A relatively consistent body of research supports an inverse graded relationship between socioeconomic status (SES) and cardiovascular disease (CVD). More recently, researchers have proposed various life course SES hypotheses, which posit that the combination, accumulation, and/or interactions of different environments and experiences throughout life can affect adult risk of CVD. Different life course designs have been utilized to examine the impact of SES throughout the life course. This systematic review describes the four most common life course hypotheses, categorizes the studies that have examined the associations between life course SES and CVD according to their life course design, discusses the strengths and weaknesses of the different designs, and summarizes the studies' findings.This research reviewed 49 observational studies in the biomedical literature that included socioeconomic measures at a time other than adulthood as independent variables, and assessed subclinical CHD, incident CVD morbidity and/or mortality, and/or the prevalence of traditional CVD risk factors as their outcomes. Studies were categorized into four groups based upon life course design and analytic approach. The study authors' conclusions and statistical tests were considered in summarizing study results.Study results suggest that low SES throughout the life course modestly impacts CVD risk factors and CVD risk. Specifically, studies reviewed provided moderate support for the role of low early-life SES and elevated levels of CVD risk factors and CVD morbidity and mortality, little support for a unique influence of social mobility on CVD, and consistent support for the detrimental impact of the accumulation of negative SES experiences/conditions across the life course on CVD risk.While the basic life course SES study designs have various methodologic and conceptual limitations, they provide an important approach from which to examine the influence of social factors on CVD development. Some limitations may be addressed through the analysis of study cohorts followed from childhood, the evaluation of CVD risk factors in early and middle adulthood, and the use of multiple SES measures and multiple life course analysis approaches in each life course study. The adult behavioral lifestyle theory of cardiovascular disease (CVD) describes an adult's lifestyle choices and levels of physiologic risk factors as the primary predictors of CVD risk -3. This A force behind the interest in the impact of early-life conditions on adult health is the fetal origins (Barker) hypothesis. This hypothesis, which has been met with considerable support and criticism, posits that poor nutrition during fetal and early infant development  can increase risks for adult disease. While the dominance of the adult lifestyle model of chronic disease development may not be threatened by evidence for the fetal origins hypothesis, a reconsideration of the primary importance of adult behaviors and risk factors is underway.A life course approach to chronic disease proposes that the combination, accumulation, and/or interaction of the social environments and biological insults experienced throughout the life course impact current and future events, environments, and health conditions and thus ultimately impact adult health ,14. VariThis review describes the major groups of conceptual life course SES models, and then categorizes and summarizes studies that examine the associations between life course SES and CVD risk. Studies are grouped by the basic life course design utilized; summaries include methodologic critiques and descriptions of certain key studies. Evidence supporting each conceptual life course model is considered and future research directions are discussed.The different extant hypotheses on the influence of life course SES on CVD can be grouped into four broad conceptual models: the latent effects, pathway, social mobility, and cumulative life course models. Most studies reviewed tested the influence of life course SES on CVD outcomes via the operation of one or more of these models.The "latent effects" life course conceptual model hypothesizes that adverse early life experiences increase the risk of CVD in later life, independent of intervening SES, lifestyle, or traditional CVD risk factors. Power & Hertzman (1997) describe a latent effects model wherein certain early life events may have strong independent effects on adult health . Kuh andIn this life course model, early life events and environments influence later life experiences, opportunities, and health risk factors. Hertzman et al. 2001) propose a developmental process linking early-life psychosocial environments with adult health risk via pathway effects, wherein early experiences place an individual onto a certain "life trajectory," eventually impacting adult health . Similar01 proposWhile a pathway life course model is intuitively appealing, its operation is difficult to test empirically. Life course studies typically collect information on participants at two or three time points, which does not permit the continuous, lifelong operation of pathway effects to be observed.Social mobility theories all hypothesize that SES mobility across the life course impacts adult health, although the different proposed theories posit different health effects. Forsdahl (1978) hypothesized that deprivation in early life followed by later affluence combine to produce elevated CHD mortality risk, partly via elevation of adult cholesterol levels . Others A cumulative SES life course model hypothesizes that psychosocial and physiological experiences and environments during early and later life accumulate to influence adult disease risk. Davey-Smith et al. 2002) suggest that if factors operating at different life stages are combined, large differences in CVD risk will be observed . Kuh et 02 suggesWe conducted a MEDLINE search using keywords and MeSH terms related to SES, life course, early life, longitudinal studies, CVD and CVD risk factors. References identified and books on life course research were also examined for study citations. Inclusion criteria were: (1) publication date between January 1966 and July 2003; (2) SES or related measures as independent variables; (3) outcomes of subclinical CHD, CVD morbidity and/or mortality, or traditional CVD risk factors. Behavioral risk factors were limited to: adult diet, physical activity, alcohol consumption, and smoking. Physiological outcomes were: measures of obesity, blood pressure, intima-media thickness, fibrinogen, insulin resistance, dyslipidemia, and lung function. Ecologic studies, studies primarily evaluating the fetal origins hypothesis, and studies only examining the impact of birth weight, height, upper leg length, patterns of growth, household crowding, geographic mobility or numbers of siblings were excluded. Forty-nine studies met the inclusion criteria.Quantitative summarization of the study findings is problematic due to varied study populations, SES measures and CVD outcomes evaluated, time points evaluated, and designs utilized. Moreover, a tally approach to summarizing across studies based on statistical significance can be misleading due to the different strengths, weaknesses, power and biases of each study (p. 671) . SummariStudies were categorized by their life course study design(s) and study question(s) into four groups: early SES → outcome, early SES → risk factor, social trajectory, and cumulative SES studies. Several studies appear in multiple categories, as they employed more than one of these designs. Table These studies examined the effect of childhood and/or adolescent SES on risk of adult CVD , CVD death, incident/fatal stroke) -56 and tTable For studies with CHD and stroke outcomes limited to early-life SES (i.e., not controlling for adult SES or other risk factors) almost all included a point estimate consistent with an inverse association. However, in adjusted analyses, authors of only one study with a CHD-related outcome and fewer than half the studies of CVD mortality reported inverse adjusted associations. Thus, the existence of a "direct effect" of early-life SES after adjusting for adult risk factors was not strongly supported.To prevent statistical confounding by adult conditions, most studies employed statistical models that adjusted the association between childhood SES and adult CVD risk for adult SES and behavioral/ physiological CVD risk factors. This may be an over-adjustment, as certain risk factors (e.g., BMI, smoking) are part of the pathways through which low childhood SES may influence CVD risk. Little information is usually available on how participants' early-life SES may have influenced levels of adult CVD risk factors. Additionally, if unknown variables influence both the distribution of a CVD risk factor adjusted for and the distribution of the outcome measure, then adjustment will result in an incorrect partitioning of the total effect of early-life SES into "direct" and "indirect" components -59. ThusSome studies examined and qualitatively compared the effects of adjustment for different classes of CVD risk factors. Gliksman et al. (1995), for example, analyzed the association between father's occupational class and adult CVD in a series of models adjusting for different classes of potential mediators or confounders. This allowed for a more complete description of the impact of adult environment, behavior, and physiologic risk factors versus the latent impact of early life socioeconomic factors . StudiesLife course studies in the early SES → risk factor group evaluated the influence of early- and/or mid-life SES or living conditions on later-life behavioral or physiologic CVD risk factors ,52,61-73Table As with the early SES → outcome studies, concerns that lack of adequate adjustment or over-adjustment may lead to biased estimates are germane to this group of studies. The issue of covariate adjustment is complex in these studies, given that many CVD risk factors are considered. Certain physiologic risk factors (e.g., insulin resistance, dyslipidemia) may be linked to early-life SES through latent physiologic effects of negative childhood exposures, continuous exposure to negative physiologic stimuli, or other physiologic or life course pathway mechanisms ,63,70,74Studies evaluating the social mobility life course model typically considered the impact of inter-generational or intra-generational social mobility on CVD and CVD risk factors ,73,75-79Table Four studies examined differences in CVD risk between stable low-SES trajectory and stable high-SES trajectory individuals ,44,75,79In these studies the unit of analysis is a trajectory, permitting the impact of change over time to be examined. However, the socioeconomic trajectories in most reviewed studies were limited to two time points, and groups compared tended to share the same SES at one of these time points. These similarities may partly explain why seven of ten studies did not report an association between social mobility and risk of CVD risk factors or events. Social trajectory studies incorporating SES at three or more time points allow for the analysis of more informative trajectories than studies evaluating SES at only two points . Yet, evThese studies tested the operation of the cumulative life course conceptual model, typically by summing the number of times participants experienced unfavorable SES situations during early, middle or later life, and creating SES indices representing the accumulation of these experiences ,79,81,82Table Davey-Smith et al. (2002) employed a unique cumulative SES measure, combining early and later-life occupational class experience with the CVD risk factors of smoking and heavy alcohol consumption . They reOnly a large cohort study of Norwegians reported a statistically significant supra-multiplicative interaction between early- and later-life SES on risk of CVD . AnotherThe reported associations between cumulative