Helicase-defective RuvB(D113E) promotes RuvAB-mediated branch migration in vitro.

In Escherichia coli, the RuvA and RuvB proteins interact at Holliday junctions to promote branch migration leading to the formation of heteroduplex DNA. RuvA provides junction-binding specificity and RuvB drives ATP-dependent branch migration. Since RuvB contains sequence motifs characteristic of a DNA helicase and RuvAB exhibit helicase activity in vitro, we have analysed the role of DNA unwinding in relation to branch migration. A mutant RuvB protein, RuvB(D113E), mutated in helicase motif II (the DExx box), has been purified to homogeneity. The mutant protein forms hexameric rings on DNA similar to those formed by wild-type protein and promotes branch migration in the presence of RuvA. However, RuvB(D113E) exhibits reduced ATPase activity and is severely compromised in its DNA helicase activity. Models for RuvAB-mediated branch migration that invoke only limited DNA unwinding activity are proposed.

Co-trimoxazole prophylaxis is associated with reduced risk of incident tuberculosis in participants in the Swiss HIV Cohort Study.

Co-trimoxazole reduces mortality in HIV-infected adults with tuberculosis (TB), and in vitro data suggest potential antimycobacterial activity of co-trimoxazole. We aimed to evaluate whether prophylaxis with co-trimoxazole is associated with a decreased risk of incident TB in Swiss HIV Cohort Study (SHCS) participants. We determined the incidence of TB per 1,000 person-years from January 1992 to December 2012. Rates were analyzed separately in participants with current or no previous antiretroviral treatment (ART) using Poisson regression adjusted for CD4 cell count, sex, region of origin, injection drug use, and age. A total of 13,431 cohort participants contributed 107,549 person-years of follow-up: 182 patients had incident TB-132 (73%) before and 50 (27%) after ART initiation. The multivariable incidence rate ratios for cumulative co-trimoxazole exposure per year for persons with no previous ART and current ART were 0.70 (95% confidence interval [CI], 0.55 to 0.89) and 0.87 (95% CI, 0.74 to 1.0), respectively. Co-trimoxazole may prevent the development of TB among HIV-positive persons, especially among those with no previous ART.

Repression of flagellar genes in exponential phase by CsgD and CpxR, two crucial modulators of Escherichia coli biofilm formation.

Escherichia coli adapts its lifestyle to the variations of environmental growth conditions, swapping between swimming motility or biofilm formation. The stationary-phase sigma factor RpoS is an important regulator of this switch, since it stimulates adhesion and represses flagellar biosynthesis. By measuring the dynamics of gene expression, we show that RpoS inhibits the transcription of the flagellar sigma factor, FliA, in exponential growth phase. RpoS also partially controls the expression of CsgD and CpxR, two transcription factors important for bacterial adhesion. We demonstrate that these two regulators repress the transcription of fliA, flgM, and tar and that this regulation is dependent on the growth medium. CsgD binds to the flgM and fliA promoters around their -10 promoter element, strongly suggesting direct repression. We show that CsgD and CpxR also affect the expression of other known modulators of cell motility. We propose an updated structure of the regulatory network controlling the choice between adhesion and motility.

Bristle pattern formation in the thorax formation of Drosophila: a systems level approach

A fundamental question in developmental biology is how tissues are patterned to give rise to differentiated body structures with distinct morphologies. The Drosophila wing disc offers an accessible model to understand epithelial spatial patterning. It has been studied extensively using genetic and molecular approaches. Bristle patterns on the thorax, which arise from the medial part of the wing disc, are a classical model of pattern formation, dependent on a pre-pattern of trans-activators and –repressors. Despite of decades of molecular studies, we still only know a subset of the factors that determine the pre-pattern. We are applying a novel and interdisciplinary approach to predict regulatory interactions in this system. It is based on the description of expression patterns by simple logical relations (addition, subtraction, intersection and union) between simple shapes (graphical primitives). Similarities and relations between primitives have been shown to be predictive of regulatory relationships between the corresponding regulatory factors in other Systems, such as the Drosophila egg. Furthermore, they provide the basis for dynamical models of the bristle-patterning network, which enable us to make even more detailed predictions on gene regulation and expression dynamics. We have obtained a data-set of wing disc expression patterns which we are now processing to obtain average expression patterns for each gene. Through triangulation of the images we can transform the expression patterns into vectors which can easily be analysed by Standard clustering methods. These analyses will allow us to identify primitives and regulatory interactions. We expect to identify new regulatory interactions and to understand the basic Dynamics of the regulatory network responsible for thorax patterning. These results will provide us with a better understanding of the rules governing gene regulatory networks in general, and provide the basis for future studies of the evolution of the thorax-patterning network in particular.

