Logic programming-based Minimal Cut Sets reveal consortium-level therapeutic targets for chronic wound infections.

Minimal Cut Sets (MCSs) identify sets of reactions which, when removed from a metabolic network, disable certain cellular functions. The traditional search for MCSs within genome-scale metabolic models (GSMMs) targets cellular growth, identifies reaction sets resulting in a lethal phenotype if disrupted, and retrieves a list of corresponding gene, mRNA, or enzyme targets. Using the dual link between MCSs and Elementary Flux Modes (EFMs), our logic programming-based tool aspefm was able to compute MCSs of any size from GSMMs in acceptable run times. The tool demonstrated better performance when computing large-sized MCSs than the mixed-integer linear programming methods. We applied the new MCSs methodology to a medically-relevant consortium model of two cross-feeding bacteria, Staphylococcus aureus and Pseudomonas aeruginosa. aspefm constraints were used to bias the computation of MCSs toward exchanged metabolites that could complement lethal phenotypes in individual species. We found that interspecies metabolite exchanges could play an essential role in rescuing single-species growth, for instance inosine could complement lethal reaction knock-outs in the purine synthesis, glycolysis, and pentose phosphate pathways of both bacteria. Finally, MCSs were used to derive a list of promising enzyme targets for consortium-level therapeutic applications that cannot be circumvented via interspecies metabolite exchange.

Fine-tuning mitochondrial activity in Yarrowia lipolytica for citrate overproduction.

Yarrowia lipolytica is a non-conventional yeast with promising industrial potentials for lipids and citrate production. It is also widely used for studying mitochondrial respiration due to a respiratory chain like those of mammalian cells. In this study we used a genome-scale model (GEM) of Y. lipolytica metabolism and performed a dynamic Flux Balance Analysis (dFBA) algorithm to analyze and identify metabolic levers associated with citrate optimization. Analysis of fluxes at stationary growth phase showed that carbon flux derived from glucose is rewired to citric acid production and lipid accumulation, whereas the oxidative phosphorylation (OxPhos) shifted to the alternative respiration mode through alternative oxidase (AOX) protein. Simulations of optimized citrate secretion flux resulted in a pronounced lipid oxidation along with reactive oxygen species (ROS) generation and AOX flux inhibition. Then, we experimentally challenged AOX inhibition by adding n-Propyl Gallate (nPG), a specific AOX inhibitor, on Y. lipolytica batch cultures at stationary phase. Our results showed a twofold overproduction of citrate (20.5 g/L) when nPG is added compared to 10.9 g/L under control condition (no nPG addition). These results suggest that ROS management, especially through AOX activity, has a pivotal role on citrate/lipid flux balance in Y. lipolytica. All taken together, we thus provide for the first time, a key for the understanding of a predominant metabolic mechanism favoring citrate overproduction in Y. lipolytica at the expense of lipids accumulation.

Cancer and Alzheimer's disease: intracellular pH scales the metabolic disorders.

Alzheimer's disease (AD) and cancer have much in common than previously recognized. These pathologies share common risk factors (inflammation and aging), with similar epidemiological and biochemical features such as impaired mitochondria. Metabolic reprogramming occurs during aging and inflammation. We assume that inflammation is directly responsible of the Warburg effect in cancer cells, with a decreased oxidative phosphorylation and a compensatory highthroughput glycolysis (HTG). Similarly, the Warburg effect in cancer is thought to support an alkaline intracellular pH (pHi), a key component of unrelenting cell growth. In the brain, inflammation results in increased secretion of lactate by astrocytes. The increased uptake of lactic acid by neurons results in the inverse Warburg effect, such as seen in AD. The neuronal activity is dampened by a fall of pHi. Pronounced cytosol acidification results in decreased mitochondrial energy yield as well as apoptotic cell death. The link between AD and cancer is reinforced by the fact that treatment aiming at restoring the mitochondrial activity have been experimentally shown to be effective in both diseases. Low carb diet, lipoic acid, and/or methylene blue could then appear promising in both sets of these clinically diverse diseases.

Thermodynamic Approaches in Flux Analysis.

