Molecular Interaction Studies of HIV-1 Matrix Protein p17 and Heparin: IDENTIFICATION OF THE HEPARIN-BINDING MOTIF OF p17 AS A TARGET FOR THE DEVELOPMENT OF MULTITARGET ANTAGONISTS

Once released by HIV cells, p17 binds heparan sulfate proteoglycans (HSPGs) and CXCR1 on leukocytes causing their dysfunction. By exploiting an approach integrating computational modeling, site-directed mutagenesis of p17, chemical desulfation of heparin, and surface plasmon resonance, we characterized the interaction of p17 with heparin, a HSPG structural analog, and CXCR1. p17 binds to heparin with an affinity (Kd 190 nM) that is similar to those of other heparin-binding viral proteins. Two stretches of basic amino acids (basic motifs) are present in p17 N and C termini. Neutralization (Arg3Ala substitution) of the N-terminal, but not of the C-terminal basic motif, causes the loss of p17 heparin-binding capacity. The N-terminal heparin-binding motif of p17 partially overlaps the CXCR1-binding domain. Accordingly, its neutralization prevents also p17 binding to the chemochine receptor. Competition experiments demonstrated that free heparin and heparan sulfate (HS), but not selectively 2-O-, 6-O-, and N-O desulfated heparins, prevent p17 binding to substrate-immobilized heparin, indicating that the sulfate groups of the glycosaminoglycan mediate p17 interaction. Evaluation of the p17 antagonist activity of a panel of biotechnological heparins derived by chemical sulfation of the Escherichia coli K5 polysaccharide revealed that the highlyN,O-sulfated derivative prevents the binding of p17 to both heparin and CXCR1, thus inhibiting p17-driven chemotactic migration of human monocytes with an efficiency that is higher than those of heparin and HS. Here, we characterized at a molecular level the interaction of p17 with its cellular receptors, laying the basis for the development of heparin-mimicking p17 antagonists

