Gossypium), which contains seven allopolyploids derived from a single polyploidization event 1–2 Million years ago, to investigate deleterious mutation accumulation. We use two methods of identifying deleterious mutations at the nucleotide and amino acid level, along with whole-genome resequencing of 43 individuals spanning six allopolyploid species and their two diploid progenitors, to demonstrate that deleterious mutations accumulate faster in allopolyploids than in their diploid progenitors. We find that, unlike what would be expected under models of demographic changes alone, strongly deleterious mutations show the biggest difference between ploidy levels, and this effect diminishes for moderately and mildly deleterious mutations. We further show that the proportion of nonsynonymous mutations that are deleterious differs between the two coresident subgenomes in the allopolyploids, suggesting that homoeologous masking acts unequally between subgenomes. Our results provide a genome-wide perspective on classic notions of the significance of gene duplication that likely are broadly applicable to allopolyploids, with implications for our understanding of the evolutionary fate of deleterious mutations. Finally, we note that some measures of selection  may be biased when species of different ploidy levels are compared.Whole-genome duplication (polyploidization) is among the most dramatic mutational processes in nature, so understanding how natural selection differs in polyploids relative to diploids is an important goal. Population genetics theory predicts that recessive deleterious mutations accumulate faster in allopolyploids than diploids due to the masking effect of redundant gene copies, but this prediction is hitherto unconfirmed. Here, we use the cotton genus ( Genome duplication (polyploidy) is among the most dramatic mutational processes in nature, causing myriad saltational changes at the cellular and organismal levels , and is Novel evolutionary patterns created by polyploidy at the genic  represents one of the best studied allopolyploid systems (G. hirsutum (AD1) and G. barbadense (AD2), occur in small, isolated populations on islands or in coastal regions. Subsequent to their initial domestication 4,000–8,000 years ago in the Yucatan Peninsula (AD1) and NW S. America (AD2), respectively, the ranges of the two domesticated species rapidly expanded to encompass much of the American tropics and subtropics and then spread globally with the rise of the international cotton fiber trade , with a focus on how allopolyploidization and speciation have shaped the number and genomic distribution of deleterious mutations. We use two methods to estimate the strength of selection at the amino acid and nucleotide level and show support for a nearly century-old hypothesis that polyploids accumulate mutations faster than their diploid progenitors. We demonstrate that, in agreement with this hypothesis, polyploidy has the greatest influence on strongly, rather than moderately or mildly, deleterious mutations. We also find that deleterious mutations accumulate asymmetrically between the two coresident subgenomes in the allopolyploid nucleus, indicating that these masking effects may act unequally between the subgenomes. In total, our results support theoretical predictions that allopolyploidy can lead to a faster rate of deleterious mutation accumulation through masking of recessive deleterious variants, and that the relationship of the rate of deleterious mutation accumulation between subgenomes and their progenitor diploids is complex, even when comparing identical pairs of single-copy homoeologs among lineages.To investigate patterns of deleterious mutations, we viewed our data at three phylogenetic depths: single nucleotide mutations that occurred anywhere on the global phylogenetic tree A–D, mutaG. mustelinum  and G. darwinii  having consistently lower rates than the rest of the clade, and in both subgenomes. When viewing mutations that have accumulated since the divergence of the earliest polyploid lineage  and G. raimondii  did not differ between subgenomes, indicating that any mapping biases or erroneous SNP calls in these samples were removed by our variant filtering criteria. Gossypium raimondii (D5) contained more derived nonsynonymous mutations than did G. herbaceum (A1), and this lineage-specific difference was reflected in the Dt and At subgenomes as well. When lineage-specific effects that arose from the long, shared ancestry between the subgenomes and their progenitor diploids were removed , and the Dt subgenome contained 17–36% more than G. raimondii . These results demonstrate that even though the rates of synonymous mutation accumulation did not differ significantly between the diploids and polyploids, polyploidy significantly increases the rate of nonsynonymous substitution accumulation. Finally, when restricting our attention to only those mutations that have arisen following polyploid formation  consistently had the lowest number of derived mutations in both subgenomes. Notably, however, the Hawaiian Island endemic G. tomentosum  has a higher number of derived nonsynonymous mutations than expected based on the patterns of synonymous mutations, potentially reflecting the population bottleneck associated with its origin following long-distance dispersal to the Hawaiian Islands (see Discussion). In summary, polyploidy significantly enhances the rate of nonsynonymous mutation accumulation in all Gossypium allopolyploids, and does so asymmetrically across coresident genomes.In contrast to the relative homogeneity in rates of synonymous substitution among diploids and polyploids, rates of nonsynonymous mutation accumulation differed significantly at all phylogenetic depths. Notably, at the deepest phylogenetic depth B, estima removed F, a cleaormation J, the liGossypium reflects an increase in neutral or nearly neutral nonsynonymous mutations, or if instead this elevation is attributable to a greater accumulation of deleterious mutations. To address this, we used two approaches to estimate whether a mutation was deleterious: BAD_Mutations and GERP++. BAD_Mutations performs a likelihood ratio test from a gene-specific multispecies alignment to determine if a mutation at a particular nonsynonymous site is potentially deleterious, whereas GERP++ uses a genome-wide multiple sequence alignment  to estimate the degree of conservation at a particular site in the genome (including noncoding and synonymous sites). Notably, because one of the hallmark long-term processes following polyploidy is pseudogenization  shows the highest number of all the polyploids, and G. mustelinum  shows the fewest of all the polyploids.As shown in An interesting pattern arises when comparing estimates of the GERP load G and numAlthough deleterious mutations are accumulating faster in polyploids relative to diploids, it is not obvious whether this increased rate is different from the increased rate of accumulation of nonsynonymous mutations. To test this, we compared, among ploidy levels, the total proportion of nonsynonymous mutations that were considered deleterious by BAD_Mutations . For mutG. darwinii  from the Galapagos Islands, and largest in the Brazilian endemic and inland species G. mustelinum , indicating that asymmetries between subgenomes of the same species may vary within a single clade of allopolyploids.At shallower phylogenetic depths B, the diDemography is a potential confounding factor in estimating the rate of deleterious mutation accumulation. Shifts in demography are known to complicate inferences of the strength of selection and genetic load ; for exaPolyploidy is typically associated with a population bottleneck , but becs) of deleterious mutations is less than or equal to 1/(2 Ne), where Ne is the effective population size. The reduction of Ne during a population bottleneck would therefore allow weakly deleterious mutations to escape purifying selection , whereas strongly deleterious mutations with a selective coefficient greater than 1/(2Ne) would still be removed by purifying selection. On the other hand, as Ne increases during population expansion, mutations that are mildly deleterious are expected to be more efficiently purged from the population.Demographic shifts, including population bottlenecks and expansions, have a large influence on the accumulation of deleterious mutations. According to the nearly neutral theory , the fatIn both demographic scenarios, we expect that mildly or moderately deleterious mutations would be most differentially affected, whereas strongly deleterious mutations would consistently be removed by purifying selection. Based on this theory, if the differences in the number of deleterious mutations we see between diploids and polyploids are due to demography, then we would expect to see most of that difference reflected in mildly, rather than strongly, deleterious mutations. In contrast, if masking of deleterious alleles in polyploids is driving a higher rate of accumulation relative to diploids, this pattern will not be observed.To test if our data were consistent with changes in demography, we first asked if there was a correlation between the degree of deleteriousness of a mutation (as measured by GERP) and its relative increase in the polyploids compared with the diploids. To answer this question, we plotted the relative change of deleterious mutations in each subgenome relative to its most closely related diploid progenitor. We plotted this relative change for three different degrees of deleteriousness—strongly deleterious mutations (4 < GERP ≤ 6), moderately deleteriousness (2 < GERP ≤ 4), and mildly deleteriousness (0 < GERP ≤ 2) deleterious . We founGossypium harbor more mutations at phylogenetically conserved sites than do their closest diploid progenitors, as determined by two different methods of detecting deleterious mutations. We also find that the