Serial analysis of gene expression (SAGE) is used to obtain quantitative snapshots of the transcriptome. These profiles are count-based and are assumed to follow a Binomial or Poisson distribution. However, tag counts observed across multiple libraries  have additional variance that cannot be accommodated by this assumption alone. Several models have been proposed to account for this effect, all of which utilize a continuous prior distribution to explain the excess variance. Here, a Poisson mixture model, which assumes excess variability arises from sampling a mixture of distinct components, is proposed and the merits of this model are discussed and evaluated.The goodness of fit of the Poisson mixture model on 15 sets of biological SAGE replicates is compared to the previously proposed hierarchical gamma-Poisson  model, and a substantial improvement is seen. In further support of the mixture model, there is observed: 1) an increase in the number of mixture components needed to fit the expression of tags representing more than one transcript; and 2) a tendency for components to cluster libraries into the same groups. A confidence score is presented that can identify tags that are differentially expressed between groups of SAGE libraries. Several examples where this test outperforms those previously proposed are highlighted.The Poisson mixture model performs well as a) a method to represent SAGE data from biological replicates, and b) a basis to assign significance when testing for differential expression between multiple groups of replicates. Code for the R statistical software package is included to assist investigators in applying this model to their own data. Serial analysis of gene expression (SAGE) is a technique for obtaining a quantitative, global snapshot of the transcriptome . The mett-test) should be avoided. Indeed, such tests require tag counts be normalized by division with the total library size; this removal of library size from the set of sufficient statistics discards an informative facet of the data.As a counting technology, SAGE produces profiles consisting of a digital output that is quantitative in nature. For example, a statement can be made with reasonable certainty that a SAGE tag observed 30 times in a library of 100,000 tags corresponds to a transcript that comprises 0.03% of the total transcriptome; the same statement cannot be made reliably with analog values, like that obtained from a microarray. Accordingly, a reliable statistical model should account for the discrete, count-based nature of SAGE observations. When testing for differential expression between groups, where each group can contain multiple libraries, statistical methods that incorporate a continuous probability distribution , so the form of the Poisson and Binomial distribution is essentially the same. A fortunate characteristic of both of these distributions is that they are a function of a single parameter only, since the variance in observed data is directly calculable from the mean.viz. beta-binomial using logistic regression  r r4] refegression , tw-test ]beta1 <- fit$coefficients[[2]]# get the expression (expressed as a proportion) for each groupprop0 <- exp(beta0)prop1 <- exp(beta0+beta1)# calculate significance score for differential expression# i.e. null hypothesis that beta_1 = 0t.value <- summary(fit)$coefficients [2]p.value <- 2 * pt, fit$df.residual)In contrast to the canonical log-linear model, we assume the actual expression is distributed asθi ~ Gamma,μi = nipii is the actual expression in terms of the proportion of all expressed tags. Here, the unconditional mean and variance are E(θi) = μi and Var(θi) = μi2ϕ. Since we are now sampling from this latent Gamma distribution, the observed tag counts are conditional on this underlying expression and are distributed aswhere, as above, pi | pi,ϕ ~ Poisson(θi)Yi) = μi and Var(Yi) = μi(1+μiϕ).Now, the unconditional mean and variance are E = log(ni) + xiβlog(Yi = exp(xiβ)pβ and the overdispersion parameter (ϕ) can be performed. The MASS library ]beta1 <- fit$coefficients[[2]]# get the dispersion parameterdispersion <- 1/fit$theta# get the expression (expressed as a proportion) for each groupprop0 <- exp(beta0)prop1 <- exp(beta0+beta1)# calculate significance score for differential expression# i.e. null hypothesis that beta_1 = 0t.value <- summary(fit)$coefficients [2]p.value <- 2 * pt, fit$df.residual)A more complete discussion of this model and its application to SAGE, including significance testing, is described in .Like the canonical log-linear regression model, we assume the observed tag counts are Poisson distributed. However, the counts are conditional on the choice of a Poisson-distributed component, such thati | k ~ Poisson(μik)Yμik = nipikik is the actual expression for component k in terms of the proportion of all expressed tags. The posterior probability that an observed tag count belongs to a component k is given bywhere the component k = 1, 2, ..., K and pψ is the parameter vector containing the component means  and mixing coefficients . f(.) is the Poisson probability density function. To fit the model, one must estimate the values in ψ. This can be done by maximum likelihood estimation (MLE) using the EM algorithm ] <- fit   aic.fits [k] <- AIC(fits [k]])   if(k == 1) next   if(aic.fits [k] >= aic.fits [k-1]) break}# what number of components minimized AIC?k.optimal <- which)[1]fit <- fits [k.optimal]]# get the theta parametersthetas <- arrayfor thetas [i] <- parameters$coef# get the pi parameterspis <- attributes(fit)$prior# what is the confidence score that the fitted components# differentiate between groups?confidence <- pmm.confidenceThe confidence score, ranging from 50–100%, is explained in the Results section and code for performing the calculation is available [see Additional file SDZ conceived of, developed, and tested the presented research.Derivation of confidence score. A derivation of a confidence score for differential expression based on a Poisson mixture model fit.Click here for fileSupplementary SAGE library information. An Excel spreadsheet containing accessions, sizes, and descriptions of the libraries included in this study.Click here for fileConfidence score R function. R source code for a function to calculate the differential expression confidence score based on a Poisson mixture model fit.Click here for file


Genome-wide scans for positively selected genes (PSGs) in mammals have provided insight into the dynamics of genome evolution, the genetic basis of differences between species, and the functions of individual genes. However, previous scans have been limited in power and accuracy owing to small numbers of available genomes. Here we present the most comprehensive examination of mammalian PSGs to date, using the six high-coverage genome assemblies now available for eutherian mammals. The increased phylogenetic depth of this dataset results in substantially improved statistical power, and permits several new lineage- and clade-specific tests to be applied. Of ∼16,500 human genes with high-confidence orthologs in at least two other species, 400 genes showed significant evidence of positive selection (FDR<0.05), according to a standard likelihood ratio test. An additional 144 genes showed evidence of positive selection on particular lineages or clades. As in previous studies, the identified PSGs were enriched for roles in defense/immunity, chemosensory perception, and reproduction, but enrichments were also evident for more specific functions, such as complement-mediated immunity and taste perception. Several pathways were strongly enriched for PSGs, suggesting possible co-evolution of interacting genes. A novel Bayesian analysis of the possible “selection histories” of each gene indicated that most PSGs have switched multiple times between positive selection and nonselection, suggesting that positive selection is often episodic. A detailed analysis of Affymetrix exon array data indicated that PSGs are expressed at significantly lower levels, and in a more tissue-specific manner, than non-PSGs. Genes that are specifically expressed in the spleen, testes, liver, and breast are significantly enriched for PSGs, but no evidence was found for an enrichment for PSGs among brain-specific genes. This study provides additional evidence for widespread positive selection in mammalian evolution and new genome-wide insights into the functional implications of positive selection. Populations evolve as mutations arise in individual organisms and, through hereditary transmission, gradually become “fixed”  in the population. Many mutations have essentially no effect on organismal fitness and can become fixed only by the stochastic process of neutral drift. However, some mutations produce a selective advantage that boosts their chances of reaching fixation. Genes in which new mutations tend to be beneficial, rather than neutral or deleterious, tend to evolve rapidly and are said to be under positive selection. Genes involved in immunity and defense are a well-known example; rapid evolution in these genes presumably occurs because new mutations help organisms to prevail in evolutionary “arms races” with pathogens. Many mammalian genes show evidence of positive selection, but open questions remain about the overall impact of positive selection in