Impact of Physico-Chemical Properties of Cellulose Nanocrystal/Silver Nanoparticle Hybrid Suspensions on Their Biocidal and Toxicological Effects.

There is a demand for nanoparticles that are environmentally acceptable, but simultaneously efficient and low cost. We prepared silver nanoparticles (AgNPs) grafted on a native bio-based substrate (cellulose nanocrystals, CNCs) with high biocidal activity and no toxicological impact. AgNPs of 10 nm are nucleated on CNCs in aqueous suspension with content from 0.4 to 24.7 wt%. XANES experiments show that varying the NaBH4/AgNO3 molar ratio affects the AgNP oxidation state, while maintaining an fcc structure. AgNPs transition from 10 nm spherical NPs to 300 nm triangular-shaped AgNPrisms induced by H2O2 post-treatment. The 48 h biocidal activity of the hybrid tested on B. Subtilis is intensified with the increase of AgNP content irrespective of the Ag+/Ag0 ratio in AgNPs, while the AgNSphere-AgNPrism transition induces a significant reduction of biocidal activity. A very low minimum inhibitory concentration of 0.016 mg AgNP/mL is determined. A new long-term biocidal activity test (up to 168 h) proved efficiency favorable to the smaller AgNPs. Finally, it is shown that AgNPs have no impact on the phagocytic capacity of mammalian cells.

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 two-dimensional 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 two-dimensional gels is performed.

In Situ Formation of Silver Nanoparticles (Ag-NPs) onto Textile Fibers.

Silver nanoparticles (Ag-NPs) adhered/inserted on textile fibers have an effective antimicrobial role. However, their release due to low adherence and their fate in the natural settings have been questioned in terms of toxicity level. In order to overcome this recurrent problem of adherence, the in situ formation of Ag-NPs in five textile fibers (cotton (untreated and chemically bleached), sheep's wool, polyamide, and polyester) was assessed. Herein, the fibers were first immersed in a silver ion solution (1 g/L of AgNO3) for ion saturation at room T for 24 h followed by draining fibers and their reimmersion this time in a strong chemical reducing solution (0.25 g/L of NaBH4) at room T for 24 h. This latter step leads to the in situ formation of Ag-NPs where size (5 nm < size < 50 nm), surface covering concentration, and aggregation degree depend on the textile fiber kind as deduced from FESEM images. This simple lab chemical method allows instantaneous in situ formation of Ag-NPs onto fibers without the requirement of additional thermal treatment. Moreover, for natural fibers, the formation of Ag-NPs inside of them is also expected as confirmed from FESEM images in cotton cross sections. In complement, all textile fibers containing Ag-NPs (sheep's wool 10 mg/g > untreated cotton 2.3 mg/g > bleached cotton 1 mg/g > polyamide 0.62 mg/g > polyester 0.28 mg/g) were submitted to interact with strong oxidants in an aqueous media (7.5% v/v of H2O2, 0.5 and 0.05 M of HNO3 and ultrapure water as the control) using flow-through reactor experiments. Here, breakthrough curves reveal that the oxidative dissolution rate (given in mol/g min) of adhered Ag-NPs (ionic release) depends strongly on fiber nature, and nature and concentration of oxidant solution. In summary, this fundamental study suggests that Ag-NPs may be successfully adhered/inserted in natural fibers (wool and cotton) in a safety-design perspective with performant biocide properties as confirmed by using Bacillus subtilis.

ZnO and TiO2 nanoparticles alter the ability of Bacillus subtilis to fight against a stress.

