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633 Publications visible to you, out of a total of 633
The FAIR Guiding Principles for scientific data management and stewardship

Abstract

Not specified

Authors: Mark D. Wilkinson, Michel Dumontier, IJsbrand Jan Aalbersberg, Gabrielle Appleton, Myles Axton, Arie Baak, Niklas Blomberg, Jan-Willem Boiten, Luiz Bonino da Silva Santos, Philip E. Bourne, Jildau Bouwman, Anthony J. Brookes, Tim Clark, Mercè Crosas, Ingrid Dillo, Olivier Dumon, Scott Edmunds, Chris T. Evelo, Richard Finkers, Alejandra Gonzalez-Beltran, Alasdair J.G. Gray, Paul Groth, Carole Goble, Jeffrey S. Grethe, Jaap Heringa, Peter A.C ’t Hoen, Rob Hooft, Tobias Kuhn, Ruben Kok, Joost Kok, Scott J. Lusher, Maryann E. Martone, Albert Mons, Abel L. Packer, Bengt Persson, Philippe Rocca-Serra, Marco Roos, Rene van Schaik, Susanna-Assunta Sansone, Erik Schultes, Thierry Sengstag, Ted Slater, George Strawn, Morris A. Swertz, Mark Thompson, Johan van der Lei, Erik van Mulligen, Jan Velterop, Andra Waagmeester, Peter Wittenburg, Katherine Wolstencroft, Jun Zhao, Barend Mons

Date Published: 15th Mar 2016

Publication Type: Not specified

Evaluation of GMP-compliant culture media for in vitro expansion of human bone marrow mesenchymal stromal cells.

Abstract (Expand)

Mesenchymal stromal cells (MSCs) from human bone marrow serve as a resource for cell-based therapies in regenerative medicine. Clinical applications require standardized protocols according to good … manufacturing practice (GMP) guidelines. Donor variability as well as the intrinsic heterogeneity of MSC populations must be taken into consideration. The composition of the culture medium is a key factor in successful MSC expansion. The aim of this study was to comparatively assess the efficiency of xeno-free human platelet lysate (HPL)-based cell expansion with two commercially available media-StemPro MSC SFM CTS (for human ex vivo tissue and cell culture processing applications) and MSCGM (non-GMP-compliant, for research only)-in an academic setting as the first optimization step toward GMP-compliant manufacturing. We report the feasibility of MSC expansion up to the yielded cell number with all three media. MSCs exhibited the typical fibroblastoid morphology, with distinct differences in cell size depending on the medium. The differentiation capacity and characteristic immunophenotype were confirmed for all MSC populations. Proliferation was highest using StemPro MSC SFM CTS, whereas HPL medium was more cost-effective and its composition could be adjusted individually according to the respective needs. In summary, we present a comprehensive evaluation of GMP-compatible culture media for MSC expansion. Both StemPro and HPL medium proved to be suitable for clinical application and allowed sufficient cell proliferation. Specific differences were observed and should be considered according to the intended use. This study provides a detailed cost analysis and tools that may be helpful for the establishment of GMP-compliant MSC expansion.

Authors: P. Wuchter, M. Vetter, R. Saffrich, A. Diehlmann, K. Bieback, A. D. Ho, P. Horn

Date Published: 26th Feb 2016

Publication Type: Journal

Microcavity arrays as an in vitro model system of the bone marrow niche for hematopoietic stem cells.

Abstract (Expand)

In previous studies human mesenchymal stromal cells (MSCs) maintained the "stemness" of human hematopoietic progenitor cells (HPCs) through direct cell-cell contact in two-dimensional co-culture … systems. We establish a three-dimensional (3D) co-culture system based on a custom-made chip, the 3(D)-KITChip, as an in vitro model system of the human hematopoietic stem cell niche. This array of up to 625 microcavities, with 300 mum size in each orientation, was inserted into a microfluidic bioreactor. The microcavities of the 3(D)-KITChip were inoculated with human bone marrow MSCs together with umbilical cord blood HPCs. MSCs used the microcavities as a scaffold to build a complex 3D mesh. HPCs were distributed three-dimensionally inside this MSC network and formed ss-catenin- and N-cadherin-based intercellular junctions to the surrounding MSCs. Using RT(2)-PCR and western blots, we demonstrate that a proportion of HPCs maintained the expression of CD34 throughout a culture period of 14 days. In colony-forming unit assays, the hematopoietic stem cell plasticity remained similar after 14 days of bioreactor co-culture, whereas monolayer co-cultures showed increasing signs of HPC differentiation and loss of stemness. These data support the notion that the 3D microenvironment created within the microcavity array preserves vital stem cell functions of HPCs more efficiently than conventional co-culture systems.

Authors: P. Wuchter, R. Saffrich, S. Giselbrecht, C. Nies, H. Lorig, S. Kolb, A. D. Ho, E. Gottwald

Date Published: 3rd Feb 2016

Publication Type: Journal

Context-specific flow through the MEK/ERK module produces cell- and ligand-specific patterns of ERK single and double phosphorylation.

Abstract (Expand)

The same pathway, such as the mitogen-activated protein kinase (MAPK) pathway, can produce different cellular responses, depending on stimulus or cell type. We examined the phosphorylation dynamics of … the MAPK kinase MEK and its targets extracellular signal-regulated kinase 1 and 2 (ERK1/2) in primary hepatocytes and the transformed keratinocyte cell line HaCaT A5 exposed to either hepatocyte growth factor or interleukin-6. By combining quantitative mass spectrometry with dynamic modeling, we elucidated network structures for the reversible threonine and tyrosine phosphorylation of ERK in both cell types. In addition to differences in the phosphorylation and dephosphorylation reactions, the HaCaT network model required two feedback mechanisms, which, as the experimental data suggested, involved the induction of the dual-specificity phosphatase DUSP6 and the scaffold paxillin. We assayed and modeled the accumulation of the double-phosphorylated and active form of ERK1/2, as well as the dynamics of the changes in the monophosphorylated forms of ERK1/2. Modeling the differences in the dynamics of the changes in the distributions of the phosphorylated forms of ERK1/2 suggested that different amounts of MEK activity triggered context-specific responses, with primary hepatocytes favoring the formation of double-phosphorylated ERK1/2 and HaCaT A5 cells that produce both the threonine-phosphorylated and the double-phosphorylated form. These differences in phosphorylation distributions explained the threshold, sensitivity, and saturation of the ERK response. We extended the findings of differential ERK phosphorylation profiles to five additional cultured cell systems and matched liver tumor and normal tissue, which revealed context-specific patterns of the various forms of phosphorylated ERK.

