Publications

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Mutations in pre-mRNA processing factors (PRPFs) cause 40% of autosomal dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed PRPFs cause retinal disease. To understand the molecular basis of this phenotype, we have generated RP type 11 (PRPF31-mutated) patient-specific retinal organoids and retinal pigment epithelium (RPE) from induced pluripotent stem cells (iPSC). Impaired alternative splicing of genes encoding pre-mRNA splicing proteins occurred in patient-specific retinal cells and Prpf31+/− mouse retinae, but not fibroblasts and iPSCs, providing mechanistic insights into retinal-specific phenotypes of PRPFs. RPE was the most affected, characterised by loss of apical-basal polarity, reduced trans-epithelial resistance, phagocytic capacity, microvilli, and cilia length and incidence. Disrupted cilia morphology was observed in patient-derived-photoreceptors that displayed progressive features associated with degeneration and cell stress. In situ gene-editing of a pathogenic mutation rescued key structural and functional phenotypes in RPE and photoreceptors, providing proof-of-concept for future therapeutic strategies. eTOC PRPF31 is a ubiquitously expressed pre-mRNA processing factor that when mutated causes autosomal dominant RP. Using a patient-specific iPSC approach, Buskin and Zhu et al. show that retinal-specific defects result from altered splicing of genes involved in the splicing process itself, leading to impaired splicing, loss of RPE polarity and diminished phagocytic ability as well as reduced cilia incidence and length in both photoreceptors and RPE.

Authors: Adriana Buskin, Lili Zhu, Valeria Chichagova, Basudha Basu, Sina Mozaffari-Jovin, David Dolan, Alastair Droop, Joseph Collin, Revital Bronstein, Sudeep Mehrotra, Michael Farkas, Gerrit Hilgen, Kathryn White, Dean Hallam, Katarzyna Bialas, Git Chung, Carla Mellough, Yuchun Ding, Natalio Krasnogor, Stefan Przyborski, Jumana Al-Aama, Sameer Alharthi, Yaobo Xu, Gabrielle Wheway, Katarzyna Szymanska, Martin McKibbin, Chris F Inglehearn, David J Elliott, Susan Lindsay, Robin R Ali, David H Steel, Lyle Armstrong, Evelyne Sernagor, Eric Pierce, Reinhard Luehrmann, Sushma Nagaraja Grellscheid, Colin A Johnson, Majlinda Lako

Date Published: No date defined

Journal: Not specified

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BioRxiv preprint, 4 April 2018. Abstract: Daily light-dark cycles (LD) drive dynamic regulation of plant and algal transcriptomes via photoreceptor pathways and 24-hour, circadian rhythms. Diel regulation of protein levels and modifications has been less studied. Ostreococcus tauri, the smallest free-living eukaryote, provides a minimal model proteome for the green lineage. Here, we compare transcriptome data under LD to the algal proteome and phosphoproteome, assayed using shotgun mass-spectrometry. Under 10% of 855 quantified proteins were rhythmic but two-thirds of 860 phosphoproteins showed rhythmic modification(s). Most rhythmic proteins peaked in the daytime. Model simulations showed that light-stimulated protein synthesis largely accounts for this distribution of protein peaks. Prompted by apparently dark-stable proteins, we sampled during prolonged dark adaptation, where stable RNAs and very limited change to the proteome suggested a quiescent, cellular “dark state”. In LD, acid-directed and proline-directed protein phosphorylation sites were regulated in antiphase. Strikingly, 39% of rhythmic phospho-sites reached peak levels just before dawn. This anticipatory phosphorylation is distinct from light-responsive translation but consistent with plant phosphoprotein profiles, suggesting that a clock-regulated phospho-dawn prepares green cells for daytime functions.

Authors: Zeenat B. Noordally, Matthew M. Hindle, Sarah F. Martin, Daniel Seaton, Ian Simpson, Thierry Le Bihan, Andrew Millar

Date Published: No date defined

Journal: Not specified

Abstract

BioRxiv preprint:

Authors: Hannah A Kinmonth-Schultz, Melissa J MacEwen, Daniel Seaton, Andrew Millar, Takato Imaizumi, Soo-Hyung Kim

Date Published: No date defined

Journal: Not specified

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The recovery of 1-butanol from fermentation broth is energy-intensive since typical concentrations in fermentation broth are below 20 g L(-1). To prevent butanol inhibition and high downstream processing costs, we aimed at producing butyl esters instead of 1-butanol. It is shown that it is possible to perform simultaneously clostridial fermentation, esterification of the formed butanol to butyl butyrate, and extraction of this ester by hexadecane. The very high partition coefficient of butyl butyrate pulls the esterification towards the product side even at fermentation pH and relatively low butanol concentrations. The hexadecane extractant is a model diesel compound and is nontoxic to the cells. If butyl butyrate enriched diesel can directly be used as car fuel, no product recovery is required. A proof-of-principle experiment for the one-pot bio-ester production from glucose led to 5 g L(-1) butyl butyrate in the hexadecane phase. The principle may be extended to a wide range of esters, especially to longer chain ones.

Authors: C. van den Berg, A. S. Heeres, L. A. van der Wielen, A. J. Straathof

Date Published: No date defined

Journal: Biotechnol Bioeng

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Butyl butyrate (BB) is a valuable chemical that can be used as flavor, fragrance, extractant, and so on in various industries. Meanwhile, BB can also be used as a fuel source with excellent compatibility as gasoline, aviation kerosene, and diesel components. The conventional industrial production of BB is highly energy-consuming and generates various environmental pollutants. Recently, there have been tremendous interests in producing BB from renewable resources through biological routes. In this study, based on the fermentation using the hyper-butyrate producing strain Clostridium tyrobutyricum ATCC 25755, efficient BB production through in situ esterification was achieved by supplementation of lipase and butanol into the fermentation. Three commercially available lipases were assessed and the one from Candida sp. (recombinant, expressed in Aspergillus niger) was identified with highest catalytic activity for BB production. Various conditions that might affect BB production in the fermentation have been further evaluated, including the extractant type, enzyme loading, agitation, pH, and butanol supplementation strategy. Under the optimized conditions (5.0 g L(-1) of enzyme loading, pH at 5.5, butanol kept at 10.0 g L(-1) ), 34.7 g L(-1) BB was obtained with complete consumption of 50 g L(-1) glucose as the starting substrate. To our best knowledge, the BB production achieved in this study is the highest among the ever reported from the batch fermentation process. Our results demonstrated an excellent biological platform for renewable BB production from low-value carbon sources. Biotechnol. Bioeng. 2017;114: 1428-1437. (c) 2017 Wiley Periodicals, Inc.

Authors: Z. T. Zhang, S. Taylor, Y. Wang

Date Published: No date defined

Journal: Biotechnol Bioeng

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Thraustochytrids are unicellular, marine protists, and there is a growing industrial interest in these organisms, particularly because some species, including strains belonging to the genus Aurantiochytrium, accumulate high levels of docosahexaenoic acid (DHA). Here, we report the draft genome sequence of Aurantiochytrium sp. T66 (ATCC PRA-276), with a size of 43 Mbp, and 11,683 predicted protein-coding sequences. The data has been deposited at DDBJ/EMBL/Genbank under the accession LNGJ00000000. The genome sequence will contribute new insight into DHA biosynthesis and regulation, providing a basis for metabolic engineering of thraustochytrids.

Authors: B. Liu, H. Ertesvag, I. M. Aasen, O. Vadstein, T. Brautaset, T. M. Heggeset

Date Published: No date defined

Journal: Genom Data

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Absolute measurements of protein abundance are important in the understanding of biological processes and the precise computational modeling of biological pathways. We developed targeted LC-MS/MS assays in the selected reaction monitoring (SRM) mode to quantify over 50 mitochondrial proteins in a single run. The targeted proteins cover the tricarboxylic acid cycle, fatty acid beta-oxidation, oxidative phosphorylation, and the detoxification of reactive oxygen species. Assays used isotopically labeled concatemers as internal standards designed to target murine mitochondrial proteins and their human orthologues. Most assays were also suitable to quantify the corresponding protein orthologues in rats. After exclusion of peptides that did not pass the selection criteria, we arrived at SRM assays for 55 mouse, 52 human, and 51 rat proteins. These assays were optimized in isolated mitochondrial fractions from mouse and rat liver and cultured human fibroblasts and in total liver extracts from mouse, rat, and human. The developed proteomics approach is suitable for the quantification of proteins in the mitochondrial energy metabolic pathways in mice, rats, and humans as a basis for translational research. Initial data show that the assays have great potential for elucidating the adaptive response of human patients to mutations in mitochondrial proteins in a clinical setting.

Authors: J. C. Wolters, J. Ciapaite, K. van Eunen, K. E. Niezen-Koning, A. Matton, R. J. Porte, P. Horvatovich, Barbara Bakker, R. Bischoff, H. P. Permentier

Date Published: No date defined

Journal: J Proteome Res

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Amino acids (aa) are not only building blocks for proteins, but also signalling molecules, with the mammalian target of rapamycin complex 1 (mTORC1) acting as a key mediator. However, little is known about whether aa, independently of mTORC1, activate other kinases of the mTOR signalling network. To delineate aa-stimulated mTOR network dynamics, we here combine a computational-experimental approach with text mining-enhanced quantitative proteomics. We report that AMP-activated protein kinase (AMPK), phosphatidylinositide 3-kinase (PI3K) and mTOR complex 2 (mTORC2) are acutely activated by aa-readdition in an mTORC1-independent manner. AMPK activation by aa is mediated by Ca(2+)/calmodulin-dependent protein kinase kinase beta (CaMKKbeta). In response, AMPK impinges on the autophagy regulators Unc-51-like kinase-1 (ULK1) and c-Jun. AMPK is widely recognized as an mTORC1 antagonist that is activated by starvation. We find that aa acutely activate AMPK concurrently with mTOR. We show that AMPK under aa sufficiency acts to sustain autophagy. This may be required to maintain protein homoeostasis and deliver metabolite intermediates for biosynthetic processes.

Authors: P. Dalle Pezze, S. Ruf, A. G. Sonntag, M. Langelaar-Makkinje, P. Hall, A. M. Heberle, Patricia Razquin Navas, K. van Eunen, R. C. Tolle, J. J. Schwarz, H. Wiese, B. Warscheid, J. Deitersen, B. Stork, E. Fassler, S. Schauble, U. Hahn, P. Horvatovich, Daryl Shanley, Kathrin Thedieck

Date Published: No date defined

Journal: Nat Commun

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It is a long-standing enigma how glycogen storage disease (GSD) type I patients retain a limited capacity for endogenous glucose production despite the loss of glucose-6-phosphatase activity. Insight into the source of residual endogenous glucose production is of clinical importance given the risk of sudden death in these patients, but so far contradictory mechanisms have been proposed. We investigated glucose-6-phosphatase-independent endogenous glucose production in hepatocytes isolated from a liver-specific GSD Ia mouse model (L-G6pc(-/-) mice) and performed real-time analysis of hepatic glucose fluxes and glycogen metabolism in L-G6pc(-/-) mice using state-of-the-art stable isotope methodologies. Here we show that G6pc-deficient hepatocytes are capable of producing glucose. In vivo analysis of hepatic glucose metabolism revealed that the hepatic glucokinase flux was decreased by 95% in L-G6pc(-/-) mice. It also showed increased glycogen phosphorylase flux in L-G6pc(-/-) mice, which is coupled to the release of free glucose through glycogen debranching. Although the ex vivo activities of debranching enzyme and lysosomal acid maltase, two major hepatic alpha-glucosidases, were unaltered in L-G6pc(-/-) mice, pharmacological inhibition of alpha-glucosidase activity almost completely abolished residual glucose production by G6pc-deficient hepatocytes. CONCLUSION: Our data indicate that hepatocytes contribute to residual glucose production in GSD Ia. We show that alpha-glucosidase activity, i.e. glycogen debranching and/or lysosomal glycogen breakdown, contributes to residual glucose production by GSD Ia hepatocytes. A strong reduction in hepatic GCK flux in L-G6pc-/- mice furthermore limits the phosphorylation of free glucose synthesized by G6pc-deficient hepatocytes, allowing the release of glucose into the circulation. The almost complete abrogation of GCK flux in G6pc-deficient liver also explains the contradictory reports on residual glucose production in GSD Ia patients. (Hepatology 2017;66:2042-2054).

Authors: B. S. Hijmans, A. Boss, T. H. van Dijk, M. Soty, H. Wolters, E. Mutel, Bert Groen, T. G. J. Derks, G. Mithieux, A. Heerschap, D. J. Reijngoud, F. Rajas, M. H. Oosterveer

Date Published: No date defined

Journal: Hepatology

Abstract (Expand)

UNLABELLED: An existing detailed kinetic model for the steady-state behavior of yeast glycolysis was tested for its ability to simulate dynamic behavior. Using a small subset of experimental data, the original model was adapted by adjusting its parameter values in three optimization steps. Only small adaptations to the original model were required for realistic simulation of experimental data for limit-cycle oscillations. The greatest changes were required for parameter values for the phosphofructokinase reaction. The importance of ATP for the oscillatory mechanism and NAD(H) for inter-and intra-cellular communications and synchronization was evident in the optimization steps and simulation experiments. In an accompanying paper [du Preez F et al. (2012) FEBS J279, 2823-2836], we validate the model for a wide variety of experiments on oscillatory yeast cells. The results are important for re-use of detailed kinetic models in modular modeling approaches and for approaches such as that used in the Silicon Cell initiative. DATABASE: The mathematical models described here have been submitted to the JWS Online Cellular Systems Modelling Database and can be accessed at http://jjj.biochem.sun.ac.za/database/dupreez/index.html.

Authors: F. B. du Preez, Dawie Van Niekerk, B. Kooi, J. M. Rohwer, Jacky Snoep

Date Published: No date defined

Journal: FEBS J

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UNLABELLED: In an accompanying paper [du Preez et al., (2012) FEBS J279, 2810-2822], we adapt an existing kinetic model for steady-state yeast glycolysis to simulate limit-cycle oscillations. Here we validate the model by testing its capacity to simulate a wide range of experiments on dynamics of yeast glycolysis. In addition to its description of the oscillations of glycolytic intermediates in intact cells and the rapid synchronization observed when mixing out-of-phase oscillatory cell populations (see accompanying paper), the model was able to predict the Hopf bifurcation diagram with glucose as the bifurcation parameter (and one of the bifurcation points with cyanide as the bifurcation parameter), the glucose- and acetaldehyde-driven forced oscillations, glucose and acetaldehyde quenching, and cell-free extract oscillations (including complex oscillations and mixed-mode oscillations). Thus, the model was compliant, at least qualitatively, with the majority of available experimental data for glycolytic oscillations in yeast. To our knowledge, this is the first time that a model for yeast glycolysis has been tested against such a wide variety of independent data sets. DATABASE: The mathematical models described here have been submitted to the JWS Online Cellular Systems Modelling Database and can be accessed at http://jjj.biochem.sun.ac.za/database/dupreez/index.html.

Authors: F. B. du Preez, Dawie Van Niekerk, Jacky Snoep

Date Published: No date defined

Journal: FEBS J

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Photoperiod duration can be predicted from previous days, but irradiance fluctuates in an unpredictable manner. To investigate how allocation to starch responds to changes in these two environmental variables, Arabidopsis Col-0 was grown in a 6 h and a 12 h photoperiod at three different irradiances. The absolute rate of starch accumulation increased when photoperiod duration was shortened and when irradiance was increased. The proportion of photosynthate allocated to starch increased strongly when photoperiod duration was decreased but only slightly when irradiance was decreased. There was a small increase in the daytime level of sucrose and twofold increases in glucose, fructose and glucose 6-phosphate at a given irradiance in short photoperiods compared to long photoperiods. The rate of starch accumulation correlated strongly with sucrose and glucose levels in the light, irrespective of whether these sugars were responding to a change in photoperiod or irradiance. Whole plant carbon budget modelling revealed a selective restriction of growth in the light period in short photoperiods. It is proposed that photoperiod sensing, possibly related to the duration of the night, restricts growth in the light period in short photoperiods, increasing allocation to starch and providing more carbon reserves to support metabolism and growth in the long night.

Authors: Virginie Mengin, E. T. Pyl, T. Alexandre Moraes, R. Sulpice, N. Krohn, B. Encke, M. Stitt

Date Published: No date defined

Journal: Plant Cell Environ

Abstract

Not specified

Authors: Johanna Krahmer, Ashwin Ganpudi, Ammad Abbas, Andrew Romanowski, Karen Halliday

Date Published: No date defined

Journal: Plant Physiol

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Crop biomass and yield are tightly linked to how the light signaling network translates information about the environment into allocation of resources, including photosynthates. Once activated, the phytochrome (phy) class of photoreceptors signal and re-deploy carbon resources to alter growth, plant architecture, and reproductive timing. Most of the previous characterization of the light-modulated growth program has been performed in the reference plant Arabidopsis thaliana. Here, we use Brassica rapa as a crop model to test for conservation of the phytochrome-carbon network. In response to elevated levels of CO2, B. rapa seedlings showed increases in hypocotyl length, shoot and root fresh weight, and the number of lateral roots. All of these responses were dependent on nitrogen and polar auxin transport. In addition, we identified putative B. rapa orthologs of PhyB and isolated two nonsense alleles. BrphyB mutants had significantly decreased or absent CO2-stimulated growth responses. Mutant seedlings also showed misregulation of auxin-dependent genes and genes involved in chloroplast development. Adult mutant plants had reduced chlorophyll levels, photosynthetic rate, stomatal index, and seed yield. These findings support a recently proposed holistic role for phytochromes in regulating resource allocation, biomass production, and metabolic state in the developing plant.

Authors: Andrej Arsovski, J. E. Zemke, B. D. Haagen, S. H. Kim, Jennifer Nemhauser

Date Published: No date defined

Journal: J Exp Bot

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Bile, the central metabolic product of the liver, is transported by the bile canaliculi network. The impairment of bile flow in cholestatic liver diseases has urged a demand for insights into its regulation. Here, we developed a predictive 3D multi-scale model that simulates fluid dynamic properties successively from the subcellular to the tissue level. The model integrates the structure of the bile canalicular network in the mouse liver lobule, as determined by high-resolution confocal and serial block-face scanning electron microscopy, with measurements of bile transport by intravital microscopy. The combined experiment-theory approach revealed spatial heterogeneities of biliary geometry and hepatocyte transport activity. Based on this, our model predicts gradients of bile velocity and pressure in the liver lobule. Validation of the model predictions by pharmacological inhibition of Rho kinase demonstrated a requirement of canaliculi contractility for bile flow in vivo. Our model can be applied to functionally characterize liver diseases and quantitatively estimate biliary transport upon drug-induced liver injury.

Authors: K. Meyer, O. Ostrenko, G. Bourantas, H. Morales-Navarrete, N. Porat-Shliom, F. Segovia-Miranda, H. Nonaka, A. Ghaemi, J. M. Verbavatz, Lutz Brusch, I. Sbalzarini, Y. Kalaidzidis, R. Weigert, M. Zerial

Date Published: No date defined

Journal: Cell Syst

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Morpheus is a modeling environment for the simulation and integration of cell-based models with ordinary differential equations and reaction-diffusion systems. It allows rapid development of multiscale models in biological terms and mathematical expressions rather than programming code. Its graphical user interface supports the entire workflow from model construction and simulation to visualization, archiving and batch processing.

Authors: J. Starruss, W. de Back, Lutz Brusch, A. Deutsch

Date Published: No date defined

Journal: Bioinformatics

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We used parameter scanning to emulate changes to the limiting rate for steps in a fitted model of glucose-derepressed yeast glycolysis. Three flux-control regimes were observed, two of which were under the dominant control of hexose transport, in accordance with various experimental studies and other model predictions. A third control regime in which phosphofructokinase exerted dominant glycolytic flux control was also found, but it appeared to be physiologically unreachable by this model, and all realistically obtainable flux control regimes featured hexose transport as a step involving high flux control.

Authors: L. Pritchard, D. B. Kell

Date Published: No date defined

Journal: Eur J Biochem

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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, Ines Heiland, S. Schuster, P. Puntervoll, Mathias Ziegler

Date Published: No date defined

Journal: J Biol Chem

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Kynurenine formation by tryptophan-catabolic indoleamine-2,3-dioxygenase 1 (IDO1) plays a key role in tumor immune evasion and inhibition of IDO1 is efficacious in preclinical models of breast cancer. As the response of breast cancer to immune checkpoint inhibitors may be limited, a better understanding of the expression of additional targetable immunomodulatory pathways is of importance. We therefore investigated the regulation of IDO1 expression in different breast cancer subtypes. We identified estrogen receptor alpha (ER) as a negative regulator of IDO1 expression. Serum kynurenine levels as well as tumoral IDO1 expression were lower in patients with ER-positive than ER-negative tumors and an inverse relationship between IDO1 and estrogen receptor mRNA was observed across 14 breast cancer data sets. Analysis of whole genome bisulfite sequencing, 450k, MassARRAY and pyrosequencing data revealed that the IDO1 promoter is hypermethylated in ER-positive compared with ER-negative breast cancer. Reduced induction of IDO1 was also observed in human ER-positive breast cancer cell lines. IDO1 induction was enhanced upon DNA demethylation in ER-positive but not in ER-negative cells and methylation of an IDO1 promoter construct reduced IDO1 expression, suggesting that enhanced methylation of the IDO1 promoter suppresses IDO1 in ER-positive breast cancer. The association of ER overexpression with epigenetic downregulation of IDO1 appears to be a particular feature of breast cancer as IDO1 was not suppressed by IDO1 promoter hypermethylation in the presence of high ER expression in cervical or endometrial cancer.

