Expertise

La plateforme de génomique fonctionnelle de Nice Sophia Antipolis existe depuis 1999. Initialement orientée vers la conception, la fabrication et l'analyse de puces à ADN, elle a contribué à ouvrir cette nouvelle technologie à une large communauté, mettant à cette occasion en place un système d'information performant (Mediante), capable de gérer de grandes masses de données, et fonctionnant en production depuis plus de 20 ans.

Son activité s'est maintenant réorientée vers des services de séquencage à haut-débit (Illumina NextSeq500), offrant dans ce contexte de nombreux types d'analyses des acides nucléiques, et une capacité pour analyser de grandes collections d'échantillons, y compris au niveau de la cellule unique.

L'activité de routine concerne des applications comme le Séquençage à haut-débit sur séquenceur Illumina NextSeq 2000 et Promethion (RNA-seq, DNA-seq, ...), le profilage d'expression génomique sur cellule unique sur technologie 10xGenomics Chromium et Parse bioscience (scRNA-seq, scVDJ-seq, scATAC-seq, ...), le séquençage longue lecture (Nanopore) de librairies scRNA-seq : ScNaUmi-seq. Et plus récemment la spatiale transcriptomique Merscope (Vizgen®) et Xenium (10x Genomics®). La plateforme se compose de 5 ingénieurs wet lab et de 2 bio-informaticiens.

Equipements

1. Contrôle qualité : Nanodrop™, Bioanalyzer, Qubit®, Blue pippin
2. Analyse Single Cell : Chromium X (10x Genomics®)
3. Séquencage : NextSeq500 Illumina, MinION et PromethION Oxford Nanopore Technology®
4. Spatiale Transcriptomique : Merscope (Vizgen®), Xenium (10x Genomics®)
5. Robots : Assist plus (Integra®), Opentrons.

Les résultats sont stockés automatiquement sur le portail d'informations de la plateforme Mediante. Cela concerne notamment les fichiers .BAM d'alignement, les fichiers .BW de couverture et l'ensemble des fichiers de l'analyse secondaire et des analyses statistiques conduites en partenariat avec le collaborateur. Sur demande l'ensemble des données brutes sont également mises à disposition et une aide est fournit pour la soumission des données vers la base de données publiques GEO (Gene Expression Omnibus).

Related publications

The variant 20I/501Y.V1, associated to a higher risk of transmissibility, emerged in Nice city (Southeast of France, French Riviera) during January 2021. The pandemic has resumed late December 2020 in this area. A high incidence rate together with a fast turn-over of the main circulating variants, provided us the opportunity to analyze modifications in clinical profile and outcome traits. We performed an observational study in the University hospital of Nice from December 2020 to February 2021. We analyzed data of sequencing of SARS-CoV-2 from the sewage collector and PCR screening from all positive samples at the hospital. Then, we described the characteristics of all COVID-19 patients admitted in the emergency department (ED) (n = 1247) and those hospitalized in the infectious diseases ward or ICU (n = 232). The UK-variant was absent in this area in December, then increasingly spread in January representing 59% of the PCR screening performed mid-February. The rate of patients over 65 years admitted to the ED decreased from 63 to 50% (p = 0.001). The mean age of hospitalized patients in the infectious diseases ward decreased from 70.7 to 59.2 (p < 0.001) while the proportion of patients without comorbidity increased from 16 to 42% (p = 0.007). Spread of the UK-variant in the Southeast of France affects younger and healthier patients.

Background: Wastewater surveillance was proposed as an epidemiological tool to define the prevalence and evolution of the SARS-CoV-2 epidemics. However, most implemented SARS-CoV-2 wastewater surveillance projects were based on qPCR measurement of virus titers and did not address the mutational spectrum of SARS-CoV-2 circulating in the population. Methods: We have implemented a nanopore RNA sequencing monitoring system in the city of Nice (France, 550,000 inhabitants). Between October 2020 and March 2021, we monthly analyzed the SARS-CoV-2 variants in 113 wastewater samples collected in the main wastewater treatment plant and 20 neighborhoods. Findings: We initially detected the lineages predominant in Europe at the end of 2020 (B.1.160, B.1.177, B.1.367, B.1.474, and B.1.221). In January, a localized emergence of a variant (Spike:A522S) of the B.1.1.7 lineage occurred in one neighborhood. It rapidly spread and became dominant all over the city. Other variants of concern (B.1.351, P.1) were also detected in some neighborhoods, but at low frequency. Comparison with individual clinical samples collected during the same week showed that wastewater sequencing correctly identified the same lineages as those found in COVID-19 patients. Interpretation: Wastewater sequencing allowed to document the diversity of SARS-CoV-2 sequences within the different neighborhoods of the city of Nice. Our results illustrate how sequencing of sewage samples can be used to track pathogen sequence diversity in the current pandemics and in future infectious disease outbreaks.