mammals. For example, which key differences between species can be attributed to positive selection? How have patterns of selection changed across the mammalian phylogeny? What are the effects of population size and gene expression patterns on positive selection? Here we attempt to shed light on these and other questions in a comprehensive study of ∼16,500 genes in six mammalian genomes. Positive darwinian selection is an important source of evolutionary innovation and a major force behind the divergence of species. The Neutralist-Selectionist debate of the past 30 years has gradually given way to a general consensus that both neutral drift and positive selection play major roles in evolutionary change. Interest has therefore shifted to questions of which genes positive selection has affected, how strong was the effect, when did it occur, and what were its functional consequences. Heightening interest in these questions is a growing appreciation that methods for detecting positive selection can also be valuable tools for gaining insight into gene function Phylogenetic and population genetic methods for detecting positive selection serve as complementary tools for functional and evolutionary analysis. These methods operate at different time scales, with phylogenetic methods being best suited for detecting selection that operates over relatively long periods in evolutionary history, and population genetic methods being best suited for detecting more recent selection. Population genetic methods can potentially detect selection operating at individual sites, through the effects of linkage with flanking alleles, while phylogenetic methods generally require multiple sites to have been affected in a sequence of interest. At the same time, decay of linkage disequilibrium at longer evolutionary time scales can allow phylogenetic methods to more accurately pinpoint the specific locations of functionally important substitutions. In some cases, phylogenetic methods also allow such substitutions to be mapped to particular branches of a phylogenetic tree, thereby providing useful insights about the evolutionary histories of the sequences in question.With the availability of multiple complete genome sequences, it has become possible to apply phylogenetic methods for the detection of positive selection at a genome-wide scale. Within mammals, several genome-wide scans for positive selection on protein-coding genes have been conducted, using both phylogenetic Nevertheless, much remains to be learned about positive selection in mammalian genomes, even within protein-coding regions. The most comprehensive scans for PSGs so far Here we present a phylogenetic analysis of positive selection in the six eutherian mammalian genomes for which high-coverage, high-quality sequence assemblies are now available: the human ortholog sets (human genes and non-human orthologs) were identified by an automatic pipeline that made use of syntenic whole-genome alignments, sequence quality scores, and other data  to 400 . As in previous studies, the numbers of genes identified by the tests for individual primate branches were small, primarily due to weak power caused by low levels of inter-species divergence. The inclusion of additional non-primate mammals does not appear to have improved the power of these tests substantially, but it does allow a distinction to be made between selection on the branches to the hominids and to macaque. The tests for selection on the branch to the primates and in the primate clade also yielded fairly small numbers of PSGs, but the tests for selection in, or on the branch to, the rodents identified somewhat (nearly three-fold) larger numbers. In general, even with the larger data set, our power to detect selection on individual lineages and clades is still fairly weak, and differences in numbers of identified PSGs almost certainly reflect differences in power more than differences in the prevalence of selection. Nevertheless, these LRTs together produced a fairly large set of high-confidence PSGs, permitting a more detailed and thorough functional analysis than has previously been possible in mammals (see below).P-values (whether or not they met the significance threshold for PSGs), as determined by the Mann-Whitney U (MWU) test ; full results are shown in The identified PSGs are significantly enriched for a large number of functional categories, according to the Gene Ontology (GO) test see . Better test see  and S3. test see . In the P = 4.2×10−8), viruses (P = 3.0×10−8), wounding (P = 3.2×10−8), and acute inflammation (P = 4.7×10−11). In some cases, different categories reflect the same or very similar sets of genes , while in others they reflect quite distinct gene sets (“response to wounding” and “response to virus”) (P = 1.9×10−9) and adaptive (P = 1.5×10−5) immunity are over-represented, with many PSGs contributing to both classes. The conventional division of adaptive immunity into humoral (P = 1.6×10−7) and cellular (P = 3.5×10−7) responses is reflected in the enriched GO categories. Various mechanisms of immune response are represented, including previously identified categories for natural killer cell (P = 1.6×10−8), B-cell (P = 4.8×10−7), and T-cell (P = 1.2×10−8) mediated immunity −8) and complement-mediated immunity  . Genes iP<0.05  and receptors , but some are part of the membrane attack complex , which punctures cell membranes to initiate cell lysis. Many of these PSGs are known to interact with one another, suggesting possible co-evolution. Two of three biochemical pathways known to activate the complement system are also enriched for PSGs , as is the coagulation cascade that interacts with the complement system  show particularly strong enrichments for PSGs. For example, five of the six SIGLEC genes included in our analysis are under positive selection (see HAVCR1 (LRT P = 6.9×10−9)—revealed several sites under positive selection in its N-terminal V-like immunoglobulin (IgV) domain. Three of these sites correspond to regions of the protein believed to play critical roles in binding to viruses or in regulating the immune function of the gene , whose deficiency is associated with increased susceptibility to autoimmune and allergic diseases Several gene families of the immunoglobulin superfamily , such as the protein p53, which also regulates maternal reproduction ADAM2 (P = 2.9×10−6), which is integral to fertilization ADAM15 (P = 5.4×10−4) and ADAM29 (P = 3.4×10−4), which are strong candidates for cancer evolution driven by sexual conflict. In addition, the testes development-related gene CCDC54 (P = 3.3×10−4) is currently a target of cancer immunotherapy research The hierarchical clustering of GO categories  reveals P = 4.3×10−39) and “G-protein coupled receptor protein signaling pathway” . Previously, enrichments for such categories have been attributed primarily to olfactory receptors P = 6.9×10−36). However, eight PSGs are involved in “sensory perception of taste,” including five taste receptors (P = 1.4×10−10). Interestingly, several of these are bitter taste receptors. The sense of bitter taste is critical in allowing organisms to avoid toxic and harmful substances, and extensive gene expansion of bitter taste receptors is known to have occurred during mammalian evolution TAS2R1, TAS2R5, and a recently expanded cluster of genes at chr12p13 . Another PSG, TAS1R2, is a receptor of sweet and umami taste, and the PSG RTP3 is a transmembrane protein that is involved in the transport of taste receptors and apparently influences their expression.A smaller and somewhat less diverse group of enriched categories is associated with sensory perception. Among the most inclusive categories of this type are “sensory perception of chemical stimulus”  are dominated by olfactory and taste receptors, but they also include other types of genes. For example, TMC2 (P = 1.1×10−4) is expressed in the inner ear and is important for balance and hearing ACCN4 (P = 1.0×10−6) has been implicated in synaptic transmission, pain perception, and mechanoperception SLC6A5 (P = 3.0×10−4) is associated with hyperekplexia, a neurological disorder characterized by an excessive startle response NMUR (P = 6.1×10−4) is involved in the mammalian circadian oscillator system NTSR1 (P = 8.1×10−4) mediates hypotension, hyperglycemia, hypothermia, antinociception, and regulation of intestinal motility and secretion The PSGs in the “neurological processes” category (MGAM (P = 2.4×10−8) is essential for the small intestinal digestion of starch, giving it a critical role in human metabolism, as starches of plant origin make up two-thirds of most human diets MAN2B1 (P = 1×10−6) is involved in the cleavage of the alpha form of mannose, a sugar monomer. Defects in this gene cause lysosomal alpha-mannosidosis, a lysosomal storage disease characterized by the accumulation of unbranched oligosaccharide chains TCN1 (P = 2.9×10−31) is a major constituent of secondary granules in neutrophils and facilitates the transport of vitamin B12 into cells, which is important for the normal functioning of the brain and nervous system, and for the formation of blood P = 8.3×10−4) including genes that metabolize xenobiotics and drugs . Positive selection in these and other genes is likely to have been influenced by changes in food preferences during mammalian evolution.Similarly, the