Introduction Prox1 (Prospero homeobox 1) est un facteur de transcription essentiel dans le développement vasculaire lymphatique et les morphogenèses pancréatique et hépatique. Des polymorphismes du ...gène PROX1 sont associés à des niveaux de glycémie plus élevés, une plus faible sécrétion d’insuline (index HOMA-B) et un risque plus élevé de diabète de type 2 dans différents échantillons de population. Cependant, les mécanismes moléculaires reliant Prox1 au diabète de type 2 ne sont pas connus. Nous avons précédemment montré que Prox1 participe à la sécrétion d’insuline induite par le glucose dans les cellules INS1-E. Le but de cette étude est d’identifier les facteurs agissant en aval de Prox1 dans les cellules INS1-E. Matériels et méthodes Nous avons réalisé des expériences transcriptomiques (puce Agilent 4 * 44K Rat Gene Expression Microarrays) pour comparer le niveau d’expression des gènes des cellules INS1-E transfectées avec des siRNA anti-Prox1 ou contrôles. Les gènes différentiellement exprimés ont été filtrés pour une valeur de p ajustée < 0,05. Les données ont ensuite été intégrées en utilisant le logiciel Ingenuity Pathway Analysis afin d’identifier les principales voies métaboliques impliquées. Résultats L’analyse a révélé que 60 gènes étaient significativement sur-exprimés (0,52 < Log2 Fold Change < 2,36) et 37 gènes sous-exprimés (– 0,50 < Log2 Fold Change < -1,41) à la suite de l’inhibition de l’expression de Prox1. Nous avons vérifié la modulation de l’expression de 15 de ces 97 gènes par qRT-PCR et tous ont été confirmés. Nous avons observé qu’il y a respectivement 4 et 5 voies canoniques significativement impliquées dans le métabolisme du glucose et la sécré-tion d’insuline. Les données détaillées seront présentées lors du congrès. Conclusion Ce travail nous a permis d’identifier une centaine de gènes régulés par Prox1 dans les cellules bêta-pancréatiques qui pourraient être impliqués dans les fonctions de Prox1 et expliquer son association avec le diabète de type 2. Déclaration d’intérêt Les auteurs déclarent ne pas avoir d’intérêt direct ou indirect (financier ou en nature) avec un organisme privé, industriel ou commercial en relation avec le sujet présenté.
Prostate cancer (PCa) is one of the major public health problems in Western countries. Recently, the TMPRSS2:ERG gene fusion, which results in the aberrant expression of the transcription factor ERG, ...has been shown to be the most common gene rearrangement in PCa. Previous studies have determined the contributions of this fusion in PCa disease initiation and/or progression in vitro and in vivo. In this study on TMPRSS2:ERG regulation in PCa, we used an androgen receptor and TMPRSS2:ERG fusion double-negative PCa cell model: PC3c. In three cell clones with different TMPRSS2:ERG expression levels, ectopic expression of the fusion resulted in significant induction of cell migration and invasion in a dose-dependent manner. In agreement with this phenotype, high-throughput microarray analysis revealed that a set of genes, functionally associated with cell motility and invasiveness, were deregulated in a dose-dependent manner in TMPRSS2:ERG-expressing cells. Importantly, we identified increased MMP9 (Metalloproteinase 9) and PLXNA2 (Plexin A2) expression in TMPRSS2:ERG-positive PCa samples, and their expression levels were significantly correlated with ERG expression in a PCa cohort. In line with these findings, there was evidence that TMPRSS2:ERG directly and positively regulates MMP9 and PLXNA2 expression in PC3c cells. Moreover, PLXNA2 upregulation contributed to TMPRSS2:ERG-mediated enhancements of PC3c cell migration and invasion. Furthermore, and importantly, PLXNA2 expression was upregulated in metastatic PCa tumors compared with localized primary PCa tumors. This study provides novel insights into the role of the TMPRSS2:ERG fusion in PCa metastasis.
The only recognized genetic determinant of the common forms of Alzheimer's disease (AD) is the epsilon 4 allele of the apolipoprotein E gene (APOE). To identify new candidate genes, we recently ...performed transcriptomic analysis of 2741 genes in chromosomal regions of interest using brain tissue of AD cases and controls. From 82 differentially expressed genes, 1156 polymorphisms were genotyped in two independent discovery subsamples (n=945). Seventeen genes exhibited at least one polymorphism associated with AD risk, and following correction for multiple testing, we retained the interleukin (IL)-33 gene. We first confirmed that the IL-33 expression was decreased in the brain of AD cases compared with that of controls. Further genetic analysis led us to select three polymorphisms within this gene, which we analyzed in three independent case-control studies. These polymorphisms and a resulting protective haplotype were systematically associated with AD risk in non-APOE epsilon 4 carriers. Using a large prospective study, these associations were also detected when analyzing the prevalent and incident AD cases together or the incident AD cases alone. These polymorphisms were also associated with less cerebral amyloid angiopathy (CAA) in the brain of non-APOE epsilon 4 AD cases. Immunohistochemistry experiments finally indicated that the IL-33 expression was consistently restricted to vascular capillaries in the brain. Moreover, IL-33 overexpression in cellular models led to a specific decrease in secretion of the A beta(40) peptides, the main CAA component. In conclusion, our data suggest that genetic variants in IL-33 gene may be associated with a decrease in AD risk potentially in modulating CAA formation.