Authors: D. L. Dewi, S. R. Mohapatra, S. Blanco Cabanes, I. Adam, L. F. Somarribas Patterson, B. Berdel, M. Kahloon, L. Thurmann, S. Loth, K. Heilmann, D. Weichenhan, O. Mucke, Ines Heiland, P. Wimberger, J. D. Kuhlmann, Karl-Heinz Kellner, Sarah Schott, C. Plass, M. Platten, C. Gerhauser, S. Trump, Christiane Opitz

Date Published: No date defined

Journal: Oncoimmunology

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INTRODUCTION: Male breast cancer (MBC) is a rare and inadequately characterized disease. The aim of the present study was to characterize MBC tumors transcriptionally, to classify them into comprehensive subgroups, and to compare them with female breast cancer (FBC). METHODS: A total of 66 clinicopathologically well-annotated fresh frozen MBC tumors were analyzed using Illumina Human HT-12 bead arrays, and a tissue microarray with 220 MBC tumors was constructed for validation using immunohistochemistry. Two external gene expression datasets were used for comparison purposes: 37 MBCs and 359 FBCs. RESULTS: Using an unsupervised approach, we classified the MBC tumors into two subgroups, luminal M1 and luminal M2, respectively, with differences in tumor biological features and outcome, and which differed from the intrinsic subgroups described in FBC. The two subgroups were recapitulated in the external MBC dataset. Luminal M2 tumors were characterized by high expression of immune response genes and genes associated with estrogen receptor (ER) signaling. Luminal M1 tumors, on the other hand, despite being ER positive by immunohistochemistry showed a lower correlation to genes associated with ER signaling and displayed a more aggressive phenotype and worse prognosis. Validation of two of the most differentially expressed genes, class 1 human leukocyte antigen (HLA) and the metabolizing gene N-acetyltransferase-1 (NAT1), respectively, revealed significantly better survival associated with high expression of both markers (HLA, hazard ratio (HR) 3.6, P = 0.002; NAT1, HR 2.5, P = 0.033). Importantly, NAT1 remained significant in a multivariate analysis (HR 2.8, P = 0.040) and may thus be a novel prognostic marker in MBC. CONCLUSIONS: We have detected two unique and stable subgroups of MBC with differences in tumor biological features and outcome. They differ from the widely acknowledged intrinsic subgroups of FBC. As such, they may constitute two novel subgroups of breast cancer, occurring exclusively in men, and which may consequently require novel treatment approaches. Finally, we identified NAT1 as a possible prognostic biomarker for MBC, as suggested by NAT1 positivity corresponding to better outcome.

Authors: I. Johansson, C. Nilsson, P. Berglund, M. Lauss, M. Ringner, H. Olsson, L. Luts, E. Sim, S. Thorstensson, M. L. Fjallskog, I. Hedenfalk

Date Published: No date defined

Journal: Breast Cancer Res

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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: No date defined

Journal: Genes Chromosomes Cancer

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Atlantic salmon migrates from rivers to sea to feed, grow and develop gonads before returning to spawn in freshwater. The transition to marine habitats is associated with dramatic changes in the environment, including water salinity, exposure to pathogens, and shift in dietary lipid availability. Many changes in physiology and metabolism occur across this life-stage transition, but little is known about the molecular nature of these changes. Here we use a long term feeding experiment to study transcriptional regulation of lipid metabolism in Atlantic salmon gut and liver in both fresh- and saltwater. We find that lipid metabolism becomes significantly less plastic to differences in dietary lipid composition when salmon transitions to saltwater and experiences increased dietary lipid availability. Expression of genes in liver relating to lipogenesis and lipid transport decrease overall and become less responsive to diet, while genes for lipid uptake in gut become more highly expressed. Finally, analyses of evolutionary consequences of the salmonid specific whole-genome duplication on lipid metabolism reveals several pathways with significantly different (p<0.05) duplicate retention or duplicate regulatory conservation. We also find a limited number of cases where the whole genome duplication has resulted in an increased gene dosage. In conclusion, we find variable and pathway-specific effects of the salmonid genome duplication on lipid metabolism genes. A clear life-stage associated shift in lipid metabolism regulation is evident, and we hypothesize this to be, at least partly, driven by non-dietary factors such as the preparatory remodeling of gene regulation and physiology prior to sea migration. This article is protected by copyright. All rights reserved.

Authors: Gareth Gillard, Thomas Harvey, Arne Gjuvsland, Yang Jin, Magny Sidsel Thomassen, S. Lien, M. Leaver, Jacob Seilø Torgersen, Torgeir R. Hvidsten, Jon Olav Vik, Sandve Simen

Date Published: No date defined

Journal: Mol Ecol

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SABIO-RK (http://sabiork.h-its.org/) is a manually curated database containing data about biochemical reactions and their reaction kinetics. The data are primarily extracted from scientific literature and stored in a relational database. The content comprises both naturally occurring and alternatively measured biochemical reactions and is not restricted to any organism class. The data are made available to the public by a web-based search interface and by web services for programmatic access. In this update we describe major improvements and extensions of SABIO-RK since our last publication in the database issue of Nucleic Acid Research (2012). (i) The website has been completely revised and (ii) allows now also free text search for kinetics data. (iii) Additional interlinkages with other databases in our field have been established; this enables users to gain directly comprehensive knowledge about the properties of enzymes and kinetics beyond SABIO-RK. (iv) Vice versa, direct access to SABIO-RK data has been implemented in several systems biology tools and workflows. (v) On request of our experimental users, the data can be exported now additionally in spreadsheet formats. (vi) The newly established SABIO-RK Curation Service allows to respond to specific data requirements.

Authors: Ulrike Wittig, Maja Rey, A. Weidemann, Renate Kania, Wolfgang Müller

Date Published: No date defined

Journal: Nucleic Acids Res

Abstract (Expand)

Protein synthesis and degradation determine the cellular levels of proteins, and their control hence enables organisms to respond to environmental change. Experimentally, these are little known proteome parameters; however, recently, SILAC-based mass spectrometry studies have begun to quantify turnover in the proteomes of cell lines, yeast, and animals. Here, we present a proteome-scale method to quantify turnover and calculate synthesis and degradation rate constants of individual proteins in autotrophic organisms such as algae and plants. The workflow is based on the automated analysis of partial stable isotope incorporation with (15)N. We applied it in a study of the unicellular pico-alga Ostreococcus tauri and observed high relative turnover in chloroplast-encoded ATPases (0.42-0.58% h(-1)), core photosystem II proteins (0.34-0.51% h(-1)), and RbcL (0.47% h(-1)), while nuclear-encoded RbcS2 is more stable (0.23% h(-1)). Mitochondrial targeted ATPases (0.14-0.16% h(-1)), photosystem antennae (0.09-0.14% h(-1)), and histones (0.07-0.1% h(-1)) were comparatively stable. The calculation of degradation and synthesis rate constants k(deg) and k(syn) confirms RbcL as the bulk contributor to overall protein turnover. This study performed over 144 h of incorporation reveals dynamics of protein complex subunits as well as isoforms targeted to different organelles.

Authors: S. F. Martin, V. S. Munagapati, E. Salvo-Chirnside, L. E. Kerr, T. Le Bihan

Date Published: No date defined

Journal: J Proteome Res

Abstract (Expand)

BACKGROUND: Unicellular cyanobacteria of the genus Cyanothece are recognized for their ability to execute nitrogen (N2)-fixation in the dark and photosynthesis in the light. An understanding of these mechanistic processes in an integrated systems context should provide insights into how Cyanothece might be optimized for specialized environments and/or industrial purposes. Systems-wide dynamic proteomic profiling with mass spectrometry (MS) analysis should reveal fundamental insights into the control and regulation of these functions. RESULTS: To expand upon the current knowledge of protein expression patterns in Cyanothece ATCC51142, we performed quantitative proteomic analysis using partial ("unsaturated") metabolic labeling and high mass accuracy LC-MS analysis. This dynamic proteomic profiling identified 721 actively synthesized proteins with significant temporal changes in expression throughout the light-dark cycles, of which 425 proteins matched with previously characterized cycling transcripts. The remaining 296 proteins contained a cluster of proteins uniquely involved in DNA replication and repair, protein degradation, tRNA synthesis and modification, transport and binding, and regulatory functions. Functional classification of labeled proteins suggested that proteins involved in respiration and glycogen metabolism showed increased expression in the dark cycle together with nitrogenase, suggesting that N2-fixation is mediated by higher respiration and glycogen metabolism. Results indicated that Cyanothece ATCC51142 might utilize alternative pathways for carbon (C) and nitrogen (N) acquisition, particularly, aspartic acid and glutamate as substrates of C and N, respectively. Utilization of phosphoketolase (PHK) pathway for the conversion of xylulose-5P to pyruvate and acetyl-P likely constitutes an alternative strategy to compensate higher ATP and NADPH demand. CONCLUSION: This study provides a deeper systems level insight into how Cyanothece ATCC51142 modulates cellular functions to accommodate photosynthesis and N2-fixation within the single cell.

Authors: U. K. Aryal, J. Stockel, R. K. Krovvidi, M. A. Gritsenko, M. E. Monroe, R. J. Moore, D. W. Koppenaal, R. D. Smith, H. B. Pakrasi, J. M. Jacobs

Date Published: No date defined

Journal: BMC Syst Biol

Abstract (Expand)

The diurnal cycle strongly influences many plant metabolic and physiological processes. Arabidopsis thaliana rosettes were harvested six times during 12-h-light/12-h-dark treatments to investigate changes in gene expression using ATH1 arrays. Diagnostic gene sets were identified from published or in-house expression profiles of the response to light, sugar, nitrogen, and water deficit in seedlings and 4 h of darkness or illumination at ambient or compensation point [CO(2)]. Many sugar-responsive genes showed large diurnal expression changes, whose timing matched that of the diurnal changes of sugars. A set of circadian-regulated genes also showed large diurnal changes in expression. Comparison of published results from a free-running cycle with the diurnal changes in Columbia-0 (Col-0) and the starchless phosphoglucomutase (pgm) mutant indicated that sugars modify the expression of up to half of the clock-regulated genes. Principle component analysis identified genes that make large contributions to diurnal changes and confirmed that sugar and circadian regulation are the major inputs in Col-0 but that sugars dominate the response in pgm. Most of the changes in pgm are triggered by low sugar levels during the night rather than high levels in the light, highlighting the importance of responses to low sugar in diurnal gene regulation. We identified a set of candidate regulatory genes that show robust responses to alterations in sugar levels and change markedly during the diurnal cycle.

Authors: O. E. Blasing, Y. Gibon, M. Gunther, M. Hohne, R. Morcuende, D. Osuna, O. Thimm, B. Usadel, W. R. Scheible, M. Stitt

Date Published: No date defined

Journal: Plant Cell

Abstract (Expand)

The balance between the supply and utilization of carbon (C) changes continually. It has been proposed that plants respond in an acclimatory manner, modifying C utilization to minimize harmful periods of C depletion. This hypothesis predicts that signaling events are initiated by small changes in C status. We analyzed the global transcriptional response to a gradual depletion of C during the night and an extension of the night, where C becomes severely limiting from 4 h onward. The response was interpreted using published datasets for sugar, light, and circadian responses. Hundreds of C-responsive genes respond during the night and others very early in the extended night. Pathway analysis reveals that biosynthesis and cellular growth genes are repressed during the night and genes involved in catabolism are induced during the first hours of the extended night. The C response is amplified by an antagonistic interaction with the clock. Light signaling is attenuated during the 24-h light/dark cycle. A model was developed that uses the response of 22K genes during a circadian cycle and their responses to C and light to predict global transcriptional responses during diurnal cycles of wild-type and starchless pgm mutant plants and an extended night in wild-type plants. By identifying sets of genes that respond at different speeds and times during C depletion, our extended dataset and model aid the analysis of candidates for C signaling. This is illustrated for AKIN10 and four bZIP transcription factors, and sets of genes involved in trehalose signaling, protein turnover, and starch breakdown.

Authors: B. Usadel, O. E. Blasing, Y. Gibon, K. Retzlaff, M. Hohne, M. Gunther, M. Stitt

Date Published: No date defined

Journal: Plant Physiol

Abstract (Expand)

Plants use the circadian clock to sense photoperiod length. Seasonal responses like flowering are triggered at a critical photoperiod when a light-sensitive clock output coincides with light or darkness. However, many metabolic processes, like starch turnover, and growth respond progressively to photoperiod duration. We first tested the photoperiod response of 10 core clock genes and two output genes. qRT-PCR analyses of transcript abundance under 6, 8, 12 and 18 h photoperiods revealed 1-4 h earlier peak times under short photoperiods and detailed changes like rising PRR7 expression before dawn. Clock models recapitulated most of these changes. We explored the consequences for global gene expression by performing transcript profiling in 4, 6, 8, 12 and 18 h photoperiods. There were major changes in transcript abundance at dawn, which were as large as those between dawn and dusk in a given photoperiod. Contributing factors included altered timing of the clock relative to dawn, light signalling and changes in carbon availability at night as a result of clock-dependent regulation of starch degradation. Their interaction facilitates coordinated transcriptional regulation of key processes like starch turnover, anthocyanin, flavonoid and glucosinolate biosynthesis and protein synthesis and underpins the response of metabolism and growth to photoperiod.

Authors: A. Flis, R. Sulpice, D. D. Seaton, A. A. Ivakov, M. Liput, C. Abel, A. J. Millar, M. Stitt

Date Published: No date defined

Journal: Plant Cell Environ

Abstract (Expand)

bioRxiv preprint 2017 Plants respond to seasonal cues such as the photoperiod, to adapt to current conditions and to prepare for environmental changes in the season to come. To assess photoperiodic responses at the protein level, we quantified the proteome of the model plant Arabidopsis thaliana by mass spectrometry across four photoperiods. This revealed coordinated changes of abundance in proteins of photosynthesis, primary and secondary metabolism, including pigment biosynthesis, consistent with higher metabolic activity in long photoperiods. Higher translation rates in the day time than the night likely contribute to these changes via rhythmic changes in RNA abundance. Photoperiodic control of protein levels might be greatest only if high translation rates coincide with high transcript levels in some photoperiods. We term this proposed mechanism ‘translational coincidence’, mathematically model its components, and demonstrate its effect on the Arabidopsis proteome. Datasets from a green alga and a cyanobacterium suggest that translational coincidence contributes to seasonal control of the proteome in many phototrophic organisms. This may explain why many transcripts but not their cognate proteins exhibit diurnal rhythms.

Authors: Daniel Seaton, Alexander Graf, Katja Baerenfaller, Mark Stitt, Andrew Millar, Wilhelm Gruissem

Date Published: No date defined

Journal: Not specified

Abstract (Expand)

Predicting a multicellular organism's phenotype quantitatively from its genotype is challenging, as genetic effects must propagate up time and length scales. Circadian clocks are intracellular regulators that control temporal gene expression patterns and hence metabolism, physiology and behaviour, from sleep/wake cycles in mammals to flowering in plants1-3. Clock genes are rarely essential but appropriate alleles can confer a competitive advantage4,5 and have been repeatedly selected during crop domestication3,6. Here we quantitatively explain and predict canonical phenotypes of circadian timing in a multicellular, model organism. We used metabolic and physiological data to combine and extend mathematical models of rhythmic gene expression, photoperiod-dependent flowering, elongation growth and starch metabolism within a Framework Model for growth of Arabidopsis thaliana7-9. The model predicted the effect of altered circadian timing upon each particular phenotype in clock-mutant plants. Altered night-time metabolism of stored starch accounted for most but not all of the decrease in whole-plant growth rate. Altered mobilisation of a secondary store of organic acids quantitatively explained the remaining defect. Our results link genotype through specific processes to higher-level phenotypes, formalising our understanding of a subtle, pleiotropic syndrome at the whole-organism level, and validating the systems approach to understand complex traits starting from intracellular circuits.

Authors: Yin Hoon Chew, Daniel Seaton, Virginie Mengin, Anna Flis, Sam T. Mugford, Alison M. Smith, Mark Stitt, Andrew Millar

Date Published: No date defined

Journal: Not specified

Abstract (Expand)

Negative feedback control is a ubiquitous feature of biochemical systems, as is time delay between a signal and its response. Negative feedback in conjunction with time delay can lead to oscillations. In a cellular context, it might be beneficial to mitigate oscillatory behaviour to avoid recurring stress situations. This can be achieved by increasing the distance between the parameters of the system and certain thresholds, beyond which oscillations occur. This distance has been termed resistance. Here, we prove that in a generic three-dimensional negative feedback system the resistance of the system is modified by nested autoinhibitory feedbacks. Our system features negative feedbacks through both input-inhibition as well as output-activation, a signalling component with mass conservation and perfect adaptation. We show that these features render the system applicable to biological data, exemplified by the high osmolarity glycerol system in yeast and the mammalian p53 system. Output-activation is better supported by data than input-inhibition and also shows distinguished properties with respect to the system's stimulus. Our general approach might be useful in designing synthetic systems in which oscillations can be tuned by synthetic autoinhibitory feedbacks.

Authors: J. Schaber, A. Lapytsko, D. Flockerzi

Date Published: No date defined

Journal: J R Soc Interface

Abstract (Expand)

Atlantic salmon can synthesize polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (20:5n-3), arachidonic acid (20:4n-6) and docosahexaenoic acid (22:6n-3) via activities of very long chain fatty acyl elongases (Elovls) and fatty acyl desaturases (Fads), albeit to a limited degree. Understanding molecular mechanisms of PUFA biosynthesis and regulation is a pre-requisite for sustainable use of vegetable oils in aquafeeds as current sources of fish oils are unable to meet increasing demands for omega-3 PUFAs. By generating CRISPR-mediated elovl2 partial knockout (KO), we have shown that elovl2 is crucial for multi-tissue synthesis of 22:6n-3 in vivo and that endogenously synthesized PUFAs are important for transcriptional regulation of lipogenic genes in Atlantic salmon. The elovl2-KOs showed reduced levels of 22:6n-3 and accumulation of 20:5n-3 and docosapentaenoic acid (22:5n-3) in the liver, brain and white muscle, suggesting inhibition of elongation. Additionally, elovl2-KO salmon showed accumulation of 20:4n-6 in brain and white muscle. The impaired synthesis of 22:6n-3 induced hepatic expression of sterol regulatory element binding protein-1 (srebp-1), fatty acid synthase-b, Δ6fad-a, Δ5fad and elovl5. Our study demonstrates key roles of elovl2 at two penultimate steps of PUFA synthesis in vivo and suggests Srebp-1 as a main regulator of endogenous PUFA synthesis in Atlantic salmon.

Authors: Alex K. Datsomor, Nikola Zic, Keshuai Li, Rolf E. Olsen, Yang Jin, Jon Olav Vik, Rolf B. Edvardsen, Fabian Grammes, Anna Wargelius, Per Winge

Date Published: 1st Dec 2019

Journal: Sci Rep

Abstract (Expand)

Chiral 2-substituted 3-hydroxycarboxylic acid derivatives are valuable building blocks for the preparation of naturally occurring and synthetic biologically active molecules. Current methodologies for the preparation of these compounds are still limited for large-scale production due to the high costs, limited microbial strains, low yields, difficult downstream processing, and limited range of structures. We report an effective chemoenzymatic method for the synthesis of enantiomerically pure 2 substituted 3 hydroxycarboxylic esters. The strategy comprises: i) a stereoselective aldol addition of 2 oxoacids to methanal catalyzed by two enantiocomplementary 2 oxoacid aldolases, ii) oxidative decarboxylation, and iii) esterification. Compounds with S-configuration were obtained in 69-80% isolated yields (94-99% ee), and the R enantiomers in 57-88% (88-95% ee), using a substrate concentration range of 0.1-1.0 M. The method developed offers a versatile alternative route to this important class of chiral building blocks, and highlights the exciting opportunities available for using natural enzymes with minimal active site modification.

Authors: Roser Marín-Valls, Karel Hernández, Michael Bolte, Jesús Joglar, Jordi Bujons, Pere Clapés

Date Published: 8th Jul 2019

Journal: ACS Catal.

Abstract (Expand)

Zebrafish is a useful modeling organism for the study of vertebrate development, immune response, and metabolism. Metabolic studies can be aided by mathematical reconstructions of the metabolic network of zebrafish. These list the substrates and products of all biochemical reactions that occur in the zebrafish. Mathematical techniques such as flux-balance analysis then make it possible to predict the possible metabolic flux distributions that optimize, for example, the turnover of food into biomass. The only available genome-scale reconstruction of zebrafish metabolism is ZebraGEM. In this study, we present ZebraGEM 2.0, an updated and validated version of ZebraGEM. ZebraGEM 2.0 is extended with gene-protein-reaction associations (GPRs) that are required to integrate genetic data with the metabolic model. To demonstrate the use of these GPRs, we performed an in silico genetic screening for knockouts of metabolic genes and validated the results against published in vivo genetic knockout and knockdown screenings. Among the single knockout simulations, we identified 74 essential genes, whose knockout stopped growth completely. Among these, 11 genes are known have an abnormal knockout or knockdown phenotype in vivo (partial), and 41 have human homologs associated with metabolic diseases. We also added the oxidative phosphorylation pathway, which was unavailable in the published version of ZebraGEM. The updated model performs better than the original model on a predetermined list of metabolic functions. We also determined a minimal feed composition. The oxidative phosphorylation pathways were validated by comparing with published experiments in which key components of the oxidative phosphorylation pathway were pharmacologically inhibited. To test the utility of ZebraGEM2.0 for obtaining new results, we integrated gene expression data from control and Mycobacterium marinum-infected zebrafish larvae. The resulting model predicts impeded growth and altered histidine metabolism in the infected larvae.