proportion of all nonsynonymous mutations that are inferred to be deleterious is higher in polyploids than in their diploid progenitors and that polyploidy has the greatest effect on strongly deleterious  has been characterized by numerous long-distance dispersal events , whereas the subgenome with the lower genetic load derives from the predominantly selfing C. orientalis . In anotgenitors . It is ugenitors . In GossGossypium polyploids are less fit than their closely related diploid progenitors because they harbor more deleterious mutations in their genomes, especially mutations that have already been driven to fixation. We note, however, that the fitness effects of a mutation may change as a result of the genetic  or environmental  context in which it occurs  may be biased when comparing a polyploid to diploid because the polyploid would be expected to accumulate putatively deleterious sites more quickly (and maintain a higher genetic diversity at nonsynonymous sites) than their diploid relatives. This increased dN/dS value in allopolyploids compared with diploid progenitors was recently shown in five allopolyploid systems in addition to Gossypium , and duplicated genes associated with an ancient polyploid event in Brassica rapa were shown to contain higher amounts of genetic variation than nonduplicated genes  acting on that locus, and the dominance coefficient (h), defined as the proportion of the fitness cost that a mutation harbors when in a heterozygous state. In allopolyploids, however, the evolutionary fate of new mutations may be determined not only by allelic dominance at that locus, but also by the interaction arising from the coexistence of its homoeologous locus, a term we call “homoeologous epistatic dominance.” The relationships between this homoeologous epistatic dominance, allelic dominance, and the selection coefficient are likely complicated and potentially heavily influenced by other biological considerations such as biased expression of homoeologs, sub- or neofunctionalization, and homoeologous recombination, among others. Moreover, notwithstanding these polyploidy-specific effects, even the genome-wide relationships between two of these factors, allelic dominance and the selection coefficient, have only been modeled using genomic data in the past few years  may play an important and underrecognized role in determining how selection acts on new mutations, notwithstanding the burgeoning literature on fates of duplicate gene evolution . The evoew years .cis- and trans-effects , we nullified any variant call in which all individuals were heterozygous to remove any collapsed genomic region in the reference genome or paralogous regions that were not present in the reference genome. We treated G. stephensii and G. ekmanianum as a single species because we only sampled two individuals of G. stephensii, so removing any sites in which both individuals were heterozygous errantly removed real variants that were not due to paralogy mapping issues. All scripts for generating and filtering variant calls are located on our GitHub repository (https://github.com/conJUSTover/Deleterious-Mutations-Polyploidy).After the data were downloaded from NCBI, adapter sequence removal and quality score filtering of FASTQ reads were performed using Trimmomatic v0.36  using the genome , and reae genome  and onlye genome  before te genome  SNP CallG. hirsutum reference genome, and kept only homoeologous pairs that were less than 5% different in their total annotated CDS length. To remove homoeologous pairs that may have experienced homoeologous exchange events  could also bias the estimates of the genetic load in a way that is not related to new mutation following polyploidization or speciation. To control for positions in these non-HE homoeologs that may be influenced by gene conversion, we linked variant positions between homoeologs in the following way. We first performed pairwise alignments of the CDS sequences using MACSEv2 , 2018, wIn total, we only used those SNP sites that: 1) did not link to an indel in its homoeologous pair, 2) were biallelic and had consistently inferred ancestral states in the two subgenomes, 3) the derived allele was found in only one of the two subgenomes or their respective diploid progenitors, and 4) the derived allele was fixed in a diploid and segregating in its respective subgenome (or vice-versa).Gossypium . Genomes were aligned to the G. hirsutum reference genome using the LASTZ/MULTIZ approach used by the UCSC genome browser. Briefly, genomes were masked using Repeatmasker using a custom repeat library enriched with Gossypium TEs  as described in https://github.com/conJUSTover/Deleterious-Mutations-Polyploidy).Using these genome alignments, we used the gerp++ package  to calcuGossypium raimondii since it was sampled as part of this project.Secondly, we used the BAD_Mutations , 2018 pihttps://github.com/conJUSTover/Deleterious-Mutations-Polyploidy).We used the GERP load   and the To determine if there was a bias in the rate of deleterious mutation accumulation between the two subgenomes, we used homoeologous SNPs in which the derived allele showed a parsimony-informative position between the two subgenomes of allopolyploids and the two diploid progenitors  was calculated on a sitewise basis using vcftools (π across all genes, we summed the total sitewise π values and divided by the total length of the concatenated CDS sequences, removing any positions which did not have a null SNP call in the VCF file.For each species, vcftools . To findMolecular Biology and Evolution online.msac024_Supplementary_DataClick here for additional data file.


On this, theologians, philosophers, and biologists can agree: we are more than the sum of our genes. Biological complexity arises not from gene number but from patterns of gene expression, which change under the direction of both genetic and so-called epigenetic mechanisms. Epigenetics, broadly defined, concerns heritable changes in gene function that don't involve changes in DNA sequence. Until recently, studies of heritable traits have focused largely on mutations in DNA. But it's become increasingly clear that how DNA is packaged in the nucleus also impacts heritability.Epigenetic changes are mediated largely by proteins that shape and remodel chromatin—the association of DNA and histone proteins that condenses the genome into compact bundles inside the nucleus. Different cell types have different chromatin arrangements during development and cell differentiation that appear to regulate gene expression, which possibly accounts for the unique gene expression patterns associated with specific cell types. Such phenomena have been well-studied for specific genes or chromosomal regions, but to understand the full impact of epigenetic mechanisms on gene regulation, we need a more panoramic view of gene organization within the nucleus.In a new study, Thomas Cremer together with Andreas Bolzer and an interdisciplinary team of German physicists, bioinformaticians, and geneticists created 3D positional maps of each human chromosome simultaneously in a single nucleus to investigate the link between chromatin structure and cell-specific gene expression. Working with human fibroblasts, cultured from a skin biopsy from a two-year-old boy, the authors were able to visualize and study the order of the full genetic complement within a human nucleus.Cremer and colleagues first produced a 3D topological map of all 46 chromosomes in different cell types at key points in the cell cycle—a landmark achievement—using a fluorescent staining technique that preserves chromosome shape during visual inspection under the microscope. Next, they established that small chromosomes in quiescent (nondividing) fibroblasts hewed close to the center of the nucleus while the large chromosomes were preferentially found at the nuclear rim, regardless of their gene density. Nuclei from cells entering the prometaphase stage of the cell cycle—just before chromosomes are aligned along the center of the nucleus prior to segregation—revealed a size-correlated chromosomal distribution akin to that seen in the quiescent nuclei. Statistical modeling analyses indicated that these size correlations do not simply reflect the geometric constraints of fitting into the nucleus, but likely hint at some degree of functional order within the nucleus.Because previous studies of cells with sphere-like nuclei correlated chromosomal arrangements with gene density, the authors investigated how shape affects chromosome position along the nuclear radius. Fibroblast nuclei are somewhat flat and ellipsoidal. Chromosomes in similarly shaped amniotic fluid cells assumed the same size-related positions taken by chromosomes in fibroblast nuclei. But when the authors examined the higher-order chromatin arrangements in fibroblasts and lymphocytes, they found that, even though the cell types differ in nuclear shape and radial chromosomal arrangements, they both show a nonrandom higher-order chromatin architecture correlated with gene density. Many questions remain concerning the functional and physiological significance of these observations: Do shape changes produce changes in chromosomal arrangements and vice versa? Do shape changes produce changes in gene expression patterns?Cremer and colleagues conclude that, although nonrandom chromosome positions occur, these appear to be governed by a degree of uncertainty and more likely reflect probabilistic preferences inside the nucleus. Still, deterministic mechanisms in higher-order chromatin structure may exist—sequestering gene-rich chromatin areas in the nuclear interior, for example, protected from malevolent agents entering the nucleus. And given the coexistence of size-correlated features with gene-density-correlated features seen in this study, it may well be that both random and deterministic factors combine to create the nuclear landscape.