Authors: N. Iwamoto, L. A. D'Alessandro, S. Depner, B. Hahn, B. A. Kramer, P. Lucarelli, A. Vlasov, M. Stepath, M. E. Bohm, D. Deharde, G. Damm, D. Seehofer, W. D. Lehmann, U. Klingmuller, M. Schilling

Date Published: 2nd Feb 2016

Publication Type: Journal

Targeting glycolysis in the malaria parasite Plasmodium falciparum

Abstract (Expand)

Glycolysis is the main pathway for ATP production in the malaria parasite Plasmodium falciparum and essential for its survival. Following a sensitivity analysis of a detailed kinetic model for glycolysis … in the parasite, the glucose transport reaction was identified as the step whose activity needed to be inhibited to the least extent to result in a 50% reduction in glycolytic flux. In a subsequent inhibitor titration with cytochalasin B, we confirmed the model analysis experimentally and measured a flux control coefficient of 0.3 for the glucose transporter. In addition to the glucose transporter, the glucokinase and phosphofructokinase had high flux control coefficients, while for the ATPase a small negative flux control coefficient was predicted. In a broader comparative analysis of glycolytic models, we identified a weakness in the P. falciparum pathway design with respect to stability towards perturbations in the ATP demand.

Authors: David D. van Niekerk, Gerald P. Penkler, Francois du Toit, Jacky L. Snoep

Date Published: 1st Feb 2016

Publication Type: Not specified

A molecular arms race between host innate antiviral response and emerging human coronaviruses

Abstract (Expand)

Coronaviruses have been closely related with mankind for thousands of years. Communityacquired human coronaviruses have long been recognized to cause common cold. However, zoonotic coronaviruses are … now becoming more a global concern with the discovery of highly pathogenic severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses causing severe respiratory diseases. Infections by these emerging human coronaviruses are characterized by less robust interferon production. Treatment of patients with recombinant interferon regimen promises beneficial outcomes, suggesting that compromised interferon expression might contribute at least partially to the severity of disease. The mechanisms by which coronaviruses evade host innate antiviral response are under intense investigations. This review focuses on the fierce arms race between host innate antiviral immunity and emerging human coronaviruses. Particularly, the host pathogen recognition receptors and the signal transduction pathways to mount an effective antiviral response against SARS and MERS coronavirus infection are discussed. On the other hand, the counter-measures evolved by SARS and MERS coronaviruses to circumvent host defense are also dissected. With a better understanding of the dynamic interaction between host and coronaviruses, it is hoped that insights on the pathogenesis of newly-identified highly pathogenic human coronaviruses and new strategies in antiviral development can be derived.

Authors: Lok-Yin Roy Wong, Pak-Yin Lui, Dong-Yan Jin

Date Published: 1st Feb 2016

Publication Type: Journal

Dysregulated Type I Interferon and Inflammatory Monocyte-Macrophage Responses Cause Lethal Pneumonia in SARS-CoV-Infected Mice

Abstract (Expand)

Highly pathogenic human respiratory coronaviruses cause acute lethal disease characterized by exuberant inflammatory responses and lung damage. However, the factors leading to lung pathology are not … well understood. Using mice infected with SARS (severe acute respiratory syndrome)-CoV, we show that robust virus replication accompanied by delayed type I interferon (IFN-I) signaling orchestrates inflammatory responses and lung immunopathology with diminished survival. IFN-I remains detectable until after virus titers peak, but early IFN-I administration ameliorates immunopathology. This delayed IFN-I signaling promotes the accumulation of pathogenic inflammatory monocytemacrophages (IMMs), resulting in elevated lung cytokine/chemokine levels, vascular leakage, and impaired virus-specific T cell responses. Genetic ablation of the IFN-ab receptor (IFNAR) or IMM depletion protects mice from lethal infection, without affecting viral load. These results demonstrate that IFN-I and IMM promote lethal SARS-CoV infection and identify IFN-I and IMMs as potential therapeutic targets in patients infected with pathogenic coronavirus and perhaps other respiratory viruses.

Authors: Rudragouda Channappanavar, Anthony R. Fehr, Rahul Vijay, Matthias Mack, Jincun Zhao, David K. Meyerholz, Stanley Perlman

Date Published: 1st Feb 2016

Publication Type: Journal

ORF8-Related Genetic Evidence for Chinese Horseshoe Bats as the Source of Human Severe Acute Respiratory Syndrome Coronavirus

Abstract

Abstract. Several lineage B betacoronaviruses termed severe acute respiratory syndrome (SARS)–like CoVs (SL-CoVs) were identified from Rhinolophus bats in Chin

Authors: Zhiqiang Wu, Li Yang, Xianwen Ren, Junpeng Zhang, Fan Yang, Shuyi Zhang, Qi Jin

Date Published: 21st Jan 2016

Publication Type: Journal

cy3sabiork: A Cytoscape app for visualizing kinetic data from SABIO-RK

Abstract (Expand)

Kinetic data of biochemical reactions are essential for the creation of kinetic models of biochemical networks. One of the main resources of such information is SABIO-RK, a curated database for kinetic … data of biochemical reactions and their related information. Despite the importance for computational modelling there has been no simple solution to visualize the kinetic data from SABIO-RK. In this work, I present cy3sabiork, an app for querying and visualization of kinetic data from SABIO-RK in Cytoscape. The kinetic information is accessible via a combination of graph structure and annotations of nodes, with provided information consisting of: (I) reaction details, enzyme and organism; (II) kinetic law, formula, parameters; (III) experimental conditions; (IV) publication; (V) additional annotations. cy3sabiork creates an intuitive visualization of kinetic entries in form of a species-reaction-kinetics graph, which reflects the reaction-centered approach of SABIO-RK. Kinetic entries can be imported in SBML format from either the SABIO-RK web interface or via web service queries. The app allows for easy comparison of kinetic data, visual inspection of the elements involved in the kinetic record and simple access to the annotation information of the kinetic record. I applied cy3sabiork in the computational modelling of galactose metabolism in the human liver.

