Cryptococcus neoformans is a pathogenic yeasts that infect mostly immunocompromised people. The strain H99 belongs to the C. neoformans variety grubii group. This is the reference strain that have already been sequenced. A project initiated by the Broad Institute aims at analysing the genome of about 300 clinical isolates including 28 of our French isolates. Part of our French isolates have been screened in terms of interaction with macrophages (774). Huge variations in terms of phagocytosis and intracellular proliferation of the yeasts were observed between clinical isolates (Alanio et al. mBio 2011). Thirteen of these clinical isolates together with H99 were studied in terms of transcriptome (using RNASeq). This work was performed by MA Dillies at the PF2. We correlated the expression of about 150 genes with the phenotype of the 13 clinical isolates and H99.

In the context of the swiss consortion InfectX (www.infectx.ch), we performed siRNA and drug screens to investigate the invasion of host cells by the bacterial pathogen Listeria monocytogenes. As a primary readout for infection we detected by fluorescence microscopy the presence of the bacterially-secreted protein InlC, which accumulates in the cytoplasm of infected cells (Kühbacher et al. 2013). A first manuscript concerning the analysis of a kinome-wide siRNA library and a drug library has been published recently (Rämö et al. 2014) and the analysis of a genome-wide siRNA screen has been submitted for publication (Kühbacher et al. In Revision), in which major signaling pathways subverted by L. monocytogenes are identified. However, besides the presence of InlC, many other features have been measured in infected cells including InlC intensity, bacterial distribution in host cells, actin morphology and distribution, cellular replication state, cellular context of infected cells, etc. We intend to analyze the full set of measured parameters in order to identify global signaling cascades subverted by L. monocytogenes for infection or used by the cell to control bacterial proliferation.

Cell surface protein signatures have been successful to discriminate hematopoietic progenitor populations allowing major advances in understanding blood cell production, to define pathways in hematologic malignancies and to foster new therapeutic approaches. Limited knowledge on the phenotype of cells that participate in heart formation impairs our understanding of progenitors of the cardiac cell lineages and their eventual persistence in the adult organ. As a consequence, therapies to restore heart function after injury have been unsuccessful. A number of membrane proteins have been identified on cardiomyocytes; on cardiac fibroblasts; and on endothelial cells, however a multi-parametric analysis of the phenotype of the different cardiac cell compartments along development is still missing. We combined multi-parametric flow cytometry with transcriptional characterization, based on well-known gene expression patterns, to describe major cardiac cell-subsets. The expression of CD24, CD54, Sca-1 and CD90 allowed defining cardiac populations in the non-hematopoietic and non-endothelial cell fraction by flow cytometry. Transcriptional profiling of the sorted populations enabled the identification of cardiomyocytes, in the CD24+ population, while differential expression of CD54, Sca-1 and CD90 defined four cardiac stromal compartments. The identified subsets exhibited specific distributions in three analyzed regions (atria, auriculo-ventricular junction and ventricles). We have thus identified a panel of surface markers, some of which novel in the cardiac context, that allowed assigning surface signatures to different cellular fractions by their unique transcriptional profiles. This work is the foundation for comprehensive studies on the role of different cell fractions by their unique transcriptional profiles.

Stem cells are defined by their is their capacity for self-renewal and differentiation. Some adult tissues maintain a reservoir of stem cells, that generally reside within specialized microenvironments, known as stem cell niches, that regulate their behaviour. Skeletal muscle stem (satellite) cells are quiescent in homeostatic conditions in adults, and they are activated after muscle injury, when they re-enter the cell cycle, proliferate and differentiate into myoblasts, which will then fuse to form new muscle fibers. Satellite cells express the paired/homeodomain gene Pax7, which plays a critical role in satellite cell maintenance postnatally. Numerous experiments have shown that the skeletal muscle stem cell population is heterogeneous, therefore like many other stem cell systems, characterising the stem cell states is a major objective. In our laboratory, a reversible dormant cell state was identified, correspondent to a Pax7Hi quiescent subpopulation (top 10% of the Pax7-nGFP+ cells isolated from the transgenic mouse model Tg:Pax7-nGFP) with a lower metabolic activity and longer lag for the first cell division compared to Pax7Lo cells [1]. Muscle stem cells that survive for extended periods post-mortem are also dormant, suggesting that this property, in addition to anoxia [2] contributes to their viability. Therefore, different physiological states are associated with distinct cell states of muscle stem cells. Metabolism could play a critical role in dictating whether a cell remains quiescent, proliferates or differentiates. Stem cell metabolic plasticity in homeostasis and differentiation, as well as during cell reprogramming, is well described in different cell systems. However, unanswered questions remain regarding the metabolic regulation of satellite cell biology and skeletal muscle regeneration. In this project, we will investigate the behaviour of muscle stem cells in distinct physiological states, especially post-mortem and aging.

The PIBnet initiative is a joint effort by the above laboratories to modernize their activities, including collection management and microbial characterization approaches and technologies. Within this large concerted effort, a priority is to promote WGS as the major characterization approach of microbial agents for surveillance and outbreak investigation. Our ambition is to have shifted to WGS as a routine strain characterization method for epidemiological surveillance and outbreak investigation in the Institut Pasteur NRCs at the end of 2016. The target volume is 10 000 genomes a year. On the bioinformatics level, this requires implementing (1) fast data treatment tools and (2) Genotyping/classification schemes and methods to extract medically relevant information from genomic sequences (resistome, virulome).

