For many infectious diseases, novel treatment options are needed to address problems with cost, toxicity and resistance to current drugs. Systems biology tools can be used to gain valuable insight into pathogenic processes and aid in expediting drug discovery. In the past decade, constraint-based modeling of genome-scale metabolic networks has become widely used. Focusing on pathogen metabolic networks, we review in silico strategies to identify effective drug targets, and we highlight recent successes as well as limitations associated with such computational analyses. We further discuss how accounting for the host environment and even targeting the host may offer new therapeutic options. These systems-level approaches are beginning to provide novel avenues for drug targeting against infectious agents.

Advances in computational metabolic optimization are required to realize the full potential of new in vivo metabolic engineering technologies by bridging the gap between computational design and strain development. We present Redirector, a new Flux Balance Analysis-based framework for identifying engineering targets to optimize metabolite production in complex pathways. Previous optimization frameworks have modeled metabolic alterations as directly controlling fluxes by setting particular flux bounds. Redirector develops a more biologically relevant approach, modeling metabolic alterations as changes in the balance of metabolic objectives in the system. This framework iteratively selects enzyme targets, adds the associated reaction fluxes to the metabolic objective, thereby incentivizing flux towards the production of a metabolite of interest. These adjustments to the objective act in competition with cellular growth and represent up-regulation and down-regulation of enzyme mediated reactions. Using the iAF1260 E. coli metabolic network model for optimization of fatty acid production as a test case, Redirector generates designs with as many as 39 simultaneous and 111 unique engineering targets. These designs discover proven in vivo targets, novel supporting pathways and relevant interdependencies, many of which cannot be predicted by other methods. Redirector is available as open and free software, scalable to computational resources, and powerful enough to find all known enzyme targets for fatty acid production.

Author Summary

A deeper understanding of biological processes, along with methods in synthetic biology, is driving the frontier of metabolic engineering. In particular, a better representation of cell metabolism will enable the engineering of bacterial strains that can act as factories for valuable biochemical products, from medicines to biofuels. Models which predict the behavior of these complex biological systems enable better engineering design as well as a more comprehensive understanding of fundamental biological principles. Here we develop a new method, called Redirector, for modeling metabolic alterations, and their relationship to cell growth. This method optimizes genetic engineering changes to achieve metabolite production using a new representation of the metabolic impact of genetic manipulation, which is more biologically realistic than existing models. We discover proven and novel engineering targets to improve fatty acid production, correctly predicting how different combinations of genes build upon one another. This work demonstrates that Redirector is a powerful method for designing cell factories and improving our understanding of metabolic systems.

Metabolomics is a relatively new “omics” platform, which analyzes a discrete set of metabolites detected in bio-fluids or tissue samples of organisms. It has been used in a diverse array of studies to detect biomarkers and to determine activity rates for pathways based on changes due to disease or drugs. Recent improvements in analytical methodology and large sample throughput allow for creation of large datasets of metabolites that reflect changes in metabolic dynamics due to disease or a perturbation in the metabolic network. However, current methods of comprehensive analyses of large metabolic datasets (metabolomics) are limited, unlike other “omics” approaches where complex techniques for analyzing coexpression/coregulation of multiple variables are applied. This paper discusses the shortcomings of current metabolomics data analysis techniques, and proposes a new multivariate technique (ADEMA) based on mutual information to identify expected metabolite level changes with respect to a specific condition. We show that ADEMA better predicts De Novo Lipogenesis pathway metabolite level changes in samples with Cystic Fibrosis (CF) than prediction based on the significance of individual metabolite level changes. We also applied ADEMA's classification scheme on three different cohorts of CF and wildtype mice. ADEMA was able to predict whether an unknown mouse has a CF or a wildtype genotype with 1.0, 0.84, and 0.9 accuracy for each respective dataset. ADEMA results had up to 31% higher accuracy as compared to other classification algorithms. In conclusion, ADEMA advances the state-of-the-art in metabolomics analysis, by providing accurate and interpretable classification results.

Author Summary

Metabolomics is an experimental approach that analyzes differences in metabolite levels detected in experimental samples. It has been used in the literature to understand the changes in metabolism with respect to diseases or drugs. Unlike transcriptomics or proteomics, which analyze gene and protein expression levels respectively, the techniques that consider co-regulation of multiple metabolites are quite limited. In this paper, we propose a novel technique, called ADEMA, which computes the expected level changes for each metabolite with respect to a given condition. ADEMA considers multiple metabolites at the same time and is mutual information (MI)-based. We show that ADEMA predicts metabolite level changes for young mice with Cystic Fibrosis (CF) better than significance testing that considers one metabolite at a time. Using three different datasets that contain CF and wild-type (WT) mice, we show that ADEMA can classify an individual as being CF or WT based on the metabolic profiles (with 1.0, 0.84, and 0.9 accuracy, respectively). Compared to other well-known classification algorithms, ADEMA's accuracy is higher by up to 31%.

