budget and economy consumed by Social Security spending. Balancing Adequacy and Equity—the relative balance struck between the goals of individual equity and income adequacy. The current Social Security system’s benefit structure attempts to strike a balance between these two goals. From the beginning, Social Security benefits were set in a way that focused especially on replacing some portion of workers’ preretirement earnings. Over time other changes were made that were intended to enhance the program’s role in helping ensure adequate incomes. Retirement income adequacy, therefore, is addressed in part through the program’s progressive benefit structure, providing proportionately larger benefits to lower earners and certain household types, such as those with dependents. Individual equity refers to the relationship between contributions made and benefits received. This can be thought of as the rate of return on individual contributions. Balancing these seemingly conflicting objectives through the political process has resulted in the design of the current Social Security program and should still be taken into account in any proposed reforms. Implementing and Administering Proposed Reforms—how readily a proposal could be implemented, administered, and explained to the public. Program complexity makes implementation and administration both more difficult and harder to explain. Some degree of implementation and administrative complexity arises in virtually all proposed changes to Social Security, even those that make incremental changes in the already existing structure. Although these issues may appear technical or routine on the surface, they are important issues because they have the potential to delay—if not derail—reform if they are not considered early enough for planning purposes. Moreover, issues such as feasibility and cost can, and should, influence policy choices. Continued public acceptance of and confidence in the Social Security program require that any reforms and their implications for benefits be well understood. This means that the American people must understand why change is necessary, what the reforms are, why they are needed, how they are to be implemented and administered, and how they will affect their own retirement income. All reform proposals will require some additional outreach to the public so that future beneficiaries can adjust their retirement planning accordingly. The more transparent the implementation and administration of reform, and the more carefully such reform is phased in, the more likely it will be understood and accepted by the American people. The weight that different policymakers place on different criteria will vary, depending on how they value different attributes. For example, if offering individual choice and control is less important than maintaining replacement rates for low-income workers, then a reform proposal emphasizing adequacy considerations might be preferred. As they fashion a comprehensive proposal, however, policymakers will ultimately have to balance the relative importance they place on each of these criteria. As we have noted in the past before this committee and elsewhere, a comprehensive evaluation is needed that considers a range of effects together. Focusing on comprehensive packages of reforms will enable us to foster credibility and acceptance. This will help us avoid getting mired in the details and losing sight of important interactive effects. It will help build the bridges necessary to achieve consensus. Reform’s Potential Effects on the Social Security Program A variety of proposals have been offered to address Social Security’s financial problems. Many proposals contain reforms that would alter benefits or revenues within the structure of the current defined benefits system. Some would reduce benefits by modifying the benefit formula (such as increasing the number of years used to calculate benefits or using price indexing instead of wage indexing), reduce cost-of-living adjustments (COLA), raise the normal and/or early retirement ages, or revise dependent benefits. Some of the proposals also include measures or benefit changes that seek to strengthen progressivity (e.g., replacement rates) in an effort to mitigate the effect on low-income workers. Others have proposed revenue increases, including raising the payroll tax or expanding the Social Security taxable wage base that finances the system; increasing the taxation of benefits; or covering those few remaining workers not currently required to participate in Social Security, such as older state and local government employees. A number of proposals also seek to restructure the program through the creation of individual accounts. Under a system of individual accounts, workers would manage a portion of their own Social Security contributions to varying degrees. This would expose workers to a greater degree of risk in return for both greater individual choice in retirement investments and the possibility of a higher rate of return on contributions than available under current law. There are many different ways that an individual account system could be set up. For example, contributions to individual accounts could be mandatory or they could be voluntary. Proposals also differ in the manner in which accounts would be financed, the extent of choice and flexibility concerning investment options, the way in which benefits are paid out, and the way the accounts would interact with the existing Social Security program—individual accounts could serve either as an addition to or as a replacement for part of the current benefit structure. In addition, the timing and impact of individual accounts on the solvency, sustainability, adequacy, equity, net savings, and rate of return associated with the Social Security system varies depending on the structure of the total reform package. Individual accounts by themselves will not lead the system to sustainable solvency. Achieving sustainable solvency requires more revenue, lower benefits, or both. Furthermore, incorporating a system of individual accounts may involve significant transition costs. These costs come about because the Social Security system would have to continue paying out benefits to current and near-term retirees concurrently with establishing new individual accounts. Individual accounts can contribute to sustainability as they could provide a mechanism to prefund retirement benefits that would be immune to demographic booms and busts. However, if such accounts are funded through borrowing, no such prefunding is achieved. An additional important consideration in adopting a reform package that contains individual accounts would be the level of benefit adequacy achieved by the reform. To the extent that benefits are not adequate, it may result in the government eventually providing additional revenues to make up the difference. Also, some degree of implementation and administrative complexity arises in virtually all proposed changes to Social Security. The greatest potential implementation and administrative challenges are associated with proposals that would create individual accounts. These include, for example, issues concerning the management of the information and money flow needed to maintain such a system, the degree of choice and flexibility individuals would have over investment options and access to their accounts, investment education and transitional efforts, and the mechanisms that would be used to pay out benefits upon retirement. The federal Thrift Savings Plan (TSP) could serve as a model for providing a limited amount of options that reduce risk and administrative costs while still providing some degree of choice. However, a system of accounts that spans the entire national workforce and millions of employers would be significantly larger and more complex than TSP or any other system we have in place today. Another important consideration for Social Security reform is assessing a proposal’s effect on national saving. Individual account proposals that fund accounts through redirection of payroll taxes or general revenue do not increase national saving on a first order basis. The redirection of payroll taxes or general revenue reduces government saving by the same amount that the individual accounts increase private saving. Beyond these first order effects, the actual net effect of a proposal on national saving is difficult to estimate due to uncertainties in predicting changes in future spending and revenue policies of the government as well as changes in the saving behavior of private households and individuals. For example, the lower surpluses and higher deficits that result from redirecting payroll taxes to individual accounts could lead to changes in federal fiscal policy that would increase national saving. On the other hand, households may respond by reducing their other saving in response to the creation of individual accounts. No expert consensus exists on how Social Security reform proposals would affect the saving behavior of private households and businesses. Finally, the effort to reform Social Security is occurring as our nation’s private pension system is also facing serious challenges. Only about half of the private sector workforce is covered by a pension plan. A number of large underfunded traditional defined benefit plans—plans where the employer bears the risk of investment—have been terminated by bankrupt firms, including household names like Bethlehem Steel, US Airways, and Polaroid. These terminations have resulted in thousands of workers losing promised benefits and have saddled the Pension Benefit Guaranty Corporation, the government corporation that partially insures certain defined benefit pension benefits, with billions of dollars in liabilities that threaten its long-term solvency. Meanwhile, the number of traditional defined benefit pension plans continues to decline as employers increasingly offer workers defined contribution plans like 401(k) plans where, like individual accounts, workers face the potential of both greater return and greater risk. These challenges serve to reinforce the imperative to place Social Security on a sound financial footing which provides a foundation of certain and secure retirement income. Regardless of what type of Social Security reform package is adopted, continued confidence in the Social Security program is essential. This means that the American people must understand why change is necessary, what the reforms are, why they are needed, how they are to be implemented and administered, and how they will affect their own retirement income. All reform proposals will require some additional outreach to the public so that future beneficiaries can adjust their retirement planning accordingly. The more transparent the implementation and administration of reform, and the more carefully such reform is phased in, the more likely it will be understood and accepted by the American people. Conclusions Social Security does not face an immediate crisis but it does face a large and growing financial problem. In addition, our Social Security challenge is only part of a much broader fiscal challenge that includes, among other things, the need to reform Medicare, Medicaid, and our overall health care system. Today we have an opportunity to address Social Security as a first step toward improving the nation’s long-term fiscal outlook. Steps to reform our federal health care system are likely to be much more difficult. They are also likely to require a series of incremental actions over an extended period of time. As I have said before, the future sustainability of programs is the key issue policy makers should address—i.e., the capacity of the economy and budget to afford the commitment over time. Absent substantive reform, these important federal programs will not be sustainable. Furthermore, absent reform, younger workers will face dramatic benefit reductions or tax increases that will grow over time. Many retirees and near retirees fear cuts that would affect them in the immediate future while young people believe they will get little or no Social Security benefits in the longer term. I believe that it is possible to reform Social Security in a way that will ensure the program’s solvency, sustainability, and security while exceeding the expectations of all generations of Americans. This is a work of the U.S. government and is not subject to copyright protection in the United States. It may be reproduced and distributed in its entirety without further permission from GAO. However, because this work may contain copyrighted images or other material, permission from the copyright holder may be necessary if you wish to reproduce this material separately. | Summary: Social Security is the foundation of the nation's retirement income system, helping to protect the vast majority of American workers and their families from poverty in old age. However, it is much more than a retirement program, providing millions of Americans with disability insurance and survivors' benefits. As the baby boom generation retires and given longer life spans and lower birth rates, Social Security's financing shortfall will grow. The current gap between promised and funded benefits is $3.7 trillion and is growing daily. The Chairman of the House Budget Committee asked GAO to discuss the need for Social Security reform. This testimony addresses the nature of Social Security's long-term financing problem and why it is preferable for Congress to take action sooner rather than later. This testimony also notes the broader context in which reform proposals should be considered and the criteria that GAO has recommended as a basis for analyzing any Social Security reform proposals. Although the Social Security system is not in crisis today, it faces a serious and growing solvency and sustainability challenge that is growing as time passes. If we did nothing until 2042, achieving actuarial balance would require a 30-percent reduction in benefits or a 43-percent increase in payroll taxes. Furthermore, Social Security's problems are a subset of our nation's overall fiscal challenge. Absent reform, the nation will ultimately have to choose among escalating federal deficits and debt, huge tax increases and/or dramatic budget cuts. As GAO's long-term budget simulations show, substantive reform of Social Security and our major federal health programs (e.g., Medicare and Medicaid) is critical to saving our fiscal future. Taking action soon would also serve to reduce the amount of change needed to ensure that Social Security is solvent, sustainable, and secure for current and future generations. Acting sooner would also serve to improve the federal government's credibility with the markets and the confidence of the American people in the government's ability to address long-range challenges before they reach crisis proportions. However, financial stability should not be the only consideration when evaluating reform proposals. Other important objectives, such as balancing the adequacy and equity of the benefits structure and various administrative and operational issues need to be considered. Furthermore, any changes to Social Security should be considered in the context of the broader challenges facing our nation, such as the changing nature of the private pension system, escalating health care costs, and the need to reform Medicare and Medicaid. | 6,739 | 532 | gov_report | en |
Summarize: DESCRIPTION This invention relates to improvements in the invention disclosed in the basic U.S. patent application No. 973,719, now U.S. Pat. No. 4,247,960 relating to a device for actuating the visors of helmets in general, and in particular motorcyclists' helmets. With specific reference to motorcyclists' helmets, provided with built-in visors, these (visors) must be easy to turn over in order to expose the helmet's aperture and must be capable of being positioned, in addition to the closed and fully open position, in all positions intermediate to said limit positions, by means of controls which can be easily actuated by the user with only one hand, this being essential in the specific case of motorcyclists. This invention maintains firm the principle stated in said patent, according to which the extremities of the visor are hinged to the helmet by slider elements running in suitable guides or ways in said helmet and work in conjuction with a control means through which the operator imparts to the visor a rectilinear motion, at first making accessible the edges of the visor from the ledge of the helmet successively causing the visor to oscillate about the pins of the hinges to free the aperture of the helmet. This invention relates to improvements intended to shift and secure the visor angularly in a desired position and hold it in said position, in consideration also of the possibility to neutralize, more or less the wind action exerted on the visor while the vehicle is running. These solutions are totally based on the inventive principles set forth in the above-mentioned patent. Specially, the actuating means for the visor are provided with a pinion gear rack assembly one of the elements in which is secured to the helmet structure, the other moveable and operatively linked to the control means of the visor; a hinge is provided, so that by actuating the moveable part of the pinion gear rack assembly, the visor is subjected to rectilinear and successive angular displacement. The invention will now be explained in the description which follows in conjunction with the attached drawings, which illustrate, only by way of example, some forms of an embodiment of the device as applied on a normal motorcyclists' helmet fitted with a built in visor. In the drawings: FIG. 1 is a front elevation view of the helmet, fitted with the device according to the invention. FIG. 2 illustrates, on a larger scale, a horizontal section taken on lines II--II of FIG. 1. FIG. 3 is a vertical cross sectional view on lines III--III of FIG. 2. FIG. 4 is a front view of the device shown in FIG. 1, with parts viewed in cross section. FIGS. 5 and 6, are similar respectively to FIGS. 2 and 4 and show a variation of the device. With reference to the figures in the drawing, parts equal or equivalent to those described and illustrated in the said patent are identified by the same symbols specifically, letter A indicates the helmet and letter B the visor, the edge of which B1 runs in a ledge A1 adjacent to the edge of the aperture of the visor while letter C identifies one of the two devices of the visor operatively linked to a single control means D secured in a suitable and convenient position of the helmet, as will be described infra. With reference specifically to FIGS. 2 and 4, a bushing 22d is secured to each end of visor B, for example, by means of polygonal couplings 66; said bushing terminates at the end opposite to the securing end with a crown 68 which holds flexible gasket 70 in engagement with the ledge of a housing in the hub of pinion 26d, forming in this manner, between said parts, a friction coupling as will be explained infra. A locking means 24d engages with the hole in bushing 22d; the head of said locking means engages with an annular end of a ledge in visor B to hold this in place during its movements. Said locking means 24d may consist of an ordinary screw engaged in the threaded hole of bushing 22d; in the case illustrated, locking means 24d is of a die cast flexible material, terminating with a head 72 engaging yieldingly with a shoulder piece in bushing 22d. Bushing 10d of pinion gear 26d forms the other element of hinge 10d-12d and is rotatably held by a supporting plate 12d to form a slider jointly with the pinion, in guides 74, running longitudinally by a plate, and supporting hollow tube 16d which, together with the parts forming device C are held in a ledge 75 in the lateral part of helmet A. Hollow supporting tube 16d and hence device C is secured to the wall of helmet A by pins provided with engaging teeth 76 of flexible material in said hollow tube which engage the holes in the bottom wall of ledge 75 of helmet A. It is obvious that these parts may be replaced by equivalent parts such as screws or similar elements. The plate or hollow supporting tube 16d is provided with a rack 28d toward one of its ends opposite the bottom wall of one of said guides 74; the plate or hollow tube can thus engage pinion gear 26d projecting for a short length from said hollow tube 16d, in order to permit assembly 12d-26d to run in guides 74, at first freely and subsequently to cause engagement of pinion gear 26d of the assembly with rack 28 as will be described infra. In order to ensure the required positioning of pinion gear 26d during the linear travel determined by assembly 12d-26d, the pinion is provided, in eccentric positon, with a pin 78 (see pages 3 and 4) engaging with a slot 80 extending lengthwise along the internal part of supporting hollow tube 16d. Said groove terminates with an enlarged part 82, close to the initial portion of rack 28d. Thus, when pinion gear 26d engages said rack, pin 78 disengages from groove 80. The plate of support 12d terminates, at its free end, with a clip 52d which holds one of the ends of a flexible control means F consisting for example, of a steel wire, Bowden cable or Teleflex cable or similar. Said control means F runs slidably in a suitable sheath, secured to the internal part of helmet A its other end being secured to a manual actuating means D of helmet A in a position easily accessible to the user. The lower edge of helmet A holds, by means of pin 84, a knurled disc 36d to which is connected a pinion 85. The teeth of this pinion engage in a diametrically opposite position the other two ends of flexible control means F (pertaining to right hand and left hand actuating devices C in helmet A). Each of the ends 85, in particular in Teleflex cables has a helically wound wire the turns of which form a rack which engages the teeth of pinion 83--of course, said ends may have suitable racks so that by means of the movement of disk 36a it is possible to move the two devices C of the visor B--of course, pinion 83 may comprise a rocker, depending on the characteristics of the flexible controls F which are used. The operating mode of device C is quite evident from the above description. Considering that in FIGS. 2 and 4 of the drawing the device is shown in the condition corresponding to the lowered and retracted position of visor B, i.e. with its edge engaged with ledge A1 of helmet A; to open the visor, the user can act, for example, with the thumb of one hand on the knurled contour of disc 36d in order to rotate this. This ensures that the flexible cables F impart to assemblies 12d-26d of each device C synchronous movements, both in the direction of arrow x in FIG. 2. Specifically, supporting plates 12d are caused to run along guides 74 without causing rotation of pinion gears 26d as the latter are held in angular locked position due to the engagement of pins 78 in their respective rectilinear grooves 80. It follows that visor B is shifted horizontally in the direction of arrow X as shown in FIG. 2 and this movement continues until the teeth of pinion gear 26d engage rack 28d. In this case, any further movement in the direction of arrow X, causes upward travel of visor B, which is thus moved away from the aperture of the helmet. As a result of the presence of clutch 70, visor B can be stopped and maintained steadily in any position comprised between the two limit positions, the first being horizontal (at which the visor closes the helmet's aperture) and the other being inclined (at which the helmet's aperture is fully open). The action of clutch 70 on visor B constantly ensures required positioning of the visor, even in the presence of strong wind, since it is possible to vary the action of said clutch 70 by providing it with suitable adjusting means apt to modify the braking action on the visor B. Furthermore, the action of the clutch can be modified and controlled to provide automatic closing of the visor, utilizing the wind thrust when said visor is in a position close to its closed position. In order to facilitate operation of visor B, the two devices C are secured to helmet A by orienting the bottom of ledges 75 so as to incline these conveniently with respect to the vertical medium line of helmet A and to diverge them frontally and outwardly with respect to the helmet. This arrangement facilitates the turning over of visor B as it is sufficient to impart to it a slight rectilinear movement to disengage its edge from ledge A with consequent overturning. It is evident that by actuating knurled ring 36d in a direction opposite to that previously considered, the above mentioned movements occur in reverse succession so that visor B is lowered and caused to re-enter into ledge A of the helmet. FIGS. 5 and 6 of the drawings represent a simplified version of the device according to the invention, similar to that illustrated in FIGS. 2 and 3 of the cited patent. In this version, the plate or hollow tube 16f is provided with guides 74f for supporting plate 12f, also shaped as a bracket between the arms of which freely runs bushing 22f, bound to visor B by clutch 68f. Supporting plate 22f holds a hinged connecting rod 84 which holds, by means of pin 14f, (through complementary plate 10f) bushing 22f for visor B. Connecting rod 86 is provided at its free end with teeth 26f formed by a pinion gear which engages (as will be described infra), a toothed rack 28f in the bottom wall of hollow tube 16f. Said wall is also provided, in a suitable position, with a prong 88 shaped as an inclined plane and which can engage with the side end of connecting rod 86f. Plate 22f terminates with a clip 52f for control cable I, connected to control means D as shown in FIG. 1. Also in this case, operation of disc 36d causes assembly 22f -86 to shift in the direction of arrow x, and teeth 26f connecting rod 86 to engage rack 28f, whilst the other end of said connecting rod 86 also engages clip 88. It follows that bushing 22f and end B 1 of visor "B" become disengaged from the housing in hollow tube 16f, while the successive engagement of teeth 26f with rack 28f completes the disengagement of the edge of the visor from ledge "A" on the contour of the helmet's opening. The user then completes lifting of visor B by acting on it directly or utilizing wind action to orient the visor conveniently on the helmet. It is understood that modifications may be introduced in the device depending on requirements; for example pinion gear rack 26d-28d assembly can be substituted equivalently by a cam or eccentric, the contour of which engages on or more projections on supporting hollow tube 16d to impart to visor B, (following a rectilinear displacement), also an angular displacement around bush 22d. Control of slider 12d can be effected either by means of a fluid under pressure or by means of a piston cylinder assembly. Moreover, supporting hollow tube 16d can be located in the structure of helmet A itself, it being evident that this and other changes will not affect nor depart from the spirit and protective coverage of this patent. | Summary: A device presents a tube, fixed to the wall of a helmet and guides for a slide, which can be moved along said guides by means of a cable S. The slide keeps hinged the visor B through a clutch connection, having a pinion which engages a rack 28D fixed to a support. The movement of the slide in one direction X, at first releases the ends of the visor from the shoulder of the helmet and afterwards engages the pinion with the rack, and this induces a swinging movement of the visor B around its pivots. | 3,035 | 125 | big_patent | en |
Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Helping Home Owners Make Energy- Efficiency Residential Upgrades Now Act of 2008'' or the ``H-HOMERUN Act of 2008''. SEC. 2. RESIDENTIAL ENERGY EFFICIENCY DIRECT LOAN PROGRAM. (a) Establishment.--The Secretary of Housing and Urban Development (in this Act referred to as the ``Secretary'') shall establish and implement a program to make direct loans, to the extent amounts are provided for costs of such loans pursuant to subsection (f), for providing energy efficiency improvements for single family housing. (b) Use of Loan Funds.--A direct loan made under this section may be made for the costs of the acquisition or installation, or both the acquisition and installation, as applicable, of any energy efficiency improvement, including-- (1) a solar heating system; (2) a solar cooling system; (3) the application of a residential energy conservation measure; (4) a photovoltaic energy system; (5) a geothermal heat pump system; (6) a residential wind turbine; (7) a ``green roof'' (a roof of a building that is partially or completely covered with vegetation and soil, or a growing medium, planted over a waterproofing membrane); and (8) any cost-effective energy efficiency improvement eligible to be financed under a mortgage insured under the Energy Efficient Mortgage program established by section 513 of the Housing and Community Development Act of 1992 (42 U.S.C. 12712 note) and expanded pursuant to section 513(b) of such Act. (c) Loan Eligibility Requirements.-- (1) Contract requirement.--The Secretary may make a direct loan under this section only if the Secretary has entered into a contract with a borrower setting forth the terms of the loan. (2) Repayment requirement.--The Secretary shall require full repayment of the principal amount of the loan. (3) Interest rate.--Loans made under this section shall bear interest at a rate that is-- (A) fixed over the term of the loan; (B) at least 2 percentage points less than the average rate available from a private source for a comparable loan at the time of the making of the loan; and (C) subject to such other requirements or limitations as the Secretary may prescribe. (4) Investment requirement.--A borrower shall pay on account of the energy efficiency improvements for which the loan is made at least 5 percent of the Secretary's estimate of the cost of acquisition, installation, or both acquisition and installation, as applicable, in cash or its equivalent. (5) Credit underwriting standards.--The Secretary shall establish credit underwriting standards to evaluate the eligibility of borrowers to receive loans under this section. (6) Security for loan.--The Secretary shall determine the reasonable value of the interest in property that will serve as security for a direct loan made under this section and shall establish procedures for appraisals upon which the Secretary may base such determinations. (7) Repayment schedule.--Direct loans made under this section shall be repaid in monthly installments. (8) Principal residence requirement.--A direct loan made pursuant to this section shall be used only for providing energy efficiency improvements for the principal residence of the borrower. (9) Other terms.--Direct loans made under this section shall be subject to such other terms, conditions, and restrictions as the Secretary may require. (d) Energy Efficiency Requirements.-- (1) Cost-effective energy efficiency improvements.--The Secretary shall require, for any energy efficiency improvement for single family housing for which a direct loan is made under this section, that the total present value cost of the improvement (including any maintenance and repair expenses) is less than the total present value of the energy saved over the useful life of the improvement. (2) Energy efficiency determination.-- (A) Determination.--For purposes of paragraph (1), the cost of the improvement and an estimation of the energy savings of the improvement shall be determined pursuant to a home energy rating report based upon a physical inspection of the property by a home energy ratings system, or energy consultant, approved by the Secretary. (B) Qualified inspection and determination.--For purposes of subparagraph (A), the physical inspection shall be conducted and the determination shall be made by an individual certified by an appropriate national organization as the Secretary may provide. (e) Definition of Single Family Housing.--For the purposes of this section, the term ``single family housing'' means any residential structure consisting of from 1 to 4 dwelling units. (f) Authorization of Appropriations.-- (1) In general.--There are authorized to be appropriated to cover the costs (as such term is defined in section 502 of the Congressional Budget Act of 1974) of direct loans under this section such sums as may be necessary for each of the fiscal years 2009 to 2019. (2) Aggregate outstanding limitation.--The aggregate outstanding principal balance of direct loans made under this section shall not at any time exceed $100,000,000,000. SEC. 3. HUD ENERGY EFFICIENT MORTGAGE PROGRAM AMENDMENTS. (a) Cost of Improvements.--Subparagraph (C) of section 513(a)(2) of the Housing and Community Development Act of 1992 (42 U.S.C. 12712 note) is amended to read as follows: ``(C) Cost of improvements.--The Secretary shall not establish a maximum limitation on the cost of the cost-effective energy efficiency improvements to be financed by the energy efficient mortgage provided under the program established by this section and as expanded pursuant to subsection (b).''. (b) Investment Requirement.--Section 513(a)(2) of the Housing and Community Development Act of 1992 (42 U.S.C. 12712 note) is amended by adding at the end the following new subparagraph: ``(D) Investment requirement.--A mortgagor shall pay on account of the cost-effective energy efficiency improvements for which the mortgage is made at least 5 percent of the Secretary's estimate of the cost of acquisition, installation, or both acquisition and installation, as applicable, in cash or its equivalent.''. (c) Cost-Effective Determination.--Paragraph (2) of section 513(c) of the Housing and Community Development Act of 1992 (42 U.S.C. 12712 note) is amended-- (1) in the last sentence by-- (A) striking ``sufficient for'' and all that follows; and (B) inserting ``based upon a physical inspection of the property by a home energy ratings system, or energy consultant, approved by the Secretary.'' after ``pursuant to a home energy rating report''; and (2) by adding at the end the following new sentence: ``Such physical inspection shall be conducted and such determination shall be made by an individual certified by an appropriate national organization as the Secretary may provide.'' | Title: To establish a direct loan program for providing energy efficiency improvements for single family housing, and for other purposes Summary: Helping Home Owners Make Energy-Efficiency Residential Upgrades Now Act of 2008 or the H-HOMERUN Act of 2008 - Requires the Secretary of Housing and Urban Development to establish a program to make direct loans for energy efficiency improvements for single family housing. Sets forth loan requirements. Directs the Secretary to require that the total present value cost of such improvement is less than the total present value of the energy saved over the useful life of the improvement. Requires such cost and savings to be determined pursuant to a home energy rating report based upon a physical inspection of the property by a home energy rating system, or energy consultant, approved by the Secretary. Requires the aggregate outstanding principal balance of direct loans to not at any time exceed $100 billion. Amends the Housing and Community Development Act of 1992 to prohibit the Secretary from establishing a maximum limitation on the cost of the cost-effective energy efficiency improvements to be financed by an energy efficient mortgage provided under the Energy Efficient Mortgages Program. Requires a mortgagor to pay on account of the cost-effective energy efficiency improvements for which the mortgage is made at least 5% of the Secretary's estimate of the cost of acquisition and/or installation. | 1,614 | 284 | billsum | en |
Summarize: By. John Hall For Mailonline. Chelsea Manning is not receiving hormone therapy treatment for her gender identity condition, despite it being approved by U.S. Defense Secretary Chuck Hagel. The American Civil Liberties Union and Manning's attorney contacted defence department officials, including Hagel, to notify them that a lawsuit will be filed if treatment is not confirmed by September 4. The former intelligence analyst is currently serving a 35-year sentence at the U.S. Disciplinary Barracks, Fort Leavenworth for leaking classified documents to the WikiLeaks website. Manning, who changed her name from Bradley after her conviction, has been diagnosed with gender dysphoria - the sense of being a woman in a man's body. Denied treatment: Chelsea Manning is currently serving a 35-year sentence at the U.S. Disciplinary Barracks, Fort Leavenworth for leaking classified documents to the WikiLeaks website. Manning first sought evaluation and treatment for her gender condition after she was sent to the Fort Leavenworth prison in September 2013. She is asking for hormone therapy and to be able to live as a woman. Military doctors confirmed the gender dysphoria diagnosis and recommended a treatment plan, but she has yet to receive any treatment, according to the American Civil Liberties Union. 'The continued failure to provide Ms. Manning with this treatment is inconsistent with well-established medical protocols and basic constitutional principles,' Chase Strangio, attorney for the ACLU's Lesbian Gay Bisexual and Transgender Project, said in a statement. Strangio said refusing to treat Manning is 'cruel and unusual punishment' and contended the Army is withholding treatment for political reasons. Convicted: Chelsea Manning, who changed her name from Bradley after being jailed, has been diagnosed with gender dysphoria - the sense of being a woman in a man's body. The lack of treatment puts Manning at risk for serious long-term physical and psychological harm, her advocates said. Manning was sentenced last year for six Espionage Act violations and 14 other offenses after she gave WikiLeaks more than 700,000 secret military and U.S. State Department documents. Manning's request for treatment was the first ever made by a transgender military inmate. It conflicts with a policy that prohibits transgender people from serving in the U.S. military, but Manning can't be discharged from the service while serving her prison sentence | Summary: Manning is supposed to be receiving treatment for her gender condition. Hormone therapy was agreed by U.S. Defense Secretary Chuck Hagel. But process has not started, according to American Civil Liberties Union. Now lawyers are threatening legal action if treatment does not begin soon. Former intelligence analyst is currently serving a 35-year prison sentence. She was convicted of leaking 700,000 classified documents to WikiLeaks. | 554 | 97 | cnn_dailymail | en |
Summarize: By. Chris Greenwood. A payout of £4,500 to Milly Dowler’s killer provoked outrage among MPs and campaigners last night. Levi Bellfield was granted compensation for a prison attack when a fellow inmate attempted to blind him. He suffered only minor cuts to his face. He sued the Government for failing to protect him at high-security Wakefield Prison and won a three-year legal battle after the Ministry of Justice admitted full liability at a county court hearing. Payout: Levi Bellfield (left), who was jailed in 2011 for the murder of schoolgirl Milly Dowler (right), has been awarded £4,500 in compensation after being attacked by an inmate at Wakefield Prison in Yorkshire. It is understood that the long dispute, as civil servants tried to contest his claim, has run up a substantial legal bill which will be met by taxpayers. Bellfield has told friends he will spend the money renting a caravan in Kent for his elderly mother. Last night Labour MP Ian Austin condemned the payout as ‘disgusting’ and promised to raise it with Justice Secretary Chris Grayling. He said: ‘This is a complete and utter disgrace. I will be asking Mr Grayling how this evil child murderer could possibly be awarded such a huge sum of money. Every right-thinking person will agree this is distasteful and wrong.’ National Victims’ Association founder David Hines added: ‘It really gets us that murderers claim for being attacked and get rewards. It’s a sin.’ Bellfield was told in 2011 that he would die behind bars for the murder of the 13-year-old in Walton-on-Thames, Surrey, nine years earlier. Attack: Bellfield won the compensation after being attacked by an inmate with a makeshift weapon at Wakefield prison in Yorkshire (pictured) in 2009. Outrage: Labour MP Ian Austin, pictured, condemned the payout as 'disgusting' and promised to raise it with Justice Secretary Chris Grayling. He was already serving a whole life term handed down in 2008 for the murders of Amelie Delagrange, 22, and 19-year-old Marsha McDonnell. Bellfield lured Milly back to his flat before killing her and dumping her body in remote woodland in Hampshire where he used to walk his dog. The teenager’s skeletal remains were found six months later. A series of police blunders left Bellfield free to stalk and kill two more victims and attempt to kill a third. The compensation is likely to enrage the Dowler family, who were forced to relive their ordeal during the Old Bailey trial. Speaking after Bellfield’s conviction, her mother Sally said she hoped the killer would receive the ‘same brutality’ in prison that he used against his victims. ‘The length to which the system goes to protect his human rights seems so unfair compared to what we as a family have had to endure,’ she added. The attack took place when an inmate with a makeshift weapon tried to stab Bellfield in the eye at the West Yorkshire prison in 2009. Victims: Bellfield got a whole life term in 2008 for the murders of Amelie Delagrange (left), 22, and 19-year-old Marsha McDonnell (right) The burly former bouncer and wheel clamper only suffered a minor scrape but claimed prison guards should have protected him. Ministry of Justice lawyers spent three years trying to stop Bellfield receiving any taxpayer-funded compensation. But earlier this week he was awarded the cash at Durham County Court after they were forced to admit full liability. A source close to Bellfield’s family said: ‘Levi will transfer the funds to relatives. He wants them to use it on the caravan. ‘He’s angry that prison guards let him in that part of the prison. He says by putting him in that wing, it was just asking for an attack to happen. Another inmate waited for him outside one of the bathrooms. The inmate went for his eye but missed. It was pretty tame.’ A spokesman for the Ministry of Justice said: ‘We are hugely disappointed that Levi Bellfield was awarded £4,500 by a judge following an assault by a prisoner in 2009 at HMP Wakefield.’ | Summary: Levi Bellfield wins compensation after he was attacked by fellow inmate. Triple-killer was set upon with a makeshift weapon at Wakefield prison. Ministry of Justice fought for years to stop him receiving taxpayer's money. But he was awarded the cash as Ministry should have protected him. He plans to give the money to his elderly mother so she can rent a caravan. Bellfield kidnapped Milly Dowler in Surrey in 2002 and was jailed in 2011. He is also serving life for killing Amelie Delagrange and Marsha McDonnell. Outraged Labour MP Ian Austin condemned the payout as 'disgusting' | 989 | 146 | cnn_dailymail | en |
