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See detailModeling the effects of commonly used drugs on human metabolism
Sahoo, Swagatika UL; Haraldsdottir, Hulda UL; Fleming, Ronan MT UL et al

in FEBS Journal (2014)

Metabolism contributes significantly to the pharmacokinetics and pharmacodynamics of a drug. In addition, diet and genetics have a profound effect on cellular metabolism under health and disease. Herein ... [more ▼]

Metabolism contributes significantly to the pharmacokinetics and pharmacodynamics of a drug. In addition, diet and genetics have a profound effect on cellular metabolism under health and disease. Herein, we assembled a comprehensive, literature-based drug metabolic reconstruction of the 18 most highly prescribed drug groups including statins, antihypertensives, immunosuppressants, and analgesics. This reconstruction captures in detail our current understanding of their absorption, intra-cellular distribution, metabolism, and elimination. We combined this drug module with the most comprehensive reconstruction of human metabolism, Recon 2, yielding Recon2_DM1796, which accounts for 2803 metabolites and 8161 reactions. By defining 50 specific drug objectives that captured the overall drug metabolism of these compounds, we investigated effects of dietary composition and inherited metabolic disorders on drug metabolism and drug-drug interactions. Our main findings include (i) shift in dietary patterns significantly affect statins and acetaminophen metabolism, (ii) disturbed statin metabolism contributes to the clinical phenotype of mitochondrial energy disorders, and (iii) the interaction between statins and cyclosporine can be explained by several common metabolic and transport pathways other than the previously established CYP3A4 connection. This work holds the potential for studying adverse drug reactions and designing patient-specific therapies. [less ▲]

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See detailConsensus and conflict cards for metabolic pathway databases.
Stobbe, Miranda D.; Swertz, Morris A.; Thiele, Ines UL et al

in BMC Systems Biology (2013), 7

BACKGROUND: The metabolic network of H. sapiens and many other organisms is described in multiple pathway databases. The level of agreement between these descriptions, however, has proven to be low. We ... [more ▼]

BACKGROUND: The metabolic network of H. sapiens and many other organisms is described in multiple pathway databases. The level of agreement between these descriptions, however, has proven to be low. We can use these different descriptions to our advantage by identifying conflicting information and combining their knowledge into a single, more accurate, and more complete description. This task is, however, far from trivial. RESULTS: We introduce the concept of Consensus and Conflict Cards (C2Cards) to provide concise overviews of what the databases do or do not agree on. Each card is centered at a single gene, EC number or reaction. These three complementary perspectives make it possible to distinguish disagreements on the underlying biology of a metabolic process from differences that can be explained by different decisions on how and in what detail to represent knowledge. As a proof-of-concept, we implemented C2CardsHuman, as a web application http://www.molgenis.org/c2cards, covering five human pathway databases. CONCLUSIONS: C2Cards can contribute to ongoing reconciliation efforts by simplifying the identification of consensus and conflicts between pathway databases and lowering the threshold for experts to contribute. Several case studies illustrate the potential of the C2Cards in identifying disagreements on the underlying biology of a metabolic process. The overviews may also point out controversial biological knowledge that should be subject of further research. Finally, the examples provided emphasize the importance of manual curation and the need for a broad community involvement. [less ▲]

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See detailToward systems metabolic engineering in cyanobacteria: opportunities and bottlenecks.
Nogales, Juan; Gudmundsson, Steinn; Thiele, Ines UL

in Bioengineered (2013), 4(3), 158-163

We recently assessed the metabolism of Synechocystis sp PCC6803 through a constraints-based reconstruction and analysis approach and identified its main metabolic properties. These include reduced ... [more ▼]

We recently assessed the metabolism of Synechocystis sp PCC6803 through a constraints-based reconstruction and analysis approach and identified its main metabolic properties. These include reduced metabolic robustness, in contrast to a high photosynthetic robustness driving the optimal autotrophic metabolism. Here, we address how these metabolic features affect biotechnological capabilities of this bacterium. The search for growth-coupled overproducer strains revealed that the carbon flux re-routing, but not the electron flux, is significantly more challenging under autotrophic conditions than under mixo- or heterotrophic conditions. We also found that the blocking of the light-driven metabolism was required for carbon flux re-routing under mixotrophic conditions. Overall, our analysis, which represents the first systematic evaluation of the biotechnological capabilities of a photosynthetic organism, paradoxically suggests that the light-driven metabolism itself and its unique metabolic features are the main bottlenecks in harnessing the biotechnological potential of Synechocystis. [less ▲]