Author: Matthias König

Date Published: 2016

Publication Type: Not specified

Search for a minimal set of parameters by assessing the total optimisation potential for a dynamic model of a biochemical network

Abstract (Expand)

Selecting an efficient small set of adjustable parameters to improve metabolic features of an organism is important for a reduction of implementation costs and risks of unpredicted side effects. In … practice, to avoid the analysis of a huge combinatorial space for the possible sets of adjustable parameters, experience-, and intuition-based subsets of parameters are often chosen, possibly leaving some interesting counter-intuitive combinations of parameters unrevealed. The combinatorial scan of possible adjustable parameter combinations at the model optimization level is possible; however, the number of analyzed combinations is still limited. The total optimization potential (TOP) approach is proposed to assess the full potential for increasing the value of the objective function by optimizing all possible adjustable parameters. This seemingly unpractical combination of adjustable parameters allows assessing the maximum attainable value of the objective function and stopping the combinatorial space scanning when the desired fraction of TOP is reached and any further increase in the number of adjustable parameters cannot bring any reasonable improvement. The relation between the number of adjustable parameters and the reachable fraction of TOP is a valuable guideline in choosing a rational solution for industrial implementation. The TOP approach is demonstrated on the basis of two case studies.

Authors: Egils Stalidzans, Ivars Mozga, Jurijs Sulins, Peteris Zikmanis

Date Published: 2016

Publication Type: Not specified

The role of HPV RNA transcription, immune response-related gene expression and disruptive TP53 mutations in diagnostic and prognostic profiling of head and neck cancer.

Abstract (Expand)

Stratification of head and neck squamous cell carcinomas (HNSCC) based on HPV16 DNA and RNA status, gene expression patterns, and mutated candidate genes may facilitate patient treatment decision. We … characterize head and neck squamous cell carcinomas (HNSCC) with different HPV16 DNA and RNA (E6*I) status from 290 consecutively recruited patients by gene expression profiling and targeted sequencing of 50 genes. We show that tumors with transcriptionally inactive HPV16 (DNA+ RNA-) are similar to HPV-negative (DNA-) tumors regarding gene expression and frequency of TP53 mutations (47%, 8/17 and 43%, 72/167, respectively). We also find that an immune response-related gene expression cluster is associated with lymph node metastasis, independent of HPV16 status and that disruptive TP53 mutations are associated with lymph node metastasis in HPV16 DNA- tumors. We validate each of these associations in another large data set. Four gene expression clusters which we identify differ moderately but significantly in overall survival. Our findings underscore the importance of measuring the HPV16 RNA (E6*I) and TP53-mutation status for patient stratification and identify associations of an immune response-related gene expression cluster and TP53 mutations with lymph node metastasis in HNSCC.

Authors: G. Wichmann, M. Rosolowski, K. Krohn, M. Kreuz, A. Boehm, A. Reiche, U. Scharrer, D. Halama, J. Bertolini, U. Bauer, D. Holzinger, M. Pawlita, J. Hess, C. Engel, D. Hasenclever, M. Scholz, P. Ahnert, H. Kirsten, A. Hemprich, C. Wittekind, O. Herbarth, F. Horn, A. Dietz, M. Loeffler

Date Published: 15th Dec 2015

Publication Type: Journal

Quantitative analysis of drug effects at the whole-body level: a case study for glucose metabolism in malaria patients

Abstract (Expand)

We propose a hierarchical modelling approach to construct models for disease states at the whole-body level. Such models can simulate effects of drug-induced inhibition of reaction steps on the whole-body … physiology. We illustrate the approach for glucose metabolism in malaria patients, by merging two detailed kinetic models for glucose metabolism in the parasite Plasmodium falciparum and the human red blood cell with a coarse-grained model for whole-body glucose metabolism. In addition we use a genome-scale metabolic model for the parasite to predict amino acid production profiles by the malaria parasite that can be used as a complex biomarker.

Authors: J. L. Snoep, K. Green, J. Eicher, D. C. Palm, G. Penkler, F. du Toit, N. Walters, R. Burger, H. V. Westerhoff, D. D. van Niekerk

Date Published: 27th Nov 2015

Publication Type: Not specified

Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells.

Abstract (Expand)

The mitochondrial NAD pool is particularly important for the maintenance of vital cellular functions. Although at least in some fungi and plants, mitochondrial NAD is imported from the cytosol by carrier … proteins, in mammals, the mechanism of how this organellar pool is generated has remained obscure. A transporter mediating NAD import into mammalian mitochondria has not been identified. In contrast, human recombinant NMNAT3 localizes to the mitochondrial matrix and is able to catalyze NAD(+) biosynthesis in vitro. However, whether the endogenous NMNAT3 protein is functionally effective at generating NAD(+) in mitochondria of intact human cells still remains to be demonstrated. To modulate mitochondrial NAD(+) content, we have expressed plant and yeast mitochondrial NAD(+) carriers in human cells and observed a profound increase in mitochondrial NAD(+). None of the closest human homologs of these carriers had any detectable effect on mitochondrial NAD(+) content. Surprisingly, constitutive redistribution of NAD(+) from the cytosol to the mitochondria by stable expression of the Arabidopsis thaliana mitochondrial NAD(+) transporter NDT2 in HEK293 cells resulted in dramatic growth retardation and a metabolic shift from oxidative phosphorylation to glycolysis, despite the elevated mitochondrial NAD(+) levels. These results suggest that a mitochondrial NAD(+) transporter, similar to the known one from A. thaliana, is likely absent and could even be harmful in human cells. We provide further support for the alternative possibility, namely intramitochondrial NAD(+) synthesis, by demonstrating the presence of endogenous NMNAT3 in the mitochondria of human cells.

Authors: M. R. VanLinden, C. Dolle, I. K. Pettersen, V. A. Kulikova, M. Niere, G. Agrimi, S. E. Dyrstad, F. Palmieri, A. A. Nikiforov, K. J. Tronstad, M. Ziegler

Date Published: 13th Nov 2015

Publication Type: Not specified

Data2Dynamics: a modeling environment tailored to parameter estimation in dynamical systems.

