Nontyphoidal Salmonella strains (NTS) are among the most common foodborne enteropathogens and constitute a major cause of global morbidity and mortality, imposing a substantial burden on global health. The increasing antibiotic resistance of NTS bacteria has attracted a lot of research on understanding their modus operandi during infection. Growth in the gut lumen is a critical phase of the NTS infection. This might offer opportunities for intervention. However, the metabolic richness of the gut lumen environment and the inherent complexity and robustness of the metabolism of NTS bacteria call for modeling approaches to guide research efforts. In this study, we reconstructed a thermodynamically constrained and context-specific genome-scale metabolic model (GEM) for S. Typhimurium SL1344, a model strain well-studied in infection research. We combined sequence annotation, optimization methods and in vitro and in vivo experimental data. We used GEM to explore the nutritional requirements, the growth limiting metabolic genes, and the metabolic pathway usage of NTS bacteria in a rich environment simulating the murine gut. This work provides insight and hypotheses on the biochemical capabilities and requirements of SL1344 beyond the knowledge acquired through conventional sequence annotation and can inform future research aimed at better understanding NTS metabolism and identifying potential targets for infection prevention.

MOTIVATION: Large-scale kinetic models are an invaluable tool to understand the dynamic and adaptive responses of biological systems. The development and application of these models have been limited by the availability of computational tools to build and analyze large-scale models efficiently. The toolbox presented here provides the means to implement, parameterize and analyze large-scale kinetic models intuitively and efficiently. RESULTS: We present a Python package (SKiMpy) bridging this gap by implementing an efficient kinetic modeling toolbox for the semiautomatic generation and analysis of large-scale kinetic models for various biological domains such as signaling, gene expression and metabolism. Furthermore, we demonstrate how this toolbox is used to parameterize kinetic models around a steady-state reference efficiently. Finally, we show how SKiMpy can implement multispecies bioreactor simulations to assess biotechnological processes. AVAILABILITY AND IMPLEMENTATION: The software is available as a Python 3 package on GitHub: https://github.com/EPFL-LCSB/SKiMpy, along with adequate documentation. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

MOTIVATION: Finding biosynthetic pathways is essential for metabolic engineering of organisms to produce chemicals, biodegradation prediction of pollutants and drugs, and for the elucidation of bioproduction pathways of secondary metabolites. A key step in biosynthetic pathway design is the extraction of novel metabolic pathways from big networks that integrate known biological, as well as novel, predicted biotransformations. However, the efficient analysis and the navigation of big biochemical networks remain a challenge. RESULTS: Here, we propose the construction of searchable graph representations of metabolic networks. Each reaction is decomposed into pairs of reactants and products, and each pair is assigned a weight, which is calculated from the number of conserved atoms between the reactant and the product molecule. We test our method on a biochemical network that spans 6546 known enzymatic reactions to show how our approach elegantly extracts biologically relevant metabolic pathways from biochemical networks, and how the proposed network structure enables the application of efficient graph search algorithms that improve navigation and pathway identification in big metabolic networks. The weighted reactant-product pairs of an example network and the corresponding graph search algorithm are available online. The proposed method extracts metabolic pathways fast and reliably from big biochemical networks, which is inherently important for all applications involving the engineering of metabolic networks. AVAILABILITY AND IMPLEMENTATION: https://github.com/EPFL-LCSB/nicepath. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