Authors: L. van Steijn, F. J. Verbeek, H. P. Spaink, R. M. H. Merks

Date Published: 20th Jun 2019

Journal: Zebrafish

Abstract (Expand)

The dopaminergic effect of PAH and PFAS mixtures, prepared according to environmentally relevant concentrations, has been studied in juvenile female Atlantic cod ( Gadus morhua). Benzo[a]pyrene, dibenzothiophene, fluorene, naphthalene, phenanthrene, and pyrene were used to prepare a PAH mixture, while PFNA, PFOA, PFOS, and PFTrA were used to prepare a PFAS mixture. Cod were injected intraperitoneally twice, with either a low (1x) or high (20x) dose of each compound mixture or their combinations. After 2 weeks of exposure, levels of plasma 17beta-estradiol (E2) were significantly elevated in high PAH/high PFAS treated group. Brain dopamine/metabolite ratios (DOPAC/dopamine and HVA+DOPAC/dopamine) changed with E2 plasma levels, except for high PAH/low PFAS and low PAH/high PFAS treated groups. On the transcript levels, th mRNA inversely correlated with dopamine/metabolite ratios and gnrh2 mRNA levels. Respective decreases and increases of drd1 and drd2a after exposure to the high PAH dose were observed. Specifically, high PFAS exposure decreased both drds, leading to high plasma E2 concentrations. Other studied end points suggest that these compounds, at different doses and combinations, have different toxicity threshold and modes of action. These effects indicate potential alterations in the feedback signaling processes within the dopaminergic pathway by these contaminant mixtures.

Authors: Essa Ahsan Khan, L. B. Bertotto, Karina Dale, R. Lille-Langoy, Fekadu Yadetie, Odd André Karlsen, Anders Goksøyr, D. Schlenk, A. Arukwe

Date Published: 18th Jun 2019

Journal: Environ Sci Technol

Abstract (Expand)

We described an efficient in situ generation of hydroxypyruvate from d‐serine catalyzed by a d‐amino acid oxidase from Rhodotorula gracilis. This strategy revealed an interesting alternative to the conventional chemical synthesis of hydroxypyruvate starting from toxic bromopyruvate or to the enzymatic transamination from l‐serine requiring an additional substrate as amino acceptor. Hydroxypyruvate thus produced was used as donor substrate of transketolases from Escherichia coli or from Geobacillus stearothermophilus catalyzing the stereoselective formation of a carbon−carbon bond. The enzymatic cascade reaction was performed in one‐pot in the presence of d‐serine and appropriate aldehydes for the synthesis of valuable (3S)‐hydroxyketones, which were obtained with high enantio‐ and diastereoselectivity and in good yield. The efficiency of the process was based on the irreversibility of both reactions allowing complete conversion of d‐serine and aldehydes.

Authors: None

Date Published: 6th Jun 2019

Journal: Volume361, Issue11 Special Issue: Biocatalysis, Pages 2550-2558

Abstract (Expand)

Nitrogen heterocycles are structural motifs found in many bioactive natural products and of utmost importance in pharmaceutical drug development. In this work, a stereoselective synthesis of functionalized N‐heterocycles was accomplished in two steps, comprising the biocatalytic aldol addition of ethanal and simple aliphatic ketones such as propanone, butanone, 3‐pentanone, cyclobutanone, and cyclopentanone to N‐Cbz‐protected aminoaldehydes using engineered variants of d‐fructose‐6‐phosphate aldolase from Escherichia coli (FSA) or 2‐deoxy‐d‐ribose‐5‐phosphate aldolase from Thermotoga maritima (DERATma) as catalysts. FSA catalyzed most of the additions of ketones while DERATma was restricted to ethanal and propanone. Subsequent treatment with hydrogen in the presence of palladium over charcoal, yielded low‐level oxygenated N‐heterocyclic derivatives of piperidine, pyrrolidine and N‐bicyclic structures bearing fused cyclobutane and cyclopentane rings, with stereoselectivities of 96–98 ee and 97:3 dr in isolated yields ranging from 35 to 79%.

Authors: Raquel Roldán, Karel Hernández, Jesús Joglar, Jordi Bujons, Teodor Parella, Wolf Dieter Fessner, Pere Clapés

Date Published: 6th Jun 2019

Journal: Adv. Synth. Catal.

Abstract (Expand)

Sulfolobus solfataricus P2 grows on different carbohydrates as well as alcohols, peptides and amino acids. Carbohydrates such as D-glucose or D-galactose are degraded via the modified, branched Entner-Doudoroff (ED) pathway whereas growth on peptides requires the Embden-Meyerhof-Parnas (EMP) pathway for gluconeogenesis. As for most hyperthermophilic Archaea an important control point is established at the level of triosephophate conversion, however, the regulation at the level of pyruvate/phosphoenolpyruvate conversion was not tackled so far. Here we describe the cloning, expression, purification and characterization of the pyruvate kinase (PK, SSO0981) and the phosphoenolpyruvate synthetase (PEPS, SSO0883) of Sul. solfataricus. The PK showed only catabolic activity [catalytic efficiency (PEP): 627.95 mM(-1)s(-1), 70 degrees C] with phosphoenolpyruvate as substrate and ADP as phosphate acceptor and was allosterically inhibited by ATP and isocitrate (K i 0.8 mM). The PEPS was reversible, however, exhibited preferred activity in the gluconeogenic direction [catalytic efficiency (pyruvate): 1.04 mM(-1)s(-1), 70 degrees C] and showed some inhibition by AMP and alpha-ketoglutarate. The gene SSO2829 annotated as PEPS/pyruvate:phosphate dikinase (PPDK) revealed neither PEPS nor PPDK activity. Our studies suggest that the energy charge of the cell as well as the availability of building blocks in the citric acid cycle and the carbon/nitrogen balance plays a major role in the Sul. solfataricus carbon switch. The comparison of regulatory features of well-studied hyperthermophilic Archaea reveals a close link and sophisticated coordination between the respective sugar kinases and the kinetic and regulatory properties of the enzymes at the level of PEP-pyruvate conversion.

Authors: Patrick Haferkamp, B. Tjaden, Lu Shen, C. Brasen, Theresa Kouril, Bettina Siebers

Date Published: 30th Apr 2019

Journal: Front Microbiol

Abstract (Expand)

The aim of this study was to assess whether fish in Kollevag, a sheltered bay on the western coast of Norway, previously utilized as a waste disposal site, could be affected by environmental contaminants leaking from the waste. Farmed, juvenile Atlantic cod (Gadus morhua) were caged for six weeks at three different locations in Kollevag bay and at one reference location. Sediments and cod samples (bile and liver) were analyzed for polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), brominated flame retardants (BFRs), per-and polyfluoroalkyl substances (PFASs) and polycyclic aromatic hydrocarbon (PAH) metabolites, revealing a contamination gradient at the four stations. Furthermore, hepatosomatic index (HSI) and Fulton's condition factor (CF) were significantly lower in cod caged closest to the disposal site. Levels and activities of biomarker proteins, such as vitellogenin (Vtg), metallothionein (Mt), and biotransformation and oxidative stress enzymes, including cytochrome P450 1a and 3a (Cyp1a, Cyp3a), glutathione s-transferase (Gst) and catalase (Cat), were quantified in blood plasma and liver tissue. Hepatic Cat and Gst activities were significantly reduced in cod caged at the innermost stations in Kollevag, indicating modulation of oxidative stress responses. However, these results contrasted with reduced hepatic lipid peroxidation. Significant increases in transcript levels were observed for genes involved in lipid metabolism (fasn and acly) in cod liver, while transcript levels of ovarian steroidogenic enzyme genes such as p450scc, cyp19, 3beta-hsd and 20beta-hsd showed significant station-dependent increases. Cyp1a and Vtg protein levels were however not significantly altered in cod caged in Kollevag. Plasma levels of estradiol (E2) and testosterone (T) were determined by enzyme immunoassay (EIA) and showed elevated E2 levels, but only at the innermost station. We conclude that the bay of Kollevag did not fullfill adequate environmental condition based on environmental quality standards (EQSs) for chemicals in coastal waters. Following a six weeks caging period, environmental contaminants accumulated in cod tissues and effects were observed on biomarker responses, especially those involved in reproductive processes in cod ovary.

Authors: Karina Dale, M. B. Muller, Zhanna Tairova, Essa Ahsan Khan, K. Hatlen, M. Grung, Fekadu Yadetie, R. Lille-Langoy, Nello Blaser, H. J. Skaug, J. L. Lyche, A. Arukwe, Ketil Hylland, Odd André Karlsen, Anders Goksøyr

Date Published: 26th Feb 2019

Journal: Mar Environ Res

Abstract (Expand)

Factors affecting the establishment of the gut microbiota in animals living in marine environments remain largely unknown. In terrestrial animals, however, it is well established that the juvenile environment has a major impact on the gut microbiota later in life. Atlantic salmon Salmo salar is an anadromous fish important in aquaculture with a juvenile freshwater stage and an adult seawater stage. For wild salmon, there are major dietary changes with respect to availability of long-chain polyunsaturated n-3 fatty acids (LC-n-3 PUFA) with lower abundance in freshwater systems. The aim of our work was therefore to determine the effect of a juvenile freshwater diet with high LC-n-3 PUFA, as compared to a diet low in LC-n-3 PUFA (designed to increase the endogenous LC-n-3 PUFA production), on the transition to a seawater gut microbiota for Atlantic salmon. We found a juvenile freshwater microbiota high in Firmicutes for fish raised with low LC-n-3 PUFA, while the microbiota for fish given high LC-n-3 PUFA feed was high in Proteobacteria. One hundred days after transfer to a common sea cage, fish that were given low LC-n-3 PUFA diets in freshwater showed significantly higher (p = 0.02, Kruskal-Wallis) Mycoplasma content (90 ± 7%; mean ± SD) compared to fish raised on a high LC-n-3 PUFA diet in freshwater (25 ± 31% Mycoplasma). Shotgun metagenome sequencing from fish raised with a low LC-n-3 PUFA diet identified a salmon-associated Mycoplasma in sea, being distinct from currently known Mycoplasma. The genome sequence information indicated a mutualistic lifestyle of this bacterium. Mycoplasma has also previously been identified as dominant (>70%) in sea-living adult Atlantic salmon. Taken together, our results suggest that the juvenile freshwater diet influences the establishment of the gut microbiota in marine Atlantic salmon.

Authors: Yang Jin, Inga Leena Angell, Sandve Simen, Lars Snipen, Y Olsen, Knut Rudi

Date Published: 24th Jan 2019

Journal: Aquacult. Environ. Interact.

Abstract

Not specified

Authors: Tjasa Kumelj, Snorre Sulheim, Alexander Wentzel, Eivind Almaas

Date Published: 7th Dec 2018

Journal: Biotechnol. J.

Abstract

Not specified

Authors: Maxwell Lewis Neal, Matthias König, David Nickerson, Göksel Mısırlı, Reza Kalbasi, Andreas Dräger, Koray Atalag, Vijayalakshmi Chelliah, Michael T Cooling, Daniel L Cook, Sharon Crook, Miguel de Alba, Samuel H Friedman, Alan Garny, John H Gennari, Padraig Gleeson, Martin Golebiewski, Mike Hucka, Nick Juty, Chris Myers, Brett G Olivier, Herbert M Sauro, Martin Scharm, Jacky Snoep, Vasundra Toure, Anil Wipat, Olaf Wolkenhauer, Dagmar Waltemath

Date Published: 21st Nov 2018

Journal: Not specified

Abstract

Not specified

Author: Timon Oefelein

Date Published: 20th Nov 2018

Journal: SCS

Abstract (Expand)

Plants sense light and temperature changes to regulate flowering time. Here, we show that expression of the Arabidopsis florigen gene, FLOWERING LOCUS T (FT), peaks in the morning during spring, a different pattern than we observe in the laboratory. Providing our laboratory growth conditions with a red/far-red light ratio similar to open-field conditions and daily temperature oscillation is sufficient to mimic the FT expression and flowering time in natural long days. Under the adjusted growth conditions, key light signalling components, such as phytochrome A and EARLY FLOWERING 3, play important roles in morning FT expression. These conditions stabilize CONSTANS protein, a major FT activator, in the morning, which is probably a critical mechanism for photoperiodic flowering in nature. Refining the parameters of our standard growth conditions to more precisely mimic plant responses in nature can provide a powerful method for improving our understanding of seasonal response.

Authors: Y. H. Song, A. Kubota, M. S. Kwon, M. F. Covington, N. Lee, E. R. Taagen, D. Laboy Cintron, D. Y. Hwang, R. Akiyama, S. K. Hodge, H. Huang, N. H. Nguyen, D. A. Nusinow, A. J. Millar, K. K. Shimizu, T. Imaizumi

Date Published: 27th Sep 2018

Journal: Nat Plants

Abstract (Expand)

Caulobacter crescentus is a gram-negative bacterium that can utilize xylose as a substrate using the Weimberg pathway, which converts xylose to α-ketoglutarate in five steps without carbon loss. This is an interesting pathway for heterologous expression in other organisms in order to enable xylose utilization in biorefinery processes. C. crescentus was grown on xylose, arabinose and glucose, and maximum specific growth rates determined for the three substrates were 0.11 h−1, 0.05 h−1, and 0.15 h−1 respectively. Growth was found to be significantly inhibited at sugar concentration of 20 g L−1, shown primarily by an increased lag phase. Enzyme activity assays showed that the Weimberg pathway was active in cells grown, not only on xylose but also on arabinose. No activity was found for growth on glucose. Furthermore, substantial amounts of α-ketoglutarate—up to a yield of 0.4 g g−1—was excreted during growth on xylose, but no other extracellular intermediates in the Weimberg pathway were detected during growth on xylose. Apparently, C. crescentus is not well adapted for efficient growth on high xylose levels, and responds by an extended lag phase and secretion of α-ketoglutarate.

Authors: Henrik Almqvist, Sara Jonsdottir Glaser, Celina Tufvegren, Lisa Wasserstrom, Gunnar Liden

Date Published: 1st Jun 2018

Journal: Fermentation

Abstract (Expand)

Cell volume is an important parameter for modelling cellular processes. Temperature-induced variability of cellular size, volume, intracellular granularity, a fraction of budding cells of yeast Saccharomyces cerevisiae CEN.PK 113–7D (in anaerobic glucose unlimited batch cultures) were measured by flow cytometry and matched with the performance of the biomass growth (maximal specific growth rate (μmax), specific rate of glucose consumption, the rate of maintenance, biomass yield on glucose). The critical diameter of single cells was 7.94 μm and it is invariant at growth temperatures above 18.5°C. Below 18.5°C, it exponentially increases up to 10.2 μm. The size of the bud linearly depends on μmax, and it is between 50% at 5°C and 90% at 31°C of the averaged single cell. The intracellular granularity (side scatter channel (SSC)-index) negatively depends on μmax. There are two temperature regions (5–31°C vs. 33–40°C) where the relationship between SSC-index and various cellular parameters differ significantly. In supraoptimal temperature range (33–40°C), cells are less granulated perhaps due to a higher rate of the maintenance. There is temperature dependent passage through the checkpoints in the cell cycle which influences the μmax. The results point to the existence of two different morphological states of yeasts in these different temperature regions.

Authors: Maksim Zakhartsev, Matthias Reuss

Date Published: 26th Apr 2018

Journal: Not specified

Abstract

Not specified

Authors: Falk Schreiber, Gary D. Bader, Padraig Gleeson, Martin Golebiewski, Mike Hucka, Sarah M. Keating, Nicolas Le Novère, Chris Myers, David Nickerson, Björn Sommer, Dagmar Waltemath

Date Published: 25th Apr 2018

Journal: Not specified

Abstract (Expand)

Leptospirillum ferriphilum plays a major role in acidic, metal rich environments where it represents one of the most prevalent iron oxidizers. These milieus include acid rock and mine drainage as well as biomining operations. Despite its perceived importance, no complete genome sequence of this model species' type strain is available, limiting the possibilities to investigate the strategies and adaptations Leptospirillum ferriphilumT applies to survive and compete in its niche. This study presents a complete, circular genome of Leptospirillum ferriphilumT DSM 14647 obtained by PacBio SMRT long read sequencing for use as a high quality reference. Analysis of the functionally annotated genome, mRNA transcripts, and protein concentrations revealed a previously undiscovered nitrogenase cluster for atmospheric nitrogen fixation and elucidated metabolic systems taking part in energy conservation, carbon fixation, pH homeostasis, heavy metal tolerance, oxidative stress response, chemotaxis and motility, quorum sensing, and biofilm formation. Additionally, mRNA transcript counts and protein concentrations were compared between cells grown in continuous culture using ferrous iron as substrate and bioleaching cultures containing chalcopyrite (CuFeS2). Leptospirillum ferriphilumT adaptations to growth on chalcopyrite included a possibly enhanced production of reducing power, reduced carbon dioxide fixation, as well as elevated RNA transcripts and proteins involved in heavy metal resistance, with special emphasis on copper efflux systems. Finally, expression and translation of genes responsible for chemotaxis and motility were enhanced.

Authors: Stephan Christel, Malte Herold, Soeren Bellenberg, Mohamed El Hajjami, Antoine Buetti-Dinh, Igor Pivkin, Wolfgang Sand, Paul Wilmes, Ansgar Poetsch, Mark Dopson

Date Published: 1st Feb 2018

Journal: Appl Environ Microbiol

Abstract (Expand)

Gut microbiota associations through habitat transitions are fundamentally important yet poorly understood. One such habitat transition is the migration from freshwater to saltwater for anadromous fish, such as salmon. The aim of the current work was therefore to determine the freshwater-to-saltwater transition impact on the gut microbiota in farmed Atlantic salmon, with dietary interventions resembling freshwater and saltwater diets with respect to fatty acid composition. Using deep 16S rRNA gene sequencing and quantitative PCR, we found that the freshwater-to-saltwater transition had a major association with the microbiota composition and quantity, while diet did not show significant associations with the microbiota. In saltwater there was a 100-fold increase in bacterial quantity, with a relative increase of Firmicutes and a relative decrease of both Actinobacteria and Proteobacteria. Irrespective of an overall shift in microbiota composition from freshwater to saltwater, we identified three core clostridia and one Lactobacillus-affiliated phylotype with wide geographic distribution that were highly prevalent and co-occurring. Taken together, our results support the importance of the dominating bacteria in the salmon gut, with the freshwater microbiota being immature. Due to the low number of potentially host-associated bacterial species in the salmon gut, we believe that farmed salmon can represent an important model for future understanding of host-bacterium interactions in aquatic environments. IMPORTANCE Little is known about factors affecting the interindividual distribution of gut bacteria in aquatic environments. We have shown that there is a core of four highly prevalent and co-occurring bacteria irrespective of feed and freshwater-to-saltwater transition. The potential host interactions of the core bacteria, however, need to be elucidated further.

Authors: Knut Rudi, Inga Leena Angell, Phillip B. Pope, Jon Olav Vik, Simen Rød Sandve, Lars Snipen

Date Published: 15th Jan 2018

Journal: Appl Environ Microbiol

Abstract (Expand)

Chemical reaction networks are ubiquitous in biology, and their dynamics is fundamentally stochastic. Here, we present the software library pSSAlib, which provides a complete and concise implementation of the most efficient partial-propensity methods for simulating exact stochastic chemical kinetics. pSSAlib can import models encoded in Systems Biology Markup Language, supports time delays in chemical reactions, and stochastic spatiotemporal reaction-diffusion systems. It also provides tools for statistical analysis of simulation results and supports multiple output formats. It has previously been used for studies of biochemical reaction pathways and to benchmark other stochastic simulation methods. Here, we describe pSSAlib in detail and apply it to a new model of the endocytic pathway in eukaryotic cells, leading to the discovery of a stochastic counterpart of the cut-out switch motif underlying early-to-late endosome conversion. pSSAlib is provided as a stand-alone command-line tool and as a developer API. We also provide a plug-in for the SBMLToolbox. The open-source code and pre-packaged installers are freely available from http://mosaic.mpi-cbg.de.

Authors: Oleksandr Ostrenko, Pietro Incardona, Rajesh Ramaswamy, Lutz Brusch, Ivo F. Sbalzarini

Date Published: 4th Dec 2017

Journal: PLoS Comput Biol

Abstract (Expand)

Nucleic acids, which constitute the genetic material of all organisms, are continuously exposed to endogenous and exogenous damaging agents, representing a significant challenge to genome stability and genome integrity over the life of a cell or organism. Unrepaired DNA lesions, such as single- and double-stranded DNA breaks (SSBs and DSBs), and single-stranded gaps can block progression of the DNA replication fork, causing replicative stress and/or cell cycle arrest. However, translesion synthesis (TLS) DNA polymerases, such as Rev1, have the ability to bypass some DNA lesions, which can circumvent the process leading to replication fork arrest and minimize replicative stress. Here, we show that Rev1-deficiency in mouse embryo fibroblasts or mouse liver tissue is associated with replicative stress and mitochondrial dysfunction. In addition, Rev1-deficiency is associated with high poly(ADP) ribose polymerase 1 (PARP1) activity, low endogenous NAD+, low expression of SIRT1 and PGC1α and low adenosine monophosphate (AMP)-activated kinase (AMPK) activity. We conclude that replication stress via Rev1-deficiency contributes to metabolic stress caused by compromized mitochondrial function via the PARP-NAD+-SIRT1-PGC1α axis.

Authors: Nima Borhan Fakouri, Jon Ambæk Durhuus, Christine Elisabeth Regnell, Maria Angleys, Claus Desler, Md Mahdi Hasan-Olive, Ana Martín-Pardillos, Anastasia Tsaalbi-Shtylik, Kirsten Thomsen, Martin Lauritzen, Vilhelm A. Bohr, Niels de Wind, Linda Hildegard Bergersen, Tim Rasmussen

Date Published: 1st Dec 2017

Journal: Sci Rep

Abstract

Not specified

Authors: None

Date Published: 24th Oct 2017

Journal: Not specified

Abstract (Expand)

In (hyper)thermophilic organisms metabolic processes have to be adapted to function optimally at high temperature. We compared the gluconeogenic conversion of 3-phosphoglycerate via 1,3-bisphosphoglycerate to glyceraldehyde-3-phosphate at 30 degrees C and at 70 degrees C. At 30 degrees C it was possible to produce 1,3-bisphosphoglycerate from 3-phosphoglycerate with phosphoglycerate kinase, but at 70 degrees C, 1,3-bisphosphoglycerate was dephosphorylated rapidly to 3-phosphoglycerate, effectively turning the phosphoglycerate kinase into a futile cycle. When phosphoglycerate kinase was incubated together with glyceraldehyde 3-phosphate dehydrogenase it was possible to convert 3-phosphoglycerate to glyceraldehyde 3-phosphate, both at 30 degrees C and at 70 degrees C, however, at 70 degrees C only low concentrations of product were observed due to thermal instability of glyceraldehyde 3-phosphate. Thus, thermolabile intermediates challenge central metabolic reactions and require special adaptation strategies for life at high temperature.

