Streaming acoustique dans un puits de culture cellulaire : application à la sonoporation

Le phénomène de sonoporation est intimement lié à l'interaction entre une cellule et une bulle de cavitation. Pour obtenir une information sur le caractère spatial de la cavitation ultrasonore générée, et en vue de la transfection de cellules adhérentes dans un puits de culture biologique, cette étude vise à caractériser le champ acoustique et le streaming dans une cavité non rectangulaire. Pour ce faire, des particules fluorescentes (3µm) sont utilisées sous microscope et permettent de quantifier le champ de déplacement au voisinage de la paroi

NMR structure and ion channel activity of the p7 protein from hepatitis C virus

The small membrane protein p7 of hepatitis C virus forms oligomers and exhibits ion channel activity essential for virus infectivity. These viroporin features render p7 an attractive target for antiviral drug development. In this study, p7 from strain HCV-J (genotype 1b) was chemically synthesized and purified for ion channel activity measurements and structure analyses. p7 forms cation-selective ion channels in planar lipid bilayers and at the single-channel level by the patch clamp technique. Ion channel activity was shown to be inhibited by hexamethylene amiloride but not by amantadine. Circular dichroism analyses revealed that the structure of p7 is mainly α-helical, irrespective of the membrane mimetic medium (e.g. lysolipids, detergents, or organic solvent/water mixtures). The secondary structure elements of the monomeric form of p7 were determined by 1H and 13C NMR in trifluoroethanol/water mixtures. Molecular dynamics simulations in a model membrane were combined synergistically with structural data obtained from NMR experiments. This approach allowed us to determine the secondary structure elements of p7, which significantly differ from predictions, and to propose a three-dimensional model of the monomeric form of p7 associated with the phospholipid bilayer. These studies revealed the presence of a turn connecting an unexpected N-terminal α-helix to the first transmembrane helix, TM1, and a long cytosolic loop bearing the dibasic motif and connecting TM1 to TM2. These results provide the first detailed experimental structural framework for a better understanding of p7 processing, oligomerization, and ion channel gating mechanism.Instituto de Física La Plat

NMR structure and ion channel activity of the p7 protein from hepatitis C virus

The small membrane protein p7 of hepatitis C virus forms oligomers and exhibits ion channel activity essential for virus infectivity. These viroporin features render p7 an attractive target for antiviral drug development. In this study, p7 from strain HCV-J (genotype 1b) was chemically synthesized and purified for ion channel activity measurements and structure analyses. p7 forms cation-selective ion channels in planar lipid bilayers and at the single-channel level by the patch clamp technique. Ion channel activity was shown to be inhibited by hexamethylene amiloride but not by amantadine. Circular dichroism analyses revealed that the structure of p7 is mainly α-helical, irrespective of the membrane mimetic medium (e.g. lysolipids, detergents, or organic solvent/water mixtures). The secondary structure elements of the monomeric form of p7 were determined by 1H and 13C NMR in trifluoroethanol/water mixtures. Molecular dynamics simulations in a model membrane were combined synergistically with structural data obtained from NMR experiments. This approach allowed us to determine the secondary structure elements of p7, which significantly differ from predictions, and to propose a three-dimensional model of the monomeric form of p7 associated with the phospholipid bilayer. These studies revealed the presence of a turn connecting an unexpected N-terminal α-helix to the first transmembrane helix, TM1, and a long cytosolic loop bearing the dibasic motif and connecting TM1 to TM2. These results provide the first detailed experimental structural framework for a better understanding of p7 processing, oligomerization, and ion channel gating mechanism.Instituto de Física La Plat

Deamidation and disulfide bridge formation in human calbindin D28k with effects on calcium binding

