Sea Bass Primary Cultures versus RTgill-W1 Cell Line: Influence of Cell Model on the Sensitivity to Nanoparticles

Determination of acute toxicity to vertebrates in aquatic environments is mainly performed following OECD test guideline 203, requiring the use of a large number of fish and with mortality as endpoint. This test is also used to determine toxicity of nanomaterials in aquatic environments. Since a replacement method for animal testing in nanotoxicity studies is desirable, the feasibility of fish primary cultures or cell lines as a model for nanotoxicity screenings is investigated here. Dicentrarchus labrax primary cultures and RTgill-W1 cell line were exposed to several concentrations (0.1 to 200 ug/mL) of different nanoparticles (TiO2, polystyrene and silver), and cytotoxicity, metabolic activity and reactive oxygen species formation were investigated after 24 and 48 h of exposure. Protein corona as amount of protein bound, as well as the influence of surface modification (-COOH, -NH2), exposure media (Leibovitz’s L15 or seawater), weathering and cell type were the experimental variables included to test their influence on the results of the assays. Data from all scenarios was split based on the significance each experimental variable had in the result of the cytotoxicity tests, in an exploratory approach that allows for better understanding of the determining factors affecting toxicity. Data shows that more variables significantly influenced the outcome of toxicity tests when the primary cultures were exposed to the different nanoparticles. Toxicity tests performed in RTgill-W1 were influenced only by exposure time and nanoparticle concentration. The whole data set was integrated in a biological response index to show the overall impact of nanoparticle exposures.This study was supported by a postdoctoral grant to AJ-R (Basque Government; POSDOC program 2017–2019), Basque Country, Spain; and Federal Ministry of Education and Research, BMBF (NanoUmwelt, grant agreement Nº 030150B), Germany

Vers une meilleur compréhension des réponses cellulaires aux stimuli externes en utilisant des approches informatiques dit réseaux

Pendant mes travaux de thèse, j'ai développé et appliqué des méthodes informatiques utilisant des données de réseaux afin d'aider l'analyse des données biologiques à haut-débit. Ma thèse consiste en trois projets : L'identification de protéines supplémentaires dans des approches de protéomique différentielle à l'aide des réseaux d'interaction protéiques, l'identification de réseaux régulatoires sous-jacents aux réponses aux stress abiotiques dans arabidopsis thaliana et l'analyse de signature transcriptomique de réponse immunitaire d'hôte spécifique à différentes étapes d'infection par shigella flexneri.In the course of my Ph.D work, i have developed and applied methods making use of network information to adavance the analysis of high-throughput biological data. My thesis comprises three projects :- The identification of additional proteins in differential protemics using protein interaction networks. In this study, we developed a novel computational approach based on protein-protein interaction networks to identify a list of proteins that might have remained undetected in differential proteomic profiling experiments.- The transcriptional regulatory networks underlying responses to environmental stresses. Based on publicly available data, measuring the response of A. Thaliana to a set of abiotic stresses in a time-resolved manner, we applied two complimentary approaches to derive gene regulatory networks underlying the plant's response to the perceived stresses.- The analysis of transcriptional host immune response signatures specific for distinct stages of infection by shigella flexneri. During their host invasion process, shigella localize to different subcellular niches

Sea Bass Primary Cultures versus RTgill-W1 Cell Line: Influence of Cell Model on the Sensitivity to Nanoparticles

