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Sample records for metabolic flux analysis

  1. Principal Metabolic Flux Mode Analysis.

    Science.gov (United States)

    Bhadra, Sahely; Blomberg, Peter; Castillo, Sandra; Rousu, Juho; Wren, Jonathan

    2018-02-06

    In the analysis of metabolism, two distinct and complementary approaches are frequently used: Principal component analysis (PCA) and stoichiometric flux analysis. PCA is able to capture the main modes of variability in a set of experiments and does not make many prior assumptions about the data, but does not inherently take into account the flux mode structure of metabolism. Stoichiometric flux analysis methods, such as Flux Balance Analysis (FBA) and Elementary Mode Analysis, on the other hand, are able to capture the metabolic flux modes, however, they are primarily designed for the analysis of single samples at a time, and not best suited for exploratory analysis on a large sets of samples. We propose a new methodology for the analysis of metabolism, called Principal Metabolic Flux Mode Analysis (PMFA), which marries the PCA and stoichiometric flux analysis approaches in an elegant regularized optimization framework. In short, the method incorporates a variance maximization objective form PCA coupled with a stoichiometric regularizer, which penalizes projections that are far from any flux modes of the network. For interpretability, we also introduce a sparse variant of PMFA that favours flux modes that contain a small number of reactions. Our experiments demonstrate the versatility and capabilities of our methodology. The proposed method can be applied to genome-scale metabolic network in efficient way as PMFA does not enumerate elementary modes. In addition, the method is more robust on out-of-steady steady-state experimental data than competing flux mode analysis approaches. Matlab software for PMFA and SPMFA and data set used for experiments are available in https://github.com/aalto-ics-kepaco/PMFA. sahely@iitpkd.ac.in, juho.rousu@aalto.fi, Peter.Blomberg@vtt.fi, Sandra.Castillo@vtt.fi. Detailed results are in Supplementary files. Supplementary data are available at https://github.com/aalto-ics-kepaco/PMFA/blob/master/Results.zip.

  2. Primary Metabolic Pathways and Metabolic Flux Analysis

    DEFF Research Database (Denmark)

    Villadsen, John

    2015-01-01

    his chapter introduces the metabolic flux analysis (MFA) or stoichiometry-based MFA, and describes the quantitative basis for MFA. It discusses the catabolic pathways in which free energy is produced to drive the cell-building anabolic pathways. An overview of these primary pathways provides...... the reader who is primarily trained in the engineering sciences with atleast a preliminary introduction to biochemistry and also shows how carbon is drained off the catabolic pathways to provide precursors for cell mass building and sometimes for important industrial products. The primary pathways...... to be examined in the following are: glycolysis, primarily by the EMP pathway, but other glycolytic pathways is also mentioned; fermentative pathways in which the redox generated in the glycolytic reactions are consumed; reactions in the tricarboxylic acid (TCA) cycle, which produce biomass precursors and redox...

  3. From elementary flux modes to elementary flux vectors: Metabolic pathway analysis with arbitrary linear flux constraints

    Science.gov (United States)

    Klamt, Steffen; Gerstl, Matthias P.; Jungreuthmayer, Christian; Mahadevan, Radhakrishnan; Müller, Stefan

    2017-01-01

    Elementary flux modes (EFMs) emerged as a formal concept to describe metabolic pathways and have become an established tool for constraint-based modeling and metabolic network analysis. EFMs are characteristic (support-minimal) vectors of the flux cone that contains all feasible steady-state flux vectors of a given metabolic network. EFMs account for (homogeneous) linear constraints arising from reaction irreversibilities and the assumption of steady state; however, other (inhomogeneous) linear constraints, such as minimal and maximal reaction rates frequently used by other constraint-based techniques (such as flux balance analysis [FBA]), cannot be directly integrated. These additional constraints further restrict the space of feasible flux vectors and turn the flux cone into a general flux polyhedron in which the concept of EFMs is not directly applicable anymore. For this reason, there has been a conceptual gap between EFM-based (pathway) analysis methods and linear optimization (FBA) techniques, as they operate on different geometric objects. One approach to overcome these limitations was proposed ten years ago and is based on the concept of elementary flux vectors (EFVs). Only recently has the community started to recognize the potential of EFVs for metabolic network analysis. In fact, EFVs exactly represent the conceptual development required to generalize the idea of EFMs from flux cones to flux polyhedra. This work aims to present a concise theoretical and practical introduction to EFVs that is accessible to a broad audience. We highlight the close relationship between EFMs and EFVs and demonstrate that almost all applications of EFMs (in flux cones) are possible for EFVs (in flux polyhedra) as well. In fact, certain properties can only be studied with EFVs. Thus, we conclude that EFVs provide a powerful and unifying framework for constraint-based modeling of metabolic networks. PMID:28406903

  4. OpenFLUX: efficient modelling software for 13C-based metabolic flux analysis

    Directory of Open Access Journals (Sweden)

    Nielsen Lars K

    2009-05-01

    Full Text Available Abstract Background The quantitative analysis of metabolic fluxes, i.e., in vivo activities of intracellular enzymes and pathways, provides key information on biological systems in systems biology and metabolic engineering. It is based on a comprehensive approach combining (i tracer cultivation on 13C substrates, (ii 13C labelling analysis by mass spectrometry and (iii mathematical modelling for experimental design, data processing, flux calculation and statistics. Whereas the cultivation and the analytical part is fairly advanced, a lack of appropriate modelling software solutions for all modelling aspects in flux studies is limiting the application of metabolic flux analysis. Results We have developed OpenFLUX as a user friendly, yet flexible software application for small and large scale 13C metabolic flux analysis. The application is based on the new Elementary Metabolite Unit (EMU framework, significantly enhancing computation speed for flux calculation. From simple notation of metabolic reaction networks defined in a spreadsheet, the OpenFLUX parser automatically generates MATLAB-readable metabolite and isotopomer balances, thus strongly facilitating model creation. The model can be used to perform experimental design, parameter estimation and sensitivity analysis either using the built-in gradient-based search or Monte Carlo algorithms or in user-defined algorithms. Exemplified for a microbial flux study with 71 reactions, 8 free flux parameters and mass isotopomer distribution of 10 metabolites, OpenFLUX allowed to automatically compile the EMU-based model from an Excel file containing metabolic reactions and carbon transfer mechanisms, showing it's user-friendliness. It reliably reproduced the published data and optimum flux distributions for the network under study were found quickly ( Conclusion We have developed a fast, accurate application to perform steady-state 13C metabolic flux analysis. OpenFLUX will strongly facilitate and

  5. Metabolic flux analysis of heterotrophic growth in Chlamydomonas reinhardtii.

    Directory of Open Access Journals (Sweden)

    Nanette R Boyle

    Full Text Available Despite the wealth of knowledge available for C. reinhardtii, the central metabolic fluxes of growth on acetate have not yet been determined. In this study, 13C-metabolic flux analysis (13C-MFA was used to determine and quantify the metabolic pathways of primary metabolism in C. reinhardtii cells grown under heterotrophic conditions with acetate as the sole carbon source. Isotopic labeling patterns of compartment specific biomass derived metabolites were used to calculate the fluxes. It was found that acetate is ligated with coenzyme A in the three subcellular compartments (cytosol, mitochondria and plastid included in the model. Two citrate synthases were found to potentially be involved in acetyl-coA metabolism; one localized in the mitochondria and the other acting outside the mitochondria. Labeling patterns demonstrate that Acetyl-coA synthesized in the plastid is directly incorporated in synthesis of fatty acids. Despite having a complete TCA cycle in the mitochondria, it was also found that a majority of the malate flux is shuttled to the cytosol and plastid where it is converted to oxaloacetate providing reducing equivalents to these compartments. When compared to predictions by flux balance analysis, fluxes measured with 13C-MFA were found to be suboptimal with respect to biomass yield; C. reinhardtii sacrifices biomass yield to produce ATP and reducing equivalents.

  6. Synergy between 13C-metabolic flux analysis and flux balance analysis for understanding metabolic adaption to anaerobiosis in e. coli

    Science.gov (United States)

    Genome-based Flux Balance Analysis (FBA, constraints based flux analysis) and steady state isotopic-labeling-based Metabolic Flux Analysis (MFA) are complimentary approaches to predicting and measuring the operation and regulation of metabolic networks. Here a genome-derived model of E. coli metabol...

  7. PFA toolbox: a MATLAB tool for Metabolic Flux Analysis.

    Science.gov (United States)

    Morales, Yeimy; Bosque, Gabriel; Vehí, Josep; Picó, Jesús; Llaneras, Francisco

    2016-07-11

    Metabolic Flux Analysis (MFA) is a methodology that has been successfully applied to estimate metabolic fluxes in living cells. However, traditional frameworks based on this approach have some limitations, particularly when measurements are scarce and imprecise. This is very common in industrial environments. The PFA Toolbox can be used to face those scenarios. Here we present the PFA (Possibilistic Flux Analysis) Toolbox for MATLAB, which simplifies the use of Interval and Possibilistic Metabolic Flux Analysis. The main features of the PFA Toolbox are the following: (a) It provides reliable MFA estimations in scenarios where only a few fluxes can be measured or those available are imprecise. (b) It provides tools to easily plot the results as interval estimates or flux distributions. (c) It is composed of simple functions that MATLAB users can apply in flexible ways. (d) It includes a Graphical User Interface (GUI), which provides a visual representation of the measurements and their uncertainty. (e) It can use stoichiometric models in COBRA format. In addition, the PFA Toolbox includes a User's Guide with a thorough description of its functions and several examples. The PFA Toolbox for MATLAB is a freely available Toolbox that is able to perform Interval and Possibilistic MFA estimations.

  8. Constraining genome-scale models to represent the bow tie structure of metabolism for 13C metabolic flux analysis

    DEFF Research Database (Denmark)

    Backman, Tyler W.H.; Ando, David; Singh, Jahnavi

    2018-01-01

    Determination of internal metabolic fluxes is crucial for fundamental and applied biology because they map how carbon and electrons flow through metabolism to enable cell function. 13C Metabolic Flux Analysis (13C MFA) and Two-Scale 13C Metabolic Flux Analysis (2S-13C MFA) are two techniques used...

  9. Comparison between elementary flux modes analysis and 13C-metabolic fluxes measured in bacterial and plant cells.

    Science.gov (United States)

    Beurton-Aimar, Marie; Beauvoit, Bertrand; Monier, Antoine; Vallée, François; Dieuaide-Noubhani, Martine; Colombié, Sophie

    2011-06-20

    (13)C metabolic flux analysis is one of the pertinent ways to compare two or more physiological states. From a more theoretical standpoint, the structural properties of metabolic networks can be analysed to explore feasible metabolic behaviours and to define the boundaries of steady state flux distributions. Elementary flux mode analysis is one of the most efficient methods for performing this analysis. In this context, recent approaches have tended to compare experimental flux measurements with topological network analysis. Metabolic networks describing the main pathways of central carbon metabolism were set up for a bacteria species (Corynebacterium glutamicum) and a plant species (Brassica napus) for which experimental flux maps were available. The structural properties of each network were then studied using the concept of elementary flux modes. To do this, coefficients of flux efficiency were calculated for each reaction within the networks by using selected sets of elementary flux modes. Then the relative differences - reflecting the change of substrate i.e. a sugar source for C. glutamicum and a nitrogen source for B. napus - of both flux efficiency and flux measured experimentally were compared. For both organisms, there is a clear relationship between these parameters, thus indicating that the network structure described by the elementary flux modes had captured a significant part of the metabolic activity in both biological systems. In B. napus, the extension of the elementary flux mode analysis to an enlarged metabolic network still resulted in a clear relationship between the change in the coefficients and that of the measured fluxes. Nevertheless, the limitations of the method to fit some particular fluxes are discussed. This consistency between EFM analysis and experimental flux measurements, validated on two metabolic systems allows us to conclude that elementary flux mode analysis could be a useful tool to complement (13)C metabolic flux analysis

  10. FluxFix: automatic isotopologue normalization for metabolic tracer analysis.

    Science.gov (United States)

    Trefely, Sophie; Ashwell, Peter; Snyder, Nathaniel W

    2016-11-25

    Isotopic tracer analysis by mass spectrometry is a core technique for the study of metabolism. Isotopically labeled atoms from substrates, such as [ 13 C]-labeled glucose, can be traced by their incorporation over time into specific metabolic products. Mass spectrometry is often used for the detection and differentiation of the isotopologues of each metabolite of interest. For meaningful interpretation, mass spectrometry data from metabolic tracer experiments must be corrected to account for the naturally occurring isotopologue distribution. The calculations required for this correction are time consuming and error prone and existing programs are often platform specific, non-intuitive, commercially licensed and/or limited in accuracy by using theoretical isotopologue distributions, which are prone to artifacts from noise or unresolved interfering signals. Here we present FluxFix ( http://fluxfix.science ), an application freely available on the internet that quickly and reliably transforms signal intensity values into percent mole enrichment for each isotopologue measured. 'Unlabeled' data, representing the measured natural isotopologue distribution for a chosen analyte, is entered by the user. This data is used to generate a correction matrix according to a well-established algorithm. The correction matrix is applied to labeled data, also entered by the user, thus generating the corrected output data. FluxFix is compatible with direct copy and paste from spreadsheet applications including Excel (Microsoft) and Google sheets and automatically adjusts to account for input data dimensions. The program is simple, easy to use, agnostic to the mass spectrometry platform, generalizable to known or unknown metabolites, and can take input data from either a theoretical natural isotopologue distribution or an experimentally measured one. Our freely available web-based calculator, FluxFix ( http://fluxfix.science ), quickly and reliably corrects metabolic tracer data for

  11. Two-Scale 13C Metabolic Flux Analysis for Metabolic Engineering.

    Science.gov (United States)

