Sweet silver: A formaldehyde-free silver staining using aldoses as developing agents, with enhanced compatibility with mass spectrometry

Protein detection methods after electrophoresis have to be sensitive, homogeneous, and not to impair downstream analysis of proteins by MS. Speed, low cost, and user friendliness are also favored features. Silver staining combines many of these features, but its compatibility with MS is limited. We describe here, a new variant of silver staining that is completely formaldehyde-free. Reducing sugars in alkaline borate buffer are used as developers. While keeping the benefits of silver staining, this method is shown to afford a much better performance in terms of compatibility with MS, both in PMF by MALDI and in LC/ESI/MS/MS

Ubiquitin, a central component of selective cytoplasmic proteolysis, is linked to proteins residing at the locus of non-selective proteolysis, the vacuole

Ubiquitin, an evolutionary highly conserved protein, is known to be involved in selective proteolysis in the cytoplasm. Here we show that ubiquitin-protein conjugates are also found in the yeast vacuole. Mutants defective in the major vacuolar endopeptidases, proteinase yscA and yscB, lead to accumulation of ubiquitin-protein conjugates in this cellular organelle.

Salivary Proteins Associated with Periodontitis in Patients with Type 2 Diabetes Mellitus

The objective of this study was to investigate the salivary proteins that are associated with periodontitis in patients with Type 2 diabetes mellitus (T2DM). Volunteers for the study were patients from the Diabetic Unit, University of Malaya Medical Centre, whose periodontal status was determined. The diabetic volunteers were divided into two groups, i.e., patients with periodontitis and those who were periodontally healthy. Saliva samples were collected and treated with 10% TCA/acetone/20 mM DTT to precipitate the proteins, which were then separated using two-dimensional polyacrylamide gel electrophoresis. Gel images were scanned using the GS-800TM Calibrated Densitometer. The protein spots were analyzed and expressed in percentage volumes. The percentage volume of each protein spot was subjected to Mann-Whitney statistical analysis using SPSS software and false discovery rate correction. When the expression of the salivary proteins was compared between the T2DM patients with periodontitis with those who were periodontally healthy, seven proteins, including polymeric immunoglobulin receptor, plastin-2, actin related protein 3, leukocyte elastase inhibitor, carbonic anhydrases 6, immunoglobulin J and interleukin-1 receptor antagonist, were found to be differentially expressed (p < 0.01304). This implies that the proteins may have the potential to be used as biomarkers for the prediction of T2DM patients who may be prone to periodontitis

A Novel Universal Primer-Multiplex-PCR Method with Sequencing Gel Electrophoresis Analysis

In this study, a novel universal primer-multiplex-PCR (UP-M-PCR) method adding a universal primer (UP) in the multiplex PCR reaction system was described. A universal adapter was designed in the 5′-end of each specific primer pairs which matched with the specific DNA sequences for each template and also used as the universal primer (UP). PCR products were analyzed on sequencing gel electrophoresis (SGE) which had the advantage of exhibiting extraordinary resolution. This method overcame the disadvantages rooted deeply in conventional multiplex PCR such as complex manipulation, lower sensitivity, self-inhibition and amplification disparity resulting from different primers, and it got a high specificity and had a low detection limit of 0.1 ng for single kind of crops when screening the presence of genetically modified (GM) crops in mixture samples. The novel developed multiplex PCR assay with sequencing gel electrophoresis analysis will be useful in many fields, such as verifying the GM status of a sample irrespective of the crop and GM trait and so on

Production of a recombinant polyester-cleaving hydrolase from Thermobifida fusca in Escherichia coli

The hydrolase (Thermobifida fusca hydrolase; TfH) from T. fusca was produced in Escherichia coli as fusion protein using the OmpA leader sequence and a His(6) tag. Productivity could be raised more than 100-fold. Both batch and fed-batch cultivations yield comparable cell specific productivities whereas volumetric productivities differ largely. In the fed-batch cultivations final rTfH concentrations of 0.5 g L(−1) could be achieved. In batch cultivations the generated rTfH is translocated to the periplasm wherefrom it is completely released into the extracellular medium. In fed-batch runs most of the produced rTfH remains as soluble protein in the cytoplasm and only a fraction of about 35% is translocated to the periplasm. Migration of periplasmic proteins in the medium is obviously coupled with growth rate and this final transport step possibly plays an important role in product localization and efficacy of the Sec translocation process

Targeting of calcineurin to an NFAT-like docking site is required for the calcium-dependent activation of the background K(+)channel, TRESK

The two-pore domain K+ channel, TRESK (TWIK-related spinal cord K+ channel) is activated in response to the calcium signal by the calcium/calmodulin-dependent protein phosphatase, calcineurin. In the present study we report that calcineurin also interacts with TRESK via an NFAT-like docking site, in addition to its enzymatic action. In its intracellular loop, mouse TRESK possesses the amino acid sequence, PQIVID, which is similar to the calcineurin binding consensus motif, PXIXIT (where X denotes any amino acids), necessary for NFAT (nuclear factor of activated T cells) activation and nuclear translocation. Mutations of the PQIVID sequence of TRESK to PQIVIA, PQIVAD, or PQAVAD increasingly deteriorated the calcium-dependent activation in the listed order and correspondingly reduced the benzocaine sensitivity (a property discriminating activated channels from resting ones), when it was measured after the calcium signal in Xenopus oocytes. Microinjection of VIVIT peptide, designed to inhibit the NFAT-calcineurin interaction specifically, also eliminated TRESK activation. The intracellular loop of TRESK, expressed as a GST fusion protein, bound constitutively active calcineurin in vitro. PQAVAD mutation as well as addition of VIVIT peptide to the reaction abrogated this calcineurin binding. Wild type calcineurin was recruited to GST-TRESK-loop in the presence of calcium and calmodulin. These results indicate that the PQIVID sequence is a docking site for calcineurin, and its occupancy is required for the calcium-dependent regulation of TRESK. Immunosuppressive compounds, developed to target the NFAT binding site of calcineurin, are also expected to interfere with TRESK regulation, in addition to their desired effect on NFAT

