Publications

573 Publications visible to you, out of a total of 573

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

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

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

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

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

Abstract (Expand)

STAT5A and STAT5B are important transcription factors that dimerize and transduce activation signals of cytokine receptors directly to the nucleus. A typical cytokine that mediates STAT5 activation is erythropoietin (Epo). Differential functions of STAT5A and STAT5B have been reported. However, the extent to which phosphorylated STAT5A and STAT5B (pSTAT5A, pSTAT5B) form homo- or heterodimers is not understood, nor is how this might influence the signal transmission to the nucleus. To study this, we designed a concept to investigate the isoform-specific dimerization behavior of pSTAT5A and pSTAT5B that comprises isoform-specific immunoprecipitation (IP), measurement of the degree of phosphorylation, and isoform ratio determination between STAT5A and STAT5B. For the main analytical method, we employed quantitative label-free and -based mass spectrometry. For the cellular model system, we used Epo receptor (EpoR)-expressing BaF3 cells (BaF3-EpoR) stimulated with Epo. Three hypotheses of dimer formation between pSTAT5A and pSTAT5B were used to explain the analytical results by a static mathematical model: formation of (i) homodimers only, (ii) heterodimers only, and (iii) random formation of homo- and heterodimers. The best agreement between experimental data and model simulations was found for the last case. Dynamics of cytoplasmic STAT5 dimerization could be explained by distinct nuclear import rates and individual nuclear retention for homo- and heterodimers of phosphorylated STAT5.

Authors: M. E. Boehm, L. Adlung, M. Schilling, S. Roth, U. Klingmuller, W. D. Lehmann

Date Published: 5th Dec 2014

Publication Type: Journal

Abstract (Expand)

Kinetoplastea such as trypanosomatid parasites contain specialized peroxisomes that uniquely contain enzymes of the glycolytic pathway and other parts of intermediary metabolism and hence are called glycosomes. Their specific enzyme content can vary strongly, quantitatively and qualitatively, between different species and during the parasites’ life cycle. The correct sequestering of enzymes has great importance for the regulation of the trypanosomatids’ metabolism and can, dependent on environmental conditions, even be essential. Glycosomes also play a pivotal role in life-cycle regulation of Trypanosoma brucei, as the translocation of a protein phosphatase from the cytosol forms part of a crucial developmental control switch. Many glycosomal proteins are differentially phosphorylated in different life-cycle stages, possibly indicative for unique forms of activity regulation, whereas many kinetic activity regulation mechanisms common for glycolytic enzymes are absent in these organisms. Glycosome turnover occurs by autophagic degradation of redundant organelles and assembly of new ones. This may provide the trypanosomatids with a manner to rapidly and efficiently adapt their metabolism to the sudden, major nutritional changes often encountered during the life cycle. This could also have helped facilitating successful adaptation of kinetoplastids, at multiple occasions during evolution, to their parasitic life style.

Authors: Balázs Szöör, , Melisa Gualdrón-López, Paul AM Michels

Date Published: 1st Dec 2014

Publication Type: Not specified

Abstract (Expand)

Biomass-derived d-xylose represents an economically interesting substrate for the sustainable microbial production of value-added compounds. The industrially important platform organism Corynebacterium glutamicum has already been engineered to grow on this pentose as sole carbon and energy source. However, all currently described C. glutamicum strains utilize d-xylose via the commonly known isomerase pathway that leads to a significant carbon loss in the form of CO2, in particular, when aiming for the synthesis of alpha-ketoglutarate and its derivatives (e.g. l-glutamate). Driven by the motivation to engineer a more carbon-efficient C. glutamicum strain, we functionally integrated the Weimberg pathway from Caulobacter crescentus in C. glutamicum. This five-step pathway, encoded by the xylXABCD-operon, enabled a recombinant C. glutamicum strain to utilize d-xylose in d-xylose/d-glucose mixtures. Interestingly, this strain exhibited a tri-phasic growth behavior and transiently accumulated d-xylonate during d-xylose utilization in the second growth phase. However, this intermediate of the implemented oxidative pathway was re-consumed in the third growth phase leading to more biomass formation. Furthermore, C. glutamicum pEKEx3-xylXABCDCc was also able to grow on d-xylose as sole carbon and energy source with a maximum growth rate of mumax=0.07+/-0.01h(-1). These results render C. glutamicum pEKEx3-xylXABCDCc a promising starting point for the engineering of efficient production strains, exhibiting only minimal carbon loss on d-xylose containing substrates.