Fibrosis is a deleterious invasion of tissues associated with many pathological conditions, such as Duchenne muscular dystrophy (DMD) for which no cure is at present available for its prevention or its treatment. Fibro-adipogenic progenitors (FAPs) are resident cells in the human skeletal muscle and can differentiate into myofibroblasts, which represent the key cell population responsible for fibrosis. In this study, we delineated the pool of microRNAs (miRNAs) that are specifically modulated by TGF?1 in FAPs versus myogenic progenitors (MPs) by a global miRNome analysis. A subset of candidates, including several "FibromiRs", was found differentially expressed between FAPs and MPs and was also deregulated in DMD versus healthy biopsies. Among them, the expression of the TGF?1-induced miR-199a~214 cluster was strongly correlated with the fibrotic score in DMD biopsies. Loss-of-function experiments in FAPs indicated that a miR-214-3p inhibitor efficiently blocked expression of fibrogenic markers in both basal conditions and following TGF?1 stimulation. We found that FGFR1 is a functional target of miR-214-3p, preventing the signaling of the anti-fibrotic FGF2 pathway during FAP fibrogenesis. Overall, our work demonstrates that the « FibromiR » miR-214-3p is a key activator of FAP fibrogenesis by modulating the FGF2/FGFR1/TGF? axis, opening new avenues for the treatment of DMD.

Tissue regeneration requires coordinated and dynamic remodeling of stem and progenitor cells and the surrounding niche. Although the plasticity of epithelial cells has been well explored in many tissues, the dynamic changes occurring in niche cells remain elusive. Here, we show that, during lung repair after naphthalene injury, a population of PDGFR?+ cells emerges in the non-cartilaginous conducting airway niche, which is normally populated by airway smooth muscle cells (ASMCs). This cell population, which we term "repair-supportive mesenchymal cells" (RSMCs), is distinct from conventional ASMCs, which have previously been shown to contribute to epithelial repair. Gene expression analysis on sorted lineage-labeled cells shows that RSMCs express low levels of ASMC markers, but high levels of the pro-regenerative marker Fgf10. Organoid co-cultures demonstrate an enhanced ability for RSMCs in supporting club-cell growth. Our study highlights the dynamics of mesenchymal cells in the airway niche and has implications for chronic airway-injury-associated diseases.

Aging is a multifactorial process that results in progressive loss of regenerative capacity and tissue function while simultaneously favoring the development of a large array of age-related diseases. Evidence suggests that the accumulation of senescent cells in tissue promotes both normal and pathological aging. Oxic stress is a key driver of cellular senescence. Because symbiotic long-lived reef corals experience daily hyperoxic and hypoxic transitions, we hypothesized that these long-lived animals have developed specific longevity strategies in response to light. We analyzed transcriptome variation in the reef coral Stylophora pistillata during the day-night cycle and revealed a signature of the FoxO longevity pathway. We confirmed this pathway by immunofluorescence using antibodies against coral FoxO to demonstrate its nuclear translocation. Through qPCR analysis of nycthemeral variations of candidate genes under different light regimens, we found that, among genes that were specifically up- or downregulated upon exposure to light, human orthologs of two "light-up" genes (HEY1 and LONF3) exhibited anti-senescence properties in primary human fibroblasts. Therefore, these genes are interesting candidates for counteracting skin aging. We propose a large screen for other light-up genes and an investigation of the biological response of reef corals to light (e.g., metabolic switching) to elucidate these processes and identify effective interventions for promoting healthy aging in humans.

Light chain (LC) deposition disease (LCDD) is a rare disorder characterized by glomerular and peritubular amorphous deposits of a monoclonal immunoglobulin LC, leading to nodular glomerulosclerosis and nephrotic syndrome. We developed a transgenic model using site-directed insertion of the variable domain of a pathogenic human LC gene into the mouse immunoglobulin ? locus, ensuring its production by all plasma cells (PCs). High free LC levels were achieved after backcrossing with mice presenting increased PC differentiation and no immunoglobulin heavy chain production. Our mouse model recapitulates the characteristic features of LCDD, including progressive glomerulosclerosis, nephrotic-range proteinuria, and finally kidney failure. The variable domain of the LC bears alone the structural properties involved in its pathogenicity. RNA sequencing conducted on PCs demonstrated that LCDD LC induces endoplasmic reticulum stress, likely accounting for the high efficiency of proteasome inhibitor-based therapy. Accordingly, reduction of circulating pathogenic LC was efficiently achieved and not only preserved renal function but also partially reversed kidney lesions. Finally, transcriptome analysis of presclerotic glomeruli revealed that proliferation and extracellular matrix remodeling represented the first steps of glomerulosclerosis, paving the way for future therapeutic strategies in LCDD and other kidney diseases featuring diffuse glomerulosclerosis, particularly diabetic nephropathy.

