Structural insights into chaperone addiction of toxin-antitoxin systems

International audienceSecB chaperones assist protein export by binding both unfolded proteins and the SecA motor. Certain SecB homologs can also control toxin-antitoxin (TA) systems known to modulate bacterial growth in response to stress. In such TA-chaperone (TAC) systems, SecB assists the folding and prevents degradation of the antitoxin, thus facilitating toxin inhibition. Chaperone dependency is conferred by a C-terminal extension in the antitoxin known as chaperone addiction (ChAD) sequence, which makes the antitoxin aggregation-prone and prevents toxin inhibition. Using TAC of Mycobacterium tuberculosis, we present the structure of a SecB-like chaperone bound to its ChAD peptide. We find differences in the binding interfaces when compared to SecB–SecA or SecB-preprotein complexes, and show that the antitoxin can reach a functional form while bound to the chaperone. This work reveals how chaperones can use discrete surface binding regions to accommodate different clients or partners and thereby expand their substrate repertoire and functions

The influence of decision-making in tree ring-based climate reconstructions.

Tree-ring chronologies underpin the majority of annually-resolved reconstructions of Common Era climate. However, they are derived using different datasets and techniques, the ramifications of which have hitherto been little explored. Here, we report the results of a double-blind experiment that yielded 15 Northern Hemisphere summer temperature reconstructions from a common network of regional tree-ring width datasets. Taken together as an ensemble, the Common Era reconstruction mean correlates with instrumental temperatures from 1794-2016 CE at 0.79 (p < 0.001), reveals summer cooling in the years following large volcanic eruptions, and exhibits strong warming since the 1980s. Differing in their mean, variance, amplitude, sensitivity, and persistence, the ensemble members demonstrate the influence of subjectivity in the reconstruction process. We therefore recommend the routine use of ensemble reconstruction approaches to provide a more consensual picture of past climate variability

The insecticide resistance status of malaria vectors in the Mekong region

<p>Abstract</p> <p>Background</p> <p>Knowledge on insecticide resistance in target species is a basic requirement to guide insecticide use in malaria control programmes. Malaria transmission in the Mekong region is mainly concentrated in forested areas along the country borders, so that decisions on insecticide use should ideally be made at regional level. Consequently, cross-country monitoring of insecticide resistance is indispensable to acquire comparable baseline data on insecticide resistance.</p> <p>Methods</p> <p>A network for the monitoring of insecticide resistance, MALVECASIA, was set up in the Mekong region in order to assess the insecticide resistance status of the major malaria vectors in Cambodia, Laos, Thailand, and Vietnam. From 2003 till 2005, bioassays were performed on adult mosquitoes using the standard WHO susceptibility test with diagnostic concentrations of permethrin 0.75% and DDT 4%. Additional tests were done with pyrethroid insecticides applied by the different national malaria control programmes.</p> <p>Results</p> <p><it>Anopheles dirus s.s</it>., the main vector in forested malaria foci, was susceptible to permethrin. However, in central Vietnam, it showed possible resistance to type II pyrethroids. In the Mekong delta, <it>Anopheles epiroticus </it>was highly resistant to all pyrethroid insecticides tested. It was susceptible to DDT, except near Ho Chi Minh City where it showed possible DDT resistance. In Vietnam, pyrethroid susceptible and tolerant <it>Anopheles minimus s.l</it>. populations were found, whereas <it>An. minimus s.l</it>. from Cambodia, Laos and Thailand were susceptible. Only two <it>An. minimus s.l</it>. populations showed DDT tolerance. <it>Anopheles vagus </it>was found resistant to DDT and to several pyrethroids in Vietnam and Cambodia.</p> <p>Conclusion</p> <p>This is the first large scale, cross-country survey of insecticide resistance in <it>Anopheles </it>species in the Mekong Region. A unique baseline data on insecticide resistance for the Mekong region is now available, which enables the follow-up of trends in susceptibility status in the region and which will serve as the basis for further resistance management. Large differences in insecticide resistance status were observed among species and countries. In Vietnam, insecticide resistance was mainly observed in low or transmission-free areas, hence an immediate change of malaria vector control strategy is not required. Though, resistance management is important because the risk of migration of mosquitoes carrying resistance genes from non-endemic to endemic areas. Moreover, trends in resistance status should be carefully monitored and the impact of existing vector control tools on resistant populations should be assessed.</p

