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Glucosylceramides (GlcCer), glucose-conjugated sphingolipids, are major components of the endomembrane system and plasma membrane in most eukaryotic cells. Yet the quantitative significance and cellular functions of GlcCer are not well characterized in plants and other multi-organ eukaryotes. To address this, we examined Arabidopsis lines that were lacking or deficient in GlcCer by insertional disruption or by RNA interference (RNAi) suppression of the single gene for GlcCer synthase (GCS, At2g19880), the enzyme that catalyzes GlcCer synthesis. Null mutants for GCS (designated 'gcs-1') were viable as seedlings, albeit strongly reduced in size, and failed to develop beyond the seedling stage. Heterozygous plants harboring the insertion allele exhibited reduced transmission through the male gametophyte. Undifferentiated calli generated from gcs-1 seedlings and lacking GlcCer proliferated in a manner similar to calli from wild-type plants. However, gcs-1 calli, in contrast to wild-type calli, were unable to develop organs on differentiation media. Consistent with a role for GlcCer in organ-specific cell differentiation, calli from gcs-1 mutants formed roots and leaves on media supplemented with the glucosylated sphingosine glucopsychosine, which was readily converted to GlcCer independent of GCS. Underlying these phenotypes, gcs-1 cells had altered Golgi morphology and fewer cisternae per Golgi apparatus relative to wild-type cells, indicative of protein trafficking defects. Despite seedling lethality in the null mutant, GCS RNAi suppression lines with ≤2% of wild-type GlcCer levels were viable and fertile. Collectively, these results indicate that GlcCer are essential for cell-type differentiation and organogenesis, and plant cells produce amounts of GlcCer in excess of that required for normal development.

Background Phospholipids are important structural and signaling molecules in plant membranes. Some fluorescent dyes can stain general lipids of membranes, but labeling and visualization of specific lipid classes have yet to be developed for most components of the membrane. New techniques for visualizing membrane lipids are needed to further delineate their dynamic structural and signaling roles in plant cells. In this study we examined whether propargylcholine, a bioortholog of choline, can be used to label the major membrane lipid, phosphatidylcholine, and other choline phospholipids in plants. We established that propargylcholine is readily taken up by roots, and that its incorporation is not detrimental to plant growth. After plant tissue is harvested and fixed, a click-chemistry reaction covalently links the alkyne group of propargylcholine to a fluorescently-tagged azide, resulting in specific labeling of choline phospholipids. Results Uptake of propargylcholine, followed by click chemistry with fluorescein or Alexa Fluor 594 azide was used to visualize choline phospholipids in cells of root, leaf, stem, silique and seed tissues from Arabidopsis thaliana. Co-localization with various subcellular markers indicated coinciding fluorescent signals in cell membranes, such as the tonoplast and the ER. Among different cell types in the leaf epidermis, guard cells displayed strong labeling. Mass spectrometry-based lipidomic analysis of the various plant tissues revealed that incorporation of propargylcholine was strongest in roots with approximately 50% of total choline phospholipids being labeled, but it was also incorporated in the other tissues including seeds. Phospholipid profiling confirmed that, in each tissue analyzed, incorporation of the bioortholog had little impact on the pool of choline plus choline-like phospholipids or other lipid species. Conclusion We developed and validated a click-chemistry based method for fluorescence imaging of choline phospholipids using a bioortholog of choline, propargylcholine, in various cell-types and tissues from Arabidopsis. This click-chemistry method provides a direct way to metabolically tag and visualize specific lipid molecules in plant cells. This work paves the way for future studies addressing in situ localization of specific lipids in plants. Electronic supplementary material The online version of this article (10.1186/s13007-018-0299-2) contains supplementary material, which is available to authorized users.

The class IV homeodomain leucine zipper transcription factor GLABRA2 (GL2) acts in a complex regulatory circuit that regulates the differentiation of trichomes in Arabidopsis thaliana. We describe a genetic interaction with HOMEODOMAIN GLABROUS11 (HDG11), previously identified as a negative regulator of trichome branching. gl2 hdg11 double mutants display enhanced trichome cell-type differentiation defects. Transgenic expression of HDG11 using the GL2 promoter partially suppresses gl2 trichome phenotypes. Vice versa, expression of GL2 under the control of its native promoter partially complements hdg11 ectopic branching. Since gl2 hdg11 and gl2 myb23 double mutants and the triple mutant display similar trichome differentiation defects, we investigated a connection to the R2R3-MYB transcription factor MYB23. We show that MYB23 transcript levels are significantly reduced in shoots from gl2 mutants and that GL2 can drive the expression of a MYB23-promoter fusion to green fluorescent protein. Yeast one-hybrid, chromatin immunoprecipitation, and in planta reporter gene experiments indicate that an L1-box in the MYB23 promoter acts as a GL2 binding site. Taken together, our findings reveal a functional redundancy between GL2 and HDG11, two homeodomain leucine zipper transcription factors previously thought to mediate opposing functions in trichome morphogenesis. A model is proposed in which GL2 transcript levels are maintained through a positive feedback loop involving GL2 activation of MYB23.

Two fungal-secreted α-fucosidases and their genes were characterized. FoFCO1 was purified from culture filtrates of Fusarium oxysporum strain 0685 grown on l-fucose and its encoding gene identified in the sequenced genome of strain 4287. FoFCO1 was active on p-nitrophenyl-α-fucoside (pNP-Fuc), but did not defucosylate a nonasaccharide (XXFG) fragment of pea xyloglucan. A putative α-fucosidase gene (FgFCO1) from Fusarium graminearum was expressed in Pichia pastoris. FgFCO1 was ∼1,800 times less active on pNP-Fuc than FoFCO1, but was able to defucosylate the XXFG nonasaccharide. Although FgFCO1 and FoFCO1 both belong to Glycosyl Hydrolase family 29, they share

Based on the analysis of its genome sequence, the ectomycorrhizal (ECM) basidiomycetous fungus Laccaria bicolor was shown to be lacking many of the major classes of secreted enzymes that depolymerize plant cell wall polysaccharides. To test whether this is also a feature of other ECM fungi, we searched a survey genome database of Amanita bisporigera with the proteins found in the secretome of Trichoderma reesei (syn. Hypocrea jecorina), a biochemically well-characterized industrial fungus. Additional proteins were also used as queries to compensate for major groups of cell-wall-degrading enzymes lacking in the secretome of T. reesei and to substantiate conclusions drawn from the T. reesei collection. By MS/MS-based “shotgun” proteomics, 80 proteins were identified in culture filtrates of T. reesei strain RUTC30 grown on corn cell walls and in a commercial “cellulase” preparation, Spezyme CP. The two T. reesei enzyme preparations were qualitatively and quantitatively similar, the most striking difference being the lack of at least five major peptidases from the commercial enzyme mixture. Based on our analysis of A. bisporigera, this ECM fungus is deficient in many major classes of cell-wall-degrading enzymes, including both glycosyl hydrolases and carbohydrate esterases. By comparison, the genomes of the saprophytic basidiomycetes Coprinopsis cinerea and Galerina marginata (using a genome survey sequence approximately equivalent in depth to that of A. bisporigera) have, like T. reesei, a much more complete complement of cell-wall-degrading enzymes.

