De <i>novo </i>transcriptomes of 14 gammarid individuals for proteogenomic analysis of seven taxonomic groups

Gammarids are amphipods found worldwide distributed in fresh and marine waters. They play an important role in aquatic ecosystems and are well established sentinel species in ecotoxicology. In this study, we sequenced the transcriptomes of a male individual and a female individual for seven different taxonomic groups belonging to the two genera Gammarus and Echinogammarus: Gammarus fossarum A, G. fossarum B, G. fossarum C, Gammarus wautieri, Gammarus pulex, Echinogammarus berilloni, and Echinogammarus marinus. These taxa were chosen to explore the molecular diversity of transcribed genes of genotyped individuals from these groups. Transcriptomes were de novo assembled and annotated. High-quality assembly was confirmed by BUSCO comparison against the Arthropod dataset. The 14 RNA-Seq-derived protein sequence databases proposed here will be a significant resource for proteogenomics studies of these ecotoxicologically relevant non-model organisms. These transcriptomes represent reliable reference sequences for whole-transcriptome and proteome studies on other gammarids, for primer design to clone specific genes or monitor their specific expression, and for analyses of molecular differences between gammarid species

Protéogénomique pour la découverte de marqueurs moléculaires chez les amphipodes

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Phospholipase A de venin d'abeille et antigènes tumoraux pour la conception de vaccins contre le cancer

Les cellules tumorales sur-expriment des protéines du soi ou des protéines anormales. Ces protéines fonctionnent comme des antigènes tumoraux permettant la reconnaissance des tumeurs par le système immunitaire. Or, les cellules tumorales sont capables d inhiber celui-ci, ce qui explique le développement des cancers. Cependant, l identification des antigènes tumoraux et la compréhension des mécanismes fondamentaux de l immunité ont permis d envisager de vacciner les patients atteints de cancer. Parmi les antigènes tumoraux, on distingue la famille des antigènes cancer/testis. Ceux-ci, normalement exprimés par les cellules germinales sont retrouvés dans un grand nombre de tumeur. Ils pourraient donc entrer dans la composition de vaccins. Nous avons caractérisé le potentiel vaccinal de trois antigènes cancer / testis, NY-ESO-1, le produit d expression du gène Lage-1ORF2 (CAMEL) et le produit d expression du gène Trag-3. Ces antigènes sont reconnus par les cellules T CD4+ du système immunitaire. Un nouvel épitope, dérivé de NY-ESO-1, NY-ESO-187-111, a été identifié. Il se fixe sur plusieurs molécules HLA II couvrant 87% de la population caucasienne. Ces antigènes et les épitopes qui en sont dérivés peuvent être utilisés pour la vaccination. De nombreux vaccins basés sur l injection de peptide-épitopes ont été testés dans le cadre d études cliniques. Des régressions tumorales sont observées dans une minorité de cas. Une nouvelle stratégie de vaccination a été développée grâce à l utilisation des propriétés des cellules dendritiques comme cellules présentatrices d antigènes. Ces cellules chargées avec des protéines entières améliorent la réponse immune en élargissant l éventail de peptides présentés aux lymphocytes T. Pour maintenir la réponse anti-tumorale, les lymphocytes CD4+ et CD8+ doivent être activés. Cependant, la présentation de peptides dérivés d antigènes exogènes solubles sur les molécules du CMH de classe I est très faible. Nous proposons d augmenter la captation d antigènes par les cellules dendritiques en couplant ceux-ci à une protéine vectrice capable de lier la membrane de ces cellules. Le mutant H34Q de la phospholipase A2 de venin d abeille (bvPLA2H34Q) a été choisi pour cela. Des cellules dendritiques humaines ont été chargées avec la protéine de fusion PNY dans laquelle l antigène tumoral NY-ESO-1 a été couplé à bvPLA2H34Q. PNY augmente la présentation des épitopes de NY-ESO-1 par les molécules du CMH de classe II et favorise l activation de lymphocytes T CD8+ spécifiques de NY-ESO-1 capables de reconnaître des cellules de mélanome humain.The body s ability to develop an immune reaction against tumours was discovered with the tumour associated antigens. These proteins, present on the surface of tumour cells, can be detected by cell-mediated immunity. Different categories of tumour antigens which induce cytotoxic T lymphocyte responses in vitro and in vivo have been identified. Some of them, namely "cancer testis" antigens, are expressed in different tumours and normal testis. From the highly tissue-restricted expression profile, this expanding family of cancer/testis antigens is a potential target for cancer vaccines. In this study, three cancer/testis antigens are characterised for their potential ability to induce CD4+ T cells : NY-ESO-1, the LAGE-1 ORF2 protein and the expression product of the Trag-3 gene. A new MHC class II restricted peptide derived from NY-ESO-1, NY-ESO-187-111, was identified. The peptide binds to the MHC class II molecules covering 87% of the Caucasians. These antigens and this peptide may be promising candidates for cancer specific immunotherapy. The overall goal of tumour vaccines is to elicit, restore or augment anti-tumour immune responses. Clinical studies have shown that dendritic cell vaccines have the best potential among all other cancer vaccines to induce signals and change the immune response against tumours. Moreover, an immune response against a broad range of HLA I and HLA II epitopes is required to maximize the efficiency of vaccines. In order to increase and maintain an antigen specific tumour reactive response, without the need of restricting the target population by HLA type, the choice of dendritic cells loaded with full size tumour antigens is recommended. Here, we have demonstrated that the use of PNY (recombinant protein made of the fusion of bvPLA2 mutant H34Q with NY-ESO-1) favours the restricted HLA II peptides presentation, the priming of CD4+ T cells and promotes the cross-priming of CD8+ T cells specific for NY-ESO-1. Dendritic cells loaded with PNY are able to prime and to activate CD8+ T cells which are capable of recognizing human melanoma cellsPARIS-Museum Hist.Naturelle (751052304) / SudocSudocFranceF

