Genomic and proteomic analyses of Mycobacterium bovis BCG Mexico 1931 reveal a diverse immunogenic repertoire against tuberculosis infection

<p>Abstract</p> <p>Background</p> <p>Studies of <it>Mycobacterium bovis </it>BCG strains used in different countries and vaccination programs show clear variations in the genomes and immune protective properties of BCG strains. The aim of this study was to characterise the genomic and immune proteomic profile of the BCG 1931 strain used in Mexico.</p> <p>Results</p> <p>BCG Mexico 1931 has a circular chromosome of 4,350,386 bp with a G+C content and numbers of genes and pseudogenes similar to those of BCG Tokyo and BCG Pasteur. BCG Mexico 1931 lacks Region of Difference 1 (RD1), RD2 and N-RD18 and one copy of IS6110, indicating that BCG Mexico 1931 belongs to DU2 group IV within the BCG vaccine genealogy. In addition, this strain contains three new RDs, which are 53 (RDMex01), 655 (RDMex02) and 2,847 bp (REDMex03) long, and 55 single-nucleotide polymorphisms representing non-synonymous mutations compared to BCG Pasteur and BCG Tokyo. In a comparative proteomic analysis, the BCG Mexico 1931, Danish, Phipps and Tokyo strains showed 812, 794, 791 and 701 protein spots, respectively. The same analysis showed that BCG Mexico 1931 shares 62% of its protein spots with the BCG Danish strain, 61% with the BCG Phipps strain and only 48% with the BCG Tokyo strain. Thirty-nine reactive spots were detected in BCG Mexico 1931 using sera from subjects with active tuberculosis infections and positive tuberculin skin tests.</p> <p>Conclusions</p> <p>BCG Mexico 1931 has a smaller genome than the BCG Pasteur and BCG Tokyo strains. Two specific deletions in BCG Mexico 1931 are described (RDMex02 and RDMex03). The loss of RDMex02 (<it>fadD23</it>) is associated with enhanced macrophage binding and RDMex03 contains genes that may be involved in regulatory pathways. We also describe new antigenic proteins for the first time.</p

Plasma proteomic analysis reveals altered protein abundances in cardiovascular disease

Background: Cardiovascular disease (CVD) describes the pathological conditions of the heart and blood vessels. Despite the large number of studies on CVD and its etiology, its key modulators remain largely unknown. To this end, we performed a comprehensive proteomic analysis of blood plasma, with the scope to identify disease-associated changes after placing them in the context of existing knowledge, and generate a well characterized dataset for further use in CVD multi-omics integrative analysis. Methods: LC–MS/MS was employed to analyze plasma from 32 subjects (19 cases of various CVD phenotypes and 13 controls) in two steps: discovery (13 cases and 8 controls) and test (6 cases and 5 controls) set analysis. Following label-free quantification, the detected proteins were correlated to existing plasma proteomics datasets (plasma proteome database; PPD) and functionally annotated (Cytoscape, Ingenuity Pathway Analysis). Differential expression was defined based on identification confidence (≥ 2 peptides per protein), statistical significance (Mann–Whitney p value ≤ 0.05) and a minimum of twofold change. Results: Peptides detected in at least 50% of samples per group were considered, resulting in a total of 3796 identified proteins (838 proteins based on ≥ 2 peptides). Pathway annotation confirmed the functional relevance of the findings (representation of complement cascade, fibrin clot formation, platelet degranulation, etc.). Correlation of the relative abundance of the proteins identified in the discovery set with their reported concentrations in the PPD was significant, confirming the validity of the quantification method. The discovery set analysis revealed 100 differentially expressed proteins between cases and controls, 39 of which were verified (≥ twofold change) in the test set. These included proteins already studied in the context of CVD (such as apolipoprotein B, alpha-2-macroglobulin), as well as novel findings (such as low density lipoprotein receptor related protein 2 [LRP2], protein SZT2) for which a mechanism of action is suggested. Conclusions: This proteomic study provides a comprehensive dataset to be used for integrative and functional studies in the field. The observed protein changes reflect known CVD-related processes (e.g. lipid uptake, inflammation) but also novel hypotheses for further investigation including a potential pleiotropic role of LPR2 but also links of SZT2 to CVD

