TY - JOUR A1 - Andronic, Joseph A1 - Shirakashi, Ryo A1 - Pickel, Simone U. A1 - Westerling, Katherine M. A1 - Klein, Teresa A1 - Holm, Thorge A1 - Sauer, Markus A1 - Sukhorukov, Vladimir L. T1 - Hypotonic Activation of the Myo-Inositol Transporter SLC5A3 in HEK293 Cells Probed by Cell Volumetry, Confocal and Super-Resolution Microscopy JF - PLoS One N2 - Swelling-activated pathways for myo-inositol, one of the most abundant organic osmolytes in mammalian cells, have not yet been identified. The present study explores the SLC5A3 protein as a possible transporter of myo-inositol in hyponically swollen HEK293 cells. To address this issue, we examined the relationship between the hypotonicity-induced changes in plasma membrane permeability to myo-inositol Pino [m/s] and expression/localization of SLC5A3. Pino values were determined by cell volumetry over a wide tonicity range (100–275 mOsm) in myo-inositol-substituted solutions. While being negligible under mild hypotonicity (200–275 mOsm), Pino grew rapidly at osmolalities below 200 mOsm to reach a maximum of ∼3 nm/s at 100–125 mOsm, as indicated by fast cell swelling due to myo-inositol influx. The increase in Pino resulted most likely from the hypotonicity-mediated incorporation of cytosolic SLC5A3 into the plasma membrane, as revealed by confocal fluorescence microscopy of cells expressing EGFP-tagged SLC5A3 and super-resolution imaging of immunostained SLC5A3 by direct stochastic optical reconstruction microscopy (dSTORM). dSTORM in hypotonic cells revealed a surface density of membrane-associated SLC5A3 proteins of 200–2000 localizations/μm2. Assuming SLC5A3 to be the major path for myo-inositol, a turnover rate of 80–800 myo-inositol molecules per second for a single transporter protein was estimated from combined volumetric and dSTORM data. Hypotonic stress also caused a significant upregulation of SLC5A3 gene expression as detected by semiquantitative RT-PCR and Western blot analysis. In summary, our data provide first evidence for swelling-mediated activation of SLC5A3 thus suggesting a functional role of this transporter in hypotonic volume regulation of mammalian cells. KW - electrolytes KW - isotonic KW - membrane proteins KW - cell membranes KW - hypotonic KW - hypotonic solutions KW - tonicity KW - permeability Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126408 VL - 10 IS - 3 ER - TY - JOUR A1 - Weiß, Clemens Leonard A1 - Schultz, Jörg T1 - Identification of divergent WH2 motifs by HMM-HMM alignments JF - BMC Research Notes N2 - Background The actin cytoskeleton is a hallmark of eukaryotic cells. Its regulation as well as its interaction with other proteins is carefully orchestrated by actin interaction domains. One of the key players is the WH2 motif, which enables binding to actin monomers and filaments and is involved in the regulation of actin nucleation. Contrasting conserved domains, the identification of this motif in protein sequences is challenging, as it is short and poorly conserved. Findings To identify divergent members, we combined Hidden-Markov-Model (HMM) to HMM alignments with orthology predictions. Thereby, we identified nearly 500 proteins containing so far not annotated WH2 motifs. This included shootin-1, an actin binding protein involved in neuron polarization. Among others, WH2 motifs of ‘proximal to raf’ (ptr)-orthologs, which are described in the literature, but not annotated in genome databases, were identified. Conclusion In summary, we increased the number of WH2 motif containing proteins substantially. This identification of candidate regions for actin interaction could steer their experimental characterization. Furthermore, the approach outlined here can easily be adapted to the identification of divergent members of further domain families. KW - WH2 domain KW - spire KW - shootin-1 KW - actin nucleation KW - HHblits Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126413 VL - 8 IS - 18 ER - TY - JOUR A1 - Kuhn, Joachim A1 - Gripp, Tatjana A1 - Flieder, Tobias A1 - Dittrich, Marcus A1 - Hendig, Doris A1 - Busse, Jessica A1 - Knabbe, Cornelius A1 - Birschmann, Ingvild T1 - UPLC-MRM Mass Spectrometry Method for Measurement of the Coagulation Inhibitors Dabigatran and Rivaroxaban in Human Plasma and Its Comparison with Functional Assays JF - PLOS ONE N2 - Introduction The fast, precise, and accurate measurement of the new generation of oral anticoagulants such as dabigatran and rivaroxaban in patients' plasma my provide important information in