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See detailApplying systems biology methods to the study of human physiology in extreme environments
Edwards, Lindsay; Thiele, Ines UL

in Extreme Physiology & Medicine (2013), 2(8),

Systems biology is defined in this review as ‘an iterative process of computational model building and experimental model revision with the aim of understanding or simulating complex biological systems’ ... [more ▼]

Systems biology is defined in this review as ‘an iterative process of computational model building and experimental model revision with the aim of understanding or simulating complex biological systems’. We propose that, in practice, systems biology rests on three pillars: computation, the omics disciplines and repeated experimental perturbation of the system of interest. The number of ethical and physiologically relevant perturbations that can be used in experiments on healthy humans is extremely limited and principally comprises exercise, nutrition, infusions (e.g. Intralipid), some drugs and altered environment. Thus, we argue that systems biology and environmental physiology are natural symbionts for those interested in a system-level understanding of human biology. However, despite excellent progress in high-altitude genetics and several proteomics studies, systems biology research into human adaptation to extreme environments is in its infancy. A brief description and overview of systems biology in its current guise is given, followed by a mini review of computational methods used for modelling biological systems. Special attention is given to high-altitude research, metabolic network reconstruction and constraint-based modelling. [less ▲]

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See detailRobust flux balance analysis of multi- scale biochemical reaction networks
Sun, Y; Fleming, Ronan MT UL; Thiele, Ines UL et al

in BMC Bioinformatics (2013), 1(14), 240

Background:Biological processes such as metabolism, signaling, and macromolecular synthesis can be modeled as large networks of biochemical reactions. Large and comprehensive networks, like integrated ... [more ▼]

Background:Biological processes such as metabolism, signaling, and macromolecular synthesis can be modeled as large networks of biochemical reactions. Large and comprehensive networks, like integrated networks that represent metabolism and macromolecular synthesis, are inherently multiscale because reaction rates can vary over many orders of magnitude. They require special methods for accurate analysis because naive use of standard optimization systems can produce inaccurate or erroneously infeasible results. Results: We describe techniques enabling off-the-shelf optimization software to compute accurate solutions to the poorly scaled optimization problems arising from flux balance analysis of multiscale biochemical reaction networks. We implement lifting techniques for flux balance analysis within the openCOBRA toolbox and demonstrate our techniques using the first integrated reconstruction of metabolism and macromolecular synthesis for E. coli. Conclusion:Our techniques enable accurate flux balance analysis of multiscale networks using off-the-shelf optimization software. Although we describe lifting techniques in the context of flux balance analysis, our methods can be used to handle a variety of optimization problems arising from analysis of multiscale network reconstructions. [less ▲]

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See detailA community-driven global reconstruction of human metabolism.
Thiele, Ines UL; Swainston, N.; Fleming, Ronan MT UL et al

in Nature Biotechnology (2013), 31

Multiple models of human metabolism have been reconstructed, but each represents only a subset of our knowledge. Here we describe Recon 2, a community-driven, consensus ‘metabolic reconstruction’, which ... [more ▼]

Multiple models of human metabolism have been reconstructed, but each represents only a subset of our knowledge. Here we describe Recon 2, a community-driven, consensus ‘metabolic reconstruction’, which is the most comprehensive representation of human metabolism that is applicable to computational modeling. Compared with its predecessors, the reconstruction has improved topological and functional features, including ~2× more reactions and ~1.7× more unique metabolites. Using Recon 2 we predicted changes in metabolite biomarkers for 49 inborn errors of metabolism with 77% accuracy when compared to experimental data. Mapping metabolomic data and drug information onto Recon 2 demonstrates its potential for integrating and analyzing diverse data types. Using protein expression data, we automatically generated a compendium of 65 cell type–specific models, providing a basis for manual curation or investigation of cell-specific metabolic properties. Recon 2 will facilitate many future biomedical studies and is freely available at http://humanmetabolism.org/. [less ▲]