A study of salt tolerance in Arabidopsis thaliana and Hordeum vulgare

ABSTRACT The original objective of this work was to compare the cellular processes in salt tolerant and salt sensitive plants cells to gain insight into the mechanisms that confer halotolerance. Halotolerant and salt sensitive cell lines were derived from the model glycophyte Arabidopsis thaliana; in addition cell suspension cultures from the dicot halophytes Beta vulgaris and Atriplex halimus were also generated. Unfortunately, severe disruptions were encountered following a serious fire; persistent power failures, and failures of new equipment hampered progress with this work. For this reason, only comparisons between the Arabidopsis cell lines were completed. The halotolerant (HHS) cell lines survival strategy is to prevent Na accumulation when grown in < 100 mM NaCl. Wild type (WT) cells grow faster than HHS cells in the absence of NaCl, but rapidly take up Na in 50 mM NaCl where their growth is severely affected, and fail to grow completely above 100 mM NaCl. No evidence was found to suggest this growth impairment arose from osmotic stress or nutrient ion deficiencies. Protein profiling of HHS cells identified a number of proteins whose abundance is regulated by salt stress. These included proteins involved in ion transport, central metabolism, and general stress responses. The implications of these findings are discussed. In a separate project, a whole plant approach was taken to establish the physiological mechanisms that account for the reported difference in halotolerance between two commercially grown barley lines originating from China. Measurements on growth and development, plant water status, tissue ion profiling, photosynthesis rates, and transpiration rates suggested the tolerant line (Zhou 1) enters the reproductive phase of its life cycle approximately one week earlier than the sensitive line (Zhou 85), and this critical period allows floral development resulting in improved yields. This early ii flowering is not associated with the well characterized PpD-H1 locus controlling early flowering in cereals. The main conclusion from this study is that for glycophytes that do not complete a full life cycle above 100 mM NaCl (which includes all of the world’s major crops), it is the ionic component of salinity stress that impairs growth and yield. Further research on salinity stress in crops should focus on understanding the processes that control ionic balance rather than osmoregulation

Combined immunodeficiency and hypoglycemia associated with mutations in hypoxia upregulated 1

Non peer reviewe

Enhanced whole genome sequence and annotation of Clostridium stercorarium DSM8532T using RNA-seq transcriptomics and high-throughput proteomics

BACKGROUND: Growing interest in cellulolytic clostridia with potential for consolidated biofuels production is mitigated by low conversion of raw substrates to desired end products. Strategies to improve conversion are likely to benefit from emerging techniques to define molecular systems biology of these organisms. Clostridium stercorarium DSM8532(T) is an anaerobic thermophile with demonstrated high ethanol production on cellulose and hemicellulose. Although several lignocellulolytic enzymes in this organism have been well-characterized, details concerning carbohydrate transporters and central metabolism have not been described. Therefore, the goal of this study is to define an improved whole genome sequence (WGS) for this organism using in-depth molecular profiling by RNA-seq transcriptomics and tandem mass spectrometry-based proteomics. RESULTS: A paired-end Roche/454 WGS assembly was closed through application of an in silico algorithm designed to resolve repetitive sequence regions, resulting in a circular replicon with one gap and a region of 2 kilobases with 10 ambiguous bases. RNA-seq transcriptomics resulted in nearly complete coverage of the genome, identifying errors in homopolymer length attributable to 454 sequencing. Peptide sequences resulting from high-throughput tandem mass spectrometry of trypsin-digested protein extracts were mapped to 1,755 annotated proteins (68% of all protein-coding regions). Proteogenomic analysis confirmed the quality of annotation and improvement pipelines, identifying a missing gene and an alternative reading frame. Peptide coverage of genes hypothetically involved in substrate hydrolysis, transport and utilization confirmed multiple pathways for glycolysis, pyruvate conversion and recycling of intermediates. No sequences homologous to transaldolase, a central enzyme in the pentose phosphate pathway, were observed by any method, despite demonstrated growth of this organism on xylose and xylan hemicellulose. CONCLUSIONS: Complementary omics techniques confirm the quality of genome sequence assembly, annotation and error-reporting. Nearly complete genome coverage by RNA-seq likely indicates background DNA in RNA extracts, however these preps resulted in WGS enhancement and transcriptome profiling in a single Illumina run. No detection of transaldolase by any method despite xylose utilization by this organism indicates an alternative pathway for sedoheptulose-7-phosphate degradation. This report combines next-generation omics techniques to elucidate previously undefined features of substrate transport and central metabolism for this organism and its potential for consolidated biofuels production from lignocellulose. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1471-2164-15-567) contains supplementary material, which is available to authorized users

