Non-Metabolic Membrane Tubulation and Permeability Induced by Bioactive Peptides

BACKGROUND: Basic cell-penetrating peptides are potential vectors for therapeutic molecules and display antimicrobial activity. The peptide-membrane contact is the first step of the sequential processes leading to peptide internalization and cell activity. However, the molecular mechanisms involved in peptide-membrane interaction are not well understood and are frequently controversial. Herein, we compared the membrane activities of six basic peptides with different size, charge density and amphipaticity: Two cell-penetrating peptides (penetratin and R9), three amphipathic peptides and the neuromodulator substance P. METHODOLOGY/PRINCIPAL FINDINGS: Experiments of X ray diffraction, video-microscopy of giant vesicles, fluorescence spectroscopy, turbidimetry and calcein leakage from large vesicles are reported. Permeability and toxicity experiments were performed on cultured cells. The peptides showed differences in bilayer thickness perturbations, vesicles aggregation and local bending properties which form lipidic tubular structures. These structures invade the vesicle lumen in the absence of exogenous energy. CONCLUSIONS/SIGNIFICANCE: We showed that the degree of membrane permeabilization with amphipathic peptides is dependent on both peptide size and hydrophobic nature of the residues. We propose a model for peptide-induced membrane perturbations that explains the differences in peptide membrane activities and suggests the existence of a facilitated “physical endocytosis,” which represents a new pathway for peptide cellular internalization

Development and Validation of an Internationally-Standardized, High-Resolution Capillary Gel-Based Electrophoresis PCR-Ribotyping Protocol for Clostridium difficile

PCR-ribotyping has been adopted in many laboratories as the method of choice for C. difficile typing and surveillance. However, issues with the conventional agarose gel-based technique, including inter-laboratory variation and interpretation of banding patterns have impeded progress. The method has recently been adapted to incorporate high-resolution capillary gel-based electrophoresis (CE-ribotyping), so improving discrimination, accuracy and reproducibility. However, reports to date have all represented single-centre studies and inter-laboratory variability has not been formally measured or assessed. Here, we achieved in a multi-centre setting a high level of reproducibility, accuracy and portability associated with a consensus CE-ribotyping protocol. Local databases were built at four participating laboratories using a distributed set of 70 known PCR-ribotypes. A panel of 50 isolates and 60 electronic profiles (blinded and randomized) were distributed to each testing centre for PCR-ribotype identification based on local databases generated using the standard set of 70 PCR-ribotypes, and the performance of the consensus protocol assessed. A maximum standard deviation of only ±3.8bp was recorded in individual fragment sizes, and PCR-ribotypes from 98.2% of anonymised strains were successfully discriminated across four ribotyping centres spanning Europe and North America (98.8% after analysing discrepancies). Consensus CE-ribotyping increases comparability of typing data between centres and thereby facilitates the rapid and accurate transfer of standardized typing data to support future national and international C. difficile surveillance programs

Simulations de spectres de résonance paramagnétique électronique avec ajustement automatique des paramètres spectroscopiques et du temps de corrélation de réorientation

La détermination précise du tenseur de couplage hyperfin A et du tenseur g peut être réalisée par ajustement automatique des paramètres recherchés, en minimisant la somme des carrés des écarts entre les spectres de RPE expérimental et calculé, au moyen de l'algorithme de Levenberg-Marquardt. Cette démarche est illustrée par l'exemple d'un gel de poly 4-vinylepyridine réticulé par des ions vanadyles, rigide à basses températures.Les mouvements de réorientation de la sonde paramagnétique modifient la forme des spectres comme on peut le constater pour ce gel au-dessus de 260 K. Le temps de corrélation τR pour un modèle de réorientation brownienne isotrope est pris comme paramètre ajustable des simulations spectrales, en utilisant l'algorithme de minimisation de la suite de Fibonacci

Some recent applications of computer simulations of ESR spectra to the dynamics of spin probes in gels and membranes

The ESR of S=1/2 paramagnetic species is one of the most convenient methods for probing microheterogeneous structures in gels, lyotropic liquid crystals or membranes. The interpretation of ESR spectra in term of dynamics of the spin probes generally requires computer simulations whose principles are briefly described. Some examples are given of simulations applied to the determination of the flexibility of the tridimensional network in polymer gels crosslinked by Cu2+ and VO2+ as well as to the dynamics of nitroxide spin probes in gels and in phospholipid model membranes

Genetic Context of Plasmid-Carried blaCMY-2-Like Genes in Enterobacteriaceae▿ †

Analysis of 15 European clinical Enterobacteriaceae isolates showed that differences in the genetic context of blaCMY-2-like genes reflected the replicon type, usually IncA/C or IncI1. These blaCMY-2 loci may originate from the same ISEcp1-mediated mobilization from the Citrobacter freundii chromosome as structures described in earlier studies