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See detailInferring the metabolism of human orphan metabolites from their metabolic network context affirms human gluconokinase activity.
Rolfsson, Ottar; Paglia, Giuseppe; Magnusdottir, Manuela et al

in Biochemical Journal (2013), 449(2), 427-435

Metabolic network reconstructions define metabolic information within a target organism and can therefore be used to address incomplete metabolic information. In the present study we used a computational ... [more ▼]

Metabolic network reconstructions define metabolic information within a target organism and can therefore be used to address incomplete metabolic information. In the present study we used a computational approach to identify human metabolites whose metabolism is incomplete on the basis of their detection in humans but exclusion from the human metabolic network reconstruction RECON 1. Candidate solutions, composed of metabolic reactions capable of explaining the metabolism of these compounds, were then identified computationally from a global biochemical reaction database. Solutions were characterized with respect to how metabolites were incorporated into RECON 1 and their biological relevance. Through detailed case studies we show that biologically plausible non-intuitive hypotheses regarding the metabolism of these compounds can be proposed in a semi-automated manner, in an approach that is similar to de novo network reconstruction. We subsequently experimentally validated one of the proposed hypotheses and report that C9orf103, previously identified as a candidate tumour suppressor gene, encodes a functional human gluconokinase. The results of the present study demonstrate how semi-automatic gap filling can be used to refine and extend metabolic reconstructions, thereby increasing their biological scope. Furthermore, we illustrate how incomplete human metabolic knowledge can be coupled with gene annotation in order to prioritize and confirm gene functions. [less ▲]

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See detailA systems biology approach to studying the role of microbes in human health.
Thiele, Ines UL; Heinken, Almut Katrin UL; Fleming, Ronan MT UL

in Current Opinion in Biotechnology (2013), 24(1), 4-12

Host-microbe interactions play a crucial role in human health and disease. Of the various systems biology approaches, reconstruction of genome-scale metabolic networks combined with constraint-based ... [more ▼]

Host-microbe interactions play a crucial role in human health and disease. Of the various systems biology approaches, reconstruction of genome-scale metabolic networks combined with constraint-based modeling has been particularly successful at in silico predicting the phenotypic characteristics of single organisms. Here, we summarize recent studies, which have applied this approach to investigate microbe-microbe and host-microbe metabolic interactions. This approach can be also expanded to investigate the properties of an entire microbial community, as well as single organisms within the community. We illustrate that the constraint-based modeling approach is suitable to model host-microbe interactions at molecular resolution and will enable systematic investigation of metabolic links between the human host and its microbes. Such host-microbe models, combined with experimental data, will ultimately further our understanding of how microbes influence human health. [less ▲]

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See detailPredicting the impact of diet and enzymopathies on human small intestinal epithelial cells
Sahoo, Swagatika UL; Thiele, Ines UL

in Human Molecular Genetics (2013), 22(13), 2705-22

Small intestinal epithelial cells (sIECs) have a significant share in whole body metabolism as they perform enzymatic digestion and absorption of nutrients. Furthermore, the diet plays a key role in a ... [more ▼]

Small intestinal epithelial cells (sIECs) have a significant share in whole body metabolism as they perform enzymatic digestion and absorption of nutrients. Furthermore, the diet plays a key role in a number of complex diseases including obesity and diabetes. The impact of diet and altered genetic backgrounds on human metabolism may be studied by using computational modeling. A metabolic reconstruction of human sIECs was manually assembled using the literature. The resulting sIEC model was subjected to two different diets to obtain condition-specific metabolic models. Fifty defined metabolic tasks evaluated the functionalities of these models, along with the respective secretion profiles, which distinguished between impacts of different dietary regimes. Under the average American diet, the sIEC model resulted in higher secretion flux for metabolites implicated in metabolic syndrome. In addition, enzymopathies were analyzed in the context of the sIEC metabolism. Computed results were compared with reported gastrointestinal (GI) pathologies and biochemical defects as well as with biomarker patterns used in their diagnosis. Based on our simulations, we propose that (i) sIEC metabolism is perturbed by numerous enzymopathies, which can be used to study cellular adaptive mechanisms specific for such disorders, and in the identification of novel co-morbidities, (ii) porphyrias are associated with both heme synthesis and degradation and (iii) disturbed intestinal gamma-aminobutyric acid synthesis may be linked to neurological manifestations of various enzymopathies. Taken together, the sIEC model represents a comprehensive, biochemically accurate platform for studying the function of sIEC and their role in whole body metabolism. [less ▲]