Metabolic flux is frequently rerouted through cellular metabolism in response to dynamic changes in the intra- and extra-cellular environment. Capturing the mechanisms underlying these metabolic transitions in quantitative and predictive models is a prominent challenge in systems biology. Progress in this regard has been made by integrating high-throughput gene expression data into genome-scale stoichiometric models of metabolism. Here, we extend previous approaches to perform a Temporal Expression-based Analysis of Metabolism (TEAM). We apply TEAM to understanding the complex metabolic dynamics of the respiratorily versatile bacterium Shewanella oneidensis grown under aerobic, lactate-limited conditions. TEAM predicts temporal metabolic flux distributions using time-series gene expression data. Increased predictive power is achieved by supplementing these data with a large reference compendium of gene expression, which allows us to take into account the unique character of the distribution of expression of each individual gene. We further propose a straightforward method for studying the sensitivity of TEAM to changes in its fundamental free threshold parameter θ, and reveal that discrete zones of distinct metabolic behavior arise as this parameter is changed. By comparing the qualitative characteristics of these zones to additional experimental data, we are able to constrain the range of θ to a small, well-defined interval. In parallel, the sensitivity analysis reveals the inherently difficult nature of dynamic metabolic flux modeling: small errors early in the simulation propagate to relatively large changes later in the simulation. We expect that handling such “history-dependent” sensitivities will be a major challenge in the future development of dynamic metabolic-modeling techniques.

Author Summary

Understanding the dynamic response of microorganisms to environmental changes is a major challenge in systems biology. In many cases, these responses manifest themselves through changes in gene transcription, which then propagate to adjust flow through metabolism. Here, we implement a Temporal Expression-based Analysis of Metabolism (TEAM) by dynamically integrating a genome-scale model of the metabolism of S. oneidensis with high-throughput measurements of gene expression and growth data. TEAM recapitulates the complex cascade of secretion and re-uptake of intermediary carbon sources that S. oneidensis exhibits in the experimental data. We show that these complicated metabolic behaviors are best captured when TEAM explicitly accounts for each gene's unique transcriptional signature. Furthermore, by way of a newly proposed sensitivity analysis, we reveal and study the inherent difficulty of dynamic metabolic flux modeling: small changes early in a simulation can easily spread and lead to significant changes towards the end of it. We expect that further development of robust dynamic flux balance methods will need to overcome such “history-dependent” sensitivities in order to achieve increased predictive accuracy.

Genome-scale metabolic networks provide a comprehensive structural framework for modeling genotype-phenotype relationships through flux simulations. The solution space for the metabolic flux state of the cell is typically very large and optimization-based approaches are often necessary for predicting the active metabolic state under specific environmental conditions. The objective function to be used in such optimization algorithms is directly linked with the biological hypothesis underlying the model and therefore it is one of the most relevant parameters for successful modeling. Although linear combination of selected fluxes is widely used for formulating metabolic objective functions, we show that the resulting optimization problem is sensitive towards stoichiometry representation of the metabolic network. This undesirable sensitivity leads to different simulation results when using numerically different but biochemically equivalent stoichiometry representations and thereby makes biological interpretation intrinsically subjective and ambiguous. We hereby propose a new method, Minimization of Metabolites Balance (MiMBl), which decouples the artifacts of stoichiometry representation from the formulation of the desired objective functions, by casting objective functions using metabolite turnovers rather than fluxes. By simulating perturbed metabolic networks, we demonstrate that the use of stoichiometry representation independent algorithms is fundamental for unambiguously linking modeling results with biological interpretation. For example, MiMBl allowed us to expand the scope of metabolic modeling in elucidating the mechanistic basis of several genetic interactions in Saccharomyces cerevisiae.

Author Summary

One of the challenging tasks in systems biology is to quantitatively predict the metabolic behavior of the cell under given genetic and environmental constraints. To this end, genome-scale metabolic reconstructions and simulation tools are indispensable. The choice of the objective function to be used for simulating genome-scale metabolic models is dependent on the biological context and one of the most relevant parameters for successful modeling. Formulation of the intended objective function often requires the use of multiple fluxes, e.g. the sum of fluxes through ATP-producing reactions. We demonstrate that the existing tools confound biological interpretation of the simulations due to undesired dependence on the representation of stoichiometry and propose a new tool – Minimization of Metabolites Balance (MiMBl). MiMBl allows casting of the desired biological objective functions into linear optimization models and gives consistent simulation results when using numerically different but biochemically equivalent stoichiometry representations. We demonstrate relevance of MiMBl for addressing biological questions through improved predictions of genetic interactions within the yeast metabolic network. Genetic interactions imply functional relationship between the genes and therefore allow assessing different hypotheses for the underlying biological principles. MiMBl explains several of the genetic interactions as outcome of flux re-routing for minimal metabolite turnover adjustments.

Cellular metabolism continuously processes an enormous range of external compounds into endogenous metabolites and is as such a key element in human physiology. The multifaceted physiological role of the metabolic network fulfilling the catalytic conversions can only be fully understood from a whole-body perspective where the causal interplay of the metabolic states of individual cells, the surrounding tissue and the whole organism are simultaneously considered. We here present an approach relying on dynamic flux balance analysis that allows the integration of metabolic networks at the cellular scale into standardized physiologically-based pharmacokinetic models at the whole-body level. To evaluate our approach we integrated a genome-scale network reconstruction of a human hepatocyte into the liver tissue of a physiologically-based pharmacokinetic model of a human adult. The resulting multiscale model was used to investigate hyperuricemia therapy, ammonia detoxification and paracetamol-induced toxication at a systems level. The specific models simultaneously integrate multiple layers of biological organization and offer mechanistic insights into pathology and medication. The approach presented may in future support a mechanistic understanding in diagnostics and drug development.

