Dopexamine and norepinephrine versus epinephrine on gastric perfusion in patients with septic shock: a randomized study [NCT00134212]

INTRODUCTION: Microcirculatory blood flow, and notably gut perfusion, is important in the development of multiple organ failure in septic shock. We compared the effects of dopexamine and norepinephrine (noradrenaline) with those of epinephrine (adrenaline) on gastric mucosal blood flow (GMBF) in patients with septic shock. The effects of these drugs on oxidative stress were also assessed. METHODS: This was a prospective randomized study performed in a surgical intensive care unit among adults fulfilling usual criteria for septic shock. Systemic and pulmonary hemodynamics, GMBF (laser-Doppler) and malondialdehyde were assessed just before catecholamine infusion (T(0)), as soon as mean arterial pressure (MAP) reached 70 to 80 mmHg (T(1)), and 2 hours (T(2)) and 6 hours (T(3)) after T(1). Drugs were titrated from 0.2 μg kg(-1 )min(-1 )with 0.2 μg kg(-1 )min(-1 )increments every 3 minutes for epinephrine and norepinephrine, and from 0.5 μg kg(-1 )min(-1 )with 0.5 μg kg(-1 )min(-1 )increments every 3 minutes for dopexamine. RESULTS: Twenty-two patients were included (10 receiving epinephrine, 12 receiving dopexamine–norepinephrine). There was no significant difference between groups on MAP at T(0), T(1), T(2), and T(3). Heart rate and cardiac output increased significantly more with epinephrine than with dopexamine–norepinephrine, whereas. GMBF increased significantly more with dopexamine–norepinephrine than with epinephrine between T(1 )and T(3 )(median values 106, 137, 133, and 165 versus 76, 91, 90, and 125 units of relative flux at T(0), T(1), T(2 )and T(3), respectively). Malondialdehyde similarly increased in both groups between T(1 )and T(3). CONCLUSION: In septic shock, at doses that induced the same effect on MAP, dopexamine–norepinephrine enhanced GMBF more than epinephrine did. No difference was observed on oxidative stress

Transcription-coupled deposition of histone modifications during MHC class II gene activation

Posttranslational histone modifications associated with actively expressed genes are generally believed to be introduced primarily by histone-modifying enzymes that are recruited by transcription factors or their associated co-activators. We have performed a comprehensive spatial and temporal analyses of the histone modifications that are deposited upon activation of the MHC class II gene HLA-DRA by the co-activator CIITA. We find that transcription-associated histone modifications are introduced during two sequential phases. The first phase precedes transcription initiation and is characterized exclusively by a rapid increase in histone H4 acetylation over a large upstream domain. All other modifications examined, including the acetylation and methylation of several residues in histone H3, are restricted to short regions situated at or within the 5′ end of the gene and are established during a second phase that is concomitant with ongoing transcription. This second phase is completely abrogated when elongation by RNA polymerase II is blocked. These results provide strong evidence that transcription elongation can play a decisive role in the deposition of histone modification patterns associated with inducible gene activatio

Transcription-coupled deposition of histone modifications during MHC class II gene activation

Posttranslational histone modifications associated with actively expressed genes are generally believed to be introduced primarily by histone-modifying enzymes that are recruited by transcription factors or their associated co-activators. We have performed a comprehensive spatial and temporal analyses of the histone modifications that are deposited upon activation of the MHC class II gene HLA-DRA by the co-activator CIITA. We find that transcription-associated histone modifications are introduced during two sequential phases. The first phase precedes transcription initiation and is characterized exclusively by a rapid increase in histone H4 acetylation over a large upstream domain. All other modifications examined, including the acetylation and methylation of several residues in histone H3, are restricted to short regions situated at or within the 5′ end of the gene and are established during a second phase that is concomitant with ongoing transcription. This second phase is completely abrogated when elongation by RNA polymerase II is blocked. These results provide strong evidence that transcription elongation can play a decisive role in the deposition of histone modification patterns associated with inducible gene activation

Specificity determinants for the two tRNA substrates of the cyclodipeptide synthase AlbC from Streptomyces noursei

International audienceCyclodipeptide synthases (CDPSs) use two aminoacyl-tRNA substrates in a sequential ping-pong mechanism to form a cyclodipeptide. The crystal structures of three CDPSs have been determined and all show a Rossmann-fold domain similar to the catalytic domain of class-I aminoacyl-tRNA synthetases (aaRSs). Structural features and mutational analyses however suggest that CDPSs and aaRSs interact differently with their tRNA substrates. We used AlbC from Streptomyces noursei that mainly produces cyclo(l-Phe-l-Leu) to investigate the interaction of a CDPS with its substrates. We demonstrate that Phe-tRNA Phe is the first substrate accommodated by AlbC. Its binding to AlbC is dependent on basic residues located in the helix ␣4 that form a basic patch at the surface of the protein. AlbC does not use all of the Leu-tRNA Leu isoacceptors as a second substrate. We show that the G 1-C 72 pair of the acceptor stem is essential for the recognition of the second substrate. Substitution of D163 located in the loop ␣6–␣7 or D205 located in the loop ␤6–␣8 affected Leu-tRNA Leu isoacceptors specificity, suggesting the involvement of these residues in the binding of the second substrate. This is the first demonstration that the two substrates of CDPSs are accommodated in different binding sites

Production, secretion and purification of a correctly folded staphylococcal antigen in Lactococcus lactis.

