arXiv subjects

Thierry Rabilloud

Publications and source records attributed to Thierry Rabilloud.

At least 19 recordsLinked to original sources

arXiv2019

A toxicology-informed, safer by design approach for the fabrication of transparent electrodes based on silver nanowires

Fabrication of silver nanowires (AgNWs) with fine and independent control of both the diameter (from 30 to 120 nm) and length (from 5 to 120 $μ$m) by concomitant addition of co-nucleants and temperature control is demonstrated, and used for the preparation of size standards. Percolating random networks were fabricated using these standards and their optoelectronic properties were measured and compared with regard to the nanowire dimensions. The transparent electrodes appear suitable for various applications and exhibit excellent performances (e.g. 16 ohm sq --1 at 93% transparency), with haze values varying from 1.6 to 26.2%. Besides, in vitro toxicological studies carried out on murine macrophages with the same size standards revealed that AgNWs are weakly toxic (no toxicity observed below 50 $μ$g mL --1 Ag), in particular compared to other silver nanoparticles. Short AgNWs (4 $μ$m) appeared to be slightly more toxic than longer AgNWs (10 and 20 $μ$m). Conversely, long AgNWs (20 $μ$m) induced a more prolonged pro-inflammatory response in murine macrophages. These results contribute, in a safer by design approach, to promoting the use of short AgNWs. The global knowledge dealing with the combination of nanowire dimensions associated with optoelectronic performances and related toxicity should encourage the rational use of AgNWs, and guide the choice of the most adequate AgNW dimensions in an integrated approach.

physics.app-ph↗

arXiv2018

Zinc oxide induces the stringent response and major reorientations in the central metabolism of Bacillus subtilis

Microorganisms, such as bacteria, are one of the first targets of nanoparticles in the environment. In this study, we tested the effect of two nanoparticles, ZnO and TiO2, with the salt ZnSO4 as the control, on the Gram-positive bacterium Bacillus subtilis by 2D gel electrophoresis-based proteomics. Despite a significant effect on viability (LD50), TiO2 NPs had no detectable effect on the proteomic pattern, while ZnO NPs and ZnSO4 significantly modified B. subtilis metabolism. These results allowed us to conclude that the effects of ZnO observed in this work were mainly attributable to Zn dissolution in the culture media. Proteomic analysis highlighted twelve modulated proteins related to central metabolism: MetE and MccB (cysteine metabolism), OdhA, AspB, IolD, AnsB, PdhB and YtsJ (Krebs cycle) and XylA, YqjI, Drm and Tal (pentose phosphate pathway). Biochemical assays, such as free sulfhydryl, CoA-SH and malate dehydrogenase assays corroborated the observed central metabolism reorientation and showed that Zn stress induced oxidative stress, probably as a consequence of thiol chelation stress by Zn ions. The other patterns affected by ZnO and ZnSO4 were the stringent response and the general stress response. Nine proteins involved in or controlled by the stringent response showed a modified expression profile in the presence of ZnO NPs or ZnSO4: YwaC, SigH, YtxH, YtzB, TufA, RplJ, RpsB, PdhB and Mbl. An increase in the ppGpp concentration confirmed the involvement of the stringent response during a Zn stress. All these metabolic reorientations in response to Zn stress were probably the result of complex regulatory mechanisms including at least the stringent response via YwaC.

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arXiv2018

Differential proteomics highlights macrophage-specific responses to amorphous silica nanoparticles

The technological and economic benefits of engineered nanomaterials may be offset by their adverse effects on living organisms. One of the highly produced nanomaterials under such scrutiny is amorphous silica nanoparticles, which are known to have an appreciable, although reversible, inflammatory potential. This is due to their selective toxicity toward macrophages, and it is thus important to study the cellular responses of this cell type to silica nanoparticles to better understand the direct or indirect adverse effects of nanosilica. We have here studied the responses of the RAW264.7 murine macrophage cells and of the control MPC11 plasma cells to subtoxic concentrations of nanosilica, using a combination of pro-teomic and targeted approaches. This allowed us to document alterations in the cellular cytoskeleton, in the phagocytic capacity of the cells as well as their ability to respond to bacterial stimuli. More surprisingly, silica nanoparticles also induce a greater sensitivity of macrophages to DNA alkylating agents, such as styrene oxide, even at doses which do not induce any appreciable cell death.

q-bio.GN↗

arXiv2016

A single step protein assay that is both detergent and reducer compatible: The cydex blue assay