Author Summary

Cellular metabolism is a key element in human physiology. Ideally the metabolic network needs to be considered within the context of the surrounding tissue and organism since the various levels of biological organization are mutually influencing each other. To mechanistically describe the interplay between intracellular space and extracellular environment, we here integrate the genome-scale metabolic network model HepatoNet1 at the cellular scale into physiologically-based pharmacokinetic models at the whole-body level. The resulting multiscale model allows the quantitative description of metabolic behavior in the context of time-resolved metabolite concentration profiles in the body and the surrounding liver tissue. The model has been applied to three case studies covering fundamental aspects of medicine and pharmacology: drug administration, biomarker identification and drug-induced toxication. Most notably, our multiscale approach fosters an improved quantitative understanding of drug action and the impact of metabolic disorders at an organism level, based on a genome-scale representation of cellular metabolism. Computational models such as the one presented include various aspects of human physiology and may therefore significantly support rational approaches in medical diagnostics and pharmaceutical drug development in the future.

Actin is an abundant protein that constitutes a main component of the eukaryotic cytoskeleton. Its polymerization and depolymerization are regulated by a variety of actin-binding proteins. Their functions range from nucleation of actin polymerization to sequestering G-actin in 1∶1 complexes. The kinetics of forming these complexes, with rate constants varying at least three orders of magnitude, is critical to the distinct regulatory functions. Previously we have developed a transient-complex theory for computing protein association mechanisms and association rate constants. The transient complex refers to an intermediate in which the two associating proteins have near-native separation and relative orientation but have yet to form short-range specific interactions of the native complex. The association rate constant is predicted as ka = ka0 , where ka0 is the basal rate constant for reaching the transient complex by free diffusion, and the Boltzmann factor captures the bias of long-range electrostatic interactions. Here we applied the transient-complex theory to study the association kinetics of seven actin-binding proteins with G-actin. These proteins exhibit three classes of association mechanisms, due to their different molecular shapes and flexibility. The 1000-fold ka variations among them can mostly be attributed to disparate electrostatic contributions. The basal rate constants also showed variations, resulting from the different shapes and sizes of the interfaces formed by the seven actin-binding proteins with G-actin. This study demonstrates the various ways that actin-binding proteins use physical properties to tune their association mechanisms and rate constants to suit distinct regulatory functions.

Author Summary

Actin polymerization and depolymerization drive cell motility and are regulated by a variety of actin-binding proteins. The widely-varying rate constants (ka) of the actin-binding proteins associating with G-actin, spanning at least three orders of magnitude, appear to be tuned for their distinct regulatory functions. Here we applied our previously developed transient-complex theory to study the association kinetics of seven actin-binding proteins with G-actin. These proteins exhibit three classes of association mechanisms, due to their different molecular shapes and flexibility. The 1000-fold ka variations among them can mostly be attributed to disparate inter-protein electrostatic interactions. By computing the association mechanisms and quantifying the physical determinants of association rate constants, the present study reveals critical links between the structure and function of the actin-binding proteins.

Although all brain cells bear in principle a comparable potential in terms of energetics, in reality they exhibit different metabolic profiles. The specific biochemical characteristics explaining such disparities and their relative importance are largely unknown. Using a modeling approach, we show that modifying the kinetic parameters of pyruvate dehydrogenase and mitochondrial NADH shuttling within a realistic interval can yield a striking switch in lactate flux direction. In this context, cells having essentially an oxidative profile exhibit pronounced extracellular lactate uptake and consumption. However, they can be turned into cells with prominent aerobic glycolysis by selectively reducing the aforementioned parameters. In the case of primarily oxidative cells, we also examined the role of glycolysis and lactate transport in providing pyruvate to mitochondria in order to sustain oxidative phosphorylation. The results show that changes in lactate transport capacity and extracellular lactate concentration within the range described experimentally can sustain enhanced oxidative metabolism upon activation. Such a demonstration provides key elements to understand why certain brain cell types constitutively adopt a particular metabolic profile and how specific features can be altered under different physiological and pathological conditions in order to face evolving energy demands.

Author Summary

In an environment with appropriate oxygen levels (normoxia), most eukaryotic cells produce energy by oxidizing glucose into carbon dioxide and water. In this process, glucose is transformed into pyruvate, which then fuels oxidative phosphorylation in the mitochondria. Interestingly, Otto Warburg reported back in the 1920's that some eukaryotic cells prominently process glucose-derived pyruvate into lactate, hence “avoiding" the mitochondrial oxidation despite adequate oxygen concentrations. This phenomenon was termed aerobic glycolysis and was first observed in cancer cells. Since then, it has also been described in several normal tissues including the central nervous system. The biochemical basis of aerobic glycolysis has remained elusive until now. Taking advantage of a modeling approach, we unraveled the main metabolic characteristics that determine whether a cell will be strictly oxidative or rather will exhibit aerobic glycolysis. When applied in the context of the central nervous system, our findings not only provide a theoretical demonstration of why neurons and astrocytes differ in terms of metabolic profile, but also suggest that such complementarity forms the basis for metabolic cooperation between the two cell types.

Constraint-based models of metabolism have been used in a variety of studies on drug discovery, metabolic engineering, evolution, and multi-species interactions. These genome-scale models can be generated for any sequenced organism since their main parameters (i.e., reaction stoichiometry) are highly conserved. Their relatively low parameter requirement makes these models easy to develop; however, these models often result in a solution space with multiple possible flux distributions, making it difficult to determine the precise flux state in the cell. Recent research efforts in this modeling field have investigated how additional experimental data, including gene expression, protein expression, metabolite concentrations, and kinetic parameters, can be used to reduce the solution space. This mini-review provides a summary of the data-driven computational approaches that are available for reducing the solution space and thereby improve predictions of intracellular fluxes by constraint-based models.

