Interpretation of two-dimensional electrophoresis gels

The study of human protein polymorphism presents a major advantage in clinical chemistry to discover forms associated with risks or involved in well specified pathological states. If this polymorphism corresponds to structural differences reflected by physical property modifications it can be visualized on a map obtained by 2-dimensional electrophoretic technique. This study deals with apolipoproteins A-I, A-II, A-IV, C2, C3, and E that can be found on 2-dimensional gels. Each of these proteins appears on images under the form of a constellation of several spots. An expertise was extracted on these apolipoproteins in order to itemize all their known variants and their geometrical representations. This knowledge was implemented in a system to recognize what are the constellations we are actually dealing with, on a gel. A management system handles specific techniques taken from image processing to extract parameters from 2-dimensional gel images and artificial intelligence. Knowledge-based expert systems which perform the matching, allowing a partial interpretation of the gel. The accuracy of quantity measurement and quality control are greatly improved by adding calibration proteins. This same system will allow us to further implement conceptual clustering techniques to identify relevant relations in apolipoprotein metabolism. Apolipoprotein polymorphism is used in this work as a model which could be extended to other protein constellations in the future

Taxonomical composition and functional analysis of biofilms sampled from a nuclear storage pool

Sampling small amounts of biofilm from harsh environments such as the biofilm present on the walls of a radioactive material storage pool offers few analytical options if taxonomic characterization and estimation of the different biomass contributions are the objectives. Although 16S/18S rRNA amplification on extracted DNA and sequencing is the most widely applied method, its reliability in terms of quantitation has been questioned as yields can be species-dependent. Here, we propose a tandem-mass spectrometry proteotyping approach consisting of acquiring peptide data and interpreting then against a generalist database without any a priori. The peptide sequence information is transformed into useful taxonomical information that allows to obtain the different biomass contributions at different taxonomical ranks. This new methodology is applied for the first time to analyze the composition of biofilms from minute quantities of material collected from a pool used to store radioactive sources in a nuclear facility. For these biofilms, we report the identification of three genera, namely Sphingomonas, Caulobacter, and Acidovorax, and their functional characterization by metaproteomics which shows that these organisms are metabolic active. Differential expression of Gene Ontology GOslim terms between the two main microorganisms highlights their metabolic specialization

Apport de la simulation numérique et de l'expérience pour la compréhension des phénomènes de frottement en emboutissage et hydroformage