Determination of protein concentration in often an absolute pre-requisite in preparing samples for biochemical and proteomic analyses. However, current protein assay methods are not compatible with both reducers and detergents, which are however present simultaneously in most denaturing extraction buffers used in proteomics and electrophoresis, and in particular in SDS electrophoresis. We found that inclusion of cyclodextrins in a Coomassie blue-based assay made it compatible with detergents, as cyclodextrins complex detergents in a 1:1 molecular ratio. As this type of assay is intrinsically resistant to reducers, we have thus developed a single step assay that is both detergent and reducer compatible. Depending on the type and concentration of detergents present in the sample buffer, either beta-cyclodextrin or alpha-cyclodextrin can be used, the former being able to complex a wider range of detergents and the latter being able to complex higher amounts of detergents due to its greater solubility in water. Cyclodextrins are used at final concentrations of 2-10 mg/mL in the assay mix. This typically allows to measure samples containing as little as 0.1 mg/mL protein, in the presence of up to 2% detergent and reducers such as 5 % mercaptoethanol or 50 mM DTT in a single step with a simple spectrophotometric assay. This article is protected by copyright. All rights reserved.

q-bio.GN↗

arXiv2014

Paleoproteomics explained to youngsters: how did the wedding of two-dimensional electrophoresis and protein sequencing spark proteomics on: Let there be light

Taking the opportunity of the 20th anniversary of the word "proteomics", this young adult age is a good time to remember how proteomics came from enormous progress in protein separation and protein microanalysis techniques, and from the conjugation of these advances into a high performance and streamlined working setup. However, in the history of the almost three decades that encompass the first attempts to perform large scale analysis of proteins to the current high throughput proteomics that we can enjoy now, it is also interesting to underline and to recall how difficult the first decade was. Indeed when the word was cast, the battle was already won. This recollection is mostly devoted to the almost forgotten period where proteomics was being conceived and put to birth, as this collective scientific work will never appear when searched through the keyword "proteomics". BIOLOGICAL SIGNIFICANCE: The significance of this manuscript is to recall and review the two decades that separated the first attempts of performing large scale analysis of proteins from the solid technical corpus that existed when the word "proteomics" was coined twenty years ago. This recollection is made within the scientific historical context of this decade, which also saw the blossoming of DNA cloning and sequencing. This article is part of a Special Issue entitled: 20 years of Proteomics in memory of Viatliano Pallini. Guest Editors: Luca Bini , Juan J. Calvete, Natacha Turck, Denis Hochstrasser and Jean-Charles Sanchez.

q-bio.GN↗

arXiv2014

Analysis of cellular responses of macrophages to zinc ions and zinc oxide nanoparticles: a combined targeted and proteomic approach

Two different zinc oxide nanoparticles, as well as zinc ions, are used to study the cellular responses of the RAW 264 macrophage cell line. A proteomic screen is used to provide a wide view of the molecular effects of zinc, and the most prominent results are cross-validated by targeted studies. Furthermore, the alteration of important macrophage functions (e.g. phagocytosis) by zinc is also investigated. The intracellular dissolution/uptake of zinc is also studied to further characterize zinc toxicity. Zinc oxide nanoparticles dissolve readily in the cells, leading to high intracellular zinc concentrations, mostly as protein-bound zinc. The proteomic screen reveals a rather weak response in the oxidative stress response pathway, but a strong response both in the central metabolism and in the proteasomal protein degradation pathway. Targeted experiments confirm that carbohydrate catabolism and proteasome are critical determinants of sensitivity to zinc, which also induces DNA damage. Conversely, glutathione levels and phagocytosis appear unaffected at moderately toxic zinc concentrations.

q-bio.GN↗

arXiv2014

The proteomic to biology inference, a frequently overlooked concern in the interpretation of proteomic data: A plea for functional validation

Proteomics will celebrate its 20th year in 2014. In this relatively short period of time, it has invaded most areas of biology and its use will probably continue to spread in the future. These two decades have seen a considerable increase in the speed and sensitivity of protein identification and characterization, even from complex samples. Indeed, what was a challenge twenty years ago is now little more than a daily routine. Although not completely over, the technological challenge now makes room to another challenge, which is the best possible appraisal and exploitation of proteomic data to draw the best possible conclusions from a biological point of view. The point developed in this paper is that proteomic data are almost always fragmentary. This means in turn that although better than an mRNA level, a protein level is often insufficient to draw a valid conclusion from a biological point of view, especially in a world where PTMs play such an important role. This means in turn that transformation of proteomic data into biological data requires an important intermediate layer of functional validation, i.e. not merely the confirmation of protein abundance changes by other methods, but a functional appraisal of the biological consequences of the protein level changes highlighted by the proteomic screens.