Comprehensive LC-MS/MS analysis of the saliva metabolome in association to oral health status: a population-based study

Periodontitis is one of the most prevalent oral diseases worldwide caused by multifactorial interactions between host and oral bacteria. Altered cellular metabolism of host and microbes releases a number of intermediary end-products known as metabolites. Recently, there is an increasing interest in identifying metabolites from oral fluids like saliva to widen the understanding of the complex pathogenesis of periodontitis. It is believed, that some metabolites might serve as indicators toward early detection and screening of periodontitis and perhaps even for monitoring its prognosis in the future. Because contemporary periodontal screening methods are deficient, there is an urgent need for novel approaches in periodontal screening procedures. To this end we associated oral parameters (clinical attachment level, periodontal probing depth, supragingival plaque, supragingival calculus, number of missing teeth, and removable denture) with a large set of salivary metabolites (n=383) obtained by mass spectrometry among a subsample (n=909) of non-diabetic participants of the Study of Health in Pomerania (SHIP-Trend-0). Linear regression analyses were performed in age-stratified groups and adjusted for potential confounders. A multifaceted image of associated metabolites (n=107) with considerable differences according to age groups was revealed. In the young (20-39 years) and middle-aged groups (40-59 years), we found metabolites predominantly associated with periodontal variables; whereas among the older subjects (60 + years), tooth loss was strongly associated with metabolite levels. Metabolites associated with periodontal variables were clearly linked to tissue destruction, host- defence mechanisms and bacterial metabolism. Across all age groups, the bacterial metabolite phenylacetate was significantly associated with periodontal variables. Our results revealed alterations of the salivary metabolome in association with age and oral health status. Among our comprehensive panel of metabolites, periodontitis was significantly associated with the bacterial metabolite phenylacetate, a promising substance for further biomarker research.Parodontitis zählt zu den hochprävalenten oralen Erkrankungen und betrifft Millionen von Menschen auf der ganzen Welt. Sie beeinträchtigt die Lebensqualität der Betroffenen negativ durch Einschränkungen in verschiedenen funktionellen und ästhetischen Aspekten. Darüber hinaus besteht der Verdacht, dass Parodontitis ein Risikofaktor im Zusammenhang mit der Entstehung von weit verbreiteten systemischen Erkrankungen darstellt. Bis jetzt gibt es noch keine zuverlässigen, reproduzierbaren Screeningtests für den klinischen Alltag. Parodontitis wird noch immer anhand von eindeutigen, klinisch sichtbaren (gerötetes Zahnfleisch, Zahnfleischblutung, gelockerte Zähne, Zahnfleischrückgang), messbaren (vergrößerte Sondierungstiefen) oder röntgenologischen Befunden diagnostiziert, welche in der Regel in bereits fortgeschrittenen Stadien der Erkrankung zu finden sind. Aufgrund dieser Defizite in der Früherkennung von Parodontitis beschäftigt sich