Sex chromosome complement contributes to sex differences in coxsackievirus B3 but not influenza A virus pathogenesis

<p>Abstract</p> <p>Background</p> <p>Both coxsackievirus B3 (CVB3) and influenza A virus (IAV; H1N1) produce sexually dimorphic infections in C57BL/6 mice. Gonadal steroids can modulate sex differences in response to both viruses. Here, the effect of sex chromosomal complement in response to viral infection was evaluated using four core genotypes (FCG) mice, where the <it>Sry </it>gene is deleted from the Y chromosome, and in some mice is inserted into an autosomal chromosome. This results in four genotypes: XX or XY gonadal females (XXF and XYF), and XX or XY gonadal males (XXM and XYM). The FCG model permits evaluation of the impact of the sex chromosome complement independent of the gonadal phenotype.</p> <p>Methods</p> <p>Wild-type (WT) male and female C57BL/6 mice were assigned to remain intact or be gonadectomized (Gdx) and all FCG mice on a C57BL/6 background were Gdx. Mice were infected with either CVB3 or mouse-adapted IAV, A/Puerto Rico/8/1934 (PR8), and monitored for changes in immunity, virus titers, morbidity, or mortality.</p> <p>Results</p> <p>In CVB3 infection, mortality was increased in WT males compared to females and males developed more severe cardiac inflammation. Gonadectomy suppressed male, but increased female, susceptibility to CVB3. Infection with IAV resulted in greater morbidity and mortality in WT females compared with males and this sex difference was significantly reduced by gonadectomy of male and female mice. In Gdx FCG mice infected with CVB3, XY mice were less susceptible than XX mice. Protection correlated with increased CD4+ forkhead box P3 (FoxP3)+ T regulatory (Treg) cell activation in these animals. Neither CD4+ interferon (IFN)γ (T helper 1 (Th1)) nor CD4+ interleukin (IL)-4+ (Th2) responses differed among the FCG mice during CVB3 infection. Infection of Gdx FCG mice revealed no effect of sex chromosome complement on morbidity or mortality following IAV infection.</p> <p>Conclusions</p> <p>These studies indicate that sex chromosome complement can influence pathogenicity of some, but not all, viruses.</p

Natural growth rates in Antarctic krill (Euphausia superba): II. Predictive models based on food, temperature, body length, sex, and maturity stage

We used the instantaneous growth rate method to determine the effects of food, temperature, krill length, sex, and maturity stage on in situ summer growth of krill across the southwest Atlantic sector of the Southern Ocean. The main aims were to examine the separate effects of each variable and to generate a predictive model of growth based on satellite-derivable environmental data. Both growth increments in length on moulting (GIs) and daily growth rates (DGRs, mm d-1) ranged greatly among the 59 swarms, from 0.58–15% and 0.013–0.32 mm d-1. However, all swarms maintained positive mean growth, even those in the low chlorophyll a (Chl a) zone of the central Scotia Sea. Among a suite of indices of food quantity and quality, large-scale monthly Chl a values from SeaWiFS predicted krill growth the best. Across our study area, the great contrast between bloom and nonbloom regions was a major factor driving variation in growth rates, obscuring more subtle effects of food quality. GIs and DGRs decreased with increasing krill length and decreased above a temperature optimum of 0.5°C. This probably reflects the onset of thermal stress at the northern limit of krill’s range. Thus, growth rates were fastest in the ice edge blooms of the southern Scotia Sea and not at South Georgia as previously suggested. This reflects both the smaller size of the krill and the colder water in the south being optimum for growth. Males tended to have higher GIs than females but longer intermoult periods, leading to similar DGRs between sexes. DGRs of equivalent-size krill tended to decrease with maturity stage, suggesting the progressive allocation of energy toward reproduction rather than somatic growth. Our maximum DGRs are higher than most literature values, equating to a 5.7% increase in mass per day. This value fits within a realistic energy budget, suggesting a maximum carbon ration of ~20% d-1. Over the whole Scotia Sea/South Georgia area, the gross turnover of krill biomass was ~1% d-1

Direct and Absolute Quantification of over 1800 Yeast Proteins via Selected Reaction Monitoring