Abstract (Expand)

UNLABELLED: Modeling of dynamical systems using ordinary differential equations is a popular approach in the field of systems biology. Two of the most critical steps in this approach are to construct … dynamical models of biochemical reaction networks for large datasets and complex experimental conditions and to perform efficient and reliable parameter estimation for model fitting. We present a modeling environment for MATLAB that pioneers these challenges. The numerically expensive parts of the calculations such as the solving of the differential equations and of the associated sensitivity system are parallelized and automatically compiled into efficient C code. A variety of parameter estimation algorithms as well as frequentist and Bayesian methods for uncertainty analysis have been implemented and used on a range of applications that lead to publications. AVAILABILITY AND IMPLEMENTATION: The Data2Dynamics modeling environment is MATLAB based, open source and freely available at http://www.data2dynamics.org. CONTACT: andreas.raue@fdm.uni-freiburg.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Authors: A. Raue, B. Steiert, M. Schelker, C. Kreutz, T. Maiwald, H. Hass, J. Vanlier, C. Tonsing, L. Adlung, R. Engesser, W. Mader, T. Heinemann, J. Hasenauer, M. Schilling, T. Hofer, E. Klipp, F. Theis, U. Klingmuller, B. Schoberl, J. Timmer

Date Published: 1st Nov 2015

Publication Type: Journal

Current and Future Chemotherapy for Chagas Disease.

Abstract (Expand)

Human American trypanosomiasis, commonly called Chagas disease, is one of the most neglected illnesses in the world and remains one of the most prevalent chronic infectious diseases of Latin America … with thousands of new cases every year. The only treatments available have been introduced five decades ago. They have serious, undesirable side effects and disputed benefits in the chronic stage of the disease - a characteristic and debilitating cardiomyopathy and/or megavisceras. Several laboratories have therefore focused their efforts in finding better drugs. Although recent years have brought new clinical trials, these are few and lack diversity in terms of drug mechanism of action, thus resulting in a weak drug discovery pipeline. This fragility has been recently exposed by the failure of two candidates; posaconazole and E1224, to sterilely cure patients in phase 2 clinical trials. Such setbacks highlight the need for continuous, novel and high quality drug discovery and development efforts to discover better and safer treatments. In this article we will review past and current findings on drug discovery for Trypanosoma cruzi made by academic research groups, industry and other research organizations over the last half century. We also analyze the current research landscape that is now better placed than ever to deliver alternative treatments for Chagas disease in the near future.

Authors: L. Gaspar, C. B. Moraes, L. H. Freitas-Junior, S. Ferrari, L. Costantino, M. P. Costi, R. P. Coron, T. K. Smith, J. L. Siqueira-Neto, J. H. McKerrow, A. Cordeiro-da-Silva

Date Published: 20th Oct 2015

Publication Type: Journal

Defining the robust behaviour of the plant clock gene circuit with absolute RNA timeseries and open infrastructure

Abstract (Expand)

Our understanding of the complex, transcriptional feedback loops in the circadian clock mechanism has depended upon quantitative, timeseries data from disparate sources. We measure clock gene RNA … profiles in Arabidopsis thaliana seedlings, grown with or without exogenous sucrose, or in soil-grown plants and in wild-type and mutant backgrounds. The RNA profiles were strikingly robust across the experimental conditions, so current mathematical models are likely to be broadly applicable in leaf tissue. In addition to providing reference data, unexpected behaviours included co-expression of PRR9 and ELF4, and regulation of PRR5 by GI. Absolute RNA quantification revealed low levels of PRR9 transcripts (peak approx. 50 copies cell(-1)) compared with other clock genes, and threefold higher levels of LHY RNA (more than 1500 copies cell(-1)) than of its close relative CCA1. The data are disseminated from BioDare, an online repository for focused timeseries data, which is expected to benefit mechanistic modelling. One data subset successfully constrained clock gene expression in a complex model, using publicly available software on parallel computers, without expert tuning or programming. We outline the empirical and mathematical justification for data aggregation in understanding highly interconnected, dynamic networks such as the clock, and the observed design constraints on the resources required to make this approach widely accessible.

Authors: A. Flis, A. P. Fernandez, T. Zielinski, V. Mengin, R. Sulpice, K. Stratford, A. Hume, A. Pokhilko, M. M. Southern, D. D. Seaton, H. G. McWatters, M. Stitt, K. J. Halliday, A. J. Millar

Date Published: 16th Oct 2015

Publication Type: Not specified

Severe Acute Respiratory Syndrome (SARS) Coronavirus ORF8 Protein Is Acquired from SARS-Related Coronavirus from Greater Horseshoe Bats through Recombination

Abstract (Expand)