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See detailSystems-level characterization of a host-microbe metabolic symbiosis in the mammalian gut.
Heinken, Almut Katrin UL; Sahoo, Swagatika UL; Fleming, Ronan MT UL et al

in Gut microbes (2013), 4(1), 28-40

The human gut microbiota consists of ten times more microorganisms than there are cells in our body, processes otherwise indigestible nutrients, and produces important energy precursors, essential amino ... [more ▼]

The human gut microbiota consists of ten times more microorganisms than there are cells in our body, processes otherwise indigestible nutrients, and produces important energy precursors, essential amino acids, and vitamins. In this study, we assembled and validated a genome-scale metabolic reconstruction of Bacteroides thetaiotaomicron (iAH991), a prominent representative of the human gut microbiota, consisting of 1488 reactions, 1152 metabolites, and 991 genes. To create a comprehensive metabolic model of host-microbe interactions, we integrated iAH991 with a previously published mouse metabolic reconstruction, which was extended for intestinal transport and absorption reactions. The two metabolic models were linked through a joint compartment, the lumen, allowing metabolite exchange and providing a route for simulating different dietary regimes. The resulting model consists of 7239 reactions, 5164 metabolites, and 2769 genes. We simultaneously modeled growth of mouse and B. thetaiotaomicron on five different diets varying in fat, carbohydrate, and protein content. The integrated model captured mutually beneficial cross-feeding as well as competitive interactions. Furthermore, we identified metabolites that were exchanged between the two organisms, which were compared with published metabolomics data. This analysis resulted for the first time in a comprehensive description of the co-metabolism between a host and its commensal microbe. We also demonstrate in silico that the presence of B. thetaiotaomicron could rescue the growth phenotype of the host with an otherwise lethal enzymopathy and vice versa. This systems approach represents a powerful tool for modeling metabolic interactions between a gut microbe and its host in health and disease. [less ▲]

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See detailIntracellular metabolite profiling of platelets: evaluation of extraction processes and chromatographic strategies.
Paglia, Giuseppe; Magnusdottir, Manuela; Thorlacius, Steinunn et al

in Journal of Chromatography. B : Analytical Technologies in the Biomedical & Life Sciences (2012), 898

An extraction method for intracellular metabolite profiling should ideally be able to recover the broadest possible range of metabolites present in a sample. However, the development of such methods is ... [more ▼]

An extraction method for intracellular metabolite profiling should ideally be able to recover the broadest possible range of metabolites present in a sample. However, the development of such methods is hampered by the diversity of the physico-chemical properties of metabolites as well as by the specific characteristics of samples and cells. In this study, we report the optimization of an UPLC-MS method for the metabolite analysis of platelet samples. The optimal analytical protocol was determined by testing seven different extraction methods as well as by employing two different LC-MS methods, in which the metabolites were separated by using hydrophilic interaction liquid chromatography (HILIC) and reversed phase liquid chromatography (RPLC). The optimal conditions were selected using the coverage of the platelets' metabolome, the response of the identified metabolites, the reproducibility of the analytical method, and the time of the analysis as main evaluation criteria. Our results show that methanol-water (7:3) extraction coupled with HILIC-MS method provides the best compromise, allowing identification of 107 metabolites in a platelet cell extract sample, 91% of them with a RSD% lower than 20. A higher number of metabolites could be detected when analyzing the platelet samples with two different LC-MS methods or when using complementary extraction methods in parallel. [less ▲]

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See detailAn in silico re-design of the metabolism in Thermotoga maritima for increased biohydrogen production
Nogales, Juan E; Gudmunsson, Steinn; Thiele, Ines UL

in International Journal of Hydrogen Energy (2012), 37(17), 1220512218

Microbial hydrogen production is currently hampered by lack of efficiency. We examine how hydrogen production in the hyperthermophilic bacterium Thermotoga maritima can be increased in silico. An updated ... [more ▼]

