A forward genetic screen to identify hydrogenase-deficient mutants in the unicellular green alga Chlamydomonas reinhardtii

The ability of the unicellular green alga Chlamydomonas reinhardtii to evolve molecular hydrogen (H2) is due to the presence of oxygen-sensitive Fe-hydrogenases (HydA1/2), expressed in anoxic conditions that drive the photosynthetic electron flow to reduce protons into H2. In order to identify new players involved in H2 photoproduction in Chlamydomonas, an insertion mutant library was generated using cassettes conferring resistance to hygromycin or paromomycin. Hydrogenase activity is physiologically relevant during a transition from dark anoxia to light. In dark anoxic conditions, the cellular redox poise is high and the photosynthetic electron transport chain is fully reduced. Upon illumination, hydrogenase activity allows the reoxidation of photosynthetic intersystem electron carriers until oxic conditions and carbon fixation ability are restored. We thus designed an in vivo fluorescence imaging screen based on the different kinetics of photosynthesis induction between wild type and hydrogenase-deficient mutants [1]. At this stage, three putative hydrogenase mutants have been identified on 10,000 transformants. Molecular characterization of the insertion site of the resistance cassette by TAIL-PCR and genetic analyses of the linkage between the antibiotic resistance and the fluorescence phenotype showed that one mutant was untagged with the resistance while two tagged mutants were deficient for the HydG assembly factor

Expression and function of human hemokinin-1 in human and guinea pig airways

<p>Abstract</p> <p>Background</p> <p>Human hemokinin-1 (hHK-1) and endokinins are peptides of the tachykinin family encoded by the <it>TAC4 </it>gene. <it>TAC4 </it>and hHK-1 expression as well as effects of hHK-1 in the lung and airways remain however unknown and were explored in this study.</p> <p>Methods</p> <p>RT-PCR analysis was performed on human bronchi to assess expression of tachykinin and tachykinin receptors genes. Enzyme immunoassay was used to quantify hHK-1, and effects of hHK-1 and endokinins on contraction of human and guinea pig airways were then evaluated, as well as the role of hHK-1 on cytokines production by human lung parenchyma or bronchi explants and by lung macrophages.</p> <p>Results</p> <p>In human bronchi, expression of the genes that encode for hHK-1, tachykinin NK₁-and NK₂-receptors was demonstrated. hHK-1 protein was found in supernatants from explants of human bronchi, lung parenchyma and lung macrophages. Exogenous hHK-1 caused a contractile response in human bronchi mainly through the activation of NK₂-receptors, which blockade unmasked a NK₁-receptor involvement, subject to a rapid desensitization. In the guinea pig trachea, hHK-1 caused a concentration-dependant contraction mainly mediated through the activation of NK₁-receptors. Endokinin A/B exerted similar effects to hHK-1 on both human bronchi and guinea pig trachea, whereas endokinins C and D were inactive. hHK-1 had no impact on the production of cytokines by explants of human bronchi or lung parenchyma, or by human lung macrophages.</p> <p>Conclusions</p> <p>We demonstrate endogenous expression of <it>TAC4 </it>in human bronchi, the encoded peptide hHK-1 being expressed and involved in contraction of human and guinea pig airways.</p

CHRONIC TENDINOPATHIES AND PLATELET-RICH PLASMA TREATMENT: HOW TO IMPROVE THE EFFICIENCY?

peer reviewedIntroduction/Background Platelet-rich plasma (PRP) is blood plasma with a high concentration of autologous platelets which constitute an immense reservoir of growth factors. The clinical use of PRP is widespread in various medical applications. Material and Method In a literature review, we take a closer look at eight parameters which may influence the quality of PRP: 1) anticoagulants used to preserve the best platelet function, 2) the speed of centrifugation used to extract the platelets, 3) the platelet concentrations obtained, 4) the impact of the concentration of red and while blood cells on PRP actions, 5) platelet activators encouraging platelet degranulation and, hence, the release of growth factors, and 6) the use or nonuse of local anesthetics when carrying out infiltration. In addition to these parameters, it may be interesting to analyze other variables such as 7) the use of ultrasound guidance during the injection with a view to determining the influence they have on potential recovery. Results Analysis of the 59 studies shows that a majority use ACD-A as an anticoagulant during sampling. The best results have been obtained in studies which use no platelet activator. In terms of the speed of centrifugation, analysis of all these studies appears to confirm the existence of a great number of protocols in the literature. It is difficult to draw any conclusions when these different variables are unknown (volume of initial sample, equipment used to obtain the PRP). Although it would appear inadvisable to administer a local anesthetic Finally, it appears to be advisable to carry out infiltration under ultrasound guidance. Conclusion Our study helped identify features of PRP recommended a platelet concentration lower than 5 times the baseline (from 3 to 4), and avoiding leukocytes and erythrocytes. We recommend leveraging this information about PRP for future studies