Write a title and summarize: High-throughput data generation and genome-scale stoichiometric models have greatly facilitated the comprehensive study of metabolic networks. The computation of all feasible metabolic routes with these models, given stoichiometric, thermodynamic, and steady-state constraints, provides important insights into the metabolic capacities of a cell. How the feasible metabolic routes emerge from the interplay between flux constraints, optimality objectives, and the entire metabolic network of a cell is, however, only partially understood. We show how optimal metabolic routes, resulting from flux balance analysis computations, arise out of elementary flux modes, constraints, and optimization objectives. We illustrate our findings with a genome-scale stoichiometric model of Escherichia coli metabolism. In the case of one flux constraint, all feasible optimal flux routes can be derived from elementary flux modes alone. We found up to 120 million of such optimal elementary flux modes. We introduce a new computational method to compute the corner points of the optimal solution space fast and efficiently. Optimal flux routes no longer depend exclusively on elementary flux modes when we impose additional constraints; new optimal metabolic routes arise out of combinations of elementary flux modes. The solution space of feasible metabolic routes shrinks enormously when additional objectives---e. g. those related to pathway expression costs or pathway length---are introduced. In many cases, only a single metabolic route remains that is both feasible and optimal. This paper contributes to reaching a complete topological understanding of the metabolic capacity of organisms in terms of metabolic flux routes, one that is most natural to biochemists and biotechnologists studying and engineering metabolism. Research in biotechnology and medicine benefits from understanding the metabolic capacity of organisms, including their sensitivities to genetic and environmental changes. Genome-scale stoichiometric models of metabolism [1,2] and the availability of annotated genome sequences have greatly accelerated metabolic research. The combined use of high-throughput metabolomics data, comprehensive protocols [3], and (automated) reconstruction tools [4] has resulted in an explosion in the number and size of genome-scale stoichiometric metabolic models [5,6]. Constraint-based modeling has become an indispensable tool to deal with these large models, used in biotechnology [7,8] and medicine [9,10]. The most common constraint-based modeling method is Flux Balance Analysis (FBA) [11,12], which—given certain capacity constraints on fluxes—optimizes an objective function, e. g. the biomass production flux [13]. The accuracy of FBA predictions depends on the availability of realistic flux constraints, which can be derived from experimental data. Generally, there are insufficient flux constraints to obtain a single unique solution and a large space of optimal flux distributions results [14–16]. These alternative flux distributions give an impression of the robustness of a metabolic network [17], but not every alternative is equally favorable for the organism. In some environments organisms are strongly selected for yield, almost regardless of the protein burden, while in other environments the protein burden has a significant impact. The solution space can be analyzed further with secondary objectives [18–22], e. g. minimization of the number of active fluxes [23] or the sum of absolute fluxes [24], which have been used as proxies for maximization of the protein expression efficiency and minimization of the protein burden, respectively. Analyzing the solution space and optimizing secondary objectives requires adequate mathematical and computations methods. Several approaches were proposed to give insight into the geometry of the optimal solution space [14,15,25–28], which is mathematically represented by a polyhedron [29]. Flux Variability Analysis (FVA) [14] and Flux Coupling Analysis (FCA) [25] provide valuable information on the boundaries of the solution space, but do not give understanding in terms of metabolic routes. Such an understanding would be extremely helpful, as most biologists intuitively think in terms of metabolic routes. Characterization of the optimal solution space provides valuable insight into how our limited knowledge of constraints affects the prediction of a metabolic state of an organism. The recently developed method, CoPE-FBA (Comprehensive Polyhedron Enumeration FBA) [16], enumerates the vertices, the corner points of the optimal solution space. The number of vertices originates from the feasible, alternative metabolic routes through a small number of subnetworks, consisting only of reactions with correlated flux variability (Fig. 1). This method provides the structural insights that FVA and FCA lack, and explains the typical combinatorial explosion of the vertices; the optimal solution space can easily have millions of vertices that arise from independent combinations of alternative flux routes through only a few, small segments of the metabolic network. However, CoPE-FBA suffers from computational difficulties, it is slow, and—perhaps more important—the provided solution does not yield all non-decomposable flux routes in the optimum, limiting the use of CoPE-FBA. For instance, it cannot be used to assess the influence of secondary objectives on the solution space. We aim to obtain a better understanding of the interplay between constraints, objectives, and optimality for genome-scale stoichiometric models. We uniquely characterize the optimal solution space by adjusting CoPE-FBA to split each reversible reaction into two irreversible reactions; this yields all non-decomposable flux routes in the optimum. We start by illustrating the differences between the CoPE-FBA outcomes of metabolic models with and without reversible-reaction splitting. Next, we explain the relationship between these vertices and elementary flux modes (EFMs) with an optimal substrate-product yield. Finally, we show that secondary objectives typically collapse the optimal solution space to a unique solution (a vertex) or to a small set of vertices, using the iAF1260 genome-scale model of Escherichia coli metabolism. Enumerating all non-decomposable flux routes in the optimum requires a more efficient computational method, which we also present in the Methods section of this work. This results in CoPE-FBA 2. 0, our tool of choice for analyzing the optimal solution space in terms of network topology. We developed the toy network shown in Fig. 2A to illustrate: (i) the characterization of the optimal solution space of an FBA in terms of metabolic flux routes, (ii) that reversible-reaction splitting guarantees finding all non-decomposable metabolic flux routes in the optimum, (iii) the relationship between vertices and optimal-yield EFMs, and (iv) the optimization of secondary objectives over the optimal solution space. Our toy network consists of 18 metabolites and reactions where the source metabolite X and sink metabolite Y are considered boundary metabolites. All reactions, besides the reactions where ATP and ADP act as cofactors, are isomerization (uni-uni) reactions and reversible reactions are illustrated by two headed arrows. For our FBA model, we selected maximization of the flux through reaction R18 as our objective function, Zobj. To constrain the solution space we used one inequality constraint, J1 ≤ 2. Throughout this work, we call this type of (inequality) constraint a restricting non-zero flux constraint. The resulting FBA is formulated as the linear program: Maximize Z o b j = J 18 subject to, NJ = 0 - ∞ ≤ J r ≤ ∞ J r ∈ reversible reactions 0 ≤ J i ≤ ∞ J i ∈ irreversible reactions 0 ≤ J 1 ≤ 2 (1) where NJ = 0 is the steady-state constraint with N as stoichiometric matrix and J as flux vector (or flux pathway). Simple metabolic models can be optimized by hand, but linear programming is required for the solution of any realistic genome-scale model. FBA optimization confirmed that, for this set of capacity constraints, maximization of our objective function gives J18 = 1. We analyzed the realistic genome-scale stoichiometric model iAF1260 of Escherichia coli (E. coli) metabolism [34]. By modifying the oxygen uptake constraint, we constructed three different FBA models of iAF1260 that depict aerobic, aerobic restricted, and anaerobic growth (for details see Methods). We set maximization of biomass production rate as the objective function. For these growth conditions, general CoPE-FBA results for both the model with and without reversible-reaction splitting are shown in Table 1. With splitting we found for each growth condition many more vertices (up to 120 × 106). Since we also used an ATP maintenance demand constraint of 8. 39 mmol gDW−1 h−1, our vertices are not instances of EFMs. Without this constraint, all vertices in both the aerobic and anaerobic growth condition are instances of their corresponding EFMs (not shown). We found only a few CoPE-FBA subnetworks which together completely reveal the variability in the vertices. Most reactions are inactive after optimizing the objective function (S1 Table). In the remainder of this section, we use the results obtained from the model with splitting because this yielded all non-decomposable flux pathways in the optimum. The recently developed computational method, CoPE-FBA (Comprehensive Polyhedra Enumeration Flux Balance Analysis) [16], offers the premise of a simplified biological understanding of the optimal solution space of metabolic network models; a kind of understanding which is not possible with other popular methods such as Flux Variability Analysis [14] and Flux Coupling Analysis [25]. We further developed this method: Rather than enumerating the minimal generating set, we used reversible-reaction splitting [31,32] to enumerate all non-decomposable flux pathways in the optimum. This allows us to focus solely on the vertices for the analysis of optimal flux pathways. Enumerating all non-decomposable flux pathways in the optimum is a very demanding task compared to enumerating only a (small) subset of these flux pathways; especially for CoPE-FBA as presented by Kelk et al [16]. Therefore, we also developed an efficient computational method, CoPE-FBA 2. 0, for the (unique) characterization of the optimal solution space. We can now characterize the optimal solution space in the order of minutes for most (bacterial) genome-scale models on just an ordinary computer. CoPE-FBA 2. 0 is efficient because it first determines the subnetworks and subsequently enumerates the vertices for each subnetwork (see Methods for more details). To illustrate this, the 120 ⋅ 106 vertices enumerated for E. coli under aerobic growth conditions originate from eight subnetworks with respectively 6,3, 5184,3, 2,54,2, 2 vertices. This means that while we determined in total only 5256 vertices (the sum), we actually enumerated 120. 932. 352 vertices (the multiplication) within 15 minutes on an ordinary computer. The further development of CoPE-FBA facilitated in achieving a better understanding of how optimal flux pathways resulting from FBA arise out of EFMs, use of constraints, and optimality conditions. We recall that the vertices correspond to optimal-yield EFMs if there is only a single restricting flux constraint. Both restricting and demanding flux constraints modify the (optimal) solution space. Typically, the optimization problem remains underdetermined and an optimal solution space will continue to exist. We can get a unique solution by adding additional constraints that concern all flux values in the model (e. g. protein cost constraints). Then, the optimal state is an instance of an optimal-yield EFM if there is only a single restricting flux constraint. Alternatively, the optimal state corresponds to a convex combination of optimal-yield EFMs. For this reason, we can also use CoPE-FBA 2. 0 to quickly enumerate all optimal-yield EFMs, which can be useful because enumerating the complete set of EFMs of a genome-scale model is a laborious undertaking [37,38]. Other constraints that also concern all reactions, but not their flux values, such as minimal PL, will often lead to optimal solution spaces. While these objectives have been used frequently to find more realistic FBA outcomes [18–22], we showed that for both minimization of PL and PC, optimal solution spaces continue to exist (Table 2). This result shows that we should be careful drawing conclusions from predicted flux distributions after using a secondary objective. Using CoPE-FBA with only irreversible reactions allows for a straightforward identification of the origin of the remaining solution space. Specifically, these solution spaces originate from identically favorable pathways through CoPE-FBA subnetworks. Similarly, we can use these CoPE-FBA subnetworks to directly explain the differences after secondary optimization, as we showed for the multimodal distributions of vertex cost. In this research, we further demonstrated the use of CoPE-FBA 2. 0 for the E. coli iAF1260 genome-scale model by determining PL and PC for each enumerated vertex for different growth conditions. We found Gaussian-shaped and multimodal distributions for PL and PC, respectively. These results can be further used to deduce a hypothesis of the selection pressure if we know the flux distribution. If the objective of E. coli would be to minimize PC (or PJ), we would not expect E. coli to exploit the unique optimal solution since the difference between this optimal solution and many suboptimal solutions is almost negligible. We do, however, expect E. coli to exploit the “cheap” reactions that cause the bi- or multimodal distribution of vertex cost. Interestingly, in aerobic conditions, our analysis predicted that all cheap pathways exploit ubiquinone-8 which is also the major electron acceptor in E. coli under these conditions [35,36]. If the objective of E. coli would be to minimize PL, many different flux vectors give rise to an optimal or near-optimal solution. The multitude of optimal solutions hinders the construction of a hypothesis about PL from individual reactions. In future research we see as possible application of our method, finding the minimal flux distance between alternative optimal flux vectors in different conditions, to answer questions about how species can adapt to changing conditions. In conclusion, we present a better understanding of the principles of the optimal solution space and an efficient method to enumerate all non-decomposable flux pathways in this state. This paves the way to answer biological questions about the flexibility of organisms while growing at optimal states in a fast and straightforward manner. This work, therefore, contributes to reaching a topological understanding of metabolic functionality in the optimum in terms of metabolic flux pathways. In the future, the development of graphical maps [39] can further simplify the analysis by allowing for straightforward visualization and inspection of these metabolic flux pathways. For a metabolic network of m metabolites and r reactions, the m × r stoichiometric coefficients are often represented in the stoichiometric matrix N. The stoichiometric coefficient nij is positive if metabolite i is net produced in reaction j, negative if metabolite i is net consumed in reaction j, and otherwise zero. The representation of a metabolic network in a stoichiometric model is particularly useful for constraint-based modeling techniques like Flux Balance Analysis (FBA). By using a linear programming approach, FBA can optimize (maximize or minimize) an objective function subject to the steady-state constraint, thermodynamic constraints, and capacity constraints: Maximize or Minimize Z o b j = c T J subject to, NJ = 0 J min ≤ J ≤ J max (3) Here, c is a vector of coefficients that represent the contribution of each flux in vector J to the objective function Zobj. Next, NJ = 0 is the steady-state constraint. Finally, Jmin and Jmax specify the minimal and maximal flux values for each reaction. In addition to providing a unique optimal outcome of the objective function, FBA provides a corresponding optimal flux distribution Jopt. Most FBA models are underdetermined systems, and therefore, many corresponding Jopt exist. For more details about FBA, we refer to Orth et al. [12]. The Minkowski sum given in Equation (4) provides the description of any Jopt in terms of vertices, rays, and linealities [16,29]. J opt = ∑ k = 1 s α k φ k + ∑ k = 1 t β k φ k + ∑ k = 1 u γ k ψ k (4) Here, the vectors φk, φk, and ψk represent the vertices, rays, and linealities, respectively. Additionally, s, t, and u represent the upper boundaries of the sum functions indicating the number of vertices, rays, and linealities, respectively. Furthermore, αk, βk, and γk represent the weighting coefficient that satisfy the following constraints: ∑ k = 1 s α k = 1, αk ≥ 0, βk ≥ 0, and γk can take any value. In words, vertices can be summed by a convex combination, rays can be summed as a conical combination, and linealities can be summed as a linear combination. This Minkowski sum alters (Equation (5) ) once we split each reversible reaction into two irreversible reactions, because linealities do not exist anymore. J opt = ∑ k = 1 s α k φ k + ∑ k = 1 t β k φ k (5) Each split reversible reaction fulfills all conditions for a ray, thus many more rays are found when we split each reversible reaction. Each of these additional rays is also an EFM and an extreme pathway which are considered irrelevant because they only reformulate reversibility [32,40]. The set of vertices yields a flux space without futile cycles. CoPE-FBA subnetworks are defined within this flux space and have a fixed input-output relationship, which we can write mathematically as: N A J A = d ≠ 0 (6) where A is a vector of reactions that form the subnetwork [41]. Subsequently, NA and JA are the stoichiometric matrix and the flux vector of the subnetwork, and d is the fixed input-output relationship of the subnetwork. We can also calculate subnetworks (modules) in a flux space with futile cycles. These subnetworks are called F-modules and can be determined via FluxModules [42]. We can distinguish two types of F-modules: F-modules essential for optimality, i. e. d ≠ 0 F-modules not essential for optimality, i. e. d = 0 F-modules not essential for optimality are rays or linealities not connected to optimal flux pathways. An F-module essential for optimality can be a CoPE-FBA subnetwork, a ray or lineality connected to optimal flux pathways, or a combination of those. Kelk et al. 2012 [16] developed the CoPE-FBA pipeline developed to characterize the optimal solution space in terms of vertices, rays, and linealities. Enumeration of genome-scale models without reversible-reaction splitting can take already several days with this computational method. We developed a new pipeline, CoPE-FBA 2. 0, to make this enumeration less memory and CPU intensive. First, we preprocessed the model as also described by Kelk et al. [16]. Then, we executed the following steps: Determine F-modules and extract the fixed network. We used a Python implementation of FluxModules to quickly determine the F-modules. Determine d for each F-module. To circumvent numerical issues we used rational FBA (QSopt_EX version 2. 5. 0 [43]) to determine d. FBA output was also used to set the values of the fixed network. Reconstruct F-module models. For each F-module we reconstructed a model that consisted only of the reactions and metabolites of the F-module. We added input and output reactions to fix d of each F-module essential for optimality in the optimal solution space. Dummy species were added both the input and output reaction to guarantee use of both reactions. Perform CoPE-FBA as described in Kelk et al. 2012 [16] for each F-module. Enumeration on each F-module essential for optimality yielded all vertices. F-modules not essential for optimality were enumerated to determine the total number of rays. Reconstruct network vertices. We merged fixed parts and the enumerated vertices for all subnetworks. Enumerating the optimal solution space via CoPE-FBA 2. 0 took minutes to hours rather than days to weeks for the original pipeline developed by Kelk et al. 2012 [16]. The CoPE-FBA 2. 0 pipeline and all data files used during these study are available for download from http: //memesa-tools. sf. net. In the constructed E. coli subnetworks, the input-output relationship was the only constraint. Consequently, each enumerated subnetwork vertex should correspond to an optimal-yield EFM of the subnetwork. We successfully used the rank test [44] to show that each enumerated “subnetwork vertex” v is an instance of an (optimal-yield) EFM. First, we determined the zero indices of v. Second, from the stoichiometric matrix N of the subnetwork we eliminated all columns with a zero index in v to create a submatrix Nnz. Third, we used single value decomposition to determine the rank of Nnz. Last, we used the rank–nullity theorem to determine its nullity (Equation (7) ), the dimension of the right nullspace which should be one if v is an EFM. n u l l i t y N nz = 1 (7) Theoretically, enumerated vertices of subnetworks do not have to be instances of optimal-yield EFMs, because additional restricting flux constraints can be located inside these subnetworks. In addition, even if all vertices of all subnetworks were instances of optimal-yield EFMs, vertices describing the optimal pathways through the complete network do not have to correspond to EFMs. This is only true if there is one restricting non-zero flux constraint and no demanding flux constraints. As a secondary objective, we used, in addition to pathway length (PL) and pathway sum of absolute fluxes (PJ), also pathway cost (PC) —a proxy for the minimization of the ATP utilization in protein synthesis—to reduce the size of the solution space. P L = | { J j: J j ≠ 0 } | (8) P J = ∑ j = 1 r | J j | (9) P C = ∑ j = 1 r c j | J j | (10) The PL is identical to the number of flux carrying reactions, while the PC is identical to the sum of absolute flux values multiplied with cj, the protein cost for each individual reaction. This cost is the scaled length of the proteins that were associated to this reaction, which we used as a proxy for all costs. In other words, cj < 1 when the associated protein length is smaller than average and vice versa. We set cj = 1 when no information about associated proteins was available. Because multiple proteins can be associated to a particular reaction via AND and OR rules, different definitions of cj were used: maximum, average, minimum, and equal. An AND rule corresponds to taking the sum of protein lengths, while an OR rule corresponds to taking the maximum, average, or minimum. Using equal cost is identical to minimizing the sum of absolute fluxes, a widely-used secondary objective. Taking the maximum, average, minimum, or equal definition of cj did not effect the interpretation of our results. The aerobic restricted version (maximum O2 uptake was 18. 5 mmol gDW−1 h−1) of iAF1260 was obtained from the BiGG database [45]. Maximum glucose uptake was set to 12. 77 mmol gDW−1 h−1 and we modified the bounds on the O2 uptake reaction to create specific aerobic (no constraint on O2 uptake) and anaerobic (exchange of O2 set to zero) conditions. In all cases, the model required an ATP maintenance flux of 8. 39 mmol gDW−1 h−1. The model was edited and prepared for enumeration using PySCeS CBMPy [46,47]. All models are provided as Supplementary Dataset S1. Optimization of secondary objectives (minimization of PL, PJ, and PC) was also done with PySCeS CBMPy. We used a mixed-integer linear program to minimize PL and a linear program to minimize PJ and PC. | Title: Interplay between Constraints, Objectives, and Optimality for Genome-Scale Stoichiometric Models Summary: Organisms depend on huge networks of molecular reactions for environmental sensing, information integration, gene expression, and metabolism. The discovery of general principles of network behavior is a major ambition of systems biology and of great interest to biotechnology and medicine. We present a computational tool that calculates all optimal states of metabolism in terms of pathways, which is arguably the most intuitive and preferred approach to characterize whole-cell metabolism. We show how the space of all feasible flux distributions can be compactly described in terms of a unique set of minimal and feasible pathways, given realistic stoichiometric, thermodynamic, and optimization-objective constraints. This description clarifies the interplay between flux constraints and optimization objectives. We explain why some fluxes are variable and cross-correlate within the solution space while others do not and how multi-objective optimization shrinks the solution space. We illustrate our findings with a toy metabolic model to explain the main insights and apply it to a genome-scale stoichiometric model of Escherichia coli metabolism. | 5,628 | 246 | lay_plos | en |
Write a title and summarize: Many bacterial pathogens produce extracellular proteases that degrade the extracellular matrix of the host and therefore are involved in disease pathogenesis. Dichelobacter nodosus is the causative agent of ovine footrot, a highly contagious disease that is characterized by the separation of the hoof from the underlying tissue. D. nodosus secretes three subtilisin-like proteases whose analysis forms the basis of diagnostic tests that differentiate between virulent and benign strains and have been postulated to play a role in virulence. We have constructed protease mutants of D. nodosus; their analysis in a sheep virulence model revealed that one of these enzymes, AprV2, was required for virulence. These studies challenge the previous hypothesis that the elastase activity of AprV2 is important for disease progression, since aprV2 mutants were virulent when complemented with aprB2, which encodes a variant that has impaired elastase activity. We have determined the crystal structures of both AprV2 and AprB2 and characterized the biological activity of these enzymes. These data reveal that an unusual extended disulphide-tethered loop functions as an exosite, mediating effective enzyme-substrate interactions. The disulphide bond and Tyr92, which was located at the exposed end of the loop, were functionally important. Bioinformatic analyses suggested that other pathogenic bacteria may have proteases that utilize a similar mechanism. In conclusion, we have used an integrated multidisciplinary combination of bacterial genetics, whole animal virulence trials in the original host, biochemical studies, and comprehensive analysis of crystal structures to provide the first definitive evidence that the extracellular secreted proteases produced by D. nodosus are required for virulence and to elucidate the molecular mechanism by which these proteases bind to their natural substrates. We postulate that this exosite mechanism may be used by proteases produced by other bacterial pathogens of both humans and animals. Dichelobacter nodosus is a Gram negative, anaerobic rod that is the principal causative agent of ovine footrot, a debilitating disease of the hoof of ruminants. The disease results in significant costs to the worldwide sheep industry due to a reduction in meat and wool production and the expenditure associated with prevention and treatment programs [1], [2], [3]. Footrot is characterized by the separation of the keratinous hoof from the underlying tissue, resulting in severe lameness and loss of body condition [4], [5]. The severity of the disease can vary from benign footrot, which presents as an interdigital dermatitis that does not progress, to virulent footrot, which results in severe under-running of the horn of the hoof and the separation of the hoof from the underlying tissue [1]. Type IV fimbriae are an essential virulence factor [6], [7]; it also has been suggested that three closely-related secreted subtilisin-like proteases produced by D. nodosus may be required for virulence [8], [9]. In strains that cause virulent footrot these proteases are called acidic protease isoenzymes 2 and 5 from virulent strains (AprV2 and AprV5) and basic protease from virulent strains (BprV). In benign strains the comparable proteases are termed AprB2, AprB5 and BprB. All of these proteases are synthesised as inactive precursors with an N-terminal pre-pro-region, a serine protease domain and a C-terminal domain of unknown function. The active protease is produced by cleavage of the N-terminal pre-pro region and the C-terminal domain [10], [11], [12]. The protease domains have significant sequence identity to members of the subtilase family of serine proteases (54% identity with closest homologue from Dehalococcoides sp. VS), but sequence alignments indicate several insertions in the D. nodosus proteases [13]. Previous studies suggested that these proteases may represent the key difference between virulent and benign strains of D. nodosus; proteases secreted by virulent isolates have a greater thermostability and elastase activity (as monitored on elastin agar plates) than those of benign strains and it is postulated that this difference may relate to their in vivo activity against host tissue. These features are utilized in diagnostic tests to distinguish between virulent and benign footrot [14], [15], [16]. Comparison of the protease sequences from the virulent strain A198 with the benign strain C305 revealed that within the mature protease domain there is a single amino acid difference between AprV2 and AprB2 [12], and between AprV5 and AprB5 [11], and 96% sequence identity between BprV and BprB [17]. In this multidisciplinary study we set out to determine the role of these proteases in virulence and to determine the molecular basis for their function. We constructed isogenic protease mutants and characterized their protease activity and virulence. We showed that AprV2 was required for a virulent D. nodosus isolate to cause disease. Determination of the crystal structure of AprV2 revealed the presence of a novel exosite loop. This combined genetic and structural approach has permitted a comprehensive investigation of the secreted protease component of a pathogenic organism, and furthermore provided novel insight into how subtilisin-like proteases may have been hijacked by pathogenic microorganisms to degrade extracellular matrix components. To assess the contribution of each of the three extracellular proteases to the overall protease activity of the virulent D. nodosus isolate VCS1703A, separate chromosomal mutants of each protease gene were constructed by allelic exchange events that involved double crossovers. To confirm that the observed phenotypes resulted from these mutations, the mutants were complemented by insertion of the wild-type protease genes into the chromosome. Quantitative protease assays of culture supernatants, using azocasein as the substrate, showed that mutation of the aprV5 and aprV2 genes reduced total protease activity by 71% and 39%, respectively (Figure 1A). Complementation with the respective wild-type genes returned total protease activity to wild-type levels; however, it was also observed that the complemented aprV5 strain tended to lose protease activity upon repeated subculture. Since only a 12% reduction (P<0. 05) was observed in the bprV mutant, BprV does not appear to make a major contribution to total protease activity. These results indicate that AprV5, either directly or indirectly, makes the major contribution to total extracellular protease activity. To confirm the individual contribution of each protease gene to the overall protease activity of VCS1703A, double and triple mutants were also constructed. Only very low levels of protease activity were observed in the aprV2bprV and aprV5bprV double mutants (Figure 1B). Negligible protease activity was detected in the triple mutant and the aprV2aprV5 double mutant. The reduction in total protease activity for the double mutants was greater than the combined reduction in the total protease activity for the single mutants suggesting that the secreted proteases either act synergistically to degrade target substrates or that one or more of the proteases may be involved in the activation of the other proteases, or both. The ability of D. nodosus to digest insoluble elastin in an agar medium has been used as a diagnostic test to distinguish virulent and benign strains. Virulent isolates digest elastin within seven to ten days, while benign strains show no digestion after >21 days incubation [14]. Analysis of the wild type, the protease mutants and the complemented strains on elastin agar showed that the aprV2 mutant was unable to digest elastin, even after 30 days incubation, whereas both the aprV5 and bprV mutants were able to digest elastin at wild-type levels, showing clearing after 10 days (Figure S1A). Complementation of the aprV2 mutation restored the ability to digest elastin. In addition, the aprV5bprV mutants still had elastase activity. These results provided evidence that the AprV2 protease was responsible for the elastase activity. Note that the levels of elastase activity in culture supernatants were not high enough for detection in quantitative elastase assays. Sequence analysis of the aprV2 and aprB2 genes has shown there is only one amino acid difference (Y92R) between the two mature proteases [12]. To see what effect complementing the aprV2 mutant with the aprB2 gene would have on the protease phenotype of the resultant strain we inserted the aprB2 gene from the benign strain CS101 into the site of the disrupted aprV2 gene. Analysis of this strain in the azocasein assay showed that its total protease activity was not significantly different to the wild type (Figure 1A). However, this complemented strain had the in vitro phenotype of a benign strain since it had no elastase activity (Figure S1A), and its protease thermostability profile (Figure S1B) was that expected of a benign isolate [16]. The apparent difference in elastase activity between AprV2 and AprB2 was assessed in vitro using an Elastin-Congo Red substrate and purified recombinant proteins. Under these conditions, the ability of AprB2 to degrade the substrate was significantly less than AprV2 (p<0. 05, Figure 2). Both proteases displayed similar activity against the soluble chromogenic elastase substrate, N-Methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (AAPVn), with kinetic parameters that were of the same order of magnitude (Table 1). These results suggest that the Y92R substitution does not contribute directly to catalysis at the active site of the enzyme. To determine the role in disease of each of the proteases, virulence testing in sheep was carried out on the wild type, the aprV2, aprV5 and bprV mutants and their corresponding complemented strains, using our standard procedure [6], [7]. These experiments represented a rigorous test of the ability of these bacteria to cause disease since such pen-based trials often magnify the ability of less virulent isolates to cause disease. Comparative analysis of the footrot scores of sheep infected with the wild-type strain and the aprV2 mutant revealed a significant difference (P<0. 0001); the aprV2 mutant was effectively avirulent (Figure 3). Complementation of the aprV2 mutant with the wild-type aprV2 gene restored the wild-type virulence profile, fulfilling molecular Koch' s postulates. To our surprise, complementation of the aprV2 mutant with aprB2 also restored a virulent phenotype, indicating that AprV2-mediated elastase activity was not required for virulence. Analysis of isolates obtained from infected lesions confirmed that they had the expected phenotypic and genotypic properties. The aprV5 and bprV mutants were also avirulent (Figure 3), but complementation of these strains with the respective wild-type genes did not restore virulence, which was unexpected. Extensive sequencing of each of the protease genes in these strains showed them to be intact. However, upon subculture, protease secretion and/or elastin digestion by the aprV5 and bprV complemented strains were variable, as was their ability to undergo twitching motility, a property that is essential for virulence [7]. Therefore, we suggest that the complemented derivatives were genetically unstable and that secondary mutations were being selected in these strains. Consequently, no meaningful conclusions can be drawn from the aprV5 and bprV sheep virulence experiments. Since the elastase activity of AprV2 was not required for virulence it was of interest to determine which hoof proteins were degraded by Aprv2 and AprB2. Fragments of hoof from a disease-free sheep were exposed to recombinant AprV2 and AprB2 and solubilised proteins were identified. AprV2 degraded type I keratin, serum albumin and the beta subunit of haemoglobin (Figure 4). Importantly, the hoof digestion pattern produced by AprB2 was similar to that produced by AprV2 (Figure 4), which is consistent with results of the virulence trials. To investigate how the single amino acid difference (Y92R) between the active forms of AprV2 and AprB2 alters the substrate specificity of AprB2 we determined the crystal structures of AprV2 and AprB2 to 2. 0 Å and 1. 7 Å, respectively (Table 2; Figure 5A, B). The two structures were very similar (RMSD of 0. 28 Å for 339 Cα) (Figure 5C) and therefore we will describe the structure with reference to AprV2. AprV2 adopts a subtilisin-like fold consisting of a curved six-stranded parallel beta sheet sandwiched between two and five alpha helices (Figure 5A, B). A two stranded anti-parallel beta hairpin runs perpendicular to the plane of the central beta sheet. The proteases have two disulphide bonds, Cys89-Cys141 and Cys183-Cys220 and three calcium binding sites (Figure S2). The proposed catalytic triad (Asp41, His105 and Ser277) of AprV2 is located at the C-terminal edge of the beta sheet. The most striking feature of the substrate binding site is a large, elongated S1 binding pocket, which is lined by residues 177–180,204–208,215 and 218, and appears capable of accommodating bulky side-chains such as phenylalanine (Figure 5D). This finding is consistent with previous studies, which have shown that AprV2 preferentially cleaves after phenylalanine or leucine residues [18]. Comparison with other subtilisin-like proteases reveals several major insertions (termed I1-I4) in the loops that surround the active site cleft (Figure 6A, B; Figure S3). Most notable is the large well ordered I2 loop (residues 82–102) that is tethered to the subtilisin-like fold by a disulphide bond between Cys141 and Cys89 (Figure 6). The loop is well defined in the electron density, with low B-factors, suggesting that it has limited mobility in the crystal structure (Figure 6C). However, the apparent stability of the I2 loop is likely to arise from crystal packing and it is uncertain if this conformation would be favoured in solution. Surprisingly, the single amino acid difference between AprV2 and AprB2 (Y92R) is located at the tip of this extended loop, ∼27 Å from the active site serine (S277). A PSI-BLAST search revealed that the additional loops present in AprV2 may be conserved in other extracellular proteases (Figure S3B). We constructed an I2 loop truncation mutant, AprV2Δ83–99, but the resultant protein was not functional, therefore we investigated the role of the I2 loop using site-directed mutagenesis. We targeted residue 92 as the Y92R substitution reduced the ability of AprB2 to degrade Elastin-Congo Red (Figure 2), while maintaining its ability to degrade the elastin-like peptide AAPVn (Table 1). We used site-directed mutagenesis to convert Tyr92 to Asp, Ala, Leu or Phe and examined the ability of the resultant proteins to degrade insoluble Elastin-Congo Red. While the presence of a negative charge (Asp), positive charge (Arg) or smaller hydrophobic (Ala and Leu) side-chain decreased elastin degradation, the Phe substitution increased the elastase activity of the enzyme (Figure 2). We also examined the ability of these mutants to degrade AAPVn (Table 1), fibronectin (Figure S4) or hoof material (Figure S5). No major differences were discernable. Based on these data we conclude that an aromatic ring is required at position 92 for maximal activity against insoluble elastin, but that this activity is not related to the hoof digestion observed in a footrot lesion. To test the importance of the Cys89-Cys141 disulfide bond for proteolytic activity, we also used site-directed mutagenesis to convert Cys141 to Ser141. Although this AprV2. C141S derivative was still active against small peptide substrates (Table 1), we noted that the ability of this enzyme to degrade fibronectin, insoluble elastin and proteins from sheep hoof was reduced. Notably, wild-type AprV2 was able to break down fibronectin in 48 h, whereas at the corresponding time point AprV2. C141S-treated fibronectin was still intact (Figure 7A). The elastinolytic activity of the C141S protease was also approximately two-fold lower than wild type (Figure 2). Finally, the ability of AprV2. C141S to degrade sheep hoof material was significantly reduced compared to AprV2 and AprB2 (Figure 4). Together these data suggest that the integrity of the I2 loop and the Cys89-Cys141 disulfide bond is important for maximal AprV2 protease activity. To determine whether the C141S substitution affects the conformation/mobility of the I2 loop we determined the crystal structure of AprV2. C141S to 2. 1 Å (structure refinement statistics in Table 2). The structure of AprV2. C141S was very similar to that of AprV2, overlaying with an RMSD of 0. 21 Å for 339 Cα. The structure confirmed the absence of the Cys89-Cys141 disulfide bond and revealed that while the I2 loop had a slightly different structure to that of AprV2 there were no significant differences in the structure of the active site or primary substrate binding site (Figure 8A and S6). The average B factors for the I2 loops in AprV2 and AprV2. C141S were 14. 6 Å2 and 28. 4 Å2, respectively, indicating that the I2 loop is more mobile in the substituted protease (Figure 8B, C). Given that the conformation of the I2 loop is stabilized by crystal packing in all three structures we investigated whether the I2 loop was more mobile in solution in AprV2. C141S using intrinsic tryptophan fluorescence spectroscopy; the I2 loop contains two tryptophans. Steady state fluorescence quenching data showed that the wild-type protease was more protected from quenching by potassium iodide than the C141S enzyme (Figure 7B), confirming that the conformation of the I2 loop in AprV2. C141S is different to that in the wild type. Although the extracellular proteases of D. nodosus have been considered for many years to be potential virulence factors [9], their importance in the pathogenesis of disease had not been established. The analysis of their role in disease has always been complicated by the fact each isolate produces three very closely related proteases. The genetic approach utilized here has now provided clear evidence that the AprV2 protease is essential for virulence. This conclusion is based on data that showed that an aprV2 mutant was unable to cause footrot in sheep, unlike the isogenic wild-type strain, and that the ability to cause disease was restored to wild-type levels in the complemented derivative. No definitive conclusions could be drawn from the virulence testing of the aprV5 and bprV mutants. However, it is likely that the AprV5 and BprV proteases also play a role in disease, especially since the three secreted proteases appear to act synergistically, with the double protease mutants, aprV2aprV5, aprV2bprV, and aprV5bprV, showing lower secreted protease activity than that expected based on the secreted protease activity of the individual mutants (Figure 1B). It remains to be elucidated how this synergism arises, although it could occur at the processing, secretion or substrate level. Benign and virulent strains of D. nodosus can be differentiated by phenotypic analysis of their extracellular proteases, including analysis of their elastase activity and thermostability [14], [16], [19], [20]. We now have established that AprV2 is responsible for the elastase activity of the virulent isolate VCS1703A. Purified AprB2, which differs in sequence from AprV2 only at residue 92, is less efficient at degrading elastin. Therefore, it was important to examine the effect on virulence of complementing the aprV2 mutant with aprB2. This AprV2−AprB2+ strain was benign by the standard laboratory tests used to differentiate virulent and benign strains. Unexpectedly, it was virulent in the sheep footrot trial, producing disease that was indistinguishable in footrot severity from that caused by the isogenic wild-type strain. Since this strain still produces both AprV5 and BprV we conclude that the presence of one benign protease (AprB2) in combination with two virulent proteases (AprV5 and BprV) is not sufficient to make a strain benign even though in a laboratory diagnostic context the strain would be designated as benign. Therefore, although it appears that the properties of AprV2 and AprB2 are responsible for the differentiation of benign and virulent strains in laboratory tests, there clearly are other virulence factors, such as the other virulent proteases, that contribute to virulent disease. We have shown that AprV2 mediates the degradation of keratin (Figure 4), a component of the ovine hoof that confers physical protection and tissue integrity. This finding suggests that AprV2 has a direct role in destroying the keratin layer of the ovine hoof, a characteristic feature of virulent footrot. The initial site of D. nodosus attachment during infection is at the epidermal layer of the interdigital skin and degradation of keratin in this area by AprV2 is likely to be required to break through the skin horn junction, allowing the subsequent under-running of the horn by D. nodosus. An intriguing feature identified in the structures of AprV2 and AprB2 is the disulphide-tethered I2 loop. This loop is located next to, and partially occludes, the substrate binding site of the enzyme (Figure 6). The single amino acid difference between AprV2 and AprB2 is located at the tip of the loop. We have shown that residue 92 is a key determinant of elastase activity. Although, substitution of Tyr92 with Arg reduced the degradation of insoluble elastin, no significant differences in either KM or Vmax were observed for hydrolysis of the elastin-like peptide, AAPVn. This result suggests that residue 92 does not contribute to catalysis at the active site of the enzyme. Instead, the reduced elastinolytic activity of AprB2 is likely to arise from impaired enzyme-substrate interactions at a site distal to the active site. We therefore propose that the I2 loop functions as an exosite, mediating the formation of a stable enzyme-substrate complex. The disulphide bond tethering the I2 loop appears to be important for this function since its disruption alters the mobility of the loop, which significantly reduces the ability of the protease to degrade fibronectin, insoluble elastin and other proteins from the sheep hoof. We have identified several serine proteases that like AprV2 also appear to contain large insertions between the β1 strand and α2 helix (the location of the I1 and I2 loops) (Figure S3). These enzymes include MprA, from Burkholderia pseudomallei (the causative agent of melioidosis), TgSUB1 and TgSUB2, from Toxoplasma gondii (the causative agent of toxoplasmosis) and PfSUB1 from the malaria parasite Plasmodium falciparum. MprA degrades physiologically relevant proteins and may play a role in causing the lung damage associated with melioidosis [21], [22], however, it has only a minor role in virulence [23]. PfSUB1 and TgSUB2 appear to be critical for parasite survival [24], [25]. The presence of the I2-like insertions in these proteins suggests that the mechanism of exosite loop-mediated proteolysis used by the D. nodosus secreted proteases may represent a mechanism of substrate recognition that is utilized by other bacterial proteases. Studies investigating the structure of these proteins along with the function of their I2-like insertions will shed some light on this hypothesis and may lead to the development of improved diagnostic reagents and the identification of novel vaccine and drug targets. Strains and plasmids are detailed in Table 3. D. nodosus strains were routinely grown in an anaerobic chamber (Coy Laboratory Products Inc.) as described previously [6]. To construct the single mutants, suicide plasmids were inserted into D. nodosus strain VCS1703A by natural transformation [6]. The aprV2 and bprV mutants were complemented by transforming the mutants with the relevant plasmids, which reconstituted the disrupted gene and inserted a different resistance marker. The aprV5 mutant was complemented by inserting an intact copy of aprV5 and a kanamycin resistance marker into one of the three rrnA operons. The aprV2bprV double mutant was constructed by inserting the bprV suicide plasmid into an aprV2 mutant, and the aprV2aprV5 double mutant constructed by inserting the aprV2 suicide plasmid into an aprV5 mutant. An aprV5bprV double mutant was constructed by inserting a suicide plasmid into the wild-type strain, which disrupted both genes when a double crossover event occurred. Finally, a triple aprV2aprV5bprV mutant was constructed by inserting the aprV5bprV suicide plasmid into the aprV2 mutant. All mutants and complemented strains were confirmed by PCR and Southern hybridizations. PCR-RFLP analysis of the omp gene family was used to confirm that mutants were derived from the wild-type strain [26]. Elastase activity and protease thermostability assays for the differentiation of benign and virulent strains of D. nodosus were as described previously [14], [16], [27]. Virulence testing in sheep was performed as before [6], [7]. The sheep were randomly allocated into nine groups of eight sheep and challenged blind with the various strains. A plain agar challenge was used as the negative control. The feet of all animals were examined and scored for footrot lesions at the start of the trial and then at weekly intervals using a standard lesion scoring method [28], [29]. The total weighted foot score (TWFS) was used to provide an unambiguous overall footrot score for each animal [29]. The trial was carried out in a PC2 containment facility at Elizabeth Macarthur Agricultural Institute in accordance with the guidelines of the Australian Government Office of the Gene Technology Regulator and the Elizabeth Macarthur Agricultural Institute Animal Ethics Committee. All assays were performed in 20 mM Tris-HCl pH 8 and 5 mM CaCl2 (buffer A) with the exception of the Elastin-Congo Red elastase assay, which was performed in 25 mM Bis-Tris pH6. 5,150 mM NaCl, 5 mM CaCl2 and 5% glycerol. Quantitative determination of total protease activity or elastase activity was carried out using azocasein [26] or Elastin-Congo Red [30] assays, respectively. Degradation of AAPVn by recombinant protease (1 µM) was measured at 25°C as described [31]. KM and Vmax were determined by plotting initial velocities against AAPVn concentration and fitted by non-linear regression (Prism). Fibronectin degradation was measured by incubating human fibronectin (1 µM, BD Biosciences) with recombinant protease (0. 1 µM) at 25°C. Cleavage products were visualised by SDS-PAGE. Proteolytic degradation of hoof was determined by incubating dissected hoof material (2. 2% (w/v) in buffer A) from a disease-free sheep with recombinant protease (110 µg/ml) at 25°C. Samples were taken over a 16 hour period and degradation products were visualised by SDS-PAGE. Hoof material (14% (w/v) in buffer A) from a disease-free sheep was incubated with 100 µg of recombinant protease at 25°C for 18 h. Degradation products were separated by SDS-PAGE. The bands were excised and subjected to in-gel tryptic digestion and the digests analysed by LC-MS/MS using a HCT ULTRA ion trap mass spectrometer (BrukerDaltonics) coupled online with a 1200 series capillary HPLC (Agilent technologies). Proteins were identified by searching the LC-MS/MS data against the National Center for Biotechnology Information (NCBI) non-redundant and Swiss-Prot databases using the MASCOT search engine (version 2. 1, Matrix Science Inc.) with all taxonomy selected. AprV2 and AprB2 were purified and crystallised as before [32]. Data collection statistics have been reported [32]. The expression construct for AprV2. C141S was generated using the Quikchange site-directed mutagenesis kit (Stratagene) and pET22b. AprV2 as the template. Expression, purification and crystallisation of AprV2. C141S were as for AprV2. Data collection statistics for AprV2. C141S are in Table S1. Unless stated otherwise, all programs used for structural and crystallographic analysis were located within the CCP4 interface [33] to the CCP4 suite [34]. Manual building and maximum likelihood refinement were carried out using COOT [35] and REFMAC5 [36], respectively. The protease structures were solved by molecular replacement using PHASER [37]. A search model for AprB2 was derived from the coordinates of Bacillus Ak. 1 protease (PDB code 1DBI [38]), identified using the FFAS server [39]. The search model was generated using the SCRWL server and consisted of all conserved side-chains with the remaining non-alanine/glycine residues truncated at the Cγ atom [40]. The initial model of AprB2 was subject to several iterations of manual building and refinement. The model was then subjected to automatic building using ARP/wARP [41] before the structure was completed by more cycles of manual building and refinement. The refined AprB2 structure with the I2 loop deleted was used as the MR search model for AprV2 and AprV2. C141S. The initial models were subjected to simulated annealing using PHENIX [42], [43]. Successive rounds of manual building and refinement incorporating TLS [44] generated the final models. Water molecules were added to all models using ARP/warp v 5. 0 [45], [46]. Structure validation was carried out using MolProbity [47] and COOT [35]. Refinement statistics for the structures determined are presented in Table 2. The coordinates and structure factors are available from the Protein Data Bank (2LPA; 2LPC; 2LPC). Raw data and images are available from TARDIS (www. tardis. edu. au) [48]. Recombinant protease (0. 5 µM in buffer A) was incubated with increasing amounts of quenching solution (2 M KI and 1 mM Na2S2O3) and the change in the fluorescence emission intensity of the tryptophan residues (λex290 nm/λem340 nm) was measured using a Perkin-Elmer LS50B spectrofluorometer. The data were analysed as before [49]. The sheep virulence experiments were carried out in a PC2 containment facility at the Elizabeth Macarthur Agricultural Institute in accordance with the guidelines of the Australian Government Office of the Gene Technology Regulator and the Elizabeth Macarthur Agricultural Institute Animal Ethics Committee. These experiments were approved by the Elizabeth Macarthur Agricultural Institute Animal Ethics Committee. | Title: The Subtilisin-Like Protease AprV2 Is Required for Virulence and Uses a Novel Disulphide-Tethered Exosite to Bind Substrates Summary: Extracellular proteases are produced by many bacterial pathogens and are commonly involved in the degradation of the host extracellular matrix, facilitating invasion and colonization. One such pathogen is Dichelobacter nodosus, the causative agent of ovine footrot, a disease of major economic significance to the international sheep industry. D. nodosus secretes a number of serine proteases, which are thought to cause the tissue damage associated with virulent footrot. Our study showed that a D. nodosus mutant lacking one of the three secreted proteases, AprV2, failed to cause virulent disease in sheep. We used x-ray crystallography to solve the structure of AprV2 and the closely related protease AprB2. Our structures revealed an unusual extended disulphide-tethered loop that is located next to, but does not form part of, the primary substrate binding site. Through targeted mutagenesis studies we were able to show that this loop functions as an exosite, mediating effective enzyme-substrate interactions. Bioinformatic analyses suggest that subtilases from other pathogenic bacteria may contain this loop and may therefore utilize a similar mechanism. Our multidisciplinary research approach has provided a comprehensive understanding of the functional role of extracellular proteases in the pathogenesis of ovine footrot. | 7,568 | 322 | lay_plos | en |