Defining intracellular protein concentration is critical in molecular systems biology. Although strategies for determining relative protein changes are available, defining robust absolute values in copies per cell has proven significantly more challenging. Here we present a reference data set quantifying over 1800 Saccharomyces cerevisiae proteins by direct means using protein-specific stable-isotope labeled internal standards and selected reaction monitoring (SRM) mass spectrometry, far exceeding any previous study. This was achieved by careful design of over 100 QconCAT recombinant proteins as standards, defining 1167 proteins in terms of copies per cell and upper limits on a further 668, with robust CVs routinely less than 20%. The selected reaction monitoring-derived proteome is compared with existing quantitative data sets, highlighting the disparities between methodologies. Coupled with a quantification of the transcriptome by RNA-seq taken from the same cells, these data support revised estimates of several fundamental molecular parameters: a total protein count of ∼100 million molecules-per-cell, a median of ∼1000 proteins-per-transcript, and a linear model of protein translation explaining 70% of the variance in translation rate. This work contributes a “gold-standard” reference yeast proteome (including 532 values based on high quality, dual peptide quantification) that can be widely used in systems models and for other comparative studies. Reliable and accurate quantification of the proteins present in a cell or tissue remains a major challenge for post-genome scientists. Proteins are the primary functional molecules in biological systems and knowledge of their abundance and dynamics is an important prerequisite to a complete understanding of natural physiological processes, or dysfunction in disease. Accordingly, much effort has been spent in the development of reliable, accurate and sensitive techniques to quantify the cellular proteome, the complement of proteins expressed at a given time under defined conditions (1). Moreover, the ability to model a biological system and thus characterize it in kinetic terms, requires that protein concentrations be defined in absolute numbers (2, 3). Given the high demand for accurate quantitative proteome data sets, there has been a continual drive to develop methodology to accomplish this, typically using mass spectrometry (MS) as the analytical platform. Many recent studies have highlighted the capabilities of MS to provide good coverage of the proteome at high sensitivity often using yeast as a demonstrator system (4⇓⇓⇓⇓⇓–10), suggesting that quantitative proteomics has now “come of age” (1). However, given that MS is not inherently quantitative, most of the approaches produce relative quantitation and do not typically measure the absolute concentrations of individual molecular species by direct means. For the yeast proteome, epitope tagging studies using green fluorescent protein or tandem affinity purification tags provides an alternative to MS. Here, collections of modified strains are generated that incorporate a detectable, and therefore quantifiable, tag that supports immunoblotting or fluorescence techniques (11, 12). However, such strategies for copies per cell (cpc) quantification rely on genetic manipulation of the host organism and hence do not quantify endogenous, unmodified protein. Similarly, the tagging can alter protein levels - in some instances hindering protein expression completely (11). Even so, epitope tagging methods have been of value to the community, yielding high coverage quantitative data sets for the majority of the yeast proteome (11, 12). MS-based methods do not rely on such nonendogenous labels, and can reach genome-wide levels of coverage. Accurate estimation of absolute concentrations i.e. protein copy number per cell, also usually necessitates the use of (one or more) external or internal standards from which to derive absolute abundance (4). Examples include a comprehensive quantification of the Leptospira interrogans proteome that used a 19 protein subset quantified using selected reaction monitoring (SRM)1 to calibrate their label-free data (8, 13). It is worth noting that epitope tagging methods, although also absolute, rely on a very limited set of standards for the quantitative western blots and necessitate incorporation of a suitable immunogenic tag (11). Other recent, innovative approaches exploiting total ion signal and internal scaling to estimate protein cellular abundance (10, 14), avoid the use of internal standards, though they do rely on targeted proteomic data to validate their approach. The use of targeted SRM strategies to derive proteomic calibration standards highlights its advantages in comparison to label-free in terms of accuracy, precision, dynamic range and limit of detection and has gained currency for its reliability and sensitivity (3, 15⇓–17). Indeed, SRM is often referred to as the “gold standard proteomic quantification method,” being particularly well-suited when the proteins to be quantified are known, when appropriate surrogate peptides for protein quantification can be selected a priori, and matched with stable isotope-labeled (SIL) standards (18⇓–20). In combination with SIL peptide standards that can be generated through a variety of means (3, 15), SRM can be used to quantify low copy number proteins, reaching down to ∼50 cpc in yeast (5). However, although SRM methodology has been used extensively for S. cerevisiae protein quantification by us and others (19, 21, 22), it has not been used for large protein cohorts because of the requirement to generate the large numbers of attendant SIL peptide standards; the largest published data set is only for a few tens of proteins. It remains a challenge therefore to robustly quantify an entire eukaryotic proteome in absolute terms by direct means using targeted MS and this is the focus of our present study, the Census Of the Proteome of Yeast (CoPY). We present here direct and absolute quantification of nearly 2000 endogenous proteins from S. cerevisiae grown in steady state in a chemostat culture, using the SRM-based QconCAT approach. Although arguably not quantification of the entire proteome, this represents an accurate and rigorous collection of direct yeast protein quantifications, providing a gold-standard data set of endogenous protein levels for future reference and comparative studies. The highly reproducible SIL-SRM MS data, with robust CVs typically less than 20%, is compared with other extant data sets that were obtained via alternative analytical strategies. We also report a matched high quality transcriptome from the same cells using RNA-seq, which supports additional calculations including a refined estimate of the total protein content in yeast cells, and a simple linear model of translation explaining 70% of the variance between RNA and protein levels in yeast chemostat cultures. These analyses confirm the validity of our data and approach, which we believe represents a state-of-the-art absolute quantification compendium of a significant proportion of a model eukaryotic proteome