Authors: A. Radek, K. Krumbach, J. Gatgens, V. F. Wendisch, W. Wiechert, M. Bott, S. Noack, J. Marienhagen

Date Published: 12th Oct 2014

Publication Type: Not specified

Abstract (Expand)

For adaptation between anaerobic, micro-aerobic and aerobic conditions Escherichia coli's metabolism and in particular its electron transport chain (ETC) is highly regulated. Although it is known that the global transcriptional regulators FNR and ArcA are involved in oxygen response it is unclear how they interplay in the regulation of ETC enzymes under micro-aerobic chemostat conditions. Also, there are diverse results which and how quinones (oxidised/reduced, ubiquinone/other quinones) are controlling the ArcBA two-component system. In the following a mathematical model of the E. coli ETC linked to basic modules for substrate uptake, fermentation product excretion and biomass formation is introduced. The kinetic modelling focusses on regulatory principles of the ETC for varying oxygen conditions in glucose-limited continuous cultures. The model is based on the balance of electron donation (glucose) and acceptance (oxygen or other acceptors). Also, it is able to account for different chemostat conditions due to changed substrate concentrations and dilution rates. The parameter identification process is divided into an estimation and a validation step based on previously published and new experimental data. The model shows that experimentally observed, qualitatively different behaviour of the ubiquinone redox state and the ArcA activity profile in the micro-aerobic range for different experimental conditions can emerge from a single network structure. The network structure features a strong feed-forward effect from the FNR regulatory system to the ArcBA regulatory system via a common control of the dehydrogenases of the ETC. The model supports the hypothesis that ubiquinone but not ubiquinol plays a key role in determining the activity of ArcBA in a glucose-limited chemostat at micro-aerobic conditions.

Editor:

Date Published: 30th Sep 2014

Publication Type: Not specified

Abstract (Expand)

Multistable gene regulatory systems sustain different levels of gene expression under identical external conditions. Such multistability is used to encode phenotypic states in processes including nutrient uptake and persistence in bacteria, fate selection in viral infection, cell-cycle control and development. Stochastic switching between different phenotypes can occur as the result of random fluctuations in molecular copy numbers of mRNA and proteins arising in transcription, translation, transport and binding. However, which component of a pathway triggers such a transition is generally not known. By linking single-cell experiments on the lactose-uptake pathway in E. coli to molecular simulations, we devise a general method to pinpoint the particular fluctuation driving phenotype switching and apply this method to the transition between the uninduced and induced states of the lac-genes. We find that the transition to the induced state is not caused only by the single event of lac-repressor unbinding, but depends crucially on the time period over which the repressor remains unbound from the lac-operon. We confirm this notion in strains with a high expression level of the lac-repressor (leading to shorter periods over which the lac-operon remains unbound), which show a reduced switching rate. Our techniques apply to multistable gene regulatory systems in general and allow to identify the molecular mechanisms behind stochastic transitions in gene regulatory circuits.

Editor:

Date Published: 24th Sep 2014

Publication Type: Not specified

Abstract (Expand)

Mesenchymal stromal cells (MSCs) possess broad immunomodulatory capacities that are currently investigated for potential clinical application in treating autoimmune disorders. Third-party MSCs suppress alloantigen-induced proliferation of peripheral blood mononuclear cells providing the rationale for clinical use in graft-versus-host disease (GvHD). We confirmed that MSCs strongly inhibited proliferation of CD8(+) T cells in a mixed lymphocyte reaction. However, MSCs also suppressed proliferation of T cells specifically recognizing cytomegalovirus (CMV) and influenza virus. Inhibition was dose dependent, but independent of the culture medium. MSCs inhibited proliferation of specific CD8(+) T cells and the release of IFN-gamma by specific CD8(+) T cells for immunodominant HLA-A2- and HLA-B7- restricted antigen epitopes derived from CMV phosphoprotein 65 and influenza matrix protein. This is in contrast to a recently reported scenario where MSCs exert differential effects on alloantigen and virus-specific T cells potentially having an impact on surveillance and prophylaxis of patients treated by MSCs.