Background: Bacterial colonization in cystic fibrosis (CF) lungs has been directly associated to the loss of CFTR function, and/or secondarily linked to repetitive cycles of chronic inflammation/infection. We hypothesized that altered molecular properties of mucins could contribute to this process. Methods: Newborn CFTR+/+ and CFTR-/- were sacrificed before and 6 h after inoculation with luminescent Pseudomonas aeruginosa into the tracheal carina. Tracheal mucosa and the bronchoalveolar lavage (BAL) fluid were collected to determine the level of mucin O-glycosylation, bacteria binding to mucins and the airways transcriptome. Disturbances in mucociliary transport were determined by ex-vivo imaging of luminescent Pseudomonas aeruginosa. Results: We provide evidence of an increased sialylation of CF airway mucins and impaired mucociliary transport that occur before the onset of inflammation. Hypersialylation of mucins was reproduced on tracheal explants from non CF animals treated with GlyH101, an inhibitor of CFTR channel activity, indicating a causal relationship between the absence of CFTR expression and the sialylation of mucins. This increased sialylation was correlated to an increased adherence of P. aeruginosa to mucins. In vivo infection of newborn CF piglets by live luminescent P. aeruginosa demonstrated an impairment of mucociliary transport of this bacterium, with no evidence of pre-existing inflammation. Conclusions: Our results document for the first time in a well-defined CF animal model modifications that affect the O-glycan chains of mucins. These alterations precede infection and inflammation of airway tissues, and provide a favorable context for microbial development in CF lung that hallmarks this disease.

Most pathogenic oomycetes of the genus Phytophthora spread in water films as flagellated zoospores. Zoospores perceive and produce signals attracting other zoospores, resulting in autoaggregation in vitro or biofilm formation on plant surface. The mechanisms underlying intercellular communication and consequent attraction, adhesion and aggregation are largely unknown. In Phytophthora parasitica, the perception of a K+ gradient induces coordinated motion and aggregation. To define cellular and molecular events associated with oomycete aggregation, we combined transcriptomic and ultrastructural analyses. Results indicate involvement of electroception in K+ sensing. They establish that the transcriptome repertoire required for swimming and aggregation is already fully functional at zoospore release. At the time points analyzed, aggregates are mainly constituted of zoospores. They produce vesicular and fibrillary material discharged at cell-to-cell contacts. Consistently, the signature of transcriptome dynamics during transition to aggregates is an upregulation of genes potentially related to vesicular trafficking. Moreover, transcriptomic and functional analyses show a strong enhancement of carbonic anhydrase activity, indicating that pH homeostasis may contribute to aggregation by acting on both zoospore movement and adhesion. This study poses the molecular and cellular bases of aggregative behavior within oomycetes and expands the current knowledge of ion perception-mediated dissemination of propagules in the rhizosphere.

Melanoma patients harboring the BRAFV600E mutation are treated with vemurafenib. Almost all of them ultimately acquire resistance, leading to disease progression. Here, we find that a small molecule from a marine sponge, panicein A hydroquinone (PAH), overcomes resistance of BRAFV600E melanoma cells to vemurafenib, leading to tumor elimination in corresponding human xenograft models in mice. We report the synthesis of PAH and demonstrate that this compound inhibits the drug efflux activity of the Hedgehog receptor, Patched. Our SAR study allowed identifying a key pharmacophore responsible for this activity. We showed that Patched is strongly expressed in metastatic samples from a cohort of melanoma patients and is correlated with decreased overall survival. Patched is a multidrug transporter that uses the proton motive force to efflux drugs. This makes its function specific to cancer cells, thereby avoiding toxicity issues that are commonly observed with inhibitors of ABC multidrug transporters. Our data provide strong evidence that PAH is a highly promising lead for the treatment of vemurafenib resistant BRAFV600E melanoma.