Despite the identification of horseshoe bats as the reservoir of severe acute respiratory syndrome (SARS)-related coronaviruses (SARSr-CoVs), the origin of SARS-CoV ORF8, which contains the 29-nucleotide … signature deletion among human strains, remains obscure. Although two SARS-related Rhinolophus sinicus bat CoVs (SARSr-Rs-BatCoVs) previously detected in Chinese horseshoe bats (Rhinolophus sinicus) in Yunnan, RsSHC014 and Rs3367, possessed 95% genome identities to human and civet SARSr-CoVs, their ORF8 protein exhibited only 32.2 to 33% amino acid identities to that of human/civet SARSr-CoVs. To elucidate the origin of SARS-CoV ORF8, we sampled 348 bats of various species in Yunnan, among which diverse alphacoronaviruses and betacoronaviruses, including potentially novel CoVs, were identified, with some showing potential interspecies transmission. The genomes of two betacoronaviruses, SARSr-Rf-BatCoV YNLF_31C and YNLF_34C, from greater horseshoe bats (Rhinolophus ferrumequinum), possessed 93% nucleotide identities to human/civet SARSr-CoV genomes. Although these two betacoronaviruses displayed lower similarities than SARSr-Rs-BatCoV RsSHC014 and Rs3367 in S protein to civet SARSr-CoVs, their ORF8 proteins demonstrated exceptionally high (80.4 to 81.3%) amino acid identities to that of human/civet SARSr-CoVs, compared to SARSr-BatCoVs from other horseshoe bats (23.2 to 37.3%). Potential recombination events were identified around ORF8 between SARSr-Rf-BatCoVs and SARSr-Rs-BatCoVs, leading to the generation of civet SARSr-CoVs. The expression of ORF8 subgenomic mRNA suggested that the ORF8 protein may be functional in SARSr-Rf-BatCoVs. The high Ka/Ks ratio among human SARS-CoVs compared to that among SARSr-BatCoVs supported that ORF8 is under strong positive selection during animal-to-human transmission. Molecular clock analysis using ORF1ab showed that SARSr-Rf-BatCoV YNLF_31C and YNLF_34C diverged from civet/human SARSr-CoVs in approximately 1990. SARS-CoV ORF8 originated from SARSr-CoVs of greater horseshoe bats through recombination, which may be important for animal-to-human transmission., IMPORTANCE Although horseshoe bats are the primary reservoir of SARS-related coronaviruses (SARSr-CoVs), it is still unclear how these bat viruses have evolved to cross the species barrier to infect civets and humans. Most human SARS-CoV epidemic strains contain a signature 29-nucleotide deletion in ORF8, compared to civet SARSr-CoVs, suggesting that ORF8 may be important for interspecies transmission. However, the origin of SARS-CoV ORF8 remains obscure. In particular, SARSr-Rs-BatCoVs from Chinese horseshoe bats (Rhinolophus sinicus) exhibited \textless40% amino acid identities to human/civet SARS-CoV in the ORF8 protein. We detected diverse alphacoronaviruses and betacoronaviruses among various bat species in Yunnan, China, including two SARSr-Rf-BatCoVs from greater horseshoe bats that possessed ORF8 proteins with exceptionally high amino acid identities to that of human/civet SARSr-CoVs. We demonstrated recombination events around ORF8 between SARSr-Rf-BatCoVs and SARSr-Rs-BatCoVs, leading to the generation of civet SARSr-CoVs. Our findings offer insight into the evolutionary origin of SARS-CoV ORF8 protein, which was likely acquired from SARSr-CoVs of greater horseshoe bats through recombination.

Authors: Susanna K. P. Lau, Yun Feng, Honglin Chen, Hayes K. H. Luk, Wei-Hong Yang, Kenneth S. M. Li, Yu-Zhen Zhang, Yi Huang, Zhi-Zhong Song, Wang-Ngai Chow, Rachel Y. Y. Fan, Syed Shakeel Ahmed, Hazel C. Yeung, Carol S. F. Lam, Jian-Piao Cai, Samson S. Y. Wong, Jasper F. W. Chan, Kwok-Yung Yuen, Hai-Lin Zhang, Patrick C. Y. Woo

Date Published: 22nd Sep 2015

Publication Type: Journal

Copy number profiling by array comparative genomic hybridization identifies frequently occurring BRCA2-like male breast cancer.

Abstract (Expand)

Genomic aberrations can be used to subtype breast cancer. In this study, we investigated DNA copy number (CN) profiles of 69 cases of male breast cancer (MBC) by array comparative genomic hybridization … (aCGH) to detect recurrent gains and losses in comparison with female breast cancers (FBC). Further, we classified these profiles as BRCA1-like, BRCA2-like or non-BRCA-like profiles using previous classifiers derived from FBC, and correlated these profiles with pathological characteristics. We observed large CN gains on chromosome arms 1q, 5p, 8q, 10p, 16p, 17q, and chromosomes 20 and X. Large losses were seen on chromosomes/chromosome arms 1p, 6p, 8p, 9, 11q, 13, 14q, 16q, 17p, and 22. The pattern of gains and losses in estrogen receptor positive (ER+) MBC was largely similar to ER+ FBC, except for gains on chromosome X in MBC, which were uncommon in FBC. Out of 69 MBC patients, 15 patients (22%) had a BRCA2-like profile, of which 2 (3%) were also BRCA1-like. One patient (1%) was only BRCA1-like; the remaining 53 (77%) patients were classified as non-BRCA-like. BRCA2-like cases were more often p53 accumulated than non-BRCA-like cases (P = 0.014). In conclusion, the pattern of gains and losses in ER+ MBC was largely similar to that of its ER+ FBC counterpart, except for gains on chromosome X in MBC, which are uncommon in FBC. A significant proportion of MBC has a BRCA2-like aCGH profile, pointing to a potentially hereditary nature, and indicating that they could benefit from a drug regimen targeting BRCA defects as in FBC.

Authors: H. D. Biesma, P. C. Schouten, M. M. Lacle, J. Sanders, W. Brugman, R. Kerkhoven, I. Mandjes, P. van der Groep, P. J. van Diest, S. C. Linn

Date Published: 11th Sep 2015

Publication Type: Journal

Metabolic efficiency in yeast Saccharomyces cerevisiae in relation to temperature dependent growth and biomass yield

Abstract (Expand)

Canonized view on temperature effects on growth rate of microorganisms is based on assumption of protein denaturation, which is not confirmed experimentally so far. We develop an alternative concept, … which is based on view that limits of thermal tolerance are based on imbalance of cellular energy allocation. Therefore, we investigated growth suppression of yeast Saccharomyces cerevisiae in the supraoptimal temperature range (30–40 °C), i.e. above optimal temperature (Topt). The maximal specific growth rate (μmax) of biomass, its concentration and yield on glucose (Yx/glc) were measured across the whole thermal window (5–40 °C) of the yeast in batch anaerobic growth on glucose. Specific rate of glucose consumption, specific rate of glucose consumption for maintenance (mglc), true biomass yield on glucose (View the MathML source), fractional conservation of substrate carbon in product and ATP yield on glucose (Yatp/glc) were estimated from the experimental data. There was a negative linear relationship between ATP, ADP and AMP concentrations and specific growth rate at any growth conditions, whilst the energy charge was always high (~0.83). There were two temperature regions where mglc differed 12-fold, which points to the existence of a ‘low’ (within 5–31 °C) and a ‘high’ (within 33–40 °C) metabolic mode regarding maintenance requirements. The rise from the low to high mode occurred at 31–32 °C in step-wise manner and it was accompanied with onset of suppression of μmax. High mglc at supraoptimal temperatures indicates a significant reduction of scope for growth, due to high maintenance cost. Analysis of temperature dependencies of product formation efficiency and Yatp/glc revealed that the efficiency of energy metabolism approaches its lower limit at 26–31 °C. This limit is reflected in the predetermined combination of View the MathML source, elemental biomass composition and degree of reduction of the growth substrate. Approaching the limit implies a reduction of the safety margin of metabolic efficiency. We hypothesize that a temperature increase above Topt (e.g. >31 °C) triggers both an increment in mglc and suppression of μmax, which together contribute to an upshift of Yatp/glc from the lower limit and thus compensate for the loss of the safety margin. This trade-off allows adding 10 more degrees to Topt and extends the thermal window up to 40 °C, sustaining survival and reproduction in supraoptimal temperatures. Deeper understanding of the limits of thermal tolerance can be practically exploited in biotechnological applications.

