PAX3 Confers Functional Heterogeneity in Skeletal Muscle Stem Cell Responses to Environmental Stress

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High-resolution nuclear magnetic resonance spectroscopy studies of polysaccharides crosslinked by sodium trimetaphosphate: a proposal for the reaction mechanism

An NMR spectroscopy study ((31)P, (1)H, (13)C) of the postulated crosslinking mechanism of sodium trimetaphosphate (STMP) on polysaccharides is reported using methyl alpha-D-glucopyranoside as a model. In a first step, reaction of STMP with Glc-OMe gives grafted sodium tripolyphosphate (STPP(g)). On the one hand, STTP(g) can react with a second alcohol functionality to give a crosslinked monophosphate. On the other hand, a monophosphate (grafted phosphate) could be obtained by alkaline degradation of STPP(g). NMR spectroscopy allows to detect the various species formed and to obtain the crosslinking density of STMP-polysaccharides hydrogels.     

Propagation of fluorescent viruses in growing plaques

To study virus propagation, we have developed a method by which the propagation of the Lambda bacteriophage can be observed and quantified. This is done by creating a fusion protein of the capsid protein gpD and the enhanced yellow fluorescent protein (EYFP). We show that this fusion allows capsid formation and that the modified viruses propagate on a surface covered with host bacteria thus forming fluorescent plaques. The intensity of fluorescence in a growing plaque determines the distribution of phages. This provides a new tool to study the propagation of infection at the microscopic level.     

Interleukin-6 induces keratin expression in intestinal epithelial cells: potential role of keratin-8 in interleukin-6-induced barrier function alterations

Keratin 8 (K8) and keratin-18 (K18) are the major intermediate filament proteins in the intestinal epithelia. The regulation and function of keratin in the intestinal epithelia is largely unknown. In this study we addressed the role and regulation of K8 and K18 expression by interleukin 6 (IL-6). Caco2-BBE cell line and IL-6 null mice were used to study the effect of IL-6 on keratin expression. Keratin expression was studied by Northern blot, Western blot, and confocal microscopy. Paracellular permeability was assessed by apical-to-basal transport of a fluorescein isothiocyanate dextran probe (FD-4). K8 was silenced using the small interfering RNA approach. IL-6 significantly up-regulated mRNA and protein levels of K8 and K18. Confocal microscopy showed a reticular pattern of intracellular keratin localized to the subapical region after IL-6 treatment. IL-6 also induced serine phosphorylation of K8. IL-6 decreased paracellular flux of FD-4 compared with vehicle-treated monolayers. K8 silencing abolished the decrease in paracellular permeability induced by IL-6. Administration of dextran sodium sulfate (DSS) significantly increased intestinal permeability in IL-6-/- mice compared with wild type mice given DSS. Collectively, our data demonstrate that IL-6 regulates the colonic expression of K8 and K18, and K8/K18 mediates barrier protection by IL-6 under conditions where intestinal barrier is compromised. Thus, our data uncover a novel function of these abundant cytoskeletal proteins, which may have implications in intestinal disorders such as inflammatory bowel disease wherein barrier dysfunction underlies the inflammatory response.     

Polysialylation increases lateral diffusion of neural cell adhesion molecule in the cell membrane

Polysialic acid (PSA) is a polymer of N-acetylneuraminic acid residues added post-translationally to the membrane-bound neural cell adhesion molecule (NCAM). The large excluded volume created by PSA polymer is thought to facilitate cell migration by decreasing cell adhesion. Here we used live cell imaging (spot fluorescence recovery after photobleaching and fluorescence correlation spectroscopy) combined with biochemical approaches in an attempt to uncover a link between cell motility and the impact of polysialylation on NCAM dynamics. We show that PSA regulates specifically NCAM lateral diffusion and this is dependent on the integrity of the cytoskeleton. However, whereas the glial-derivative neurotrophic factor chemotactic effect is dependent on PSA, the molecular dynamics of PSA-NCAM is not directly affected by glial-derivative neurotrophic factor. These findings reveal a new intrinsic mechanism by which polysialylation regulates NCAM dynamics and thereby a biological function like cell migration.     

ADAM-15/metargidin mediates homotypic aggregation of human T lymphocytes and heterotypic interactions of T lymphocytes with intestinal epithelial cells

Intestinal epithelial cells (IEC) play an immunoregulatory role in the intestine. This role involves cell-cell interactions with intraepithelial lymphocytes that may also play a role in some enteropathies. The discovery of the RGD motif-containing Protein ADAM-15 (a disintegrin and metalloprotease-15) raises the question of its involvement in these cell-cell interactions. Cell adhesion assays were performed using the Jurkat E6.1 T cell line as a model of T lymphocytes and Caco2-BBE monolayers as a model of intestinal epithelia. Our results show that an anti-ADAM-15 ectodomain antibody inhibited the attachment of Jurkat cells on Caco2-BBE monolayers. Overexpression of ADAM-15 in Caco2-BBE cells enhanced Jurkat cell binding, and overexpression of ADAM-15 in Jurkat cells enhanced their aggregation. Mutagenesis experiments showed that both the mutation of ADAM-15 RGD domain or the deletion of its cytoplasmic tail decreased these cell-cell interactions. Moreover, wound-healing experiments showed that epithelial ADAM-15-mediated Jurkat cell adhesion to Caco2-BBE cells enhances the mechanisms of wound repair. We also found that ADAM-15-mediated aggregation of Jurkat cells increases the expression of tumor necrosis factor-alpha mRNA. These results demonstrate the following: 1) ADAM-15 is involved in heterotypic adhesion of intraepithelial lymphocytes to IEC as well as in homotypic aggregation of T cells; 2) both the RGD motif and the cytoplasmic tail of ADAM-15 are involved for these cell-cell interactions; and 3) ADAM-15-mediated cell-cell interactions are involved in mechanisms of epithelial restitution and production of pro-inflammatory mediators. Altogether these findings point to ADAM-15 as a possible therapeutic target for prevention of inappropriate T cell activation involved in some pathologies.     