La mesure du CODB : un index du potentiel de reviviscence bactérienne des eaux

La mesure de la matière organique biodégradable dans l'eau est déterminée à partir de tests biologiques qui reposent sur deux concepts.Le premier est basé sur le suivi de la croissance de souches pures ou d'une population bactérienne mixte dans un échantillon d'eau. Le maximum de croissance obtenu est converti en Carbone Organique facilement Assimilable (COA) et exprimé en µg de C eq. acétate/l en tenant compte du rendement de croissance de ces bactéries dans des solutions d'acétate de sodium.Le second repose sur le suivi de la décroissance du Carbone Organique Dissous (COD) dans un échantillon d'eau ensemencé par une flore bactérienne indigène des eaux (flore en suspension ou flore fixée sur des particules de sable). La matière organique biadégradée est exprimée sous forme de Carbone Organique Dissous Biodégradable (CODB).Des essais ont été réalisés sur différents types d'eau (eaux de rivière de la Seine, de l'Oise et de la Marne, eaux en cours de traitement de.potabilisation, eaux distribuées et eaux distillées) afin de mettre en évidence la relation existant entre la mesure du CODB en présence de bactéries fixées sur du sable et le maximum de croissance bactérienne enregistré dans les mêmes échantillons stérilisés puis réensemencés par des souches pures (Pseudomonas fluorescens P17, Pseudomonas fluorescens P17 + Spirillum NOX) ou par un inoculum mixte de bactéries indigènes de l'eau.Les résultats de cette étude mettent en évidence :- une relation entre le CODB et le maximum de croissance (Pseudomonas fluorescens P17) médiocre (r = 0,716 ; n = 28) pour des échantillons d'eau ensemencés par Pseudomonas fluorescens P17 seul (dénombrement en gélose);- une relation entre le CODB et le maximum de croissance (Pseudomonas fluorescens P17) améliorée (r = 0,850, n = 31) pour des échantillons ensemencés simultanément avec un mélange de Pseudomonas fluorescens P17 et Spirillum NOX (dénombrement en gélose);- une relation entre le CODB et le maximum de croissance (Spirillum NOX) très faible (r = 0,264 n = 31; corrélation non significative) pour des échantillons ensemencés simultanément avec un mélange de P17 + NOX (dénombrement en gélose);- le coefficient de corrélation entre le CODB et le COA (Pseudomonas fluorescens P17 + Spirillum NOX) est de 0.769 (n = 31) avec une équivalence de 140 µg de COA (eq. acétate) par mg de CODE lorsque P17 est utilisé isolément et 90 µg de COA (eq. acétate) par mg de CODB lorsque P17 et NOX sont utilisés simultanément;- la relation entre le CODB et le maximum de croissance (flore naturelle mixte) est par contre très satisfaisante (r = 0,943; e = 30) lorsque les dénombrements bactériens sont effectués par microscopie en épifluorescence (coloration à l'acridine orange).Le rendement de croissance est alors de 1,7.109 cellules pour 1 mg de CODB mesuré en présence de sable biologique.En conclusion, la mesure du CODB au moyen de bactéries fixées, originellement décrite pour évaluer l'efficacité des filières de traitement de potabilisation vis-à-vis de l'élimination de la Matière Organique Biodégradable permet aussi de prédire le potentiel de recroissance bactérienne (bactéries indigènes) de différents types d'eau.Some attempts have been made to obtain an assessment of either the easily Assimilable Organic Carbon (AOC) or the total amount of Biodegradabte Dissolved Organic Carbon (BDOC) in drinking water.The first approach was developed in several methods.One of these methods consists of seeding pasteurized samples of water with pure cultures of bacteria (Pseudomonas fluorescens P17, P17 + Spirillum NOX). The growth of bacteria is monitored by a spread plate technique. AOC is expressed as equivalent amount of carbon (µg C eq. acetate/l) by using known yield coefficients of these strains in acetate or in oxalate.In the second approach, the BDOC content of water is evaluated by the reduction of Dissolved Organic Carton (DOC) in a water sample incubated up to 30 days with suspended indigenous bacteria or 5-7 days with bacteria fixed on sand-particles.This work was undertaken to determine the existing relationship between values of BDOC recorded by using fixed bacteria on sand and :- the maximum growth of a pure strain of Pseudomonas fluorescens P17. Water samples (60 ml) were pasteurized and inoculated with a subculture of Pseudomonas fluorescens (initial concentration = 500 CFU/ml). The maximum growth M 20 °C ± 1 °C was recorded by a pour plate method in PCA agar alter 3-4 days of incubation.- the maximum growth of strains P17 and NOX inoculated simultaneously in pasteurized water samples (40 ml) (500-1000 CFU/ml for each species). Each maximum growth at 20 °C ± 1 °C after seven, eight and vine days of incubation was recorded (spread plate method on R2A agar) and converted in AOC (growth constants : 4.1 106 CFU per µg C for P17, 1.2 107 CFU per µg C for NOX).- the maximum growth of a mixed indigenous population of bacteria. Water samples (500 ml) were filtered (0,2 µm filter) and reinoculated with one percent of river water. The maximum growth was recorded alter 3-4 days of incubation at 20 °C ± 1 °C by microscopic epifluorescent counts (acridin orange coloration).Comparisons were done with different types of water including river water (Seine, Oise and Marne), partially treated water (conventional treatments), fully treated water, ground water, mineral water and distilled water.The study bas demonstrated :- a pour correlation (r = 0.716; n = 28) between BDOC values and the maximum growth of Pseudomonas fluorescens. This observation can be explained by the limited spectrum of activity and the low affinity of Pseudomonas fluorescens for organic molecules. Under these conditions, the growth yield was 5.9 108 CFU per mg C (BDOC).- a better correlation (r = 0,850; n = 31) between BDOC and the maximum growth of P17 for water samples seeded simultaneously with P17 and NOX. Under these conditions, no correlation (r = 0.264 n = 31) was found between values of BDOC and maximum growth of NOX, demonstrating varying affinities of the NOX strain for ozonation by-products. In spite of this limit, the relation between BDOC and AOC values remained relatively good (r = 0.769; n = 31) with a factor of conversion of 1 mg BDOC for 90 µg C eq. acetate.- a significative correlation (r = 0.943; n = 30) between values of BDOC (fixed bacteria on sand) and the maximum growth of indigenous bacteria by direct microscopic examination epifluorescent counts. The growth yield was then 1,7 109 cells per mg of BDOC.In conclusion, the rapid estimation of BDOC by the technique using indigenous bacteria fixed on sand particles can be considered as a good predictor of the potential bacteria regrowth for different types of water

Regulation of caspase-3 processing by cIAP2 controls the switch between pro-inflammatory activation and cell death in microglia.

