Induction of a chemoattractant transcriptional response by a Campylobacter jejuni boiled cell extract in colonocytes

<p>Abstract</p> <p>Background</p> <p><it>Campylobacter jejuni</it>, the commonest cause of bacterial diarrhoea worldwide, can also induce colonic inflammation. To understand how a previously identified heat stable component contributes to pro-inflammatory responses we used microarray and real-time quantitative PCR to investigate the transcriptional response to a boiled cell extract of <it>Campylobacter jejuni </it>NCTC 11168.</p> <p>Results</p> <p>RNA was extracted from the human colonocyte line HCA-7 (clone 29) after incubation for 6 hours with <it>Campylobacter jejuni </it>boiled cell extract and was used to probe the Affymetrix Human Genome U133A array. Genes differentially affected by <it>Campylobacter jejuni </it>boiled cell extract were identified using the Significance Score algorithm of the Bioconductor software suite and further analyzed using the Ingenuity Pathway Analysis program. The chemokines CCL20, CXCL3, CXCL2, Interleukin 8, CXCL1 and CXCL6 comprised 6 of the 10 most highly up-regulated genes, all with Significance Scores ≥ 10. Members of the Tumor Necrosis Factor α/Nuclear Factor-κB super-family were also significantly up-regulated and involved in the most significantly regulated signalling pathways (Death receptor, Interleukin 6, Interleukin 10, Toll like receptor, Peroxisome Proliferator Activated Receptor-γ and apoptosis). Ingenuity Pathway Analysis also identified the most affected functional gene networks such as cell movement, gene expression and cell death. In contrast, down-regulated genes were predominantly concerned with structural and metabolic functions.</p> <p>Conclusion</p> <p>A boiled cell extract of <it>Campylobacter jejuni </it>has components that can directly switch the phenotype of colonic epithelial cells from one of resting metabolism to a pro-inflammatory one, particularly characterized by increased expression of genes for leukocyte chemoattractant molecules.</p

Caractérisation du protéome vascuolaire de la plante modèle Arabidopsis thaliana et étude de son rôle dans la détoxication du cadmium

To better understand the mechanisms governing cellular traffic, transport process of substrates across the tonoplast, storage of various metabolites and their ultimate degradation, a comprehensive and thorough analysis of Arabidopsis thaliana vacuolar proteome was performed. Protein subcellular localization knowledge is an important step toward assigning functions of organelles and plant metabolism compartmentation. But confident description of proteome organelle content requires clear identification of the true resident proteins of the studied compartment. This task involves pitfalls and requires that either organelle preparations are free of contaminants or that techniques are used to discriminate between genuine organelle residents and contaminating proteins. To achieve this, vacuoles purification protocol from protoplasts on a Ficoll density gradient has been improved. The combination of several proteomic approaches attempt to present soluble and membrane vacuolar proteins in a quantitative and functional manner. Different approaches have thus shown associations and complex molecular mechanisms that govern the various vacuolar activities. The constitute proteins library provides references to study the vacuolar proteome dynamics in response to different stresses including heavy metals. Many methods without a priori or targeted were proposed to study the impact of cadmium on the vacuole, the key cell compartment of detoxification. Proteomics provides powerful tools for characterizing the protein contents of vacuoles during cadmium stress.Afin de mieux comprendre les mécanismes du trafic cellulaire, les processus de transport des substrats vacuolaires à travers le tonoplaste, le stockage des métabolites et leur dégradation, une analyse globale et exhaustive du protéome vacuolaire d'Arabidopsis thaliana a été réalisée. La connaissance de la localisation subcellulaire des protéines permet de mieux comprendre la fonction des organelles et la compartimentation du métabolisme des plantes. Mais la description précise du protéome d'un organite nécessite d'identifier clairement les véritables protéines résidantes du compartiment étudié. Une tâche si précise est complexe puisqu'elle nécessite la mise en place d'une préparation d'organites purs et homogènes. Pour y parvenir, un protocole de purification de vacuoles à partir de protoplastes isolés de cellules en culture sur un gradient de densité de Ficoll a été amélioré. La combinaison de plusieurs approches de protéomique a permis d'identifier les protéines présentes dans les fractions vacuolaires soluble et membranaire de façon quantitative et fonctionnelle. Les différentes approches ont ainsi mis en évidence des associations et mécanismes moléculaires complexes qui régissent les différentes activités vacuolaires. Cette protéothèque de référence constitue une base pour étudier la dynamique du protéome vacuolaire en réponse à plusieurs stress incluant les métaux lourds. Plusieurs méthodes sans a priori et ciblée ont été proposé afin d'étudier l'impact du cadmium sur la vacuole, ce compartiment cellulaire clé de la détoxication

Caractérisation du protéome vascuolaire de la plante modèle Arabidopsis thaliana et étude de son rôle dans la détoxication du cadmium