A novel chemolithoautotrophic and hyperthermophilic member of the genus Ignicoccus was isolated from a submarine hydrothermal system at the Kolbeinsey Ridge, to the north of Iceland. The new isolate showed high similarity to the two species described to date, Ignicoccus islandicus and Ignicoccus pacificus, in its physiological properties as well as in its unique cell architecture. However, phylogenetic analysis and investigations on the protein composition of the outer membrane demonstrated that the new isolate was clearly distinct from I. islandicus and I. pacificus. Furthermore, it is the only organism known so far which is able to serve as a host for ‘Nanoarchaeum equitans’, the only cultivated member of the ‘Nanoarchaeota’. Therefore, the new isolate represents a novel species of the genus Ignicoccus, which we name Ignicoccus hospitalis sp. nov. (type strain KIN4/IT=DSM 18386T=JCM 14125T).

Human papillomavirus (HPV) was detected by using two sets of deoxyribonucleotide primers for differentiating between 'low-risk' types (HPV11 and HPV6) and 'high-risk' (hri) types (HPV16, HPV18 and HPV33). A new application of the Chemiprobe method for labeling DNA was used to detect products of the polymerase chain reaction (PCR) from 36 cervical biopsies. This method, first demonstrated by Uchimura et al. (submitted), is based on the sulfonation of a polycytidylic acid tail of 5-20 monomers attached to the 5' end of either one or both of the PCR primers. This procedure can increase the sensitivity of detection of PCR products more than 100-fold with respect to ethidium bromide (EtdBr) staining. Various methods were used to detect hri HPV DNA in the 36 clinical samples. The number of positive results obtained was as follows, two by Southern-blot hybridization; five by PCR amplification followed by electrophoresis and detection of products by EtdBr staining; six by PCR amplification using one or two sulfonated C-tailed primers followed by electroblotting and immunoenzymatic visualization; and five by hybridization of sulfonated genomic viral recombinant with a PCR product immobilized on a membrane. The yield of the PCR product was significantly greater when one of the primers was C-tailed than when both or neither of the primers were C-tailed. PCR employing sulfonated C-tailed oligo primers is very specific and sensitive, and the entire procedure can be employed as a nonradioactive substitute for radioactive dot-blot or Southern-blot hybridization procedures, routinely used for detection of HPV in clinical samples.

International audience; The proper tissue-specific regulation of gene expression is essential for development and homeostasis in metazoans. However, the illegitimate expression of normally tissue-restricted genes-like testis- or placenta-specific genes-is frequently observed in tumors; this promotes transformation, but also allows immunotherapy. Two important questions are: how is the expression of these genes controlled in healthy cells? And how is this altered in cancer? To address these questions, we used an unbiased approach to test the ability of 350 distinct genetic or epigenetic perturbations to induce the illegitimate expression of over 40 tissue-restricted genes in primary human cells. We find that almost all of these genes are remarkably resistant to reactivation by a single alteration in signaling pathways or chromatin regulation. However, a few genes differ and are more readily activated; one is the placenta-expressed gene ADAM12, which promotes invasion. Using cellular systems, an animal model, and bioinformatics, we find that a non-canonical but druggable TGF-β/KAT2A/TAK1 axis controls ADAM12 induction in normal and cancer cells. More broadly, our data show that illegitimate gene expression in cancer is an heterogeneous phenomenon, with a few genes activatable by simple events, and most genes likely requiring a combination of events to become reactivated.

International audience; To better study and manage chestnut trees and species, we identified nuclear single nucleotide polymorphism (SNP) markers using restriction-associated DNA sequencing. Out of 343 loci tested, 68 SNP markers were selected that withhold stringent quality criteria such as quasi-systematic amplification across species and Mendelian segregation in both purebred and hybrid individuals. They provide sufficient power for species, hybrids and backcross characterization as well as for clonal identification, as shown by a comparison with single sequenced repeat (SSR) loci.

DNA barcoding and metabarcoding is increasingly used to effectively and precisely assess and monitor biodiversity in aquatic ecosystems. As these methods rely on data availability and quality of barcode reference libraries, it is important to develop and follow best practices to ensure optimal quality and traceability of the metadata associated with the reference barcodes used for identification. Sufficient metadata, as well as vouchers, corresponding to each reference barcode must be available to ensure reliable barcode library curation and, thereby, provide trustworthy baselines for downstream molecular species identification. This document (1) specifies the data and metadata required to ensure the relevance, the accessibility and traceability of DNA barcodes and (2) specifies the recommendations for DNA harvesting and for the storage of both voucher specimens/samples and barcode data, (undefined)

Background Multidrug-resistant (MDR) Mycobacterium tuberculosis complex strains not detected by commercial molecular drug susceptibility testing (mDST) assays due to the RpoB I491F resistance mutation are threatening the control of MDR tuberculosis (MDR-TB) in Eswatini. Methods We investigate the evolution and spread of MDR strains in Eswatini with a focus on bedaquiline (BDQ) and clofazimine (CFZ) resistance using whole-genome sequencing in two collections ((1) national drug resistance survey, 2009–2010; (2) MDR strains from the Nhlangano region, 2014–2017). Results MDR strains in collection 1 had a high cluster rate (95%, 117/123 MDR strains) with 55% grouped into the two largest clusters (gCL3, n = 28; gCL10, n = 40). All gCL10 isolates, which likely emerged around 1993 (95% highest posterior density 1987–1998), carried the mutation RpoB I491F that is missed by commercial mDST assays. In addition, 21 (53%) gCL10 isolates shared a Rv0678 M146T mutation that correlated with elevated minimum inhibitory concentrations (MICs) to BDQ and CFZ compared to wild type isolates. gCL10 isolates with the Rv0678 M146T mutation were also detected in collection 2. Conclusion The high clustering rate suggests that transmission has been driving the MDR-TB epidemic in Eswatini for three decades. The presence of MDR strains in Eswatini that are not detected by commercial mDST assays and have elevated MICs to BDQ and CFZ potentially jeopardizes the successful implementation of new MDR-TB treatment guidelines. Measures to limit the spread of these outbreak isolates need to be implemented urgently.