Cohort Profile: The Malawi Longitudinal Study of Families and Health (MLSFH)

The Malawi Longitudinal Study of Families and Health (MLSFH) is one of very few long-standing publicly-available longitudinal cohort studies in a sub-Saharan African (SSA) context. It provides a rare record of more than a decade of demographic, socioeconomic and health conditions in one of the world\u27s poorest countries. With data collection rounds in 1998, 2001, 2004, 2006, 2008, 2010 and 2012 for up to 4,000 individuals, the MLSFH permits researchers to investigate the multiple influences that contribute to HIV risks in sexual partnerships, the variety of ways that people manage risk within and outside of marriage, the possible effects of HIV prevention policies and programs, and the mechanisms through which poor rural individuals, families, households, and communities cope with the impacts of high morbidity and mortality that are often---but not always---related to HIV/AIDS. The MLSFH been used to document (i) the influence of social networks on HIV-related behaviors and perceptions, (ii) the HIV prevention strategies employed by individuals in rural high-HIV prevalence contexts, (iii) the relationship between life-course transitions and HIV infection risks, (iv) the acceptability of HIV testing and counseling (HTC) and the consequences of HTC on subsequent behaviors, and (v) the health and well-being across the life-course of individuals facing multiple challenges resulting from high disease burdens and widespread poverty

Mutation of the Diamond-Blackfan Anemia Gene Rps7 in Mouse Results in Morphological and Neuroanatomical Phenotypes

The ribosome is an evolutionarily conserved organelle essential for cellular function. Ribosome construction requires assembly of approximately 80 different ribosomal proteins (RPs) and four different species of rRNA. As RPs co-assemble into one multi-subunit complex, mutation of the genes that encode RPs might be expected to give rise to phenocopies, in which the same phenotype is associated with loss-of-function of each individual gene. However, a more complex picture is emerging in which, in addition to a group of shared phenotypes, diverse RP gene-specific phenotypes are observed. Here we report the first two mouse mutations (Rps7(Mtu) and Rps7(Zma)) of ribosomal protein S7 (Rps7), a gene that has been implicated in Diamond-Blackfan anemia. Rps7 disruption results in decreased body size, abnormal skeletal morphology, mid-ventral white spotting, and eye malformations. These phenotypes are reported in other murine RP mutants and, as demonstrated for some other RP mutations, are ameliorated by Trp53 deficiency. Interestingly, Rps7 mutants have additional overt malformations of the developing central nervous system and deficits in working memory, phenotypes that are not reported in murine or human RP gene mutants. Conversely, Rps7 mouse mutants show no anemia or hyperpigmentation, phenotypes associated with mutation of human RPS7 and other murine RPs, respectively. We provide two novel RP mouse models and expand the repertoire of potential phenotypes that should be examined in RP mutants to further explore the concept of RP gene-specific phenotypes.This research was supported in part by the Intramural Research Program of NHGRI, NIH, and the Wellcome Trust and by NHMRC Australia grant 366746. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Lipidomic analysis of variation in response to simvastatin in the Cholesterol and Pharmacogenetics Study

Statins are commonly used for reducing cardiovascular disease risk but therapeutic benefit and reductions in levels of low-density lipoprotein cholesterol (LDL-C) vary among individuals. Other effects, including reductions in C-reactive protein (CRP), also contribute to treatment response. Metabolomics provides powerful tools to map pathways implicated in variation in response to statin treatment. This could lead to mechanistic hypotheses that provide insight into the underlying basis for individual variation in drug response. Using a targeted lipidomics platform, we defined lipid changes in blood samples from the upper and lower tails of the LDL-C response distribution in the Cholesterol and Pharmacogenetics study. Metabolic changes in responders are more comprehensive than those seen in non-responders. Baseline cholesterol ester and phospholipid metabolites correlated with LDL-C response to treatment. CRP response to therapy correlated with baseline plasmalogens, lipids involved in inflammation. There was no overlap of lipids whose changes correlated with LDL-C or CRP responses to simvastatin suggesting that distinct metabolic pathways govern statin effects on these two biomarkers. Metabolic signatures could provide insights about variability in response and mechanisms of action of statins