different clinical circumstances such as in the case of suspicion of overdose, when patients switch from existing oral anticoagulant, in patients with hepatic or renal impairment, by concomitant use of interaction drugs, or to assess anticoagulant concentration in patients' blood before major surgery. Methods Here, we describe a quick and precise method to measure the coagulation inhibitors dabigatran and rivaroxaban using ultra-performance liquid chromatography electrospray ionization-tandem mass spectrometry in multiple reactions monitoring (MRM) mode (UPLC-MRM MS). Internal standards (ISs) were added to the sample and after protein precipitation; the sample was separated on a reverse phase column. After ionization of the analytes the ions were detected using electrospray ionization-tandem mass spectrometry. Run time was 2.5 minutes per injection. Ion suppression was characterized by means of post-column infusion. Results The calibration curves of dabigatran and rivaroxaban were linear over the working range between 0.8 and 800 mu g/L (r > 0.99). Limits of detection (LOD) in the plasma matrix were 0.21 mu g/L for dabigatran and 0.34 mu g/L for rivaroxaban, and lower limits of quantification (LLOQ) in the plasma matrix were 0.46 mu g/L for dabigatran and 0.54 mu g/L for rivaroxaban. The intraassay coefficients of variation (CVs) for dabigatran and rivaroxaban were < 4% and 6%; respectively, the interassay CVs were < 6% for dabigatran and < 9% for rivaroxaban. Inaccuracy was < 5% for both substances. The mean recovery was 104.5% (range 83.8-113.0%) for dabigatran and 87.0%(range 73.6-105.4%) for rivaroxaban. No significant ion suppressions were detected at the elution times of dabigatran or rivaroxaban. Both coagulation inhibitors were stable in citrate plasma at -20 degrees C, 4 degrees C and even at RT for at least one week. A method comparison between our UPLC-MRM MS method, the commercially available automated Direct Thrombin Inhibitor assay (DTI assay) for dabigatran measurement from CoaChrom Diagnostica, as well as the automated anti-Xa assay for rivaroxaban measurement from Chromogenix both performed by ACL-TOP showed a high degree of correlation. However, UPLC-MRM MS measurement of dabigatran and rivaroxaban has a much better selectivity than classical functional assays measuring activities of various coagulation factors which are susceptible to interference by other coagulant drugs. Conclusions Overall, we developed and validated a sensitive and specific UPLC-MRM MS assay for the quick and specific measurement of dabigatran and rivaroxaban in human plasma. KW - LC-MS/MS KW - validation KW - serum KW - quantification KW - apixaban KW - diagnostic accuracy KW - performance liquid chromatography KW - factor XA inhibitor KW - direct oral anticoagulants KW - direct thrombin inhibitor Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-136023 VL - 10 IS - 12 ER - TY - JOUR A1 - Singh, Amit K. A1 - Kingston, Joseph J. A1 - Gupta, Shishir K. A1 - Batra, Harsh V. T1 - Recombinant Bivalent Fusion Protein rVE Induces CD4+ and CD8+ T-Cell Mediated Memory Immune Response for Protection Against Yersinia enterocolitica Infection JF - Frontiers in Microbiology N2 - Studies investigating the correlates of immune protection against Yersinia infection have established that both humoral and cell mediated immune responses are required for the comprehensive protection. In our previous study, we established that the bivalent fusion protein (rVE) comprising immunologically active regions of Y pestis LcrV (100-270 aa) and YopE (50-213 aa) proteins conferred complete passive and active protection against lethal Y enterocolitica 8081 challenge. In the present study, cohort of BALB/c mice immunized with rVE or its component proteins rV, rE were assessed for cell mediated immune responses and memory immune protection against Y enterocolitica 8081 rVE immunization resulted in extensive proliferation of both CD4 and CD8 T cell subsets; significantly high antibody titer with balanced IgG1: IgG2a/IgG2b isotypes (1:1 ratio) and up regulation of both Th1 (INF-\(\alpha\), IFN-\(\gamma\), IL 2, and IL 12) and Th2 (IL 4) cytokines. On the other hand, rV immunization resulted in Th2 biased IgG response (11:1 ratio) and proliferation of CD4+ T-cell; rE group of mice exhibited considerably lower serum antibody titer with predominant Th1 response (1:3 ratio) and CD8+ T-cell proliferation. Comprehensive protection with superior survival (100%) was observed among rVE immunized