Due to the physicochemical properties of nanoparticles, the use of nanomaterials increases over time in industrial and medical processes. We herein report the negative impact of nanoparticles, using solid growth conditions mimicking a biofilm, on the ability of Bacillus subtilis to fight against a stress. Bacteria have been exposed to sublethal doses of nanoparticles corresponding to conditions that bacteria may meet in their natural biotopes, the upper layer of soil or the gut microbiome. The analysis of the proteomic data obtained by shotgun mass spectrometry have shown that several metabolic pathways are affected in response to nanoparticles, n-ZnO or n-TiO2, or zinc salt: the methyglyoxal and thiol metabolisms, the oxidative stress and the stringent responses. Nanoparticles being embedded in the agar medium, these impacts are the consequence of a physiological adaptation rather than a physical cell injury. Overall, these results show that nanoparticles, by altering bacterial physiology and especially the ability to resist to a stress, may have profound influences on a "good bacteria", Bacillus subtilis, in its natural biotope and moreover, on the global equilibrium of this biotope.

What Room for Two-Dimensional Gel-Based Proteomics in a Shotgun Proteomics World?

Two-dimensional gel electrophoresis was instrumental in the birth of proteomics in the late 1980s. However, it is now often considered as an outdated technique for proteomics-a thing of the past. Although this opinion may be true for some biological questions, e.g., when analysis depth is of critical importance, for many others, two-dimensional gel electrophoresis-based proteomics still has a lot to offer. This is because of its robustness, its ability to separate proteoforms, and its easy interface with many powerful biochemistry techniques (including western blotting). This paper reviews where and why two-dimensional gel electrophoresis-based proteomics can still be profitably used. It emerges that, rather than being a thing of the past, two-dimensional gel electrophoresis-based proteomics is still highly valuable for many studies. Thus, its use cannot be dismissed on simple fashion arguments and, as usual, in science, the tree is to be judged by the fruit.

The poly-gamma-glutamate of Bacillus subtilis interacts specifically with silver nanoparticles.

For many years, silver nanoparticles, as with other antibacterial nanoparticles, have been extensively used in manufactured products. However, their fate in the environment is unclear and raises questions. We studied the fate of silver nanoparticles in the presence of bacteria under growth conditions that are similar to those found naturally in the environment (that is, bacteria in a stationary phase with low nutrient concentrations). We demonstrated that the viability and the metabolism of a gram-positive bacteria, Bacillus subtilis, exposed during the stationary phase is unaffected by 1 mg/L of silver nanoparticles. These results can be partly explained by a physical interaction of the poly-gamma-glutamate (PGA) secreted by Bacillus subtilis with the silver nanoparticles. The coating of the silver nanoparticles by the secreted PGA likely results in a loss of the bioavailability of nanoparticles and, consequently, a decrease of their biocidal effect.

Author Correction: Impact of nanoparticles on the Bacillus subtilis (3610) competence.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Impact of nanoparticles on the Bacillus subtilis (3610) competence.

Due to the physicochemical properties of nanoparticles, the use of nanomaterials increases every year in industrial and medical processes. At the same time, the increasing number of bacteria becoming resistant to many antibiotics, mostly by a horizontal gene transfer process, is a major public health concern. We herein report, for the first time, the role of nanoparticles in the physiological induction of horizontal gene transfer in bacteria. Besides the most well-known impacts of nanoparticles on bacteria, i.e. death or oxidative stress, two nanoparticles, n-ZnO and n-TiO2, significantly and oppositely impact the transformation efficiency of Bacillus subtilis in biofilm growth conditions, by modification of the physiological processes involved in the induction of competence, the first step of transformation. This effect is the consequence of a physiological adaptation rather than a physical cell injury: two oligopeptide ABC transporters, OppABCDF and AppDFABC, are differentially expressed in response to nanoparticles. Interestingly, a third tested nanoparticle, n-Ag, has no significant effect on competence in our experimental conditions. Overall, these results show that nanoparticles, by altering bacterial physiology and especially competence, may have profound influences in unsuspected areas, such as the dissemination of antibiotic resistance in bacteria.

Impact of a Model Soil Microorganism and of Its Secretome on the Fate of Silver Nanoparticles.