The sheet metal forming industry uses more and more the numerical simulation computer codes in order to minimize the development time and the cost of trial and error loops. Friction which has a major influence on the product quality is usually described by a constant Coulomb friction coefficient. The validity of this very simplified description of the real frictional behaviour is discussed in this thesis. The first part focuses on the stamping process. A new frictional device is developed based on a flat/flat tribometer. It allows measuring either static or dynamic friction coefficient. Experiments on coated and non coated steel sheet demonstrate that the static friction coefficient is significantly higher than the dynamic one on the whole range of contact pressures. The influence of a local and time variable friction coefficient on the result of the stamping operation is discussed using several friction models based on tribometry results and implemented in two numerical codes (FORGE2® et ABAQUS®): The static to dynamic transition of the friction coefficient described as a function of the sliding length underlines the role of the static coefficient of friction on the beginning of sliding under the blank holder and also on the thickness distribution under the punch where small relative sliding occurs. A pressure and sliding speed friction model describes well the influence of the heterogeneity of these quantities. For higher punch speeds, the difference of the results obtained with a constant and a variable friction coefficient decreases. The use of two constant friction coefficients, the punch friction being higher than the blank-holder friction, improves significantly the results.The Devine's model which describes the influence of the microscopic history of the contact (microplasticity, hydrodynamic effects) has been improved. It is an interesting alternative model which provides also an estimation of the evolution of friction with the plateaux upsetting and the onset of galling.The second part is dedicated to the friction in hydroforming of tubes. We develop a frictional device and apply it to AA 6060 tubes. The Coulomb friction coefficient increase from typical values of 0,03 for greases to 0,3 for other lubricants (oil, solid lubricants). Finite elements calculations show the huge influence of the friction coefficient on the thickness distribution in the case of plane strain forming.L'industrie de la mise en forme utilise de plus en plus les codes de simulation numérique afin de minimiser les temps de conception et les coûts de mise au point des outillages. Le frottement, qui influence fortement le résultat de la mise en forme, est généralement modélisé par un coefficient de frottement de Coulomb constant. La validité de cette description sommaire du comportement tribologique du triplet tôle-lubrifiant-outils est discutée dans ce mémoire. Dans une première partie relative au procédé d'emboutissage, l'utilisation du tribomètre plan-plan est étendue à la caractérisation de la transition statique-dynamique. Cette nouvelle procédure d'essai met en évidence un niveau de frottement statique plus élevé que le frottement dynamique dans toute la gamme de pressions de contact explorée et pour des tôles d'aciers nus et revêtus. L'influence du frottement statique et plus généralement d'un frottement local et évolutif sur le résultat de la mise en forme est discutée à travers différentes modélisations calées sur les résultats de tribométrie et implémentées dans des codes de calculs par éléments finis (FORGE2® et ABAQUS®) : Une modélisation de la transition statique-dynamique comme fonction de la longueur de glissement, met en évidence l'influence du coefficient de frottement statique sur le retard à l'avalement du flan sous le serre-flan et sur la répartition des épaisseurs dans les zones de faibles glissements relatifs sous le poinçon. Une modélisation du frottement comme fonction de la vitesse de glissement et de la pression de contact permet de prendre en compte de manière réaliste l'influence de l'hétérogénéité de ces paramètres du contact. Pour des vitesses d'emboutissage croissantes, l'écart entre les résultats à frottement constant et variable décroît. La discrétisation du frottement par zone, le frottement sous le poinçon étant plus élevé que sous le serre-flan, permet une réduction sensible de l'erreur commise avec l'utilisation d'un coefficient de frottement constant pour tous les outillages. Le modèle de Devine qui décrit l'histoire microscopique du contact (interactions microplastiques, aspects hydrodynamiques) a été généralisé. C'est un modèle alternatif intéressant qui en outre permet d'estimer l'évolution du frottement local avec l'arasement des plateaux et une approche de l'avarie de contact. La seconde partie est dédiée à l'étude du frottement en hydroformage de tubes. Nous développons un essai de caractérisation du frottement et l'appliquons à des tubes en alliage d'aluminium 6060. Le coefficient de frottement de Coulomb varie entre de très faibles valeurs (0,03) pour des graisses à des valeurs beaucoup plus élevées (0,3) pour des huiles ou des lubrifiant solides. Des simulations par éléments finis mettent en évidence l'influence majeure de cette gamme de coefficient de frottement sur la distribution d'épaisseur en déformation plane

Apport de la simulation numérique et de l expérience pour la compréhension des phénomènes de frottement en emboutissage et hydroformage

PARIS-MINES ParisTech (751062310) / SudocSOPHIA ANTIPOLIS-Mines ParisTech (061522302) / SudocSudocFranceF

Insights from next-generation proteomics of bacterial endophytes response to chickpea root exudates and GacS role on plant growth-promoting traits regulation