Determination of acute toxicity to vertebrates in aquatic environments is mainly performed following OECD test guideline 203, requiring the use of a large number of fish and with mortality as endpoint. This test is also used to determine toxicity of nanomaterials in aquatic environments. Since a replacement method for animal testing in nanotoxicity studies is desirable, the feasibility of fish primary cultures or cell lines as a model for nanotoxicity screenings is investigated here. Dicentrarchus labrax primary cultures and RTgill-W1 cell line were exposed to several concentrations (0.1 to 200 ug/mL) of different nanoparticles (TiO2, polystyrene and silver), and cytotoxicity, metabolic activity and reactive oxygen species formation were investigated after 24 and 48 h of exposure. Protein corona as amount of protein bound, as well as the influence of surface modification (-COOH, -NH2), exposure media (Leibovitz’s L15 or seawater), weathering and cell type were the experimental variables included to test their influence on the results of the assays. Data from all scenarios was split based on the significance each experimental variable had in the result of the cytotoxicity tests, in an exploratory approach that allows for better understanding of the determining factors affecting toxicity. Data shows that more variables significantly influenced the outcome of toxicity tests when the primary cultures were exposed to the different nanoparticles. Toxicity tests performed in RTgill-W1 were influenced only by exposure time and nanoparticle concentration. The whole data set was integrated in a biological response index to show the overall impact of nanoparticle exposures.This study was supported by a postdoctoral grant to AJ-R (Basque Government; POSDOC program 2017–2019), Basque Country, Spain; and Federal Ministry of Education and Research, BMBF (NanoUmwelt, grant agreement Nº 030150B), Germany

Bacterial Internalization, Localization, and Effectors Shape the Epithelial Immune Response during Shigella flexneri Infection

International audienceIntracellular pathogens are differentially sensed by the compartmentalized host immune system. Nevertheless, gene expression studies of infected cells commonly average the immune responses, neglecting the precise pathogen localization. To overcome this limitation, we dissected the transcriptional immune response to Shigella flexneri across different infection stages in bulk and single cells. This identified six distinct transcriptional profiles characterizing the dynamic, multilayered host response in both bystander and infected cells. These profiles were regulated by external and internal danger signals, as well as whether bacteria were membrane bound or cytosolic. We found that bacterial internalization triggers a complex, effector-independent response in bystander cells, possibly to compensate for the undermined host gene expression in infected cells caused by bacterial effectors, particularly OspF. Single-cell analysis revealed an important bacterial strategy to subvert host responses in infected cells, demonstrating that OspF disrupts concomitant gene expression of proinflammatory, apoptosis, and stress pathways within cells. This study points to novel mechanisms through which bacterial internalization, localization, and injected effectors orchestrate immune response transcriptional signatures

Trends in characteristics of 24-h urine samples and their relevance for human biomonitoring studies: 20 years of experience in the German Environmental Specimen Bank

To document trends in human exposure to environmental pollutants, the German Environmental Specimen Bank (ESB) has been routinely collecting and archiving 24-h urine samples from young adults at four sampling sites in Germany on an annual basis. For the purpose of normalizing measured analyte concentrations, urinary creatinine (UC), specific gravity (SG), conductivity (CON), and total urine volume (UVtot) of 24-h urine samples have also been recorded. These parameters are however susceptible to variation over time, as well as within/among participants and normalization against them can thus affect the interpretation of data regarding exposure to environmental pollutants. To evaluate the influence of normalization against these parameters, we first sought to determine variations of these parameters with regard to differences between sexes and trends over time. We analysed data from 8619 urine samples collected from 1997 to 2016. We observed an inverse relation between UVtot and UC, SG, and CON. We also found differences between sexes for UC, SG and CON, but not UVtot. UC, SG, and CON showed significant decreasing trends over time in both sexes. In contrast, a significant increase of over 30% in UVtot, independent of participant age and BMI, was revealed. This increase in UVtot and the concomitant sample dilution is likely to have an impact on measured analyte concentrations in 24-h urine samples. Hence, normalization of urinary concentrations is warranted when interpreting time trends of human exposure. Next, urinary calcium (Ca2+) concentrations of ESB participants were used to demonstrate the effects of normalization against each of the four urine parameters. From 1997 to 2016, measured Ca2+ concentrations showed a statistically significant but scientifically implausible decrease. Normalization of Ca2+ concentrations against UVtot (by calculating the total daily excretion), UC, or CON, but not SG, eliminated this decrease. Consistent with previous work, Ca2+ concentrations in urine and total daily Ca2+ excretion were higher for males than females. Normalization against UC, SG, or CON, however, attenuated this difference. Thus, to avoid misinterpretation in trend analysis and sex-specific excretion in 24-h urine samples, the calculation of the total daily excretion is recommended