Microbial hydrogen production is currently hampered by lack of efficiency. We examine how hydrogen production in the hyperthermophilic bacterium Thermotoga maritima can be increased in silico. An updated genome-scale metabolic model of T. maritima was used to i) describe in detail the H2 metabolism in this bacterium, ii) identify suitable carbon sources for enhancing H2 production, and iii) to design knockout strains, which increased the in silico hydrogen production up to 20%. A novel synthetic oxidative module was further designed, which connects the cellular NADPH and ferredoxin pools by inserting into the model a NADPH-ferredoxin reductase. We then combined this in silico knock-in strain with a knockout strain design, resulting in an in silico production strain with a predicted 125% increase in hydrogen yield. The in silico strains designs presented here may serve as blueprints for future metabolic engineering efforts of T. maritima. [less ▲]

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See detailQuantitative Assignment of Reaction Directionality in a Multicompartmental Human Metabolic Reconstruction
Haraldsdottir, Hulda UL; Thiele, Ines UL; Fleming, Ronan MT UL

in Biophysical Journal (2012), 102(8), 17031711

Reaction directionality is a key constraint in the modeling of genome-scale metabolic networks. We thermodynamically constrained reaction directionality in a multicompartmental genome-scale model of human ... [more ▼]

Reaction directionality is a key constraint in the modeling of genome-scale metabolic networks. We thermodynamically constrained reaction directionality in a multicompartmental genome-scale model of human metabolism, Recon 1, by calculating, in vivo, standard transformed reaction Gibbs energy as a function of compartment-specific pH, electrical potential, and ionic strength. We show that compartmental pH is an important determinant of thermodynamically determined reaction directionality. The effects of pH on transport reaction thermodynamics are only seen to their full extent when metabolites are represented as pseudoisomer groups of multiple protonated species. We accurately predict the irreversibility of 387 reactions, with detailed propagation of uncertainty in input data, and manually curate the literature to resolve conflicting directionality assignments. In at least half of all cases, a prediction of a reversible reaction directionality is due to the paucity of compartment-specific quantitative metabolomic data, with remaining cases due to uncertainty in estimation of standard reaction Gibbs energy. This study points to the pressing need for 1), quantitative metabolomic data, and 2), experimental measurement of thermochemical properties for human metabolites. [less ▲]

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See detailMass conserved elementary kinetics is sufficient for the existence of a non-equilibrium steady state concentration.
Fleming, Ronan MT UL; Thiele, Ines UL

in Journal of Theoretical Biology (2012), 314

Living systems are forced away from thermodynamic equilibrium by exchange of mass and energy with their environment. In order to model a biochemical reaction network in a non-equilibrium state one ... [more ▼]

Living systems are forced away from thermodynamic equilibrium by exchange of mass and energy with their environment. In order to model a biochemical reaction network in a non-equilibrium state one requires a mathematical formulation to mimic this forcing. We provide a general formulation to force an arbitrary large kinetic model in a manner that is still consistent with the existence of a non-equilibrium steady state. We can guarantee the existence of a non-equilibrium steady state assuming only two conditions; that every reaction is mass balanced and that continuous kinetic reaction rate laws never lead to a negative molecule concentration. These conditions can be verified in polynomial time and are flexible enough to permit one to force a system away from equilibrium. With expository biochemical examples we show how reversible, mass balanced perpetual reaction(s), with thermodynamically infeasible kinetic parameters, can be used to perpetually force various kinetic models in a manner consistent with the existence of a steady state. Easily testable existence conditions are foundational for efforts to reliably compute non-equilibrium steady states in genome-scale biochemical kinetic models. [less ▲]

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See detailMultiscale modeling of metabolism and macromolecular synthesis in E. coli and its application to the evolution of codon usage.
Thiele, Ines UL; Fleming, Ronan MT UL; Que, Richard et al

in PLoS ONE (2012), 7(9), 45635

Biological systems are inherently hierarchal and multiscale in time and space. A major challenge of systems biology is to describe biological systems as a computational model, which can be used to derive ... [more ▼]

