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Abstract Background The behaviour of biological systems can be deduced from their mathematical models. However, multiple sources of data in diverse forms are required in the construction of a model in order to define its components and their biochemical reactions, and corresponding parameters. Automating the assembly and use of systems biology models is dependent upon data integration processes involving the interoperation of data and analytical resources. Results Taverna workflows have been developed for the automated assembly of quantitative parameterised metabolic networks in the Systems Biology Markup Language (SBML). A SBML model is built in a systematic fashion by the workflows which starts with the construction of a qualitative network using data from a MIRIAM-compliant genome-scale model of yeast metabolism. This is followed by parameterisation of the SBML model with experimental data from two repositories, the SABIO-RK enzyme kinetics database and a database of quantitative experimental results. The models are then calibrated and simulated in workflows that call out to COPASIWS, the web service interface to the COPASI software application for analysing biochemical networks. These systems biology workflows were evaluated for their ability to construct a parameterised model of yeast glycolysis. Conclusions Distributed information about metabolic reactions that have been described to MIRIAM standards enables the automated assembly of quantitative systems biology models of metabolic networks based on user-defined criteria. Such data integration processes can be implemented as Taverna workflows to provide a rapid overview of the components and their relationships within a biochemical system.

Introduction: Cleft palate is among the most common birth abnormalities. The success of primary surgery in the early months of life is crucial for successful feeding, speech, hearing, dental development and facial growth. Over recent decades, age at palatal surgery in infancy has reduced. This has led to palatal closure in one-stage procedures being carried out around the age of 12 months, but in some cases as early as 6 months. The primary objective of the Timing Of Primary Surgery for Cleft Palate (TOPS)trial is to determine whether surgery for cleft palate performed at 6 or 12 months of age is most beneficial for speech outcomes., Methods and Analysis: Infants with a diagnosis of non-syndromic isolated cleft palate will be randomised to receive standardised primary surgery (Sommerlad technique) for closure of the cleft at either 6 months or 12 months, corrected for gestational age. The primary outcome will be perceived insufficient velopharyngeal function at 5 years of age. Secondary outcomes measured across 12 months, 3 years and 5 years will include growth, safety of the procedure, dentofacial development, speech, hearing level and middle ear function. Video and audio recordings of speech will be collected in a standardised age-appropriate manner and analysed independently by multiple speech and language therapists. The trial aims to recruit and follow-up 300 participants per arm. Data will be analysed according to the intention-to-treat principle using a 5% significance level. All analyses will be prespecified within a full and detailed statistical analysis plan., Ethics and Dissemination: Ethical approval has been sought in each participating country according to country-specific procedures. Trial results will be presented at conferences, published in peer-reviewed journals and disseminated through relevant patient support groups., Trial Registration Number: NCT00993551; Pre-results., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

We present an experimental and computational pipeline for the generation of kinetic models of metabolism, and demonstrate its application to glycolysis in Saccharomyces cerevisiae. Starting from an approximate mathematical model, we employ a "cycle of knowledge" strategy, identifying the steps with most control over flux. Kinetic parameters of the individual isoenzymes within these steps are measured experimentally under a standardised set of conditions. Experimental strategies are applied to establish a set of in vivo concentrations for isoenzymes and metabolites. The data are integrated into a mathematical model that is used to predict a new set of metabolite concentrations and reevaluate the control properties of the system. This bottom-up modelling study reveals that control over the metabolic network most directly involved in yeast glycolysis is more widely distributed than previously thought., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