PSGs associated with diet include but are not limited to taste and olfactory receptors. For example, P-values for genes of certain functional categories than for others. Interestingly, these categories were dramatically different for the primate- and rodent-clade LRTs, with nearly all of the primate categories relating to sensory perception, and nearly all of the rodent categories relating to immunity and defense  and color vision , and receptors involved in immunity . The PSGs identified by the rodent-clade test include few such genes, but they include many genes involved in responses to wounding, inflammation, and stress, as well as genes involved in complement activation and innate immunity. Thus, we find little evidence that genes directly involved in brain development and function have (as a group) been driven by positive selection in primates, but many genes that provide sensory information to the brain do appear to have experienced positive selection. These changes in sensory perception could conceivably have been brought on by, or could have contributed to, increased brain size and complexity in primates.Few functional enrichments were evident for the PSGs identified by the branch- and clade-specific LRTs, primarily because these sets were quite small in size. However, the more powerful LRTs, such as those for the primate and rodent clades , did pro defense . Indeed,ω0<1, ω0 = 1, or ω2>1) or absence of positive selection (each site evolves with ω0<1 or ω0 = 1). The model allows a posterior distribution over selection histories to be inferred for each gene, and it allows for estimates of the number of genes under positive selection on individual branches and clades that consider uncertainty about selection histories. Unlike the branch- and clade-specific LRTs—which are simple one-sided hypothesis tests and are necessarily conservative about rejection of the null hypothesis—this model considers all candidate histories symmetrically, and allows for “soft” (probabilistic), rather than absolute, choices of history at each gene.To gain further insight into the patterns of positive selection that have shaped present-day mammalian genes, we devised a model that allows for probabilistic inferences about the selection histories of individual genes. A selection history is defined as an assignment to each branch of the phylogeny of one of two evolutionary modes: positive selection (each site evolves with 9−1 = 511 possible histories for the nine-branch (unrooted) phylogeny. To reduce computational cost, the inference of selection histories was conditioned on the maximum likelihood estimates of the parameters of the codon models , suggesting longer-term selective pressure that have also affected nonhuman primates. A third example is CGA, which encodes the alpha subunit of the four human glycoprotein hormones . This gene shows strong evidence of positive selection specific to the primate clade (PP = 0.82), consistent with the proposal that relatively recent adaptations in pregnancy and development have played a critical role in the evolution of the human endocrine system CGB1 and CGB2 (which encode two of the six beta subunits of chorionic gonadotropin) are thought to have originated by gene duplication in the common ancestor of humans and great apes CGA. Finally, the complement components C7 and C8B, which encode proteases in the membrane attack complex, are predicted with high probability to be under selection in rodents only . Differences in complement proteases are thought to explain certain differences in the immune responses of humans and rodents In addition to being useful in a bulk statistical analysis of all PSGs, the Bayesian framework can be used to identify the single most likely selection history for each gene. In some cases, these histories are consistent with known functional differences between species, and help to shed light on the evolutionary basis of these differences. For example, the sweet receptor P<4×10−4 in all tissues for PSGs defined by the all-branch test, one-sided FET). Moreover, among genes expressed above background, expression levels are significantly lower for PSGs than for non-PSGs . In addition, we observed a positive correlation of ω with tissue bias, as measured by τ (ρ = 0.24) ω.) Unlike in previous studies, however, we were able to examine these correlations separately for positively and non-positively selected genes, using the set of PSGs identified by the all-branches LRT. Interestingly, the correlations of ω with expression level τ are much stronger within the non-PSGs than within the PSGs, indicating that the observed correlations are primarily driven by negative rather than positive selection ; and 45 of 715 (6.3%) testes-specific genes were PSGs, compared with 2.1% of other genes (P = 8.2×10−10). There were also significant, but weaker, enrichments for PSGs among liver-specific (P = 9.1×10−3) and breast-specific (P = 1.0×10−2) genes. Not surprisingly, the spleen-specific PSGs generally appear to be immune-related, and many of the testes-specific PSGs are involved in spermatogenesis or sperm adhesion  on all branches of the phylogeny, but that estimates of ω vary considerably within the mammals. These estimates are largest for the hominids (ω≈0.25), smallest for the non-primate mammals (0.12<ω≤0.14), and intermediate for non-hominid primates (0.17<ω<0.21). It is thought that increased estimates of ω in hominids primarily result from weakened purifying selection, owing to reduced effective population sizes While positive selection was our primary focus, our data set also provides an opportunity to compare the average rates of protein evolution in various mammalian lineages. We estimated a separate nonsynonymous-synonymous rate ratio log sets . Consistω and population size further, we made use of a theoretical relationship between ω and the scaled selection coefficient γ  were roughly the same (hN = cN) mω in macaque to hω in human/chimpanzee from our 10,980 human-chimpanzee-macaque ortholog trios. Our estimate of mω / hω = 0.732 implies an estimate for the ratio of the macaque to human ancestral population sizes of mN / hN = 1.41 [bootstrapping 95% CI ]. In comparison, the ancestral macaque population size has been estimated at ∼73,000 mN / hN = 1.41 , suggesting that a genome-wide scan will tend to identify only the most extreme cases of positive selection. If a rigorous correction for multiple testing is applied, a test based on hominids only has essentially no power, even for fairly long genes under strong selection (ω among lineages and among sites), and they must be interpreted cautiously. However, estimates of relative power—which will be less sensitive to these simplifying assumptions—indicate a substantial improvement is achieved by the addition of the three non-primate mammals.To compare the power of our LRTs with the power of previous tests based on hominid or primate genomes, we simulated data sets under a range of parameter values and measured the fraction of cases in which positive selection was predicted . These eelection . The sitelection . The absDrosophilaSince it first became possible to compare the sequences of complete mammalian genomes about five years ago, a number of genome-wide scans for positively selected genes (PSGs) have been conducted using phylogenetic methods FAS/p53 apoptotic pathway . Various explanations have been proposed for the observed decrease in ω among genes expressed at high levels and/or expressed broadly across tissues, including selection for translational efficiency, selection against misfolding, or increased selection due to pleiotropy ω with expression level and τ hold strongly within non-PSGs, but are much less pronounced within PSGs. Thus, expression levels and patterns are strongly correlated with both the strength of negative selection and the likelihood of positive selection, but they are only weakly correlated with the strength of positive selection. It appears that genes may be more likely to come under positive selection if they are in a state of evolutionary flexibility brought on by reduced or tissue-specific expression, but once positive selection has taken hold their subsequent evolutionary course is not strongly dependent on their expression patterns.Our finding that PSGs are expressed at lower levels and in a more tissue-specific manner than non-PSGs is consistent with a well-known negative correlation As additional mammalian genomes become available, the statistical power to detect positive selection will improve. However, most forthcoming genomes are being sequenced at low coverage, and will inevitably exhibit increased levels of error in base calls, genome assemblies, ortholog identification (due to short contigs), and alignment—all of which can lead to spurious signals for positive selection.  