BACKGROUND: Significant efforts have been made in building large-scale kinetic models of cellular metabolism in the past two decades. However, most kinetic models published to date, remain focused around central carbon pathways or are built around ad hoc reduced models without clear justification on their derivation and usage. Systematic algorithms exist for reducing genome-scale metabolic reconstructions to build thermodynamically feasible and consistently reduced stoichiometric models. However, it is important to study how network complexity affects conclusions derived from large-scale kinetic models built around consistently reduced models before we can apply them to study biological systems. RESULTS: We reduced the iJO1366 Escherichia Coli genome-scale metabolic reconstruction systematically to build three stoichiometric models of different size. Since the reduced models are expansions around the core subsystems for which the reduction was performed, the models are nested. We present a method for scaling up the flux profile and the concentration vector reference steady-states from the smallest model to the larger ones, whilst preserving maximum equivalency. Populations of kinetic models, preserving similarity in kinetic parameters, were built around the reference steady-states and their metabolic sensitivity coefficients (MSCs) were computed. The MSCs were sensitive to the model complexity. We proposed a metric for measuring the sensitivity of MSCs to these structural changes. CONCLUSIONS: We proposed for the first time a workflow for scaling up the size of kinetic models while preserving equivalency between the kinetic models. Using this workflow, we demonstrate that model complexity in terms of networks size has significant impact on sensitivity characteristics of kinetic models. Therefore, it is essential to account for the effects of network complexity when constructing kinetic models. The presented metric for measuring MSC sensitivity to structural changes can guide modelers and experimentalists in improving model quality and guide synthetic biology and metabolic engineering. Our proposed workflow enables the testing of the suitability of a kinetic model for answering certain study-specific questions. We argue that the model-based metabolic design targets that are common across models of different size are of higher confidence, while those that are different could be the objective of investigations for model improvement.

The metabolic capabilities of the species and the local environment shape the microbial interactions in a community either through the exchange of metabolic products or the competition for the resources. Cells are often arranged in close proximity to each other, creating a crowded environment that unevenly reduce the diffusion of nutrients. Herein, we investigated how the crowding conditions and metabolic variability among cells shape the dynamics of microbial communities. For this, we developed CROMICS, a spatio-temporal framework that combines techniques such as individual-based modeling, scaled particle theory, and thermodynamic flux analysis to explicitly incorporate the cell metabolism and the impact of the presence of macromolecular components on the nutrients diffusion. This framework was used to study two archetypical microbial communities (i) Escherichia coli and Salmonella enterica that cooperate with each other by exchanging metabolites, and (ii) two E. coli with different production level of extracellular polymeric substances (EPS) that compete for the same nutrients. In the mutualistic community, our results demonstrate that crowding enhanced the fitness of cooperative mutants by reducing the leakage of metabolites from the region where they are produced, avoiding the resource competition with non-cooperative cells. Moreover, we also show that E. coli EPS-secreting mutants won the competition against the non-secreting cells by creating less dense structures (i.e. increasing the spacing among the cells) that allow mutants to expand and reach regions closer to the nutrient supply point. A modest enhancement of the relative fitness of EPS-secreting cells over the non-secreting ones were found when the crowding effect was taken into account in the simulations. The emergence of cell-cell interactions and the intracellular conflicts arising from the trade-off between growth and the secretion of metabolites or EPS could provide a local competitive advantage to one species, either by supplying more cross-feeding metabolites or by creating a less dense neighborhood.

Microorganisms are frequently organized into crowded structures that affect the nutrients diffusion. This reduction in metabolite diffusion could modify the microbial dynamics, meaning that computational methods for studying microbial systems need accurate ways to model the crowding conditions. We previously developed a computational framework, termed CROMICS, that incorporates the effect of the (time-dependent) crowding conditions on the spatio-temporal modeling of microbial communities, and we used it to demonstrate the crowding influence on the community dynamics. To further identify scenarios where crowding should be considered in microbial modeling, we herein applied and extended CROMICS to simulate several environmental conditions that could potentially boost or dampen the crowding influence in biofilms. We explore whether the nutrient supply (rich- or low-nutrient media), the cell-packing configuration (square or hexagonal spherical cell arrangement), or the cell growing conditions (planktonic state or biofilm) modify the crowding influence on the growth of Escherichia coli. Our results indicate that the growth rate, the abundance and appearance time of different cell phenotypes as well as the amount of by-products secreted to the medium are sensitive to some extent to the local crowding conditions in all scenarios tested, except in rich-nutrient media. Crowding conditions enhance the formation of nutrient gradient in biofilms, but its effect is only appreciated when cell metabolism is controlled by the nutrient limitation. Thus, as soon as biomass (and/or any other extracellular macromolecule) accumulates in a region, and cells occupy more than 14% of the volume fraction, the crowding effect must not be underestimated, as the microbial dynamics start to deviate from the ideal/expected behaviour that assumes volumeless cells or when a homogeneous (reduced) diffusion is applied in the simulation. The modeling and simulation of the interplay between the species diversity (cell shape and metabolism) and the environmental conditions (nutrient quality, crowding conditions) can help to design effective strategies for the optimization and control of microbial systems.