With the advent of high-throughput technologies, the field of systems biology has amassed an abundance of “omics” data, quantifying thousands of cellular components across a variety of scales, ranging from mRNA transcript levels to metabolite quantities. Methods are needed to not only integrate this omics data but to also use this data to heighten the predictive capabilities of computational models. Several recent studies have successfully demonstrated how flux balance analysis (FBA), a constraint-based modeling approach, can be used to integrate transcriptomic data into genome-scale metabolic network reconstructions to generate predictive computational models. In this review, we summarize such FBA-based methods for integrating expression data into genome-scale metabolic network reconstructions, highlighting their advantages as well as their limitations.

Melanomas depend on autocrine signals for proliferation and survival; however, no systematic screen of known RTKs has been performed to identify which autocrine signaling pathways are activated in melanoma. Here we performed a comprehensive analysis of 42 receptor tyrosine kinases (RTKs) in 6 individual human melanoma tumor specimens as well as 17 melanoma cell lines, some of which were derived from the tumor specimens. We identified 5 RTKs that were active in almost every one of the melanoma tissue specimens and cell lines, including two previously unreported receptors, IGF1R and MSPR, in addition to three receptors (VEGFR, FGFR and HGFR) known to be autocrine activated in melanoma. We show by real time quantitative PCR that all melanoma cell lines expressed genes for the RTK ligands HGF, IGF1 and MSP. Addition of antibodies to either IGF1 or HGF, but not to MSP, to the culture medium blocked melanoma cell proliferation, and even caused net loss of melanoma cells. Antibody addition deactivated IGF1R and HGFR receptors, as well as MAPK signaling. Thus, IGF1 is a new growth factor for autocrine driven proliferation of human melanoma in vitro. Our results suggest that IGF1-IGF1R autocrine pathway in melanoma is a possible target for therapy in human melanomas.

Despite the crucial role of the liver in glucose homeostasis, a detailed mathematical model of human hepatic glucose metabolism is lacking so far. Here we present a detailed kinetic model of glycolysis, gluconeogenesis and glycogen metabolism in human hepatocytes integrated with the hormonal control of these pathways by insulin, glucagon and epinephrine. Model simulations are in good agreement with experimental data on (i) the quantitative contributions of glycolysis, gluconeogenesis, and glycogen metabolism to hepatic glucose production and hepatic glucose utilization under varying physiological states. (ii) the time courses of postprandial glycogen storage as well as glycogen depletion in overnight fasting and short term fasting (iii) the switch from net hepatic glucose production under hypoglycemia to net hepatic glucose utilization under hyperglycemia essential for glucose homeostasis (iv) hormone perturbations of hepatic glucose metabolism. Response analysis reveals an extra high capacity of the liver to counteract changes of plasma glucose level below 5 mM (hypoglycemia) and above 7.5 mM (hyperglycemia). Our model may serve as an important module of a whole-body model of human glucose metabolism and as a valuable tool for understanding the role of the liver in glucose homeostasis under normal conditions and in diseases like diabetes or glycogen storage diseases.

Author Summary

Glucose is an indispensable fuel for all cells and organs, but at the same time leads to problems at high concentrations. As a consequence, blood glucose is controlled in a narrow range to guarantee constant supply and on the other hand avoid damages associated with elevated glucose levels. The liver is the main organ controlling blood glucose by (i) releasing newly synthesized or stored glucose in the blood stream when blood glucose is low (ii) using and storing glucose when blood glucose is elevated. These processes are regulated by hormones, in particular insulin, glucagon and epinephrine. We developed the first detailed kinetic model of this crucial metabolic system integrated with its hormonal control and validated the model based on a multitude of experimental data. Our model enables for the first time to simulate hepatic glucose metabolism in depth. Our results show how due to the hormonal control of key enzymes the liver metabolism can be switched between glucose production and utilization. We provide an essential model to analyze glucose regulation in the normal state and diseases associated with defects in glucose homeostasis like diabetes.

Considering cells as biofactories, we aimed to optimize its internal processes by using the same engineering principles that large industries are implementing nowadays: lean manufacturing. We have applied reverse engineering computational methods to transcriptomic, metabolomic and phenomic data obtained from a collection of tomato recombinant inbreed lines to formulate a kinetic and constraint-based model that efficiently describes the cellular metabolism from expression of a minimal core of genes. Based on predicted metabolic profiles, a close association with agronomic and organoleptic properties of the ripe fruit was revealed with high statistical confidence. Inspired in a synthetic biology approach, the model was used for exploring the landscape of all possible local transcriptional changes with the aim of engineering tomato fruits with fine-tuned biotechnological properties. The method was validated by the ability of the proposed genomes, engineered for modified desired agronomic traits, to recapitulate experimental correlations between associated metabolites.

Author Summary

Considering cells as biofactories, we aimed to optimize their internal processes by using existing design principles acquired from engineering. Herein, we present a synthetic biology approach based on experimental and computational methodology that integrates genomic, transcriptomic, metabolomic and phenomic data to formulate a kinetic and constraint based model of tomato agronomic and fruit quality characteristics. The model has been used for exploring the landscape of all possible local transcriptional changes with the aim of engineering tomato fruits with improved biotechnological properties. The methodology was validated by the ability of the proposed engineered genomes with modified desired agronomic traits, to recapitulate correlations between associated metabolites that are found experimentally in a number of examples.