Background-\ud \ud The limited available literature suggests that sleep problems are linked to an increased risk of mild cognitive impairment (MCI). However, this association has been little studied to date in low-income settings.\ud \ud Objective-\ud \ud To investigate the association between sleep problems and MCI in a large sample of adults from six low-and middle-income countries (LMICs).\ud \ud Design-\ud \ud Cross-sectional.\ud \ud Setting-\ud \ud Study on Global Ageing and Adult Health (SAGE).\ud \ud Subjects-\ud \ud 32,715 individuals aged ≥50 years with preservation in functional abilities [age range 50–114 years; 51.7% females].\ud \ud Methods-\ud \ud MCI was defined using the National Institute on Aging-Alzheimer's Association criteria. Sleep problems were assessed by the question “Overall in the last 30 days, how much of a problem did you have with sleeping, such as falling asleep, waking up frequently during the night or waking up too early in the morning?” and categorized as “None”, “Mild”, “Moderate”, “Severe/Extreme”. Multivariable logistic regression analysis and meta-analysis were conducted.\ud \ud Results-\ud \ud Compared to no sleep problems, mild, moderate, and severe/extreme sleep problems were associated with significant 1.40, 1.83, and 2.69 times higher odds for MCI with similar associations being observed between age groups and sex. Severe/extreme sleep problems were positively associated with MCI (i.e., OR > 1) in the six countries studied with the overall estimate being OR = 1.80 (95% CI = 1.50–2.16), and a low level of between-country heterogeneity was observed (I2 = 28.2%).\ud \ud Conclusions-\ud \ud Sleep problems were associated with higher odds for MCI. Interventions to improve sleep quality among middle-aged and older adults in LMICs may be an effective strategy in reducing risk of MCI and dementia.

Yellow fever is a vector-borne disease endemic in tropical regions of Africa, where 90% of the global burden occurs, and Latin America. It is notoriously under-reported with uncertainty arising from a complex transmission cycle including a sylvatic reservoir and non-specific symptom set. Resulting estimates of burden, particularly in Africa, are highly uncertain. We examine two established models of yellow fever transmission within a Bayesian model averaging framework in order to assess the relative evidence for each model’s assumptions and to highlight possible data gaps. Our models assume contrasting scenarios of the yellow fever transmission cycle in Africa. The first takes the force of infection in each province to be static across the observation period; this is synonymous with a constant infection pressure from the sylvatic reservoir. The second model assumes the majority of transmission results from the urban cycle; in this case, the force of infection is dynamic and defined through a fixed value of R0 in each province. Both models are coupled to a generalised linear model of yellow fever occurrence which uses environmental covariates to allow us to estimate transmission intensity in areas where data is sparse. We compare these contrasting descriptions of transmission through a Bayesian framework and trans-dimensional Markov chain Monte Carlo sampling in order to assess each model’s evidence given the range of uncertainty in parameter values. The resulting estimates allow us to produce Bayesian model averaged predictions of yellow fever burden across the African endemic region. We find strong support for the static force of infection model which suggests a higher proportion of yellow fever transmission occurs as a result of infection from an external source such as the sylvatic reservoir. However, the model comparison highlights key data gaps in serological surveys across the African endemic region. As such, conclusions concerning the most prevalent transmission routes for yellow fever will be limited by the sparsity of data which is particularly evident in the areas with highest predicted transmission intensity. Our model and estimation approach provides a robust framework for model comparison and predicting yellow fever burden in Africa. However, key data gaps increase uncertainty surrounding estimates of model parameters and evidence. As more mathematical models are developed to address new research questions, it is increasingly important to compare them with established modelling approaches to highlight uncertainty in structures and data., Author summary Yellow fever (YF) is notoriously under reported due to non-specific symptom spectrum and the true burden is highly uncertain as a result of a complex transmission cycle. As such, estimates surrounding YF burden are highly uncertain and the mechanisms behind transmission are often unclear. We assess these mechanisms and the resulting uncertainty by estimating two existing models of YF transmission within a product space framework. This allows us to produce updated estimates of transmission intensity and to compare the relative support for each model given the data. We find strong support for a model assuming a static force of infection, approximating the constant infection pressure from the sylvatic reservoir of YF. We also highlight areas where data is sparse, often the same areas estimated to have especially high transmission intensity. This is the first robust multi-model approach to applied YF modelling and provides a framework that could be extended to other disease models.

International audience; Macrophages are key cells of innate immune response and serve as the first line of defense against bacteria. Transcription profiling of bacteria-infected macrophages could provide important insights on the pathogenicity and host defense mechanisms during infection. We have examined transcription profiles of bovine monocyte-derived macrophages (bMDMs) isolated from the blood of 12 animals and infected in vitro with two strains of Streptococcus agalactiae. Illumina sequencing of RNA from 36 bMDMs cultures exposed in vitro to either one of two sequence types of S. agalactiae (ST103 or ST12) for 6 h and unchallenged controls was performed. Analyses of over 1,656 million high-quality paired-end sequence reads revealed 5,936 and 6,443 differentially expressed genes (p < 0.05) in bMDMs infected with ST103 and ST12, respectively, versus unchallenged controls. Moreover, 588 genes differentially expressed between bMDMs infected with ST103 versus ST12 were identified. Ingenuity pathway analysis of the differentially up-regulated genes in the bMDMs infected with ST103 revealed significant enrichment for granulocyte adhesion and diapedesis, while significant enrichment for the phagosome formation pathway was found among down-regulated genes. Moreover, Ingenuity pathway analysis of the differentially up-regulated genes in the bMDMs infected with ST12 showed significant enrichment for type 1/type 2 T helper cell activation, while the complement activation pathway was overrepresented in the down-regulated genes. Our study identified pathogen-induced regulation of key genes and pathways involved in the immune response of macrophages against infection but also likely involved in bacterial evasion of the host immune system. These results may contribute to better understanding of the mechanisms underlying subclinical infection such as bovine streptococcal mastitis.

A thorough understanding of the heritability, genetic correlations and additive and non-additive variance components of tree growth and wood properties is a requisite for effective tree breeding. This knowledge is essential to maximize genetic gain, that is, the amount of increase in trait performance achieved annually through directional selection. Understanding the genetic attributes of traits targeted by breeding is also important to sustain decade-long genetic progress, that is, the progress made by increasing the average genetic value of the offspring as compared to that of the parental generation. In this study, we report quantitative genetic parameters for fifteen growth, wood chemical and physical traits for the world-famous Eucalyptus urograndis hybrid (E. grandis × E. urophylla). These traits directly impact the optimal use of wood for cellulose pulp, paper, and energy production. A population of 1,000 trees sampled in a progeny trial was phenotyped directly or following the development and use of near-infrared spectroscopy calibration models. Trees were genotyped with 33,398 SNPs and 24,001 DArT-seq genome-wide markers and genomic realized relationship matrices (GRM) were used for parameter estimation with an individual-tree additive-dominant mixed model. Wood chemical properties and wood density showed stronger genetic control than growth, cellulose and fiber traits. Additive effects are the main drivers of genetic variation for all traits, but dominance plays an equally or more important role for growth, singularly in this hybrid. GRM´s with >10,000 markers provided stable relationships estimates and more accurate parameters than pedigrees by capturing the full genetic relationships among individuals and disentangling the non-additive from the additive genetic component. Low correlations between growth and wood properties indicate that simultaneous selection for wood traits can be applied with minor effects on genetic gain for growth. Conversely, moderate to strong correlations between wood density and chemical traits exist, likely due to their interdependency on cell wall structure such that responses to selection will be connected for these traits. Our results illustrate the advantage of using genomewide marker data to inform tree breeding in general and have important consequences for operational breeding of eucalypt urograndis hybrids. Fil: de Lima, Bruno Marco. Universidade de Sao Paulo; Brasil. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; Brasil Fil: Cappa, Eduardo Pablo. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación de Recursos Naturales. Instituto de Recursos Biológicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Silva Junior, Orzenil B.. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; Brasil. Universidade Católica de Brasília; Brasil Fil: Garcia, Carla. No especifíca; Fil: Mansfield, Shawn D.. University of British Columbia; Canadá Fil: Grattapaglia, Dario. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; Brasil. Universidade Católica de Brasília; Brasil