International audienceLactococcus lactis, a lactic acid bacterium traditionally used to ferment milk and manufacture cheeses, is also, in the biotechnology field, an interesting host to produce proteins of medical interest, as it is "Generally Recognized As Safe". Furthermore, as L. lactis naturally secretes only one major endogenous protein (Usp45), the secretion of heterologous proteins in this species facilitates their purification from a protein-poor culture medium. Here, we developed and optimized protein production and secretion in L. lactis to obtain proteins of high quality, both correctly folded and pure to a high extent. As proteins to be produced, we chose the two transmembrane members of the HtrA protease family in Staphylococcus aureus, an important extra-cellular pathogen, as these putative surface-exposed antigens could constitute good targets for vaccine development. A recombinant ORF encoding a C-terminal, soluble, proteolytically inactive and tagged form of each staphylococcal HtrA protein was cloned into a lactococcal expression-secretion vector. After growth and induction of recombinant gene expression, L. lactis was able to produce and secrete each recombinant rHtrA protein as a stable form that accumulated in the culture medium in similar amounts as the naturally secreted endogenous protein, Usp45. L. lactis growth in fermenters, in particular in a rich optimized medium, led to higher yields for each rHtrA protein. Protein purification from the lactococcal culture medium was easily achieved in one step and allowed recovery of highly pure and stable proteins whose identity was confirmed by mass spectrometry. Although rHtrA proteins were monomeric, they displayed the same secondary structure content, thermal stability and chaperone activity as many other HtrA family members, indicating that they were correctly folded. rHtrA protein immunogenicity was established in mice. The raised polyclonal antibodies allowed studying the expression and subcellular localization of wild type proteins in S. aureus: although both proteins were expressed, only HtrA1 was found to be, as predicted, exposed at the staphylococcal cell surface suggesting that it could be a better candidate for vaccine development. In this study, an efficient process was developed to produce and secrete putative staphylococcal surface antigens in L. lactis and to purify them to homogeneity in one step from the culture supernatant. This allowed recovering fully folded, stable and pure proteins which constitute promising vaccine candidates to be tested for protection against staphylococcal infection. L. lactis thus proved to be an efficient and competitive cell factory to produce proteins of high quality for medical applications

A saturated SSR/DArT linkage map of Musa acuminata addressing genome rearrangements among bananas

Background: The genus Musa is a large species complex which includes cultivars at diploid and triploid levels. These sterile and vegetatively propagated cultivars are based on the A genome from Musa acuminata, exclusively for sweet bananas such as Cavendish, or associated with the B genome (Musa balbisiana) in cooking bananas such as Plantain varieties. In M. acuminata cultivars, structural heterozygosity is thought to be one of the main causes of sterility, which is essential for obtaining seedless fruits but hampers breeding. Only partial genetic maps are presently available due to chromosomal rearrangements within the parents of the mapping populations. This causes large segregation distortions inducing pseudo-linkages and difficulties in ordering markers in the linkage groups. The present study aims at producing a saturated linkage map of M. acuminata, taking into account hypotheses on the structural heterozygosity of the parents. Results: An F1 progeny of 180 individuals was obtained from a cross between two genetically distant accessions of M. acuminata, 'Borneo' and 'Pisang Lilin' (P. Lilin). Based on the gametic recombination of each parent, two parental maps composed of SSR and DArT markers were established. A significant proportion of the markers (21.7%) deviated (p < 0.05) from the expected Mendelian ratios. These skewed markers were distributed in different linkage groups for each parent. To solve some complex ordering of the markers on linkage groups, we associated tools such as tree-like graphic representations, recombination frequency statistics and cytogenetical studies to identify structural rearrangements and build parsimonious linkage group order. An illustration of such an approach is given for the P. Lilin parent. Conclusions: We propose a synthetic map with 11 linkage groups containing 489 markers (167 SSRs and 322 DArTs) covering 1197 cM. This first saturated map is proposed as a "reference Musa map" for further analyses. We also propose two complete parental maps with interpretations of structural rearrangements localized on the linkage groups. The structural heterozygosity in P. Lilin is hypothesized to result from a duplication likely accompanied by an inversion on another chromosome. This paper also illustrates a methodological approach, transferable to other species, to investigate the mapping of structural rearrangements and determine their consequences on marker segregation. (Résumé d'auteur