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

Background: The behaviour of biological systems can be deduced from their mathematical models. However, multiple sources of data in diverse forms are required in the construction of a model in order to define its components and their biochemical reactions, and corresponding parameters. Automating the assembly and use of systems biology models is dependent upon data integration processes involving the interoperation of data and analytical resources., Results: Taverna workflows have been developed for the automated assembly of quantitative parameterised metabolic networks in the Systems Biology Markup Language (SBML). A SBML model is built in a systematic fashion by the workflows which starts with the construction of a qualitative network using data from a MIRIAM-compliant genome-scale model of yeast metabolism. This is followed by parameterisation of the SBML model with experimental data from two repositories, the SABIO-RK enzyme kinetics database and a database of quantitative experimental results. The models are then calibrated and simulated in workflows that call out to COPASIWS, the web service interface to the COPASI software application for analysing biochemical networks. These systems biology workflows were evaluated for their ability to construct a parameterised model of yeast glycolysis., Conclusions: Distributed information about metabolic reactions that have been described to MIRIAM standards enables the automated assembly of quantitative systems biology models of metabolic networks based on user-defined criteria. Such data integration processes can be implemented as Taverna workflows to provide a rapid overview of the components and their relationships within a biochemical system.

NOD2, a cytosolic receptor for the bacterial proteoglycan fragment muramyl dipeptide (MDP), plays an important role in the recognition of intracellular pathogens. Variants in the bacterial sensor domain of NOD2 are genetically associated with an increased risk for the development of Crohn disease, a human chronic inflammatory bowel disease. In the present study, global protein expression changes after MDP stimulation were analyzed by two-dimensional PAGE of total protein extracts of human cultured cells stably transfected with expression constructs encoding for wild type NOD2 (NOD2(WT)) or the disease-associated NOD2 L1007fsinsC (NOD2(SNP13)) variant. Differentially regulated proteins were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) peptide mass fingerprinting and MALDI MS/MS. The limited overlap in the responses of the NOD2-overexpressing cell lines to MDP included a down-regulation of heat shock 70-kDa protein 4. A complex pro-inflammatory program regulated by NOD2(WT) that encompasses a regulation of key genes involved in protein folding, DNA repair, cellular redox homeostasis, and metabolism was observed both under normal growth conditions and after stimulation with MDP. By using the comparison of NOD2(WT) and disease-associated NOD2(SNP13) variant, we have identified a proteomic signature pattern that may further our understanding of the influence of genetic variations in the NOD2 gene in the pathophysiology of chronic inflammatory bowel disease.

The Escherichia coli starvation-induced DNA protection protein Dps was observed to be degraded rapidly during exponential growth. This turnover is dependent on the clpP and clpX genes. The clpA gene is not required for Dps proteolysis, suggesting that Dps is a substrate for ClpXP protease but not for ClpAP protease. Dps proteolysis was found to be highly regulated. Upon carbon starvation, Dps is stabilized, which together with increased Dps synthesis allows strong accumulation of Dps in the stationary phase. The addition of glucose to starving cells results in rapid resumption of Dps proteolysis by ClpXP. Oxidative stress also leads to efficient stabilization of Dps. After hyperosmotic shift, however, proteolysis remains unaffected. Thus, regulated proteolysis of Dps strongly contributes to controlling Dps levels under very specific stress conditions. In contrast to the regulated degradation of RpoS by ClpXP, Dps proteolysis is independent of the recognition factor RssB. In addition, during starvation, clpP and, to a somewhat lesser extent, clpA are involved in maintaining ongoing Dps synthesis (acting at the level of Dps translation), which is required for strong Dps accumulation in long-term stationary phase cells. In summary, both ClpXP and ClpAP exert significant control of Dps levels by affecting log phase stability and stationary phase synthesis of Dps respectively.

To elucidate the involvement of proteolysis in the regulation of stationary-phase adaptation, the clpA, clpX, and clpP protease mutants of Escherichia coli were subjected to proteome analysis during growth and during carbon starvation. For most of the growth-phase-regulated proteins detected on our gels, the clpA, clpX, or clpP mutant failed to mount the growth-phase regulation found in the wild type. For example, in the clpP and clpA mutant cultures, the Dps protein, the WrbA protein, and the periplasmic lysine-arginine-ornithine binding protein ArgT did not display the induction typical for late-stationary-phase wild-type cells. On the other hand, in the protease mutants, a number of proteins accumulated to a higher degree than in the wild type, especially in late stationary phase. The proteins affected in this manner include the LeuA, TrxB, GdhA, GlnA, and MetK proteins and alkyl hydroperoxide reductase (AhpC). These proteins may be directly degraded by ClpAP or ClpXP, respectively, or their expression could be modulated by a protease-dependent mechanism. From our data we conclude that the levels of most major growth-phase-regulated proteins in E. coli are at some point controlled by the activity of at least one of the ClpP, ClpA, and ClpX proteins. Cultures of the strains lacking functional ClpP or ClpX also displayed a more rapid loss of viability during extended stationary phase than the wild type. Therefore, regulation by proteolysis seems to be more important, especially in resting cells, than previously suspected.

