hnRNP I/PTB can antagonize the splicing repressor activity of SRp30c

The control of alternative pre-mRNA splicing often requires the participation of factors displaying synergistic or antagonistic activities. In the hnRNP A1 pre-mRNA, three elements promote the exclusion of alternative exon 7B, while a fourth intron element (CE9) represses splicing of exon 7B to the downstream exon. We have shown previously that the 5' portion of the 38-nucleotide-long CE9 element is bound by SRp30c, and that this interaction is important for repression in vitro. To determine whether SRp30c alone can impose repression, we tested a high-affinity SRp30c binding site that we identified using the SELEX protocol. We find that multiple high-affinity SRp30c sites are required to replicate the level of repression obtained with CE9, and that both the 5' and the 3' portions of CE9 contribute to SRp30c binding. Performing RNA affinity chromatography with the complete CE9 element recovered hnRNP I/PTB. Surprisingly however, His-tagged PTB reduced the binding of SRp30c to CE9 in a nuclear extract, stimulated splicing to a downstream 3' splice site, and relieved the CE9-mediated splicing repression in vitro. Our in vivo results are consistent with the notion that increasing PTB levels alleviates the repression imposed by CE9 to a downstream 3' splice site. Thus, PTB can function as an anti-repressor molecule to counteract the splicing inhibitory activity of SRp30c.     

A rapid assay for chloroplast-encoded triazine resistance in higher plants

We have designed a simple and rapid assay for chloroplast-based triazine resistance in higher plants using PCR amplification of thepsbA gene coupled toMaeI digestion of the amplified product to distinguish triazine resistant from sensitive biotypes. Our assay is universal and avoids the need of lengthy procedures of previously published assays, which either required spraying of seedlings in a controlled environment, quantification of chlorophyll fluorescence of leaf discs after incubation in triazine solution, DNA sequencing of thepsbA gene, or Southern-blot analysis. Our diagnostic system is qualitative, reliable, fast and simple. More than 100 seedlings taken directly from the field can be analyzed in one day. This system has a direct application towards a more rational use of herbicides in production fields. It also represents a valuable tool to monitor spreading of resistant biotypes through time and space and can serve as a model system applicable to other gene monitoring needs.     

Design of a specific colonic mucus marker using a human commensal bacterium cell surface domain

Imaging living cells and organs requires innovative, specific, efficient, and well tolerated fluorescent markers targeting cellular components. Such tools will allow proceeding to the dynamic analysis of cells and the adaptation of tissues to environmental cues. In this study, we have identified and synthesized a novel non-toxic fluorescent marker allowing a specific fluorescent staining of the human colonic mucus. Our strategy to identify a molecule able to specifically bind to the human colonic mucus was on the basis of the mucus adhesion properties of commensal bacteria. We identified and characterized the mucus-binding property of a 70-amino acid domain (MUB(70)) expressed on the surface of Lactobacillus strains. The chemical synthesis of MUB(70) was achieved using the human commensal bacterium Lactobacillus reuteri AF120104 protein as a template. The synthesized Cy5-conjugated MUB(70) marker specifically stained the colonic mucus on fixed human, rabbit, and guinea pig tissues. Interestingly, murine tissue was not stained, suggesting significant differences in the composition of the murine colonic mucus. In addition, this marker stained the mucus of living cultured human colonic cells (HT29-MTX) and human colonic tissue explants. Using a biotinylated derivative of MUB(70), we demonstrated that this peptide binds specifically to Muc2, the most abundant secreted mucin, through its glycosylated moieties. Hence, Cy5-MUB(70) is a novel and specific fluorescent marker for mammalian colonic mucus. It may be used for live imaging analysis but also, as demonstrated in this study, as a marker for the diagnosis and the prognosis of colonic mucinous carcinomas.     

3D analysis from micro-MRI during in situ compression on cancellous bone

A mini-compression jig was built to perform in situ tests on bovine trabecular bone monitored by micro-MRI. The MRI antenna provided an isotropic resolution of 78 μm that allows for a volume correlation method to be used. Three-dimensional displacement fields are then evaluated within the bone sample during the compression test. The performances of the correlation method are evaluated and discussed to validate the technique on trabecular bone. By considering correlation residuals and estimates of acquisition noise, the measured results are shown to be trustworthy. By analyzing average strain levels for different interrogation volumes along the loading direction, it is shown that the sample size is less than that of a representative volume element. This study shows the feasibility of the 3D-displacement and strain field analyses from micro-MRI images. Other biological tissues could be considered in future work.     

Régulation de la biosynthèse de l'aspartate-ammonium lyase (aspartase) de Pseudomonas fluorescens

Variations in aspartasic activity in various media are due to aspartate-ammonium lyase induction and to regulation of the biosynthesis of this enzyme. Evidence for neosynthesis of the enzyme is provided by labelling and separation of the protein. The inducer appears to be aspartic acid. The biosynthesis is subject to pronounced catabolic repression. The physiological function of aspartate-ammonium lyase is discussed.     