    Ando, David; Garcia Martin, Hector

    2018-01-01

    Accelerating the Design-Build-Test-Learn (DBTL) cycle in synthetic biology is critical to achieving rapid and facile bioengineering of organisms for the production of, e.g., biofuels and other chemicals. The Learn phase involves using data obtained from the Test phase to inform the next Design phase. As part of the Learn phase, mathematical models of metabolic fluxes give a mechanistic level of comprehension to cellular metabolism, isolating the principle drivers of metabolic behavior from the peripheral ones, and directing future experimental designs and engineering methodologies. Furthermore, the measurement of intracellular metabolic fluxes is specifically noteworthy as providing a rapid and easy-to-understand picture of how carbon and energy flow throughout the cell. Here, we present a detailed guide to performing metabolic flux analysis in the Learn phase of the DBTL cycle, where we show how one can take the isotope labeling data from a 13 C labeling experiment and immediately turn it into a determination of cellular fluxes that points in the direction of genetic engineering strategies that will advance the metabolic engineering process.For our modeling purposes we use the Joint BioEnergy Institute (JBEI) Quantitative Metabolic Modeling (jQMM) library, which provides an open-source, python-based framework for modeling internal metabolic fluxes and making actionable predictions on how to modify cellular metabolism for specific bioengineering goals. It presents a complete toolbox for performing different types of flux analysis such as Flux Balance Analysis, 13 C Metabolic Flux Analysis, and it introduces the capability to use 13 C labeling experimental data to constrain comprehensive genome-scale models through a technique called two-scale 13 C Metabolic Flux Analysis (2S- 13 C MFA) [1]. In addition to several other capabilities, the jQMM is also able to predict the effects of knockouts using the MoMA and ROOM methodologies. The use of the jQMM library is

  12. WUFlux: an open-source platform for 13C metabolic flux analysis of bacterial metabolism.

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    He, Lian; Wu, Stephen G; Zhang, Muhan; Chen, Yixin; Tang, Yinjie J

    2016-11-04

    Flux analyses, including flux balance analysis (FBA) and 13 C-metabolic flux analysis ( 13 C-MFA), offer direct insights into cell metabolism, and have been widely used to characterize model and non-model microbial species. Nonetheless, constructing the 13 C-MFA model and performing flux calculation are demanding for new learners, because they require knowledge of metabolic networks, carbon transitions, and computer programming. To facilitate and standardize the 13 C-MFA modeling work, we set out to publish a user-friendly and programming-free platform (WUFlux) for flux calculations in MATLAB ® . We constructed an open-source platform for steady-state 13 C-MFA. Using GUIDE (graphical user interface design environment) in MATLAB, we built a user interface that allows users to modify models based on their own experimental conditions. WUFlux is capable of directly correcting mass spectrum data of TBDMS (N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide)-derivatized proteinogenic amino acids by removing background noise. To simplify 13 C-MFA of different prokaryotic species, the software provides several metabolic network templates, including those for chemoheterotrophic bacteria and mixotrophic cyanobacteria. Users can modify the network and constraints, and then analyze the microbial carbon and energy metabolisms of various carbon substrates (e.g., glucose, pyruvate/lactate, acetate, xylose, and glycerol). WUFlux also offers several ways of visualizing the flux results with respect to the constructed network. To validate our model's applicability, we have compared and discussed the flux results obtained from WUFlux and other MFA software. We have also illustrated how model constraints of cofactor and ATP balances influence fluxome results. Open-source software for 13 C-MFA, WUFlux, with a user-friendly interface and easy-to-modify templates, is now available at http://www.13cmfa.org /or ( http://tang.eece.wustl.edu/ToolDevelopment.htm ). We will continue

  13. Finding elementary flux modes in metabolic networks based on flux balance analysis and flux coupling analysis: application to the analysis of Escherichia coli metabolism.

    Science.gov (United States)

    Tabe-Bordbar, Shayan; Marashi, Sayed-Amir

    2013-12-01

    Elementary modes (EMs) are steady-state metabolic flux vectors with minimal set of active reactions. Each EM corresponds to a metabolic pathway. Therefore, studying EMs is helpful for analyzing the production of biotechnologically important metabolites. However, memory requirements for computing EMs may hamper their applicability as, in most genome-scale metabolic models, no EM can be computed due to running out of memory. In this study, we present a method for computing randomly sampled EMs. In this approach, a network reduction algorithm is used for EM computation, which is based on flux balance-based methods. We show that this approach can be used to recover the EMs in the medium- and genome-scale metabolic network models, while the EMs are sampled in an unbiased way. The applicability of such results is shown by computing “estimated” control-effective flux values in Escherichia coli metabolic network.

  14. Metabolic flux analysis of the halophilic archaeon Haladaptatus paucihalophilus

    International Nuclear Information System (INIS)

    Liu, Guangxiu; Zhang, Manxiao; Mo, Tianlu; He, Lian; Zhang, Wei; Yu, Yi; Zhang, Qi; Ding, Wei

    2015-01-01

    This work reports the 13 C-assisted metabolic flux analysis of Haladaptatus paucihalophilus, a halophilic archaeon possessing an intriguing osmoadaption mechanism. We showed that the carbon flow is through the oxidative tricarboxylic acid (TCA) cycle whereas the reductive TCA cycle is not operative in H. paucihalophilus. In addition, both threonine and the citramalate pathways contribute to isoleucine biosynthesis, whereas lysine is synthesized through the diaminopimelate pathway and not through the α-aminoadipate pathway. Unexpected, the labeling patterns of glycine from the cells grown on [1- 13 C]pyruvate and [2- 13 C]pyruvate suggest that, unlike all the organisms investigated so far, in which glycine is produced exclusively from the serine hydroxymethyltransferase (SHMT) pathway, glycine biosynthesis in H. paucihalophilus involves different pathways including SHMT, threonine aldolase (TA) and the reverse reaction of glycine cleavage system (GCS), demonstrating for the first time that other pathways instead of SHMT can also make a significant contribution to the cellular glycine pool. Transcriptional analysis confirmed that both TA and GCS genes were transcribed in H. paucihalophilus, and the transcriptional level is independent of salt concentrations in the culture media. This study expands our understanding of amino acid biosynthesis and provides valuable insights into the metabolism of halophilic archaea. - Highlights: • Serine hydroxymethyltransferase, threonine aldolase, and glycine cleavage system all contribute to the glycine pool of H. paucihalophilus. • Threonine and the citramalate pathways contribute equally to the isoleucine biosynthesis in H. paucihalophilus. • Lysine in H. paucihalophilus is synthesized through the diaminopimelate pathway and not through the α-aminoadipate pathway. • Glycine biosynthesis is likely unrelated to the cell osmoadaption mechanism.

  15. Metabolic flux analysis of the halophilic archaeon Haladaptatus paucihalophilus

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Guangxiu; Zhang, Manxiao [Key Laboratory of Desert and Desertification, Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou, 730000 (China); Key Laboratory of Extreme Environmental Microbial Resources and Engineering, Gansu Province, Lanzhou, 730000 (China); Mo, Tianlu [Department of Chemistry, Fudan University, Shanghai, 200433 (China); He, Lian [Key Laboratory of Combinatory Biosynthesis and Drug Discovery (Ministry of Education), School of Pharmaceutical Sciences, Wuhan University, Wuhan, 430071 (China); Zhang, Wei [Key Laboratory of Desert and Desertification, Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou, 730000 (China); Key Laboratory of Extreme Environmental Microbial Resources and Engineering, Gansu Province, Lanzhou, 730000 (China); Yu, Yi, E-mail: yu_yi@whu.edu.cn [Key Laboratory of Combinatory Biosynthesis and Drug Discovery (Ministry of Education), School of Pharmaceutical Sciences, Wuhan University, Wuhan, 430071 (China); Zhang, Qi, E-mail: qizhang@sioc.ac.cn [Department of Chemistry, Fudan University, Shanghai, 200433 (China); Ding, Wei, E-mail: dingw@lzu.edu.cn [Key Laboratory of Desert and Desertification, Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou, 730000 (China); Key Laboratory of Extreme Environmental Microbial Resources and Engineering, Gansu Province, Lanzhou, 730000 (China); Department of Chemistry, Fudan University, Shanghai, 200433 (China)

    2015-11-27

    This work reports the {sup 13}C-assisted metabolic flux analysis of Haladaptatus paucihalophilus, a halophilic archaeon possessing an intriguing osmoadaption mechanism. We showed that the carbon flow is through the oxidative tricarboxylic acid (TCA) cycle whereas the reductive TCA cycle is not operative in H. paucihalophilus. In addition, both threonine and the citramalate pathways contribute to isoleucine biosynthesis, whereas lysine is synthesized through the diaminopimelate pathway and not through the α-aminoadipate pathway. Unexpected, the labeling patterns of glycine from the cells grown on [1-{sup 13}C]pyruvate and [2-{sup 13}C]pyruvate suggest that, unlike all the organisms investigated so far, in which glycine is produced exclusively from the serine hydroxymethyltransferase (SHMT) pathway, glycine biosynthesis in H. paucihalophilus involves different pathways including SHMT, threonine aldolase (TA) and the reverse reaction of glycine cleavage system (GCS), demonstrating for the first time that other pathways instead of SHMT can also make a significant contribution to the cellular glycine pool. Transcriptional analysis confirmed that both TA and GCS genes were transcribed in H. paucihalophilus, and the transcriptional level is independent of salt concentrations in the culture media. This study expands our understanding of amino acid biosynthesis and provides valuable insights into the metabolism of halophilic archaea. - Highlights: • Serine hydroxymethyltransferase, threonine aldolase, and glycine cleavage system all contribute to the glycine pool of H. paucihalophilus. • Threonine and the citramalate pathways contribute equally to the isoleucine biosynthesis in H. paucihalophilus. • Lysine in H. paucihalophilus is synthesized through the diaminopimelate pathway and not through the α-aminoadipate pathway. • Glycine biosynthesis is likely unrelated to the cell osmoadaption mechanism.

  16. Constraining Genome-Scale Models to Represent the Bow Tie Structure of Metabolism for 13C Metabolic Flux Analysis

    Directory of Open Access Journals (Sweden)

    Tyler W. H. Backman

    2018-01-01

    Full Text Available Determination of internal metabolic fluxes is crucial for fundamental and applied biology because they map how carbon and electrons flow through metabolism to enable cell function. 13 C Metabolic Flux Analysis ( 13 C MFA and Two-Scale 13 C Metabolic Flux Analysis (2S- 13 C MFA are two techniques used to determine such fluxes. Both operate on the simplifying approximation that metabolic flux from peripheral metabolism into central “core” carbon metabolism is minimal, and can be omitted when modeling isotopic labeling in core metabolism. The validity of this “two-scale” or “bow tie” approximation is supported both by the ability to accurately model experimental isotopic labeling data, and by experimentally verified metabolic engineering predictions using these methods. However, the boundaries of core metabolism that satisfy this approximation can vary across species, and across cell culture conditions. Here, we present a set of algorithms that (1 systematically calculate flux bounds for any specified “core” of a genome-scale model so as to satisfy the bow tie approximation and (2 automatically identify an updated set of core reactions that can satisfy this approximation more efficiently. First, we leverage linear programming to simultaneously identify the lowest fluxes from peripheral metabolism into core metabolism compatible with the observed growth rate and extracellular metabolite exchange fluxes. Second, we use Simulated Annealing to identify an updated set of core reactions that allow for a minimum of fluxes into core metabolism to satisfy these experimental constraints. Together, these methods accelerate and automate the identification of a biologically reasonable set of core reactions for use with 13 C MFA or 2S- 13 C MFA, as well as provide for a substantially lower set of flux bounds for fluxes into the core as compared with previous methods. We provide an open source Python implementation of these algorithms at https://github.com/JBEI/limitfluxtocore.

  17. Flux balance analysis of genome-scale metabolic model of rice ...

    Indian Academy of Sciences (India)

    Here, we analyse a genome-scale metabolic model of rice leaf using Flux Balance Analysis to investigate whether it has potential metabolic flexibility to increase the biosynthesis of any of the biomass components. We initially simulate the metabolic responses under an objective to maximize the biomass components.

  18. Flux balance analysis of genome-scale metabolic model of rice ...

    Indian Academy of Sciences (India)

    2015-09-28

    Sep 28, 2015 ... genome-scale metabolic model of rice leaf using Flux Balance Analysis to investigate whether it has potential .... is number of reactions. In steady state. S.v = 0. (2) where v is the flux vector of reactions (Kauffman et al. 2003). Objective function is. Z = w.v. (3). 820 .... All the reactions are not included.

  19. COMPLETE-MFA: complementary parallel labeling experiments technique for metabolic flux analysis.

    Science.gov (United States)

    Leighty, Robert W; Antoniewicz, Maciek R

    2013-11-01

    We have developed a novel approach for measuring highly accurate and precise metabolic fluxes in living cells, termed COMPLETE-MFA, short for complementary parallel labeling experiments technique for metabolic flux analysis. The COMPLETE-MFA method is based on combined analysis of multiple isotopic labeling experiments, where the synergy of using complementary tracers greatly improves the precision of estimated fluxes. In this work, we demonstrate the COMPLETE-MFA approach using all singly labeled glucose tracers, [1-(13)C], [2-(13)C], [3-(13)C], [4-(13)C], [5-(13)C], and [6-(13)C]glucose to determine precise metabolic fluxes for wild-type Escherichia coli. Cells were grown in six parallel cultures on defined medium with glucose as the only carbon source. Mass isotopomers of biomass amino acids were measured by gas chromatography-mass spectrometry (GC-MS). The data from all six experiments were then fitted simultaneously to a single flux model to determine accurate intracellular fluxes. We obtained a statistically acceptable fit with more than 300 redundant measurements. The estimated flux map is the most precise flux result obtained thus far for E. coli cells. To our knowledge, this is the first time that six isotopic labeling experiments have been successfully integrated for high-resolution (13)C-flux analysis. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. Metabolic Flux Analysis of Shewanella spp. Reveals Evolutionary Robustness in Central Carbon Metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Yinjie J.; Martin, Hector Garcia; Dehal, Paramvir S.; Deutschbauer, Adam; Llora, Xavier; Meadows, Adam; Arkin, Adam; Keasling, Jay D.