Authors: Theresa Kouril, J. J. Eicher, Bettina Siebers, Jacky Snoep

Date Published: 7th Oct 2017

Journal: Microbiology

Abstract (Expand)

Sulfolobus solfataricus is a thermoacidophilic Archaeon that thrives in terrestrial hot springs (solfatares) with optimal growth at 80 degrees C and pH 2-4. It catabolizes specific carbon sources, such as D-glucose, to pyruvate via the modified Entner-Doudoroff (ED) pathway. This pathway has two parallel branches, the semi-phosphorylative and the non-phosphorylative. However, the strategy of S.solfataricus to endure in such an extreme environment in terms of robustness and adaptation is not yet completely understood. Here, we present the first dynamic mathematical model of the ED pathway parameterized with quantitative experimental data. These data consist of enzyme activities of the branched pathway at 70 degrees C and 80 degrees C and of metabolomics data at the same temperatures for the wild type and for a metabolic engineered knockout of the semi-phosphorylative branch. We use the validated model to address two questions: 1. Is this system more robust to perturbations at its optimal growth temperature? 2. Is the ED robust to deletion and perturbations? We employed a systems biology approach to answer these questions and to gain further knowledge on the emergent properties of this biological system. Specifically, we applied deterministic and stochastic approaches to study the sensitivity and robustness of the system, respectively. The mathematical model we present here, shows that: 1. Steady state metabolite concentrations of the ED pathway are consistently more robust to stochastic internal perturbations at 80 degrees C than at 70 degrees C; 2. These metabolite concentrations are highly robust when faced with the knockout of either branch. Connected with this observation, these two branches show different properties at the level of metabolite production and flux control. These new results reveal how enzyme kinetics and metabolomics synergizes with mathematical modelling to unveil new systemic properties of the ED pathway in S.solfataricus in terms of its adaptation and robustness.

Authors: Sofia Figueiredo, Theresa Kouril, D. Esser, Patrick Haferkamp, Patricia Wieloch, Dietmar Schomburg, Peter Ruoff, Bettina Siebers, Jörg Schaber

Date Published: 12th Jul 2017

Journal: PLoS One

Abstract (Expand)

Tissues use feedback circuits in which cells send signals to each other to control their growth and survival. We show that such feed- back circuits are inherently unstable to mutants that misread the signal level: Mutants have a growth advantage to take over the tissue, and cannot be eliminated by known cell-intrinsic mecha- nisms. To resolve this, we propose that tissues have biphasic responses in which the signal is toxic at both high and low levels, such as glucotoxicity of beta cells, excitotoxicity in neurons, and toxicity of growth factors to T cells. This gives most of these mutants a frequency-dependent selective disadvantage, which leads to their elimination. However, the biphasic mechanisms create a new unstable fixed point in the feedback circuit beyond which runaway processes can occur, leading to risk of diseases such as diabetes and neurodegenerative disease. Hence, glucotoxicity, which is a dangerous cause of diabetes, may have a protective anti- mutant effect. Biphasic responses in tissues may provide an evolu- tionary stable strategy that avoids invasion by commonly occurring mutants, but at the same time cause vulnerability to disease.

Authors: Omer Karin, Uri Alon

Date Published: 26th Jun 2017

Journal: Mol Syst Biol

Abstract (Expand)

Constraint based methods, such as the Flux Balance Analysis, are widely used to model cellular growth processes without relying on extensive information on the regulatory features. The regulation is instead substituted by an optimization problem usually aiming at maximal biomass accumulation. A recent extension to these methods called the dynamic enzyme-cost Flux Balance Analysis (deFBA) is a fully dynamic modeling method allowing for the prediction of necessary enzyme levels under changing environmental conditions. However, this method was designed for deterministic settings in which all dynamics, parameters, etc. are exactly known. In this work, we present a theoretical framework extending the deFBA to handle uncertainties and provide a robust solution. We use the ideas from multi-stage nonlinear Model Predictive Control (MPC) and its feature to represent the evolution of uncertainties by an exponentially growing scenario tree. While this representation is able to construct a deterministic optimization problem in the presence of uncertainties, the computational cost also increases exponentially. We counter this by using a receding prediction horizon and reshape the standard deFBA to the short-time deFBA (sdeFBA). This leads us, along with further simplification of the scenario tree, to the robust deFBA (rdeFBA). This framework is capable of handling the uncertainties in the model itself as well as uncertainties experienced by the modeled system. We applied these algorithms to two case-studies: a minimal enzymatic nutrient uptake network, and the abstraction of the core metabolic process in bacteria.

Authors: Henning Lindhorst, Sergio Lucia, Rolf Findeisen, Steffen Waldherr

Date Published: 13th Jun 2017

Journal: Not specified

Abstract (Expand)

Adaptive Laboratory Evolution (ALE) is increasingly being used as a technique for untargeted strain optimization. This work aimed at developing an automated and miniaturized ALE approach based on repetitive batch cultivations in microtiter plates. The new method is applied to the recently published strain Corynebacterium glutamicum pEKEx3-xylXABCDCc, which is capable of utilizing d-xylose via the Weimberg (WMB) pathway. As a result, the significantly improved strain WMB2evo was obtained, showing a specific growth rate of 0.26h-1 on d-xylose as sole carbon and energy source. WMB2evo grows stable during lab-scale bioreactor operation, demonstrating the high potential of this strain for future biorefinery applications. Genome sequencing of cell samples from two different ALE processes revealed potential key mutations, e.g. in the gene cg0196 (encoding for the transcriptional regulator IolR of the myo-inositol metabolism). These findings open up new perspectives for the rational engineering of C. glutamicum towards improved d-xylose utilization.

Authors: A. Radek, Niklas Tenhaef, M. F. Muller, Christian Brüsseler, W. Wiechert, Jan Marienhagen, T. Polen, Stephan Noack

Date Published: 30th May 2017

Journal: Bioresour Technol

Abstract (Expand)

The thermoacidophilic Crenarchaeon Sulfolobus solfataricus is a model organism for archaeal adaptation to extreme environments and renowned for its ability to degrade a broad variety of substrates. It has been well characterised concerning the utilisation of numerous carbohydrates as carbon source. However, its amino acid metabolism, especially the degradation of single amino acids, is not as well understood. In this work, we performed metabolic modelling as well as metabolome, transcriptome and proteome analysis on cells grown on caseinhydrolysate as carbon source in order to draw a comprehensive picture of amino acid metabolism in S. solfataricus P2. We found that 10 out of 16 detectable amino acids are imported from the growth medium. Overall, uptake of glutamate, methionine, leucine, phenylalanine and isoleucine was the highest of all observed amino acids. Our simulations predict an incomplete degradation of leucine and tyrosine to organic acids, and in accordance with this, we detected the export of branched-chain and aromatic organic acids as well as amino acids, ammonium and trehalose into the culture supernatants. The branched-chain amino acids as well as phenylalanine and tyrosine are degraded to organic acids via oxidative Stickland reactions. Such reactions are known for prokaryotes capable of anaerobic growth, but so far have never been observed in an obligate aerobe. Also, 3-methyl-2-butenoate and 2-methyl-2-butenoate are for the first time found as products of modified Stickland reactions for the degradation of branched-chain amino acids. This work presents the first detailed description of branched-chain and aromatic amino acid catabolism in S. solfataricus.

Authors: Helge Stark, Jacqueline Wolf, Andreas Albersmeier, Trong Khoa Pham, Julia D. Hofmann, Bettina Siebers, Jörn Kalinowski, Phil Wright, Meina Neumann-Schaal, Dietmar Schomburg

Date Published: 29th May 2017

Journal: FEBS J

Abstract (Expand)

PCB 153 is one of the most abundant PCB congeners detected in biological samples. It is a persistent compound that is still present in the environment despite the ban on production and use of PCBs in the late 1970s. It has strong tendencies to bioaccumulate and biomagnify in biota, and studies have suggested that it is an endocrine and metabolic disruptor. In order to study mechanisms of toxicity, we exposed Atlantic cod (Gadus morhua) to various doses of PCB 153 (0, 0.5, 2 and 8 mg/kg body weight) for two weeks and examined the effects on expression of liver proteins using label-free quantitative proteomics. Label-free liquid chromatography-mass spectrometry analysis of the liver proteome resulted in the quantification of 1272 proteins, of which 78 proteins were differentially regulated in the PCB 153-treated dose groups compared to the control group. Functional enrichment analysis showed that pathways significantly affected are related to the lipid metabolism, cytoskeletal remodeling, cell cycle and cell adhesion. Importantly, the main effects appear to be on lipid metabolism, with up-regulation of enzymes in the de novo fatty acid synthesis pathway, consistent with previous transcriptomics results. Increased plasma triglyceride levels were also observed in the PCB 153 treated fish, in agreement with the induction of the lipogenic genes and proteins. The results suggest that PCB 153 perturbs lipid metabolism in the Atlantic cod liver. Elevated levels of lipogenic enzymes and plasma triglycerides further suggest increased synthesis of fatty acids and triglycerides.

Author: Fekadu Yadetie, Eystein Oveland, Anne Døskeland, Frode Berven Anders Goksøyr, Odd André Karlsen

Date Published: 1st Apr 2017

Journal: Aquatic Toxicology

Abstract (Expand)

The transketolase from Geobacillus stearothermophilus (TKGst) is a thermostable enzyme with notable high activity and stability at elevated temperatures, but it accepts non‐α‐hydroxylated aldehydes only with low efficiency. Here we report a protein engineering study of TKGst based on double‐site saturation mutagenesis either at Leu191 or at Phe435 in combination with Asp470; these are the residues responsible for substrate binding in the active site. Screening of the mutagenesis libraries resulted in several positive variants with activity towards propanal up to 7.4 times higher than that of the wild type. Variants F435L/D470E and L191V/D470I exhibited improved (73 % ee, 3S) and inverted (74 % ee, 3R) stereoselectivity, respectively, for propanal. L191V, L382F/E, F435L, and D470/D470I were concluded to be positive mutations at Leu191, Leu382, Phe435, and Asp470 both for activity and for stereoselectivity improvement. These results should benefit further engineering of TKGst for various applications in asymmetric carboligation.

Authors: Chaoqiang Zhou, Thangavelu Saravanan, Marion Lorillière, Dongzhi Wei, Franck Charmantray, Laurence Hecquet, Wolf Dieter Fessner, Dong Yi

Date Published: 2nd Mar 2017

Journal: ChemBioChem

Abstract

Not specified

Authors: Wolfgang Müller, Meik Bittkowski, Martin Golebiewski, Renate Kania, Maja Rey, Andreas Weidemann, Ulrike Wittig

Date Published: 1st Mar 2017

Journal: Datenbank Spektrum

Abstract (Expand)

Standards are essential to the advancement of science and technology. In systems and synthetic biology, numerous standards and associated tools have been developed over the last 16 years. This special issue of the Journal of Integrative Bioinformatics aims to support the exchange, distribution and archiving of these standards, as well as to provide centralised and easily citable access to them.

Authors: F. Schreiber, G. D. Bader, P. Gleeson, Martin Golebiewski, M. Hucka, N. Le Novere, C. Myers, D. Nickerson, B. Sommer, Dagmar Waltemath

Date Published: 12th Feb 2017

Journal: J Integr Bioinform

Abstract (Expand)

Signaling through the AKT and ERK pathways controls cell proliferation. However, the integrated regulation of this multistep process, involving signal processing, cell growth and cell cycle progression, is poorly understood. Here, we study different hematopoietic cell types, in which AKT and ERK signaling is triggered by erythropoietin (Epo). Although these cell types share the molecular network topology for pro-proliferative Epo signaling, they exhibit distinct proliferative responses. Iterating quantitative experiments and mathematical modeling, we identify two molecular sources for cell type-specific proliferation. First, cell type-specific protein abundance patterns cause differential signal flow along the AKT and ERK pathways. Second, downstream regulators of both pathways have differential effects on proliferation, suggesting that protein synthesis is rate-limiting for faster cycling cells while slower cell cycles are controlled at the G1-S progression. The integrated mathematical model of Epo-driven proliferation explains cell type-specific effects of targeted AKT and ERK inhibitors and faithfully predicts, based on the protein abundance, anti-proliferative effects of inhibitors in primary human erythroid progenitor cells. Our findings suggest that the effectiveness of targeted cancer therapy might become predictable from protein abundance.

Authors: Lorenz Adlung, S. Kar, Marie-Christine Wagner, B. She, Sajib Chakraborty, J. Bao, S. Lattermann, M. Boerries, Hauke Busch, P. Wuchter, A. D. Ho, J. Timmer, Marcel Schilling, T. Hofer, U. Klingmuller

Date Published: 27th Jan 2017

Journal: Mol Syst Biol

Abstract (Expand)

Gram-positive Streptomyces bacteria produce thousands of bioactive secondary metabolites, including antibiotics. To systematically investigate genes affecting secondary metabolism, we developed a hyperactive transposase-based Tn5 transposition system and employed it to mutagenize the model species Streptomyces coelicolor, leading to the identification of 51,443 transposition insertions. These insertions were distributed randomly along the chromosome except for some preferred regions associated with relatively low GC content in the chromosomal core. The base composition of the insertion site and its flanking sequences compiled from the 51,443 insertions implied a 19-bp expanded target site surrounding the insertion site, with a slight nucleic acid base preference in some positions, suggesting a relative randomness of Tn5 transposition targeting in the high-GC Streptomyces genome. From the mutagenesis library, 724 mutants involving 365 genes had altered levels of production of the tripyrrole antibiotic undecylprodigiosin (RED), including 17 genes in the RED biosynthetic gene cluster. Genetic complementation revealed that most of the insertions (more than two-thirds) were responsible for the changed antibiotic production. Genes associated with branched-chain amino acid biosynthesis, DNA metabolism, and protein modification affected RED production, and genes involved in signaling, stress, and transcriptional regulation were overrepresented. Some insertions caused dramatic changes in RED production, identifying future targets for strain improvement.IMPORTANCE High-GC Gram-positive streptomycetes and related actinomycetes have provided more than 100 clinical drugs used as antibiotics, immunosuppressants, and antitumor drugs. Their genomes harbor biosynthetic genes for many more unknown compounds with potential as future drugs. Here we developed a useful genome-wide mutagenesis tool based on the transposon Tn5 for the study of secondary metabolism and its regulation. Using Streptomyces coelicolor as a model strain, we found that chromosomal insertion was relatively random, except at some hot spots, though there was evidence of a slightly preferred 19-bp target site. We then used prodiginine production as a model to systematically survey genes affecting antibiotic biosynthesis, providing a global view of antibiotic regulation. The analysis revealed 348 genes that modulate antibiotic production, among which more than half act to reduce production. These might be valuable targets in future investigations of regulatory mechanisms, for strain improvement, and for the activation of silent biosynthetic gene clusters.

Authors: Z. Xu, Y. Wang, K. F. Chater, H. Y. Ou, H. H. Xu, Z. Deng, M. Tao

Date Published: 8th Jan 2017

Journal: Appl Environ Microbiol

Abstract (Expand)

Organisms use circadian clocks to generate 24-h rhythms in gene expression. However, the clock can interact with other pathways to generate shorter period oscillations. It remains unclear how these different frequencies are generated. Here, we examine this problem by studying the coupling of the clock to the alternative sigma factor sigC in the cyanobacterium Synechococcus elongatus. Using single-cell microscopy, we find that psbAI, a key photosyn- thesis gene regulated by both sigC and the clock, is activated with two peaks of gene expression every circadian cycle under constant low light. This two-peak oscillation is dependent on sigC, without which psbAI rhythms revert to one oscillatory peak per day. We also observe two circadian peaks of elongation rate, which are dependent on sigC, suggesting a role for the frequency doubling in modulating growth. We propose that the two-peak rhythm in psbAI expression is generated by an incoherent feedforward loop between the clock, sigC and psbAI. Modelling and experiments suggest that this could be a general network motif to allow frequency doubling of outputs.

Authors: Bruno MC Martins, Arijit K Das, Liliana Antunes, James CW Locke

Date Published: 22nd Dec 2016

Journal: Mol Syst Biol

Abstract (Expand)

BACKGROUND: Defects in genes involved in mitochondrial fatty-acid oxidation (mFAO) reduce the ability of patients to cope with metabolic challenges. mFAO enzymes accept multiple substrates of different chain length, leading to molecular competition among the substrates. Here, we combined computational modeling with quantitative mouse and patient data to investigate whether substrate competition affects pathway robustness in mFAO disorders. RESULTS: First, we used comprehensive biochemical analyses of wild-type mice and mice deficient for medium-chain acyl-CoA dehydrogenase (MCAD) to parameterize a detailed computational model of mFAO. Model simulations predicted that MCAD deficiency would have no effect on the pathway flux at low concentrations of the mFAO substrate palmitoyl-CoA. However, high concentrations of palmitoyl-CoA would induce a decline in flux and an accumulation of intermediate metabolites. We proved computationally that the predicted overload behavior was due to substrate competition in the pathway. Second, to study the clinical relevance of this mechanism, we used patients' metabolite profiles and generated a humanized version of the computational model. While molecular competition did not affect the plasma metabolite profiles during MCAD deficiency, it was a key factor in explaining the characteristic acylcarnitine profiles of multiple acyl-CoA dehydrogenase deficient patients. The patient-specific computational models allowed us to predict the severity of the disease phenotype, providing a proof of principle for the systems medicine approach. CONCLUSION: We conclude that substrate competition is at the basis of the physiology seen in patients with mFAO disorders, a finding that may explain why these patients run a risk of a life-threatening metabolic catastrophe.

Authors: Karen Van Eunen, C. M. Volker-Touw, A. Gerding, A. Bleeker, J. C. Wolters, W. J. van Rijt, A. M. Martines, K. E. Niezen-Koning, R. M. Heiner, H. Permentier, A. K. Groen, D. J. Reijngoud, Terry G.J. Derks, Barbara Bakker

Date Published: 7th Dec 2016

Journal: BMC Biol

Abstract (Expand)

Pseudomonas is a highly versatile genus containing species that can be harmful to humans and plants while others are widely used for bioengineering and bioremediation. We analysed 432 sequenced Pseudomonas strains by integrating results from a large scale functional comparison using protein domains with data from six metabolic models, nearly a thousand transcriptome measurements and four large scale transposon mutagenesis experiments. Through heterogeneous data integration we linked gene essentiality, persistence and expression variability. The pan-genome of Pseudomonas is closed indicating a limited role of horizontal gene transfer in the evolutionary history of this genus. A large fraction of essential genes are highly persistent, still non essential genes represent a considerable fraction of the core-genome. Our results emphasize the power of integrating large scale comparative functional genomics with heterogeneous data for exploring bacterial diversity and versatility.

Authors: J. J. Koehorst, J. C. van Dam, Ruben Van Heck, E. Saccenti, V. A. Dos Santos, M. Suarez-Diez, P. J. Schaap

Date Published: 7th Dec 2016

Journal: Sci Rep

Abstract (Expand)

The FAIRDOMHub is a repository for publishing FAIR (Findable, Accessible, Interoperable and Reusable) Data, Operating procedures and Models (https://fairdomhub.org/) for the Systems Biology community. It is a web-accessible repository for storing and sharing systems biology research assets. It enables researchers to organize, share and publish data, models and protocols, interlink them in the context of the systems biology investigations that produced them, and to interrogate them via API interfaces. By using the FAIRDOMHub, researchers can achieve more effective exchange with geographically distributed collaborators during projects, ensure results are sustained and preserved and generate reproducible publications that adhere to the FAIR guiding principles of data stewardship.

Authors: Katy Wolstencroft, Olga Krebs, Jacky Snoep, Natalie Stanford, Finn Bacall, Martin Golebiewski, Rostyslav Kuzyakiv, Quyen Nguyen, Stuart Owen, S. Soiland-Reyes, Jakub Straszewski, D. D. van Niekerk, Alan Williams, L. Malmstrom, Bernd Rinn, Wolfgang Müller, Carole Goble

Date Published: 3rd Dec 2016

Journal: Nucleic Acids Res

Abstract (Expand)

Sucrose translocation between plant tissues is crucial for growth, development and reproduction of plants. Systemic analysis of these metabolic and underlying regulatory processes allow a detailed understanding of carbon distribution within the plant and the formation of associated phenotypic traits. Sucrose translocation from ‘source’ tissues (e.g. mesophyll) to ‘sink’ tissues (e.g. root) is tightly bound to the proton gradient across the membranes. The plant sucrose transporters are grouped into efflux exporters (SWEET family) and proton-symport importers (SUC, STP families). To better understand regulation of sucrose export from source tissues and sucrose import into sink tissues, there is a need for a metabolic model that takes in account the tissue organisation of Arabidopsis thaliana with corresponding metabolic specificities of respective tissues in terms of sucrose and proton production/utilization. An ability of the model to operate under different light modes (‘light’ and ‘dark’) and correspondingly in different energy producing modes is particularly important in understanding regulatory modules.

Authors: Maksim Zakhartsev, Irina Medvedeva, Yuriy Orlov, Ilya Akberdin, Olga Krebs, Waltraud Schulze

Date Published: 1st Dec 2016

Journal: BMC Plant Biol

Abstract (Expand)

Ataxia telangiectasia (A-T) is a rare autosomal recessive disease characterized by progressive neurodegeneration and cerebellar ataxia. A-T is causally linked to defects in ATM, a master regulator of the response to and repair of DNA double-strand breaks. The molecular basis of cerebellar atrophy and neurodegeneration in A-T patients is unclear. Here we report and examine the significance of increased PARylation, low NAD+, and mitochondrial dysfunction in ATM-deficient neurons, mice, and worms. Treatments that replenish intracellular NAD+ reduce the severity of A-T neuropathology, normalize neuromuscular function, delay memory loss, and extend lifespan in both animal models. Mechanistically, treatments that increase intracellular NAD+ also stimulate neuronal DNA repair and improve mitochondrial quality via mitophagy. This work links two major theories on aging, DNA damage accumulation, and mitochondrial dysfunction through nuclear DNA damage-induced nuclear-mitochondrial signaling, and demonstrates that they are important pathophysiological determinants in premature aging of A-T, pointing to therapeutic interventions.

