Effect of rocket (Eruca sativa) extract on MRSA growth and proteome: Metabolic adjustments in plant-based media

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) in food has provoked a great concern about the presence of MRSA in associated foodstuff. Although MRSA is often detected in various retailed meat products, it seems that food handlers are more strongly associated with this type of food contamination. Thus, it can be easily postulated that any food could be contaminated with this pathogen in an industrial environment or in household and cause food poisoning. To this direction, the effect of rocket (Eruca sativa) extract on MRSA growth and proteome was examined in the present study. This goal was achieved with the comparative study of the MRSA strain COL proteome, cultivated in rocket extract versus the standard Luria-Bertani growth medium. The obtained results showed that MRSA was able to grow in rocket extract. In addition, proteome analysis using 2-DE method showed that MRSA strain COL is taking advantage of the sugar-, lipid-, and vitamin-rich substrate in the liquid rocket extract, although its growth was delayed in rocket extract compared to Luria-Bertani medium. This work could initiate further research about bacterial metabolism in plant-based media and defense mechanisms against plant-derived antibacterials

Proteomics-based confirmation of protein expression and correction of annotation errors in the Brucella abortus genome

<p>Abstract</p> <p>Background</p> <p>Brucellosis is a major bacterial zoonosis affecting domestic livestock and wild mammals, as well as humans around the globe. While conducting proteomics studies to better understand <it>Brucella abortus </it>virulence, we consolidated the proteomic data collected and compared it to publically available genomic data.</p> <p>Results</p> <p>The proteomic data was compiled from several independent comparative studies of <it>Brucella abortus </it>that used either outer membrane blebs, cytosols, or whole bacteria grown in media, as well as intracellular bacteria recovered at different times following macrophage infection. We identified a total of 621 bacterial proteins that were differentially expressed in a condition-specific manner. For 305 of these proteins we provide the first experimental evidence of their expression. Using a custom-built protein sequence database, we uncovered 7 annotation errors. We provide experimental evidence of expression of 5 genes that were originally annotated as non-expressed pseudogenes, as well as start site annotation errors for 2 other genes.</p> <p>Conclusions</p> <p>An essential element for ensuring correct functional studies is the correspondence between reported genome sequences and subsequent proteomics studies. In this study, we have used proteomics evidence to confirm expression of multiple proteins previously considered to be putative, as well as correct annotation errors in the genome of <it>Brucella abortus </it>strain 2308.</p

Differential attenuation of Marek's disease virus-induced tumours and late-Marek's disease virus-induced immunosuppression

Marek's disease virus (MDV) is a herpesvirus that induces lymphoma and a variety of non-neoplastic syndromes in chickens. Furthermore, very virulent plus (vv+) MDVs induce a form of immunosuppression (late-MDV-IS) that might involve both neoplastic and non-neoplastic mechanisms. The objective of this study was to evaluate whether the attenuation of MDV-induced tumours and late-MDV-IS occurs simultaneously or can be dissociated. The immunosuppressive ability of three viruses derived from vv+ MDV strain 686 (wild-type 686, the somewhat attenuated molecular clone 686-BAC, and the nononcogenic molecular clone lacking the two copies of the oncogene meq 686-BACΔMEQ) was evaluated. Late-MDV-IS was evaluated indirectly by assessing the negative effect of MDV strains on the protection conferred by infectious laryngotracheitis (ILT) vaccines. Our results showed that the ability to induce late-MDV-IS was attenuated before the ability to induce tumours. Strain 686 induced both tumours and late-MDV-IS, 686-BAC induced tumours but did not induce late-MDV-IS and 686-BACΔMEQ did not induce either tumours or late-MDV-IS. Further comparison of strains 686 and 686-BAC revealed that strain 686 reduced the humoral immune responses to ILTV (1132 vs 2167) more severely, showed higher levels of meq transcripts (2.1E+09 vs 4.98E+8) and higher expression of MDV microRNAs (mdv1-miR-M4-5p and mdv1-miR-M2-3p) in the spleen, and further reduced the percentage of CD45+-MHC-I+splenocytes (13 vs32 %) compared to molecular clone 686-BAC. This study suggests that the immunosuppressive ability of MDV might follow a continuous spectrum and only the most virulent MDVs can overcome a certain threshold level and induce clinical MDV-IS in the ILT model