With the advent of modern high throughput genomics, there is a significant need for genome-scale analysis techniques that can assist in complex systems analysis. Metabolic genome-scale network reconstructions (GENREs) paired with constraint-based modeling are an efficient method to integrate genomics, transcriptomics, and proteomics to conduct organism-specific analysis. This text explains key steps in the GENRE construction process and several methods of constraint-based modeling that can help elucidate basic life processes and development of disease treatment, bioenergy solutions, and industrial bioproduction applications.

Background

Tumor cells in vivo encounter diverse types of microenvironments both at the site of the primary tumor and at sites of distant metastases. Understanding how the various mechanical properties of these microenvironments affect the biology of tumor cells during disease progression is critical in identifying molecular targets for cancer therapy.

Methodology/Principal Findings

This study uses flexible polyacrylamide gels as substrates for cell growth in conjunction with a novel proteomic approach to identify the properties of rigidity-dependent cancer cell lines that contribute to their differential growth on soft and rigid substrates. Compared to cells growing on more rigid/stiff substrates (>10,000 Pa), cells on soft substrates (150–300 Pa) exhibited a longer cell cycle, due predominantly to an extension of the G1 phase of the cell cycle, and were metabolically less active, showing decreased levels of intracellular ATP and a marked reduction in protein synthesis. Using stable isotope labeling of amino acids in culture (SILAC) and mass spectrometry, we measured the rates of protein synthesis of over 1200 cellular proteins under growth conditions on soft and rigid/stiff substrates. We identified cellular proteins whose syntheses were either preferentially inhibited or preserved on soft matrices. The former category included proteins that regulate cytoskeletal structures (e.g., tubulins) and glycolysis (e.g., phosphofructokinase-1), whereas the latter category included proteins that regulate key metabolic pathways required for survival, e.g., nicotinamide phosphoribosyltransferase, a regulator of the NAD salvage pathway.

Conclusions/Significance

The cellular properties of rigidity-dependent cancer cells growing on soft matrices are reminiscent of the properties of dormant cancer cells, e.g., slow growth rate and reduced metabolism. We suggest that the use of relatively soft gels as cell culture substrates would allow molecular pathways to be studied under conditions that reflect the different mechanical environments encountered by cancer cells upon metastasis to distant sites.

Background

Systems biology holds promise as a new approach to drug target identification and drug discovery against neglected tropical diseases. Genome-scale metabolic reconstructions, assembled from annotated genomes and a vast array of bioinformatics/biochemical resources, provide a framework for the interrogation of human pathogens and serve as a platform for generation of future experimental hypotheses. In this article, with the application of selection criteria for both Leishmania major targets (e.g. in silico gene lethality) and drugs (e.g. toxicity), a method (MetDP) to rationally focus on a subset of low-toxic Food and Drug Administration (FDA)-approved drugs is introduced.

Results

This metabolic network-driven approach identified 15 L. major genes as high-priority targets, 8 high-priority synthetic lethal targets, and 254 FDA-approved drugs. Results were compared to previous literature findings and existing high-throughput screens. Halofantrine, an antimalarial agent that was prioritized using MetDP, showed noticeable antileishmanial activity when experimentally evaluated in vitro against L. major promastigotes. Furthermore, synthetic lethality predictions also aided in the prediction of superadditive drug combinations. For proof-of-concept, double-drug combinations were evaluated in vitro against L. major and four combinations involving the drug disulfiram that showed superadditivity are presented.

Conclusions

A direct metabolic network-driven method that incorporates single gene essentiality and synthetic lethality predictions is proposed that generates a set of high-priority L. major targets, which are in turn associated with a select number of FDA-approved drugs that are candidate antileishmanials. Additionally, selection of high-priority double-drug combinations might provide for an attractive and alternative avenue for drug discovery against leishmaniasis.

Circadian clocks provide an internal measure of external time allowing organisms to anticipate and exploit predictable daily changes in the environment. Rhythms driven by circadian clocks have a temperature compensated periodicity of approximately 24 hours that persists in constant conditions and can be reset by environmental time cues. Computational modelling has aided our understanding of the molecular mechanisms of circadian clocks, nevertheless it remains a major challenge to integrate the large number of clock components and their interactions into a single, comprehensive model that is able to account for the full breadth of clock phenotypes. Here we present a comprehensive dynamic model of the Neurospora crassa circadian clock that incorporates its key components and their transcriptional and post-transcriptional regulation. The model accounts for a wide range of clock characteristics including: a periodicity of 21.6 hours, persistent oscillation in constant conditions, arrhythmicity in constant light, resetting by brief light pulses, and entrainment to full photoperiods. Crucial components influencing the period and amplitude of oscillations were identified by control analysis. Furthermore, simulations enabled us to propose a mechanism for temperature compensation, which is achieved by simultaneously increasing the translation of frq RNA and decreasing the nuclear import of FRQ protein.

Author Summary

Circadian clocks are internal timekeepers that integrate signals from the environment and orchestrate cellular events to occur at the most favourable time of day. Circadian clocks in animals, plants, fungi and bacteria have similar characteristic properties and molecular architecture. They have a periodicity of approximately 24 hours, persist in constant conditions and can be reset by environmental time cues such as light and temperature. Another essential property, whose molecular basis is poorly understood, is that the period is temperature compensated i.e. it remains the same over a range of temperatures. Computational modelling has become a valuable tool to predict and understand the underlying mechanisms of such complex molecular systems, but existing clock models are often restricted in the scope of molecular reactions they cover and in the breadth of conditions they are able to reproduce. We therefore built a comprehensive model of the circadian clock of the fungus Neurospora crassa, which encompasses existing knowledge of the biochemistry of the Neurospora clock. We validated this model against a wide range of experimental phenotypes and then used the model to investigate possible molecular explanations of temperature compensation. Our simulations suggest that temperature compensation of period is achieved by changing the abundance and cellular localisation of a key clock protein.

