Fluorescence-Based Rapid Detection of Microbiological Contaminants in Water Samples

Microbiological contamination of process waters is a current issue for pharmaceutical industries. Traditional methods require several days to obtain results; therefore, rapid microbiological methods are widely requested to shorten time-to-result. Milliflex Quantum was developed for the rapid detection and enumeration of microorganisms in filterable samples. It combines membrane filtration to universal fluorescent staining of viable microorganisms. This new alternative method was validated using European and United States Pharmacopeia definitions, with sterile water and/or sterile water artificially contaminated with microorganisms. The Milliflex Quantum method was demonstrated to be reliable, robust, specific, accurate, and linear over the whole range of assays following these guidelines. The Milliflex Quantum system was challenged to detect natural contaminants in different types of pharmaceutical purified process waters. Milliflex Quantum was demonstrated to detect accurately contaminants 3- to 7-fold faster than traditional membrane filtration method. The staining procedure is nondestructive allowing downstream identification following a positive result. The Milliflex Quantum offers a fast, sensitive, and robust alternative to the compendial membrane filtration method

Nod2 Mediates Susceptibility to Yersinia pseudotuberculosis in Mice

Nucleotide oligomerisation domain 2 (NOD2) is a component of the innate immunity known to be involved in the homeostasis of Peyer patches (PPs) in mice. However, little is known about its role during gut infection in vivo. Yersinia pseudotuberculosis is an enteropathogen causing gastroenteritis, adenolymphitis and septicaemia which is able to invade its host through PPs. We investigated the role of Nod2 during Y. pseudotuberculosis infection. Death was delayed in Nod2 deleted and Crohn's disease associated Nod2 mutated mice orogastrically inoculated with Y. pseudotuberculosis. In PPs, the local immune response was characterized by a higher KC level and a more intense infiltration by neutrophils and macrophages. The apoptotic and bacterial cell counts were decreased. Finally, Nod2 deleted mice had a lower systemic bacterial dissemination and less damage of the haematopoeitic organs. This resistance phenotype was lost in case of intraperitoneal infection. We concluded that Nod2 contributes to the susceptibility to Y. pseudotuberculosis in mice

<em>Yersinia pseudotuberculosis</em> infection disrupts the intestinal barrier and enables systemic translocation of <em>Yersinia</em> and bacteria from the gut flora in mice

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The NOD2-RICK complex signals from the plasma membrane

International audienceNOD2 plays an important role in the innate immunity of the intestinal tract. By sensing the muramyl dipeptide (MDP), a bacterial wall component, NOD2 triggers the NF-kappaB signaling pathway and promotes the release of proinflammatory cytokines such as interleukin-8. Mutations in Nod2 (1007FS, R702W, G908R) impinge on NOD2 functions and are associated with the pathogenesis of Crohn disease, a chronic inflammatory bowel disease. Although NOD2 is usually described as a cytosolic receptor for MDP, the protein is also localized at the plasma membrane, and the 1007FS mutation delocalizes NOD2 to the cytoplasm (Barnich, N., Aguirre, J. E., Reinecker, H. C., Xavier, R., and Podolsky, D. K. (2005) J. Cell Biol. 170, 21-26; McDonald, C., Chen, F. F., Ollendorff, V., Ogura, Y., Marchetto, S., Lecine, P., Borg, J. P., and Nunez, G. (2005) J. Biol. Chem. 280, 40301-40309). In this study, we demonstrate that membrane-bound versions of NOD2 and Crohn disease-associated mutants R702W and G908R are capable of responding to MDP and activating the NF-kappaB pathway from this location. In contrast, the 1007FS mutant remains unable to respond to MDP from the plasma membrane. We also show that NOD2 promotes the membrane recruitment of RICK, a serine-threonine kinase involved in NF-kappaB activation downstream of NOD2. Furthermore, the artificial attachment of RICK at the plasma membrane provokes a constitutive and strong activation of the NF-kappaB pathway and secretion of interleukin-8 showing that optimal RICK activity depends upon its subcellular localization. Finally, we show that endogenous RICK localizes at the plasma membrane in the THP1 cell line. Thus, our data suggest that NOD2 is responsible for the membrane recruitment of RICK to induce a regulated NF-kappaB signaling and production of proinflammatory cytokines

Yersinia pseudotuberculosis Effector YopJ Subverts the Nod2/RICK/TAK1 Pathway and Activates Caspase-1 to Induce Intestinal Barrier Dysfunction

International audienceYersinia pseudotuberculosis is an enteropathogenic bacteria that disrupts the intestinal barrier and invades its host through gut-associated lymphoid tissue and Peyer's patches (PP). We show that the Y. pseudotuberculosis effector YopJ induces intestinal barrier dysfunction by subverting signaling of the innate immune receptor Nod2, a phenotype that can be reversed by pretreating with the Nod2 ligand muramyl-dipeptide. YopJ, but not the catalytically inactive mutant YopJ(C172A), acetylates critical sites in the activation loops of the RICK and TAK1 kinases, which are central mediators of Nod2 signaling, and decreases the affinity of Nod2 for RICK. Concomitantly, Nod2 interacts with and activates caspase-1, resulting in increased levels of IL-1β. Finally, IL-1β within PP plays an essential role in inducing intestinal barrier dysfunction. Thus, YopJ alters intestinal permeability and promotes the dissemination of Yersinia as well as commensal bacteria by exploiting the mucosal inflammatory response

Is autism curable?

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental disorder of multifactorial origin. Today, ASD is generally not curable, although it is treatable to a varying degree to prevent worse outcomes. Some reports indicate the possibility of major improvements or even recovery in ASD. However, these studies are based on scientific shortcomings, and the lack of a clear definition of 'cure' in ASD further compromises interpretation of research findings. The development of animal models and decreasing costs of genome sequencing provide new options for treatment research and individualized medicine in ASD. This article briefly reviews several issues related to the question whether there is recovery from ASD, starting with a short overview of the presumed aetiologies