Authors: G. Malcherek, N. Jin, A. G. Huckelhoven, J. Mani, L. Wang, U. Gern, A. Diehlmann, P. Wuchter, A. Schmitt, B. Chen, A. D. Ho, M. Schmitt

Date Published: 18th Sep 2014

Publication Type: Journal

Abstract (Expand)

Understanding how dynamic molecular networks affect whole-organism physiology, analogous to mapping genotype to phenotype, remains a key challenge in biology. Quantitative models that represent processes at multiple scales and link understanding from several research domains can help to tackle this problem. Such integrated models are more common in crop science and ecophysiology than in the research communities that elucidate molecular networks. Several laboratories have modeled particular aspects of growth in Arabidopsis thaliana, but it was unclear whether these existing models could productively be combined. We test this approach by constructing a multiscale model of Arabidopsis rosette growth. Four existing models were integrated with minimal parameter modification (leaf water content and one flowering parameter used measured data). The resulting framework model links genetic regulation and biochemical dynamics to events at the organ and whole-plant levels, helping to understand the combined effects of endogenous and environmental regulators on Arabidopsis growth. The framework model was validated and tested with metabolic, physiological, and biomass data from two laboratories, for five photoperiods, three accessions, and a transgenic line, highlighting the plasticity of plant growth strategies. The model was extended to include stochastic development. Model simulations gave insight into the developmental control of leaf production and provided a quantitative explanation for the pleiotropic developmental phenotype caused by overexpression of miR156, which was an open question. Modular, multiscale models, assembling knowledge from systems biology to ecophysiology, will help to understand and to engineer plant behavior from the genome to the field.

Authors: Y. H. Chew, B. Wenden, A. Flis, V. Mengin, J. Taylor, C. L. Davey, C. Tindal, H. Thomas, H. J. Ougham, P. de Reffye, M. Stitt, M. Williams, R. Muetzelfeldt, K. J. Halliday, A. J. Millar

Date Published: 10th Sep 2014

Publication Type: Not specified

Abstract (Expand)

Transcription by RNA polymerase may be interrupted by pauses caused by backtracking or misincorporation that can be resolved by the conserved bacterial Gre-factors. However, the consequences of such pausing in the living cell remain obscure. Here, we developed molecular biology and transcriptome sequencing tools in the human pathogen Streptococcus pneumoniae and provide evidence that transcription elongation is rate-limiting on highly expressed genes. Our results suggest that transcription elongation may be a highly regulated step of gene expression in S. pneumoniae. Regulation is accomplished via long-living elongation pauses and their resolution by elongation factor GreA. Interestingly, mathematical modeling indicates that long-living pauses cause queuing of RNA polymerases, which results in 'transcription traffic jams' on the gene and thus blocks its expression. Together, our results suggest that long-living pauses and RNA polymerase queues caused by them are a major problem on highly expressed genes and are detrimental for cell viability. The major and possibly sole function of GreA in S. pneumoniae is to prevent formation of backtracked elongation complexes.

Authors: , , M. Herber, L. Attaiech, , , S. Klumpp, ,

Date Published: 6th Sep 2014

Publication Type: Not specified

Abstract (Expand)

African trypanosomes are an excellent system for quantitative modelling of post-transcriptional mRNA control. Transcription is constitutive and polycistronic; individual mRNAs are excised by trans splicing and polyadenylation. We here measure mRNA decay kinetics in two life cycle stages, bloodstream and procyclic forms, by transcription inhibition and RNASeq. Messenger RNAs with short half-lives tend to show initial fast degradation, followed by a slower phase; they are often stabilized by depletion of the 5'-3' exoribonuclease XRNA. Many longer-lived mRNAs show initial slow degradation followed by rapid destruction: we suggest that the slow phase reflects gradual deadenylation. Developmentally regulated mRNAs often show regulated decay, and switch their decay pattern. Rates of mRNA decay are good predictors of steady state levels for short mRNAs, but mRNAs longer than 3 kb show unexpectedly low abundances. Modelling shows that variations in splicing and polyadenylation rates can contribute to steady-state mRNA levels, but this is completely dependent on competition between processing and co-transcriptional mRNA precursor destruction.

