Loss of PopZ At activity in Agrobacterium tumefaciens by Deletion or Depletion Leads to Multiple Growth Poles, Minicells, and Growth Defects.

Agrobacterium tumefaciens grows by addition of peptidoglycan (PG) at one pole of the bacterium. During the cell cycle, the cell needs to maintain two different developmental programs, one at the growth pole and another at the inert old pole. Proteins involved in this process are not yet well characterized. To further characterize the role of pole-organizing protein A. tumefaciens PopZ (PopZ At ), we created deletions of the five PopZ At domains and assayed their localization. In addition, we created a popZAt deletion strain (ΔpopZAt ) that exhibited growth and cell division defects with ectopic growth poles and minicells, but the strain is unstable. To overcome the genetic instability, we created an inducible PopZ At strain by replacing the native ribosome binding site with a riboswitch. Cultivated in a medium without the inducer theophylline, the cells look like ΔpopZAt cells, with a branching and minicell phenotype. Adding theophylline restores the wild-type (WT) cell shape. Localization experiments in the depleted strain showed that the domain enriched in proline, aspartate, and glutamate likely functions in growth pole targeting. Helical domains H3 and H4 together also mediate polar localization, but only in the presence of the WT protein, suggesting that the H3 and H4 domains multimerize with WT PopZ At , to stabilize growth pole accumulation of PopZ AtIMPORTANCEAgrobacterium tumefaciens is a rod-shaped bacterium that grows by addition of PG at only one pole. The factors involved in maintaining cell asymmetry during the cell cycle with an inert old pole and a growing new pole are not well understood. Here we investigate the role of PopZ At , a homologue of Caulobacter crescentus PopZ (PopZ Cc ), a protein essential in many aspects of pole identity in C. crescentus We report that the loss of PopZ At leads to the appearance of branching cells, minicells, and overall growth defects. As many plant and animal pathogens also employ polar growth, understanding this process in A. tumefaciens may lead to the development of new strategies to prevent the proliferation of these pathogens. In addition, studies of A. tumefaciens will provide new insights into the evolution of the genetic networks that regulate bacterial polar growth and cell division

Cryptomelane formation from nanocristalline vernadite precursor.

International audienceVernadite is a nanocristalline and turbostratic phyllomanganate which is ubiquitous in the environment. Its layers, built of MnO6 octahedra connected through their edges, contain vacancies and (or) isomorphic substitutions, both creating a layer charge deficit that can exceed 1 valence unit per layer octahedron. In addition, vernadite has a high affinity for many trace metals (e.g., Co, Ni and Zn) and frequently contain heterovalent Mn cations which provides this mineral with the capacity to oxidize redox-sensitive trace elements (e.g., As, Se) and organic pollutants. As a result of these exceptional properties, vernadite controls the fate of many trace elements in soils and sediments. In the environment, vernadite is often found associated with tectomanganates (“tunnel”-like structures) such as cryptomelane, of which it is thought to be the precursor. A sound description of the vernadite-to-cryptomelane transformation, at the atomic scale, is mandatory to be able to understand and thus model the fate of metals initially present in vernadite structure. To contribute to a better understanding of this transformation, we have synthesized vernadite samples having various Mn4+/Mn3+ ratios (and thus various layer charge) and we have monitored their transformation, under conditions analogous to those prevailing in soils (dry state and ambient conditions, in the dark) over a time scale of ~10 years [1-2]. Initial samples were characterized using a combination of chemistry, thermogravimetric analyses and powder X-ray diffraction. Samples structural formula ranged between Na+0.06(H2O)0.30Mn3+0.19[Mn3+0.12Mn4+0.71Vac0.17O2] (where species under brackets form the layer – “Vac” stands for “layer vacancies”, and species on the left are in the interlayer space) and Na+0.27(H2O)0.30Mn3+0.10[Mn3+0.10Mn4+0.76Vac0.14O2]. Transformation was monitored using high-energy X-ray scattering (with both Bragg-rod and pair distribution function formalisms) and transmission electron microscopy (TEM and STEM). With time, layer Mn3+ was found to migrate in the interlayer, probably to reduce strains induced by its Jahn-Teller distorted coordination sphere. When the abundance of interlayer Mn3+ reached ~0.3 per layer octahedron, interlayer Mn3+ from adjacent layers were found to share their hydration sphere and to form cryptomelane domains

Pre-failure behaviour of an unstable limestone cliff from displacement and seismic data

We monitored the displacement and seismic activity of an unstable vertical rock slice in a natural limestone cliff of the southeast Vercors massif, southeast France, during the months preceding its collapse. Displacement measurements showed an average acceleration of the movement of its top, with clear increases in the displacement velocity and in the discrete seismic event production rate during periods where temperature falls, with more activity when rainfall or frost occurs. Crises of discrete seismic events produce high amplitudes in periodograms, but do not change the high frequency base noise level rate. We infer that these crises express the critical crack growth induced by water weakening (from water vapor condensation or rain) of the rock strength rather than to a rapid change in applied stresses. Seismic noise analysis showed a steady increase in the high frequency base noise level and the emergence of spectral modes in the signal recorded by the sensor installed on the unstable rock slice during the weeks preceding the collapse. High frequency seismic noise base level seems to represent subcritical crack growth. It is a smooth and robust parameter whose variations are related to generalized changes in the rupture process. Drop of the seismic noise amplitude was concomitant with the emergence of spectral modes – that are compatible with high-order eigenmodes of the unstable rock slice – during the later stages of its instability. Seismic noise analysis, especially high frequency base noise level analysis may complement that of inverse displacement velocity in early-warning approaches when strong displacement fluctuations occur

