Double trouble:Bacillus depends on a functional Tat machinery to avoid severe oxidative stress and starvation upon entry into a NaCl-depleted environment

The widely conserved twin-arginine translocases (Tat) allow the transport of fully folded cofactor-containing proteins across biological membranes. In doing so, these translocases serve different biological functions ranging from energy conversion to cell division. In the Gram-positive soil bacterium Bacillus subtilis, the Tat machinery is essential for effective growth in media lacking iron or NaCl. It was previously shown that this phenomenon relates to the Tat-dependent export of the heme-containing peroxidase EfeB, which converts Fe2+ to Fe3+ at the expense of hydrogen peroxide. However, the reasons why the majority of tat mutant bacteria perish upon dilution in NaCl-deprived medium and how, after several hours, a sub-population adapts to this condition was unknown. Here we show that, upon growth in the absence of NaCl, the bacteria face two major problems, namely severe oxidative stress at the membrane and starvation leading to death. The tat mutant cells can overcome these challenges if they are fed with arginine, which implies that severe arginine depletion is a major cause of death and resumed arginine synthesis permits their survival. Altogether, our findings show that the Tat system of B. subtilis is needed to preclude severe oxidative stress and starvation upon sudden drops in the environmental Na+ concentration as caused by flooding or rain

Improved Wound Healing of Airway Epithelial Cells Is Mediated by Cold Atmospheric Plasma: A Time Course-Related Proteome Analysis

The promising potential of cold atmospheric plasma (CAP) treatment as a new therapeutic option in the field of medicine, particularly in Otorhinolaryngology and Respiratory medicine, demands primarily the assessment of potential risks and the prevention of any direct and future cell damages. Consequently, the application of a special intensity of CAP that is well tolerated by cells and tissues is of particular interest. Although improvement of wound healing by CAP treatment has been described, the underlying mechanisms and the molecular influences on human tissues are so far only partially characterized. In this study, human S9 bronchial epithelial cells were treated with cold plasma of atmospheric pressure plasma jet that was previously proven to accelerate the wound healing in a clinically relevant extent. We studied the detailed cellular adaptation reactions for a specified plasma intensity by time-resolved comparative proteome analyses of plasma treated vs. nontreated cells to elucidate the mechanisms of the observed improved wound healing and to define potential biomarkers and networks for the evaluation of plasma effects on human epithelial cells. K-means cluster analysis and time-related analysis of fold-change factors indicated concordantly clear differences between the short-term (up to 1 h) and long-term (24-72 h) adaptation reactions. Thus, the induction of Nrf2-mediated oxidative and endoplasmic reticulum stress response, PPAR-alpha/RXR activation as well as production of peroxisomes, and prevention of apoptosis already during the first hour after CAP treatment are important cell strategies to overcome oxidative stress and to protect and maintain cell integrity and especially microtubule dynamics. After resolving of stress, when stress adaptation was accomplished, the cells seem to start again with proliferation and cellular assembly and organization. The observed strategies and identification of marker proteins might explain the accelerated wound healing induced by CAP, and these indicators might be subsequently used for risk assessment and quality management of application of nonthermal plasma sources in clinical settings. Copyright © 2019 Christian Scharf et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited

Adaptive immune response to lipoproteins of Staphylococcus aureus in healthy subjects

Staphylococcus aureus is a frequent commensal but also a dangerous pathogen, causing many forms of infection ranging from mild to life-threatening conditions. Among its virulence factors are lipoproteins, which are anchored in the bacterial cell membrane. Lipoproteins perform various functions in colonization, immune evasion, and immunomodulation. These proteins are potent activators of innate immune receptors termed Toll-like receptors 2 and 6. This study addressed the specific B-cell and T-cell responses directed to lipoproteins in human S. aureus carriers and non-carriers. 2D immune proteomics and ELISA approaches revealed that titers of antibodies (IgG) binding to S. aureus lipoproteins were very low. Proliferation assays and cytokine profiling data showed only subtle responses of T cells; some lipoproteins did not elicit proliferation. Hence, the robust activation of the innate immune system by S. aureus lipoproteins does not translate into a strong adaptive immune response. Reasons for this may include inaccessibility of lipoproteins for B cells as well as ineffective processing and presentation of the antigens to T cells.</p