Bioinformatique et protéomique, une alliance à haute valeur ajoutée pour mieux explorer la variabilité biologique intra-population et interespèces

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Discriminating sub-population responses of a mixture of human cell lines by proteogenomics

International audienceMonitoring proteome dynamics from different human cell types present concomitantly in a given sample is of great interest and could be applied to ultra-precise molecular characterization of complex tissues. Here, we propose a proteogenomics-based strategy to point at cell line molecular signatures. For this, the proteome is analyzed by high-throughput shotgun mass spectrometry and specific bioinformatics search are performed. First, mRNA from chondrosarcoma cells (SW1353 cell line) and immortalized chondrocytes (T/C28A2 cell line) were sequenced by RNAseq for establishing the most appropriate protein sequence database. For this an innovative cascade search allows to conciliate de novo and mapping RNAseq assemblies and the Human swissprot databases (Cogne et al., 2018). A set of 2 million of discriminating peptide sequences of the two cell lines are then identified. From them, 480 peptide sequences were detected and monitored based on extracted ion chromatogram (XIC) signals recorded by tandem mass spectrometry. A list of 55 peptides were used for quantitating the ratio of each cell type in a given co-culture sample with high precision selected with cell lines mixed at 2:1, 1:1; and 1:2 ratio. This new methodology was used to analyze the bystander effect generated by irradiated chondrosarcoma cells (SW1353 cell line) on immortalized chondrocytes (T/C28A2 cell line) in co-culture conditions. Such strategy could be applied to investigate intercellular interactions between different cell types, paving the way to new insights into the molecular mechanisms of crosstalk between human cells

Proteogénomique pour le developpement des biomarqueurs pour la surveillance ecotoxicologique des environnements aquatiques

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Proteogénomique pour le developpement des biomarqueurs pour la surveillance ecotoxicologique des environnements aquatiques

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P47. Proteomic analysis of the Radiation-Induced Bystander Effects between low-dose irradiated Chondrosarcoma cells and Bystander Chondrocytes

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The alliance of proteogenomics & multiplexed targeted ecotoxicoproteomics for environmental monitoring of river water quality

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