SES and CVD risk are more consistent than those reported in the other life course study designs. However, three of the seven studies evaluated were based on the same cohort ,81,82. CCertain general limitations and assumptions of life course SES studies should be considered. First of all, SES is a theoretical construct operationalized using various measures (e.g., income, occupation, education) that tap into different components of this construct. Despite several proposed SES evaluation schemes -85, therMost life course studies used retrospective cohorts or a case-control design, relying on participants' recall of early life SES. There has been little systematic evaluation of the validity of recalled early life circumstances or of the potential for such recall errors to bias associations. In studies directly comparing the impact of childhood and adult SES on adult CVD risk, greater error in childhood (vs. adult) SES measures may underestimate the true impact of child SES ,87. AddiMost studies were limited to white males. While the number of studies including women has increased in recent years, minority groups remain underrepresented in most life course studies. These groups may interact differently with the economic and/or social systems of the majority group; there are also established associations between minority status, low SES, and increased CVD risk ,84,85. TThe majority of the early SES → outcome group studies' results describe an inverse association between early life SES and risk of adult CHD, non-fatal MI, and CVD mortality. Similarly most early SES → risk factor studies suggest that low early-life SES negatively impacts levels of adult CVD risk factors. Most studies reported relationships between lower early-life SES and elevated alcohol intake, BMI/WHR, and insulin resistance, as well as decreased leisure physical activity. Together, the early SES → outcome and early SES → risk factor studies support the hypothesis that low early-life SES is inversely associated with adult risk of CVD. However, support for the hypothesis of a "direct effect" of early-life SES on risk of CVD mortality is equivocal as findings were less consistent after risk factor adjustment. Additionally, reported associations between early-life SES and adult health behaviors detract from the evidence supporting a latent effects life course model, as they suggest that CVD risk may be driven by learned behaviors.The operation of social chains of risk or life trajectories described by a pathway life course model are difficult to observe or test, since almost all early SES → risk factor studies consider only two points during the life course. However, significant associations between early-life SES and the adult risk factors suggest behavioral, psychosocial or environmental links that may be best explained as a pathway effect. As described below, the pathway model will need further examination through the use of series of linked studies.While nine of 10 social trajectory studies reported some relationship between inter- or intra-generational social mobility and CVD-related outcomes, only four were identified as providing evidence in favor of a social mobility hypothesis. Conversely, most studies comparing stable low- and high-SES trajectory groups reported markedly increased CVD mortality among the low-SES group, with many socially mobile groups having a CVD risk between the stable high and low groups. Determining whether these associations are a function of social mobility, or whether they are due to exposure to low SES at some point in the life course and not to mobility, is problematic. Future studies should focus on differentiating between the influences of cumulative SES and social mobility.Of the conceptual models discussed, the cumulative life course model was the most consistently supported. However, the weight of these findings is limited as much of the data came from one cohort ,60,61. AResearchers have noted the limitations of life course SES study designs examining only one potential pathway linking SES and CVD. Accordingly, recent studies tend to use a combination of SES measures, CVD risk factor and/or outcomes and life course study designs ,52,53,68As modern life course cohorts are followed from childhood, longer prospective studies or series of cross-sectional studies that evaluate associations between early-life experiences and risk factor levels at several time points may provide the opportunity to observe the operation of pathway life course effects. For example, two studies in the early SES → risk factor group ,90 compaIndividuals' socioeconomic environments may impact their CVD risk factor levels and CVD risk. Community socioeconomic conditions may influence individuals' ability to engage in leisure physical activity or eat a healthy diet -97. SomeLife course SES studies have generally failed to consider the length of exposure to the various socioeconomic conditions measured. As this may influence the impact of negative SES experiences on adult health, future cumulative life course studies may benefit from evaluating the effect of length of exposure into their indices.The wide range of populations, analysis designs, exposures, and outcomes used in the life course studies reviewed precludes a simple, quantitative analysis of the impact of life course SES on CVD risk. Nevertheless, the results thus far modestly support the existence of life course SES effects on risk of adult CVD. The cumulative life course model is more consistently supported by extant studies than other models. However, the different methodologic issues of each study design make direct comparisons of the relative support for each conceptual model difficult. Analyses utilizing multiple life course designs within the same study offer the best approach to testing which theories best describe the links between life course SES and CVD risk. The inclusion of minority participants, different SES measures, and data from early and middle adulthood in large, prospective, mixed-design studies will allow more informed and generalizable statements to be made about the impact of life course SES on adult disease risk. While this area of research needs methodologic refinement, it offers a promising and informative perspective from which to understand the development of chronic disease.The author(s) declare that they have no competing interests.R. A. Pollitt conducted the literature search, reviewed and categorized the articles and had primary responsibility for writing the manuscript. K. M. Rose assisted in the categorization and summarization of the papers reviewed, and helped to revise the manuscript in response to the reviewer's comments. All authors participated in interpreting the studies' results and preparing the methodologic criticism, provided input on the various drafts, and read and approved the final manuscript.The pre-publication history for this paper can be accessed here:SES – CVD life course studies using an early SES → outcome designClick here for fileSES – CVD life course studies using an early SES → risk factor designClick here for fileSES – CVD life course studies using a social trajectory designClick here for fileSES – CVD life course studies using a cumulative SES designClick here for file


DNA mutates, and it's a good thing it does. If it didn't, there could only be one kind of life, not the millions there are today, and species could not adapt to new challenges. This is because mutations in genes—the coding portion of DNA—are the raw material for evolution. However, genes make up a surprisingly small fraction of our DNA. If the genome were a cookbook, its 30,000-odd genetic recipes would be scattered among millions of pages of apparently meaningless nonsense.Mutations affect all DNA, not just the genes, and this provides population geneticists with a veritable toolbox of methods useful, for example, in DNA profiling. Importantly, all these methods rely on the idea of a “molecular clock,” the notion that mutations rain down on noncoding DNA like a fine drizzle, so constantly that genetic similarity is a good measure of evolutionary time. Thus, if orangutans diverged from humans twice as long ago as did chimpanzees, on any given piece of DNA we would find twice as many differences between the orangutan sequence and the human sequence as between humans and chimps. The mutations are marking time.If the molecular clock works, scientists can do wonderful things like estimating how long ago it was that the common ancestor of all humans lived, or when birds evolved from dinosaurs. The clock assumes that mutations occur independently of each other and at a constant rate. By analyzing thousands of noncoding DNA sequences scattered throughout the human genome, Edward Vowles and William Amos have found that the clock is anything but constant. Instead, a mutation in one spot in the genome affects the chance of getting another mutation nearby.Not all noncoding DNA is made up of benign tracts of random letters. Some sequences appear to be more difficult to copy than others, and these trouble spots can give rise to alphabetic stuttering. DNA is made up of four component chemical units, called nucleotides, which are often referred to by their initial letters: A, C, G, and T. Stuttering occurs when the same pairs or triplets of letters occur together, for example ACACAC. Such regions are called microsatellites, and instead of mutating by swapping one letter for another, as most nucleotides do, these sequences evolve mainly by gaining and losing triplets or pairs like “AC.”In this study, Vowles and Amos used the published sequence of the human genome to track down and compare thousands upon thousands of microsatellites. If the molecular clock ran smoothly, they would expect to find no similarity at all between the DNA sequences surrounding any pair of unrelated microsatellites. To their surprise, they found the complete reverse, with entirely unrelated microsatellites showing widespread and obvious similarities in their flanking DNA. This meant that mutations near microsatellites were not random, but favored certain letters in certain positions. Just as a new shipwreck will attract its own special community of marine life, so microsatellites appear gradually to change the surrounding DNA towards a common pattern. The result is convergent evolution, an unusual state of affairs where, as time goes by, DNA sequences become more similar, not less.As yet, the exact mechanisms remain unclear, though it probably has something to do with how comfortably different combinations of letters sit next to each other. In English, “U” always follows “Q” and “B” never follows “V.” Similar rules may apply to DNA, albeit on a much subtler level. For example, if a microsatellite contains alternating As and Cs, the flanking regions also tend to have As at alternate positions, in phase with the As in the microsatellite. It is as if the DNA prefers the pattern in the microsatellite to extend into the flanking DNA, rather than abruptly stopping at the end of the microsatellite.These findings suggest that it may be wise to take the notion of a molecular clock at face value. With a perfect clock, two or three identical mutations would be highly unlikely, but we now know that this may be possible near microsatellites. Vowles and Amos estimate that as much as 30% of the genome may show evidence of convergent evolution, simply because microsatellites are so common. These mutation biases probably exist to a lesser extent in most sequences. Once scientists understand more fully how and where these biases operate, they may be able to estimate more accurately the risk of any given mutation occurring, be it one that causes human disease or makes a virus more virulent. These findings represent yet another windfall from the Human Genome Project, and act as a powerful reminder that unexpected results always lurk around the corner as we delve deeper into the secret world of the genome.