Each year in the United States, an estimated 40,000 children are born with congenital cytomegalovirus (CMV) infection, causing an estimated 400 deaths and leaving approximately 8000 children with permanent disabilities such as hearing or vision loss, or mental retardation. More children are affected by serious CMV-related disabilities than by several better-known childhood maladies, including Down syndrome, fetal alcohol syndrome, and spina bifida.i.e., they will work if consistently followed) and the American College of Obstetricians and Gynecologists recommends that physicians counsel pregnant women about preventing CMV acquisition through careful attention to hygiene. However, because of concerns about effectiveness , the medical and public health communities appear reluctant to embrace primary CMV prevention via improved hygienic practices, and educational interventions are rare. Current data on the effectiveness of such measures in preventing CMV infection are promising, but limited. There is strong evidence, however, that educational interventions can prevent other infectious diseases with similar transmission modes, suggesting that effective interventions can also be found for CMV. Until a CMV vaccine becomes available, effective educational interventions are needed to inform women about congenital CMV prevention.Congenital CMV is a prime target for prevention not only because of its substantial disease burden but also because the biology and epidemiology of CMV suggest that there are ways to reduce viral transmission. Because exposure to the saliva or urine of young children is a major cause of CMV infection among pregnant women, it is likely that good personal hygiene, especially hand-washing, can reduce the risk of CMV acquisition. Experts agree that such measures are likely to be efficacious (Perhaps no single cause of birth defects and developmental disabilities in the United States currently provides greater opportunity for improved outcomes in more children than congenital CMV. Given the present state of knowledge, women deserve to be informed about how they can reduce their risk of CMV infection during pregnancy, and trials are needed to identify effective educational interventions. Haemophilus influenzae meningitis . Pe. Pe21]. Of all the ways to fight congenital CMV disease, the development of a vaccine is viewed as the most promising. Considerable progress has been made over the last 30 years, but insufficient interest by vaccine manufacturers  and technical challenges make it is uncertain when a vaccine will become available . AvenuesA number of experts have suggested that women be educated about hygienic practices for preventing CMV transmission from young children, and there is little dispute over what the prevention guidelines should entail Figure  7,11,2929-32. Thefficacy of hygienic practices  but, instead, the effectiveness of interventions to promote them .Why the ambivalence toward hygienic practices? Studies have shown that transmission of CMV via the urine and saliva of children is a major cause of infection among pregnant women ,20. In aIt is important to recognize the implications of the consensus that hygienic practices are efficacious for preventing CMV transmission. Individual women have the right to know that, under ideal conditions, risk of child-to-mother CMV transmission can be reduced by proper hygienic practices. This is equivalent to the ethical obligation to inform individuals that, under ideal conditions, safer sexual practices will reduce the risk of acquiring HIV. This obligation is independent of whether any particular educational program or intervention is effective. All women of childbearing age, whether they are CMV seropositive or seronegative, carry some risk of new CMV infection during pregnancy and thus should be informed of hygienic practices that reduce that risk. As Revello and Gerna aptly remind us, "...withholding information on possible medical interventions is unethical " . The terAs there is consensus on the efficacy of hygienic practices in preventing CMV transmission, the next step is to evaluate the effectiveness of educational interventions in preventing CMV transmission. Current evidence of effectiveness is promising, but limited. In one study, after non-pregnant women were educated about CMV prevention, hygienic practices improved . In a smMore conclusive evidence of effectiveness can be found in the literature on community-based interventions for the prevention of other infectious diseases with similar transmission modes. For example, a meta-analysis found that community intervention trials that encouraged washing hands with soap reduced the risk of diarrheal diseases by 47% . Hand-waAlthough all women of childbearing age deserve to be informed about CMV, interventions for preventing CMV transmission are most likely to be effective for pregnant women, who tend to be highly motivated, often changing behavior to protect the health of their developing fetuses. As a case in point, 25% of low-income smokers spontaneously quit smoking during pregnancy ; this peIn sum, the evidence to date gives every indication that effective interventions can be found for preventing CMV infection among pregnant women. Thus, the paradigm must shift from wondering whether such interventions will be effective to developing and evaluating interventions until effective ones are identified.)  is that we accept that, under exceptional circumstances, common sense might be applied when considering the potential risks and benefits of interventions [our emphasis]."." Common sAlthough an effective CMV vaccine would be ideal and vaccine development deserves increased support, hope for a vaccine tomorrow should not stand in the way of a vigorous educational message today. Prevention through improved hygienic practices will not be easy, but washing our hands of the problem by staying the present course guarantees that each year in the United States, hundreds of children will die and thousands of others will swell the ranks of CMV-affected children growing up with serious disabilities. Just as the medical and public health communities are successfully meeting the challenges posed by polio, rubella, HIV, and neural tube defects, now is the time to meet the congenital CMV challenge head on by making awareness and prevention high priorities.• Each year in the United States, congenital CMV- causes an estimated 400 deaths- leaves more than 8000 children with permanent disabilities such as hearing or vision loss, or mental retardation• Exposure to the saliva or urine of young children is a major cause of CMV infection among pregnant women.• Risk of CMV infection is likely to be reduced by careful attention to good personal hygiene, especially hand-washing.• Women should be informed about how to reduce their risk of CMV infection during pregnancy.• Trials are needed to identify educational interventions that are effective in preventing CMV infection.The author(s) declare that they have no competing interests.MJC and KFD contributed equally to the drafting and critical revision of the manuscript. Both authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The advent of standardized classification and assessment of psychiatric disorders, and considerable joint efforts among many countries has led to the reporting of international rates of psychiatric disorders, and inevitably, their comparison between different racial groups.In neurologic diseases with defined genetic etiologies, the same genetic cause has different phenotypes in different racial groups.We suggest that genetic differences between races mean that diagnostic criteria refined in one racial group, may not be directly and simply applicable to other racial groups and thus more effort needs to be expended on defining diseases in other groups. Cross-racial confounds  make the interpretation of rates in different groups even more hazardous than seems to have been appreciated. The DSM  and ICD Despite this derivation, cross-racial comparisons of disorder rates are a frequent topic of investigation and interpretation ,8. WhileWhile clinical neurological diagnoses are essentially made by the same operational process of clinical consensus, these clinical diagnoses have traditionally been validated through neuropathological examination. In many cases, this biological validation can be made since the genetic etiologies of many neurological diseases are now known. In the limited number of times this biological validation has been compared retrospectively against the clinical (and pathologic) characterization of neurological disease, it has become clear that the clinical characterization holds up poorly in different racial groups. For example, the phenotypes of both Spinocerebellar Ataxia types 2 and 3 (SCA2 and SCA3) are different in Eastern Asians and Sub-Saharan-Africans  respectively from their phenotypes in Caucasians -13. DentGiven this precedent, it seems ill advised to interpret any differences in disorder rates between different racial groups as being indicative of different rates of exposures to risk factors. This potential for confounding genetic variability is in addition to the more widely recognized, culturally derived issues inherent in the interpretation of most "risk factors" in psychiatric disorders, such as parenting styles, low socioeconomic status, temperament, or psychological "trauma." Since the diagnostic criteria have been imperfectly tuned to ensure, as far as possible, that everyone with mental illness in a Caucasian population receives a diagnosis , it should not be surprising to find that the rates in non-Caucasians would be different. Therefore, even given identical exposures, one should expect lower rates of DSM/ICD categories in non-Caucasians since less people in those groups would fit comfortably a diagnostic category. The limited data available suggest this is generally the case . This doDSM – Diagnostic and Statistical Manual of Mental Disorders; ICD – International Classification of Diseases.The author(s) declare that they have no competing interests.JH and NL conceived of the manuscript. JH drafted the initial version and both participated in multiple revisions until the final version.The pre-publication history for this paper can be accessed here:


One hundred and seventeen patients with cerebral glioma (Kernohan grades III and IV) were treated with adjuvant chemotherapy using procarbazine (PCB), CCNU and vincristine (VCR) following whole head irradiation. Cell cultures were prepared from 40 patients in this series and their sensitivity to each cytotoxic drug was assessed in a mictotitration assay with 35 S-methionine incorporation as the end point. Twenty-two of forty (55%) patients responded to PCB and/or CCNU in vitro, and sensitivity to these drugs was linked with increased RFI, whilst sensitivity to VCR was not. The RFI of patients who had responded to PCB or CCNU in vitro was significantly longer than the RFI of patients whose tumours failed to respond in vitro or patients who had not been tested. There was no difference in sex ratio, extent of operation, radiation dose and degree of steroid cover between responders, non-responders and untested groups. Grade III tumours tended to be more sensitive in vitro than grade IV tumours. The age of patients also influenced in vitro chemosensitivity. Patients with chemosensitive tumours in vitro tended to be younger than patients with insensitive tumours in vitro. Further statistical analysis, taking into account these prognostic factors, indicated an association between chemosensitivity in vitro and RFI.