Effect of Therapeutic Hypothermia Initiated After 6 Hours of Age on Death or Disability Among Newborns With Hypoxic-Ischemic Encephalopathy: A Randomized Clinical Trial

Importance: Hypothermia initiated at less than 6 hours after birth reduces death or disability for infants with hypoxic-ischemic encephalopathy at 36 weeks' or later gestation. To our knowledge, hypothermia trials have not been performed in infants presenting after 6 hours. Objective: To estimate the probability that hypothermia initiated at 6 to 24 hours after birth reduces the risk of death or disability at 18 months among infants with hypoxic-ischemic encephalopathy. Design, Setting, and Participants: A randomized clinical trial was conducted between April 2008 and June 2016 among infants at 36 weeks' or later gestation with moderate or severe hypoxic-ischemic encephalopathy enrolled at 6 to 24 hours after birth. Twenty-one US Neonatal Research Network centers participated. Bayesian analyses were prespecified given the anticipated limited sample size. Interventions: Targeted esophageal temperature was used in 168 infants. Eighty-three hypothermic infants were maintained at 33.5°C (acceptable range, 33°C-34°C) for 96 hours and then rewarmed. Eighty-five noncooled infants were maintained at 37.0°C (acceptable range, 36.5°C-37.3°C). Main Outcomes and Measures: The composite of death or disability (moderate or severe) at 18 to 22 months adjusted for level of encephalopathy and age at randomization. Results: Hypothermic and noncooled infants were term (mean [SD], 39 [2] and 39 [1] weeks' gestation, respectively), and 47 of 83 (57%) and 55 of 85 (65%) were male, respectively. Both groups were acidemic at birth, predominantly transferred to the treating center with moderate encephalopathy, and were randomized at a mean (SD) of 16 (5) and 15 (5) hours for hypothermic and noncooled groups, respectively. The primary outcome occurred in 19 of 78 hypothermic infants (24.4%) and 22 of 79 noncooled infants (27.9%) (absolute difference, 3.5%; 95% CI, -1% to 17%). Bayesian analysis using a neutral prior indicated a 76% posterior probability of reduced death or disability with hypothermia relative to the noncooled group (adjusted posterior risk ratio, 0.86; 95% credible interval, 0.58-1.29). The probability that death or disability in cooled infants was at least 1%, 2%, or 3% less than noncooled infants was 71%, 64%, and 56%, respectively. Conclusions and Relevance: Among term infants with hypoxic-ischemic encephalopathy, hypothermia initiated at 6 to 24 hours after birth compared with noncooling resulted in a 76% probability of any reduction in death or disability, and a 64% probability of at least 2% less death or disability at 18 to 22 months. Hypothermia initiated at 6 to 24 hours after birth may have benefit but there is uncertainty in its effectiveness

Management of rare inherited bleeding disorders: Proposals of the French Reference Centre on Haemophilia and Rare Coagulation Disorders

Introduction: The rare coagulation disorders may present significant difficulties in diagnosis and management. In addition, considerable inter-individual variation in bleeding phenotype is observed amongst affected individuals, making the bleeding risk difficult to assess in affected individuals. The last international recommendations on rare inherited bleeding disorders (RIBDs) were published by the United Kingdom Haemophilia Centre Doctors' Organisation in 2014. Since then, new drugs have been marketed, news studies on surgery management in patients with RIBD have been published, and new orphan diseases have been described. Aim: Therefore, the two main objectives of this review, based on the recent recommendations published by the French Reference Centre on Haemophilia and Rare Bleeding Disorders, are: (i) to briefly describe RIBD (clinical presentation and diagnostic work-up) to help physicians in patient screening for the early detection of such disorders; and (ii) to focus on the current management of acute haemorrhages and long term prophylaxis, surgical interventions, and pregnancy/delivery in patients with RIBD.</p