HIV-1 replication requires the integration of the viral genome into the cell genome. A viral-encoded enzyme, integrase (IN), performs this critical step of infection and is a promising target for anti-viral therapeutics. If the catalytic properties of INs are well characterized, the mechanisms responsible for their site selectivity are still under investigation. Several cellular proteins, such as the LEDFGF/p75 transcription co-activator, the RNA polymerase II machinery, nuclear pore proteins and specific modified histones have been proposed to be involved in IN selectivity at a genomic level but the underlying molecular mechanisms remain to be demonstrated. In addition, structural parameters of the target DNA helix (curvature, flexibility, topology) are proposed to regulate IN selectivity at a local level. Our aims are to study the role of these different parameters of IN selectivity, using both in vitro and in vivo approaches. In vitro, we will map integration sites on various target DNA substrates (naked DNA or chromatin, minicircles, plasmids with different topologies, transcribed templates) and will test the effect of purified proteins suspected to regulate IN selectivity. In vivo, integration sites will be mapped in cells depleted of these suspected regulators or in cells incubated with drugs targeting enzymes involved in transcription, DNA topology or histone modifications. Integration sites will be mapped using published or “home-made” protocols and the sites will be compared with DNA structural parameters, nucleosome positions, histone modifications or transcriptional parameters (published maps). Bio-informatics tools are crucial for these correlative and statistical analyses of integration sites. Our project relies on complementary in vivo, in vitro and in silico approaches. It should establish molecular and mechanistic rules of HIV-1 integration selectivity that could serve in the development of new antiviral strategies and of safer gene therapy vectors.

Grâce à la plateforme PF2 à Pasteur, nous avons réalisé une étude de microarray ( Affymetrix Genechip Mouse Gene 2.0 ST Array) avec les fibroblastes de souris. Pourriez vous SVP nous aider à analyser ces données?

Molecular markers of cervix lesions linked to HPV infections.

Les cyanobactéries sont des microorganismes qui prolifèrent dans de nombreux plans d’eau et perturbent leurs fonctionnements et leurs usages car elles sont capables de produire des toxines dangereuses pour la santé humaine et animale. Si la réglementation sanitaire est basée, pour l’instant, sur la surveillance d’une seule toxine, il est désormais connu que ces microorganismes sont capables d’en synthétiser un grand nombre qu’il conviendrait de mieux prendre en compte dans le futur. C’est pourquoi, dans le but de mieux connaître le potentiel toxique des cyanobactéries, ma thèse s'applique, par des études sur leur génome et par une approche de chimie, à caractériser les gènes impliqués dans la synthèse de ces métabolites ainsi que les métabolites produits par ces gènes, à déterminer sur des souches de culture et dans des échantillons naturels provenant de plans d’eau d’Ile de France quel est le potentiel de production de ces métabolites et à mieux comprendre les facteurs environnementaux qui favorisent cette production. Deux équipes de Paris (Pasteur et iEES) sont associées sur ce travail qui implique également des collaborations étrangères. S'il est désormais bien connu qu'une part importante du métabolisme des cyanobactéries qui sont des microorganismes photosynthétiques, est régulée en fonction des phases de lumière et d'obscurité, les connaissances disponibles sur la synthèse des métabolites secondaires sont en revanche beaucoup plus limitées. Ces métabolites ont pourtant un double intérêt puisque certains sont toxiques pour l'Homme alors que d'autres ont un intérêt pharmaceutique potentiel. Leur synthèse repose sur l'expression de clusters de gènes pouvant être de très grande taille (jusqu’à 100 kb par région).

The PIBnet initiative is a joint effort by 15 National Reference Centers (NRC), 8 Collaborative Centers of World Health Organization, the Collection de l’Institut Pasteur & Cyanobacteria collection and the CIBU to modernize their activities, including collection management and microbial characterization approaches and technologies. Within this large concerted effort, a priority is to promote whole genome sequencing (WGS) as the major characterization approach of microbial agents for surveillance and outbreak investigation.   Our ambition is to have shifted to WGS as a routine strain characterization method for epidemiological surveillance and outbreak investigation in the Institut Pasteur at the end of 2016. The target volume is 10,000 genomes a year. On the bioinformatics level, this requires implementing fast data treatment tools, databases, genotyping schemes and methods to extract medically relevant information from genomic sequences (resistome, virulome).

Human macrophages are the main cell target of Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis. Once phagocytosed, bacilli escape bactericidal functions of macrophages and they multiply inside the cell. Understanding interactions between bacilli and human phagocytes is primordial to develop new strategies to combat tuberculosis. We propose a project aiming at deciphering cell-cell communications between infected cells and non-infected cells. We will use the transcriptomic and the proteomic approaches to discriminate the global response of these differents sub-populations. To our knowledge, this work represents the first study that combines two different levels for the identification of a specific response of infected-cells and neighboring non-infected cells. This study would determine cellular markers of a Mtb signature.

Cryptococcus neoformans is a sugar-coated yeast that is able to interact closely with numerous organisms in the environment including amebae, paramecium of nematodes. The interaction with these organisms probably shaped its virulence. The ability to survive nutrient starvation, oxidative stress, desiccation, both in the environment and in humans, indicates a high level of physiological and metabolic plasticity of the yeast. In humans, after primary infection during childhood, the yeast is able to survive within the host for years before reactivation, leading to a deadly disseminated fungal infection. This phenomenon, called dormancy / quiescence is one of the main biological features of this fungus in relation with disease pathogenesis. It is known in bacteria, parasites and other fungi. There is no consensus on the definition of dormancy. Most often, dormant cells are characterized by a low metabolic activity sometimes undetectable under normal laboratory conditions and the ability to be resuscitated by adequate stimuli. In C. neoformans, dormancy has only been demonstrated epidemiologically in our laboratory but not experimentally so far. We developed an assay where yeasts cells exhibiting characteristics of potentially dormant cells were generated. Our current project aims at exploring the conditions leading to, the biology of the entry in and the mechanisms sustaining dormancy.