Thus, careful data quality controls will be needed to take advantage of these data. In addition, when considering the impact of additional sequences on statistical power, it is useful to distinguish between positive selection that acts continuously (or in recurrent episodes) over a long evolutionary period, and positive selection that acts transiently or in a lineage-specific manner. Deep phylogenetic sequencing should generally improve detection power for continuous or recurrent positive selection, but power for transient selection depends strongly on the sequenced species and the lineages of interest. For example, the genome sequences of a dozen non-primate mammals will likely have little effect on the power to detect human-specific selection, while the gorilla and neanderthal genomes could help considerably. There are fundamental limitations in the detection of weak, transient, or highly localized positive selection that will not be overcome by any amount of genome sequencing. Nevertheless, the availability of several new primate genomes, including those of the orangutan, marmoset, and gorilla, may significantly improve power for PSGs in primates.HAVCR1 gene. Nevertheless, they provide a valuable starting point for experimental follow-up. At the same time, more can be done to incorporate non-sequence data—such as structural and expression data—into computational methods for detecting positive selection. Thus, improvements in both computational and experimental methods will be needed to establish deeper and more informative connections between evolutionary dynamics and molecular function.Our ability to connect positive selection with function remains rudimentary, but gradual progress is being made. As additional sequence data becomes available, it will become possible to associate selection with individual residues of proteins with greater accuracy. At the same time, more data is becoming available on the specific functional roles of individual amino acids, for example, from structural or mutagenesis studies. As a result, it will increasingly become possible to find direct links between selection and function. Often these links will initially be tentative, as in our site-specific analysis of the The latest human (hg18), chimpanzee (panTro2), rhesus macaque (rheMac2), mouse (mm8), rat (rn4), and dog (canFam2) genome assemblies were obtained from the University of California, Santa Cruz (UCSC) Genome Browser. Human-referenced whole-genome alignments were constructed from syntenic pairwise alignments with human (the “syntenic nets”) using the UCSC/MULTIZ alignment pipeline A starting gene set was composed from of the human RefSeq To establish 1∶1 orthology, each human gene and putative non-human ortholog was examined for evidence of an inparalog  for each G+C class using evolverμ, ω0 and p0 were drawn from the empirical distribution defined by the real alignments (using estimates obtained under the null model), and the remaining parameters were fixed at global estimates for each G+C class. Log likelihood ratios (LLRs) were then computed exactly as for the real data. The nominal P -value for a LLR of r was defined as the fraction of all simulated alignments with LLR≥r, unless the number of such alignments was <10, in which case we assumed P-values, according to the simulation experiments). The method of Benjamini and Hochberg P-value threshold for a false discovery rate of <0.05. For the lineage- and clade-specific LRTs, P-values were computed assuming the null distribution was a 50∶50 mixture of a For the LRT for selection on any branch, zero see .X1,…, NX) be the alignment data, with iX denoting the alignment for the i th gene , and let Z = be the set of selection histories, with iZ denoting the selection history for the i th gene . Recall that a selection history is defined as a pattern of presence or absence of positive selection on the branches of the unrooted phylogeny. Let ibZ ∈ {0,1} indicate the selective mode (with 1 representing positive selection) for branch b ∈ {1,…,B} (here B = 9) under history iZ. The parameters of the switching model, denoted θ, are defined below. The model assumes independence of genes and independence of histories, and conditional independence of X and θ given Z. Thus, the complete data likelihood is given by:Let X = (P(iZ|θ), is a function of the set of switches in selective mode required to explain the history parsimoniously. For each history to be explained parsimoniously, switches must be allowed to occur early (near the ancestor) or late (near the descendant) on each internal branch, as well as (early) on each external branch  correspond to four possible scenarios at nbP—gain of selection , loss of selection , absence of gain , or absence of loss . The probabilities of these scenarios  are defined by a parameter for gains (nbGθ) and a parameter for losses (nbLθ) at each point. In addition, the prior probability of selection at the root of the tree is given by a parameter θ0. . The prior probability for each element of θ is defined by a (conjugate) Beta distribution with parameters α and β . Because these elements are independent in the prior,The switching model effectively defines a prior distribution over histories, which tends to favor simpler histories over more complex ones  in equation 1 is simply the likelihood at gene i of a branch-site codon model that assumes selection history iZ. A full Bayesian approach would integrate over the parameters of these codon models, but this would be computationally prohibitive. Instead, we make the Empirical Bayes simplification of conditioning the analysis on maximum likelihood estimates of the parameters of the codon models. The maximized log likelihoods ijL for all genes i and histories j are precomputed using existing software  and stored in an N×M matrix, which is then used in the inference of selection histories.The term θ are unobserved, and the goal is to infer their joint posterior distribution,The variables Z and iZ conditional on iX and a previously sampled θ, and sampling each element of θ conditional on a previously sampled Z. It is straightforward to derive the required conditional distributions and to sample from them  from P. Estimated posterior expected values of interest were obtained by averaging these samples or functions of these samples, and Bayesian 95% confidence intervals were obtained by taking the 0.025 and 0.975 quantiles of the sampled values. For example, the posterior expected number of genes under selection on branch k (see T is the number of samples and the function kf(Z) counts the number of genes under selection on branch k in a set of histories Z.After an appropriate burn-in period, each iteration of the Gibbs sampler produces a sample  P-value for independence of rows and columns was computed by Fisher's exact test. In addition, the distributions of LRT P -values among the genes assigned to C and not assigned to C were compared by a (one-sided) Mann-Whitney U (MWU) test. (Notice that S is not considered in this case.) Nominal P -values computed by the FET and MWU tests were corrected for multiple comparisons using the method of Holm Each gene was assigned categories from the GO http://www.affymetrix.com/support/technical/sample_data/exon_array_data.affx). The RMA-based probeset summaries The analysis of gene expression was based on the publicly available “Tissues+Mixtures” sample data set for the Affymetrix GeneChip Human Exon 1.0 ST Array  were combined using Fisher's method P<0.001. Similarly, an estimated expression intensity for each gene×tissue was calculated by first taking the median over the three replicates of each RMA-based probeset summary, then taking the median of these values over all probesets associated with the gene. The analysis of expression intensities was restricted to genes significantly expressed above background so that genes expressed at or near the background level did not drive the results.To calculate a τγ, defined as γ = maxtγt, where tγ is the squared cosine of the angle between the expression vector and the coordinate axis associated with t  the statistic th t see . In defiω for each branch were obtained using the codeml program in the PAML software package κ (fix_kappa = 0) and a single ω across sites per branch . The tree topology shown in Maximum likelihood estimates of ω should be given by γ = 2Ns. Therefore, γ can be estimated as f−1(ω), where ω = f(γ) denotes the function above. , multiple alignments of orthologous gene sets, GO and PANTHER category assignments, detailed results of the LRTs and the Bayesian analysis, and other resources. In addition, the candidate genes and predicted PSGs are displayed as a track in the UCSC Genome Browser .A website is available at Figure S1Complement component and coagulation pathways.(0.70 MB TIF)Click here for additional data file.Figure S2Boxplot of marginal posterior distributions.(0.17 MB TIF)Click here for additional data file.Figure S3Full power results.(0.34 MB TIF)Click here for additional data file.Figure S4Apoptosis pathway.(0.47 MB TIF)Click here for additional data file.Figure S5p53 signaling pathway.(0.45 MB TIF)Click here for additional data file.Figure S6GO similarity.(0.29 MB TIF)Click here for additional data file.Figure S7Expression results for all tissues.(0.50 MB TIF)Click here for additional data file.Table S1Minimum species configurations required for likelihood ratio tests.(0.02 MB PDF)Click here for additional data file.Table S2GO categories over-represented among predicted PSGs.(0.11 MB PDF)Click here for additional data file.Table S3PANTHER categories over-represented among predicted PSGs.(0.10 MB PDF)Click here for additional data file.Text S1Supplementary methods with complete set of supplementary figures and tables (including some not referenced in main article).(2.86 MB PDF)Click here for additional data file.