Erratum in : Correction for Adam et al., Evolutionary history of carbon monoxide dehydrogenase/acetyl-CoA synthase, one of the oldest enzymatic complexes. [Proc Natl Acad Sci U S A. 2018]; International audience; Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ ACS) is a five-subunit enzyme complex responsible for the carbonyl branch of the Wood-Ljungdahl (WL) pathway, considered one of the most ancient metabolisms for anaerobic carbon fixation, but its origin and evolutionary history have been unclear. While traditionally associated with methanogens and acetogens, the presence of CODH/ACS homologs has been reported in a large number of un-cultured anaerobic lineages. Here, we have carried out an exhaustive phylogenomic study of CODH/ACS in over 6,400 archaeal and bacterial genomes. The identification of complete and likely functional CODH/ACS complexes in these genomes significantly expands its distribution in microbial lineages. The CODH/ACS complex displays astounding conservation and vertical inheritance over geological times. Rare intradomain and interdomain transfer events might tie into important functional transitions, including the acquisition of CODH/ACS in some archaeal methanogens not known to fix carbon, the tinkering of the complex in a clade of model bacterial acetogens, or emergence of archaeal-bacterial hybrid complexes. Once these transfers were clearly identified, our results allowed us to infer the presence of a CODH/ACS complex with at least four subunits in the last universal common ancestor (LUCA). Different scenarios on the possible role of ancestral CODH/ACS are discussed. Despite common assumptions, all are equally compatible with an autotrophic, mixotrophic, or heterotrophic LUCA. Functional characterization of CODH/ACS from a larger spectrum of bacterial and archaeal lineages and detailed evolutionary analysis of the WL methyl branch will help resolve this issue.

International audience; The conservation and sustainable use of forests in the twenty-first century pose huge challenges for forest management and policy. Society demands that forests provide a wide range of ecosystem services, from timber products, raw materials and renewable energy to sociocultural amenities and habitats for nature conservation. Innovative management and policy approaches need to be developed to meet these often-conflicting demands in a context of environmental change of uncertain magnitude and scale. Genetic diversity is a key component of resilience and adaptability. Overall, forest tree populations are genetically very diverse, conferring them an enormous potential for genetic adaptation via the processes of gene flow and natural selection. Here, we review the main challenges facing our forests in the coming century and focus on how recent progress in genetics can contribute to the development of appropriate practical actions that forest managers and policy makers can adopt to promote forest resilience to climate change. Emerging knowledge will inform and clarify current controversies relating to the choice of appropriate genetic resources for planting, the effect of silvicultural systems and stand tending on adaptive potential and the best ways to harness genetic diversity in breeding and conservation programs. Gaps in our knowledge remain, and we identify where additional information is needed (e.g., the adaptive value of peripheral populations or the genetic determinism of key adaptive traits) and the types of studies that are required to provide this key understanding.

Estimating the rates at which bacterial genomes evolve is critical to understanding major evolutionary and ecological processes such as disease emergence, long-term host-pathogen associations, and short-term transmission patterns. The surge in bacterial genomic data sets provides a new opportunity to estimate these rates and reveal the factors that shape bacterial evolutionary dynamics. For many organisms estimates of evolutionary rate display an inverse association with the time-scale over which the data are sampled. However, this relationship remains unexplored in bacteria due to the difficulty in estimating genome-wide evolutionary rates, which are impacted by the extent of temporal structure in the data and the prevalence of recombination. We collected 36 whole genome sequence data sets from 16 species of bacterial pathogens to systematically estimate and compare their evolutionary rates and assess the extent of temporal structure in the absence of recombination. The majority (28/36) of data sets possessed sufficient clock-like structure to robustly estimate evolutionary rates. However, in some species reliable estimates were not possible even with “ancient DNA” data sampled over many centuries, suggesting that they evolve very slowly or that they display extensive rate variation among lineages. The robustly estimated evolutionary rates spanned several orders of magnitude, from 10−6 to 10−8 nucleotide substitutions site-1 year-1. This variation was largely attributable to sampling time, which was strongly negatively associated with estimated evolutionary rates, with this relationship best described by an exponential decay curve. To avoid potential estimation biases such time-dependency should be considered when inferring evolutionary time-scales in bacteria.