Cell Death and Disease is an open-access journal published by Nature Publishing Group. This work is licensed under a Creative Commons Attribution 4.0 International Licence. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons licence, users will need to obtain permission from the licence holder to reproduce the material.The activation of microglia, resident immune cells of the central nervous system, and inflammation-mediated neurotoxicity are typical features of neurodegenerative diseases, for example, Alzheimer's and Parkinson's diseases. An unexpected role of caspase-3, commonly known to have executioner role for apoptosis, was uncovered in the microglia activation process. A central question emerging from this finding is what prevents caspase-3 during the microglia activation from killing those cells? Caspase-3 activation occurs as a two-step process, where the zymogen is first cleaved by upstream caspases, such as caspase-8, to form intermediate, yet still active, p19/p12 complex; thereafter, autocatalytic processing generates the fully mature p17/p12 form of the enzyme. Here, we show that the induction of cellular inhibitor of apoptosis protein 2 (cIAP2) expression upon microglia activation prevents the conversion of caspase-3 p19 subunit to p17 subunit and is responsible for restraining caspase-3 in terms of activity and subcellular localization. We demonstrate that counteracting the repressive effect of cIAP2 on caspase-3 activation, using small interfering RNA targeting cIAP2 or a SMAC mimetic such as the BV6 compound, reduced the pro-inflammatory activation of microglia cells and promoted their death. We propose that the different caspase-3 functions in microglia, and potentially other cell types, reside in the active caspase-3 complexes formed. These results also could indicate cIAP2 as a possible therapeutic target to modulate microglia pro-inflammatory activation and associated neurotoxicity observed in neurodegenerative disorders

Teaching Fluid Mechanics for Undergraduate Students in Applied Industrial Biology: from Theory to Atypical Experiments

EBI is a further education establishment which provides education in applied industrial biology at level of MSc engineering degree. Fluid mechanics at EBI was considered by students as difficult who seemed somewhat unmotivated. In order to motivate them, we applied a new play-based pedagogy. Students were asked to draw inspiration from everyday life situations to find applications of fluid mechanics and to do experiments to verify and validate some theoretical results obtained in course. In this paper, we present an innovative teaching/learning pedagogy which includes the concept of learning through play and its implications in fluid mechanics for engineering. Examples of atypical experiments in fluid mechanics made by students are presented. Based on teaching evaluation by students, it is possible to know how students feel the course. The effectiveness of this approach to motivate students is presented through an analysis of students' teaching assessment. Learning through play proved a great success in fluid mechanics where course evaluations increased substantially. Fluid mechanics has been progressively perceived as interesting, useful, pleasant and easy to assimilate. It is shown that this pedagogy which includes educational gaming presents benefits for students. These experiments seem therefore to be a very effective tool for improving teaching/learning activities in higher education

A search-based technique for testing from extended finite state machine model

Extended finite state machines (EFSMs), and languages such as state-charts that are similar to EFSMs, are widely used to model state-based systems. When testing from an EFSM M it is common to aim to produce a set of test sequences (input sequences) that satisfies a test criterion that relates to the transition paths (TPs) of M that are executed by the test sequences. For example, we might require that the set of TPs triggered includes all of the transitions of M. One approach to generating such a set of test sequences is to split the problem into two stages: choosing a set of TPs that achieves the test criterion and then producing test sequences to trigger these TPs. However, the EFSM may contain infeasible TPs and the problem of generating a test sequence to trigger a given feasible TP (FTP) is generally uncomputable. In this paper we present a search-based approach that uses two techniques: (1) A TP fitness metric based on our previous work that estimates the feasibility of a given transition path; and (2) A fitness function to guide the search for a test sequence to trigger a given FTP. We evaluated our approach on five EFSMs: A simple in-flight safety system; a class II transport protocol; a lift system; an ATM; and the Inres initiator. In the experiments the proposed approach successfully tested approximately 96.75 % of the transitions and the proposed test sequence generation technique triggered all of the generated FTPs

Pinto Bandeira no caminho da indicação geográfica de vinhos.

bitstream/item/146614/1/Tonietto-BonVivant-v8-n95-p17-2007.pd