Evaluation of concrete structures by combining non-destructive testing methods (SENSO project)

The management and maintenance of the built heritage is one of the main interests of the owners of concrete structures. The engineers wish to obtain quantitative information about concrete properties and their variability. Non-destructive testing (NDT) is very popular in this context as it quickly provides relevant information on the integrity and evolution of the material, but several kinds of indicators representative of the concrete condition need to be evaluated. A French Project, named SENSO, aims to develop methods for the non-destructive evaluation of concrete based on a multi-techniques approach. Several families of techniques are concerned (ultrasonic, electromagnetic, electrical, etc.). The main objective is to define the sensitivity of the techniques and the variability of the evaluation for each indicator concerned. To achieve this, a large experimental programme, involving a representative range of concretes and several indicators, has been carried out. A large database, linking the NDT observables and the indicators, allows the different observables to be distinguished in terms of quality (linked to the variability) and in terms of relevance for the characterisation of each indicator. The improvement of the indicator evaluation by means of technique combinatio

Automated protein resonance assignments of magic angle spinning solid-state NMR spectra of β1 immunoglobulin binding domain of protein G (GB1)

Magic-angle spinning solid-state NMR (MAS SSNMR) represents a fast developing experimental technique with great potential to provide structural and dynamics information for proteins not amenable to other methods. However, few automated analysis tools are currently available for MAS SSNMR. We present a methodology for automating protein resonance assignments of MAS SSNMR spectral data and its application to experimental peak lists of the β1 immunoglobulin binding domain of protein G (GB1) derived from a uniformly 13C- and 15N-labeled sample. This application to the 56 amino acid GB1 produced an overall 84.1% assignment of the N, CO, CA, and CB resonances with no errors using peak lists from NCACX 3D, CANcoCA 3D, and CANCOCX 4D experiments. This proof of concept demonstrates the tractability of this problem

Structure of a Wbl protein and implications for NO sensing by M. tuberculosis

Mycobacterium tuberculosis causes pulmonary tuberculosis (TB) and claims ~1.8 million human lives per annum. Host nitric oxide (NO) is important in controlling TB infection. M. tuberculosis WhiB1 is a NO-responsive Wbl protein (actinobacterial iron-sulfur proteins first identified in the 1970s). Until now, the structure of a Wbl protein has not been available. Here a NMR structural model of WhiB1 reveals that Wbl proteins are four-helix bundles with a core of three α-helices held together by a [4Fe-4S] cluster. The iron-sulfur cluster is required for formation of a complex with the major sigma factor (σA) and reaction with NO disassembles this complex. The WhiB1 structure suggests that loss of the iron-sulfur cluster (by nitrosylation) permits positively charged residues in the C-terminal helix to engage in DNA binding, triggering a major reprogramming of gene expression that includes components of the virulence-critical ESX-1 secretion system

NMR methods to monitor the enzymatic depolymerization of heparin

Heparin and the related glycosaminoglycan, heparan sulfate, are polydisperse linear polysaccharides that mediate numerous biological processes due to their interaction with proteins. Because of the structural complexity and heterogeneity of heparin and heparan sulfate, digestion to produce smaller oligosaccharides is commonly performed prior to separation and analysis. Current techniques used to monitor the extent of heparin depolymerization include UV absorption to follow product formation and size exclusion or strong anion exchange chromatography to monitor the size distribution of the components in the digest solution. In this study, we used 1H nuclear magnetic resonance (NMR) survey spectra and NMR diffusion experiments in conjunction with UV absorption measurements to monitor heparin depolymerization using the enzyme heparinase I. Diffusion NMR does not require the physical separation of the components in the reaction mixture and instead can be used to monitor the reaction solution directly in the NMR tube. Using diffusion NMR, the enzymatic reaction can be stopped at the desired time point, maximizing the abundance of larger oligosaccharides for protein-binding studies or completion of the reaction if the goal of the study is exhaustive digestion for characterization of the disaccharide composition. In this study, porcine intestinal mucosa heparin was depolymerized using the enzyme heparinase I. The unsaturated bond formed by enzymatic cleavage serves as a UV chromophore that can be used to monitor the progress of the depolymerization and for the detection and quantification of oligosaccharides in subsequent separations. The double bond also introduces a unique multiplet with peaks at 5.973, 5.981, 5.990, and 5.998 ppm in the 1H-NMR spectrum downfield of the anomeric region. This multiplet is produced by the proton of the C-4 double bond of the non-reducing end uronic acid at the cleavage site. Changes in this resonance were used to monitor the progression of the enzymatic digestion and compared to the profile obtained from UV absorbance measurements. In addition, in situ NMR diffusion measurements were explored for their ability to profile the different-sized components generated over the course of the digestion

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