Collect statistics on variants found in different strains of influenza virus. Determine variant effects to the protein sequence.

An experiment of RNAseq was performed in triplicate with the strain A of Clostridium tetani in TGY (low production of toxin) and MS (high production of toxin) at 24 and 48 h of culture.  This RNAseq was performed in the Pasteur platform PF2. Different comparisons were performed: MS-48h vs MS-24h, TGY-48h vs TGY-24h, TGY-24h vs MS-24h and TGY-48h vs MS-48h. Excel files with differentially expressed genes were provided. For each comparison there are three excel files: complete.xls (contains results for all features), up.xls (contain results for significantly up-regulated features. Features are ordered from de most significant adjusted p-value to the less significant one) and down.xls  (contain results for significantly down-regulated features. Features are ordered from de most significant adjusted p-value to the less significant one). We would like to perform GO term enrichment analysis on the set of genes showing differential expression patterns in the considered contrasts. State of art tools to identify functional enrichments (e.g. DAVID knowledge) do not recognize Clostridium tetani protein and gene identifiers. One possibility is that such tools do not include Clostridium tetani entries in their databases.  The aim of this project is to develop an alternative approach to carry out the functional enrichment analysis on the differentially expressed genes.

Analysis of the immune response to yellow fever 17D vaccination

Aim : In vitro infection of innate immune cells by L. amazonensis (L.am.) seems to be associated to an increase in resistance to cell death of infected cells. This project aims at deciphering the impact of in vitro L. amazonensis (L.am.) amastigotes infection on cell death processes including apoptosis, autophagy, pyroptosis and necroptosis in different host cells, i.e. Bone-marrow- derived macrophages (BMDMs) and dendritic cells (BMDCs) after one day of infection. Material : RNA samples were obtained from control and infected BMDMs (BALB/c mice) or BMDCs (BALB/c, C57BL/6 and DBA/2 mice) after their sorting by Fluorescence Activated- Cell sorting. For BALB/c BMDCs, amastigotes were also added to cells in presence of immune serum to trigger their opsonization. Samples were analysed a few years ago at the « Plateforme Transcriptome et Epigénome » with the Affymetrix technology. RNAs from BMDMs and BMDCs were analysed with the Affymetrix Mouse430_2 GeneChips and the Affymetrix Mouse Gene ST 1.0 arrays respectively.  

The objective of this study is to evaluate how the different programming of distinct CD4+ T cell subsets affected their susceptibility to HIV infection and the survival of infected cells. Dr. Saez-Cirion's Team evaluates how the metabolic state of CD4⁺ T cells and the regulation of cellular factors shape the distribution of the HIV reservoir in distinct CD4⁺ T cell subset. This project uses new technologies and reagents to analyze cells from uninfected donors, and from HIV patients in acute infection, chronic infection and HIV remission.

The Transcriptome & Epigenome Platform is dedicated to the development and use of high throughput approaches for transcriptomics and epigenomics studies. The platform is accessible to any research team from the Pasteur Institute (80% of the projects) as well as from outside. It is involved (most often as collaborator) in several projects funded by the ANR, AVIESAN and by the Pasteur Institute in the framework of the PTR programs. Next Generation Sequencing (NGS) based on the Illumina technology (HiSeq 2000/2500 sequencers) is used to perform RNA-sequencing experiments for which a large amount of data is generated. After a first step involving bioinformatics, specific statistical methods must be used be analyze rigorously the data. These analyses are most often performed by the statistician(s) of the platform. They are also in charge of bibliographical survey activity.

The Transcriptome & Epigenome Platform is dedicated to the development and use of high throughput approaches for transcriptomics and epigenomics studies. The platform is accessible to any research team from the Pasteur Institute (80% of the projects) as well as from outside. It is involved (most often as collaborator) in several projects funded by the ANR, Microbes and Brain, ERANET and by the Pasteur Institute in the framework of the PTR programs. Next Generation Sequencing (NGS) based on the Illumina technology (HiSeq 2000/2500 sequencers) is used to perform RNA-sequencing experiments for which a large amount of data is generated. After a first step involving bioinformatics, specific statistical methods must be used be analyze rigorously the data. These analyses are most often performed by the statistician(s) of the platform. They are also in charge of bibliographical survey activity.

  Shigella is an enteroinvasive bacterium that induces bacillary dysentery. It invades human intestinal epithelial cells causing the inflammatory destruction of the colonic epithelium. Our previous work reported that Shigella inhibits the secretion of its host cell (Mounier et al Cell Host Microbes 2012) via several virulent proteins injected by the bacterium such as IpaJ and VirA. These two effectors may have redundant action. Thus, to address the effect of Shigella on the secretome of its host cell and in particular define the precise action of IpaJ and VirA, we conducted a mass spectrometry (MS) analysis on apical (A) and basal (B) secretion of polarized infected human intestinal cells using several strains: wild type (WT), avirulent strain (mxiD) and mutated strains either on virA, ipaJ or both (virAipaJ) and compared to non-infected cells (NI). We have in total 6 infection conditions and we collected apical and basal secretion medium (total 12 MS data). Most of the conditions were done in triplicate. We have now these quantitative MS data (excel file) that gave between 300 (basal secretion) to 1000 proteins (apical secretion) secreted differently among the different conditions. We need your collaboration to analyze our data to: Check whether our protein candidates are in databases of secreted factors Obtain protein clusters of our hits among the 6 conditions used (12 datasets)

assess the faisability of the chip analysis to our samples from laser capture microdissection of the mouse intestine, of diverse quality and preparation levels