die Forschung zunehmend mit der Suche nach passenden Biomarkern, welche als diagnostische Werkzeuge dienlich sein könnten. Dabei rückt zunehmend neben der Genomik und Proteomik auch die Metabolomik in den Fokus der wissenschaftlichen Untersuchungen. Parodontitis wird durch multifaktorielle Interaktionen zwischen dem Menschen und bestimmten pathogenen Keimen der Mundhöhle verursacht. Dabei produzieren Wirt und Bakterium verschiedene Zwischen- oder Endprodukte, sogenannte Metabolite. Es wird angenommen, dass einige dieser Metaboliten als Indikatoren für die Früherkennung und das Screening von Parodontitis eignen könnten. In diesem Zusammenhang assoziierten wir orale Parameter (klinisches Attachment-Level, Parodontal-Sondierungstiefe, supragingivale Plaque, supragingivale Zahnstein, Anzahl der fehlenden Zähne und herausnehmbaren Zahnersatz) mit einer großen Menge von Speichelmetaboliten (n=383), die durch Massenspektrometrie aus einer Probandengruppe (n=909) von nicht-diabetischen Teilnehmern der Gesundheitsstudie in Pommern (SHIP-Trend-0) gewonnen wurden. Lineare Regressionsanalysen wurden in nach Alter geschichteten Gruppen durchgeführt und mögliche Störfaktoren bereinigt. Insgesamt fanden wir 107 Metabolite, die zu unseren untersuchten oralen Variablen assoziiert waren. Dabei zeigten sich erheblichen Unterschieden zwischen den Altersgruppen. In der Gruppe der Jüngeren (20-39 Jahre) und der Gruppe mittleren Alters (40-59 Jahre) fanden wir Metabolite, die überwiegend mit parodontalen Variablen assoziiert waren. Bei den älteren Probanden (>60 Jahre) war jedoch der Zahnverlust stark mit den gefundenen Metaboliten assoziiert. Die mit Parodontitis assoziierten Metaboliten standen eindeutig im Zusammenhang mit Gewebezerstörung, Wirtsabwehrmechanismen und bakteriellem Stoffwechsel. In allen drei Altersgruppen war der bakterielle Metabolit Phenylacetat signifikant positiv mit den Parodontitis-Variablen assoziiert. Um zu spezifizieren, ob unsere gefundenen Metabolite aus dem lokalen Stoffwechsel innerhalb der Mundhöhle stammen oder es sich um Stoffe aus dem Blutplasma handelt (die über die Sulcusflüssigkeit in den Speichel gelangt sind oder bei der Speichelentstehung bereits translatiert wurden) führten wir zusätzlich eine umfangreiche Plasmametabolomanalyse bei den Probanden durch. Anhand der Ergebnisse lies sich keine Übereinstimmung finden, was für einen Ursprung der mit Parodontitis assoziierten Metabolite aus dem lokalen oralen Stoffwechsel spricht. Unsere Ergebnisse zeigten deutlich die Veränderungen des Speichelmetaboloms im Zusammenhang mit Alter und dem allgemeinen Gesundheitszustand der Mundhöhle. Durch die Kombination mit Plasma-Metabolomik-Analysen können wir außerdem die systemischen Auswirkungen unserer Ergebnisse beurteilen. Nicht zuletzt waren die Level des Speichelmetaboliten Phenylacetate über alle Altersbereiche hinweg konsistent mit Variablen der Parodontitis assoziiert wodurch dieser einen potenziellen Marker für das Screening darstellt. Unsere umfassende Arbeit dient als Referenz für zukünftige Forschungen in Fall-Kontroll-Studien zu Parodontitis und liefert wichtige Hinweise für die Exploration von Speichelproben für die Biomarker-Forschung im Allgemeinen