Insights into Substrate Modification by Dehydratases from Type I Polyketide Synthases

International audienceDehydration reactions play a crucial role in the de novo biosynthesis of fatty acids and a wide range of pharmacologically active polyketide natural products with strong emphasis on human medicine. The type I polyketide synthase PpsC from Mycobacterium tuberculosis catalyzes key biosynthetic steps of lipid virulence factors phthiocerol dimycocerosates and phenolic glycolipids. Given the insolubility of the natural C28-C30 fatty acyl substrate of the PpsC dehydratase (DH) domain, we investigated its structure-function relationships in the presence of shorter surrogate substrates. Since most enzymes belonging to the (R)-specific enoyl hydratase/hydroxyacyl dehydratase family conduct the reverse hydration reaction in vitro, we have determined the X-ray structures of the PpsC DH domain, both unliganded (apo) and in complex with trans-but-2-enoyl-CoA or trans-dodec-2-enoyl-CoA derivatives. This study provides for the first time a snapshot of dehydratase-ligand interactions following a hydration reaction. Our structural analysis allowed us to identify residues essential for substrate binding and activity. The structural comparison of the two complexes also sheds light on the need for long acyl chains for this dehydratase to carry out its function, consistent with both its in vitro catalytic behavior and the physiological role of the PpsC enzyme

Caractérisation de la variabilité sérologique du virus Y de la pomme de terre (PVY) : impact sur le diagnostic

Bgpi : équipe 6absen

Phthiocerol Dimycocerosates From Mycobacterium tuberculosis Increase the Membrane Activity of Bacterial Effectors and Host Receptors

International audienceMycobacterium tuberculosis (Mtb) synthesizes a variety of atypical lipids that are exposed at the cell surface and help the bacterium infect macrophages and escape elimination by the cell's immune responses. In the present study, we investigate the mechanism of action of one family of hydrophobic lipids, the phthiocerol dimycocerosates (DIM/PDIM), major lipid virulence factors. DIM are transferred from the envelope of Mtb to host membranes during infection. Using the polarity-sensitive fluorophore C-Laurdan, we visualized that DIM decrease the membrane polarity of a supported lipid bilayer put in contact with mycobacteria, even beyond the site of contact. We observed that DIM activate the complement receptor 3, a predominant receptor for phagocytosis of Mtb by macrophages. DIM also increased the activity of membrane-permeabilizing effectors of Mtb, among which the virulence factor EsxA. This is consistent with previous observations that DIM help Mtb disrupt host cell membranes. Taken together, our data show that transferred DIM spread within the target membrane, modify its physical properties and increase the activity of host cell receptors and bacterial effectors, diverting in a non-specific manner host cell functions. We therefore bring new insight into the molecular mechanisms by which DIM increase Mtb's capability to escape the cell's immune responses

Effet de la consommation des pâtes alimentaires mixtes blé-légumineuses sur le métabolisme protéique du rat âgé