Eukaryotic whole genome sequences are accumulating at an impressive rate. Effective methods for comparing multiple whole eukaryotic genomes on a large scale are needed. Most attempted solutions involve the production of large scale alignments, and many of these require a high stringency pre-screen for putative orthologs in order to reduce the effective size of the dataset and provide a reasonably high but unknown fraction of correctly aligned homologous sites for comparison. As an alternative, highly efficient methods that do not require the pre-alignment of operationally defined orthologs are also being explored.A non-alignment method based on the Singular Value Decomposition (SVD) was used to compare the predicted protein complement of nine whole eukaryotic genomes ranging from yeast to man. This analysis resulted in the simultaneous identification and definition of a large number of well conserved motifs and gene families, and produced a species tree supporting one of two conflicting hypotheses of metazoan relationships.Our SVD-based analysis of the entire protein complement of nine whole eukaryotic genomes suggests that highly conserved motifs and gene families can be identified and effectively compared in a single coherent definition space for the easy extraction of gene and species trees. While this occurs without the explicit definition of orthologs or homologous sites, the analysis can provide a basis for these definitions. Several methods have been developed for the detailed global comparison of multiple whole genomes and the production of global phylogenies. Most of these methods require the prior identification and selection of a reasonably small subset of putative orthologs within which individual homologous sites are identified with some degree of confidence using alignment -7. FrequThough generally less developed, many non-alignment methods, considered initially by Blaisdell , are curThe nine eukaryotic genomes compared in this analysis are listed in Table All the proteins in the dataset were recoded as overlapping tetrapeptide frequency vectors and the resulting data matrix was decomposed by the SVD. A total of 437 singular triplets were obtained as output. The "protein" vectors provided in the "right" factor matrix are known to provide reduced dimensional definitions for all proteins in the dataset as linear combinations of the orthogonal "right" singular vectors (rsv's). Conversely, the right singular vectors themselves frequently represent "ideal" versions of proteins defining a given gene family ,14 Proteth singular vector). Those provided by negative vector values are followed by an "a" (e.g. 277a). Its worth noting at this point that protein family definitions provided by the SVD necessarily account for not only "what is there" (tetrapeptides that form the motifs that define the family), but also "what is not there" (tetrapeptides excluded by that family of proteins, but likely to form anti-correlated motifs within other families of proteins).Each right singular vector can potentially define two distinct gene families. In this case, the highest positive elemental values within a vector identify proteins associated with one protein family, while the highest negative values identify proteins associated with an anti-correlated family (i.e. proteins that rarely share the same tetrapeptides). Frequently, however, strong family definitions are provided for just one protein family. In this case, the anti-correlated proteins are seen to be derived from a mixture of two or more families. Since the choice of sign is arbitrary, strong family definitions are equally likely to be provided by either the positive or the negative values within a vector. Family definitions provided by positive vector values are denoted below using the simple vector index  comprising a given singular triplet. The lsv's describe these copep motifs as linear combinations of the 160,000 possible tetrapeptides. Those with high positive values identify peptides found with high probability in the conserved motif of a given subfamily, while those with a high negative value identify peptides excluded with high probability. Therefore, like the rsv's, the lsv's frequently describe two distinct anti-correlated entities (in this case motifs rather than protein families) using either positive or negative values within the vector. Using essentially the same procedure described above for any given rsv, the tetrapeptides having the largest positive or largest negative projections on any given lsv were identified in order to provide a focused summary of the motifs described by that vector. For motif extraction, however, an arbitrary cut-off value  was used to identify dominant tetrapeptides.In most cases, it is possible to cluster the resulting short list of dominant tetrapeptides into several uninterrupted copep strings formed by tetrapeptides that overlap in 3 of 4 consecutive amino acid positions. Using this procedure, one long copep string was identified for each of the singular triplets listed in Table Figure Instead of simply providing restricted motif summaries using the most dominant elements of the left singular vectors, we have also attempted to examine entire vectors in order to gain a better understanding of the motifs (and associated protein families) they describe. A reasonably efficient method for depicting left singular vectors is presented in Figure The detailed comparative information contained within the hundreds of singular vectors and their corresponding motifs and gene families was subsequently used to build a species phylogeny by summing all the SVD-derived right protein vectors separately for each organism and then comparing the relative orientation of the resulting species vectors . Figure Use of the "successive, delete-one" jackknife procedure as a species tree branch statistic is justified by the fact that SVD provides singular triplets in order of their "dominance" in explaining the data set . MathemaWhile our SVD-based analysis technically considers all proteins present within all nine genomes of the data set, it is likely that accurate vector definitions are provided for only a small fraction of these proteins. Theoretically, the 437 singular triplets could effectively describe as many as 2 × 437 = 874 protein families. However, many of these vectors appear to best describe particular subfamilies of larger groups of closely related proteins. Thus, the 58 protein families listed in Table 3 proteins removed) resulted in 22% bootstrap and 100% modified jackknife support for the coelomate lineage, but 0% support for the ecdysozoan lineage. Removal of a much higher fraction of poorly described proteins (cut-off = .05, about 105 proteins removed) produced an equivalent result. Hence, poorly described proteins contribute little to the support that our analysis provides for the coelomate model.In an attempt to increase the fraction of well described proteins used to define species, proteins having poor projections on all 437 right singular vectors were ignored during the summation process. Arbitrary vector magnitude cut-off values of 0.005 or 0.05 were applied to reduce the number of poorly described proteins used to build species trees. Even though the highest and most stringent cut-off value removed the majority of proteins during summation, both new species trees had identical topologies to that of the tree shown in Figure As demonstrated above, an SVD-based analysis of multiple genomes automatically interprets proteins from input genomes as potential members of a limited list of hierarchically defined protein families and subfamilies. Each subfamily is defined in detail by one or more singular vectors as linear combinations of a large number of peptides (160,000 tetrapeptides, in this case). Potentially, a large number of proteomes lacking annotation can be directly interpreted using this method, assuming a sufficient number of annotated proteomes are included in the analysis. Although most of the genomes used in the present analysis were already accompanied by detailed protein annotations, formal annotations of the Frub and Agam proteins were not readily available. Nevertheless, our SVD-based analysis was able to provide precise protein motif descriptions and subfamily affiliations, not only for the six Frub or Agam proteins shown in Figure Our method bears partial resemblance to a recently described graph-theoretic method for rapidly clustering massive datasets of whole genome protein sequence . In thisAlthough our descriptive analysis of singular triplets , three output matrices were obtained, consisting of 437 singular triplets . Each left singular vector produced by the SVD defines one or two conserved motifs within the dataset as particular linear combinations of tetrapeptides [Each protein sequence in the dataset was recoded as a high dimensional vector containing raw frequencies for each of the 160,000 possible tetrapeptides. Previous work has established that although tripeptides work well for estimating similarities between highly divergent proteins contained within small sets of viral genomes , tetrapepeptides ,14. Simi"Dominant" vector elements  were extracted from the left singular vectors and summarized using the C++ program "Copepx" . These vDistance matrices were derived by summing all the SVD-derived right protein vectors for a given organism and then comparing the relative orientation of the resulting species vectors using the program Cosdist ,14. SpecHomo Sapiens (Hsap), Mus musculus (Mmus), Rattus Norvegicus (Rnov), Anopheles gambiae (Agam), Drosophila melanogaster (Dmel), Caenorhabditis elegans (Cele), Plasmodium falciparum , and Saccharomyces cerevisiae (Scer), Fugu rubripes (Frub), correlated peptide (copep), correlated protein (coprot), right singular vector (rsv), left singular vector (lsv).GS conceived the study, gathered the input data, provided primary interpretation of the output, and drafted the manuscript. MB wrote and adapted software, performed computational analysis on the input data, and provided manuscript modifications. All authors read and approved the final manuscript.Copep Motifs. Long copep strings identified within the left singular vectors of a given s-triplet.Click here for file