Biological systems are inherently hierarchal and multiscale in time and space. A major challenge of systems biology is to describe biological systems as a computational model, which can be used to derive novel hypothesis and drive experiments leading to new knowledge. The constraint-based reconstruction and analysis approach has been successfully applied to metabolism and to the macromolecular synthesis machinery assembly. Here, we present the first integrated stoichiometric multiscale model of metabolism and macromolecular synthesis for Escherichia coli K12 MG1655, which describes the sequence-specific synthesis and function of almost 2000 gene products at molecular detail. We added linear constraints, which couple enzyme synthesis and catalysis reactions. Comparison with experimental data showed improvement of growth phenotype prediction with the multiscale model over E. coli's metabolic model alone. Many of the genes covered by this integrated model are well conserved across enterobacters and other, less related bacteria. We addressed the question of whether the bias in synonymous codon usage could affect the growth phenotype and environmental niches that an organism can occupy. We created two classes of in silico strains, one with more biased codon usage and one with more equilibrated codon usage than the wildtype. The reduced growth phenotype in biased strains was caused by tRNA supply shortage, indicating that expansion of tRNA gene content or tRNA codon recognition allow E. coli to respond to changes in codon usage bias. Our analysis suggests that in order to maximize growth and to adapt to new environmental niches, codon usage and tRNA content must co-evolve. These results provide further evidence for the mutation-selection-drift balance theory of codon usage bias. This integrated multiscale reconstruction successfully demonstrates that the constraint-based modeling approach is well suited to whole-cell modeling endeavors. [less ▲]

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See detailA systems biology approach to drug targets in Pseudomonas aeruginosa biofilm.
Sigurdsson, Gunnar; Fleming, Ronan MT UL; Heinken, Almut Katrin UL et al

in PLoS ONE (2012), 7(4), 34337

Antibiotic resistance is an increasing problem in the health care system and we are in a constant race with evolving bacteria. Biofilm-associated growth is thought to play a key role in bacterial ... [more ▼]

Antibiotic resistance is an increasing problem in the health care system and we are in a constant race with evolving bacteria. Biofilm-associated growth is thought to play a key role in bacterial adaptability and antibiotic resistance. We employed a systems biology approach to identify candidate drug targets for biofilm-associated bacteria by imitating specific microenvironments found in microbial communities associated with biofilm formation. A previously reconstructed metabolic model of Pseudomonas aeruginosa (PA) was used to study the effect of gene deletion on bacterial growth in planktonic and biofilm-like environmental conditions. A set of 26 genes essential in both conditions was identified. Moreover, these genes have no homology with any human gene. While none of these genes were essential in only one of the conditions, we found condition-dependent genes, which could be used to slow growth specifically in biofilm-associated PA. Furthermore, we performed a double gene deletion study and obtained 17 combinations consisting of 21 different genes, which were conditionally essential. While most of the difference in double essential gene sets could be explained by different medium composition found in biofilm-like and planktonic conditions, we observed a clear effect of changes in oxygen availability on the growth performance. Eight gene pairs were found to be synthetic lethal in oxygen-limited conditions. These gene sets may serve as novel metabolic drug targets to combat particularly biofilm-associated PA. Taken together, this study demonstrates that metabolic modeling of human pathogens can be used to identify oxygen-sensitive drug targets and thus, that this systems biology approach represents a powerful tool to identify novel candidate antibiotic targets. [less ▲]

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See detailContextualization procedure and modeling of monocyte specific TLR signaling.
Aurich, Maike Kathrin UL; Thiele, Ines UL

in PLoS ONE (2012), 7(12), 49978

Innate immunity is the first line of defense against invasion of pathogens. Toll-like receptor (TLR) signaling is involved in a variety of human diseases extending far beyond immune system-related ... [more ▼]

Innate immunity is the first line of defense against invasion of pathogens. Toll-like receptor (TLR) signaling is involved in a variety of human diseases extending far beyond immune system-related diseases, affecting a number of different tissues and cell-types. Computational models often do not account for cell-type specific differences in signaling networks. Investigation of these differences and its phenotypic implications could increase understanding of cell signaling and processes such as inflammation. The wealth of knowledge for TLR signaling has been recently summarized in a stoichiometric signaling network applicable for constraint-based modeling and analysis (COBRA). COBRA methods have been applied to investigate tissue-specific metabolism using omics data integration. Comparable approaches have not been conducted using signaling networks. In this study, we present ihsTLRv2, an updated TLR signaling network accounting for the association of 314 genes with 558 network reactions. We present a mapping procedure for transcriptomic data onto signaling networks and demonstrate the generation of a monocyte-specific TLR network. The generated monocyte network is characterized through expression of a specific set of isozymes rather than reduction of pathway contents. While further tailoring the network to a specific stimulation condition, we observed that the quantitative changes in gene expression due to LPS stimulation affected the tightly connected set of genes. Differential expression influenced about one third of the entire TLR signaling network, in particular, NF-kappaB activation. Thus, a cell-type and condition-specific signaling network can provide functional insight into signaling cascades. Furthermore, we demonstrate the energy dependence of TLR signaling pathways in monocytes. [less ▲]