Summary: pyTFA and matTFA are the first published implementations of the original TFA paper. Specifically, they include explicit formulation of Gibbs energies and metabolite concentrations, which enables straightforward integration of metabolite concentration measurements. Motivation: High-throughput analytic technologies provide a wealth of omics data that can be used to perform thorough analyses for a multitude of studies in the areas of Systems Biology and Biotechnology. Nevertheless, most studies are still limited to constraint-based Flux Balance Analyses (FBA), neglecting an important physicochemical constraint: thermodynamics. Thermodynamics-based Flux Analysis (TFA) in metabolic models enables the integration of quantitative metabolomics data to study their effects on the net-flux directionality of reactions in the network. In addition, it allows us to estimate how far each reaction operates from thermodynamic equilibrium, which provides critical information for guiding metabolic engineering decisions. Results: We present a Python package (pyTFA) and a Matlab toolbox (matTFA) that implement TFA. We show an example of application on both a reduced and a genome-scale model of E. coli., and demonstrate TFA and data integration through TFA reduce the feasible flux space with respect to FBA. Availability and implementation: Documented implementation of TFA framework both in Python (pyTFA) and Matlab (matTFA) are available on www.github.com/EPFL-LCSB/. Supplementary information: Supplementary data are available at Bioinformatics online.

Natural soil is characterized as a complex habitat with patchy hydrated islands and spatially variable nutrients that is in a constant state of change due to wetting-drying dynamics. Soil microbial activity is often concentrated in sparsely distributed hotspots that contribute disproportionally to macroscopic biogeochemical nutrient cycling and greenhouse gas emissions. The mechanistic representation of such dynamic hotspots requires new modeling approaches capable of representing the interplay between dynamic local conditions and the versatile microbial metabolic adaptations. We have developed IndiMeSH (Individual-based Metabolic network model for Soil Habitats) as a spatially explicit model for the physical and chemical microenvironments of soil, combined with an individual-based representation of bacterial motility and growth using adaptive metabolic networks. The model uses angular pore networks and a physically based description of the aqueous phase as a backbone for nutrient diffusion and bacterial dispersal combined with dynamic flux balance analysis to calculate growth rates depending on local nutrient conditions. To maximize computational efficiency, reduced scale metabolic networks are used for the simulation scenarios and evaluated strategically to the genome scale model. IndiMeSH was compared to a well-established population-based spatiotemporal metabolic network model (COMETS) and to experimental data of bacterial spatial organization in pore networks mimicking soil aggregates. IndiMeSH was then used to strategically study dynamic response of a bacterial community to abrupt environmental perturbations and the influence of habitat geometry and hydration conditions. Results illustrate that IndiMeSH is capable of representing trophic interactions among bacterial species, predicting the spatial organization and segregation of bacterial populations due to oxygen and carbon gradients, and provides insights into dynamic community responses as a consequence of environmental changes. The modular design of IndiMeSH and its implementation are adaptable, allowing it to represent a wide variety of experimental and in silico microbial systems.

