Dirk Repsilber

The Metabolic Response of Heterotrophic Arabidopsis Cells to Oxidative Stress

To cope with oxidative stress, the metabolic network of plant cells must be reconfigured either to bypass damaged enzymes or to support adaptive responses. To characterize the dynamics of metabolic change during oxidative stress, heterotrophic Arabidopsis (Arabidopsis thaliana) cells were treated with menadione and changes in metabolite abundance and 13C-labeling kinetics were quantified in a time series of samples taken over a 6 h period. Oxidative stress had a profound effect on the central metabolic pathways with extensive metabolic inhibition radiating from the tricarboxylic acid cycle and including large sectors of amino acid metabolism. Sequential accumulation of metabolites in specific pathways indicated a subsequent backing up of glycolysis and a diversion of carbon into the oxidative pentose phosphate pathway. Microarray analysis revealed a coordinated transcriptomic response that represents an emergency coping strategy allowing the cell to survive the metabolic hiatus. Rather than attempt to replace inhibited enzymes, transcripts encoding these enzymes are in fact down-regulated while an antioxidant defense response is mounted. In addition, a major switch from anabolic to catabolic metabolism is signaled. Metabolism is also reconfigured to bypass damaged steps (e.g. induction of an external NADH dehydrogenase of the mitochondrial respiratory chain). The overall metabolic response of Arabidopsis cells to oxidative stress is remarkably similar to the superoxide and hydrogen peroxide stimulons of bacteria and yeast (Saccharomyces cerevisiae), suggesting that the stress regulatory and signaling pathways of plants and microbes may share common elements.

Human gene expression profiles of susceptibility and resistance in tuberculosis

Tuberculosis (TB) still poses a profound burden on global health, owing to significant morbidity and mortality worldwide. Although a fully functional immune system is essential for the control of Mycobacterium tuberculosis infection, the underlying mechanisms and reasons for failure in part of the infected population remain enigmatic. Here, whole-blood microarray gene expression analyses were performed in TB patients and in latently as well as uninfected healthy controls to define biomarkers predictive of susceptibility and resistance. Fc gamma receptor 1B (FCGRIB)was identified as the most differentially expressed gene, and, in combination with four other markers, produced a high degree of accuracy in discriminating TB patients and latently infected donors. We determined differentially expressed genes unique for active disease and identified profiles that correlated with susceptibility and resistance to TB. Elevated expression of innate immune-related genes in active TB and higher expression of particular gene clusters involved in apoptosis and natural killer cell activity in latently infected donors are likely to be the major distinctive factors determining failure or success in controlling M. tuberculosis infection. The gene expression profiles defined in this study provide valuable clues for better understanding of progression from latent infection to active disease and pave the way for defining predictive correlates of protection in TB.

Candidate biomarkers for discrimination between infection and disease caused by Mycobacterium tuberculosis.

Infection with Mycobacterium tuberculosis is controlled by an efficacious immune response in about 90% of infected individuals who do not develop disease. Although essential mediators of protection, e.g., interferon-γ, have been identified, these factors are insufficient to predict the outcome of M. tuberculosis infection. As a first step to determine additional biomarkers, we compared gene expression profiles of peripheral blood mononuclear cells from tuberculosis patients and M. tuberculosis-infected healthy donors by microarray analysis. Differentially expressed candidate genes were predominantly derived from monocytes and comprised molecules involved in the antimicrobial defense, inflammation, chemotaxis, and intracellular trafficking. We verified differential expression for alpha-defensin 1, alpha-defensin 4, lactoferrin, Fcγ receptor 1A (cluster of differentiation 64 [CD64]), bactericidal permeability-increasing protein, and formyl peptide receptor 1 by quantitative polymerase chain reaction analysis. Moreover, we identified increased protein expression of CD64 on monocytes from tuberculosis patients. Candidate biomarkers were then assessed for optimal study group discrimination. Using a linear discriminant analysis, a minimal group of genes comprising lactoferrin, CD64, and the Ras-associated GTPase 33A was sufficient for classification of (1) tuberculosis patients, (2) M. tuberculosis-infected healthy donors, and (3) noninfected healthy donors.