Identification of additional proteins in differential proteomics using protein interaction networks

International audienceIn this study, we developed a novel computational approach based on protein-protein interaction networks to identify a list of proteins that might have remained undetected in differential proteomic profiling experiments. We tested our computational approach on two sets of human smooth muscle cell protein extracts that were affected differently by DNase I treatment. Differential proteomic analysis by saturation DIGE resulted in the identification of 41 human proteins. The application of our approach to these 41 input proteins consisted of four steps: (i) Compilation of a human protein-protein interaction network from public databases; (ii) calculation of interaction scores based on functional similarity; (iii) determination of a set of candidate proteins that are needed to efficiently and confidently connect the 41 input proteins; and (iv) ranking of the resulting 25 candidate proteins. Two of the three highest-ranked proteins, beta-arrestin 1, and beta-arrestin 2, were experimentally tested, revealing that their abundance levels in human smooth muscle cell samples were indeed affected by DNase I treatment. These proteins had not been detected during the experimental proteomic analysis. Our study suggests that our computational approach may represent a simple, universal, and cost-effective means to identify additional proteins that remain elusive for current 2D gel-based proteomic profiling techniques

Towards Harmonized Biobanking for Biomonitoring: A Comparison of Human Biomonitoring-Related and Clinical Biorepositories

Human biomonitoring (HBM) depends on high-quality human samples to identify status and trends in exposure and ensure comparability of results. In this context, much effort has been put into the development of standardized processes and quality assurance for sampling and chemical analysis, while effects of sample storage and shipment on sample quality have been less thoroughly addressed. To characterize the currently applied storage and shipment procedures within the consortium of the European Human Biomonitoring Initiative (HBM4EU), which aims at harmonization of HBM in Europe, a requirement analysis based on data from an online survey was conducted. In addition, the online survey was addressed to professionals in clinical biobanking represented by members of the European, Middle Eastern and African Society for Biopreservation and Biobanking (ESBB) to identify the current state-of-the-art in terms of sample storage and shipment. Results of this survey conducted in these two networks were compared to detect processes with potential for optimization and harmonization. In general, many similarities exist in sample storage and shipment procedures applied by ESBB members and HBM4EU partners and many requirements for ensuring sample quality are already met also by HBM4EU partners. Nevertheless, a need for improvement was identified for individual steps in sample storage, shipment, and related data management with potential impact on sample and data quality for HBM purposes. Based on these findings, recommendations for crucial first steps to further strengthen sample quality, and thus foster advancement in HBM on a pan-European level are given

Transcriptional and spatial profiling of the kidney allograft unravels a central role for FcyRIII+ innate immune cells in rejection

Abstract Rejection remains the main cause of premature graft loss after kidney transplantation, despite the use of potent immunosuppression. This highlights the need to better understand the composition and the cell-to-cell interactions of the alloreactive inflammatory infiltrate. Here, we performed droplet-based single-cell RNA sequencing of 35,152 transcriptomes from 16 kidney transplant biopsies with varying phenotypes and severities of rejection and without rejection, and identified cell-type specific gene expression signatures for deconvolution of bulk tissue. A specific association was identified between recipient-derived FCGR3A+ monocytes, FCGR3A + NK cells and the severity of intragraft inflammation. Activated FCGR3A+ monocytes overexpressed CD47 and LILR genes and increased paracrine signaling pathways promoting T cell infiltration. FCGR3A + NK cells overexpressed FCRL3, suggesting that antibody-dependent cytotoxicity is a central mechanism of NK-cell mediated graft injury. Multiplexed immunofluorescence using 38 markers on 18 independent biopsy slides confirmed this role of FcγRIII+ NK and FcγRIII+ nonclassical monocytes in antibody-mediated rejection, with specificity to the glomerular area. These results highlight the central involvement of innate immune cells in the pathogenesis of allograft rejection and identify several potential therapeutic targets that might improve allograft longevity