High-Resolution Analysis of Parent-of-Origin Allelic Expression in the Arabidopsis Endosperm

Genomic imprinting is an epigenetic phenomenon leading to parent-of-origin specific differential expression of maternally and paternally inherited alleles. In plants, genomic imprinting has mainly been observed in the endosperm, an ephemeral triploid tissue derived after fertilization of the diploid central cell with a haploid sperm cell. In an effort to identify novel imprinted genes in Arabidopsis thaliana, we generated deep sequencing RNA profiles of F1 hybrid seeds derived after reciprocal crosses of Arabidopsis Col-0 and Bur-0 accessions. Using polymorphic sites to quantify allele-specific expression levels, we could identify more than 60 genes with potential parent-of-origin specific expression. By analyzing the distribution of DNA methylation and epigenetic marks established by Polycomb group (PcG) proteins using publicly available datasets, we suggest that for maternally expressed genes (MEGs) repression of the paternally inherited alleles largely depends on DNA methylation or PcG-mediated repression, whereas repression of the maternal alleles of paternally expressed genes (PEGs) predominantly depends on PcG proteins. While maternal alleles of MEGs are also targeted by PcG proteins, such targeting does not cause complete repression. Candidate MEGs and PEGs are enriched for cis-proximal transposons, suggesting that transposons might be a driving force for the evolution of imprinted genes in Arabidopsis. In addition, we find that MEGs and PEGs are significantly faster evolving when compared to other genes in the genome. In contrast to the predominant location of mammalian imprinted genes in clusters, cluster formation was only detected for few MEGs and PEGs, suggesting that clustering is not a major requirement for imprinted gene regulation in Arabidopsis

Death and the Societies of Late Antiquity

Ce volume bilingue, comprenant un ensemble de 28 contributions disponibles en français et en anglais (dans leur version longue ou abrégée), propose d’établir un état des lieux des réflexions, recherches et études conduites sur le fait funéraire à l’époque tardo-antique au sein des provinces de l’Empire romain et sur leurs régions limitrophes, afin d’ouvrir de nouvelles perspectives sur ses évolutions possibles. Au cours des trois dernières décennies, les transformations considérables des méthodologies déployées sur le terrain et en laboratoire ont permis un renouveau des questionnements sur les populations et les pratiques funéraires de l’Antiquité tardive, période marquée par de multiples changements politiques, sociaux, démographiques et culturels. L’apparition de ce qui a été initialement désigné comme une « Anthropologie de terrain », qui fut le début de la démarche archéothanatologique, puis le récent développement d’approches collaboratives entre des domaines scientifiques divers (archéothanatologie, biochimie et géochimie, génétique, histoire, épigraphie par exemple) ont été décisives pour le renouvellement des problématiques d’étude : révision d’anciens concepts comme apparition d’axes d’analyse inédits. Les recherches rassemblées dans cet ouvrage sont articulées autour de quatre grands thèmes : l’évolution des pratiques funéraires dans le temps, l’identité sociale dans la mort, les ensembles funéraires en transformation (organisation et topographie) et les territoires de l’empire (du cœur aux marges). Ces études proposent un réexamen et une révision des données, tant anthropologiques qu’archéologiques ou historiques sur l’Antiquité tardive, et révèlent, à cet égard, une mosaïque de paysages politiques, sociaux et culturels singulièrement riches et complexes. Elles accroissent nos connaissances sur le traitement des défunts, l’emplacement des aires funéraires ou encore la structure des sépultures, en révélant une diversité de pratiques, et permettent au final de relancer la réflexion sur la manière dont les sociétés tardo-antiques envisagent la mort et sur les éléments permettant d’identifier et de définir la diversité des groupes qui les composent. Elles démontrent ce faisant que nous pouvons véritablement appréhender les structures culturelles et sociales des communautés anciennes et leurs potentielles transformations, à partir de l’étude des pratiques funéraires.This bilingual volume proposes to draw up an assessment of the recent research conducted on funerary behavior during Late Antiquity in the provinces of the Roman Empire and on their borders, in order to open new perspectives on its possible developments. The considerable transformations of the methodologies have raised the need for a renewal of the questions on the funerary practices during Late Antiquity, a period marked by multiple political, social, demographic and cultural changes. The emergence field anthropology, which was the beginning of archaeothanatology, and then the recent development of collaborative approaches between various scientific fields (archaeothanatology, biochemistry and geochemistry, genetics, history, epigraphy, for example), have been decisive. The research collected in this book is structured around four main themes: Evolution of funerary practices over time; Social identity through death; Changing burial grounds (organisation and topography); Territories of the Empire (from the heart to the margins). These studies propose a review and a revision of the data, both anthropological and archaeological or historical on Late Antiquity, and reveal a mosaic of political, social, and cultural landscapes singularly rich and complex. In doing so, they demonstrate that we can truly understand the cultural and social structures of ancient communities and their potential transformations, based on the study of funerary practices