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arXiv2013

How to use 2D gel electrophoresis in plant proteomics

Two-dimensional electrophoresis has nurtured the birth of proteomics. It is however no longer the exclusive setup used in proteomics, with the development of shotgun proteomics techniques that appear more fancy and fashionable nowadays.Nevertheless, 2D gel-based proteomics still has valuable features, and sometimes unique ones, which make it often an attractive choice when a proteomics strategy must be selected. These features are detailed in this chapter, as is the rationale for selecting or not 2D gel-based proteomics as a proteomic strategy.

q-bio.GN↗

arXiv2013

When 2D is not enough, go for an extra dimension

The use of an extra SDS separation in a different buffer system provide a technique for deconvoluting 2D gel spots made of several proteins (Colignon et al. Proteomics, 2013, 13, 2077-2082). This technique keeps the quantitative analysis of the protein amounts and combines it with a strongly improved identification process by mass spectrometry, removing identification ambiguities in most cases. In some favorable cases, posttranslational variants can be separated by this procedure. This versatile and easy to use technique is anticipated to be a very valuable addition to the toolbox used in 2D gel-based proteomics.

q-bio.GN↗

arXiv2013

Multidimensional separation prior to mass spectrometry: Getting closer to the bottom of the iceberg

While prefractionation has previously been shown to improve results in MS analysis, a novel combination provides an additional dimension of separation: protein fractionation by SDS-PAGE followed by IEF of tryptic peptides before separation by RP-LC [Atanassov and Urlaub, Proteomics 2013, 13, 2947-2955]. This three-step separation procedure prior to MS/MS substantially increases proteome coverage and represents a further step toward a more comprehensive analysis of complex proteomes.

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arXiv2013

Molecular responses of mouse macrophages to copper and copper oxide nanoparticles inferred from proteomic analyses

The molecular responses of macrophages to copper-based nanoparticles have been investigated via a combination of proteomic and biochemical approaches, using the RAW264.7 cell line as a model. Both metallic copper and copper oxide nanoparticles have been tested, with copper ion and zirconium oxide nanoparticles used as controls. Proteomic analysis highlighted changes in proteins implicated in oxidative stress responses (superoxide dismutases and peroxiredoxins), glutathione biosynthesis, the actomyosin cytoskeleton, and mitochondrial proteins (especially oxidative phosphorylation complex subunits). Validation studies employing functional analyses showed that the increases in glutathione biosynthesis and in mitochondrial complexes observed in the proteomic screen were critical to cell survival upon stress with copper-based nanoparticles; pharmacological inhibition of these two pathways enhanced cell vulnerability to copper-based nanoparticles, but not to copper ions. Furthermore, functional analyses using primary macrophages derived from bone marrow showed a decrease in reduced glutathione levels, a decrease in the mitochondrial transmembrane potential, and inhibition of phagocytosis and of lipopolysaccharide-induced nitric oxide production. However, only a fraction of these effects could be obtained with copper ions. In conclusion, this study showed that macrophage functions are significantly altered by copper-based nanoparticles. Also highlighted are the cellular pathways modulated by cells for survival and the exemplified cross-toxicities that can occur between copper-based nanoparticles and pharmacological agents.

q-bio.GN↗

arXiv2013

Two-dimensional SDS-PAGE fractionation of biological samples for biomarker discovery

Two-dimensional electrophoresis is still a very valuable tool in proteomics, due to its reproducibility and its ability to analyze complete proteins. However, due to its sensitivity to dynamic range issues, its most suitable use in the frame of biomarker discovery is not on very complex fluids such as plasma, but rather on more proximal, simpler fluids such as CSF, urine, or secretome samples. Here, we describe the complete workflow for the analysis of such dilute samples by two-dimensional electrophoresis, starting from sample concentration, then the two-dimensional electrophoresis step per se, ending with the protein detection by fluorescence.

q-bio.GN↗

arXiv2012

Improved proteomic analysis of nuclear proteins, as exemplified by the comparison of two myeloïd cell lines nuclear proteomes