Networks of reactions inside the cell are constrained by the laws of mass and energy balance. Constrained-based modelling (CBM) is the most used method to describe the mass balance of metabolic network. The main key concepts in CBM are stoichiometric analysis such as elementary flux mode analysis or flux balance analysis. Some of these methods have focused on adding thermodynamics constraints to eliminate non-physical fluxes or inconsistencies in the metabolic system. Here, we review the main different approaches and how they tackle the different class of problems.

A kinetic metabolic study of lipid production in Chlorella protothecoides under heterotrophic condition.

BACKGROUND: Microalgae have been proposed as potential platform to produce lipid-derived products, such as biofuels. Knowledge on the intracellular carbon flow distribution may identify key metabolic processes during lipid synthesis thus refining culture/genetic strategies to maximize cell lipid productivity. A kinetic metabolic model simulating cell metabolic behavior and lipid production was first applied in the microalgae platform Chlorella protothecoides under heterotrophic condition. It combines both physiology and flux information in a kinetic approach. Cell nutrition, growth, lipid production and almost 30 metabolic intermediates covering central carbon metabolism were included and simulated. RESULTS: Model simulations were shown to adequately agree with experimental data, which is suggesting that the proposed model copes with Chlorella protothecoides cells' biology. The dynamic metabolic flux analysis using the model showed a reversible starch flux from accumulation to decomposing when glucose reached depletion, while net lipid flux shows a quasi-constant rate. The sensitive flux parameters on starch and lipid metabolism suggested that starch synthesis is the major competing pathway that affects lipid accumulation in C. protothecoides. Flux analysis also demonstrated that high lipid yield under heterotrophic condition is accompanied with high lipid flux and low TCA activity. Meanwhile, the dynamic flux distribution also suggests a relatively constant ratio of glucose distributed to biomass, lipid, starch, nucleotides as well as pentose phosphate pathway. CONCLUSION: The model described not only experimental data, but also unraveled intracellular carbon flow distribution and identify key metabolic processes during lipid synthesis. Most of the metabolic kinetics also showed statistical significance for metabolic mechanism. Therefore, this study unravels the mechanisms of the glucose impact on the dynamic carbon flux distribution, thus improving our understanding of the links between carbon fluxes and lipid metabolism in C. protothecoides.

Metabolic therapies inhibit tumor growth in vivo and in silico.

In the recent years, cancer research succeeded with sensitive detection methods, targeted drug delivery systems, and the identification of a large set of genes differently expressed. However, although most therapies are still based on antimitotic agents, which are causing wide secondary effects, there is an increasing interest for metabolic therapies that can minimize side effects. In the early 20th century, Otto Warburg revealed that cancer cells rely on the cytoplasmic fermentation of glucose to lactic acid for energy synthesis (called "Warburg effect"). Our investigations aim to reverse this effect in reprogramming cancer cells' metabolism. In this work, we present a metabolic therapy specifically targeting the activity of specific enzymes of central carbon metabolism, combining the METABLOC bi-therapeutic drugs combination (Alpha Lipoic Acid and Hydroxycitrate) to Metformin and Diclofenac, for treating tumors implanted in mice. Furthermore, a dynamic metabolic model describing central carbon metabolism as well as fluxes targeted by the drugs allowed to simulate tumors progression in both treated and non-treated mice, in addition to draw hypotheses on the effects of the drugs on tumor cells metabolism. Our model predicts metabolic therapies-induced reversed Warburg effect on tumor cells.

Thermodynamic constraints for identifying elementary flux modes.

Metabolic pathway analysis is a key method to study metabolism and the elementary flux modes (EFMs) is one major concept allowing one to analyze the network in terms of minimal pathways. Their practical use has been hampered by the combinatorial explosion of their number in large systems. The EFMs give the possible pathways at steady state, but the real pathways are limited by biological constraints. In this review, we display three different methods that integrate thermodynamic constraints in terms of Gibbs free energy in the EFMs computation.

Identifying Biomarkers of Wharton's Jelly Mesenchymal Stromal Cells Using a Dynamic Metabolic Model: The Cell Passage Effect.

