Genome-Scale, Constraint-Based Modeling of Nitrogen Oxide Fluxes during Coculture of Nitrosomonas europaea and Nitrobacter winogradskyi.

Nitrification, the aerobic oxidation of ammonia to nitrate via nitrite, emits nitrogen (N) oxide gases (NO, NO2, and N2O), which are potentially hazardous compounds that contribute to global warming. To better understand the dynamics of nitrification-derived N oxide production, we conducted culturing experiments and used an integrative genome-scale, constraint-based approach to model N oxide gas sources and sinks during complete nitrification in an aerobic coculture of two model nitrifying bacteria, the ammonia-oxidizing bacterium Nitrosomonas europaea and the nitrite-oxidizing bacterium Nitrobacter winogradskyi. The model includes biotic genome-scale metabolic models (iFC578 and iFC579) for each nitrifier and abiotic N oxide reactions. Modeling suggested both biotic and abiotic reactions are important sources and sinks of N oxides, particularly under microaerobic conditions predicted to occur in coculture. In particular, integrative modeling suggested that previous models might have underestimated gross NO production during nitrification due to not taking into account its rapid oxidation in both aqueous and gas phases. The integrative model may be found at https://github.com/chaplenf/microBiome-v2.1. IMPORTANCE Modern agriculture is sustained by application of inorganic nitrogen (N) fertilizer in the form of ammonium (NH4+). Up to 60% of NH4+-based fertilizer can be lost through leaching of nitrifier-derived nitrate (NO3-), and through the emission of N oxide gases (i.e., nitric oxide [NO], N dioxide [NO2], and nitrous oxide [N2O] gases), the latter being a potent greenhouse gas. Our approach to modeling of nitrification suggests that both biotic and abiotic mechanisms function as important sources and sinks of N oxides during microaerobic conditions and that previous models might have underestimated gross NO production during nitrification.

Genome scale metabolic reconstruction of Chlorella variabilis for exploring its metabolic potential for biofuels.

A compartmentalized genome scale metabolic network was reconstructed for Chlorella variabilis to offer insight into various metabolic potentials from this alga. The model, iAJ526, was reconstructed with 1455 reactions, 1236 metabolites and 526 genes. 21% of the reactions were transport reactions and about 81% of the total reactions were associated with enzymes. Along with gap filling reactions, 2 major sub-pathways were added to the model, chitosan synthesis and rhamnose metabolism. The reconstructed model had reaction participation of 4.3 metabolites per reaction and average lethality fraction of 0.21. The model was effective in capturing the growth of C. variabilis under three light conditions (white, red and red+blue light) with fair agreement. This reconstructed metabolic network will serve an important role in systems biology for further exploration of metabolism for specific target metabolites and enable improved characteristics in the strain through metabolic engineering.

Effects of selected electron transport chain inhibitors on 24-h hydrogen production by Synechocystis sp. PCC 6803.

One factor limiting biosolar hydrogen (H(2)) production from cyanobacteria is electron availability to the hydrogenase enzyme. In order to optimize 24-h H(2) production this study used Response Surface Methodology and Q2, an optimization algorithm, to investigate the effects of five inhibitors of the photosynthetic and respiratory electron transport chains of Synechocystis sp. PCC 6803. Over 3 days of diurnal light/dark cycling, with the optimized combination of 9.4 mM KCN (3.1 µmol 10(10) cells(-1)) and 1.5 mM malonate (0.5 µmol 10(10) cells(-1)) the H(2) production was 30-fold higher, in EHB-1 media previously optimized for nitrogen (N), sulfur (S), and carbon (C) concentrations (Burrows et al., 2008). In addition, glycogen concentration was measured over 24 h with two light/dark cycling regimes in both standard BG-11 and EHB-1 media. The results suggest that electron flow as well as glycogen accumulation should be optimized in systems engineered for maximal H(2) output.

Optimization of pH and nitrogen for enhanced hydrogen production by Synechocystis sp. PCC 6803 via statistical and machine learning methods.

The nitrogen (N) concentration and pH of culture media were optimized for increased fermentative hydrogen (H(2)) production from the cyanobacterium, Synechocystis sp. PCC 6803. The optimization was conducted using two procedures, response surface methodology (RSM), which is commonly used, and a memory-based machine learning algorithm, Q2, which has not been used previously in biotechnology applications. Both RSM and Q2 were successful in predicting optimum conditions that yielded higher H(2) than the media reported by Burrows et al., Int J Hydrogen Energy. 2008;33:6092-6099 optimized for N, S, and C (called EHB-1 media hereafter), which itself yielded almost 150 times more H(2) than Synechocystis sp. PCC 6803 grown on sulfur-free BG-11 media. RSM predicted an optimum N concentration of 0.63 mM and pH of 7.77, which yielded 1.70 times more H(2) than EHB-1 media when normalized to chlorophyll concentration (0.68 +/- 0.43 micromol H(2) mg Chl(-1) h(-1)) and 1.35 times more when normalized to optical density (1.62 +/- 0.09 nmol H(2) OD(730) (-1) h(-1)). Q2 predicted an optimum of 0.36 mM N and pH of 7.88, which yielded 1.94 and 1.27 times more H(2) than EHB-1 media when normalized to chlorophyll concentration (0.77 +/- 0.44 micromol H(2) mg Chl(-1) h(-1)) and optical density (1.53 +/- 0.07 nmol H(2) OD(730) (-1) h(-1)), respectively. Both optimization methods have unique benefits and drawbacks that are identified and discussed in this study.

Signal transduction pathway modeling using sequences of chromatophore images.