Lung cancer is the leading cause of cancer death worldwide, with poor prognosis and a high rate of recurrence despite early surgical removal. Hypoxic regions within tumors represent sources of aggressiveness and resistance to therapy. Although long non-coding RNAs (lncRNAs) are increasingly recognized as major gene expression regulators, their regulation and function following hypoxic stress are still largely unexplored. Combining profiling studies on early-stage lung adenocarcinoma (LUAD) biopsies and on A549 LUAD cell lines cultured in normoxic or hypoxic conditions, we identified a subset of lncRNAs that are both correlated with the hypoxic status of tumors and regulated by hypoxia in vitro. We focused on a new transcript, NLUCAT1, which is strongly upregulated by hypoxia in vitro and correlated with hypoxic markers and poor prognosis in LUADs. Full molecular characterization showed that NLUCAT1 is a large nuclear transcript composed of six exons and mainly regulated by NF-?B and NRF2 transcription factors. CRISPR-Cas9-mediated invalidation of NLUCAT1 revealed a decrease in proliferative and invasive properties, an increase in oxidative stress and a higher sensitivity to cisplatin-induced apoptosis. Transcriptome analysis of NLUCAT1-deficient cells showed repressed genes within the antioxidant and/or cisplatin-response networks. We demonstrated that the concomitant knockdown of four of these genes products, GPX2, GLRX, ALDH3A1, and PDK4, significantly increased ROS-dependent caspase activation, thus partially mimicking the consequences of NLUCAT1 inactivation in LUAD cells. Overall, we demonstrate that NLUCAT1 contributes to an aggressive phenotype in early-stage hypoxic tumors, suggesting it may represent a new potential therapeutic target in LUADs.

Diffuse large B cell lymphoma (DLBCL) is a heterogeneous disease treated with anti-CD20-based immuno-chemotherapy (R-CHOP). We identified that low levels of GAPDH predict a poor response to R-CHOP treatment. Importantly, we demonstrated that GAPDHlow lymphomas use OxPhos metabolism and rely on mTORC1 signaling and glutaminolysis. Consistently, disruptors of OxPhos metabolism (phenformin) or glutaminolysis (L-asparaginase) induce cytotoxic responses in GAPDHlow B cells and improve GAPDHlow B cell-lymphoma-bearing mice survival, while they are low or not efficient on GAPDHhigh B cell lymphomas. Ultimately, we selected four GAPDHlow DLBCL patients, who were refractory to all anti-CD20-based therapies, and targeted DLBCL metabolism using L-asparaginase (K), mTOR inhibitor (T), and metformin (M) (called KTM therapy). Three out of the four patients presented a complete response upon one cycle of KTM. These findings establish that the GAPDH expression level predicts DLBCL patients' response to R-CHOP treatment and their sensitivity to specific metabolic inhibitors.

RATIONALE: Given the paucity of effective treatments for Idiopathic Pulmonary Fibrosis (IPF), new insights into the deleterious mechanisms controlling lung fibroblast activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies. Transforming growth factor ? (TGF-?) is the main pro-fibrotic factor, but its inhibition is associated with severe side effects due to its pleiotropic role. OBJECTIVES: We hypothesized that downstream non-coding effectors of TGF-? in fibroblasts may represent new effective therapeutic targets whose modulation may be well-tolerated. METHODS: We investigated the whole non-coding fraction of TGF-?-stimulated lung fibroblast transcriptome to identify new genomic determinants of lung fibroblast differentiation into myofibroblast. Differential expression of the long non-coding RNA DNM3OS and its associated miRNAs was validated in a murine model of pulmonary fibrosis and in IPF tissue samples. Distinct and complementary antisense oligonucleotide-based strategies aiming at interfering with DNM3OS were used to elucidate the role of DNM3OS and its associated miRNAs in IPF pathogenesis. MEASUREMENTS AND MAIN RESULTS: We identified DNM3OS as a fibroblast-specific critical downstream effector of TGF-?-induced lung myofibroblast activation. Mechanistically, DNM3OS regulates this process in trans by giving rise to three distinct profibrotic mature miRNAs (i.e. miR-199a-5p/3p and miR-214-3p), which influence both SMAD and non-SMAD components of TGF-? signaling in a multifaceted way. In vivo, we showed that interfering with DNM3OS function not only prevents lung fibrosis but also improves established pulmonary fibrosis. CONCLUSION: Pharmacological approaches aiming at interfering with DNM3OS may represent new effective therapeutic strategies in IPF.