Peroxisomal and microsomal lipid pathways associated with resistance to hepatic steatosis and reduced pro-inflammatory state.

Accumulation of fat in the liver increases the risk to develop fibrosis and cirrhosis and is associated with development of the metabolic syndrome. Here, to identify genes or gene pathways that may underlie the genetic susceptibility to fat accumulation in liver, we studied A/J and C57Bl/6 mice that are resistant and sensitive to diet-induced hepatosteatosis and obesity, respectively. We performed comparative transcriptomic and lipidomic analysis of the livers of both strains of mice fed a high fat diet for 2, 10, and 30 days. We found that resistance to steatosis in A/J mice was associated with the following: (i) a coordinated up-regulation of 10 genes controlling peroxisome biogenesis and β-oxidation; (ii) an increased expression of the elongase Elovl5 and desaturases Fads1 and Fads2. In agreement with these observations, peroxisomal β-oxidation was increased in livers of A/J mice, and lipidomic analysis showed increased concentrations of long chain fatty acid-containing triglycerides, arachidonic acid-containing lysophosphatidylcholine, and 2-arachidonylglycerol, a cannabinoid receptor agonist. We found that the anti-inflammatory CB2 receptor was the main hepatic cannabinoid receptor, which was highly expressed in Kupffer cells. We further found that A/J mice had a lower pro-inflammatory state as determined by lower plasma levels and IL-1β and granulocyte-CSF and reduced hepatic expression of their mRNAs, which were found only in Kupffer cells. This suggests that increased 2-arachidonylglycerol production may limit Kupffer cell activity. Collectively, our data suggest that genetic variations in the expression of peroxisomal β-oxidation genes and of genes controlling the production of an anti-inflammatory lipid may underlie the differential susceptibility to diet-induced hepatic steatosis and pro-inflammatory state.

A repeated GGA motif is critical for the activity and stability of the riboregulator RsmY of Pseudomonas fluorescens.

The riboregulator RsmY of Pseudomonas fluorescens strain CHA0 is an example of small regulatory RNAs belonging to the global Rsm/Csr regulatory systems controlling diverse cellular processes such as glycogen accumulation, motility, or formation of extracellular products in various bacteria. By binding multiple molecules of the small regulatory protein RsmA, RsmY relieves the negative effect of RsmA on the translation of several target genes involved in the biocontrol properties of strain CHA0. RsmY and functionally related riboregulators have repeated GGA motifs predicted to be exposed in single-stranded regions, notably in the loops of hairpins. The secondary structure of RsmY was corroborated by in vivo cleavage with lead acetate. RsmY mutants lacking three or five (out of six) of the GGA motifs showed reduced ability to derepress the expression of target genes in vivo and failed to bind the RsmA protein efficiently in vitro. The absence of GGA motifs in RsmY mutants resulted in reduced abundance of these transcripts and in a shorter half-life (< or = 6 min as compared with 27 min for wild type RsmY). These results suggest that both the interaction of RsmY with RsmA and the stability of RsmY strongly depend on the GGA repeats and that the ability of RsmY to interact with small regulatory proteins such as RsmA may protect this RNA from degradation.