Summarize: Photograph by Robyn Beck/AFP/Getty Images. I confess that when Lady Gaga began talking about her commitment to stop bullying, I was skeptical. She first spoke out after the suicide of 14-year-old Jamey Rodemeyer, who killed himself after he was teased about being gay. (Before taking his life, he’d posted on Facebook a Gaga lyric, “Don't forget me when I come crying to heaven’s door.”) Gaga’s response to Jamey’s death on Twitter started with an understandable swell of emotion. “The past days I've spent reflecting, crying, and yelling. I have so much anger. It is hard to feel love when cruelty takes someones life,” she wrote, linking to the “It Gets Better” video Jamey made before he died. Then Gaga latched onto a bad idea: “Bullying must become illegal,” she tweeted. “It is a hate crime.” Emily Bazelon Emily Bazelon is a staff writer at the New York Times Magazine and the author of Sticks and Stones. Maybe that’s an understandable reaction to a tragedy, too, but no thoughtful educator, psychologist, or bullying prevention trainer I’ve met thinks criminalizing bullying makes sense. Bullying, after all, usually involves kids taunting each other. However cruel and hurtful that taunting may be, do we really want to start hauling them before a judge for it? The law doesn’t normally treat nonviolent meanness as a crime. We shouldn’t start by making kids our guinea pigs. Thankfully, a few months later Gaga dropped the hate crime idea and announced her plan to launch the Born This Way Foundation, which would aim “to reach youth and create a new culture of kindness, bravery, acceptance and empowerment." I hate the word empowerment: It’s meaningless. But the rest of the Gaga’s ideals sounded lofty but good. And Gaga’s new foundation has been working with the MacArthur Foundation, the Harvard Graduate School of Education, and the Berkman Center for Internet & Society. It sounded like an intriguing combination: weighty institutional partners plus Gaga’s fame and credibility with kids. Advertisement But what truly won me over to Gaga’s anti-bullying work was the video she made about her own experience of being bullied. With candor that crosses the line into self-exposure—in a good way—Gaga describes being thrown into a trash can by a bunch of kids in high school. She remembers how she laughed nervously in some weak attempt to please her tormentors, and how other kids saw through this pose. Gaga didn’t tell her parents or other adults what had happened. Nor did she allow herself to think about it much either, until the experiences of her teenage fans forced her to. “I think it took me to get to know my fans and to see similar struggles in them to access that wound in myself,” she says to the camera. It’s believable and it makes me think she just might stick with this foundation thing. The next question, then, is what the Born This Way Foundation will actually do. I spent the day at a launch event for the foundation at Harvard, and the answer is: They don’t know yet. But they’re taking a smart approach to figuring it out, by bringing together some of the best thinkers about kids and conflict. The materials this effort has produced so far, which I’m looking forward to reading, are posted here. Gaga has donated $1.2 million of her own money to the foundation, but her greatest contribution may be her star power. That power can accomplish a lot, as was clear in Cambridge on Wednesday.* She might not have been necessary to bring out the academics in attendance, but surely her cachet helped round up the unlikely combination of Oprah Winfrey, Deepak Chopra, and Kathleen Sebelius, who were also on hand. (Gaga’s participation also caught the attention of New York Times columnist Nicholas Kristof.) It was the best mix of high, middle, and low I’d seen in—pretty much forever. After Harvard president Drew Gilpin Faust introduced Oprah to a standing ovation, before a not-quite-capacity crowd of about 1,000 in the university’s Sanders Theater, Oprah boomed “HAAARRVVAARRD!” Gaga laughed. “This might be one of the best days of my life!” she said. Oprah leaned in for one of her trademark moments of intimacy, and I worried she might lose an eye to one of the black bat wings of Gaga’s headdress. (Oh yes, the pop star, as ever, was in costume.) The mostly adult audience was pleased and relieved to hear Gaga say later on, “There’s all this focus on the victims but victims and bullies are on the same playing field. They both need our help. So how do we not just save the victim but save the bully too?” That dovetails nicely with the expert consensus that bullying prevention shouldn’t be largely punitive. It suggested Gaga was absorbing the wisdom she is marshaling with her foundation. Oprah had left the stage by this point, and Harvard law professor Charles Ogletree was now monitoring a panel that included, Gaga, Chopra, Sebelius, University of Nebraska psychologist Susan Swearer, Alyssa Rodemeyer, the sister of Jamey, and a father whose kids had been bullied. When Gaga spoke, I listened for clues to her evolving thinking on bullying. I liked how she boldly repeated “We don’t have the answer” in response to various impossible how-will-you-fix-everything questions. I winced when she talked about how the Columbine school shooters were bullied, since that’s a myth long busted. And I was cheered again when she shouted out to the researchers in the audience, with a kick of her insanely high-heeled shoe: “Give me all your information! Use me. If you discover something tremendous help us figure out what we can do to spread the message.” Gaga dissented from the gathered experts, however, when Swearer talked about the important role adults play in helping kids deal with bullying. “Can I push back on that?” Gaga said urgently. “I don’t think that works. I don’t think teachers give a shit a lot of the time. I want Alyssa and other youth like her to intervene. This problem has just been going on so long and what you’re trying isn’t working.” This suggested to me that the Born This Way Foundation won’t be working closely with schools, which would be too bad, since schools could use the help. But it might also be inevitable: Just as the pop star is skeptical of adults, surely many educators would be skeptical that the girl in the meat dress can help them prevent violence in their hallways. But if Gaga’s edgy appeal has its limits, it’s also crucial for her crusade. She’s not trying to be mainstream, and that could be a good thing, if her foundation figures out how to deploy her at the right moments and in front of the right audiences. Gaga’s best moment of the day came in response to a question from a teenager named Zachary, who told her he was transitioning from male to female, and wanted to know what she would do to support programs that are already in place, “like the really strong Gay-Straight Alliance at my school.” Gaga said she and her foundation would do everything they could to support and learn from such efforts. “And by the way,” she added, with a melting smile, “You look great.” When she was in high school, Lady Gaga says, she was thrown into a trash can. The culprits were boys down the block, she told me in an interview on Wednesday in which she spoke — a bit reluctantly — about the repeated cruelty of peers during her teenage years. “I was called really horrible, profane names very loudly in front of huge crowds of people, and my schoolwork suffered at one point,” she said. “I didn’t want to go to class. And I was a straight-A student, so there was a certain point in my high school years where I just couldn’t even focus on class because I was so embarrassed all the time. I was so ashamed of who I was.” Searching for ways to ease the trauma of adolescence for other kids, Lady Gaga came to Harvard University on Wednesday for the formal unveiling of her Born This Way Foundation, meant to empower kids and nurture a more congenial environment in and out of schools. Lady Gaga is on to something important here. Experts from scholars to Education Secretary Arne Duncan are calling for more focus on bullying not only because it is linked to high rates of teen suicide, but also because it is an impediment to education. A recent study from the University of Virginia suggests that when a school has a climate of bullying, it’s not just the targeted kids who suffer — the entire school lags academically. A British scholar found that children who simply witness bullying are more likely to skip school or abuse alcohol. American studies have found that children who are bullied are much more likely to contemplate suicide and to skip school. The scars don’t go away, Lady Gaga says. “To this day,” she told me, “some of my closest friends say, ‘Gaga, you know, everything’s great. You’re a singer; your dreams have come true.’ But, still, when certain things are said to you over and over again as you’re growing up, it stays with you and you wonder if they’re true.” Any self-doubt Lady Gaga harbors should have been erased by the huge throngs that greeted her at Harvard. “This might be one of the best days of my life,” she told the cheering crowd. The event was an unusual partnership between Lady Gaga and Harvard University in trying to address teen cruelty. Oprah Winfrey showed up as well, along with Kathleen Sebelius, the secretary of health and human services. Kathleen McCartney, dean of the Graduate School of Education here at Harvard, said that she and her colleagues invited Lady Gaga because they had been searching for ways to address bullying as a neglected area of education — and as a human rights issue. As many as one-fifth of children feel bullied, she said, adding: “If you don’t feel safe as a child, you can’t learn.” Lady Gaga describes her foundation as her “new love affair,” and said that, initially, she thought about focusing on a top-down crackdown on bullying. But, over time, she said, she decided instead to use her followers to start a bottom-up movement to try to make it cooler for young people to be nice. I asked Lady Gaga if people won’t be cynical about an agenda so simple and straightforward as kindling kindness. Exceptionally articulate, she seemed for the first time at a loss for words. “That cynicism is exactly what we’re trying to change,” she finally said. Bullying isn’t, of course, just physical violence. Lady Gaga’s mother, Cynthia Germanotta, who will serve as president of the Born This Way Foundation, says that one of the most hurtful episodes in her daughter’s childhood came when schoolmates organized a party and deliberately excluded Lady Gaga. Lady Gaga was reluctant to talk too much about her own experiences as a teenager for fear that her foundation would seem to be solely about bullying. Her aim is a far broader movement to change the culture and create a more supportive and tolerant environment. “It’s more of a hippie approach,” she explained. “The Born This Way Foundation is not restitution or revenge for my experiences,” Lady Gaga told me. “I want to make that clear. This is: I am now a woman, I have a voice in the universe, and I want to do everything I can to become an expert in social justice and hope I can make a difference and mobilize young people to change the world.” Yes, that sounds grandiose and utopian, but I’m reluctant to bet against one of the world’s top pop stars and the person with the most Twitter followers in the world. In any case, she’s indisputably right about one point: Bullying and teenage cruelty are human rights abuses that need to be higher on our agenda. | Summary: Lady Gaga launched her anti-bullying Born This Way Foundation yesterday with typical fanfare and grousing from skeptics. But don't scoff just yet, write Nicholas Kristof at the New York Times and Emily Bazelon at Slate. Gaga is working with the MacArthur Foundation and the Harvard Graduate School of Education, among others, and she seems genuinely sincere, especially in recounting her own days as a bullied teen in high school. Kristof: Her plan of "kindling kindness" and getting youths themselves to change things might sound "grandiose and utopian, but I'm reluctant to bet against one of the world's top pop stars and the person with the most Twitter followers in the world," he writes. "In any case, she's indisputably right about one point: Bullying and teenage cruelty are human rights abuses that need to be higher on our agenda." Bazelon: Gaga has thankfully moved on from her wrongheaded idea to make bullying a hate crime, and the foundation is a good next step. So what will it actually do? "I spent the day at a launch event for the foundation at Harvard, and the answer is: They don't know yet. But they're taking a smart approach to figuring it out, by bringing together some of the best thinkers about kids and conflict." | 2,730 | 288 | multi_news | en |
Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Help Separated Families Act of 2012''. SEC. 2. IMMIGRATION STATUS ALONE NOT A DISQUALIFICATION FROM BEING A PLACEMENT FOR A FOSTER CHILD. Section 471(a)(19) of the Social Security Act (42 U.S.C. 671(a)(19)) is amended-- (1) by striking ``(19) provides that the State'' and inserting the following: ``(19) provides that-- ``(A) the State''; and (2) by adding after and below the end the following: ``(B) such standards shall ensure that the immigration status alone of a parent, legal guardian, or relative shall not disqualify the parent, legal guardian, or relative from being a placement for a child;''. SEC. 3. STATE PLAN REQUIREMENT TO ACCEPT CERTAIN DOCUMENTS ISSUED BY FOREIGN ENTITIES AS SUFFICIENT IDENTIFICATION FOR PURPOSES OF INITIATING A CRIMINAL RECORDS CHECK OR A FINGERPRINT-BASED CHECK. Section 471(a)(20) of the Social Security Act (42 U.S.C. 671(a)(20)) is amended-- (1) in subparagraph (A), by inserting ``which procedures shall require the State (including the State agency, the child welfare agency of any county or other political subdivision of the State, and caseworkers and supervisors of any such agency) to accept a foreign consulate identification card, a foreign passport, or such other foreign identification document as may be allowed in regulations prescribed by the Secretary, as sufficient identification for purposes of initiating a criminal records check or a fingerprint-based check,'' before ``including procedures''; and (2) in subparagraph (C), by inserting ``, which procedures shall require the State (including the State agency, the child welfare agency of any county or other political subdivision of the State, and caseworkers and supervisors of any such agency) to accept a foreign consulate identification card, a foreign passport, or such other foreign identification document as may be allowed in regulations prescribed by the Secretary, as sufficient identification for purposes of initiating a criminal records check or a fingerprint-based check'' before the semicolon. SEC. 4. STATE CHILD WELFARE AGENCIES ENCOURAGED TO GRANT WAIVERS OF REQUIREMENTS THAT WOULD PREVENT A CHILD FROM BEING PLACED WITH A RELATIVE ON THE BASIS OF A MINOR LEGAL INFRACTION BY THE RELATIVE. It is the sense of the Congress that the child welfare agency of a State, or of any county or other political subdivision of a State, should grant a waiver of any requirement which would prevent the placement of a child with a relative of the child, on the basis of a minor legal infraction, if the relative would otherwise be considered eligible for such a placement. SEC. 5. STATE PLAN REQUIREMENT TO NOTIFY RELATIVES SEEKING PLACEMENT OF A CHILD THAT THEIR IMMIGRATION STATUS WILL NOT BE QUESTIONED. Section 471(a)(29) of the Social Security Act (42 U.S.C. 671(a)(29)) is amended-- (1) by striking ``and'' at the end of subparagraph (C); (2) by adding ``and'' at the end of subparagraph (D); and (3) by adding at the end the following: ``(E) the immigration status of any such relative seeking placement of the child with the relative shall not be questioned, except to the extent necessary in determining eligibility for relevant services or programs;''. SEC. 6. PROHIBITION ON STATE FILING FOR TERMINATION OF PARENTAL RIGHTS IN FOSTER CARE CASES IN WHICH OTHERWISE FIT AND WILLING PARENT OR RELATIVE HAS BEEN DEPORTED OR IS INVOLVED IN AN IMMIGRATION PROCEEDING, UNLESS CERTAIN CONDITIONS HAVE BEEN MET. Section 475(5)(E) of the Social Security Act (42 U.S.C. 675(5)(E)) is amended by adding after and below the end the following flush text: ``except that the State, and a county or other political subdivision of the State, shall not file (or join in the filing of such a petition) based on the removal of the parent from the United States or the involvement of the parent in (including detention pursuant to) an immigration proceeding, unless (I) the State (or the county or other political subdivision of the State, as the case may be) has made reasonable efforts to identify, locate, and contact any parent of the child, who has been removed from the United States, and any adult relative of the child, referred to in section 471(a)(29), including through the diplomatic or consular offices of the country to which the parent was removed, to notify such a parent or relative of the intent of the State (or the county or other political subdivision of the State, as the case may be) to file (or join in the filing of) such a petition, and to reunify the child with any such parent or relative; or (II) the parent is unfit or unwilling to be a parent of the child;''. SEC. 7. EFFECTIVE DATE. (a) In General.--The amendments made by this Act shall take effect on the 1st day of the 1st fiscal year beginning on or after the date of the enactment of this Act, and shall apply to payments under part E of title IV of the Social Security Act for calendar quarters beginning on or after such date. (b) Delay Permitted if State Legislation Required.--If the Secretary of Health and Human Services determines that State legislation (other than legislation appropriating funds) is required in order for a State plan approved under part E of title IV of the Social Security Act to meet the additional requirements imposed by the amendments made by this Act, the plan shall not be regarded as failing to meet any of the additional requirements before the 1st day of the 1st calendar quarter beginning after the 1st regular session of the State legislature that begins after the date of the enactment of this Act. For purposes of the preceding sentence, if the State has a 2-year legislative session, each year of the session is deemed to be a separate regular session of the State legislature. | Title: To amend part E of title IV of the Social Security Act to ensure that immigration status alone does not disqualify a parent, legal guardian, or relative from being a placement for a foster child, to prohibit a State, county, or other political subdivision of a State from filing for termination of parental rights in foster care cases in which an otherwise fit and willing parent or legal guardian has been deported or is involved in (including detention pursuant to) an immigration proceeding, unless certain conditions have been met, and for other purposes Summary: Help Separated Families Act of 2012 - Amends part E (Foster Care and Adoption Assistance) of title IV of the Social Security Act to: (1) require state child protection standards to ensure that the immigration status alone of a parent, legal guardian, or relative shall not disqualify the parent, legal guardian, or relative from being a placement for a child; and (2) require the state procedures for criminal records checks to require the state to accept foreign identification documents as sufficient identification for purposes of initiating a criminal records check or a fingerprint-based check. Expresses the sense of Congress that the child welfare agency of a state, or of any county or other political subdivision of a state, should grant a waiver of any requirement which would prevent the placement of a child with a relative of the child, on the basis of a minor legal infraction, if the relative would otherwise be considered eligible for such a placement. Requires the state plan for foster care and adoption assistance to notify relatives seeking placement of a child that their immigration status will not be questioned, except to the extent necessary in determining eligibility for relevant services or programs. Prohibits a state or local government agency from filing for termination of parental rights in foster care cases based on the removal of the parent from the United States or the parent's involvement in an immigration proceeding, unless: (1) the state (or local agency) has made reasonable efforts to notify of the intention to file such a petition any parent of the child who has been removed from the United States, and any adult relative of the child, including through the diplomatic or consular offices of the country to which the parent was removed, and to reunify the child with any such parent or relative; or (2) the parent is unfit or unwilling to be a parent of the child. | 1,494 | 505 | billsum | en |
Summarize: By. Reuters. and Daily Mail Reporter. The Winter Olympics city of Sochi, Russia used to be a haven for gays during the Soviet-era, but the community there has mostly fled the country following President Vladimir Putin's ban on 'gay propaganda'. Mayak Caberet is one of the last gay clubs left, and doesn't even really function as a bar for homosexuals anymore since most of the customers are straight couples who come to watch the nightly drag show. Club owner Roman Kochagov told Reuters that on any given night, only a third of his customers are gay men, far fewer than when he opened the club nine years ago. He says that it's a myth that there's still a gay community left in Sochi. VIDEO Scroll down for video. The last bar in town: Mayak Cabaret is one of the last vestiges of the gay community in Sochi, Russia. Gone: The gay community in Sochi has mostly fled the seaside resort since President Putin has cracked down on homosexuality in the country. Above, a woman walks past the anonymous exterior of the club. No one left: Most of the customers at the club these days are straight couples who come to watch the nightly drag show. 'The number of gays has dropped for years. Ever year there have been fewer and fewer...now they have almost all disappeared,' he said, adding that he himself is looking to leave the country. Sochi's gay scene has been shrinking since Russia won the right to host the 2014 Winter Games, and the decline has continued since President Vladimir Putin signed a law this year banning the spread of 'gay propaganda' among minors. The new law has focused attention on Sochi, which will host many foreigners as well as Russians during the Olympics. Some gay activists question its legality and others have called for a boycott of the Games in protest. Many members of the city's lesbian, gay, bisexual and transgender (LGBT) community have left the country. They worry Sochi's reputation as a city of tolerance will decline further. Putin's increasingly conservative social agenda in his third term as president has boosted the role of the Russian Orthodox Church, whose leader has suggested homosexuality is one of Russia's biggest threats, and given more air time to anti-homosexual rhetoric on media outlets. Outlawing homosexuality: In his third term as president, Vladimir Putin has become increasingly conservative, making the views of the Russian Orthodox Church the law of the land. Above, Putin at his annual press conference yesterday. Kochagov and his partner Andrei Tanichev opened Mayak in a darkly lit one-story building on a promenade overlooking the Black Sea. They had finished a successful run with a gay hotel and wanted to try something more ambitious. The building housing the Mayak Cabaret has only one outside light, shining over a brown steel door and doorbell. Except for the music that it pumps out after 10:00 p.m. every evening, it is barely recognizable as a club. Youths who have learned it is a gay club have ripped the sign off so many times, Kochagov said, that he has stopped putting it back up. Fights sometimes break out between his customers and clients of a working-class bar next door, but the level of violence barely compares with what high school student Vladislav Slavsky says he puts up with on a regular basis. His club: Mayak's co-owner Andrey Tanichev poses for a photograph in his venue. He and Roman Kochagov started the club after opening up a successful gay hotel. Losing customers: On any given night, Kochagov says only a third of his customers are gay men. Many straight couples turn out to watch the nightly drag show. Above, a performer gets ready back stage. Sochi's past: The resort town on the Black Sea became known as a more liberal-minded city during Soviet times. Gay men knew of it as a place to cruise for flings on the beach. At Mayak they employ singers and. dancers - all men - and Kochagov says he draws a profit every night from. crowds among the city's well-heeled who prefer to avoid the resort's. tourist bars. By Russian standards, the city has a marked liberal feel. 'People. here don't care who I sleep with. I walk down the street with my. boyfriend, people may know I'm gay, but no one pays any attention,' said. Marcel Aflin, 30, who worked in the northern oil city of Salekhard to. earn money to come and enjoy the sun and the beach of Sochi. But. Sochi is much less tolerant than many Western European cities. Many in. Sochi's gay community have left, lured abroad by the gay scenes in. cities such as Berlin or Barcelona. During. Soviet times, Sochi had gained a reputation for tolerance, especially. after it became a top tourism spot at a time when Soviet regulations. stipulated that husbands and wives must vacation away from each other. and their children. The city became the background for many summertime romances among married and unmarried people. Health. spa regulations also demanded that visitors were divided by sex and. that strangers were put up in the same room during their stay in the. region. Homosexuality,. which was a crime in the Soviet Union, was decriminalized only in 1993. By the time the Soviet Union crumbled, however, Sochi's beaches had. earned a reputation as a place for gay men to pick up partners. The future: Many in Russia are still unsure what the new gay propaganda law will mean for the LGBT community in the country - but fear it will lead to violence. What to do? Putin's gay propaganda law has become a point of issue with many countries sending athletes to Russia for the Winter Olympics. Above, a man strips onstage during a performance at Mayak. 'Many, many gay men know about it and went there,' said David Tuller, who wrote a book about gay life in Russia which was published in 1996. 'It always had a reputation as a city where you could go and cruise on the beach,' he said. Now locals say they can feel Putin's increasingly conservative political course, which has galvanized his support among more traditional parts of the country. The nationwide bill that outlaws gay 'propaganda' gives little detail on what exactly is banned and gay activists fear the possible proximity of children could be used to ban gay rights rally or even punish displays of affection. Response: Ice skater Brian Boitano came out this week ahead of his trip to Sochi as a part of the U.S. delegation. He was the third American to make such a public statement in recent days. Holding hands or kissing a same-sex partner in public, they say, might be enough to be hit with a fine equivalent to $170. Russian lawmakers say the law is a reflection of the country's social mores, and that it is needed to protect minors. Putin has condemned gay unions for failing to produce children as Russia battles a demographic crisis. But the legislation has brought calls for a boycott of the Olympics from activists such as British comedian Stephen Fry, who visited Russia this year to draw attention to the bill. Gay activists have also asked International Olympic Committee chief Thomas Bach to launch an investigation into the Russian law and the implications it would hold for visitors during the Olympics. Protest Sochi: Some, like British comedian Stehpeh Fry, said the games should be boycotted to protest Putin's new law. Gay rights activist Vladislav Slavsky (right) poses with his unidentified boyfriend on Sochi's Black Sea promenade. Russia's sports minister, Vitaly Mutko, said this month that the government should have waited until after the Games to implement the law banning gay propaganda. 'It would have been possible to calculate what kind of reaction this would have caused in the West, especially on the eve of the Olympics,' he said in an interview with business daily RBK. In an attempt to burnish Russia's image ahead of the Games, Putin has warned Russians against homophobia and said gay people would be welcome in Sochi during the Olympics. Many in Russia's gay community say the law sends out a signal for people to single them out for discrimination and in some cases violence | Summary: Mayak Caberet is one of the last gay bars left in the Black Sea resort town of Sochi, Russia which will host the 2014 Winter Olympics. Sochi was known as a haven for gays in the Soviet-era but the LGBT community there has mostly left the country. Russia has become an increasingly hostile country towards gays in President Vladimir Putin's third term. Putin recently passed a ban on 'gay propaganda' among minors. Many are calling for the games to be protested, while some Olympians have publicly come out in order to take make a stance against the law. | 1,863 | 127 | cnn_dailymail | en |
Summarize: Online survey shows 29% would support possible takeover while 41% said they could not imagine supporting such an event Almost a third of Americans could imagine supporting a military coup against their own government, according to a new poll. The YouGov survey showed 29% of Americans could imagine supporting a coup. Yet, 41% said they could not imagine supporting such an event. YouGov, which conducts internet polls about “politics, public affairs, products, brands and other topics of general interest”, surveyed 1,000 people online on the issue. They found that 43% of Republicans would support a military coup in certain instances, while only 20% of Democrats and 29% of independents would. The overall numbers increased when participants were “asked whether they would hypothetically support the military stepping in to take control from a civilian government which is beginning to violate the constitution”. 43% said yes to this, and 29% said no. Abraham Wyner, director of the undergraduate program in statistics at the Wharton School of the University of Pennsylvania, said that online polls were “worse than just about any other way you can put together a poll” because they were prone to selection bias, meaning proper randomization was not achieved and the sample was not representative of the population – since people can choose to participate. “People who are participating in an online poll are generally attracted to that poll because of some variable, some characteristic which is connected typically to one outcome or the other in that poll,” he said. In an attempt to avoid selection bias, YouGov obtains responses from a panel of internet users and weighs the responses to be nationally representative. This distinguishes them from the average internet poll where anybody can choose to respond to a question on a website. •The story was amended on 14 September to include information on how YouGov weighs online polls. Do you have any family members who have ever been incarcerated? Is walking to school a fair punishment for bullying? Would you be interested in a new weight-loss pill? Do you have any family members who have ever been incarcerated? Is walking to school a fair punishment for bullying? Would you be… | Summary: Americans in general trust their military-and 29% of them trust so much that they can imagine supporting a military takeover of the government, according to a YouGov poll. That rose to 43% among Republicans, while just 20% of Democrats said they could envision supporting a military coup in the US. When respondents were asked if they could potentially support the military "stepping in to take control from a civilian government which is beginning to violate the Constitution," the overall number saying yes rose to 43%, though a military coup itself wouldn't exactly be constitutional. The same poll found that most people have a lot more respect for military officers than they do for civilian leaders: Some 71% said military officers put the interests of the country ahead of their own interests, while just 12% thought the same about members of Congress. But there's probably no need to worry about tanks rolling down Pennsylvania Avenue or a Main Street near you anytime soon: The Pentagon hasn't shown much interest in taking over the nation, and a University of Pennsylvania expert tells the Guardian that the YouGov poll, like any online poll, isn't likely to be truly representative of the US population. | 489 | 260 | multi_news | en |
Summarize: Mr. Vidal loved conspiracy theories of all sorts, especially the ones he imagined himself at the center of, and he was a famous feuder; he engaged in celebrated on-screen wrangles with Mailer, Capote and William F. Buckley Jr. Mr. Vidal did not lightly suffer fools — a category that for him comprised a vast swath of humanity, elected officials particularly — and he was not a sentimentalist or a romantic. “Love is not my bag,” he said. By the time he was 25, he had already had more than 1,000 sexual encounters with both men and women, he boasted in his memoir “Palimpsest.” Mr. Vidal tended toward what he called “same-sex sex,” but frequently declared that human beings were inherently bisexual, and that labels like gay (a term he disliked) or straight were arbitrary and unhelpful. For 53 years, he had a live-in companion, Howard Austen, a former advertising executive, but the secret of their relationship, he often said, was that they did not sleep together. Mr. Vidal sometimes claimed to be a populist — in theory, anyway — but he was not convincing as one. Both by temperament and by birth he was an aristocrat. A Child on the Senate Floor Eugene Luther Gore Vidal Jr. was born on Oct. 3, 1925, at the United States Military Academy at West Point, where his father, Eugene, had been an All-American football player and a track star and had returned as a flying instructor and assistant football coach. An aviation pioneer, Eugene Vidal Sr. went on to found three airlines, including one that became T.W.A. He was director of the Bureau of Air Commerce under President Franklin D. Roosevelt. Mr. Vidal’s mother, Nina, was an actress and socialite and the daughter of Thomas Pryor Gore, a Democratic senator from Oklahoma. Mr. Vidal, who once said he had grown up in “the House of Atreus,” detested his mother, whom he frequently described as a bullying, self-pitying alcoholic. She and Mr. Vidal’s father divorced in 1935, and she married Hugh D. Auchincloss, the stepfather of Jacqueline Kennedy Onassis — a connection that Mr. Vidal never tired of bringing up. After her remarriage, Mr. Vidal lived with his mother at Merrywood, the Auchincloss family estate in Virginia, but his fondest memories were of the years the family spent at his maternal grandfather’s sprawling home in the Rock Creek Park neighborhood of Washington. He loved to read to his grandfather, who was blind, and sometimes accompanied him onto the Senate floor. Mr. Vidal’s lifelong interest in politics began to stir back then, and from his grandfather, an America Firster, he probably also inherited his unwavering isolationist beliefs. Mr. Vidal attended St. Albans School in Washington, where he lopped off his Christian names and became simply Gore Vidal, which he considered more literary-sounding. Though he shunned sports himself, he formed an intense romantic and sexual friendship — the most important of his life, he later said — with Jimmie Trimble, one of the school’s best athletes. Advertisement Continue reading the main story Trimble was his “ideal brother,” his “other half,” Mr. Vidal said, the only person with whom he ever felt wholeness. Jimmie’s premature death at Iwo Jima in World War II at once sealed off their relationship in a glow of A. E. Housman-like early perfection, and seemingly made it impossible for Mr. Vidal ever to feel the same way about anyone else. After leaving St. Albans in 1939, Mr. Vidal spent a year at the Los Alamos Ranch School in New Mexico before enrolling at Phillips Exeter Academy in New Hampshire. He contributed stories and poems to the Exeter literary magazine, but he was an indifferent student who excelled mostly at debating. A classmate, the writer John Knowles, later used him as the model for Brinker Hadley, the know-it-all conspiracy theorist in “A Separate Peace,” his Exeter-based novel. Mr. Vidal graduated from Exeter at 17 — only by cheating on virtually every math exam, he later admitted — and enlisted in the Army, becoming first mate on a freight supply ship in the Aleutian Islands. He began work on “Williwaw,” a novel set on a troopship and published in 1946, while he was an associate editor at the publishing company E. P. Dutton, a job he soon gave up. Written in a pared-down, Hemingway-like style, “Williwaw” (the title is a meteorological term for a sudden wind out of the mountains) won some admiring reviews but gave little clue to the kind of writer Mr. Vidal would become. Neither did his second book, “In a Yellow Wood” (1947), about a brokerage clerk and his wartime Italian mistress. Mr. Vidal later said it was so bad, he couldn’t bear to reread it. He nevertheless became a glamorous young literary figure, pursued by Anaïs Nin and courted by Christopher Isherwood and Tennessee Williams. In 1948 Mr. Vidal published “ The City and the Pillar,” which was dedicated to J. T. (Jimmie Trimble). It is what would now be called a coming-out story, about a handsome, athletic young Virginia man who gradually discovers that he is homosexual. By today’s standards it is tame and discreet, but at the time it caused a scandal and was denounced as corrupt and pornographic. Mr. Vidal later claimed that the literary and critical establishment, The New York Times especially, had blacklisted him because of the book, and he may have been right. He had such trouble getting subsequent novels reviewed that he turned to writing mysteries under the pseudonym Edgar Box and then, for a time, gave up novel-writing altogether. To make a living he concentrated on writing for television, then for the stage and the movies. Politics Onstage, and for Real Work was plentiful. He wrote for most of the shows that presented hourlong original dramas in the 1950s, including “Studio One,” “Philco Television Playhouse” and “Goodyear Playhouse.” He became so adept, he could knock off an adaptation in a weekend and an original play in a week or two. He turned “Visit to a Small Planet,” his 1955 television drama about an alien who comes to earth to study the art of war, into a Broadway play. His most successful play was “The Best Man,” about two contenders for the presidential nomination. It ran for 520 performances on Broadway before it, too, became a well-received film, in 1964, with a cast headed by Henry Fonda and a screenplay by Mr. Vidal. It was revived on Broadway in 2000 and is now being revived there again as “Gore Vidal’s The Best Man.” Photo Mr. Vidal’s reputation as a script doctor was such that in 1956 MGM hired him as a contract writer; among other projects he helped rewrite the screenplay of “Ben-Hur,” though he was denied an official credit. He also wrote the screenplay for the movie adaptation of his friend Tennessee Williams’s play “Suddenly, Last Summer.” By the end of the ’50s, though, Mr. Vidal, at last financially secure, had wearied of Hollywood and turned to politics. He had purchased Edgewater, a Greek Revival mansion in Dutchess County, N.Y., and it became his headquarters for his 1960 run for Congress. He was encouraged by Eleanor Roosevelt, a neighbor who had become a friend and adviser. The 29th Congressional District was a Republican stronghold, and though Mr. Vidal, running as Eugene Gore on a platform that included taxing the wealthy, lost, he received more votes in running for the seat than any Democrat in 50 years. And he never tired of pointing out he did better in the district than the Democratic presidential candidate that year, John F. Kennedy. Newsletter Sign Up Continue reading the main story Please verify you're not a robot by clicking the box. Invalid email address. Please re-enter. You must select a newsletter to subscribe to. Sign Up You will receive emails containing news content, updates and promotions from The New York Times. You may opt-out at any time. You agree to receive occasional updates and special offers for The New York Times's products and services. Thank you for subscribing. An error has occurred. Please try again later. View all New York Times newsletters. Mr. Vidal also returned to writing novels in the ’60s and published three books in fairly quick succession: “Julian” (1964), “Washington, D.C.” (1967) and “Myra Breckinridge” (1968). “Julian,” which some critics still consider Mr. Vidal’s best, was a painstakingly researched historical novel about the fourth-century Roman emperor who tried to convert Christians back to paganism. (Mr. Vidal himself never had much use for religion, Christianity especially, which he once called “intrinsically funny.”) “Washington, D.C.” was a political novel set in the 1940s. “Myra Breckinridge,” Mr. Vidal’s own favorite among his books, was a campy black comedy about a male homosexual who has sexual reassignment surgery. (A 1970 film version, with Raquel Welch and Mae West, proved to be a disaster.) Advertisement Continue reading the main story Perhaps without intending it, Mr. Vidal had set a pattern. In the years to come he found his greatest successes with historical novels, notably what became known as his American Chronicles: “Washington, D.C.,” “Burr” (1973), “1876” (1976), “Lincoln” (1984), “Empire” (1987), “Hollywood” (1990) and “The Golden Age” (2000). He turned out to have a gift for this kind of writing. These novels were learned and scrupulously based on fact, but also witty and contemporary-feeling, full of gossip and shrewd asides. Harold Bloom wrote that Mr. Vidal’s imagination of American politics “is so powerful as to compel awe.” Writing in The Times, Christopher Lehmann-Haupt said, “Mr. Vidal gives us an interpretation of our early history that says in effect that all the old verities were never much to begin with.” But Mr. Vidal also persisted in writing books like “Myron” (1974), a sequel to “Myra,” and “Live From Golgotha: The Gospel According to Gore Vidal” (1992), which were clearly meant as provocations. “Live From Golgotha,” for example, rewrites the Gospels, with Saint Paul as a huckster and pederast and Jesus a buffoon. John Rechy said of it in The Los Angeles Times Book Review, “If God exists and Jesus is his son, then Gore Vidal is going to hell.” In the opinion of many critics, though, Mr. Vidal’s ultimate reputation is apt to rest less on his novels than on his essays, many of them written for The New York Review of Books. His collection “The Second American Revolution” won the National Book Critics Circle Award for criticism in 1982. About a later collection, “ United States : Essays 1952-1992,” R. W. B. Lewis wrote in The New York Times Book Review that Vidal the essayist was “so good that we cannot do without him,” adding, “He is a treasure of state.” Mr. Vidal’s essays were literary, resurrecting the works of forgotten writers like Dawn Powell and William Dean Howells, and also political, taking on issues like sexuality and cultural mores. The form suited him ideally: he could be learned, funny, stylish, show-offy and incisive all at once. Even Jason Epstein, Mr. Vidal’s longtime editor at Random House, once admitted that he preferred the essays to the novels, calling Mr. Vidal “an American version of Montaigne.” “I always thought about Gore that he was not really a novelist,” Mr. Epstein wrote, “that he had too much ego to be a writer of fiction because he couldn’t subordinate himself to other people the way you have to as a novelist.” Vidal vs. Buckley (and Mailer) Success did not mellow Mr. Vidal. In 1968, while covering the Democratic National Convention on television, he called William F. Buckley a “crypto-Nazi.” Buckley responded by calling Mr. Vidal a “queer,” and the two were in court for years. In a 1971 essay he compared Norman Mailer to Charles Manson, and a few months later Mailer head-butted him in the green room while the two were waiting to appear on the Dick Cavett show. They then took their quarrel on the air in a memorable exchange that ended with Mr. Cavett’s telling Mailer to take a piece of paper on the table in front of them and “fold it five ways and put it where the moon don’t shine.” In 1975 he sued Truman Capote for libel after Capote wrote that Mr. Vidal had been thrown out of the Kennedy White House. Mr. Vidal won a grudging apology. Some of his political positions were similarly quarrelsome and provocative. Mr. Vidal was an outspoken critic of Israel ’s treatment of the Palestinians, and once called Norman Podhoretz, the editor of Commentary, and his wife, the journalist Midge Decter, “Israeli fifth columnists.” In the 1990s he wrote sympathetically about Timothy McVeigh, who was executed for the Oklahoma City bombing. And after the Sept. 11 terrorist attacks, he wrote an essay for Vanity Fair arguing that America had brought the attacks upon itself by maintaining imperialist foreign policies. In another essay, for The Independent, he compared the attacks to the Japanese raid on Pearl Harbor, arguing that both Presidents Franklin D. Roosevelt and George W. Bush knew of them in advance and exploited them to advance their agendas. Advertisement Continue reading the main story As for literature, it was more or less over, he declared more than once, and he had reached a point where he no longer much cared. He became a sort of connoisseur of decline, in fact. America is “rotting away at a funereal pace,” he told The Times of London in 2009. “We’ll have a military dictatorship pretty soon, on the basis that nobody else can hold everything together.” In 2003 Mr. Vidal and his companion, Mr. Austen, who was ill, left their cliffside Italian villa La Rondinaia (the Swallow’s Nest) on the Gulf of Salerno and moved to the Hollywood Hills to be closer to Cedars-Sinai Medical Center. Mr. Austen died that year, and in “Point to Point Navigation,” his second volume of memoirs, Mr. Vidal recalled that Mr. Austen asked from his deathbed, “Didn’t it go by awfully fast?” “Of course it had,” Mr. Vidal wrote. “We had been too happy and the gods cannot bear the happiness of mortals.” Mr. Austen was buried in Washington in a plot Mr. Vidal had purchased in Rock Creek Cemetery. The gravestone was already inscribed with their names side by side. Besides his nephew, Burr Steers, Mr. Vidal’s survivors include his sister, Nina Gore Auchincloss Straight. After Mr. Austen’s death, Mr. Vidal lived alone in declining health himself. He was increasingly troubled by a knee injury he suffered in the war, and used a wheelchair to get around. In November 2009 he made a rare public appearance to attend the National Book Awards in New York, where he was given a lifetime achievement award. He had evidently not prepared any remarks, and instead delivered a meandering impromptu speech that was sometimes funny and sometimes a little hard to follow. At one point he even seemed to speak fondly of Buckley, his old nemesis. It sounded like a summing up. “Such fun, such fun,” he said. Celebrated American author, playwright, politician and commentator Gore Vidal has died, aged 86, at his home in the Hollywood Hills. Gore Vidal wrote 25 novels, including the bestselling Burr and Myra Breckenridge, more than 200 essays and several plays. He also ran for political office twice and was a well-known commentator. Held in high regard for his independent thoughts, Vidal was better known for opposing wars including Vietnam and Iraq, and mocking of religion and prudery. On word of his death this morning, Channel 4 news reporter Krishnan Guru-Murthy said: "I imagine Gore Vidal will get to heaven and find it terribly bland. I hope he somehow sends us back a review." Comedian Ricky Gervais tweeted one of Vidal's many famous quotes: "... to rid one's self of this consciousness... nothing wrong with that.." Gore Vidal RIP. "The writer Gore Vidal was thoughtful, ideologically consistent, extremely committed and an American isolationist," said Australia's Foreign Minister Bob Carr. Actress Joan Collins paid tribute, saying, "So sad my friend the brilliant Gore Vidal has died, he was a total original & genius." Actor James Urbaniak tweeted: "Aw, Gore Vidal. RIP. Who will carry on the mid-Atlantic accent? It's up to me and Kelsey Grammer now." Exiled writer Taslima Nasreen paid tributes, quoting Vidal, "Democracy is supposed to give you the feeling of choice, like Painkiller X and Painkiller Y. But they're both just aspirin." Congratulating Vidal in 2009 at the National Book Awards ceremony, writer, editor and publisher Dave Eggers said: "He meant everything to me when I was learning how to write and learning how to read." "His words, his intellect, his activism, his ability and willingness to always speak up and hold his government accountable, especially has been so inspiring to me I can't articulate it." Scissor Sister Jake Shears tweeted, “’Camp is nothing but another word for someone who hasn’t got any talent.’ RIP Gore Vidal. A truly great American original. What a legacy.” Courtney Love paid tribute, “Style is knowing who you are, what you want to say and not giving a damn.’ Quoted by Gore Vidal… you will be missed, rest in peace Gore.” Michael Moore tweeted one of Vidal’s famous quotations, “Half of the American people have never read a newspaper. Half never voted for President. One hopes it is the same half." British actor Nicholas Pegg shared a personal favourite, "Norman Mailer punched Gore Vidal at a party after a bad review. Still on the floor, Vidal declared: 'Once again, words fail Norman Mailer.'" David Niose, president of the American Humanist Association, spoke of Vidal’s contribution as honorary president: “The progressive and humanist values Gore Vidal repeatedly espoused moved the culture in a positive direction…He spent his life pointing out the places in society that needed the most attention without worrying who might be embarrassed or upset by his opinions.” American actor and writer Frank Conniff tweeted, “Gore Vidal dreaded the idea of an afterlife, because it would mean he'd have to see Norman Mailer again. Rest In Peace.” Gore Vidal, a celebrated writer, cultural gadfly and occasional political candidate, died July 31 at his home in Hollywood Hills, Calif., at 86. He had complications from pneumonia, his nephew, the actor, director and screenwriter Burr Steers, told the Associated Press. Known for his urbanity and wit — “Every time a friend succeeds, I die a little”; “A narcissist is someone better looking than you are” — Mr. Vidal enjoyed a literary career that spanned more than 60 years, and he once said that he hoped to be remembered as “the person who wrote the best sentences of his time.” Mr. Vidal was an astonishingly versatile man of letters and nearly the last major writer of the modern era to have served in World War II. Having resolved at age 20 to live by his pen, he wrote plays for television and Broadway, including the classic political drama “The Best Man”; helped script such movies as “Ben-Hur,” the 1959 epic starring Charlton Heston; and gained notoriety for the campy 1968 novel “Myra Breckinridge,” about a transsexual film enthusiast. Mr. Vidal also won plaudits from scholars, critics and ordinary readers for historical novels such as the best-selling “Julian” (1964), “Burr” (1973) and “Lincoln” (1984). “United States,” which gathers Mr. Vidal’s essays on art, politics and himself, received the 1993 National Book Award. In print or on television — he was a frequent talk-show guest — the worldly Mr. Vidal provoked controversy with his laissez-faire attitude toward every sort of sexuality, his well-reasoned disgust with what he called American imperialism and his sophisticated cynicism about love, religion, patriotism and other sacred cows. He took an acerbic view of American leadership. “Today’s public figures can no longer write their own speeches or books,” he once quipped, “and there is some evidence they cannot read them either.” As a boy, a thirst for learning Mr. Vidal was born Oct. 3, 1925, at West Point, N.Y., where his father, Eugene Vidal, was teaching aeronautics at the military academy. His mother, Nina, was the socialite daughter of Sen. T.P. Gore of Oklahoma. Christened Eugene Luther Gore Vidal, the future writer later lopped off the first two names “for political as well as for aesthetic reasons.” Young Gore spent much of his childhood in Washington and was particularly attached to his grandfather. The senator was blind, so the boy passed many hours reading to him aloud, thus inaugurating his own lifelong passion for learning and books. “The first grown-up book that I read on my own was a nineteenth-century edition of ‘Tales from Livy’ that I’d found in my grandfather’s library,” he once wrote. By 14, he added, “I wanted to know the entire history of the entire world.” Mr. Vidal attended the private St. Albans School in Washington, where he fell in love with a fellow student named Jimmie Trimble, who was killed in combat on Iwo Jima during World War II. In his memoirs “Palimpsest” (1995) and “Point to Point Navigation” (2006), Mr. Vidal makes clear that this youthful passion marked his entire life: He never truly loved anyone again, although he would enjoy hundreds of sexual encounters, many of them with anonymous strangers, in which he took pleasure but, as he repeatedly insisted, never gave any except inadvertently. Although Mr. Vidal maintained a more than 50-year partnership with his companion, Howard Austen, he constantly underscored that the secret of its longevity was “no sex.” Austen died in 2003. As a teenager, Mr. Vidal was sent to boarding school at Phillips Exeter Academy in New Hampshire, from which he graduated in 1943. Rather than go on to Harvard, he enlisted in the Army, serving as first mate on a small supply ship in the Aleutians. That experience inspired his widely praised first novel, “Williwaw,” which appeared in 1946, when the author was 20. Influence on stage and screen After his discharge, Mr. Vidal decided to bag college and live in New York as a full-time writer. Before long, he became an intimate confidant of diarist Anais Nin and a friend of playwright Tennessee Williams. He also brought out two more novels, including “The City and the Pillar” (1948), an account of two all-American boys and what was — at that time — “the love that dare not speak its name.” Although that book is now viewed as a pioneering work of gay literature, its casual acceptance of homosexual impulses offended some contemporary critics — and Mr. Vidal’s subsequent seven novels went unnoticed by Time magazine, Newsweek and the New York Times. Because most of his fiction of the 1950s — even now admired works such as “Messiah” (1954), the study of a religious cult — proved commercially lackluster, Mr. Vidal decided to earn his living by writing TV dramas, Broadway plays and movie scripts. He also cranked out three mysteries under the pen name Edgar Box. With the money from his commercial writing, Mr. Vidal paid the mortgage on a grandly pillared Greek Revival manse called Edgewater, located on the banks of the Hudson River near Rhinecliff, N.Y. There, he threw parties attended by rising literary notables such as Saul Bellow and Norman Mailer, critics Lionel and Diana Trilling, and movie stars Joanne Woodward and Paul Newman (who became his close friends). Although Mr. Vidal enjoyed a varied social and sexual life, he nonetheless worked hard. On Broadway he hit pay dirt with “Visit to a Small Planet” (1957), in which an alien named Kreton lands on Earth and announces that human beings are his hobby. It deftly skewers contemporary mores and Cold War anxieties. The even more highly regarded political drama “The Best Man” (1960), which was nominated for a Tony Award as best play, presented a behind-the-scenes look at the wheeling and dealing between two men competing for the Democratic presidential nomination. Both plays became films and have periodically been revived on stage. As a screenwriter in the 1950s and occasionally afterward, Mr. Vidal contributed to many films, often without screen credit. He wrote a teleplay that inspired “The Left-Handed Gun”(with Newman as Billy the Kid); he was called in to doctor “Ben-Hur”(and tweaked the script to suggest a homosexual subtext to explain the relationship between the epic’s hero and his enemy Messala); and he worked with Williams on the hothouse melodrama “Suddenly, Last Summer.” In his later years, Mr. Vidal appeared with some frequency in films, notably as himself in Federico Fellini’s “Roma” (1972) and as a U.S. senator in Tim Robbins’s “Bob Roberts” (1992). By the early 1960s, the not-yet-40-year-old Mr. Vidal had achieved his goal of financial independence. For the next three decades, he spent much of his life in Italy, his villa a lodestone for literati and glitterati. There he carefully researched his novel “Julian” — about the apostate Roman emperor — and its success relaunched his moribund career as a fiction writer. In general, the older Mr. Vidal published three kinds of fiction: historical novels set in the ancient world, such as “Julian” and 1981’s “Creation” (which features Socrates, Zoroaster, the Buddha and Confucius); campy fantasies that mock American prejudices and conventionalities, the most famous of which is “Myra Breckinridge”; and the so-called American Chronicle, a series of seven novels — the best known are “Burr” and “Lincoln” — detailing the secret political history of the United States. In these years, Mr. Vidal also grew more prominent as a pundit, on TV and in the pages of the New York Review of Books and other periodicals. Although he had published essays and reviews since the 1950s, Mr. Vidal increasingly cast himself as a modern-day Voltaire, commenting on the nation’s follies with waspish asperity and wit. “There is no human problem which could not be solved if people would simply do as I advise,” he said. He insisted that everyone is really bisexual: “There is no such thing as a homosexual or a heterosexual person. There are only homo- or heterosexual acts. Most people are a mixture of impulses if not practices.” Political elections, he observed, were “held at great cost without issues and with interchangeable candidates.” Most famously, during the heated days of the national conventions of 1968, Mr. Vidal squared off on the issue of freedom of speech with conservative pundit William F. Buckley Jr. The pair ended up trading insults: “Crypto-Nazi,” Mr. Vidal said. “You queer,” Buckley shot back. Mr. Vidal collected the best of his discursive prose in “United States” (1993), a mammoth volume of literary essays, political polemics and autobiographical reminiscences for which he received the National Book Award. Although it included a scandalously frank account of the Kennedys entitled “The Holy Family,” Mr. Vidal’s finest pieces were not his attacks but his appreciations. He produced exemplary reappraisals — composed in ballpoint pen on yellow legal pads — of his favorite childhood authors (notably L. Frank Baum, creator of the “Oz” books) and of many once-undervalued figures, such as Dawn Powell, Italo Calvino, William Dean Howells, Thomas Love Peacock and Logan Pearsall Smith. That some of these writers continued to be neglected only supports Mr. Vidal’s frequent lament: The age of the reader is passing, and we are living through its twilight’s last gleaming. The occasional politician Although Mr. Vidal found success as a writer and intellectual, he failed in his attempts to win political office. He twice ran unsuccessfully in elections, campaigning for Congress in 1960 and then for the Senate in 1982. While politics was in his view always corrupt, he also felt it had grown more so since World War II. “I date the end of the old republic and the birth of the empire to the invention, in the late thirties, of air conditioning,” he said. Before air conditioning, “the politicians would abandon Washington in the summer; now they stay around all through the year, making mischief.” In his later years, Mr. Vidal grew less witty and increasingly vehement in his political views, speaking out against American policy after the Sept. 11, 2001, terrorist attacks and denouncing the 2003 invasion of Iraq. Mr. Vidal once pointed out that his literary genealogy included “Petronius, Juvenal, Apuleius — then Shakespeare — then Peacock, Meredith, James, Proust.” Like those cultivated and polished writers, Mr. Vidal cast a cold eye on the society of his time and resolutely upheld the values of urbanity and pleasure against the onslaughts of the barbarian, the puritan and the philistine. “Always a godfather, never a god,” he once remarked at a christening. style@washpost.com Dirda reviews books each Thursday in Style and conducts a book discussion for The Post at wapo.st/reading-room. | Summary: Gore Vidal has passed away, and that means everyone is contemplating the life of one of America's most caustic, controversial men of letters. Of course, given that Vidal once said he hoped to be remembered as "the person who wrote the best sentences of his time," the best many can do is quote him. Vidal was "an Augustan figure who believed himself to be the last of a breed," writes the New York Times, "and he was probably right." Vidal Quote: "I'm exactly as I appear. There is no warm, lovable person inside. Beneath my cold exterior, once you break the ice, you find cold water." The Nation recalls his tumultuous history writing for the magazine, in which he took some controversial stances-like declaring certain Jewish pundits "an Israeli Fifth Column Division" and supporting a version of the 9/11 truther conspiracy-but also crafted gems like, "We are the United States of Amnesia. We learn nothing because we remember nothing.... The withered Bill of Rights, like a dead trumpet vine, clings to our pseudo-Roman columns." "Vidal could be guaranteed to say something entertainingly outrageous about American imperialism or contemporary sexual mores," the Washington Post recalls. "Everything and anything could be skewered with stiletto-like finesse, and nothing was sacred." Vidal Quote: "There is no human problem which could not be solved if people would simply do as I advise." The Telegraph rounds up a number of notable celebrity responses to Vidal's death, almost all of which are quotes of his. Including: "Style is knowing who you are, what you want to say and not giving a damn." | 7,108 | 375 | multi_news | en |
Summarize: House Majority Leader Eric Cantor spoke to an FBI whistle-blower two weeks ago who accused then-CIA Director David Petraeus of having an extramarital affair and potentially jeopardizing the security of classified information, according to a news report. Cantor’s chief of staff, Steve Stombres, later spoke with FBI officials to pass on the accusations from the whistle-blower, the New York Times reported on Saturday night. Text Size - + reset King: FBI obligated to tell Obama Hayen: Timing ‘mysterious’ (Also on POLITICO: 6 questions on Petraeus’s exit) Cantor’s involvement in the Petraeus scandal is the first indication that anyone outside of the FBI knew Petraeus was under scrutiny for an extramarital relationship or potentially leaking classified information. The FBI had been investigating Petraeus for several months after his alleged mistress, author Paula Broadwell, was suspected of sending “harassing” e-mails to another woman close to Petraeus. (Also on POLITICO: Reports: Probe began with 2 women) During their probe of Broadwell’s activities, FBI agents uncovered information about a reputed romantic relationship between Broadwell and Petraeus. Broadwell is the author of a flattering biography on Petraeus’s extraordinary military career that was released early in 2012. There were also concerns that Broadwell may have obtained sensitive information via her ties to Petraeus. (PHOTOS: Gen. David Petraeus’s career) But the revelation of Cantor’s role in the scandal - and the emergence of an FBI whistle-blower - raises dramatic new questions about how the bureau conducted what was clearly a hugely sensitive problem, both in terms of the FBI’s relationship with the CIA and what it could mean for the highly esteemed Petraeus. It also raises the stakes for the political fallout surrounding the scandal. Top CIA officials have been asked to brief members of the House Intelligence Committee next week on what happened and how the case unfolded. “I was contacted by an F.B.I. employee concerned that sensitive, classified information may have been compromised and made certain [FBI] Director Mueller was aware of these serious allegations and the potential risk to our national security,” Cantor said in a statement. Petraeus resigned on Friday after 14 months atop the CIA while admitting to an extramarital affair. The announcement stunned Washington, as lawmakers, Obama administration officials and the press scrambled to find out the reasons behind his abrupt departure. James Clapper, director of National Intelligence, only learned about the FBI probe on Tuesday, according to news reports. Following discussions with Petraeus, Clapper told the CIA director he should resign. Petraeus then met with President Barack Obama on Thursday to inform him of that decision. The informant was brought to Cantor’s attention by Rep. David Reichert (R-Wash.). Reichert declined to comment on his role in the scandal. A Cantor aide said Stombres spoke with FBI officials on Oct. 31 to pass on the allegations about Petraeus. Cantor’s office declined to provide more information beyond saying that the New York Times report “was accurate.” It is unclear if Cantor told Speaker John Boehner (R-Ohio) or other top House Republicans - including Intelligence Committee Chairman Mike Rogers (Mich.) - of the allegations surrounding Petraeus. Eric Cantor, the House majority leader, said Saturday an F.B.I. employee whom his staff described as a whistle-blower told him about Mr. Petraeus’s affair and a possible security breach in late October, which was after the investigation had begun. “I was contacted by an F.B.I. employee concerned that sensitive, classified information may have been compromised and made certain Director Mueller was aware of these serious allegations and the potential risk to our national security,” Mr. Cantor said in a statement. Mr. Cantor talked to the person after being told by Representative Dave Reichert, Republican of Washington, that a whistle-blower wanted to speak to someone in the Congressional leadership about a national security concern. On Oct. 31, his chief of staff, Steve Stombres, called the F.B.I. to tell them about the call. “They took the information,” said Doug Heye, Mr. Cantor’s deputy chief of staff, “and gave the standard answer: they were not able to confirm or deny any investigation, but said that all necessary steps were being taken to make sure no confidential information was at risk.” White House officials said they were informed on Wednesday night that Mr. Petraeus was considering resigning because of an extramarital affair. On Thursday morning, just before a staff meeting at the White House, President Obama was told. That afternoon, Mr. Petraeus went to see him and informed him that he strongly believed he had to resign. Mr. Obama did not accept his resignation right away, but on Friday, he called Mr. Petraeus and accepted it. Mr. Petraeus, 60, said in a statement that he was resigning after 14 months as head of the Central Intelligence Agency because he had shown “extremely poor judgment” in engaging in the affair. He has been married for 38 years. Ms. Broadwell, 40, is also married. She and her husband have two children and live in Charlotte, N.C. On Saturday, the two government officials who had been briefed on the case dismissed a range of media speculation that the F.B.I. inquiry might have focused on leaks of classified information to the news media or even foreign spying. “People think that because it’s the C.I.A. director, it must involve bigger issues,” one official said. “Think of a small circle of people who know each other.” The F.B.I. investigators were not pursuing evidence of Mr. Petraeus’s marital infidelity, which would not be a criminal matter, the official said. But their examination of his e-mails, most or all of them sent from a personal account and not from his C.I.A. account, raised the possibility of security breaches that needed to be addressed directly with him. “Alarms went off on larger security issues,” the official said. As a result, F.B.I. agents spoke with the C.I.A. director about two weeks ago, and Mr. Petraeus learned in the discussion, if he was not already aware, that they knew of his affair with Ms. Broadwell, the official said. | Summary: Deep in the New York Times report about "harassing" emails that Paula Broadwell allegedly sent another woman lies this coda in the downfall of David Petraeus: An FBI whistleblower reached out to House Majority Leader Eric Cantor two weeks ago to alert him to the affair. "I was contacted by an FBI employee concerned that sensitive, classified information may have been compromised and made certain Director Mueller was aware of these serious allegations and the potential risk to our national security," said Cantor yesterday in a statement. Cantor's chief of staff passed along the information to the FBI itself on Oct. 31; it was nearly a week later before Petraeus' own boss learned of the allegations. Cantor's office isn't elaborating, other than to call the Times report "accurate." Politico notes that the reveal raises questions about how the investigation unfolded, not to mention the FBI's relationship with the CIA. CIA honchos are slated to brief the House Intelligence Committee next week on the investigation. | 1,427 | 222 | multi_news | en |
Summarize: Big deal or big hype? We’ll find out Saturday night when Floyd Mayweather meets Conor McGregor in a super welterweight boxing match in Las Vegas. >> Read more trending news Mayweather is 49-0 and has won 12 world titles in five weight classes. He is coming out of two years of inactivity to fight McGregor, the UFC star who is 21-3 as a professional. Here are some last-minute numbers to crunch as the opening bell draws near: 8 oz. -- The size of the boxing gloves the two fighters will use. Normally, boxers use 10 oz. gloves in fights sanctioned by the Nevada State Athletic Commission. 12 rounds -- The scheduled length of the bout. "It won't go the distance, mark my words," Mayweather said. 26 -- The number of knockouts in Mayweather’s pro career. 40 -- Mayweather’s age. It could be a factor against McGregor, who is 29. 74 inches – McGregor’s reach when throwing punches – he will hold a two-inch advantage over Mayweather. $99.95 – The cost to order a high-definition broadcast of the fight on pay-per-view. 154 pounds – The official weight limit in the fight. At Friday’s weigh-in, Mayweather tipped the scales at 149.5 pounds, CBS Sports reported. McGregor, making his pro boxing debut, weighed in at 153 pounds. 3,360 – The number of diamonds included on “The Money Belt,” which will be awarded to the fight’s winner. The belt also will include 600 sapphires and 300 emeralds. 20,000 -- The seating capacity of Las Vegas’s T-Mobile Arena, where the bout will be held. 4.9 million – The number of pay-per-view orders that UFC chief Dana White expects the fight to draw, which would be a record. $300 million – The amount of money Mayweather said he will earn from the match. “Just being real. They call me ‘Money Mayweather’ for a reason,” he told Fox Business in an email interview. McGregor is set to earn anywhere from $50 million to $100 million, according to various estimates. Both fighters approved a confidential agreement on how the purse will be split, Rare reported. Mayweather is guaranteed $100 million. $700 million – Projected revenue for Mayweather vs. McGregor from pay-per-view buys, ticket sales and other sources of income,. By comparison, Mayweather’s record-breaking match with Manny Pacquiao in 2015 yielded an estimated $600 million. Conor McGregor doesn't think Floyd Mayweather can keep up with him. (0:38) LAS VEGAS -- For the second time in three fights, Floyd Mayweather will earn a guaranteed purse of $100 million. Mayweather, coming out of a two-year retirement, will earn that sum as a minimum for his junior middleweight boxing match against UFC star Conor McGregor on Saturday (Showtime PPV, 9 p.m. ET) at the T-Mobile Arena, according to contract figures released on Friday night by the Nevada State Athletic Commission. Mayweather also earned a guaranteed $100 million for his 2015 megafight with Manny Pacquiao, though he made around $250 million because of his share of the enormous profits from the record-shattering event. The fight with McGregor, 29, is largely expected to break the Mayweather-Pacquiao records, which would give Mayweather (49-0, 26 KOs), 40, another payday well in excess of $200 million. McGregor's minimum purse is $30 million, which dwarfs any he has made for his fights in the Octagon during his mixed martial arts career. But the Irishman will wind up making tens of millions of dollars more than his minimum because of his share of the promotion. His take is likely to exceed $100 million. Gervonta Davis (18-0, 17 KOs), who was stripped of his junior lightweight world title for weighing in at 132 pounds, two more than the division limit, will make $600,000 for his co-featured bout against Francisco Fonseca (19-0-1, 13 KOs) because he was overweight. Fonseca will earn $35,000, though a portion of Davis' purse is likely to go to him because Davis missed his weight. Secondary light heavyweight titlist Nathan Cleverly (30-3, 16 KOs), of Wales, will make $100,000 for his defense against Badou Jack, though Cleverly is likely to earn more from British television. Jack (20-1-3, 12 KOs), fighting for the first time since vacating his super middleweight title to move up in weight, will earn $750,000. Cruiserweights Andrew Tabiti (14-0, 12 KOs) and Steve Cunningham (29-8-1, 13 KOs), a former two-time world titleholder, will make $100,000 apiece for their pay-per-view opening bout. Welterweight Thomas Dulorme will make $75,000, and Yordenis Ugas will be paid $50,000 for their 10-round bout, which headlines the Fox-televised portion of the undercard. Floyd Mayweather and Conor McGregor has always been about the money. The two fighters, who will make history on Saturday night when they square off in Las Vegas on Showtime PPV, seemingly took this match to rake in one of the biggest financial gains from a sporting event ever. Now, we have a rough idea of just how much money the fighters will be making. According to contract details from the Nevada State Athletic Commission, Mayweather will earn a disclosed purse of $100 million while McGregor will bring in $30 million, according to ESPN. It is the same disclosed purse Mayweather received for his fight against Manny Pacquiao in 2015. Meanwhile, McGregor will make a minimum of $30 million, which is nearly 10 times what he made from his last UFC against Eddie Alvarez at UFC 205 -- his last previous high salary. Here's how the fight card breaks down with disclosed salaries for each of the main card fighters. Fighter Fighter Weightclass Floyd Mayweather $100 million Conor McGregor $30 million Junior middleweight Gervonta Davis $600,000 Francisco Fonseca $35,000 Junior lightweight Badou Jack $750,000 Nathan Cleverly $100,000 Light heavyweight Andrew Tabiti $100,000 Steve Cunningham $100,000 Cruiserweight The numbers are just insane, but the craziest part is how much they can still be inflated depending on how many PPV buys, ticket prices and other expenditures add up from the event. | Summary: Whether or not it'll be a good fight (experts tend to agree "not"), Saturday's match between Floyd Mayweather and Conor McGregor is certain to be profitable-potentially historically so-for both fighters. ESPN reports Mayweather is guaranteed a minimum purse of $100 million and McGregor a minimum purse of $30 million. It may seem like McGregor is getting the short end of the stick, but that $30 million is nearly 10 times what he earned in his last UFC fight, according to CBS Sports. The purse amounts were released Friday night by the Nevada State Athletic Commission. While those numbers are eye-popping enough, both fighters are expected to take home far more than the minimum thanks to ticket sales, pay-per-view orders, and more. UFC head Dana White expects a record 4.9 million people will watch the fight on pay-per-view, and the fight's total revenue is projected to be $700 million, that Atlanta Journal-Constitution reports. That's $100 million more than Mayweather's record-setting 2015 fight against Manny Pacquiao. ESPN believes McGregor will end up taking home more than $100 million and Mayweather more than $200 million. But the latter is predicting his payday will be at least $300 million. Those sky-high numbers make sense, as CBS states both fighters "seemingly took this match to rake in one of the biggest financial gains from a sporting event ever." | 1,624 | 335 | multi_news | en |
Summarize: CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority from U.S. Provisional Patent Application Ser. No. 61/993,383, filed on May 15, 2014, the entirety of which is expressly incorporated herein by reference. FIELD OF THE INVENTION [0002] The present invention relates generally to items used in surgical procedures, and more particularly to a process for molding exterior coatings on those items and the items formed by the molding process. BACKGROUND OF THE INVENTION [0003] There are many types of devices that are used in surgical procedures. The devices enable a physician to perform the multitude of tasks required to successfully complete the procedure. Oftentimes, the procedure that the physician needs to perform requires the use of items, implements or other tools that require a certain amount of rigidity in the tool in order for the tool to effective in its particular use in the procedure. As such, many of these items or tools are formed of a generally rigid material, such as a metal, that provides the desired amount of rigidity. [0004] However, with these tools formed at least partially of metal, the nature of the metal creates problems with regard to the re-use of the tool. The reason for this is that the metal, as well as any coating applied to the exterior of the metal, such as an anodized coating which is necessary for implements that are formed of titanium, must be sterilized after each use. With certain metals and coatings, the sterilization process can be problematic, as the metals and/or coating can become brittle or otherwise damaged upon sterilization after an initial use. Any damage done to the metal and/or coating can cause issues with the stability or integrity of the implement during subsequent uses which consequently can endanger the patient. [0005] Thus, it is desirable to develop implements that are formed of metal and a material that enables the implement/tool incorporating the metal to be sterilized and reused in multiple procedures without detrimentally affecting the tool and/or the metal component(s) of the tool. SUMMARY OF THE INVENTION [0006] Briefly described, one aspect of the present disclosure provides an implement or tool formed of a substantially rigid, but optionally somewhat flexible core material that is enclosed within an inert material. The inert material provide a protective barrier around the core material and is capable of being sterilized after use without degrading the protective properties of the inert material to enable the implement to be reused. The inert material is molded over the core material to conform to the shape of the actual implement to provide the appropriate size and shape for the implement or tool when used by a physician in the procedure. Once used, the implement can be removed and subsequently sterilized, such as in an autoclave, for additional uses. [0007] According to another aspect of the present disclosure, the inert material is flexible and stretchable to accommodate any required flexibility of the core material while maintaining the core enclosed within the inert material. Thus, the implement can be bent in order to accurately conform to the proper location and configuration of for the implement when positioned within the body of the patient during the procedure and the inert material will maintain its conformance with the shape of the core. [0008] Numerous other aspects, features, and advantages of the present invention will be made apparent from the following detailed description together with the drawings figures. BRIEF DESCRIPTION OF THE DRAWINGS [0009] The drawings illustrate the best mode currently contemplated of practicing the present invention. [0010] In the drawings: [0011] FIG. 1 is an isometric view of one embodiment of an implement core constructed according to the present disclosure; [0012] FIG. 2 is a side elevation view of the core of FIG. 1 ; [0013] FIG. 3 is a cross-sectional view of the core of FIG. 2 ; [0014] FIG. 4 is an isometric view of the core of FIG. 1 after a first molding step; [0015] FIG. 5 is a side elevation view of the core of FIG. 4 ; [0016] FIG. 6 is a cross-sectional view of the core of FIG. 5 ; [0017] FIG. 7 is an isometric view of the core/implement of FIG. 1 after a second molding step; [0018] FIG. 8 is a side elevation view of the core/implement of FIG. 7 ; [0019] FIG. 9 is a cross-sectional view of the core/implement of FIG. 8 ; [0020] FIG. 10 is an isometric view of a second embodiment of an implement core constructed according to the present disclosure; [0021] FIG. 11 is a side elevation view of the implement core of FIG. 10 ; [0022] FIG. 12 is an isometric view of the implement core of FIG. 10 after a first molding step; [0023] FIG. 13 is a side elevation view of the implement core of FIG. 12 ; [0024] FIG. 14 is an isometric view of the implement core of FIG. 10 after a second molding step; [0025] FIG. 15 is a side elevation view of the implement core of FIG. 14. DETAILED DESCRIPTION OF THE INVENTION [0026] Referring now in detail to the drawing figures, wherein like reference numerals represent like parts throughout the several views, one exemplary embodiment of an implement constructed according to the present disclosure is illustrated generally at 100 in FIG. 7. As best shown in FIGS. 1-9, the illustrated exemplary embodiment of the implement 100 is formed as a rod template having a central core 12 and an enclosure 14 disposed around the core 12 formed of a first component or portion(s) 16 and a second component or portion(s) 18. In the illustrated exemplary embodiment, the rod template 100 is formed to be generally straight, though other curved, looped or other configurations for the rod template 100 are also contemplated as being within the scope of the disclosure of the present invention. [0027] Though any suitable shape for the core 12 can be utilized, in the illustrated embodiment the core 12 is formed with a generally flat rectangular or cylindrical cross-sectional shape with a first end 20 and a second end 22 joined by opposed sides 24, though any suitable cross-sectional shape can be utilized to impart the desired amount of flexibility to the core 12. The core 12 is shaped in any suitable machine and/or process to provide the desired shape for the core 12, which may include apertures or other features therein, as desired. [0028] The material forming the core 12 is selected to be a generally rigid, but flexible material that can be altered in shape by applying a physical force to the core 12. Once the force is removed, the core 12 remains in the shape to which it was altered by the applied force. In one exemplary embodiment of the core 12, the core 12 is formed of a shape memory material, such as a shape memory metal alloy, including the materials marketed under the trade name Nitinol® by Nitinol Devices & Components, Inc. of Fremont, Calif. [0029] The enclosure 14 is disposed around the core 12 and each portion 16 and 18 joined together to form the enclosure 14 is formed of a biologically inert and flexible material that can conform to the shape of the core 12 in any configuration for the core 12. in one embodiment, the material forming the portions 16 and 18 of the enclosure 14 is a silicone, such as a silicone rubber, including a high consistence rubber (HCR). [0030] The portions 16 and 18 of the enclosure 14 are formed with any features (not shown) desired to enhance the utility of the implement 100 when utilized within the body of the patient. The features can include apertures 110, notches (not shown), raised or depressed tactile portions, or printed indicia, among others. The apertures can extend completely through the respective portions 16 and 18 without intersecting the core 12, thereby preserving the integrity of the enclosure 14 around the core 12. Further, the shape of the portions 16 and 18 forming the enclosure 14 can be shaped as desired. Also, the shape of the portions 16 and 18 can be selected independently of the shape of the core 12 to facilitate the operation or use of the implement 100, or to conform to the shape of the core 12, as desired. [0031] In one embodiment, the implement 100 is formed by initially forming the core 12 of the desired material in any suitable manner, such as by extruding or molding the material into the desired shape for the core 12, as shown in FIGS. 1-3. The core 12 is then placed within a suitable mold (not shown) to enable the material selected to form the first portion 16 to be introduced into the mold containing the core 12 and form a portion of the enclosure 14 on or over the core 12 that contains the desired features within the portion 16. Any suitable molding process can be utilized to form the first portion 16 around the core 12, such as those shown in commonly owned U.S. Pat. No. 8,641,955 and its related applications, each of which are expressly incorporated by reference herein in their entirety. In the illustrated embodiment best shown in FIGS. 4-6, the first portion 16 constitutes a number of spaced sections 102 disposed along the length of the core 12. [0032] Subsequently, the core 12 and the first portion 16 that has been molded onto or over the core 12 are removed or transferred from the first mold and placed within a separate or second mold (not shown) used to form the other of the second portion 18 on or over the core 12 in connection with the first portion 16 and with the desired features. The material selected to form the second portion 18 can be selected to be the same or different in one or more respects or attributes than the material used to form the first portion 16, in order to provide the desired attributes to the enclosure 14 and the implement 100, so long as the materials forming the first portion 16 and second portion 18 are capable of mating, co-mingling or otherwise joining to one another in the molding process used to form the enclosure 14 around the core 12, which can be the same or different that the process used to form the first section 16. Additionally, suitable materials can be applied to one or both of the portions 16 and/or 18 to properly affix the portions 16 and 18 to one another, either during molding of the portions 16 and 18 to one another, or when affixing pre-molded portions 16 and 18 to one another around the core 12. [0033] In alternative exemplary embodiments, the portions 16 and 18 can be formed subsequently or simultaneously within a single mold in any suitable molding process. In the illustrated embodiment, the second portion 18 includes a number of spaced sections 104 disposed along the length of the core 12 and joining the sections 102 to form the enclosure 14. In this embodiment, as shown in FIGS. 7-9, the sections 102 and 104 form a seamless enclosure 14 around the core 12 complete with end caps 106 disposed over each end 20, 22 of the core 12. The seamless enclosure 14 moves, stretches and/or flexes with the core 12 to retain the core 12 encased within the enclosure 14, such that the sterilization of the implement 100 does not contact the core 12. [0034] In a second embodiment of the implement 200 shown in FIGS. 10-15 illustrates the implement 200 as a flex driver. The implement 200 includes a suitably shaped core 212 with a pair of opposed ends 220 and 222. The ends 220 and 222 define a central section 224 therebetween, as best shown in FIGS. 10-13. In the embodiment shown in FIGS. 12 and 13, the first portion 216, which can be formed similarly to the first portion 16 in the prior embodiment, is molded onto the core 212 in a first mold (not shown) in a first molding step over at least approximately one half of the central section 224 in a suitable process, such as those cited as examples for the molding of the first portion 16 in the prior embodiment. In this process, however, the ends 220 and 222 can function as stops for the flow of the material forming the first portion 216 at each end 220 and 222. [0035] Subsequently, the core 212 can be removed from the first mold for positioning in a second mold (not shown), or simply rotated within the first mold to expose the uncovered portion 226 of the central section 224 within the second mold. Once properly positioned, the second portion 218 can be formed over the uncovered section 226 to form the enclosure 214 over the central section 224 with the first portion 216 and without end caps, leaving the ends 220, 222 exposed. [0036] In alternative exemplary embodiments for either embodiment of the implement 100, 200, the process for molding the first portion 16, 216 and/or second portion 18, 218 can be performed in any number of separate molding steps in order to form the enclosure 14, 214 on the core 12, 212 with the desired appearance, attributes or other characteristics with any desired number and/or types of different materials forming the portions 16, 216 and/or 18, 218. [0037] Various other embodiments of the present disclosure are contemplated as being within the scope of the filed claims particularly pointing out and distinctly claiming the subject matter regarded as the invention. | Summary: A reusable surgical implement is provided that is formed of a core positioned within an enclosure. The core is formed of a suitable rigid, and optionally flexible material to enable the implant to conform to the desired use for the implement in a surgical procedure. The material forming the enclosure is also stretchable and flexible to accommodate the configuration and/or any flexing of the core, and is biologically inert to enable the implant to be sterilized after use for use in subsequent surgical procedures while protecting the material forming the core. The enclosure can be molded around the core in separate portions or components using multiple molding steps to form an enclosure with the desired attributes. | 3,457 | 140 | big_patent | en |