Others and we have shown that T cells have an important role in hippocampal synaptic plasticity, including neurogenesis in the dentate gyrus, spinogenesis, and glutamatergic synaptic function in the CA of the hippocampus. Hippocampus plasticity is particularly involved in the brain effects of the enriched environment (EE), and interestingly CD4+ and CD8+ T cells play essential and differential roles in these effects. However, the precise mechanisms by which they act on the brain remain elusive. OBJECTIVES: We searched for a putative mechanism of action by which CD4+ T cells could influence brain plasticity and hypothesized that they could regulate protein transport at the level of the blood-CSF barrier in the choroid plexus. METHOD: We compared mice housed in EE and deprived of CD4+ T cells using a depleting antibody with a control group injected with the control isotype. We analyzed in the hippocampus the gene expression profiles using the Agilent system, and the expression of target proteins in plasma, CSF, and the choroid plexus using ELISA. RESULTS: We show that CD4+ T cells may influence EE-induced hippocampus plasticity via thyroid hormone signaling by regulating in the choroid plexus the expression of transthyretin, the major transporter of thyroxine (T4) to the brain parenchyma. CONCLUSIONS: Our study highlights the contribution of close interactions between the immune and neuroendocrine systems in brain plasticity and function.

Root-knot nematodes (RKN), genus Meloidogyne, are plant parasitic worms that have the ability to transform root vascular cylinder cells into hypertrophied, multinucleate and metabolically over-active feeding cells. Redifferentiation into feeding cells is the result of a massive transcriptional reprogramming of root cells targeted by RKN. Since RKN are able to induce similar feeding cells in roots of thousands of plant species, these worms are thought to manipulate essential and conserved plant molecular pathways. Small non-coding RNAs of uninfected roots and infected root galls induced by M. incognita from Arabidopsis thaliana were sequenced by high throughput sequencing. SiRNA populations were analysed by using the Shortstack algorithm. We identified siRNA clusters that are differentially expressed in infected roots and evidenced an over-representation of the 23-24?nt siRNAs in infected tissue. This size corresponds to heterochromatic siRNAs (hc-siRNAs) which are known to regulate expression of transposons and genes at the transcriptional level, mainly by inducing DNA methylation. Correlation of siRNA clusters expression profile with transcriptomic data identified several protein coding genes that are candidates to be regulated by siRNAs at the transcriptional level by RNA directed DNA methylation (RdDM) pathway either directly or indirectly via silencing of neighbouring transposable elements.

Multiciliated cells (MCCs) harbor dozens to hundreds of motile cilia, which generate hydrodynamic forces important in animal physiology. In vertebrates, MCC differentiation involves massive centriole production by poorly characterized structures called deuterosomes. Here, single-cell RNA sequencing reveals that human deuterosome stage MCCs are characterized by the expression of many cell cycle-related genes. We further investigated the uncharacterized vertebrate-specific cell division cycle 20B (CDC20B) gene, which hosts microRNA-449abc. We show that CDC20B protein associates to deuterosomes and is required for centriole release and subsequent cilia production in mouse and Xenopus MCCs. CDC20B interacts with PLK1, a kinase known to coordinate centriole disengagement with the protease Separase in mitotic cells. Strikingly, over-expression of Separase rescues centriole disengagement and cilia production in CDC20B-deficient MCCs. This work reveals the shaping of deuterosome-mediated centriole production in vertebrate MCCs, by adaptation of canonical and recently evolved cell cycle-related molecules.

Fragile X syndrome (FXS), the most common form of inherited intellectual disability (ID) and a leading cause of autism, results from the loss of expression of the Fmr1 gene which encodes the RNA-binding protein Fragile X Mental Retardation Protein (FMRP). Among the thousands mRNA targets of FMRP, numerous encode regulators of ion homeostasis. It has also been described that FMRP directly interacts with Ca2+ channels modulating their activity. Collectively these findings suggest that FMRP plays critical roles in Ca2+ homeostasis during nervous system development. We carried out a functional analysis of Ca2+ regulation using a calcium imaging approach in Fmr1-KO cultured neurons and we show that these cells display impaired steady state Ca2+ concentration and an altered entry of Ca2+ after KCl-triggered depolarization. Consistent with these data, we show that the protein product of the Cacna1a gene, the pore-forming subunit of the Cav2.1 channel, is less expressed at the plasma membrane of Fmr1-KO neurons compared to wild-type (WT). Thus, our findings point out the critical role that Cav2.1 plays in the altered Ca2+ flux in Fmr1-KO neurons, impacting Ca2+ homeostasis of these cells. Remarkably, we highlight a new phenotype of cultured Fmr1-KO neurons that can be considered a novel cellular biomarker and is amenable to small molecule screening and identification of new drugs to treat FXS.