PLoS Medicine that shows an association between apoE levels and cardiovascular disease mortality.Reilly and Rader discuss a new study in Apolipoprotein E (apoE) has effects on lipoprotein metabolism and the vasculature that have long been considered to be anti-atherogenic . In the ApoE is the main ligand for clearance of triglyceride rich particles via the low density lipoprotein receptor (LDLR) and related receptors in the liver, thereby regulating plasma levels of these lipoproteins and thus also of plasma cholesterol and triglyceride levels. In mice, apoE gene deletion results in severe hypercholesterolemia, due to accumulation of VLDL and remnant particles, and in accelerated atherosclerosis. In humans, there are two common apoE polymorphisms, apoE2 and apoE4, that affect its function compared with the wild-type apoE3 and that have been extensively studied (sidebar) [ApoE is believed to have additional properties beyond regulation of lipoprotein remnant clearance that have been historically considered anti-atherogenic in nature . For exaPLoS Medicine that should stimulate more intense examination of the role of apoE in human atherosclerosis [In this context, Mooijaart and colleagues present novel data inlerosis . They reThe study provides clear evidence for the direct association of apoE levels with cardiovascular disease (CVD) mortality in this cohort, and also shows that this association is independent of apoE genotype. The study also highlights the remarkable paucity of data on the relationship between plasma apoE levels and CVD and outcomes in humans and suggests that this issue should be studied in additional observational studies and even in interventional clinical trials.However, Mooijaart and colleagues's study leaves several unanswered questions that need to be addressed in future studies. It is possible that plasma apoE levels may have been simply a surrogate for plasma levels of atherogenic remnant lipoprotein particles, which were not measured as part of this study. Several other studies have shown that direct measures of remnant particles provide independent value in predicting CVD beyond traditional lipoprotein measures . FurtherThe authors suggest that apoE may actually be pro-atherogenic in humans and thus causally linked to the increased CVD mortality in people with higher apoE levels. This runs counter to current dogma that apoE is on balance anti-atherogenic. However, a specific pro-inflammatory role for apoE has been suggested by recent work. Elzen and colleagues  recentlyMooijaart et al. showed that baseline apoE levels were directly associated with change in C-reactive protein (CRP) levels over time and suggested that this provided evidence for apoE in promoting inflammation in a causal relationship with CVD. However, use of a low-sensitivity CRP assay, with almost 20% of levels below the detection for this assay, combined with potential bias in reporting change in CRP levels only in the lowest CRP quartile, suggest the need for more rigorous assessment of this claim. Furthermore, plasma levels of both apoE and CRP are largely liver derived and could be regulated in a similar fashion by hepatic-directed factors .Measurement of diverse and more specific measures of innate and adaptive immunity as well as additional inflammatory markers in the full cohort would have provided more robust evidence for a link to inflammation. However, expansion of such correlative data would not address issues of causality, as the relationship of plasma apoE with inflammatory markers may purely reflect its association with remnant atherogenic lipoproteins rather than any causal relationship. Experimental studies of the administration of apoE to humans, perhaps in specific settings of modest evoked inflammation, will provide the strongest evidence for pro- or anti-inflammatory effects of plasma apoE in humans.These recent papers by Mooijaart et al. and Elzen et al. provide new impetus for addressing several key questions relating to apoE function in atherosclerosis and as a plasma biomarker of CVD risk . In summApoE2: exhibitsreduced affinity for the LDLR, with reduced clearance of apoE-containing remnant lipoproteins. Individuals homozygous for apoE2/E2 have higher plasma apoE levels, often develop hyperlipidemia due to accumulation of remnant lipoproteins, and are at risk for premature atherosclerotic disease.ApoE4: hasincreased LDLR affinity with more rapid clearance of apoE-containing remnant lipoproteins. Carriers of apoE4 have lower plasma apoE levels, but increased LDL cholesterol levels and increased cardiovascular risk.Are plasma apoE levels independent positive predictors of CVD events in other cohorts?Could apoE levels be a predictor of “residual risk” in statin-treated patients?Does plasma apoE simply serve as a surrogate for atherogenic remnant particles?Does apoE in the HDL fraction have a different relationship to CVD than total plasma apoE levels?Does apoE have distinct lipoprotein-independent functions in humans in vivo?What is the impact of apoE's lipid antigen-presenting functions in atherosclerosis?What is the net impact of macrophage apoE in modulating atherosclerosis?What is the relevance of emerging studies that show a role for apoE in promoting adult obesity and insulin resistance ?


Education and public policies are largely failing to encourage people to exercise. Could our knowledge of exercise biology lead to pharmaceutical treaments that could confer the same benefits as exercise? Regular physical activity has been recognized to confer health benefits since antiquity . HoweverA large body of epidemiological literature consistently documents greater longevity in persons who are physically active on a near-daily basis, and reveals inverse relationships between levels of daily exercise and incidence of major chronic disorders such as obesity , hyperteHowever, knowledge of the likely health benefits accruing to the physically active so far has not been a sufficient stimulus to promote sustained changes in behavior for most of the American population. If education and public policies are insufficient to promote behavioral changes to increase physical activity among most people, can advances in biotechnology confer such benefits to individuals unable or unwilling to perform the necessary physical effort?Greater knowledge of how cells and tissues are modified in response to recurring bouts of exercise provides a basis for more precise recommendations as to the mode, intensity, and amount of exercise required to produce specific health benefits  and metabolic (glycolytic versus oxidative) properties. At one extreme, the fastest glycolytic fibers have high levels of enzymes that generate ATP via glycolysis but few mitochondria (approximately 1% of cell volume). At the other end of the spectrum, slow oxidative fibers generate force with slower kinetics but are capable of long periods of repeated contraction without fatigue. They are rich in mitochondria (3%–10% of cell volume). Other myofibers, called fast oxidative, are both relatively fast and resistant to fatigue, and are rich in mitochondria (like the slow oxidative fibers). Muscles composed primarily of fast glycolytic fibers are needed for rapid movements  but fatigue when sustained periods of activity are required .Most human muscles exhibit a mosaic pattern of different fiber types , with a Patterning of skeletal muscle fiber composition is initially determined during embryonic development, but can be partially or completely overturned by stimuli applied to fully mature adult myofibers: by hormonal influences , but most importantly by different patterns of motor nerve activity and contractile work. Myofibers that experience phasic patterns of contractile work—brief bursts of activity interspersed within long periods of inactivity—will assume the fast glycolytic phenotype. Myofibers subjected to tonic patterns of work activity—sustained periods of repetitive contraction on a habitual basis—will take on fast oxidative or slow oxidative properties. Under experimental conditions in laboratory animals, it is possible to transform muscles completely from one myofiber phenotype to another in a reversible manner, solely by altering the pattern of neural stimulation. We know that having a high proportion of oxidative muscle fibers conveys health benefits, and the possibility to control fiber composition through therapeutic intervention is promising.At a cellular and molecular level, how does a fast glycolytic myofiber sense a tonic pattern of contractile activity and transduce that information to transform itself into a cell with fast oxidative or slow oxidative properties? We know that such signals must be transduced to the nucleus, activating certain genes and suppressing others, for myofiber plasticity to occur. We know the identities of some of the nuclear transcription factors that carry these signals, and of other proteins that regulate the function of these transcription factors .Quite a variety of intracellular messengers have been proposed to provide the proximate signals in exercising muscles to stimulate activity-dependent gene regulation. This discussion will focus on a signaling cascade mediated by calcineurin, a calcium-regulated protein phosphatase that signals to the nucleus via transcription factors of the nuclear factor of activated T cells (NFAT) family. Upon receipt of the appropriate calcium signal, calcineurin is activated and removes phosphate groups from NFAT, thereby permitting translocation of NFAT to the nucleus. Within the nucleus, NFAT binds DNA and activates transcription (in concert with other transcription factors) of relevant downstream target genes that encode proteins necessary for fast oxidative or slow oxidative myofiber phenotypes.Calcineurin and NFAT proteins are abundant in skeletal myofibers, and several lines of evidence support the viewpoint that the calcineurin–NFAT pathway plays a role in mediating activity-dependent gene regulation in muscle ,37,38,39Muscle contractions are initiated under the influence of the motor nerve by release of calcium from the sarcoplasmic reticulum, which triggers actin–myosin crossbridge cycling . CalciumTo explain this cellular memory, we propose that, as the bursts of contractile activity are sustained over time , a second source of calcium is mobilized from outside of the cell and enters via a class of calcium channels that are called “store-operated” or “non-voltage-dependent.” This second source of calcium is not required for muscle contractions, but is required to sustain calcium-dependent signaling to the nucleus. Phasic patterns of contractile activity do not promote calcium entry via store-operated channels. Tonic patterns of activity, in contrast, would not only promote the mobilization of extracellular calcium but also increase the number of store-operated calcium channels with each bout of exercise. Myofibers would thereby grow progressively more responsive to tonic activity. Consistent with this model, we know that daily running increases the expression of a putative store-operated calcium channel called TRPC3. Moreover, increasing the abundance of TRPC3 in cultured myotubes prolongs the period in which intracellular calcium is elevated following a depolarizing stimulus, sustains the transcription factor NFAT within the nucleus, and augments expression of NFAT-dependent target genes .A great deal of additional research remains to be done before we have a comprehensive understanding of how habitual physical activity promotes changes in gene expression in skeletal muscles, and in turn improves fitness and reduces risk for diabetes, hypertension, dyslipidemia, and coronary artery disease. However, studies of the relationships between the proteins of calcium metabolism and calcium-regulated signaling pathways—as described here in a simplified manner with respect to TRPC3, calcineurin, and NFAT proteins—are illustrative of progress in this field. Other notable findings point to additional signaling proteins  and transcription factors  active in pathways that intersect with calcineurin–NFAT signaling ,46,47,48Long the province of physiologists who have contributed valuable insights in past decades, exercise science more recently has attracted the attention of molecular biologists, who have recognized the biological interest and medical importance of this field. Biotechnology and pharmaceutical companies also are beginning to take interest.This review has focused on adaptive responses of skeletal muscle to changing patterns of physical activity, and on the role of the calcium–calcineurin–NFAT signaling cascade in controlling gene expression in skeletal myofibers. Further advances in our understanding of signaling mechanisms that govern activity-dependent gene regulation in skeletal muscle could lead to drugs, gene therapy, or devices that can, at least in part, substitute for daily exercise. Although it is unlikely that such technologies would fully recapitulate exercise-induced adaptations that affect other tissues of the body, beneficial effects on work performance and whole-body metabolism have been demonstrated using gene transfer techniques to alter skeletal muscles in animal models. If it proves possible to drive similar effects in skeletal muscles in humans, the interventions capable of providing such effects would almost certainly find broad clinical application.