This study tested the effectiveness of a COI barcode in discriminating bird species, one of the largest and best-studied vertebrate groups. We determined COI barcodes for 260 species of North American birds and found that distinguishing species was generally straightforward. All species had a different COI barcode(s), and the differences between closely related species were, on average, 18 times higher than the differences within species. Our results identified four probable new species of North American birds, suggesting that a global survey will lead to the recognition of many additional bird species. The finding of large COI sequence differences between, as compared to small differences within, species confirms the effectiveness of COI barcodes for the identification of bird species. This result plus those from other groups of animals imply that a standard screening threshold of sequence difference (10× average intraspecific difference) could speed the discovery of new animal species. The growing evidence for the effectiveness of DNA barcodes as a basis for species identification supports an international exercise that has recently begun to assemble a comprehensive library of COI sequences linked to named specimens.Short DNA sequences from a standardized region of the genome provide a DNA barcode for identifying species. Compiling a public library of DNA barcodes linked to named specimens could provide a new master key for identifying species, one whose power will rise with increased taxon coverage and with faster, cheaper sequencing. Recent work suggests that sequence diversity in a 648-bp region of the mitochondrial gene, cytochrome  c oxidase I might have this potentialCan a single gene be used to identify all different animal species? An analysis of 255 North American birds shows that cytochrome  Archaea, and subsequently to a redrawing of the tree of life. More recently, the polymerase chain reaction has allowed sequence diversity in any gene to be examined. Genes that evolve slowly, like rRNA, often do not differ among closely related organisms, but they are indispensable in recovering ancient relationships, providing insights as far back as the origin of cellular life  has been widely employed in phylogenetic studies of animals because it evolves much more rapidly than nuclear DNA, resulting in the accumulation of differences between closely related species . In factThe general concordance of mtDNA trees with species trees implies that, rather than analyzing DNA from morphologically identified specimens, it could be used the other way around, namely to identify specimens by analyzing their DNA. Past applications of DNA-based species identification range from reconstructing food webs by identifying fragments in stomachs  to recogc oxidase I (COI), might serve as a DNA barcode for the identification of animal species. This gene region is easily recovered and it provides good resolution, as evidenced by the fact that deep sequence divergences were the rule between 13,000 closely related pairs of animal species (If a short region of mtDNA that consistently differentiated species could be found and accepted as a standard, a library of sequences linked to vouchered specimens would make this sequence an identifier for species, a “DNA barcode” . Recent  species . The pre species .All 260 bird species had a different COI sequence(s); none was shared between species. COI sequences in the 130 species represented by two or more individuals were either identical or most similar to other sequences of the same species. Furthermore, with a few interesting exceptions discussed below, COI sequence differences between closely related species were far higher than differences within species  .Tringa solitaria, Solitary Sandpiper; Sturnella magna, Eastern Meadowlark; Cisthorus palustris, Marsh Wren; and Vireo gilvus, Warbling Vireo). COI sequences in each of these polytypic species separated into pairs of divergent clusters in the NJ tree. The intraspecific K2P distances in these exceptional species were 3.7%–7.2%, 9- to 17-fold higher than the average distance (In most cases the neighbor-joining (NJ) tree showed shallow intraspecific and deep interspecific divergences . Howeverdistance .Larus argentatus, L. canus, L. delawarensis, L. glaucoides, L. hyperboreus, L. marinus, and L. thayeri (Herring Gull, Mew Gull, Ring-billed Gull, Iceland Gull, Glaucous Gull, Great Black-Backed Gull, and Thayer's Gull); Haematopus bachmani and H. palliatus (Black Oystercatcher and American Oystercatcher); Corvus brachyrhynchos and C. caurinus (American Crow and Northwestern Crow); and Anas platyrhynchos and A. rubripes   .Although species were the focus of this study, we noted that the NJ tree of COI sequences generally matched avian classifications at higher levels, with most genera, families, and orders appearing as nested monophyletic lineages concordant with current taxonomy  and S1.The simplest test of species identification by DNA barcode is whether any sequences are found in two species; none was in this study. Although sequences were not shared by species, sequence variation did occur in some species. Thus the second test is whether the differences within species are much less than those among species. In this study we found that COI differences among most of the 260 North American bird species far exceeded those within species.In order to conservatively test the effectiveness of COI barcodes as an identification tool, our sample must not have underestimated variability within species or have overestimated it among species. Our measures of intraspecific variation could be underestimates if members of a species show sequence divergence across their distribution that our study failed to adequately register. The two to three representatives of the 130 species used to examine this issue were collected from sites that were, on average, approximately 1,080 km apart, suggesting adequate representation of genetic diversity across their ranges. However, to further investigate this issue, we compared sequence differences within species to geographic distances between the collection points for their specimens and found these were unrelated . Based oOn the other hand, our discovery of four polytypic species within a sample of 130 makes it likely there are other North American birds with divergent populations that may represent hidden species. Recent studies have identified marked mtDNA divergences within North American populations of Common Ravens , Fox SpaCalidris mauri and C. pusilla, Fraternicula arctica and F. corniculata, and Empidonax traillii and E. virescens. There were sufficient data to perform this analysis on three of the four polytypic species and on 70 of the 126 remaining species  are split into two by some taxonomists , and theThe importance of sampling multiple individuals within each species is highlighted by a recent review which found evidence of species-level paraphyly or polyphyly in 23% of 2,319 animal species, including 16.7% of 331 bird species . This reT. solitaria PS-1 and T. solitaria PS-2, highlighting a need for further taxonomic study.We expect that the assembly of a comprehensive barcode library will help to initiate taxonomic investigations that will ultimately lead to the recognition of many new avian species. This process will begin with the discovery of novel COI barcodes. Some of these cases will simply represent the first barcode records for described but previously unanalyzed species, but taxonomic study will confirm that others derive from new species. We propose that specimens with barcodes diverging deeply from known taxa should be known by a “provisional species” designation that links them to the nearest established taxon. For example, the divergent clusters of Solitary Sandpiper specimens might be called What threshold might be appropriate for flagging genetically divergent specimens as provisional species? This threshold should certainly be high enough to separate only specimens that very likely belong to different species. Because patterns of intraspecific and interspecific variation in COI appear similar in various animal groups  in the stomachs of predators. A DNA barcode could similarly identify fragments of birds that strike aircraft  and recoThe two essential components for an effective DNA barcode system (and thus a new master key to the encyclopedia of life ) are stab and COI, are rivals for the largest number of animal sequence records greater than 600 bp in GenBank (4,791 and 3,009 species, respectively). However, COI coverage for birds is modest; 173 species share COI sequences with 600-bp overlap. As these records derive from a global avifauna of 10,000 species, they provide a limited basis to evaluate the utility of a COI-based identification system for any continental fauna, impelling us to gather new sequences.Existing data can only yield limited new insights into the effectiveness of a DNA-based identification system for birds. Two mitochondrial genes, cyt http://www.barcodinglife.com). Taxonomic assignments follow the latest North American checklist . In cases where this primer pair failed, an alternate reverse primer (BirdR2-ACTACATGTGAGATGATTCCGAATCCAG) was generally combined with BirdF1 to generate a 751-bp product, but a third reverse primer (BirdR3-AGGAGTTTGCTAGTACGATGCC) was used for two species of Falco. The 50-μl PCR reaction mixes included 40 μl of ultrapure water, 1.0 U of Taq polymerase, 2.5 μl of MgCl2, 4.5 μl of 10× PCR buffer, 0.5 μl of each primer (0.1 mM), 0.25 μl of each dNTP (0.05 mM), and 0.5–3.0 μl of DNA. The amplification regime consisted of 1 min at 94 °C followed by 5 cycles of 1 min at 94 °C, 1.5 min at 45 °C, and 1.5 min at 72 °C, followed in turn by 30 cycles of 1 min at 4 °C, 1.5 min at 51 °C, and 1.5 min at 72 °C, and a final 5 min at 72 °C. PCR products were visualized in a 1.2% agarose gel. All PCR reactions that generated a single, circa 750-bp, product were then cycle sequenced, while gel purification was used to recover the target gene product in cases where more than one band was present. Sequencing reactions, carried out using Big Dye v3.1 and the BirdF1 primer, were analyzed on an ABI 377 sequencer. The electropherogram and sequence for each specimen are in the “Birds of North America” file, but all sequences have also been deposited in GenBank .Click here for additional data file.Sequences described in