En dehors des aspects écologiques, l’augmentation de la part des produits végétaux dans notre alimentation peut contribuer à prévenir les risques pour la santé associés à une consommation excessive d'aliments d'origine animale. De plus, les produits végétaux notamment les légumineuses sont riches en protéines (24-28%), en fibres alimentaires (12-31%) et en micro-constituants et pauvres en lipides (̴ 2%), et présentent, de ce fait, un réel intérêt pour l’alimentation humaine, en particulier celle des séniors. En effet, au cours du vieillissement, il est nécessaire de contrôler la consommation de graisses saturées, d’améliorer l'apport en micro-constituants et en fibres et d’optimiser la qualité des apports protéiques. Cependant, les légumineuses comme la plupart des sources végétales sont composées de protéines déficientes en certains acides aminés essentiels, notamment les acides aminés soufrés. Les protéines de légumineuses présentent un profil en acides aminés essentiels complémentaire à celui du blé. L’association blé-légumineuses permet ainsi d’améliorer l’équilibre en acides aminés essentiels. Il a été démontré dans la littérature que l’incorporation de la farine de fèverole à hauteur de 35 à 70% dans les pâtes alimentaires permet d’obtenir un profil équilibré en acides aminés essentiels par rapport aux besoins de l’organisme mais aussi d’augmenter la teneur en protéines des pâtes sèches (de 13 à 17-20%). Le premier objectif de notre travail était de produire des pâtes alimentaires caractérisées par une composition optimale en acides aminés essentiels en associant de la semoule de blé avec des farines de légumineuses (soit de la fèverole, de la lentille ou du pois) à un ratio blé/légumineuse d’environ (30/70). La composition en acides aminés essentiels de ces pâtes alimentaires a été étudiée et comparée au profil recommandé par l’Anses (2007).Outre la composition en acides aminés essentiels, la digestibilité des protéines est un paramètredéterminant de sa valeur nutritionnelle. Ce paramètre peut varier en fonction de la structure du réseauprotéique. Le second objectif de notre travail était d’évaluer les modifications structurales du réseauprotéique à l’échelle moléculaire induites par l’incorporation des différentes farines de légumineuses dans les pâtes alimentaires et leurs répercussions sur la digestibilité in vitro des protéines. Enfin, l’effet de la consommation de ces pâtes alimentaires mixtes sur le métabolisme protéique des rats âgés a été étudié en comparaison avec des protéines animales de référence (caséine et lactosérum).Les résultats montrent que toutes les pâtes alimentaires mixtes produites répondent aux besoins en acides aminés essentiels tels que définis pour le sujet adulte sain. De plus, nous avons montré que la différence de la structure protéique des pâtes produites pourrait influencer leurs digestibilités in vitro et in vivo. En effet, la pâte mixte blé/fèverole plus riche en protéines liées par des interactions faibles (électrostatiques, hydrophobes et hydrophiles) présentait une meilleure digestibilité protéique in vitro et in vivo que la pâte mixte blé/lentille plus riche en pont covalents de type disulfure. Chez le rat âgé, l’utilisation nette des protéines ainsi que la synthèse protéique musculaire suite à la consommation des pâtes aux légumineuses étaient équivalentes à celles de la caséine. Cependant, la consommation de lactosérum était caractérisée par une utilisation nette des protéines et une synthèse protéique musculaire 43 et 50% plus élevées que les pâtes aux légumineuses en relation avec l’effet anabolique important du lactosérum, notamment sa richesse en leucine et la rapidité de son assimilation.Ces pâtes alimentaires mixtes blé-légumineuses, riches en protéines et équilibrées en acides aminésessentiels permettent d’optimiser l’apport protéique chez les rats âgés. Elles pourraient être proposéescomme une alternative possible à des protéines animales destinées aux personnes âgées

Assay development for identifying inhibitors of the mycobacterial FadD32 activity

FadD32, a fatty acyl-AMP ligase (FAAL32) involved in the biosynthesis of mycolic acids, major and specific lipid components of the mycobacterial cell envelope, is essential for the survival of Mycobacterium tuberculosis, the causative agent of tuberculosis. The protein catalyzes the conversion of fatty acid to acyl-adenylate (acyl-AMP) in the presence of adenosine triphosphate and is conserved in all the mycobacterial species sequenced so far, thus representing a promising target for the development of novel antituberculous drugs. Here, we describe the optimization of the protein purification procedure and the development of a high-throughput screening assay for FadD32 activity. This spectrophotometric assay measuring the release of inorganic phosphate was optimized using the Mycobacterium smegmatis FadD32 as a surrogate enzyme. We describe the use of Tm (melting temperature) shift assay, which measures the modulation of FadD32 thermal stability, as a tool for the identification of potential ligands and for validation of compounds as inhibitors. Screening of a selected library of compounds led to the identification of five novel classes of inhibitors