The most common cause of nongenetic mental retardation in the Western world is fetal alcohol syndrome (FAS). About one in 1,000 United States children is born with FAS, which is caused by prenatal exposure to alcohol. Children with FAS typically have abnormal facial features and reduced growth. They also have central nervous system abnormalities that lead to impaired learning and memory skills, hyperactivity, and other behavioral problems. These neurological disabilities arise because ethanol disrupts the formation and survival of neurons in the developing brain, particularly in the last trimester of pregnancy and in the first few years of postnatal life when brain development is particularly active.There is no cure for FAS, but it is 100% preventable. Public health officials recommend that women planning pregnancy and sexually active women who do not use effective birth control avoid alcohol—there is no safe dose of alcohol or safe time to drink it during pregnancy. Sadly, this advice is often ignored. In the US, one in 12 pregnant women admits to drinking alcohol, and one in 30 reports binge drinking (five or more drinks at one time). Given such figures, ways to prevent or attenuate the effects of alcohol on the developing brain are badly needed. Alessandro Ieraci and Daniel Herrera now report that nicotinamide (the amide form of vitamin B3) can prevent some of the deleterious effects of ethanol on developing mice brains, and suggest that nicotinamide might be suitable as a preventative therapy for FAS.Nicotinamide is the precursor of β-nicotinamide adenine dinucleotide, which enhances the action of many enzymes and is therefore essential for cellular function. Nicotinamide and other forms of vitamin B3 have been used for many years as dietary supplements to treat and prevent pellagra, a vitamin deficiency disease. Large oral doses of nicotinamide have also been used over extended periods of time in clinical trials to treat type I diabetes and bullous pemphigoid . In addition, recent animal data indicate that nicotinamide is also neuroprotective. In rat models of stroke, for example, it improves neurological outcomes by inhibiting the neuronal apoptosis caused by oxygen deprivation. In apoptosis (a highly organized form of cell death), mitochondrial breakdown releases the protein cytochrome-c, which activates enzymes known as initiator caspases. These activate effector caspases (including caspase-3), which digest other cellular substrates and kill the cell without releasing any potentially harmful molecules. Nicotinamide acts as a neuroprotectant in part by inhibiting cytochrome-c release and caspase-3 activation.As induction of apoptosis is one known mechanism by which ethanol harms neurons, Ieraci and Herrera asked whether nicotinamide could reduce the effects of ethanol in a mouse model of FAS. Subcutaneous injection of ethanol triggers widespread neurodegeneration in seven-day-old mice—whose brain development is comparable to that of human fetuses in the third trimester of pregnancy. The researchers report that an ethanol injection sufficient to raise blood ethanol levels to those that a human fetus would be exposed to if its mother indulged in binge-like drinking activated caspase-3 and induced the release of cytochrome-c. They then show that nicotinamide injected up to eight hours after the ethanol reduced caspase-3 activation and cytochrome-c release without altering blood or brain ethanol levels.As the “normalizing” effects of nicotinamide were strongest when it was administered zero to two hours after alcohol exposure, Ieraci and Herrera next investigated whether nicotinamide given two hours after alcohol injection could prevent ethanol-induced neuronal death. To look for early signs of neuronal injury, they stained brain sections with Fluoro-Jade B soon after exposing the mice to ethanol. Most damage occurred in brain regions particularly sensitive to ethanol at this age—namely, the anterior cingulate cortex (which is involved in cognition), the hippocampus (a region needed for learning and memory), and the thalamus (which relays messages from the outside world to other brain regions); nicotinamide treatment reduced this damage. When the researchers stained brain sections for NeuN, a marker of mature neurons, several days after ethanol exposure, they found reduced numbers of neurons (compared with control brains) in similar brain regions, and, again, nicotinamide treatment reduced ethanol's effects. Finally, the researchers used three standard behavioral assays to test whether the reduction in ethanol-induced neuronal death produced by nicotinamide affected the behavior of adult mice. They report that nicotinamide reversed the increase in hyperactivity and the decrease in fear caused by ethanol exposure, and prevented the impairment in learning and memory induced by ethanol.In mice, then, these results show that nicotinamide can reverse the molecular, cellular, and behavioral effects of ethanol exposure on developing brains. While the beneficial effects observed were most pronounced when nicotinamide was given at the same time or shortly after alcohol exposure, the study suggests that there is a time window of a few hours during which treatment with nicotinamide might be effective. More studies are needed to determine exactly how nicotinamide protects neurons against alcohol-induced damage, but the data raise the possibility that nicotinamide treatment may provide a way to prevent some human cases of FAS.


