Complex shape forming of a flax woven fabric; analysis of the tow buckling and misalignment defect

With the view to minimise the impact on the environment and to produce structural parts with a goodproduction-rate/cost-ratio, the sheet forming of woven flax based fabric was investigated in this study. Aflax fibre plain-weave fabric has been used to form a complex tetrahedron shape. This shape is of partic-ular interest as it contains several geometric singularities required by many automotive parts such asdouble or triple curvature and low-curvature edges. Globally, the complex tetrahedron shape wasobtained, but tow buckling (out of plane bending of tows) was observed in specific zones of the shape.The main mechanism at the origin of this defect has been defined. A reduction of the tow buckle sizewas obtained by increasing the membrane tension. The influence of fabric architecture at the mesoscopicand macroscopic scales on the appearance of the tow buckles was demonstrated and discussed. Solutionsto prevent the appearance of this defect based on the design of the fabric architecture at the tow or fabricscales were successfully proposed. As a consequence, when sheet forming of complex shapes is consid-ered, specific fabric architectures should be chosen to prevent the appearance of the buckling defec

Bloom’s Syndrome and PICH Helicases Cooperate with Topoisomerase IIα in Centromere Disjunction before Anaphase

Centromeres are specialized chromosome domains that control chromosome segregation during mitosis, but little is known about the mechanisms underlying the maintenance of their integrity. Centromeric ultrafine anaphase bridges are physiological DNA structures thought to contain unresolved DNA catenations between the centromeres separating during anaphase. BLM and PICH helicases colocalize at these ultrafine anaphase bridges and promote their resolution. As PICH is detectable at centromeres from prometaphase onwards, we hypothesized that BLM might also be located at centromeres and that the two proteins might cooperate to resolve DNA catenations before the onset of anaphase. Using immunofluorescence analyses, we demonstrated the recruitment of BLM to centromeres from G2 phase to mitosis. With a combination of fluorescence in situ hybridization, electron microscopy, RNA interference, chromosome spreads and chromatin immunoprecipitation, we showed that both BLM-deficient and PICH-deficient prometaphase cells displayed changes in centromere structure. These cells also had a higher frequency of centromeric non disjunction in the absence of cohesin, suggesting the persistence of catenations. Both proteins were required for the correct recruitment to the centromere of active topoisomerase IIα, an enzyme specialized in the catenation/decatenation process. These observations reveal the existence of a functional relationship between BLM, PICH and topoisomerase IIα in the centromere decatenation process. They indicate that the higher frequency of centromeric ultrafine anaphase bridges in BLM-deficient cells and in cells treated with topoisomerase IIα inhibitors is probably due not only to unresolved physiological ultrafine anaphase bridges, but also to newly formed ultrafine anaphase bridges. We suggest that BLM and PICH cooperate in rendering centromeric catenates accessible to topoisomerase IIα, thereby facilitating correct centromere disjunction and preventing the formation of supernumerary centromeric ultrafine anaphase bridges

In vivo investigation of Lcr35® anti-candidiasis properties in Caenorhabditis elegans reveals the involvement of highly conserved immune pathways

International audienceLactic acid bacteria, including the microorganisms formerly designated as Lactobacillus, are the major representatives of Live Biotherapeutic Microorganisms (LBM) when used for therapeutic purposes. However, in most cases, the mechanisms of action remain unknown. The antifungal potential of LBM has already been demonstrated using preclinical models (cell cultures, laboratory animals). Understanding their mechanisms of action is strategic for the development of new therapeutics for humans. Here, Caenorhabditis elegans was used as an in vivo model to analyze pro-longevity, anti-aging and anti-candidiasis effects of the LBM Lacticaseibacillus rhamnosus (formerly Lactobacillus rhamnosus) Lcr35®. A high-throughput transcriptomic analysis revealed a specific response of C. elegans depending on whether it is in the presence of the LBM L. rhamnosus Lcr35 (structural response), the yeast Candida albicans (metabolic response) or both (structural and metabolic responses) in a preventive and a curative conditions. Studies on C. elegans mutants demonstrated that the p38 MAPK (sek-1, skn-1) and the insulin-like (daf-2, daf-16) signaling pathways were involved in the extended lifespan provided by L. rhamnosus Lcr35® strain whereas the JNK pathway was not involved (jnk-1). In addition, the anti C. albicans effect of the bacterium requires the daf-16 and sek-1 genes while it is independent of daf-2 and skn-1. Moreover, the anti-aging effect of Lcr35®, linked to the extension of longevity, is not due to protection against oxidative stress (H2O2). Taken together, these results formally show the involvement of the p38 MAP kinase and insulin- like signaling pathways for the longevity extension and anti-Candida albicans properties of Lcr35 with, however, differences in the genes involved. Overall, these findings provide new insight for understanding the mechanisms of action of a probiotic strain with antimicrobial potential

