Temperature measurement of sub-micrometric ICs by scanning thermal microscopy

Surface temperature measurements were performed with a Scanning Thermal Microscope mounted with a thermoresistive wire probe of micrometrSurface temperature measurements were performed with a Scanning Thermal Microscope mounted with a thermoresistive wire probe of micrometric size. A CMOS device was designed with arrays of resistive lines 0.35µm in width. The array periods are 0.8 µm and 10µm to study the spatial resolution of the SThM. Integrated Circuits with passivation layers of micrometric and nanometric thicknesses were tested. To enhance signal-to-noise ratio, the resistive lines were heated with an AC current. The passivation layer of nanometric thickness allows us to distinguish the lines when the array period is 10μm. The results raise the difficulties of the SThM measurement due to the design and the topography of ICs on one hand and the size of the thermal probe on the other hand.ic size. A CMOS device was designed with arrays of resistive lines 0.35µm in width. The array periods are 0.8 µm and 10µm to study the spatial resolution of the SThM. Integrated Circuits with passivation layers of micrometric and nanometric thicknesses were tested. To enhance signal-to-noise ratio, the resistive lines were heated with an AC current. The passivation layer of nanometric thickness allows us to distinguish the lines when the array period is 10μm. The results raise the difficulties of the SThM measurement due to the design and the topography of ICs on one hand and the size of the thermal probe on the other hand

Le site aurignacien de plein-air de Combemenue à Brignac-la-Plaine (Corrèze) : apport de la géochéologie et de l’étude de l’industrie lithique à la compréhension des processus taphonomiques

Le site paléolithique de Combemenue en Corrèze a livré un niveau d’occupation d’Aurignacien récent enfoui à faible profondeur, sur un replat près du sommet d’un versant. Le contexte de faible enfouissement laissant suspecter des perturbations significatives du niveau archéologique dues à une longue exposition aux agents naturels en surface ou en subsurface du sol, une étude taphonomique détaillée a été entreprise. Différents points ont été examinés : la distribution spatiale du matériel, la disposition des objets (fabrique), leur granulométrie, leur état de surface ainsi que les remontages. Les résultats obtenus ont été confrontés de manière à proposer un scénario qui rende compte au mieux de l’ensemble des observations faites sur le site. Cette étude indique que l’assemblage lithique originel a subi un appauvrissement sélectif en petits éléments sous l’action du ruissellement. Simultanément, il est possible qu’une partie du matériel de plus grande taille initialement présent ait été soustrait du site par les mécanismes sédimentaires. Ces modifications ont eu des répercussions sur la distribution spatiale des vestiges. Celle-ci se caractérise par une absence de concentration claire, tant lorsqu’on considère la répartition de l’ensemble du matériel que celle de catégories particulières d’objets. Une diffusion progressive des vestiges par le ruissellement ou les phénomènes périglaciaires rend bien compte des transformations observées. En revanche, les déplacements n’ont pas entraîné d’altération physique importante des pièces, dont la majorité ne porte pas de stigmate postérieur à sa production ou son utilisation par les Aurignaciens. Les transformations décrites ici pour le site de Combemenue sont probablement représentatives de celles subies par un grand nombre de sites paléolithiques localisés sur une pente négligeable dans un contexte géomorphologique peu favorable à un enfouissement rapide.An Upper Aurignacian level located on a hilltop flat surface has been found at Combemenue (Brignac-la-Plaine, Corrèze, France) during field surveys along the A89 highway. Geomorphological evidence, and particularly the shallow depth of the level, strongly suggests that it has undergone significant perturbation due to a long-lasting exposure to sedimentary and pedological processes at the soil surface or subsurface. Therefore, a detailed taphonomic study has been made to evaluate the impact of natural processes in site patterning. It involves examination of the spatial distribution of the artefacts, their orientation and dip (fabric), their size distribution, their alteration and refits. The results have been compared to the data derived from the techno-economical analysis. This study indicates that the lithic assemblage has undergone a selective impoverishment in small-sized artefacts as the result of surface wash. A part of the coarser artefacts may have been also removed from the site by the same processes. The lack of any clear artefact concentration indicates that the spatial distribution has been modified significantly. This can be adequately described by a model of progressive diffusion of the artefacts by surface wash or periglacial processes. By contrast, the movements did not provoke strong physical alteration of the pieces. The Combemenue site is thought to be representative of a number of palaeolithic sites located in similar geomorphological contexts, which are characterized by low sedimentation rates

A new role for complement C3: regulation of antigen processing through an inhibitory activity.