Adaptations for the maintenance of water balance by three species of Antarctic mites

Three species of Antarctic mites, Alaskozetes antarcticus, Hydrogamasellus antarcticus and Rhagidia gerlachei, are abundant in the vicinity of Palmer Station, Antarctica. No single mechanism for reducing water stress was shared by all three species. A. antarcticus and R. gerlachei (both ca. 200 μg) are over twice as large as H. antarcticus (ca. 90 μg), but all had similar body water content (67%) and tolerated a loss of up to 35% of their body water before succumbing to dehydration. All imbibed free water and had the capacity to reduce water loss behaviorally by forming clusters. Alaskozetes antarcticus was distinct in that it relied heavily on water conservation (xerophilic classification) that was largely achieved by its thick cuticular armor, a feature shared by all members of this suborder (Oribatida), and abundant cuticular hydrocarbons. In comparison to the other two species, A. antarcticus was coated with 2–3× the amount of cuticular hydrocarbons, had a 20-fold reduction in net transpiration rate, and had a critical transition temperature (CTT) that indicates a pronounced suppression in activation energy (E _a) at temperatures below 25°C. In contrast, H. antarcticus and R. gerlachei lack a CTT, have lower amounts of cuticular hydrocarbons and have low E _as and high net transpiration rates, classifying them as hydrophilic. Only H. antarcticus was capable of utilizing water vapor to replenish its water stores, but it could do so only at relative humidities close to saturation (95–98 %RH). Thus, H. antarcticus and R. gerlachei require wet habitats and low temperature to counter water loss, and replace lost water behaviorally through predation. Compared to mites from the temperate zone, all three Antarctic species had a lower water content, a feature that commonly enhances cold tolerance.     

Role of Cattle Movements in Bovine Tuberculosis Spread in France between 2005 and 2014

Live animal movements are a major transmission route for the spread of infectious agents such as Mycobacterium bovis, the main agent of bovine Tuberculosis (bTB). France became officially bTB-free in 2001, but M. bovis is still circulating in the cattle population, with about a hundred of outbreaks per year, most located in a few geographic areas. The aim of this study was to analyse the role of cattle movements in bTB spread in France between 2005 and 2014, using social network analysis and logistic regression models. At a global scale, the trade network was studied to assess the association between several centrality measures and bTB infection though a case-control analysis. The bTB infection status was associated with a higher in-degree (odds-ratio [OR] = 2.4 [1.1–5.4]) and with a higher ingoing contact chain (OR = 2.2 [1.0–4.7]). At a more local scale, a second case-control analysis was conducted to estimate the relative importance of cattle movements and spatial neighbourhood. Only direct purchase from infected herds was shown to be associated with bTB infection (OR = 2.9 [1.7–5.2]), spatial proximity to infected herds being the predominant risk factor, with decreasing ORs when distance increases. Indeed, the population attributable fraction was 12% [5%–18%] for cattle movements and 73% [68%–78%] for spatial neighbourhood. Based on these results, networks of potential effective contacts between herds were built and analysed for the three major spoligotypes reported in France. In these networks, the links representing cattle movements were associated with higher edge betweenness than those representing the spatial proximity between infected herds. They were often links connecting distinct communities and sometimes distinct geographical areas. Therefore, although their role was quantitatively lower than the one of spatial neighbourhood, cattle movements appear to have been essential in the French bTB dynamics between 2005 and 2014.     

Topological Analysis of the Aerobic Membrane-Bound Formate Dehydrogenase of Escherichia coli

Besides formate dehydrogenase N (FDH-N), which is involved in the major anaerobic respiratory pathway in the presence of nitrate, Escherichia coli synthesizes a second isoenzyme, called FDH-O, whose physiological role is to ensure rapid adaptation during a shift from aerobiosis to anaerobiosis. FDH-O is a membrane-bound enzyme complex composed of three subunits, α (FdoG), β (FdoH), and γ (FdoI), which exhibit high sequence similarity to the equivalent polypeptides of FDH-N. The topology of these three subunits has been studied by using blaM (β-lactamase) gene fusions. A collection of 47 different randomly generated Fdo-BlaM fusions, 4 site-specific fusions, and 3 sandwich fusions were isolated along the entire sequence of the three subunits. In contrast to previously reported predictions from sequence analysis, our data suggested that the αβ catalytic dimer is located in the cytoplasm, with a C-terminal anchor for β protruding into the periplasm. As expected, the γ subunit, which specifies cytochrome b, was shown to cross the cytoplasmic membrane four times, with the N and C termini exposed to the cytoplasm. Protease digestion studies of the ³⁵S-labelled FDH-O heterotrimer in spheroplasts add further support to this model. Consistently, prior studies regarding the bioenergetic function of formate dehydrogenase provided evidence for a mechanism in which formate is oxidized in the cytoplasm.     

Proteins associated with the exon junction complex also control the alternative splicing of apoptotic regulators

Several apoptotic regulators, including Bcl-x, are alternatively spliced to produce isoforms with opposite functions. We have used an RNA interference strategy to map the regulatory landscape controlling the expression of the Bcl-x splice variants in human cells. Depleting proteins known as core (Y14 and eIF4A3) or auxiliary (RNPS1, Acinus, and SAP18) components of the exon junction complex (EJC) improved the production of the proapoptotic Bcl-x(S) splice variant. This effect was not seen when we depleted EJC proteins that typically participate in mRNA export (UAP56, Aly/Ref, and TAP) or that associate with the EJC to enforce nonsense-mediated RNA decay (MNL51, Upf1, Upf2, and Upf3b). Core and auxiliary EJC components modulated Bcl-x splicing through different cis-acting elements, further suggesting that this activity is distinct from the established EJC function. In support of a direct role in splicing control, recombinant eIF4A3, Y14, and Magoh proteins associated preferentially with the endogenous Bcl-x pre-mRNA, interacted with a model Bcl-x pre-mRNA in early splicing complexes, and specifically shifted Bcl-x alternative splicing in nuclear extracts. Finally, the depletion of Y14, eIF4A3, RNPS1, SAP18, and Acinus also encouraged the production of other proapoptotic splice variants, suggesting that EJC-associated components are important regulators of apoptosis acting at the alternative splicing level.