Kinetics of crystal growth and equilibrium domains in eclogite of the Sesia Zone, Western Alps

Kinetics of crystal growth and equilibrium domains in eclogite of the Sesia Zone, Western Alps Darbellay Bastien Institut de Minéralogie et Géochimie Résumé grand public Comme toute matière, la roche est sensible à son environnement et cherche à s'adapter pour acquérir un état stable (état d'équilibre). Les changements des conditions physiques (température et pression) vont ainsi impliquer des modifications dans la roche. Le métamorphisme est l'étude de ces changements. Les minéraux qui constituent la roche peuvent modifier, leur structure, leur chimie ou être remplacer par d'autres minéraux plus stables. Il est ainsi crucial de déterminer les processus responsables et limitant de la croissance minérale. Trois processus permettent la croissance ; (1) la dissolution des éléments du réactant, (2) le transport de ces éléments vers le site de croissance, (3) l'incorporation de ces éléments dans la nouvelle structure. Cette thèse se focalise sur les structures des minéraux de haute pression (forme, zonation chimique, structure interne) pour essayer de déterminer les facteurs importants à l'origine de leur état final. Les zones d'étude se situent dans la zone de Sésia. La première partie traite de la problématique liée à l'incorporation d'un élément dans une structure minérale. A l'image de la croissance humaine, les irrégularités minéralogiques permettent de mettre en lumière un dysfonctionnement de la croissance due à un excès ou à une carence d'un élément. Bien dosé, cet élément est cependant essentiel à la croissance. Les zoisites (épidotes) des métabasites de la région de Cima di Bonze montrent une zonation chimique en sablier. Dans cette zonation la teneur en fer excède la capacité maximum que peut contenir la structure orthorhombique de la zoisite. Des défauts de structure permettent l'accommodation de cet excès. La zoisite peut ainsi adapter sa structure pour permettre l'incorporation d'une relativement grande quantité de fer. Les études précédentes montraient, pour des conditions similaires, la formation de deux épidotes distinctes. La deuxième partie se penche sur la compétition entre le minéral qui fait sa croissance et les minéraux (réactants) qui l'entourent. Les métapélites de la région du Monte Mucrone contiennent des grenats atollaires. Des études détaillées de la texture et de la zonation chimique du grenat ainsi qu'une modélisation thermodynamique ont permis de mieux cerner les facteurs importants responsables de la forme atollaire. Cette structure est obtenue par un changement du comportement de la croissance du grenat le long d'un chemin P-T hercynien. Dans un premier stade, le grenat croît rapidement et consume peu le quartz de la matrice. La croissance se fait ainsi le long des jointures des grains de quartz. Dans un second temps, les changements de conditions PT donnent une croissance lente du grenat et une forte consommation du quartz. Le grenat peut ainsi développer sa forme dodécaédrale classique. La troisième partie s'intéresse aux distances de transport par diffusion d'un élément (ici l'argon) durant la haute pression. Pour ce faire, un profile d'âges 40Ar/39Ar sur biotite a été mesuré depuis un veine de haute pression riche en argon jusque dans son encaissant (granitoïd du Monte Mucrone). Le profile montre une répartition des âges suivant une courbe de diffusion. Le transport se fait sur une longueur de deux centimètre avec l'aide d'un fluide. Il est réduit à une échelle millimétrique quand la phase fluide disparaît. Cette étude montre ainsi les difficultés de transport des éléments durant la haute pression ne permettant pas un rééquilibrage de la roche à grande échelle. Kinetics of crystal growth and equilibrium domains in eclogite of the Sesia Zone, Western Alps Darbellay Bastien Institut de Minéralogie et Géochimie Résumé de thèse Les processus de croissance (diffusion des éléments et les réactions d'interface) et les conditions dans lesquelles les minéraux grandissent (température, pression, fluide, composition chimique de la roche), déterminent la texture ainsi que la zonation des minéraux. Cette thèse se focalise, par le biais de textures peu communes, sur trois différents processus impliqués dans la croissance minérale à haute pression (Zone de Sésia, Alpes de l'Ouest, Italie). L'incorporation d'un élément dans une structure minérale ne peut se faire que dans des sites en accord avec la taille et la charge ionique de l'élément. De plus, la balance de charge doit être maintenue dans le minéral. La régularité de la structure cristalline fixe ainsi une limite maximum de concentration d'un élément donné. Les zoisites provenant des métabasites de la région de Cima di Bonze montrent des zonations en sablier caractérisées par une concentration anormale en fer. La zonation se marque par une différente teinte de biréfringence et par un plus grand angle d'extinction que le reste de la zoisite. Une inter-croissance de clinozoisite à l'intérieur de la structure orthorhombique de la zoisite peut ainsi être suspectée. Les analyses XRD (diffraction des rayons x) ainsi que les analyses Raman ne confirment pas cette suspicion. Seules les analyses TEM (microscope à électrons transmis) montrent des défauts de structure pouvant être interprétés comme des modules de clinozoisite. Ils ne peuvent cependant pas être considérés comme une phase thermodynamique. Un nouveau trou d'immiscibilité entre deux zoisite (X ep= 0.1 and Xep = 0.15) a ainsi pu être établi. Dans les métapélites la région