International audienceIncreasing evidence underlines the involvement of complement component C3 in the establishment of acquired immunity which appears to play a complex role and to act at different levels. As antigen proteolysis by antigen presenting cells is a key event in the control of antigen presentation efficiency, and consequently in the quality of the immune response, we investigated whether C3 could modulate this step. Our results demonstrate for the first time that C3 can interfere with antigen proteolysis: (i) proteolysis of tetanus toxin (TT) by the lysosomal fraction from a human monocytic cell line (U937) is impaired in the presence of C3, (ii) this effect is C3-specific and involves the C3c fragment of the protein, (iii) C3c is effective even after disulfide disruption, but none of its three constitutive peptides is individually accountable for this inhibitory effect and (iv) the target-protease(s) exhibit(s) a serine-protease activity. The physiological relevance of our results is demonstrated by experiments showing a subcellular colocalisation of TT and C3 after their uptake by U937 and the reduction of TT proteolysis once internalised together with C3. These results highlight a novel role for C3 that broadens its capacity to modulate acquired immune response

Injection of Pseudomonas aeruginosa Exo Toxins into Host Cells Can Be Modulated by Host Factors at the Level of Translocon Assembly and/or Activity

Pseudomonas aeruginosa type III secretion apparatus exports and translocates four exotoxins into the cytoplasm of the host cell. The translocation requires two hydrophobic bacterial proteins, PopB and PopD, that are found associated with host cell membranes following infection. In this work we examined the influence of host cell elements on exotoxin translocation efficiency. We developed a quantitative flow cytometry based assay of translocation that used protein fusions between either ExoS or ExoY and the ß-lactamase reporter enzyme. In parallel, association of translocon proteins with host plasma membranes was evaluated by immunodetection of PopB/D following sucrose gradient fractionation of membranes. A pro-myelocytic cell line (HL-60) and a pro-monocytic cell line (U937) were found resistant to toxin injection even though PopB/D associated with host cell plasma membranes. Differentiation of these cells to either macrophage- or neutrophil-like cell lines resulted in injection-sensitive phenotype without significantly changing the level of membrane-inserted translocon proteins. As previous in vitro studies have indicated that the lysis of liposomes by PopB and PopD requires both cholesterol and phosphatidyl-serine, we first examined the role of cholesterol in translocation efficiency. Treatment of sensitive HL-60 cells with methyl-ß-cyclodextrine, a cholesterol-depleting agent, resulted in a diminished injection of ExoS-Bla. Moreover, the PopB translocator was found in the membrane fraction, obtained from sucrose-gradient purifications, containing the lipid-raft marker flotillin. Examination of components of signalling pathways influencing the toxin injection was further assayed through a pharmacological approach. A systematic detection of translocon proteins within host membranes showed that, in addition to membrane composition, some general signalling pathways involved in actin polymerization may be critical for the formation of a functional pore. In conclusion, we provide new insights in regulation of translocation process and suggest possible cross-talks between eukaryotic cell and the pathogen at the level of exotoxin translocation

Biallelic NAA60 variants with impaired n-terminal acetylation capacity cause autosomal recessive primary familial brain calcifications

Primary familial brain calcification (PFBC) is characterized by calcium deposition in the brain, causing progressive movement disorders, psychiatric symptoms, and cognitive decline. PFBC is a heterogeneous disorder currently linked to variants in six different genes, but most patients remain genetically undiagnosed. Here, we identify biallelic NAA60 variants in ten individuals from seven families with autosomal recessive PFBC. The NAA60 variants lead to loss-of-function with lack of protein N-terminal (Nt)-acetylation activity. We show that the phosphate importer SLC20A2 is a substrate of NAA60 in vitro. In cells, loss of NAA60 caused reduced surface levels of SLC20A2 and a reduction in extracellular phosphate uptake. This study establishes NAA60 as a causal gene for PFBC, provides a possible biochemical explanation of its disease-causing mechanisms and underscores NAA60-mediated Nt-acetylation of transmembrane proteins as a fundamental process for healthy neurobiological functioning

Etude de la susceptibilité des cellules eucaryotes à l'injection de toxines par le système de sécrétion de type 3 de Pseudomonas aeruginosa