Summarize: Background Increasing computer interconnectivity—most notably growth in the use of the Internet—has revolutionized the way that our government, our nation, and much of the world communicate and conduct business. While this interconnectivity offers us huge benefits, without proper safeguards it also poses significant risks to the government’s computer systems and, more importantly, to the critical operations and infrastructures they support. We reported in 2005 that while federal agencies showed improvement in addressing information security, they also continued to have significant control weaknesses in federal computer systems that put federal operations and assets at risk of inadvertent or deliberate misuse, financial information at risk of unauthorized modification or destruction, sensitive information at risk of inappropriate disclosure, and critical operations at the risk of disruption. The significance of these weaknesses led us to conclude in the audit of the federal government’s fiscal year 2005 financial statements that information security was a material weakness. Our audits also identified instances of similar types of weaknesses in non-financial systems. FISMA Authorized and Streng Enacted into law on December 17, 2002, as title III of the E- Government Act of 2002, FISMA authorized and strengthened information security program, evaluation, and reporting requirements. The Act assigns specific responsibilities to agency heads, chief information officers, and IGs. It also assigns responsibilities to OMB, which include developing and overseeing the implementation of policies, principles, standards, and guidelin on information security and reviewing at least annually, and approving or disapproving, agency information security programs. Overall, FISMA requires each agency (including agencies with national security systems) to develop, document, and implement an agencywide information security program. This program should provide security for the information and information systems that support the operations and assets of the agency, including those provided or managed by another agency, contractor, or other source. Specifically, this program is to include ● periodic assessments of the risk and magnitude of harm tha se, disclosure, could result from the unauthorized access, u disruption, modification, or destruction of information or information systems; risk-based policies and procedures that cost-effectively red information security risks to an acceptable level and ensure that information security is addressed throughout the life cycle of each information system, including minimally acceptable system configuration requirements; uce subordinate plans for providing adequate information security for networks, facilities, and systems or groups of information systems; security awareness training for agency personnel, including contractors and other users of information systems that support the operations and assets of the agency; periodic evaluation of the effectiveness of information security policies, procedures, and practices, performed with a frequency depending on risk, but no less than annually, and that includes testing of management, operational, and technical controls for every system identified in the agency’s required inventory of major information systems; a process for planning, implementing, evaluating, and documenting remedial action information security policies, procedures, and practice agency; to address any deficiencies in the procedures for detecting, reporting, and responding to security incidents; and ● plans and procedures to ensure continuity of operations for information systems that support the operations and assets of th agency. FISMA also established a requirement that each agency develop, maintain, and annually update an inventory of major information systems (including major national security systems) that are operated by the agency or under its control. This inventory is to include an identification of the interfaces between each system an all other systems or networks, including those not operated by o r under the control of the agency. Each agency is also required to have an annual independent evaluation of its information security program and practices, including control testing and compliance assessment. Evaluations of non-national security systems are to be performed by the agency IG or by an independent external auditor, while evaluations related to national security systems are to be performed only by an entity designated by the agency head. The agencies are to report annually to OMB, selected congressional committees, and the Comptroller General on the adequacy of information security policies, procedures, practices, and compliance with FISMA requirements. In addition, agency heads are required to make annual reports of the results of their independent evaluations to OMB. OMB must submit a report to Congress no later than March 1 of each year on agency compliance, including a summary of the findings of agencies’ independent evaluations. Other major provisions direct that the National Institute of Standards and Technology (NIST) develop, for systems other than national security systems: (1) standards to be used by all agencies to categorize all their information and information systems based on the objectives of providing appropriate levels of information security according to a range of risk levels; (2) guidelines recommending the types of information and information systems to be included in each category; and (3) minimum information security requirements for information and information systems in each category. NIST must also develop a definition of and guidelines concerning detection and handling of information security incidents and guidelines. OMB Reporting Instructions a OMB provides instructions to the agencies and their IGs on the annual FISMA reporting requirements. OMB’s fiscal year 2005 reporting instructions, similar to the 2004 instructions, ha focus on performance measures. OMB has developed performan measures in the following areas: testing of security controls, ● agency systems and contractor systems reviewed annually, ● testing of contingency plans, incident reporting, ● annual security awareness training for employees and contractors, ● annual specialized training for ● minimally acceptable configuration requirements. Further, OMB has provided instructions for continued agency reporting on the statu action and milestones. Required for all programs an a weaknesses and show estimated resource needs or other challe to resolving them, key milestones and completion dates, and th status of corrective actions. The plans are to be submitted twice a year to OMB. In addition, agencies are to submit quarterly up dates that indicate the number of weaknesses for which corrective action has been completed as originally scheduled, or has been delayed, as well as the number of new weaknesses discovered since the last update. s of remediation efforts through plans of n IT security weakness has been found, these plans list the The annual IGs’ reports requested by OMB are to be based on the results of their independent evaluations, including work performed throughout the reporting period (such as work performed as part of the annual financial audits of the agencies). While OMB asked the IGs to respond to some of the same questions as the agencies, it al asked them to assess whether their agency had developed, implemented, and was managing an agencywide plan of actions and milestones. Further, OMB asked the IGs to assess the quality of the certification and accreditation process at their agencies, as well as the status of their agency’s inventory of major information systems. OMB did not request that the IGs validate agency responses to the performance measures. Instead, as part of their independent evaluations of a subset of agency systems, IGs were asked to assess the reliability of the data for those systems that they evaluated. OMB’s Report to Congress Noted Improvements and Weaknesses In its March 2006 report to Congress on fiscal year 2005 FISMA implementation, OMB emphasized that the federal government has made progress in meeting key performance measures for IT security; however, uneven implementation of security efforts leaves weaknesses in several areas. OMB determined through its assessment of FISMA reports that advances have occurred at a governmentwide level in the following areas of IT security: ● Systems certification and accreditation. Agencies recorded a 19 percent increase in the total number of IT systems and reported that the percentage of certified and accredited systems rose from 77 percent in fiscal year 2004 to 85 percent in 2005. Moreover, OMB noted that 88 percent of systems assessed as high-risk have been certified and accredited. ● Assessed quality of the certification and accreditation process. OMB’s analysis of reports from the IGs revealed an increase in agencies with a certification process rated as “satisfactory” or higher, from 15 in 2004 to 17 in 2005. ● Plans of action and milestone process. OMB noted that out of 25 agencies that it reviewed in detail, 19 IGs report that their agencies have effective remediation processes, compared to 18 in 2004. In addition to these areas of improvement, OMB detected areas with continuing weaknesses: ● Contractor systems oversight. IGs for 6 of 24 agencies (one agency IG did not respond) rated agency oversight of contractor systems in the “rarely” range, while 3 others rated this oversight in the next lowest range, “sometimes.” ● Security controls testing. Agencies tested the security controls on a lower percentage of systems, dropping from 76 percent in fiscal year 2004 to 72 percent in 2005. OMB noted a better rate of testing for high-risk systems, with a governmentwide total of 83 percent. ● Incident reporting. OMB stated that some agencies continue to report security incidents to the Department of Homeland Security only sporadically and that others report notably low levels of incidents. ● Agencywide plans of action and milestones. While IGs for 19 agencies reported effective POA&M processes, 6 others reported ineffective processes. ● Certification and accreditation process. OMB commented that while no IG rated the certification and accreditation process for its agency as failing, eight rated the process as “poor.” The OMB report also discusses a plan of action to improve performance, assist agencies in their information security activities, and promote compliance with statutory and policy requirements. OMB has set a goal for agencies to have 90 percent of their systems certified and accredited and their certification and accreditation process rated as “satisfactory” or better by their IGs. Agency 2005 FISMA Reports Show Mixed Results In their FISMA-mandated reports for fiscal year 2005, the 24 major agencies reported both improvements and weaknesses in major performance indicators. The following key measures showed increased performance and/or continuing challenges: ● percentage of systems certified and accredited; ● percentage of agencies with an agencywide minimally acceptable ● percentage of agency systems reviewed annually; ● percentage of contractor systems reviewed annually; ● percentage of employees and contractors receiving annual security percentage of employees with significant security respo receiving specialized security training annually; and nsibilities ● percentage of contingency plans tested. Figure 1 illustrates that the major agencies have made steady progress in fiscal year 2005 certifying and accrediting their systems, although they have made mixed progress in meeting other key performance measures compared with the previous two fiscal years. Summaries of the results for specific measures follow. Certification and Accreditati Included in OMB’s policy for federal information security is a requirement that agency management officials formally authorize their information systems to process information and, thereby accept the risk associated with their operation. This management authorization (accreditation) is to be supported by a formal technical evaluation (certification) of the management, operationa and technical controls established in an information system’s security plan. For FISMA reporting, OMB requires agencies to repo the number of systems authorized for processing after completing certification and accreditation. Configuration Management FISMA requires each agency to have policies and procedures that ensure compliance with minimally acceptable system configuration requirements, as determined by the agency. In fiscal year 2004, for the first time, agencies reported on the degree to which they had security configurations for specific operating systems and software applications. Our analysis of the 2005 agency FISMA reports foun d that all 24 major agencies reported that they had agencywide policies containing system configurations, an increase from the 20 agencies who reported having them in 2004. However, implementation of these requirements at the system level continues to be uneven. Specifically, 14 agencies reported having configuration policies, but they did not always implement them on their systems. Annual Review of Agency Sys FISMA periodi security policies, procedures, and practices to be performed with a frequency that depends on risk, but no less than annually. This effo is to include testing of management, operational, and technical controls of every information system identified in the FISMA- required inventory of major systems. Periodically evaluating the effectiveness of security policies and controls and acting to address any identified weaknesses are fundamental activities that allow an organization to manage its information security risks cost- effectively, rather than reacting to individual problems ad hoc only after a violation has been detected or an audit finding has been reported. In order to measure the performance of security programs OMB requires that agencies report the number and percentage of systems that they have reviewed during the year. requires that agency information security programs include c testing and evaluation of the effectiveness of information Agencies reported a decrease in the percentage of underwent an annual review in 2005, after reporting major gains in this performance measure in 2004. In the 2005 reports, agencies stated that 84 percent of their systems had been reviewed in the last their systems that year, as compared to 96 percent in 2004. While 23 agencies repor that they had reviewed 90 percent or more of their systems in 2004, 19 agencies reported this achievement in 2005, as shown in figure 3. Annual Review of Contractor Systems Under FISM information security p maintained by or on behalf of the agency and information s used or operated by an agency or by a contractor. As OMB emphasized in its fiscal year 2005 FISMA reporting guidance, agenc or use IT security programs apply to all organizations that possess federal information or that operate, use, or have access to federal information systems on behalf of a federal agency. Such other organizations may include contractors, grantees, state and local governments, and industry partners. According to longstanding OMB policy concerning sharing government information and interconnecting systems, federal security requirements continue apply, and the agency is responsible for ensuring appropriate security controls. The key performance measure of annual review of contractor systems by agencies decreased from 83 percent in 2004 to 74 percent in 2005, reducing the rate of reviews performed to below 2003 levels. However, the number of agencies that reported reviewing over 90 percent of their contractor systems has incr from 10 in 2004 to 17 in 2005. A breakdown of the percentages for fiscal year 2005 is provided in figure 4. Although agencies reported that 74 percent of their contractor systems were reviewed in 2005, they only reviewed 51 percent of the contractor systems assessed as high-risk, as opposed to 89 percent of moderate-risk systems and 84 percent of low-risk systems. Without adequate contractor review, agencies cannot be assured that federal information held and processed by contractors is secure. Security Awareness Training FISMA requires agencies to provide security awareness training. This training should inform personnel, including contractors and other users of information systems supporting the operations and assets of an agency, of information security risks associated with their activities and of the agency’s responsibilities in complying w policies and procedures designed to reduce these risks. Our studie of best practices at leading organizations have shown that such organizations took steps to ensure that personnel involved in various aspects of information security programs had the skills and knowledge they needed. Specialized Security Training Under FISMA, agencies are required to provide training in information security to personnel with significant security responsibilities. As previously noted, our study of best practices at leading organizations has shown that such organizations recognized that staff expertise needed to be updated frequently to keep security employees current on changes in threats, vulnerabilities, software, technologies, security techniques, and security monitoring tools. OMB directs agencies to report on the percentage of their employees with significant security responsibilities who have received specialized training. Agencies reported varying levels of compliance in providing specialized training to employees with significant security responsibilities. Of the 24 agencies that we reviewed, 12 reported that they had provided specialized security training for 90 percent or more of these employees. (see fig. 6). Although there was a gain of one point in the percentage of employees who received specialized security training for fiscal year 2005 (82 percent) over 2004 (81 percent), both of these years show a decrease from the level reported in 2003 (85 percent). Given the rapidly changing threats in information security, agencies need to keep their IT security employees up to date on changes in technology. Otherwise, agencies may face increased risk of security breaches. Testing of Contingency Plans Contingency plans provide specific instructions for restoring critical systems, including such elements as arrangements for alternative processing facilities in case the usual facilities are significantly damaged or cannot be accessed due to unexpected events such as a temporary power failure, the accidental loss of files, or a major disaster. It is important that these plans be clearly documented, communicated to potentially affected staff, and updated to reflect current operations. The testing of contingency plans is essential to determining whether the plans will function as intended in an emergency, and the frequency of plan testing will vary depending on the criticality of the entity’s operations. The most useful tests involve simulating a disaster to test overall service continuity. Such a test includes testing whether the alternative data processing site will function as intended and whether critical computer data and programs to be recovered from off-site storage will be accessible and current. In executing the plan, managers are able to identify weaknesses and make changes accordingly. Moreover, such tests assess how well employees have been trained to carry out their roles and responsibilities during a disaster. To show the status of implementing this requirement, OMB specifies that agencies report the number of systems with tested contingency plans. Overall, agencies continued to report that they have not tested a significant number of their contingency plans with only 61 percent of systems with tested plans. Although this number continues to show small increases each year since 2003, figure 7 illustrates that 5 agencies reported less than 50 percent of their systems had tested contingency plans. In addition, agencies do not appear to be appropriately prioritizing testing of contingency plans by system risk level, with high-risk systems having the lowest rate of systems with tested plans of the three risk levels. Without testing, agencies can have limited assurance that they will be able to recover mission critical applications, business processes, and information in the event of an unexpected interruption. Inventory of Major Systems FISMA requires that agencies develop, maintain, and annually update an inventory of major information systems operated by the agency, or under its control. The total number of agency systems is a key element in OMB’s performance measures, in that agency progress is indicated by the percentage of total systems that meet specific information security requirements. For the 2005 reports, OMB required agencies to report the number of major systems and asked the IGs about the status and accuracy of their agencies’ inventories. In 2005, agencies reported 10,261 systems, composed of 9,175 agency systems and 1,094 contractor systems. However, only 13 IGs reported that their agencies’ inventories were substantially complete. A complete inventory of major information systems is a key element of managing the agency’s IT resources, including the security of those resources. Without reliable information on agencies’ inventories, the agencies, the administration, and Congress cannot be fully assured of agencies’ progress in implementing FISMA. Risk Assessments FISMA mandates that agencies assess the risk and magnitude of harm that could result from the unauthorized access, use, disclosure disruption, modification, or destruction of their information and information systems. The Federal Information Processing Standard (FIPS) 199, Standards for Security Categorization of Federal Information and Information Systems, and related NIST guidance provide a common framework for categorizing systems according to risk. The framework establishes three levels of potential impact on organizational operation, assets, or individuals should a breach of security occur—high (severe or catastrophic), moderate (serious), and low (limited)—and is used to determine the impact for each of the FISMA-specified security objectives of confidentiality, integrity, and availability. Once determined, security categories are to be used in conjunction with vulnerability and threat information in assessing the risk to an organization. OMB’s fiscal year 2005 reporting instructions included the new requirement that agencies report their systems and certain performance measures using FIPS 199 risk levels. If agencies did not categorize systems, or used a method other than FIPS 199 to determine risk level, they were required to explain why in their FISMA reports. For the first time, in the 2005 reporting, agencies reported the risk levels for their agency and contractor systems, as illustrated in table 1. Agencies reported that 9 percent of their systems were not categorized by risk level. The majority of systems without risk levels assigned were found at 4 agencies. One agency did not categorize 77 percent of its systems. Without assigned risk levels, agencies cannot make risk-based decisions on the security needs of their information and information systems. Actions are Needed to Improve FISMA Reporting and Underlying Information Security Weaknesses There are actions that OMB and the agencies can take to improve FISMA reporting and compliance and to address underlying weaknesses in information security controls. In our July 2005 report, we evaluated the adequacy and effectiveness of agencies’ information security policies and practices and the federal government’s implementation of FISMA requirements. We recommended that the Director of OMB take actions in revising future FISMA reporting instructions to increase the usefulness of the agencies’ annual reports to oversight bodies by: ● requiring agencies to report FISMA data by risk category; ● reviewing guidance to ensure the clarity of instructions; ● requesting the IGs report on the quality of additional agency processes, such as the annual system reviews. These recommendations were designed to strengthen reporting under FISMA by encouraging more complete information on the implementation of agencies’ information security programs. Consistent with our recommendation, OMB required agencies to report certain performance measures by system risk level for the first time in fiscal year 2005. As a result, we were able to identify potential areas of concern in the agencies’ implementation of FISMA. For example, agencies do not appear to be prioritizing certain information security control activities, such as annual review of contractor systems or testing of contingency plans, based on system risk levels. For both of these activities, federal implementation of the control is lower for high-risk systems than it is for moderate or low-risk systems. OMB has also taken steps to increase the clarity of instructions in their annual guidance. It has removed several questions from prior years that could have been subject to differing interpretations by the IGs and the agencies. Those questions related to agency inventories and to plans of actions and milestones. In addition, OMB clarified reporting instructions for minimally acceptable configuration requirements. The resulting reports are more consistent and, therefore, easier to analyze and compare. However, opportunities still exist to enhance reporting on the quality of the agencies’ information security-related processes. The qualitative assessments of the certification and accreditation process and the plans of actions and milestones have greatly enhanced Congress’, OMB’s, and our understanding of the implementation of these requirements at the agencies. Additional information on the quality of agencies’ processes for annually reviewing or testing systems, for example, could improve understanding of these processes by examining whether federal guidance is applied correctly, or whether weaknesses discovered during the review or test are tracked for remediation. Extending qualitative assessments to additional agency processes could improve the information available on agency implementation of information security requirements. Federal Agencies Need to Take Actions to Increase FISMA Compliance and Address Already Identified Information Security Weaknesses Agencies need to take action to implement the information security management program mandated by FISMA and use that program to address their outstanding information security weaknesses. An agencywide security program provides a framework and continuing cycle of activities for managing risk, developing security policies, assigning responsibilities, and monitoring the adequacy of the entity’s computer-related controls. Without a well-designed program, security controls may be inadequate; responsibilities may be unclear, misunderstood, or improperly implemented; and controls may be inconsistently applied. Such conditions may lead to insufficient protection of sensitive or critical resources and disproportionately high expenditures for controls over low-risk resources. As we have previously reported, none of the 24 major agencies has fully implemented agencywide information security programs as required by FISMA. Agencies often did not adequately assess risks, develop sufficient risk-based policies or procedures for information security, ensure that existing policies and procedures were implemented effectively, or monitor operations to ensure compliance and determine the effectiveness of existing controls. Moreover, as demonstrated by the 2005 FISMA reports, many agencies still do not have complete and accurate inventories of their major systems. Until agencies effectively and fully implement agencywide information security programs, federal data and systems will not be adequately safeguarded against unauthorized use, disclosure, and modification. Agencies need to take action to implement and strengthen their information security management programs. Such actions should include completing and maintaining an accurate, complete inventory of major systems, and prioritizing information security efforts based on system risk levels. Strong incident procedures are necessary to detect, report, and respond to security incidents effectively. Agencies also should implement strong remediation processes that include processes for planning, implementing, evaluating, and documenting remedial actions to address any identified information security weaknesses. Finally, agencies need to implement risk-based policies and procedures that efficiently and effectively reduce information security risks to an acceptable level. Even as federal agencies are working to implement information security management programs, they continue to have significant control weaknesses in their computer systems that threaten the integrity, reliability, and availability of federal information and systems. In addition, these weaknesses place financial information at risk of unauthorized modification or destruction, sensitive information at risk of inappropriate disclosure, and critical operations at risk of disruption. The weaknesses appear in both access controls and other information security controls defined in our audit methodology for performing information security evaluations and audits. These areas are (1) access controls, which ensure that only authorized individuals can read, alter, or delete data; (2) software change controls, which provide assurance that only authorized software programs are implemented; (3) segregation of duties, which reduces the risk that one individual can independently perform inappropriate actions without detection; (4) continuity of operations planning, which provides for the prevention of significant disruptions of computer-dependent operations, and (5) an agencywide security program, which provides the framework for ensuring that risks are understood and that effective controls are selected and properly implemented. In the 24 major agencies’ fiscal year 2005 reporting regarding their financial systems, 6 reported information security as a material weakness and 14 reported it as a reportable condition. Our audits also identified similar weaknesses in nonfinancial systems. In our prior reports, we have made specific recommendations to the agencies to mitigate identified information security weaknesses. The IGs have also made specific recommendations as part of their information security review work. Agencies would benefit from addressing common weaknesses in access controls. As we have previously reported, the majority of the 24 major agencies had access control weaknesses. A basic management control objective for any organization is to protect data supporting its critical operations from unauthorized access, which could lead to improper modification, disclosure, or deletion of the data. Based on our previous work performing information security audits, agencies can take steps to enhance the four basic areas of access controls: ● User identification and authentication. To enable a computer system to identify and differentiate users so that activities on the system can be linked to specific individuals, agencies assign unique user accounts to specific users, a process called identification. Authentication is the method or methods by which a system establishes the validity of a user’s claimed identity. Agencies need to implement strong user identification and authentication controls. ● User access rights and file permissions. The concept of “least privileged” is a basic underlying principle for security computer systems and data. It means that users are only granted those access rights and file permissions that they need to do their work. Agencies would benefit from establishing the concept of least privilege as the basis for all user rights and permissions. ● Network services and devices. Sensitive programs and information are stored on networks, which are collections of interconnected computer systems and devices that allow users to share resources. Organizations secure their networks, in part, by installing and configuring networks devices that permit authorized requests and limit services that are available. Agencies need to put in place strong controls that ensure only authorized access to their networks. ● Audit and monitoring of security-related events. To establish individual accountability, monitor compliance with security policies, and investigate security violations, it is crucial that agencies implement system or security software that provides an audit trail that they can use to determine the source of a transaction, or to monitor the activities of users on the agencies’ systems. To detect and prevent unauthorized activity, agencies should have strong monitoring and auditing capabilities. In addition to electronic access controls, other important controls should be in place to ensure the security and reliability of an agency’s data. ● Software change controls. Counteracting identified weaknesses in software change controls would help agencies ensure that software was updated correctly and that changes to computer systems were properly approved. Software change controls ensure that only authorized and fully tested software is placed in operation. These controls -- which also limit and monitor access to powerful programs and sensitive files associated with computer operations -- are important in providing reasonable assurance that access controls are not compromised and that the system will not be impaired. These policies, procedures, and techniques help to ensure that all programs and program modifications are properly authorized, tested, and approved. Failure to implement these controls increases the risk that unauthorized programs or changes could be -- inadvertently or deliberately -- placed into operation. ● Segregation of duties. Agencies have opportunities to implement effective segregation of duties to address the weaknesses identified in this area. Segregation of duties refers to the policies, procedures, and organizational structure that help to ensure that one individual cannot independently control all key aspects of a process or computer-related operation and thereby conduct unauthorized actions or gain unauthorized access to assets or records. Proper segregation of duties is achieved by dividing responsibilities among two or more individuals or organizational groups. For example, agencies need to segregate duties to ensure that individuals cannot add fictitious users to a system, assign them elevated access privileges, and perform unauthorized activities without detection. Without adequate segregation of duties, there is an increased risk that erroneous or fraudulent transactions can be processed, improper program changes implemented, and computer resources damaged or destroyed. ● Continuity of operations. The majority of agencies could benefit from having adequate continuity of operations planning. An organization must take steps to ensure that it is adequately prepared to cope with the loss of operational capabilities due to earthquake, fire, accident, sabotage, or any other disruption. An essential element in preparing for such catastrophes is an up-to-date, detailed, and fully tested continuity of operations plan. To ensure that the plan is complete and fully understood by all key staff, it should be tested, including surprise tests, and test plans and results documented to provide a basis for improvement. Among the aspects of continuity planning that agencies need to address should be: (1) ensuring that plans contain adequate contact information for emergency communications; (2) documenting the location of all vital records for the agencies and methods of updating those records in an emergency; (3) conducting tests, training, or exercises frequently enough to have assurance that the plan would work in an emergency. Losing the capability to process, retrieve, and protect information that is maintained electronically can significantly affect an agency’s ability to accomplish its mission. ● Physical security. Physical security controls are important for protecting computer facilities and resources from espionage, sabotage, damage, and theft. These controls restrict physical access to computer resources, usually by limiting access to the buildings and rooms in which the resources are housed. With inadequate physical security, there is increased risk that unauthorized individuals could gain access to sensitive computing resources and data and inadvertently or deliberately misuse or destroy them. In summary, through the continued emphasis of information security by Congress, the administration, agency management, and the accountability community, the federal government has seen improvements in its information security. However, despite the advances shown by increases in key performance measures, progress remains mixed. If information security is to continue to improve, agency management must remain committed to the implementation of FISMA and the information security management program it mandates. Only through the development of strong IT security management can the agencies address the persistent, long- standing weaknesses they face in information security controls. Mr. Chairman, this concludes my statement. I would be happy to answer any questions that you or members of the Committee may have at this time. Should you have any questions about this testimony, please contact me at (202) 512-6244. I can also be reached by e-mail at wilshuseng@gao.gov. Individuals making key contributions to this testimony include Suzanne Lightman, Assistant Director, Larry Crosland, Joanne Fiorino, and Mary Marshall. This is a work of the U.S. government and is not subject to copyright protection in the United States. It may be reproduced and distributed in its entirety without further permission from GAO. However, because this work may contain copyrighted images or other material, permission from the copyright holder may be necessary if you wish to reproduce this material separately. | Summary: For many years, GAO has reported that ineffective information security is a widespread problem that has potentially devastating consequences. In its reports to Congress since 1997, GAO has identified information security as a governmentwide high-risk issue--most recently in January 2005. Concerned with accounts of attacks on commercial systems via the Internet and reports of significant weaknesses in federal computer systems that make them vulnerable to attack, Congress passed the Federal Information Security Management Act of 2002 (FISMA), which permanently authorized and strengthened the federal information security program, evaluation, and reporting requirements established for federal agencies. This testimony discusses the federal government's progress and challenges in implementing FISMA, as reported by the Office of Management and Budget (OMB), the agencies, and the Inspectors General (IGs), and actions needed to improve FISMA reporting and address underlying information security weaknesses. In its fiscal year 2005 report to Congress, OMB discusses progress in implementing key information security requirements, but at the same time cites challenging weaknesses that remain. The report notes several governmentwide findings, such as the varying effectiveness of agencies' security remediation processes and the inconsistent quality of agencies' certification and accreditation (the process of authorizing operation of a system, including the development and implementation of risk assessments and security controls). Nevertheless, fiscal year 2005 data reported by 24 major agencies, compared with data reported for the previous 2 fiscal years, show that these agencies have made steady progress in certifying and accrediting systems, although they reported mixed progress in meeting other key statutory information security requirements. For example, agencies reported that only 61 percent of their systems had tested contingency plans, thereby reducing assurance that agencies will be able to recover from the disruption of those systems with untested plans. Federal entities can act to improve the usefulness of the annual FISMA reporting process and to mitigate underlying information security weaknesses. OMB has taken several actions to improve FISMA reporting--such as requiring agencies to provide performance information based on the relative importance or risk of the systems--and can further enhance the reliability and quality of reported information. Agencies also can take actions to fully implement their FISMA-mandated programs and address the weaknesses in their information security controls. Such actions include completing and maintaining accurate inventories of major systems, prioritizing information security efforts based on system risk levels, and strengthening controls that are to prevent, limit, and detect access to the agencies' information and information systems. | 7,250 | 544 | gov_report | en |
Summarize: From a 'health and safety' tree decorated with hazard tape and a traffic cone fairy to one made entirely of rubbish bags, are these Britain's worst Christmas trees? The dodgy Christmas trees, from towns and villages up and down the country, have been ridiculed as being the worst in the country for their poor decoration and non-traditional, and occasionally bizarre design. One town's tree was so bad that it even had it's own Facebook page - The Embarrassment of Mottram Christmas Tree! - set up by locals in the Greater Manchester village in protest over its spindly, sparse branches and lack of decorations. Scroll down for videos. In Mier, Stoke-on-Trent, their town Christmas tree was literally rubbish: Plastic bags collected by local schoolchildren were stacked high to create the bizarre festive offering (pictured) In Bradford-on-Avon, Wiltshire, the choice of decorations was described 'as something you'd find after an explosion in a rag factory' (left) while in Streatham, London, locals were left with a conical piece of white plastic with lights topped with a star and decorated with one solitary piece of tinsel and a few baubles (right) Based on a lonely patch of grass at a crossroads, the tree nicknamed 'twiggy' was decorated with a solitary string of fairy lights until residents decided to decorate it themselves. But at least theirs was actually a tree. In Mier, Stoke-on-Trent, their town Christmas tree was literally rubbish. Plastic bags collected by local schoolchildren were stacked high to create the bizarre festive offering. In some areas, the trees were considered such a health and safety risk they were fenced off from the public. This health-and-safety-conscious tree in north Oxford was decorated with bows made from hazard warning tape and plastic and instead of an angel on top, sits a plastic traffic cone (pictured) This unusual Christmas tree in Dymchurch, Kent, was described as a 'great big weed growing out of a stick' Locals in Mottram, Greater Manchester, set up a Facebook page in protest against their town tree - The Embarrassment of Mottram Christmas Tree! - until they decide to decorate it themselves (right) while this conical tree (left) was dubbed one of the worst in the country when it appeared in Stockton-on-Tees High Street last year and has now been donated to a local charity. Christmas trees in Derby, in the Midlands, Stockton-on-Tees in Middlesborough and Liscard in Merseyside were all surrounded by metal fences, leading to Liscard residents branding their town's festive feature an 'absolute joke' and launching a campaign to jazz up their 'depressing' tree. But when it came to red tape, it was hard to beat the health-and-safety-conscious tree in north Oxford. Surrounded by plastic fencing, it was decorated with bows made from hazard warning tape and plastic. Instead of an angel on top, sits a plastic traffic cone. Other criticism came over trees being too bare and under decorated, non-traditional shapes and design and - in the case of one in Dymchurch, Kent, the fact it allegedly looked like 'a great big weed growing out of a stick'. In Bradford-on-Avon, Wiltshire, the choice of decorations was described 'as something you'd find after an explosion in a rag factory' while in Streatham, London, locals were left with a conical piece of white plastic with lights topped with a star and decorated with one solitary piece of tinsel and a few baubles. And a conical tree, dubbed one of the worst in the country when it was placed on Stockton-on-Tees High Street last year, was so hated it has not returned but has been donated to a local charity. People complained trees had been fenced off from the public by a health and safety perimeter such as this conical festive offering in Derby. Brighton and Hove City Council's administration commissioned two'recycled' trees, the first - made from old ring-binders (left) and the second - an old library shelf, filled with flotsam and jetsam found on the beach, including a rubber glove as a 'fairy' (right) While residents in Liscard, Merseyside, had their festive spirit dashed after seeing the underwhelming tree that has no lights and is surrounded by metal fencing on concrete blocks (pictured) Finally, in Haddenham, Cambridgeshire locals gathered round their bizarre'star' tree made of metal poles wrapped in tinsel | Summary: The trees across the country have been ridiculed for their poor decoration and non-traditional- even bizarre- design. They include a 'rubbish' Christmas tree made entirely from plastic bags collected by children in Mier, Stoke-on-Trent. And a health-and-safety-conscious tree in Oxford was decorated with hazard tape and topped with a traffic cone. One tree was so bad locals set up its own Facebook page - The Embarrassment of Mottram Christmas Tree. | 1,035 | 106 | cnn_dailymail | en |