A message from the founders of the Public Library of Science The Internet is awash with medical information. Eight hundred million people have direct access to the Internet , and in But something is conspicuously missing. The most reliable medical information on the Internet—the contents of peer-reviewed medical journals—is hidden from the public and most of the world's physicians. Although most medical journals are available online, their publishers limit access to those who choose, and can afford, to pay for access. This should not, and need not, be so.The Lancet,The New England Journal of Medicine, and The Journal of the American Medical Association on cheap paper and selling them to subscribers at a few pennies a copy. For more than a century, printed journals like these were the dominant means of conveying medical knowledge around the world.In the 19th and early 20th centuries independent physicians and small medical societies, interested in making the best new medical knowledge available to doctors, students, and the public, began to publish general medical journals containing case reports, ideas for new treatments, and the results of medical experiments. These pioneers took advantage of the best available technology for disseminating information, printing titles like PLoS Medicine, we are embracing this opportunity.But technology has changed. The Internet is now the most economical and efficient conduit for the delivery of information to most places. Publishers of medical journals realize this—when the Internet took off, they took their journals online. But while they adapted their means of distribution to the 21st century, they left their business model in the 19th century, continuing to charge readers for access just as they had done for their printed journals. This has been good for business—medical publishing has never been more profitable—but it comes at a huge cost. The established medical publishers have turned their back on the opportunity to make the latest and best medical information available to anyone with an Internet connection. With the launch of Everything we publish is immediately, freely available online throughout the world, with no restrictions on distribution, copying, printing, or legitimate use.PLoS Medicine is immediately freely available online throughout the world, with no restrictions on distribution, copying, printing, or legitimate use. Of course, it costs us money to publish this journal, and we must cover our expenses. But the fee-for-access business model that made perfect sense for the printed journal is no longer consistent with the mission of medical publishing because it needlessly limits the reach of the medical literature. And so we have adopted a new model. Instead of charging readers for access to our journal, we ask the authors of accepted research articles to pay a publication fee to cover the costs of peer review, editorial oversight, and production. This “open access” business model ensures our financial health as a publisher while allowing us to convey everything we publish to the widest possible audience.Everything published in could benefit from their research can benefit from it.Of course, we do not expect authors to cover publication costs personally—rather, we expect the government agencies, companies, foundations, research institutions, hospitals, or universities that sponsor the research to pay the fee. These organizations have always considered the wide dissemination of the results of the research they support to be an integral part of their mission. Virtually every leading sponsor of medical research has announced its willingness to pay for open-access publication, the costs of which average less than one percent of the cost of the research itself—a small price to pay to ensure that everyone who We realize that not everyone with something important to convey in a medical journal has access to such funds. To ensure that we don't replace a barrier to access with barriers to publication, we've raised money to cover the publication costs of articles whose authors are unable to pay them. And, for every PLoS journal, an author's ability to pay will never be a consideration in our decision to publish an article.Despite its obvious benefits, open-access publication has met with fierce opposition. Established medical publishers—now businesses more than forces for change—see open-access not as an opportunity to fulfill a mission of public service but as a threat to their lucrative businesses. They contend that their journals still serve the community well, and object that open access threatens their very existence. This is nonsense!It is our responsibility as publishers and members of the medical community not only to give patients access to the medical literature, but to provide them with tools to use it wisely.The Wellcome Trust, the world's largest charitable sponsor of biomedical research, seeking to ensure that the results of the science it funds are “disseminated widely and freely available to all,” recently commissioned a thorough analysis of the scientific and medical publishing industry . It concPLoS Biology, and it is thriving—not only as a destination for the best research in all areas of biology, but also as a resource for students, teachers, and members of the public who have never before had direct access to the product of scientific inquiry (see for yourself at www.plosbiology.org). We are now bringing this success and this spirit to medicine.We know firsthand that the Wellcome Trust is right. In October 2003, we launched our first journal, PLoS Medicine is here to challenge the status quo. We are first and foremost an open-access publisher working to ensure that everyone has access to the latest medical research and expertise. But we aim to be more than just an open-access alternative to established general medical journals. We are determined to make PLoS Medicine the best medical journal in the world by providing outstanding original research and new ideas; thought-provoking, educational, and imaginative features for readers; and the fastest, fairest, and most rigorous peer review for authors.The world of medical journals needs a fresh infusion of idealism. All of today's leading medical journals are more than 70 years old, and As an open-access journal, we see our audience differently than do the conventional medical journals: our audience is composed of medical researchers, physicians, and other health-care providers, patients and their advocates, students, and the public around the world. It will be a great challenge to create a journal that will serve such a diverse audience—we welcome this challenge. We will make it possible for the results of advanced research on infectious diseases to guide treatment in remote clinics thousands of miles away. We will make the results of a clinical trial of a new drug accessible and understandable both to doctors who might prescribe it and to people who might start taking it. We will make research on rare diseases accessible to general practitioners and patients so that they can work together to recognize and treat them.Whereas some would argue that medical journals should not be accessible to patients because patients are unable to use the information effectively, we believe it is our responsibility as publishers and members of the medical community not only to give patients access, but to provide them with tools to use the medical literature wisely. Medical research is a partnership between medical scientists and millions of voluntary human participants, conducted largely with public funds. 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The pharmacological action of specific immunosuppressants is mediated by immunophilins. While cyclosporin A binds to cyclophilins, FK506/tacrolimus, rapamycin, and others bind to FK506 binding proteins (FKBPs). Different physiological actions of immunophilins were described but their genuine function, however, remains elusive and is still under investigation. A yeast two-hybrid screen was performed using the FK506 binding protein 13 kDa (FKBP13) as a bait and a fetal liver expression library as a prey.The C-chain of complement C1q (C1q-C) was detected to interact with FKBP13 in the yeast two-hybrid system and in a protein complementation assay. Neither FKBP12, FKBP25, FKBP52 nor the unrelated immunophilin CypA did react with C1q-C in the yeast system stressing the specificity of the interaction. Binding of C1q-C to FKBP13 could not be prevented in the presence of FK506, demonstrating that possibly other regions than the binding pocket of the drug are responsible for the interaction of the two proteins.It is concluded that exclusively FKBP13 but no other FKBPs tested so far interact with the C-chain of complement C1q in the two different assays and further work will be initiated to investigate the physiological relevance of the interaction. The drug immunophilin complex binds to and inhibits the protein phosphatase calcineurin thus preventing signal transduction in activated T cells [ectively . FKBP52 ectively . Anotherectively . Finallyectively ,9.The