Authors: Maksim Zakhartsev, Xuelian Yang, Matthias Reuss, Hans Otto Pörtner

Date Published: 1st Aug 2015

Publication Type: Not specified

Entrainment of heterogeneous glycolytic oscillations in single cells

Abstract (Expand)

Cell signaling, gene expression, and metabolism are affected by cell-cell heterogeneity and random changes in the environment. The effects of such fluctuations on cell signaling and gene expression … have recently been studied intensively using single-cell experiments. In metabolism heterogeneity may be particularly important because it may affect synchronisation of metabolic oscillations, an important example of cell-cell communication. This synchronisation is notoriously difficult to describe theoretically as the example of glycolytic oscillations shows: neither is the mechanism of glycolytic synchronisation understood nor the role of cell-cell heterogeneity. To pin down the mechanism and to assess its robustness and universality we have experimentally investigated the entrainment of glycolytic oscillations in individual yeast cells by periodic external perturbations. We find that oscillatory cells synchronise through phase shifts and that the mechanism is insensitive to cell heterogeneity (robustness) and similar for different types of external perturbations (universality).

Authors: Anna-Karin Gustavsson, Caroline B. Adiels, Bernhard Mehlig, Mattias Goksör

Date Published: 1st Aug 2015

Publication Type: Not specified

Energy Conservation Associated with Ethanol Formation from H2 and CO2 in Clostridium autoethanogenum Involving Electron Bifurcation.

Abstract (Expand)

UNLABELLED: Most acetogens can reduce CO2 with H2 to acetic acid via the Wood-Ljungdahl pathway, in which the ATP required for formate activation is regenerated in the acetate kinase reaction. However, … a few acetogens, such as Clostridium autoethanogenum, Clostridium ljungdahlii, and Clostridium ragsdalei, also form large amounts of ethanol from CO2 and H2. How these anaerobes with a growth pH optimum near 5 conserve energy has remained elusive. We investigated this question by determining the specific activities and cofactor specificities of all relevant oxidoreductases in cell extracts of H2/CO2-grown C. autoethanogenum. The activity studies were backed up by transcriptional and mutational analyses. Most notably, despite the presence of six hydrogenase systems of various types encoded in the genome, the cells appear to contain only one active hydrogenase. The active [FeFe]-hydrogenase is electron bifurcating, with ferredoxin and NADP as the two electron acceptors. Consistently, most of the other active oxidoreductases rely on either reduced ferredoxin and/or NADPH as the electron donor. An exception is ethanol dehydrogenase, which was found to be NAD specific. Methylenetetrahydrofolate reductase activity could only be demonstrated with artificial electron donors. Key to the understanding of this energy metabolism is the presence of membrane-associated reduced ferredoxin:NAD(+) oxidoreductase (Rnf), of electron-bifurcating and ferredoxin-dependent transhydrogenase (Nfn), and of acetaldehyde:ferredoxin oxidoreductase, which is present with very high specific activities in H2/CO2-grown cells. Based on these findings and on thermodynamic considerations, we propose metabolic schemes that allow, depending on the H2 partial pressure, the chemiosmotic synthesis of 0.14 to 1.5 mol ATP per mol ethanol synthesized from CO2 and H2. IMPORTANCE: Ethanol formation from syngas (H2, CO, and CO2) and from H2 and CO2 that is catalyzed by bacteria is presently a much-discussed process for sustainable production of biofuels. Although the process is already in use, its biochemistry is only incompletely understood. The most pertinent question is how the bacteria conserve energy for growth during ethanol formation from H2 and CO2, considering that acetyl coenzyme A (acetyl-CoA), is an intermediate. Can reduction of the activated acetic acid to ethanol with H2 be coupled with the phosphorylation of ADP? Evidence is presented that this is indeed possible, via both substrate-level phosphorylation and electron transport phosphorylation. In the case of substrate-level phosphorylation, acetyl-CoA reduction to ethanol proceeds via free acetic acid involving acetaldehyde:ferredoxin oxidoreductase (carboxylate reductase).

Authors: J. Mock, Y. Zheng, A. P. Mueller, S. Ly, L. Tran, S. Segovia, S. Nagaraju, M. Kopke, P. Durre, R. K. Thauer

Date Published: 8th Jul 2015

Publication Type: Journal

An Ultrasensitive Mechanism Regulates Influenza Virus-Induced Inflammation

Abstract (Expand)

Influenza viruses present major challenges to public health, evident by the 2009 influenza pandemic. Highly pathogenic influenza virus infections generally coincide with early, high levels of inflammatory … cytokines that some studies have suggested may be regulated in a strain-dependent manner. However, a comprehensive characterization of the complex dynamics of the inflammatory response induced by virulent influenza strains is lacking. Here, we applied gene co-expression and nonlinear regression analysis to time-course, microarray data developed from influenza-infected mouse lung to create mathematical models of the host inflammatory response. We found that the dynamics of inflammation-associated gene expression are regulated by an ultrasensitive-like mechanism in which low levels of virus induce minimal gene expression but expression is strongly induced once a threshold virus titer is exceeded. Cytokine assays confirmed that the production of several key inflammatory cytokines, such as interleukin 6 and monocyte chemotactic protein 1, exhibit ultrasensitive behavior. A systematic exploration of the pathways regulating the inflammatoryassociated gene response suggests that the molecular origins of this ultrasensitive response mechanism lie within the branch of the Toll-like receptor pathway that regulates STAT1 phosphorylation. This study provides the first evidence of an ultrasensitive mechanism regulating influenza virus-induced inflammation in whole lungs and provides insight into how different virus strains can induce distinct temporal inflammation response profiles. The approach developed here should facilitate the construction of gene regulatory models of other infectious diseases.