Extensive research is being done in order to use chromatophore cells as biosensors for various substances. In this paper, a link between the biological aspect of chromatophores and digital image/video processing techniques used for chromatophore characterization is established for this purpose. A model of the Gs--AC--PKA--granule motion-image feature signal transduction pathway is proposed, starting from the concentration of the input ligand and ending in the pigment area extracted from a microscope image. The model extends an existing system biology differential equation based model of the Gs--AC--PKA transduction pathway obtained from the Database of Quantitative Cellular Signaling (DQCS). Examples are presented to demonstrate the effectiveness of the proposed system model.

Stimulation of insulin release by glyceraldehyde may not be similar to glucose.

Glyceraldehyde (GA) has been used to study insulin secretion for decades and it is widely assumed that beta-cell metabolism of GA after its phosphorylation by triokinase is similar to metabolism of glucose; that is metabolism through distal glycolysis and oxidation in mitochondria. New data supported by existing information indicate that this is true for only a small amount of GA's metabolism and also suggest why GA is toxic. GA is metabolized at 10-20% the rate of glucose in pancreatic islets, even though GA is a more potent insulin secretagogue. GA also inhibits glucose metabolism to CO2 out of proportion to its ability to replace glucose as a fuel. This study is the first to measure methylglyoxal (MG) in beta-cells and shows that GA causes large increases in MG in INS-1 cells and d-lactate in islets but MG does not mediate GA-induced insulin release. GA severely lowers NAD(P) and increases NAD(P)H in islets. High NADH combined with GA's metabolism to CO2 may initially hyperstimulate insulin release, but a low cytosolic NAD/NADH ratio will block glycolysis at glyceraldehyde phosphate (GAP) dehydrogenase and divert GAP toward MG and D-lactate formation. Accumulation of D-lactate and 1-phosphoglycerate may explain why GA makes the beta-cell acidic. Reduction of both GA and MG by abundant beta-cell aldehyde reductases will lower the cytosolic NADPH/NADP ratio, which is normally high.

Analysis of Methylglyoxal Metabolism in CHO Cells Grown in Culture.

Recent evidence suggests that several unknown or ill-characterized factors strongly influence cell growth and function in culture. Isolating these factors is necessary in order to maximize culture productivities. Methylglyoxal (MG), a potent protein and nucleic acid modifying agent, has been identified as a player in the signaling pathways associated with cell death and is known to be detrimental to cultured cells. This compound is produced in all mammalian systems by spontaneous phosphate elimination from glycolytic pathway intermediates. A kinetic model that qualitatively describes the cellular distribution of protein-associated MG in the absence of enzymatic adduct formation predicted far lower levels of reversibly bound MG than measured in cultured CHO cells. This suggests that the targeted modification of proteins through enzymatically mediated mechanisms is a significant sink for cellular methylglyoxal. The model was validated with measurements of carbon flux through the glyoxalase pathway to d-lactic acid, a unique end product of MG metabolism in mammalian systems. Fluxes to d-lactic acid of up to 16.8 mmol ml-packed cells(-1) day(-1) were measured with CHO cells grown in batch culture or 100-fold more than found in normal tissues.

Fish chromatophores as cytosensors in a microscale device: detection of environmental toxins and bacterial pathogens.

Fish chromatophores from Betta splendens are used as the cytosensor element in the development of a portable microscale device capable of detecting certain environmental toxins and bacterial pathogens by monitoring changes in pigment granule distribution. The adaptation of chromatophores to a microscale environment has required the development of enabling technologies to produce miniaturized culture chambers, to integrate microfluidics for sample delivery, to miniaturize image capture, and to design new statistical methods for image analyses. Betta splendens chromatophores were selected as the cytosensor element because of their moderate size, their toleration of close contact, and most importantly, for their responses to a broad range of chemicals and pathogenic bacteria. A miniaturized culture chamber has been designed that supports chromatophore viability for as long as 3 months, and that can be easily transported without damage to the cells. New statistical methods for image analyses have been developed that increase sensitivity and also decrease the time required for detection of significant changes in pigment granule distribution. Betta chromatophores have been tested for their responses to selected pathogenic bacteria and chemical agents. We discuss in detail the aggregation of pigment granules seen when chromatophores are incubated with Bacillus cereus, a common cause of food poisoning. Also described are the more subtle responses of chromatophores to a class of environmental chemical toxins, polynuclear aromatic hydrocarbons. We show that the chromatophores are able to detect the presence of certain polynuclear aromatic hydrocarbons at concentrations lower than the Environment Protection Agency (EPA) 550.1 standards.

Tumor necrosis factor-induced modulation of glyoxalase I activities through phosphorylation by PKA results in cell death and is accompanied by the formation of a specific methylglyoxal-derived AGE.

Tumor necrosis factor (TNF)-induced cell death in the fibrosarcoma cell line L929 is a caspase-independent process that is characterized by increased production of reactive oxygen species (ROS) in the mitochondria. To elucidate this ROS-dependent cell death pathway, a comparative study of the phosphoproteins present in TNF-treated and control cells was performed. Here we report that TNF induces an increased phosphorylation of glyoxalase I that is mediated by protein kinase A and required for cell death. We also show that TNF induces a substantial increase in intracellular levels of methylglyoxal (MG) that leads to the formation of a specific MG-derived advanced glycation end product and that this formation occurs as a consequence of increased ROS production. These data indicate that MG modification of proteins is a targeted process and that MG may thus function as a signal molecule during the regulation of cell death. Furthermore, we provide evidence that the TNF-induced phosphorylation of glyoxalase I is not involved in detoxification of MG by means of the glyoxalase system, but that phosphorylated glyoxalase I is on the pathway leading to the formation of a specific MG-derived advanced glycation end product.