In line with the pathophysiological continuum described between nose and bronchus in allergic respiratory diseases, we assessed whether nasal epithelium could mirror the Th2 status of bronchial epithelium.Nasal and bronchial cells were collected by brushings from patients with allergic rhinitis and asthma (AR, n=12), isolated allergic rhinitis (R, n=14) and healthy controls (C, n=13). Cellular composition was assessed by flow cytometry. Gene expression was analysed by RNA sequencing. Th2, Th17 and interferon signatures were derived from the literature.Infiltration by polymorphonuclear neutrophils in nose excluded 30% of the initial cohort. All bronchial samples from AR group were Th2-high. Nasal samples gene expression profile from the AR group correctly predicted the paired bronchial sample Th2 status in 71% of cases. Nevertheless, nasal cells did not appear as a reliable surrogate of the Th2 response, in particular due to a more robust influence of the interferon response in 14/26 nasal samples. Th2 scores correlated with mast cells counts (p<0.001) and numbers of sensitizations (p=0.006 and 0.002), while Th17 scores correlated with PMN counts (p<0.014).The large variability in nasal cell composition and type of inflammation restricts its use as a surrogate for assessing bronchial Th2 inflammation in AR patients.

Despite constant progress in prognostic risk stratification, children with acute myeloid leukemia (AML) still relapse. Treatment failure and subsequent relapse have been attributed to acute myeloid leukemia-initiating cells (LSC), which harbor stem cell properties and are inherently chemoresistant. Although pediatric and adult AML represent two genetically very distinct diseases, we reasoned that common LSC gene expression programs are shared and consequently, the highly prognostic LSC17 signature score recently developed in adults may also be of clinical interest in childhood AML. Here, we demonstrated prognostic relevance of the LSC17 score in pediatric non-core-binding factor AML using Nanostring technology (ELAM02) and RNA-seq data from the NCI (TARGET-AML). AML were stratified by LSC17 quartile groups (lowest 25%, intermediate 50% and highest 25%) and children with low LSC17 score had significantly better event-free survival (EFS: HR?=?3.35 (95%CI?=?1.64-6.82), P?
19. Effect of mutant variants of the KRAS gene on PD-L1 expression and on the immune microenvironment and association with clinical outcome in lung adenocarcinoma patients, Lung Cancer. 2018 Jul;121:70-75. (Pubmed: 29858030)
Falk AT, Yazbeck N, Guibert N, Chamorey E, Paquet A, Ribeyre L, Bence C, Zahaf K, Leroy S, Marquette CH, Cohen C, Mograbi B, Maziès J, Hofman V, Brest P, Hofman P, Ilié

OBJECTIVES: The effect of anti-PD-1/PD-L1 inhibitors on lung adenocarcinomas (LADCs) with KRAS mutations is debatable. We examined the association between specific mutant KRAS proteins and the immune infiltrates with the outcome of patients with LADCs. PATIENTS AND METHODS: In 219 LADCs harboring either wild-type (WT) or mutated KRAS gene, we quantified the density of several immune markers by immunohistochemistry followed by automated digital image analysis. Data were correlated to clinicopathological parameters and outcome of patients. RESULTS: Tumors harboring mutant KRAS-G12?V had a significantly higher PD-L1 expression compared to other tumors (p?=?0.044), while mutant KRAS-G12D tumors showed an increase in the density of CD66b+ cells (p?=?0.001). High PD-L1 expression in tumor cells was associated to improved overall survival (OS) in KRAS mutant patients (p?=?0.012), but not in the WT population (p?=?0.385), whereas increased PD-L1 expression in immune cells correlated to poor OS of KRAS-WT patients (p?=?0.025), with no difference in patients with KRAS mutations. CONCLUSIONS: KRAS mutational status can affect the immune microenvironment and survival of LADC patients in a heterogeneous way, implying that specific mutant KRAS variants expressed by the tumor should be considered when stratifying patients for immunotherapy.

Living in an enriched environment (EE) benefits health by acting synergistically on various biological systems including the immune and the central nervous systems. The dialog between the brain and the immune cells has recently gained interest and is thought to play a pivotal role in beneficial effects of EE. Recent studies show that T lymphocytes have an important role in hippocampal plasticity, learning, and memory, although the precise mechanisms by which they act on the brain remain elusive. Using a mouse model of EE, we show here that CD4+ T cells are essential for spinogenesis and glutamatergic synaptic function in the CA of the hippocampus. However, CD4+ lymphocytes do not influence EE-induced neurogenesis in the DG of the hippocampus, by contrast to what we previously demonstrated for CD8+ T cells. Importantly, CD4+ T cells located in the choroid plexus have a specific transcriptomic signature as a function of the living environment. Our study highlights the contribution of CD4+ T cells in the brain plasticity and function.

