Development of a new bicistronic retroviral vector with strong IRES activity

BACKGROUND: Internal Ribosome Entry Site (IRES)-based bicistronic vectors are important tools in today's cell biology. Among applications, the expression of two proteins under the control of a unique promoter permits the monitoring of expression of a protein whose biological function is being investigated through the observation of an easily detectable tracer, such as Green Fluorescent Protein (GFP). However, analysis of published results making use of bicistronic vectors indicates that the efficiency of the IRES-controlled expression can vary widely from one vector to another, despite their apparent identical IRES sequences. We investigated the molecular basis for these discrepancies. RESULTS: We observed up to a 10 fold difference in IRES-controlled expression from distinct bicistronic expression vectors harboring the same apparent IRES sequences. We show that the insertion of a HindIII site, in place of the initiating AUG codon of the wild type EMCV IRES, is responsible for the dramatic loss of expression from the second cistron, whereas expression from the first cistron remains unaffected. Thus, while the replacement of the authentic viral initiating AUG by a HindIII site results in the theoretical usage of the initiation codon of the HindIII-subcloned cDNA, the subsequent drop of expression dramatically diminishes the interest of the bicistronic structure. Indeed, insertion of the HindIII site has such a negative effect on IRES function that detection of the IRES-controlled product can be difficult, and sometimes even below the levels of detection. It is striking to observe that this deleterious modification is widely found in available IRES-containing vectors, including commercial ones, despite early reports in the literature stating the importance of the integrity of the initiation codon for optimal IRES function. CONCLUSION: From these observations, we engineered a new vector family, pPRIG, which respects the EMCV IRES structure, and permits easy cloning, tagging, sequencing, and expression of any cDNA in the first cistron, while keeping a high level of expression from its IRES-dependent second cistron (here encoding eGFP)

Characterization of human mesenchymal stem cell secretome at early steps of adipocyte and osteoblast differentiation

<p>Abstract</p> <p>Background</p> <p>It is well established that adipose tissue plays a key role in energy storage and release but is also a secretory organ and a source of stem cells. Among different lineages, stem cells are able to differentiate into adipocytes and osteoblasts. As secreted proteins could regulate the balance between both lineages, we aimed at characterizing the secretome of human multipotent adipose-derived stem cell (hMADS) at an early step of commitment to adipocytes and osteoblasts.</p> <p>Results</p> <p>A proteomic approach, using mono-dimensional electrophoresis and tandem mass spectrometry, allowed us to identify a total of 73 proteins at day 0 and day 3 of adipocyte and osteoblast differentiation. Analysis of identified proteins showed that 52 % corresponded to classical secreted proteins characterized by a signal peptide, that 37 % previously described in the extracellular compartment were devoid of signal peptide and that 11 % neither exhibited a signal peptide nor had been previously described extracellularly. These proteins were classified into 8 clusters according to their function. Quantitative analysis has been performed for 8 candidates: PAI-1, PEDF, BIGH3, PTX3, SPARC, ENO1, GRP78 and MMP2. Among them, PAI-1 was detected at day 0 and day 3 of osteoblast differentiation but never in adipocyte secretome. Furthermore we showed that PAI-1 mRNA was down-regulated in the bone of ovariectomized mice.</p> <p>Conclusion</p> <p>Given its regulation during the early events of hMADS cell differentiation and its status in ovariectomized mice, PAI-1 could play a role in the adipocyte/osteoblast balance and thus in bone diseases such as osteoporosis.</p

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements

Inflammation, tissu adipeux et obésité (participation des voies TLR4 et CD40)

L obésité s accompagne d un état micro-inflammatoire chronique systémique et tissulaire. De nombreuses études montrent que l inflammation contribue à l état de résistance à l insuline, au diabète de type 2, à la stéatose hépatique et à l athérothrombose. Les mécanismes par lesquels l inflammation altère la signalisation insulinique et induit un état de résistance sont, à l heure actuelle, assez bien connus, mais peu de données sont disponibles quant aux mécanismes initiateurs de l inflammation au cours de l obésité. Le contact de l organisme avec des pathogènes, véhiculés par l air, l eau ou les aliments, est un moyen commun d initiation de l inflammation. Nous avons étudié l importance des principaux récepteurs de l immunité innée impliqués dans la signalisation du LPS, le CD14 et le TLR4, dans le développement de l obésité et de ses complications métaboliques. Nous montrons qu au cours d un régime riche en lipides les taux circulants de LPS augmentent et définissent l endotoxémie métabolique (50 fois plus faible que celle observée en phase septique). La perfusion de doses métaboliques de LPS mime les effets obtenus lors d un régime gras en induisant : une inflammation, une prise de poids et une résistance à l insuline hépatique. Ces effets sont prévenus en absence de CD14. Lors d un régime gras, l absence de CD14 ou de TLR4 fonctionnel réduit l état inflammatoire, améliore la sensibilité à l insuline du tissu adipeux et protège de la stéatose hépatique. A coté des voies d initiation de l inflammation impliquant les récepteurs de l immunité innée, les lymphocytes T peuvent contribuer à l initiation de l état inflammatoire au cours de l obésité. Nous avons étudié la contribution des lymphocytes T et de la voie du CD40/CD40L à l état inflammatoire du tissu adipeux et le rôle qu ils pourraient jouer dans le développement des complications de l obésité. Nous montrons l augmentation du nombre de lymphocytes T dans le tissu adipeux de sujets obèses par comparaison à des sujets de poids normal et dans un système de coculture, une coopération entre les lymphocytes T et les adipocytes humains conduisant à la production de cytokines inflammatoires. Cet effet est du à la fois aux produits sécrétés par les lymphocytes T et au contact entre les deux types cellulaires. Le système CD40/40L pourrait être impliqué dans ces phénomènes. En effet, pour la première fois, nous montrons que l adipocyte possède un récepteur CD40 fonctionnel dont la stimulation induit une inflammation et altère le métabolisme adipocytaire. L ensemble de ces résultats indique que la voie de réponse aux pathogènes (LPS) contribue au développement de l état inflammatoire et aux complications métaboliques associées à l obésité. Les lymphocytes T, tout comme cela a été montré pour les macrophages,contribuent à l état inflammatoire du tissu adipeux et pourraient, via le système CD40/CD40L, altérer le métabolisme adipocytaire.AIX-MARSEILLE2-BU Méd/Odontol. (130552103) / SudocSudocFranceF

Les activités d'élevage porcin en Corse. Des difficultés de la transition entre économie souterraine et économie formelle

International audienc

Modulation of T Cell Activation in Obesity

International audienceSIGNIFICANCE: Immune T cells are present in adipose tissues (AT), and the stoichiometry of the different T cell subsets is altered during diet-induced obesity (DIO). T cells contribute to the early steps of AT inflammation during DIO. Recent Advances: Many factors could potentially be responsible for this altered pro-inflammatory versus anti-inflammatory T cell balance.CRITICAL ISSUES: T cells are potentially activated in AT, which vitamin D might contribute to, as will be discussed in this article. In addition, we will review the different possible contributors to T cell activation in AT, such as the CD28 and CD154 T cell costimulatory molecules in AT.FUTURE DIRECTIONS: The potential antigen presentation capacities of adipocytes should be further investigated. Moreover, the properties of these AT resident (or migrating to AT) T cells must be further assessed. Antioxid. Redox Signal. 26, 489-500