du Monte Mucrone, des grenats fortement zonés montrent une évolution texturale singulière. Ils présentent une forme initiale de `champignon' qui se développe pour former une structure atollaire finale. L'étude conjuguée de la structure 3D et des zonations, ainsi que l'établissement d'un model thermodynamique, indiquent que ces structures proviennent de deux épisodes de croissances : (1) La croissance du grenat durant un chemin prograde hercynien (de 525 °C et 6.2 kbar à 640 °C et 9 kbar) permet la formation des textures atollaires. Elles sont le résultat d'une croissance poecilitique initiale suivie d'une croissance idiomorphique du grenat. (2) La structure est rendue plus complexe par la cristallisation d'un grenat homogène tout autour ainsi qu'à l'intérieur du grenat hercynien durant la haute pression alpine (550 °C and 20 kbar). L'arrivée de l'eau durant la haute pression facilite le transport d'éléments et permet une cristallisation rapide du grenat. La diffusion peut être un facteur limitant de la croissance minéralogique. Elle a aussi une grande importance pour la géochronologie. Une veine de haute pression à l'intérieur du granitoïde du Monte Mucrone a été étudiée dans le but de déterminer la distance de diffusion de l'argon. Le profile d'âges 40Ar/39Ar sur biotites, établi de la veine vers le métagranitoïde, suit une courbe de diffusion. Les âges sont élevés proche de la veine (800 Ma) puis décroissent jusqu'à des âges homogènes (170-150 Ma) à deux centimètres de la veine. La présence de fluide, marqué par de hautes concentrations en chlore, permet une diffusion centimétrique. Cependant, la distance est réduite à une échelle millimétrique quand le fluide est absent. Les très faibles distances de diffusion préservent les âges pré-alpins et impliquent un événement géologique pour les âges de 170-150 Ma. Kinetics of crystal growth and equilibrium domains in eclogite of the Sesia Zone, Western Alps Darbellay Bastien Institut de Minéralogie et Géochimie Thesis abstract Rock textures and zonings are the consequence of growth processes (element diffusion and interface reaction) steered by the environment in which they grew (pressure, differential stress, temperature, fluid and rock composition). The thesis presented here focuses on three different topics, each of it dealing with aspects of mineral growth processes during subduction, in a high-pressure environment. All studies were conducted in the Sesia Zone of the Western European Alps, Italy. The first study addresses the crystallography and geochemistry of element incorporation in zoisite, one of the major hydrous minerals found in subduction zone rocks. Elements can be incorporated into a mineral structure only on crystallographic sites that offer enough space for the ion and the overall charge balance has to be maintained. Element concentrations are hence limited. Incorporation of some elements produces complex zoning, including hourglass like patterns, which are the focus of the first contribution. Zoisites from Cima di Bonze (Sesia Zone) show spectacular hourglass zoning defined by Fe-content variations. The hourglass zones have a distinct birefringence and a different extinction angle than the regular part of the zoisite. We show by detailed XRD (X-ray diffraction) and confocal Raman analyses that the high Fe-zones are nevertheless zoisite, and not clinozoisite as one might expect. High resolution TEM (transmission electron microscopy) analyses show planar defects on (100) that can be interpreted as small-scale clinozoisite modules. However, these clinozoisites cannot be interpreted as a distinctive thermodynamic phase and the entire mineral has to be considered as zoisite. The miscibility gap between two zoisites (Xep = 0.1 and Xep = 0.15) can be then definite at 550 ± 50°C and 14 to 20 Kbar. Strongly zoned garnets in quartz rich metapelite from the Monte Mucrone area (Sesia Zone) show evolution form 3D mushroom to atoll structure. The second contribution presents textural investigations, garnet zoning and thermodynamic modeling that demonstrate that atoll garnets are the result of two distinctive growth events. (1) Garnet atoll structure is already formed during a prograde Hercynian path from 525 °C and 6.2 kbar to 640 °C and 9 kbar. It results in an initial poikilitic growth followed by a final idiomorphic growth event. (2) Alpine HP garnet are homogenous (550 °C and 20 kbar) and grew around and also inside the Hercynian garnet. Lack of prograde Alpine garnet and fast growth of the HP garnet is explained by the absence of water during much of the prograde path. Water saturation was only observed towards the end, close towards the peak metamorphic conditions. Diffusion could be a limiting factor for crystal growth. It has also a great importance in geochronology. HP vein inside the metagranitoide of the Monte Mucrone (~300 Ma) was investigated to determine argon diffusion scales during high-pressure metamorphism. 40Ar/39Ar biotite ages profile from the vein toward the metagranodiorite show a diffusion curve: old ages (800 Ma) located close to the vein decrease until homogenous 170-150 Ma ages are obtained, two centimeter away from the vein. Centimeter-scale diffusion occurs with help of a fluid phase marked by high chlorine concentrations. Argon diffusion is reduced to a millimeter scale when free fluid is absent. Very short diffusion distance permits to preserve pre-Alpine ages. The 170-150 Ma ages are considered to be geologic meaningful, probably resulting from the extensional tectonics linked to opening of the Tethian ocean.