Besides the beneficial association that legumes establish with rhizobia, these plants are also colonized by other endophytic bacteria. Although it is believed that these bacteria also have an important role on plant fitness, the molecular mechanisms of how non-rhizobial bacteria respond to the host-derived signals are poorly understood compared to the well-characterized N2 -fixing legumes-rhizobia symbioses. Here, a high-throughput proteomic analyses of two endophytic bacteria detected proteins involved in metabolism, cell envelope biosynthetic process, stress response, defense against oxidative stress, chemotaxis, nitrogen metabolic process, type iv secretion system and transmembrane transport as response to chickpea root exudates. One of the genes highly upregulated in cellular proteome of Pseudomonas sp. Q1 after exposition to chickpea root exudates is the gene on locus 4453. This gene (2,754 bp) encodes the GacS (global activator of antibiotic and cyanide synthesis), a membrane-bound sensor hybrid histidine kinase, member of the Gac two-component signal transduction system. To investigate the phenotypic traits under the regulation of the Pseudomonas sp. Q1 GacS protein, a knockout mutant for gacS gene was obtained. The swarming and swimming motility, biofilm production or phosphate solubilization were not affected by gacS gene deletion on ∆gacS mutant strain. On the other hand, the production and secretion of siderophores by ∆gacS mutant was reduced around 8.8% compared with the wild-type strain but the biosynthesis of antimicrobial metabolites in vitro was not affected. Our results showed that GacS of Q1 strain is only partially involved in the regulation of the mainly plant beneficial traits of Pseudomonas sp. Q1, suggesting that in this strain the role of this protein can be fulfilled by one or more additional two-component signal transduction systems. However, further studies are necessary to clarify the role of GacS in the regulation of surface motility, siderophore production and antifungal activity of Q1 strain

Reply to comment on Fisichella et al. (2012), “Intestinal toxicity evaluation of TiO₂ degraded surface-treated nanoparticles: a combined physico-chemical and toxicogenomics approach in Caco-2 cells” by Faust et al.

Abstract In this response, we discuss the major differences that clearly distinguish our results from those mentioned by Faust et al. In particular, the experiments have been conducted on nanoparticles of different nature, what mainly explains the observed discrepancies. This is a reply to http://www.particleandfibretoxicology.com/content/pdf/1743-8977-9-39.pdf.</p

Table_1_Taxonomical composition and functional analysis of biofilms sampled from a nuclear storage pool.XLSX

Sampling small amounts of biofilm from harsh environments such as the biofilm present on the walls of a radioactive material storage pool offers few analytical options if taxonomic characterization and estimation of the different biomass contributions are the objectives. Although 16S/18S rRNA amplification on extracted DNA and sequencing is the most widely applied method, its reliability in terms of quantitation has been questioned as yields can be species-dependent. Here, we propose a tandem-mass spectrometry proteotyping approach consisting of acquiring peptide data and interpreting then against a generalist database without any a priori. The peptide sequence information is transformed into useful taxonomical information that allows to obtain the different biomass contributions at different taxonomical ranks. This new methodology is applied for the first time to analyze the composition of biofilms from minute quantities of material collected from a pool used to store radioactive sources in a nuclear facility. For these biofilms, we report the identification of three genera, namely Sphingomonas, Caulobacter, and Acidovorax, and their functional characterization by metaproteomics which shows that these organisms are metabolic active. Differential expression of Gene Ontology GOslim terms between the two main microorganisms highlights their metabolic specialization.</p

Image_1_Taxonomical composition and functional analysis of biofilms sampled from a nuclear storage pool.JPEG

Sampling small amounts of biofilm from harsh environments such as the biofilm present on the walls of a radioactive material storage pool offers few analytical options if taxonomic characterization and estimation of the different biomass contributions are the objectives. Although 16S/18S rRNA amplification on extracted DNA and sequencing is the most widely applied method, its reliability in terms of quantitation has been questioned as yields can be species-dependent. Here, we propose a tandem-mass spectrometry proteotyping approach consisting of acquiring peptide data and interpreting then against a generalist database without any a priori. The peptide sequence information is transformed into useful taxonomical information that allows to obtain the different biomass contributions at different taxonomical ranks. This new methodology is applied for the first time to analyze the composition of biofilms from minute quantities of material collected from a pool used to store radioactive sources in a nuclear facility. For these biofilms, we report the identification of three genera, namely Sphingomonas, Caulobacter, and Acidovorax, and their functional characterization by metaproteomics which shows that these organisms are metabolic active. Differential expression of Gene Ontology GOslim terms between the two main microorganisms highlights their metabolic specialization.</p

