Focused Proteomics Analysis of Habu Snake (Protobothrops flavoviridis) Venom Using Antivenom-Based Affinity Chromatography Reveals Novel Myonecrosis-Enhancing Activity of Thrombin-Like Serine Proteases

Snakebites are one of the major causes of death and long-term disability in the developing countries due to the presence of various bioactive peptides and proteins in snake venom. In Japan, the venom of the habu snake (Protobothrops flavoviridis) causes severe permanent damage due to its myonecrotic toxins. Antivenom immunoglobulins are an effective therapy for snakebites, and antivenom was recently developed with effective suppressive activity against myonecrosis induced by snake venom. To compare the properties of an antivenom having anti-myonecrotic activity with those of conventional antivenom with no anti-myonecrotic activity, this study applied focused proteomics analysis of habu venom proteins using 2D gel electrophoresis. As a target protein for antivenom immunoglobulins with anti-myonecrotic activity, we identified a thrombin-like serine protease, TLSP2 (TLf2), which was an inactive proteolytic isoform due to the replacement of the active site, His43 with Arg. Additionally, we identified the unique properties and a novel synergistic function of pseudoenzyme TLf2 as a myonecrosis-enhancing factor. To our knowledge, this is the first report of a function of a catalytically inactive snake serine protease.

Changes in Porcine Corpus Luteum Proteome Associated with Development, Maintenance, Regression, and Rescue during Estrous Cycle and Early Pregnancy

Corpus luteum (CL), a transitory gland, undergoes rapid growth in a limited time to produce progesterone (P4) followed by its regression. A complex molecular signaling is involved in controlling luteal P4 production. In the present study, 2D gel electrophoresis-based proteomics and in silico functional analysis were used to identify changes in key proteins and pathways in CL along the different stages of the estrous cycle as its development progresses from early (Day 3) to mid-luteal phase (Day 9), effective functioning (Day 12) followed by regression (Day 15) or, in the case of pregnancy, rescue of function (Day 15). A total of 273 proteins were identified by MALDI-MS/MS analysis that showed significant changes in abundances at different stages of CL development or regression and rescue. Functional annotation of differentially abundant proteins suggested enrichment of several important pathways and functions during CL development and function maintenance including cell survival, endocytosis, oxidative stress response, estradiol metabolism, and angiogenesis. On the other hand, differentially abundant proteins during CL regression were associated with decreased steroid synthesis and metabolism and increased apoptosis, necrosis, and infiltration of immune cells. Establishment of pregnancy rescues CL from regression by maintaining the expression of proteins that support steroidogenesis as pathways such as the super-pathway of cholesterol biosynthesis, RhoA signaling, and functions such as fatty acid metabolism and sterol transport were enriched in CL of pregnancy. In this study, some novel proteins were identified along CL development that advances our understanding of CL survival and steroidogenesis.

Polymicrobial Synergy Stimulates Porphyromonas Gingivalis Survival And Gingipain Expression In A Multispecies Subgingival Community

Background Dysbiosis in subgingival microbial communities, resulting from increased inflammatory transudate from the gingival tissues, is an important factor in initiation and development of periodontitis. Dysbiotic communities are characterized by increased numbers of bacteria that exploit the serum-like transudate for nutrients, giving rise to a proteolytic community phenotype. Here we investigate the contribution of interactions between members of a sub-gingival community to survival and development of virulence in a serum environment - modelling that in the subgingival pocket. Methods Growth and proteolytic activity of three P. gingivalis strains in nutrient-rich broth or a serum environment were assessed using A600 and a fluorescent protease substrate, respectively. Adherence of P. gingivalis strains to serum-coated surfaces was studied with confocal microscopy and 2D-gel electrophoresis of bacterial supernatants used to investigate extracellular proteins. A model multi-species sub-gingival community containing Fusobacterium nucleatum, Streptococcus constellatus, Parvimonas micra with wild type or isogenic mutants was then created and growth and proteolytic activity in serum assessed as above. Community composition over time was monitored using culture techniques and qPCR. Results The P. gingivalis strains showed different growth rates in nutrient-rich broth related to the level of proteolytic activity (largely gingipains) in the cultures. Despite being able to adhere to serum-coated surfaces, none of the strains was able to grow alone in a serum environment. In the subgingival consortium however, all the included species were able to grow in the serum environment and the community adopted a proteolytic phenotype. Inclusion of P. gingivalis strains lacking gingipains in the consortium revealed that the ability of the community to grow was largely due to Rgp gingipain. Conclusions In the multi-species consortium, growth was facilitated by the wild-type and Rgp-expressing strains of P. gingivalis, suggesting that Rgp is involved in delivery of nutrients to the whole community through degradation of complex serum substrates. Whereas they are constitutively expressed by P. gingivalis in nutrient-rich broth, gingipain expression in the model periodontal pocket environment (serum) appears to be orchestrated through signaling to P. gingivalis from other members of the community, a phenomenon which can then promote growth of the whole community.

