Development of neutralizing scFv-Fc against botulinum neurotoxin A light chain from a macaque immune library

Botulinum toxins (BoNTs) are among the most toxic substances on earth, with serotype A toxin being the most toxic substance known. They are responsible for human botulism, a disease characterized by flaccid muscle paralysis that occurs naturally through food poisoning or the colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNT has been classified as a category A agent by the Centers for Disease Control, and it is one of six agents with the highest potential risk of use as bioweapons. Human or human-like neutralizing antibodies are thus required for the development of anti-botulinum toxin drugs to deal with this possibility. In this study, Macaca fascicularis was hyperimmunized with a recombinant light chain of BoNT/A. An immune phage display library was constructed and, after multistep panning, several scFv with nanomolar affinities that inhibited the endopeptidase activity of BoNT/A1 in vitro as scFv-Fc, with a molar ratio (ab binding site:toxin) of up to 1:1, were isolated. The neutralization of BoNT/A-induced paralysis by the SEM120-IID5, SEM120-IIIC1 and SEM120-IIIC4 antibodies was demonstrated in mouse phrenic nerve-hemidiaphragm preparations with the holotoxin. The neutralization observed is the strongest ever measured in the phrenic nerve-hemidiaphragm assay for BoNT/A1 for a monoclonal antibody. Several scFv-Fc inhibiting the endopeptidase activity of botulinum neurotoxin A were isolated. For SEM120-IID5, SEM120-IIIC1, and SEM120-IIIC4, inhibitory effects in vitro and protection against the toxin ex vivo were observed. The human-like nature of these antibodies makes them promising lead candidates for further development of immunotherapeutics for this disease.     

To team up or not: single versus team driving in European road freight transport

Flexible Services and Manufacturing Journal - The last decades have seen a tremendous amount of research being devoted to effectively managing vehicle fleets and minimizing empty mileage. However,...     

The adaptor molecule Lnk negatively regulates tumor necrosis factor-alpha-dependent VCAM-1 expression in endothelial cells through inhibition of the ERK1 and -2 pathways

Lnk, with APS and SH2-B (Src homology 2-B), belongs to a family of SH2-containing proteins with potential adaptor functions. Lnk regulates growth factor and cytokine receptor-mediated pathways implicated in lymphoid, myeloid, and platelet homeostasis. We have previously shown that Lnk is expressed and up-regulated in vascular endothelial cells (ECs) in response to tumor necrosis factor-alpha (TNFalpha). In this study, we have shown that, in ECs, Lnk down-regulates the expression, at both mRNA and protein levels, of the proinflammatory molecules VCAM-1 and E-selectin induced by TNFalpha. Mechanistically, our data indicated that, in response to TNFalpha, NFkappaB/p65 phosphorylation and translocation as well as IkappaBalpha phosphorylation and degradation were unchanged, suggesting that Lnk does not modulate NFkappaB activity. However, Lnk activates phosphatidylinositol 3-kinase (PI3K) as reflected by Akt phosphorylation. Our results identify endothelial nitric-oxide synthase as a downstream target of Lnk-mediated activation of the PI3K/Akt pathway and HO-1 as a new substrate of Akt. We found that sustained Lnk-mediated activation of PI3K in TNFalpha-activated ECs correlated with the inhibition of ERK1/2 phosphorylation, whereas phosphorylation of p38 and c-Jun NH(2)-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs) was unchanged. ERK1/2 inhibition decreases VCAM-1 expression in TNFalpha-treated ECs. Collectively, our results identify the adaptor Lnk as a negative regulator in the TNFalpha-signaling pathway mediating ERK inhibition and suggest a role for Lnk in the interplay between PI3K and ERK triggered by TNFalpha in ECs.     

Ras Regulates Rb via NORE1A

Mutations in the Ras oncogene are one of the most frequent events in human cancer. Although Ras regulates numerous growth-promoting pathways to drive transformation, it can paradoxically promote an irreversible cell cycle arrest known as oncogene-induced senescence. Although senescence has clearly been implicated as a major defense mechanism against tumorigenesis, the mechanisms by which Ras can promote such a senescent phenotype remain poorly defined. We have shown recently that the Ras death effector NORE1A plays a critical role in promoting Ras-induced senescence and connects Ras to the regulation of the p53 tumor suppressor. We now show that NORE1A also connects Ras to the regulation of a second major prosenescent tumor suppressor, the retinoblastoma (Rb) protein. We show that Ras induces the formation of a complex between NORE1A and the phosphatase PP1A, promoting the activation of the Rb tumor suppressor by dephosphorylation. Furthermore, suppression of Rb reduces NORE1A senescence activity. These results, together with our previous findings, suggest that NORE1A acts as a critical tumor suppressor node, linking Ras to both the p53 and the Rb pathways to drive senescence.     