The Bloom Syndrome Protein Limits the Lethality Associated with RAD51 Deficiency

International audienceLittle is known about the functional interaction between the Bloom's syndrome protein (BLM) and the recombinase RAD51 within cells. Using RNA interference technology, we provide the first demonstration that RAD51 acts upstream from BLM to prevent anaphase bridge formation. RAD51 downregulation was associated with an increase in the frequency of BLM-positive anaphase bridges, but not of BLM-associated ultrafine bridges. Time-lapse live microscopy analysis of anaphase bridge cells revealed that BLM promoted cell survival in the absence of Rad51. Our results directly implicate BLM in limiting the lethality associated with RAD51 deficiency through the processing of anaphase bridges resulting from the RAD51 defect. These findings provide insight into the molecular basis of some cancers possibly associated with variants of the RAD51 gene family

BLM-deficient cells and PICH-downregulated display non disjunction of centromeres and impaired recruitment of active Topo IIα to centromeres.

<p>(A) GFP-BLM and BS cells were transfected for 72 hours with Rad21 siRNAs and transfected either with control siRNAs or with PICH siRNAs. BLM, PICH and Rad21 proteins levels were assessed by immunoblotting, probing the same membrane with anti-BLM (ab-476), anti-PICH and anti-Rad21 antibodies and with anti-β actin antibody, as a loading control (lower left panel). Chromosome spreads were performed and sorted on the basis of their phenotype: X-shapes, incomplete disjunction or complete disjunction. The scale bars indicate 5 µm (upper left panel). This classification is based on the intensity profiles of centromeres (upper right panel). We analyzed 500 spreads from three independent experiments for each cell line. The frequency of each phenotype, in each of the three cell lines, is shown in the histogram (lower right panel). Bars represent SD. (B) GFP-BLM, BS and GFP-I841T cells were transfected for 72 hours with Rad21 siRNAs and the same experiments as in (A) (right panels) were carried out. We checked the levels of BLM and Rad21 proteins by western blotting (left panels).</p

BLM localizes to centromeres in mitotic cells.

<p>(A) Endogenous BLM localizes to centromeres in HeLa cells. Wide-field microscopy after immunofluorescence staining on chromosome spreads obtained by cytocentrifugation of BLM siRNA-transfected or control siRNA-transfected HeLa S3 cells. Staining is shown for BLM (green), CREST (red) and chromosomes (blue). Single chromosome magnifications are shown (upper panels). Proteins levels were assessed by immunoblotting, probing the same membrane with anti-BLM (ab-476) antibody and then with anti-β actin antibody, as a loading control (lower left panel). Quantification of centromeric fluorescence signals for BLM (normalized according to the CREST signal) for a total of 20 centromeres in control cells (siCtrl) and 24 centromeres in BLM-depleted cells (siBLM) from two independent experiments, demonstrate the specificity of ab476 BLM antibodies (lower right panel). Data are means and SD normalized with respect to controls. Scale bar = 5 µm. (B) Centromeres detected with CREST serum (red) from all the chromosomes of HeLa siCtrl cells and HeLa siBLM cells from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033905#pone-0033905-g002" target="_blank">Figure 2A</a> were analyzed for BLM signals (green). Chromosomes were visualized by DAPI staining (blue) (left panels). The same analysis was performed on a total of 9 HeLa siCtrl cells and 9 HeLa siBLM cells from two independent experiments: the percentage of centromeres giving a BLM signal is shown (right panel).</p