    2009-08-19

    Shewanella spp. are a group of facultative anaerobic bacteria widely distributed in marine and fresh-water environments. In this study, we profiled the central metabolic fluxes of eight recently sequenced Shewanella species grown under the same condition in minimal med-ium with [3-13C] lactate. Although the tested Shewanella species had slightly different growth rates (0.23-0.29 h31) and produced different amounts of acetate and pyruvate during early exponential growth (pseudo-steady state), the relative intracellular metabolic flux distributions were remarkably similar. This result indicates that Shewanella species share similar regulation in regard to central carbon metabolic fluxes under steady growth conditions: the maintenance of metabolic robustness is not only evident in a single species under genetic perturbations (Fischer and Sauer, 2005; Nat Genet 37(6):636-640), but also observed through evolutionary related microbial species. This remarkable conservation of relative flux profiles through phylogenetic differences prompts us to introduce the concept of metabotype as an alternative scheme to classify microbial fluxomics. On the other hand, Shewanella spp. display flexibility in the relative flux profiles when switching their metabolism from consuming lactate to consuming pyruvate and acetate.

  1. Flux analysis uncovers key role of functional redundancy in formaldehyde metabolism.

    Directory of Open Access Journals (Sweden)

    Christopher J Marx

    2005-02-01

    Full Text Available Genome-scale analysis of predicted metabolic pathways has revealed the common occurrence of apparent redundancy for specific functional units, or metabolic modules. In many cases, mutation analysis does not resolve function, and instead, direct experimental analysis of metabolic flux under changing conditions is necessary. In order to use genome sequences to build models of cellular function, it is important to define function for such apparently redundant systems. Here we describe direct flux measurements to determine the role of redundancy in three modules involved in formaldehyde assimilation and dissimilation in a bacterium growing on methanol. A combination of deuterium and (14C labeling was used to measure the flux through each of the branches of metabolism for growth on methanol during transitions into and out of methylotrophy. The cells were found to differentially partition formaldehyde among the three modules depending on the flux of methanol into the cell. A dynamic mathematical model demonstrated that the kinetic constants of the enzymes involved are sufficient to account for this phenomenon. We demonstrate the role of redundancy in formaldehyde metabolism and have uncovered a new paradigm for coping with toxic, high-flux metabolic intermediates: a dynamic, interconnected metabolic loop.

  2. Isotopically nonstationary metabolic flux analysis (INST-MFA) of photosynthesis and photorespiration in plants

    Science.gov (United States)

    Photorespiration is a central component of photosynthesis; however to better understand its role it should be viewed in the context of an integrated metabolic network rather than a series of individual reactions that operate independently. Isotopically nonstationary 13C metabolic flux analysis (INST...

  3. Evaluation of 13C isotopic tracers for metabolic flux analysis in mammalian cells.

    Science.gov (United States)

    Metallo, Christian M; Walther, Jason L; Stephanopoulos, Gregory

    2009-11-01

    (13)C metabolic flux analysis (MFA) is the most comprehensive means of characterizing cellular metabolic states. Uniquely labeled isotopic tracers enable more focused analyses to probe specific reactions within the network. As a result, the choice of tracer largely determines the precision with which one can estimate metabolic fluxes, especially in complex mammalian systems that require multiple substrates. Here we have experimentally determined metabolic fluxes in a tumor cell line, successfully recapitulating the hallmarks of cancer cell metabolism. Using these data, we computationally evaluated specifically labeled (13)C glucose and glutamine tracers for their ability to precisely and accurately estimate fluxes in central carbon metabolism. These methods enabled us to identify the optimal tracer for analyzing individual fluxes, specific pathways, and central carbon metabolism as a whole. [1,2-(13)C(2)]glucose provided the most precise estimates for glycolysis, the pentose phosphate pathway, and the overall network. Tracers such as [2-(13)C]glucose and [3-(13)C]glucose also outperformed the more commonly used [1-(13)C]glucose. [U-(13)C(5)]glutamine emerged as the preferred isotopic tracer for the analysis of the tricarboxylic acid (TCA) cycle. These results provide valuable, quantitative information on the performance of (13)C-labeled substrates and can aid in the design of more informative MFA experiments in mammalian cell culture.

  4. Metabolic flux balance analysis and the in silico analysis of Escherichia coli K-12 gene deletions

    Directory of Open Access Journals (Sweden)

    Edwards Jeremy S

    2000-07-01

    Full Text Available Abstract Background Genome sequencing and bioinformatics are producing detailed lists of the molecular components contained in many prokaryotic organisms. From this 'parts catalogue' of a microbial cell, in silico representations of integrated metabolic functions can be constructed and analyzed using flux balance analysis (FBA. FBA is particularly well-suited to study metabolic networks based on genomic, biochemical, and strain specific information. Results Herein, we have utilized FBA to interpret and analyze the metabolic capabilities of Escherichia coli. We have computationally mapped the metabolic capabilities of E. coli using FBA and examined the optimal utilization of the E. coli metabolic pathways as a function of environmental variables. We have used an in silico analysis to identify seven gene products of central metabolism (glycolysis, pentose phosphate pathway, TCA cycle, electron transport system essential for aerobic growth of E. coli on glucose minimal media, and 15 gene products essential for anaerobic growth on glucose minimal media. The in silico tpi-, zwf, and pta- mutant strains were examined in more detail by mapping the capabilities of these in silico isogenic strains. Conclusions We found that computational models of E. coli metabolism based on physicochemical constraints can be used to interpret mutant behavior. These in silica results lead to a further understanding of the complex genotype-phenotype relation. Supplementary information: http://gcrg.ucsd.edu/supplementary_data/DeletionAnalysis/main.htm

  5. SUMOFLUX: A Generalized Method for Targeted 13C Metabolic Flux Ratio Analysis.

    Directory of Open Access Journals (Sweden)

    Maria Kogadeeva

    2016-09-01

    Full Text Available Metabolic fluxes are a cornerstone of cellular physiology that emerge from a complex interplay of enzymes, carriers, and nutrients. The experimental assessment of in vivo intracellular fluxes using stable isotopic tracers is essential if we are to understand metabolic function and regulation. Flux estimation based on 13C or 2H labeling relies on complex simulation and iterative fitting; processes that necessitate a level of expertise that ordinarily preclude the non-expert user. To overcome this, we have developed SUMOFLUX, a methodology that is broadly applicable to the targeted analysis of 13C-metabolic fluxes. By combining surrogate modeling and machine learning, we trained a predictor to specialize in estimating flux ratios from measurable 13C-data. SUMOFLUX targets specific flux features individually, which makes it fast, user-friendly, applicable to experimental design and robust in terms of experimental noise and exchange flux magnitude. Collectively, we predict that SUMOFLUX's properties realistically pave the way to high-throughput flux analyses.

  6. Multiobjective flux balancing using the NISE method for metabolic network analysis.

    Science.gov (United States)

    Oh, Young-Gyun; Lee, Dong-Yup; Lee, Sang Yup; Park, Sunwon

    2009-01-01

    Flux balance analysis (FBA) is well acknowledged as an analysis tool of metabolic networks in the framework of metabolic engineering. However, FBA has a limitation for solving a multiobjective optimization problem which considers multiple conflicting objectives. In this study, we propose a novel multiobjective flux balance analysis method, which adapts the noninferior set estimation (NISE) method (Solanki et al., 1993) for multiobjective linear programming (MOLP) problems. NISE method can generate an approximation of the Pareto curve for conflicting objectives without redundant iterations of single objective optimization. Furthermore, the flux distributions at each Pareto optimal solution can be obtained for understanding the internal flux changes in the metabolic network. The functionality of this approach is shown by applying it to a genome-scale in silico model of E. coli. Multiple objectives for the poly(3-hydroxybutyrate) [P(3HB)] production are considered simultaneously, and relationships among them are identified. The Pareto curve for maximizing succinic acid production vs. maximizing biomass production is used for the in silico analysis of various combinatorial knockout strains. This proposed method accelerates the strain improvement in the metabolic engineering by reducing computation time of obtaining the Pareto curve and analysis time of flux distribution at each Pareto optimal solution. (c) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009.

  7. Genome-scale metabolic flux analysis of Streptomyces lividans growing on a complex medium.

    Science.gov (United States)

    D'Huys, Pieter-Jan; Lule, Ivan; Vercammen, Dominique; Anné, Jozef; Van Impe, Jan F; Bernaerts, Kristel

    2012-09-15

    Constraint-based metabolic modeling comprises various excellent tools to assess experimentally observed phenotypic behavior of micro-organisms in terms of intracellular metabolic fluxes. In combination with genome-scale metabolic networks, micro-organisms can be investigated in much more detail and under more complex environmental conditions. Although complex media are ubiquitously applied in industrial fermentations and are often a prerequisite for high protein secretion yields, such multi-component conditions are seldom investigated using genome-scale flux analysis. In this paper, a systematic and integrative approach is presented to determine metabolic fluxes in Streptomyces lividans TK24 grown on a nutritious and complex medium. Genome-scale flux balance analysis and randomized sampling of the solution space are combined to extract maximum information from exometabolome profiles. It is shown that biomass maximization cannot predict the observed metabolite production pattern as such. Although this cellular objective commonly applies to batch fermentation data, both input and output constraints are required to reproduce the measured biomass production rate. Rich media hence not necessarily lead to maximum biomass growth. To eventually identify a unique intracellular flux vector, a hierarchical optimization of cellular objectives is adopted. Out of various tested secondary objectives, maximization of the ATP yield per flux unit returns the closest agreement with the maximum frequency in flux histograms. This unique flux estimation is hence considered as a reasonable approximation for the biological fluxes. Flux maps for different growth phases show no active oxidative part of the pentose phosphate pathway, but NADPH generation in the TCA cycle and NADPH transdehydrogenase activity are most important in fulfilling the NADPH balance. Amino acids contribute to biomass growth by augmenting the pool of available amino acids and by boosting the TCA cycle, particularly

  8. Synergizing metabolic flux analysis and nucleotide sugar metabolism to understand the control of glycosylation of recombinant protein in CHO cells

    LENUS (Irish Health Repository)

    Burleigh, Susan C

    2011-10-18

    Abstract Background The glycosylation of recombinant proteins can be altered by a range of parameters including cellular metabolism, metabolic flux and the efficiency of the glycosylation process. We present an experimental set-up that allows determination of these key processes associated with the control of N-linked glycosylation of recombinant proteins. Results Chinese hamster ovary cells (CHO) were cultivated in shake flasks at 0 mM glutamine and displayed a reduced growth rate, glucose metabolism and a slower decrease in pH, when compared to other glutamine-supplemented cultures. The N-linked glycosylation of recombinant human chorionic gonadotrophin (HCG) was also altered under these conditions; the sialylation, fucosylation and antennarity decreased, while the proportion of neutral structures increased. A continuous culture set-up was subsequently used to understand the control of HCG glycosylation in the presence of varied glutamine concentrations; when glycolytic flux was reduced in the absence of glutamine, the glycosylation changes that were observed in shake flask culture were similarly detected. The intracellular content of UDP-GlcNAc was also reduced, which correlated with a decrease in sialylation and antennarity of the N-linked glycans attached to HCG. Conclusions The use of metabolic flux analysis illustrated a case of steady state multiplicity, where use of the same operating conditions at each steady state resulted in altered flux through glycolysis and the TCA cycle. This study clearly demonstrated that the control of glycoprotein microheterogeneity may be examined by use of a continuous culture system, metabolic flux analysis and assay of intracellular nucleotides. This system advances our knowledge of the relationship between metabolic flux and the glycosylation of biotherapeutics in CHO cells and will be of benefit to the bioprocessing industry.

  9. Integration of a constraint-based metabolic model of Brassica napus developing seeds with 13C-Metabolic Flux Analysis

    Directory of Open Access Journals (Sweden)

    Jordan eHay

    2014-12-01

    Full Text Available The use of large-scale or genome-scale metabolic reconstructions for modeling and simulation of plant metabolism and integration of those models with large-scale omics and experimental flux data is becoming increasingly important in plant metabolic research. Here we report an updated version of bna572, a bottom-up reconstruction of oilseed rape (Brassica napus L.; Brassicaceae developing seeds with emphasis on representation of biomass-component biosynthesis. New features include additional seed-relevant pathways for isoprenoid, sterol, phenylpropanoid, flavonoid, and choline biosynthesis. Being now based on standardized data formats and procedures for model reconstruction, bna572+ is available as a COBRA-compliant Systems Biology Markup Language (SBML model and conforms to the Minimum Information Requested in the Annotation of Biochemical Models (MIRIAM standards for annotation of external data resources. Bna572+ contains 966 genes, 671 reactions, and 666 metabolites distributed among 11 subcellular compartments. It is referenced to the Arabidopsis thaliana genome, with gene-protein-reaction associations resolving subcellular localization. Detailed mass and charge balancing and confidence scoring were applied to all reactions. Using Brassica napus seed specific transcriptome data, expression was verified for 78% of bna572+ genes and 97% of reactions. Alongside bna572+ we also present a revised carbon centric model for 13C-Metabolic Flux Analysis (13C-MFA with all its reactions being referenced to bna572+ based on linear projections. By integration of flux ratio constraints obtained from 13C-MFA and by elimination of infinite flux bounds around thermodynamically infeasible loops based on COBRA loopless methods, we demonstrate improvements in predictive power of Flux Variability Analysis (FVA. Using this combined approach we characterize the difference in metabolic flux of developing seeds of two Brassica napus genotypes contrasting in starch and

  10. Dynamic metabolic flux analysis using B-splines to study the effects of temperature shift on CHO cell metabolism

    Directory of Open Access Journals (Sweden)