BACKGROUND: The biological mechanisms by which cleaning products and disinfectants-an emerging risk factor-affect respiratory health remain incompletely evaluated. Studying genes by environment interactions (G × E) may help identify new genes related to adult-onset asthma. OBJECTIVES: We identified interactions between genetic polymorphisms of a large set of genes involved in the response to oxidative stress and occupational exposures to low molecular weight (LMW) agents or irritants on adult-onset asthma. METHODS: Our data came from three large European cohorts: Epidemiological Family-based Study of the Genetics and Environment of Asthma (EGEA), Swiss Cohort Study on Air Pollution and Lung and Heart Disease in Adults (SAPALDIA), and European Community Respiratory Health Survey in Adults (ECRHS). A candidate pathway-based strategy identified 163 genes involved in the response to oxidative stress and potentially related to exposures to LMW agents/irritants. Occupational exposures were evaluated using an asthma job-exposure matrix and job-specific questionnaires for cleaners and healthcare workers. Logistic regression models were used to detect G × E interactions, adjusted for age, sex, and population ancestry, in 2,599 adults (mean age, 47 years; 60% women, 36% exposed, 18% asthmatics). p-Values were corrected for multiple comparisons. RESULTS: Ever exposure to LMW agents/irritants was associated with current adult-onset asthma [OR = 1.28 (95% CI: 1.04, 1.58)]. Eight single nucleotide polymorphism (SNP) by exposure interactions at five loci were found at p < 0.005: PLA2G4A (rs932476, chromosome 1), near PLA2R1 (rs2667026, chromosome 2), near RELA (rs931127, rs7949980, chromosome 11), PRKD1 (rs1958980, rs11847351, rs1958987, chromosome 14), and PRKCA (rs6504453, chromosome 17). Results were consistent across the three studies and after accounting for smoking. CONCLUSIONS: Using a pathway-based selection process, we identified novel genes potentially involved in adult asthma by interaction with occupational exposure. These genes play a role in the NF-κB pathway, which is involved in inflammation. Citation: Rava M, Ahmed I, Kogevinas M, Le Moual N, Bouzigon E, Curjuric I, Dizier MH, Dumas O, Gonzalez JR, Imboden M, Mehta AJ, Tubert-Bitter P, Zock JP, Jarvis D, Probst-Hensch NM, Demenais F, Nadif R. 2017. Genes interacting with occupational exposures to low molecular weight agents and irritants on adult-onset asthma in three European studies. Environ Health Perspect 125:207-214; http://dx.doi.org/10.1289/EHP376. We thank all study members and staff involved in data collections in each cohort: EGEA: We thank the Epidemiological Study on Genetics and Environment of Asthma (EGEA) cooperative group members as follows. Coordination: V. Siroux [epidemiology, principal investigator (PI) since 2013]; F. Demenais (genetics); I. Pin (clinical aspects); R. Nadif (biology); F. Kauffmann (PI 1992–2012). Respiratory epidemiology: Inserm U 700, Paris: M. Korobaeff (EGEA1), F. Neukirch (EGEA1); Inserm U 707, Paris: I. Annesi-Maesano (EGEA1–2); Inserm U1168 (ex-CESP/U 1018), Villejuif: F. Kauffmann, N. Le Moual, R. Nadif, M.P. Oryszczyn (EGEA1–2), R. Varraso; Inserm U 823, Grenoble: V. Siroux. Genetics: Inserm U 393, Paris: J. Feingold; Inserm U 946, Paris: E. Bouzigon, F. Demenais, M.H. Dizier; CNG, Evry: I. Gut (now CNAG, Barcelona, Spain), M. Lathrop (now McGill University, Montreal, Canada). Clinical centers: Grenoble: I. Pin, C. Pison; Lyon: D. Ecochard (EGEA1), F. Gormand, Y. Pacheco; Marseille: D. Charpin (EGEA1), D. Vervloet (EGEA1–2); Montpellier: J. Bousquet; Paris Cochin: A. Lockhart (EGEA1), R. Matran (now in Lille); Paris Necker: E. Paty (EGEA1–2), P. Scheinmann (EGEA1–2); Paris Trousseau: A. Grimfeld (EGEA1–2), J. Just. Data and quality management: Inserm ex-U155 (EGEA1): J. Hochez; Inserm U1168 (ex-CESP/U 1018), Villejuif: N. Le Moual; Inserm ex-U780: C. Ravault (EGEA1–2); Inserm ex-U794: N. Chateigner (EGEA1–2); Grenoble: J. Quentin-Ferran (EGEA1–2). SAPALDIA: We thank the team of the Swiss study on Air Pollution and Lung and Heart Diseases in Adults (SAPALDIA). Study directorate: N.M. Probst-Hensch (PI; e/g); T. Rochat (p), C. Schindler (s), N. Künzli (e/exp), J.M. Gaspoz (c). Scientific team: J.C. Barthélémy (c), W. Berger (g), R. Bettschart (p), A. Bircher (a), C. Brombach (n), P.O. Bridevaux (p), L. Burdet (p), D. Felber Dietrich (e), M. Frey (p), U. Frey (pd), M.W. Gerbase (p), D. Gold (e), E. de Groot (c), W. Karrer (p), F. Kronenberg (g), B. Martin (pa), A. Mehta (e), D. Miedinger (o), M. Pons (p), F. Roche (c), T. Rothe (p), P. Schmid- Grendelmeyer (a), D. Stolz (p), A. Schmidt-Trucksäss (pa), J. Schwartz (e), A. Turk (p), A. von Eckardstein (cc), E. Zemp Stutz (e). Scientific team at coordinating centers: M. Adam (e), I. Aguilera (exp), S. Brunner (s), D. Carballo (c), S. Caviezel (pa), I. Curjuric (e), A. Di Pascale (s), J. Dratva (e), R. Ducret (s), E. Dupuis Lozeron (s), M. Eeftens (exp), I. Eze (e), E. Fischer (g), M. Foraster (e), M. Germond (s), L. Grize (s), S. Hansen (e), A. Hensel (s), M. Imboden (g), A. Ineichen (exp), A. Jeong (g), D. Keidel (s), A. Kumar (g), N. Maire (s), A. Mehta (e), R. Meier (exp), E. Schaffner (s), T. Schikowski (e), M. Tsai (exp). Abbreviations: (a) allergology, (c) cardiology, (cc) clinical chemistry, (e) epide - miology, (exp) exposure, (g) genetic and molecular biology, (m) meteorology, (n) nutrition, (o) occupational health, (p) pneumology, (pa) physical activity, (pd) pediatrics, (s) statistics. The study could not have been done without the help of the study partici - pants, technical and administrative support and the medical teams and field workers at the local study sites. Local fieldworkers: Aarau: S. Brun, G. Giger, M. Sperisen, M. Stahel; Basel: C. Bürli, C. Dahler, N. Oertli, I. Harreh, F. Karrer, G. Novicic, N. Wyttenbacher; Davos: A. Saner, P. Senn, R. Winzeler; Geneva: F. Bonfils, B. Blicharz, C. Landolt, J. Rochat; Lugano: S. Boccia, E. Gehrig, M.T. Mandia, G. Solari, B. Viscardi; Montana: A.P. Bieri, C. Darioly, M. Maire; Payerne: F. Ding, P. Danieli, A. Vonnez; Wald: D. Bodmer, E. Hochs, R. Kunz, C. Meier, J. Rakic, U. Schafroth, A. Walder. Administrative staff: N. Bauer Ott, C. Gabriel, R. Gutknecht. ECRHS: The ECRHS data incorporated in this analysis would not have been available without the collaboration of the following individuals and their research teams. ECRHS Co-ordinating Centre. P. Burney, D. Jarvis, S. Chinn, J. Knox (ECRHS II), C. Luczynska†, J. Potts. Steering Committee for ECRHS II. P. Burney, D. Jarvis, S. Chinn, U. Ackermann-Liebrich, J.M. Anto, I. Cerveri, R. deMarco†, T. Gislason, J. Heinrich, C. Janson, N. Kunzli, B. Leynaert, F. Neukirch, J. Schouten, J. Sunyer; C. Svanes, P. Vermeire†, M. Wjst. Principal Investigators and Senior Scientific Teams for ECRHS II centers within this analysis: Estonia: Tartu (R. Jogi, A. Soon); France: Paris (F. Neukirch, B. Leynaert, R. Liard, M. Zureik), Grenoble (I. Pin, J. Ferran-Quentin); Germany: Erfurt (J. Heinrich, M. Wjst, C. Frye, I. Meyer), Hamburg (K. Richter, D. Nowak); Norway: Bergen (A. Gulsvik, E. Omenaas, C. Svanes, B. Laerum); Spain: Barcelona (J.M. Anto, J. Sunyer, M. Kogevinas, J.P. Zock, X. Basagana, A. Jaen, F. Burgos), Huelva (J. Maldonado, A. Pereira, J.L. Sanchez), Albacete (J. Martinez-Moratalla Rovira, E. Almar), Galdakao (N. Muniozguren, I. Urritia), Oviedo (F. Payo); Sweden: Uppsala (C. Janson, G. Boman, D. Norback, M. Gunnbjornsdottir), Umeå (E. Norrman, M. Soderberg, K. Franklin, B. Lundback, B. Forsberg, L. Nystrom); Switzerland: Basel (N. Kunzli, B. Dibbert, M. Hazenkamp, M. Brutsche, U. Ackermann-Liebrich); United Kingdom: Norwich (D. Jarvis, B. Harrison), Ipswich (D. Jarvis, R. Hall, D. Seaton). Sí