phylogenetically ancient complement system composed of more than 30 proteins is part of the immunogenic system including a cascade of interacting proteins called C1 to C9. Activation finally leads to lysis of marked cells. C1, the first protein in this cascade, is composed of one C1q and two C1r and C1s proteins. While C1q binds the Fc regions of two IgGs or one IgM molecule, C1r and C1s are known to activate other components of the pathway, namely C4 and C2.C1q contains 18 polypeptide chains (six A-, six B-, and six C-chains) and is composed of six globular heads linked via six collagen like stalks to a fibril-like central region . Each ofIn addition C1q seems to play an important role as an immunmodulator in its own and has pathophysiological impact. C1q deficient individuals develop a systemic lupus erythematodes (SLE)-like disease  and C1q In this work interaction of the C-chain of complement C1q (C1q-C) with a member of the FK506 binding protein (FKBP) family was detected and the specificity of the interaction was further investigated.6 independent clones) were screened and selected for histidine prototrophy. Among 216 colonies isolated as His+, 21 were found to display beta-galactosidase  activity. Plasmids were isolated from each of these clones and were used for retransformation of yeast L40 pretransformed with pBTM-FKBP13w/oS. Six clones remained positive (clone A3, B10, B31, B55, C8, C30) and turned out to code for five different cDNAs after sequence analysis. One of the clones (A3) representing the cDNA of the C-chain of complement C1 corresponding to amino acids 122 to 217 was selected for the present study. The interaction of a second clone with FKBPs was described elsewhere [The physiological function of immunophilins in endocrine systems is rarely characterized. A commercially available fetal human liver cDNA expression library was used to identify proteins that interact with the immunophilin FKBP13 which is localized in the endoplasmic reticulum. Plasmids encoding FKBP13 without signal peptide fused to the LexA-DBD (pBTM-FKBP13w/oS) and the liver cDNA library were sequentially transformed into yeast L40. Ten million yeast double transformants  and different plasmids each of them coding for FKBP12, FKBP13w/oS, FKBP25, and FKBP52 fused in frame to the DBD of LexA (pBTM-FKBP12, 13w/oS, 25, and 52). Cotransformants were checked for histidine prototrophy and β-gal activity. Only FKBP13 interacted with the C1q-Cw/oS protein in the yeast two-hybrid system whereas FKBP12, 25, and 52 failed in this respect.Cyclophilins are the second family of immunophilins. To check for cross reactivity, binding of C1q-Cw/oS to the prototype of cyclophilins, cyclophilin A (CypA), was investigated in the same system. Yeast L40 was cotransformed with a plasmid expressing LexA-CypA hybrids (pBTM-CypA) and pGAD-C1q-Cw/oS and was tested for β-gal activity and histidine prototrophy. C1q-Cw/oS and CypA failed to interact in the yeast two-hybrid system.In order to verify the results obtained with the yeast two-hybrid screen, the GST pull-down approach was used first. As the expression of C1q-Cw/oS either in a coupled reticulocyte lysate system or in bacteria failed, the interaction of C1q-Cw/oS with FKBP13w/oS was analyzed in a protein complementation assay. INS-1 cells were cotransformed with a plasmid coding for FKBP13w/oS fused in frame via a 15 amino acid peptide linker to aa 198 to 287 of β-lactamase (pCDNA-FKBP13w/oS-Bla2) and a plasmid coding for β-lactamase aa 21 to 196 fused in frame via a 15 amino acid peptide linker to C1q-Cw/oS (pCMVSPORT-Bla[1]M182TlinkCENP-A). As shown in Fig. The crystal structure of FK506/FKBP complexes predicts that the recognition site in the endogenous ligand(s) equivalent to FK506 would best be emulated by a Iso-Pro or Leu-Pro motif . FK506 mA yeast two-hybrid screen was carried out using the FK506 binding protein 13 kDa (FKBP13) as a bait and a human liver expression library as a prey. The screen revealed the C-chain of complement C1q (C1q-C) to interact with FKBP13. Three other FKBPs, namely FKBP12, FKBP25 and FKBP52 failed to interfere with C1q-C under same conditions indicating specificity for the positive result in the yeast system. The interaction of C1q-C with FKBP13 was proved by using additionally a protein complementation assay (PCA), demonstrating that the interaction of C1q-C with the FKBP13 was not an artifact in the yeast two-hybrid system. The interaction of C1q-C with FKBP13 in the PCA seems to be comparable to the interaction of the two GCN4 leucine zippers (Zip) which were used as a positive control. The intensity of the interaction might depend on the efficiency of the cell transfection, cell density, cell growth, and other factors but hydrolysis rates of nitrocefin as a marker of the interaction are clearly different from the negative control which can be best seen after 270 min of incubation . They can be divided into FKBPs that bind for example FK506/tacrolimus or rapamycin and cyclophilins that mediate the pharmacological action of cyclosporin A (CsA). Cyclophilin A (CypA) can be regarded as the prototype of the cyclophilins. Unlike the aforementioned FKBP13, the immunophilin CypA does not interfere with C1q-C in the yeast two-hybrid system underlining the specificity of the interaction of C1q-C with FKBP13.The immunosuppressive drug FK506 is not able to decrease the affinity of C1q-C to FKBP13 in the yeast two-hybrid system even when used in a concentration of 100 micromolar. The common binding motif of FKBPs for FK506 is, therefore, less likely to be uniquely involved in the C1q-C FKBP13 interaction. On the other hand, at low micromolar concentrations FK506 seems to „strengthen" the interaction of C1q-C with FKBP13. Possibly, C1q-C, when produced artificially in yeast, binds to yeast FKBP as well and has to be liberated from these interfering positions by FK506, then contributing to the interaction with the FKBP13-AD hybrid protein. An excess of FK506 should further diminish the C1q-C FKBP13 interaction but the drug cannot be tested in the milimolar range because of its toxicity. The influence of FK506 on the interaction of the two proteins was not investigated in a mammalian cell line because the influence in the yeast system is more likely comparable to other investigations e.g. ,18]. Add. Add18]]Using two different experimental approaches, the C-chain of complement C1q was shown to interact with FKBP13. FKBP13, initially used as the bait for the yeast two-hybrid screen in this work, is regarded as an endoplasmic reticulum (ER) protein. It might act as a chaperone but specifically interacts with other proteins like a homologue of the erythrocyte membrane cytoskleletal protein 4.1 or the FKBP associated protein 48 (FAP48), too. FKBP13 was detected in membrane preparations from erythrocytes as well, demonstrating that its expression and maintenance is not restricted to the ER . The C-cThe cDNA encoding FKBP13 amino acids 16–142 was amplified from pBluescript-FKBP13 (a gift from Dr. S. Burakoff, Dana-Faber Cancer Institute, Boston, MA) using specific primers CGCCGGAATTCATGCTGAGCGCCGTG and CGGCTGGATCCGAACAGTCTGGTC. The cDNA encoding full-length FKBP25 was amplified from pBluescript-FKBP25 (a gift from Dr. S. Burakoff, Dana-Faber Cancer Institute, Boston, MA) using the specific primers CGCGCGAATTCAACA GTCTGGTCCCTGATG and GGCGTAGGATCCGGGGTTGACTCCGGGGGC. The cDNA encoding full-length cDNA of CypA was amplified from I.M.A.G.E clone 5264185 (ResGen, Huntsville, AL) by using the specific primers GGTCCGGAATTC ATGGTCAACCCCACCGTGTTC and GGCAGCTGGATCCACAAGTCA AAC TTATTCGAG. After restriction digest, cDNAs were fused to the DNA binding domain of LexA by inserting the EcoRI/BamHI fragments into pBTM116  to give HIS3 and LacZ, was sequentially transformed with pBTM-FKBP13w/oS and the cDNA library using the lithium acetate method [+ colonies were patched and assayed for β-gal activity. Positive clones, inserted into the pACT2 vector, were rescued and tested for specificity by retransformation into yeast L40 with pBTM-FKBP13w/oS. Different clones, that expressed detectable β-gal activity within four hours, were used for further analysis. All assays of β-gal activity and histidine prototrophy were performed according to standard procedures [The human fetal liver MATCHMAKER cDNA library  was a gift from Dr. C. Sorg . The yeast reporter strain L40, containing two reporter genes, e method . Double ocedures .6 INS-1 cells, an insulinoma cell line [The protein complementation assay (PCA) was performed as described elsewhere . Brieflyell line , were coThe authors contributed equally to this work