mice when compared to the significantly lower survival rates among rE (37.5%) and rV (25%) groups when IP challenged with Y enterocolitica 8081 after 120 days of immunization. Findings in this and our earlier studies define the bivalent fusion protein rVE as a potent candidate vaccine molecule with the capability to concurrently stimulate humoral and cell mediated immune responses and a proof of concept for developing efficient subunit vaccines against Gram negative facultative intracellular bacterial pathogens. KW - I-tasser KW - Yersinia enterocolitica KW - memory immune responses KW - cytokine profiling KW - CD8+T cells KW - CD4+T cells KW - recombinant protein rVE KW - resistance KW - pneumonic plague KW - pestis infection KW - nonhuman-primates KW - III secretion KW - V-antigen KW - mice KW - vaccine Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-136114 VL - 6 IS - 1407 ER - TY - JOUR A1 - Appel, Mirjam A1 - Scholz, Claus-Jürgen A1 - Müller, Tobias A1 - Dittrich, Marcus A1 - König, Christian A1 - Bockstaller, Marie A1 - Oguz, Tuba A1 - Khalili, Afshin A1 - Antwi-Adjei, Emmanuel A1 - Schauer, Tamas A1 - Margulies, Carla A1 - Tanimoto, Hiromu A1 - Yarali, Ayse T1 - Genome-Wide Association Analyses Point to Candidate Genes for Electric Shock Avoidance in Drosophila melanogaster JF - PLoS ONE N2 - Electric shock is a common stimulus for nociception-research and the most widely used reinforcement in aversive associative learning experiments. Yet, nothing is known about the mechanisms it recruits at the periphery. To help fill this gap, we undertook a genome-wide association analysis using 38 inbred Drosophila melanogaster strains, which avoided shock to varying extents. We identified 514 genes whose expression levels and/or sequences covaried with shock avoidance scores. We independently scrutinized 14 of these genes using mutants, validating the effect of 7 of them on shock avoidance. This emphasizes the value of our candidate gene list as a guide for follow-up research. In addition, by integrating our association results with external protein-protein interaction data we obtained a shock avoidance- associated network of 38 genes. Both this network and the original candidate list contained a substantial number of genes that affect mechanosensory bristles, which are hairlike organs distributed across the fly's body. These results may point to a potential role for mechanosensory bristles in shock sensation. Thus, we not only provide a first list of candidate genes for shock avoidance, but also point to an interesting new hypothesis on nociceptive mechanisms. KW - functional analysis KW - disruption project KW - natural variation KW - complex traits KW - networks KW - behavior KW - flies KW - temperature KW - genetics KW - painful Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-152006 VL - 10 IS - 5 ER - TY - JOUR A1 - Karl, Stefan A1 - Dandekar, Thomas T1 - Jimena: Efficient computing and system state identification for genetic regulatory networks JF - BMC Bioinformatics N2 - Background: Boolean networks capture switching behavior of many naturally occurring regulatory networks. For semi-quantitative modeling, interpolation between ON and OFF states is necessary. The high degree polynomial interpolation of Boolean genetic regulatory networks (GRNs) in cellular processes such as apoptosis or proliferation allows for the modeling of a wider range of node interactions than continuous activator-inhibitor models, but suffers from scaling problems for networks which contain nodes with more than ~10 inputs. Many GRNs from literature or new gene expression experiments exceed those limitations and a new approach was developed. Results: (i) As a part of our new GRN simulation framework Jimena we introduce and setup Boolean-tree-based data structures; (ii) corresponding algorithms greatly expedite the calculation of the polynomial interpolation in almost all cases, thereby expanding the range of networks which can be simulated by this model in reasonable time. (iii) Stable states for discrete models are efficiently counted and identified using binary decision diagrams. As application example, we show how system states can now be sampled efficiently in small up to large scale hormone disease networks (Arabidopsis thaliana development and immunity, pathogen Pseudomonas syringae and modulation by cytokinins and plant hormones). Conclusions: Jimena simulates currently available GRNs about 10-100 times faster than the previous implementation of the