Authors: , M. Ryten, D. Droll, , V. Farber, , C. Merce, , ,

Date Published: 26th Aug 2014

Publication Type: Not specified

Abstract (Expand)

Data-based mathematical modeling of biochemical reaction networks, e.g., by nonlinear ordinary differential equation (ODE) models, has been successfully applied. In this context, parameter estimation and uncertainty analysis is a major task in order to assess the quality of the description of the system by the model. Recently, a broadened eigenvalue spectrum of the Hessian matrix of the objective function covering orders of magnitudes was observed and has been termed as sloppiness. In this work, we investigate the origin of sloppiness from structures in the sensitivity matrix arising from the properties of the model topology and the experimental design. Furthermore, we present strategies using optimal experimental design methods in order to circumvent the sloppiness issue and present nonsloppy designs for a benchmark model.

Authors: Christian Tönsing, Jens Timmer, Clemens Kreutz

Date Published: 1st Aug 2014

Publication Type: Not specified

Abstract (Expand)

An essential function of innate immunity is to distinguish self from non-self and receptors have evolved to specifically recognize viral components and initiate the expression of antiviral proteins to restrict viral replication. Coronaviruses are RNA viruses that replicate in the host cytoplasm and evade innate immune sensing in most cell types, either passively by hiding their viral signatures and limiting exposure to sensors or actively, by encoding viral antagonists to counteract the effects of interferons. Since many cytoplasmic viruses exploit similar mechanisms of innate immune evasion, mechanistic insight into the direct interplay between viral RNA, viral RNA-processing enzymes, cellular sensors and antiviral proteins will be highly relevant to develop novel antiviral targets and to restrict important animal and human infections.

Authors: Eveline Kindler, Volker Thiel

Date Published: 1st Aug 2014

Publication Type: Journal

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Authors: Karl Fogelmark, Carl Troein

Date Published: 17th Jul 2014

Publication Type: Not specified

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Authors: Karl Fogelmark, Carl Troein

Date Published: 17th Jul 2014

Publication Type: Journal

Abstract (Expand)

Solvent screening for in situ liquid extraction of products from acetone-butanol-ethanol (ABE) fermentation was carried out, taking into account biological parameters (biocompatibility, bioavailability, and product yield) and ex-traction performance (partition coefficient and selectivity) determined in real fermentation broth. On the basis of different solvent characteristics obtained from literature, 16 compounds from different chemical families were selected and experimentally evaluated for their extraction capabilities in a real ABE fermentation broth system. From these compounds, nine potential solvents were also tested for their biocompatibility towards Clostridium acetobutylicum. Moreover, bioavailability and differences in substrate consumption and total n-butanol production with respect to solvent-free fermentations were quantified for each biocompatible solvent. Product yield was enhanced in the presence of organic solvents having higher affinity for butanol and butyric acid. Applying this methodology, it was found that the Guerbet alcohol 2-butyl-1-octanol presented the best extracting characteristics (the highest partition coefficient (6.76) and the third highest selectivity (644)), the highest butanol yield (27.4 %), and maintained biocompatibility with C. acetobutylicum.

Authors: H. González-Peñas, T. A. Lu-Chau, M. T. Moreira, J. M. Lema

Date Published: 1st Jul 2014

Publication Type: Journal

Abstract (Expand)

BACKGROUND: Polychlorinated biphenyls (PCBs) are persistent organic pollutants (POPs) with harmful effects in animals and humans. Although PCB 153 is one of the most abundant among PCBs detected in animal tissues, its mechanism of toxicity is not well understood. Only few studies have been conducted to explore genes and pathways affected by PCB 153 by using high throughput transcriptomics approaches. To obtain better insights into toxicity mechanisms, we treated juvenile Atlantic cod (Gadus morhua) with PCB 153 (0.5, 2 and 8 mg/kg body weight) for 2 weeks and performed gene expression analysis in the liver using oligonucleotide arrays. RESULTS: Whole-genome gene expression analysis detected about 160 differentially regulated genes. Functional enrichment, interactome, network and gene set enrichment analysis of the differentially regulated genes suggested that pathways associated with cell cycle, lipid metabolism, immune response, apoptosis and stress response were among the top significantly enriched. Particularly, genes coding for proteins in DNA replication/cell cycle pathways and enzymes of lipid biosynthesis were up-regulated suggesting increased cell proliferation and lipogenesis, respectively. CONCLUSIONS: PCB 153 appears to activate cell proliferation and lipogenic genes in cod liver. Transcriptional up-regulation of marker genes for lipid biosynthesis resembles lipogenic effects previously reported for persistent organic pollutants (POPs) and other environmental chemicals. Our results provide new insights into mechanisms of PCB 153 induced toxicity.