Perfusion was performed by cannulation of the femoral artery and vein in rats bearing DS-sarcoma on the hind foot dorsum. Perfusion rate was adjusted to maintain a perfusion pressure of 100–140 mmHg throughout the experiment. Following equilibration, nifedipine was continuously infused for 30 min (8.3 μg min−1 kg−1 BW). During constant-pressure isolated limb perfusion, nifedipine can significantly increase perfusion rate (+100%) and RBC flux (+60%) through experimental leg tumours. ‘Steal phenomena’ in favour of the surrounding normal tissue and oedema formation were not observed. Despite the increased oxygen availability (+63%) seen upon application of this calcium channel blocker, nifedipine does not result in a substantial reduction of tumour hypoxia, most probably due to an increase in O2 uptake with rising O2 supply to the tumour-bearing hind limb. Nifedipine application during isolated limb perfusion can enhance tumour microcirculation and may therefore promote the delivery (pharmacokinetics) of anti-cancer drugs to the tumour and by this improve the efficacy of pressure-controlled isolated limb perfusion.Isolated limb perfusion allows the direct application of therapeutic agents to a tumour-bearing extremity. The present study investigated whether the dihydropyridine-type CaBritish Journal of Cancer (2002) 87, 1462–1469. doi:10.1038/sj.bjc.6600611www.bjcancer.com© 2002 Cancer Research UK Isolated limb perfusion (ILP) is a treatment modality for malignancies of the extremities in which the tumour-bearing limb is isolated from the patient's circulatory system and perfused separately. This procedure allows the administration of anti-cancer agents to the tumour at high doses with only a minimum risk of systemic toxicity. High-dose regional chemotherapy was one of the first concepts in ILP which was complemented more recently by the application of cytokines and immune-modulators frequently in combination with hyperthermia .The response to chemo- or cytokine-therapy may however be compromised by ‘biological’ factors such as tumour blood flow and microcirculation, tissue oxygenation or pH distribution. The individual tumour microenvironment is a paramount determinant in the outcome of non-surgical treatments . For exaOne group of drugs that might achieve these goals (and by this might increase the efficacy of ILP) are calcium channel blockers (CCB). In experimental tumour therapy, CCBs have been of broad interest because of their ability to modify tumour blood flow and improve oxygenation status.2 supply, CCBs also seem to be able to improve the O2 status directly by reducing oxygen consumption  have been shown to improve tumour blood flow, oxygen availability , 1989, tsumption .in vitro and in vivo by CCBs of the dihydropyridine type  into the dorsum of the hind foot. Tumours were used when they reached a volume of between 0.8 and 1.3 ml, 4 to 9 days after tumour implantation corresponding to a tumour mass of 0.5% of the body weight  housed in our animal care facility were used in this study. They received a standard diet and acidified water −1. This stock solution was added continuously at a rate of 0.5 ml h−1 kg−1 body weight  to the perfusion medium via a catheter placed in the femoral artery resulting in a nifedipine dose of 8.3 μg nifedipine min−1kg−1 body weight. An equivalent volume of the vehicle was infused into control animals.Under protection from direct light, 2 mg nifedipine  was dissolved in 10 ml 96% ethanol. Further dilution was made with isotonic saline resulting in a concentration of 0.1 mg mlsingle-pass ILP of the tumour-bearing extremity was performed  obtained from donor rats. Prior to use, blood was diluted to a haematocrit of 25% with bicarbonate-buffered oxypolygelatine , an isotonic colloidal blood plasma substitute solution which is used in the clinical setting. By using this drug for haemodilution an isotonic and isooncotic perfusate was obtained. Mean haemoglobin concentration (cHb) was 77 g l−1. Mean glucose and lactate levels were 6.1 and 6.9 mM, respectively. Blood was oxygenated with a humidified gas mixture containing 6% CO2, 36% O2 and 58% N2 (v v−1).A −1 i.p., Narcoren™, Merial, Hallbergmoos, Germany) and polyethylene catheters were surgically placed into the thoracic aorta via the left common carotid artery and into the right external jugular vein. Mean arterial blood pressure (MABP) was continually monitored through the connection of the arterial catheter to a Statham pressure transducer . Arterial blood gas analysis was performed before and during ILP experiments using a pH/blood gas analyser . Since the anaesthetic agent used is known to induce respiratory depression in high concentrations, the depth of anaesthesia was assessed by monitoring arterial blood pressure and blood gas status to ensure that these parameters remained within the physiological range throughout the experimental period.When tumours had reached the target volume, animals underwent general anaesthesia  was injected intravenously to prevent coagulation. An incision was made in one groin and the femoral artery and vein were exposed. After ligation of accessible collaterals, the femoral vessels were cannulated , flushed with 2 ml of warmed oxypolygelatine and then connected to the perfusion equipment. Blood flow in deep-seated collaterals was restricted by a groin tourniquet which was tightened upon commencement of the perfusion. Perfusate flow rate was varied (range: 0.27–2.63 ml min−1) to achieve a constant perfusion pressure (PP) of approximately 100 to 140 mmHg in order to maintain an adequate tissue perfusion which might otherwise be impaired with PP approximately 15 mmHg below the systemic MABP  was used to measure red blood cell flux (RBC flux). Details of this method have been described earlier by Although, attempts were made to maintain the PP at a constant level during ILP, slight pressure changes (±5–10 mmHg) occurred. In order to assess whether changes in RBC flux were due to variations in PP or the result of nifedipine-induced vasodilation, the relative tumour vascular resistance (TVR) was calculated as a measure of the resistance to flow. The TVR was defined by the ratio of the MABP (or PP) and the RBC flux. This parameter is suitable for assessment of changes in the vascular diameter from variations in tumour blood flow.2) was assessed polarographically using a flexible O2-sensitive catheter electrode  which was inserted into the centre of the tumour for continuous monitoring of tumour pO2. Before each experiment, the pO2 electrode was calibrated with room air in a chamber with constant temperature, taking the ambient barometric pressure into account.Mean tumour oxygen partial pressure , 15, and 30 min to assess pH/blood gas status  as well as glucose and lactate concentrations which were determined enzymatically using standard test kits .After the surgical procedure, animals were allowed to stabilise and measurements commenced once constant baseline readings for PP, RBC flux and tumour oxygen tension were obtained for at least 20 min . Thereafter baseline values for blood flow rate, PP, RBC flux and tumour pOM perchloric acid, centrifuged and the supernatant neutralised with 2 M potassium hydroxide and diluted 1 : 2 with the mobile HPLC-phase. Concentrations were then determined using reversed-phase high-performance liquid chromatography (HPLC) and UV-detection at 254 nm. The isocratic separation was performed by a Superspher RP 18 end-capped column  and a guard cartridge system (5×4 mm). The mobile phase consisted of 0.05 M ammonium dihydrogen phospate, 0.01 M tetrabutylammonium hydroxide and 11.5% acetonitrile (v v−1), adjusted to pH 6.4. The flow rate was 0.9 ml min−1 and the sample size 40 μl. Concentrations of all metabolites are expressed as μmol g−1 tissue wet weight. Wet weight was estimated from tissue samples as follows: tumours (perfused for 1 h) were excised from the hind foot dorsum, skin was removed and wet weight recorded. Tumours were dried at 60°C until constant weight readings were attained. The tissue water content was the same in perfused and in untreated tumours (82.1±0.2% vs 82.0±0.1%) of comparable volume. Oedema formation during ILP can thus be excluded.In an additional series of experiments, the tumour of the isolated perfused leg and of the contralateral hind limbs of the anaesthetised animals were surgically removed and rapidly frozen in liquid nitrogen immediately following termination of the perfusion procedure (30 min of equilibration plus 30 min of nifedipine or vehicle infusion) and the tumours subsequently removed. The tumours were ground to a fine powder and subsequently freeze-dried. Thereafter, glucose and lactate concentrations were assayed enzymatically using standard test kits . Concentrations of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) were determined by high-performance liquid chromatography  to 81% of the baseline value. Thereafter however, the decrease was intentionally compensated by the increased perfusate flow rate resulting in a constant PP of between 130 and 135 mmHg. During nifedipine application, the perfusate flow rate had to be increased by almost 100% in order to maintain a constant PP  remained almost constant  were taken together, a linear correlation (r2=0.606) was seen between the O2 supply to the tumour-bearing leg and the O2 uptake into the tumour , and higher lactate levels in the isolated perfused limb tumours . However, the higher glycolytic rate during ILP did not have a strong impact on the bioenergetic status. In isolated perfused tumours (without nifedipine) the ATP levels were 0.66±0.08 compared to 1.18±0.13 μmol g−1 in the contralateral non-isolated-perfused tumours. Although the oxygen supply was restricted during ILP per se (due to the reduced haematocrit of the perfusate which results in a lower oxygen transport capacity), a stable bioenergetic status was maintained.As a result of the reduced haematocrit (25%) used in the perfusate, the oxygen supply during ILP −1 during nifedipine application vs 0.59±0.09 μmol g−1 in ILP tumours without nifedipine). However, since the oxygenation status was not improved by nifedipine neither the lactate levels (25.7±2.2 vs 27.0±3.5 μmol g−1) nor the ATP concentration (0.79±0.11 vs 0.66±0.08 μmol g−1) markedly changed during nifedipine treatment compared to tumours during ILP without nifedipine. Obviously, the increase in tumour blood flow had practically no impact on the metabolic or bioenergetic status of the tumour.With nifedipine infusion during ILP, tumour perfusion substantially increased and resulted in a considerably higher nutrient supply which was reflected by a higher  tumour glucose concentration  of chemotherapeutic agents can be expected. In addition, the deterioration and heterogeneity of tumour blood flow is responsible for hypoxia in tumours which in turn reduces the efficacy of oxygen-dependent chemotherapeutic agents. For this reason, a supportive treatment modality  which leads to a reduction of heterogeneity or an improvement of tumour blood flow and/or a reduction of hypoxia might be of clinical interest.−1 led to a two-fold increase in melphalan concentration in the tumour tissue. The effect of CCBs on tumour perfusion has been investigated extensively over the last two decades. 2 values measured in tumours of isolated perfused limbs were lower  than in non-perfused contralateral control tumours with comparable volumes  (2-supply conditions were restricted (compared to control conditions in non-perfused limbs). This is partially due to the lower cHb of the perfusate (74±2 g l−1) compared to whole blood. Control measurements in the normal subcutis of perfused hind limbs (non-tumour bearing) and non-artificially perfused legs also showed poorer oxygenation during ILP (median pO2=37 mmHg in subcutis during ILP and 49 mmHg in control legs). In many experimental ILP studies  . These d2-values of 30% would only be abolished if the flow rate were to be increased by a factor of 4 or more. An improvement in tumour oxygenation was reported in two studies by 2 by approximately 50% in the tumour center . 2 rose only slightly by 2–3 mmHg  the O2-uptake by the tumour increased linearly (2 consumption is dependent on the oxygen availability as long as a ‘saturation level’ of the O2 supply is not reached. An improvement in the convective O2 transport (by increased perfusion) will only reduce tumour hypoxia if the O2 supply greatly exceeds the consumption rate. Due to the reduced haemoglobin level during ILP, the oxygen transport capacity of the perfusate is diminished (0.11 ml O2 per ml blood during ILP vs 0.2 ml O2 per ml blood in controls) resulting in a restricted supply situation. Obviously, the O2 supply in the present study was markedly lower than the ‘saturation level’ and the convective O2 transport during ILP did not meet the demands of the tumour tissue. During ILP therefore, where the O2 transport capacity is reduced, an improvement in the perfusion rate of 50–100% is probably not sufficient to bring about a significant increase in the median tumour pO2.Since the oxygenation status of a tissue results from a dynamic steady-state between the oxygen supply and the cellular Olinearly . PreviouAs a result of the restricted oxygen supply during ILP (due to the reduced haematocrit of the perfusate), the lactate levels in tumours of isolated perfused limbs were significantly higher than in tumours of contralateral control limbs, indicating a much higher glycolytic rate. However, the higher glycolytic rate had only a minor impact on the bioenergetic status resulting in ATP levels which are comparable to those found previously in untreated tumours  and in tIn conclusion, nifedipine can significantly improve tumour perfusion during pressure-controlled ILP. ‘Steal phenomena’ in favour of the surrounding normal tissue and oedema formation were not observed. Nifedipine can enhance tumour microcirculation and may therefore promote the delivery (pharmacokinetics) of anti-cancer agents. Although the application of this calcium channel blocker increases oxygen availability to the tumour the improvement of perfusion by nifedipine does not result in a substantial reduction of tumour hypoxia. On the basis of these results, nifedipine application during ILP can be expected to increase the delivery of anti-cancer drugs to the tumour and by this improve the efficacy of pressure-controlled ILP.