Sulfidation is a key process for silver nanoparticles released from consumer products in the environment. This study focuses on the impact of a model soil microorganism, Bacillus subtilis, on the fate of pristine and already sulfidized Ag-NPs. The nanoparticles were incubated with the initial growth medium, isolated secretome, and living bacteria, and characterized for their size and morphology, agglomeration state, structure, and Ag speciation. No Ag internalization or sorption on the cell wall was detected. A partial sulfidation, leading to an Ag-Ag2S core-shell structure, was observed in the presence of the secretome, and the rate limiting step of the reaction was the oxidation of Ag0, and it was favored near the crystal dislocations. The sulfidation was complete in the presence of the living bacteria and followed an indirect pathway. Both crystalline Ag2S and amorphous Ag2S and/or Ag-thiol were identified. At the opposite, the bacteria had no impact on Ag2S. These results suggest that microorganisms participate in the sulfidation of Ag-NPs in aerobic systems such as unsaturated soils, and thus affect the bioavailability of Ag. It is important to take these transformations into account during exposure experiments, since they drastically change the exposure conditions. Finally, the secretome of B. subtilis might be used for the green synthesis of Ag-Ag2S core-shell nanoparticles.

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.

Preparing protein extracts for quantitative two-dimensional gel comparison.

This unit describes basic protocols for efficient and reproducible protein solubilization from a variety of biological samples, including cultured animal cells and tissues, plant cells and tissues, bacteria, nuclei, other subcellular organelles, plasma, serum, and other biological fluids. The optimized extraction process is strongly sample dependent and cannot be described for every type of sample. Instead, typical protocols are provided as general guidelines and illustrate good starting points for optimization of sample preparation. These solubilization procedures take into account the constraints imposed by two-dimensional electrophoresis and are thus well suited for proteomic approaches.

Improved proteomic analysis of nuclear proteins, as exemplified by the comparison of two myeloid 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 the 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. Delta2D 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 the observed changes are due to post-translational modifications, thereby 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.

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).

Two-dimensional gel electrophoresis in proteomics: Past, present and future.

Two-dimensional gel electrophoresis has been instrumental in the birth and developments of proteomics, although it is no longer the exclusive separation tool used in the field of proteomics. In this review, a historical perspective is made, starting from the days where two-dimensional gels were used and the word proteomics did not even exist. The events that have led to the birth of proteomics are also recalled, ending with a description of the now well-known limitations of two-dimensional gels in proteomics. However, the often-underestimated advantages of two-dimensional gels are also underlined, leading to a description of how and when to use two-dimensional gels for the best in a proteomics approach. Taking support of these advantages (robustness, resolution, and ability to separate entire, intact proteins), possible future applications of this technique in proteomics are also mentioned.

Silver staining of proteins in 2DE gels.

Silver staining detects proteins after electrophoretic separation on polyacrylamide gels. Its main positive features are its excellent sensitivity (in the low nanogram range) and the use of very simple and cheap equipment and chemicals. The sequential phases of silver staining are protein fixation, then sensitization, then silver impregnation, and finally image development. Several variants of silver staining are described here, which can be completed in a time range from 2 h to 1 day after the end of the electrophoretic separation. Once completed, the stain is stable for several weeks.

Power and limitations of electrophoretic separations in proteomics strategies.

Proteomics can be defined as the large-scale analysis of proteins. Due to the complexity of biological systems, it is required to concatenate various separation techniques prior to mass spectrometry. These techniques, dealing with proteins or peptides, can rely on chromatography or electrophoresis. In this review, the electrophoretic techniques are under scrutiny. Their principles are recalled, and their applications for peptide and protein separations are presented and critically discussed. In addition, the features that are specific to gel electrophoresis and that interplay with mass spectrometry (i.e., protein detection after electrophoresis, and the process leading from a gel piece to a solution of peptides) are also discussed.

Fully denaturing two-dimensional electrophoresis of membrane proteins: a critical update.