Authors: Evandro Fei Fang, Henok Kassahun, Deborah L. Croteau, Morten Scheibye-Knudsen, Krisztina Marosi, Huiming Lu, Raghavendra A. Shamanna, Sumana Kalyanasundaram, Ravi Chand Bollineni, Mark A. Wilson, Wendy B. Iser, Bradley N. Wollman, Marya Morevati, Jun Li, Jesse S. Kerr, Qiping Lu, Tyler B. Waltz, Jane Tian, David A. Sinclair, Mark P. Mattson, Marianne B. Nilsen, Vilhelm A. Bohr

Date Published: 1st Oct 2016

Journal: Cell Metabolism

Abstract (Expand)

Archaea are characterised by a complex metabolism with many unique enzymes that differ from their bacterial and eukaryotic counterparts. The thermoacidophilic archaeon Sulfolobus solfataricus is known for its metabolic versatility and is able to utilize a great variety of different carbon sources. However, the underlying degradation pathways and their regulation are often unknown. In this work, we analyse growth on different carbon sources using an integrated systems biology approach. The comparison of growth on L-fucose and D-glucose allows first insights into the genome-wide changes in response to the two carbon sources and revealed a new pathway for L-fucose degradation in S. solfataricus. During growth on L-fucose we observed major changes in the central carbon metabolic network, as well as an increased activity of the glyoxylate bypass and the 3-hydroxypropionate/4-hydroxybutyrate cycle. Within the newly discovered pathway for L-fucose degradation the following key reactions were identified: (i) L-fucose oxidation to L-fuconate via a dehydrogenase, (ii) dehydration to 2-keto-3-deoxy-L-fuconate via dehydratase, (iii) 2-keto-3-deoxy-L-fuconate cleavage to pyruvate and L-lactaldehyde via aldolase and (iv) L-lactaldehyde conversion to L-lactate via aldehyde dehydrogenase. This pathway as well as L-fucose transport shows interesting overlaps to the D-arabinose pathway, representing another example for pathway promiscuity in Sulfolobus species. This article is protected by copyright. All rights reserved.

Authors: Jacqueline Wolf, Helge Stark, K. Fafenrot, Andreas Albersmeier, Trong Khoa Pham, K. B. Muller, B. Meyer, L. Hoffmann, L. Shen, Stefan Albaum, Theresa Kouril, K. Schmidt-Hohagen, M. Neumann-Schaal, C. Brasen, J. Kalinowski, Phil Wright, Sonja-Verena Albers, Dietmar Schomburg, Bettina Siebers

Date Published: 10th Sep 2016

Journal: Mol Microbiol

Abstract (Expand)

In systems biology, one of the major tasks is to tailor model complexity to information content of the data. A useful model should describe the data and produce well-determined parameter estimates and predictions. Too small of a model will not be able to describe the data whereas a model which is too large tends to overfit measurement errors and does not provide precise predictions. Typically, the model is modified and tuned to fit the data, which often results in an oversized model. To restore the balance between model complexity and available measurements, either new data has to be gathered or the model has to be reduced. In this manuscript, we present a data-based method for reducing non-linear models. The profile likelihood is utilised to assess parameter identifiability and designate likely candidates for reduction. Parameter dependencies are analysed along profiles, providing context-dependent suggestions for the type of reduction. We discriminate four distinct scenarios, each associated with a specific model reduction strategy. Iterating the presented procedure eventually results in an identifiable model, which is capable of generating precise and testable predictions. Source code for all toy examples is provided within the freely available, open-source modelling environment Data2Dynamics based on MATLAB available at http://www.data2dynamics.org/, as well as the R packages dMod/cOde available at https://github.com/dkaschek/. Moreover, the concept is generally applicable and can readily be used with any software capable of calculating the profile likelihood.

Authors: T. Maiwald, H. Hass, Bernhard Steiert, Joep Vanlier, R. Engesser, A. Raue, F. Kipkeew, H. H. Bock, D. Kaschek, C. Kreutz, Jens Timmer

Date Published: 3rd Sep 2016

Journal: PLoS One

Abstract (Expand)

MOTIVATION: A major goal of drug development is to selectively target certain cell types. Cellular decisions influenced by drugs are often dependent on the dynamic processing of information. Selective responses can be achieved by differences between the involved cell types at levels of receptor, signaling, gene regulation or further downstream. Therefore, a systematic approach to detect and quantify cell type-specific parameters in dynamical systems becomes necessary. RESULTS: Here, we demonstrate that a combination of nonlinear modeling with L1 regularization is capable of detecting cell type-specific parameters. To adapt the least-squares numerical optimization routine to L1 regularization, sub-gradient strategies as well as truncation of proposed optimization steps were implemented. Likelihood-ratio tests were used to determine the optimal regularization strength resulting in a sparse solution in terms of a minimal number of cell type-specific parameters that is in agreement with the data. By applying our implementation to a realistic dynamical benchmark model of the DREAM6 challenge we were able to recover parameter differences with an accuracy of 78%. Within the subset of detected differences, 91% were in agreement with their true value. Furthermore, we found that the results could be improved using the profile likelihood. In conclusion, the approach constitutes a general method to infer an overarching model with a minimum number of individual parameters for the particular models. AVAILABILITY AND IMPLEMENTATION: A MATLAB implementation is provided within the freely available, open-source modeling environment Data2Dynamics. Source code for all examples is provided online at http://www.data2dynamics.org/ CONTACT: bernhard.steiert@fdm.uni-freiburg.de.

Authors: Bernhard Steiert, Jens Timmer, C. Kreutz

Date Published: 3rd Sep 2016

Journal: Bioinformatics

Abstract (Expand)

BACKGROUND: Methylmecury (MeHg) is a widely distributed environmental pollutant with considerable risk to both human health and wildlife. To gain better insight into the underlying mechanisms of MeHg-mediated toxicity, we have used label-free quantitative mass spectrometry to analyze the liver proteome of Atlantic cod (Gadus morhua) exposed in vivo to MeHg (0, 0.5, 2 mg/kg body weight) for 2 weeks. RESULTS: Out of a toltal of 1143 proteins quantified, 125 proteins were differentially regulated between MeHg-treated samples and controls. Using various bioinformatics tools, we performed gene ontology, pathway and network enrichment analysis, which indicated that proteins and pathways mainly related to energy metabolism, antioxidant defense, cytoskeleton remodeling, and protein synthesis were regulated in the hepatic proteome after MeHg exposure. Comparison with previous gene expression data strengthened these results, and further supported that MeHg predominantly affects many energy metabolism pathways, presumably through its strong induction of oxidative stress. Some enzymes known to have functionally important oxidation-sensitive cysteine residues in other animals are among the differentially regulated proteins, suggesting their modulations by MeHg-induced oxidative stress. Integrated analysis of the proteomics dataset combined with previous gene expression dataset showed a more pronounced effect of MeHg on amino acid, glucose and fatty acid metabolic pathways, and suggested possible interactions of the cellular energy metabolism and antioxidant defense pathways. CONCLUSIONS: MeHg disrupts mainly redox homeostasis and energy generating metabolic pathways in cod liver. The energy pathways appear to be modulated through MeHg-induced oxidative stress, possibly mediated by oxidation sensitive enzymes.

Authors: Fekadu Yadetie, S. Bjorneklett, H. K. Garberg, E. Oveland, F. Berven, A. Goksoyr, O. A. Karlsen

Date Published: 9th Aug 2016

Journal: BMC Genomics

Abstract (Expand)

Lung cancer, with its most prevalent form non-small-cell lung carcinoma (NSCLC), is one of the leading causes of cancer-related deaths worldwide, and is commonly treated with chemotherapeutic drugs such as cisplatin. Lung cancer patients frequently suffer from chemotherapy-induced anemia, which can be treated with erythropoietin (EPO). However, studies have indicated that EPO not only promotes erythropoiesis in hematopoietic cells, but may also enhance survival of NSCLC cells. Here, we verified that the NSCLC cell line H838 expresses functional erythropoietin receptors (EPOR) and that treatment with EPO reduces cisplatin-induced apoptosis. To pinpoint differences in EPO-induced survival signaling in erythroid progenitor cells (CFU-E, colony forming unit-erythroid) and H838 cells, we combined mathematical modeling with a method for feature selection, the L1 regularization. Utilizing an example model and simulated data, we demonstrated that this approach enables the accurate identification and quantification of cell type-specific parameters. We applied our strategy to quantitative time-resolved data of EPO-induced JAK/STAT signaling generated by quantitative immunoblotting, mass spectrometry and quantitative real-time PCR (qRT-PCR) in CFU-E and H838 cells as well as H838 cells overexpressing human EPOR (H838-HA-hEPOR). The established parsimonious mathematical model was able to simultaneously describe the data sets of CFU-E, H838 and H838-HA-hEPOR cells. Seven cell type-specific parameters were identified that included for example parameters for nuclear translocation of STAT5 and target gene induction. Cell type-specific differences in target gene induction were experimentally validated by qRT-PCR experiments. The systematic identification of pathway differences and sensitivities of EPOR signaling in CFU-E and H838 cells revealed potential targets for intervention to selectively inhibit EPO-induced signaling in the tumor cells but leave the responses in erythroid progenitor cells unaffected. Thus, the proposed modeling strategy can be employed as a general procedure to identify cell type-specific parameters and to recommend treatment strategies for the selective targeting of specific cell types.

Authors: R. Merkle, Bernhard Steiert, F. Salopiata, S. Depner, A. Raue, N. Iwamoto, M. Schelker, H. Hass, M. Wasch, M. E. Bohm, O. Mucke, D. B. Lipka, C. Plass, W. D. Lehmann, C. Kreutz, J. Timmer, Marcel Schilling, U. Klingmuller

Date Published: 6th Aug 2016

Journal: PLoS Comput Biol

Abstract (Expand)

Plants sense the light environment through an ensemble of photoreceptors. Members of the phytochrome class of light receptors are known to play a critical role in seedling establishment, and are among the best-characterized plant signaling components. Phytochromes also regulate adult plant growth; however, our knowledge of this process is rather fragmented. This study demonstrates that phytochrome controls carbon allocation and biomass production in the developing plant. Phytochrome mutants have a reduced CO2 uptake, yet overaccumulate daytime sucrose and starch. This finding suggests that even though carbon fixation is impeded, the available carbon resources are not fully used for growth during the day. Supporting this notion, phytochrome depletion alters the proportion of day:night growth. In addition, phytochrome loss leads to sizeable reductions in overall growth, dry weight, total protein levels, and the expression of CELLULOSE SYNTHASE-LIKE genes. Because cellulose and protein are major constituents of plant biomass, our data point to an important role for phytochrome in regulating these fundamental components of plant productivity. We show that phytochrome loss impacts core metabolism, leading to elevated levels of tricarboxylic acid cycle intermediates, amino acids, sugar derivatives, and notably the stress metabolites proline and raffinose. Furthermore, the already growth-retarded phytochrome mutants are less responsive to growth-inhibiting abiotic stresses and have elevated expression of stress marker genes. This coordinated response appears to divert resources from energetically costly biomass production to improve resilience. In nature, this strategy may be activated in phytochrome-disabling, vegetation-dense habitats to enhance survival in potentially resource-limiting conditions.

Authors: Deyue Yang, Daniel D. Seaton, Johanna Krahmer, Karen J. Halliday

Date Published: 5th Jul 2016

Journal: Proc Natl Acad Sci USA

Abstract (Expand)

Whole-cell (WC) modeling is a promising tool for biological research, bioengineering, and medicine. However, substantial work remains to create accurate, comprehensive models of complex cells. METHODS: We organized the 2015 Whole-Cell Modeling Summer School to teach WC modeling and evaluate the need for new WC modeling standards and software by recoding a recently published WC model in SBML. RESULTS: Our analysis revealed several challenges to representing WC models using the current standards. CONCLUSION: We, therefore, propose several new WC modeling standards, software, and databases. SIGNIFICANCE: We anticipate that these new standards and software will enable more comprehensive models.

Authors: D. Waltemath, J. Karr, F. Bergmann, V. Chelliah, M. Hucka, M. Krantz, W. Liebermeister, P. Mendes, C. Myers, P. Pir, B. Alaybeyoglu, N. Aranganathan, K. Baghalian, A. Bittig, P. Burke, M. Cantarelli, Y. Chew, R. Costa, J. Cursons, T. Czauderna, A. Goldberg, H. Gomez, J. Hahn, T. Hameri, D. Gardiol, D. Kazakiewicz, I. Kiselev, V. Knight-Schrijver, C. Knupfer, Matthias König, D. Lee, A. Lloret-Villas, N. Mandrik, J. Medley, B. Moreau, H. Naderi-Meshkin, S. Palaniappan, D. Priego-Espinosa, M. Scharm, M. Sharma, K. Smallbone, N. Stanford, J. H. Song, T. Theile, M. Tokic, N. Tomar, V. Toure, J. Uhlendorf, T. Varusai, L. Watanabe, F. Wendland, M. Wolfien, J. Yurkovich, Y. Zhu, A. Zardilis, A. Zhukova, F. Schreiber

Date Published: 16th Jun 2016

Journal: IEEE Trans Biomed Eng

Abstract (Expand)

Wild-type Corynebacterium glutamicum has no endogenous metabolic activity for utilizing the lignocellulosic pentose d-xylose for cell growth. Therefore, two different engineering approaches have been pursued resulting in platform strains harbouring a functional version of either the Isomerase (ISO) or the Weimberg (WMB) pathway for d-xylose assimilation. In a previous study we found for C. glutamicum WMB by-product formation of xylitol during growth on d-xylose and speculated that the observed lower growth rates are due to the growth inhibiting effect of this compound. Based on a detailed phenotyping of the ISO, WMB and the wild-type strain of C. glutamicum, we here show that this organism has a natural capability to synthesize xylitol from d-xylose under aerobic cultivation conditions. We furthermore observed the intracellular accumulation of xylitol-5-phosphate as a result of the intracellular phosphorylation of xylitol, which was particularly pronounced in the C. glutamicum ISO strain. Interestingly, low amounts of supplemented xylitol strongly inhibit growth of this strain on d-xylose, d-glucose and d-arabitol. These findings demonstrate that xylitol is a suitable substrate of the endogenous xylulokinase (XK, encoded by xylB) and its overexpression in the ISO strain leads to a significant phosphorylation of xylitol in C. glutamicum. Therefore, in order to circumvent cytotoxicity by xylitol-5-phosphate, the WMB pathway represents an interesting alternative route for engineering C. glutamicum towards efficient d-xylose utilization.

Authors: A. Radek, M. F. Muller, J. Gatgens, L. Eggeling, K. Krumbach, J. Marienhagen, Stephan Noack

Date Published: 15th Jun 2016

Journal: J Biotechnol

Abstract

Not specified

Authors: Antoine Buetti-Dinh, Olga Dethlefsen, Ran Friedman, Mark Dopson

Date Published: 26th May 2016

Journal: Not specified

Abstract (Expand)

OBJECTIVE: The aim of this study was to assess the relationship between fluorine-18 fluorodeoxyglucose (F-FDG) uptake and molecular biological markers in esophageal squamous cell carcinoma (ESCC) patients. METHODS: Our patient population included 51 patients who underwent F-FDG PET/computed tomography before surgery. Excised tumor tissue was analyzed immunohistochemically using monoclonal antibodies for glucose transporter-1 (GLUT-1), GLUT-3, CD34 [microvessel density (MVD) marker], CD68 (macrophage marker), and CD163 (tumor-associated macrophage marker). The relationships among pathological factors [pathological T stage (p-T stage), pathological lymph node status (p-N status), pathological stage (p-stage), and pathological tumor length], the maximum standardized uptake value (SUVmax), and these molecular biological markers were evaluated using Spearman's rank test and the Kruskal-Wallis test. RESULTS: GLUT-1, GLUT-3, CD34, and CD163 significantly correlated with SUVmax (r=0.547, P<0.001 for GLUT-1; r=0.569, P<0.001 for GLUT-3; r=0.463, P=0.001 for CD34, r=0.455, P=0.001 for CD163), whereas SUVmax, GLUT-1, GLUT-3, CD34, and CD163 significantly correlated with p-T stage (r=0.552, P<0.001 for SUVmax, r=0.307, P=0.03 for GLUT-1, r=0.349, P=0.013 for GLUT-3, r=0.313, P=0.027 for CD34, r=0.526 for CD163, P<0.001), but not with p-N status. CD68 levels showed no significant correlation with SUVmax, p-T stage, p-stage, or p-N status. CONCLUSION: SUVmax, GLUT-1 expression, GLUT-3 expression, MVD, and TAMs show a relationship with the tumor stage and extent of ESCC. GLUT-1, GLUT-3, MVD, and TAMs are associated with the mechanism of F-FDG uptake in ESCC.

Authors: Y. Hirose, H. Kaida, A. Kawahara, S. Matono, T. Tanaka, S. Kurata, M. Kage, M. Ishibashi, T. Abe

Date Published: 25th May 2016

Journal: Nucl Med Commun

Abstract (Expand)

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in industrialized countries and is increasing in prevalence. The pathomechanisms, however, are poorly understood. This study assessed the unexpected role of the Hedgehog pathway in adult liver lipid metabolism. Using transgenic mice with conditional hepatocyte-specific deletion of Smoothened in adult mice, we showed that hepatocellular inhibition of Hedgehog signaling leads to steatosis by altering the abundance of the transcription factors GLI1 and GLI3. This steatotic 'Gli-code' caused the modulation of a complex network of lipogenic transcription factors and enzymes, including SREBP1 and PNPLA3, as demonstrated by microarray analysis and siRNA experiments and could be confirmed in other steatotic mouse models as well as in steatotic human livers. Conversely, activation of the Hedgehog pathway reversed the "Gli-code" and mitigated hepatic steatosis. Collectively, our results reveal that dysfunctions in the Hedgehog pathway play an important role in hepatic steatosis and beyond.

Authors: Madlen Matz-Soja, Christiane Rennert, Kristin Schönefeld, Susanne Aleithe, Jan Boettger, Wolfgang Schmidt-Heck, Thomas S Weiss, Amalya Hovhannisyan, Sebastian Zellmer, Nora Klöting, Angela Schulz, Jürgen Kratzsch, Reinhardt Guthke, Rolf Gebhardt

Date Published: 17th May 2016

Journal: Not specified

Abstract (Expand)

The whole-genome duplication 80 million years ago of the common ancestor of salmonids (salmonid-specific fourth vertebrate whole-genome duplication, Ss4R) provides unique opportunities to learn about the evolutionary fate of a duplicated vertebrate genome in 70 extant lineages. Here we present a high-quality genome assembly for Atlantic salmon (Salmo salar), and show that large genomic reorganizations, coinciding with bursts of transposon-mediated repeat expansions, were crucial for the post-Ss4R rediploidization process. Comparisons of duplicate gene expression patterns across a wide range of tissues with orthologous genes from a pre-Ss4R outgroup unexpectedly demonstrate far more instances of neofunctionalization than subfunctionalization. Surprisingly, we find that genes that were retained as duplicates after the teleost-specific whole-genome duplication 320 million years ago were not more likely to be retained after the Ss4R, and that the duplicate retention was not influenced to a great extent by the nature of the predicted protein interactions of the gene products. Finally, we demonstrate that the Atlantic salmon assembly can serve as a reference sequence for the study of other salmonids for a range of purposes.

Authors: S. Lien, B. F. Koop, S. R. Sandve, J. R. Miller, M. P. Kent, T. Nome, Torgeir R. Hvidsten, J. S. Leong, D. R. Minkley, A. Zimin, Fabian Grammes, H. Grove, Arne Gjuvsland, B. Walenz, R. A. Hermansen, K. von Schalburg, E. B. Rondeau, A. Di Genova, J. K. Samy, J. Olav Vik, M. D. Vigeland, L. Caler, U. Grimholt, S. Jentoft, D. Inge Vage, P. de Jong, T. Moen, M. Baranski, Y. Palti, D. R. Smith, J. A. Yorke, A. J. Nederbragt, A. Tooming-Klunderud, K. S. Jakobsen, X. Jiang, D. Fan, Y. Hu, D. A. Liberles, R. Vidal, P. Iturra, S. J. Jones, I. Jonassen, A. Maass, S. W. Omholt, W. S. Davidson

Date Published: 18th Apr 2016

Journal: Nature

Abstract (Expand)

Reconstructing and understanding the Human Physiome virtually is a complex mathematical problem, and a highly demanding computational challenge. Mathematical models spanning from the molecular level through to whole populations of individuals must be integrated, then personalized. This requires interoperability with multiple disparate and geographically separated data sources, and myriad computational software tools. Extracting and producing knowledge from such sources, even when the databases and software are readily available, is a challenging task. Despite the difficulties, researchers must frequently perform these tasks so that available knowledge can be continually integrated into the common framework required to realize the Human Physiome. Software and infrastructures that support the communities that generate these, together with their underlying standards to format, describe and interlink the corresponding data and computer models, are pivotal to the Human Physiome being realized. They provide the foundations for integrating, exchanging and re-using data and models efficiently, and correctly, while also supporting the dissemination of growing knowledge in these forms. In this paper, we explore the standards, software tooling, repositories and infrastructures that support this work, and detail what makes them vital to realizing the Human Physiome.