In our complex and multifaceted world, our senses are continuously bombarded with much more information than our brains can fully process. Attention is a critical cognitive function that allows us to select for detailed analysis only the most relevant information at any given moment. The crucial nature of attention is clearly evident in the game “Where's Waldo.” Picking out the odd, bespectacled man wearing the familiar red-and-white striped shirt and hat amidst an array of distracting objects that contain similar colors and patterns is astonishingly difficult. Only by directing our attention or our eyes sequentially to many different spatial locations can we eventually find Waldo. However, the basic neural mechanisms that allow us to focus our attention on specific locations in our visual field, even in uncluttered scenes, are not well understood.Previous functional neuroimaging studies have revealed a network of frontal and parietal brain regions that, along with the visual cortex, show enhanced activity during visual-spatial attention. It has been hypothesized that this frontal-parietal network controls attention by sending “top-down” signals that “bias” activity in specific regions of visual cortex. This biasing activity is, in turn, thought to facilitate the accurate, rapid processing of information in attended regions of space. However, these neuroimaging studies were unable to elucidate the temporal cascade of neural events associated with the control of attention—critical information for understanding how different brain regions coordinate their actions as succeeding stages of attentional control unfold.In this study, Tineke Grent-‘t-Jong and Marty Woldorff set out to discover both the timing and location of brain activations in the attentional control network that underlie our ability to direct our visual spatial attention. To do this, the authors combined two complementary methods: event-related potentials (ERPs), which measure electrical changes in the brain with scalp electrodes and thus provide very precise timing information on brain activity triggered by sensory or cognitive events, and functional magnetic resonance imaging (fMRI), which localizes event-triggered brain activations by measuring associated changes in blood flow. This innovative study surmounted previous obstacles to the study of attentional control by including important control conditions and by using the exact same experimental paradigm and task contrasts (comparisons of patterns of activation elicited by different tasks) for both methodologies. This multimethodological approach enabled the authors to obtain measures of cognitive brain activity with good temporal and spatial resolution and thereby gain a more complete understanding of the neural mechanisms underlying top-down attentional control.The authors used a spatial cueing paradigm in which participants kept their visual gaze directed toward the center of a computer screen. A series of 4-second trials were presented, with an instructional letter cue displayed at the beginning of each trial. On most trials, the letter cue (“L” or “R”) instructed them to covertly attend  to either the lower left or the lower right portion of the screen to try to detect the possible brief appearance of a faint dot at the cued location. However, the authors included an important control condition: on some trials, the cue (“P”) instructed the subject to not orient their attention to a particular location, as no faint dot would appear during that trial. By incorporating both of these interpret-cue trials and attention-orienting trials, Grent-‘t-Jong and Woldorff were able to separate the activations related to spatial orienting from those associated with general cue processing that were not related to orienting.Both the ERP and fMRI results revealed that some activations were elicited by both the interpret cues and the attentional-orienting cues, and some were associated only with attentional orienting. More specifically, the ERPs showed that during the first 400 ms, the attend-cue and interpret-cue trials produced similar patterns of activity, which the fMRI indicated was associated with the more lateral regions of frontal and parietal cortex. After 400 ms, the response to the interpret cues quickly returned to baseline, whereas the attend cues elicited additional, widespread waves of orienting-specific activity that continued for hundreds of milliseconds. Moreover, the fMRI confirmed that this orienting-related activity was associated with greater activation specifically in the more medial portions of the frontal and parietal cortex. These results indicate that lateral parts of the frontal-parietal network play a role in the initial general cue processing and interpretation, whereas the more medial frontal-parietal regions function more specifically in the subsequent orienting of spatial attention.The authors then set out to determine the relative timing of the medial frontal and parietal activations underlying this attentional-orienting process. To do this, the authors used modeling procedures to relate these orienting-specific regions delineated by the fMRI to the corresponding orienting-specific ERP activity. This analysis indicated that the initial part of this ERP orienting activity (from 400–700 ms) derived mainly from the medial frontal regions. Starting at around 700 ms, however, the analyses indicated that both the frontal and parietal medial regions contributed to the ERP activity patterns, indicating that the orienting-related frontal activation preceded the parietal activation by several hundred milliseconds. These frontal and parietal activations were then followed shortly after by enhanced, biasing-related activity in specific regions of the visual cortex, before the arrival of any target stimuli in the attended region of space.Altogether, these results reveal the systems-level timing and sequence of neural events underlying the voluntary orienting of spatial attention. More specifically, these results indicate that voluntary attentional orienting is initiated by the more medial portions of the frontal cortex, which then recruit medial parietal areas a few hundred milliseconds later. These regions then act in concert to sustain attention and provide a biasing signal to the specific regions of the visual cortex that process stimuli in the attended region of space. This biasing-related activity is in turn thought to reflect a neurophysiological process that facilitates the processing in the visual cortex of visual stimuli in the attended location.


Cells make no exception and indeed any discontinuity in the DNA double helix is promptly detected, triggering an alteration of cell proliferation and an attempt to repair. Yet, linear chromosomes bear DNA ends that are compatible with normal cell proliferation and they escape, under normal conditions, any repair. How telomeres, the chromosomes tips, achieve that is not fully understood. We recently observed that the Rad9/Hus1/Rad1 (911) complex, previously known for its functions in DNA metabolism and DNA damage responses, is constitutively associated with telomeres and plays an important role in their maintenance. Here, we summarize the available data and discuss the potential mechanisms of 911 action at telomeres. Any discontinuity within the DNA double helix is perceived by the cell as a threat to its genetic integrity. Consequently, cells respond promptly to the generation of DNA interruptions by mounting a coordinated set of actions collectively known as the DNA-damage response (DDR) with the intent of arresting cell cycle progression and initiating DNA repair. In higher eukaryotes, the large protein kinases Atm and Atr play central roles in initiating the DDR . Atm resThe 911 trimer resembles the toroidal sliding clamp proliferating cell nuclear antigen (PCNA) and is loaded onto DNA by a clamp loader composed of Rad17 and replication factor C (RFC) subunits . While mSaccharomyces cerevisiae in which an internal tract of telomeric repeats inhibits DDR signalling [Telomeres, the ends of linear chromosomes, are exceptional, as they are DNA ends that do not normally trigger a robust DDR and are compatible with normal cellular proliferation in checkpoint proficient cells. They contain long stretches of DNA tandem repeats (TTAGGG in vertebrates) and terminate in a 3' protruding single-stranded DNA overhang. Due to the inability of the standard DNA replication machinery to fully replicate DNA ends, many cells maintain their telomeres by the action of telomerase (Tert), a specialized reverse transcriptase that uses its associated RNA component (Terc) as a template to elongate chromosome ends (see  for a hignalling . This angnalling -24; convgnalling .Despite a tight inhibitory control of DDR at telomeres, mammalian chromosome ends are nevertheless a physiological substrate of the DDR apparatus as the inactivation of genes encoding certain DDR factors involved in DSB repair such as DNA-PK -30, ParpCaenorhabditis elegans strains lacking HUS-1 or MRT-2, a functional ortholog of mammalian Rad1, display progressive telomere shortening and loss of germ-line immortality [Schizosaccharomyces pombe orthologues of this complex have been reported to associate with telomeric repeats and are required for telomere length maintenance [Saccharomyces cerevisiae causes mild telomere length changes, although some effects appear to be laboratory or strain specific [Recently, we identified an important role for 911 at telomeres in mammals . We repoortality ,45. In Sntenance . The delspecific -49.C. elegans [in vitro. These results suggest a unique functional role for 911 in the regulation of telomerase activity. Meanwhile, additional DDR proteins further contribute to telomere homeostasis through other important functions such as promoting T-loop formation and suppressing chromosomal end joining as well as sister telomere exchanges [A key question raised by the above-mentioned studies centres precisely on how the 911 complex participates in telomere maintenance. To determine whether 911 affected the function of telomerase, the enzyme that catalyzes telomere extension, we compared telomerase activity in wild type cells and cells lacking Hus1 or other 911 components. Excitingly, we discovered that lack of 911 leads to reduced telomerase activity without affecting the expression of the core components of the telomerase complex. We therefore conclude that 911 controls telomerase activity. The notion that 911 promotes telomerase activity is consistent with findings indicating that defects in 911 and Tert have equivalent and non-additive effects on telomere length in  elegans . Interesxchanges ,40.The structural similarity that the 911 trimer shares with PCNA provides a useful starting point for considering how the 911 complex might influence telomerase activity. Best known for its role as a processivity factor for DNA polymerases during DNA synthesis, PCNA functions as a molecular scaffold that recruits replication and repair proteins to DNA . Evidencin vitro experiments suggest that 5'-recessed DNA ends are preferential sites for loading of the 911 complex, making telomeres a natural candidate substrate in vivo as well [When applied to the issue of telomere maintenance, this model would suggest that the 911 complex might act by directly binding and regulating the activity of telomerase. Consistent with this possibility, in a number of tests, 911 biochemically associates with catalytically active telomerase complex. The 911 complex is appropriately situated for such an interaction, as  as well ,54. An a as well .Although the available data are consistent with a model in which 911 directly interacts with and stimulates telomerase (as shown in figure Notably, the rate of telomere shortening that we observe in Hus1-null cells is higher than that reported for mice lacking telomerase, suggesting that additional mechanisms of telomere shortening might be at play in the absence of the 911 complex. Rapid telomere shortening could be caused by nucleolytic degradation or aberrant recombination events at uncapped telomeres. The 911 complex has been implicated previously in telomeric recombination. In ALT cells, telomeres are maintained by a recombinational mechanism in which short telomeres invade homologous telomeric sequences and use this as a template for lengthening . InteresFinally, it is worth considering that the presence of a poorly functioning telomerase on DNA might be more detrimental to telomere stability than not having telomerase at all. A poorly processive polymerase on DNA can cause DNA replication fork stalling and such an event in checkpoint-deficient cells has been shown to lead to fork reversal and hyper-recombination . In agreS. pombe and C. elegans, loss of 911 components leads to telomere shortening and end-to-end chromosome fusions. Moreover, the 911 complex can be co-immunoprecipitated with telomerase activity and is required for optimal telomerase activity in in vitro assays. The precise molecular function of 911 at telomeres, which may involve stimulation of telomerase directly through a physical interaction or indirectly through effects on the telomeric DNA substrate, has yet to be completely resolved. This and other aspects of telomere maintenance undoubtedly will continue to be areas of intense interest given the principal role of telomeres as determinants of cellular lifespan and genome stability.The linear structure of eukaryotic chromosomes creates a requirement for special mechanisms to replicate and protect chromosome ends. Over the past several years, a seemingly unlikely caretaker of telomeres has emerged. DDR proteins, which normally act to eradicate free DNA ends, have proven to be critical to telomere homeostasis. As reviewed here, the 911 complex is the latest example of a mammalian DDR factor that localizes to telomeres and acts to maintain telomere integrity. Consistent with results from