The obligate intracellular pathogenic bacterium, Ehrlichia ruminantium, is the causal agent of heartwater, a fatal disease in ruminants transmitted by Amblyomma ticks. So far, three strains have been attenuated by successive passages in mammalian cells. The attenuated strains have improved capacity for growth in vitro, whereas they induced limited clinical signs in vivo and conferred strong protection against homologous challenge. However, the mechanisms of pathogenesis and attenuation remain unknown. In order to improve knowledge of E. ruminantium pathogenesis, we performed a comparative transcriptomic analysis of two distant strains of E. ruminantium, Gardel and Senegal, and their corresponding attenuated strains. Overall, our results showed an upregulation of gene expression encoding for the metabolism pathway in the attenuated strains compared to the virulent strains, which can probably be associated with higher in vitro replicative activity and a better fitness to the host cells. We also observed a significant differential expression of membrane protein-encoding genes between the virulent and attenuated strains. A major downregulation of map1-related genes was observed for the two attenuated strains, whereas upregulation of genes encoding for hypothetical membrane proteins was observed for the four strains. Moreover, CDS_05140, which encodes for a putative porin, displays the highest gene expression in both attenuated strains. For the attenuated strains, the significant downregulation of map1-related gene expression and upregulation of genes encoding other membrane proteins could be important in the implementation of efficient immune responses after vaccination with attenuated vaccines. Moreover, this study revealed an upregulation of gene expression for 8 genes encoding components of Type IV secretion system and 3 potential effectors, mainly in the virulent Gardel strain. Our transcriptomic study, supported by previous proteomic studies, provides and also confirms new information regarding the characterization of genes involved in E. ruminantium virulence and attenuation mechanisms.

Fragile X syndrome (FXS), the most common form of inherited intellectual disability, is due to the functional deficiency of the fragile X mental retardation protein (FMRP), an RNA-binding protein involved in translational regulation of many messenger RNAs, playing key roles in synaptic morphology and plasticity. To date, no effective treatment for FXS is available. We searched for FMRP targets by HITS-CLIP during early development of multiple mouse brain regions (hippocampus, cortex and cerebellum) at a time of brain development when FMRP is most highly expressed and synaptogenesis reaches a peak. We identified the largest dataset of mRNA targets of FMRP available in brain and we defined their cellular origin. We confirmed the G-quadruplex containing structure as an enriched motif in FMRP RNA targets. In addition to four less represented motifs, our study points out that, in the brain, CTGKA is the prominent motif bound by FMRP, which recognizes it when not engaged in Watson-Crick pairing. All of these motifs negatively modulated the expression level of a reporter protein. While the repertoire of FMRP RNA targets in cerebellum is quite divergent, the ones of cortex and hippocampus are vastly overlapping. In these two brain regions, the Phosphodiesterase 2a (Pde2a) mRNA is a prominent target of FMRP, which modulates its translation and intracellular transport. This enzyme regulates the homeostasis of cAMP and cGMP and represents a novel and attractive therapeutic target to treat FXS.

Large-scale population genomic surveys are essential to explore the phenotypic diversity of natural populations. Here we report the whole-genome sequencing and phenotyping of 1,011 Saccharomyces cerevisiae isolates, which together provide an accurate evolutionary picture of the genomic variants that shape the species-wide phenotypic landscape of this yeast. Genomic analyses support a single 'out-of-China' origin for this species, followed by several independent domestication events. Although domesticated isolates exhibit high variation in ploidy, aneuploidy and genome content, genome evolution in wild isolates is mainly driven by the accumulation of single nucleotide polymorphisms. A common feature is the extensive loss of heterozygosity, which represents an essential source of inter-individual variation in this mainly asexual species. Most of the single nucleotide polymorphisms, including experimentally identified functional polymorphisms, are present at very low frequencies. The largest numbers of variants identified by genome-wide association are copy-number changes, which have a greater phenotypic effect than do single nucleotide polymorphisms. This resource will guide future population genomics and genotype-phenotype studies in this classic model system.