Project context and summary : Cryptococcus neoformans is a sugar-coated yeast that is able to interact closely with numerous organisms in the environment including amebae, paramecium of nematodes. The interaction with these organisms probably shaped its virulence. The ability to survive nutrient starvation, oxidative stress, desiccation, both in the environment and in humans, indicates a high level of physiological and metabolic plasticity of the yeast. In humans, after primary infection during childhood, the yeast is able to survive within the host for years before reactivation, leading to a deadly disseminated fungal infection. This phenomenon, called dormancy / quiescence is one of the main biological features of this fungus in relation with disease pathogenesis. It is known in bacteria, parasites and other fungi. There is no consensus on the definition of dormancy. Most often, dormant cells are characterized by a low metabolic activity sometimes undetectable under normal laboratory conditions and the ability to be resuscitated by adequate stimuli. In C. neoformans, dormancy has only been demonstrated epidemiologically in our laboratory but not experimentally so far. We developed an assay where yeasts cells exhibiting characteristics of potentially dormant cells were generated. Our current project aims at exploring the conditions leading to, the biology of the entry in and the mechanisms sustaining dormancy.

Listeria monocytogenes is a gram-positive bacterium responsible for the food-borne disease listeriosis. This pathogen can invade and replicate in the cytoplasm of both macrophages and non-professional phagocytes. In order to better characterize the host response to Listeria, we are using microarrays to identify genes and cytokines up or downregulated during infection.

It has been found in the past that the peptidoglycan (PGN) degradation fragments DAP-containing muramyl tripeptide (M-triDAP) and muramyl di-peptide (MDP) stimulate innate immune receptors Nod1 and Nod2. However, it remains to be clarified how the fragments reach Nod1 and Nod2, since these receptors are intracellular. The aim of the project is thus to investigate novel factors in peptidoglycan signalling, using gene trap mutagenesis in human near-haploid cells to randomly knock out genes and do a genome wide screen to establish a library. There is a pressing need to find novel therapeutic strategies, and it has been shown in the past that the above described technique of finding genes works well for this (see Carrette et al., 2011). Results will be validated in relevant mouse models and epithelial cell lines using CRISPR-Cas.

Integration of the viral reverse-transcribed genome into the genome of infected cells is an essential step of retroviral replication and is performed by a viral-encoded enzyme, named integrase (IN). In the case of HIV-1, IN is a new and efficient anti-viral target. The selectivity of this enzyme for its cellular genomic sites is also a major parameter of HIV replication and is regulated by several cellular parameters. One of them is chromatin, and different levels of this nucleoprotein complex are involved in the regulation of IN selectivity. Using in vitro integration assays, established by our team and collaborators, we have studied this regulation at two levels of chromatin architecture: large poly-nucleosome templates (Botbol et al., 2008; Lesbats et al., 2011; Benleulmi et al., 2015; Naughtin et al., 2015) or nucleosome-induced DNA curvature mimicked by DNA minicircles (Pasi et al., 2016). Our present project is to study IN selectivity into mononucleosomes (MN). These MNs will be used as target substrates of integration and the role of MN structure, histone modifications and IN cofactors will be studied. Results obtained in vitro, will be confronted to structural data obtained by molecular modeling and to integration sites observed in infected cells. This project will benefit from our expertise in integration in chromatin templates and a previous collaboration with the C3BI on the analysis of integration sites (Pasi, M., Mornico, D., S. Volant, S., et al., 2016). This project is funded by the ANRS.

Over the last years, innate lymphoid cells (ILC) have been increasingly investigated. Despite the absence of antigen specific receptors, they belong to the lymphoid lineage and represent important sentinels for tissue homeostasis and inflammation. They contribute to numerous homeostatic and pathophysiological situations via specific cytokine production. ILC are currently divided into three groups based on the expression of specific transcription factors and secretion of cytokines. We focus this study on fetal ILC3 development. We have observed that contrary to lymphocytes, ILC can migrate toward lymphoid organs, tissues and mucosal sites as lymphoid precusors and terminate their developmental program in situ. In the fetal spleen, we observe different stages of ILC3 with precursors that are already RORgt+ but could still give rise to other ILC fate. Hence, these splenic ILC3 precursors were sorted and analyzed by microarrays. The identification of gene expression differences was used to design a single cell transcriptomic assay. The single cell transcriptomic assay is based on this specific selection of primers for transcription factors and cytokine receptors. We evaluate their differential expression in single cells at different stages of their plasticity. The aim is to decipher the progression from an ILC precursor stage to an another in one cell. We are also using the new polaris technology to detect and evaluate at different early timepoints the sequence of molecular events for changing ILC cell fate. In this case, we chose to use the sc RNAseq technology. The single cell transcriptomic will be analyzed and bioinformatic programs will be applied in order to organize the sequential molecular events and to build a hierarchical developmental model in case of ILC cell fate decisions.