    Verónica S. Martínez

    2015-12-01

    Full Text Available Metabolic flux analysis (MFA is widely used to estimate intracellular fluxes. Conventional MFA, however, is limited to continuous cultures and the mid-exponential growth phase of batch cultures. Dynamic MFA (DMFA has emerged to characterize time-resolved metabolic fluxes for the entire culture period. Here, the linear DMFA approach was extended using B-spline fitting (B-DMFA to estimate mass balanced fluxes. Smoother fits were achieved using reduced number of knots and parameters. Additionally, computation time was greatly reduced using a new heuristic algorithm for knot placement. B-DMFA revealed that Chinese hamster ovary cells shifted from 37 °C to 32 °C maintained a constant IgG volume-specific productivity, whereas the productivity for the controls peaked during mid-exponential growth phase and declined afterward. The observed 42% increase in product titer at 32 °C was explained by a prolonged cell growth with high cell viability, a larger cell volume and a more stable volume-specific productivity. Keywords: Dynamic, Metabolism, Flux analysis, CHO cells, Temperature shift, B-spline curve fitting

  11. Optimization of 13C isotopic tracers for metabolic flux analysis in mammalian cells.

    Science.gov (United States)

    Walther, Jason L; Metallo, Christian M; Zhang, Jie; Stephanopoulos, Gregory

    2012-03-01

    Mammalian cells consume and metabolize various substrates from their surroundings for energy generation and biomass synthesis. Glucose and glutamine, in particular, are the primary carbon sources for proliferating cancer cells. While this combination of substrates generates static labeling patterns for use in (13)C metabolic flux analysis (MFA), the inability of single tracers to effectively label all pathways poses an obstacle for comprehensive flux determination within a given experiment. To address this issue we applied a genetic algorithm to optimize mixtures of (13)C-labeled glucose and glutamine for use in MFA. We identified tracer combinations that minimized confidence intervals in an experimentally determined flux network describing central carbon metabolism in tumor cells. Additional simulations were used to determine the robustness of the [1,2-(13)C(2)]glucose/[U-(13)C(5)]glutamine tracer combination with respect to perturbations in the network. Finally, we experimentally validated the improved performance of this tracer set relative to glucose tracers alone in a cancer cell line. This versatile method allows researchers to determine the optimal tracer combination to use for a specific metabolic network, and our findings applied to cancer cells significantly enhance the ability of MFA experiments to precisely quantify fluxes in higher organisms. Copyright © 2011 Elsevier Inc. All rights reserved.

  12. Computational analysis of storage synthesis in developing Brassica napus L. (oilseed rape) embryos: Flux variability analysis in relation to 13C-metabolic flux analysis

    Energy Technology Data Exchange (ETDEWEB)

    Hay, J.; Schwender, J.

    2011-08-01

    Plant oils are an important renewable resource, and seed oil content is a key agronomical trait that is in part controlled by the metabolic processes within developing seeds. A large-scale model of cellular metabolism in developing embryos of Brassica napus (bna572) was used to predict biomass formation and to analyze metabolic steady states by flux variability analysis under different physiological conditions. Predicted flux patterns are highly correlated with results from prior 13C metabolic flux analysis of B. napus developing embryos. Minor differences from the experimental results arose because bna572 always selected only one sugar and one nitrogen source from the available alternatives, and failed to predict the use of the oxidative pentose phosphate pathway. Flux variability, indicative of alternative optimal solutions, revealed alternative pathways that can provide pyruvate and NADPH to plastidic fatty acid synthesis. The nutritional values of different medium substrates were compared based on the overall carbon conversion efficiency (CCE) for the biosynthesis of biomass. Although bna572 has a functional nitrogen assimilation pathway via glutamate synthase, the simulations predict an unexpected role of glycine decarboxylase operating in the direction of NH4+ assimilation. Analysis of the light-dependent improvement of carbon economy predicted two metabolic phases. At very low light levels small reductions in CO2 efflux can be attributed to enzymes of the tricarboxylic acid cycle (oxoglutarate dehydrogenase, isocitrate dehydrogenase) and glycine decarboxylase. At higher light levels relevant to the 13C flux studies, ribulose-1,5-bisphosphate carboxylase activity is predicted to account fully for the light-dependent changes in carbon balance.

  13. Metabolic network reconstruction, growth characterization and 13C-metabolic flux analysis of the extremophile Thermus thermophilus HB8.

    Science.gov (United States)

    Swarup, Aditi; Lu, Jing; DeWoody, Kathleen C; Antoniewicz, Maciek R

    2014-07-01

    Thermus thermophilus is an extremely thermophilic bacterium with significant biotechnological potential. In this work, we have characterized aerobic growth characteristics of T. thermophilus HB8 at temperatures between 50 and 85°C, constructed a metabolic network model of its central carbon metabolism and validated the model using (13)C-metabolic flux analysis ((13)C-MFA). First, cells were grown in batch cultures in custom constructed mini-bioreactors at different temperatures to determine optimal growth conditions. The optimal temperature for T. thermophilus grown on defined medium with glucose was 81°C. The maximum growth rate was 0.25h(-1). Between 50 and 81°C the growth rate increased by 7-fold and the temperature dependence was described well by an Arrhenius model with an activation energy of 47kJ/mol. Next, we performed a (13)C-labeling experiment with [1,2-(13)C] glucose as the tracer and calculated intracellular metabolic fluxes using (13)C-MFA. The results provided support for the constructed network model and highlighted several interesting characteristics of T. thermophilus metabolism. We found that T. thermophilus largely uses glycolysis and TCA cycle to produce biosynthetic precursors, ATP and reducing equivalents needed for cells growth. Consistent with its proposed metabolic network model, we did not detect any oxidative pentose phosphate pathway flux or Entner-Doudoroff pathway activity. The biomass precursors erythrose-4-phosphate and ribose-5-phosphate were produced via the non-oxidative pentose phosphate pathway, and largely via transketolase, with little contribution from transaldolase. The high biomass yield on glucose that was measured experimentally was also confirmed independently by (13)C-MFA. The results presented here provide a solid foundation for future studies of T. thermophilus and its metabolic engineering applications. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  14. Metabolic flux analysis of Cyanothece sp. ATCC 51142 under mixotrophic conditions.

    Science.gov (United States)

    Alagesan, Swathi; Gaudana, Sandeep B; Sinha, Avinash; Wangikar, Pramod P

    2013-11-01

    Cyanobacteria are a group of photosynthetic prokaryotes capable of utilizing solar energy to fix atmospheric carbon dioxide to biomass. Despite several "proof of principle" studies, low product yield is an impediment in commercialization of cyanobacteria-derived biofuels. Estimation of intracellular reaction rates by (13)C metabolic flux analysis ((13)C-MFA) would be a step toward enhancing biofuel yield via metabolic engineering. We report (13)C-MFA for Cyanothece sp. ATCC 51142, a unicellular nitrogen-fixing cyanobacterium, known for enhanced hydrogen yield under mixotrophic conditions. Rates of reactions in the central carbon metabolism under nitrogen-fixing and -non-fixing conditions were estimated by monitoring the competitive incorporation of (12)C and (13)C from unlabeled CO2 and uniformly labeled glycerol, respectively, into terminal metabolites such as amino acids. The observed labeling patterns suggest mixotrophic growth under both the conditions, with a larger fraction of unlabeled carbon in nitrate-sufficient cultures asserting a greater contribution of carbon fixation by photosynthesis and an anaplerotic pathway. Indeed, flux analysis complements the higher growth observed under nitrate-sufficient conditions. On the other hand, the flux through the oxidative pentose phosphate pathway and tricarboxylic acid cycle was greater in nitrate-deficient conditions, possibly to supply the precursors and reducing equivalents needed for nitrogen fixation. In addition, an enhanced flux through fructose-6-phosphate phosphoketolase possibly suggests the organism's preferred mode under nitrogen-fixing conditions. The (13)C-MFA results complement the reported predictions by flux balance analysis and provide quantitative insight into the organism's distinct metabolic features under nitrogen-fixing and -non-fixing conditions.

  15. 13C Metabolic Flux Analysis for systematic metabolic engineering of S. cerevisiae for overproduction of fatty acids.

    Directory of Open Access Journals (Sweden)

    Amit Ghosh

    2016-10-01

    Full Text Available Efficient redirection of microbial metabolism into the abundant production of desired bioproducts remains non-trivial. Here we used flux-based modeling approaches to improve yields of fatty acids in S. cerevisiae. We combined 13C labeling data with comprehensive genome-scale models to shed light onto microbial metabolism and improve metabolic engineering efforts. We concentrated on studying the balance of acetyl-CoA, a precursor metabolite for the biosynthesis of fatty acids. A genome-wide acetyl-CoA balance study showed ATP citrate lyase from Y. lipolytica as a robust source of cytoplasmic acetyl-CoA and malate synthase as a desirable target for down-regulation in terms of acetyl-CoA consumption. These genetic modifications were applied to S. cerevisiae WRY2, a strain that is capable of producing 460 mg L of free fatty acids. With the addition of ATP citrate lyase and down-regulation of malate synthase the engineered strain produced 26 per cent more free fatty acids. Further increases in free fatty acid production of 33 per cent were obtained by knocking out the cytoplasmic glycerol-3-phosphate dehydrogenase, which flux analysis had shown was competing for carbon flux upstream with the carbon flux through the acetyl-CoA production pathway in the cytoplasm. In total, the genetic interventions applied in this work increased fatty acid production by 70 per cent.

  16. To be certain about the uncertainty: Bayesian statistics for 13 C metabolic flux analysis.

    Science.gov (United States)

    Theorell, Axel; Leweke, Samuel; Wiechert, Wolfgang; Nöh, Katharina

    2017-11-01

    13 C Metabolic Fluxes Analysis ( 13 C MFA) remains to be the most powerful approach to determine intracellular metabolic reaction rates. Decisions on strain engineering and experimentation heavily rely upon the certainty with which these fluxes are estimated. For uncertainty quantification, the vast majority of 13 C MFA studies relies on confidence intervals from the paradigm of Frequentist statistics. However, it is well known that the confidence intervals for a given experimental outcome are not uniquely defined. As a result, confidence intervals produced by different methods can be different, but nevertheless equally valid. This is of high relevance to 13 C MFA, since practitioners regularly use three different approximate approaches for calculating confidence intervals. By means of a computational study with a realistic model of the central carbon metabolism of E. coli, we provide strong evidence that confidence intervals used in the field depend strongly on the technique with which they were calculated and, thus, their use leads to misinterpretation of the flux uncertainty. In order to provide a better alternative to confidence intervals in 13 C MFA, we demonstrate that credible intervals from the paradigm of Bayesian statistics give more reliable flux uncertainty quantifications which can be readily computed with high accuracy using Markov chain Monte Carlo. In addition, the widely applied chi-square test, as a means of testing whether the model reproduces the data, is examined closer. © 2017 Wiley Periodicals, Inc.

  17. 13CFLUX2--high-performance software suite for (13)C-metabolic flux analysis.

    Science.gov (United States)

    Weitzel, Michael; Nöh, Katharina; Dalman, Tolga; Niedenführ, Sebastian; Stute, Birgit; Wiechert, Wolfgang

    2013-01-01

    (13)C-based metabolic flux analysis ((13)C-MFA) is the state-of-the-art method to quantitatively determine in vivo metabolic reaction rates in microorganisms. 13CFLUX2 contains all tools for composing flexible computational (13)C-MFA workflows to design and evaluate carbon labeling experiments. A specially developed XML language, FluxML, highly efficient data structures and simulation algorithms achieve a maximum of performance and effectiveness. Support of multicore CPUs, as well as compute clusters, enables scalable investigations. 13CFLUX2 outperforms existing tools in terms of universality, flexibility and built-in features. Therewith, 13CFLUX2 paves the way for next-generation high-resolution (13)C-MFA applications on the large scale. 13CFLUX2 is implemented in C++ (ISO/IEC 14882 standard) with Java and Python add-ons to run under Linux/Unix. A demo version and binaries are available at www.13cflux.net.

  18. Identifying model error in metabolic flux analysis - a generalized least squares approach.

    Science.gov (United States)

    Sokolenko, Stanislav; Quattrociocchi, Marco; Aucoin, Marc G

    2016-09-13

    The estimation of intracellular flux through traditional metabolic flux analysis (MFA) using an overdetermined system of equations is a well established practice in metabolic engineering. Despite the continued evolution of the methodology since its introduction, there has been little focus on validation and identification of poor model fit outside of identifying "gross measurement error". The growing complexity of metabolic models, which are increasingly generated from genome-level data, has necessitated robust validation that can directly assess model fit. In this work, MFA calculation is framed as a generalized least squares (GLS) problem, highlighting the applicability of the common t-test for model validation. To differentiate between measurement and model error, we simulate ideal flux profiles directly from the model, perturb them with estimated measurement error, and compare their validation to real data. Application of this strategy to an established Chinese Hamster Ovary (CHO) cell model shows how fluxes validated by traditional means may be largely non-significant due to a lack of model fit. With further simulation, we explore how t-test significance relates to calculation error and show that fluxes found to be non-significant have 2-4 fold larger error (if measurement uncertainty is in the 5-10 % range). The proposed validation method goes beyond traditional detection of "gross measurement error" to identify lack of fit between model and data. Although the focus of this work is on t-test validation and traditional MFA, the presented framework is readily applicable to other regression analysis methods and MFA formulations.