Authors: F. Yadetie, O. A. Karlsen, M. Eide, C. Hogstrand, A. Goksoyr

Date Published: 19th Jun 2014

Publication Type: Not specified

Abstract

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Authors: Anna-Karin Gustavsson, David D. van Niekerk, Caroline B. Adiels, Bob Kooi, Mattias Goksör, Jacky L. Snoep

Date Published: 1st Jun 2014

Publication Type: Not specified

Abstract (Expand)

BACKGROUND AIMS: Human mesenchymal stem or stromal cells (MSCs) represent a potential resource not only for regenerative medicine but also for immunomodulatory cell therapies. The application of different MSC culture protocols has significantly hampered the comparability of experimental and clinical data from different laboratories and has posed a major obstacle for multicenter clinical trials. Manufacturing of cell products for clinical application in the European Community must be conducted in compliance with Good Manufacturing Practice and requires a manufacturing license. In Germany, the Paul-Ehrlich-Institut as the Federal Authority for Vaccines and Biomedicines is critically involved in the approval process. METHODS: This report summarizes a consensus meeting between researchers, clinicians and regulatory experts on standard quality requirements for MSC production. RESULTS: The strategy for quality control testing depends on the product's cell composition, the manufacturing process and the indication and target patient population. Important quality criteria in this sense are, among others, the immunophenotype of the cells, composition of the culture medium and the risk for malignant transformation, as well as aging and the immunosuppressive potential of the manufactured MSCs. CONCLUSIONS: This position paper intends to provide relevant information to interested parties regarding these criteria to foster the development of scientifically valid and harmonized quality standards and to support approval of MSC-based investigational medicinal products.

Authors: P. Wuchter, K. Bieback, H. Schrezenmeier, M. Bornhauser, L. P. Muller, H. Bonig, W. Wagner, R. Meisel, P. Pavel, T. Tonn, P. Lang, I. Muller, M. Renner, G. Malcherek, R. Saffrich, E. C. Buss, P. Horn, M. Rojewski, A. Schmitt, A. D. Ho, R. Sanzenbacher, M. Schmitt

Date Published: 27th May 2014

Publication Type: Journal

Abstract (Expand)

The African trypanosome, Trypanosoma brucei, is a unicellular parasite causing African Trypanosomiasis (sleeping sickness in humans and nagana in animals). Due to some of its unique properties, it has emerged as a popular model organism in systems biology. A predictive quantitative model of glycolysis in the bloodstream form of the parasite has been constructed and updated several times. The Silicon Trypanosome is a project that brings together modellers and experimentalists to improve and extend this core model with new pathways and additional levels of regulation. These new extensions and analyses use computational methods that explicitly take different levels of uncertainty into account. During this project, numerous tools and techniques have been developed for this purpose, which can now be used for a wide range of different studies in systems biology.