Fibronectin distribution in the chick embryo during formation of the blastula.

The distribution of the fibronectin-rich extracellular matrix (ECM) in the chick embryo during formation of the blastula has been evaluated semiquantitatively using an electron microscopical immunogold staining technique. During the first 10 h of postlaying development, fibronectin was found in both embryonic area pellucida and extra-embryonic area opaca of the blastoderm. In the area pellucida, the fibronectin was (1) associated with the basal lamina of the epiblast, (2) present between epiblastic and hypoblastic cells and (3) occasionally internalized in hypoblastic cells. Along the embryonic axis, a transient and high density of ECM was associated with the front of the anteriorly and rapidly expanding hypoblast. Very high density of fibronectin was observed in the marginal zone of the area pellucida, where the epiblastic and deeper cell layers show contacts and intense re-arrangements. In the area opaca, fibronectin was at first found only sporadically between contacting cells, but its density increased steadily and markedly during the first day of development. These rapid and significant changes in the regional distribution of fibronectin-rich ECM are discussed with respect to the early morphogenesis of the chick embryo.

Cannabinoid withdrawal syndrome is reduced in pre-proenkephalin knock-out mice

The functional interactions between the endogenous cannabinoid and opioid systems were evaluated in pre-proenkephalin-deficient mice. Antinociception induced in the tail-immersion test by acute Delta9-tetrahydrocannabinol was reduced in mutant mice, whereas no difference between genotypes was observed in the effects induced on body temperature, locomotion, or ring catalepsy. During a chronic treatment with Delta9-tetrahydrocannabinol, the development of tolerance to the analgesic responses induced by this compound was slower in mice lacking enkephalin. In addition, cannabinoid withdrawal syndrome, precipitated in Delta9-tetrahydrocannabinol-dependent mice by the injection of SR141716A, was significantly attenuated in mutant mice. These results indicate that the endogenous enkephalinergic system is involved in the antinociceptive responses of Delta9-tetrahydrocannabinol and participates in the expression of cannabinoid abstinence.