Authors: D. Nickerson, K. Atalag, B. de Bono, J. Geiger, Carole Goble, S. Hollmann, J. Lonien, Wolfgang Müller, B. Regierer, Natalie Stanford, Martin Golebiewski, P. Hunter

Date Published: 7th Apr 2016

Journal: Interface Focus

Abstract (Expand)

There is an urgent need to improve the infrastructure supporting the reuse of scholarly data. A diverse set of stakeholders-representing academia, industry, funding agencies, and scholarly publishers-have come together to design and jointly endorse a concise and measureable set of principles that we refer to as the FAIR Data Principles. The intent is that these may act as a guideline for those wishing to enhance the reusability of their data holdings. Distinct from peer initiatives that focus on the human scholar, the FAIR Principles put specific emphasis on enhancing the ability of machines to automatically find and use the data, in addition to supporting its reuse by individuals. This Comment is the first formal publication of the FAIR Principles, and includes the rationale behind them, and some exemplar implementations in the community.

Authors: M. D. Wilkinson, M. Dumontier, I. J. Aalbersberg, G. Appleton, M. Axton, A. Baak, N. Blomberg, J. W. Boiten, L. B. da Silva Santos, P. E. Bourne, J. Bouwman, A. J. Brookes, T. Clark, M. Crosas, I. Dillo, O. Dumon, S. Edmunds, C. T. Evelo, R. Finkers, A. Gonzalez-Beltran, A. J. Gray, P. Groth, Carole Goble, J. S. Grethe, J. Heringa, P. A. 't Hoen, R. Hooft, T. Kuhn, R. Kok, J. Kok, S. J. Lusher, M. E. Martone, A. Mons, A. L. Packer, B. Persson, P. Rocca-Serra, M. Roos, R. van Schaik, S. A. Sansone, E. Schultes, T. Sengstag, T. Slater, G. Strawn, M. A. Swertz, M. Thompson, J. van der Lei, E. van Mulligen, J. Velterop, A. Waagmeester, P. Wittenburg, Katy Wolstencroft, J. Zhao, B. Mons

Date Published: 16th Mar 2016

Journal: Sci Data

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

Journal: Sci. Data

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, Rainer Saffrich, A. Diehlmann, K. Bieback, A. D. Ho, P. Horn

Date Published: 19th Feb 2016

Journal: Exp Hematol

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, Marcel Schilling

Date Published: 4th Feb 2016

Journal: Sci Signal

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: Dawie Van Niekerk, Gerald P. Penkler, Francois du Toit, Jacky Snoep

Date Published: 1st Feb 2016

Journal: FEBS J

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, Rainer Saffrich, S. Giselbrecht, C. Nies, H. Lorig, S. Kolb, A. D. Ho, E. Gottwald

Date Published: 30th Jan 2016

Journal: Cell Tissue Res

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

Journal: IEEE/ACM Trans. Comput. Biol. and Bioinf.

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

Journal: F1000Res

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: Jacky Snoep, Kathleen Green, J. Eicher, D. C. Palm, G. Penkler, F. du Toit, N. Walters, R. Burger, H. V. Westerhoff, Dawie Van Niekerk

Date Published: 27th Nov 2015

Journal: Biochemical Society Transactions

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, Marc Niere, G. Agrimi, S. E. Dyrstad, F. Palmieri, A. A. Nikiforov, K. J. Tronstad, Mathias Ziegler

Date Published: 13th Nov 2015

Journal: J Biol Chem

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, Andrew Millar

Date Published: 16th Oct 2015

Journal: Open Biol

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

Journal: Sci Rep

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

Journal: Journal of Thermal Biology

Abstract (Expand)

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, Bernhard Steiert, M. Schelker, C. Kreutz, T. Maiwald, H. Hass, J. Vanlier, Christian Tönsing, Lorenz Adlung, R. Engesser, W. Mader, T. Heinemann, J. Hasenauer, Marcel Schilling, T. Hofer, E. Klipp, F. Theis, U. Klingmuller, B. Schoberl, J. Timmer

Date Published: 3rd Jul 2015

Journal: Bioinformatics

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, Marcel Schilling, J. Timmer, S. Klamt, U. Klingmuller

Date Published: 23rd Apr 2015

Journal: PLoS Comput Biol

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

Journal: Metabolomics

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, Dawie Van Niekerk, Jacky Snoep

Date Published: 1st Apr 2015

Journal: FEBS J

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: Natalie Stanford, P. Millard, N. Swainston

Date Published: 24th Mar 2015

Journal: Front Cell Dev Biol

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

Date Published: 21st Jan 2015

Journal: Mol Syst Biol

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, Lars Kaderali, F. S. Domingues, M. Albrecht, V. Lohmann, H. Erfle, Ralf Bartenschlager

Date Published: 8th Jan 2015

Journal: PLoS Pathog

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: Nadine Veith, Margrete Solheim, Koen Van Grinsven, B. G. Olivier, Jennifer Levering, R. Grosseholz, Jeroen Hugenholtz, Helge Holo, Ingolf Nes, Bas Teusink, Ursula Kummer

Date Published: 19th Dec 2014

Journal: Appl Environ Microbiol

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, Jurgen Haanstra, Melisa Gualdrón-López, Paul AM Michels

Date Published: 1st Dec 2014

Journal: Current Opinion in Microbiology

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, Lorenz Adlung, Marcel Schilling, S. Roth, U. Klingmuller, W. D. Lehmann

Date Published: 7th Nov 2014

Journal: J Proteome Res

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, Volker Wendisch, W. Wiechert, M. Bott, Stephan Noack, J. Marienhagen

Date Published: 12th Oct 2014

Journal: J Biotechnol

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.

Authors: None

Date Published: 30th Sep 2014

Journal: PLoS One

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.

Authors: None

Date Published: 24th Sep 2014

Journal: Nucleic Acids Res

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: 17th Sep 2014

Journal: Leukemia

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: Yin Hoon Chew, B. Wenden, A. Flis, Virginie Mengin, J. Taylor, C. L. Davey, C. Tindal, H. Thomas, H. J. Ougham, P. de Reffye, M. Stitt, M. Williams, Robert Muetzelfeldt, Karen Halliday, Andrew Millar

Date Published: 10th Sep 2014

Journal: Proc Natl Acad Sci U S A

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: Yulia Yuzenkova, Pamela Gamba, M. Herber, L. Attaiech, Sulman Shafeeq, Oscar Kuipers, S. Klumpp, Nikolay Zenkin, Jan-Willem Veening

Date Published: 6th Sep 2014

Journal: Nucleic Acids Res

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: Abeer Fadda, M. Ryten, D. Droll, Federico Rojas, V. Farber, Jurgen Haanstra, C. Merce, Barbara Bakker, Keith Matthews, Christine Clayton

Date Published: 26th Aug 2014

Journal: Mol Microbiol

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

Journal: Phys. Rev. E

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

Journal: BMC Genomics

Abstract

Not specified

Authors: Anna-Karin Gustavsson, Dawie Van Niekerk, Caroline B. Adiels, Bob Kooi, Mattias Goksör, Jacky Snoep

Date Published: 1st Jun 2014

Journal: FEBS J

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, Rainer Saffrich, E. C. Buss, P. Horn, M. Rojewski, A. Schmitt, A. D. Ho, R. Sanzenbacher, M. Schmitt

Date Published: 20th May 2014

Journal: Cytotherapy

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: Katja Bettenbrock, Hao Bai, Michael Ederer, Jeff Green, Klaas Hellingwerf, Michael Holcombe, S. Kunz, Matthew Rolfe, Guido Sanguinetti, Oliver Sawodny, Poonam Sharma, Sonja Steinsiek, Robert Poole

Date Published: 7th May 2014

Journal: Adv Microb Physiol

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: Fiona Achcar, Abeer Fadda, Jurgen Haanstra, Eduard Kerkhoven, Dong-Hyun Kim, Alejandro Leroux, T. Papamarkou, Federico Rojas, Barbara Bakker, Mike Barrett, Christine Clayton, Mark Girolami, Luise Krauth-Siegel, Keith Matthews, Rainer Breitling

Date Published: 7th May 2014

Journal: Adv Microb Physiol

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: Hao Bai, Matthew Rolfe, Wenjing Jia, S. Coakley, Robert Poole, Jeff Green, Michael Holcombe

Date Published: 24th Apr 2014

Journal: PLoS Comput Biol

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.

Authors: None

Date Published: 27th Mar 2014

Journal: Front Microbiol

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, Carole Goble, Martin Golebiewski, Michael Hucka, Nick Juty, Olga Krebs, 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

Journal: Stand. Genomic Sci.

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.

Authors: None

Date Published: 27th Jan 2014

Journal: PLoS One

Abstract

Not specified

Authors: Anna-Karin Gustavsson, Dawie Van Niekerk, Caroline B. Adiels, Mattias Goksör, Jacky Snoep

Date Published: 3rd Jan 2014

Journal: Not specified

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 andd 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: Joaquin Ariño, Ebru Aydar, Samuel Drulhe, Daniel Ganser, Jesus Jorrin, Matthias Kahm, Falko Krause, Silvia Petrezselyova, Lynne Yenush, Olga Zimmermannová, G. Paul H. van Heusden, Maik Kschischo, Jost Ludwig, Chris Palmer, Jose Ramos, Hana Sychrova

Date Published: 2014

Journal: Advances in Microbial Systems Biology

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: Daniel Seaton, O. Ebenhoh, Andrew Millar, A. Pokhilko

Date Published: 18th Dec 2013

Journal: J R Soc Interface

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, Rainer Saffrich, M. Hanke, V. Eckstein, A. D. Ho, M. Grunze, A. Rosenhahn

Date Published: 14th Dec 2013

Journal: Cell Tissue Res

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: Eduard Kerkhoven, Fiona Achcar, V. P. Alibu, R. J. Burchmore, I. H. Gilbert, M. Trybilo, N. N. Driessen, D. Gilbert, Rainer Breitling, Barbara Bakker, Mike Barrett

Date Published: 5th Dec 2013

Journal: PLoS Comput Biol

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: 22nd Nov 2013

Journal: Biomaterials

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, Rainer Saffrich, V. Eckstein, T. Bruckner, W. Wagner, A. D. Ho, P. Wuchter

Date Published: 10th Oct 2013

Journal: Cytotherapy

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

Journal: PLoS One

Abstract

Not specified

Authors: M. Schmitt, L. P. Muller, G. Keysser, H. M. Lorenz, A. D. Ho, P. Wuchter

Date Published: 4th Sep 2013

Journal: Dtsch Med Wochenschr

Abstract (Expand)

Amplified marker-gene sequences can be used to understand microbial community structure, but they suffer from a high level of sequencing and amplification artifacts. The UPARSE pipeline reports operational taxonomic unit (OTU) sequences with </=1% incorrect bases in artificial microbial community tests, compared with >3% incorrect bases commonly reported by other methods. The improved accuracy results in far fewer OTUs, consistently closer to the expected number of species in a community.

Author: R. C. Edgar

Date Published: 18th Aug 2013

Journal: Nat Methods

Abstract (Expand)

Green fluorescent protein (GFP) offers efficient ways of visualizing promoter activity and protein localization in vivo, and many different variants are currently available to study bacterial cell biology. Which of these variants is best suited for a certain bacterial strain, goal, or experimental condition is not clear. Here, we have designed and constructed two "superfolder" GFPs with codon adaptation specifically for Bacillus subtilis and Streptococcus pneumoniae and have benchmarked them against five other previously available variants of GFP in B. subtilis, S. pneumoniae, and Lactococcus lactis, using promoter-gfp fusions. Surprisingly, the best-performing GFP under our experimental conditions in B. subtilis was the one codon optimized for S. pneumoniae and vice versa. The data and tools described in this study will be useful for cell biology studies in low-GC-rich Gram-positive bacteria.

Authors: W. Overkamp, K. Beilharz, R. Detert Oude Weme, A. Solopova, H. Karsens, A. Kovacs, J. Kok, Oscar Kuipers, Jan-Willem Veening

Date Published: 16th Aug 2013

Journal: Appl Environ Microbiol

Abstract

Not specified

Authors: None

Date Published: 25th Jul 2013

Journal: PLoS Comput Biol

Abstract (Expand)

Four enzymes of the gluconeogenic pathway in Sulfolobus solfataricus were purified and kinetically characterized. The enzymes were reconstituted in vitro to quantify the contribution of temperature instability of the pathway intermediates to carbon loss from the system. The reconstituted system, consisting of phosphoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, triose phosphate isomerase and the fructose 1,6-bisphosphate aldolase/ phosphatase maintained a constant consumption rate of 3-phosphoglycerate and production of fructose 6-phosphate over a 1 hour period. Cofactors ATP and NADPH were regenerated via pyruvate kinase and glucose dehydrogenase. A mathematical model was constructed on the basis of the kinetics of the purified enzymes and the measured half-life times of the pathway intermediates. The model quantitatively predicted the systems uxes and metabolite concentrations. Relative enzyme concentrations were chosen such that half the carbon in the system was lost due to degradation of the thermolabile intermediates dihydroxyacetone phosphate, glyceraldehyde 3-phosphate and 1,3 bisphosphoglycerate, indicating that intermediate instability at high temperature can significantly affect pathway efficiency. This article is protected by copyright. All rights reserved.

Authors: Theresa Kouril, Dominik Esser, Julia Kort, Hans Westerhoff, Bettina Siebers, Jacky Snoep

Date Published: 20th Jul 2013

Journal: FEBS J.

Abstract (Expand)

We present an experimental and computational pipeline for the generation of kinetic models of metabolism, and demonstrate its application to glycolysis in Saccharomyces cerevisiae. Starting from an approximate mathematical model, we employ a "cycle of knowledge" strategy, identifying the steps with most control over flux. Kinetic parameters of the individual isoenzymes within these steps are measured experimentally under a standardised set of conditions. Experimental strategies are applied to establish a set of in vivo concentrations for isoenzymes and metabolites. The data are integrated into a mathematical model that is used to predict a new set of metabolite concentrations and reevaluate the control properties of the system. This bottom-up modelling study reveals that control over the metabolic network most directly involved in yeast glycolysis is more widely distributed than previously thought.

Authors: K. Smallbone, H. L. Messiha, K. M. Carroll, C. L. Winder, N. Malys, W. B. Dunn, E. Murabito, N. Swainston, J. O. Dada, F. Khan, P. Pir, E. Simeonidis, I. Spasic, J. Wishart, D. Weichart, N. W. Hayes, D. Jameson, D. S. Broomhead, S. G. Oliver, S. J. Gaskell, J. E. McCarthy, N. W. Paton, H. V. Westerhoff, D. B. Kell, P. Mendes

Date Published: 9th Jul 2013

Journal: FEBS Lett

Abstract (Expand)

In pathogenic trypanosomes, trypanothione synthetase (TryS) catalyzes the synthesis of both glutathionylspermidine (Gsp) and trypanothione [bis(glutathionyl)spermidine, T(SH)2]. Here we present a thorough kinetic analysis of Trypanosoma brucei TryS in a newly developed phosphate buffer system at pH 7.0 and 37 °C, mimicking the physiological environment of the enzyme in the cytosol of bloodstream parasites. Under these conditions, TryS displays Km-values for GSH, ATP, spermidine and Gsp of 34, 18, 687, and 32 μM, respectively, as well as Ki-values for GSH and T(SH)2 of 1 mM and 360 μM, respectively. As Gsp hydrolysis has a Km-value of 5.6 mM, the in vivo amidase activity is probably negligible. To obtain a deeper insight in the molecular mechanism of TryS, we have formulated alternative kinetic models, with elementary reaction steps represented by linear kinetic equations. The model parameters were fitted to the extensive matrix of steady-state data obtained for different substrate/product combinations under the in vivo-like conditions. The best model describes the full kinetic profile and is able to predict time course data that were not used for fitting. This systems biology approach to enzyme kinetics led us to conclude that (i) TryS follows a ter-reactant mechanism, (ii) the intermediate Gsp dissociates from the enzyme between the two catalytic steps and (iii) T(SH)2 inhibits the enzyme by remaining bound at its product site and, as does the inhibitory GSH, by binding to the activated enzyme complex. The newly detected concerted substrate and product inhibition suggests that TryS activity is tightly regulated.

Authors: None

Date Published: 3rd Jul 2013

Journal: J. Biol. Chem.

Abstract (Expand)

Streptococcus pyogenes (group A Streptococcus, GAS) is an important human pathogen causing mild superficial infections of skin and mucous membranes, but also life-threatening systemic diseases. S. pyogenes and other prokaryotic organisms use the arginine deiminase system (ADS) for survival in acidic environments. In this study, the arginine deiminase (AD), and carbamate kinase (CK) from S. pyogenes M49 strain 591 were heterologously expressed in E. coli DH5α, purified, and kinetically characterized. AD and CK from S. pyogenes M49 share high amino acid sequence similarity with the respective enzymes from Lactococcus lactis subsp. lactis IL1403 (45.6% and 53.5% identical amino acids) and Enterococcus faecalis V583 (66.8% and 66.8% identical amino acids). We found that the arginine deiminase of S. pyogenes is not allosterically regulated by the intermediates and products of the arginine degradation (E. g, ATP, citrulline, carbamoyl phosphate). The Km and Vmax values for arginine were 1.13±0.12 mM (mean ± SD) and 1.51±0.07 μmol/min/mg protein. The carbamate kinase is inhibited by ATP but unaffected by arginine and citrulline. The Km and Vmax values for ADP were 0.72±0.08 mM and 1.10±0.10 μmol/min/mg protein and the Km for carbamoyl phosphate was 0.65±0.07 mM. The optimum pH and temperature for both enzymes were 6.5 and 37°C, respectively.

Authors: None

Date Published: 1st Jul 2013

Journal: Protein Expression and Purification

Abstract

Not specified

Authors: Anna Feldman-Salit, Silvio Hering, H. Messiha, Nadine Veith, Vlad Cojocaru, Antje Sieg, Hans Westerhoff, Bernd Kreikemeyer, Rebecca Wade, Tomas Fiedler

Date Published: 17th May 2013

Journal: Journal of Biological Chemistry

Abstract (Expand)

In response to changing extracellular pH levels, phosphate-limited continuous cultures of Clostridium acetobutylicum reversibly switches its metabolism from the dominant formation of acids to the prevalent production of solvents. Previous experimental and theoretical studies have revealed that this pH-induced metabolic switch involves a rearrangement of the intracellular transcriptomic, proteomic and metabolomic composition of the clostridial cells. However, the influence of the population dynamics on the observations reported has so far been neglected. Here, we present a method for linking the pH shift, clostridial growth and the acetone-butanol-ethanol fermentation metabolic network systematically into a model which combines the dynamics of the external pH and optical density with a metabolic model. Furthermore, the recently found antagonistic expression pattern of the aldehyde/alcohol dehydrogenases AdhE1/2 and pH-dependent enzyme activities have been included into this combined model. Our model predictions reveal that the pH-induced metabolic shift under these experimental conditions is governed by a phenotypic switch of predominantly acidogenic subpopulation towards a predominantly solventogenic subpopulation. This model-driven explanation of the pH-induced shift from acidogenesis to solventogenesis by population dynamics casts an entirely new light on the clostridial response to changing pH levels. Moreover, the results presented here underline that pH-dependent growth and pH-dependent specific enzymatic activity play a crucial role in this adaptation. In particular, the behaviour of AdhE1 and AdhE2 seems to be the key factor for the product formation of the two phenotypes, their pH-dependent growth, and thus, the pH-induced metabolic switch in C. acetobutylicum.

Authors: None

Date Published: 3rd May 2013

Journal: Appl. Microbiol. Biotechnol.

Abstract (Expand)

Systems metabolomics, the identification and quantification of cellular metabolites and their integration with genomics and proteomics data, promises valuable functional insights into cellular biology. However, technical constraints, sample complexity issues and the lack of suitable complementary quantitative data sets prevented accomplishing such studies in the past. Here, we present an integrative metabolomics study of the genome-reduced bacterium Mycoplasma pneumoniae. We experimentally analysed its metabolome using a cross-platform approach. We explain intracellular metabolite homeostasis by quantitatively integrating our results with the cellular inventory of proteins, DNA and other macromolecules, as well as with available building blocks from the growth medium. We calculated in vivo catalytic parameters of glycolytic enzymes, making use of measured reaction velocities, as well as enzyme and metabolite pool sizes. A quantitative, inter-species comparison of absolute and relative metabolite abundances indicated that metabolic pathways are regulated as functional units, thereby simplifying adaptive responses. Our analysis demonstrates the potential for new scientific insight by integrating different types of large-scale experimental data from a single biological source.

Authors: T. Maier, J. Marcos, J. A. Wodke, B. Paetzold, M. Liebeke, R. Gutierrez-Gallego, Luis Serrano

Date Published: 20th Apr 2013

Journal: Mol Biosyst

Abstract (Expand)

Mycoplasma pneumoniae, a threatening pathogen with a minimal genome, is a model organism for bacterial systems biology for which substantial experimental information is available. With the goal of understanding the complex interactions underlying its metabolism, we analyzed and characterized the metabolic network of M. pneumoniae in great detail, integrating data from different omics analyses under a range of conditions into a constraint-based model backbone. Iterating model predictions, hypothesis generation, experimental testing, and model refinement, we accurately curated the network and quantitatively explored the energy metabolism. In contrast to other bacteria, M. pneumoniae uses most of its energy for maintenance tasks instead of growth. We show that in highly linear networks the prediction of flux distributions for different growth times allows analysis of time-dependent changes, albeit using a static model. By performing an in silico knock-out study as well as analyzing flux distributions in single and double mutant phenotypes, we demonstrated that the model accurately represents the metabolism of M. pneumoniae. The experimentally validated model provides a solid basis for understanding its metabolic regulatory mechanisms.