A Proteomics Approach to Identify Possible Biomarkers of Early and Late Stages of E. coli-induced Urinary Tract Infections

As one of the most common bacterial infections globally, urinary tract infections (UTI)s affect the bladder and kidneys of many the bladders and kidneys of many. Along with gram-negative bacteria, Escherichia coli (E. coli) causes nearly 40% of nosocomial UTIs, 25% of recurrent infections, and between 80 to 90% of community-acquired infections. Proteomics, commonly used to study changes in protein expression of organisms, can be used to explore candidate biomarkers useful for the diagnosis of pathological conditions. Here, protein profiles of samples from patients diagnosed with E. coli-induced UTI were compared to identify distinctive proteins. Extracted proteins from bacteria from patients’ urine samples were separated into excisable spots using 2D-gel electrophoresis. The gels were then analyzed using Progenesis SameSpot software to select uniquely expressed protein spots, excised, and analyzed by LC/MS. The results were then compared against a database of known proteins. We identified two proteins, outer membrane protein A (OmpA) and RNA polymerase-binding transcription factor (DksA), involved in the survival of E. coli in the harsh environment of the host. We suggest their use as a part of a battery of possible biomarkers proteins for E. coli-induced UTI, and suggest that their overexpression is possibly associated with the stage of infection, early or late.

Purification and Characterization of Antibodies Directed against the α-Gal Epitope

The α-Gal epitope is an immunogen trisaccharide structure consisting of N-acetylglucosamine (GlcNAc)β1,4-galactose (Gal)α1,3-Gal. It is presented as part of complex-type glycans on glycoproteins or glycolipids on cell surfaces of non-primate mammalians. About 1% of all antibodies in human sera are specific toward α1,3-Gal and are therefore named as anti-α-Gal antibodies. This work comprises the purification and characterization of anti-α-Gal antibodies from human immunoglobulin G (IgG). A synthetically manufactured α Gal epitope affinity resin was used to enrich anti-α-Gal antibodies. Selectivity experiments with purified antibodies were carried out using enzyme-linked immunosorbent assays (ELISA), Western blotting, and erythrocyte agglutination. Furthermore, binding affinities toward α-Gal were determined by surface plasmon resonance (SPR) and the IgG distribution of anti α Gal antibodies (83% IgG2, 14% IgG1, 2% IgG3, 1% IgG4) was calculated applying ELISA and immunodiffusion. A range of isoelectric points from pH 6 to pH 8 was observed in 2D gel electrophoresis. Glycan profiling of anti α Gal antibodies revealed complex biantennary structures with high fucosylation grades (86%). Additionally, low amounts of bisecting GlcNAc (15%) and sialic acids (13%) were detected. The purification of anti-α-Gal antibodies from human IgG was successful, and their use as detection antibodies for α Gal-containing structures was evaluated.

Translational Proteomics for Transfusion Medicine: Resolution of the IVIG Proteomes of Different Geographically Sourced and Prepared IVIG Immunotherapies

Intravenous Immunoglobulin’s (IVIG’s) are prepared from thousands of donor plasmas and as a result comprise an extreme broad mix and depth of Antibody (Ab) specificities. IVIG formulations available in Australia are from both local and overseas donor sources and extracted using a variety of purification methods and immunoglobulin purities, with the Australia-derived and prepared IVIG listed at >98% and the overseas-derived preparations at ≥ 95%. Because of these differences it was predicted that the formulations might individually vary in composition and that together with obvious genetic and geographic antigenic (Ag) environment differences may result in notable variability between formulations. Hence a focussed comparative proteomic profiling of IVIG formulations was undertaken to identify notable similarities and differences across products. Comparisons between formulations did reveal marked qualitative differences in 2D-gel Antibody profiling that included parameters of isoelectric charge (pI), as well as immunoglobulin (Ig) monomer to dimer ratio variability between products, including high molecular weight (MW) immunoglobulin multimers for some. These notable differences were in part quite likely a product of the respective purification methods used, and capacity to select (or de-select) for antibodies of such different properties. Furthermore, for identification of non-Ig proteins carried over from plasma through purifications Mass spectrometry was performed. This identified a few such ancillary proteins, and their identities, in general, differed between formulations. Proteins detected included the most abundant protein of plasma, albumin, as well as other mostly large and abundant proteins; RAG1 - V(D)J recombination activating protein1, gelsolin, complement Factor-B, serotransferrin, tetranectin, NADH ubiquinone oxidoreductase, caspase 3 and VEGFR1. An alternate strategy used commercial Multiplex xMAP assay to detect cytokines, which are small and present in plasma at trace but highly active quantities. This revealed various different cytokine profiles across the formulations studied. The identification of additional proteins, and especially cytokines in IVIGs, is particularly notable, and the positive, negative or null biological relevance for clinical use, needs resolution. Collectively these findings reveal marked differences between Australian and overseas-derived (non-Australian) IVIGs in immunoglobulin composition and biochemical characteristics, and presence of additional carry-over proteins from plasma. These findings prompt the need for further evaluation of the micro-compositions of individual formulations. Perhaps detailed mining and improved comparative understanding of each IVIG formulation, may enable highly tailored and strategic clinical use of certain formulations that are personalised best-fit treatments for particular conditions. Such as in treatment of a neuropathy, as compared to another formulation, more suited for treating a particular infectious disease. The most salient and overarching study conclusion is need for caution in attributing equivalence across IVIGs.