  19. Metabolic flux analysis during the exponential growth phase of Saccharomyces cerevisiae in wine fermentations.

    Science.gov (United States)

    Quirós, Manuel; Martínez-Moreno, Rubén; Albiol, Joan; Morales, Pilar; Vázquez-Lima, Felícitas; Barreiro-Vázquez, Antonio; Ferrer, Pau; Gonzalez, Ramon

    2013-01-01

    As a consequence of the increase in global average temperature, grapes with the adequate phenolic and aromatic maturity tend to be overripe by the time of harvest, resulting in increased sugar concentrations and imbalanced C/N ratios in fermenting musts. This fact sets obvious additional hurdles in the challenge of obtaining wines with reduced alcohols levels, a new trend in consumer demands. It would therefore be interesting to understand Saccharomyces cerevisiae physiology during the fermentation of must with these altered characteristics. The present study aims to determine the distribution of metabolic fluxes during the yeast exponential growth phase, when both carbon and nitrogen sources are in excess, using continuous cultures. Two different sugar concentrations were studied under two different winemaking temperature conditions. Although consumption and production rates for key metabolites were severely affected by the different experimental conditions studied, the general distribution of fluxes in central carbon metabolism was basically conserved in all cases. It was also observed that temperature and sugar concentration exerted a higher effect on the pentose phosphate pathway and glycerol formation than on glycolysis and ethanol production. Additionally, nitrogen uptake, both quantitatively and qualitatively, was strongly influenced by environmental conditions. This work provides the most complete stoichiometric model used for Metabolic Flux Analysis of S. cerevisiae in wine fermentations employed so far, including the synthesis and release of relevant aroma compounds and could be used in the design of optimal nitrogen supplementation of wine fermentations.

  20. Metabolic flux analysis during the exponential growth phase of Saccharomyces cerevisiae in wine fermentations.

    Directory of Open Access Journals (Sweden)

    Manuel Quirós

    Full Text Available As a consequence of the increase in global average temperature, grapes with the adequate phenolic and aromatic maturity tend to be overripe by the time of harvest, resulting in increased sugar concentrations and imbalanced C/N ratios in fermenting musts. This fact sets obvious additional hurdles in the challenge of obtaining wines with reduced alcohols levels, a new trend in consumer demands. It would therefore be interesting to understand Saccharomyces cerevisiae physiology during the fermentation of must with these altered characteristics. The present study aims to determine the distribution of metabolic fluxes during the yeast exponential growth phase, when both carbon and nitrogen sources are in excess, using continuous cultures. Two different sugar concentrations were studied under two different winemaking temperature conditions. Although consumption and production rates for key metabolites were severely affected by the different experimental conditions studied, the general distribution of fluxes in central carbon metabolism was basically conserved in all cases. It was also observed that temperature and sugar concentration exerted a higher effect on the pentose phosphate pathway and glycerol formation than on glycolysis and ethanol production. Additionally, nitrogen uptake, both quantitatively and qualitatively, was strongly influenced by environmental conditions. This work provides the most complete stoichiometric model used for Metabolic Flux Analysis of S. cerevisiae in wine fermentations employed so far, including the synthesis and release of relevant aroma compounds and could be used in the design of optimal nitrogen supplementation of wine fermentations.

  1. A minimal growth medium for the basidiomycetePleurotus sapidusfor metabolic flux analysis.

    Science.gov (United States)

    Fraatz, Marco A; Naeve, Stefanie; Hausherr, Vanessa; Zorn, Holger; Blank, Lars M

    2014-01-01

    Pleurotus sapidus secretes a huge enzymatic repertoire including hydrolytic and oxidative enzymes and is an example for higher basidiomycetes being interesting for biotechnology. The complex growth media used for submerged cultivation limit basic physiological analyses of this group of organisms. Using undefined growth media, only little insights into the operation of central carbon metabolism and biomass formation, i.e. , the interplay of catabolic and anabolic pathways, can be gained. The development of a chemically defined growth medium allowed rapid growth of P. sapidus in submerged cultures. As P. sapidus grew extremely slow in salt medium, the co-utilization of amino acids using 13 C-labelled glucose was investigated by gas chromatography-mass spectrometry (GC-MS) analysis. While some amino acids were synthesized up to 90% in vivo from glucose ( e.g., alanine), asparagine and/or aspartate were predominantly taken up from the medium. With this information in hand, a defined yeast free salt medium containing aspartate and ammonium nitrate as a nitrogen source was developed. The observed growth rates of P. sapidus were well comparable with those previously published for complex media. Importantly, fast growth could be observed for 4 days at least, up to cell wet weights (CWW) of 400 g L -1 . The chemically defined medium was used to carry out a 13 C-based metabolic flux analysis, and the in vivo reactions rates in the central carbon metabolism of P. sapidus were investigated. The results revealed a highly respiratory metabolism with high fluxes through the pentose phosphate pathway and TCA cycle. The presented chemically defined growth medium enables researchers to study the metabolism of P. sapidus , significantly enlarging the analytical capabilities. Detailed studies on the production of extracellular enzymes and of secondary metabolites of P. sapidus may be designed based on the reported data.

  2. Metabolic flux analysis of the hydrogen production potential in Synechocystis sp. PCC6803

    Energy Technology Data Exchange (ETDEWEB)

    Navarro, E. [Departamento de Lenguajes y Ciencias de la Computacion, Campus de Teatrinos, Universidad de Malaga, 29071 Malaga (Spain); Montagud, A.; Fernandez de Cordoba, P.; Urchueguia, J.F. [Instituto Universitario de Matematica Pura y Aplicada, Universidad Politecnica de Valencia, Camino de Vera 14, 46022 Valencia (Spain)

    2009-11-15

    Hydrogen is a promising energy vector; however, finding methods to produce it from renewable sources is essential to allow its wide-scale use. In that line, biological hydrogen production, although it is considered as a possible alternative, requires substantial improvements to overcome its present low yields. In that direction, genetic manipulation probably will play a central role and from that point of view metabolic flux analysis (MFA) constitutes an important tool to guide a priori most suitable genetic modifications oriented to a hydrogen yield increase. In this work MFA has been applied to analyze hydrogen photoproduction of Synechocystis sp. PCC6803. Flux analysis was carried out based on literature data and several basic fluxes were estimated in different growing conditions of the system. From this analysis, an upper limit for hydrogen photoproduction has been determined indicating a wide margin for improvement. MFA was also used to find a feasible operating space for hydrogen production, which avoids oxygen inhibition, one of the most important limitations to make hydrogen production cost effective. In addition, a set of biotechnological strategies are proposed that would be consistent with the performed mathematical analysis. (author)

  3. Multi-objective experimental design for (13)C-based metabolic flux analysis.

    Science.gov (United States)

    Bouvin, Jeroen; Cajot, Simon; D'Huys, Pieter-Jan; Ampofo-Asiama, Jerry; Anné, Jozef; Van Impe, Jan; Geeraerd, Annemie; Bernaerts, Kristel

    2015-10-01

    (13)C-based metabolic flux analysis is an excellent technique to resolve fluxes in the central carbon metabolism but costs can be significant when using specialized tracers. This work presents a framework for cost-effective design of (13)C-tracer experiments, illustrated on two different networks. Linear and non-linear optimal input mixtures are computed for networks for Streptomyces lividans and a carcinoma cell line. If only glucose tracers are considered as labeled substrate for a carcinoma cell line or S. lividans, the best parameter estimation accuracy is obtained by mixtures containing high amounts of 1,2-(13)C2 glucose combined with uniformly labeled glucose. Experimental designs are evaluated based on a linear (D-criterion) and non-linear approach (S-criterion). Both approaches generate almost the same input mixture, however, the linear approach is favored due to its low computational effort. The high amount of 1,2-(13)C2 glucose in the optimal designs coincides with a high experimental cost, which is further enhanced when labeling is introduced in glutamine and aspartate tracers. Multi-objective optimization gives the possibility to assess experimental quality and cost at the same time and can reveal excellent compromise experiments. For example, the combination of 100% 1,2-(13)C2 glucose with 100% position one labeled glutamine and the combination of 100% 1,2-(13)C2 glucose with 100% uniformly labeled glutamine perform equally well for the carcinoma cell line, but the first mixture offers a decrease in cost of $ 120 per ml-scale cell culture experiment. We demonstrated the validity of a multi-objective linear approach to perform optimal experimental designs for the non-linear problem of (13)C-metabolic flux analysis. Tools and a workflow are provided to perform multi-objective design. The effortless calculation of the D-criterion can be exploited to perform high-throughput screening of possible (13)C-tracers, while the illustrated benefit of multi

  4. Comprehensive analysis of glucose and xylose metabolism in Escherichia coli under aerobic and anaerobic conditions by13C metabolic flux analysis.

    Science.gov (United States)

    Gonzalez, Jacqueline E; Long, Christopher P; Antoniewicz, Maciek R

    2017-01-01

    Glucose and xylose are the two most abundant sugars derived from the breakdown of lignocellulosic biomass. While aerobic glucose metabolism is relatively well understood in E. coli, until now there have been only a handful of studies focused on anaerobic glucose metabolism and no 13 C-flux studies on xylose metabolism. In the absence of experimentally validated flux maps, constraint-based approaches such as MOMA and RELATCH cannot be used to guide new metabolic engineering designs. In this work, we have addressed this critical gap in current understanding by performing comprehensive characterizations of glucose and xylose metabolism under aerobic and anaerobic conditions, using recent state-of-the-art techniques in 13 C metabolic flux analysis ( 13 C-MFA). Specifically, we quantified precise metabolic fluxes for each condition by performing parallel labeling experiments and analyzing the data through integrated 13 C-MFA using the optimal tracers [1,2- 13 C]glucose, [1,6- 13 C]glucose, [1,2- 13 C]xylose and [5- 13 C]xylose. We also quantified changes in biomass composition and confirmed turnover of macromolecules by applying [U- 13 C]glucose and [U- 13 C]xylose tracers. We demonstrated that under anaerobic growth conditions there is significant turnover of lipids and that a significant portion of CO 2 originates from biomass turnover. Using knockout strains, we also demonstrated that β-oxidation is critical for anaerobic growth on xylose. Quantitative analysis of co-factor balances (NADH/FADH 2 , NADPH, and ATP) for different growth conditions provided new insights regarding the interplay of energy and redox metabolism and the impact on E. coli cell physiology. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  5. Metabolic flux analysis of Saccharomyces cerevisiae in a sealed winemaking fermentation system.

    Science.gov (United States)

    Li, Hua; Su, Jing; Ma, Wen; Guo, Anque; Shan, Zuhua; Wang, Hua

    2015-03-01

    A sealed fermentation (SF) system and an anaerobic fermentation (AF) system (under normal atmospheric pressure conditions) were employed to study the influence of endogenous carbon dioxide (CO2) on the metabolism of Saccharomyces cerevisiae. The results showed that the fermentation stopped when 82.0 g L(-1) glucose was consumed and the endogenously produced CO2: pressure reached to 14.3 MPa in SF system, while the sugar was used up during AF. The total yeast viable count in the end of AF was higher than that of SF. It was also observed that the ethanol yield in AF and SF was similar, the glycerol yield in AF was 1.26 times higher than that in SF, while the succinic acid and acetic acid yields in SF were 24.7 and 26 times higher than that in AF, respectively. Additionally, this work provides a stoichiometric model used for metabolic flux analysis of S. cerevisiae to compare the flux distribution in SF and AF. The results showed that CO2 had an important effect on the pathways of oxaloacetic acid formation from pyruvic acid and ribose-5-phosphate formation from glucose-6-phosphate. However, the pathway of ethanol formation from pyruvic acid (decarboxylation reaction), catalyzed by pyruvate decarboxylase, was insensitive to CO2. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

  6. Metabolic flux ratio analysis and cell staining suggest the existence of C4 photosynthesis in Phaeodactylum tricornutum.

    Science.gov (United States)

    Huang, A; Liu, L; Zhao, P; Yang, C; Wang, G C

    2016-03-01

    Mechanisms for carbon fixation via photosynthesis in the diatom Phaeodactylum tricornutum Bohlin were studied recently but there remains a long-standing debate concerning the occurrence of C4 photosynthesis in this species. A thorough investigation of carbon metabolism and the evidence for C4 photosynthesis based on organelle partitioning was needed. In this study, we identified the flux ratios between C3 and C4 compounds in P. tricornutum using (13)C-labelling metabolic flux ratio analysis, and stained cells with various cell-permeant fluorescent probes to investigate the likely organelle partitioning required for single-cell C4 photosynthesis. Metabolic flux ratio analysis indicated the C3/C4 exchange ratios were high. Cell staining indicated organelle partitioning required for single-cell C4 photosynthesis might exist in P. tricornutum. The results of (13)C-labelling metabolic flux ratio analysis and cell staining suggest single-cell C4 photosynthesis exists in P. tricornutum. This study provides insights into photosynthesis patterns of P. tricornutum and the evidence for C4 photosynthesis based on (13)C-labelling metabolic flux ratio analysis and organelle partitioning. © 2015 The Society for Applied Microbiology.