Authors: Jason E. Shoemaker, Satoshi Fukuyama, Amie J. Eisfeld, Dongming Zhao, Eiryo Kawakami, Saori Sakabe, Tadashi Maemura, Takeo Gorai, Hiroaki Katsura, Yukiko Muramoto, Shinji Watanabe, Tokiko Watanabe, Ken Fuji, Yukiko Matsuoka, Hiroaki Kitano, Yoshihiro Kawaoka

Date Published: 5th Jun 2015

Publication Type: Journal

Disentangling the Complexity of HGF Signaling by Combining Qualitative and Quantitative Modeling.

Abstract (Expand)

Signaling pathways are characterized by crosstalk, feedback and feedforward mechanisms giving rise to highly complex and cell-context specific signaling networks. Dissecting the underlying relations … is crucial to predict the impact of targeted perturbations. However, a major challenge in identifying cell-context specific signaling networks is the enormous number of potentially possible interactions. Here, we report a novel hybrid mathematical modeling strategy to systematically unravel hepatocyte growth factor (HGF) stimulated phosphoinositide-3-kinase (PI3K) and mitogen activated protein kinase (MAPK) signaling, which critically contribute to liver regeneration. By combining time-resolved quantitative experimental data generated in primary mouse hepatocytes with interaction graph and ordinary differential equation modeling, we identify and experimentally validate a network structure that represents the experimental data best and indicates specific crosstalk mechanisms. Whereas the identified network is robust against single perturbations, combinatorial inhibition strategies are predicted that result in strong reduction of Akt and ERK activation. Thus, by capitalizing on the advantages of the two modeling approaches, we reduce the high combinatorial complexity and identify cell-context specific signaling networks.

Authors: L. A. D'Alessandro, R. Samaga, T. Maiwald, S. H. Rho, S. Bonefas, A. Raue, N. Iwamoto, A. Kienast, K. Waldow, R. Meyer, M. Schilling, J. Timmer, S. Klamt, U. Klingmuller

Date Published: 24th Apr 2015

Publication Type: Journal

Construction and validation of a detailed kinetic model of glycolysis in Plasmodium falciparum

Abstract (Expand)

The enzymes in the Embden–Meyerhof–Parnas pathway of Plasmodium falciparum trophozoites were kinetically characterized and their integrated activities analyzed in a mathematical model. For validation … of the model, we compared model predictions for steady-state fluxes and metabolite concentrations of the hexose phosphates with experimental values for intact parasites. The model, which is completely based on kinetic parameters that were measured for the individual enzymes, gives an accurate prediction of the steady-state fluxes and intermediate concentrations. This is the first detailed kinetic model for glucose metabolism in P. falciparum, one of the most prolific malaria-causing protozoa, and the high predictive power of the model makes it a strong tool for future drug target identification studies. The modelling workflow is transparent and reproducible, and completely documented in the SEEK platform, where all experimental data and model files are available for download.

Authors: Gerald Penkler, Francois du Toit, Waldo Adams, Marina Rautenbach, Daniel C. Palm, David D. van Niekerk, Jacky L. Snoep

Date Published: 1st Apr 2015

Publication Type: Not specified

Fast sampling for quantitative microbial metabolomics: new aspects on cold methanol quenching: metabolite co-precipitation

Abstract (Expand)

The intra- and extracellular concentrations of 16 metabolites were measured in chemostat (D = 0.1 h−1) anaerobic cultures of the yeast Saccharomyces cerevisiae CEN.PK-113-7D growing on minimal medium. … Two independent sampling workflows were employed: (i) conventional cold methanol quenching and (ii) a differential approach. Metabolites were quantified in different sample fractions (total, extracellular, quenching supernatant, methanol/water extract and pellet) in order to derive their mass balance. The differential method in combination with absolute metabolite quantification by gas-chromatography with isotope dilution mass spectrometry (GC–IDMS) was used as a benchmark to assess quality of the cold methanol quenching procedure. Quantitative comparison of metabolite concentrations in all fractions collected by different quenching techniques indicates asystematic loss of the total mass of various metabolites in course of the cold methanol quenching. Pellet resulting from the cold methanol quenching besides biomass contains considerable amounts of precipitated inorganic salts from the fermentation media. Quantitative analysis has revealed significant co-precipitation of polar extracellular metabolites together with these salts. This phenomenon is especially significant for metabolites with large extracellular mass-fraction. We report that the co-precipitation is a hitherto neglected phenomenon and concluded that its degree strongly linked to culturing conditions (i.e. media composition) and chemical properties of the particular metabolite. Thus, intracellular metabolite levels measured from samples collected by cold methanol quenching might be uncertain and variably biased due to corruption by described phenomena.

Authors: Maksim Zakhartsev, Oliver Vielhauer, Thomas Horn, Xuelian Yang, Matthias Reuss

Date Published: 1st Apr 2015

Publication Type: Not specified

Incorporation of Spike and Membrane Glycoproteins into Coronavirus Virions

Abstract (Expand)

The envelopes of coronaviruses (CoVs) contain primarily three proteins; the two major glycoproteins spike (S) and membrane (M), and envelope (E), a non-glycosylated protein. Unlike other enveloped … viruses, CoVs bud and assemble at the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC). For efficient virion assembly, these proteins must be targeted to the budding site and to interact with each other or the ribonucleoprotein. Thus, the efficient incorporation of viral envelope proteins into CoV virions depends on protein trafficking and protein–protein interactions near the ERGIC. The goal of this review is to summarize recent findings on the mechanism of incorporation of the M and S glycoproteins into the CoV virion, focusing on protein trafficking and protein–protein interactions.

