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Basic Regulatory Principles of Escherichia coli's Electron Transport Chain for Varying Oxygen Conditions

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

What makes the lac-pathway switch: identifying the fluctuations that trigger phenotype switching in gene regulatory systems

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

Mesenchymal stromal cells inhibit proliferation of virus-specific CD8(+) T cells.

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

Multiscale digital Arabidopsis predicts individual organ and whole-organism growth

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

Control of transcription elongation by GreA determines rate of gene expression in Streptococcus pneumoniae

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

Transcriptome-wide analysis of trypanosome mRNA decay reveals complex degradation kinetics and suggests a role for co-transcriptional degradation in determining mRNA levels

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

Cause and cure of sloppiness in ordinary differential equation models

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

To sense or not to sense viral RNA—essentials of coronavirus innate immune evasion

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

Rethinking Transcriptional Activation in the Arabidopsis Circadian Clock

Abstract

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Authors: Karl Fogelmark, Carl Troein

Date Published: 17th Jul 2014

Publication Type: Not specified

Rethinking Transcriptional Activation in the Arabidopsis Circadian Clock

Abstract

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Authors: Karl Fogelmark, Carl Troein

Date Published: 17th Jul 2014

Publication Type: Journal

Solvent screening methodology for in situ ABE extractive fermentation

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

Liver transcriptome analysis of Atlantic cod (Gadus morhua) exposed to PCB 153 indicates effects on cell cycle regulation and lipid metabolism

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

Allosteric regulation of phosphofructokinase controls the emergence of glycolytic oscillations in isolated yeast cells

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

Standardization of Good Manufacturing Practice-compliant production of bone marrow-derived human mesenchymal stromal cells for immunotherapeutic applications.

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

The silicon trypanosome: a test case of iterative model extension in systems biology

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

Towards a Systems Level Understanding of the Oxygen Response of Escherichia coli

Abstract (Expand)

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

Agent-based modeling of oxygen-responsive transcription factors in Escherichia coli

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

A mathematical model of metabolism and regulation provides a systems-level view of how Escherichia coli responds to oxygen

Abstract (Expand)

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

Meeting report from the fourth meeting of the Computational Modeling in Biology Network (COMBINE)

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

Analysis of Escherichia coli mutants with a linear respiratory chain

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

Genome-scale metabolic modelling of hepatocytes reveals serine deficiency in patients with non-alcoholic fatty liver disease.

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

Heterogeneity of glycolytic oscillatory behaviour in individual yeast cells

Abstract

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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

Systems Biology of Monovalent Cation Homeostasis in Yeast

Abstract (Expand)

Maintenance of monovalent cation homeostasis (mainly K(+) and Na(+)) is vital for cell survival, and cation toxicity is at the basis of a myriad of relevant phenomena, such as salt stress in crops and …d diverse human diseases. Full understanding of the importance of monovalent cations in the biology of the cell can only be achieved from a systemic perspective. Translucent is a multinational project developed within the context of the SysMO (System Biology of Microorganisms) initiative and focussed in the study of cation homeostasis using the well-known yeast Saccharomyces cerevisiae as a model. The present review summarize how the combination of biochemical, genetic, genomic and computational approaches has boosted our knowledge in this field, providing the basis for a more comprehensive and coherent vision of the role of monovalent cations in the biology of the cell.

Authors: , Ebru Aydar, Samuel Drulhe, Daniel Ganser, , , , , Lynne Yenush, Olga Zimmermannová, G. Paul H. van Heusden, , , Chris Palmer, ,

Date Published: 2014

Publication Type: Not specified

Regulatory principles and experimental approaches to the circadian control of starch turnover

Abstract (Expand)

In many plants, starch is synthesized during the day and degraded during the night to avoid carbohydrate starvation in darkness. The circadian clock participates in a dynamic adjustment of starch … turnover to changing environmental condition through unknown mechanisms. We used mathematical modelling to explore the possible scenarios for the control of starch turnover by the molecular components of the plant circadian clock. Several classes of plausible models were capable of describing the starch dynamics observed in a range of clock mutant plants and light conditions, including discriminating circadian protocols. Three example models of these classes are studied in detail, differing in several important ways. First, the clock components directly responsible for regulating starch degradation are different in each model. Second, the intermediate species in the pathway may play either an activating or inhibiting role on starch degradation. Third, the system may include a light-dependent interaction between the clock and downstream processes. Finally, the clock may be involved in the regulation of starch synthesis. We discuss the differences among the models' predictions for diel starch profiles and the properties of the circadian regulators. These suggest additional experiments to elucidate the pathway structure, avoid confounding results and identify the molecular components involved.

Authors: D. D. Seaton, O. Ebenhoh, A. J. Millar, A. Pokhilko

Date Published: 18th Dec 2013

Publication Type: Not specified

Plerixafor induces the rapid and transient release of stromal cell-derived factor-1 alpha from human mesenchymal stromal cells and influences the migration behavior of human hematopoietic progenitor cells.