RCPSP et temps de montage dépendant de la séquence pour la modélisation et la résolution d'un problème de livraison collecte

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Développement de méthodes analytiques par LC-MS/MS pour la caractérisation de l’activité et de l’expression des CYP450s chez l’humain

Ce projet de recherche comporte deux parties principales qui possèdent comme lien unificateur l’amélioration des méthodes et techniques utilisées actuellement pour évaluer aussi bien l’activité que l’expression des cytochromes P450 (CYP450s) et menant par la suite à leur application en clinique. Le premier volet de ce projet de recherche porte sur le développement de méthodes LC-MS/MS pour un cocktail de 7 substrats marqueurs des CYP450s. Notre objectif est de développer et valider une méthode LC-MS/MS spécifique et sensible permettant l’évaluation des activités des CYP1A2, 2B6, 2C9, 2C19, 2D6, 3A4/5 et 2E1 suivant l’administration orale et à faible dose d’un cocktail de substrats marqueurs chez des patients et sujets sains. Les méthodes développées peuvent être utilisées pour évaluer les mécanismes de variabilité interindividuelle comme l’impact de polymorphismes génétiques, de facteurs environnementaux et de maladies dans le processus de métabolisme et d’élimination des médicaments, mais également pour investiguer les interactions médicamenteuses. Ce cocktail a été appliqué avec succès, dans un projet clinique portant sur l’évaluation des effets du diabète sur la capacité métabolique par les CYP450s. Le deuxième volet de ce projet de recherche vise à développer des méthodes analytiques par LC-HRMS afin de caractériser et quantifier les CYP1A1, 1A2, 1B1, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2, 3A4, 3A5, 3A7 et 4F2 dans l’intestin grêle humain. Notre hypothèse suggère que les CYP450s retrouvés le long de l’intestin grêle peuvent affecter significativement l’effet de premier passage de certains médicaments administrés par voie orale et influencer leurs concentrations plasmatiques et conséquemment, leurs effets pharmacologiques et/ou toxiques. Ma participation à ce projet a permis d’identifier des peptides protéotypiques par digestion in silico et in vitro et de développer des méthodes de quantification absolue par LC-HRMS. Ce projet est une première étape dans la caractérisation des CYP450s majeurs le long de l'intestin grêle. Il permettra de mieux comprendre les mécanismes de variabilité interindividuelle dans la réponse aux médicaments associés au processus d'absorption intestinal et de mieux prédire la variabilité dans la biodisponibilité des médicaments et de développer des modèles pharmacocinétiques plus complexes.This research project is divided into two sections, both aiming at the development of sensitive and specific LC-MS methods to evaluate activity and expression of CYP450 and finally, looking at their clinical application. The first section of this research project focuses on the development of analytical methods by LC-MS/MS for a seven CYP450 probe-drug cocktail. Although these cocktails have shown value they also suffer from many limitations. Our objective was to develop and validate highly sensitive and selective LC-MS/MS assays allowing the determination of CYP1A2, 2B6, 2C9, 2C19, 2D6, 3A4/5 and 2E1 activities following administration of low oral doses of a modified CYP450 probe-drug cocktail in patients. These methods can be used to phenotype CYP450 activities, evaluate inter-individual variabilities, study the impact of pathological conditions on drug metabolism and elimination, and evaluate drug-drug interactions. Our CYP450 cocktail assays have been successfully applied to phenotype CYP450 activities in type 2 diabetic patients. The second section of this project aims at the development of a LC-HRMS method for the characterization and absolute quantification of CYP1A1, 1A2, 1B1, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2, 3A4, 3A5, 3A7 and 4F2 in the human small intestine. Our hypothesis suggests that CYP450 isoenzymes found along the small intestine can significantly affect the first-pass effect of certain drugs administered orally and thus influence their pharmacological and/or toxic effects. My participation in this project allowed to identify proteotypic peptides by in silico and in vitro digestion and to develop LC-HRMS methods allowing the absolute quantification of CYP450. This project is a first step in the characterization of the main CYP450 along the small intestine. This project will allow a better understanding of inter-individual variability in drug response associated with intestinal absorption of drugs, a better prediction of variability in drug bioavailability and to develop more complex pharmacokinetic models

Crystal structure of Ni-sorbed synthetic vernadite: A powder X-ray diffraction study