Authors: Judith Wodke, J. Puchalka, M. Lluch-Senar, J. Marcos, Eva Yus, M. Godinho, R. Gutierrez-Gallego, V. A. dos Santos, Luis Serrano, Edda Klipp, T. Maier

Date Published: 4th Apr 2013

Journal: Mol Syst Biol

Abstract (Expand)

In systems biology, quantitative experimental data is the basis of building mathematical models. In most of the cases, they are stored in Excel files and hosted locally. To have a public database for collecting, retrieving and citing experimental raw data as well as experimental conditions is important for both experimentalists and modelers. However, the great effort needed in the data handling procedure and in the data submission procedure becomes the crucial limitation for experimentalists to contribute to a database, thereby impeding the database to deliver its benefit. Moreover, manual copy and paste operations which are commonly used in those procedures increase the chance of making mistakes. Excemplify, a web-based application, proposes a flexible and adaptable template-based solution to solve these problems. Comparing to the normal template based uploading approach, which is supported by some public databases, rather than predefining a format that is potentiall impractical, Excemplify allows users to create their own experiment-specific content templates in different experiment stages and to build corresponding knowledge bases for parsing. Utilizing the embedded knowledge of used templates, Excemplify is able to parse experimental data from the initial setup stage and generate following stages spreadsheets automatically. The proposed solution standardizes the flows of data traveling according to the standard procedures of applying the experiment, cuts down the amount of manual effort and reduces the chance of mistakes caused by manual data handling. In addition, it maintains the context of meta-data from the initial preparation manuscript and improves the data consistency. It interoperates and complements RightField and SEEK as well.

Authors: L. Shi, L. Jong, Ulrike Wittig, P. Lucarelli, Markus Stepath, S. Mueller, L. A. D'Alessandro, Ursula Klingmüller, Wolfgang Müller

Date Published: 4th Apr 2013

Journal: J Integr Bioinform

Abstract (Expand)

In a continuous culture under phosphate limitation the metabolism of Clostridium acetobutylicum depends on the external pH level. By comparing seven steady-state conditions between pH 5.7 and pH 4.5 we show that the switch from acidogenesis to solventogenesis occurs between pH 5.3 and pH 5.0 with an intermediate state at pH 5.1. Here, an integrative study is presented investigating how a changing external pH level affects the clostridial acetone–butanol–ethanol (ABE) fermentation pathway. This is of particular interest as the biotechnological production of n-butanol as biofuel has recently returned into the focus of industrial applications. One prerequisite is the furthering of the knowledge of the factors determining the solvent production and their integrative regulations. We have mathematically analysed the influence of pH-dependent specific enzyme activities of branch points of the metabolism on the product formation. This kinetic regulation was compared with transcriptomic regulation regarding gene transcription and the proteomic profile. Furthermore, both regulatory mechanisms were combined yielding a detailed projection of their individual and joint effects on the product formation. The resulting model represents an important platform for future developments of industrial butanol production based on C. acetobutylicum.

Authors: None

Date Published: 1st Feb 2013

Journal: Microbial Biotechnology

Abstract (Expand)

BACKGROUND AND METHODOLOGY: Recently, we reported on a new class of naphthoquinone derivatives showing a promising anti-trypanosomatid profile in cell-based experiments. The lead of this series (B6, 2-phenoxy-1,4-naphthoquinone) showed an ED(50) of 80 nM against Trypanosoma brucei rhodesiense, and a selectivity index of 74 with respect to mammalian cells. A multitarget profile for this compound is easily conceivable, because quinones, as natural products, serve plants as potent defense chemicals with an intrinsic multifunctional mechanism of action. To disclose such a multitarget profile of B6, we exploited a chemical proteomics approach. PRINCIPAL FINDINGS: A functionalized congener of B6 was immobilized on a solid matrix and used to isolate target proteins from Trypanosoma brucei lysates. Mass analysis delivered two enzymes, i.e. glycosomal glycerol kinase and glycosomal glyceraldehyde-3-phosphate dehydrogenase, as potential molecular targets for B6. Both enzymes were recombinantly expressed and purified, and used for chemical validation. Indeed, B6 was able to inhibit both enzymes with IC(50) values in the micromolar range. The multifunctional profile was further characterized in experiments using permeabilized Trypanosoma brucei cells and mitochondrial cell fractions. It turned out that B6 was also able to generate oxygen radicals, a mechanism that may additionally contribute to its observed potent trypanocidal activity. CONCLUSIONS AND SIGNIFICANCE: Overall, B6 showed a multitarget mechanism of action, which provides a molecular explanation of its promising anti-trypanosomatid activity. Furthermore, the forward chemical genetics approach here applied may be viable in the molecular characterization of novel multitarget ligands.

Authors: S. Pieretti, Jurgen Haanstra, M. Mazet, R. Perozzo, C. Bergamini, F. Prati, R. Fato, G. Lenaz, G. Capranico, R. Brun, Barbara Bakker, P. A. Michels, L. Scapozza, M. L. Bolognesi, A. Cavalli

Date Published: 17th Jan 2013

Journal: PLoS Negl Trop Dis

Abstract

Not specified

Authors: Pablo I. Nikel, Victor De Lorenzo

Date Published: 2013

Journal: Metabolic Engineering

Abstract (Expand)

Research in Systems Biology involves integrating data and knowledge about the dynamic processes in biological systems in order to understand and model them. Semantic web technologies should be ideal for exploring the complex networks of genes, proteins and metabolites that interact, but much of this data is not natively available to the semantic web. Data is typically collected and stored with free-text annotations in spreadsheets, many of which do not conform to existing metadata standards and are often not publically released. Along with initiatives to promote more data sharing, one of the main challenges is therefore to semantically annotate and extract this data so that it is available to the research community. Data annotation and curation are expensive and undervalued tasks that have enormous benefits to the discipline as a whole, but fewer benefits to the individual data producers. By embedding semantic annotation into spreadsheets, however, and automatically extracting this data into RDF at the time of repository submission, the process of producing standards-compliant data, that is available for semantic web querying, can be achieved without adding additional overheads to laboratory data management. This paper describes these strategies in the context of semantic data management in the SEEK. The SEEK is a web-based resource for sharing and exchanging Systems Biology data and models that is underpinned by the JERM ontology (Just Enough Results Model), which describes the relationships between data, models, protocols and experiments. The SEEK was originally developed for SysMO, a large European Systems Biology consortium studying micro-organisms, but it has since had widespread adoption across European Systems Biology.

Authors: None

Date Published: 2013

Journal: The Semantic Web – ISWC 2013

Abstract (Expand)

Background The stressosome is a bacterial signalling complex that responds to environmental changes by initiating a protein partner switching cascade, which leads to the release of the alternative sigma factor, sigmaB. Stress perception increases the phosphorylation of the stressosome sensor protein, RsbR, and the scaffold protein, RsbS, by the protein kinase RsbT. Subsequent dissociation of RsbT from the stressosome activates the sigmaB cascade. However, the sequence of physical events that occur in the stressosome during signal transduction is insufficiently understood. Results Here, we use computational modelling to correlate the structure of the stressosome with the efficiency of the phosphorylation reactions that occur upon activation by stress. In our model, the phosphorylation of any stressosome protein is dependent upon its nearest neighbours and their phosphorylation status. We compare different hypotheses about stressosome activation and find that only the model representing the allosteric activation of the kinase RsbT, by phosphorylated RsbR, qualitatively reproduces the experimental data. Conclusions Our simulations and the associated analysis of published data support the following hypotheses: (i) a simple Boolean model is capable of reproducing stressosome dynamics, (ii) different stressors induce identical stressosome activation patterns, and we also confirm that (i) phosphorylated RsbR activates RsbT, and (ii) the main purpose of RsbX is to dephosphorylate RsbS-P.

Authors: Ulf Liebal, Thomas Millat, Jon Marles-Wright, Rick Lewis, Olaf Wolkenhauer

Date Published: 2013

Journal: BMC Syst Biol

Abstract

Not specified

Authors: Adrienne Zaprasis, J. Brill, M. Thuring, G. Wunsche, M. Heun, H. Barzantny, Tamara Hoffmann, Erhard Bremer

Date Published: 28th Dec 2012

Journal: Applied and Environmental Microbiology

Abstract (Expand)

The Twin-arginine Translocation (Tat) pathway is known to translocate fully folded proteins across bacterial, archaeal and organellar membranes. To date, the mechanisms involved in processing, proofreading and quality control of Tat substrates have remained largely elusive. Bacillus subtilis is an industrially relevant Gram-positive model bacterium. The Tat pathway in B. subtilis differs from that of other well-studied organisms in that it is composed of two complexes operating in parallel. To obtain a better understanding of this pathway in B. subtilis and to identify Tat-associated proteins, the B. subtilis 'Tat proteome' was investigated by quantitative proteomics. Metabolically labeled proteins from cytoplasmic, membrane and extracellular fractions were analyzed by LC-MS/MS. Changes in the amounts of identified peptides allowed for quantitative comparisons of their abundance in tat mutant strains. The observed differences were suggestive of indirect or direct protein-protein relationships. The rich data set generated was then approached in hypothesis-driving and hypothesis-driven manners. The hypothesis-driving approach led to the identification of a novel delayed biofilm phenotype of certain tat mutant strains, whereas the hypothesis-driven approach identified the membrane protein QcrA as a new Tat substrate of B. subtilis. Thus, our quantitative proteomics analyses have unveiled novel Tat pathway-dependent phenotypes in Bacillus.

Authors: Vivianne J Goosens, Andreas Otto, Corinna Glasner, Carmine G Monteferrante, René van der Ploeg, Michael Hecker, Dörte Becher, Jan Maarten Van Dijl

Date Published: 22nd Dec 2012

Journal: J. Proteome Res.

Abstract (Expand)

Glycine betaine is an effective osmoprotectant for Bacillus subtilis. Its import into osmotically stressed cells led to the build-up of large pools, whose size was sensitively determined by the degree of the imposed osmotic stress. The amassing of glycine betaine caused a repression in the formation of an osmostress-adaptive pool of proline, the only osmoprotectant that B. subtilis can synthesize de novo. The ABC transporter OpuA is the main glycine betaine uptake system of B. subtilis. Expression of opuA was up-regulated in response to both sudden and sustained increases in the external osmolarity. Non-ionic osmolytes exerted a stronger inducing effect on transcription than ionic osmolytes, and this was reflected in the development of corresponding OpuA-mediated glycine betaine pools. Primer extension analysis and site-directed mutagenesis pinpointed the osmotically controlled opuA promoter. Deviations from the consensus sequence of SigA-type promoters serve to keep the transcriptional activity of the opuA promoter low in the absence of osmotic stress. Expression of opuA was down regulated in a finely tuned manner in response to increases in the intracellular glycine betaine pool, regardless whether this osmoprotectant was imported or newly synthesized from choline. Such an effect was also exerted by carnitine, an effective osmoprotectant for B. subtilis that is not a substrate for the OpuA transporter. opuA expression was up-regulated in a B. subtilis mutant unable to synthesize proline in response to osmotic stress. Collectively, our data suggest that the intracellular solute pool is a key determinant for the osmotic control of opuA expression.

Authors: Tamara Hoffmann, Annette Wensing, Margot Brosius, Leif Steil, Uwe Voelker, Erhard Bremer

Date Published: 24th Nov 2012

Journal: J. Bacteriol.

Abstract (Expand)

DEAD-box RNA helicases play important roles in remodeling RNA molecules and in facilitating a variety of RNA-protein interactions that are key to many essential cellular processes. In spite of the importance of RNA, our knowledge about RNA helicases is only limited. In this study we investigated the role of the four DEAD-box RNA helicases in Gram positive model-organism Bacillus subtilis. A strain deleted of all RNA helicases is able to grow at 37°C but not at lower temperatures. Especially the deletion of cshA, cshB or yfmL lead to cold-sensitive phenotypes. Moreover, these mutant strains exhibit unique defects in ribosome biogenesis suggesting distinct functions for the individual enzymes in this process. Based on protein accumulation, severity of the cold-sensitive phenotype and the interaction with components of the RNA degradosome, CshA is the major RNA helicase of B. subtilis. To unravel the functions of CshA in addition to ribosome biogenesis we conducted microarray analysis and identified the ysbAB and frlBONMD mRNAs as targets that are strongly affected by the deletion of the cshA gene. Our findings suggest that the different helicases make distinct contributions to the physiology of B. subtilis. Ribosome biogenesis and RNA degradation are two of their major tasks in B. subtilis.

Authors: Martin Lehnik-Habrink, Leonie Rempeters, Akos T Kovács, Christoph Wrede, Claudia Baierlein, Heike Krebber, Oscar Kuipers, Joerg Stuelke

Date Published: 24th Nov 2012

Journal: J. Bacteriol.

Abstract (Expand)

ABSTRACT: BACKGROUND: With increased experimental availability and accuracy of bio-molecular networks, tools for their comparative and evolutionary analysis are needed. A key component for such studies is the alignment of networks. RESULTS: We introduce the Bioconductor package GraphAlignment for pairwise alignment of bio-molecular networks. The alignment incorporates information both from network vertices and network edges and is based on an explicit evolutionary model, allowing inference of all scoring parameters directly from empirical data. We compare the performance of our algorithm to an alternative algorithm, Graemlin 2.0.On simulated data, GraphAlignment outperforms Graemlin 2.0 in several benchmarks except for computational complexity. When there is little or no noise in the data, GraphAlignment is slower than Graemlin 2.0. It is faster than Graemlin 2.0 when processing noisy data containing spurious vertex associations. Its typical case complexity grows approximately as O(N^2.6). On empirical bacterial protein-protein interaction networks (PIN) and gene co-expression networks, GraphAlignment outperforms Graemlin 2.0 with respect to coverage and specificity, albeit by a small margin. On large eukaryotic PIN, Graemlin 2.0 outperforms GraphAlignment. CONCLUSIONS: The GraphAlignment algorithm is robust to spurious vertex associations, correctly resolves paralogs, and shows very good performance in identification of homologous vertices defined by high vertex and/or interaction similarity.

Authors: Michal Kolar, Jörn Meier, Ville Mustonen, Michael Lässig, Johannes Berg

Date Published: 21st Nov 2012

Journal: BMC Syst Biol

Abstract (Expand)

The high osmolarity glycerol (HOG) pathway in yeast serves as a prototype signalling system for eukaryotes. We used an unprecedented amount of data to parameterise 192 models capturing different hypotheses about molecular mechanisms underlying osmo-adaptation and selected a best approximating model. This model implied novel mechanisms regulating osmo-adaptation in yeast. The model suggested that (i) the main mechanism for osmo-adaptation is a fast and transient non-transcriptional Hog1-mediated activation of glycerol production, (ii) the transcriptional response serves to maintain an increased steady-state glycerol production with low steady-state Hog1 activity, and (iii) fast negative feedbacks of activated Hog1 on upstream signalling branches serves to stabilise adaptation response. The best approximating model also indicated that homoeostatic adaptive systems with two parallel redundant signalling branches show a more robust and faster response than single-branch systems. We corroborated this notion to a large extent by dedicated measurements of volume recovery in single cells. Our study also demonstrates that systematically testing a model ensemble against data has the potential to achieve a better and unbiased understanding of molecular mechanisms.

Authors: J. Schaber, R. Baltanas, A. Bush, E. Klipp, A. Colman-Lerner

Date Published: 15th Nov 2012

Journal: Mol Syst Biol

Abstract (Expand)

Plant and microbial metabolic engineering is commonly used in the production of functional foods and quality trait improvement. Computational model-based approaches have been used in this important endeavour. However, to date, fish metabolic models have only been scarcely and partially developed, in marked contrast to their prominent success in metabolic engineering. In this study we present the reconstruction of fully compartmentalised models of the Danio rerio (zebrafish) on a global scale. This reconstruction involves extraction of known biochemical reactions in D. rerio for both primary and secondary metabolism and the implementation of methods for determining subcellular localisation and assignment of enzymes. The reconstructed model (ZebraGEM) is amenable for constraint-based modelling analysis, and accounts for 4,988 genes coding for 2,406 gene-associated reactions and only 418 non-gene-associated reactions. A set of computational validations (i.e., simulations of known metabolic functionalities and experimental data) strongly testifies to the predictive ability of the model. Overall, the reconstructed model is expected to lay down the foundations for computational-based rational design of fish metabolic engineering in aquaculture.

Author: M. Bekaert

Date Published: 14th Nov 2012

Journal: PLoS One

Abstract (Expand)

The Gram-positive soil bacterium Bacillus subtilis uses glucose and malate as the preferred carbon sources. In the presence of either glucose or malate, the expression of genes and operons for the utilization of secondary carbon sources is subject to carbon catabolite repression. While glucose is a preferred substrate in many organisms from bacteria to man, the factors that contribute to the preference for malate have so far remained elusive. In this work, we have studied the contribution of the different malate-metabolizing enzymes in B. subtilis, and we have elucidated their distinct functions. The malate dehydrogenase and the phosphoenolpyruvate carboxykinase are both essential for malate utilization; they introduce malate into gluconeogenesis. The NADPH-generating malic enzyme YtsJ is important to establish the cellular pools of NADPH for anabolic reactions. Finally, the NADH-generating malic enzymes MaeA, MalS, and MleA are involved in keeping the ATP levels high. Together, this unique array of distinct activities makes malate a preferred carbon source for B. subtilis.

Authors: Frederik M Meyer, Joerg Stuelke

Date Published: 10th Nov 2012

Journal: FEMS Microbiol. Lett.

Abstract (Expand)

Methylmercury (MeHg) is a widely distributed contaminant polluting many aquatic environments, with health risks to humans exposed mainly through consumption of seafood. The mechanisms of toxicity of MeHg are not completely understood. In order to map the range of molecular targets and gain better insights into the mechanisms of toxicity, we prepared Atlantic cod (Gadus morhua) 135k oligonucleotide arrays and performed global analysis of transcriptional changes in the liver of fish treated with MeHg (0.5 and 2 mg/kg of body weight) for 14 days. Inferring from the observed transcriptional changes, the main pathways significantly affected by the treatment were energy metabolism, oxidative stress response, immune response and cytoskeleton remodeling. Consistent with known effects of MeHg, many transcripts for genes in oxidative stress pathways such as glutathione metabolism and Nrf2 regulation of oxidative stress response were differentially regulated. Among the differentially regulated genes, there were disproportionate numbers of genes coding for enzymes involved in metabolism of amino acids, fatty acids and glucose. In particular, many genes coding for enzymes of fatty acid beta-oxidation were up-regulated. The coordinated effects observed on many transcripts coding for enzymes of energy pathways may suggest disruption of nutrient metabolism by MeHg. Many transcripts for genes coding for enzymes in the synthetic pathways of sulphur containing amino acids were also up-regulated, suggesting adaptive responses to MeHg toxicity. By this toxicogenomics approach, we were also able to identify many potential biomarker candidate genes for monitoring environmental MeHg pollution. These results based on changes on transcript levels, however, need to be confirmed by other methods such as proteomics.

Authors: Fekadu Yadetie, O. A. Karlsen, A. Lanzen, K. Berg, P. Olsvik, C. Hogstrand, A. Goksoyr

Date Published: 30th Oct 2012

Journal: Aquat Toxicol

Abstract (Expand)

In Escherichia coli several systems are known to transport glucose into the cytoplasm. The main glucose uptake system under batch conditions is the glucose phosphoenolpyruvate:carbohydrate phosphotransferase system (glucose-PTS), but also the mannose-PTS, as well as the galactose and maltose transporters can translocate glucose. Mutant strains which lack the EIIBC protein of the glucose-PTS have been previously investigated because their lower rate of acetate formation offers advantages in industrial applications. Nevertheless, a systematic study to analyze the impact of the different glucose uptake systems has not been undertaken. Specifically, how the bacteria cope with the deletion of the major glucose uptake system and which alternative transporters react to compensate for this deficit has not been studied in detail. Therefore, a series of mutant strains were analyzed in aerobic and anaerobic batch cultures, as well as in glucose limited continuous cultivations. Deletion of EIIBC, disturbs glucose transport severely. cAMP-CRP levels rise, induction of the mgl-operon occurs. Nevertheless mgl transcription is not essential, as deletion of this transporter did not affect growth rate; the activities of the remaining transporters seems to be sufficient by induction of the galactose and maltose transporters. Despite the strong up-regulation of mgl under glucose limitations, deletion of this transport-system did not lead to further changes.

Authors: None

Date Published: 8th Oct 2012

Journal: Journal of Bacteriology

Abstract (Expand)

How cells dynamically respond to fluctuating environmental conditions depends on the architecture and noise of the underlying genetic circuits. Most work characterizing stress pathways in the model bacterium Bacillus subtilis has been performed on bulk cultures using ensemble assays. However, investigating the single cell response to stress is important since noise might generate significant phenotypic heterogeneity. Here, we study the stress response to carbon source starvation and compare both population and single cell data. Using a top-down approach, we investigate the transcriptional dynamics of various stress-related genes of B. subtilis in response to carbon source starvation and to increased cell density. Our data reveal that most of the tested gene-regulatory networks respond highly heterogeneously to starvation and cells show a large degree of variation in gene expression. The level of highly dynamic diversification within B. subtilis populations under changing environments reflects the necessity to study cells at the single cell level.

Authors: None

Date Published: 4th Oct 2012

Journal: Environ. Microbiol.

Abstract (Expand)

The increase in volume and complexity of biological data has led to increased requirements to reuse that data. Consistent and accurate metadata is essential for this task, creating new challenges in semantic data annotation and in the constriction of terminologies and ontologies used for annotation. The BioSharing community are developing standards and terminologies for annotation, which have been adopted across bioinformatics, but the real challenge is to make these standards accessible to laboratory scientists. Widespread adoption requires the provision of tools to assist scientists whilst reducing the complexities of working with semantics. This paper describes unobtrusive ‘stealthy’ methods for collecting standards compliant, semantically annotated data and for contributing to ontologies used for those annotations. Spreadsheets are ubiquitous in laboratory data management. Our spreadsheet-based RightField tool enables scientists to structure information and select ontology terms for annotation within spreadsheets, producing high quality, consistent data without changing common working practices. Furthermore, our Populous spreadsheet tool proves effective for gathering domain knowledge in the form of Web Ontology Language (OWL) ontologies. Such a corpus of structured and semantically enriched knowledge can be extracted in Resource Description Framework (RDF), providing further means for searching across the content and contributing to Open Linked Data (http://linkeddata.org/)

Authors: Katy Wolstencroft, Stuart Owen, Matthew Horridge, Simon Jupp, Olga Krebs, Jacky Snoep, Franco Du Preez, Wolfgang Müller, Robert Stevens, Carole Goble

Date Published: 1st Oct 2012

Journal: Concurrency Computat.: Pract. Exper.