polynomial interpolation model and even greater gains are achieved for large scale-free networks. This speed-up also facilitates a much more thorough sampling of continuous state spaces which may lead to the identification of new stable states. Mutants of large networks can be constructed and analyzed very quickly enabling new insights into network robustness and behavior. KW - Boolean function KW - genetic regulatory network KW - interpolation KW - stable state KW - binary decision diagram KW - Boolean tree Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-128671 VL - 14 ER - TY - JOUR A1 - Zirkel, J. A1 - Cecil, A. A1 - Schäfer, F. A1 - Rahlfs, S. A1 - Ouedraogo, A. A1 - Xiao, K. A1 - Sawadogo, S. A1 - Coulibaly, B. A1 - Becker, K. A1 - Dandekar, T. T1 - Analyzing Thiol-Dependent Redox Networks in the Presence of Methylene Blue and Other Antimalarial Agents with RT-PCR-Supported in silico Modeling JF - Bioinformatics and Biology Insights N2 - BACKGROUND: In the face of growing resistance in malaria parasites to drugs, pharmacological combination therapies are important. There is accumulating evidence that methylene blue (MB) is an effective drug against malaria. Here we explore the biological effects of both MB alone and in combination therapy using modeling and experimental data. RESULTS: We built a model of the central metabolic pathways in P. falciparum. Metabolic flux modes and their changes under MB were calculated by integrating experimental data (RT-PCR data on mRNAs for redox enzymes) as constraints and results from the YANA software package for metabolic pathway calculations. Several different lines of MB attack on Plasmodium redox defense were identified by analysis of the network effects. Next, chloroquine resistance based on pfmdr/and pfcrt transporters, as well as pyrimethamine/sulfadoxine resistance (by mutations in DHF/DHPS), were modeled in silico. Further modeling shows that MB has a favorable synergism on antimalarial network effects with these commonly used antimalarial drugs. CONCLUSIONS: Theoretical and experimental results support that methylene blue should, because of its resistance-breaking potential, be further tested as a key component in drug combination therapy efforts in holoendemic areas. KW - methylene blue KW - malaria KW - elementary mode analysis KW - drug KW - resistance KW - combination therapy KW - pathway KW - metabolic flux Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-123751 N1 - This is an open access article. Unrestricted non-commercial use is permitted provided the original work is properly cited. VL - 6 ER - TY - JOUR A1 - Krueger, Beate A1 - Friedrich, Torben A1 - Förster, Frank A1 - Bernhardt, Jörg A1 - Gross, Roy A1 - Dandekar, Thomas T1 - Different evolutionary modifications as a guide to rewire two-component systems JF - Bioinformatics and Biology Insights N2 - Two-component systems (TCS) are short signalling pathways generally occurring in prokaryotes. They frequently regulate prokaryotic stimulus responses and thus are also of interest for engineering in biotechnology and synthetic biology. The aim of this study is to better understand and describe rewiring of TCS while investigating different evolutionary scenarios. Based on large-scale screens of TCS in different organisms, this study gives detailed data, concrete alignments, and structure analysis on three general modification scenarios, where TCS were rewired for new responses and functions: (i) exchanges in the sequence within single TCS domains, (ii) exchange of whole TCS domains; (iii) addition of new components modulating TCS function. As a result, the replacement of stimulus and promotor cassettes to rewire TCS is well defined exploiting the alignments given here. The diverged TCS examples are non-trivial and the design is challenging. Designed connector proteins may also be useful to modify TCS in selected cases. KW - histidine kinase KW - connector KW - Mycoplasma KW - engineering KW - promoter KW - sensor KW - response regulator KW - synthetic biology KW - sequence alignment Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-123647 N1 - This is an open access article. Unrestricted non-commercial use is permitted provided the original work is properly cited. VL - 6 ER - TY - JOUR A1 - Merget, Benjamin A1 - Koetschan, Christian A1 - Hackl, Thomas A1 - Förster, Frank A1 - Dandekar, Thomas A1 - Müller, Tobias A1 - Schultz, Jörg A1 - Wolf, Matthias T1 - The ITS2 Database JF - Journal of Visual Expression N2 - The internal