Our recent analyses suggest that the genetic determinants of human neuroanatomical diversity are massively polygenic. Like other quantitative traits such as height – but also IQ or ASD risk – neuroanatomical diversity seems to result from the aggregated effect of thousands of frequent variants, each of small effect. GWAS should then require populations of hundreds of thousands of individuals to start to detect the individual variants. GCTA (genomic complex trait analysis) offers an alternative approach to obtain valuable neurogenetic information despite the current impossibility to detect enough individual variants to explaining any substantial part of the variability. We are currently pooling together neuroimaging genomics data from multiple international projects (in particular, IMAGEN, ENIGMA, UK Biobank) to replicate and extend our earlier analyses. We aim to: (1) Compute the amount of variance captured by genome-wide SNPs (SNP-heritability) for the several brain regions: ICV, BV, Hip, Th, Ca, Pa, Pu, Amy and Acc, (2) Compute the matrix of SNP-based genetic correlation among structures, (3) Partition the variance captured by SNPs among structural and functional sets: per chromosome, genic vs non-genic, low/medium/high minor-allele frequency, positive/negative selection, involved or not in neurodevelopment, etc. (4) Compare our results with those obtained using GWAS-based estimations (for example, those used in ENIGMA2). GCTA requires the computation of matrices of genetic relationship among all individuals, and thus, direct access to the genotyping data. Once the matrices are computed, the genotyping data is no longer required, and it is not possible to reconstruct an individual's genome from the matrices. Our analysis of the IMAGEN cohort was based on 1,765 Individuals, which gave us sufficient statistical power (80%) to detect only strong heritabilities (h2~45%), and the estimations had very large standard errors (~20%). A cohort of 4,000 subjects should allow us to decrease the standard error to ~8% (80% power to detect h2=22%), and a cohort of 8,000 subjects should decrease it to ~4% (80% power to detect h2=11%). In this way, we could obtain more accurate estimates, but also detect eventually more subtle effects related to functional genomic partitions.   References Yang et al (2010) Common SNPs explain a large proportion of heritability for human height. Nature Genetics, doi: 10.1038/ng.608 Davies et al (2011) Genome-wide association studies establish that human intelligence is highly heritable and polygenic. Molecular Psychiatry, doi: 10.1038/mp.2011.85 Gaugler et al (2014) Most genetic risk for autism resides with common variation. Nature Genetics, doi: 10.1038/ng.3039 Wood et al (2014) Defining the role of common variation in the genomic and biological architecture of adult human height, doi: 10.1038/ng.3097

After the behavioural characterisation of the Shank3 KO cohort, we extracted and dissected different region of the brain: cortex, hippocampus, striatum, cerebellum.

These regions were selected according to the expression level of Shank3 (Peça et al. 2011). Using QUIAGEN miRNAeasy with DNAse, miRNA-enriched RNA was extracted from the brain regions in order to perform RNAseq.

We will ask 3 questions:

• What is the pattern of Shank3 isoforms in different brain regions?

• What are the genes/pathways differentially expressed in wild-type and Shank3 knock-out mice?

• What are the genes/pathways associated with the severity of the self grooming behaviour ?

Beside data driven eperiments, we will test candidate genes/pathways such as glutamatergic receptors and <

See below  

Nous avons créer un programme sous R pur l'analyse non supervisé de fichier de cytométrie et nous avons besoin d'aide pour optimiser ce programme. Nous avons également besoin de conseils pour optimiser le clustering. Nous avons déjà rencontré Hugo Varet.

In recent years, large genome-wide association studies (GWAS) have been successful in identifying thousands of significant genetic associations for multiple traits and diseases1. In the course of this endeavor, sample size has proven to be the key factor for identifying new variants. For example, GWAS of body mass index (BMI), now including up to 350,000 individuals from more than 100 cohorts, have been able to identify genetic variant that explain as low as 0.02% of BMI variance2. While standard approaches for detecting new genetic variants associated with traits and diseases will go on as sample size increases, multivariate analyses have been proposed as an alternative strategy for both improving detection of new variants and exploring the multidimensional components of complex traits and diseases. Intuitively, multivariate analysis can be used to improve detection of variants displaying a pleiotropic effect3 by accumulating moderate evidence of association across multiple traits and diseases. Several recent examples have been published about not only GWAS hit overlap across related traits4, but also of genome-wide shared genetic effect5. Multivariate analyses of GWAS have also proven useful to understand shared genetics between diseases5, and potential causal relationship between phenotypes using Mendelian randomization (MR)6. Importantly, most of existing multivariate methods are based on GWAS summary statistics, while approaches based on individual-level data have been seldom considered because of major practical and ethical issues. In the continuity of ongoing work on multi-phenotype analysis (Aschard et al 20147, Aschard et al 20158), we developed an effective and robust multivariate approach of GWAS summary statistics that addresses the major barriers of existing approaches, i.e. the presence of correlation between studies that would exists when GWAS analyzed share sample9-16. Our approach consists in a robust omnibus multivariate test of GWAS summary statis

Nous avons pu montrer grâce au projet de recherche clinique VOYAG’R (PHRC AOR 11101- IP Pr S. Matheron) que les voyages dans une zone tropicale constituaient un risque d’acquérir une Entérobactérie multi-résistante (EMR). Pendant 15 mois, la présence d’EMR a été recherchée chez 574 sujets avant et après leur voyage en zone tropicale. Les voyageurs porteurs au retour étaient suivis sur une période de 12 mois (1, 2, 3, 6 et 12 mois ou jusqu’à négativation). Au cours de leur séjour, la moitié des voyageurs ont acquis une EMR. Nous avons également observé que trois mois après leur retour, 95% des voyageurs avaient éliminé leur EMR. Cependant 11 sujets ont conservé leur(s) souche(s) d’EMR pendant au moins 6 mois. Ces souches persistantes pourraient avoir des capacités pour perdurer au sein du tube digestif