A hallmark of severe sepsis is systemic inflammation which activates leukocytes and can result in their misdirection. This leads to both impaired migration to the locus of infection and increased infiltration into healthy tissues. In order to better understand the pathophysiologic mechanisms involved, we developed a coarse-grained phenomenological model of the acute inflammatory response in CLP (cecal ligation and puncture)-induced sepsis in rats. This model incorporates distinct neutrophil kinetic responses to the inflammatory stimulus and the dynamic interactions between components of a compartmentalized inflammatory response. Ensembles of model parameter sets consistent with experimental observations were statistically generated using a Markov-Chain Monte Carlo sampling. Prediction uncertainty in the model states was quantified over the resulting ensemble parameter sets. Forward simulation of the parameter ensembles successfully captured experimental features and predicted that systemically activated circulating neutrophils display impaired migration to the tissue and neutrophil sequestration in the lung, consequently contributing to tissue damage and mortality. Principal component and multiple regression analyses of the parameter ensembles estimated from survivor and non-survivor cohorts provide insight into pathologic mechanisms dictating outcome in sepsis. Furthermore, the model was extended to incorporate hypothetical mechanisms by which immune modulation using extracorporeal blood purification results in improved outcome in septic rats. Simulations identified a sub-population (about of the treated population) that benefited from blood purification. Survivors displayed enhanced neutrophil migration to tissue and reduced sequestration of lung neutrophils, contributing to improved outcome. The model ensemble presented herein provides a platform for generating and testing hypotheses in silico, as well as motivating further experimental studies to advance understanding of the complex biological response to severe infection, a problem of growing magnitude in humans.

Author Summary

The pathophysiology of sepsis is complex and our mechanistic understanding remains incomplete. Mathematical models of the inflammatory response have been providing intellectual frameworks to reason about the complexity of sepsis. Due to an incompletely understood system along with very limited data, our approach focuses on building simplified, falsifiable and predictive models, and offers a means to quantify parametric uncertainty. Based on the construct that deterministic ensemble models exhibit population-like behavior, we developed a population-based computational framework that incorporates dysregulated neutrophil hyperactivity as a cellular dysfunction in septic processes. We hypothesize that probability distributions of physiological parameters conditional on population observations can characterize the range of possible physiologic responses in a population. Comparing the parameter ensembles from different phenotypes reveals some factors that play an important role in the expression of such phenotypes, such as sepsis survival. This framework can serve as an effective tool to gain insight into the pathophysiology of severe sepsis and generate testable hypotheses that guide future experiments. Our approach holds promise as a tool for integrating domain knowledge and experimental data into a quantitative assessment of population dynamics.

One of the most obvious phenotypes of a cell is its metabolic activity, which is defined by the fluxes in the metabolic network. Although experimental methods to determine intracellular fluxes are well established, only a limited number of fluxes can be resolved. Especially in eukaryotes such as yeast, compartmentalization and the existence of many parallel routes render exact flux analysis impossible using current methods. To gain more insight into the metabolic operation of S. cerevisiae we developed a new computational approach where we characterize the flux solution space by determining elementary flux modes (EFMs) that are subsequently classified as thermodynamically feasible or infeasible on the basis of experimental metabolome data. This allows us to provably rule out the contribution of certain EFMs to the in vivo flux distribution. From the 71 million EFMs in a medium size metabolic network of S. cerevisiae, we classified 54% as thermodynamically feasible. By comparing the thermodynamically feasible and infeasible EFMs, we could identify reaction combinations that span the cytosol and mitochondrion and, as a system, cannot operate under the investigated glucose batch conditions. Besides conclusions on single reactions, we found that thermodynamic constraints prevent the import of redox cofactor equivalents into the mitochondrion due to limits on compartmental cofactor concentrations. Our novel approach of incorporating quantitative metabolite concentrations into the analysis of the space of all stoichiometrically feasible flux distributions allows generating new insights into the system-level operation of the intracellular fluxes without making assumptions on metabolic objectives of the cell.

Author Summary

Fluxes in metabolic pathways are a highly informative aspect of an organism's phenotype. The experimental determination of such fluxes is well established and has proven very useful. To address some of the limitations of experimental flux analysis, such as when the cell is divided in multiple compartments, stoichiometric modeling provides a valuable addition. The approach that we take is based on stoichiometric modeling where we consider the thermodynamic feasibility of many different possible routes through the metabolic network of Saccharomyces cerevisiae using experimentally determined metabolite concentrations. We show that next to conclusions on single biochemical reactions in the metabolic network, we obtain system-level insights on thermodynamically infeasible flux patterns. We found that the compartmental concentrations of and NADH are the causes for the system-level infeasibilities. With the current advances in quantitative metabolomics and biochemical thermodynamics, we envision that the presented method will help gaining more insight into complex metabolic systems.