In March 2003, the French Ministry of Health implemented a program on preparedness and response to a biological attack using smallpox as weapon. This program included the establishment of a preoutbreak national team that could be revaccinated against smallpox.To identify demographic and clinical factors associated with vaccination success defined as the presence of a pustule at the inoculation site at day 8 (days 7–9), with an undiluted vaccinia virus derived from a Lister strain among preimmunized volunteers.From March 2003 to November 2006, we have studied prospectively 226 eligible volunteers. Demographic data were recorded for each volunteer . Smallpox vaccine adverse reactions were diagnosed on the basis of clinical examination performed at days 0, 7, 14, 21 and 28 after revaccination.A total of 226 volunteers (sex ratio H/F = 2.7) were revaccinated. Median age was 45 years (range: 27–63 yrs). All volunteers completed follow-up. Median number of vaccinations before revaccination was 2 (range: 1–8). The median delay between time of the study and the last vaccination was 29 years . Sixty-one volunteers (27%) experienced one (n = 40) or more (n = 21) minor side effects during the 2–14 days after revaccination. Successful vaccination was noted in 216/226 volunteers (95.6%) at day 8 and the median of the pustule diameter was 5 mm (range: 1–20 mm). Size of the pustule at day 8 was correlated with age (p = 0.03) and with the presence of axillary adenopathy after revaccination (p = 0.007). Sex, number of prior vaccinations, delay between the last vaccination and revaccination, and local or systemic side effects with the exception of axillary adenopathy, were not correlated with the size of the pustule at day 8.Previously vaccinated volunteers can be successfully revaccinated with the Lister strain. The events of 2001 in the United States had worldwide repercussions on the awareness of bioterrorism as well as on the development of plans to counteract bioterrorism amongst many countries. Governments and international entities with responsibilities related to maintenance of peace, security, safety and health protection reviewed urgently their political, economic, diplomatic, military and legal means to face up to such attacks and embarked upon major efforts to increase their preparedness. Individual nation states have developed plans to fight against bioterrorism and have united in order to resist this threat. More than 150 pathogens have been reported as potential agents for bioterrorism In March 2003, the French Ministry of Health implemented a program on preparedness and response to a biological attack using smallpox as weapon The objective of this study was to identify demographic and clinical factors associated with vaccination success, defined as the presence of a pustule at the inoculation site eight days post vaccination and measured by the size of the pustule among 226 eligible preimmunized volunteers.From March 2003 to November 2006, we have studied prospectively 226 eligible volunteers selected for the French national team against smallpox. They have received at least one dose of smallpox vaccine in the past, without major side effects, and have evidence of vaccine “take” as evidenced by the presence of a scar resulting from previous smallpox vaccination or notification on a notebook vaccination. Reimmunization for this team was performed with the Lister strain (Pourquier® vaccine). Screening to identify and exclude subjects with contraindications for them or household close contacts to smallpox immunization was performed . An HIV 7.7 pfu/mL. This lyophilized vaccine, product prepared from calf lymph, was reconstituted by adding sterile diluent to the powder and administered into the dermis by using the multiple-puncture technique with a presterilized bifurcated needle (10–15 punctures in an area of about 5 mm in diameter as recommended for revaccination over the insertion of the left deltoid muscle). The same physician performed all the vaccinations to exclude technical problems associated with the vaccination procedure. The vaccination site was covered with gauze in combination with a semipermeable membrane. Dressings were changed and the vaccination sites were assessed every seven days (d0–d28) until the lesions dried and an eschar formed. Following revaccination, vaccination success was measured by the development of a vaccination site take, defined as the presence of a pustule at the inoculation site 7 to 9 days post vaccination.There is currently two licensed smallpox vaccine in France using the Lister strain. There are approximately 72 millions doses of vaccine potentially available. The vaccine used (Pourquier® vaccine) contains live unattenuated vaccinia virus derived from the Lister strain which produces cross immunity against variola major and minor. The titer of vaccinia virus was 10Demographic data were recorded for each volunteer . There were no restrictions on work activities following revaccination. Smallpox vaccine adverse reactions were diagnosed on the basis of clinical examination performed at days 0, 7, 14, 21 and 28 after revaccination. Volunteers were also questioned at each follow-up visit for the presence of any vaccine-related adverse events such as local and systemic symptoms for at least 4 weeks after revaccination. Fever was defined as temperature ≥37.8°C.The quantitative variables  are not normally distributed. By consequence, the non parametric tests using exact calculation rather than asymptotic ones have been applied by using StatXact® software version 6 (Cytel Studio). Without any certitude regarding the hypothetic direction of the relation, all tests have been interpreted based on a two-tails p-value. The α risk has been chosen at 5%. The Bonferoni correction has been taken into account according to the number of tests done. The quantitative variables have been crossed by using the Mann-Whitney exact test. The Spearman rank correlation test has been used to test the correlation between quantitative variables.A total of 226 volunteers (sex ration H/F = 2.70) were revaccinated and included in this study . Median Among the 226 volunteers, 61 (27%) experienced one (n = 40) or more (n = 21) minor side effects during the 2–14 days after revaccination. No side effect was reported during the third and the fourth week after revaccination. No serious adverse effect was notified and no adverse event was reported in their household close contacts. Moreover, no cardiac complication following revaccination was reported in this study. Local symptoms were: local pruritis (n = 17) (8%), axillary lymphadenopathy (n = 7) (3%), and large vaccination reaction (robust take) >7.5 cm in diameter (n = 2) (1%), and systemic symptoms were: fever >37.8°C (n = 27) (12%), fatigue (n = 14) (6%), headache (n = 6) (3%), myalgia (n = 6) (3%), nausea (n = 2) (1%), diarrhea (n = 2) (1%) and cough (n = 2) (1%) .Successful vaccination was noted in 216/226 volunteers (95.6%) at day 8 (days 7–9). Median of the pustule diameter in these 216 volunteers, at day 8 after revaccination was 5 mm (range: 1–22 mm): median age was 45.5 years (range: 27–63 years) and median delay between time of the study and the last vaccination was 29 years (range: 18–60 years). Among volunteers, 10 did not develop any clinical take (pustule diameter = 0) at the vaccination site (median age was 41 years (range: 33–49 years) and median delay between time of the study and the last vaccination was 33.5 years (range: 20–45 years)). We did not find any difference between these two groups of volunteers with or without pustule.A larger size of the pustule at day 8 was statistically correlated with a greater age (p = 0.03) and with the presence of axillary adenopathy after revaccination (p = 0.007). Sex, number of prior vaccinations, delay between the last vaccination and revaccination, and local or systemic side effects with the exception of axillary adenopathy, were not associated with the size of the pustule at day 8.The data of this study shows that volunteers previously immunized with smallpox vaccine are successfully revaccinated with an undiluted Lister strain.Successful vaccination was noted in 216 of the 226 volunteers (95.6%). This result is in accordance with other studies which used an undiluted Lister strain It has been usually reported that compared with vaccinia-naïve subjects, prevaccinated patients had significantly smaller pustule lesion In the literature, local lymphadenopathy is reported in 7 to 88% of cases It has been also reported that the 1∶5 and 1∶10 dilutions of smallpox vaccine in adults who had not been previously immunized, were associated with a smaller incidence of adenopathy than those reported in naïve subjects given undiluted vaccine In our study, only 61 patients (27%) experienced at least one minor side effect during the 2–14 days after revaccination with the Lister strain. These side effects included only moderate local and/or systemic adverse events. No serious adverse effect, such as postvaccinal encephalitis, progressive vaccinia, eczema vaccinatum, generalized vaccinia, inadvertent inoculation, cardiac complication or death was notified in the volunteers and their household close contacts Previously vaccinated volunteers can be successfully revaccinated with the Lister strain. Large studies are needed to analyse second and third generations vaccines