International audience; A scarcity of favourable habitats and introgression from exotic cultivars are two major threats to black poplars (Populus nigra L.) in Europe. Natural vegetative propagation contributes to maintenance of the species in areas where seedling recruitment is limited. Exhaustive sampling of all mature trees in a natural P. nigra stand (413 individuals at recorded positions), genotyping at 11 SSR loci, and a standardized analysis framework resulted in a precise description of clonality in terms of (a) frequency, (b) spatial growth form, and (c) impacts on the overall spatial genetic structure (SGS). The high proportion of replicated genotypes detected resulted in a genotypic richness (R) of 0.47. Up to 18 ramets were found per multilocus lineage (MLL), but 95% of MLLs contained fewer than five ramets (Pareto index β = 1.07). No significant difference in vegetative propagation potential was found between genders. Uneven spatial distribution of ramets, with clustering of clonal ramets (aggregation index A c = 0.62) and near-zero intermingling between MLLs (clonal dominance index D c = 0.99), resulted in a ‘phalanx’ clonal growth form, explaining most of the SGS observed over short distances (0–20 m, Sp = 0.0324). Although they did not exhibit the typical columnar shape of the Lombardy poplar (P. nigra var. italica), five trees were found to be probable F1 hybrids of this old and widely distributed cultivar.

International audience; Many different environmental factors (nutrients, pollutants, chemicals, physical activity, lifestyle, physical and mental stress) can modulate epigenetic markers in the developing and adult organism. Epigenetics, in turn, can cause and is associated with several neurodegenerative and aging-dependent human diseases. Alzheimer's disease certainly represents one of the most relevant neurodegenerative disorders due to its incidence and its huge socio-economic impact. Therefore, it is easy to understand why recent literature focuses on the epigenetic modifications associated with Alzheimer's disease and other neurodegenerative disorders. One of the most intriguing and, at the same time, worrying evidence is that even "mild" environmental factors (such as behavioral or physical stress) as well as the under-threshold exposure to pollutants and chemicals, can be effective. Finally, even mild nutrients disequilibria can result in long-lasting and functional alterations of many epigenetic markers, although they don't have an immediate acute effect. Therefore, we will probably have to re-define the current risk threshold for many factors, molecules and stresses. Among the many different environmental factors affecting the epigenome, nutrition represents one of the most investigated fields; the reasons are probably that each person interacts with nutrients and that, in turn, nutrients can modulate at molecular level the epigenetic biochemical pathways. The role that nutrition can exert in modulating epigenetic modifications in Alzheimer's disease will be discussed with particular emphasis on the role of B vitamins and DNA methylation.

Background Leptospirosis is a worldwide zoonosis that is endemic in tropical areas, such as Reunion Island. The species Leptospira interrogans is the primary agent in human infections, but other pathogenic species, such as L. kirschner and L. borgpetersenii, are also associated with human leptospirosis. Methods and Findings In this study, a melting curve analysis of the products that were amplified with the primer pairs lfb1 F/R and G1/G2 facilitated an accurate species classification of Leptospira reference strains. Next, we combined an unsupervised high resolution melting (HRM) method with a new statistical approach using primers to amplify a two variable-number tandem-repeat (VNTR) for typing at the subspecies level. The HRM analysis, which was performed with ScreenClust Software, enabled the identification of genotypes at the serovar level with high resolution power (Hunter-Gaston index 0.984). This method was also applied to Leptospira DNA from blood samples that were obtained from Reunion Island after 1998. We were able to identify a unique genotype that is identical to that of the L. interrogans serovars Copenhageni and Icterohaemorrhagiae, suggesting that this genotype is the major cause of leptospirosis on Reunion Island. Conclusions Our simple, rapid, and robust genotyping method enables the identification of Leptospira strains at the species and subspecies levels and supports the direct genotyping of Leptospira in biological samples without requiring cultures.

International audience; The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values 1. 'How to use PSA' remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer 2 or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application 3,4. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer 5,6 and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer 7-10. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).

Background Drug susceptible clinical isolates of Candida albicans frequently become highly tolerant to drugs during chemotherapy, with dreadful consequences to patient health. We used RNA sequencing (RNA-seq) to analyze the transcriptomes of a CDR (Candida Drug Resistance) strain and its isogenic drug sensitive counterpart. Results RNA-seq unveiled differential expression of 228 genes including a) genes previously identified as involved in CDR, b) genes not previously associated to the CDR phenotype, and c) novel transcripts whose function as a gene is uncharacterized. In particular, we show for the first time that CDR acquisition is correlated with an overexpression of the transcription factor encoding gene CZF1. CZF1 null mutants were susceptible to many drugs, independently of known multidrug resistance mechanisms. We show that CZF1 acts as a repressor of β-glucan synthesis, thus negatively regulating cell wall integrity. Finally, our RNA-seq data allowed us to identify a new transcribed region, upstream of the TAC1 gene, which encodes the major CDR transcriptional regulator. Conclusion Our results open new perspectives of the role of Czf1 and of our understanding of the transcriptional and post-transcriptional mechanisms that lead to the acquisition of drug resistance in C. albicans, with potential for future improvements of therapeutic strategies.

By transactivating expression of miRNAs that repress expression of the ZEB1 and ZEB2 transcription factors, p53 inhibits the epithelial–mesenchymal transition., p53 suppresses tumor progression and metastasis. Epithelial–mesenchymal transition (EMT) is a key process in tumor progression and metastasis. The transcription factors ZEB1 and ZEB2 promote EMT. Here, we show that p53 suppresses EMT by repressing expression of ZEB1 and ZEB2. By profiling 92 primary hepatocellular carcinomas (HCCs) and 9 HCC cell lines, we found that p53 up-regulates microRNAs (miRNAs), including miR-200 and miR-192 family members. The miR-200 family members transactivated by p53 then repress ZEB1/2 expression. p53-regulated miR-192 family members also repress ZEB2 expression. Inhibition or overexpression of the miRNAs affects p53-regulated EMT by altering ZEB1 and ZEB2 expression. Our findings indicate that p53 can regulate EMT, and that p53-regulated miRNAs are critical mediators of p53-regulated EMT.