Plasma proteome profiling identifies changes associated to AD but not to FTD

Background Frontotemporal dementia (FTD) is caused by frontotemporal lobar degeneration (FTLD), characterized mainly by inclusions of Tau (FTLD-Tau) or TAR DNA binding43 (FTLD-TDP) proteins. Plasma biomarkers are strongly needed for specific diagnosis and potential treatment monitoring of FTD. We aimed to identify specific FTD plasma biomarker profiles discriminating FTD from AD and controls, and between FTD pathological subtypes. In addition, we compared plasma results with results in post-mortem frontal cortex of FTD cases to understand the underlying process. Methods Plasma proteins (n = 1303) from pathologically and/or genetically confirmed FTD patients (n = 56; FTLD-Tau n = 16; age = 58.2 +/- 6.2; 44% female, FTLD-TDP n = 40; age = 59.8 +/- 7.9; 45% female), AD patients (n = 57; age = 65.5 +/- 8.0; 39% female), and non-demented controls (n = 148; 61.3 +/- 7.9; 41% female) were measured using an aptamer-based proteomic technology (SomaScan). In addition, exploratory analysis in post-mortem frontal brain cortex of FTD (n = 10; FTLD-Tau n = 5; age = 56.2 +/- 6.9, 60% female, and FTLD-TDP n = 5; age = 64.0 +/- 7.7, 60% female) and non-demented controls (n = 4; age = 61.3 +/- 8.1; 75% female) were also performed. Differentially regulated plasma and tissue proteins were identified by global testing adjusting for demographic variables and multiple testing. Logistic lasso regression was used to identify plasma protein panels discriminating FTD from non-demented controls and AD, or FTLD-Tau from FTLD-TDP. Performance of the discriminatory plasma protein panels was based on predictions obtained from bootstrapping with 1000 resampled analysis. Results Overall plasma protein expression profiles differed between FTD, AD and controls (6 proteins; p = 0.005), but none of the plasma proteins was specifically associated to FTD. The overall tissue protein expression profile differed between FTD and controls (7-proteins; p = 0.003). There was no difference in overall plasma or tissue expression profile between FTD subtypes. Regression analysis revealed a panel of 12-plasma proteins discriminating FTD from AD with high accuracy (AUC: 0.99). No plasma protein panels discriminating FTD from controls or FTD pathological subtypes were identified. Conclusions We identified a promising plasma protein panel as a minimally-invasive tool to aid in the differential diagnosis of FTD from AD, which was primarily associated to AD pathophysiology. The lack of plasma profiles specifically associated to FTD or its pathological subtypes might be explained by FTD heterogeneity, calling for FTD studies using large and well-characterize cohorts

Binding to medium and long chain fatty acyls is a common property of HEAT and ARM repeat modules.

Covalent post-translational modification (PTM) of proteins with acyl groups of various carbon chain-lengths regulates diverse biological processes ranging from chromatin dynamics to subcellular localization. While the YEATS domain has been found to be a prominent reader of acetylation and other short acyl modifications, whether additional acyl-lysine reader domains exist, particularly for longer carbon chains, is unclear. Here, we employed a quantitative proteomic approach using various modified peptide baits to identify reader proteins of various acyl modifications. We discovered that proteins harboring HEAT and ARM repeats bind to lysine myristoylated peptides. Recombinant HEAT and ARM repeats bind to myristoylated peptides independent of the peptide sequence or the position of the myristoyl group. Indeed, HEAT and ARM repeats bind directly to medium- and long-chain free fatty acids (MCFA and LCFA). Lipidomic experiments suggest that MCFAs and LCFAs interact with HEAT and ARM repeat proteins in mammalian cells. Finally, treatment of cells with exogenous MCFAs and inhibitors of MCFA-CoA synthases increase the transactivation activity of the ARM repeat protein β-catenin. Taken together, our results suggest an unappreciated role for fatty acids in the regulation of proteins harboring HEAT or ARM repeats