Summarize: CROSS REFERENCE TO RELATED APPLICATIONS This application is a divisional application of U.S. patent application Ser. No. 13/247,180, entitled GRAFT INTRODUCER, filed Sep. 28, 2011, which is incorporated herein by reference. BACKGROUND This application relates to tissue manipulation instruments, and more particularly to instruments for implantation of graft tissue into a bone hole. In certain surgical procedures, such as tenodesis, a graft tissue is attached to a bone. For instance, in tenodesis a biceps tendon is detached from its attachment to the glenoid and is reattached to the humerus. In one popular method of reattachment a bone tunnel is created on the humerus and the detached tendon is pushed into the tunnel and then held in place via an interference bone screw implanted into the tunnel. Positioning the tendon in the tunnel can be tricky. In one method, the graft tissue is externalized from the patient and whip stitched to make a stiff construct at the termination of the tissue. The stiff construct may be pushed directly into tunnel using graspers or the like. A length of suture at the distal portion of the whip stitch may be used to pull the graft tissue into the tunnel. It is desirable in many cases, however, to perform the entire operation with the graft tissue internalized within the patient. Thus, producing the whip stitch is difficult for the surgeon. In another method, the graft tissue is folded near the location of tunnel and pushed into the tunnel at the fold. When using instrumentation of prior art, the graft tissue has a natural tendency to compress against the instrument. Should the frictional contact between the graft tissue and instrument be greater than the frictional contact between the graft tissue and tunnel, it is likely that the tissue will move from its desired position as the instrument is retracted from the tunnel. SUMMARY OF THE INVENTION The present invention overcomes these and other limitations of the prior art in a simple and elegant design. An instrument according to the present invention provides for implanting a graft into a bone hole. The instrument comprises an elongated handle having a forked distal termination having a first tine and a second tine defining a space therebetween. A flexible member spans the space between the first tine and the second tine so that the graft can be received within the space against the flexible member and thereby manipulated into the bone hole. Preferably, the first tine comprises an open ended first notch at its distal end, the flexible member being received within the first notch. Preferably, the flexible member having a first section between the first tine and second tine, and a second section extending from the first notch proximally along the handle where it preferably, is releasably attached to the handle so that a user can release the flexible member from the handle to relax tension in the flexible member. Preferably, the second tine comprises an open ended second notch with the flexible member being received in both the first notch and second notch and spanning the space therebetween. In one aspect of the invention, one or both of the first tine and second tine are flexible, with the space between the first tine and second tine being adjustable by tension on the flexible member. In one aspect of the invention, the handle is cannulated having a longitudinal cannulation opening to the space between the first tine and second tine. In one aspect of the invention, a guide wire is provided which is passable through the cannulation and which is adapted to be fixed into the bone hole whereby the instrument can be passed down to the bone hole over the guide wire. Preferably, the first tine and second tine are curved about a central longitudinal axis of the instrument to accommodate to the bone hole. Preferably, the instrument is provided sterile and packaged in a bacteria proof package. A method according to the present invention provides for implanting a graft into a bone hole. The method comprises the steps of: positioning the graft between a first tine and a second tine at a distal end of a shaft of a surgical instrument and against a flexible member spanning a space defined between the first tine and second tine; manipulating the first and second tines with the graft therebetween into a bone tunnel; pushing the graft via the flexible member into the bone tunnel; and releasing tension in the flexible member to allow its movement relative to at least one of the first tine and second tine and removing the first and second tines from the bone tunnel, leaving the graft positioned therein. Preferably, the graft is folded upon itself over the flexible member. In one aspect of the invention, the flexible member is received in an open distally facing notch on the first tine. In one aspect of the invention, the step of releasing tension on the flexible member includes allowing it to fall outwardly of an open distally facing notch on the first tine. In one aspect of the invention, at least one of the first tine and second tines are flexible a distance between them is controlled via tension on the flexible member. In an aspect of the invention, the first tine and second tines are directed toward the bone tunnel by passing a guide wire leading from the bone tunnel through a cannulation through the shaft. After the graft has been positioned in the bone tunnel an anchor can be passed into the tunnel to fix the graft therein, preferably by passing the anchor over the guide wire. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a side elevation view of a graft implantation tool according to the present invention; FIG. 2 is a side elevation view of a graft anchor for use with the tool of FIG. 1 ; FIG. 3 is a side elevation view of the tool of FIG. 1 shown adjacent a bone tunnel and a graft ready for implantation into the tunnel; FIG. 4 is a side elevation view of the tool of FIG. 1 shown initially capturing the graft and entering the tunnel; FIG. 5 is a side elevation view of the tool of FIG. 1 shown fully inserted into the tunnel; FIG. 6 is a side elevation view of the tool of FIG. 1 shown retracting from the tunnel leaving the graft in the tunnel; FIG. 7 is a side elevation view of the tool of FIG. 1 oriented to show the graft entering the tunnel from the rear of this view and an anchor being implanted into the tunnel; FIG. 8 is a side elevation view of the tunnel and graft of FIG. 7 oriented to show the graft entering the tunnel from the left side and illustrating a completed implantation of the graft; FIG. 9 is a side elevation view of an alternative embodiment of a graft implantation tool according to the present invention showing flexible tines in a relaxed state; FIG. 10 is a side elevation view of the tool of FIG. 9 showing the tines in a collapsed state; FIG. 11A is a side elevation view of a tine of FIG. 1 showing a suture capture notch; FIG. 11B is a side elevation view of a tine of an alternative embodiment of a graft implantation tool according to the present invention showing a suture capture hole; FIG. 11C is a side elevation view of a tine of an alternative embodiment of a graft implantation tool according to the present invention showing an elongated suture capture hole; and FIG. 11D is a side elevation view of a tine of an alternative embodiment of a graft implantation tool according to the present invention showing an alternative suture capture notch. FIG. 11E is a side elevation view of a tine of an alternative embodiment of a graft implantation tool according to the present invention; FIG. 11F is a side elevation view of a tine of an alternative embodiment of a graft implantation tool according to the present invention; FIG. 11G is a side elevation view of a tine of an alternative embodiment of a graft implantation tool according to the present invention; and FIG. 11H is a side elevation view of a tine of an alternative embodiment of a graft implantation tool according to the present invention. DETAILED DESCRIPTION FIG. 1 depicts a graft implantation tool 10 according to the present invention. It comprises an elongated cannulated shaft 12 with a forked distal end 14. The distal end 14 comprises a first tine 16 and second tine 18 defining a space 20 therebetween. Each of the tines 16 and 18 has a distal terminal end 22 with a distal terminal notch 24. A length of suture 26 or other flexible material with suitable tensile strength spans the space 20 between the notches 24. It has a first end 28 affixed to the shaft 12 where the second tine 18 meets the shaft 12. From there it extends down along an exterior surface 30 of the second tine 18 enters the second tine notch 24, spans the space 20, enters the first tine notch 24 and then extends up the shaft 14 where it is secured in a suture retainer 32, which is shown for illustrative purposes as a simple cleat but any suitable retention can be employed as will be appreciated by those of skill in the art. A cannulation 34 extends axially through the shaft 12 and opens into the space 20 between the tines 16 and 18. The cannulation 34 is wide enough to pass an interference anchor 36 (see FIG. 2 ). The tines 16 and 18 are curved on their exterior surfaces 30 and interior surfaces 38 to fit snugly into a bone tunnel (not shown in FIG. 1 ) and to pass the anchor 36. One or both of the tines 16 and 18 can be flexible with their spacing being controlled by tension in the suture 26 spanning the space 20. In such event their relaxed position is preferably slightly spread from parallel as they extend distally. This allows a more open presentation to allow easier loading of a graft (not shown in FIG. 1 ) into the space 20. Turning also now to FIG. 2, the anchor 36 comprises an elongated body 40 having exterior threads 42, a narrow distal tip 44, a proximal tool recess 46, such as for receipt of a hex driver, and an axial cannulation 47 for passage of a guide wire (not shown in FIGS. 1 and 2 ). Other configurations can be employed as will be appreciated by those of skill in the art. One suitable anchor is the MILAGRO interference screw available from DePuy Mitek, Inc. of Raynham, Mass. The tool 10 can be fabricated from any biocompatible materials or combinations thereof providing adequate strength for constructing the cannulated shaft 12 and having adequate elastic properties to provide the flexibility of one or both tines 16, 18 to accommodate variations in the distance across the space 20. Metallic materials that can be used to manufacture the instrument of the present invention include stainless steel, titanium, alloys of nickel and titanium, or other biocompatible metallic materials. It can also be formed of polyethylene, polypropylene, PEEK, or other biocompatible non-absorbable polymers. Turning also to FIGS. 3 to 6, use of the tool 10 will now be described. FIG. 3 shows a biceps tendon 48 which has been removed from its placement on the glenoid (not shown) and is placed adjacent to a bone tunnel 50 which has been prepared in a humerus bone 52. A guide wire 54 extends from the tunnel 50. Options for creation of the bone tunnel 50 and placement of the guide wire 54 will be apparent to those of skill in the art. The tool 10 has been passed down over the guide wire 54 and is positioned adjacent to the tunnel 50. The tendon 48 is positioned over the tunnel 50 with the suture 26 orthogonal to the tendon 48. As the tines 16 and 18 are pressed into the tunnel 50 ( FIG. 4 ) the tendon 48 is received between the tines 16 and 18 and caught upon the suture 26 causing the tendon 48 to fold upon itself. The tendon 48 is then pressed down into the bottom of the tunnel 50 as illustrated in FIG. 5. At this time the suture 26 is released from the suture retainer 32 releasing tension in the suture 26 and allowing removal of the tines 16 and 18 without the suture hanging up on the tendon 48 and affecting its implantation in the tunnel 50 as illustrated in FIG. 6. The anchor 36 can then be implanted, preferably over the guide wire 54 employing techniques as may be known or become known to those of skill in the art. For instance, FIG. 7 shows the anchor 36 being passed down a cannula 51 via a cannulated driver 53 and being threaded into the tunnel 50 to trap the tendon 48 therein. FIG. 8 illustrates the completed repair. FIGS. 9 and 10 illustrate an alternative embodiment of a graft implantation tool 56 according to the present invention. It has flexible first and second tines 58 and 60, respectively, and a suture 62 passing from the second tine 60 through a distal notch 64 therein across a space 66 between the tines 58 and 60, through a distal notch 64 in the first tine 58. Under slack tension in the suture 62 distal ends 68 of the tines 58 and 60 spread open allowing easy entry of a graft 66 into the space 66. Tension on the suture 62 causes the tines 58 and 60 to collapse inwardly toward each other grasping the graft 66. The narrowing of the tine spacing may also ease its entry into a bone tunnel. Although shown with tines 16 and 18 which are axially aligned with the shaft 12 they could be angled with respect to the shaft 12. Also the shaft could be curved. Various depths of the notches 24 into the tines 16 and 18 may be employed for positioning the graft tendon 48 at different axial positions along the tines 16 and 18. The tension on the suture 26 can also affect such placement with a bit of slack in it between the notches 24 allowing the suture 26 to bow proximally as the tendon 48 is engaged. Turning also now to FIGS. 11 A to H, the axial notch 24 as disclosed in FIG. 1 and FIG. 11A is preferred in the first tine 16 for easy release of the suture 26 from the tendon 48 after its implantation without the suture 26 catching on tendon 48 or the tine 16. Other designs may enhance temporary holding of the suture 26 so that it does not fall out of place. For instance a closed circular hole 70, elongated hole 72 or elongated notch 74 with a capture leg 76 or notch 80 and more aggressive capture leg 82 may be substituted especially in the second tine 18. A notch 84 having an expanded capture leg 86 and a restriction 88 leading into the capture leg 86 allows suture to slip in easily but not slip back out. A notch 89 with a restriction 90 provides some measure of capture but still allows the suture to be extracted from the notch 89 if desired. A notch 92 can be provided with a tortuous path such as in inward spiral 94. These designs limit suture 26 from falling out of the notch inadvertently yet still allow free sliding of the suture 26 therethrough so that it will not catch on the tendon 48 as the tines 16 and 18 are removed from the tunnel 50. The invention has been described with reference to the preferred embodiments. Obviously, modifications and alterations will occur to others upon reading and understanding the preceding detailed description. It is intended that the invention be construed as including all such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof. | Summary: An instrument for placing a graft into a bone tunnel comprises an elongated shaft having a forked distal end comprising a pair of tines. A suture spans a space defined between the tines whereby the graft may be positioned between the tines and against the suture so as to be manipulated into the bone tunnel. The tendon folds about the suture, the suture having a releasable tension such that the instrument can be removed from the bone, leaving the tendon behind without the tendon hanging up on the suture. | 3,800 | 118 | big_patent | en |
Summarize: By. Joshua Gardner and Rachel Quigley. PUBLISHED:. 17:53 EST, 26 October 2013. |. UPDATED:. 17:56 EST, 26 October 2013. One month after a kind-hearted police officer gave the starving mother and her family a chance, Jessica Robles' life has taken a dramatic turn. Not only does she have food for her children, but she has a steady job and invitations to appear on shows like Good Morning America and Inside Edition. The mother-of-three owes her recent good fortune to officer Vicki Thomas who, when called to deal with a shoplifter at a Miami Publix, decided to help instead of arresting her after Robles admitted she would probably steal again because her kids were hungry. Thomas sent her home with a misdemeanor citation and $100 worth of groceries. So grateful: Jessica Robles made the desperate decision to steal food for her hungry family. But getting caught has helped her turn over a new leaf. Kindness of strangers: Miami-Dade police officer Vicki Thomas chose to buy a penniless mom some of the groceries she was caught trying to steal instead of arresting her. 'I made the decision to buy. her some groceries because arresting her wasn't going to solve the. problem with her children being hungry,'she said. 'To see them go through the bags when we brought them in, it was like Christmas,' Thomas said. 'That $100 to me was worth it' Since. news of Thomas's spontaneous act of kindness spread, good will offers of money,. food, and even a job poured into the Robles household. Miami-Dade police have also been swamped with calls from TV. personalities like Ellen DeGeneres and Steve Harvey wanting the Vicki Thomas to. appear on their show. Speaking about the life-changing moment from her home this week, Robles, 30, told the Miami Herald: 'I knew when I left home that day that I had to come back with something to eat for my kids. I had to do it.' She told Officer Thomas on the day she tried to walk out of Publix with $300 worth of shopping in a cart: 'I wish I could tell you I will never do this again. But I can’t because my children are hungry, and I don’t know what I will do.' Robles’s boyfriend had lost his job and, because of a paperwork issue, the federal assistance had stopped. Officer Thomas thought of her own grandchildren and zipped around the grocery aisles at the store grabbing rice, pasta and food she knew her grandchildren loved. 'The public has been amazing and overwhelming,' the officer of 23 years said on Friday of the aftermath of her kind actions. She told Robles that all she wanted her to do was help someone else when she was back on her feet. 'And she said she would,' Officer Thomas said. [With] my brother in the dirt hungry, asking for food, and we have to tell him, "There is nothing here."'Robles' 12-year-old daughter Anais opened up about3 the family's dire situation. Generosity: Since news of her story has spread, Robles was given $700 more to buy groceries with from concerned community members and got to keep the leftover cash. John Challenor of phonedoctor.com invited Robles in to see her resume and hired her as a customer service rep on the spot. 'There's. no words,' Robles told Challenor through tears, 'how grateful I am that. you took your time and helped somebody out. Especially somebody like. me.' | Summary: Instead of hauling Jessica Robles off to jail, Miami-Dade County Officer Vicki Thomas decided to help her out. 'Arresting her wasn't going to solve the problem with her children being hungry,' said Thomas, who instead gave her a misdemeanor citation. Since then, the story has inspired residents and businesses of Miami to help Robles out. Ellen DeGeneres and Steve Harvey want Officer Thomas on their shows. | 858 | 97 | cnn_dailymail | en |
Summarize: Gov. Bob McDonnell, R-Va., has a resume that should put him in good company with other 2016 Republican presidential contenders: Swing-state governor, retired military officer, and national recognition in Republican circles from his time chairing the Republican Governors' Association. But he's now embroiled in a controversy that has the potential to derail any future ambitions for higher office. The Washington Post reported this week that a Virginia businessman gave $145,000 to McDonnell and members of his family in the form of loans, donations, and gifts between 2011 and 2012. It's the latest development in the scandal involving the McDonnells' relationship to Jonnie R. Williams, Sr., the head of the nutritional supplement company Star Scientific. The McDonnells have helped promote the company, including hosting an event at the Executive Mansion in Richmond to launch a new product. McDonnell: No "special treatment" to Williams or other donors In a few public statements on the matter, McDonnell has said he disclosed all donations as required by Virginia law and that Star Scientific did not receive special treatment. Aides to the governor point out that under Virginia law, elected officials do not need to disclose loans given to businesses or gifts to family members. "Star Scientific and Jonnie Williams have not received any board appointments, economic development grants, targeted tax incentives or government contracts during this administration," McDonnell press secretary Taylor Thornley Keeney said in a written statement. "The governor has been diligent over the years in making his financial disclosures." That hasn't stopped the Post editors from excoriating McDonnell, calling his personal life an example of "profligacy, irresponsibility and entitlement" in a blistering editorial. Two Democratic state legislators have called on McDonnell to resign. And the scandal is threatening to embroil the Republicans' gubernatorial nominee, Ken Cuccinelli, who also has a relationship with Williams. "What we've all been seeing is very painful for Virginia, and it's completely inconsistent with Virginia's very reserved traditions," Cuccinelli said in a statement that seemed to distance himself from McDonnell. "All of this emphasizes the need for clearer and faster disclosures that cover the whole family, as well as a cap on the size and types of gifts." During the 2012 campaign, McDonnell was mentioned as a possible vice presidential candidate or future cabinet secretary for Republican nominee Mitt Romney. Virginia governors are limited to one term, so McDonnell will leave office in January 2014 with two years to plot his political future. He does have accomplishments from his time in office, including a steadily falling unemployment rate that stood at 5.3 percent in May and a a large transportation overhaul enacted this spring. But the Star Scientific scandal could dog him. "It'll be a question or questions that he would have to answer down the road," says one prominent Republican strategist. "But depending on how it plays, it doesn't mean it's disqualifying in the future. He's been a great governor, he's a very attractive candidate, he's articulate. Depending on how this plays out, people could be in a very forgiving mood." How the controversy plays out will also depend on both the FBI and Virginia state prosecutors. Both are reportedly investigating whether any of Williams' payments to the McDonnells were illegal. "It's done," another Washington-based Republican said about McDonnell's 2016 changes. "That conversation doesn't even exist. The conversation now is if there is going to be a resignation." That's an ignominious position to be in for someone once seen as a rising star in the Republican Party. Here's what else the potential 2016 candidates have been up to this week, courtesy of CBS News' Jaclyn Peiser: Sen. Rand Paul, R-Ky.: The Washington Free Beacon reported on Tuesday that a co-author of Paul's book "The Tea Party Goes to Washington" and close aide to the senator had previously expressed admiration for the Confederacy. Paul hired Jack Hunter to assist on his book in 2010 and then later made Hunter his social media director. Hunter had a radio program, "The Southern Avenger," in which he openly discussed racial pride, pro-secession ideals, and praised John Wilkes Booth for assassinating President Abraham Lincoln. Paul responded in an interview with the Huffington Post on Thursday saying that he doesn't agree with all of Hunter's past statements but he still stood by his aide saying that Hunter "is incredibly talented." Gov. Chris Christie, R-N.J.: Democratic gubernatorial candidate Barbara Buono released an online attack ad on Tuesday accusing Christie of focusing too much on the 2016 campaign rather than on the issues facing New Jersey. The Democratic candidate even made a jab at Christie's trips to Iowa, saying, "There's no fried butter in Newark, just 13 percent unemployment." Gov. Scott Walker, R-Wis.: A Wisconsin federal judge this week blocked a law that increases restrictions on doctors performing abortions. Walker signed the bill last Friday and it took effect on Monday. The judge ruled that banning doctors who don't have admitting privileges to local hospitals from performing abortions is unconstitutional and violates due process. Gov. Rick Perry, R-Texas: Rick Perry announced on Monday that he will not seek a fourth term as governor of Texas. Perry held a private gathering for close friends and supporters in San Antonio to announce his political future. Although he did not officially declare that he will attempt another run for president in 2016, he said in a Fox News interview that it is "certainly" an option. Former Sen. Rick Santorum, R-Pa.: Santorum went to Austin on Thursday to show his support for the Texas abortion bill. The former senator held a press conference with Lt. Gov. David Dewhurst, R-Texas, state Sen. Ken Paxton, R-Texas, and other conservative activists to praise Texas lawmakers for "standing up for life." Former Secretary of State Hillary Clinton: Hillary Clinton received a library of her own in Little Rock on Monday. The former first lady of Arkansas celebrated the event by reading "The Very Hungry Caterpillar" in her newly dedicated Hillary Rodham Clinton Children's Library and Learning Center. That evening Clinton helped honor Oscar de la Renta at the William J. Clinton Presidential Center where one of her infamous pantsuits and many other de la Renta pieces are on display. Gov. Andrew Cuomo, D-N.Y.: At an event in Buffalo on Tuesday, Cuomo told the press that despite his continued efforts, he is not optimistic about receiving federal funds to help with the flooding in upstate New York. And when asked about his opinion on both disgraced congressman Anthony Weiner and disgraced Gov. Eliot Spitzer running for office in New York City, Cuomo responded, "I am just watching the theater that is going on in New York City in this political season." Biden: Fallen Arizona Hotshots heroes "long before we knew their names" Vice President Joe Biden: The vice president gave a eulogy on Tuesday at the funeral for the 19 firefighters who died in the Arizona wildfire last month. Biden noted that the Granite Mountain Hotshots were "an elite unit in every sense of that phrase" and "they saw their jobs not as jobs, but as a duty, a duty to their fellow citizens." Biden was joined by Sen. John McCain, R-Ariz., Sen. Jeff Flake, R-Ariz., and Gov. Jan Brewer, R-Ariz. Gov. Martin O'Malley, D-Md.: The Maryland governor attended a fundraiser on Wednesday for Virginia Democrat and gubernatorial hopeful Terry McAuliffe. O'Malley, accompanied by Lt. Gov. Anthony Brown, headlined the event in Annapolis at the home of former ambassador to Sweden Thomas L. Siebert. In 2010, the political world pegged Bob McDonnell as a president in the making. Last year, they put him on every VP list. As recently as May, they called the popular Virginia governor a political model for his would-be successors in Richmond, Democrat and Republican alike. And now – well, now nobody’s sure what to call Bob McDonnell. Text Size - + reset McDonnell refutes allegations Virginia's dirty politics Suddenly under legal and political siege, McDonnell is the subject of one of the swiftest downfalls in recent memory: once known as a spotlessly clean, law-and-order politician, the governor stands accused of questionable financial dealings that range from the tacky to the jaw-dropping. (PHOTOS: Scandal pols: Where are they now?) The McDonnell saga has gripped Richmond – and increasingly Washington – as a cascade of embarrassing disclosures have buffeted the governor and his wife, Maureen. A series of Washington Post stories have documented their cozy relationship with a donor, Star Scientific CEO Jonnie Williams, who gave financial gifts to the McDonnell family including a cumulative $120,000 in the form of a check to Maureen McDonnell and a cash infusion to a family company. With a federal investigation well underway, downcast McDonnell allies say they see little hope that the governor’s reputation will recover, and some privately express doubt that he’ll be able to serve out his term. They describe a pervasive mood of shock and gloom throughout the governor’s extended political family. McDonnell himself is said to be frustrated and distraught, in a state of disbelief but not denial about the gravity of his predicament. His friends say that McDonnell firmly believes that he has done nothing illegal, and he told the Richmond station WTVR that in his 37 years of adult life, “No one’s raised questions about my integrity or my character.” (Also on POLITICO: Va. poll: No on McDonnell 2016) The question everyone’s raising now is: How on earth did McDonnell let this happen? How did a famously disciplined politician set himself up for this operatic dive from the peak of American political life? The most popular theory at the moment is that the governor, having overextended himself in the housing market prior to the 2008 crash, averted his gaze when Williams started propping up his family. McDonnell wouldn’t be the first politician to try and sustain a lifestyle beyond his means, and then sink into deeper trouble by getting bailed out. In that view, the Virginia tale is a case study in the personal disorientation that stems from ascending to great political heights – the inevitable blurring of lines between one’s donors and friends, and the loss of perspective that comes with winning an office of immense power and limited financial reward. But the truth is that nobody outside McDonnell’s family, and perhaps no one aside from the governor himself, knows precisely what blind spot or lapse in judgment set him on the path to self-immolation. (WATCH: Virginia Gov. McDonnell refutes scandal allegations) “This is not the Bob I know,” said former Virginia Rep. Tom Davis, who called the whole grim story wildly at odds with the larger arc of McDonnell’s career. “He is one of the most ethical guys I’ve ever worked with.” Said Davis: “I’ve seen Bob in situations where a lot of other politicians would cross lines, and Bob has never gotten close to them. If what is reported is accurate, it is not in keeping with what I’ve seen in 20 years of knowing him.” Democratic state Sen. Creigh Deeds, who ran unsuccessfully against McDonnell in statewide elections for both governor and attorney general, was just as incredulous. “I’ve run two campaigns against the guy [and] I can tell you, I’m totally surprised. I’m just shocked,” Deeds said. “He had the world in his hands. He had everything in the palm of his hands and a very bright future. And now – now I don’t know.” (Also on POLITICO: Glenn Thrush's politics week in review) McDonnell allies continue to emphasize that much of what’s been reported may not run afoul of Virginia’s laws governing political gift-giving. There’s still no public evidence that Williams, for all his largesse, received anything from the government in return that would represent quid pro quo. There are two swift routes to political downfall. One is sex. The other is money. The first is humiliating but survivable. The second tends to be terminal, even criminal. Today’s topic is the second, in the form of Virginia Gov. Bob McDonnell (R) and the now mountainous evidence that — whether he technically complied with Virginia’s Swiss cheese disclosure laws or not in accepting thousands of dollars in gifts from a wealthy businessman — he has no business continuing in office. Ruth Marcus is a columnist and editorial writer for The Post, specializing in American politics and domestic policy. View Archive The sordid McDonnell details in a bit, but first the comparisons between politicians and illicit sex and politicians and illicit money. They are linked to the twin delusions of the erring politician: his (I use the male form intentionally) sense of entitlement and his conviction of invulnerability. I work so hard, the politician tells himself. I deserve a little (insert specific failing). No one will find out, the politician tells himself. I was smart enough to get elected (governor/president/senator). 1 of 102 Full Screen Autoplay Close Skip Ad × Tom Toles goes local View Photos A collection of cartoons on D.C., Maryland and Virginia. Caption A collection of cartoons on D.C., Maryland and Virginia. Buy Photo Wait 1 second to continue. Wrong, wrong, wrong. There are differences, as well, between the politician tripped up by sex and the one felled by greed. The former can argue that he was not thinking with... well, he was not thinking. He is hardly the first to do something dumb in the grip of lust, love, whatever. Yet he most likely has a wife and family, collateral damage in his sexual escapades. Points off for that — and more off if he has his wife by his side at the confessional news conference. The greedy pol is blameworthy in a different way, again both heightening and lessening his guilt. On the negative side, he was not swept away by the passion of the moment; he calculated that he could accept the money, the Rolex, whatever, and get away with it. On the plus side — and this is explanation, not excuse — he may have been acting under familial pressure, and in what he conceived as the best interests of his family, rather than against it, as the straying spouse certainly has. Much modern political corruption, especially of the penny-ante sort, can be explained by the yawning gap between the relatively paltry income of the politician and the wealth of the private-sector types fluttering around him. The politician feels aggrieved, which in turn feeds his sense of entitlement. The political spouse sees her friends driving fancier cars, wearing fancier clothes — all this while her husband is probably working longer hours, to the detriment of his family. You can understand, although not excuse, the husband whose ethical judgment is warped by marital guilt, the wife whose judgment is warped by marital resentment. Which brings us to the McDonnells, and the flagrant, repeated misconduct exposed by The Post’s Rosalind Helderman. The story began with relatively trivial, if astonishingly morally obtuse, bits of graft and back-scratching: ●The $15,000 check that businessman Jonnie R. Williams Sr. gave to help cover the catering bill at the McDonnells’ daughter’s wedding — an event that took place three days after Virginia first lady Maureen McDonnell flew to Florida, where she touted a dietary supplement made by Williams’s company, Star Scientific Inc. Three months later, Star Scientific used the governor’s mansion for a luncheon, attended by the governor, to promote the supplement. ●The $6,500 Rolex, complete with engraved inscription, “71st Governor of Virginia,” that Williams bought for the governor at Maureen McDonnell’s behest. She allegedly requested the bauble moments before a meeting she had arranged for Williams to pitch a top state health official on the supplement. ● Maureen McDonnell’s reported $15,000 spree at Bergdorf Goodman, again on Williams’s tab — this a year after a staffer foiled McDonnell’s bid for a Williams-underwritten Oscar de la Renta inaugural gown. Now comes reporting that raises the story to a new level of outrage: Williams last year gave $70,000 — supposedly a loan — to a corporation owned by McDonnell and his sister; plus $50,000 to Maureen McDonnell in 2011, and $10,000 as a wedding present this year to another McDonnell daughter. As astonishing is the governor’s technocratic defense: that he is complying with the letter of Virginia disclosure rules, which do not require reporting of gifts to family members. “To, after the fact, impose some new requirements on an official,” McDonnell told a Norfolk radio show, “obviously wouldn’t be fair.” But gifts and entanglements like these are simply wrong, a violation of the governor’s duty to citizens, whatever the rules. That McDonnell doesn’t get this basic point makes him unfit for office. Obviously. Read more from Ruth Marcus’s archive, follow her on Twitter or subscribe to her updates on Facebook. Republican gubernatorial nominee Ken Cuccinelli is facing unwelcome spillover from the drip-drip of revelations about money and gifts flowing from a Virginia businessman to Gov. Bob McDonnell. Jonnie Williams, who heads nutritional supplement manufacturer Star Scientific, has given $145,000 to the governor and his wife, including expensive clothing, a Rolex watch and checks to defray the costs of their daughters' weddings, according to the latest report in the Washington Post. The FBI is investigating. Cuccinelli also has close ties to Williams, accepting vacations at his home and airplane flights and investing more than $10,000 in stock in his company. The attorney general tried to remedy the situation by amending his financial disclosures and recusing himself from the state's $1.7 million tax dispute with Star Scientific. At the same time, Cuccinelli has kept an arm's length from the governor, breaking with him on a major transportation deal, campaigning without him at the state Republican convention and initiating the state probe into McDonnell's relationship with Williams. “What we’ve all been seeing has been very painful for Virginia, and it’s been completely inconsistent with Virginia’s very reserved traditions," Cuccinelli said in a statement clearly trying to distance himself from the scandal. His campaign also sought to turn the tables on his Democratic opponent on Wednesday by alleging, "In the history of American politics, there has never been a gubernatorial candidate more embroiled in political scandal and questionable financial dealings than Terry McAuliffe." The anti-McAuliffe screed did not include anything new, rehashing accusations dating back to his fundraising for former President Clinton, and some Cuccinelli supporters remain worried about fallout from the rapidly unfolding scandal surrounding McDonnell. "Right now it's a case of a terrible optics, and it's essentially handed a McAuliffe an arsenal of political ammunition," said Eric Odom, a prominent Virginia tea party activist. "He probably has ten campaign ads against Cuccinelli lined up and ready to go." Indeed, McAuliffe's allies from the Democratic National Committee to ProgressVA to the Democratic Governors Association on Wednesday all circulated timelines of the relationship between Cuccinelli and Williams. "Cuccinelli has tried to deny his involvement in the scandal but he can't hide from the facts," declared the DNC. At a campaign stop in Alexandria Wednesday afternoon, Cuccinelli wouldn't answer whether he thought McDonnell should step down. "The other discussion takes away from what Virginians care about: jobs," he said. Cuccinelli said that he's never offered Williams any favors – "only thing he's gotten from my office is opposition" – and called for increased disclosure and stricter caps on gift-giving to Virginia politicians. "Right now there are two investigations running, one of which began with my referral, and we need to let those play out; however, all of this emphasizes the need for clearer and faster disclosures that cover the whole family, as well as a cap on the size and types of gifts," he said in a follow-up statement. "This situation also demonstrates why transparency is so important in our system. That’s why I’ve released eight years of my tax returns, and I will put them out each of the next four years while I am governor." McAuliffe has called for a ban on gifts under $100. He has released three years of tax returns. Odom urged Cuccinelli to be pro-active and lay out clear guidelines for legal, ethical behavior by elected officials. "He needs to go on the offensive, blaze his own path and show some leadership," he said. That could be sticky for Cuccinelli, since he simultaneously wears two hats as the state's top lawyer and the Republican nominee. Nearly every past attorney general who ran for governor stepped down from his state post. Cuccinelli is also treading carefully to protect his conservative base while reaching out to the moderate Republicans and Democrats who helped elect McDonnell in 2009. The transportation deal is the line in the sand between these two constituencies, with tea party activists fuming over the subsequent tax hike and the political establishment and business community reveling over future road improvements. "To be brutally honest, Bob McDonnell looks like a fourth-string quarterback fumbling the handoff to Cuccinelli, first with the tax increase and then these revelations" about his relationship with Williams, said Keith Appell, a conservative political consultant. While Cuccinelli's opposition to the $600 million in transportation funding appeases the conservative wing of his party, it's widely viewed as potential impediment in a general election. At this point, however, the disagreement with the beleaguered gornor could be helpful for Cuccinelli's public image. Even before the transportation law passed, no one would have mistaken Cuccinelli -- who has crusaded against climate change science, gay marriage and abortion -- for a clone of the governor. "He is definitely his own man with a very principled outlook on government and politics," said Morton Blackwell, the Republican national committeeman from Virginia. "Ken's position on the transportation deal was based on principle, not politics, but I suppose there are some good deeds that are rewarded." The Republican National Committee rallied behind Cuccinelli on Wednesday, announcing that Chairman Reince Priebus would attend openings of campaign offices the next day in Hanover and North Chesterfield. Another prominent Virginia conservative, Richard Viguerie, said, "I haven't seen any evidence that McDonnell's problems have leaked over to the governor's race. It's not a help obviously, but every election is about the future, and I think that's going to be true of this race. The fact that the present governor messed up and made mistakes won't affect who people vote for." Cuccinelli's campaign is eager to steer the debate to what it called McAuliffe's "well-established record of trading access for cash and questionable business deals." A memo on Wednesday from his top political adviser, Chris LaCivita, accused the former Democratic National Committee chairman of selling access to the White House and noted that he put up $1.35 million in collateral to finance Clinton's mortgage on a home in Westchester, N.Y. Cuccinelli's campiagn also mocked McAuliffe's unfulfilled promises to create thousands of jobs through an electric car company and renewable energy project. "McAuliffe is going to have his own problems, so I think it will all be a wash if there's negative fallout from McDonnell on Ken Cuccinelli," said Jamie Radtke, founder of the Virginia Tea Party Federation. "Everything is predictive on Cuccinelli being able to get his message out." Cameron Smith contributed to this report. Correction: A previous version of this story misstated who is being investigated over a relationship with Jonnie Williams. It is Virginia Gov. Bob McDonnell. | Summary: Bob McDonnell's response to revelations that he and his family accepted $120,000 in cash from a major donor, the latest in a string of such reports, was "astonishing" writes Ruth Marcus at the Washington Post: McDonnell calmly asserted that he hadn't broken the letter of the law. Marcus doesn't care. "Whether he technically complied with Virginia's Swiss cheese disclosure laws or not," she writes, "he has no business continuing in office," and indeed, his very failure to grasp the wrongness of what he'd done makes it worse. The entire affair has quickly derailed the career of a guy who was recently on Mitt Romney's VP shortlist. "It's a colossal fall from grace," one Virginia Republican tells Politico. As for McDonnell's 2016 presidential hopes, "that conversation doesn't even exist" anymore, one Republican tells CBS. "The conversation now is if there is going to be a resignation." What's more, the scandal could ensnare McDonnell's would-be successor, Ken Cuccinelli, who is also close to the Star Scientific exec who's been so generous with McDonnell, the National Journal reports. | 5,553 | 274 | multi_news | en |
Summarize: Introduction On August 22, 2008, Federal Reserve Board Chairman Ben S. Bernanke spoke about systemic risk and raised the issue of having the authority to conduct macroprudential oversight. Similarly, Timothy F. Geithner, while president of the Federal Reserve Bank of New York, also spoke about the need for expanding current prudential supervision of individual financial institutions. Both Federal Reserve officials spoke in the context of growing discussions among academics and central bankers concerning the adoption of a macroprudential policy perspective. Systemic risk may be defined as risk that cannot be avoid ed through diversification. Systemic risk may also be defined in a similar manner to contagion, in which liquidity and payment problems that affect one or a few financial entities will spread and disrupt financial activity more widely in the system. Systemic risk can increase as a result of financial innovation that enhances capital mobility and provides access to additional sources of capital outside of the confines of regulated financial institutions. Although the financial system increases its capacity to provide credit, market expectations grow in importance, which may increase the fragility of the system. Systemic events occur with sudden shifts in the expectations and subsequent reactions of financial market participants. A panic, liquidity disruption, or decline in asset prices may cause sudden and unpredictable reactions by market participants that overwhelm even those regulated financial institutions with sound risk management practices. Consequently, a popular characterization of systemic risk as financial institutions that are "too-big-to-fail" is misleading because it fails to capture the importance of impulsive reactions to various financial events that can be magnified into systemic risk events. Macroprudential supervision or oversight refers to the monitoring of the entire financial system and its vulnerability to systemic risk. Macroprudential oversight arguably complements regulatory structures for individual financial institutions. In addition to monitoring for systemic risk, other tasks fall under the scope of macroprudential oversight. Administrators would be responsible for the development of early warning systems of financial distress, such as a composite index of leading indicators. System-wide stress-testing exercises, which involve introducing some extreme financial disruption into a model of the financial system or various components to evaluate the impact on asset portfolios, would be conducted. Finally, macroprudential policy administrators would also be able to provide advice to other regulatory agencies on matters related to financial stability. This report begins by briefly summarizing how recent innovations in finance, while increasing the capacity to borrow and lend, also resulted in a large volume of banking transactions occurring outside of traditional banking institutions. Monitoring these institutions for safety and soundness, which is referred to as microprudential oversight, does not directly address the challenges posed by systemic risk. Hence, the benefits and limitations of macroprudential policy will be discussed. Intermediation and Financial Innovation Financial intermediation is the process of matching borrowers with lenders. The typical intermediation transaction made by banks consists of providing loans to borrowers at higher rates than it costs banks to borrow the funds from savers, who are the ultimate lenders. In other words, long-term loans that banks originate to borrowers are funded by short-term loans made to banks, usually in the form of savings deposits. Banks profit from the spread between the rates they receive and the rates they pay. Banks also earn income from various fees and service charges. The intermediation transaction carries a variety of risks. Banks face the risk of borrower default on the long-term loans. Banks face liquidity and interest rate risks on the short-term funding side of the transaction. Until the long-term loan is fully repaid, banks must continue to attract short-term savers. A bank must continuously be able to roll over or renew its short-term funding (loans) because the funds used to originate the long-term loan have been disbursed to the borrower. It is possible that banks could find themselves low on deposits, perhaps due to a sudden demand for cash or changes in economic conditions. Financial market conditions could also change such that short-term rates rise higher than long-term rates, and continued funding of long-term loans becomes costly. Given the default and funding liquidity risks associated with the intermediation transaction, banks and other financial institutions are always looking for innovative ways to reduce risk, which ultimately facilitates the expansion of intermediation and credit availability. Although the intermediation transaction remains the same conceptually over time, its means of execution has evolved and diversified, which is considered financial innovation. Financial innovations include securitization, growth of the commercial paper market, automated underwriting, derivative markets, and nontraditional mortgage products, which allow the long-term borrower and lender to share the risk of fluctuating long rates. These developments arguably facilitated intermediation in terms of reducing or managing the risks associated with supplying credit, which increased lending capacity. In fact, financial innovation in the mortgage market during the 1990s arguably enhanced homeownership by reducing loan origination costs and increasing the array and variety of mortgage products that could be offered to borrowers. Such financial innovation also allowed aspects of the intermediation transactions to occur outside of what is considered traditional banking institutions. For example, some businesses, rather than obtaining traditional short-term loans from depository institutions, may issue their own debt in the form of commercial paper. Commercial paper issuances are typically unsecured, short-term promissory notes or bonds that investors (savers) can hold in their portfolios and, upon maturity, roll the proceeds into newer commercial paper issuances. Hedge funds, pension funds, and other financial entities may decide to purchase long-term, less liquid assets with funds obtained from their issuances of commercial paper. The commercial paper is the liability of the issuing firms, and the long-term assets (loans) acquired were not funded by liabilities (deposits) of a traditional depository institution. Securitization is another example of a financial innovation that allowed aspects of the intermediation transaction to occur off bank balance sheets. Securitizers are entities that purchase long-term loans, then use the payment streams to create short-term securities (similar in nature to commercial paper with a variety of risk-return options) to investors, which fund the loan purchases. During the 2000s, many subprime loans were originated by non-bank lenders that did not hold deposits. These loans were then funded via the securitization process with funds raised from commercial paper or similar issuances. Consequently, intermediation transactions were no longer limited to taking place inside traditional banks; they could now occur in the broader financial markets. Microprudential Oversight and Limitations A microprudential regulatory approach focuses on the safety and soundness of individual financial institutions. This regulatory approach monitors lending by supervised institutions and attempts to encourage prudent behavior. Microprudential regulators evaluate bank data against Basel I and II capital ratios and CAMELS rating criteria. The objective of microprudential oversight is to increase the protection of the deposits in these institutions. The microprudential approach to regulation, however, cannot completely prevent bank failures. The market value of bank assets or loans can suddenly decline with sudden increases in unemployment, which is indicative of future repayment problems, even though the loans were prudently originated before the shift in local financial conditions. This vulnerability applies especially to small banks whose loans are tied to the local economy. Larger banks may be less susceptible to regional economic conditions if they are geographically diversified, but they remain vulnerable to systemic risk, which falls outside the scope of microprudential regulation. Financial innovation may expand the financial system such that more intermediation transactions can take place outside of more traditional banking institutions, but the risks have not disappeared. The risks, rather than being borne by a particular institution, are spread among a multitude of financial market participants. Furthermore, the transfer of risk to financial markets may increase the interconnectivity among all financial market participants, causing the entire financial system to be vulnerable to unanticipated payments disruptions or sudden declines in asset values. For example, suppose banking institutions used derivatives instruments to reduce the default and interest rate risks associated with the intermediation transaction. These instruments would have transferred these risks to another counterparty, perhaps outside of the banking system, willing to sell protection against such risks. If, however, the counterparty finds itself having to make higher than anticipated payments following some unforeseen event, all financial market participants may question the ability of other market participants to repay commitments. Such erosion of confidence may be considered systemic and have harmful consequences on other participants even though only one counterparty was late or completely defaulted on a payment. Hence, the transfer of risk led to an increase in the interconnectivity of financial market participants. Moreover, microprudential oversight, which is limited to the regulation of the risk management practices of individual institutions, would not eliminate the systemic risk in this scenario largely because financial market expectations are impossible to regulate. Macroprudential Oversight and Limitations Central banks are generally tasked with "lender-of-last-resort" responsibilities to ensure that regulated depository institutions reliably have access to short-term loans. This access ensures that healthy but illiquid banks