Direct Meta-Analyses Reveal Unexpected Microbial Life in the Highly Radioactive Water of an Operating Nuclear Reactor Core

International audienceThe pools of nuclear reactor facilities constitute harsh environments for life, bathed with ionizing radiation, filled with demineralized water and containing toxic radioactive elements. The very few studies published to date have explored water pools used to store spent nuclear fuels. Due to access restrictions and strong handling constraints related to the high radioactivity level, nothing is presently known about life in water pools that directly cool nuclear cores. In this work, we investigated the microbial communities in the cooling pool of the French Osiris nuclear reactor using direct meta-omics approaches, namely, DNA metabarcoding and proteotyping based on 16S ribosomal RNA gene sequencing and on peptide analysis, respectively. We identified 25 genera in the highly radioactive core water supply during operation with radionuclide activity higher than 3 × 10 9 Bq/m 3. The prevailing genera Variovorax and Sphingomonas at operation were supplanted by Methylobacterium, Asanoa, and Streptomyces during shutdown. Variovorax might use dihydrogen produced by water radiolysis as an energy source

Striking the current metaproteomics dogma for deeper characterization of microbiota

International audienceIntroduction: Metaproteomics is the analysis of complex samples to describe how they function. The data may give novel taxonomical information based on peptide information. The current dogma in metaproteomics is i) to rely only on taxon-specific peptides for extracting the taxonomical information, and ii) to interpret MS/MS data with metagenomics data acquired on the same sample. We propose a new metaproteomics pipeline that allows a quick identification of any microorganism present in the sample based on the whole dataset, without need of additional costly metagenomics information. Methods: We developed in python an in-house pipeline to assign taxonomical information to each detected peptide against a generalist database and for the deconvolution of this complex signal. In parallel, we developed a procedure to regularly update and curate the database to avoid taxonomic misassignment. We also optimized the sample preparation for extracting proteins of any organisms and the tandem mass spectrometry acquisition to maximize the results.Results: We discovered the principle of a mathematical signature describing the number of peptide sequences shared with all other organisms calculated by modeling and phylogenetic relationships. This principle allows deciphering the precise content of a sample by the linear combination of such signatures applied on any experimental metaproteomic dataset. A sample can thus be described by its peptide-specified taxa and their respective relative ratios defined from the global peptide information. Its efficiency is exemplified with artificial mixtures. We developed the informatic pipeline to interpret quickly MS/MS datasets in terms of taxonomy, obtain response linearity regarding the label-free quantitation of biomass contributions, and establish the most appropriate protein sequence database for each sample. Several examples will be commented such as a deep characterization of human feces and a comparative analysis of sentinel animal intestines. Conclusions: This methodology paves the way to accurate label-free quantitative metaproteomics without the need of metagenomic information. It has been proved robust with artificial mixtures of microorganisms and has been applied to a large panel of samples of interest in terms of clinics, biotechnology, and environment. Novel Aspect: A new procedure for interpreting MS/MS metaproteomic dataset allows characterizing any sample in terms of taxonomy and improve the functional characterization.References:•Pible & Armengaud (2015) Improving the quality of genome, protein sequence, and taxonomy databases: a prerequisite for microbiome meta-omics 2.0. Proteomics 15(20):3418-23. •Grenga et al. (2019). Pathogen proteotyping: a rapidly developing application of mass spectrometry to address clinical concerns. Clinical Mass Spectrometry, In press.•Pible et al (2019) Improving relative quantitation of the biomass contributions of microorganisms present in a mixture by tandem mass spectrometry and the phylopeptidomics concept. Submitted.•Gouveia et al (2019) A new metaproteomics approach reveals the composition and protein function of the intestinal microbiota in a crustacean sentinel species. Submitted.•Hayoun et al. (2019) Evaluation of sample preparation methods for fast proteotyping of microorganisms by tandem mass spectrometry. Submitted