Background Reverse engineering in systems biology entails inference of gene regulatory networks from observational data. This data typically include gene expression measurements of wild type and mutant cells in response to a given stimulus. It has been shown that when more than one type of experiment is used in the network inference process the accuracy is higher. Therefore the development of generally applicable and effective methodologies that embed multiple sources of information in a single computational framework is a worthwhile objective. Results This paper presents a new method for network inference, which uses multi-objective optimisation (MOO) to integrate multiple inference methods and experiments. We illustrate the potential of the methodology by combining ODE and correlation-based network inference procedures as well as time course and gene inactivation experiments. Here we show that our methodology is effective for a wide spectrum of data sets and method integration strategies. Conclusions The approach we present in this paper is flexible and can be used in any scenario that benefits from integration of multiple sources of information and modelling procedures in the inference process. Moreover, the application of this method to two case studies representative of bacteria and vertebrate systems has shown potential in identifying key regulators of important biological processes.

International audience; We investigated space-use patterns of the West African savanna buffalo (Syncerus caffer brachyceros), a little-studied subspecies occurring at the northern limit of the African buffalo's geographical range. This buffalo generally ranges in small herds (about 45 individuals) and has a low body mass (approximately 400 kg) relative to the Cape buffalo (S. c. caffer). We monitored the movements of 7 breeding herds in W Regional Park (Burkina Faso, Benin, Niger) using global positioning system collars and activity data loggers. Habitat selection was analyzed at both large (interseasonal) and small (intraseasonal) scales in a context where resources are segregated spatially at some times of year. Both biotic (primary production and vegetation types) and abiotic (timing of rainfall and surface water) covariates, and the extent to which neighboring herds shared space, were considered. In the dry season buffalo herds ranged close (within 5.3 +/- 2.0 km, mean +/- SD) to segments of permanent rivers. At the onset of the monsoon all herds but 1 (which had year-round access to suitable resources) performed a large (35 +/- 10 km) directional movement in response to a large-scale gradient of primary production. Spatiotemporal dynamics of forage and water resources thus jointly stimulated interseasonal directional movements and shaped large (335 +/- 167 km(2)) annual home ranges. Furthermore, the establishment of home ranges in the wet season appears to be conditioned by a threshold (about 10%) in the availability of perennial grasses. Habitat-selection analysis at intraseasonal scale also underlines the key role played by perennial grasses for buffaloes. The spatial arrangements of home ranges of neighboring herds also suggest that interherd behavioral avoidance is a high-level constraint on foraging processes. The ability of the African buffalo to cope with contrasting environmental conditions throughout most sub-Saharan ecosystems highlights the high behavioral plasticity of this species.

The use of morphological characters for the classification of cyanobacteria has often led to ambiguous strain assignment. In the past two decades, the availability of sequences, such as those of the 16S rRNA,nif,cpcandrpoC1genes, and the use of metagenomics, has steadily increased and has made the reconstruction of evolutionary relationships of some cyanobacterial groups possible in addition to improving strain assignment. Conserved indels (insertions/deletions) are present in all cyanobacterial RpoB (βsubunit of RNA polymerase) sequences presently available in public databases. These indels are located in the Rpb2_6 domain of RpoB, which is involved in DNA binding and DNA-directed RNA polymerase activity. They are variable in length (6–44 aa) and sequence, and form part of what appears to be a longer signature sequence (43–81 aa). Indeed, a number of these sequences turn out to be distinctive among several strains of a given genus and even among strains of a given species. These signature sequences can thus be used to identify cyanobacteria at a subgenus level and can be useful molecular markers to establish the taxonomic positions of cyanobacterial isolates in laboratory cultures, and/or to assess cyanobacterial biodiversity in space and time in natural ecosystems.

International audience; Laboratory mice are well known to be highly susceptible to virulent strains of Yersinia pestis in experimental models of bubonic plague. We have found that Mus spretus-derived SEG/Pas (SEG) mice are exceptionally resistant to virulent CO92 and 6/69 wild type strains. Upon subcutaneous injection of 10(2) colony-forming units (CFU), 90% of females and 68% of males survived, compared with only an 8% survival rate for both male and female C57BL/6 mice. Furthermore, half of the SEG mice survived a challenge of up to 10(7) CFU. The time required for mortality was similar between B6 and SEG, suggesting that survival is dependent on early rather than late processes. The analysis of 322 backcross mice identified three significant quantitative trait loci (QTLs) on chromosomes 3, 4 and 6, with dominant SEG protective alleles. Each QTL increased the survival rate by approximately 20%. The three QTLs function additively, thereby accounting for 67% of the difference between the parental phenotypes. Mice heterozygous for the three QTLs were just as resistant as SEG mice to Y. pestis challenge. The SEG strain therefore offers an invaluable opportunity to unravel mechanisms and underlying genetic factors of resistance against Y. pestis infection.

International audience; Chrysomyxa rusts are fungal pathogens widely present in the boreal forest. Taxonomic delimitation and precise species identification are difficult within this genus because several species display similar morphological features. We applied a DNA barcode system based on the ribosomal internal transcribed spacer region (ITS), large subunit (28S) ribosomal RNA gene, mitochondrial cytochrome oxidase 1 (CO1) and mitochondrial NADH dehydrogenase subunit 6 (NAD6) in 86 strains from 16 different Chrysomyxa species, including members of the Chrysomyxa ledi species complex. The nuclear ITS and 28S loci revealed higher resolving power than the mitochondrial genes. Amplification of the full CO1 barcode region failed due to the presence of introns limiting the dataset obtained with this barcode. In most cases the ITS barcodes were in agreement with taxonomic species based on phenotypic characters. Nevertheless we observed genetically distinct (different DNA barcodes) lineages within Chrysomyxa pyrolae and Chrysomyxa rhododendri, providing some evidence for allopatric speciation within these morphologically defined species. This finding, together with the observed pattern of host specificities of the studied rust fungi, suggest that species diversification within the C. ledi species complex might be governed by a set of factors such as specialisation to certain Ericaceae species as telial hosts and to a lesser extent specialization to different spruce species as aecial hosts. Moreover allopatric speciation by geographic disruption of species also seems to take place. When our data were integrated into a broader phylogenetic framework the Chrysomyxa genus unexpectedly was not resolved as a monophyletic group. Indeed the spruce cone rusts C. pyrolae and C. monesis coalesced with the pine needle rusts belonging to the genus Coleosporium, whereas the microcyclic species Chrysomyxa weirii was embedded within a clade comprising the genus Melampsora.