Proteomic Analysis of Whole Human Saliva Detects Enhanced Expression of Interleukin-1 Receptor Antagonist, Thioredoxin and Lipocalin-1 in Cigarette Smokers Compared to Non-Smokers

A gel-based proteomics approach was used to screen for proteins of differential abundance between the saliva of smokers and those who had never smoked. Subjecting precipitated proteins from whole human saliva of healthy non-smokers to two-dimensional electrophoresis (2-DE) generated typical profiles comprising more than 50 proteins. While 35 of the proteins were previously established by other researchers, an additional 22 proteins were detected in the 2-DE saliva protein profiles generated in the present study. When the 2-DE profiles were compared to those obtained from subjects considered to be heavy cigarette smokers, three saliva proteins, including interleukin-1 receptor antagonist, thioredoxin and lipocalin-1, showed significant enhanced expression. The distribution patterns of lipocalin-1 isoforms were also different between cigarette smokers and non-smokers. The three saliva proteins have good potential to be used as biomarkers for the adverse effects of smoking and the risk for inflammatory and chronic diseases that are associated with it

Characterization of the Arabidopsis thaliana 2-Cys peroxiredoxin interactome

This document is the Accepted Manuscript of the following article: Delphine Cerveau, et al, ‘Characterization of the Arabidopsis thaliana 2-Cys peroxiredoxin interactome’, Plant Science, Vol. 252, pp. 30-41, July 2016, doi: https://doi.org/10.1016/j.plantsci.2016.07.003. This manuscript version is distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License CC BY NC-ND 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.Peroxiredoxins are ubiquitous thiol-dependent peroxidases for which chaperone and signaling roles havebeen reported in various types of organisms in recent years. In plants, the peroxidase function of thetwo typical plastidial 2-Cys peroxiredoxins (2-Cys PRX A and B) has been highlighted while the otherfunctions, particularly in ROS-dependent signaling pathways, are still elusive notably due to the lack ofknowledge of interacting partners. Using an ex vivo approach based on co-immunoprecipitation of leafextracts from Arabidopsis thaliana wild-type and mutant plants lacking 2-Cys PRX expression followedby mass spectrometry-based proteomics, 158 proteins were found associated with 2-Cys PRXs. Alreadyknown partners like thioredoxin-related electron donors (Chloroplastic Drought-induced Stress Proteinof 32 kDa, Atypical Cysteine Histidine-rich Thioredoxin 2) and enzymes involved in chlorophyll synthe-sis (Protochlorophyllide OxidoReductase B) or carbon metabolism (Fructose-1,6-BisPhosphatase) wereidentified, validating the relevance of the approach. Bioinformatic and bibliographic analyses allowedthe functional classification of the identified proteins and revealed that more than 40% are localized inplastids. The possible roles of plant 2-Cys PRXs in redox signaling pathways are discussed in relation withthe functions of the potential partners notably those involved in redox homeostasis, carbon and aminoacid metabolisms as well as chlorophyll biosynthesis.Peer reviewe

Assessment of Virally Vectored Autoimmunity as a Biocontrol Strategy for Cane Toads

BACKGROUND: The cane toad, Bufo (Chaunus) marinus, is one of the most notorious vertebrate pests introduced into Australia over the last 200 years and, so far, efforts to identify a naturally occurring B. marinus-specific pathogen for use as a biological control agent have been unsuccessful. We explored an alternative approach that entailed genetically modifying a pathogen with broad host specificity so that it no longer caused disease, but carried a gene to disrupt the cane toad life cycle in a species specific manner. METHODOLOGY/PRINCIPAL FINDINGS: The adult beta globin gene was selected as the model gene for proof of concept of autoimmunity as a biocontrol method for cane toads. A previous report showed injection of bullfrog tadpoles with adult beta globin resulted in an alteration in the form of beta globin expressed in metamorphs as well as reduced survival. In B. marinus we established for the first time that the switch from tadpole to adult globin exists. The effect of injecting B. marinus tadpoles with purified recombinant adult globin protein was then assessed using behavioural (swim speed in tadpoles and jump length in metamorphs), developmental (time to metamorphosis, weight and length at various developmental stages, protein profile of adult globin) and genetic (adult globin mRNA levels) measures. However, we were unable to detect any differences between treated and control animals. Further, globin delivery using Bohle iridovirus, an Australian ranavirus isolate belonging to the Iridovirus family, did not reduce the survival of metamorphs or alter the form of beta globin expressed in metamorphs. CONCLUSIONS/SIGNIFICANCE: While we were able to show for the first time that the switch from tadpole to adult globin does occur in B. marinus, we were not able to induce autoimmunity and disrupt metamorphosis. The short development time of B. marinus tadpoles may preclude this approach