Statistical analysis of PI survey for the scientific direction

Background: Dyslexia is characterized by difficulty with learning to read fluently and with accurate comprehension despite normal intelligence. It affects 5–10% of school-age children. Familial studies repeatedly showed that first-degree relatives of affected individuals have a 30–50% risk of developing the disorder. Twin studies showed that heritability was approximately 50% with a higher concordance rate for monozygotic twins compared to dizygotic twins. Although genetic factors contribute to dyslexia, very little is known on the genes associated with the condition. Preliminary data: Our project consists in the complementary analysis of (i) a cohort of 209 patients with dyslexia, 89 relatives and 95 very well phenotyped controls and (ii) an extended pedigree (Nantaise family) with 12 members diagnosed with dyslexia in three generations. For all the individuals of the project, we genotyped >600K SNPs in order to detect SNP association and copy-number variants (CNVs). For the extended pedigree, we also used linkage analysis and whole genome sequence (WGS). Our preliminary results indicate that a single region on chromosome 7q36 is segregating with dyslexia in the Nantaise family. The region is located within CNTNAP2, a gene previously proposed as a susceptibility gene, but without formal proof of its association. The WGS data of three affected and three unaffected individuals of the pedigree was performed to detect all the variants in the linkage region. Project: We proposed to use this unique resource in France to characterize the genetic profile of patients with dyslexia. We will (i) detect the CNVs present in the patients and (ii) detect the variants in the linkage region.

Mood disorders such as bipolar and major depressive disorders affects 2-5% of the population worldwide. They are relapsing, major psychiatric illnesses with poorly understood neurobiology, typically incomplete treatment responses, and often unsatisfactory clinical outcomes, with a high risk of premature mortality due to suicide and clinical comorbidities. Its complex manifestations include marked disturbances of emotional regulation, sensory-perception as well as major changes in mood, thinking, and many aspects of behavior. It is of crucial importance to be able to discriminate mood states in clinical practice, as pharmacological and psychological treatments as well as course of illness and outcome differ across the different mood states. By applying advanced mathematical approaches (with statistical/computational advice from a C3BI’s colleagues) this study aims to explore how sociodemographic, psychosocial and behavioral predictors may contribute for better characterizing patients with mood disorders. The study also aims to identify biological predictors of different mood states as well as of treatment response in mood disorders. Having a deeper understanding of phenomenology and pathophysiological mechanisms of mood disorders may help clinicians to provide more personalized interventions for individuals at-risk or suffering from mood disorders.

Blood cells were collected in the field from patients admited in the Ebola treatment center of Macenta (Guinea). After red cells lysis, blood cells were frozen and RNA was extracted after the shipment of samples in France. Despite poor RNA quality, Affimetrix chips have been performed and transcriptomic data are available. The aim of this study is to compare the transcriptomic data from patients who survived and those who died and also to perform a kinetic analysis of the infection in the two groups of patients.  Samples from febrile patients who were not infected with Ebola virus have been used as negative controls.

Molecular markers of cervix lesions linked to HPV infections.

Innate lymphoid cells (ILCs) are the most recently identified components of the innate immune system. ILCs colonize different tissue sites and react promptly to microenvironmental perturbations. Due to their high plasticity, ILCs can shape their functional output in response to local cues. As such, ILCs play roles under homeostatic conditions and in the context of infection, chronic inflammation, metabolic diseases and cancer. Diverse ILC subsets (NK cells, ILC2) have been shown to regulate the metabolic homeostasis. Metabolic states affect cellular functions and have been shown to play an important role in the regulation of adaptive immunity. In contrast, almost nothing is known about innate lymphocytes metabolism and the importance of energy regulation for ILC function. This project will study metabolic profiles in human ILC subsets under diverse environmental conditions. Enhancing or interfering with ILC activity could ultimately represent a novel useful therapy for chronic inflammatory diseases.

Lyme borreliosis is the most common tick-borne disease in the northern hemisphere. In Europe, it is transmitted by Ixodes ticks that carries bacteria belonging to the Borrelia burgdorferi sensu lato complex. Our study was focused on peri-urban forests of Île-de-France. These forests are frequented by many visitors and the risk of exposure to tick bites is high. One of them, the Sénart forest, is located 30 km south of Paris (in the Île-de-France region) and has a large number of visitors (3 million per year in the late 1990s). This forest has the characteristics of being partly invaded by chipmunks (Tamias sibiricus). The chipmunk has been introduced from Eurasia, particularly Siberia, China and Korea. The first individuals were released by their owners at the western end of the Sénart forest, in the 1970s. The northeastern part of the forest was colonized recently. Our current study aims to evaluate the evolution of the infection of Ixodes ricinus by Borrelia burgdorferi sl. by comparing the results obtained during 3 years and to determine the consequences of the proliferation of this non-native rodent species, Tamias sibiricus, on the risk of transmission of Lyme borreliosis. For this purpose, we analyzed the rate of infection and the density of infected ticks during 2008, 2009 and 2011 in several locations of the Sénart forest. These results were compared to those obtained for ticks collected in 2009 in two other peri-urban forests of Île-de-France (Rambouillet and Notre-Dame) that have not yet been colonized by these rodents. The density of nymphs, adults as well as the infected density of nymphs and adults were compared according to several factors: location of tick collection in the forest,  presence or absence of chipmunks, type of vegetation, temperature and humidity.