Validation of ELISA-based detection of L. monocytogenes and E. coli O157:H7 in fresh cut vegetables

Innovative diagnostic methods were developed for the detection and quantification of Listeria monocytogenes and Escherichia coli O157:H7 in minimally processed fresh cut fruits and vegetables. The aim of the present study was to validate the technical efficiency of these methods and evaluate their efficacy and viability for routine analysis. To this purpose, ready-to-eat fresh fruits and vegetables were collected throughout the production chain. A multidisciplinary approach, including a newly developed ELISA method compared to ISO procedures, was applied to detect the pathogenic bacteria after harvesting, processing and shelf-life. Results obtained exhibited the technical efficiency of the developed methods showing similar sensitivity, specificity, negative predictive values and negative likelihood ratios

Immunopeptidomic Data Integration to Artificial Neural Networks Enhances Protein-Drug Immunogenicity Prediction

Recombinant DNA technology has, in the last decades, contributed to a vast expansion of the use of protein drugs as pharmaceutical agents. However, such biological drugs can lead to the formation of anti-drug antibodies (ADAs) that may result in adverse effects, including allergic reactions and compromised therapeutic efficacy. Production of ADAs is most often associated with activation of CD4 T cell responses resulting from proteolysis of the biotherapeutic and loading of drug-specific peptides into major histocompatibility complex (MHC) class II on professional antigen-presenting cells. Recently, readouts from MHC-associated peptide proteomics (MAPPs) assays have been shown to correlate with the presence of CD4 T cell epitopes. However, the limited sensitivity of MAPPs challenges its use as an immunogenicity biomarker. In this work, MAPPs data was used to construct an artificial neural network (ANN) model for MHC class II antigen presentation. Using Infliximab and Rituximab as showcase stories, the model demonstrated an unprecedented performance for predicting MAPPs and CD4 T cell epitopes in the context of protein-drug immunogenicity, complementing results from MAPPs assays and outperforming conventional prediction models trained on binding affinity data.Fil: Barra, Carolina. Technical University of Denmark; DinamarcaFil: Ackaert, Chloe. No especifíca;Fil: Reynisson, Birkir. Technical University of Denmark; DinamarcaFil: Schockaert, Jana. No especifíca;Fil: Jessen, Leon Eyrich. Technical University of Denmark; DinamarcaFil: Watson, Mark. No especifíca;Fil: Jang, Anne. No especifíca;Fil: Comtois Marotte, Simon. No especifíca;Fil: Goulet, Jean Philippe. No especifíca;Fil: Pattijn, Sofie. No especifíca;Fil: Paramithiotis, Eustache. No especifíca;Fil: Nielsen, Morten. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentin

Synthetic prions generated in vitro are similar to a newly identified subpopulation of PrPSc from sporadic Creutzfeldt-Jakob disease

In recent studies, the amyloid form of recombinant prion protein (PrP) encompassing residues 89–230 (rPrP 89-230) produced in vitro induced transmissible prion disease in mice. These studies showed that unlike “classical” PrPSc produced in vivo, the amyloid fibrils generated in vitro were more proteinase-K sensitive. Here we demonstrate that the amyloid form contains a proteinase K-resistant core composed only of residues 152/153–230 and 162–230. The PK-resistant fragments of the amyloid form are similar to those observed upon PK digestion of a minor subpopulation of PrPSc recently identified in patients with sporadic Creutzfeldt-Jakob disease (CJD). Remarkably, this core is sufficient for self-propagating activity in vitro and preserves a β-sheet-rich fibrillar structure. Full-length recombinant PrP 23-230, however, generates two subpopulations of amyloid in vitro: One is similar to the minor subpopulation of PrPSc, and the other to classical PrPSc. Since no cellular factors or templates were used for generation of the amyloid fibrils in vitro, we speculate that formation of the subpopulation of PrPSc with a short PK-resistant C-terminal region reflects an intrinsic property of PrP rather than the influence of cellular environments and/or cofactors. Our work significantly increases our understanding of the biochemical nature of prion infectious agents and provides a fundamental insight into the mechanisms of prions biogenesis