Kinetic models of metabolism can be constructed to predict cellular regulation and devise metabolic engineering strategies, and various promising computational workflows have been developed in recent years for this. Due to the uncertainty in the kinetic parameter values required to build kinetic models, these workflows rely on ensemble modeling (EM) principles for sampling and building populations of models describing observed physiologies. Sensitivity coefficients from metabolic control analysis (MCA) of kinetic models can provide important insight about cellular control around a given physiological steady state. However, despite considering populations of kinetic models and their model outputs, current approaches do not provide adequate tools for statistical inference. To derive conclusions from model outputs, such as MCA sensitivity coefficients, it is necessary to rank/compare populations of variables with each other. Currently existing workflows consider confidence intervals (CIs) that are derived independently for each comparable variable. Hence, it is important to derive simultaneous CIs for the variables that we wish to rank/compare. Herein, we used an existing large-scale kinetic model of Escherichia Coli metabolism to present how univariate CIs can lead to incorrect conclusions, and we present a new workflow that applies three different multivariate statistical approaches. We use the Bonferroni and the exact normal methods to build symmetric CIs using the normality assumptions. We then suggest how bootstrapping can compute asymmetric CIs whilst relaxing this normality assumption. We conclude that the Bonferroni and the exact normal methods can provide simple and efficient ways for constructing reliable CIs, with the exact normal method favored over the Bonferroni when the compared variables present dependencies. Bootstrapping, despite its significantly higher computational cost, is recommended when comparing non-normal distributions of variables. Additionally, we show how the Bonferroni method can readily be used to estimate required sample numbers to attain a certain CI size.

A persistent obstacle for constructing kinetic models of metabolism is uncertainty in the kinetic properties of enzymes. Currently, available methods for building kinetic models can cope indirectly with uncertainties by integrating data from different biological levels and origins into models. In this study, we use the recently proposed computational approach iSCHRUNK (in Silico Approach to Characterization and Reduction of Uncertainty in the Kinetic Models), which combines Monte Carlo parameter sampling methods and machine learning techniques, in the context of Bayesian inference. Monte Carlo parameter sampling methods allow us to exploit synergies between different data sources and generate a population of kinetic models that are consistent with the available data and physicochemical laws. The machine learning allows us to data-mine the a priori generated kinetic parameters together with the integrated datasets and derive posterior distributions of kinetic parameters consistent with the observed physiology. In this work, we used iSCHRUNK to address a design question: can we identify which are the kinetic parameters and what are their values that give rise to a desired metabolic behavior? Such information is important for a wide variety of studies ranging from biotechnology to medicine. To illustrate the proposed methodology, we performed Metabolic Control Analysis, computed the flux control coefficients of the xylose uptake (XTR), and identified parameters that ensure a rate improvement of XTR in a glucose-xylose co-utilizing S. cerevisiae strain. Our results indicate that only three kinetic parameters need to be accurately characterized to describe the studied physiology, and ultimately to design and control the desired responses of the metabolism. This framework paves the way for a new generation of methods that will systematically integrate the wealth of available omics data and efficiently extract the information necessary for metabolic engineering and synthetic biology decisions.

Nonalcoholic fatty liver disease (NAFLD) is associated with metabolic syndromes spanning a wide spectrum of diseases, from simple steatosis to the more complex nonalcoholic steatohepatitis. To identify the deregulation that occurs in metabolic processes at the molecular level that give rise to these various NAFLD phenotypes, algorithms such as pathway enrichment analysis (PEA) can be used. These analyses require the use of predefined pathway maps, which are composed of reactions describing metabolic processes/subsystems. Unfortunately, the annotation of the metabolic subsystems can differ depending on the pathway database used, making these approaches subject to biases associated with different pathway annotations, and these methods cannot capture the balancing of cofactors and byproducts through the complex nature and interactions of genome-scale metabolic networks (GEMs). Here, we introduce a framework entitled Minimum Network Enrichment Analysis (MiNEA) that is applied to GEMs to generate all possible alternative minimal networks (MiNs), which are possible and feasible networks composed of all the reactions pertaining to various metabolic subsystems that can synthesize a target metabolite. We applied MiNEA to investigate deregulated MiNs and to identify key regulators in different NAFLD phenotypes, such as a fatty liver and liver inflammation, in both humans and mice by integrating condition-specific transcriptomics data from liver samples. We identified key deregulations in the synthesis of cholesteryl esters, cholesterol, and hexadecanoate in both humans and mice, and we found that key regulators of the hydrogen peroxide synthesis network were regulated differently in humans and mice. We further identified which MiNs demonstrate the general and specific characteristics of the different NAFLD phenotypes. MiNEA is applicable to any GEM and to any desired target metabolite, making MiNEA flexible enough to study condition-specific metabolism for any given disease or organism.