Activity of the δ-Opioid Receptor Is Partially Reduced, Whereas Activity of the κ-Receptor Is Maintained in Mice Lacking the μ-Receptor

Previous pharmacological studies have indicated the possible existence of functional interactions between μ-, δ- and κ-opioid receptors in the CNS. We have investigated this issue using a genetic approach. Here we describe in vitro and in vivo functional activity of δ- and κ-opioid receptors in mice lacking the μ-opioid receptor (MOR). Measurements of agonist-induced [35S]GTPγS binding and adenylyl cyclase inhibition showed that functional coupling of δ- and κ-receptors to G-proteins is preserved in the brain of mutant mice. In the mouse vas deferens bioassay, deltorphin II and cyclic[d-penicillamine2,d-penicillamine5] enkephalin exhibited similar potency to inhibit smooth muscle contraction in both wild-type and MOR −/− mice. δ-Analgesia induced by deltorphin II was slightly diminished in mutant mice, when the tail flick test was used. Deltorphin II strongly reduced the respiratory frequency in wild-type mice but not in MOR −/− mice. Analgesic and respiratory responses produced by the selective κ-agonist U-50,488H were unchanged in MOR-deficient mice. In conclusion, the preservation of δ- and κ-receptor signaling properties in mice lacking μ-receptors provides no evidence for opioid receptor cross-talk at the cellular level. Intact antinociceptive and respiratory responses to the κ-agonist further suggest that the κ-receptor mainly acts independently from the μ-receptor in vivo. Reduced δ-analgesia and the absence of δ-respiratory depression in MOR-deficient mice together indicate that functional interactions may take place between μ-receptors and central δ-receptors in specific neuronal pathways.

Influence of physico-chemical material characteristics on staphylococcal biofilm formation--a qualitative and quantitative in vitro analysis of five different calcium phosphate bone grafts.

Various compositions of synthetic calcium phosphates (CaP) have been proposed and their use has considerably increased over the past decades. Besides differences in physico-chemical properties, resorption and osseointegration, artificial CaP bone graft might differ in their resistance against biofilm formation. We investigated standardised cylinders of 5 different CaP bone grafts (cyclOS, chronOS (both β-TCP (tricalcium phosphate)), dicalcium phosphate (DCP), calcium-deficient hydroxyapatite (CDHA) and α-TCP). Various physico-chemical characterisations e.g., geometrical density, porosity, and specific surface area were investigated. Biofilm formation was carried out in tryptic soy broth (TSB) and human serum (SE) using Staphylococcus aureus (ATCC 29213) and S. epidermidis RP62A (ATCC 35984). The amount of biofilm was analysed by an established protocol using sonication and microcalorimetry. Physico-chemical characterisation showed marked differences concerning macro- and micropore size, specific surface area and porosity accessible to bacteria between the 5 scaffolds. Biofilm formation was found on all scaffolds and was comparable for α-TCP, chronOS, CDHA and DCP at corresponding time points when the scaffolds were incubated with the same germ and/or growth media, but much lower for cyclOS. This is peculiar because cyclOS had an intermediate porosity, mean pore size, specific surface area, and porosity accessible to bacteria. Our results suggest that biofilm formation is not influenced by a single physico-chemical parameter alone but is a multi-step process influenced by several factors in parallel. Transfer from in vitro data to clinical situations is difficult; thus, advocating the use of cyclOS scaffolds over the four other CaP bone grafts in clinical situations with a high risk of infection cannot be clearly supported based on our data.

Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica

The inhalation of airborne pollutants, such as asbestos or silica, is linked to inflammation of the lung, fibrosis, and lung cancer. How the presence of pathogenic dust is recognized and how chronic inflammatory diseases are triggered are poorly understood. Here, we show that asbestos and silica are sensed by the Nalp3 inflammasome, whose subsequent activation leads to interleukin-1beta secretion. Inflammasome activation is triggered by reactive oxygen species, which are generated by a NADPH oxidase upon particle phagocytosis. (NADPH is the reduced form of nicotinamide adenine dinucleotide phosphate.) In a model of asbestos inhalation, Nalp3-/- mice showed diminished recruitment of inflammatory cells to the lungs, paralleled by lower cytokine production. Our findings implicate the Nalp3 inflammasome in particulate matter-related pulmonary diseases and support its role as a major proinflammatory "danger" receptor