The quality and ease of proteomics analysis depends on the performance of the analytical tools used, and thus of the performances of the protein separation tools used to deconvolute complex protein samples. Among protein samples, membrane proteins are one of the most difficult sample classes, because of their hydrophobicity and embedment in the lipid bilayers. This review deals with the recent progresses and advances made in the separation of membrane proteins by 2-DE separating only denatured proteins. Traditional 2-D methods, i.e., methods using IEF in the first dimension are compared to methods using only zone electrophoresis in both dimensions, i.e., electrophoresis in the presence of cationic or anionic detergents. The overall performances and fields of application of both types of method is critically examined, as are future prospects for this field.

Ultrafast coelectrophoretic fluorescent staining of proteins with carbocyanines.

Protein detection on SDS gels or on 2-D gels must combine several features, such as sensitivity, homogeneity from one protein to another, speed, low cost, and user-friendliness. For some applications, it is also interesting to have a nonfixing stain, so that proteins can be mobilized from the gel for further use (electroelution, blotting). We show here that coelectrophoretic staining by fluorophores of the oxacarbocyanine family, and especially diheptyloxacarbocyanine, offers several positive features. The sensitivity is intermediate between the one of colloidal CBB and the one of fluorescent ruthenium complexes. Detection is achieved within 1 h after the end of the electrophoretic process and does not use any fixing or toxic agent. The fluorescent SDS-carbocyanine-protein complexes can be detected either with a laser scanner with an excitation wavelength of 488 nm or with a UV table operating at 302 nm. Excellent sequence coverage in subsequent MS analysis of proteolytic peptides is also achieved with this detection method.

Mutual regulation of Crl and Fur in Escherichia coli W3110.

The small regulatory protein Crl controls the expression of curli. Recently we have shown that Crl interacts directly with one of the most global regulators of Escherichia coli, the stress-related sigma factor RpoS, suggesting a more global role for Crl. We show here by a proteomics analysis that the expression of at least nine cellular proteins was considerably modified when Crl was overexpressed. We assessed the part of transcriptional and post-transcriptional regulation for five of these genes. The results showed that Crl regulates the expression of another global regulator, the central regulator of iron homeostasis, Fur. A molecular analysis revealed that Crl and Fur affect their own and each other's expression. We provide physical evidence for the binding of Fur to the crl and fur promoter regions. Crl modulated the affinity of Fur at the fur promoter but not at the crl promoter. The triad RpoS-Crl-Fur may thus represent the centerpiece of a global regulatory system of response to different stresses.

The Crl-RpoS regulon of Escherichia coli.

The RpoS subunit of RNA polymerase controls the expression of numerous genes involved in stationary phase and in response to different stress conditions. The regulatory protein Crl increases the activity of RpoS by direct interaction with the RpoS holoenzyme. To define the extent of the Crl regulon, we used two-dimensional SDS-PAGE to measure the role of Crl in regulating the expression of the Escherichia coliproteome in stationary phase at 30 degrees C. By comparing the proteome of four strains (wild type, crl(-), rpoS(-), and crl(-)rpoS(-)), we observed that the intensity of 74 spots was modified in at least one mutant context. 62 spots were identified by mass spectrometry and correspond to 40 distinct proteins. They were classified in four main categories: DNA metabolism, central metabolism, response to environmental modifications, and miscellaneous. Three proteins were specifically involved in quorum sensing: TnaA (the tryptophanase that converts tryptophan to indole), WrbA (Trp repressor-binding protein), and YgaG (homologous to LuxS, autoinducer-2 synthase). Because little is known about the regulation of Crl expression, we investigated the influence of diffusible molecules on the expression of Crl. Using Western blotting experiments, we showed that, at 30 degrees C, a diffusible molecule(s) produced during the transition phase between the exponential and stationary phases induces a premature expression of Crl. Indole was tested as one of the potential candidates: at 37 degrees C, it is present in the extracellular medium at a constant concentration, but at 30 degrees C, its concentration peaks during the transition phase. When indole was added to the culture medium, it also induced prematurely the expression of Crl at both the transcriptional and translational levels in a Crl-dependent manner. Crl may thus be considered a new environmental sensor via the indole concentration.