The ever-increasing availability of transcriptomic and metabolomic data can be used to deeply analyze and make ever-expanding predictions about biological processes, as changes in the reaction fluxes through genome-wide pathways can now be tracked. Currently, constraint-based metabolic modeling approaches, such as flux balance analysis (FBA), can quantify metabolic fluxes and make steady-state flux predictions on a genome-wide scale using optimization principles. However, relating the differential gene expression or differential metabolite abundances in different physiological states to the differential flux profiles remains a challenge. Here we present a novel method, named REMI (Relative Expression and Metabolomic Integrations), that employs genome-scale metabolic models (GEMs) to translate differential gene expression and metabolite abundance data obtained through genetic or environmental perturbations into differential fluxes to analyze the altered physiology for any given pair of conditions. REMI allows for gene-expression, metabolite abundance, and thermodynamic data to be integrated into a single framework, then uses optimization principles to maximize the consistency between the differential gene-expression levels and metabolite abundance data and the estimated differential fluxes and thermodynamic constraints. We applied REMI to integrate into the Escherichia coli GEM publicly available sets of expression and metabolomic data obtained from two independent studies and under wide-ranging conditions. The differential flux distributions obtained from REMI corresponding to the various perturbations better agreed with the measured fluxomic data, and thus better reflected the different physiological states, than a traditional model. Compared to the similar alternative method that provides one solution from the solution space, REMI was able to enumerate several alternative flux profiles using a mixed-integer linear programming approach. Using this important advantage, we performed a high-frequency analysis of common genes and their associated reactions in the obtained alternative solutions and identified the most commonly regulated genes across any two given conditions. We illustrate that this new implementation provides more robust and biologically relevant results for a better understanding of the system physiology.

Genome-scale metabolic reconstructions have proven to be valuable resources in enhancing our understanding of metabolic networks as they encapsulate all known metabolic capabilities of the organisms from genes to proteins to their functions. However the complexity of these large metabolic networks often hinders their utility in various practical applications. Although reduced models are commonly used for modeling and in integrating experimental data, they are often inconsistent across different studies and laboratories due to different criteria and detail, which can compromise transferability of the findings and also integration of experimental data from different groups. In this study, we have developed a systematic semi-automatic approach to reduce genome-scale models into core models in a consistent and logical manner focusing on the central metabolism or subsystems of interest. The method minimizes the loss of information using an approach that combines graph-based search and optimization methods. The resulting core models are shown to be able to capture key properties of the genome-scale models and preserve consistency in terms of biomass and by-product yields, flux and concentration variability and gene essentiality. The development of these "consistently-reduced" models will help to clarify and facilitate integration of different experimental data to draw new understanding that can be directly extendable to genome-scale models.