The US will spend $1.79 trillion on health care in 2004, yet 44 million Americans remain uninsured. What the country needs, argues McCanne, is publicly funded universal health coverage The country must abandon its fragmented system The total projected spending on health care in the United States for 2004 is $1.79 trillion—15.5% of its gross domestic product [First, I should reveal my personal bias. Physicians should be well represented in the forefront of reform. As we look back on the past half century of failed health policy decisions, we see that the dominant physicians' organization in the United States, the American Medical Association (AMA), has opposed most reform measures that would result in an equitable, affordable system for everyone. Instead, the AMA has supported an agenda that promotes physicians' freedom to maximize their personal financial reward, even though those policies may deprive tens of millions of Americans access to affordable care. The AMA agenda has contributed significantly to the current high costs of American health care and to our failure to adequately address the mediocrity that characterizes health care in the United States.www.pnhp.org) [Many American physicians—including myself—believe that the funding infrastructure should be redesigned to maximize heath care resource allocation for the primary benefit of patients. Because of the failure of organized medicine to advocate on behalf of our patients, we decided that a new organization was needed. We established Physicians for a National Health Program (nhp.org) ,3.There are 45 million Americans with no health care coverage, and not surprisingly, lack of insurance is associated with worse health outcomes [Even the insured are inadequately covered. Employers and individuals who purchase coverage are rebelling at the high price of insurance premiums. To maintain competitive premiums, insurers are designing products that reduce the benefits they pay out by increasing the out-of-pocket portion that patients are required to pay for services received. Insured patients may have to pay cash for care until a designated amount is reached (the deductible)—which could be thousands of dollars. In addition, patients are often required to pay a dollar amount (co-payment) or a percentage of the charges (coinsurance) each time services are received.Insurers may also exclude specified services from coverage, such as maternity benefits or mental health services. Most insurance plans now use lists of contracted physicians and hospitals, and impose severe financial penalties for using health care providers that are not contracted. All of these measures reduce the value of insurance by shifting costs from the insurers to the patients who actually need care.Inadequate insurance coverage is making average-income Americans poorer. A recent study found that for 29% of individuals who had average or greater-than-average incomes and were continually insured, medical bills had caused significant financial problems . For thoThere is a widespread belief that the high spending in the United States means that high quality care is being delivered to the majority, who can afford both comprehensive coverage and the attendant out-of-pocket expenses. But international comparisons of industrialized nations have shown that the United States is in the bottom quartile of population health indicators such as life expectancy and infant mortality . And in In 2000, the World Health Organization rated the United States first in its health expenditures per capita, but 37th in its overall health system performance, below most industrialized nations . The UniSome contend that the poor performance of the United States system is due to the funding of health care in the private sector, and that all would be well if the government would just take over funding. But it is not quite that simple. The greater part of health care in the United States—59%—is already funded by the tax system. On a per capita basis, the public, taxpayer-funded health care expenditures alone total more than the health care spending of every other nation's public and private funding combined  .How can the United States spend as much as it does and end up with such mediocre health care? Of the many reasons that exist, two are particularly important. The United States has a highly flawed system of funding health care and a flawed system of allocating its health care resources.In the United States, a multitude of private health plans cover the lucrative sector of society—low cost, healthy workers and their healthy families. But public programs must cover the higher costs of the elderly, individuals with permanent disabilities, and some low-income individuals. Since the uninsured are frequently unable to pay for the care they receive, the costs for their care are shifted to government programs or private plans, or to the charity of providers, even if unintended. The costly administrative excesses of private health plans, especially when contrasted to government programs, have been well documented . This frAnd what is wrong with the way that the United States allocates its resources? Many studies have confirmed that supporting a strong primary care base provides better outcomes at a lower cost . But in http://www.cms.hhs.gov/schip), the accomplishments of these incremental health reform measures have been unimpressive.What has been the response to these deficiencies in the United States health care system? In the 1990s, the Clinton administration attempted to introduce a comprehensive system of funding universal health care. The system would have used marketplace principles in a program of managed competition, but their complicated idea pleased no one, and it was never even brought to a vote. Because of this miserable political failure, policymakers decided that any comprehensive approach should be avoided, and that reform must take place in incremental steps. To date, with the notable exception of the State Children's Health Insurance Program  the expansion of our current system of public and private programs, (2) the establishment of a national health service with government ownership of the system, or (3) the replacement of all current funding with a single, publicly administered, publicly funded program of social insurance that does not alter the existing ownership status of the delivery system.The greatest political support today is for incremental expansions of our current programs, which, theoretically, would eventually result in universal coverage. There are innumerable variations of this approach. Most would increase the affordability of insurance premiums for private group and individual plans by providing financial assistance through tax policies and by modifying the benefits and coverage of the plans. Some policy analysts recommend that employers be mandated to offer coverage to their employees. Others recommend that individuals be required to purchase their own coverage. Since some individuals would be left without coverage, a public program, such as the existing Medicaid program for low-income individuals, would be used to cover everyone else. Many simulation studies have shown that these approaches could be effective in covering almost everyone, but they are the most expensive models of reform since they leave in place the administrative excesses of the fragmented system of funding care . Also, tIn contrast, simulation of both the national health service and public social insurance models of reform have shown that they would provide truly comprehensive benefits for everyone, and that they are the least expensive models . By inteThe political threshold for adopting a government-owned health service model in the United States is very high, since most citizens fear the specter of “socialized medicine.” In contrast, the Medicare program, an insurance program for the retired and for those with long-term disabilities, is very popular. There is an increasing public perception that we may need to accept a greater government role in health insurance if we are to adequately address the deteriorating status of our health care system. Correcting the flaws in Medicare and then using the program to cover everyone may be a concept that can gain political traction in the United States.Our political process is currently dominated by those who are enticed by the siren song of the market theorists and turn a deaf ear to the health policy scientists who plead for health care justice. The debate needs to focus on defining the best role for government in ensuring that people receive the best health care value. That debate needs to be guided by a thorough understanding and diligent application of sound health policy science.The continuing deterioration of affordability, coverage, and quality in health care makes it imperative that United States policymakers broaden their reform efforts beyond the ineffectual tinkering of incrementalism. A universal, single-payer, publicly funded and publicly administered program of social insurance would ensure access to affordable, comprehensive, high-quality health care for all. It should be the standard by which any other proposals are judged. If a better proposal can be crafted, now is the time to do it. People are dying while we delay.


The structural maintenance of chromosome proteins SMC1 and SMC3 play an important role in the maintenance of chromosomal integrity by preventing the premature separation of the sister chromatids at the onset of anaphase. The two proteins are constitutive components of the multimeric complex cohesin and form dimers by interacting at their central globular regions.In order to identify proteins that by binding to SMC3 may interfere with the protein dimerization process, a human cDNA library was screened by the yeast two-hybrid system by using the hinge region of SMC3 as bait. This has lead to the identification of Hinderin, a novel five domains protein including two coiled-coil motifs and sharing a strikingly structural similarity to the SMC family of proteins. Hinderin is ubiquitously expressed in human tissues. Orthologue forms of the protein are present in other vertebrates but not in lower organisms. A mapping of the interaction sites revealed that the N- and C-terminal globular domains mediate the binding of Hinderin to SMC3. Hinderin/SMC3 complexes could be recovered by immunoprecipitation from cell lysates using an anti-SMC3 antibody, thus demonstrating that the two proteins interact in vivo. On the contrary, Hinderin did not interact with SMC1. In vivo the rate of SMC1/SMC3 interaction was decreased by the ectopic expression of Hinderin.Hinderin is a novel binding partner of SMC3. Based on its ability to modulate SMC1/SMC3 interaction we postulate that Hinderin affects the availability of SMC3 to engage in the formation of multimeric protein complexes. The structural maintenance of chromosome (SMC) proteins are involved in several aspects of chromosomal dynamic, in DNA recombination and in DNA repairs -3. Two S465 to Gln807) was used as bait in a yeast two-hybrid system to identify interacting proteins expressed by a human fetal brain Matchmaker two-hybrid cDNA library (Clontech). About 3 × 106 library clones