Antigen-presenting cell (APC) activation is enhanced by vaccine adjuvants. Most vaccines are based on the assumption that adjuvant activity of Toll-like receptor (TLR) agonists depends on direct, ...functional activation of APCs. Here, we sought to establish whether TLR stimulation in non-hematopoietic cells contributes to flagellin's mucosal adjuvant activity. Nasal administration of flagellin enhanced T-cell-mediated immunity, and systemic and secretory antibody responses to coadministered antigens in a TLR5-dependent manner. Mucosal adjuvant activity was not affected by either abrogation of TLR5 signaling in hematopoietic cells or the presence of flagellin-specific, circulating neutralizing antibodies. We found that flagellin is rapidly degraded in conducting airways, does not translocate into lung parenchyma and stimulates an early immune response, suggesting that TLR5 signaling is regionalized. The flagellin-specific early response of lung was regulated by radioresistant cells expressing TLR5 (particularly the airway epithelial cells). Flagellin stimulated the epithelial production of a small set of mediators that included the chemokine CCL20, which is known to promote APC recruitment in mucosal tissues. Our data suggest that (i) the adjuvant activity of TLR agonists in mucosal vaccination may require TLR stimulation of structural cells and (ii) harnessing the effect of adjuvants on epithelial cells can improve mucosal vaccines.
We demonstrate non-intrusive, in situ detection of ammonia (NH sub(3)) in reactive hot gas flows at atmospheric pressure using mid-infrared degenerate four-wave mixing (IR-DFWM). IR-DFWM excitation ...scans were performed in the v sub(2)+v sub(3) and v sub(1)+v sub(2) vibrational bands of NH sub(3) around 2.3 mu m for gas flow temperatures of 296, 550 and 820K. Simulations based on spectroscopic parameters from the HITRAN database have been compared with the measurements in order to identify the spectral lines, and an absorption spectrum at 296K has also been measured to compare with the IR-DFWM spectra. The signal-to-noise ratio of the IR-DFWM measurement was found to be higher than that of the absorption measurement. Some spectral lines in the measured IR-DFWM and absorption spectra had no matching lines in the HITRAN simulation. The detection limit of NH sub(3) diluted in N sub(2) with IR-DFWM in this spectral range was estimated at 296, 550 and 820K to be 1.36, 4.87 and 7.0610 super(16)molecules/cm super(3). The dependence of the NH sub(3) IR-DFWM signal on the quenching properties of the buffer gas flow was investigated by comparing the signals for gas flows of N sub(2), Ar and CO sub(2) with small admixtures of NH sub(3). It was found that the signal dependence on buffer gas was large at room temperature but decreased at elevated temperatures. These results show the potential of IR-DFWM for detection of NH sub(3) in combustion environments. The potential of infrared degenerate four-wave mixing (IR-DFWM) as a tool for non-intrusive in situ spatially resolved detection of ammonia (NH sub(3)) in combustion environments is investigated. IR-DFWM excitation scans have been successfully recorded, by probing ro-vibrational transitions belonging to the v sub(2)+v sub(3) and v sub(1)+v sub(2) combination bands around 2.3 mu m, in gas flows of NH sub(3) diluted in nitrogen at atmospheric pressure and elevated temperatures up to 820K.
In the present study, we aimed to determine whether the concentrations of somatic coliphages, infectious enteroviruses or the detection of enterovirus genomes were associated with the detection of ...human pathogenic viruses in surface water. Four French rivers were sampled monthly or semimonthly for the quantitative detection of somatic coliphages, infectious enteroviruses and the qualitative RT-PCR detection of enterovirus, hepatitis A virus, Norwalk I viruses, Norwalk II viruses, astrovirus and rotavirus genomes over 12 months. All the 68 water samples tested were positive for the quantitative detection of somatic coliphages (range of concentrations: 4×10
2 to 1.6×10
5
PFU
l
−1). Infectious enteroviruses were isolated by a cell culture system in only two (3%) of the 68 concentrated water samples tested, whereas enterovirus genomes were detectable in 60 (88%) of the same samples. A positive RT-PCR detection of the genome of hepatitis A virus, Norwalk-like virus genogroup II, astrovirus, rotavirus and Norwalk-like virus genogroup I was demonstrated, respectively, in 1.5% (1/68), 1.5% (1/68), 3% (2/68), 0% and 0% of the 68 concentrated water samples tested. All of these four water samples were positive for the detection of enterovirus genomes, whereas only one of them was positive for the isolation of enteroviruses on cell culture. Moreover, the genomic detection of human pathogenic viruses appeared not to be statistically associated with the concentration levels of somatic coliphages in the 68 concentrated water samples tested (Wilcoxon rank test;
P=0.14). Taken together, our findings indicate that the quantitative detection of somatic coliphages and the isolation of enteroviruses on cell culture are not suitable parameters for the control of the viral contamination in surface water, whereas the detection of enterovirus genomes may be useful for predicting the presence of waterborne viruses.