Two Chikungunya isolates from the outbreak of La Reunion (Indian Ocean) exhibit different patterns of infection in the mosquito, Aedes albopictus

Background

A Chikungunya (CHIK) outbreak hit La Réunion Island in 2005–2006. The implicated vector was Aedes albopictus. Here, we present the first study on the susceptibility of Ae. albopictus populations to sympatric CHIKV isolates from La Réunion Island and compare it to other virus/vector combinations.

Methodology and Findings

We orally infected 8 Ae. albopictus collections from La Réunion and 3 from Mayotte collected in March 2006 with two Chikungunya virus (CHIKV) from La Réunion: (i) strain 05.115 collected in June 2005 with an Alanine at the position 226 of the glycoprotein E1 and (ii) strain 06.21 collected in November 2005 with a substitution A226V. Two other CHIKV isolates and four additional mosquito strains/species were also tested. The viral titer of the infectious blood-meal was 10⁷ plaque forming units (pfu)/mL. Dissemination rates were assessed by immunofluorescent staining on head squashes of surviving females 14 days after infection. Rates were at least two times higher with CHIKV 06.21 compared to CHIKV 05.115. In addition, 10 individuals were analyzed every day by quantitative RT-PCR. Viral RNA was quantified on (i) whole females and (ii) midguts and salivary glands of infected females. When comparing profiles, CHIKV 06.21 produced nearly 2 log more viral RNA copies than CHIKV 05.115. Furthermore, females infected with CHIKV 05.115 could be divided in two categories: weakly susceptible or strongly susceptible, comparable to those infected by CHIKV 06.21. Histological analysis detected the presence of CHIKV in salivary glands two days after infection. In addition, Ae. albopictus from La Réunion was as efficient vector as Ae. aegypti and Ae. albopictus from Vietnam when infected with the CHIKV 06.21.

Conclusions

Our findings support the hypothesis that the CHIK outbreak in La Réunion Island was due to a highly competent vector Ae. albopictus which allowed an efficient replication and dissemination of CHIKV 06.21.     

Proteome analysis of Rickettsia felis highlights the expression profile of intracellular bacteria

The proteome of Rickettsia felis, an obligate intracellular bacterium responsible for spotted fever, was analyzed using two complementary proteomic approaches: 2-DE coupled with MALDI-TOF, and SDS-PAGE with nanoLC-MS/MS. This strategy allowed identification of 165 proteins and helped to answer some questions raised by the genome sequence of this bacterium. We successfully identified potential virulence factors including two putative adhesins, four proteins of the type IV secretion system, four Sca autotransporters, four components of ABC transporters, some R. felis-specific proteins, and one antitoxin of the toxin-antitoxin system. Notably, the antitoxin was the first to be identified in intracellular bacteria. Only one protein containing rickettsia palindromic repeats was found, whereas none of the split genes, transposases, or tetratricopeptide/ankyrin repeats were detectably expressed. Comparison of the protein expression profiles of R. felis and 23 other bacterial species according to functional categories showed that intracellular bacteria express more proteins related to translation, especially ribosomal proteins. However, the remaining bacteria express more proteins related to energy production and carbohydrate/amino acid metabolism. In conclusion, this study reveals R. felis virulence factor expression and highlights the unique protein expression profile of intracellular bacteria.     

Reductive genome evolution from the mother of Rickettsia

The Rickettsia genus is a group of obligate intracellular α-proteobacteria representing a paradigm of reductive evolution. Here, we investigate the evolutionary processes that shaped the genomes of the genus. The reconstruction of ancestral genomes indicates that their last common ancestor contained more genes, but already possessed most traits associated with cellular parasitism. The differences in gene repertoires across modern Rickettsia are mainly the result of differential gene losses from the ancestor. We demonstrate using computer simulation that the propensity of loss was variable across genes during this process. We also analyzed the ratio of nonsynonymous to synonymous changes (Ka/Ks) calculated as an average over large sets of genes to assay the strength of selection acting on the genomes of Rickettsia, Anaplasmataceae, and free-living γ-proteobacteria. As a general trend, Ka/Ks were found to decrease with increasing divergence between genomes. The high Ka/Ks for closely related genomes are probably due to a lag in the removal of slightly deleterious nonsynonymous mutations by natural selection. Interestingly, we also observed a decrease of the rate of gene loss with increasing divergence, suggesting a similar lag in the removal of slightly deleterious pseudogene alleles. For larger divergence (Ks > 0.2), Ka/Ks converge toward similar values indicating that the levels of selection are roughly equivalent between intracellular α-proteobacteria and their free-living relatives. This contrasts with the view that obligate endocellular microorganisms tend to evolve faster as a consequence of reduced effectiveness of selection, and suggests a major role of enhanced background mutation rates on the fast protein divergence in the obligate intracellular α-proteobacteria.