The general stress-induced sigma subunit sigma s of Escherichia coli RNA polymerase is closely related to the vegetative sigma factor sigma 70. In view of their very similar promoter specificity in vitro, it is unclear how sigma factor selectivity in the expression of sigma s-dependent genes is generated in vivo. The csiD gene is such a strongly sigma s-dependent gene. In contrast to sigma s, which is induced in response to many different stresses, csiD, whose expression is driven from a single promoter, is induced by carbon starvation only. To our knowledge, the csiD promoter is the first characterized promoter which is not only exclusively dependent on sigma s-containing RNA polymerase (E sigma s), but also requires an activator, cAMP-CRP. In addition, leucine-responsive regulatory protein (Lrp) acts as a positive modulator of csiD expression. Also in vitro, E sigma s is more efficient than E sigma 70 in csiD promoter binding, open complex formation and run-off transcription, which might be due to the poor match of the csiD -35 region to the sigma 70 consensus and to transcription by E sigma s being less dependent on contacts in this region. By DNase I protection experiments, a cAMP-CRP binding site centered at -68.5 nucleotides upstream of the csiD transcriptional start site was identified. While cAMP-CRP stimulates E sigma 70 binding, it does not promote open complex formation by E sigma 70, but does so in conjunction with E sigma s. With linear templates, cAMP-CRP significantly stimulates E sigma s-mediated in vitro transcription, whereas transcription by E sigma 70 is negligible and hardly stimulated by cAMP-CRP. These findings may reflect different or less stringent positional requirements for an activator site for E sigma s than for E sigma 70, and indicate that cAMP-CRP contributes to sigma factor selectivity at the csiD promoter. In vitro transcription experiments with super-coiled templates, however, revealed significant cAMP-CRP-stimulated transcription also by E sigma 70. Yet, under these conditions, H-NS was found to restore E sigma s specificity by strongly interfering with cAMP-CRP/E sigma 70-dependent transcription. Lrp strongly and cooperatively binds to multiple sites located between positions -14 and -102 (in a way that suggests DNA wrapping around multiple Lrp molecules) and moderately stimulates in vitro transcription, especially with E sigma s. In summary, we conclude that the csiD promoter has an intrinsic preference for E sigma s, but that also protein factors such as cAMP-CRP, Lrp and probably H-NS as well as DNA conformation contribute to its strong E sigma s selectivity. Furthermore, this strong E sigma s preference in combination with a requirement for high concentrations of the essential activator cAMP-CRP ensures csiD expression under conditions of carbon starvation, but not other stress conditions.

In microbiology the terms 'viability' and 'culturability' are often equated. However, in recent years the apparently self-contradictory expression 'viable-but-nonculturable' ('VBNC') has been applied to cells with various and often poorly defined physiological attributes but which, nonetheless, could not be cultured by methods normally appropriate to the organism concerned. These attributes include apparent cell integrity, the possession of some form of measurable cellular activity and the apparent capacity to regain culturability. We review the evidence relating to putative VBNC cells and stress our view that most of the reports claiming a return to culturability have failed to exclude the regrowth of a limited number of cells which had never lost culturability. We argue that failure to differentiate clearly between use of the terms 'viability' and 'culturability' in an operational versus a conceptual sense is fuelling the current debate, and conclude with a number of proposals that are designed to help clarify the major issues involved. In particular, we suggest an alternative operational terminology that replaces 'VBNC' with expressions that are internally consistent.