The Wiskott-Aldrich Syndrome protein (WASP) is an adaptor protein that is essential for podosome formation in hematopoietic cells  Cell migration requires the rearrangement of adhesions and cytoskeleton in response to extracellular stimuli to form a leading edge that allows directed locomotion. In most cell types, migration-related adhesive structures are composed of integrin heterodimers and intracellular associated proteins clustered in plaques termed focal complexes and their more mature variant, focal adhesions WASP is an essential component of podosomes, where it activates the Arp2/3 complex inducing actin polymerization −/− DCs by confocal microscopy. As expected, podosomes were grouped in clusters in wild-type DCs  after the expression of eGFP-WASP (Surprisingly, the recovery of WASP levels in WIPructures A. In add −/− DCs C. This cGFP-WASP B, bottomGFP-WASP D. These −/− DCs, which express normal levels of WIP, also lack podosomes and assemble large focal contacts and disorganized actin clumps instead Our data show that WIP by itself is not sufficient to promote podosome assembly because WASPIn summary, we have shown that in DCs WIP is essential for podosome formation and cell polarity by preventing the extensive degradation of WASP by calpain and by facilitating the recruitment of WASP to discrete foci to form the core of podosomes. The tight coupling between WASP and WIP is therefore a key contributor to normal podosome dynamics and likely of major importance to the cell trafficking behavior of DCs


Tyrosine kinases of the epidermal growth factor receptor (EGFR) family are frequently mutated in human cancers. Mutations in the tyrosine kinase domain of EGFR (encoded by exons 18–24) have mostly been found in lung cancers. Some, but not all, lung cancers carrying such mutations are responsive to treatment with small-molecule EGFR inhibitors, including the two FDA-approved drugs erlotinib and gefitinib.Matthew Meyerson and colleagues undertook a systematic study of the different classes of EGFR mutations found in lung cancers in order to understand their roles in tumorigenesis on one hand, and their relation to drug sensitivity on the other. They introduced different altered EGFR versions into fibroblast and lung epithelial cells, and found that all of the mutant proteins transformed both cell types in an EGF-independent manner. Transformation was associated with constitutive kinase activity and with the activation of known downstream signaling pathways.While the various mutant receptors had similar transforming capabilities, cells expressing them differed in their response to EGFR inhibitors. Transformation of cells expressing mutations in exons 18, 19, and 21 was inhibited by 100 nM erlotinib or gefitinib, whereas no significant inhibitory effect on cells expressing an exon 20 insertion mutation was seen even at much higher concentrations of either drug. This result is consistent with the lack of clinical responses to erlotinib or gefitinib in three lung cancer patients with exon 20 mutations. In contrast, when the researchers tested another experimental EGFR inhibitor called CL-387,785, they found cells expressing the exon 20 insertion mutation to be sensitive, consistent with previous studies that had found similar patterns with other EGFR exon 20 mutations. These results highlight the problems and the possibilities of individualized cancer therapy. One drug is unlikely to fit all tumors, not even all tumors with mutations in a specific oncogene. On the other hand, having a collection of drugs against a particular target increases chances that one of them will prove effective, and that alternatives exist when tumors develop resistance. Developing such a collection and selecting the right drug for the right patient is a challenge not only scientifically but also economically.


An Oriental and Colourpoint Shorthair cat pedigree segregating for albinism was analysed for association with TYR by linkage and sequence analyses. Microsatellite FCA931, which is closely linked to TYR and TYR sequence variants were tested for segregation with the albinism phenotype. Sequence analysis of genomic DNA from wild-type and albino cats identified a cytosine deletion in TYR at position 975 in exon 2, which causes a frame shift resulting in a premature stop codon nine residues downstream from the mutation. The deletion mutation in TYR and an allele of FCA931 segregated concordantly with the albino phenotype. Taken together, our results suggest that the TYR gene corresponds to the colour locus in cats and its alleles, from dominant to recessive, are as follows: C (full colour) > cb (burmese) ≥ cs (siamese) > c .Albino phenotypes are documented in a variety of species including the domestic cat. As albino phenotypes in other species are associated with  To confirm that albinism is a TYR allele in cats, an analysis of an Oriental and Colourpoint Shorthair cat pedigree that segregates for albinism .Tyrosinase exons were sequenced as previously described , which was confirmed by sequence and restriction fragment length polymorphism (RFLP) analyses , burmese (cb) and siamese (cs), suggesting the allelic series C > cb ≥ cs > c based on mutations in TYR. However, sufficient breeding studies have not been performed to confirm the allelic series in cats, specifically, the interaction of full colour and burmese with the albino allele.In our previous study  and in cattle  albinism, but the difference in the tapetal reflex suggests that the single report of a red-eyed albino cat may be in error.Albino cats have been reported in the literature (TYR at position 975 in exon 2 is associated with albinism in cats. This mutation could be used in a DNA test to detect carriers and assist with breeding programmes. This finding also supports the use of the cat as a model for human TYR-associated albinisms.In conclusion, we propose that a cytosine deletion in


In recent years, clinical assisted reproduction techniques (ART) are increasingly being practiced worldwide, which in turn has led to an accumulated surplus of cryopreserved embryos within fertility clinics ,2. NeverAlthough such institutions are not permitted to directly profit from the transaction of cryopreserved embryos between donor and recipient, it must be remembered that there is still considerable opportunity for profiteering in medical fees arising from laboratory and clinical services rendered to the recipient. It is easy to disguise the 'sale' of altruistically donated human embryos through substantially increased medical fees, particularly in a private practice setting. Firstly, patients seeking treatment at privately run IVF programs are already prepared to pay higher medical fees for the supposedly 'better' reputation of the fertility doctor and 'enhanced' medical facilities available. Secondly, it is often the case that patients seeking donated embryos at a privately-run fertility clinic have already been exasperated by the long waiting list of government-funded IVF programs, and therefore would have no qualms with paying higher medical fees. Lastly, a privately run IVF program would usually treat the medical fees charged to individual patients as confidential information. Hence, it is difficult for patients to discern whether there is any disparity in medical fees for a self- and donor-freeze/thaw embryo transfer cycle, which could, in fact, mask 'opportunistic profiteering' by medical professionals.Therefore, the pertinent question that arises is, what would constitute a fair and honest profit margin for the medical professional and health institution in question? A suitable benchmark would obviously be the level of medical fees normally charged to patients for a self-freeze/thaw embryo transfer cycle, after initial failure at their first attempt in ART. This is because the level of medical expertise, clinical and laboratory services required for a donor- and self-freeze/thaw embryo transfer cycle should, in theory, be about the same , althougThere is nothing deplorable about profit-making in healthcare services – after all, medical professionals ought to make a decent living after numerous years of expensive education, coupled with their heavy investment in medical equipment and facilities. Nevertheless, it is imperative that they should always work towards a fair and honest profit margin, rather than exploit the short supply of legally donated human material for financial gain. It must always be remembered that the embryo donor has presumably been altruistically, rather than financially, motivated to help the recipient; such opportunistic profiteering by medical professionals would be an extreme breach of medical ethics and professional code of conduct, particularly if they are encouraging their former patients to altruistically donate supernumerary embryos for the sake of infertile couples. It must be noted that many childless couples feel a sense of exhilaration and euphoric joy upon attaining success in clinical assisted reproduction. This altered emotional state could render them particularly vulnerable to the coaxing of medical professionals, to whom they feel they owe a debt of gratitude .Another solution for greater transparency would be to establish a government-controlled national or regional register of prospective embryo donors and recipients, one successful example being CECOS in France . This wo


EHP 114:1703–1712; Kisby et al.][.Neurodevelopmental disorders such as learning disabilities, mental retardation, and autism spectrum disorders affect an estimated 5–10% of the 4 million babies born in the United States annually. In a report released in 2000, the National Research Council concluded that 3% of these disorders are the direct result of environmental exposures to neurotoxicants, with another 25% arising from the interaction between such exposures and genetic susceptibility. Investigators have shown that many of these long-term adverse outcomes can be attributed to genetic damage to immature neurons in the developing brain related to exposure to genotoxicants, chemicals that disrupt the complex, delicate cellular process that regulates development of a fully functional brain. Although the precise mechanisms involved are still poorly understood, scientists are now starting to unravel the molecular ravages caused by genotoxicants As reported in this month’s issue, a research group exposed cultures of immature neurons known as granule cells and the more developed and more abundant astrocytes to sublethal doses of two well-characterized alkylating genotoxicants: methylazoxymethanol (MAM), a highly toxic compound synthesized from the poison found in plants called cycads, and nitrogen mustard (HN2), a chemotherapeutic agent. The team then analyzed the cultures for cell viability, DNA damage, markers of apoptosis, and corresponding gene expression patterns. The intent of the research is ultimately to identify the key molecular networks that are targeted by genotoxicants, in order to understand how such agents influence brain development.Results showed that granule cells were much more sensitive to the genotoxicants than astrocytes. The exposures caused dose-dependent DNA lesions that persisted and accumulated, apparently because, unlike astrocytes, granule cells lack the ability to repair DNA damage. In other words, once the exposure has wreaked havoc on the developing neurons, the damage is done, and its impact is felt throughout the process of neuronal development, leading to long-term impairment. The authors speculate that these events “could explain why the developing cerebellum is a prime target in several human neurodevelopmental disorders.”The team also discovered that the two genotoxicants affected distinctly different sets and functional types of genes. MAM targeted differentiation, stress and immune response, cell signaling, and transcriptional regulation genes, whereas HN2 targeted apoptosis and protein synthesis gene expression. This preferential targeting suggests that different genotoxicants probably cause completely different effects in the developing brain. With a significant proportion of neurodevelopmental disabilities thought to stem directly from early exposures to DNA-damaging agents or gene–environment interactions related to such exposures, further investigation of the molecular networks involved in these effects is clearly needed.