Morphological and Proteomic Evaluation of Zea Mays in Response to Osmotic Stress

Introduction: Drought is the main abiotic stress responsible for crop loss worldwide. Maize (Zea mays L.) is a widely grown drought-sensitive crop used as a staple food by the growing population. Therefore, it is imperative to assess the molecular mechanisms behind drought response and tolerance in maize. Transcriptomic profiling of abiotic stress responsive pathways in various crops appeared to be an unreliable approach due to post-transcriptional modifications, while there is limited published data on molecular mechanisms of osmotic-stress response in maize. Hence our study aimed at profiling osmotic stress responsive proteins augmented by their associated morphological features in Z. mays. Materials and Methods: In this regard, morphological and proteomic investigations were carried out on 16-day maize seedlings exposed to 5% (w/v) and 10% (w/v) polyethylene glycol(PEG) to induce osmotic-stress. Proteomics approach (one-dimensional (1D) and two-dimensional (2D) gel electrophoresis) compared differential protein abundance between controls and the osmotic stressed maize plants. Results: Morphological parameters such as plant growth, height, shoot diameter, leaf area, and colour were highly affected with PEG treatment as compared to the untreated ones. Molecular evaluation by 1D gel electrophoresis revealed that the separated protein patterns were highly expressed in the experiments than the controls. Using 2D gel electrophoresis, a total of seven and eight protein spots were revealed in experimental plants under 5% (w/v) and 10% (w/v) PEG treatment respectively while the control plants only expressed one protein. Increased drought stress resulted in a greater number of proteins with differential abundance. Conclusion: This study has successfully profiled the total osmotic stress responsive proteins and revealed the efficiency of proteomic tools in the qualitative detection of differential proteins from maize.

The Role of Subinhibitory Concentrations of Daptomycin and Tigecycline in Modulating Virulence in Staphylococcus aureus

Staphylococcus aureus (S. aureus) infections are notoriously complicated by the ability of the organism to grow in biofilms and are difficult to eradicate with antimicrobial therapy. The purpose of the current study was to clarify the influence of sub-inhibitory concentrations (sub-MICs) of daptomycin and tigecycline antibiotics on biofilm adhesion factors and exoproteins expressions by S. aureus clinical isolates. Six clinical isolates representing positive biofilm S. aureus clones (3 methicillin-sensitive S. aureus (MSSA) and 3 methicillin-resistant S. aureus (MRSA)) were grown with sub-MICs (0.5 MIC) of two antibiotics (daptomycin and tigecycline) for 12 h of incubation. RNA extracted from culture pellets was used via relative quantitative real-time-PCR (qRT-PCR) to determine expression of specific adhesion (fnbA, fnbB, clfA, clfB, fib, ebps, cna, eno) and biofilm (icaADBC) genes. To examine the effect of sub-MIC of these antibiotics on the expression of extracellular proteins, samples from the culture supernatants of six isolates were collected after 12 h of treatment with or without tigecycline in order to profile protein production via 2D gel sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D gel-SDS-PAGE). Sub-MIC treatment of all clinical MRSA and MSSA strains with daptomycin or tigecycline dramatically induced or suppressed fnbA, fnbB, clfA, clfB, fib, ebps, cna, eno, and icaADBC gene expression. Furthermore, sub-MIC use of tigecycline significantly reduced the total number of separated protein spots across all the isolates, as well as decreasing production of certain individual proteins. Collectively, this study showed very different responses in terms of both gene expression and protein secretion across the various isolates. In addition, our results suggest that sub-MIC usage of daptomycin and tigecycline could signal virulence induction by S. aureus via the regulation of biofilm adhesion factor genes and exoproteins. If translating findings to the clinical treatment of S. aureus, the therapeutic regimen should be adapted depending on antibiotic, the virulence factor and strain type.

chGRP78 is Required for the Activation and Phosphorylation of AKT1 at Serine 478 in Chicken Sells

Background: Chicken serum-mediated proliferation regulates chGRP78 to prevent apoptosis in chicken cells via chGRP78-mediated anti-apoptosis. However, the precise molecular mechanisms underlying the chGRP78-mediated protection against apoptosis remain undefined. In an earlier study, we have shown that chGRP78 is critical for chicken embryo fibroblast (CEF) and DF-1 cell proliferation.Methods: In this experiment, we highlight AKT1 as a key target of GRP78 during apoptosis. We used 2D gel-based proteomics and bioinformatics prediction analysis for our studies. Result: Here, we detected chGRP78 binding sites in AKT1-rgulated proteins. chGRP78 promoted AKT1 activation and chGRP78 silencing decreased AKT1 levels. Taken together, we suggest that the AKT1-mediated signaling pathway plays a critical role in GRP78-stimulated fibroblast survival and anti-apoptosis. Our findings have important implications for the maintenance of chicken fibroblast cells via the inhibition of apoptosis.