Because of their unique ability to modulate the immune system, mesenchymal stromal cells (MSCs) are widely studied to develop cell therapies for detrimental immune and inflammatory disorders. However, controlling the final cell phenotype and determining immunosuppressive function following cell amplification in vitro often requires prolonged cell culture assays, all of which contribute to major bottlenecks, limiting the clinical emergence of cell therapies. For instance, the multipotent Wharton's Jelly mesenchymal stem/stromal cells (WJMSC), extracted from human umbilical cord, exhibit immunosuppressive traits under pro-inflammatory conditions, in the presence of interferon-γ (IFNγ), and tumor necrosis factor-α (TNFα). However, WJMSCs require co-culture bioassays with immune cells, which can take days, to confirm their immunomodulatory function. Therefore, the establishment of robust cell therapies would benefit from fast and reliable characterization assays. To this end, we have explored the metabolic behaviour of WJMSCs in in vitro culture, to identify biomarkers that are specific to the cell passage effect and the loss of their immunosuppressive phenotype. We clearly show distinct metabolic behaviours comparing WJMSCs at the fourth (P4) and the late ninth (P9) passages, although both P4 and P9 cells do not exhibit significant differences in their low immunosuppressive capacity. Metabolomics data were analysed using an in silico modelling platform specifically adapted to WJMSCs. Of interest, P4 cells exhibit a glycolytic metabolism compared to late passage (P9) cells, which show a phosphorylation oxidative metabolism, while P4 cells show a doubling time of 29 h representing almost half of that for P9 cells (46 h). We also clearly show that fourth passage WJMSCs still express known immunosuppressive biomarkers, although, this behaviour shows overlapping with a senescence phenotype.

A dynamic metabolic flux analysis of ABE (acetone-butanol-ethanol) fermentation by Clostridium acetobutylicum ATCC 824, with riboflavin as a by-product.

The present study reveals that supplementing sodium acetate (NaAc) strongly stimulates riboflavin production in acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum ATCC 824 with xylose as carbon source. Riboflavin production increased from undetectable concentrations to ∼0.2 g L-1 (0.53 mM) when supplementing 60 mM NaAc. Of interest, solvents production and biomass yield were also promoted with fivefold acetone, 2.6-fold butanol, and 2.4-fold biomass adding NaAc. A kinetic metabolic model, developed to simulate ABE biosystem, with riboflavin production, revealed from a dynamic metabolic flux analysis (dMFA) simultaneous increase of riboflavin (ribA) and GTP (precursor of riboflavin) (PurM) synthesis flux rates under NaAc supplementation. The model includes 23 fluxes, 24 metabolites, and 72 kinetic parameters. It also suggested that NaAc condition has first stimulated the accumulation of intracellular metabolite intermediates during the acidogenic phase, which have then fed the solventogenic phase leading to increased ABE production. In addition, NaAc resulted in higher intracellular levels of NADH during the whole culture. Moreover, lower GTP-to-adenosine phosphates (ATP, ADP, AMP) ratio under NaAc supplemented condition suggests that GTP may have a minor role in the cell energetic metabolism compared to its contribution to riboflavin synthesis.

How important is thermodynamics for identifying elementary flux modes?

We present a method for computing thermodynamically feasible elementary flux modes (tEFMs) using equilibrium constants without need of internal metabolite concentrations. The method is compared with the method based on a binary distinction between reversible and irreversible reactions. When all reactions are reversible, adding the constraints based on equilibrium constants reduces the number of elementary flux modes (EFMs) by a factor of two. Declaring in advance some reactions as irreversible, based on reliable biochemical expertise, can in general reduce the number of EFMs by a greater factor. But, even in this case, computing tEFMs can rule out some EFMs which are biochemically irrelevant. We applied our method to two published models described with binary distinction: the monosaccharide metabolism and the central carbon metabolism of Chinese hamster ovary cells. The results show that the binary distinction is in good agreement with biochemical observations. Moreover, the suppression of the EFMs that are not consistent with the equilibrium constants appears to be biologically relevant.

The Redox Status of Cancer Cells Supports Mechanisms behind the Warburg Effect.