The term "Electrophysiological remodeling" defines the changes of atrial electrophysiologic properties taking place in atrial myocites during atrial fibrillation and/or following periods of sustained atrial fibrillation (AF). Early research was prompted by clinical observations: many patients were seen to get through increasingly frequent and longer paroxisms of AF to persistent AF, until chronic AF eventually ensued, without significant changes of underlying heart disease.Indeed, clinical electrophysiology investigations reported peculiar differences between AF patients and patients without atrial arrhythmias. It was felt that the electrophysiologic milieu was the cause of AF ,2, untilIn his pioneering work, Wijffels kept goats fibrillating by an implanted device, while measuring atrial refractoriness and its rate adaptation as spontaneous restoration of sinus rhythm occurred. He found that, after several self terminating short bouts of AF, the arrhythmia became considerably longer until a duration of days or weeks was reached. The electrophysiologic companionship of AF pattern transformation consisted of significant shortening of atrial fibrillation cycle, which was paralleled by shortening of atrial ERP and loss of ERP adaptation to heart rate: either flat (no change with shortening cycle length) or inverted  ERP relationships to cycle length were observed.This findings had been already observed in AF patients ,2; the nBy the light of this observations, AF may be seen as self-maintaining by creating a functional electrophysiologic substratum, despite the coexistence of only minor structural heart disease or occasional triggers. This principle has been summarised by Maurits Allessie in the aphorism "AF begets AF".Following this observations, several studies have addressed the issue of the mechanisms by which atrial electrophysiologic remodeling occurs in different settings: basic electrophysiology, experimental electrophysiology, and clinical electrophysiology. It is nowadays believed that, beyond structural heart disease, changes of the atrial electrophysiologic substratum are responsible for AF recurrence and/or perpetuation.The aim of this review will be to assess the extent at which atrial remodeling occurs in the experimental animal and in humans, and its relevance to clinical practice.Reviewing literature, one has to consider carefully the investigational setting of reported studies, for significant differences exist in electrophysiologic properties and vulnerability to atrial arrhythmias among species , so that concepts must lean on solid evidence before moving from experimental to clinical electrophysiology, and eventually to clinical practice.On the other hand, the settings of experimental and basic electrophysiology allow precisely controlled conditions, and a deeper knowledge of the mechanisms, up to the ionic/genomic level.It is well demonstrated that atrial remodeling is a time-dependent process that develops as an adaptive regulation of cardiac myocites to maintain cell homeostasis against external stressors . The typThe most common "stressors" of atrial myocites are: tachycardia (high rate of cell depolarisation), and volume/pressure overload (heart failure syndrome).The adaptive changes at the ionic level are quite different in the tachycardia-dependent compared to the heart failure setting, as demonstrated by basic electrophysiology, hence we need to focus on different types of "ionic remodeling".High atrial rate causes calcium overload. Chronic calcium overload may cause ultrastructural changes that resemble myocardial ischemia or hibernation, and may ultimately lead to irreversible cell damage. To prevent dangerous calcium overload, atrial myocites decrease L-type Ca++ current (ICa) at first by a short-term adaptation , in the long term by downregulation of ICa -7. DecreThe process leading to ICa downregulation implies transcriptional changes at the genomic level, but the signal coupling calcium overload to changes in ion channel expression is unknown. It also remain to be understood whether calcium overload is the only initiating signal in this setting.The observations by Wijffels et al  in the eIn a series of 28 patients, Manios et al  similarlOwing to this knowledge, a considerable amount of work has been done to test the hypothesis that preventing calcium overload by Icablockers would prevent tachycardia-dependent remodeling also. Experimental studies found that pre-treatment with verapamil could attenuate  or preveThis hypothesis fits poorly with the electrophysiologic findings by Manios  who demoLee et al  have perWhile demonstrating ICa reduction by downregulation of L-type Ca++ channels, Yue et al  observedIn a similar study on dogs, Fareh et al  also demThere is considerable evidence that, in tachycardia-dependent remodeling, calcium overload plays an important role either at short or at long term if high atrial rates persist. In the clinical setting, small benefits may be observed at short term by verapamil, whereas T-type Ca++ channel blockers seem promising to prevent long term remodeling during persistent AF.These data support the concept that new therapies should target the development of the substrate for AF, as opposed to "traditional" antiarrhythmic therapy which aims to modify the product of the arrhythmic substrate.Heart failure (HF) is the most important clinical syndrome associated to AF . Heart fHeavy alterations in calcium handling are prominent in ventricular myocites: the sarcoplasmic carrier of Ca++ is downregulated in failing myocites , leadingDanshi li et al.  created Thus, a peculiar type of remodeling with an attenuated rate-adaptation of atrial refractoriness and long APD at high rates is observed in experimental HF. This is quite different from atrial tachycardia-dependent remodeling, which shows decreased APD at any cycle length and flat or reversed adaptation of refractoriness to heart rate.The mechanisms of AF onset in the setting of HF must be very different. Structural changes  related to underlying disease cause conduction abnormalities that predispose to reentrant arrhythmias, increased NCX may promote DADs by transient inward depolarising current, which may be enhanced by high cathecolamine levels and by decreased Iks. It seems then likely that AF may be initiated by triggered activity, in the HF setting.If a different sort of atrial remodeling occurs in HF, peculiar interventions for targeting this arrhythmogenic substrate should be sought.Danshi Li and co-workers  performeThis finding was related to the increased dependency of atrial refractoriness on Ikr in HF dogs, due to downregulation of Iks occurring in HF dogs.Significant clinical results were indeed observed by Torp-Pedersen et al  investigNonetheless, AF incidence increases with the severity and the duration of HF despite therapeutic interventions.Shinigawa et al  studied Interestingly, in the same experimental canine model of HF, Danshi Li et al  reportedThese two studies carry the important notion of targeting the development of the arrhythmogenic substrate understanding the pathophysiologic mechanisms of the disease or of the clinical syndrome.It is interesting to note that, either in atrial tachycardia-dependent or in HF-dependent remodeling, both reversible and irreversible changes occur. In either situation, the abnormalities due to ionic remodeling  appear to be reversible when the "stressor" is removed, but in the same time structural changes at the cellular level have occurred, which are irreversible. This "structural remodeling" adds slowing and heterogeneity of conduction velocity to the abnormalities of refractoriness in both situations, and may explain the reduced efficacy of medical interventions in patients with persistent or permanent AF.Experimental research has clearly demonstrated that "electrophysiologically" different arrhythmias share a common electrocardiographic picture of atrial fibrillation, and that the underlying differences imply peculiar therapeutic interventions.All these investigations have brought in the clinical arena a new way of thinking antiarrhythmic therapy: development of new drugs should be guided by the principle of targeting the specific ionic currents depending on the pathophysiologic mechanisms of the arrhythmia, to achieve the optimal ratio between efficacy and safety. Also, the concept of preventing structural remodeling of atrial myocites should be investigated and promptly brought into clinical practice.