Kinetic models of metabolism require detailed knowledge of kinetic parameters. However, due to measurement errors or lack of data this knowledge is often uncertain. The model of glycolysis in the parasitic protozoan Trypanosoma brucei is a particularly well analysed example of a quantitative metabolic model, but so far it has been studied with a fixed set of parameters only. Here we evaluate the effect of parameter uncertainty. In order to define probability distributions for each parameter, information about the experimental sources and confidence intervals for all parameters were collected. We created a wiki-based website dedicated to the detailed documentation of this information: the SilicoTryp wiki (http://silicotryp.ibls.gla.ac.uk/wiki/Glycolysis). Using information collected in the wiki, we then assigned probability distributions to all parameters of the model. This allowed us to sample sets of alternative models, accurately representing our degree of uncertainty. Some properties of the model, such as the repartition of the glycolytic flux between the glycerol and pyruvate producing branches, are robust to these uncertainties. However, our analysis also allowed us to identify fragilities of the model leading to the accumulation of 3-phosphoglycerate and/or pyruvate. The analysis of the control coefficients revealed the importance of taking into account the uncertainties about the parameters, as the ranking of the reactions can be greatly affected. This work will now form the basis for a comprehensive Bayesian analysis and extension of the model considering alternative topologies.

Author Summary

An increasing number of mathematical models are being built and analysed in order to obtain a better understanding of specific biological systems. These quantitative models contain parameters that need to be measured or estimated. Because of experimental errors or lack of data, our knowledge about these parameters is uncertain. Our work explores the effect of including these uncertainties in model analysis. Therefore, we studied a particularly well curated model of the energy metabolism of the parasite Trypanosoma brucei, responsible for African sleeping sickness. We first collected all the information we could find about how the model parameters were defined on a website, the SilicoTryp wiki (http:///silicotryp.ibls.gla.ac.uk/wiki/). From this information, we were able to quantify our uncertainty about each parameter, thus allowing us to analyse the model while explicitly taking these uncertainties into account. We found that, even though the model was well-defined and most of its parameters were experimentally measured, taking into account the remaining uncertainty allows us to gain more insight into model behavior. We were able to identify previously unrecognised fragilities of the model, leading to new hypotheses amenable to experimental testing.

Background

Toxins A and B (TcdA and TcdB) are Clostridium difficile's principal virulence factors, yet the pathways by which they lead to inflammation and severe diarrhea remain unclear. Also, the relative role of either toxin during infection and the differences in their effects across cell lines is still poorly understood. To better understand their effects in a susceptible cell line, we analyzed the transciptome-wide gene expression response of human ileocecal epithelial cells (HCT-8) after 2, 6, and 24 hr of toxin exposure.

Results

We show that toxins elicit very similar changes in the gene expression of HCT-8 cells, with the TcdB response occurring sooner. The high similarity suggests differences between toxins are due to events beyond transcription of a single cell-type and that their relative potencies during infection may depend on differential effects across cell types within the intestine. We next performed an enrichment analysis to determine biological functions associated with changes in transcription. Differentially expressed genes were associated with response to external stimuli and apoptotic mechanisms and, at 24 hr, were predominately associated with cell-cycle control and DNA replication. To validate our systems approach, we subsequently verified a novel G1/S and known G2/M cell-cycle block and increased apoptosis as predicted from our enrichment analysis.

Conclusions

This study shows a successful example of a workflow deriving novel biological insight from transcriptome-wide gene expression. Importantly, we do not find any significant difference between TcdA and TcdB besides potency or kinetics. The role of each toxin in the inhibition of cell growth and proliferation, an important function of cells in the intestinal epithelium, is characterized.

Living systems are capable of processing multiple sources of information simultaneously. This is true even at the cellular level, where not only coexisting signals stimulate the cell, but also the presence of fluctuating conditions is significant. When information is received by a cell signaling network via one specific input, the existence of other stimuli can provide a background activity –or chatter– that may affect signal transmission through the network and, therefore, the response of the cell. Here we study the modulation of information processing by chatter in the signaling network of a human cell, specifically, in a Boolean model of the signal transduction network of a fibroblast. We observe that the level of external chatter shapes the response of the system to information carrying signals in a nontrivial manner, modulates the activity levels of the network outputs, and effectively determines the paths of information flow. Our results show that the interactions and node dynamics, far from being random, confer versatility to the signaling network and allow transitions between different information-processing scenarios.

Author Summary

Far from being silent and static, the habitat of a cell is usually composed by multiple and simultaneous signals. We can consider nutrients, hormones, temperature, light, and other stimuli as elements building a default environment in which cells grow, divide and die. This environment, which has an intrinsically fluctuating nature, is the setting in which cells process all incoming stimuli. Here we examine the role that this background activity –or signaling chatter– plays in the transmission of information in a typical human cell. We address this question using a cellular model of signal transduction that we simulate using both random and periodic stimuli. We find that the level of background chatter determines the response of the whole signaling network to external stimuli. Different areas of the network are activated by specific levels of background activity, routing the information through chatter-dependent paths. In this way, different levels of chatter allow the network to select between different responses, given the same stimulus. These features depend on the architecture and functional connectivity of a truly biological network, since we find that randomized versions of the model are incapable of showing this behavior.

The increasing abundance of large-scale, high-throughput datasets for many closely related organisms provides opportunities for comparative analysis via the simultaneous biclustering of datasets from multiple species. These analyses require a reformulation of how to organize multi-species datasets and visualize comparative genomics data analyses results. Recently, we developed a method, multi-species cMonkey, which integrates heterogeneous high-throughput datatypes from multiple species to identify conserved regulatory modules. Here we present an integrated data visualization system, built upon the Gaggle, enabling exploration of our method's results (available at http://meatwad.bio.nyu.edu/cmmr.html). The system can also be used to explore other comparative genomics datasets and outputs from other data analysis procedures – results from other multiple-species clustering programs or from independent clustering of different single-species datasets. We provide an example use of our system for two bacteria, Escherichia coli and Salmonella Typhimurium. We illustrate the use of our system by exploring conserved biclusters involved in nitrogen metabolism, uncovering a putative function for yjjI, a currently uncharacterized gene that we predict to be involved in nitrogen assimilation.