Authors: Makoto Ujike, Fumihiro Taguchi

Date Published: 1st Apr 2015

Publication Type: Journal

RobOKoD: microbial strain design for (over)production of target compounds

Abstract (Expand)

Sustainable production of target compounds such as biofuels and high-value chemicals for pharmaceutical, agrochemical, and chemical industries is becoming an increasing priority given their current … dependency upon diminishing petrochemical resources. Designing these strains is difficult, with current methods focusing primarily on knocking-out genes, dismissing other vital steps of strain design including the overexpression and dampening of genes. The design predictions from current methods also do not translate well-into successful strains in the laboratory. Here, we introduce RobOKoD (Robust, Overexpression, Knockout and Dampening), a method for predicting strain designs for overproduction of targets. The method uses flux variability analysis to profile each reaction within the system under differing production percentages of target-compound and biomass. Using these profiles, reactions are identified as potential knockout, overexpression, or dampening targets. The identified reactions are ranked according to their suitability, providing flexibility in strain design for users. The software was tested by designing a butanol-producing Escherichia coli strain, and was compared against the popular OptKnock and RobustKnock methods. RobOKoD shows favorable design predictions, when predictions from these methods are compared to a successful butanol-producing experimentally-validated strain. Overall RobOKoD provides users with rankings of predicted beneficial genetic interventions with which to support optimized strain design.

Authors: N. J. Stanford, P. Millard, N. Swainston

Date Published: 24th Mar 2015

Publication Type: Not specified

Linked circadian outputs control elongation growth and flowering in response to photoperiod and temperature

Abstract (Expand)

Clock-regulated pathways coordinate the response of many developmental processes to changes in photoperiod and temperature. We model two of the best-understood clock output pathways in Arabidopsis, … which control key regulators of flowering and elongation growth. In flowering, the model predicted regulatory links from the clock to cycling DOF factor 1 (CDF1) and flavin-binding, KELCH repeat, F-box 1 (FKF1) transcription. Physical interaction data support these links, which create threefold feed-forward motifs from two clock components to the floral regulator FT. In hypocotyl growth, the model described clock-regulated transcription of phytochrome-interacting factor 4 and 5 (PIF4, PIF5), interacting with post-translational regulation of PIF proteins by phytochrome B (phyB) and other light-activated pathways. The model predicted bimodal and end-of-day PIF activity profiles that are observed across hundreds of PIF-regulated target genes. In the response to temperature, warmth-enhanced PIF4 activity explained the observed hypocotyl growth dynamics but additional, temperature-dependent regulators were implicated in the flowering response. Integrating these two pathways with the clock model highlights the molecular mechanisms that coordinate plant development across changing conditions.

Authors: D. D. Seaton, R. W. Smith, Y. H. Song, D. R. MacGregor, K. Stewart, G. Steel, J. Foreman, S. Penfield, T. Imaizumi, A. J. Millar, K. J. Halliday

Date Published: 21st Jan 2015

Publication Type: Not specified

Identification of HNRNPK as regulator of hepatitis C virus particle production

Abstract (Expand)

Hepatitis C virus (HCV) is a major cause of chronic liver disease affecting around 130 million people worldwide. While great progress has been made to define the principle steps of the viral life cycle, … detailed knowledge how HCV interacts with its host cells is still limited. To overcome this limitation we conducted a comprehensive whole-virus RNA interference-based screen and identified 40 host dependency and 16 host restriction factors involved in HCV entry/replication or assembly/release. Of these factors, heterogeneous nuclear ribonucleoprotein K (HNRNPK) was found to suppress HCV particle production without affecting viral RNA replication. This suppression of virus production was specific to HCV, independent from assembly competence and genotype, and not found with the related Dengue virus. By using a knock-down rescue approach we identified the domains within HNRNPK required for suppression of HCV particle production. Importantly, HNRNPK was found to interact specifically with HCV RNA and this interaction was impaired by mutations that also reduced the ability to suppress HCV particle production. Finally, we found that in HCV-infected cells, subcellular distribution of HNRNPK was altered; the protein was recruited to sites in close proximity of lipid droplets and colocalized with core protein as well as HCV plus-strand RNA, which was not the case with HNRNPK variants unable to suppress HCV virion formation. These results suggest that HNRNPK might determine efficiency of HCV particle production by limiting the availability of viral RNA for incorporation into virions. This study adds a new function to HNRNPK that acts as central hub in the replication cycle of multiple other viruses.

Authors: M. Poenisch, P. Metz, H. Blankenburg, A. Ruggieri, J. Y. Lee, D. Rupp, I. Rebhan, K. Diederich, L. Kaderali, F. S. Domingues, M. Albrecht, V. Lohmann, H. Erfle, R. Bartenschlager

Date Published: 8th Jan 2015

Publication Type: Not specified

Using a Genome-Scale Metabolic Model of Enterococcus faecalis V583 To Assess Amino Acid Uptake and Its Impact on Central Metabolism

Abstract (Expand)

Increasing antibiotic resistance in pathogenic bacteria necessitates the development of new medication strategies. Interfering with the metabolic network of the pathogen can provide novel drug targets … but simultaneously requires a deeper and more detailed organism-specific understanding of the metabolism, which is often surprisingly sparse. In light of this, we reconstructed a genome-scale metabolic model of the pathogen Enterococcus faecalis V583. The manually curated metabolic network comprises 642 metabolites and 706 reactions. We experimentally determined metabolic profiles of E. faecalis grown in chemically defined medium in an anaerobic chemostat setup at different dilution rates and calculated the net uptake and product fluxes to constrain the model. We computed growth-associated energy and maintenance parameters and studied flux distributions through the metabolic network. Amino acid auxotrophies were identified experimentally for model validation and revealed seven essential amino acids. In addition, the important metabolic hub of glutamine/glutamate was altered by constructing a glutamine synthetase knockout mutant. The metabolic profile showed a slight shift in the fermentation pattern toward ethanol production and increased uptake rates of multiple amino acids, especially l-glutamine and l-glutamate. The model was used to understand the altered flux distributions in the mutant and provided an explanation for the experimentally observed redirection of the metabolic flux. We further highlighted the importance of gene-regulatory effects on the redirection of the metabolic fluxes upon perturbation. The genome-scale metabolic model presented here includes gene-protein-reaction associations, allowing a further use for biotechnological applications, for studying essential genes, proteins, or reactions, and the search for novel drug targets.

Authors: N. Veith, M. Solheim, K. W. van Grinsven, B. G. Olivier, J. Levering, R. Grosseholz, J. Hugenholtz, H. Holo, I. Nes, B. Teusink, U. Kummer

Date Published: 19th Dec 2014

Publication Type: Not specified

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