La pathogénicité de P. aeruginosa (P. a) repose sur de nombreux facteurs de virulence dont le système de sécrétion de type III (SST3). Ce complexe multiprotéique est constitué d'une aiguille se terminant par un translocon composé des protéines PopB et PopD. En s'insérant dans les membranes plasmiques, le translocon permet le passage des exotoxines dans le cytoplasme de la cellule cible. L'induction de la synthèse et de la sécrétion des exotoxines est dépendante d'un contact entre P. a et la cellule cible. Dans ce travail, nous avons examiné l'influence de facteurs cellulaires sur l'efficacité de translocation des toxines. L'utilisation d'un système rapporteur fluorescent CCF2/b-lactamase a permis de visualiser l'injection de toxine. En parallèle, l'association des protéines du translocon avec la membrane de la cellule hôte a été évaluée par immunodétection de PopB/D après fractionnement des membranes sur gradient de sucrose. Les cellules promyelocytaires HL-60 et promonocytaires U937 sont résistantes à l'injection de toxine, bien que PopB et PopD soient associées à la membrane. Après différenciation en neutrophiles, or monocytes/macrophages, ces cellules deviennent sensibles à l'injection sans que l'on détecte de variation notable de la quantité de protéines du translocon insérées dans la membrane. Le traitement des cellules HL-60 sensibles avec un agent déplétant le cholestérol, entraine une diminution de l'injection de toxine. De plus, la protéine PopB est retrouvée dans la fraction membranaire, obtenue par purification sur gradient de sucrose, contenant le marqueur des radeaux lipidiques flotilline. Par une approche pharmacologique, nous apportons la preuve que, en plus de la composition de la membrane, des voies de signalisation intracellulaires impliquées dans la polymérisation de l'actine sont essentielles pour la formation d'un pore fonctionnel.The pathogenesis of Pseudomonas aeruginosa (P.a) implies multiple virulence factors among which the type III secretion system (T3SS). This multiprotein complex is composed of a needle through which four exotoxins are exported. The protein PopB and PopD form an oligomeric structure (translocon) at the end of the needle that inserts into the host cell membrane and translocates the exotoxins into the cytoplasm. Synthesis and toxin secretion is induced on contact with eukaryotic cell. In this work, we examined the influence of host cell elements on exotoxin translocation efficiency. The delivery of T3SS toxins was investigated using a CCF2/b-lactamase fluorescent reporter system In parallel, the association of translocon proteins with host plasma membranes was evaluated by immunodetection of PopB/D following sucrose gradient fractionation of membranes. Promyelocytic HL-60 cells and promonocytic U937 cells were found to be resistant to toxin injection even though PopB/D associated with host cell plasma membranes. Differentiation of these cells to neutrophil- or macrophage-like cells resulted in an injection-sensitive phenotype without any significant change in the level of membrane-inserted translocon proteins. Treatment of sensitive HL-60 cells with a cholesterol-depleting agent, resulted in a diminished injection of toxin. Moreover, the PopB translocator was found in the membrane fraction obtained from sucrose-gradient purifications and containing lipid-raft marker flotillin. Through a pharmacological approach, we brought evidence that, in addition to membrane composition, some general signalling pathways involved in actin polymerization may be critical for the formation of a functional pore.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

Flow electrode capacitive deionization for water desalination: optimizing ion removal capacity and flowing properties of activated carbon suspensions

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Pore-forming activity of the Pseudomonas aeruginosa type III secretion system translocon alters the host epigenome.

International audienceRecent studies highlight that bacterial pathogens can reprogram target cells by influencing epigenetic factors. The type III secretion system (T3SS) is a bacterial nanomachine that resembles a syringe on the bacterial surface. The T3SS 'needle' delivers translocon proteins into eukaryotic cell membranes, subsequently allowing injection of bacterial effectors into the cytosol. Here we show that Pseudomonas aeruginosa induces early T3SS-dependent dephosphorylation and deacetylation of histone H3 in eukaryotic cells. This is not triggered by any of the P. aeruginosa T3SS effectors, but results from the insertion of the PopB-PopD translocon into the membrane. This suggests that the P. aeruginosa translocon is a genuine T3SS effector acting as a pore-forming toxin. We visualized the translocon plugged into the host cell membrane after the bacterium has left the site of contact, and demonstrate that subsequent ion exchange through this pore is responsible for histone H3 modifications and host cell subversion

Molecular Mechanisms Involved in Pseudomonas aeruginosa Bacteremia

International audienceBloodstream infections (BSI) with Pseudomonas aeruginosa account for 8.5% of all BSIs, their mortality rate, at about 40%, is the highest among causative agents. For this reason and due to its intrinsic and acquired resistance to antibiotics, P. aeruginosa represents a threat to public health systems. From the primary site of infection, often the urinary and respiratory tracts, P. aeruginosa uses its arsenal of virulence factors to cross both epithelial and endothelial barriers, ultimately reaching the bloodstream. In this chapter, we review the main steps involved in invasion and migration of P. aeruginosa into blood vessels, and the molecular mechanisms governing bacterial survival in blood. We also review the lifestyle of P. aeruginosa "on" and "in" host cells. In the context of genomic and phenotypic diversity of laboratory strains and clinical isolates, we underline the need for more standardized and robust methods applied to host-pathogen interaction studies, using several representative strains from distinct phylogenetic groups before drawing general conclusions. Finally, our literature survey reveals a need for further studies to complete our comprehension of the complex interplay between P. aeruginosa and the immune system in the blood, specifically in relation to the complement system cascade(s) and the Membrane Attack Complex (MAC), which play crucial roles in counteracting P. aeruginosa BSI