Exercise Training Prevents Diaphragm Contractile Dysfunction in Heart Failure

Purpose: Patient studies have demonstrated the efficacy of exercise training in attenuating respiratory muscle weakness in chronic heart failure (HF), yet direct assessment of muscle fiber contractile function together with data on the underlying intracellular mechanisms remains elusive. The present study, therefore, used a mouse model of HF to assess whether exercise training could prevent diaphragm contractile fiber dysfunction by potentially mediating the complex interplay between intracellular oxidative stress and proteolysis. Methods: Mice underwent sham operation (n = 10) or a ligation of the left coronary artery and were randomized to sedentary HF (n = 10) or HF with aerobic exercise training (HF + AET; n = 10). Ten weeks later, echocardiography and histological analyses confirmed HF. Results: In vitro diaphragm fiber bundles demonstrated contractile dysfunction in sedentary HF compared with sham mice that was prevented by AET, with maximal force 21.0 ± 0.7 versus 26.7 ± 1.4 and 25.4 ± 1.4 N·cm−2, respectively (P < 0.05). Xanthine oxidase enzyme activity and MuRF1 protein expression, markers of oxidative stress and protein degradation, were ~20% and ~70% higher in sedentary HF compared with sham mice (P < 0.05) but were not different when compared with the HF + AET group. Oxidative modifications to numerous contractile proteins (i.e., actin and creatine kinase) and markers of proteolysis (i.e., proteasome and calpain activity) were elevated in sedentary HF compared with HF + AET mice (P < 0.05); however, these indices were not significantly different between sedentary HF and sham mice. Antioxidative enzyme activities were also not different between groups. Conclusion: Our findings demonstrate that AET can protect against diaphragm contractile fiber dysfunction induced by HF, but it remains unclear whether alterations in oxidative stress and/or protein degradation are primarily responsible

Extensive alterations of the whole-blood transcriptome are associated with body mass index: results of an mRNA profiling study involving two large population-based cohorts

Background: Obesity, defined as pathologically increased body mass index (BMI),is strongly related to an increased risk for numerous common cardiovascular and metabolic diseases. It is particularly associated with insulin resistance, hyperglycemia, and systemic oxidative stress and represents the most important risk factor for type 2 diabetes (T2D). However, the pathophysiological mechanisms underlying these associations are still not completely understood. Therefore, in order to identify potentially disease-relevant BMI-associated gene expression signatures, a transcriptome-wide association study (TWAS) on BMI was performed. Methods: Whole-blood mRNA levels determined by array-based transcriptional profiling were correlated with BMI in two large independent population-based cohort studies (KORA F4 and SHIP-TREND) comprising a total of 1977 individuals. Results: Extensive alterations of the whole-blood transcriptome were associated with BMI: More than 3500 transcripts exhibited significant positive or negative BMI-correlation. Three major whole-blood gene expression signatures associated with increased BMI were identified. The three signatures suggested: i) a ratio shift from mature erythrocytes towards reticulocytes, ii) decreased expression of several genes essentially involved in the transmission and amplification of the insulin signal, and iii) reduced expression of several key genes involved in the defence against reactive oxygen species (ROS). Conclusions: Whereas the first signature confirms published results, the other two provide possible mechanistic explanations for well-known epidemiological findings under conditions of increased BMI, namely attenuated insulin signaling and increased oxidative stress. The putatively causative BMI-dependent down-regulation of the expression of numerous genes on the mRNA level represents a novel finding. BMI-associated negative transcriptional regulation of insulin signaling and oxidative stress management provide new insights into the pathogenesis of metabolic syndrome and T2D

The transcriptional landscape of age in human peripheral blood

Disease incidences increase with age, but the molecular characteristics of ageing that lead to increased disease susceptibility remain inadequately understood. Here we perform a whole-blood gene expression meta-analysis in 14,983 individuals of European ancestry (including replication) and identify 1,497 genes that are differentially expressed with chronological age. The age-associated genes do not harbor more age-associated CpG-methylation sites than other genes, but are instead enriched for the presence of potentially functional CpG-methylation sites in enhancer and insulator regions that associate with both chronological age and gene expression levels. We further used the gene expression profiles to calculate the 'transcriptomic age' of an individual, and show that differences between transcriptomic age and chronological age are associated with biological features linked to ageing, such as blood pressure, cholesterol levels, fasting glucose, and body mass index. The transcriptomic prediction model adds biological relevance and complements existing epigenetic prediction models, and can be used by others to calculate transcriptomic age in external cohorts.Peer reviewe

Charakterisierung der Anpassung von 'Bacillus subtillis' an verschiedene Umweltstresse mittels Transkriptom- und Proteomanalysen

