PAX4 preserves endoplasmic reticulum integrity preventing beta cell degeneration in a mouse model of type 1 diabetes mellitus

Mellado-Gil, José Manuel et al.[Aims/hypothesis]: A strategy to enhance pancreatic islet functional beta cell mass (BCM) while restraining inflammation, through the manipulation of molecular and cellular targets, would provide a means to counteract the deteriorating glycaemic control associated with diabetes mellitus. The aims of the current study were to investigate the therapeutic potential of such a target, the islet-enriched and diabetes-linked transcription factor paired box 4 (PAX4), to restrain experimental autoimmune diabetes (EAD) in the RIP-B7.1 mouse model background and to characterise putative cellular mechanisms associated with preserved BCM. [Methods]: Two groups of RIP-B7.1 mice were genetically engineered to: (1) conditionally express either PAX4 (BPTL) or its diabetes-linked mutant variant R129W (mutBPTL) using doxycycline (DOX); and (2) constitutively express luciferase in beta cells through the use of RIP. Mice were treated or not with DOX, and EAD was induced by immunisation with a murine preproinsulin II cDNA expression plasmid. The development of hyperglycaemia was monitored for up to 4 weeks following immunisation and alterations in the BCM were assessed weekly by non-invasive in vivo bioluminescence intensity (BLI). In parallel, BCM, islet cell proliferation and apoptosis were evaluated by immunocytochemistry. Alterations in PAX4- and PAX4R129W-mediated islet gene expression were investigated by microarray profiling. PAX4 preservation of endoplasmic reticulum (ER) homeostasis was assessed using thapsigargin, electron microscopy and intracellular calcium measurements. [Results]: PAX4 overexpression blunted EAD, whereas the diabetes-linked mutant variant PAX4R129W did not convey protection. PAX4-expressing islets exhibited reduced insulitis and decreased beta cell apoptosis, correlating with diminished DNA damage and increased islet cell proliferation. Microarray profiling revealed that PAX4 but not PAX4R129W targeted expression of genes implicated in cell cycle and ER homeostasis. Consistent with the latter, islets overexpressing PAX4 were protected against thapsigargin-mediated ER-stress-related apoptosis. Luminal swelling associated with ER stress induced by thapsigargin was rescued in PAX4-overexpressing beta cells, correlating with preserved cytosolic calcium oscillations in response to glucose. In contrast, RNA interference mediated repression of PAX4-sensitised MIN6 cells to thapsigargin cell death. [Conclusions/interpretation]: The coordinated regulation of distinct cellular pathways particularly related to ER homeostasis by PAX4 not achieved by the mutant variant PAX4R129W alleviates beta cell degeneration and protects against diabetes mellitus. The raw data for the RNA microarray described herein are accessible in the Gene Expression Omnibus database under accession number GSE62846.This work was funded by grants from the Consejeria de Salud, Fundacion Publica Andaluza Progreso y Salud, Junta de Andalucia (PI-0727-2010 to BRG and PI-0085-2013 to PIL), Consejeria de Economia, Innovacion y Ciencia (P10.CTS.6359 to BRG), Ministerio de Ciencia e Innovacion (BFU2013-42789-P to IQ) and the Ministerio de Economia y Competidividad, Instituto de Salud Carlos III co-funded by Fondos FEDER (PI10/00871 and PI13/00593 to BRG). NC-V is supported by a JDRF subsidy (17-2013-372 to BRG.). AM-M is a recipient of a Miguel Servet grant (CP14/00105) from the Instituto de Salud Carlos III co-funded by Fondos FEDER and EF-M is a recipient of a Juan de la Cierva Fellowship. PM is supported by Swiss National Science Foundation grant 310030-141162, and the European Union grant IMIDIA, C2008-T7. BOB is supported by grants from the Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Republic of Singapore.Peer Reviewe

Détection des polluants métalliques particulaires dans les eaux par spectroscopie de plasma induit par laser (LIBS : Laser-Induced Breakdown Spectroscopy)

International audienceA l'échelle européenne comme sur le plan national, les pollutions de l'air et des eaux sont des préoccupations majeures. Dans le but de mieux protéger et d'améliorer la qualité des eaux, la communauté européenne s'est dotée d'une directive cadre sur l'eau (DCE). Celle-ci vise d'ici 2015 à veiller à la non-dégradation et au maintien d'un bon état général des eaux. La directive vise une liste de polluants et parmi ceux-ci, les métaux lourds. Elle ne fait pas encore mention des nanoparticules manufacturées considérées comme polluants émergents. Néanmoins, ces dernières doivent être surveillées. Aujourd'hui l'INERIS engage des actions de recherches axées sur la métrologie de l'environnement dans le but de contribuer à l'amélioration de la mesure des concentrations de polluants dans les eaux, avec comme applications le monitoring environnemental, le contrôle des effluents d'un procédé industriel, etc. Réduire la pollution implique le suivi de ces polluants à l'état particulaire avec des outils adéquats. Les contraintes imposées par des réglementations de plus en plus strictes sur les métaux lourds et l'émergence de nouveaux polluants (nanoparticules) impliquent le développement de nouveaux outils aptes à répondre aux problématiques de mesurage posées et permettant une analyse in-situ, automatique et en temps réel. La technique LIBS (Laser-Induced Breakdown Spectroscopy) a été retenue pour la conception de cet outil. Elle permet l'identification chimique élémentaire de l'eau in situ et en temps réel. Elle permet également la détermination de la concentration en masse du polluant aussi bien à l'état dissout qu'à l'état particulair

Round robin tests on laser ablation and quantitative analysis by LIBS

Referring to the “Seven ages of an analytical method” proposed by Laitinen in 19731, LIBS may be presently considered at the threshold of the sixth one, when the number of applications of the technique has significantly increased, and the need of standardization is simultaneously growing. While the preferred scope of LIBS applications is onsite, online or in situ analysis, the diversity of experimental configurations required to address such issues is somewhat contradictory with a standardization approach. An alternative way to make LIBS progress to become a fully mature analytical technique could be the determination of reference performances of different instruments or experimental setups for a given analytical case. Such an approach would have several advantages: 1) Newcomers, both researchers and industrials, could better evaluate if the technique could meet their needs; 2) It would be a solid basis to which enhancement approaches could be compared (e.g. hyphenated techniques, microwave assisted LIBS, etc.); 3) It could help to define good experimental configurations and analytical practices that could be of general interest for any application. For that purpose, two round robin tests have been organized since 2012 within the French LIBS community. The first one involved five laboratories and was focused on the characterization of laser ablation of iron. Very consistent results on the craters volume were obtained, but no correlation was found between the repeatability of laser ablation and that of the LIBS signal. The second one was devoted to quantitative analysis of glass samples. Eleven teams contributed to the work. In spite of the dispersion in the experimental setups and protocols, the results obtained on unknowns showed an acceptable bias, whereas the measurement uncertainty was much more variable among the participants. Overall, the results of those two preliminary round robin tests show that a good reproducibility level is reachable between independent LIBS measurements and, in this sense, are very encouraging for the future