C DNA content using two highly sensitive methods. In contrast to a previous report, AdV DNA was not detected at a higher frequency among neonates who later developed leukaemia, when compared with controls.Archived neonatal blood cards (Guthrie cards) from children who later contracted leukaemia and matched normal controls were assayed for adenovirus (AdV)  Greaves Molecular screening for specific candidate viruses or virus families has produced negative results (as reviewed by C class virus), and little is known about the oncogenic potential of other members of the AdV family. Recently, the first evidence for the possible contribution of AdV to the multistep process of tumour pathogenesis was shown in some paediatric tumour entities but not leukaemia (Human adenoviruses (AdVs) cause various common diseases in adults and children such as conjunctivitis, respiratory infections and diarrhoea . AlthougA provocative recent report  according to the manufacturer's instructions. This represents approximately 150 ng of DNA or 25 000 cells. The samples were quantified using PicoGreen DNA quantification reagent .A one-eighth portion of a 1.5 cmhexon gene as described previously , 0.2 μM probes, 0.3 or 0.9 μM of primers and 20 or 50 ng of template DNA. The TaqMan probes used were labelled with 6-carboxyfluorescein at the 5′-end and with blackhole quencher at the 3′-end . Amplifications were carried out using the ABI Prism 7900 HT (Applied Biosystems) for a total of 50 cycles. After an initial denaturation step for 10 min at 95°C, each cycle consisted of denaturation for 15 s at 95°C and annealing and primer extension for 60 s at 60°C. Strict precautions were undertaken to prevent contamination of PCRs with exogenous products. To reduce the risk of false-positive results due to contamination with PCR products, dTTP was partially replaced by dUTP in the reaction master mixture and a dUTP glycosylase step was performed before each PCR. Each DNA patient sample was analysed in duplicate, standard dilutions of AdV-5 were run in quadruplicates and negative controls were included in each assay. DNA derived from AdV type 5 (AdV-5)  served as a quantitated positive control. For the quantification of virus copies, external standard curves were established using 10-fold serial dilutions of quantified virus DNA preparations corresponding to defined virus particle equivalents. As a separate internal control, we used an Alu element-based assay, which amplifies the Intra-Yd6 sequence of human DNA  was screened in Guthrie card samples by using semi-nested PCR. The PCR amplifications were carried out in a final volume of 50 μl consisting of 1 × PCR buffer, 2.25 mM MgCl2, 0.2 mM each deoxynucleosidetriphosphate (Applied Biosystems), 2.5 U of Taq Gold polymerase (Applied Biosystems), 150 nM each primer  prepared using quantified viral DNA derived from the reference strain (AdV-5). Reproducible quantification of virus copies was obtained by RQ–PCR in the presence of 1–104 particles per reaction  was run in duplicate. Then, a second set was run, with 50 ng of each DNA sample (n=78) also in duplicate. None of the samples contained detectable AdV DNA, and all samples amplified for Yd6 control gene at a level consistent with the amount of DNA added  nor from healthy controls (n=99), except for one normal control sample. These results demonstrate a lower rate of positivity (∼0.5%) than that found for AdV in amniocentesis samples from the second trimester of normal pregnancies  when compared with controls (6%). Unless the Swedish mothers are exposed to very different environmental and genetic factors that influence transmission of infection to a fetus and detection in blood, when compared with pregnant woman in California, there is no obvious explanation for this discrepancy. As sero- and molecular positivity to AdV C are not significantly higher in Swedish compared with USA populations (It has been suggested that infection Speculations concerning a link between infections and childhood leukaemia have been published for decades and, despite a multitude of epidemiological correlations, a definitive mechanistic explanation remains elusive. The absence of any viral DNA in Guthrie cards does not constitute negative evidence of the role of infections in leukaemia aetiology if the critical exposure is post-natal. The early postnatal period is the time period with the strongest epidemiological evidence concerning a link between infectious disease history and childhood leukaemia . The abs


The model predicted age-related pathway alterations underlying the diminished bone formation at senescence, and hence identified critical deficits that were promising targets for therapy. Based upon model predictions, we implemented an in vivo intervention and show for the first time that supplementing mechanical stimuli with low-dose Cyclosporin A can completely rescue loading induced bone formation in the senescent skeleton. These pre-clinical data provide the rationale to consider this approved pharmaceutical alongside mild physical exercise as an inexpensive, yet potent therapy to augment bone mass in the elderly. Our analyses suggested that real-time cellular signaling strongly influences downstream bone adaptation to mechanical stimuli, and quantification of these otherwise inaccessible, transient events in silico yielded a novel intervention with clinical potential.The increasing incidence of osteoporosis worldwide requires anabolic treatments that are safe, effective, and, critically, inexpensive given the prevailing overburdened health care systems. While vigorous skeletal loading is anabolic and holds promise, deficits in mechanotransduction accrued with age markedly diminish the efficacy of readily complied, exercise-based strategies to combat osteoporosis in the elderly. Our approach to explore and counteract these age-related deficits was guided by cellular signaling patterns across hierarchical scales and by the insight that cell responses initiated during transient, rare events hold potential to exert high-fidelity control over temporally and spatially distant tissue adaptation. Here, we present an agent-based model of real-time Ca Post-menopausal and age-related osteoporosis afflicts large segments of the population and can markedly increase skeletal fragility. Bone fractures that occur as a consequence substantially increase health care expenditures and raise levels of morbidity. While strategies that prevent further loss of bone exist, options that compensate for bone loss accrued over age are less numerous. Physical exercise holds promise in this realm. However, in part due to deficits in how cells within bone respond to skeletal loading, readily complied exercise has proved ineffective in enhancing bone mass in the elderly. In this study, we examined whether the ability of physical exercise to increase bone mass can be restored at advanced age. To this end, we developed a computational model describing how a specific aspect (or pathway) activated in bone cells by skeletal loading may be altered with age. Our model proved successful in describing age-related pathway alterations and identified specific deficits that were amenable to therapeutic manipulation. We subsequently discovered that when an extremely inexpensive, currently approved pharmaceutical is used as a supplement, bone response to skeletal loading was completely rescued in aged animals. We believe that this result provides the rationale to consider this approach as a means to increasing bone mass in the elderly. Mechanical stimuli are anabolic for bone and hold promise to counteract skeletal fragility associated with bone loss pathologies during brief stimuli are focal events that guide unique downstream bone adaptation. A variety of second messengers  are acutely activated by mechanical stimuli. Of these, the Ca2+ ion second messenger system may be unique in its specificity given that all important aspects of mechanical