To better understand the energetic status of proliferating cells, we have measured the intracellular pH (pHi) and concentrations of key metabolites, such as adenosine triphosphate (ATP), nicotinamide adenine dinucleotide (NAD), and nicotinamide adenine dinucleotide phosphate (NADP) in normal and cancer cells, extracted from fresh human colon tissues. Cells were sorted by elutriation and segregated in different phases of the cell cycle (G0/G1/S/G2/M) in order to study their redox (NAD, NADP) and bioenergetic (ATP, pHi) status. Our results show that the average ATP concentration over the cell cycle is higher and the pHi is globally more acidic in normal proliferating cells. The NAD+/NADH and NADP+/NADPH redox ratios are, respectively, five times and ten times higher in cancer cells compared to the normal cell population. These energetic differences in normal and cancer cells may explain the well-described mechanisms behind the Warburg effect. Oscillations in ATP concentration, pHi, NAD+/NADH, and NADP+/NADPH ratios over one cell cycle are reported and the hypothesis addressed. We also investigated the mitochondrial membrane potential (MMP) of human and mice normal and cancer cell lines. A drastic decrease of the MMP is reported in cancer cell lines compared to their normal counterparts. Altogether, these results strongly support the high throughput aerobic glycolysis, or Warburg effect, observed in cancer cells.

Cell cycle progression is regulated by intertwined redox oscillators.

The different phases of the eukaryotic cell cycle are exceptionally well-preserved phenomena. DNA decompaction, RNA and protein synthesis (in late G1 phase) followed by DNA replication (in S phase) and lipid synthesis (in G2 phase) occur after resting cells (in G0) are committed to proliferate. The G1 phase of the cell cycle is characterized by an increase in the glycolytic metabolism, sustained by high NAD+/NADH ratio. A transient cytosolic acidification occurs, probably due to lactic acid synthesis or ATP hydrolysis, followed by cytosolic alkalinization. A hyperpolarized transmembrane potential is also observed, as result of sodium/potassium pump (NaK-ATPase) activity. During progression of the cell cycle, the Pentose Phosphate Pathway (PPP) is activated by increased NADP+/NADPH ratio, converting glucose 6-phosphate to nucleotide precursors. Then, nucleic acid synthesis and DNA replication occur in S phase. Along with S phase, unpublished results show a cytosolic acidification, probably the result of glutaminolysis occurring during this phase. In G2 phase there is a decrease in NADPH concentration (used for membrane lipid synthesis) and a cytoplasmic alkalinization occurs. Mitochondria hyperfusion matches the cytosolic acidification at late G1/S transition and then triggers ATP synthesis by oxidative phosphorylation. We hypothesize here that the cytosolic pH may coordinate mitochondrial activity and thus the different redox cycles, which in turn control the cell metabolism.

Elementary flux modes analysis of functional domain networks allows a better metabolic pathway interpretation.

Metabolic network analysis is an important step for the functional understanding of biological systems. In these networks, enzymes are made of one or more functional domains often involved in different catalytic activities. Elementary flux mode (EFM) analysis is a method of choice for the topological studies of these enzymatic networks. In this article, we propose to use an EFM approach on networks that encompass available knowledge on structure-function. We introduce a new method that allows to represent the metabolic networks as functional domain networks and provides an application of the algorithm for computing elementary flux modes to analyse them. Any EFM that can be represented using the classical representation can be represented using our functional domain network representation but the fine-grained feature of functional domain networks allows to highlight new connections in EFMs. This methodology is applied to the tricarboxylic acid cycle (TCA cycle) of Bacillus subtilis, and compared to the classical analyses. This new method of analysis of the functional domain network reveals that a specific inhibition on the second domain of the lipoamide dehydrogenase (pdhD) component of pyruvate dehydrogenase complex leads to the loss of all fluxes. Such conclusion was not predictable in the classical approach.

Global network reorganization during dynamic adaptations of Bacillus subtilis metabolism.

Adaptation of cells to environmental changes requires dynamic interactions between metabolic and regulatory networks, but studies typically address only one or a few layers of regulation. For nutritional shifts between two preferred carbon sources of Bacillus subtilis, we combined statistical and model-based data analyses of dynamic transcript, protein, and metabolite abundances and promoter activities. Adaptation to malate was rapid and primarily controlled posttranscriptionally compared with the slow, mainly transcriptionally controlled adaptation to glucose that entailed nearly half of the known transcription regulation network. Interactions across multiple levels of regulation were involved in adaptive changes that could also be achieved by controlling single genes. Our analysis suggests that global trade-offs and evolutionary constraints provide incentives to favor complex control programs.