Isolement et caractérisation de mutants affectés dans le transport d'électrons photosynthétique en anoxie chez la microalgue verte Chlamydomonas reinhardtii. Photoproduction d'hydrogène et Voie de biosynthèse de la phylloquinone.

La microalgue verte Chlamydomonas reinhardtii, qui a contribué de façon remarquable à la compréhension de nombreux mécanismes biologiques tels que la photosynthèse, intéresse aujourd’hui plus particulièrement le secteur des énergies renouvelables pour sa capacité à produire de l’hydrogène moléculaire (H2) à la lumière. Cette photoproduction d’H2 est liée à la présence en anoxie d’hydrogénases qui sont connectées via la ferrédoxine à la chaîne de transport d’électrons photosynthétique. On distingue deux voies de transfert d’électrons: la première dépendant de la photolyse de l’eau par le PSII (voie PSII-dépendante), la seconde de l’action d’une NAD(P)H déshydrogénase de type II (NDA2, voie PSII-indépendante). En raison de la sensibilité des hydrogénases à l’oxygène, cette production n’est que transitoire. Bien que ce processus puisse potentiellement réduire notre dépendance aux énergies fossiles, il est à l’heure actuelle économiquement inexploitable compte tenu de sa faible productivité. L’amélioration génétique des microalgues pourrait partiellement remédier à ce problème mais cette stratégie nécessite toutefois de posséder une connaissance fondamentale de toutes les voies métaboliques impliquées dans ce processus spécifique de l’anoxie. Afin d’identifier de nouvelles enzymes clés contrôlant la photoproduction d’H2 et de manière plus générale le métabolisme anaérobique, chez l’algue modèle Chlamydomonas reinhardtii, nous avons cherché dans un premier temps à comprendre la fonction de la protéine NDA3, une NAD(P)H déshydrogénase de type II chloroplastique. A l’instar de NDA2, celle-ci pourrait potentiellement participer à la réduction non-photochimique du pool de plastoquinones. Dans un deuxième temps, nous avons contribué au développement d’une nouvelle technique de criblage «haut débit» basée sur l’enregistrement de la cinétique d’induction de fluorescence des algues lors d’une transition de l’obscurité à la lumière en anoxie. Grâce à l’efficacité et à la rapidité de cette méthode nous avons pu identifier au sein d’une large collection de mutants insertionnels (~ 23 000) quatre mutants déficients pour le facteur d’assemblage HYDG (essentiel à la maturation des hydrogénases) et quatre autres mutants déficients pour la voie de biosynthèse de la phylloquinone. La caractérisation subséquente de ces mutants par des méthodes génétiques, moléculaires, biochimiques et biophysiques, nous a ainsi permis de comprendre in fine l’impact négatif de la perte du facteur HYDG ou de la phylloquinone sur le transfert d’électrons photosynthétique en anoxie

Optimization of poly(2-(dimethylamino)ethyl methacrylate)-co-poly(ethyleneglycol)/DNA complexes designed for cell transfection

Cell transfection relies upon the delivery of genomic material within cells in order to express selected DNA sequences. Non-viral gene delivery systems are preferable over viral vectors for several reasons: biosafety, low immunogenicity, higher loading capacity, and easiness of production. Their major drawbacks actually are their limited efficiency in vivo compared to viruses, their cytotoxicity and the fact that they are rapidly cleared up from the bloodstream. The aim of our study was to better characterize the physico-chemical behavior of the polycations based on poly(2-(dimethylamino)ethyl methacrylate)-co-poly(ethyleneglycol) and to control the formulation step to produce the polyelectrolyte complexes.Nouveaux polycations et vectorisation d’ADN dans les cellules de parois vasculaires” Convention Région Wallonne n° 14612, année 2001-2004 dans le cadre du concours Initiative 200