transcribed spacer 2 (ITS2) has been used as a phylogenetic marker for more than two decades. As ITS2 research mainly focused on the very variable ITS2 sequence, it confined this marker to low-level phylogenetics only. However, the combination of the ITS2 sequence and its highly conserved secondary structure improves the phylogenetic resolution1 and allows phylogenetic inference at multiple taxonomic ranks, including species delimitation. The ITS2 Database presents an exhaustive dataset of internal transcribed spacer 2 sequences from NCBI GenBank accurately reannotated. Following an annotation by profile Hidden Markov Models (HMMs), the secondary structure of each sequence is predicted. First, it is tested whether a minimum energy based fold (direct fold) results in a correct, four helix conformation. If this is not the case, the structure is predicted by homology modeling. In homology modeling, an already known secondary structure is transferred to another ITS2 sequence, whose secondary structure was not able to fold correctly in a direct fold. The ITS2 Database is not only a database for storage and retrieval of ITS2 sequence-structures. It also provides several tools to process your own ITS2 sequences, including annotation, structural prediction, motif detection and BLAST search on the combined sequence-structure information. Moreover, it integrates trimmed versions of 4SALE and ProfDistS for multiple sequence-structure alignment calculation and Neighbor Joining tree reconstruction. Together they form a coherent analysis pipeline from an initial set of sequences to a phylogeny based on sequence and secondary structure. In a nutshell, this workbench simplifies first phylogenetic analyses to only a few mouse-clicks, while additionally providing tools and data for comprehensive large-scale analyses. KW - homology modeling KW - molecular systematics KW - internal transcribed spacer 2 KW - alignment KW - genetics KW - secondary structure KW - ribosomal RNA KW - phylogenetic tree KW - phylogeny Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-124600 VL - 61 IS - e3806 ER - TY - JOUR A1 - Staiger, Christine A1 - Cadot, Sidney A1 - Kooter, Raul A1 - Dittrich, Marcus A1 - Müller, Tobias A1 - Klau, Gunnar W. A1 - Wessels, Lodewyk F. A. T1 - A Critical Evaluation of Network and Pathway-Based Classifiers for Outcome Prediction in Breast Cancer JF - PLoS One N2 - Recently, several classifiers that combine primary tumor data, like gene expression data, and secondary data sources, such as protein-protein interaction networks, have been proposed for predicting outcome in breast cancer. In these approaches, new composite features are typically constructed by aggregating the expression levels of several genes. The secondary data sources are employed to guide this aggregation. Although many studies claim that these approaches improve classification performance over single genes classifiers, the gain in performance is difficult to assess. This stems mainly from the fact that different breast cancer data sets and validation procedures are employed to assess the performance. Here we address these issues by employing a large cohort of six breast cancer data sets as benchmark set and by performing an unbiased evaluation of the classification accuracies of the different approaches. Contrary to previous claims, we find that composite feature classifiers do not outperform simple single genes classifiers. We investigate the effect of (1) the number of selected features; (2) the specific gene set from which features are selected; (3) the size of the training set and (4) the heterogeneity of the data set on the performance of composite feature and single genes classifiers. Strikingly, we find that randomization of secondary data sources, which destroys all biological information in these sources, does not result in a deterioration in performance of composite feature classifiers. Finally, we show that when a proper correction for gene set size is performed, the stability of single genes sets is similar to the stability of composite feature sets. Based on these results there is currently no reason to prefer prognostic classifiers based on composite features over single genes classifiers for predicting outcome in breast cancer. KW - modules KW - protein-interaction networks KW - expression signature KW - classification KW - set KW - metastasis KW - stability KW - survival KW - database KW - markers Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-131323 VL - 7 IS - 4 ER -