Abstract (Expand)

The interaction between the stromal cell-derived factor-1 alpha (SDF-1alpha, CXCL12) and its chemokine receptor CXCR4 has been reported to regulate stem cell migration, mobilization and homing. The … CXCR4 antagonist plerixafor is highly efficient in mobilizing hematopoietic progenitor cells (HPCs). However, the precise regulatory mechanisms governing the CXCR4/SDF-1alpha axis between the bone marrow niche and HPCs remain unclear. In this study, we quantify the impact of plerixafor on the interaction between human bone marrow derived mesenchymal stromal cells (MSCs) and human CD34+ HPCs. An assessment of SDF-1alpha levels in the supernatant of MSC cultures revealed that exposure to plerixafor led to a transient increase but had no long-term effect. In Transwell experiments, we observed that the addition of SDF-1alpha significantly stimulated HPC migration; this stimulation was almost completely antagonized by the addition of plerixafor, confirming the direct impact of the CXCR4/SDF-1alpha interaction on the migration capacity of HPCs. We also developed a new microstructural niche model to determine the chemotactic sensitivity of HPCs. Time-lapse microscopy demonstrated that HPCs migrated actively along an SDF-1alpha gradient within the microchannels and the quantitative assessment of the required minimum gradient initiating this chemotaxis revealed a surprisingly high sensitivity of HPCs. These data demonstrate the fine-tuned balance of the CXCR4/SDF-1alpha axis and the synergistic effects of plerixafor on HPCs and MSCs, which most likely represent the key mechanisms for the consecutive mobilization of HPCs from the bone marrow niche into the circulating blood.

Authors: P. Wuchter, C. Leinweber, R. Saffrich, M. Hanke, V. Eckstein, A. D. Ho, M. Grunze, A. Rosenhahn

Date Published: 17th Dec 2013

Publication Type: Journal

Handling uncertainty in dynamic models: the pentose phosphate pathway in Trypanosoma brucei

Abstract (Expand)

Dynamic models of metabolism can be useful in identifying potential drug targets, especially in unicellular organisms. A model of glycolysis in the causative agent of human African trypanosomiasis, … Trypanosoma brucei, has already shown the utility of this approach. Here we add the pentose phosphate pathway (PPP) of T. brucei to the glycolytic model. The PPP is localized to both the cytosol and the glycosome and adding it to the glycolytic model without further adjustments leads to a draining of the essential bound-phosphate moiety within the glycosome. This phosphate "leak" must be resolved for the model to be a reasonable representation of parasite physiology. Two main types of theoretical solution to the problem could be identified: (i) including additional enzymatic reactions in the glycosome, or (ii) adding a mechanism to transfer bound phosphates between cytosol and glycosome. One example of the first type of solution would be the presence of a glycosomal ribokinase to regenerate ATP from ribose 5-phosphate and ADP. Experimental characterization of ribokinase in T. brucei showed that very low enzyme levels are sufficient for parasite survival, indicating that other mechanisms are required in controlling the phosphate leak. Examples of the second type would involve the presence of an ATP:ADP exchanger or recently described permeability pores in the glycosomal membrane, although the current absence of identified genes encoding such molecules impedes experimental testing by genetic manipulation. Confronted with this uncertainty, we present a modeling strategy that identifies robust predictions in the context of incomplete system characterization. We illustrate this strategy by exploring the mechanism underlying the essential function of one of the PPP enzymes, and validate it by confirming the model predictions experimentally.

Authors: , , V. P. Alibu, R. J. Burchmore, I. H. Gilbert, M. Trybilo, N. N. Driessen, D. Gilbert, , ,

Date Published: 5th Dec 2013

Publication Type: Not specified

Model of tryptophan metabolism, readily scalable using tissue-specific gene expression data.

Abstract (Expand)

Tryptophan is utilized in various metabolic routes including protein synthesis, serotonin, and melatonin synthesis and the kynurenine pathway. Perturbations in these pathways have been associated with … neurodegenerative diseases and cancer. Here we present a comprehensive kinetic model of the complex network of human tryptophan metabolism based upon existing kinetic data for all enzymatic conversions and transporters. By integrating tissue-specific expression data, modeling tryptophan metabolism in liver and brain returned intermediate metabolite concentrations in the physiological range. Sensitivity and metabolic control analyses identified expected key enzymes to govern fluxes in the branches of the network. Combining tissue-specific models revealed a considerable impact of the kynurenine pathway in liver on the concentrations of neuroactive derivatives in the brain. Moreover, using expression data from a cancer study predicted metabolite changes that resembled the experimental observations. We conclude that the combination of the kinetic model with expression data represents a powerful diagnostic tool to predict alterations in tryptophan metabolism. The model is readily scalable to include more tissues, thereby enabling assessment of organismal tryptophan metabolism in health and disease.

Authors: A. K. Stavrum, I. Heiland, S. Schuster, P. Puntervoll, M. Ziegler

Date Published: 29th Nov 2013

Publication Type: Journal

Differences between healthy hematopoietic progenitors and leukemia cells with respect to CD44 mediated rolling versus adherence behavior on hyaluronic acid coated surfaces.