In the post-genomic era, Genome-scale metabolic networks (GEMs) have emerged as invaluable tools to understand metabolic capabilities of organisms. Different parts of these metabolic networks are defined as subsystems/pathways, which are sets of functional roles to implement a specific biological process or structural complex, such as glycolysis and TCA cycle. Subsystem/pathway definition is also employed to delineate the biosynthetic routes that produce biomass building blocks. In databases, such as MetaCyc and SEED, these representations are composed of linear routes from precursors to target biomass building blocks. However, this approach cannot capture the nested, complex nature of GEMs. Here we implemented an algorithm, lumpGEM, which generates biosynthetic subnetworks composed of reactions that can synthesize a target metabolite from a set of defined core precursor metabolites. lumpGEM captures balanced subnetworks, which account for the fate of all metabolites along the synthesis routes, thus encapsulating reactions from various subsystems/pathways to balance these metabolites in the metabolic network. Moreover, lumpGEM collapses these subnetworks into elementally balanced lumped reactions that specify the cost of all precursor metabolites and cofactors. It also generates alternative subnetworks and lumped reactions for the same metabolite, accounting for the flexibility of organisms. lumpGEM is applicable to any GEM and any target metabolite defined in the network. Lumped reactions generated by lumpGEM can be also used to generate properly balanced reduced core metabolic models.

Novel antimalarial therapies are urgently needed for the fight against drug-resistant parasites. The metabolism of malaria parasites in infected cells is an attractive source of drug targets but is rather complex. Computational methods can handle this complexity and allow integrative analyses of cell metabolism. In this study, we present a genome-scale metabolic model (iPfa) of the deadliest malaria parasite, Plasmodium falciparum, and its thermodynamics-based flux analysis (TFA). Using previous absolute concentration data of the intraerythrocytic parasite, we applied TFA to iPfa and predicted up to 63 essential genes and 26 essential pairs of genes. Of the 63 genes, 35 have been experimentally validated and reported in the literature, and 28 have not been experimentally tested and include previously hypothesized or novel predictions of essential metabolic capabilities. Without metabolomics data, four of the genes would have been incorrectly predicted to be non-essential. TFA also indicated that substrate channeling should exist in two metabolic pathways to ensure the thermodynamic feasibility of the flux. Finally, analysis of the metabolic capabilities of P. falciparum led to the identification of both the minimal nutritional requirements and the genes that can become indispensable upon substrate inaccessibility. This model provides novel insight into the metabolic needs and capabilities of the malaria parasite and highlights metabolites and pathways that should be measured and characterized to identify potential thermodynamic bottlenecks and substrate channeling. The hypotheses presented seek to guide experimental studies to facilitate a better understanding of the parasite metabolism and the identification of targets for more efficient intervention.

Cellular functions are largely regulated by reversible post-translational modifications of proteins which act as switches. Amongst these, S-palmitoylation is unique in that it confers hydrophobicity. Due to technical difficulties, the understanding of this modification has lagged behind. To investigate principles underlying dynamics and regulation of palmitoylation, we have here studied a key cellular protein, the ER chaperone calnexin, which requires dual palmitoylation for function. Apprehending the complex inter-conversion between single-, double- and non-palmitoylated species required combining experimental determination of kinetic parameters with extensive mathematical modelling. We found that calnexin, due to the presence of two cooperative sites, becomes stably acylated, which not only confers function but also a remarkable increase in stability. Unexpectedly, stochastic simulations revealed that palmitoylation does not occur soon after synthesis, but many hours later. This prediction guided us to find that phosphorylation actively delays calnexin palmitoylation in resting cells. Altogether this study reveals that cells synthesize 5 times more calnexin than needed under resting condition, most of which is degraded. This unused pool can be mobilized by preventing phosphorylation or increasing the activity of the palmitoyltransferase DHHC6.

Toxoplasma gondii is a human pathogen prevalent worldwide that poses a challenging and unmet need for novel treatment of toxoplasmosis. Using a semi-automated reconstruction algorithm, we reconstructed a genome-scale metabolic model, ToxoNet1. The reconstruction process and flux-balance analysis of the model offer a systematic overview of the metabolic capabilities of this parasite. Using ToxoNet1 we have identified significant gaps in the current knowledge of Toxoplasma metabolic pathways and have clarified its minimal nutritional requirements for replication. By probing the model via metabolic tasks, we have further defined sets of alternative precursors necessary for parasite growth. Within a human host cell environment, ToxoNet1 predicts a minimal set of 53 enzyme-coding genes and 76 reactions to be essential for parasite replication. Double-gene-essentiality analysis identified 20 pairs of genes for which simultaneous deletion is deleterious. To validate several predictions of ToxoNet1 we have performed experimental analyses of cytosolic acetyl-CoA biosynthesis. ATP-citrate lyase and acetyl-CoA synthase were localised and their corresponding genes disrupted, establishing that each of these enzymes is dispensable for the growth of T. gondii, however together they make a synthetic lethal pair.