Autism Spectrum Disorders (ASD) are heterogeneous neurodevelopmental disorders with a complex genetic architecture. They are characterized by impaired social communication, stereotyped behaviors and restricted interests and are frequently associated with comorbidities such as intellectual disability, epilepsy and severe sleep disorders. Hyperserotonemia and low melatonin levels are among the most replicated endophenotypes reported in ASD, but the genetic causes of such alterations remain largely unknown. Based on the biochemical profile of 717 individuals including 213 children with ASD, 128 unaffected siblings and 376 parents and other relatives, we aim to estimate the heritability of five quantitative traits associated with the melatonin synthesis pathway: whole-blood serotonin, platelet N-acetylserotonin (NAS) and plasma melatonin levels, as well as the two enzymes AANAT and ASMT activity measured in platelets.

Rationnel. Le mode de présentation clinique du sepsis est très polymorphe. Chez les patients septiques consultants dans les services des urgences, la présence d’une hyperthermie ou d’autres critères du syndrome de réponse inflammatoire systémique (SIRS) n’est pas suffisante pour aider au diagnostic de sepsis. De nombreux efforts de recherche ont abouti à la proposition d’innombrables biomarqueurs de sepsis essentiellement étudiés en soins intensifs. Même si certains, comme la procalcitonine (PCT) ont atteint un relativement bon degré de prédiction aux urgences, leur usage en routine demeure controversé. Compte tenu de la physiopathologie complexe du sepsis, une approche combinatoire pourrait permettre d’atteindre des performances difficilement envisageables avec un biomarqueur seul. Objectif primaire. Etudier les performances statistiques d’un panel de biomarqueurs d’intérêt, individuellement et en association, pour le diagnostic de sepsis aux urgences. Objectifs secondaires. Etudier les performances statistiques d’un panel de biomarqueurs d’intérêt, individuellement et en association, pour le diagnostic d’état septique grave (sepsis sévère et choc septique) et la stratification du risque (prédiction de l’admission en soins intensifs et/ou du décès). Type d’étude. Etude de cohorte monocentrique prospective non-interventionnelle Patients et critères d’inclusion. 300 patients consultant dans le service des urgences ayant une suspicion de sepsis + 30 sujets sains. Critères de non inclusion. Patient mineur de moins de 18 ans, femme enceinte, conditions de vie rendant impossible le suivi à 28 jours, refus de participer à l’étude. Mesures. Pour chaque patient, lors du bilan sanguin initial, prélèvement de 3 tubes pour le dosage a posteriori d’un panel de biomarqueurs d’intérêt explorant les différentes voies biologiques activées au cours du sepsis.  

Chronic inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, spondyloarthritis (SpA) and psoriasis cause significant morbidity and are a substantial burden for the affected individuals and the society. An important obstacle to early diagnosis and the development of more specific and effective therapies is the very limited understanding of the pathogenesis of these diseases. In the past years genome-wide association studies have identified many genes that were not known to be involved in pathogenesis, and have linked several genes in immune pathways to inflammatory diseases, indicating that the immune system plays an essential role in the pathogenesis of these diseases. The current challenge is to correlate these genetic variants with the effector mechanisms implicated in pathogenesis, to allow translation of the genetic data into relevant diagnostics and innovative treatment strategies. To meet this challenge, we have designed a clinical study that examines the immune signaling pathways, the transcriptional networks and the genotype in the same SpA patient, in order to establish a link between genetic variation, cellular phenotype and function, and pathology. This approach will advance our understanding of the pathogenic mechanisms, and identify novel and relevant diagnostic tools, therapeutic targets and biomarkers. The long-term outcome of this strategy will be the rational design of specific therapies tailored to the genotype of the patient.

The interferon-induced transmembrane (IFITM) proteins protect cells from diverse virus infections, including Influenza, HIV and Zika viruses, by inhibiting virus-cell fusion. We showed that IFITM proteins act additively in both productively infected cells and uninfected target cells to inhibit HIV-1 spread, potentially conferring these proteins with greater breadth and potency against enveloped viruses. We also reported that amino-terminal mutants of IFITM3 preventing ubiquitination or endocytosis are more abundantly incorporated into virions and exhibit enhanced inhibition of HIV-1 fusion. An analysis of primate genomes revealed that IFITM3 is the most ancient antiviral family member of the IFITM locus and has undergone a repeated duplication in independent host lineages. Some IFITM3 genes in nonhuman primates, including those that arose following gene duplication, carry amino-terminal mutations that modify protein localization and function. Our aim is to analyze the RNA levels of the various members of the IFITM family, in various normal or pathological human or animal tissues.

It has been shown that methylation can act as a kind of memory of the immune system. For patients with co-infections, it is of particular importance to know when to begin an anti-retroviral therapy, especially if they are already infected with tuberculosis. The goal of this study is to find hyper or hypo methylated loci related to the reaction of HIV patients (co-infected or not) to different kind of treatments.