Author Summary

Advancing high-throughput experimental technologies are providing access to genome-wide measurements for multiple related species on multiple information levels (e.g. mRNA, protein, interactions, functional assays, etc.). We present a biclustering algorithm and an associated visualization system for generating and exploring regulatory modules derived from analysis of integrated multi-species genomics datasets. We use multi-species-cMonkey, an algorithm of our own construction that can integrate diverse systems-biology datatypes from multiple species to form biclusters, or condition-dependent regulatory modules, that are conserved across both the multiple species analyzed and biclusters that are specific to subsets of the processed species. Our resource is an integrated web and java based system that allows biologists to explore both conserved and species-specific biclusters in the context of the data, associated networks for both species, and existing annotations for both species. Our focus in this work is on the use of the integrated system with examples drawn from exploring modules associated with nitrogen metabolism in two Gram-negative bacteria, E. coli and S. Typhimurium.

Modularity analysis offers a route to better understand the organization of cellular biochemical networks as well as to derive practically useful, simplified models of these complex systems. While there is general agreement regarding the qualitative properties of a biochemical module, there is no clear consensus on the quantitative criteria that may be used to systematically derive these modules. In this work, we investigate cyclical interactions as the defining characteristic of a biochemical module. We utilize a round trip distance metric, termed Shortest Retroactive Distance (ShReD), to characterize the retroactive connectivity between any two reactions in a biochemical network and to group together network components that mutually influence each other. We evaluate the metric on two types of networks that feature feedback interactions: (i) epidermal growth factor receptor (EGFR) signaling and (ii) liver metabolism supporting drug transformation. For both networks, the ShReD partitions found hierarchically arranged modules that confirm biological intuition. In addition, the partitions also revealed modules that are less intuitive. In particular, ShReD-based partition of the metabolic network identified a ‘redox’ module that couples reactions of glucose, pyruvate, lipid and drug metabolism through shared production and consumption of NADPH. Our results suggest that retroactive interactions arising from feedback loops and metabolic cycles significantly contribute to the modularity of biochemical networks. For metabolic networks, cofactors play an important role as allosteric effectors that mediate the retroactive interactions.

Author Summary

Mathematical models are powerful tools to understand and predict the behavior of complex systems. However, the complexity presents many challenges in developing such models. In the case of a biological cell, a fully detailed and comprehensive model of a major function such as signaling and metabolism remains out of reach, due to the very large number of interdependent biochemical reactions that are required to carry out the function. In this regard, one practical approach is to develop simplified models that nevertheless preserve the essential features of the cell as a complex system by better understanding the chemical organization of the cell, or the layout of the biochemical network. In this work, we describe a computational method to systematically identify closely interacting groups of biochemical reactions by recognizing the modular hierarchy inherent in biochemical networks. We focus on cyclical interactions based on the rationale that reactions that mutually influence each other belong in the same group. We demonstrate our method on a signaling and metabolic network and show that the results confirm biological intuition as well as provide new insights into the coordination of biochemical pathways. Prospectively, our modularization method could be used to systematically derive simplified and practically useful models of complex biological networks.

Although the metabolic networks of the three domains of life consist of different constituents and metabolic pathways, they exhibit the same scale-free organization. This phenomenon has been hypothetically explained by preferential attachment principle that the new-recruited metabolites attach preferentially to those that are already well connected. However, since metabolites are usually small molecules and metabolic processes are basically chemical reactions, we speculate that the metabolic network organization may have a chemical basis. In this paper, chemoinformatic analyses on metabolic networks of Kyoto Encyclopedia of Genes and Genomes (KEGG), Escherichia coli and Saccharomyces cerevisiae were performed. It was found that there exist qualitative and quantitative correlations between network topology and chemical properties of metabolites. The metabolites with larger degrees of connectivity (hubs) are of relatively stronger polarity. This suggests that metabolic networks are chemically organized to a certain extent, which was further elucidated in terms of high concentrations required by metabolic hubs to drive a variety of reactions. This finding not only provides a chemical explanation to the preferential attachment principle for metabolic network expansion, but also has important implications for metabolic network design and metabolite concentration prediction.

Author Summary

The metabolic networks of the three domains of life exhibit the same scale-free organization, which has been hypothetically explained in terms of preferential attachment principle. Here we reveal that the scale-free organization of metabolic networks may have a chemical basis. Through a chemoinformatic analysis on metabolic networks of Kyoto Encyclopedia of Genes and Genomes (KEGG), Escherichia coli and Saccharomyces cerevisiae, it was found that the metabolites with higher degrees of connectivity (hubs) are of relatively stronger polarity. The reason underlying this phenomenon is that to drive a variety of reactions, metabolic hubs have to be highly concentrated. Since the intracellular environments are hydrophilic, metabolic hubs have to be strong-polar to reach high concentrations. This finding has direct implications for metabolic network design and provides a chemical explanation to the preferential attachment principle, which has been validated by numerical simulations of metabolic network expansion. In addition, the correlations between metabolite concentration, metabolic network topology and metabolite chemical properties also suggest that we can use chemical and topological properties of metabolites to predict their intracellular concentrations. A support vector regression model has been successfully established to predict the metabolite concentrations for Escherichia coli.