continue funding long-term loans without disruption. Under a traditional banking model where regulated institutions originate long-term loans and fund them with short-term deposits, a central bank may assist banks in a short-term funding crunch, perhaps because of a bank run, as part of its normal course of activities. Consequently, central banks may be considered systemic risk managers for supervised banks, and they have typically been involved in macroprudential policy oversight, even if that role has not been formalized by legislation. Macroprudential oversight structures have typically been set up inside of central banks. H.R. 4173, the Wall Street Reform and Consumer Protection Act of 2009 (Representative Barney Frank), proposes to establish a Financial Services Oversight Council (FSOC), which would consist of the heads of the federal financial regulatory agencies, to provide macroprudential oversight of the U.S. financial system. A formal approach to macroprudential oversight arguably complements existing microprudential oversight to reduce systemic risks. Suppose an agency conducting macroprudential oversight were to ask microprudential supervisors to require increased transparency for a specific intermediation activity from their supervised institutions. Disclosure of such information to the entire financial system may increase overall confidence. Furthermore, if such policy actions were taken under normal conditions when financial markets are not in distress, then implementation may be more likely to boost rather than erode the confidence of financial market participants. H.R. 4173 would require the FSOC to facilitate information sharing and coordination among its members with respect to financial services policy development, rulemakings, examinations, reporting requirements, and enforcement actions. A regulatory body specifically tasked with macroprudential oversight would try to guard against bubbles and overleveraging. The regulator would also monitor stress-testing activities as well as the extent to which financial market participants are becoming more interconnected by risks. When asset values are rising, the balance sheets of financial institutions are likely to appear healthier. Financial institutions may decide to increase their lending in such an environment. As long as financial institutions maintain capital risk ratios at or above safety thresholds, the increase in lending activity would still fall within the guidelines of safety and soundness set by microprudential oversight. A rise in asset market prices, however, may be indicative of a speculative bubble. An agency responsible for macroprudential oversight may warn or even attempt to curtail the level of risk taking activity when related asset prices appear to rise rapidly at what may be considered an unsustainable pace. The macroprudential objective would be to build up safety buffers during good times that can be used as a cushion during unstable times. A limitation or drawback of macroprudential policy is its countercyclical nature, which means the policy impact will dampen business cycle activity and restrain the economy during boom times. As stated in the previous example, procyclical microprudential policy would be less likely to curtail lending activities for banking institutions when collateral asset values are rising. Macroprudential administrators, however, may perceive rising asset prices as evidence of a bubble and call for banks to raise capital or reduce lending. Although this macroprudential response may help reduce the likelihood or impact of a systemic risk event, it may also restrain short-run banking profits, economic activity, and economic growth. Consider the debate about the existence of a housing market bubble. The substantial decline in mortgage interest rates during the 1990s, resulting in part from securitization reducing the funding costs of lending, helped lower the cost of homeownership relative to renting. Changing demographic trends also affected changes in housing demand. On the other hand, the rise in home prices and use of mortgage credit levels was interpreted by some as evidence of a bubble in the housing market. Consequently, it would have been problematic to determine with certainty whether the rise in the demand for homes, which would be reflected in house price increases, was due to a rise in underlying fundamentals or speculative behavior, since both occurred simultaneously. Even when the market is characterized by speculation, speculative trading helps to provide liquidity for assets. Speculation increases the number of transactions, which makes it easier to price and sell assets. Hence, identifying bubbles would still present a challenge for macroprudential oversight. Not only would it be difficult to determine how much financial market activity would be the result of speculation, but it would also be difficult to determine how much speculation can be reduced without compromising overall asset market liquidity. Furthermore, as financial markets become more globalized, it becomes more challenging for a macroprudential regulator in one country to track and influence global expectations. Despite the problems of identifying and managing speculative behavior, macroprudential regulators would be tasked with responding to conditions that suggest the presence of a bubble given the systemic risks to financial markets when it deflates. A macroprudential response, however, may be at odds with other policy goals in which the primary focus is not safety and soundness. Some policy goals are aimed at increasing credit access to low and moderate income households and households living in underserved areas. It may be easier for financial institutions to facilitate more lending to those individuals covered by policy goals at times when collateral assets are rising and there is expanded lending capacity. Macroprudential oversight, however, might encourage a reduction of lending at a time when asset values are rising and financial conditions would be better suited to facilitate the achievement of other policy goals. A macroprudential oversight regulator may also find itself in the middle of occasional conflicts between regulators. For example, consider a case that occurred in the mid-1990s. A regulator concerned with the adequate capitalization of banks preferred that banks adopt conservative accounting practices that would result in the reporting of higher loan loss allowances. Higher loan loss allowances would indicate the ability of banks to avoid severe financial distress when unexpected loan losses occur. Another regulator, concerned with the accurate reporting of income to investors, preferred adoption of accounting practices that would reduce the reported amount of loan-loss allowances. Overstatement of loan loss allowances reduce bank net income and retained earnings on paper. Investors may interpret large loan-loss allowances as evidence of high-risk lending practices, which may reduce bank profitability, and that could translate into a decline in the bank's value or stock price. In addition, a reporting of higher loan-loss allowances may result in an initial understatement of assets and overstatement of bank income in future periods, which would translate into overly optimistic information being reported to investors in subsequent periods. A macroprudential regulator may be more inclined to support regulations that foster increased safety and soundness. Given that regulators can always require banks to provide any critical information, such disputes may be settled in favor of investor needs. If, in the particular case above, a regulator responsible for macroprudential oversight combined efforts with the regulator in favor of more conservative accounting practices, the collective efforts possibly may have had a greater influence on the outcome. In H.R. 4173, the FSOC would be given the task of resolving disputes that might arise among federal financial regulatory agencies. | Summary: Recent innovations in finance, while increasing the capacity to borrow and lend, resulted in a large volume of banking transactions occurring outside of traditional banking institutions. Also, even though existing regulators supervise individual banks for safety and soundness, there are risks that do not reside with those institutions but may still adversely affect the banking system as a whole. Macroprudential policy refers to a variety of tasks designed to defend the broad financial system against threats to its stability. Responsibilities include monitoring the system for systemic risk vulnerabilities; developing early warning systems of financial distress; conducting stress-testing exercises; and advising other regulatory agencies on matters related to financial stability. H.R. 4173, the Wall Street Reform and Consumer Protection Act of 2009 (Representative Barney Frank), establishes a Financial Services Oversight Council with macroprudential regulatory responsibilities. On March 22, 2010, the Senate Banking Committee ordered reported the Restoring American Financial Stability Act of 2010 (Senator Christopher Dodd), which also would establish a Financial Services Oversight Council with similar responsibilities. This report provides background and discusses the potential benefits and limitations of macroprudential policy efforts. This report will be updated as events warrant. | 3,550 | 274 | gov_report | en |
Write a title and summarize: SECTION 1. SHORT TITLE; PURPOSE. (a) Short Title.--This Act may be cited as the ``America Rx Act of 2010''. (b) Purpose.--The purpose of this Act is to establish an America Rx program that utilizes manufacturer rebates and pharmacy discounts to reduce prescription drug prices to those residents who are without access to discounted prices for outpatient prescription drugs. SEC. 2. AMERICA RX PROGRAM. (a) Establishment.-- (1) In general.--Not later than 1 year after the date of the enactment of this Act, the Secretary of Health and Human Services shall establish a program (in this section referred to as the ``America Rx program'') consistent with the provisions of this section to provide qualified residents with access to discounted prices for outpatient prescription drugs through rebate agreements that the Secretary has negotiated with prescription drug manufacturers. (2) Outreach.--The Secretary shall provide for-- (A) outreach efforts to build public awareness of the program and maximize enrollment of qualified residents; and (B) simplified eligibility procedures and uniform eligibility standards for qualified residents. (b) Rebate Agreements With Manufacturers.-- (1) In general.--Under the America Rx program the Secretary shall negotiate with manufacturers of outpatient prescription drugs rebate agreements with respect to drugs offered under the program to qualified residents. (2) Minimum amount of rebates.--In negotiating the amount of such a rebate under paragraph (1), the Secretary shall take into consideration the amount of the rebate calculated under the Medicaid program, the average manufacturer price of prescription drugs, and other information on prescription drug prices and price discounts. The Secretary shall negotiate the amount of such rebates in a manner so that the rebates on average are comparable to the average percentage rebate obtained in outpatient prescription drugs provided under section 1927(c) of the Social Security Act (42 U.S.C. 1396r- 8(c)). (3) Discounted prices.--The Secretary shall specify the method for computing and applying such discounts, including a method for computing and applying discounts on a uniform, average percentage basis. (4) Payment.--Rebates under such agreements shall be payable to the Secretary according to a schedule (not less often than quarterly) negotiated with manufacturers and shall be paid, directly or through States, to participating pharmacies that provide discounts to qualified residents. (5) Incentive.--In order to induce manufacturers of outpatient prescription drugs to enter into such rebate agreements to carry out the purpose described in section 1(b), the Secretary shall provide, in the case of a manufacturer that has not entered into such an agreement, for a denial of a deduction under chapter 1 of the Internal Revenue Code of 1986 for the amount of expenses of the manufacturer for advertising and marketing of drugs of the manufacturer, other than expenses for free samples of drugs subject to section 503(b)(1) of the Federal Food Drug, and Cosmetic Act intended to be distributed to patients. (6) Application of rebates.--Amounts received by the Secretary as rebates under this subsection shall be placed into an appropriate account in the Treasury and shall be available in advance of appropriations to the Secretary for the payment of discounts and other costs of participating pharmacies in carrying out the America Rx program and for the payment of administrative costs in carrying out the program. Under the America Rx program, participating pharmacies shall be ensured reasonable and timely payment of discounts they provide to qualified residents under the program. (c) Role of States in Administering Program.-- (1) In general.--Under the America Rx program the Secretary may enter into appropriate arrangements with States under which States provide for the administration of the program in return for payment of the reasonable administrative expenses associated with such administration. (2) Administrative functions.--Such administration functions may include-- (A) determinations of eligibility of qualified residents; (B) arrangements with participating pharmacies; and (C) such other functions as the Secretary determines appropriate. (3) Contractual authority.--In carrying out responsibilities under this section, the Secretary and States may enter into agreements with pharmacy benefit managers and other third parties. (d) Program Eligibility.-- (1) Qualified resident defined.--For purposes of this section, the term ``qualified resident'' means an individual who-- (A) is a citizen or national of the United States or is lawfully present in the United States (as determined in accordance with section 1411 of the Patient Protection and Affordable Care Act); and (B) as determined under regulations of the Secretary, is not covered under any public or private program that provides substantial benefits (which may be discounted prices) towards the purchase of outpatient prescription drugs. (2) Screening.-- (A) For other governmental programs.--Individuals who apply for benefits under the America Rx program shall be screened for eligibility under the Medicaid program and other applicable Governmental health care programs and, if found eligible, shall be enrolled in such program or programs. (B) Screening by health insurance exchanges.--In the case of individuals enrolling in a qualified health plan through a Health Insurance Exchange under title I of the Patient Protection and Affordable Care Act, the Exchange shall provide (at the time of enrollment) for a screening of the individual to determine if the individual is a qualified resident for purposes of the America Rx program. (3) Relation to other coverage.--In order not to discourage employers and health insurance issuers from providing comprehensive prescription drug coverage through a group health plan or health insurance coverage, no such employer or issuer may participate in a Health Insurance Exchange if the Secretary determines that the prescription drug benefits under the plan or coverage have been reduced as a result of the America Rx program. (4) Protection of the low income.--Nothing in this section shall be construed as reducing the prescription drug benefits available to low-income individuals, as well as senior citizens and the disabled, under the Medicare or Medicaid programs as a result of the implementation of the America Rx program. (e) Arrangements With Participating Pharmacies.-- (1) In general.--Under the America Rx program arrangements are made with pharmacies for the provision of prescription drugs at discounted prices to qualified residents in a reasonably accessible manner. Such arrangements shall provide that-- (A) each participating pharmacy shall-- (i) provide discounts on prices for outpatient prescription drugs for qualified residents in return for prompt reimbursement of the amount of such discounts and a reasonable dispensing fee; (ii) not charge qualified residents more (before such discounts) for outpatient prescription drugs than the amount that individuals who are not qualified residents are charged for such drugs; and (iii) report to the Secretary (or the Secretary's designee) information regarding the discounts provided and fees incurred; and (B) the program shall-- (i) ensure the reasonable and timely payment to a participating retail pharmacy on a prompt basis (no less promptly than as provided under the Medicare program) for discounted prices provided to qualified residents under the program and for reasonable dispensing fees; and (ii) not impose any additional fees on such pharmacies in connection with participation in the program. (2) Discounted prices.--The amount of the discount provided to enrolled qualifying residents shall reflect the amount of rebates obtained, reduced by expenses relating to administrative costs of the Federal and State governments and of participating pharmacies. (f) Definitions.--For purposes of this section: (1) The term ``Health Insurance Exchange'' means such an Exchange as established and operated under part 3 of subtitle D of title I of the Patient Protection and Affordable Care Act. (2) The term ``manufacturer'' has the meaning given such term in section 1927(k)(5) of the Social Security Act (42 U.S.C. 1396r-8(k)(5)). (3) The term ``Medicaid program'' means a State program under title XIX of the Social Security Act, including such a program operating under a Statewide waiver under section 1115 of such Act. (4) The term ``outpatient prescription drug'' has the meaning given the term ``covered outpatient drug'' in section 1927(k)(2) of the Social Security Act (42 U.S.C. 1396r- 8(k)(2)). (5) The term ``Secretary'' means the Secretary of Health and Human Services. (6) The term ``State'' has the meaning given such term for purposes of title XIX of the Social Security Act. | Title: To establish an America Rx program to establish fairer pricing for prescription drugs for individuals without access to prescription drugs at discounted prices Summary: America Rx Act of 2010 - Requires the Secretary of Health and Human Services (HHS) to establish the America Rx program to provide qualified residents with access to discounted prices for outpatient prescription drugs through rebate agreements that the Secretary negotiates with prescription drug manufacturers. Makes eligible only those residents that are not covered under any public or private program that provides substantial benefits towards the purchase of outpatient prescription drugs. Requires rebates to be payable to the Secretary at least quarterly and to be paid, directly or through states, to participating pharmacies that provide discounts to qualified residents. Denies manufacturers who do not participate in the rebate program a tax deduction for advertising and marketing expenses of drugs. | 1,906 | 178 | billsum | en |
Write a title and summarize: Multicellular organisms evolved via repeated functional divergence of transcriptionally related sister cell types, but the mechanisms underlying sister cell type divergence are not well understood. Here, we study a canonical pair of sister cell types, retinal photoreceptors and bipolar cells, to identify the key cis-regulatory features that distinguish them. By comparing open chromatin maps and transcriptomic profiles, we found that while photoreceptor and bipolar cells have divergent transcriptomes, they share remarkably similar cis-regulatory grammars, marked by enrichment of K50 homeodomain binding sites. However, cell class-specific enhancers are distinguished by enrichment of E-box motifs in bipolar cells, and Q50 homeodomain motifs in photoreceptors. We show that converting K50 motifs to Q50 motifs represses reporter expression in bipolar cells, while photoreceptor expression is maintained. These findings suggest that partitioning of Q50 motifs within cell type-specific cis-regulatory elements was a critical step in the evolutionary divergence of the bipolar transcriptome from that of photoreceptors. Complex tissues require the coordinated activity of a wide array of specialized cell types. It has been proposed that cellular diversity arises in the course of evolution through a ‘division of labor’ process, in which a multifunctional ancestral cell type gives rise to descendant cell types with divergent and novel functions (Arendt et al., 2009; Brunet and King, 2017). Such descendants are often referred to as ‘sister’ cell types, and typically share a range of morphological, functional, and transcriptional features while at the same time displaying key differences (Arendt, 2003; Arendt et al., 2016). A canonical example of sister cell types are mammalian retinal photoreceptors and bipolar cells (BCs) (Arendt, 2008; Lamb, 2013). In a typical vertebrate retina, photoreceptors synapse onto bipolar cells, which, in turn, synapse onto retinal ganglion cells that send their axons to the brain. Bipolar cells therefore constitute the central interneuronal cell class in the vertebrate retina. In mice, an array of 15 distinct bipolar cell types, broadly categorized as ON and OFF based on their response to light onset/offset, serve as a scaffold upon which the complex information-processing circuitry of the retina is built (Euler et al., 2014). In this paper, we refer to photoreceptors and bipolar cells as cell ‘classes’, since they each comprise multiple distinct cell types. During retinal development, photoreceptors and bipolar cells arise from the same population of OTX2-expressing progenitor cells (Koike et al., 2007; Wang et al., 2014; Emerson et al., 2013), share a similar elongate morphology (Lamb, 2013), and possess the molecular machinery required for ribbon synapse formation, a structure not found in any other retinal cell class (tom Dieck and Brandstätter, 2006). In some vertebrate species, a subset of bipolar cells exhibit additional photoreceptor-like features, including localization of their cell bodies in the outer nuclear layer and the presence of an inner segment-like structure known as Landolt’s club, which extends from the dendrite to the outer limiting membrane and contains a 9+0 cilium (Tauchi, 1990; Hendrickson, 1966; Locket, 1970; Quesada and Génis-Gálvez, 1985). These ‘transitional’ cell types point to the evolutionary origin of bipolar cells from photoreceptors (Arendt, 2008; Lamb, 2013). Both shared and divergent features of sister cell types are mediated by the transcriptional regulatory networks that govern gene expression in each cell type. In vertebrates, photoreceptors and bipolar cells express the paired-type homeodomain (HD) TFs CRX and OTX2, which are master regulators of gene expression in both cell classes (Omori et al., 2011; Nishida et al., 2003; Hennig et al., 2008; Corbo et al., 2010). A third paired-type HD TF, VSX2, is expressed specifically in bipolar cells and is required for bipolar fate (Livne-Bar et al., 2006; Liu et al., 1994). Paired-type homeodomains recognize a core ‘TAAT’ motif, with additional specificity conferred by amino acids in positions 47,50, and 54 of the homeodomain (Treisman et al., 1989; Noyes et al., 2008; Berger et al., 2008) In particular, a lysine at position 50 (K50, as found in CRX and OTX2) favors recognition of TAATCC, whereas a glutamine (Q50, as found in VSX2) favors recognition of TAATTA/G. Thus, substitution of a single amino acid in the HD toggles the TF’s binding preference for the nucleotides 3’ of the TAAT core (Treisman et al., 1989). Various bHLH TFs, which recognize E-box motifs (CANNTG), also play important roles in photoreceptor and bipolar cell gene expression programs (Tomita et al., 2000; Akagi et al., 2004). For example, NEUROD1 is required for photoreceptor survival, and BHLHE22 and BHLHE23 are required for development of OFF cone bipolar cells and rod bipolar cells, respectively (Bramblett et al., 2004; Feng et al., 2006; Huang et al., 2014; Pennesi et al., 2003). Our lab has previously shown that the cis-regulatory elements (CREs; i. e., enhancers and promoters) of mouse rods and cones are strongly enriched for K50 HD motifs as well as moderately enriched for Q50 HD and E-box motifs (Hughes et al., 2017). In addition, we recently used a massively parallel reporter assay to analyze the activity of thousands of photoreceptor enhancers identified by CRX ChIP-seq and found that both K50 HD and E-box motifs are positively correlated with enhancer activity in photoreceptors while Q50 HD motifs have a weakly negative correlation with enhancer activity (Hughes et al., 2018). In contrast, studies of individual reporters have shown that Q50 HD motifs mediate weak activation of expression via RAX, and that RAX can either enhance or suppress the transactivation activity of CRX, depending upon RAX expression levels (Irie et al., 2015; Kimura et al., 2000). Thus, Q50 HD motifs appear to have both positive and negative effects of photoreceptor enhancer activity, depending on context. In contrast, Q50 motifs in bipolar cells appear to be strongly repressive when bound by VSX2, which has been proposed to inhibit the expression of photoreceptor genes in bipolar cells (Livne-Bar et al., 2006; Dorval et al., 2006; Clark et al., 2008). The opposing effects on transcriptional activity mediated by K50 and Q50 motifs suggest that even subtle changes in HD binding sites may mediate major differences in gene expression. Indeed, a recent study in Drosophila showed that single base pair substitutions that interconvert Q50 and K50 half-sites within dimeric motifs are sufficient to switch the specificity of opsin expression within photoreceptor sub-types (Rister et al., 2015). Photoreceptors and bipolar cells offer an attractive model system in which to examine the mechanisms of cis-regulatory divergence in evolution and development, but the cis-regulatory landscape of bipolar cells is currently unknown. To elucidate the cis-regulatory grammar of bipolar cells we isolated specific bipolar cell populations from mouse retina and obtained profiles of open chromatin and gene expression. By comparing these datasets to matching data from mouse rod and cone photoreceptors we identified differential enrichment of Q50 motifs in photoreceptor-specific enhancers and a corresponding enrichment of E-boxes in bipolar-specific enhancers. We propose that the differential partitioning of Q50 motifs in photoreceptor and bipolar enhancers was a key evolutionary innovation contributing to transcriptomic divergence between the two cell classes. To obtain cell class-specific transcriptome profiles of mouse bipolar cells, we used fluorescence-activated cell sorting (FACS) to purify bipolar cell populations from adult mice. We first isolated all bipolar cells using Otx2-GFP mice. This line harbors a GFP cassette knocked into the endogenous Otx2 locus (Fossat et al., 2007). Adult Otx2-GFP mice display high-level GFP in bipolar cells and low-level expression in photoreceptors (Figure 1B). To purify ON and OFF bipolar cells separately, we crossed Otx2-GFP mice with a Grm6-YFP line, in which YFP is driven by the Grm6 promoter and expressed only in ON bipolar cells (Morgan et al., 2011). In the double transgenic mice (Otx2-GFP+; Grm6-YFP+), ON bipolar cells co-express GFP and YFP and can be separated from OFF bipolar cells based on fluorescence intensity (Figure 1B). We subjected OFF bipolar cells to a second round of sorting to maximize purity from the adjacent weakly fluorescent photoreceptor population. Purity of bipolar cell populations was confirmed by RT-qPCR which showed enrichment of ON- and OFF-specific genes in the appropriate populations and depletion of markers for other retinal cell classes as compared to whole retina (Figure 1C and Figure 1—figure supplement 2). We then used RNA-seq to profile gene expression in purified populations of bipolar cells, obtaining highly reproducible profiles across biological replicates (Figure 1—figure supplement 1). To define the transcriptional differences between photoreceptor and bipolar cells we compared RNA-seq data from bipolar cells to similar data from wild-type rods and Nrl-/- photoreceptors previously generated in our lab (Hughes et al., 2017). We used Nrl-/- photoreceptors as a surrogate for blue cones (i. e., Opn1sw-expressing cones) since mouse photoreceptors lacking Nrl transdifferentiate into blue cones during development (Daniele et al., 2005; Nikonov et al., 2005). We identified a total of 5259 genes with at least a two-fold difference in expression between bipolar cells and either rods or blue cones (FDR < 0. 05) (Supplementary file 5). Despite the large number of differentially expressed genes, published single-cell RNA-seq profiles indicate that the bipolar cell transcriptome is more similar to that of photoreceptors than to that of any other retinal cell class (Macosko et al., 2015). To evaluate the functional differences between photoreceptor and bipolar cell gene expression programs we compared the top ~ 30% most differentially expressed genes in each cell class (832 bipolar cell, 818 photoreceptor) using the gene ontology (GO) analysis tool, PANTHER (Thomas et al., 2003). Top bipolar-enriched GO terms were typical of many neuronal cell types and related to aspects of synaptic transmission, while photoreceptor-enriched GO terms mainly related to light-sensing (Supplementary file 6). Next, we compared the list of differentially expressed genes to a database of mouse TFs (AnimalTFDB3. 0; Hu et al., 2019), which revealed that 394 of the differentially expressed genes encode putative transcriptional regulators (Supplementary file 5). These include TFs known to be responsible for controlling gene expression in rods (Nrl, Nr2e3, Neurod1), cones (Thrb), bipolar cells (Vsx2, Neurod4), or both cell classes (Crx). Nearly one-third (176) of differentially expressed TFs are members of the zinc finger (ZF) family, many of which are more highly expressed in rods compared to bipolar cells but not in blue cone compared to bipolar cells. Conversely, of the top 10% differentially expressed TFs, the majority (25 of 35) are more highly expressed in bipolar cells compared to either rods or cones, and of these, most (16 of 25) are classified as HD, ZF or bHLH. Thus, the transcriptomes of bipolar cells and photoreceptors are significantly divergent, despite various functional and morphological similarities between the two cell classes. In contrast, comparison of the transcriptomes of ON and OFF bipolar cells identified only 680 genes that were differentially expressed by at least two-fold (317 ON- and 363 OFF-enriched at FDR < 0. 05; Supplementary file 5). This figure is less than half of the number of differentially expressed genes identified between rods and blue cones (1,471), indicating that the transcriptomes of the two categories of bipolar cells are quite similar. Of note, a recent study by Shekhar et al. (2016), described single-cell expression profiles for bipolar cell types using Drop-seq. To compare our results with this study, we pooled data from Shekhar et al. across inferred cell types to generate ‘pseudo-bulk’ ON, OFF, and pan bipolar cell gene expression profiles. We found that expression estimates from bulk and pooled pseudo-bulk single-cell RNA-seq are well correlated (Pearson correlation coefficients range from 0. 82 to 0. 85, Figure 2—figure supplement 1A–C). Similarly, pairwise comparisons showed that bulk ON, OFF, and pan bipolar cell populations were most strongly correlated with their pseudo-bulk counterparts, suggesting that the two approaches yield similar cell type-specific expression profiles (Figure 2—figure supplement 1D). Shekhar et al. also identified an ON bipolar cell type (BC5D) that expresses low levels of Grm6 and could thus have been incorrectly sorted into our OFF population. To evaluate this possibility, we examined RNA-seq data for Lrrtm1, a gene specific to BC5D bipolar cells (Shekhar et al., 2016). We observed low read counts for Lrrtm1 in both ON and OFF bipolar populations, suggesting that BC5D cells were not sorted into either group. To investigate further, we repeated the FACS experiments, this time also collecting cells exhibiting fluorescence levels in between those of the ON and OFF populations (termed YFP-low). We found that the YFP-low population expressed both the ON BC marker Isl1 and the BC5D marker Lrrtm1, while the ON and OFF populations did not express Lrrtm1 (Figure 1—figure supplement 2). These results indicate that low levels of Grm6-YFP expression caused BC5D cells to be excluded from both the ON and OFF bipolar populations. Given the rarity of BC5D cells, which constituted 2. 3% of cells identified as bipolar cells by Shekhar et al., we believe their absence does not materially impact our analysis. To gain insight into gene expression among individual bipolar cell types, we compared the list of genes differentially expressed between ON and OFF bipolar cells with the data of Shekhar et al. Overall, we found a strong correlation between the results of bulk ON and OFF bipolar cell expression profiling and single-cell transcriptome analysis (Figure 2; Figure 2—figure supplement 1E and Figure 2—source data 1). Additionally, 50 of the 680 differentially expressed genes found in our bulk analysis were not present in the Drop-seq data (Supplementary file 5). These 50 genes are generally expressed at low levels, even in the cell population in which they are enriched. This finding suggests that bulk RNA-seq of purified populations can provide information complementary to that obtained by single-cell profiling. Taken together, these data indicate that despite their sister cell type relationship, photoreceptor and bipolar cells have markedly distinct transcriptional profiles, while ON and OFF bipolar cells are more similar at the transcriptome level than rods and cones. To compare chromatin accessibility between photoreceptor and bipolar cells, we used ATAC-seq (Assay for Transposase-Accessible Chromatin by sequencing) to generate open chromatin profiles from FACS-purified bipolar cells (Buenrostro et al., 2013). Similar to our RNA-seq data, ATAC-seq generated highly reproducible profiles across biological replicates (Pearson correlation 0. 95–1. 00, Figure 1—figure supplement 1). For the purpose of our bioinformatic analyses, we define ‘promoters’ as those ATAC-seq peaks that occur between 1000 bp upstream and 100 bp downstream of a transcription start site (TSS); we refer to ATAC-seq peaks outside of this range as ‘enhancers’ or ‘TSS-distal’ elements. To compare chromatin accessibility across tissues, we combined ATAC-seq peaks from bipolar cells with previously generated ATAC-seq data from purified mouse rods, blue cones, and ‘green’ cones (i. e., Opn1mw-expressing cones) as well as DNase-seq data from whole retina, brain, heart, and liver to obtain a list of > 345,000 open chromatin regions (Hughes et al., 2017; ENCODE Project Consortium, 2012). Hierarchical clustering of chromatin accessibility profiles at enhancers showed that photoreceptors, bipolar cells, and whole retina cluster separately from other tissues (Figure 3A). Thus, the sister cell type relationship between photoreceptors and bipolar cells is reflected by the similarity of genome-wide patterns of enhancer chromatin accessibility. We found that whole retina DNase-seq clustered with bipolar cell samples (Figure 3A). This finding was unexpected given that rod photoreceptors constitute ~ 80% of all cells in the mouse retina (Figure 1A) (Jeon et al., 1998) and indicates that the open chromatin profile of a complex tissue is not necessarily dominated by the most abundant cell type. This is in line with our previous comparison of genome-wide patterns of chromatin accessibility in rods, cones and whole retina, which showed that more than half of the whole retina peaks did not overlap with photoreceptor peaks, suggesting that they derive from non-photoreceptor retinal cell types (Hughes et al., 2017). This finding may be a consequence of the distinctive pattern of global chromatin closure that we previously described in rod photoreceptors (Hughes et al., 2017). We next set out to examine patterns of chromatin accessibility within the retinal cell types in greater detail. We combined TSS-distal ATAC-seq peaks from bipolar cells and photoreceptors with DNase-seq peaks from whole retina to create a list of 99,684 retinal open chromatin regions. Clustering these regions based on chromatin accessibility across retinal and non-retinal cell types offers a broad view of cell class- and cell type-specific regions of open chromatin (Figure 3B). We found a large subset of bipolar-enriched peaks, many of which were also accessible in whole retina and brain (Figure 3B, green box). Conversely, a smaller subset of peaks showed selective accessibility in photoreceptors, with lower levels of accessibility in bipolar cells, and even less in other cell types (Figure 3B, red box). While pan BCs and ON-BCs showed nearly identical open chromatin profiles, OFF-BC open chromatin patterns were somewhat divergent, with slightly more accessibility in the photoreceptor-enriched subset (red box) and less in the bipolar-enriched subset (green box) compared to ON-BC. Overall, the sum of photoreceptor and bipolar cell ATAC-seq peaks accounted for 83 percent of whole-retina DNase-seq peaks, with the remainder presumably deriving from other inner retinal cell classes. These data suggest that relatively few regions of open chromatin are truly photoreceptor-specific, and that regions enriched in bipolar cells are more likely to be accessible in other tissues, particularly in brain. For instance, compared to photoreceptors, a greater proportion of bipolar cell peaks corresponded to DNase-seq peaks in brain (47% vs 37%). Finally, direct comparison of ATAC-seq peaks from photoreceptor and bipolar cells identified 55,402 differentially accessible regions (FDR < 0. 05), 75% of which are more accessible in bipolar cells (Supplementary file 7). While we observed significant differences in global chromatin accessibility between photoreceptors and bipolar cells, the open chromatin profiles of ON and OFF bipolar cells were largely similar, consistent with the high degree of similarity between their transcriptomes. Specifically, only 4263 peaks were differentially accessible between ON and OFF bipolar cells. Of note, 79% (3359) of these differential peaks were more accessible in ON bipolar cells. When examining loci surrounding genes expressed in both ON and OFF bipolar cells, we found that ON and OFF subclasses typically had similar open chromatin profiles (e. g., Vsx2, Figure 3C). In contrast, some ON- or OFF-specific genes exhibit cell subclass-specific patterns of chromatin accessibility (e. g., Isl1 and Esam, Figure 3D–E). We found an overall correlation between accessibility and associated gene expression (Figure 5—figure supplement 1) but did not see this pattern at all gene loci (e. g., Grik1, Figure 3F). Having compared global accessibility between photoreceptor and bipolar cells, we next sought to compare them in terms of ‘cis-regulatory grammar’, which we define as the number, affinity, spacing and orientation of TF binding sites within the open chromatin regions of a given cell type or class. To begin, we assessed all 319 TF binding site motifs from the HOMER database for enrichment within bipolar cell open chromatin regions (Heinz et al., 2010). The most highly enriched motifs within enhancers corresponded to CTCF, K50 HD, E-box, nuclear receptor, and MADS box motifs (Supplementary file 8). All of these motifs were previously shown to be among the most enriched motifs in photoreceptor ATAC-seq peaks as well (Hughes et al., 2017). The similarity in the patterns of TF binding site enrichment between photoreceptors and bipolar cells can be better understood in the context of known patterns of TF expression in these cell classes. Specifically, the K50 HD TFs OTX2 and CRX are master regulators of gene expression programs in photoreceptor and bipolar cells. Likewise, bHLH TFs (which recognize E-box motifs) play roles in fate specification and maintenance of both photoreceptor and bipolar cell gene expression programs, as described in the Introduction. Finally, enrichment of ZF motifs recognized by CTCF in both cell classes is in line with reports that CTCF motifs often lie in ubiquitously accessible chromatin regions, where CTCF recruitment is thought to mediate interactions between promoters and enhancers, among other functions (Splinter et al., 2006; Rao et al., 2014; Song et al., 2011). Despite the overall similarity between the cis-regulatory grammars of bipolar cells and photoreceptors, there are notable quantitative differences in motif enrichment between the two cell classes. To systematically identify these differences, we compared the proportion of peaks containing each of the 319 motifs between pan-bipolar cells and each photoreceptor cell type (Supplementary file 8). The most differentially enriched motifs corresponded to those with the highest enrichment in each cell class and are summarized in Figure 4. Although both photoreceptors and bipolar cells showed marked enrichment for K50 HD motifs, these motifs were more enriched in photoreceptors (Figure 4). Conversely, E-box motifs were more enriched in bipolar cells than photoreceptors. The most striking difference in TF binding site enrichment between the two cell classes was the enrichment of both monomeric and dimeric Q50 HD motifs in photoreceptor open chromatin regions and their lack of enrichment in bipolar regions (Figure 4). The most well-characterized Q50 HD TF expressed in photoreceptors is RAX, which is required for cone gene expression and survival (Irie et al., 2015). In contrast, bipolar cells express multiple Q50 HD TFs (VSX2, VSX1, ISL1, LXH3, LHX4, AND SEBOX) (Shekhar et al., 2016). VSX2 is required for bipolar cell development and is expressed in all mouse bipolar cell types throughout development and into adulthood (Livne-Bar et al., 2006; Liu et al., 1994). The paradoxical absence of Q50 HD motif enrichment in bipolar open chromatin regions despite the presence of multiple Q50 HD TFs in this cell class may be explained by the observation that VSX2 acts as a repressor of photoreceptor CREs (Dorval et al., 2006). The lack of Q50 motif enrichment in bipolar cells could be due to selective repression of photoreceptor-specific open chromatin regions by VSX2, which, in turn, prevents ectopic expression of photoreceptor genes in bipolar cells, a possibility that we will return to in the final section of the Results. To further compare the cis-regulatory grammars of bipolar cells and photoreceptors, we examined TF binding site co-occurrence and spacing within each cell class. For this analysis we compared a combined list of enhancer regions from rod and cones to that of bipolar cells. As expected, motif pairs enriched in specific peak sets tended to include motifs that showed the highest individual enrichment in the same cell type. Likewise, differentially enriched motif pairs included one or more differentially enriched motifs, such as K50 and Q50 HD motifs in photoreceptors and bHLH motifs in bipolar cells (Figure 4—figure supplement 1A–B). Of note, while we identified specific motif pairs enriched in each cell type, the frequencies of these pairs are consistent with an independent model (i. e., the number of occurrences of motif pairs is approximately what would be expected given the number of occurrences of each individual motif and assuming a random distribution of motifs across peaks). As with chromatin accessibility, enrichment of motif pairs is highly similar between ON and OFF bipolar cells, with nominally differentially enriched pairs showing similar proportions (Figure 4—figure supplement 1C). Finally, to investigate preferences in spacing and orientation between pairs of motifs, we plotted the density of highly enriched motifs (those depicted in Figure 4) centered on regions flanking K50 and Q50 HD motifs in each peak set. As described previously for photoreceptors, spacing and orientation preferences in bipolar open chromatin regions were minimal (Figure 4—figure supplement 2) (Hughes et al., 2017). Thus, the primary differences in the cis-regulatory grammar of the two cell classes appears to be the degree of HD and E-box motif enrichment. We next sought to determine the extent to which photoreceptor- and bipolar-enriched open chromatin regions correlate with cell type-specific gene expression. To this end, we assigned each of the 55,402 regions identified as differentially accessible between photoreceptor and bipolar cells to a candidate target gene based on proximity to the nearest transcription start site and compared mean RNA-seq expression values for the assigned genes. As described in previous studies, we observed a modest correlation between enhancer accessibility and gene expression, and a more robust correlation between promoter accessibility and gene expression (Figure 5—figure supplement 1) (Hughes et al., 2017; Pastor et al., 2014; Ampuja et al., 2017; de la Torre-Ubieta et al., 2018). Our analysis of global chromatin accessibility suggested that many of the differentially accessible peaks were also open in other tissues, especially those enriched in bipolar cells compared to photoreceptors (Figure 3E). To gain a better understanding of the cell type-specific open chromatin regions that drive gene expression differences between these two cell classes, we refined our analysis to exclude peaks shared with non-retinal cell types. We identified 8435 enhancer regions which are accessible either in photoreceptors or bipolar cells, but not accessible in brain, liver or splenic B cells (Figure 5A). This set includes 1291 regions that are open in both photoreceptors and bipolar cells, and 7144 regions that are differentially accessible between the two cell classes (Figure 5A). We found that ~ 46% (3,270) of the differentially accessible peaks were more open in bipolar cells. Thus, most of the bipolar cell-enriched regions identified in the previous section were also accessible in one or more non-retinal tissues. As with the enhancer regions from the unfiltered list, assigning genes to this more retina-specific set of differentially accessible regions also shows a correlation between accessibility and gene expression (Figure 5B). To visualize this association and identify the peaks that underly it, we plotted all 8435 peaks according to fold-change differences in accessibility and gene expression between bipolar cells and rods (Figure 5C) and between bipolar cells and blue cones (Figure 5—figure supplement 1D). We then selected for further analysis those peaks that exhibited correlated accessibility and gene expression in photoreceptors (highlighted in red or blue in Figure 5C and Figure 5—figure supplement 1D; n = 901) or bipolar cells (highlighted in green in Figure 5C and Figure 5—figure supplement 1D; n = 833). These differentially enriched peaks represent strong candidates for CREs that mediate the gene expression differences between the two cell classes. Indeed, the photoreceptor peak set contains known enhancers responsible for driving cell type-specific expression of Rhodopsin (Rho) and components of the rod-specific phototransduction cascade (Corbo et al., 2010; Nie et al., 1996), while the bipolar peak set contains a known enhancer that drives Vsx2 expression in bipolar cells (Kim et al., 2008). To gain insight into the possible biological functions of these peaks we used GREAT to assign biological annotations based on nearby genes (McLean et al., 2010). As was found with the unfiltered datasets, photoreceptor peaks are linked with genes associated with light sensation, whereas bipolar peaks are linked to genes involved in more generic neuronal functions (Figure 5—figure supplement 2). Next, we asked whether the patterns of TF binding site enrichment observed with aggregate sets of ATAC-seq peaks from each cell class would be preserved within the retina-specific peak sets associated with correlated gene expression. We compared photoreceptor-enriched regions, bipolar-enriched regions, and regions that share accessibility between the two cell classes which were either specific to the retina (Figure 5A, ‘shared retina’ n = 1,291), or unfiltered (‘shared all’, n = 47,550). We found that K50 HD motifs were enriched in both shared and cell class-selective