Authors: , , , , , , T. Papamarkou, , , , , , , ,

Date Published: 7th May 2014

Publication Type: Not specified

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Escherichia coli is a facultatively anaerobic bacterium. With glucose if no external electron acceptors are available, ATP is produced by substrate level phosphorylation. The intracellular redox balance is maintained by mixed-acid fermentation, that is, the production and excretion of several organic acids. When oxygen is available, E. coli switches to aerobic respiration to achieve redox balance and optimal energy conservation by proton translocation linked to electron transfer. The switch between fermentative and aerobic respiratory growth is driven by extensive changes in gene expression and protein synthesis, resulting in global changes in metabolic fluxes and metabolite concentrations. This oxygen response is determined by the interaction of global and local genetic regulatory mechanisms, as well as by enzymatic regulation. The response is affected by basic physical constraints such as diffusion, thermodynamics and the requirement for a balance of carbon, electrons and energy (predominantly the proton motive force and the ATP pool). A comprehensive systems level understanding of the oxygen response of E. coli requires the integrated interpretation of experimental data that are pertinent to the multiple levels of organization that mediate the response. In the pan-European venture, Systems Biology of Microorganisms (SysMO) and specifically within the project Systems Understanding of Microbial Oxygen Metabolism (SUMO), regulator activities, gene expression, metabolite levels and metabolic flux datasets were obtained using a standardized and reproducible chemostat-based experimental system. These different types and qualities of data were integrated using mathematical models. The approach described here has revealed a much more detailed picture of the aerobic-anaerobic response, especially for the environmentally critical microaerobic range that is located between unlimited oxygen availability and anaerobiosis.

Authors: , , , , , , S. Kunz, , , , , ,

Date Published: 7th May 2014

Publication Type: Not specified

Abstract (Expand)

In the presence of oxygen (O2) the model bacterium Escherichia coli is able to conserve energy by aerobic respiration. Two major terminal oxidases are involved in this process - Cyo has a relatively low affinity for O2 but is able to pump protons and hence is energetically efficient; Cyd has a high affinity for O2 but does not pump protons. When E. coli encounters environments with different O2 availabilities, the expression of the genes encoding the alternative terminal oxidases, the cydAB and cyoABCDE operons, are regulated by two O2-responsive transcription factors, ArcA (an indirect O2 sensor) and FNR (a direct O2 sensor). It has been suggested that O2-consumption by the terminal oxidases located at the cytoplasmic membrane significantly affects the activities of ArcA and FNR in the bacterial nucleoid. In this study, an agent-based modeling approach has been taken to spatially simulate the uptake and consumption of O2 by E. coli and the consequent modulation of ArcA and FNR activities based on experimental data obtained from highly controlled chemostat cultures. The molecules of O2, transcription factors and terminal oxidases are treated as individual agents and their behaviors and interactions are imitated in a simulated 3-D E. coli cell. The model implies that there are two barriers that dampen the response of FNR to O2, i.e. consumption of O2 at the membrane by the terminal oxidases and reaction of O2 with cytoplasmic FNR. Analysis of FNR variants suggested that the monomer-dimer transition is the key step in FNR-mediated repression of gene expression.

Authors: , , , S. Coakley, , ,

Date Published: 24th Apr 2014

Publication Type: Not specified

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The efficient redesign of bacteria for biotechnological purposes, such as biofuel production, waste disposal or specific biocatalytic functions, requires a quantitative systems-level understanding of energy supply, carbon, and redox metabolism. The measurement of transcript levels, metabolite concentrations and metabolic fluxes per se gives an incomplete picture. An appreciation of the interdependencies between the different measurement values is essential for systems-level understanding. Mathematical modeling has the potential to provide a coherent and quantitative description of the interplay between gene expression, metabolite concentrations, and metabolic fluxes. Escherichia coli undergoes major adaptations in central metabolism when the availability of oxygen changes. Thus, an integrated description of the oxygen response provides a benchmark of our understanding of carbon, energy, and redox metabolism. We present the first comprehensive model of the central metabolism of E. coli that describes steady-state metabolism at different levels of oxygen availability. Variables of the model are metabolite concentrations, gene expression levels, transcription factor activities, metabolic fluxes, and biomass concentration. We analyze the model with respect to the production capabilities of central metabolism of E. coli. In particular, we predict how precursor and biomass concentration are affected by product formation.

Editor:

Date Published: 27th Mar 2014

Publication Type: Not specified

Abstract (Expand)

The Computational Modeling in Biology Network (COMBINE) is an initiative to coordinate the development of community standards and formats in computational systems biology and related fields. This report summarizes the topics and activities of the fourth edition of the annual COMBINE meeting, held in Paris during September 16-20 2013, and attended by a total of 96 people. This edition pioneered a first day devoted to modeling approaches in biology, which attracted a broad audience of scientists thanks to a panel of renowned speakers. During subsequent days, discussions were held on many subjects including the introduction of new features in the various COMBINE standards, new software tools that use the standards, and outreach efforts. Significant emphasis went into work on extensions of the SBML format, and also into community-building. This year’s edition once again demonstrated that the COMBINE community is thriving, and still manages to help coordinate activities between different standards in computational systems biology.