Abstract (Expand)

We previously demonstrated that leukemia cell lines expressing CD44 and hematopoietic progenitor cells (HPC) from umbilical cord blood (CB) showed rolling on hyaluronic acid (HA)-coated surfaces under … physiological shear stress. In the present study, we quantitatively assessed the interaction of HPC derived from CB, mobilized peripheral blood (mPB) and bone marrow (BM) from healthy donors, as well as primary leukemia blasts from PB and BM of patients with acute myeloid leukemia (AML) with HA. We have demonstrated that HPC derived from healthy donors showed relative homogeneous rolling and adhesion to HA. In contrast, highly diverse behavioral patterns were found for leukemia blasts under identical conditions. The monoclonal CD44 antibody (clone BU52) abrogated the shear stress-induced rolling of HPC and leukemia blasts, confirming the significance of CD44 in this context. On the other hand, the immobile adhesion of leukemia blasts to the HA-coated surface was, in some cases, not or incompletely inhibited by BU52. The latter property was associated with non-responsiveness to induction chemotherapy and subsequently poor clinical outcome.

Authors: M. Hanke, I. Hoffmann, C. Christophis, M. Schubert, V. T. Hoang, A. Zepeda-Moreno, N. Baran, V. Eckstein, P. Wuchter, A. Rosenhahn, A. D. Ho

Date Published: 26th Nov 2013

Publication Type: Journal

Functional potentials of human hematopoietic progenitor cells are maintained by mesenchymal stromal cells and not impaired by plerixafor.

Abstract (Expand)

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) resemble an essential component of the bone marrow niche for maintenance of stemness of hematopoietic progenitor cells (HPCs). Perturbation of the C-X-C … chemokine receptor type 4 (CXCR4)/stromal cell-derived factor-1alpha (SDF-1alpha) axis by plerixafor (AMD3100) mobilizes HPCs from their niche; however, little is known about how plerixafor affects interaction of HPCs and MSCs in vitro. METHODS: We monitored cell division kinetics, surface expression of CD34 and CXCR4, migration behavior and colony-forming frequency of HPCs on co-culture with MSCs either with or without exposure to plerixafor. RESULTS: Co-culture with MSCs significantly accelerated cell division kinetics of HPCs. Despite this, the proportion of CD34(+) cells was significantly increased on co-culture, whereas the expression of CXCR4 was reduced. In addition, co-culture with MSCs led to significantly higher colony-forming capacity and enhanced migration rate of HPCs compared with mono-culture conditions. The composition of MSC sub-populations-and conversely their hematopoiesis supportive functions-may be influenced by culture conditions. We compared the stromal function of MSCs isolated with three different culture media. Overall, the supporting potentials of these MSC preparations were quite similar. Perturbation by the CXCR4-antagonist plerixafor reduced the cell division kinetics of HPCs on co-culture with MSCs. However, the progenitor cell potential of the HPCs as reflected by colony-forming capacity was not affected by plerixafor. CONCLUSIONS: These results support the notion that the CXCR4/SDF-1alpha axis is critical for HPC-MSC interaction with regard to migration, adhesion and regulation of proliferation but not for maintenance of primitive progenitor cells.

Authors: A. Ludwig, R. Saffrich, V. Eckstein, T. Bruckner, W. Wagner, A. D. Ho, P. Wuchter

Date Published: 15th Oct 2013

Publication Type: Journal

Kinase activity of ArcB from Escherichia coli is subject to regulation by both ubiquinone and demethylmenaquinone

Abstract (Expand)

Expression of the catabolic network in Escherichia coli is predominantly regulated, via oxygen availability, by the two-component system ArcBA. It has been shown that the kinase activity of ArcB is … controlled by the redox state of two critical pairs of cysteines in dimers of the ArcB sensory kinase. Among the cellular components that control the redox state of these cysteines of ArcB are the quinones from the cytoplasmic membrane of the cell, which function in 'respiratory' electron transfer. This study is an effort to understand how the redox state of the quinone pool(s) is sensed by the cell via the ArcB kinase. We report the relationship between growth, quinone content, ubiquinone redox state, the level of ArcA phosphorylation, and the level of ArcA-dependent gene expression, in a number of mutants of E. coli with specific alterations in their set of quinones, under a range of physiological conditions. Our results provide experimental evidence for a previously formulated hypothesis that not only ubiquinone, but also demethylmenaquinone, can inactivate kinase activity of ArcB. Also, in a mutant strain that only contains demethylmenaquinone, the extent of ArcA phosphorylation can be modulated by the oxygen supply rate, which shows that demethylmenaquinone can also inactivate ArcB in its oxidized form. Furthermore, in batch cultures of a strain that contains ubiquinone as its only quinone species, we observed that the ArcA phosphorylation level closely followed the redox state of the ubiquinone/ubiquinol pool, much more strictly than it does in the wild type strain. Therefore, at low rates of oxygen supply in the wild type strain, the activity of ArcB may be inhibited by demethylmenaquinone, in spite of the fact that the ubiquinones are present in the ubiquinol form.

Authors: P. Sharma, S. Stagge, M. Bekker, K. Bettenbrock, K. J. Hellingwerf

Date Published: 7th Oct 2013

Publication Type: Not specified

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