stimuli that influence bone formation in vivo  have been observed to provoke highly specific real-time Ca2+ oscillations in bone cells in vitro 2+ signaling disrupts mechano-responsive gene expression 2+ signaling within the bone cell network 2+ ion system, activation of a variety of transcription factors  provides putative links between cell responses on the order of seconds to cell function over successive days of the week. Of these, the nuclear factor of activated T-cell family of transcription factors (NFAT c1–c4) may be unique. Specifically, NFAT activation dynamics (within minutes) is remarkably specific to Ca2+ amplitudes and frequencies known to influence distinct downstream cell functions, including proliferation, differentiation and apoptosis 2+/NFAT signaling and age-related alterations in bone cells in vivo may prove useful. However, it is not currently possible to experimentally quantify real-time activation of Ca2+/NFAT signaling in situ within bone.To begin to explore underlying potential for age-related deficits in mechanotransduction function, we first focused upon observations that a single bout of mechanical stimuli (∼100 s) is sufficient to influence bone matrix secretion up to a week later 2+ signaling induced in simple networks of osteocytic cells subject to mechanical stimuli using a numerical/computational technique called agent-based modeling (ABM) 2+ signaling in cell ensembles). Based in part upon our original framework 2+/NFAT signaling in situ within bone's cellular syncytium and its relation to osteoblastic relative mineral apposition rates induced by mechanical loading  induced in vivo by a variety of mechanical stimuli in young and senescent female C57BL/6 mice 2+/NFAT pathway incorporated sufficient parametric complexity to simulate rp.BFR induced by a variety of loading protocols in young adult 2+ oscillations in individual cells . Post-hoc analysis of ABM simulations suggested that ‘knock-out’ of the remaining parameters de-couples Ca2+ oscillation from induced strains (Tεx = 0), inhibits secondary Ca2+ transients (R0 = 0), decreases Ca2+ induced dephosphorylation of NFAT (α = 0) and renders mineral apposition rates into a binary ‘on/off’ state in osteoblasts . ‘Knock-out’ of parameters resulted in simulations that were significantly different from both the experimental rp.BFR data p≤0.02;  [12], , i(t), emerged via an interplay between Ca2+ influx due to loading induced strains at the location of the osteocytic cell (εi(t)), cell-cell gap junctional exchange of Ca2+ ions and efflux from ER Ca2+ stores as follows:εxT is the parameterized threshold strain magnitude required to maximize the amplitude of Ca2+ oscillations in mechanosensory osteocytes , in is the number of cells (osteocytes and/or precursor cells) functionally coupled to the ith cell, j is an element of the cell set in, CnT represents threshold ‘incoming’ Ca2+ ion levels required to initiate oscillations in the recipient cells (specified via preliminary simulations to be 2.5%), and 0ER and 0R are the parameterized maximal Ca2+ store capacity and maximal store recovery rates, respectively. The specific mathematical formulations for osteocyte sigmoidal dose-response relation to strain (eqn 1b) were based upon observations of Ca2+ oscillations induced in bone cells by mechanical stimuli 2+ ion propagation between cells based upon reports 2+ store depletion (eqn 1d) is similar to previously 2+ store recovery upon cellular quiescence (eqn 1d), models the action of the SERCA pump working against a Ca2+ ion concentration gradient and is similar to literature reports The ABM is a biophysical model that relies on experimentally identified ‘building blocks’ to algorithmically describe how activation of the Caeoblasts . In the 2+ oscillations in osteocytic networks hold potential to regulate surface osteoblastic cells 2+ oscillations upon NFAT transcription factor dynamics. Specifically, downstream of increased intracellular Ca2+ oscillations, cytoplasmic NFAT dephosphorylation and nuclear translocation in surface precursor cells was modeled to be reflective of a ‘memory’ of Ca2+ oscillation characteristics such as amplitude, frequency and durations, and included an implicit consideration of negative feedback elements 2+ oscillations. The quantity of NFAT translocated into the ith cell's nucleus (boutt) was modeled simply as:2+ signaling and the NFAT pathway code for highly specific cell proliferation, differentiation and apoptotic events nxNFAT is the maximal nuclear NFAT DNA binding capacity, and xr.MAR, the maximal relative mineral apposition rate by osteoblasts. The mathematical form (eqn 3) reflects considerations that transcriptional regulation is likely to be a threshold-driven process Given observations that CaεxT, 0ER, 0R, α, nxNFAT, xr.MAR prior to execution of the simulations). To initialize the ABM (at t = 0), strains induced by loading at osteocyte cell locations were 0  = 0), and the stores of all cells were assumed to be filled to capacity  = ER0). Based upon preliminary studies, we simulated intracellular Ca2+ oscillations for an extra 200 seconds beyond cessation of a given loading bout to permit the unfolding of cell-cell cross talk, and NFAT dephosphorylation for an extra 100 s beyond cessation of Ca2+ oscillations to more fully account for prior Ca2+ oscillation histories.Simulations with the ABM involved solving equations (1–3), given osteocyte level strains derived over each second of a loading bout in the context of the ABM's 6 independent parameters  at the initiation of the simulations then predicts mean values i(θμ) = rp.BFRi for each loading protocol ‘i’. Under the normal error model, the log-likelihood function for the ABM is then ij’‘y is the rp.BFR measured in the jth animal for the ith loading protocol, μ(θ) is the vector of means σ2 is the vector of population variances protocolN’ is the number of independent loading protocols, ‘ain’ is the number of animals that underwent the ith loading protocol, ‘an’ is the total number of experimental observations. The maximum likelihood estimate (MLE), for the vector of means, μ(θ) and variances σ2, is simply the set of model parameters that maximizes L(μ(θ), σ2). For a given vector of mean parameters μ(θ) of σ2, maximization with respect to the variances σ2 is achieved by setting:2). Using this procedure, separate ABM models were calibrated to the rp.BFR data obtained from young animals subjected to 10 protocols and senescent animals subjected to 7 protocols  Is there evidence against the null hypothesis that each of the ABM parameters takes value zero ? (d) Is there evidence in the observed data against the null hypothesis that the population values of the ABM parameters are the same for young and senescent animals? (e) If null hypothesis in (d) is rejected, is there evidence against the null hypotheses that specific model parameters or combination of parameters are unchanged by age? (f) Do the calibrated ABMs make predictions about the results of hypothetical interventions that would restore specific ABM parameters in senescent animals to their young values? (g) Do ABM simulations of loading induced rp.BFR, upon complete restoration of parameters significantly degraded by aging, predict data from senescent animals subject to loading supplemented with CsA? We examined these questions using a series of likelihood ratio tests (within MATLAB) and factorial ANOVAs (within SPSS) where appropriate; please see supporting information for additional details  received mechanical loading using the noninvasive murine tibia loading device Protocol S1Consolidated (‘zipped’) file that includes a ‘readme’, c++ code and necessary input files for compiling and executing the code representing the developed model, and executable files compiled for both Macintosh OSX (Leopard) and windows PC based computer platforms.(5.17 MB ZIP)Click here for additional data file.Table S1Animal specific peak longitudinal normal strains (mean+S.E.) and rp.BFR (mean+S.E.) induced in vivo in young adult animals by 10 independent loading protocols, and in senescent animals by 7 independent loading protocols. Note that these data represent mean values after removal of outliers (mean ± 2×S.D.). This resulted in the removal of 1 data point in young adult animals (from n = 70) and of 4 data points in senescent animals (from n = 56).(0.07 MB DOC)Click here for additional data file.Table S2Definitions for notations and symbols used in manuscript.(0.09 MB DOC)Click here for additional data file.Text S1Methods describing previous data set used to calibrate model and statistical analysis.(0.12 MB DOC)Click here for additional data file.