Objectives: Our objective was, through whole genome sequencing, to establish a comprehensive repertoire of the non-synonymous polymorphisms (natural polymorphisms and/or mutations of resistance) in genes involved in resistance to azoles and echinocandins. Methods: Two collections of C. albicans clinical isolates were used. The first one consists of 151 epidemiologically-unrelated strains susceptible to antifungal agents. The second one consists of 9 isolates resistant to fluconazole and 1 to fluconazole and caspofungin. The whole genome sequencing was performed on an Illumina HiSeq 2000, generating 100 bp reads (roughly 100X coverage on average). The reads were mapped to the SC5314 reference genome (Assembly 22) using the BWA alignment tool. Then SNPs were detected by GATK and selected with the recommended filters. Using homemade scripts we analyzed the sequences of 5 genes involved in the resistance to azoles (ERG11, TAC1, MRR1 and UPC2) and echinocandins (FKS1) and compared them to the sequences of reference strain SC5314. Results: Among the 151 antifungal susceptible strains we identified 126 distinct natural amino-acid substitutions. Using this repertoire, we identified from the 10 resistant strains, 22 amino-acid substitutions in addition to some of the above-mentioned natural polymorphisms. Among them, 10 have already been associated to azoles or echinocandins resistance. The remaining 12 substitutions are novel putative azoles resistance mutations affecting ERG11 (n=4/11), TAC1 (n=6/7) and UPC2 (n=2/3).

Ataxia-telangiectasia (AT) is an autosomal recessive disorder characterized by a biallelic mutation of the Ataxia Telangiectasia Mutated (ATM) gene. The ATM protein is involved in a very large number of cellular functions (over 700 substrates), mainly DNA repair, cell cycle regulation, mitochondrial and oxidative metabolism, as well as regulation of gene expression. The majority of hematological malignancies occurring in AT patients are non-Hodgkin's B lymphomas and Hodgkin's lymphomas. The association with the Epstein-Barr virus (EBV) is found in 50 to 100% of these cases according to the histological subtype. EBV is a widespread virus in human populations with a prevalence greater than 90% and is associated with the development of lymphomas in immunocompromised patients. We will study the transcriptome of EBV-infected cells in ATM patients, in order to determine the impact of ATM on EBV infection control.

This project aims at identifying novel genetic traits that regulate the anti-tumor activities of NK cells and homeostasis of NK cells and other ILC using an unique mouse resource, the Collaborative Cross (CC). CC is a panel of recombinant inbred mice derived from randomized breeding of eight laboratory inbred strains combining high genetic diversity with the advantage of inbred mouse strains. We have identified CC strains that diverge in their anti-tumor immune responses and are characterizing the molecular mechanisms responsible for these differences. Ultimately, these diverse genetic traits may lead to the development of novel therapies for cancer.

Concerns are growing over urban drinking-water quality due to an inefficient management infrastructure in most low-income countries. Outbreaks of waterborne were linked to extreme climatic events, des

Chronic inflammatory systemic diseases (CIDs) are a burden to humans because of life-long debilitating illness, increased mortality and high therapy costs. CIDs’ increasing prevalence in western countries has indeed placed them at the third rank of morbi-mortality causes. Unfortunately, available treatments are poorly targeted and non-curative. That is partly linked to a complex and largely ununderstood pathophysiology. Genetic susceptibility clearly plays a role. Genes linked to the immune system have been identified, but causal genes remain mostly unknown and other factors such as intestinal microbiota have also been implicated. The complexity of CIDs’ pathophysiology suggests that a holistic approach is the most susceptible to help make significant progress. Our project intends to take advantage of recent technical progress and development of informatics tools to set up a transversal approach. High-resolution sequencing technology indeed quickly produces large amounts of accurate data. Besides, new integrative informatics tools allowing storage and integrative analysis of this resulting high amount of data are now available. We intend to set-up a CID’s network allowing the gathering and extensive analysis of data related to immuno-genetic determinants, immune repertoire and microbiota from individuals suffering from one of the three major interlinked CIDs, namely Hidradenitis Suppurativa (HS), Crohn’s disease (CD) and Spondyloarthropathy (SpA) as compared to healthy volunteers.

It has been shown that methylation can act as a kind of memory of the immune system. For patients with co-infections, it is of particular importance to know when to begin an anti-retroviral therapy, especially if they are already infected with tuberculosis. The goal of this study is to find hyper or hypo methylated loci related to the reaction of HIV patients (co-infected or not) to different kind of treatments.

This project aims at identifying novel genetic traits that regulate the anti-tumor activities of NK cells and homeostasis of NK cells and other ILC using an unique mouse resource, the Collaborative Cross (CC). CC is a panel of recombinant inbred mice derived from randomized breeding of eight laboratory inbred strains combining high genetic diversity with the advantage of inbred mouse strains. We have identified CC strains that diverge in their anti-tumor immune responses and are characterizing the molecular mechanisms responsible for these differences. Ultimately, these diverse genetic traits may lead to the development of novel therapies for cancer.

Taking advantage of a murine model of controlled microbiota composed of 12 strains, we evaluated the activity of a cocktail of three virulent bacteriophages to target a murine Escherichia coli strain

In this project we study ecological diversification of Klebsiella pneumoniae and closely related species. Using comparative genomics we want to identify the pattern of genome adaptation to different e

Anti-TNF therapy has revolutionized treatment of many chronic inflammatory diseases, including rheumatoid arthritis, Crohn’s disease and spondyloarthritis (SpA). However, clinical efficacy of TNF-inhi

Mitochondria are double-membrane bound organelles that are essential in every tissue of the body. They are metabolic hubs and signalling platforms that are deeply integrated into cellular homeostasis.

Our goal is to have a bioinformatic tool that performs the 2 x 2 comparison of matrices of numbers in matrix batches. Each matrix corresponds to the use of gene fragments (V or J) to create a re-arran

This project is integrated in the analysis of the gut ecosystem of children implicated in the AFRIBIOTA project, a translational project performed within a consortium of researchers and medical doctor