Mode of Inheritance of Primary Metabolic Traits in Tomato

To evaluate components of fruit metabolic composition, we have previously metabolically phenotyped tomato (Solanum lycopersicum) introgression lines containing segmental substitutions of wild species chromosome in the genetic background of a cultivated variety. Here, we studied the hereditability of the fruit metabolome by analyzing an additional years harvest and evaluating the metabolite profiles of lines heterozygous for the introgression (ILHs), allowing the evaluation of putative quantitative trait locus (QTL) mode of inheritance. These studies revealed that most of the metabolic QTL (174 of 332) were dominantly inherited, with relatively high proportions of additively (61 of 332) or recessively (80 of 332) inherited QTL and a negligible number displaying the characteristics of overdominant inheritance. Comparison of the mode of inheritance of QTL revealed that several metabolite pairs displayed a similar mode of inheritance of QTL at the same chromosomal loci. Evaluation of the association between morphological and metabolic traits in the ILHs revealed that this correlation was far less prominent, due to a reduced variance in the harvest index within this population. These data are discussed in the context of genomics-assisted breeding for crop improvement, with particular focus on the exploitation of wide biodiversity.

MicroRNA signatures characterize diffuse large B-cell lymphomas and follicular lymphomas

MicroRNAs (miRNA, miR) are negative regulators of gene expression that play an important role in diverse biological processes such as development, cell growth, apoptosis and haematopoiesis, suggesting their association with cancer. Here we analysed the expression signatures of 157 miRNAs in 58 diffuse large B‐cell lymphoma (DLBCL), 46 follicular lymphoma (FL) and seven non‐neoplastic lymph nodes (LN). Comparison of the possible combinations of DLBCL‐, FL‐ and LN resulted in specific DLBCL‐ and FL‐signatures, which include miRNAs with previously published function in haematopoiesis (MIRN150 and MIRN155) or tumour development (MIRN210, MIRN10A, MIRN17‐5P and MIRN145). As compared to LN, some miRNAs are differentially regulated in both lymphoma types (MIRN155, MIRN210, MIRN106A, MIRN149 and MIRN139). Conversely, some miRNAs show lymphoma‐specific aberrant expression, such as MIRN9/9*, MIRN301, MIRN338 and MIRN213 in FL and MIRN150, MIRN17‐5P, MIRN145, MIRN328 and others in DLBCL. A classification tree was computed using four miRNAs (MIRN330, MIRN17‐5P, MIRN106a and MIRN210) to correctly identify 98% of all 111 cases that were analysed in this study. Finally, eight miRNAs were found to correlate with event‐free and overall survival in DLBCL including known tumour suppressors (MIRN21, MIRN127 and MIRN34a) and oncogenes (MIRN195 and MIRNLET7G).

Identification of T-Cell Antigens Specific for Latent Mycobacterium Tuberculosis Infection

Background T-cell responses against dormancy-, resuscitation-, and reactivation-associated antigens of Mycobacterium tuberculosis are candidate biomarkers of latent infection in humans. Methodology/Principal Findings We established an assay based on two rounds of in vitro restimulation and intracellular cytokine analysis that detects T-cell responses to antigens expressed during latent M. tuberculosis infection. Comparison between active pulmonary tuberculosis (TB) patients and healthy latently M. tuberculosis-infected donors (LTBI) revealed significantly higher T-cell responses against 7 of 35 tested M. tuberculosis latency-associated antigens in LTBI. Notably, T cells specific for Rv3407 were exclusively detected in LTBI but not in TB patients. The T-cell IFNγ response against Rv3407 in individual donors was the most influential factor in discrimination analysis that classified TB patients and LTBI with 83% accuracy using cross-validation. Rv3407 peptide pool stimulations revealed distinct candidate epitopes in four LTBI. Conclusions Our findings further support the hypothesis that the latency-associated antigens can be exploited as biomarkers for LTBI.

Assessment of the immune capacity of mammary epithelial cells: comparison with mammary tissue after challenge with Escherichia coli

We examined the repertoire and extent of inflammation dependent gene regulation in a bovine mammary epithelial cell (MEC) model, to better understand the contribution of the MEC in the immune defence of the udder. We challenged primary cultures of MEC from cows with heat inactivated Escherichia coli pathogens and used Affymetrix DNA-microarrays to profile challenge related alterations in their transcriptome. Compared to acute mastitis, the most prominently activated genes comprise those encoding chemokines, interleukins, beta-defensins, serum amyloid A and haptoglobin. Hence, the MEC exert sentinel as well as effector functions of innate immune defence. E. coli stimulated a larger fraction of genes (30%) in the MEC belonging to the functional category Inflammatory Response than we recorded with the same microarrays during acute mastitis in the udder (17%). This observation underscores the exquisite immune capacity of MEC. To more closely examine the adequacy of immunological regulation in MEC, we compared the inflammation dependent regulation of factors contributing to the complement system between the udder versus the MEC. In the MEC we observed only up regulation of several complement factor-encoding genes. Mastitis, in contrast, in the udder strongly down regulates such genes encoding factors contributing to both, the classical pathway of complement activation and the Membrane Attack Complex, while the expression of factors contributing to the alternative pathway may be enhanced. This functionally polarized regulation of the complex complement pathway is not reflected in the MEC models.