Abstract (Expand)

The respiratory chain of Escherichia coli contains three quinones. Menaquinone and demethylmenaquinone have low midpoint potentials and are involved in anaerobic respiration, while ubiquinone, which has a high midpoint potential, is involved in aerobic and nitrate respiration. Here, we report that demethylmenaquinone plays a role not only in trimethylaminooxide-, dimethylsulfoxide- and fumarate-dependent respiration, but also in aerobic respiration. Furthermore, we demonstrate that demethylmenaquinone serves as an electron acceptor for oxidation of succinate to fumarate, and that all three quinol oxidases of E. coli accept electrons from this naphtoquinone derivative.

Authors: Poonam Sharma, Joost Teixeira De Mattos, Klaas J. Hellingwerf, Martijn Bekker

Date Published: 1st Sep 2012

Journal: Not specified

Abstract (Expand)

We develop a strategic ‘domino’ approach that starts with one key feature of cell function and the main process providing for it, and then adds additional processes and components only as necessary to explain provoked experimental observations. The approach is here applied to the energy metabolism of yeast in a glucose limited chemostat, subjected to a sudden increase in glucose. The puzzles addressed include (i) the lack of increase in ATP upon glucose addition, (ii) the lack of increase in ADP when ATP is hydrolyzed, and (iii) the rapid disappearance of the ‘A’ (adenine) moiety of ATP. Neither the incorporation of nucleotides into new biomass, nor steady de novo synthesis of AMP explains. Cycling of the ‘A’ moiety accelerates when the cell's energy state is endangered, another essential domino among the seven required for understanding of the experimental observations. This new domino analysis shows how strategic experimental design and observations in tandem with theory and modeling may identify and resolve important paradoxes. It also highlights the hitherto unexpected role of the ‘A’ component of ATP.

Authors: None

Date Published: 1st Sep 2012

Journal: Biochimica et Biophysica Acta (BBA) - Bioenergetics

Abstract (Expand)

In Bacillus subtilis and its relatives carbon catabolite control, a mechanism enabling to reach maximal efficiency of carbon and energy sources metabolism, is achieved by the global regulator CcpA (carbon catabolite protein A). CcpA in a complex with HPr-Ser-P (seryl-phosphorylated form of histidine-containing protein, HPr) binds to operator sites called catabolite responsive elements, cre. Depending on the cre box position relative to the promoter, the CcpA/HPr-Ser-P complex can either act as a positive or a negative regulator. The cre boxes are highly degenerate semi-palindromes with a lowly conserved consensus sequence. So far, studies aimed at revealing how CcpA can bind such diverse sites were focused on the analysis of single cre boxes. In this study, a genome-wide analysis of cre sites was performed in order to identify differences in cre sequence and position, which determine their binding affinity.

Authors: Bogumila Marciniak, Monika Pabijaniak, Anne de Jong, Robert Dűhring, Gerald Seidel, Wolfgang Hillen, Oscar Kuipers

Date Published: 17th Aug 2012

Journal: BMC Genomics

Abstract (Expand)

Understanding gene regulation requires knowledge of changes in transcription factor (TF) activities. Simultaneous direct measurement of numerous TF activities is currently impossible. Nevertheless, statistical approaches to infer TF activities have yielded non-trivial and verifiable predictions for individual TFs. Here, global statistical modelling identifies changes in TF activities from transcript profiles of Escherichia coli growing in stable (fixed oxygen availabilities) and dynamic (changing oxygen availability) environments. A core oxygen-responsive TF network, supplemented by additional TFs acting under specific conditions, was identified. The activities of the cytoplasmic oxygen-responsive TF, FNR, and the membrane-bound terminal oxidases implied that, even on the scale of the bacterial cell, spatial effects significantly influence oxygen-sensing. Several transcripts exhibited asymmetrical patterns of abundance in aerobic to anaerobic and anaerobic to aerobic transitions. One of these transcripts, ndh, encodes a major component of the aerobic respiratory chain and is regulated by oxygen-responsive TFs ArcA and FNR. Kinetic modelling indicated that ArcA and FNR behaviour could not explain the ndh transcript profile, leading to the identification of another TF, PdhR, as the source of the asymmetry. Thus, this approach illustrates how systematic examination of regulatory responses in stable and dynamic environments yields new mechanistic insights into adaptive processes.

Authors: Matthew Rolfe, Andrea Ocone, Melanie R Stapleton, Simon Hall, Eleanor W Trotter, Robert Poole, Guido Sanguinetti, Jeff Green

Date Published: 8th Aug 2012

Journal: Open Biol

Abstract

Not specified

Authors: Anna-Karin Gustavsson, Dawie Van Niekerk, Caroline B. Adiels, Franco B. du Preez, Mattias Goksör, Jacky Snoep

Date Published: 1st Aug 2012

Journal: Not specified

Abstract (Expand)

The respiratory chain of Escherichia coli contains three different cytochrome oxidases. Whereas the cytochrome bo oxidase and the cytochrome bd-I oxidase are well characterized and have been shown to contribute to proton translocation, physiological data suggested a nonelectrogenic functioning of the cytochrome bd-II oxidase. Recently, however, this view was challenged by an in vitro biochemical analysis that showed that the activity of cytochrome bd-II oxidase does contribute to proton translocation with an H(+)/e(-) stoichiometry of 1. Here, we propose that this apparent discrepancy is due to the activities of two alternative catabolic pathways: the pyruvate oxidase pathway for acetate production and a pathway with methylglyoxal as an intermediate for the production of lactate. The ATP yields of these pathways are lower than those of the pathways that have so far always been assumed to catalyze the main catabolic flux under energy-limited growth conditions (i.e., pyruvate dehydrogenase and lactate dehydrogenase). Inclusion of these alternative pathways in the flux analysis of growing E. coli strains for the calculation of the catabolic ATP synthesis rate indicates an electrogenic function of the cytochrome bd-II oxidase, compatible with an H(+)/e(-) ratio of 1. This analysis shows for the first time the extent of bypassing of substrate-level phosphorylation in E. coli under energy-limited growth conditions.

Authors: Poonam Sharma, Klaas J Hellingwerf, Maarten J Teixeira de Mattos, Martijn Bekker

Date Published: 27th Jul 2012

Journal: Appl. Environ. Microbiol.

Abstract (Expand)

The active center of multi-subunit RNA polymerase consists of two modules, the Mg(2+) module, holding the catalytic Mg(2+) ion, and a module made of a flexible domain, the Trigger Loop. Uniquely, the TL module can be substituted by alternative modules, thus changing the catalytic properties of the active center.

Authors: Yulia Yuzenkova, Mohammad Roghanian, Nikolay Zenkin

Date Published: 10th Jul 2012

Journal: Transcription

Abstract (Expand)

An existing detailed kinetic model for the steady-state behavior of yeast glycolysis was tested for its ability to simulate dynamic behavior. Using a small subset of experimental data, the original model was adapted by adjusting its parameter values in three optimization steps. Only small adaptations to the original model were required for realistic simulation of experimental data for limit-cycle oscillations. The greatest changes were required for parameter values for the phosphofructokinase reaction. The importance of ATP for the oscillatory mechanism and NAD(H) for inter-and intra-cellular communications and synchronization was evident in the optimization steps and simulation experiments. In an accompanying paper [du Preez F et al. (2012) FEBS J doi:10.1111/j.1742-4658.2012.08658.x], we validate the model for a wide variety of experiments on oscillatory yeast cells. The results are important for re-use of detailed kinetic models in modular modeling approaches and for approaches such as that used in the Silicon Cell initiative. Database The mathematical models described here have been submitted to the JWS Online Cellular Systems Modelling Database and can be accessed at http://jjj.biochem.sun.ac.za/database/dupreez/index.html.

Authors: Franco Du Preez, David D van Niekerk, Bob Kooi, Johann M Rohwer, Jacky Snoep

Date Published: 21st Jun 2012

Journal: The FEBS journal

Abstract (Expand)

In an accompanying paper [du Preez et al., (2012) FEBS J doi: 10.1111/j.1742-4658.2012.08665.x], we adapt an existing kinetic model for steady-state yeast glycolysis to simulate limit-cycle oscillations. Here we validate the model by testing its capacity to simulate a wide range of experiments on dynamics of yeast glycolysis. In addition to its description of the oscillations of glycolytic intermediates in intact cells and the rapid synchronization observed when mixing out-of-phase oscillatory cell populations (see accompanying paper), the model was able to predict the Hopf bifurcation diagram with glucose as the bifurcation parameter (and one of the bifurcation points with cyanide as the bifurcation parameter), the glucose- and acetaldehyde-driven forced oscillations, glucose and acetaldehyde quenching, and cell-free extract oscillations (including complex oscillations and mixed-mode oscillations). Thus, the model was compliant, at least qualitatively, with the majority of available experimental data for glycolytic oscillations in yeast. To our knowledge, this is the first time that a model for yeast glycolysis has been tested against such a wide variety of independent data sets. Database The mathematical models described here have been submitted to the JWS Online Cellular Systems Modelling Database and can be accessed at http://jjj.biochem.sun.ac.za/database/dupreez/index.html.

Authors: Franco Du Preez, David D van Niekerk, Jacky Snoep

Date Published: 13th Jun 2012

Journal: The FEBS journal

Abstract (Expand)

BACKGROUND: Systems biology approaches to study metabolic switching in Streptomyces coelicolor A3(2) depend on cultivation conditions ensuring high reproducibility and distinct phases of culture growth and secondary metabolite production. In addition, biomass concentrations must be sufficiently high to allow for extensive time-series sampling before occurrence of a given nutrient depletion for transition triggering. The present study describes for the first time the development of a dedicated optimized submerged batch fermentation strategy as the basis for highly time-resolved systems biology studies of metabolic switching in S. coelicolor A3(2). RESULTS: By a step-wise approach, cultivation conditions and two fully defined cultivation media were developed and evaluated using strain M145 of S. coelicolor A3(2), providing a high degree of cultivation reproducibility and enabling reliable studies of the effect of phosphate depletion and L-glutamate depletion on the metabolic transition to antibiotic production phase. Interestingly, both of the two carbon sources provided, D-glucose and L-glutamate, were found to be necessary in order to maintain high growth rates and prevent secondary metabolite production before nutrient depletion. Comparative analysis of batch cultivations with (i) both L-glutamate and D-glucose in excess, (ii) L-glutamate depletion and D-glucose in excess, (iii) L-glutamate as the sole source of carbon and (iv) D-glucose as the sole source of carbon, reveal a complex interplay of the two carbon sources in the bacterium's central carbon metabolism. CONCLUSIONS: The present study presents for the first time a dedicated cultivation strategy fulfilling the requirements for systems biology studies of metabolic switching in S. coelicolor A3(2). Key results from labelling and cultivation experiments on either or both of the two carbon sources provided indicate that in the presence of D-glucose, L-glutamate was the preferred carbon source, while D-glucose alone appeared incapable of maintaining culture growth, likely due to a metabolic bottleneck at the oxidation of pyruvate to acetyl-CoA.

Authors: A. Wentzel, P. Bruheim, A. Overby, O. M. Jakobsen, H. Sletta, W. A. Omara, D. A. Hodgson, T. E. Ellingsen

Date Published: 9th Jun 2012

Journal: BMC Syst Biol

Abstract (Expand)

Bacillus subtilis synthesizes large amounts of the compatible solute proline as a cellular defense against high osmolarity to ensure a physiologically appropriate level of hydration of the cytoplasm and turgor. It also imports proline for this purpose via the osmotically inducible OpuE transport system. Unexpectedly, an opuE mutant was at a strong growth disadvantage in high-salinity minimal media lacking proline. Appreciable amounts of proline were detected in the culture supernatant of the opuE mutant strain, and they rose concomitantly with increases in the external salinity. We found that the intracellular proline pool of severely salinity-stressed cells of the opuE mutant was considerably lower than that of its opuE(+) parent strain. This loss of proline into the medium and the resulting decrease in the intracellular proline content provide a rational explanation for the observed salt-sensitive growth phenotype of cells lacking OpuE. None of the known MscL- and MscS-type mechanosensitive channels of B. subtilis participated in the release of proline under permanently imposed high-salinity growth conditions. The data reported here show that the OpuE transporter not only possesses the previously reported role for the scavenging of exogenously provided proline as an osmoprotectant but also functions as a physiologically highly important recapturing device for proline that is synthesized de novo and subsequently released by salt-stressed B. subtilis cells. The wider implications of our findings for the retention of compatible solutes by osmotically challenged microorganisms and the roles of uptake systems for compatible solutes are considered.

Authors: Tamara Hoffmann, Carsten von Blohn, Agnieszka Stanek, Susanne Moses, Helena Barzantny, Erhard Bremer

Date Published: 8th Jun 2012

Journal: Appl. Environ. Microbiol.

Abstract (Expand)

Encouraging more broad and inclusive data sharing in today's world will involve concerted community efforts to overcome technical barriers and human foibles. Vivien Marx investigates. (includess comments from Carole Goble, and mentions SysMO, SEEK and RightField).

Author: Vivien Marx

Date Published: 7th Jun 2012

Journal: Nat Biotechnol

Abstract (Expand)

Yeast glycolytic oscillations have been studied since the 1950s in cell-free extracts and intact cells. For intact cells, sustained oscillations have so far only been observed at the population level, i.e. for synchronized cultures at high biomass concentrations. Using optical tweezers to position yeast cells in a microfluidic chamber, we were able to observe sustained oscillations in individual isolated cells. Using a detailed kinetic model for the cellular reactions, we simulated the heterogeneity in the response of the individual cells, assuming small differences in a single internal parameter. This is the first time that sustained limit-cycle oscillations have been demonstrated in isolated yeast cells. Database The mathematical model described here has been submitted to the JWS Online Cellular Systems Modelling Database and can be accessed at http://jjj.biochem.sun.ac.za/database/gustavsson/index.html free of charge.

Authors: Anna-Karin Gustavsson, David D van Niekerk, Caroline B Adiels, Franco Du Preez, Mattias Goksör, Jacky Snoep

Date Published: 23rd May 2012

Journal: The FEBS journal

Abstract (Expand)

Our quantitative knowledge of carbon fluxes in the long slender bloodstream form (BSF) Trypanosoma brucei is mainly based on non-proliferating parasites, isolated from laboratory animals and kept in buffers. In this paper we present a carbon balance for exponentially growing bloodstream form trypanosomes. The cells grew with a doubling time of 5.3h, contained 46 mu mol of carbon (10(8) cells)(-1) and had a glucose consumption flux of 160 nmol min(-1) (10(8) cells)(-1). The molar ratio of pyruvate excreted versus glucose consumed was 2.1. Furthermore, analysis of the (13)C label distribution in pyruvate in (13)C-glucose incubations of exponentially growing trypanosomes showed that glucose was the sole substrate for pyruvate production. We conclude that the glucose metabolised in glycolysis was hardly, if at all, used for biosynthetic processes. Carbon flux through glycolysis in exponentially growing trypanosomes was 10 times higher than the incorporation of carbon into biomass. This biosynthetic carbon is derived from other precursors present in the nutrient rich growth medium. Furthermore, we found that the glycolytic flux was unaltered when the culture went into stationary phase, suggesting that most of the ATP produced in glycolysis is used for processes other than growth.

Authors: Jurgen Haanstra, A. van Tuijl, J. van Dam, W. van Winden, A. G. Tielens, J. J. van Hellemond, Barbara Bakker

Date Published: 8th May 2012

Journal: Int J Parasitol

Abstract (Expand)

RNA processing and degradation are key processes in the control of transcript accumulation and thus in the control of gene expression. In Escherichia coli, the underlying mechanisms and components of RNA decay are well characterized. By contrast, Gram-positive bacteria do not possess several important players of E. coli RNA degradation, most notably the essential enzyme RNase E. Recent research on the model Gram-positive organism, Bacillus subtilis, has identified the essential RNases J1 and Y as crucial enzymes in RNA degradation. While RNase J1 is the first bacterial exoribonuclease with 5'-to-3' processivity, RNase Y is the founding member of a novel class of endoribonucleases. Both RNase J1 and RNase Y have a broad impact on the stability of B. subtilis mRNAs; a depletion of either enzyme affects more than 25% of all mRNAs. RNases J1 and Y as well as RNase J2, the polynucleotide phosphorylase PNPase, the RNA helicase CshA and the glycolytic enzymes enolase and phosphofructokinase have been proposed to form a complex, the RNA degradosome of B. subtilis. This review presents a model, based on recent published data, of RNA degradation in B. subtilis. Degradation is initiated by RNase Y-dependent endonucleolytic cleavage, followed by processive exoribonucleolysis of the generated fragments both in 3'-to-5' and in 5'-to-3' directions. The implications of these findings for pathogenic Gram-positive bacteria are also discussed.

Authors: Martin Lehnik-Habrink, Rick Lewis, Ulrike Mäder, Joerg Stuelke

Date Published: 8th May 2012

Journal: Mol. Microbiol.

Abstract (Expand)

The origin of translation and the genetic code is one of the major mysteries of evolution. The advantage of templated protein synthesis could have been achieved only when the translation apparatus had already become very complex. This means that the translation machinery, as we know it today, must have evolved towards some different essential function that subsequently sub-functionalised into templated protein synthesis. The hypothesis presented here proposes that translation originated as the result of evolution of a primordial RNA helicase, which has been essential for preventing dying out of the RNA organism in sterile double-stranded form. This hypothesis emerges because modern ribosome possesses RNA helicase activity that likely dates back to the RNA world. I hypothesise that codon-anticodon interactions of tRNAs with mRNA evolved as a mechanism used by RNA helicase, the predecessor of ribosomes, to melt RNA duplexes. In this scenario, peptide bond formation emerged to drive unidirectional movement of the helicase via a molecular ratchet mechanism powered by Brownian motion. I propose that protein synthesis appeared as a side product of helicase activity. The first templates for protein synthesis were functional RNAs (ribozymes) that were unwound by the helicase, and the first synthesised proteins were of random or non-sense sequence. I further suggest that genetic code emerged to avoid this randomness. The initial genetic code thus emerged as an assignment of amino acids to codons according to the sequences of the pre-existing RNAs to take advantage of the side products of RNA helicase function.

Authors: None

Date Published: 28th Apr 2012

Journal: J. Mol. Evol.

Abstract (Expand)

The Bacillus subtilis catabolite control protein A (CcpA) is a global transcriptional regulator which is controlled by interactions with the phosphoproteins HPrSer46P and CrhP and with low molecular weight effectors depending on the availability of preferred carbon sources like glucose. Distinct point mutations in CcpA abolish regulation of some but not all target genes suggesting additional interactions of CcpA. Therefore, in vivo crosslinking and mass spectrometry were applied to identify CcpA complexes active in repression and activation. To compensate for the excess of promoters only repressed by CcpA, this experiment was accomplished with cells with multiple copies of the activated ackA promoter. Among the identified proteins HPr, RNA polymerase (RNAP) subunits and the global regulator CodY were observed. Bacterial two-hybrid assays combining each RNAP subunit with CcpA localized CcpA binding at the α-subunit (RpoA). In vivo crosslinking combined with immunoblot analyses revealed CcpA-RpoA complexes in cultures with or without glucose whereas CcpA-HPr and CcpA-CodY complexes occurred only or predominantly in cultures with glucose. Surface plasmon resonance (SPR) analyses confirmed binding of CcpA to the N- (αNTD) and C-terminal domains (αCTD) of RpoA as well as to CodY. Furthermore, interactions of CodY with the αNTD and the αCTD were detected by SPR. The K(D) values of complexes of CcpA or CodY with the αNTD or the αCTD are between 5 and 8μM. CcpA and CodY form a loose complex with a K(D) of 60μM. These data were combined to propose a model for a transcription initiation complex at the ackA promoter.

Authors: Andrea Wünsche, Elke Hammer, Maike Bartholomae, Uwe Voelker, Andreas Burkovski, Gerald Seidel, Wolfgang Hillen

Date Published: 20th Apr 2012

Journal: The FEBS journal

Abstract (Expand)

The circadian clocks that drive daily rhythms in animals are tightly coupled among the cells of some tissues. The coupling profoundly affects cellular rhythmicity and is central to contemporary understanding of circadian physiology and behavior. In contrast, studies of the clock in plant cells have largely ignored intercellular coupling, which is reported to be very weak or absent. We used luciferase reporter gene imaging to monitor circadian rhythms in leaves of Arabidopsis thaliana plants, achieving resolution close to the cellular level. Leaves grown without environmental cycles for up to 3 wk reproducibly showed spatiotemporal waves of gene expression consistent with intercellular coupling, using several reporter genes. Within individual leaves, different regions differed in phase by up to 17 h. A broad range of patterns was observed among leaves, rather than a common spatial distribution of circadian properties. Leaves exposed to light-dark cycles always had fully synchronized rhythms, which could desynchronize rapidly. After 4 d in constant light, some leaves were as desynchronized as leaves grown without any rhythmic input. Applying light-dark cycles to such a leaf resulted in full synchronization within 2-4 d. Thus, the rhythms of all cells were coupled to external light-dark cycles far more strongly than the cellular clocks were coupled to each other. Spontaneous desynchronization under constant conditions was limited, consistent with weak intercellular coupling among heterogeneous clocks. Both the weakness of coupling and the heterogeneity among cells are relevant to interpret molecular studies and to understand the physiological functions of the plant circadian clock.

Authors: B. Wenden, D. L. Toner, S. K. Hodge, R. Grima, Andrew Millar

Date Published: 13th Apr 2012

Journal: Proc Natl Acad Sci U S A