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See detailMonitoring metabolites consumption and secretion in cultured cells using ultra-performance liquid chromatography quadrupole-time of flight mass spectrometry (UPLC-Q-ToF-MS).
Paglia, Giuseppe; Hrafnsdottir, Sigrun; Magnusdottir, Manuela et al

in Analytical and Bioanalytical Chemistry (2012), 402(3), 1183-98

Here we present an ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) method for extracellular measurements of known and unexpected metabolites in parallel. The method was developed by ... [more ▼]

Here we present an ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) method for extracellular measurements of known and unexpected metabolites in parallel. The method was developed by testing 86 metabolites, including amino acids, organic acids, sugars, purines, pyrimidines, vitamins, and nucleosides, that can be resolved by combining chromatographic and m/z dimensions. Subsequently, a targeted quantitative method was developed for 80 metabolites. The presented method combines a UPLC approach using hydrophilic interaction liquid chromatography (HILIC) and MS detection achieved by a hybrid quadrupole-time of flight (Q-ToF) mass spectrometer. The optimal setup was achieved by evaluating reproducibility and repeatability of the analytical platforms using pooled quality control samples to minimize the drift in instrumental performance over time. Then, the method was validated by analyzing extracellular metabolites from acute lymphoblastic leukemia cell lines (MOLT-4 and CCRF-CEM) treated with direct (A-769662) and indirect (AICAR) AMP activated kinase (AMPK) activators, monitoring uptake and secretion of the targeted compound over time. This analysis pointed towards a perturbed purine and pyrimidine catabolism upon AICAR treatment. Our data suggest that the method presented can be used for qualitative and quantitative analysis of extracellular metabolites and it is suitable for routine applications such as in vitro drug screening. [less ▲]

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See detailDetailing the optimality of photosynthesis in cyanobacteria through systems biology analysis.
Nogales, Juan; Gudmundsson, Steinn; Knight, Eric M. et al

in Proceedings of the National Academy of Sciences of the United States of America (2012), 109(7), 2678-2683

Photosynthesis has recently gained considerable attention for its potential role in the development of renewable energy sources. Optimizing photosynthetic organisms for biomass or biofuel production will ... [more ▼]

Photosynthesis has recently gained considerable attention for its potential role in the development of renewable energy sources. Optimizing photosynthetic organisms for biomass or biofuel production will therefore require a systems understanding of photosynthetic processes. We reconstructed a high-quality genome-scale metabolic network for Synechocystis sp. PCC6803 that describes key photosynthetic processes in mechanistic detail. We performed an exhaustive in silico analysis of the reconstructed photosynthetic process under different light and inorganic carbon (Ci) conditions as well as under genetic perturbations. Our key results include the following. (i) We identified two main states of the photosynthetic apparatus: a Ci-limited state and a light-limited state. (ii) We discovered nine alternative electron flow pathways that assist the photosynthetic linear electron flow in optimizing the photosynthesis performance. (iii) A high degree of cooperativity between alternative pathways was found to be critical for optimal autotrophic metabolism. Although pathways with high photosynthetic yield exist for optimizing growth under suboptimal light conditions, pathways with low photosynthetic yield guarantee optimal growth under excessive light or Ci limitation. (iv) Photorespiration was found to be essential for the optimal photosynthetic process, clarifying its role in high-light acclimation. Finally, (v) an extremely high photosynthetic robustness drives the optimal autotrophic metabolism at the expense of metabolic versatility and robustness. The results and modeling approach presented here may promote a better understanding of the photosynthetic process. They can also guide bioengineering projects toward optimal biofuel production in photosynthetic organisms. [less ▲]

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