  7. iMS2Flux – a high–throughput processing tool for stable isotope labeled mass spectrometric data used for metabolic flux analysis

    Directory of Open Access Journals (Sweden)

    Poskar C Hart

    2012-11-01

    Full Text Available Abstract Background Metabolic flux analysis has become an established method in systems biology and functional genomics. The most common approach for determining intracellular metabolic fluxes is to utilize mass spectrometry in combination with stable isotope labeling experiments. However, before the mass spectrometric data can be used it has to be corrected for biases caused by naturally occurring stable isotopes, by the analytical technique(s employed, or by the biological sample itself. Finally the MS data and the labeling information it contains have to be assembled into a data format usable by flux analysis software (of which several dedicated packages exist. Currently the processing of mass spectrometric data is time-consuming and error-prone requiring peak by peak cut-and-paste analysis and manual curation. In order to facilitate high-throughput metabolic flux analysis, the automation of multiple steps in the analytical workflow is necessary. Results Here we describe iMS2Flux, software developed to automate, standardize and connect the data flow between mass spectrometric measurements and flux analysis programs. This tool streamlines the transfer of data from extraction via correction tools to 13C-Flux software by processing MS data from stable isotope labeling experiments. It allows the correction of large and heterogeneous MS datasets for the presence of naturally occurring stable isotopes, initial biomass and several mass spectrometry effects. Before and after data correction, several checks can be performed to ensure accurate data. The corrected data may be returned in a variety of formats including those used by metabolic flux analysis software such as 13CFLUX, OpenFLUX and 13CFLUX2. Conclusion iMS2Flux is a versatile, easy to use tool for the automated processing of mass spectrometric data containing isotope labeling information. It represents the core framework for a standardized workflow and data processing. Due to its flexibility

  8. Reconstruction and flux analysis of coupling between metabolic pathways of astrocytes and neurons: application to cerebral hypoxia

    Directory of Open Access Journals (Sweden)

    Akιn Ata

    2007-12-01

    Full Text Available Abstract Background It is a daunting task to identify all the metabolic pathways of brain energy metabolism and develop a dynamic simulation environment that will cover a time scale ranging from seconds to hours. To simplify this task and make it more practicable, we undertook stoichiometric modeling of brain energy metabolism with the major aim of including the main interacting pathways in and between astrocytes and neurons. Model The constructed model includes central metabolism (glycolysis, pentose phosphate pathway, TCA cycle, lipid metabolism, reactive oxygen species (ROS detoxification, amino acid metabolism (synthesis and catabolism, the well-known glutamate-glutamine cycle, other coupling reactions between astrocytes and neurons, and neurotransmitter metabolism. This is, to our knowledge, the most comprehensive attempt at stoichiometric modeling of brain metabolism to date in terms of its coverage of a wide range of metabolic pathways. We then attempted to model the basal physiological behaviour and hypoxic behaviour of the brain cells where astrocytes and neurons are tightly coupled. Results The reconstructed stoichiometric reaction model included 217 reactions (184 internal, 33 exchange and 216 metabolites (183 internal, 33 external distributed in and between astrocytes and neurons. Flux balance analysis (FBA techniques were applied to the reconstructed model to elucidate the underlying cellular principles of neuron-astrocyte coupling. Simulation of resting conditions under the constraints of maximization of glutamate/glutamine/GABA cycle fluxes between the two cell types with subsequent minimization of Euclidean norm of fluxes resulted in a flux distribution in accordance with literature-based findings. As a further validation of our model, the effect of oxygen deprivation (hypoxia on fluxes was simulated using an FBA-derivative approach, known as minimization of metabolic adjustment (MOMA. The results show the power of the

  9. Flux Balance Analysis of Cyanobacterial Metabolism.The Metabolic Network of Synechocystis sp. PCC 6803

    Czech Academy of Sciences Publication Activity Database

    Knoop, H.; Gründel, M.; Zilliges, Y.; Lehmann, R.; Hoffmann, S.; Lockau, W.; Steuer, Ralf

    2013-01-01

    Roč. 9, č. 6 (2013), e1003081-e1003081 ISSN 1553-7358 R&D Projects: GA MŠk(CZ) EE2.3.20.0256 Institutional support: RVO:67179843 Keywords : SP STRAIN PCC-6803 * SP ATCC 51142 * photoautotrophic metabolism * anacystis-nidulans * reconstructions * pathway * plants * models * growth Subject RIV: EI - Biotechnology ; Bionics Impact factor: 4.829, year: 2013

  10. Investigation of useful carbon tracers for 13C-metabolic flux analysis of Escherichia coli by considering five experimentally determined flux distributions.

    Science.gov (United States)

    Maeda, Kousuke; Okahashi, Nobuyuki; Toya, Yoshihiro; Matsuda, Fumio; Shimizu, Hiroshi

    2016-12-01

    The 13 C-MFA experiments require an optimal design since the precision or confidence intervals of the estimated flux levels depends on factors such as the composition of 13 C-labeled carbon sources, as well as the metabolic flux distribution of interest. In this study, useful compositions of 13 C-labeled glucose for 13 C-metabolic flux analysis ( 13 C-MFA) of Escherichia coli are investigated using a computer simulation of the stable isotope labeling experiment. Following the generation of artificial mass spectra datasets of amino acid fragments using five literature-reported flux distributions of E. coli , the best fitted flux distribution and the 95% confidence interval were estimated by the 13 C-MFA procedure. A comparison of the precision scores showed that [1, 2- 13 C]glucose and a mixture of [1- 13 C] and [U- 13 C]glucose at 8:2 are one of the best carbon sources for a precise estimation of flux levels of the pentose phosphate pathway, glycolysis and the TCA cycle. Although the precision scores of the anaplerotic and glyoxylate pathway reactions were affected by both the carbon source and flux distribution, it was also shown that the mixture of non-labeled, [1- 13 C], and [U- 13 C]glucose at 4:1:5 was specifically effective for the flux estimation of the glyoxylate pathway reaction. These findings were confirmed by wet 13 C-MFA experiments.

  11. Analysis of polyhydroxybutyrate flux limitations by systematic genetic and metabolic perturbations.

    Science.gov (United States)

    Tyo, Keith E J; Fischer, Curt R; Simeon, Fritz; Stephanopoulos, Gregory

    2010-05-01

    Poly-3-hydroxybutyrate (PHB) titers in Escherichia coli have benefited from 10+ years of metabolic engineering. In the majority of studies, PHB content, expressed as percent PHB (dry cell weight), is increased, although this increase can be explained by decreases in growth rate or increases in PHB flux. In this study, growth rate and PHB flux were quantified directly in response to systematic manipulation of (1) gene expression in the product-forming pathway and (2) growth rates in a nitrogen-limited chemostat. Gene expression manipulation revealed acetoacetyl-CoA reductase (phaB) limits flux to PHB, although overexpression of the entire pathway pushed the flux even higher. These increases in PHB flux are accompanied by decreases in growth rate, which can be explained by carbon diversion, rather than toxic effects of the PHB pathway. In chemostats, PHB flux was insensitive to growth rate. These results imply that PHB flux is primarily controlled by the expression levels of the product forming pathway and not by the availability of precursors. These results confirm prior in vitro measurements and metabolic models and show expression level is a major affecter of PHB flux. 2009 Elsevier Inc. All rights reserved.

  12. Producing Acetic Acid of Acetobacter pasteurianus by Fermentation Characteristics and Metabolic Flux Analysis.

    Science.gov (United States)

    Wu, Xuefeng; Yao, Hongli; Liu, Qing; Zheng, Zhi; Cao, Lili; Mu, Dongdong; Wang, Hualin; Jiang, Shaotong; Li, Xingjiang

    2018-03-19

    The acetic acid bacterium Acetobacter pasteurianus plays an important role in acetic acid fermentation, which involves oxidation of ethanol to acetic acid through the ethanol respiratory chain under specific conditions. In order to obtain more suitable bacteria for the acetic acid industry, A. pasteurianus JST-S screened in this laboratory was compared with A. pasteurianus CICC 20001, a current industrial strain in China, to determine optimal fermentation parameters under different environmental stresses. The maximum total acid content of A. pasteurianus JST-S was 57.14 ± 1.09 g/L, whereas that of A. pasteurianus CICC 20001 reached 48.24 ± 1.15 g/L in a 15-L stir stank. Metabolic flux analysis was also performed to compare the reaction byproducts. Our findings revealed the potential value of the strain in improvement of industrial vinegar fermentation.

  13. Optimal tracers for parallel labeling experiments and13C metabolic flux analysis: A new precision and synergy scoring system.

    Science.gov (United States)

    Crown, Scott B; Long, Christopher P; Antoniewicz, Maciek R

    2016-11-01

    13 C-Metabolic flux analysis ( 13 C-MFA) is a widely used approach in metabolic engineering for quantifying intracellular metabolic fluxes. The precision of fluxes determined by 13 C-MFA depends largely on the choice of isotopic tracers and the specific set of labeling measurements. A recent advance in the field is the use of parallel labeling experiments for improved flux precision and accuracy. However, as of today, no systemic methods exist for identifying optimal tracers for parallel labeling experiments. In this contribution, we have addressed this problem by introducing a new scoring system and evaluating thousands of different isotopic tracer schemes. Based on this extensive analysis we have identified optimal tracers for 13 C-MFA. The best single tracers were doubly 13 C-labeled glucose tracers, including [1,6- 13 C]glucose, [5,6- 13 C]glucose and [1,2- 13 C]glucose, which consistently produced the highest flux precision independent of the metabolic flux map (here, 100 random flux maps were evaluated). Moreover, we demonstrate that pure glucose tracers perform better overall than mixtures of glucose tracers. For parallel labeling experiments the optimal isotopic tracers were [1,6- 13 C]glucose and [1,2- 13 C]glucose. Combined analysis of [1,6- 13 C]glucose and [1,2- 13 C]glucose labeling data improved the flux precision score by nearly 20-fold compared to widely use tracer mixture 80% [1- 13 C]glucose +20% [U- 13 C]glucose. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  14. Metabolic flux ratio analysis and multi-objective optimization revealed a globally conserved and coordinated metabolic response of E. coli to paraquat-induced oxidative stress.

    Science.gov (United States)

    Shen, Tie; Rui, Bin; Zhou, Hong; Zhang, Ximing; Yi, Yin; Wen, Han; Zheng, Haoran; Wu, Jihui; Shi, Yunyu

    2013-01-27

    The ability of a microorganism to adapt to changes in the environment, such as in nutrient or oxygen availability, is essential for its competitive fitness and survival. The cellular objective and the strategy of the metabolic response to an extreme environment are therefore of tremendous interest and, thus, have been increasingly explored. However, the cellular objective of the complex regulatory structure of the metabolic changes has not yet been fully elucidated and more details regarding the quantitative behaviour of the metabolic flux redistribution are required to understand the systems-wide biological significance of this response. In this study, the intracellular metabolic flux ratios involved in the central carbon metabolism were determined by fractional (13)C-labeling and metabolic flux ratio analysis (MetaFoR) of the wild-type E. coli strain JM101 at an oxidative environment in a chemostat. We observed a significant increase in the flux through phosphoenolpyruvate carboxykinase (PEPCK), phosphoenolpyruvate carboxylase (PEPC), malic enzyme (MEZ) and serine hydroxymethyltransferase (SHMT). We applied an ε-constraint based multi-objective optimization to investigate the trade-off relationships between the biomass yield and the generation of reductive power using the in silico iJR904 genome-scale model of E. coli K-12. The theoretical metabolic redistribution supports that the trans-hydrogenase pathway should not play a direct role in the defence mounted by E. coli against oxidative stress. The agreement between the measured ratio and the theoretical redistribution established the significance of NADPH synthesis as the goal of the metabolic reprogramming that occurs in response to oxidative stress. Our work presents a framework that combines metabolic flux ratio analysis and multi-objective optimization to investigate the metabolic trade-offs that occur under varied environmental conditions. Our results led to the proposal that the metabolic response of E

  15. Metabolic engineering of Synechocystis sp. PCC 6803 for enhanced ethanol production based on flux balance analysis.

    Science.gov (United States)

    Yoshikawa, Katsunori; Toya, Yoshihiro; Shimizu, Hiroshi

    2017-05-01

    Synechocystis sp. PCC 6803 is an attractive host for bio-ethanol production due to its ability to directly convert atmospheric carbon dioxide into ethanol using photosystems. To enhance ethanol production in Synechocystis sp. PCC 6803, metabolic engineering was performed based on in silico simulations, using the genome-scale metabolic model. Comprehensive reaction knockout simulations by flux balance analysis predicted that the knockout of NAD(P)H dehydrogenase enhanced ethanol production under photoautotrophic conditions, where ammonium is the nitrogen source. This deletion inhibits the re-oxidation of NAD(P)H, which is generated by ferredoxin-NADP + reductase and imposes re-oxidation in the ethanol synthesis pathway. The effect of deleting the ndhF1 gene, which encodes NADH dehydrogenase subunit 5, on ethanol production was experimentally evaluated using ethanol-producing strains of Synechocystis sp. PCC 6803. The ethanol titer of the ethanol-producing ∆ndhF1 strain increased by 145%, compared with that of the control strain.

  16. C-13 Tracer experiments and metabolite balancing for metabolic flux analysis

    DEFF Research Database (Denmark)

    Schmidt, Karsten; Marx, A.; de Graaf, A. A.

    1998-01-01

    through the pentose phosphate pathway. Hence, wrong assumptions on the presence or activity of transhydrogenation reactions will result in wrong estimations of the intracellular flux distribution. Using C-13 tracer experiments and NMR analysis, flux analysis can be performed on the basis of only well...

  17. Resuscitation of ischemic donor livers with normothermic machine perfusion: a metabolic flux analysis of treatment in rats.

    Directory of Open Access Journals (Sweden)

    Maria-Louisa Izamis

    Full Text Available Normothermic machine perfusion has previously been demonstrated to restore damaged warm ischemic livers to transplantable condition in animal models. However, the mechanisms of recovery are unclear, preventing rational optimization of perfusion systems and slowing clinical translation of machine perfusion. In this study, organ recovery time and major perfusate shortcomings were evaluated using a comprehensive metabolic analysis of organ function in perfusion prior to successful transplantation. Two groups, Fresh livers and livers subjected to 1 hr of warm ischemia (WI received perfusion for a total preservation time of 6 hrs, followed by successful transplantation. 24 metabolic fluxes were directly measured and 38 stoichiometrically-related fluxes were estimated via a mass balance model of the major pathways of energy metabolism. This analysis revealed stable metabolism in Fresh livers throughout perfusion while identifying two distinct metabolic states in WI livers, separated at t = 2 hrs, coinciding with recovery of oxygen uptake rates to Fresh liver values. This finding strongly suggests successful organ resuscitation within 2 hrs of perfusion. Overall perfused livers regulated metabolism of perfusate substrates according to their metabolic needs, despite supraphysiological levels of some metabolites. This study establishes the first integrative metabolic basis for the dynamics of recovery during perfusion treatment of marginal livers. Our initial findings support enhanced oxygen delivery for both timely recovery and long-term sustenance. These results are expected to lead the optimization of the treatment protocols and perfusion media from a metabolic perspective, facilitating translation to clinical use.

  18. Integrating flux balance analysis into kinetic models to decipher the dynamic metabolism of Shewanella oneidensis MR-1.

