Host Genes Related to Paneth Cells and Xenobiotic Metabolism Are Associated with Shifts in Human Ileum-Associated Microbial Composition

The aim of this study was to integrate human clinical, genotype, mRNA microarray and 16 S rRNA sequence data collected on 84 subjects with ileal Crohn’s disease, ulcerative colitis or control patients without inflammatory bowel diseases in order to interrogate how host-microbial interactions are perturbed in inflammatory bowel diseases (IBD). Ex-vivo ileal mucosal biopsies were collected from the disease unaffected proximal margin of the ileum resected from patients who were undergoing initial intestinal surgery. Both RNA and DNA were extracted from the mucosal biopsy samples. Patients were genotyped for the three major NOD2 variants (Leufs1007, R702W, and G908R) and the ATG16L1T300A variant. Whole human genome mRNA expression profiles were generated using Agilent microarrays. Microbial composition profiles were determined by 454 pyrosequencing of the V3–V5 hypervariable region of the bacterial 16 S rRNA gene. The results of permutation based multivariate analysis of variance and covariance (MANCOVA) support the hypothesis that host mucosal Paneth cell and xenobiotic metabolism genes play an important role in host microbial interactions

Norovirus Regulation of the Innate Immune Response and Apoptosis Occurs via the Product of the Alternative Open Reading Frame 4

Small RNA viruses have evolved many mechanisms to increase the capacity of their short genomes. Here we describe the identification and characterization of a novel open reading frame (ORF4) encoded by the murine norovirus (MNV) subgenomic RNA, in an alternative reading frame overlapping the VP1 coding region. ORF4 is translated during virus infection and the resultant protein localizes predominantly to the mitochondria. Using reverse genetics we demonstrated that expression of ORF4 is not required for virus replication in tissue culture but its loss results in a fitness cost since viruses lacking the ability to express ORF4 restore expression upon repeated passage in tissue culture. Functional analysis indicated that the protein produced from ORF4 antagonizes the innate immune response to infection by delaying the upregulation of a number of cellular genes activated by the innate pathway, including IFN-Beta. Apoptosis in the RAW264.7 macrophage cell line was also increased during virus infection in the absence of ORF4 expression. In vivo analysis of the WT and mutant virus lacking the ability to express ORF4 demonstrated an important role for ORF4 expression in infection and virulence. STAT1-/- mice infected with a virus lacking the ability to express ORF4 showed a delay in the onset of clinical signs when compared to mice infected with WT virus. Quantitative PCR and histopathological analysis of samples from these infected mice demonstrated that infection with a virus not expressing ORF4 results in a delayed infection in this system. In light of these findings we propose the name virulence factor 1, VF1 for this protein. The identification of VF1 represents the first characterization of an alternative open reading frame protein for the calicivirus family. The immune regulatory function of the MNV VF1 protein provide important perspectives for future research into norovirus biology and pathogenesis

Protein misfolding and dysregulated protein homeostasis in autoinflammatory diseases and beyond.

Cells have a number of mechanisms to maintain protein homeostasis, including proteasome-mediated degradation of ubiquitinated proteins and autophagy, a regulated process of ‘self-eating’ where the contents of entire organelles can be recycled for other uses. The unfolded protein response prevents protein overload in the secretory pathway. In the past decade, it has become clear that these fundamental cellular processes also help contain inflammation though degrading pro-inflammatory protein complexes such as the NLRP3 inflammasome. Signaling pathways such as the UPR can also be co-opted by toll-like receptor and mitochondrial reactive oxygen species signaling to induce inflammatory responses. Mutations that alter key inflammatory proteins, such as NLRP3 or TNFR1, can overcome normal protein homeostasis mechanisms, resulting in autoinflammatory diseases. Conversely, Mendelian defects in the proteasome cause protein accumulation, which can trigger interferon-dependent autoinflammatory disease. In non-Mendelian inflammatory diseases, polymorphisms in genes affecting the UPR or autophagy pathways can contribute to disease, and in diseases not formerly considered inflammatory such as neurodegenerative conditions and type 2 diabetes, there is increasing evidence that cell intrinsic or environmental alterations in protein homeostasis may contribute to pathogenesis

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Influence of the temperature on lifetime reinforcement of a filled NR

International audienceFatigue of crystallizable rubbers has been widely investigated since the pioneer work by Cadwell et al. in 1940 (Cadwell et al., Ind Eng Chem 12:19–23, 1940). This study revealed the significant influence of the mean strain on natural rubber lifetime: for non-relaxing loading conditions (i.e. R > 0), strong lifetime reinforcement was observed and attributed strain-induced crystallization (SIC). Better understanding how SIC reinforces fatigue life is therefore a key point to improve the durability of rubbers. Surprisingly, few studies investigated the effect of temperature on the lifetime, while SIC exhibits a high thermo-sensitivity (Lindley, Rubber Chem Technol 47:1253–1264, 1974; Lu Etude du comportement mécanique et des mécanismes d’endommagement des élastomères en fatigue et en fissuration par fatigue. PhD Thesis, Conservatoire National des Arts et Métiers, 1991). The present study aims therefore at investigating how temperature affects the fatigue life reinforcement due to SIC under non-relaxing loading conditions. First of all, fatigue tests are carried out at 23°C. Damage leading to the end-of-life is investigated at both the macroscopic and the microscopic scales for loading ratios from −0.25 to 0.35. As expected, NR exhibits a reinforcement for positive loading ratios R at 23°C. Damage, striations due to SIC and number of cycles at crack initiation are mapped using the Haigh diagram. At 90°C, the fatigue lifetime reinforcement is lower and the signature of SIC on the failure surface disappears. The competition between non-relaxing loading effect and temperature is finally discussed

A post mortem analysis of the strain-induced crystallization effects on fatigue of elastomers

International audienceNatural rubber (NR) is the most commonly used elastomer in the automotive industry thanks to its outstanding fatigue resistance. Strain-induced crystallization (SIC) is found to play a role of paramount importance in the great crack growth resistance of NR (Lindley, Int J Fracture 9:449–462, 1973). Typically, NR exhibits a lifetime reinforcement for non-relaxing loadings (Cadwell et al., 1940; Ruellan et al., 2019). At the microscopic scale, fatigue striations were observed on the fracture surface of Diabolo samples tested in fatigue. They are the signature of SIC (Cadwell et al., 1940; Le Cam et al., 2013; Le Cam et al., 2004). In order to provide additional information on the role of SIC in the fatigue crack growth resistance of NR, striations are investigated through post-mortem analysis after fatigue experiments using loading ranging from −0.25 to 0.25. No striation was observed in the case of tests performed at 90 °C. This confirms that the formation of striation requires a certain crystallinity level in the material. At 23 °C, two striation regimes were identified: small striation patches with different orientations (Regime 1) and zones with large and well-formed striations (Regime 2). Since fatigue striations are observed for all the loading ratios applied, they are therefore not the signature of the reinforcement. Nevertheless, increasing the minimum value of the strain amplified the striation phenomenon and the occurrence of Regime 2