Protein synthesis is a template polymerization process composed by three main steps: initiation, elongation, and termination. During translation, ribosomes are engaged into polysomes whose size is used for the quantitative characterization of translatome. However, simultaneous transcription and translation in the bacterial cytosol complicates the analysis of translatome data. We established a procedure for robust estimation of the ribosomal density in hundreds of genes from Lactococcus lactis polysome size measurements. We used a mechanistic model of translation to integrate the information about the ribosomal density and for the first time we estimated the protein synthesis rate for each gene and identified the rate limiting steps. Contrary to conventional considerations, we find significant number of genes to be elongation limited. This number increases during stress conditions compared to optimal growth and proteins synthesized at maximum rate are predominantly elongation limited. Consistent with bacterial physiology, we found proteins with similar rate and control characteristics belonging to the same functional categories. Under stress conditions, we found that synthesis rate of regulatory proteins is becoming comparable to proteins favored under optimal growth. These findings suggest that the coupling of metabolic states and protein synthesis is more important than previously thought.

Time-delays are common in many physical and biological systems and they give rise to complex dynamic phenomena. The elementary processes involved in template biopolymerization, such as mRNA and protein synthesis, introduce significant time delays. However, there is not currently a systematic mapping between the individual mechanistic parameters and the time delays in these networks. We present here the development of mathematical, time-delay models for protein translation, based on PDE models, which in turn are derived through systematic approximations of first-principles mechanistic models. Theoretical analysis suggests that the key features that determine the time-delays and the agreement between the time-delay and the mechanistic models are ribosome density and distribution, i.e., the number of ribosomes on the mRNA chain relative to their maximum and their distribution along the mRNA chain. Based on analytical considerations and on computational studies, we show that the steady-state and dynamic responses of the time-delay models are in excellent agreement with the detailed mechanistic models, under physiological conditions that correspond to uniform ribosome distribution and for ribosome density up to 70%. The methodology presented here can be used for the development of reduced time-delay models of mRNA synthesis and large genetic networks. The good agreement between the time-delay and the mechanistic models will allow us to use the reduced model and advanced computational methods from nonlinear dynamics in order to perform studies that are not practical using the large-scale mechanistic models.

BACKGROUND: Proteins are the primary regulatory agents of transcription even though mRNA expression data alone, from systems like DNA microarrays, are widely used. In addition, the regulation process in genetic systems is inherently non-linear in nature, and most studies employ a time-course analysis of mRNA expression. These considerations should be taken into account in the development of methods for the inference of regulatory interactions in genetic networks. RESULTS: We use an S-system based model for the transcription and translation process. We propose an optimization-based regulatory network inference approach that uses time-varying data from DNA microarray analysis. Currently, this seems to be the only model-based method that can be used for the analysis of time-course "relative" expressions (expression ratios). We perform an analysis of the dynamic behavior of the system when the number of experimental samples available is varied, when there are different levels of noise in the data and when there are genes that are not considered by the experimenter. Our studies show that the principal factor affecting the ability of a method to infer interactions correctly is the similarity in the time profiles of some or all the genes. The less similar the profiles are to each other the easier it is to infer the interactions. We propose a heuristic method for resolving networks and show that it displays reasonable performance on a synthetic network. Finally, we validate our approach using real experimental data for a chosen subset of genes involved in the sporulation cascade of Bacillus anthracis. We show that the method captures most of the important known interactions between the chosen genes. CONCLUSION: The performance of any inference method for regulatory interactions between genes depends on the noise in the data, the existence of unknown genes affecting the network genes, and the similarity in the time profiles of some or all genes. Though subject to these issues, the inference method proposed in this paper would be useful because of its ability to infer important interactions, the fact that it can be used with time-course DNA microarray data and because it is based on a non-linear model of the process that explicitly accounts for the regulatory role of proteins.