    Directory of Open Access Journals (Sweden)

    Xueyang Feng

    2012-02-01

    Full Text Available Shewanella oneidensis MR-1 sequentially utilizes lactate and its waste products (pyruvate and acetate during batch culture. To decipher MR-1 metabolism, we integrated genome-scale flux balance analysis (FBA into a multiple-substrate Monod model to perform the dynamic flux balance analysis (dFBA. The dFBA employed a static optimization approach (SOA by dividing the batch time into small intervals (i.e., ∼400 mini-FBAs, then the Monod model provided time-dependent inflow/outflow fluxes to constrain the mini-FBAs to profile the pseudo-steady-state fluxes in each time interval. The mini-FBAs used a dual-objective function (a weighted combination of "maximizing growth rate" and "minimizing overall flux" to capture trade-offs between optimal growth and minimal enzyme usage. By fitting the experimental data, a bi-level optimization of dFBA revealed that the optimal weight in the dual-objective function was time-dependent: the objective function was constant in the early growth stage, while the functional weight of minimal enzyme usage increased significantly when lactate became scarce. The dFBA profiled biologically meaningful dynamic MR-1 metabolisms: 1. the oxidative TCA cycle fluxes increased initially and then decreased in the late growth stage; 2. fluxes in the pentose phosphate pathway and gluconeogenesis were stable in the exponential growth period; and 3. the glyoxylate shunt was up-regulated when acetate became the main carbon source for MR-1 growth.

  19. Flux analysis of central metabolic pathways in Geobactermetallireducens during reduction of solubleFe(III)-NTA

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Yinjie J.; Chakraborty, Romy; Garcia-Martin, Hector; Chu,Jeannie; Hazen, Terry C.; Keasling, Jay D.

    2007-01-01

    We analyzed the carbon fluxes in the central metabolism ofGeobacter metallireducens strain GS-15 using 13C isotopomer modeling.Acetate labeled in the 1st or 2nd position was the sole carbon source,and Fe-NTA was the sole terminal electron acceptor. The measured labeledacetate uptake rate was 21 mmol/gdw/h in the exponential growth phase.The resulting isotope labeling pattern of amino acids allowed an accuratedetermination of the in vivo global metabolic reaction rates (fluxes)through the central metabolic pathways using a computational isotopomermodel. The tracer experiments showed that G. metallireducens containedcomplete biosynthesis pathways for essential metabolism, and this strainmight also have an unusual isoleucine biosynthesis route (usingacetyl-CoA and pyruvate as the precursors). The model indicated that over90 percent of the acetate was completely oxidized to CO2 via a completetricarboxylic acid (TCA) cycle while reducing iron. Pyruvate carboxylaseand phosphoenolpyruvate carboxykinase were present under theseconditions, but enzymes in the glyoxylate shunt and malic enzyme wereabsent. Gluconeogenesis and the pentose phosphate pathway were mainlyemployed for biosynthesis and accounted for less than 3 percent of totalcarbon consumption. The model also indicated surprisingly highreversibility in the reaction between oxoglutarate and succinate. Thisstep operates close to the thermodynamic equilibrium possibly becausesuccinate is synthesized via a transferase reaction, and the conversionof oxoglutarate to succinate is a rate limiting step for carbonmetabolism. These findings enable a better understanding of therelationship between genome annotation and extant metabolic pathways inG. metallireducens.

  20. Quantitative Metabolomics and Instationary 13C-Metabolic Flux Analysis Reveals Impact of Recombinant Protein Production on Trehalose and Energy Metabolism in Pichia pastoris

    NARCIS (Netherlands)

    Jorda, J.; Cueto Rojas, H.F.; Carnicer, M.; Wahl, S.A.; Ferrer, P.; Albiol, J.

    2014-01-01

    Pichia pastoris has been recognized as an effective host for recombinant protein production. In this work, we combine metabolomics and instationary 13C metabolic flux analysis (INST 13C-MFA) using GC-MS and LC-MS/MS to evaluate the potential impact of the production of a Rhizopus oryzae lipase (Rol)

  1. Rerouting of carbon flux in a glycogen mutant of cyanobacteria assessed via isotopically non-stationary13C metabolic flux analysis.

    Science.gov (United States)

    Hendry, John I; Prasannan, Charulata; Ma, Fangfang; Möllers, K Benedikt; Jaiswal, Damini; Digmurti, Madhuri; Allen, Doug K; Frigaard, Niels-Ulrik; Dasgupta, Santanu; Wangikar, Pramod P

    2017-10-01

    Cyanobacteria, which constitute a quantitatively dominant phylum, have attracted attention in biofuel applications due to favorable physiological characteristics, high photosynthetic efficiency and amenability to genetic manipulations. However, quantitative aspects of cyanobacterial metabolism have received limited attention. In the present study, we have performed isotopically non-stationary 13 C metabolic flux analysis (INST- 13 C-MFA) to analyze rerouting of carbon in a glycogen synthase deficient mutant strain (glgA-I glgA-II) of the model cyanobacterium Synechococcus sp. PCC 7002. During balanced photoautotrophic growth, 10-20% of the fixed carbon is stored in the form of glycogen via a pathway that is conserved across the cyanobacterial phylum. Our results show that deletion of glycogen synthase gene orchestrates cascading effects on carbon distribution in various parts of the metabolic network. Carbon that was originally destined to be incorporated into glycogen gets partially diverted toward alternate storage molecules such as glucosylglycerol and sucrose. The rest is partitioned within the metabolic network, primarily via glycolysis and tricarboxylic acid cycle. A lowered flux toward carbohydrate synthesis and an altered distribution at the glucose-1-phosphate node indicate flexibility in the network. Further, reversibility of glycogen biosynthesis reactions points toward the presence of futile cycles. Similar redistribution of carbon was also predicted by Flux Balance Analysis. The results are significant to metabolic engineering efforts with cyanobacteria where fixed carbon needs to be re-routed to products of interest. Biotechnol. Bioeng. 2017;114: 2298-2308. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  2. Flux balance analysis of genome-scale metabolic model of rice ...

    Indian Academy of Sciences (India)

    2015-09-28

    Sep 28, 2015 ... producing vitamin A–enriched golden rice (Beyer et al. 2002). On the other hand, metabolic engineering has been proven as a powerful technique for over or less production of specific metabolites in microbes (Peralta-Yahya et al. 2012). Modelling and analysis of the cellular metabolism is a key step in the ...

  3. Optimal flux patterns in cellular metabolic networks

    Energy Technology Data Exchange (ETDEWEB)

    Almaas, E

    2007-01-20

    The availability of whole-cell level metabolic networks of high quality has made it possible to develop a predictive understanding of bacterial metabolism. Using the optimization framework of flux balance analysis, I investigate metabolic response and activity patterns to variations in the availability of nutrient and chemical factors such as oxygen and ammonia by simulating 30,000 random cellular environments. The distribution of reaction fluxes is heavy-tailed for the bacteria H. pylori and E. coli, and the eukaryote S. cerevisiae. While the majority of flux balance investigations have relied on implementations of the simplex method, it is necessary to use interior-point optimization algorithms to adequately characterize the full range of activity patterns on metabolic networks. The interior-point activity pattern is bimodal for E. coli and S. cerevisiae, suggesting that most metabolic reaction are either in frequent use or are rarely active. The trimodal activity pattern of H. pylori indicates that a group of its metabolic reactions (20%) are active in approximately half of the simulated environments. Constructing the high-flux backbone of the network for every environment, there is a clear trend that the more frequently a reaction is active, the more likely it is a part of the backbone. Finally, I briefly discuss the predicted activity patterns of the central-carbon metabolic pathways for the sample of random environments.

  4. (13)C-metabolic flux analysis of lipid accumulation in the oleaginous fungus Mucor circinelloides.

    Science.gov (United States)

    Zhao, Lina; Zhang, Huaiyuan; Wang, Liping; Chen, Haiqin; Chen, Yong Q; Chen, Wei; Song, Yuanda

    2015-12-01

    The oleaginous fungus Mucor circinelloides is of industrial interest because it can produce high levels of polyunsaturated fatty acid γ-linolenic acid. M. circinelloides CBS 277.49 is able to accumulate less than 15% of cell dry weight as lipids, while M. circinelloides WJ11 can accumulate lipid up to 36%. In order to better understand the mechanisms behind the differential lipid accumulation in these two strains, tracer experiments with (13)C-glucose were performed with the growth of M. circinelloides and subsequent gas chromatography-mass spectrometric detection of (13)C-patterns in proteinogenic amino acids was carried out to identify the metabolic network topology and estimate intracellular fluxes. Our results showed that the high oleaginous strain WJ11 had higher flux of pentose phosphate pathway and malic enzyme, lower flux in tricarboxylic acid cycle, higher flux in glyoxylate cycle and ATP: citrate lyase, together, it might provide more NADPH and substrate acetyl-CoA for fatty acid synthesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Semi-automated curation of metabolic models via flux balance analysis: a case study with Mycoplasma gallisepticum.

    Directory of Open Access Journals (Sweden)

    Eddy J Bautista

    Full Text Available Primarily used for metabolic engineering and synthetic biology, genome-scale metabolic modeling shows tremendous potential as a tool for fundamental research and curation of metabolism. Through a novel integration of flux balance analysis and genetic algorithms, a strategy to curate metabolic networks and facilitate identification of metabolic pathways that may not be directly inferable solely from genome annotation was developed. Specifically, metabolites involved in unknown reactions can be determined, and potentially erroneous pathways can be identified. The procedure developed allows for new fundamental insight into metabolism, as well as acting as a semi-automated curation methodology for genome-scale metabolic modeling. To validate the methodology, a genome-scale metabolic model for the bacterium Mycoplasma gallisepticum was created. Several reactions not predicted by the genome annotation were postulated and validated via the literature. The model predicted an average growth rate of 0.358±0.12[Formula: see text], closely matching the experimentally determined growth rate of M. gallisepticum of 0.244±0.03[Formula: see text]. This work presents a powerful algorithm for facilitating the identification and curation of previously known and new metabolic pathways, as well as presenting the first genome-scale reconstruction of M. gallisepticum.

  6. Metabolic Flux Analysis of the Synechocystis sp. PCC 6803 ΔnrtABCD Mutant Reveals a Mechanism for Metabolic Adaptation to Nitrogen-Limited Conditions.

    Science.gov (United States)

    Nakajima, Tsubasa; Yoshikawa, Katsunori; Toya, Yoshihiro; Matsuda, Fumio; Shimizu, Hiroshi

    2017-03-01

    Metabolic flux redirection during nitrogen-limited growth was investigated in the Synechocystis sp. PCC 6803 glucose-tolerant (GT) strain under photoautotrophic conditions by isotopically non-stationary metabolic flux analysis (INST-MFA). A ΔnrtABCD mutant of Synechocystis sp. PCC 6803 was constructed to reproduce phenotypes arising during nitrogen starvation. The ΔnrtABCD mutant and the wild-type GT strain were cultured under photoautotrophic conditions by a photobioreactor. Intracellular metabolites were labeled over a time course using NaH13CO3 as a carbon source. Based on these data, the metabolic flux distributions in the wild-type and ΔnrtABCD cells were estimated by INST-MFA. The wild-type GT and ΔnrtABCD strains displayed similar distribution patterns, although the absolute levels of metabolic flux were lower in ΔnrtABCD. Furthermore, the relative flux levels for glycogen metabolism, anaplerotic reactions and the oxidative pentose phosphate pathway were increased in ΔnrtABCD. This was probably due to the increased expression of enzyme genes that respond to nitrogen depletion. Additionally, we found that the ratio of ATP/NADPH demand increased slightly in the ΔnrtABCD mutant. These results indicated that futile ATP consumption increases under nitrogen-limited conditions because the Calvin-Benson cycle and the oxidative pentose phosphate pathway form a metabolic futile cycle that consumes ATP without CO2 fixation and NADPH regeneration. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  7. Analysis of metabolic flux phenotypes for two Arabidopsis mutants with severe impairment in seed storage lipid synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Lonien, J.; Schwender, J.

    2009-11-01

    Major storage reserves of Arabidopsis (Arabidopsis thaliana) seeds are triacylglycerols (seed oils) and proteins. Seed oil content is severely reduced for the regulatory mutant wrinkled1 (wri1-1; At3g54320) and for a double mutant in two isoforms of plastidic pyruvate kinase (pkp{beta}{sub 1}pkp{alpha}; At5g52920 and At3g22960). Both already biochemically well-characterized mutants were now studied by {sup 13}C metabolic flux analysis of cultured developing embryos based on comparison with their respective genetic wild-type backgrounds. For both mutations, in seeds as well as in cultured embryos, the oil fraction was strongly reduced while the fractions of proteins and free metabolites increased. Flux analysis in cultured embryos revealed changes in nutrient uptakes and fluxes into biomass as well as an increase in tricarboxylic acid cycle activity for both mutations. While in both wild types plastidic pyruvate kinase (PK{sub p}) provides most of the pyruvate for plastidic fatty acid synthesis, the flux through PK{sub p} is reduced in pkp{beta}{sub 1}pkp{alpha} by 43% of the wild-type value. In wri1-1, PK{sub p} flux is even more reduced (by 82%), although the genes PKp{beta}{sub 1} and PKp{alpha} are still expressed. Along a common paradigm of metabolic control theory, it is hypothesized that a large reduction in PK{sub p} enzyme activity in pkp{beta}{sub 1}pkp{alpha} has less effect on PK{sub p} flux than multiple smaller reductions in glycolytic enzymes in wri1-1. In addition, only in the wri1-1 mutant is the large reduction in PK{sub p} flux compensated in part by an increased import of cytosolic pyruvate and by plastidic malic enzyme. No such limited compensatory bypass could be observed in pkp{beta}{sub 1}pkp{alpha}.