Afin de mieux comprendre les mécanismes du trafic cellulaire, les processus de transport des substrats vacuolaires à travers le tonoplaste, le stockage des métabolites et leur dégradation, une analyse globale et exhaustive du protéome vacuolaire d'Arabidopsis thaliana a été réalisée. La connaissance de la localisation subcellulaire des protéines permet de mieux comprendre la fonction des organelles et la compartimentation du métabolisme des plantes. Mais la description précise du protéome d'un organite nécessite d'identifier clairement les véritables protéines résidantes du compartiment étudié. Une tâche si précise est complexe puisqu'elle nécessite la mise en place d'une préparation d'organites purs et homogènes. Pour y parvenir, un protocole de purification de vacuoles à partir de protoplastes isolés de cellules en culture sur un gradient de densité de Ficoll a été amélioré. La combinaison de plusieurs approches de protéomique a permis d'identifier les protéines présentes dans les fractions vacuolaires soluble et membranaire de façon quantitative et fonctionnelle. Les différentes approches ont ainsi mis en évidence des associations et mécanismes moléculaires complexes qui régissent les différentes activités vacuolaires. Cette protéothèque de référence constitue une base pour étudier la dynamique du protéome vacuolaire en réponse à plusieurs stress incluant les métaux lourds. Plusieurs méthodes sans a priori et ciblée ont été proposé afin d'étudier l'impact du cadmium sur la vacuole, ce compartiment cellulaire clé de la détoxication.To better understand the mechanisms governing cellular traffic, transport process of substrates across the tonoplast, storage of various metabolites and their ultimate degradation, a comprehensive and thorough analysis of Arabidopsis thaliana vacuolar proteome was performed. Protein subcellular localization knowledge is an important step toward assigning functions of organelles and plant metabolism compartmentation. But confident description of proteome organelle content requires clear identification of the true resident proteins of the studied compartment. This task involves pitfalls and requires that either organelle preparations are free of contaminants or that techniques are used to discriminate between genuine organelle residents and contaminating proteins. To achieve this, vacuoles purification protocol from protoplasts on a Ficoll density gradient has been improved. The combination of several proteomic approaches attempt to present soluble and membrane vacuolar proteins in a quantitative and functional manner. Different approaches have thus shown associations and complex molecular mechanisms that govern the various vacuolar activities. The constitute proteins library provides references to study the vacuolar proteome dynamics in response to different stresses including heavy metals. Many methods without a priori or targeted were proposed to study the impact of cadmium on the vacuole, the key cell compartment of detoxification. Proteomics provides powerful tools for characterizing the protein contents of vacuoles during cadmium stress.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

IDENTIFYING THE MOLECULAR MECHANISMS OF EARLY CACHEXIA USING WHOLE TRANSCRIPTOME SEQUENCING IN MUSCLE AND FAT BIOPSIES FROM CANCER PATIENTS

Cancer cachexia is responsible for one third of cancer–related deaths and contributes to the death of many others. More than 80% of cancer patients are cachectic towards the end of life. Despite intensive research, the mechanisms of cancer cachexia are still poorly understood. It is our hypothesis that identification of early changes in gene expression in cachexia will lead to an improved understanding of the mechanism that trigger this important problem in cancer patients. Thus, to shed light on the mechanisms involved in the major cachexia target tissues, we investigated the entire transcriptome in muscle and fat to identify altered expression of genes in cancer patients with and without cachexia. Samples of rectus abdominis muscle and visceral fat were collected at surgery from patients exhibiting 5-10% weight loss prior to surgery, compared with stable-weight patients. Analysis of all expressed genes was carried out using next generation sequencing (Illumina HiSeq 2500). Also, selected differentially expressed genes were confirmed using real time RT-PCR. In muscle, 30 genes showed highly significant changes in expression (25 downregulated and 5 upregulated: P In visceral fat, expression of 6 genes were downregulated and 10 upregulated with high statistical significance (

Mitochondrial Dynamics Controls T Cell Fate Through Metabolic Programming

Activated effector T (TE) cells augment anabolic pathways of metabolism, such as aerobic glycolysis, while memory T (TM) cells engage catabolic pathways, like fatty acid oxidation (FAO). However, signals that drive these differences remain unclear. Mitochondria are metabolic organelles that actively transform their ultrastructure. Therefore, we questioned whether mitochondrial dynamics controls T cell metabolism. We show that TE cells have punctate mitochondria, while TM cells maintain fused networks. The fusion protein Opa1 is required for TM, but not TE cells after infection, and enforcing fusion in TE cells imposes TM cell characteristics and enhances antitumor function. Our data suggest that, by altering cristae morphology, fusion in TM cells configures electron transport chain (ETC) complex associations favoring oxidative phosphorylation (OXPHOS) and FAO, while fission in TE cells leads to cristae expansion, reducing ETC efficiency and promoting aerobic glycolysis. Thus mitochondrial remodeling is a signaling mechanism that instructs T cell metabolic programming

Proteostasis in T cell aging

Aging leads to a decline in immune cell function, which leaves the organism vulnerable to infections and age-related multimorbidities. One major player of the adaptive immune response are T cells, and recent studies argue for a major role of disturbed proteostasis contributing to reduced function of these cells upon aging. Proteostasis refers to the state of a healthy, balanced proteome in the cell and is influenced by synthesis (translation), maintenance and quality control of proteins, as well as degradation of damaged or unwanted proteins by the proteasome, autophagy, lysosome and cytoplasmic enzymes. This review focuses on molecular processes impacting on proteostasis in T cells, and specifically functional or quantitative changes of each of these upon aging. Importantly, we describe the biological consequences of compromised proteostasis in T cells, which range from impaired T cell activation and function to enhancement of inflamm-aging by aged T cells. Finally, approaches to improve proteostasis and thus rejuvenate aged T cells through pharmacological or physical interventions are discussed