A novel, robust peptidyl-lys metalloendopeptidase from Trametes coccinea recombinantly expressed in Komagataella phaffii

A novel peptidyl-lys metalloendopeptidase (Tc-LysN) from Tramates coccinea was recombinantly expressed in Komagataella phaffii using the native pro-protein sequence. The peptidase was secreted into the culture broth as zymogen (~38 kDa) and mature enzyme (~19.8 kDa) simultaneously. The mature Tc-LysN was purified to homogeneity with a single step anion-exchange chromatography at pH 7.2. N-terminal sequencing using TMTpro Zero and mass spectrometry of the mature Tc-LysN indicated that the pro-peptide was cleaved between the amino acid positions 184 and 185 at the Kex2 cleavage site present in the native pro-protein sequence. The pH optimum of Tc-LysN was determined to be 5.0 while it maintained ≥60% activity between pH values 4.5—7.5 and ≥30% activity between pH values 8.5—10.0, indicating its broad applicability. The temperature maximum of Tc-LysN was determined to be 60 °C. After 18 h of incubation at 80 °C, Tc-LysN still retained ~20% activity. Organic solvents such as methanol and acetonitrile, at concentrations as high as 40% (v/v), were found to enhance Tc-LysN’s activity up to ~100% and ~50%, respectively. Tc-LysN’s thermostability, ability to withstand up to 8 M urea, tolerance to high concentrations of organic solvents, and an acidic pH optimum make it a viable candidate to be employed in proteomics workflows in which alkaline conditions might pose a challenge. The nano-LC-MS/MS analysis revealed bovine serum albumin (BSA)’s sequence coverage of 84% using Tc-LysN which was comparable to the sequence coverage of 90% by trypsin peptides

Vibrio natriegens as Host for Expression of Multisubunit Membrane Protein Complexes

Escherichia coli is a convenient host for the expression of proteins, but the heterologous production of large membrane protein complexes often is hampered by the lack of specific accessory genes required for membrane insertion or cofactor assembly. In this study we introduce the non-pathogenic and fast-growing Vibrio natriegens as a suitable expression host for membrane-bound proteins from Vibrio cholerae. We achieved production of the primary Na+ pump, the NADH:quinone oxidoreductase (NQR), from V. cholerae in an active state, as indicated by increased overall NADH:quinone oxidoreduction activity of membranes from the transformed V. natriegens, and the sensitivity toward Ag+, a specific inhibitor of the NQR. Complete assembly of V. cholerae NQR expressed in V. natriegens was demonstrated by BN PAGE followed by activity staining. The secondary transport system Mrp from V. cholerae, another membrane-bound multisubunit complex, was also produced in V. natriegens in a functional state, as demonstrated by in vivo Li+ transport. V. natriegens is a promising expression host for the production of membrane protein complexes from Gram-negative pathogens

Climate-Based Models for Understanding and Forecasting Dengue Epidemics

Dengue fever is a major public health problem in the tropics and subtropics. Since no vaccine exists, understanding and predicting outbreaks remain of crucial interest. Climate influences the mosquito-vector biology and the viral transmission cycle. Its impact on dengue dynamics is of growing interest. We analyzed the epidemiology of dengue in Noumea (New Caledonia) from 1971 to 2010 and its relationships with local and remote climate conditions using an original approach combining a comparison of epidemic and non epidemic years, bivariate and multivariate analyses. We found that the occurrence of outbreaks in Noumea was strongly influenced by climate during the last forty years. Efficient models were developed to estimate the yearly risk of outbreak as a function of two meteorological variables that were contemporaneous (explicative model) or prior (predictive model) to the outbreak onset. Local threshold values of maximal temperature and relative humidity were identified. Our results provide new insights to understand the link between climate and dengue outbreaks, and have a substantial impact on dengue management in New Caledonia since the health authorities have integrated these models into their decision making process and vector control policies. This raises the possibility to provide similar early warning systems in other countries

Atelier théâtre en français langue étrangère auprès d'adolescents russes

The French theatre workshop provides a playful and experimental space to the teenagers who are learning French as a foreign language in Russia. They can exchange and interact with the other members of the group in order to realise a common project, namely, a theatrical performance in front of an audience. Requiring body language and verbal language, the French theatre workshop allows learners to mobilise all the resources at their disposal during their learning. In this way, they can overcome the linguistic gaps or insecurities they feel when they have to produce in French. Through his interpretation, the learner acts in various French-speaking contexts, fictional or real, becoming a social actor in the target language.L’atelier théâtre en français offre un espace ludique et expérimental à l’apprenant adolescent de FLE en Russie. Il peut échanger et interagir avec les autres membres de la troupe dans l’objectif de concrétiser un projet commun : une représentation théâtrale face à un public. Requérant le langage corporel et langagier, l’atelier théâtre en français permet aux apprenants de mobiliser toutes les ressources à disposition dans leur apprentissage. Ainsi, il peut palier aux lacunes ou à l’insécurité linguistique qu’il ressent lorsqu’il doit produire en français langue étrangère. A travers son interprétation, l’apprenant agit dans divers contextes francophones, fictifs ou réels, devenant un acteur social dans la langue cible

Bayreuth Productivity Analysis -a method for ascertaining and improving the holistic service productivity of acute care hospitals

Abstract The healthcare sector is lacking a method with which hospitals can measure the extent to which they achieve their goals in terms of aggregate productivity from both patients' and employees' perspectives. The Bayreuth Productivity Analysis (BPA) provides a solution to this problem because it uses two standardized questionnaires-one for patients and one for employees-to ascertain productivity at hospitals. These questionnaires were developed in several steps according to the principles of classical test theory, and they consist of six dimensions (information, organization, climate, methods, infrastructure and equipment) of five items each. One item describes a factual situation relevant to productivity and services so that it makes a contribution to the overall productivity of a hospital. After individualized evaluation of these items, the dimensions are subjectively weighted in the two questionnaires. The productivity index thus ascertained can be considered "holistic" when all patients and employees in a hospital make a differentiated assessment and weigh off each of the dimensions. In conclusion, the BPA constitutes a simple yet practicable method to ascertain and improve the holistic service productivity of hospitals