We demonstrate via simulation that hybridization of DNA molecules can be used as a similarity criterion for retrieving digital signals encoded and stored in a synthesized DNA database. After introducing some necessary DNA terminology, we briefly explain how digital signals are transformed to DNA sequences. Since retrieval is achieved through hybridization of query and data carrying DNA molecules, we present a mathematical model to estimate hybridization efficiency (also known as selectivity annealing). We show that selectivity annealing is inversely proportional to the mean squared error (MSE) of the encoded signal values. In addition, we show that the concentration of the molecules plays the same role as the decision threshold employed in digital signal matching algorithms. Finally, similar to the digital domain, we define a DNA signal-to-noise ratio (SNR) measure to assess the performance of the DNA-based retrieval scheme. Simulations are presented to validate our arguments

MOTIVATION: Metabolism, the network of chemical reactions that make life possible, is one of the most complex processes in nature. We describe here the development of a computational approach for the identification of every possible biochemical reaction from a given set of enzyme reaction rules that allows the de novo synthesis of metabolic pathways composed of these reactions, and the evaluation of these novel pathways with respect to their thermodynamic properties. RESULTS: We applied this framework to the analysis of the aromatic amino acid pathways and discovered almost 75,000 novel biochemical routes from chorismate to phenylalanine, more than 350,000 from chorismate to tyrosine, but only 13 from chorismate to tryptophan. Thermodynamic analysis of these pathways suggests that the native pathways are thermodynamically more favorable than the alternative possible pathways. The pathways generated involve compounds that exist in biological databases, as well as compounds that exist in chemical databases and novel compounds, suggesting novel biochemical routes for these compounds and the existence of biochemical compounds that remain to be discovered or synthesized through enzyme and pathway engineering. AVAILABILITY: Framework will be available via web interface at http://systemsbiology.northwestern.edu/BNICE (site under construction). CONTACT: [email protected] or [email protected] SUPPLEMENTARY INFORMATION: http://systemsbiology.northwestern.edu/BNICE/publications.

MOTIVATION: Identification of the regulatory structures in genetic networks and the formulation of mechanistic models in the form of wiring diagrams is one of the significant objectives of expression profiling using DNA microarray technologies and it requires the development and application of identification frameworks. RESULTS: We have developed a novel optimization framework for identifying regulation in a genetic network using the S-system modeling formalism. We show that balance equations on both mRNA and protein species led to a formulation suitable for analyzing DNA-microarray data whereby protein concentrations have been eliminated and only mRNA relative concentrations are retained. Using this formulation, we examined if it is possible to infer a set of possible genetic regulatory networks consistent with observed mRNA expression patterns. Two origins of changes in mRNA expression patterns were considered. One derives from changes in the biophysical properties of the system that alter the molecular-interaction kinetics and/or message stability. The second is due to gene knock-outs. We reduced the identification problem to an optimization problem (of the so-called mixed-integer non-linear programming class) and we developed an algorithmic procedure for solving this optimization problem. Using simulated data generated by our mathematical model, we show that our method can actually find the regulatory network from which the data were generated. We also show that the number of possible alternate genetic regulatory networks depends on the size of the dataset (i.e. number of experiments), but this dependence is different for each of the two types of problems considered, and that a unique solution requires fewer datasets than previously estimated in the literature. This is the first method that also allows the identification of every possible regulatory network that could explain the data, when the number of experiments does not allow identification of unique regulatory structure.