An increase in dietary n-3 fatty acids decreases a marker of bone resorption in humans

Human, animal, and in vitro research indicates a beneficial effect of appropriate amounts of omega-3 (n-3) polyunsaturated fatty acids (PUFA) on bone health. This is the first controlled feeding study in humans to evaluate the effect of dietary plant-derived n-3 PUFA on bone turnover, assessed by serum concentrations of N-telopeptides (NTx) and bone-specific alkaline phosphatase (BSAP). Subjects (n = 23) consumed each diet for 6 weeks in a randomized, 3-period crossover design: 1) Average American Diet (AAD; [34% total fat, 13% saturated fatty acids (SFA), 13% monounsaturated fatty acids (MUFA), 9% PUFA (7.7% LA, 0.8% ALA)]), 2) Linoleic Acid Diet (LA; [37% total fat, 9% SFA, 12% MUFA, 16% PUFA (12.6% LA, 3.6% ALA)]), and 3) α-Linolenic Acid Diet (ALA; [38% total fat, 8% SFA, 12% MUFA, 17% PUFA (10.5% LA, 6.5% ALA)]). Walnuts and flaxseed oil were the predominant sources of ALA. NTx levels were significantly lower following the ALA diet (13.20 ± 1.21 nM BCE), relative to the AAD (15.59 ± 1.21 nM BCE) (p < 0.05). Mean NTx level following the LA diet was 13.80 ± 1.21 nM BCE. There was no change in levels of BSAP across the three diets. Concentrations of NTx were positively correlated with the pro-inflammatory cytokine TNFα for all three diets. The results indicate that plant sources of dietary n-3 PUFA may have a protective effect on bone metabolism via a decrease in bone resorption in the presence of consistent levels of bone formation

Connexin-43 in the osteogenic BM niche regulates its cellular composition and the bidirectional traffic of hematopoietic stem cells and progenitors

Connexin-43 (Cx43), a gap junction protein involved in control of cell proliferation, differentiation and migration, has been suggested to have a role in hematopoiesis. Cx43 is highly expressed in osteoblasts and osteogenic progenitors (OB/P). To elucidate the biologic function of Cx43 in the hematopoietic microenvironment (HM) and its influence in hematopoietic stem cell (HSC) activity, we studied the hematopoietic function in an in vivo model of constitutive deficiency of Cx43 in OB/P. The deficiency of Cx43 in OB/P cells does not impair the steady state hematopoiesis, but disrupts the directional trafficking of HSC/progenitors (Ps) between the bone marrow (BM) and peripheral blood (PB). OB/P Cx43 is a crucial positive regulator of transstromal migration and homing of both HSCs and progenitors in an irradiated microenvironment. However, OB/P Cx43 deficiency in nonmyeloablated animals does not result in a homing defect but induces increased endosteal lodging and decreased mobilization of HSC/Ps associated with proliferation and expansion of Cxcl12-secreting mesenchymal/osteolineage cells in the BM HM in vivo. Cx43 controls the cellular content of the BM osteogenic microenvironment and is required for homing of HSC/Ps in myeloablated animal

Genome-wide Analysis of Simultaneous GATA1/2, RUNX1, FLI1, and SCL Binding in Megakaryocytes Identifies Hematopoietic Regulators

SummaryHematopoietic differentiation critically depends on combinations of transcriptional regulators controlling the development of individual lineages. Here, we report the genome-wide binding sites for the five key hematopoietic transcription factors—GATA1, GATA2, RUNX1, FLI1, and TAL1/SCL—in primary human megakaryocytes. Statistical analysis of the 17,263 regions bound by at least one factor demonstrated that simultaneous binding by all five factors was the most enriched pattern and often occurred near known hematopoietic regulators. Eight genes not previously appreciated to function in hematopoiesis that were bound by all five factors were shown to be essential for thrombocyte and/or erythroid development in zebrafish. Moreover, one of these genes encoding the PDZK1IP1 protein shared transcriptional enhancer elements with the blood stem cell regulator TAL1/SCL. Multifactor ChIP-Seq analysis in primary human cells coupled with a high-throughput in vivo perturbation screen therefore offers a powerful strategy to identify essential regulators of complex mammalian differentiation processes