Analysis of the variability of human normal urine by 2D-GE reveals a "public" and a "private" proteome.

The characterization of the normal urinary proteome is steadily progressing and represents a major interest in the assessment of clinical urinary biomarkers. To estimate quantitatively the variability of the normal urinary proteome, urines of 20 healthy people were collected. We first evaluated the impact of the sample conservation temperature on urine proteome integrity. Keeping the urine sample at RT or at +4°C until storage at -80°C seems the best way for long-term storage of samples for 2D-GE analysis. The quantitative variability of the normal urinary proteome was estimated on the 20 urines mapped by 2D-GE. The occurrence of the 910 identified spots was analysed throughout the gels and represented in a virtual 2D gel. Sixteen percent of the spots were found to occur in all samples and 23% occurred in at least 90% of urines. About 13% of the protein spots were present only in 10% or less of the samples, thus representing the most variable part of the normal urinary proteome. Twenty proteins corresponding to a fraction of the fully conserved spots were identified by mass spectrometry. In conclusion, a "public" urinary proteome, common to healthy individuals, seems to coexist with a "private" urinary proteome, which is more specific to each individual.

BioNetCAD: design, simulation and experimental validation of synthetic biochemical networks.

MOTIVATION: Synthetic biology studies how to design and construct biological systems with functions that do not exist in nature. Biochemical networks, although easier to control, have been used less frequently than genetic networks as a base to build a synthetic system. To date, no clear engineering principles exist to design such cell-free biochemical networks. RESULTS: We describe a methodology for the construction of synthetic biochemical networks based on three main steps: design, simulation and experimental validation. We developed BioNetCAD to help users to go through these steps. BioNetCAD allows designing abstract networks that can be implemented thanks to CompuBioTicDB, a database of parts for synthetic biology. BioNetCAD enables also simulations with the HSim software and the classical Ordinary Differential Equations (ODE). We demonstrate with a case study that BioNetCAD can rationalize and reduce further experimental validation during the construction of a biochemical network. AVAILABILITY AND IMPLEMENTATION: BioNetCAD is freely available at http://www.sysdiag.cnrs.fr/BioNetCAD. It is implemented in Java and supported on MS Windows. CompuBioTicDB is freely accessible at http://compubiotic.sysdiag.cnrs.fr/.

Computing biological functions using BioPsi, a formal description of biological processes based on elementary bricks of actions.

MOTIVATION: In the available databases, biological processes are described from molecular and cellular points of view, but these descriptions are represented with text annotations that make it difficult to handle them for computation. Consequently, there is an obvious need for formal descriptions of biological processes. RESULTS: We present a formalism that uses the BioPsi concepts to model biological processes from molecular details to networks. This computational approach, based on elementary bricks of actions, allows us to calculate on biological functions (e.g. process comparison, mapping structure-function relationships, etc.). We illustrate its application with two examples: the functional comparison of proteases and the functional description of the glycolysis network. This computational approach is compatible with detailed biological knowledge and can be applied to different kinds of systems of simulation. AVAILABILITY: www.sysdiag.cnrs.fr/publications/supplementary-materials/BioPsi_Manager/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A new method for 2D gel spot alignment: application to the analysis of large sample sets in clinical proteomics.

BACKGROUND: In current comparative proteomics studies, the large number of images generated by 2D gels is currently compared using spot matching algorithms. Unfortunately, differences in gel migration and sample variability make efficient spot alignment very difficult to obtain, and, as consequence most of the software alignments return noisy gel matching which needs to be manually adjusted by the user. RESULTS: We present Sili2DGel an algorithm for automatic spot alignment that uses data from recursive gel matching and returns meaningful Spot Alignment Positions (SAP) for a given set of gels. In the algorithm, the data are represented by a graph and SAP by specific subgraphs. The results are returned under various forms (clickable synthetic gel, text file, etc.). We have applied Sili2DGel to study the variability of the urinary proteome from 20 healthy subjects. CONCLUSION: Sili2DGel performs noiseless automatic spot alignment for variability studies (as well as classical differential expression studies) of biological samples. It is very useful for typical clinical proteomic studies with large number of experiments.