Although Tacrolimus is an immunosuppressive drug widely used in renal transplantation, its chronic use paradoxically induces nephrotoxic effects, in particular renal fibrosis, which is responsible for chronic allograft dysfunction and represents a major prognostic factor of allograft survival. As molecular pathways and mechanisms involved in Tacrolimus-induced fibrogenic response are poorly elucidated, we assessed whether miRNAs are involved in the nephrotoxic effects mediated by Tacrolimus. Treatment of CD-1 mice with Tacrolimus (1 mg/kg/d for 28 days) resulted in kidney injury and was associated with alteration of a gene expression signature associated with cellular stress, fibrosis and inflammation. Tacrolimus also affected renal miRNA expression, including miRNAs previously involved in fibrotic and inflammatory processes as "fibromirs" such as miR-21-5p, miR-199a-5p and miR-214-3p. In agreement with in vivo data, Renal Proximal Tubular Epithelial cells exposed to Tacrolimus (25 and 50 µM) showed upregulation of miR-21-5p and the concomitant induction of epithelial phenotypic changes, inflammation and oxidative stress. In conclusion, this study suggests for the first time that miRNAs, especially fibromiRs, participate to Tacrolimus-induced nephrotoxic effects. Therefore, targeting miRNAs may be a new therapeutic option to counteract Tacrolimus deleterious effects on kidney.

Root-knot nematodes, Meloidogyne spp., are obligate endoparasites that maintain a biotrophic relationship with their hosts. They infect roots as microscopic vermiform second-stage juveniles, and establish specialized feeding structures called 'giant-cells', from which they withdraw water and nutrients. The nematode effector proteins secreted in planta are key elements in the molecular dialogue of parasitism. Here, we compared Illumina RNA-seq transcriptomes for M. incognita obtained at various points in the lifecycle, and identified 31 genes more strongly expressed in parasitic stages than in preparasitic juveniles. We then selected candidate effectors for functional characterization. Quantitative real-time PCR and in situ hybridizations showed that the validated differentially expressed genes are predominantly specifically expressed in oesophageal glands of the nematode. We also soaked the nematodes in siRNA to silence these genes and to determine their role in pathogenicity. The silencing of the dorsal gland specific-Minc18876 and its paralogues resulted in a significant, reproducible decrease in the number of mature females with egg masses, demonstrating a potentially important role for the small glycine- and cysteine-rich effector MiSGCR1 in early stages of plant-nematode interaction. Finally, we report that MiSGCR1 suppresses plant cell death induced by bacterial or oomycete triggers of plant defense.

Enriched environment (EE) induces plasticity changes in the brain. Recently, CD4+ T cells have been shown to be involved in brain plasticity processes. Here, we show that CD8+ T cells are required for EE-induced brain plasticity in mice, as revealed by measurements of hippocampal volume, neurogenesis in the DG of the hippocampus, spinogenesis and glutamatergic synaptic function in the CA of the hippocampus. As a consequence, EE-induced behavioral benefits depend, at least in part, on CD8+ T cells. In addition, we show that spleen CD8+ T cells from mice housed in standard environment (SE) and EE have different properties in terms of 1) TNF? release after in vitro CD3/CD28 or PMA/Iono stimulation 2) in vitro proliferation properties 3) CD8+ CD44+ CD62Llow and CD62Lhi T cells repartition 4) transcriptomic signature as revealed by RNA sequencing. CD8+ T cells purified from the choroid plexus of SE and EE mice also exhibit different transcriptomic profiles as highlighted by single-cell mRNA sequencing. We show that CD8+ T cells are essential mediators of beneficial EE effects on brain plasticity and cognition. Additionally, we propose that EE differentially primes CD8+ T cells leading to behavioral improvement.

Root knot nematodes (RKN) are root parasites that induce the genetic reprogramming of vascular cells into giant feeding cells and the development of root galls. MicroRNAs (miRNAs) regulate gene expression during development and plant responses to various stresses. Disruption of post-transcriptional gene silencing in Arabidopsis ago1 or ago2 mutants decrease the infection rate of RKN suggesting a role for this mechanism in the plant-nematode interaction. By sequencing small RNAs from uninfected Arabidopsis roots and from galls 7 and 14 d post infection with Meloidogyne incognita, we identified 24 miRNAs differentially expressed in gall as putative regulators of gall development. Moreover, strong activity within galls was detected for five miRNA promoters. Analyses of nematode development in an Arabidopsis miR159abc mutant had a lower susceptibility to RKN, suggesting a role for the miR159 family in the plant response to M. incognita. Localization of mature miR159 within the giant and surrounding cells suggested a role in giant cell and gall. Finally, overexpression of miR159 in galls at 14 d post inoculation was associated with the repression of the miR159 target MYB33 which expression is restricted to the early stages of infection. Overall, these results implicate the miR159 in plant responses to RKN.