To more rapidly identify candidate genes located within chromosomal regions of interest defined by genome scan studies in Alzheimer's disease (AD), we have developed a customized microarray ...containing all the ORFs (n=2741) located within nine of these regions. Levels of gene expression were assessed in total RNA from brain tissue of 12 controls and 12 AD patients. Of all genes showing differential expression, we focused on the ornithine transcarbamylase (OTC) gene on Xp21.1., a key enzyme of the urea cycle which we found to be expressed in AD brains but not in controls, as confirmed by RT-PCR. We also detected mRNA expression of all the other urea cycle enzymes in AD brains. Immunochemistry experiments revealed that the OTC expression was strictly restricted to vascular endothelial cells in brain. Furthermore, OTC activity was 880% increased in the CSF of probable AD cases compared with controls. We analysed the association of the OTC -389 G/A and -241 A/G promoter polymorphisms with the risk of developing AD. We observed that rare haplotypes may be associated with the risk of AD through a possible modulation of the methylation of the OTC promoter. In conclusion, our results suggest the involvement of a new pathway in AD brains involving the urea cycle.
In order to distinguish chilling and freezing tolerance mechanisms in pea, responses to cold exposure were compared between the freezing tolerant line Champagne and the sensitive line Terese. Global ...gene expression was considered in the two lines and associated with morphological, histological and biochemical approaches. The chilling tolerance in both lines was related to responses of the CBF, COR and LEA genes belonging to the CBF regulon, with greater earliness of expression in the Champagne genotype. The freezing tolerance, only observed in Champagne, was associated with acclimation processes such as cellular osmotic stabilization, photosynthesis modifications, antioxidants production, modifications in hormone metabolism, cell wall composition and dynamics.
► Cold-responses in pea were identified by transcriptome analyses, histological and biochemical observations. ► Chilling tolerance mechanisms were detected in both freezing tolerant and sensitive pea lines. ► Freezing tolerance mechanisms were observed in tolerant line as acclimation processes.
Bug proteins form a large family of periplasmic solute-binding proteins well represented in β-proteobacteria. They adopt a characteristic Venus flytrap fold with two globular domains bisected by a ...ligand-binding cleft. The structures of two liganded Bug proteins have revealed that the family is specific for carboxylated solutes, with a characteristic mode of binding involving two highly conserved β strand-β turn-α helix motifs originating from each domain. These two motifs form hydrogen bonds with a carboxylate group of the ligand, both directly and
via conserved water molecules, and have thus been termed the carboxylate pincers. In both crystallized Bug proteins, the ligands were found enclosed between the two domains and inaccessible to solvent, suggesting an inter-domain hinge-bending motion upon ligand binding. We report here the first structures of an open, unliganded Bug protein and of the same protein with a citrate ion bound in the open cavity. One of the ligand carboxylate groups is bound to one half of the carboxylate pincers by the β strand-β turn-α helix motif from domain 1, and the citrate ion forms several additional interactions with domain 1. The ligand is accessible to solvent and has very few contacts with domain 2. In this open, liganded structure, the second part of the carboxylate pincers originating from domain 2 is not stabilized by ligand binding, and a loop replaces the β turn. In the unliganded structure, both motifs of the carboxylate pincers are highly mobile, and neither of the two β turns is formed. Thus, ligand recognition is performed by domain 1, with the carboxylate group serving as an initial anchoring point. Stabilization of the closed conformation requires proper interactions to be established with domain 2, and thus domain 2 discriminates between productively and non-productively bound ligands.
Antibiotic resistance is one of the biggest threats to human health globally. Alarmingly, multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis have now spread worldwide. Some ...key antituberculosis antibiotics are prodrugs, for which resistance mechanisms are mainly driven by mutations in the bacterial enzymatic pathway required for their bioactivation. We have developed drug-like molecules that activate a cryptic alternative bioactivation pathway of ethionamide in M. tuberculosis, circumventing the classic activation pathway in which resistance mutations have now been observed. The first-of-its-kind molecule, named SMARt-420 (Small Molecule Aborting Resistance), not only fully reverses ethionamide-acquired resistance and clears ethionamide-resistant infection in mice, it also increases the basal sensitivity of bacteria to ethionamide.
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