Authors: Dagmar Waltemath, Frank T. Bergmann, Claudine Chaouiya, Tobias Czauderna, Padraig Gleeson, , Martin Golebiewski, Michael Hucka, Nick Juty, , Nicolas Le Novère, Huaiyu Mi, Ion I. Moraru, Chris J. Myers, David Nickerson, Brett G. Olivier, Nicolas Rodriguez, Falk Schreiber, Lucian Smith, Fengkai Zhang, Eric Bonnet

Date Published: 15th Mar 2014

Publication Type: Journal

Abstract (Expand)

The respiratory chain of E. coli is branched to allow the cells' flexibility to deal with changing environmental conditions. It consists of the NADH:ubiquinone oxidoreductases NADH dehydrogenase I and II, as well as of three terminal oxidases. They differ with respect to energetic efficiency (proton translocation) and their affinity to the different quinone/quinol species and oxygen. In order to analyze the advantages of the branched electron transport chain over a linear one and to assess how usage of the different terminal oxidases determines growth behavior at varying oxygen concentrations, a set of isogenic mutant strains was created, which lack NADH dehydrogenase I as well as two of the terminal oxidases, resulting in strains with a linear respiratory chain. These strains were analyzed in glucose-limited chemostat experiments with defined oxygen supply, adjusting aerobic, anaerobic and different microaerobic conditions. In contrast to the wild-type strain MG1655, the mutant strains produced acetate even under aerobic conditions. Strain TBE032, lacking NADH dehydrogenase I and expressing cytochrome bd-II as sole terminal oxidase, showed the highest acetate formation rate under aerobic conditions. This supports the idea that cytochrome bd-II terminal oxidase is not able to catalyze the efficient oxidation of the quinol pool at higher oxygen conditions, but is functioning mainly under limiting oxygen conditions. Phosphorylation of ArcA, the regulator of the two-component system ArcBA, besides Fnr the main transcription factor for the response towards different oxygen concentrations, was studied. Its phosphorylation pattern was changed in the mutant strains. Dephosphorylation and therefore inactivation of ArcA started at lower aerobiosis levels than in the wild-type strain. Notably, not only the micro- and aerobic metabolism was affected by the mutations, but also the anaerobic metabolism, where the respiratory chain should not be important.

Editor:

Date Published: 27th Jan 2014

Publication Type: Not specified

Abstract (Expand)

Several liver disorders result from perturbations in the metabolism of hepatocytes, and their underlying mechanisms can be outlined through the use of genome-scale metabolic models (GEMs). Here we reconstruct a consensus GEM for hepatocytes, which we call iHepatocytes2322, that extends previous models by including an extensive description of lipid metabolism. We build iHepatocytes2322 using Human Metabolic Reaction 2.0 database and proteomics data in Human Protein Atlas, which experimentally validates the incorporated reactions. The reconstruction process enables improved annotation of the proteomics data using the network centric view of iHepatocytes2322. We then use iHepatocytes2322 to analyse transcriptomics data obtained from patients with non-alcoholic fatty liver disease. We show that blood concentrations of chondroitin and heparan sulphates are suitable for diagnosing non-alcoholic steatohepatitis and for the staging of non-alcoholic fatty liver disease. Furthermore, we observe serine deficiency in patients with NASH and identify PSPH, SHMT1 and BCAT1 as potential therapeutic targets for the treatment of non-alcoholic steatohepatitis.

Authors: A. Mardinoglu, R. Agren, C. Kampf, A. Asplund, M. Uhlen, J. Nielsen

Date Published: 15th Jan 2014

Publication Type: Journal

Abstract

Not specified

Authors: Anna-Karin Gustavsson, David D. van Niekerk, Caroline B. Adiels, Mattias Goksör, Jacky L. Snoep

Date Published: 3rd Jan 2014

Publication Type: Not specified

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