One of the challenges of the proteomic analysis by 2D-gel is to visualize the low abundance proteins, particularly those localized in organelles. An additional problem with nuclear proteins lies in their strong interaction with nuclear acids. Several experimental procedures have been tested to increase, in the nuclear extract, the ratio of nuclear proteins compared to contaminant proteins, and also to obtain reproducible conditions compatible with 2D-gel electrophoresis. The NaCl procedure has been chosen. To test the interest of this procedure, the nuclear protein expression profiles of macrophages and dendritic cells have been compared with a proteomic approach by 2D-gel electrophoresis. Delta 2D software and mass spectrometry analyses have allowed pointing out some proteins of interest. We have chosen some of them, involved in transcriptional regulation and/or chromatin structure for further validations. The immunoblotting experiments have shown that most of observed changes are due to post-translational modifications, thereby a exemplifying the interest of the 2D gel approach. Finally, this approach allowed us to reach not only high abundance nuclear proteins but also lower abundance proteins, such as the HP1 proteins and reinforces the interest of using 2DE-gel in proteomics because of its ability to visualize intact proteins with their modifications.

q-bio.GN↗

arXiv2012

About the mechanism of interference of silver staining with peptide mass spectrometry

The mechanism by which silver staining of proteins in polyacrylamide gels interferes with mass spectrometry of peptides produced by proteolysis has been investigated. It was demonstrated that this interference increases with time between silver staining and gel processing, although the silver image is constant. This suggested an important role of the formaldehyde used in silver staining development in this interference process. Consequently, a formaldehyde-free staining protocol has been devised, using carbohydrazide as the developing agent. This protocol showed much increased peptide coverage and retained the sensitivity of silver staining. These results were however obtained at the expense of an increased background in the stained gels and of a reduced staining homogeneity.

q-bio.GN↗

arXiv2012

The Whereabouts of 2D Gels in Quantitative Proteomics

Two-dimensional gel electrophoresis has been instrumental in the development of proteomics. Although it is no longer the exclusive scheme used for proteomics, its unique features make it a still highly valuable tool, especially when multiple quantitative comparisons of samples must be made, and even for large samples series. However, quantitative proteomics using 2D gels is critically dependent on the performances of the protein detection methods used after the electrophoretic separations. This chapter therefore examines critically the various detection methods (radioactivity, dyes, fluorescence, and silver) as well as the data analysis issues that must be taken into account when quantitative comparative analysis of 2D gels is performed.

q-bio.GN↗

arXiv2012

Silver Staining of 2D Electrophoresis Gels

Silver staining is used to detect proteins after electrophoretic separation on polyacrylamide gels. It -combines excellent sensitivity (in the low nanogram range) with the use of very simple and cheap equipment and chemicals. For its use in proteomics, two important additional features must be considered, compatibility with mass spectrometry and quantitative response. Both features are discussed in this chapter, and optimized silver staining protocols are proposed.

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arXiv2011

Two-dimensional gel electrophoresis in proteomics: A tutorial

Two-dimensional electrophoresis of proteins has preceded, and accompanied, the birth of proteomics. Although it is no longer the only experimental scheme used in modern proteomics, it still has distinct features and advantages. The purpose of this tutorial paper is to guide the reader through the history of the field, then through the main steps of the process, from sample preparation to in-gel detection of proteins, commenting the constraints and caveats of the technique. Then the limitations and positive features of two-dimensional electrophoresis are discussed (e.g. its unique ability to separate complete proteins and its easy interfacing with immunoblotting techniques), so that the optimal type of applications of this technique in current and future proteomics can be perceived. This is illustrated by a detailed example taken from the literature and commented in detail. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP 2).

q-bio.GN↗

arXiv2011

Effects of nanoparticles on murine macrophages

Metallic nanoparticles are more and more widely used in an increasing number of applications. Consequently, they are more and more present in the environment, and the risk that they may represent for human health must be evaluated. This requires to increase our knowledge of the cellular responses to nanoparticles. In this context, macrophages appear as an attractive system. They play a major role in eliminating foreign matter, e.g. pathogens or infectious agents, by phagocytosis and inflammatory responses, and are thus highly likely to react to nanoparticles. We have decided to study their responses to nanoparticles by a combination of classical and wide-scope approaches such as proteomics. The long term goal of this study is the better understanding of the responses of macrophages to nanoparticles, and thus to help to assess their possible impact on human health. We chose as a model system bone marrow-derived macrophages and studied the effect of commonly used nanoparticles such as TiO2 and Cu. Classical responses of macrophage were characterized and proteomic approaches based on 2D gels of whole cell extracts were used. Preliminary proteomic data resulting from whole cell extracts showed different effects for TiO2-NPs and Cu-NPs. Modifications of the expression of several proteins involved in different pathways such as, for example, signal transduction, endosome-lysosome pathway, Krebs cycle, oxidative stress response have been underscored. These first results validate our proteomics approach and open a new wide field of investigation for NPs impact on macrophages

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