Biomarkers of Inflammation, Immunosuppression and Stress with Active Disease Are Revealed by Metabolomic Profiling of Tuberculosis Patients

Although tuberculosis (TB) causes more deaths than any other pathogen, most infected individuals harbor the pathogen without signs of disease. We explored the metabolome of >400 small molecules in serum of uninfected individuals, latently infected healthy individuals and patients with active TB. We identified changes in amino acid, lipid and nucleotide metabolism pathways, providing evidence for anti-inflammatory metabolomic changes in TB. Metabolic profiles indicate increased activity of indoleamine 2,3 dioxygenase 1 (IDO1), decreased phospholipase activity, increased abundance of adenosine metabolism products, as well as indicators of fibrotic lesions in active disease as compared to latent infection. Consistent with our predictions, we experimentally demonstrate TB-induced IDO1 activity. Furthermore, we demonstrate a link between metabolic profiles and cytokine signaling. Finally, we show that 20 metabolites are sufficient for robust discrimination of TB patients from healthy individuals. Our results provide specific insights into the biology of TB and pave the way for the rational development of metabolic biomarkers for TB.

Functional correlations of pathogenesis-driven gene expression signatures in tuberculosis.

Tuberculosis remains a major health threat and its control depends on improved measures of prevention, diagnosis and treatment. Biosignatures can play a significant role in the development of novel intervention measures against TB and blood transcriptional profiling is increasingly exploited for their rational design. Such profiles also reveal fundamental biological mechanisms associated with the pathology of the disease. We have compared whole blood gene expression in TB patients, as well as in healthy infected and uninfected individuals in a cohort in The Gambia, West Africa and validated previously identified signatures showing high similarities of expression profiles among different cohorts. In this study, we applied a unique combination of classical gene expression analysis with pathway and functional association analysis integrated with intra-individual expression correlations. These analyses were employed for identification of new disease-associated gene signatures, identifying a network of Fc gamma receptor 1 signaling with correlating transcriptional activity as hallmark of gene expression in TB. Remarkable similarities to characteristic signatures in the autoimmune disease systemic lupus erythematosus (SLE) were observed. Functional gene clusters of immunoregulatory interactions involving the JAK-STAT pathway; sensing of microbial patterns by Toll-like receptors and IFN-signaling provide detailed insights into the dysregulation of critical immune processes in TB, involving active expression of both pro-inflammatory and immunoregulatory systems. We conclude that transcriptomics (i) provides a robust system for identification and validation of biosignatures for TB and (ii) application of integrated analysis tools yields novel insights into functional networks underlying TB pathogenesis.

Formation of metastable RNA structures by sequential folding during transcription: time-resolved structural analysis of potato spindle tuber viroid (-)-stranded RNA by temperature-gradient gel electrophoresis.

A model of functional elements critical for replication and infectivity of the potato spindle tuber viroid (PSTVd) was proposed earlier: a thermodynamically metastable structure containing a specific hairpin (HP II) in the (-)-strand replication intermediate is essential for template activity during (+)-strand synthesis. We present here a detailed kinetic analysis on how PSTVd (-)-strands fold during synthesis by sequential folding into a variety of metastable structures that rearrange only slowly into the structure distribution of the thermodynamic equilibrium. Synthesis of PSTVd (-)-strands was performed by T7-RNA-polymerase; the rate of synthesis was varied by altering the concentration of nucleoside triphosphates to mimic the in vivo synthesis rate of DNA-dependent RNA polymerase II. With dependence on rate and duration of the synthesis, the structure distributions were analyzed by temperature-gradient gel electrophoresis (TGGE). Metastable structures are generated preferentially at low transcription rates--similar to in vivo rates--or at short transcription times at higher rates. Higher transcription rates or longer transcription times lead to metastable structures in low or undetectable amounts. Instead different structures do gradually appear having a more rod-like shape and higher thermodynamic stability, and the thermodynamically optimal rod-like structure dominates finally. It is concluded that viroids are able to use metastable as well as stable structures for their biological functions.