Background Although bivalves are among the most-studied marine organisms because of their ecological role and economic importance, very little information is available on the genome sequences of oyster species. This report documents three large-scale cDNA sequencing projects for the Pacific oyster Crassostrea gigas initiated to provide a large number of expressed sequence tags that were subsequently compiled in a publicly accessible database. This resource allowed for the identification of a large number of transcripts and provides valuable information for ongoing investigations of tissue-specific and stimulus-dependant gene expression patterns. These data are crucial for constructing comprehensive DNA microarrays, identifying single nucleotide polymorphisms and microsatellites in coding regions, and for identifying genes when the entire genome sequence of C. gigas becomes available. Description In the present paper, we report the production of 40,845 high-quality ESTs that identify 29,745 unique transcribed sequences consisting of 7,940 contigs and 21,805 singletons. All of these new sequences, together with existing public sequence data, have been compiled into a publicly-available Website http://public-contigbrowser.sigenae.org:9090/Crassostrea_gigas/index.html. Approximately 43% of the unique ESTs had significant matches against the SwissProt database and 27% were annotated using Gene Ontology terms. In addition, we identified a total of 208 in silico microsatellites from the ESTs, with 173 having sufficient flanking sequence for primer design. We also identified a total of 7,530 putative in silico, single-nucleotide polymorphisms using existing and newly-generated EST resources for the Pacific oyster. Conclusion A publicly-available database has been populated with 29,745 unique sequences for the Pacific oyster Crassostrea gigas. The database provides many tools to search cleaned and assembled ESTs. The user may input and submit several filters, such as protein or nucleotide hits, to select and download relevant elements. This database constitutes one of the most developed genomic resources accessible among Lophotrochozoans, an orphan clade of bilateral animals. These data will accelerate the development of both genomics and genetics in a commercially-important species with the highest annual, commercial production of any aquatic organism.

International audience; Background : Many large secreted gel-forming mucins contain multiple copies of a "naked" cysteine-enriched domain (CYS domain) that interrupt or are adjacent to mucin domains (Ser/Thr/Pro regions). There is less similarity of homologous CYS sequences between species than there is across CYS repeats within species. These CYS domains are likely implicated in reversible mucin-mucin interactions that play a central role in mucus properties. Results : The use of multiple genome sequences in a comparative analysis of CYS sequences identified new mucins containing the CYS domain in various species. Furthermore, analysis showed that this domain is at least 650 million-year old and that CYS sequences evolved under strong selective pressure in concerted fashion in all species examined by two mechanisms that never coexist within a given gene: either multiplication of exons encoding CYS modules or successive intra-exonic duplications. Conclusion : The CYS sequences appear to have undergone concerted evolution with a high selective pressure to conserve cysteine and other amino acid residues. This domain is the best-conserved domain in secreted mucins pointing toward an important common function in many different organisms.

Shigella flexneri 2a is known to express the H-NS nucleoid-structuring protein and the paralogous protein StpA. Using bioinformatic analysis we have now discovered a third member of the H-NS protein family, Sfh (S higella f lexneri H-NS-like protein), in strain 2457T. This protein is encoded by the sfh gene, which is located on a high-molecular-mass plasmid that is closely related to the self-transmissible plasmid R27. When expressed in Escherichia coli, the Sfh protein can complement an hns null mutation, restoring wild-type Bgl, porin protein, and mucoidy phenotypes, and wild-type expression of the fliC and proU genes. While a knockout mutation in the sfh gene alone had no effect on the expression of virulence genes in S. flexneri, an additive effect on virulence gene derepression was seen when the sfh lesion was combined with a mutation in hns. Over-expression of the sfh gene repressed expression of the VirB virulence regulatory protein and transcription of a VirB-dependent structural gene promoter. The purified Sfh protein bound specifically to DNA sequences containing the promoters of the virF and virB virulence regulatory genes. These findings show that Sfh has the ability to influence genetic events beyond the genetic element that encodes it, including the expression of the S. flexneri virulence genes. They raise the possibility of a triangular relationship among three closely related proteins with broad consequences for genetic events in the bacterium that harbours them.

Phlebotomine sandflies are vectors of phleboviruses that cause sandfly fever or meningitis with significant implications for public health. Although several strains of these viruses had been isolated in Iran in the late 1970's, there was no recent data about the present situation at the outset of this study. Entomological investigations performed in 2009 and 2011 in Iran collected 4,770 sandflies from 10 different regions. Based on morphological identification, they were sorted into 315 pools according to species, sex, trapping station and date of capture. A phlebovirus, provisionally named Dashli virus (DASHV), was isolated from one pool of Sergentomyia spp, and subsequently DASHV RNA was detected in a second pool of Phlebotomus papatasi. Genetic and phylogenetic analyses based on complete coding genomic sequences indicated that (i) DASHV is most closely related to the Iranian isolates of Sandfly fever Sicilian virus [SFSV], (ii) there is a common ancestor to DASHV, Sandfly fever Sicilian- (SFS) and SFS-like viruses isolated in Italy, India, Turkey, and Cyprus (lineage I), (iii) DASHV is more distantly related with Corfou and Toros viruses (lineage II) although common ancestry is supported with 100% bootstrap, (iii) lineage I can be subdivided into sublineage Ia including all SFSV, SFCV and SFTV except those isolated in Iran which forms sublineage Ib (DASHV). Accordingly, we suggest to approve Sandfly fever Sicilian virus species consisting of the all aforementioned viruses. Owing that most of these viruses have been identified in human patients with febrile illness, DASHV should be considered as a potential human pathogen in Iran., Author summary Phlebotomine sandflies are vectors of phleboviruses that cause sandfly fever or meningitis with significant implications for public health. Although several strains of these viruses had been isolated in Iran in the late 1970's, there was no recent data about the present situation at the outset of this study. Entomological investigations performed in 2009 and 2011 in Iran collected 4,770 sandflies from 10 different regions. A phlebovirus, provisionally named Dashli virus (DASHV), was isolated / detected in two pools. DASHV strain was isolated in cell culture and complete genome sequence was determined. Sequence analysis indicated that (i) DASHV is most closely related to the Iranian isolates of Sandfly fever Sicilian virus [SFSV], a virus that is known to cause self-resolutive incapacitating febrile illness in humans, (ii) there is a common ancestor to DASHV and all other variants of SFSV isolated in Italy, India, Turkey, and Cyprus (lineage I), (iii) DASHV is more distantly related with Corfou and Toros viruses (lineage II) although common ancestry is supported with 100% bootstrap, (iii) lineage I can be subdivided into sublineage Ia including all SFSV strains, whereas Iranian viruses are most closely related and should be individualized as DASHV (sublineage Ib). Although discovered first in the 1940's, SFSV is still listed as "tentative species" by the International Committee for Taxonomy of Viruses. Based on the results described in this study, we propose to approve Sandfly fever Sicilian virus species. Owing that most of these viruses have been identified in human patients with febrile illness, DASHV should be considered as a potential human pathogen in Iran.