Calbindin D28k (calbindin) is a cytoplasmic protein expressed in the central nervous system, which is implied in Ca2+ homeostasis and enzyme regulation. A combination of biochemical methods and mass spectrometry has been used to identify post-translational modifications of human calbindin. The protein was studied at 37°C or 50°C in the presence or absence of Ca2+. One deamidation site was identified at position 203 (Asn) under all conditions. Kinetic experiments show that deamidation of Asn 203 occurs at a rate of 0.023 h−1 at 50°C for Ca2+-free calbindin. Deamidation is slower for the Ca2+-saturated protein. The deamidation process leads to two Asp iso-forms, regular Asp and iso-Asp. The form with regular Asp 203 binds four Ca2+ ions with high affinity and positive cooperativity, i.e., in a very similar manner to non-deamidated protein. The form with β-aspartic acid (or iso-Asp 203) has reduced affinity for two or three sites leading to sequential Ca2+ binding, i.e., the Ca2+-binding properties are significantly perturbed. The status of the cysteine residues was also assessed. Under nonreducing conditions, cysteines 94 and 100 were found both in reduced and oxidized form, in the latter case in an intramolecular disulfide bond. In contrast, cysteines 187, 219, and 257 were not involved in any disulfide bonds. Both the reduced and oxidized forms of the protein bind four Ca2+ ions with high affinity in a parallel manner and with positive cooperativity

Validating a French-language version of Health Communication Assessment Tool: Content validity, test-retest reliability, interrater agreement, and implications for further development

Introduction The assessment of the communication skills of nurses in clinical settings bears both practical and educational significance. Despite the significance of communication skills to realize high-quality patient-care, the development and validation of such an instrument applicable in different cultural contexts is still lacking and faces certain challenges. Additionally, the assessment of nurse-patient communication, which is dynamic and context-bounded, requires a fine-grained instrument, while not compromising the complexities of the skills under assessment. The aim of the present study was to test a French-language version of the Health Communication Assessment Tool (HCAT) (Campbell et al., 2013), on a French-speaking sample in Belgium against different measures of content relevance, validity, and reliability. Based on cognitive fluency perspective, we developed three simulated scenarios registering three levels of skill performance, including low, medium, and high demonstration. Thus, the psychometric properties of the HCAT instrument can be thoroughly examined. Methods Ten experts in communication and 52 nurse educators participated in the study. Firstly, the instrument was translated from English to French using back-translation technique. After semantic equivalence was agreed upon, the experts rated the relevance of the 22 items making up the HCAT, resulting in the removal of one culturally-irrelevant item. Subsequently, the experts and nurse educators rated the communication skills of the nurse in three scenarios. To ensure test-retest reliability, the nurse educators rated the scenarios twice within a four-week interval. The experts’ ratings were used as the gold standard when examining the reliability of the nurse educators’ ratings. Results & Discussion Exploratory and confirmatory factor analyses showed that the instrument could distinguish three aspects of communication skills, i.e. professional presentation, empathy, and trust building. Two-way random intraclass correlation coefficients (ICCs), ranging from 0.917 to 0.982, revealed that interrater agreement was excellent for consistency and absolute average-measures. However, at absolute single-measure, an ICC=0.425 suggested adequate interrater reliability for the medium-performed scenario, which decreased in low-performed (ICC=0.348) and high-performed (ICC=0.176) scenarios. Further, more than half of the items displayed poor test-retest reliability

The Performance of the Health Communication Assessment Tool© (HCAT-f) in Calibrating Different Levels of Nurse Communication Skills in a French-Speaking Context: Evidence and Clinical Implications

Communication skills training is essential in nurse education. Miscommunication may lead to adverse events and unsafe healthcare. To date, valid and reliable instruments to serve both communication training and assessment purposes across different cultural contexts are scarce. The present study empirically tested a French-language version of the Health Communication Assessment Tool© (HCAT-f) across different levels of communication skills performance to establish its reliability and validity through a cognitive fluency framework. Ten experts in communication and 52 nurse educators rated three videos simulating conversations between a nurse and a patient scheduled for lumpectomy. Each video captured a different level of communication skills performed by the nurse: High, medium, and low. Three distinct constructs were identified, i.e., professional presentation, empathy, and trust building. At absolute single-measure, an ICC = .43 suggested adequate interrater reliability of the whole scale for the medium-performed scenario, which decreased in low-performed (ICC = .35) and high-performed (ICC = .18) scenarios. The HCAT-f fulfils the criteria of linguistic equivalence, contextual relevance, and demonstrates acceptable construct validity. It can be used as a summative assessment tool after prior training on scale calibration is in place because interrater agreement was difficult to be established in high and low performance scenarios.