The estuarine, human-pathogenic bacterium Vibrio vulnificus responds to low temperature by the formation of viable but nonculturable (VBNC) cells, while starvation at moderate temperatures allows for maintenance of culturability of this organism. Recovery of cold-incubated populations of V. vulnificus was restricted to the culturable fraction in slide cultures and most probable number assays. These populations, however, gave between 1.1- and 8-fold higher c.f.u. counts on soft agar plates than on ordinary agar plates, indicating that a small and variable fraction of the cell population was injured rather than nonculturable. Thus, the population of cold-incubated cells is composed of culturable, injured and nonculturable cells, with the numbers of the culturable and injured cells rapidly decreasing during cold incubation. Recovery of nonculturable cells of the organism, however, could not be obtained by any combination of temperature and nutrient shifts in any of the assays. VBNC cells of the organism were assessed with regard to their persistence and stress resistance in comparison to growing and starved cells. The sonication resistance of VBNC cells was initially similar to that of growing cells, but increased during prolonged cold incubation. The final resistance of cold-incubated VBNC cells was equal to the markedly increased resistance of starving cells, which also displayed increased resistance against exposure to ethanol and mechanical stress. Our results indicate that in spite of the apparent absence of recovery under a wide range of laboratory conditions, VBNC cells of V. vulnificus undergo changes at low temperature which potentially allow them to persist for extended periods.

During transition into stationary phase a large set of proteins is induced in Escherichia coli. Only a minority of the corresponding genes has been identified so far. Using the λplacMu system and a plate screen for carbon starvation-induced fusion activity, a series of chromosomal lacZ fusions (csi::lacZ) was isolated. In complex medium these fusions were induced either during late exponential phase or during entry into stationary phase. csi::lacZ expression in minimal media in response to starvation for carbon, nitrogen and phosphate sources and the roles of global regulators such as the alternative sigma factor sigma; S (encoded by rpoS), cAMP/CRP and the relA gene product were investigated. The results show that almost every fusion exhibits its own characteristic pattern of expression, suggesting a complex control of stationary phase-inducible genes that involves various combinations of regulatory mechanisms for different genes. All fusions were mapped to the E. coli chromosome. Using fine mapping by Southern hybridization, cloning, sequencing and/or phenotypic analysis, csi-5, csi-17, and csi-18 could be localized in osmY (encoding a periplasmic protein), glpD (aerobic glycerol-3-phosphate dehydrogenase) and glgA (glycogen synthase), respectively. The other fusions seem to specify novel genes now designated csiA through to csiF. csi-17(glpD)::lacZ was shown to produce its own glucose-starvation induction, thus illustrating the Intricacies of gene-fusion technology when applied to the study of gene regulation.

Non-differentiating bacteria adapt to starvation induced growth arrest by a complex turn-on/turn-off pattern of protein synthesis. This response shows distinct similarities with those of spore formation in differentiating organisms. A substantial amount of information on the non-growth biology of non-differentiating bacteria can be derived from studies on Vibrio strains. One important result is that carbon rather than nitrogen or phosphorus starvation leads to the development of a starvation and stress resistant cell in these organisms. Hence, we have attempted to characterize the carbon starvation stimulon. By the use of two-dimensional gel electrophoresis of pulse-labelled cells and transposon mutagenesis, using reporter gene constructs, the identity and function of some members of the carbon starvation stimulon have been elucidated. Moreover, regulatory genes of the starvation response have been identified with these techniques. Current studies primarily address the identity and function of these genes. The role of transcript modification and stability for both long term persistence during starvation as well as the efficient recovery of cells which occurs upon nutrient addition is also addressed. It is suggested that an understanding of the functionality of the translational machinery is essential for the understanding of these adaptive pathways. This contribution also discusses the diversity of the differentiation-like response to starvation in different bacteria and whether a general starvation induced programme exists.

Vibrio vulnificus cells progressively lose culturability during incubation at 5 degrees C. This process is accelerated by the addition of supernatants from non-culturable cells obtained by incubation at 5 degrees C for 17 days. Thus the organism apparently produces a factor upon cold incubation which is triggering or causing the decline in culturability. Reversing the temperature shift can restore a culturable population comparable in numbers to the original population, but this process is largely due to regrowth. A few cells retaining the ability to grow apparently utilize the substrates released by the moribund cells, thus mimicking resuscitation of the whole population.