Brachypodium distachyon as a model plant toward improved biofuel crops: Search for secreted proteins involved in biogenesis and disassembly of cell wall polymers

Polysaccharides make up about 75% of plant cell walls and can be broken down to produce sugar substrates (saccharification) from which a whole range of products can be obtained, including bioethanol. Cell walls also contain 5–10% of proteins, which could be used to tailor them for agroindustrial uses. Here we present cell wall proteomics data of Brachypodium distachyon, a model plant for temperate grasses. Leaves and culms were analyzed during active growth and at mature stage. Altogether, 559 proteins were identified by LC‐MS/MS and bioinformatics, among which 314 have predicted signal peptides. Sixty‐three proteins were shared by two organs at two developmental stages where they could play housekeeping functions. Differences were observed between organs and stages of development, especially at the level of glycoside hydrolases and oxidoreductases. Differences were also found between the known cell wall proteomes of B. distachyon, Oryza sativa, and the Arabidopsis thaliana dicot. Three glycoside hydrolases could be immunolocalized in cell walls using polyclonal antibodies against proteotypic peptides. Organ‐specific expression consistent with proteomics results could be observed as well as cell‐specific localization. Moreover, the high number of proteins of unknown function in B. distachyon cell wall proteomes opens new fields of research for monocot cell walls.     

Differential Gene Expression Profiling of Dystrophic Dog Muscle after MuStem Cell Transplantation

BackgroundSeveral adult stem cell populations exhibit myogenic regenerative potential, thus representing attractive candidates for therapeutic approaches of neuromuscular diseases such as Duchenne Muscular Dystrophy (DMD). We have recently shown that systemic delivery of MuStem cells, skeletal muscle-resident stem cells isolated in healthy dog, generates the remodelling of muscle tissue and gives rise to striking clinical benefits in Golden Retriever Muscular Dystrophy (GRMD) dog. This global effect, which is observed in the clinically relevant DMD animal model, leads us to question here the molecular pathways that are impacted by MuStem cell transplantation. To address this issue, we compare the global gene expression profile between healthy, GRMD and MuStem cell treated GRMD dog muscle, four months after allogenic MuStem cell transplantation.ResultsIn the dystrophic context of the GRMD dog, disease-related deregulation is observed in the case of 282 genes related to various processes such as inflammatory response, regeneration, calcium ion binding, extracellular matrix organization, metabolism and apoptosis regulation. Importantly, we reveal the impact of MuStem cell transplantation on several molecular and cellular pathways based on a selection of 31 genes displaying signals specifically modulated by the treatment. Concomitant with a diffuse dystrophin expression, a histological remodelling and a stabilization of GRMD dog clinical status, we show that cell delivery is associated with an up-regulation of genes reflecting a sustained enhancement of muscle regeneration. We also identify a decreased mRNA expression of a set of genes having metabolic functions associated with lipid homeostasis and energy. Interestingly, ubiquitin-mediated protein degradation is highly enhanced in GRMD dog muscle after systemic delivery of MuStem cells.ConclusionsOverall, our results provide the first high-throughput characterization of GRMD dog muscle and throw new light on the complex molecular/cellular effects associated with muscle repair and the clinical efficacy of MuStem cell-based therapy.     

Psychological balance in high level athletes: gender-based differences and sport-specific patterns

Objectives

Few epidemiological studies have focused on the psychological health of high level athletes. This study aimed to identify the principal psychological problems encountered within French high level athletes, and the variations in their prevalence based on sex and the sport practiced.

Methods

Multivariate analyses were conducted on nationwide data obtained from the athletes'' yearly psychological evaluations.

Results

A representative sample of 13% of the French athlete population was obtained. 17% of athletes have at least one ongoing or recent disorder, generalized anxiety disorder (GAD) being the most prevalent (6%), followed by non-specific eating disorders (4.2%). Overall, 20.2% of women had at least one psychopathology, against 15.1% in men. This female predominance applied to anxiety and eating disorders, depression, sleep problems and self-harming behaviors. The highest rates of GAD appeared in aesthetic sports (16.7% vs. 6.8% in other sports for men and 38.9% vs. 10.3% for women); the lowest prevalence was found in high risk sports athletes (3.0% vs. 3.5%). Eating disorders are most common among women in racing sports (14% vs. 9%), but for men were found mostly in combat sports (7% vs. 4.8%).

Discussion

This study highlights important differences in psychopathology between male and female athletes, demonstrating that the many sex-based differences reported in the general population apply to elite athletes. While the prevalence of psychological problems is no higher than in the general population, the variations in psychopathology in different sports suggest that specific constraints could influence the development of some disorders.