regions, but to a lesser extent in regions specifically enriched in bipolar cells. The bipolar-selective regions were markedly enriched for E-box motifs but completely lacked enrichment for Q50 HD motifs (Figure 5D). Conversely, photoreceptor-selective regions were enriched for Q50 HD motifs, but lacked E-box motif enrichment. Peaks that were shared between the two cell classes showed an intermediate pattern of motif enrichment, highlighting the roles of both HD and bHLH TFs in regulating the gene expression programs of each class. Of note, CTCF enrichment was absent in all but the unfiltered peak set, suggesting that the CTCF enrichment observed in Figure 4 is attributable to ubiquitously accessible peaks. Taken together, these findings suggest that differential enrichment of Q50 HD and E-box motifs are the key features that distinguish the cis-regulatory grammars of photoreceptors and bipolar cells. Given the critical roles of HD TFs in the regulation of photoreceptor and bipolar gene expression, we further investigated the role of K50 and Q50 motifs in the cis-regulatory region upstream of Gnb3. Gnb3 encodes the β subunit of a heterotrimeric G-protein required for cone phototransduction as well as ON bipolar cell function (Dhingra et al., 2012). Gnb3 is expressed in rods, cones, and bipolar cells during early postnatal retinal development in the mouse. Selective repression of Gnb3 in rods by the nuclear receptor TF NR2E3 results in a cone + bipolar pattern after postnatal day 10 (Haider et al., 2009; Corbo and Cepko, 2005). We focused on an 820 bp region around the TSS of Gnb3 which drives robust expression in rods, cones, and bipolar cells when electroporated into early postnatal mouse retina. This region lacks Q50 motifs but contains five K50 HD motifs of varying affinity which occur in two clusters, one immediately upstream of the TSS (−65 bp) and the other more distally (−350 bp) (Figure 6A). To evaluate the role of these five K50 motifs in mediating photoreceptor and bipolar expression, we engineered reporter constructs in which each of the five motifs was individually inactivated by mutating the TAAT core to TGGT. We then introduced wild-type and mutant reporters into mouse retinal explants via electroporation and compared expression levels after 8 days. Mutations in K50 motifs 2,4 and 5 resulted in coordinate loss of expression in both photoreceptor and bipolar cells, indicating that these motifs are required for reporter expression in both cell classes (Figure 6B, D). Conversely, mutations in site 1 or 3 had no effect on expression in either cell class (Figure 6B, D). Binding site affinity did not correlate with expression, as site 3 has a higher predicted affinity than sites 4 or 5. Thus, the Gnb3 promoter region contains both essential and nonessential K50 motifs, underscoring the critical role for these shared motifs in both photoreceptors and bipolar cells. Next, we sought to determine the effect of introducing Q50 motifs into the Gnb3 promoter region. For these experiments, reporters were introduced into newborn mouse retinas via in vivo electroporation and harvested for histologic analysis after 20 days. First, we electroporated identical wild-type sequences driving both DsRed and GFP to confirm that essentially all photoreceptors and bipolar cells received both constructs (Figure 6C). Next, we compared the expression of a Gnb3 promoter containing mutations in K50 motifs 1 and 3 to that of a wild-type promoter, confirming that elimination of both of these sites has no effect on expression in either photoreceptors or bipolar cells (Figure 6C, D and Figure 6—source data 1). To test the effect of introducing Q50 motifs into the Gnb3 promoter region, we replaced K50 motifs 1 and 3 with a Q50 motif (TAATTA), both individually and in combination. Whereas introduction of a Q50 motif into site one had no apparent effect, replacement of site three caused a selective decrease in bipolar expression with no change in photoreceptor expression. When we introduced Q50 motifs into both sites, reporter expression was markedly reduced in bipolar cells, with no effect on photoreceptor expression (Figure 6C, D and Figure 6—source data 1). These data, along with previous reports of VSX2-mediated repression of photoreceptor-specific promoters and enhancers, suggest that Q50 motifs play an important role in mediating repression of photoreceptor genes in bipolar cells. In this study we generated open chromatin maps and transcriptome profiles of mouse bipolar cells, including FACS-purified ON and OFF bipolar cell populations, and compared them to analogous data from rod and cone photoreceptors. We found that photoreceptors and bipolar cells differ in the expression of thousands of genes and yet have very similar cis-regulatory grammars. The key cis-regulatory differences that distinguish the two cell classes are the preferential enrichment of Q50 HD motifs in open chromatin regions associated with photoreceptor-specific gene expression and a corresponding enrichment of E-box motifs in chromatin associated with bipolar-specific expression. The cellular features and transcriptional mechanisms shared by photoreceptors and bipolar cells have prompted speculation that these two sister cell types arose from a single ancestral photoreceptor cell type via a process of progressive cellular divergence (Arendt, 2008; Lamb, 2013). We propose that the elimination of Q50 motifs from bipolar-specific CREs likely played a key role in differentiating the bipolar transcriptome from that of photoreceptors during early stages of vertebrate retinal evolution. Alternatively, given the multiplicity of K50 HD binding sites within both photoreceptor and bipolar cell regulatory elements, a subset of photoreceptor-specific elements may have emerged through the simple conversion of K50 (TAATCC) to Q50 (TAATTA/G) sites. Prior studies of individual photoreceptor CREs showed a role for the Q50 HD TF, VSX2, in selectively repressing photoreceptor genes in bipolar cells (Livne-Bar et al., 2006; Dorval et al., 2006). Our results generalize this conclusion, suggesting that VSX2 plays a genome-wide role in silencing photoreceptor gene expression in bipolar cells. A similar role for VSX2 has recently been described in the spinal cord, where closely related progenitor cells give rise to either motor neurons or V2a interneurons (Clovis et al., 2016). VSX2 promotes V2a identity by directly repressing the motor neuron gene expression program and by competing for Q50 sites at motor neuron enhancers. Thus, in both retina and spinal cord, expression of VSX2 promotes interneuron fate at the expense of the alternative neuronal (photoreceptor or motor neuron) cell type. These parallels suggest that transcriptional repression by cell type-specific TFs such as VSX2 represent a common mechanism for differentiating the gene expression programs of two closely related cell types. In addition to differential enrichment of Q50 sites, we also observed enrichment of E-box motifs in regions associated with bipolar-specific expression (Figure 5C). The lack of corresponding enrichment in regions associated with photoreceptor-specific expression suggests that bHLH TFs also play an important role in distinguishing the gene expression programs of photoreceptor and bipolar cells. It is important to note that this finding does not contradict known roles for bHLH TFs in photoreceptors, as E-box motifs are also enriched within the complete set of photoreceptor ATAC-seq peaks (Figure 4), as well as in the subset that exhibits shared accessibility with bipolar cells (Figure 5C). The role of bHLH TFs in establishing the cellular identity of both classes is further demonstrated by multiple loss-of-function studies (Tomita et al., 2000; Bramblett et al., 2004; Feng et al., 2006; Huang et al., 2014; Pennesi et al., 2003). In contrast to the differences between photoreceptors and bipolar cells, ON and OFF bipolar cell populations displayed striking similarities in their cis-regulatory landscape and gene expression profiles. In a paper that appeared after our profiling studies had been performed, Shekhar et al. (2016) documented the single-cell expression profiles of individual bipolar cell types and showed that the transcriptomes of ON and OFF cone bipolar cells are more similar to each other than to that of rod bipolar cells (RBCs). This finding implies that the ON bipolar population analyzed in the present study represents a grouping of distinct bipolar cell types (RBC and cone ON BC), which should be separated for a more informative comparison. Despite this drawback, our analysis indicates that there are relatively few differences in chromatin accessibility between bipolar cell types. We estimate that RBCs constitute nearly 60% of the cells in our ON population (RBCs compose 56% of ON bipolar cells identified in Shekhar et al., 2016, and ~ 57% of those identified as ON BCs by Wässle et al., 2009). Thus, much of the signal in our ON bipolar RNA-seq and ATAC-seq data likely derives from RBCs. It remains to be determined whether open chromatin profiling of individual bipolar subtypes will reveal additional differences in their epigenomic landscapes beyond those reported here. We found an imperfect correlation between chromatin accessibility and gene expression in bipolar cells. For example, ATAC-seq peaks upstream of Grik1 are open in ON bipolar cells despite an absence of Grik1 expression in this cell type (Figure 3F). We observed similar instances of discordance between chromatin accessibility and gene expression in our previous analysis of rod and cone photoreceptors (Hughes et al., 2017), and other groups have documented this discrepancy in other cell types (de la Torre-Ubieta et al., 2018; Lara-Astiaso et al., 2014; Starks et al., 2019). Presumably, transcriptional activity in these instances requires expression of additional cell type-specific factors (Heinz et al., 2015), perhaps most clearly demonstrated in developmental contexts, wherein accessibility is frequently established prior to the onset of transciption (Lara-Astiaso et al., 2014). Support for the idea that bipolar cells diverged from photoreceptors via progressive partitioning of cellular function is provided by the existence of cell types in the retinas of non-mammalian vertebrates with features intermediate between those of mammalian photoreceptors and bipolar cells. In some turtle species ~ 30% of the cell bodies in the photoreceptor layer (the outer nuclear layer) belong to bipolar cells, not photoreceptors (Tauchi, 1990). These so-called ‘displaced bipolar cells’ possess an inner segment-like process that extends to the outer limiting membrane and contains abundant mitochondria and even a sensory-type (‘9 + 0’) cilium (Figure 7A, B). Thus, displaced bipolar cells closely resemble typical photoreceptors except that they lack an outer segment, possess dendrites in the outer plexiform layer, and synapse directly onto retinal ganglion cells. Another intermediate type of bipolar cell occurs in nearly all non-mammalian vertebrate classes and even in some mammalian species (Hendrickson, 1966; Locket, 1970; Quesada and Génis-Gálvez, 1985; Young and Vaney, 1990). This bipolar type has a nucleus localized to the inner nuclear layer, but retains an inner segment-like structure (Landolt’s club), which extends from the cell’s dendritic arbor to the outer limiting membrane and contains abundant mitochondria and a sensory-type cilium (Figure 7A) (Hendrickson, 1966; Locket, 1970; Quesada and Génis-Gálvez, 1985). We suggest that displaced bipolar cells and those with a Landolt’s club represent ‘transitional forms’ on the evolutionary path from photoreceptor to typical bipolar cell. The existence of these transitional forms suggests that bipolar cells may have evolved via the stepwise repression of discrete gene modules required for the development of individual cellular features, or ‘apomeres’, that are specific to photoreceptors (Arendt et al., 2016). If this evolutionary model is correct, then how can we account for the co-existence of ‘transitional’ bipolar cell types and ‘conventional’ bipolar cells in a single retina? One testable hypothesis is that VSX2 may be expressed at lower levels in transitional bipolar cell types, thereby permitting expression of additional photoreceptor gene modules and their corresponding apomeres. Alternatively, it is possible that additional activating Q50 HD TFs are expressed in transitional bipolar types, and these TFs can overcome VSX2-mediated repression of selected photoreceptor gene modules. Indeed, we and others have found that multiple Q50 HD TFs are expressed in subsets of mouse bipolar cells (Shekhar et al., 2016). In addition, there is evidence that transitional bipolar cell types with Landolt’s club may exist in the mouse (Rowan and Cepko, 2004). Thus, individual bipolar cell types may control the number of photoreceptor apomeres they express by modulating the balance of activating and repressing Q50 HD TFs in their nuclei. The evolutionary divergence of bipolar cells from photoreceptors likely required coordinated changes in both cis-regulatory grammar and HD TF expression. The Q50 HD TF, RAX, is expressed in developing vertebrate rods and cones and is required for normal activation of photoreceptor gene expression in mice (Irie et al., 2015; Rodgers et al., 2018). The expression of a RAX homolog in the photoreceptors of the tadpole larva of the protochordate, Ciona intestinalis, suggests a primordial role for this Q50 HD TF in activating photoreceptor gene expression in chordates (Cao et al., 2019). These data suggest that both K50 and Q50 motifs were present in the CREs of the ancestral vertebrate photoreceptor prior to the evolutionary emergence of bipolar cells, and that both K50 (OTX2 and CRX) and Q50 (RAX) HD TFs were required for gene activation in that ancestral cell type (Figure 7C). In this context, the emergent expression of a repressive Q50 HD TF (VSX2) in a primordial bipolar cell would have permitted selective repression of CREs containing Q50 motifs, allowing the cis-regulatory landscape of the two nascent cell types to begin to diverge. Maintaining expression of selected ‘photoreceptor’ genes in bipolar cells (e. g., Gnb3) would then have required the elimination of Q50 motifs from the cis-regulatory regions of those genes. These evolutionary considerations suggest that the modern vertebrate retina arose from an ancestral retina in which photoreceptors directly synapse onto projection neurons (i. e., ganglion cells) without an intervening layer of interneurons (left side of Figure 7C). Two lines of evidence suggest that such a retina may have existed. First, the retina of the hagfish, the most primitive extant vertebrate, reportedly has photoreceptors that directly synapse onto projection neurons (i. e., ganglion cells) (Lamb, 2013; Locket and Jørgensen, 1998). Second, some vertebrate species (including reptiles, amphibians, and larval lamprey) have an unpaired, median ‘parietal eye’ developmentally related to the pineal gland, which contains photoreceptors that directly synapse onto ganglion cells (Eakin, 1973; Dodt, 1973; Mano and Fukada, 2007; Solessio and Engbretson, 1993). It is possible that the parietal eye evolved from the midline ‘eye’ of a protochordate ancestor, akin to the present-day ascidian larva. The simple lateral eyes of a hagfish-like vertebrate ancestor may then have emerged via co-option of the gene networks required for parietal eye development. Subsequently, bipolar cells may have arisen in the lateral eyes of early vertebrates via subtle changes in cis-regulatory grammar and TF expression, paving the way for the emergence of the sophisticated interneuronal circuitry found in present-day vertebrate retinas. All animal experiments were carried out in accordance with the regulations of the IACUC at Washington University in St. Louis. Retinal dissociation and FACS were carried out using Otx2-GFP or Otx2-GFP; Grm6-YFP mice. Otx2-GFP mice were heterozygous for a GFP cassette inserted at the C-terminus of the endogenous Otx2 locus (Fossat et al., 2007). The Grm6-YFP line harbors a YFP transgene driven by the Grm6 promoter (Morgan et al., 2011). All electroporation experiments were carried out in CD-1 mice. Following dissection, retinas of mice aged 6–8 weeks, or 3 months (for one biological replicate of Figure 1—figure supplement 2) were dissociated with papain as described previously (Trimarchi et al., 2007). Briefly, two retinas were incubated in 400 µl of calcium/magnesium free Hanks’ Balanced Salt Solution (HBSS) (Thermo Fisher) containing 0. 65 mg papain (Worthington Biochem) for 10 min at 37°C. Cells were then washed in a DMEM (Thermo Fisher) solution containing 100 units DNase1 (Roche) and incubated an additional 5 min at 37°C. Cells were then resuspended in 600 µl of sorting buffer (2. 5 mM EDTA, 25 mM HEPES, 1% BSA in HBSS) and used directly for sorting. Cells were sorted on a FACS Aria-II (BD biosciences) with gates based on forward scatter, side scatter, and GFP fluorescence. OFF bipolar cell populations (Otx2-GFP+; Grm6-YFP-) were immediately sorted a second time to increase purity. An 820 bp region encompassing part of the Gnb3 5’ UTR and upstream sequence was amplified from mouse genomic DNA. Site-directed mutagenesis by overlap extension was used to modify K50 sites (Ho et al., 1989). The resultant PCR products were digested and ligated into GFP or DsRed reporter vectors derived from pCAGGS (Hsiau et al., 2007). After verification by sequencing, plasmid DNA was resuspended in PBS at a concentration of ~ 6–7 µg/µl prior to injection. All primers are listed in Supplementary file 2. In vivo subretinal injection and electroporation of newborn CD1 mice was performed as previously described (Matsuda and Cepko, 2004). Briefly, mice were first anesthetized on ice. A 30-gauge needle was then used to incise the eyelid and puncture the sclera, and a Hamilton syringe with a 33-gauge blunt-tipped needle was used to inject the DNA into the subretinal space. Tweezer electrodes placed across the head were then used to electroporate with five square pulses of 80 volts and 50 millisecond duration at 950 millisecond intervals. Explant electroporation was carried out as described previously (Hsiau et al., 2007), except that the electroporation chamber contained a solution of 0. 5 µg/µl DNA in PBS. Eyes were removed at P21, punctured with a 26-gauge needle, and incubated in 4% paraformaldehyde for 5 min before dissection to remove the cornea. The lens was removed, and eyes were then incubated for an additional 45 min in 4% paraformaldehyde. Eye cups were next washed in PBS and incubated overnight at 4°C in 30% sucrose-PBS. The following day, eye cups were incubated in a 1: 1 mixture of OCT compound (Sakura) and sucrose-PBS before being flash frozen in OCT and stored at −80°C. Retinal sections of 14 µm were cut using a cryostat (Leica CryoCut 1800), mounted on Superfrost Plus slides (Fisher), and stored at −20°C. Prior to placement of cover slips, slides were washed with PBS to remove OCT. The sections were then stained with DAPI, and coverslips were mounted using Vectashield mounting medium (Vectorlabs). Retinal sections were imaged on a Zeiss 880 laser-scanning confocal microscopy in the Washington University Center for Cellular Imaging (WUCCI) Core. Transposition and library preparation from sorted cell populations were performed as previously described (Buenrostro et al., 2015). Briefly, 30,000–100,000 sorted cells were pelleted at 500 G and washed twice in ice-cold PBS before lysis. Transposition reactions were incubated at 37°C for 30 min and purified using a Qiagen MiniElute PCR Purification kit. Libraries were amplified with Phusion High-Fidelity DNA Polymerase (NEB). Cycle number was calibrated by a parallel qPCR reaction. Gel electrophoresis was used to assess library quality, and final libraries were quantified using KAPA Library Quantification Kit (KAPA Biosystems). Equimolar concentrations of each library were pooled and run on an Illumina HiSeq2500 to obtain 50 bp paired-end reads. ATAC-seq and RNA-seq reads from bipolar cell populations were processed in an identical manner to those previously obtained from rod and cone photoreceptor cells (Hughes et al., 2017). ATAC-seq reads were aligned to the GRCm38/mm10 mouse genome assembly using Bowtie2 (v2. 3. 5) with a max fragment size of 2000 (Langmead and Salzberg, 2012). Alignments were filtered using SAMtools (v1. 9) (Li et al., 2009), PCR duplicates were removed using Picard (v2. 19. 0) (https: //broadinstitute. github. io/picard/), and nucleosome-free reads were selected by removing alignments with an insertion size greater than 150 bp. Peaks were called using MACS2 (v2. 1. 1) (Zhang et al., 2008) and annotated with HOMER (v4. 8) (Heinz et al., 2010). DNase-seq datasets generated by ENCODE (ENCODE Project Consortium, 2012) were downloaded as FASTQ files from https: //www. encodeproject. org/ and processed in the same manner as ATAC-seq data. RNA-seq reads were aligned to the GRCm38/mm10 using STAR (v2. 7. 0d) (Dobin et al., 2013), with an index prepared for 50 base-pair reads and the RefSeq gene model, and read counts were calculated using HTSeq (v1. 9) (Anders et al., 2015). All datasets are listed in Supplementary file 3. Single-cell data from Shekhar et al. were downloaded from the Gene Expression Omnibus (GSE81904) and processed as described by the authors to yield a digital expression matrix of normalized counts for each gene for each cell as well as cluster assignments for each cell. ‘Pseudo-bulk’ expression estimates were then generated by taking the weighted average of counts for each gene across cells belonging to clusters that constituted bulk populations of interest (RBC, BC5A, BC5C, BC5D, BC6, BC7, and BC8/BC9 for ON BCs; BC1A, BC1B, BC2, BC3A, BC3B, BC4 for OFF BCs; and these clusters combined for pan BCs). Finally, pseudo-bulk expression estimates were re-scaled to match bulk expression estimates. Motif enrichment, co-enrichment, and spacing analyses for ATAC-seq and DNase-seq datasets were performed as described previously using HOMER (v4. 8) (Hughes et al., 2017; Heinz et al., 2010). Differential motif enrichment was determined using a test of equal proportions (R stats v3. 5. 3) to compare each motif between pan BC and rods, blue cones or green cones. The top motifs across the three comparisons were manually filtered for redundancy and are shown in Figure 4. Motif co-occurrence analysis was performed using a list of 66 non-redundant motifs (Hughes et al., 2017) to which the motif for LHX3 (representing a Q50 HD motif) was manually added. For purposes of the analysis, peaks from rod, green cones and blue cones were merged to obtain a ‘photoreceptor’ peak list, while those from pan-, ON- and OFF-bipolar cells were merged to create a ‘bipolar cell’ peak list. Enrichment for co-occurrence was calculated by taking the log2 (observed pairs +1/expected pairs+1). Expected frequency of individual pairs was estimated from the counts for each motif within the pair (motif one count × motif two count ÷ number of total peaks). Differential enrichment between tissues was calculated with a Fisher’s exact test. Motif spacing was analyzed for the top enriched peaks shown in Figure 4. The same set of peaks for co-occurrence were centered on individual K50 or Q50 motifs, and the density of flanking secondary motifs was plotted on either strand. DESeq2 (v1. 14. 1) (Love et al., 2014) was used to test for differential expression or differential accessibility using a log2 fold-change threshold of 1 and an FDR of 0. 05. For comparison of ATAC-seq data with DNase-seq data from non-retinal tissues (Figure 5), photoreceptors were collapsed into a single level. Differentially expressed genes are listed in Supplementary file 5, and differentially accessible regions are listed in Supplementary file 7. For each comparison (i. e. ON versus OFF, pan BC versus rod), gene expression stemming from low-level contamination of bipolar cell populations with either rod or cone photoreceptors was filtered out. When comparing pan BC to photoreceptor populations, potential contaminating genes from the alternate photoreceptor type (i. e. rod genes identified as enriched in pan BC versus blue cone) were identified as those highly expressed (>16 fold) and specific to rod compared to blue cone, and also more highly expressed in rod compared to pan BC (at least four-fold). In comparing ON and OFF bipolar cells, genes enriched in each bipolar cell population were filtered for those which were also identified as highly specific to either photoreceptor population compared to the enriched bipolar cell type. For example, genes increased in OFF- compared to ON-bipolar cells were filtered for genes that were also highly enriched (>16 fold) in rods and blue cones compared to OFF bipolar cells. In total, 38 genes were filtered from those identified as enriched in pan BC compared to photoreceptors, and 39 genes were filtered from the ON versus OFF comparison (12 from the ON-enriched, 27 from the OFF-enriched). These genes include those known to be expressed at very high levels in either rod or cone photoreceptors (Corbo et al., 2007). Sorted bipolar cell populations were resuspended in 500 µl TRIzol reagent (Invitrogen), and RNA was isolated according to manufacturer’s instructions. Prior to sequencing, RNA quality was analyzed using an Agilent Bioanalyzer. cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing-HV (Clontech) per manufacturer’s instructions. cDNA was fragmented using a Covaris E210 sonicator using duty cycle 10, intensity 5, cycles/burst 200, time 180 s. cDNA was blunt-ended, an ‘A’ base added to the 3′ ends, and Illumina sequencing adapters were ligated to the ends of the cDNAs. Ligated fragments were amplified for 12 cycles using primers incorporating unique index tags. Replicate libraries from each bipolar cell population were pooled in equimolar ratios and sequenced on an Illumina HiSeq 3000 (single-end 50 bp reads). For qPCR, RNA samples were treated with TURBO DNase (Invitrogen) and cDNA was synthesized with SuperScript IV (Invitrogen) and oligo (dT) primers according to manufacturer’s instructions. For Figure 1C, expression was normalized to the average of reference genes Gapdh, Sdha, Hprt, and Pgk. For Figure 1—figure supplement 2, expression was normalized to Gapdh alone. Primers for Grm6, Gnat1, Lhx1, Pax6, Rlbp1, Slc17a6, Vsx2, Grik1, Tacr3, Isl1, and Lrrtm1 are listed in Supplementary file 2. | Title: Cis-regulatory basis of sister cell type divergence in the vertebrate retina Summary: Humans see the world through a light-sensitive tissue at the back of the eye called the retina, which is made up of three layers that each contain specific cell types. The layers form a circuit, with light-sensing photoreceptor cells in the outermost layer connected to bipolar cells in the middle layer, which connect to the brain via specialized cells in the innermost layer. Photoreceptors and bipolar cells share similar characteristics and are thought to be'sister cells' which evolved from a common ancestral cell type. However, it is not well understood how these two cells types diverged during evolution. Every cell type has a specific role, which is largely determined by the set of genes that it switches on or off. Specialized regions of DNA, called enhancers, determine whether a gene is turned on or off in a particular cell type. In each cell, DNA strands are bundled together with proteins into a coiled structure known as chromatin. In some cells, a particular enhancer may be'shut down' and rendered inactive on account of being tightly packed within chromatin. Whilst in other cells, the same enhancer may be 'open' and ready for action. For a given cell type, which genes are turned on is determined, in part, by which enhancers are open. One way to distinguish between cells is by examining how their chromatin is packaged to see which enhancers are open. Researchers have previously characterized the chromatin structure of photoreceptor cells, but the structure of chromatin in bipolar cells, and how it compares to that of photoreceptors, remained unknown. Now, Murphy et al. have examined the chromatin profile of bipolar cells from the mouse retina in order to gain a better understanding of how these two cell types may be evolutionarily related. The analysis revealed that although bipolar and photoreceptor cells switch on different sets of genes, the enhancers open in each cell type are very similar. Despite this similarity, Murphy et al. were able to detect subtle differences in short sequences of DNA, known as motifs, present in bipolar and photoreceptor enhancers. Further experiments showed that one of these motifs may be responsible for turning photoreceptor genes off in bipolar cells. This motif therefore appears to play a critical role in distinguishing photoreceptors from bipolar cells. This comparison of photoreceptor and bipolar cells has provided a possible mechanism whereby photoreceptor and bipolar cells diverged in evolution from a single common ancestral cell type. This insight may help explain how complex organisms with many cell types may have evolved from a single-cell ancestor long ago. | 14,685 | 578 | lay_elife | en |
Summarize: Mothers who sleep on their back or right-hand side on the night before giving birth are twice as likely to have a stillborn child compared with those who slept on their left, according to a study. Researchers found that the risk of stillbirth for those sleeping on the left side was 1.96 per 1,000 births, and 3.93 per 1,000 births for any other position. Tomasina Stacey, a midwifery lecturer at University of Auckland, who led the study, cautioned pregnant women not to be over-concerned by the finding. "It was an observational study, not one that can show cause and effect – all it does is show an association. It would be premature to jump up and down and say that everyone has got to sleep on their left. It's a starting point for future research." In the UK, there were more than 4,000 stillbirths in 2009; almost a third of these occurred late in the pregnancy term, after 37 weeks. "Stillbirth is a much bigger problem than most people realise, and many are unexplained," said Stacey. "In the UK and New Zealand, there has been very little change in the rate in the last 15 years." For the study, published in the British Medical Journal, she and her colleagues spoke to 155 women who had stillborn babies in Auckland between 2006 and 2009. They were asked about, among other things, their positions on going to sleep and waking up in the final weeks and month before the death of their baby. For the control group, the researchers randomly selected two women who were at the same number of weeks in their pregnancy and questioned them about their sleep in the days and month before the interview. The researchers wanted to examine the effects of sleep disorders in pregnant women, such as sleep apnea or snoring, which could be a factor in stillbirths, because these conditions can reduce the amount of oxygen getting to the baby. Instead, the study showed that sleep position on the night before birth was the decisive factor. Stacey said a possible explanation could be that when a woman does not sleep on her left side, the foetus could compress her inferior vena cava taking blood back to the heart, and a reduced flow means less oxygen gets to the mother's – and in turn the baby's – other organs. "If you've got a strong healthy baby, then the slightly reduced blood flow is fine; but if you've got a baby that is compromised in some other way, then maybe that reduction in blood flow can be the tipping point," said Stacey. The left lateral position is known in clinical settings to be the optimal position for looking after a woman in labour and is associated with foetal wellbeing in labour, she added. An accompanying editorial in the journal by Lucy Chappell of King's College London says "any simple intervention that reduces the risk of stillbirth would be extremely welcome", but argues that the finding should be treated with caution. "A forceful campaign urging pregnant women to sleep on their left side is not yet warranted," she said, adding that the study needed to be replicated to be conclusive. Alexander Heazell, of the University of Manchester School of Medicine, also pointed to a weakness – the fact that mothers were asked to recall their sleep position 25 days after a stillbirth. Stacey said her advice to those women who found it uncomfortable to sleep on their left was "not to worry about it too much". But, she added, at a population level, the associations between position and stillbirth might be important. "We could make a difference to the stillbirth rate by encouraging people to sleep on their left." By Steven Reinberg HealthDay Reporter TUESDAY, June 14 (HealthDay News) -- For pregnant women, reducing the risk for stillbirth may be as simple as sleeping on their left side, New Zealand researchers suggest. In fact, women in the study who didn't sleep on their left side had twice the risk of having a stillborn infant, the researchers noted. Overall, "the increase in risk [from right-sided sleeping] is small for the individual," stressed lead author Tomasina Stacey, a graduate student in the department of obstetrics and gynecology at the University of Auckland. However, "as many women do not sleep on their left side in late pregnancy, it may have an important impact on a population level," she reasoned. "This is a new and potentially exciting hypothesis, but further research is required before all women are advised to sleep on their left side in late pregnancy," Stacey concluded. Experts say improved blood flow to the fetus might play a role in any benefit of left-sided sleep, although that remains a theory. The report is published in the June 14 online edition of the BMJ. For the study, the researchers interviewed 155 women who delivered a stillborn baby at at least 28 weeks' gestation. They compared these women with 310 pregnant women with typical ongoing pregnancies. The women were asked about their sleep position during the last month of their pregnancy, the last week of their pregnancy, and on the night they believed the stillbirth occurred. In addition, the women were asked about snoring, daytime sleepiness, if they regularly slept during the day during the last month of pregnancy, how much sleep they got at night and how many times they got up to use the toilet at night. The researchers found no connection between snoring or daytime sleepiness and risk of late stillbirth. However, women who slept on their back or on their right side on the night before a stillbirth were twice as likely to have a late stillbirth, compared with women who slept on their left side. The absolute increase in risk of stillbirth to any one woman remained small, however: Stillbirths occurred in 1.96 per 1,000 cases among women who slept on their left side, versus 3.93 per 1,000 cases for those who slept in the other positions, the investigators found. Women who got up to use the toilet once or less on the last night of their pregnancy were also more likely to have a late stillbirth, compared with women who got up more frequently, the researchers added. And there was a significant link between sleeping regularly during the day and late stillbirth risk, and also longer-than-average sleep at night, the authors said. Still, Stacey was quick to point out that "an observational study such as this cannot determine cause-and-effect but it has identified an area that urgently requires further exploration." Lucy Chappell, a clinical senior lecturer in Maternal and Fetal Medicine in the division of women's health at King's College London and author of an accompanying journal editorial, said that "this is an interesting finding, but we should be cautious about making a large jump to saying that this is the cause of stillbirth. And we should not rush out to run a large campaign to say pregnant women should sleep on their left side." Chappell noted, however, that there are biological reasons that could support the finding. "We have long believed that blood flow to the baby, womb and placenta may be better when pregnant women are on their left," she said. Study coauthor Ed Mitchell, professor of child health in the department of pediatrics at the University of Auckland, believes the finding might help cut the number of stillbirths. "If this finding is real and there is a causal relationship between maternal sleep and stillbirths, then the rate of stillbirths might be reduced by a third, which is tremendous given that the rate of stillbirth has hardly changed over the last 20 years," he said. More information For more information on stillbirth, visit the March of Dimes. | Summary: A British study suggests that women who sleep on their left side on the last night of pregnancy have less risk of giving birth to a stillborn child, reports the Guardian. The researchers were quick to say their study in the British Medical Journal is only the "starting point for future research" and not a definitive piece of medical advice. "It would be premature to jump up and down and say that everyone has got to sleep on their left," says the lead researcher. In the study of 155 women, those who slept on their left side the night before giving birth had a risk of 1.96 per 1,000 births of having a stillborn child. Women who slept on their right side or on their back had a risk of 3.93 per 1,000 births. One theory: Sleeping on the left side allows improved blood flow to the fetus, notes HealthDay News. | 1,700 | 200 | multi_news | en |
Summarize: A woman whose fiance was killed in a motorcycle crash has extracted his sperm to fulfill their dream of having a baby, after strangers donated to pay for the procedure. Cameron Robinett, of Tucson, Arizona, was desperate to be a father. But last Friday, while house hunting in California, the 25-year-old motorcyclist crashed. Three days later he died in hospital. Devastated, Stephanie Lucas, 22, had just a small window of opportunity to extract sperm from her partner to hold on to the couple's dream of becoming parents. Stephanie Lucas has extracted sperm from her fiance Cameron Robinett, pictured, after he was killed in a motorcycle crash. After pleading for help to raise $6,000 to pay for the procedure, strangers donated. Last Friday, while house hunting in California, 25-year-old Mr Robinett crashed. Three days later he died in hospital leaving Miss Lucas devastated. Immediately she went online and urgently pleaded for funds to pay for the procedure. Touched by her appeal, strangers raised more than $11,000 (£7,000) in just one day - with several anonymous donations of $1,000 and $2,000. 'We were so excited about having a family,' Miss Lucas told MailOnline. 'Cameron was particularly excited. He said: "We don't need to be married to have a family. Let's start one now". 'He really wanted to be a daddy, so I'm just fulfilling his dream.' Mr Robinett had been viewing houses in California, where the couple planned to move to, when the accident happened on Friday. He was badly injured and passed away in Eden Hospital on Monday. 'He loved his motorcycle,' Miss Lucas recalled. 'I didn't mind him riding it. I knew he loved it, although I always told him to be careful. 'He was an adventurer. He was eccentric with a huge, open heart. He would give you the shirt off his back. I want to pass that onto our baby. We are so thankful to god we had time with him.' She said the procedure to extract the sperm cost around £5,600 ($9,000) and it will be kept in cold storage for up to a year. 'When I found out Cameron died, I was heartbroken,' she said. 'But I realised immediately I wanted to have his baby and I knew that I didn't have long. Miss Lucas, 22, had just a small window of opportunity to extract sperm from her partner to hold on to the couple's dream of becoming parents. Immediately she went online to plead for help to afford the procedure. Touched by her appeal, strangers raised more than $11,000 (£7,000) in just one day - with several anonymous donations of $1,000 and $2,000. 'Within hours I went online and pleaded for help on Go Fund Me. It was quick but the only way to do it.' Her message on the site, read: 'My fiance Cameron was in a motorcycle accident on Friday and was declared brain dead today. 'We wanted children very much and my family and his family and I have all decided we'd love to still make this happen. 'This baby would be truly loved and surrounded by an amazing family. 'Unfortunately the cost to extract the sperm is $6,000 that must be paid upfront by tomorrow evening, an amount we won't be able to come up with on our own. 'In lieu of flowers or gifts to honour Cameron, we're asking you to make a donation to actually bring a part of Cameron, the amazing, handsome, silly, guy that I loved with my whole heart, back to life.' Miss Lucas said she doesn't yet know when she'll implant it. 'The grief is very raw at the moment but I want his baby, our baby,' she said. She said a visit to where Mr Robinett was staying in California yesterday reinforced her decision to go through with the sperm extraction. After visiting the funeral home to pick up Mr Robinett's urn, Miss Lucas and his family visited the crash site and his home in California. Miss Lucas, said: 'We started to go through his clothes and found that each of his favourite things brought back a memory that made us laugh or smile. 'After all the stories were shared and laundry was sorted, we were about to leave and Cameron's mum said, "one more look". 'We tried to convince her otherwise but she was determined. And we are so glad, because buried in Cameron's laundry was a jersey, a Seahawks jersey. Miss Lucas said: 'I will never be able to say it enough - thank you for your support. It is everything' Miss Lucas said she does not know when she will have the sperm implanted, but said she knows Mr Robinett, who was desperate to become a father, will be looking down on her and their baby. 'But not just any jersey, a Marshawn Lynch jersey in size 0-3 months. Something we bought last Christmas for our future baby. 'Cameron only brought a few things with him to California, and one of the things he brought was this jersey. 'Our jaws dropped and then we all smiled and looked up to the heavens to say - "we hear you Cameron", and everyone has come together to make this happen. 'I will never be able to say it enough - thank you for your support. It is everything.' She told how she met Cameron, who ran Sweet Garlic Company, in 2011 through work, becoming engaged in December 2012. Now she has the support of her family including mother Debbie and Cameron's mother Loretta, to have their baby. 'I wasn't sure how our mums would feel but they were really supportive,' she said. 'I am beyond grateful for people's help. 'I was feeling so defeated when I found out the procedure would be so expensive and the money had to be paid upfront. 'I had lost not just my fiancé and my future, but my future family too. 'I've now been given the gift of a future. I think Cameron will be looking down on us.' A Fremont Police spokeswoman said: 'The motorcyclist collided with the garage of a residence. The motorcyclist was transported to a trauma center with life threatening injuries.' | Summary: Cameron Robinett was house hunting when he crashed on Friday. 25-year-old motorcyclist lost his battle in hospital on Monday. His fiance Stephanie Lucas pleaded for help to raise $6,000 for extraction. She had just one day to raise the money to allow the procedure to take place. Strangers donated more than $11,000 (£7,000) realising the couple's dream. Visiting Mr Robinett's California home yesterday Miss Lucas discovered a Seahawks jersey size 0-3 months that they had bought for their future child. She said: 'I've been given the gift of a future... Cameron will be looking down on us. Thank you for your support. It is everything' | 1,434 | 166 | cnn_dailymail | en |
Summarize: By. Mark Duell. The niece of a BBC weatherman who stalked him after claiming she was his daughter killed herself after a long campaign of harassment, an inquest heard yesterday. Former nursing sister Joanna Toner, 52, of Tiverton, Devon, had written letters to ex-presenter Bill Giles and she was charged with harassment - something for which she had previously been jailed. But the mother-of-two failed to turn up to court and police discovered her dead on her bed three days later when they went round to her barricaded home. Stalked: BBC weatherman Bill Giles (left) received letters from former nursing sister Joanna Toner (right), 52. A coroner yesterday ruled that Toner killed herself from a huge overdose of prescription drugs. In 2000, Toner, then 40, was jailed for 30 months over her nine year-long obsession that Mr Giles was her father when she was his niece. She made hoax bomb calls to the BBC and threatening calls to Mr Giles and his wife Maureen. Toner also poured lighter fuel over herself outside the BBC’s TV studios at Shepherd’s Bush, west London. In 1998 she was jailed for six months for similar offences against the senior BBC weatherman - and she was banned from contacting him, the BBC and the Met Office. She had stopped her campaign of harassment for a short while after going to jail - but resumed it again in 1999. In 2000 her lawyer said she intended to pursue her claim about her parenthood through legal channels. Toner’s obsession began in the late 1980s when her mother - who had a severe alcohol problem - claimed that Mr Giles was Joanna’s natural father. Target: Toner made hoax bomb calls to the BBC and threatening calls to Mr Giles (pictured) and his wife. Toner stayed with Mr Giles and his family when he was married to her aunt but the couple later divorced and he remarried in 1991. In 2000, Mr Giles said: ‘I feel very sorry for her.’ At yesterday’s inquest in Exeter, it emerged that Toner had sent more letters to Mr Giles in Oxfordshire in 2012. 'I am sure she took her own life and intended to do so' Coroner Andrew Cox. Her daughter Dawn Taylor told the coroner that she had ‘sent a letter to Bill Giles’ before she died but she had not seen any of the letters. Coroner Andrew Cox said ‘the details of suicide notes were very personal in nature’ and Miss Taylor said she would find out more details of them from the police in private rather than in the public hearing. The inquest heard Toner failed to turn up in court in September 2012 at Exeter to answer the new harassment allegations. A warrant for her arrest was issued by the court. When police went to her home it was barricaded from the inside and all the windows were locked. They forced their way in and found Toner dead next to empty packets of pills - and toxicology tests revealed she had taken a massive overdose. She also left several notes. HQ: Toner poured lighter fuel over herself outside BBC TV Centre at Shepherd's Bush in London (pictured) The coroner was told that Toner had been to see her GP but had ben abusive in the surgery and left without getting a sick note - and without that her benefits had been stopped after she ‘got angry at the Job Centre’. The inquest heard she suffered from chronic depression which dated back to the 1980s and hip and back pain. 'Various things were pressing on her. She was in court. She had been bailed for an offence of harassment, failed to attend and a warrant was issued for her arrest' Detective Sergeant Angus Cottey, Devon & Cornwall Police. Neighbours told police she was ‘unusual, really friendly but kept herself to herself’ although male friends did visit her at her home. Detective Sergeant Angus Cottey of Devon & Cornwall Police said handwritten suicide notes were found and there were no suspicious circumstances and no third party involvement in her death. He said: ‘Various things were pressing on her. She was in court. She had been bailed for an offence of harassment, failed to attend and a warrant was issued for her arrest. ‘We had to break in. She had barricaded inside and the windows would not open anyway. It was very secure. There were no signs of a struggle or assault.’ Coroner Mr Cox recorded a verdict of suicide, saying: ‘I am sure she took her own life and intended to do so.’ | Summary: Joanna Toner had written new letters to ex-BBC weatherman Bill Giles. Mother-of-two failed to turn up to court and police discovered her dead. Coroner rules Toner killed herself from overdose of prescription drugs. She was previously jailed over her nine-long obsession about Mr Giles. For confidential. support call the Samaritans in the UK on 08457 90 90 90, visit a local. Samaritans branch or click here for details. | 1,059 | 109 | cnn_dailymail | en |
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