were screened. Forty blue colonies reaching 2 mm in size after one week were collected and 21 of the isolated plasmids with inserts greater than 500 bp were sequenced. Three of the sequences matched the same region of the published cDNA Genbank clones AB037749  and AL832625 . The inserts of ~2 kb included part of the gene 3'-UTR. However the 5'-end of the coding region was not present in the retrieved clones. The issue was complicated by the fact that the sequences of AB037749 and AL832625 diverged at their 5'-end. 5'-RACE was thus employed to identify the transcriptional start site of the interacting gene by using mRNA derived from fetal kidney 293, hepatoma HepG2, and cervical HeLa human cells. All the cloned sequence coincided with that of the AL832625 clone and matched in full the putative coding sequence obtained by automated computational analysis of the human genome (Genbank XM029429). The conceptually translated sequence coded for a protein of 578 amino acids.The region of SMC3 encompassing the protein hinge domain (GlnHinderin gene spans ~400 kb of the human genome and is located on chromosome 18p11-2. The coding sequence is organized in 10 exons (fig. 441KERK) is located in exon 7 within a coiled-coil region. The Hinderin expression pattern in a panel of 16 human tissues was analyzed by semiquantitative RT-PCR . The rate of interaction between SMC3 and SMC1 is thus directly proportional to the level of luciferase that is expressed. We found that transfection of 293 cells with increasing amount of the Hinderin-V5 expression vector caused a dose-dependent decrease of luciferase activity. In cells overexpressing Hinderin, the interaction between the cohesin proteins was gradually reduced down to ~34% (n = 3, p < 0.05) (fig. In order to investigate whether Hinderin associates with SMC3 in vivo, 293 cells were transiently transfected with 1 μg/ml of Hinderin-V5 expression vector and incubated 48 h. The transfected cells displayed three-fold elevation of the ted fig. . This re05) fig. . EctopicIn eukaryotic cells, the disjunction of the sister chromatids at the onset of anaphase is required to maintain chromosomal stability and to prevent aneuploidy ,15. HumaIn summary, we have identified a novel interacting partner of SMC3. The protein, named Hinderin, specifically interacts with the hinge domain of SMC3. The protein is ubiquitously expressed in human tissues. We speculate that when in a certain context, SMC3 association with Hinderin becomes favored compared to the association to SMC1, the availability of SMC3 to engage in the cohesin complex formation is reduced. The binding of SMC3 to proteins affecting its association to functional partners represents a new modality of regulation of SMC3 activity.Pfu DNA polymerase and primers terminated with restriction sites that allowed the directional cloning of the products into the accepting vector  (see fig. Hinderin coding region was generated by RT-PCR utilizing mRNA extracted from 293 cells, and the product cloned in frame with the tag sequence in pcDNA3.1/V5-His TOPO (Invitrogen). When transfected into 293 cells, the expressed product detected with an anti-V5 monoclonal antibody had size 69 KDa, as expected for a protein encoded by the Hinderin-V5 fusion gene  and sequenced using a T7 primer. The sequence matched that of the DKFZp451C1618 clone (AL832625) and extended 5' to the published sequence of KIAA1328 (AB037749). Based on this information, the complete ene fig. .PstI or alternatively the NdeI restriction sites present in the vector multiple cloning site and at the SmaI site of the insert. The resulting linear constructs were blunt-ended and religated. pGADT7-SMC3-465/807 was generated by retriving the insert from the bait vector. SMC1-485/670 encoding the entire SMC1 hinge region, was generated by RT-PCR from 293 cells mRNA and cloned in pGADT7. Hinderin deletion constructs H-64/360 and H-360/578 were generated by restriction digestion of the pACT2-Hinderin-47/578 clone identified in the yeast two-hybrid system screening with NcoI/BglII and BglII/XhoI respectively, followed by religation of the blunt-ended vector. Hinderin in bait pGBKT7 vector was generated by retrieving the H-47/578 insert from the prey clone. H-177/360 and H-1/85 inserts were obtained respectively by PCR and by digestion of the Hinderin pcDNA3.1 expression vector with BamHI/EcoRI. Both were subsequently subcloned in pGADT7. To assess the strenght of interaction between different SMC3 and Hinderin domains, three colonies were randomly selected and grown overnight in 5 ml of selection media. After cell lysis by freeze thawing in 300 ml of 100 mM Na2HPO4 pH 7.0, 10 mM KCl, 1 mM MgSO4, buffer, β-galactosidase activity was assessed using ortho-nitrophenyl-β-D-galactopyranoside as substrate (1 mM) in the presence of 50 mM β-mercaptoethanol. After 2 h incubation at 37°C the color intensity was read at 420 nm.In order to map the SMC3 interacting site(s) a series of truncated constructs were produced in pGBKT7 vector. The inserts required for the SMC3-1/186, SMC3-976/1217, SMC3-552/807 and SMC3-711/807 constructs were produced by PCR. SMC3-465/550 and SMC3-465/716 were generated by introducing stop codons in the sequence of SMC3-465/807 using a QuikChange XL kit (Stratagene). The mutated duplex oligonucleotides used had the forward sequence: 5'-CTTTCTATACTTGTGT AAGTCACTGCTGGTAAC-3' and 5'-GACCAGTTGATGAACTAAATGCAGATAGAG-3', respectively. SMC3-465/643 and SMC3-643/807 were obtained by digesting SMC3-465/807 at the Hinderin transcript was identified by hybridization to a 335 bp 32P-labeled cDNA probe annealing to the 5'-end region of the gene. In order to examine the expression of Hinderin in different human tissues, semiquantitative RT-PCR was performed by using 0.5 μg of Marathon-ready first strand cDNA (Clontech) from 16 tissues. The PCR reaction was monitored at 20 and 30 cycles and the product analyzed on 2% agarose. In order to normalize for possible differences in mRNA content, the expression of the housekeeping gene G3PDH was analyzed in each sample.Total mRNA was extracted with TRI-reagent from subconfluent HeLa and HCT116 cells and separated on 1% agarose. After transfection to a nitrocellulose filter, the Hinderin in other species, the human protein sequence was BLASTed against the translated EST database of m. fascicularis, mouse, rat, cow, sheep, dog, zebrafish, c. elegans, drosophila, and s. cereviasie. When a homologous sequence had been identified in lower organisms, we ran the COILS program to assess whether it displayed the same secondary structure as that of the matching human sequence.The human polypeptide sequence was analyzed with the COILS program to predict globular and coiled-coil domains. The scanning window was set at 21. The homology with other known protein family was examined by querying the NCBI Conserved Domain protein database. We used PSORT to scan for nuclear and other localization signal consensus sequences. To identify orthologue forms of 3VO4, and a cocktail of protease inhibitors. The cell lysate was centrifuged at 12,000 g and the recovered supernatant preabsorbed on protein G-agarose. Aliquots (500 μl) of the cell lysate were then incubated overnight at 4°C with either 25 μg of goat anti-human SMC3 antibody or goat anti-human SMC1 antibody (Santa Cruz Biotech) or alternatively with 10 μg mouse anti-V5 antibody (Invitrogen). The immunocomplexes were captured on Protein G-agarose by incubating 1 h at 4°C. After washing in immunoprecipitation buffer containing 300 mM NaCl, the protein immunocomplex was analyzed by SDS-PAGE and the proteins transferred to nitrocellulose membranes by electroblotting. After saturation in 4% dry milk/0.1% Tween 20 in PBS, the filter was incubated 1 h at RT with primary antibody. The anti-SMC1 and anti-SMC3 immunoprecipitate filter blots were incubated with anti-V5 monoclonal antibody (200 ng/ml) whereas the V5 immunoblots were incubated with either anti SMC1 (100 ng/ml) or anti-SMC3 (100 ng/ml) antibodies. After incubation with species-specific anti IgG HRP-conjugated secondary antibody (1:10,000), the immunocomplexes were visualized by enhanced chemiluminescence reaction (ECL).293 cells were grown at ~80% confluence in 35 mm cm plates and transfected with 1 μg of Hinderin-V5 expression vector. After 48 h of incubation, the cells were washed in ice-cold phosphate-buffered saline and lysed in 1.2 ml of 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P40, 0.5% Na-deoxycholate buffer containing 100 mM NaF, 2 mM NaBamHI and SalI sites. The resulting bait and prey DNA constructs (0.25 μg/ml each) together with the Hinderin-V5 expression vector  and the reporter plasmid pG5/luc encoding the firefly luciferase (0.1 μg/ml), were co-transfected into 293 cells using Lipofectamine. Control experiments were conducted using pACT-Id and pBIND-MyoD vectors (Promega) encoding respectively GAL4:Id and VP16:MyoD fusion proteins known to interact in vivo [Firefly luciferase values were corrected for the transfection efficiency using the values of the Renilla luciferase activity encoded by the pBIND vector under the control of a strong constitutive promoter. In order to assess the effect of Hinderin on the bait and prey expression, total RNA was extracted from a group of transfected cells and the specifc transcript levels quantitated by RT-PCR using primers annealing to the ends of the fusion protein coding sequence.Inserts corresponding to the SMC3-474/702 and SMC1-474/663 hinge domains were generated by PCR and ligated in the pBIND or the pACT vectors (Promega) respectively through ligation at the  in vivo . After 4CAP carried out the two-hybrid system experiments including the immunoprecipitation studies, generated the necessary DNA constructs, and performed the gene expression analysis. GG conceived and coordinated the studies and drafted the manuscript.Primer and adapter sequences oligonucleotide sequenceClick here for file