Die hier vorgestellte Arbeit beschreibt die Nutzung globaler Transkriptom- und Proteomanalysen zur Untersuchung der Anpassung des Bodenbakteriums Bacillus subtilis an einige in seinem natürlichen Habitat vorherrschende Bedingungen. Die Ergebnisse dieser Arbeit verdeutlichen, dass mit Hilfe globaler Transkriptom- und Proteomanalysen auch für bereits intensiv untersuchte Fragestellungen neue und zum Teil unerwartete Aspekte zu entdecken sind. Im Falle der seit ca. 30 Jahren untersuchten Sporulation in B. subtilis konnte eine Reihe von neuen differenziell exprimierten Genen identifiziert werden. Ferner wurden diese Gene den vier an der Sporulation beteiligten Sigmafaktoren zugeordnet. Einige der besonders interessanten Gene wurden einer Detailanalyse unterzogen. Die Analyse der seit Anfang der 90er Jahre auf molekularer Ebene intensiv untersuchten Anpassung von B. subtilis an hohe Osmolaritat führte zur Ausweitung bereits erhaltener Befunde und zur Entdeckung neuer Facetten dieser Adaptationsstrategie. Vergleichbares gilt für die Untersuchung der Anpassung von B. subtilis an schwankende Temperaturen. Die Verwendung globaler Analysen zur Untersuchung der Kälteschockantwort und der Adaptation wachsender Zellen an Kälte und Hitze haben auch hier neue interessante Befunde geliefert. Anhand der hier vorgestellten Ergebnisse wird das Potential globaler Analysen verdeutlicht. Sie ermöglichen Einblicke in das Zusammenspiel der einzelnen Anpassungsmechanismen und liefern entscheidende Hinweise zur Entschlüsselung zellularer Regulationsnetzwerke.The work presented here describes the use of global transcriptome- and proteomeanalyses for the investigation of the adaptation of the soil bacterium Bacillus subtilis to some conditions prevailing in its natural habitat. The results of this work clarify that with the help of global transcriptome- and proteome- analyses new and partially unexpected aspects are to be discovered. In case of sporulation in B. subtilis, which is under examination for approximately 30 years, a new set of differentially expressed genes could be identified. Furthermore these genes were assigned to the four sporulation specific sigma factors. Some of the interesting genes were subjected to detailed analysis. The investigation of the adaptation of B. subtilis to high osmolarity, examined intensively since beginning of the 90's on the molecular level, led to the expansion of findings already received and to the discovery of new facets of this adaptation strategy. This is also true for the investigation of the adaptation of B. subtilis to varying temperatures. The use of global analyses for the investigation of cold shock and the adaptation of growing cells either to heat or cold also revealed new interesting findings. Considering the results presented here, the potential of global analyses is clarified. They allow insights into the possible interactions of individual adaptation mechanisms and supply crucial hints to the decoding of cellular regulatory networks

Proteomics of blood-based therapeutics: A promising tool for quality assurance in transfusion medicine

Blood-based therapeutics are cellular or plasma components derived from human blood. Their production requires appropriate selection and treatment of the donor and processing of cells or plasma proteins. In contrast to clearly defined, chemically synthesized drugs, blood-derived therapeutics are highly complex mixtures of plasma proteins or even more complex cells. Pathogen transmission by the product as well as changes in the integrity of blood constituents resulting in loss of function or immune modulation are currently important issues in transfusion medicine. Protein modifications can occur during various steps of the production process, such as acquisition, enrichment of separate components (e.g. coagulation factors, cell populations), virus inactivation, conservation, and storage. Contemporary proteomic strategies allow a comprehensive assessment of protein modifications with high coverage, offer capabilities for qualitative and even quantitative analysis, and for high-throughput protein identification. Traditionally, proteomics approaches predominantly relied on two-dimensional gel electrophoresis (2-DE). Even if 2-DE is still state of the art, it has inherent limitations that are mainly based on the physicochemical properties of the proteins analyzed; for example, proteins with extremes in molecular mass and hydrophobicity (most membrane proteins) are difficult to assess by 2-DE. These limitations have fostered the development of mass spectrometry centered on non-gel-based separation approaches, which have proven to be highly successful and are thus complementing and even partially replacing 2-DE-based approaches. Although blood constituents have been extensively analyzed by proteomics, this technology has not been widely applied to assess or even improve blood-derived therapeutics, or to monitor the production processes. As proteomic technologies have the capacity to provide comprehensive information about changes occurring during processing and storage of blood products, proteomics can potentially guide improvement of pathogen inactivation procedures and engineering of stem cells, and may also allow a better understanding of factors influencing the immunogenicity of blood-derived therapeutics. An important development in proteomics is the reduction of inter-assay variability. This now allows the screening of samples taken from the same product over time or before and after processing. Optimized preparation procedures and storage conditions will reduce the risk of protein alterations, which in turn may contribute to better recovery, reduced exposure to allogeneic proteins, and increased transfusion safety

Toward the Relevance of Platelet Subpopulations for Transfusion Medicine

Circulating platelets consist of subpopulations with different age, maturation state and size. In this review, we address the association between platelet size and platelet function and summarize the current knowledge on platelet subpopulations including reticulated platelets, procoagulant platelets and platelets exposing signals to mediate their clearance. Thereby, we emphasize the impact of platelet turnover as an important condition for platelet production in vivo. Understanding of the features that characterize platelet subpopulations is very relevant for the methods of platelet concentrate production, which may enrich or deplete particular platelet subpopulations. Moreover, the concept of platelet size being associated with platelet function may be attractive for transfusion medicine as it holds the perspective to separate platelet subpopulations with specific functional capabilities