International audienceVernadite is a nanocrystalline turbostratic phyllomanganate ubiquitous in the environment, which contains nickel in specific settings such as oceanic sediments. To improve our understanding of nickel uptake in this mineral, two series of synthetic analogs to vernadite (δ-MnO2) were prepared with Ni/Mn atomic ratios ranging from 0.002 to 0.105 at pH 4 and from 0.002 to 0.177 at pH 7, and their structures characterised using X-ray diffraction (XRD). The δ-MnO2 nano-crystals are essentially monolayers with coherent scattering domain sizes of ~10 Å perpendicular to the layer and of ~55 Å in the layer plane. The layers contain an effective proportion of ~18% vacant octahedral sites, regardless of the Ni content. At Ni/Mn ratios <1%, XRD has no sensitivity to Ni, and the layer charge deficit is apparently entirely balanced by interlayer Mn, Na, and protons. At higher Ni/Mn ratios, Ni occupies the same site as interlayer Mn above and/or below layer vacancies together with sites along the borders of the MnO2 layers, but the layer charge is balanced differently at the two pH values. At pH 4, Ni uptake is accompanied by a decrease in structural Na and protons, whereas interlayer Mn remains strongly bound to the layers. At pH 7, interlayer Mn is less strongly bound and partly replaced by Ni. The results also suggest that the number of vacant layer sites and multivalent charge-compensating interlayer species are underestimated in the current structure models for δ-MnO2

Analogue modeling of instabilities in crater lake hydrothermal systems.

International audienceWe carried out analogue experiments on two-phase boiling systems, using a porous vertical cylinder, saturated with water. The base of the cylinder was heated, and the top was cooled, as in a natural hydrothermal system. Previous work had shown that once the two-phase zone reached a certain level, thermal instabilities would develop. We made measurements of the acoustic energy related to boiling, and we found that high levels of acoustic noise were associated with the part of the cycle in which there was upward water movement. We repeated our experiments with a cooling water tank at the top of the system, representing a crater lake. This showed that periodic thermal instabilities still developed in this situation. We then compared our analogue measurements to two natural systems known to exhibit periodic behavior. There is good agreement between the thermal and acoustic cycling seen in our model and the observations made at Inferno Crater Lake in the Waimangu Geothermal area, New Zealand, whose level cycles by nearly 10 m, with a typical period of 38 days. Particularly notable is how in both systems high levels of acoustic noise are associated with rising water level. The much larger Ruapehu Crater Lake, also in New Zealand, cycled with a period of several months to a year for over a decade prior to the 1995 eruption. Strong acoustic and seismic energy usually occurred just before the lake temperature started to rise. This suggests a slightly different model, in which the increasing two-phase flow zone triggers more general convection once it reaches the base of the lake

Structure of nanocrystalline phyllomanganates produced by freshwater fungi

International audienceThe crystal structures of biogenic Mn oxides produced by three fungal strains isolated from stream pebbles were determined using chemical analyses, XANES and EXAFS spectroscopy, and powder X-ray diffraction. The fungi-mediated oxidation of aqueous Mn2+ produces layered Mn oxides analogous to vernadite, a natural nanostructured and turbostratic variety of birnessite. The crystallites have domain dimensions of ~10 nm in the layer plane (equivalent to ~35 MnO6 octahedra), and ~1.5-2.2 nm perpendicularly (equivalent to ~2-3 layers), on average. The layers have hexagonal symmetry and from 22 to 30% vacant octahedral sites. This proportion likely includes edge sites, given the extremely small lateral size of the layers. The layer charge deficit, resulting from the missing layer Mn4+ cations, is balanced mainly by interlayer Mn3+ cations in triple-corner sharing position above and/or below vacant layer octahedra. The high surface area, defective crystal structure, and mixed Mn valence confer to these bio-minerals an extremely high chemical reactivity. They serve in the environment as sorption substrate for trace elements and possess catalytic redox properties

The clinical characteristics of familial cluster headache

BACKGROUND: A positive family history predisposes to the development of cluster headache. The distinct characteristics of familial cluster headache have yet to be confirmed, however, evidence suggests a younger age of onset and higher proportion of females in this subgroup. OBJECTIVES: To assess the rate and mode of inheritance of familial cluster headache in a tertiary referral centre for headache. To describe the clinical features of familial cluster headache. METHODS: A retrospective study conducted between 2007 and 2017. Cluster headache was confirmed in probands and affected relatives. Differences in demographics, clinical characteristics, and response-to-treatment in familial cluster headache were delineated through multivariate analysis using a control cohort of 597 patients with sporadic cluster headache. RESULTS: Familial cluster headache was confirmed in 48 (7.44%) patients and predominantly reflected an autosomal dominant mode of inheritance with reduced penetrance. Familial cases were more likely to report nasal blockage (OR 4.06, 95% CI; 2.600-6.494, p < 0.001) during an attack and a higher rate of concurrent short-lasting unilateral neuralgiform headache with conjunctival injection and tearing (OR 3.76, 95% CI; 1.572-9.953, p = 0.004). CONCLUSION: These findings add to evidence suggesting a genetic component to cluster headache. Here, we demonstrated prominent nasal blockage, and a higher occurrence of concomitant short-lasting unilateral neuralgiform headache with conjunctival injection and tearing in this subgroup, further delineating the phenotype