Detection of Listeria monocytogenes in cut melon fruit using analysis of volatile organic compounds

Ready-to-eat fresh cut fruits and vegetables are increasingly popular, however due to their minimal processing there is a risk of contamination with human pathogens. Listeria monocytogenes is of particular concern as it can multiply even at the low temperatures used to store fresh cut products pre-sale. Current detection methods rely on culturing, which is time consuming and does not provide results in the time frame required. Growth of bacteria on a substrate alters its chemical composition affecting the profile of volatile organic compounds (VOCs) emitted. Use of VOCs as a detection method has been hampered by lack of sensitivity and robust sample collection methods. Here we use thermal desorption gas chromatography time of flight mass spectrometry (TD-GC-TOF-MS) followed by analysis with PerMANOVA to analyse VOC profiles. We can discriminate between fresh cut melon cubes inoculated with 6 log CFU/g of L. monocytogenes and uninoculated controls, as well as melon cubes inoculated with <1 log CFU/g of L. monocytogenes stored for 7 days at 4 °C and following equilibration for 6 h at 37 °C. This is a substantial advance in sensitivity compared to previous studies and additionally the collection method used allows remote sampling and transport of the VOCs, greatly facilitating analyses

Host Protein Biomarkers Identify Active Tuberculosis in HIV Uninfected and Co-infected Individuals

AbstractBiomarkers for active tuberculosis (TB) are urgently needed to improve rapid TB diagnosis. The objective of this study was to identify serum protein expression changes associated with TB but not latent Mycobacterium tuberculosis infection (LTBI), uninfected states, or respiratory diseases other than TB (ORD). Serum samples from 209 HIV uninfected (HIV−) and co-infected (HIV+) individuals were studied. In the discovery phase samples were analyzed via liquid chromatography and mass spectrometry, and in the verification phase biologically independent samples were analyzed via a multiplex multiple reaction monitoring mass spectrometry (MRM-MS) assay. Compared to LTBI and ORD, host proteins were significantly differentially expressed in TB, and involved in the immune response, tissue repair, and lipid metabolism. Biomarker panels whose composition differed according to HIV status, and consisted of 8 host proteins in HIV− individuals (CD14, SEPP1, SELL, TNXB, LUM, PEPD, QSOX1, COMP, APOC1), or 10 host proteins in HIV+ individuals (CD14, SEPP1, PGLYRP2, PFN1, VASN, CPN2, TAGLN2, IGFBP6), respectively, distinguished TB from ORD with excellent accuracy (AUC=0.96 for HIV− TB, 0.95 for HIV+ TB). These results warrant validation in larger studies but provide promise that host protein biomarkers could be the basis for a rapid, blood-based test for TB

PrP(Sc)-specific antibodies with the ability to immunodetect prion oligomers.

The development of antibodies with binding capacity towards soluble oligomeric forms of PrPSc recognised in the aggregation process in early stage of the disease would be of paramount importance in diagnosing prion diseases before extensive neuropathology has ensued. As blood transfusion appears to be efficient in the transmission of the infectious prion agent, there is an urgent need to develop reagents that would specifically recognize oligomeric forms of the abnormally folded prion protein, PrPSc.To that end, we show that anti-PrP monoclonal antibodies (called PRIOC mAbs) derived from mice immunised with native PrP-coated microbeads are able to immunodetect oligomers/multimers of PrPSc. Oligomer-specific immunoreactivity displayed by these PRIOC mAbs was demonstrated as large aggregates of immunoreactive deposits in prion-permissive neuroblastoma cell lines but not in equivalent non-infected or prn-p(0/0) cell lines. In contrast, an anti-monomer PrP antibody displayed diffuse immunoreactivity restricted to the cell membrane. Furthermore, our PRIOC mAbs did not display any binding with monomeric recombinant and cellular prion proteins but strongly detected PrPSc oligomers as shown by a newly developed sensitive and specific ELISA. Finally, PrioC antibodies were also able to bind soluble oligomers formed of Aβ and α-synuclein. These findings demonstrate the potential use of anti-prion antibodies that bind PrPSc oligomers, recognised in early stage of the disease, for the diagnosis of prion diseases in blood and other body fluids