  8. Quantitative Metabolomics and Instationary 13C-Metabolic Flux Analysis Reveals Impact of Recombinant Protein Production on Trehalose and Energy Metabolism in Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Joel Jordà

    2014-05-01

    Full Text Available Pichia pastoris has been recognized as an effective host for recombinant protein production. In this work, we combine metabolomics and instationary 13C metabolic flux analysis (INST 13C-MFA using GC-MS and LC-MS/MS to evaluate the potential impact of the production of a Rhizopus oryzae lipase (Rol on P. pastoris central carbon metabolism. Higher oxygen uptake and CO2 production rates and slightly reduced biomass yield suggest an increased energy demand for the producing strain. This observation is further confirmed by 13C-based metabolic flux analysis. In particular, the flux through the methanol oxidation pathway and the TCA cycle was increased in the Rol-producing strain compared to the reference strain. Next to changes in the flux distribution, significant variations in intracellular metabolite concentrations were observed. Most notably, the pools of trehalose, which is related to cellular stress response, and xylose, which is linked to methanol assimilation, were significantly increased in the recombinant strain.

  9. Pool size measurements facilitate the determination of fluxes at branching points in nonstationary metabolic flux analysis: The case of Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Robert eHeise

    2015-06-01

    Full Text Available Pool size measurements are important for the estimation of absolute intracellular fluxes in particular scenarios based on data from heavy carbon isotope experiments. Recently, steady-state fluxes estimates were obtained for central carbon metabolism in an intact illuminated rosette of Arabidopsis thaliana grown photoautotrophically (Szecowka et al., 2013; Heise et al., 2014. Fluxes were estimated therein by integrating mass-spectrometric data of the dynamics of the unlabeled metabolic fraction, data on metabolic pool sizes, partitioning of metabolic pools between cellular compartments and estimates of photosynthetically inactive pools, with a simplified model of plant central carbon metabolism. However, the fluxes were determined by treating the pool sizes as fixed parameters. Here we investigated whether and, if so, to what extent the treatment of pool sizes as parameters to be optimized in three scenarios may affect the flux estimates. The results are discussed in terms of benchmark values for canonical pathways and reactions, including starch and sucrose synthesis as well as the ribulose-1,5-bisphosphate carboxylation and oxygenation reactions. In addition, we discuss pathways emerging from a divergent branch point for which pool sizes are required for flux estimation, irrespective of the computational approach used for the simulation of the observable labelling pattern. Therefore, our findings indicate the necessity for development of techniques for accurate pool size measurements to improve the quality of flux estimates from nonstationary flux estimates in intact plant cells in the absence of alternative flux measurements.

  10. Integrated analysis of gene expression and metabolic fluxes in PHA-producing Pseudomonas putida grown on glycerol.

    Science.gov (United States)

    Beckers, Veronique; Poblete-Castro, Ignacio; Tomasch, Jürgen; Wittmann, Christoph

    2016-05-03

    Given its high surplus and low cost, glycerol has emerged as interesting carbon substrate for the synthesis of value-added chemicals. The soil bacterium Pseudomonas putida KT2440 can use glycerol to synthesize medium-chain-length poly(3-hydroxyalkanoates) (mcl-PHA), a class of biopolymers of industrial interest. Here, glycerol metabolism in P. putida KT2440 was studied on the level of gene expression (transcriptome) and metabolic fluxes (fluxome), using precisely adjusted chemostat cultures, growth kinetics and stoichiometry, to gain a systematic understanding of the underlying metabolic and regulatory network. Glycerol-grown P. putida KT2440 has a maintenance energy requirement [0.039 (mmolglycerol (gCDW h)(-1))] that is about sixteen times lower than that of other bacteria, such as Escherichia coli, which provides a great advantage to use this substrate commercially. The shift from carbon (glycerol) to nitrogen (ammonium) limitation drives the modulation of specific genes involved in glycerol metabolism, transport electron chain, sensors to assess the energy level of the cell, and PHA synthesis, as well as changes in flux distribution to increase the precursor availability for PHA synthesis (Entner-Doudoroff pathway and pyruvate metabolism) and to reduce respiration (glyoxylate shunt). Under PHA-producing conditions (N-limitation), a higher PHA yield was achieved at low dilution rate (29.7 wt% of CDW) as compared to a high rate (12.8 wt% of CDW). By-product formation (succinate, malate) was specifically modulated under these regimes. On top of experimental data, elementary flux mode analysis revealed the metabolic potential of P. putida KT2440 to synthesize PHA and identified metabolic engineering targets towards improved production performance on glycerol. This study revealed the complex interplay of gene expression levels and metabolic fluxes under PHA- and non-PHA producing conditions using the attractive raw material glycerol as carbon substrate. This

  11. Derivative processes for modelling metabolic fluxes

    Science.gov (United States)

    Žurauskienė, Justina; Kirk, Paul; Thorne, Thomas; Pinney, John; Stumpf, Michael

    2014-01-01

    Motivation: One of the challenging questions in modelling biological systems is to characterize the functional forms of the processes that control and orchestrate molecular and cellular phenotypes. Recently proposed methods for the analysis of metabolic pathways, for example, dynamic flux estimation, can only provide estimates of the underlying fluxes at discrete time points but fail to capture the complete temporal behaviour. To describe the dynamic variation of the fluxes, we additionally require the assumption of specific functional forms that can capture the temporal behaviour. However, it also remains unclear how to address the noise which might be present in experimentally measured metabolite concentrations. Results: Here we propose a novel approach to modelling metabolic fluxes: derivative processes that are based on multiple-output Gaussian processes (MGPs), which are a flexible non-parametric Bayesian modelling technique. The main advantages that follow from MGPs approach include the natural non-parametric representation of the fluxes and ability to impute the missing data in between the measurements. Our derivative process approach allows us to model changes in metabolite derivative concentrations and to characterize the temporal behaviour of metabolic fluxes from time course data. Because the derivative of a Gaussian process is itself a Gaussian process, we can readily link metabolite concentrations to metabolic fluxes and vice versa. Here we discuss how this can be implemented in an MGP framework and illustrate its application to simple models, including nitrogen metabolism in Escherichia coli. Availability and implementation: R code is available from the authors upon request. Contact: j.norkunaite@imperial.ac.uk; m.stumpf@imperial.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24578401

  12. Genealogy profiling through strain improvement by using metabolic network analysis: metabolic flux genealogy of several generations of lysine-producing corynebacteria.

    Science.gov (United States)

    Wittmann, Christoph; Heinzle, Elmar

    2002-12-01

    A comprehensive approach of metabolite balancing, (13)C tracer studies, gas chromatography-mass spectrometry, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and isotopomer modeling was applied for comparative metabolic network analysis of a genealogy of five successive generations of lysine-producing Corynebacterium glutamicum. The five strains examined (C. glutamicum ATCC 13032, 13287, 21253, 21526, and 21543) were previously obtained by random mutagenesis and selection. Throughout the genealogy, the lysine yield in batch cultures increased markedly from 1.2 to 24.9% relative to the glucose uptake flux. Strain optimization was accompanied by significant changes in intracellular flux distributions. The relative pentose phosphate pathway (PPP) flux successively increased, clearly corresponding to the product yield. Moreover, the anaplerotic net flux increased almost twofold as a consequence of concerted regulation of C(3) carboxylation and C(4) decarboxylation fluxes to cover the increased demand for lysine formation; thus, the overall increase was a consequence of concerted regulation of C(3) carboxylation and C(4) decarboxylation fluxes. The relative flux through isocitrate dehydrogenase dropped from 82.7% in the wild type to 59.9% in the lysine-producing mutants. In contrast to the NADPH demand, which increased from 109 to 172% due to the increasing lysine yield, the overall NADPH supply remained constant between 185 and 196%, resulting in a decrease in the apparent NADPH excess through strain optimization. Extrapolated to industrial lysine producers, the NADPH supply might become a limiting factor. The relative contributions of PPP and the tricarboxylic acid cycle to NADPH generation changed markedly, indicating that C. glutamicum is able to maintain a constant supply of NADPH under completely different flux conditions. Statistical analysis by a Monte Carlo approach revealed high precision for the estimated fluxes, underlining the

  13. Analysis of Metabolic Pathways and Fluxes in a Newly Discovered Thermophilic and Ethanol-Tolerant Geobacillus Strain

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Yinjie J.; Sapra, Rajat; Joyner, Dominique; Hazen, Terry C.; Myers, Samuel; Reichmuth, David; Blanch, Harvey; Keasling, Jay D.

    2009-01-20

    A recently discovered thermophilic bacterium, Geobacillus thermoglucosidasius M10EXG, ferments a range of C5 (e.g., xylose) and C6 sugars (e.g., glucose) and istolerant to high ethanol concentrations (10percent, v/v). We have investigated the central metabolism of this bacterium using both in vitro enzyme assays and 13C-based flux analysis to provide insights into the physiological properties of this extremophile and explore its metabolism for bio-ethanol or other bioprocess applications. Our findings show that glucose metabolism in G. thermoglucosidasius M10EXG proceeds via glycolysis, the pentose phosphate pathway, and the TCA cycle; the Entner?Doudoroff pathway and transhydrogenase activity were not detected. Anaplerotic reactions (including the glyoxylate shunt, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase) were active, but fluxes through those pathways could not be accuratelydetermined using amino acid labeling. When growth conditions were switched from aerobic to micro-aerobic conditions, fluxes (based on a normalized glucose uptake rate of 100 units (g DCW)-1 h-1) through the TCA cycle and oxidative pentose phosphate pathway were reduced from 64+-3 to 25+-2 and from 30+-2 to 19+-2, respectively. The carbon flux under micro-aerobic growth was directed formate. Under fully anerobic conditions, G. thermoglucosidasius M10EXG used a mixed acid fermentation process and exhibited a maximum ethanol yield of 0.38+-0.07 mol mol-1 glucose. In silico flux balance modeling demonstrates that lactate and acetate production from G. thermoglucosidasius M10EXG reduces the maximum ethanol yieldby approximately threefold, thus indicating that both pathways should be modified to maximize ethanol production.

  14. Characterization of the metabolic shift between oxidative and fermentative growth in Saccharomyces cerevisiae by comparative 13C flux analysis

    Directory of Open Access Journals (Sweden)

    Wittmann Christoph

    2005-11-01

    Full Text Available Abstract Background One of the most fascinating properties of the biotechnologically important organism Saccharomyces cerevisiae is its ability to perform simultaneous respiration and fermentation at high growth rate even under fully aerobic conditions. In the present work, this Crabtree effect called phenomenon was investigated in detail by comparative 13C metabolic flux analysis of S. cerevisiae growing under purely oxidative, respiro-fermentative and predominantly fermentative conditions. Results The metabolic shift from oxidative to fermentative growth was accompanied by complex changes of carbon flux throughout the whole central metabolism. This involved a flux redirection from the pentose phosphate pathway (PPP towards glycolysis, an increased flux through pyruvate carboxylase, the fermentative pathways and malic enzyme, a flux decrease through the TCA cycle, and a partial relocation of alanine biosynthesis from the mitochondrion to the cytosol. S. cerevisiae exhibited a by-pass of pyruvate dehydrogenase in all physiological regimes. During oxidative growth this by-pass was mainly provided via pyruvate decarboxylase, acetaldehyde dehydrogenase, acetyl-CoA synthase and transport of acetyl-CoA into the mitochondrion. During fermentative growth this route, however, was saturated due to limited enzyme capacity. Under these conditions the cells exhibited high carbon flux through a chain of reactions involving pyruvate carboxylase, the oxaloacetate transporter and malic enzyme. During purely oxidative growth the PPP alone was sufficient to completely supply NADPH for anabolism. During fermentation, it provided only 60 % of the required NADPH. Conclusion We conclude that, in order to overcome the limited capacity of pyruvate dehydrogenase, S. cerevisiae possesses different metabolic by-passes to channel carbon into the mitochondrion. This involves the conversion of cytosolic pyruvate either into acetyl CoA or oxaloacetate followed by

  15. Metabolic flux analysis of a phenol producing mutant of Pseudomonas putida S12: verification and complementation of hypotheses derived from transcriptomics.

    Science.gov (United States)

    Wierckx, Nick; Ruijssenaars, Harald J; de Winde, Johannes H; Schmid, Andreas; Blank, Lars M

    2009-08-20

    The physiological effects of genetic and transcriptional changes observed in a phenol producing mutant of the solvent-tolerant Pseudomonas putida S12 were assessed with metabolic flux analysis. The upregulation of a malate/lactate dehydrogenase encoding gene could be connected to a flux increase from malate to oxaloacetate. A mutation in the pykA gene decreased in vitro pyruvate kinase activity, which is consistent with a lower flux from phosphoenolpyruvate to pyruvate. Changes in the oprB-1, gntP and gnuK genes, encoding a glucose-selective porin, gluconokinase and a gluconate transporter respectively, altered the substrate uptake profile. Metabolic flux analysis furthermore revealed cellular events not predicted by the transcriptome analysis. Gluconeogenic formation of glucose-6-phosphate from triose-3-phosphate was abolished, in favour of increased phosphoenolpyruvate production. An increased pentose phosphate pathway flux resulted in higher erythrose-4-phosphate production. Thus, the availability of these two central phenol precursors was improved. Furthermore, metabolic fluxes were redistributed such that the overall TCA cycle flux was unaffected and energy production increased. Engineering P. putida S12 for phenol production has yielded a strain that channels carbon fluxes to previously unfavourable routes to reconcile the drain on metabolites required for phenol production, while maintaining basal flux levels through central carbon metabolism.

  16. Metabolic flux analysis of a phenol producing mutant of Pseudomonas putida S12: Verification and complementation of hypotheses derived from transcriptomics

    NARCIS (Netherlands)

    Wierckx, N.; Ruijssenaars, H.J.; Winde, J.H.de; Schmid, A.; Blank, L.M.

    2009-01-01

    The physiological effects of genetic and transcriptional changes observed in a phenol producing mutant of the solvent-tolerant Pseudomonas putida S12 were assessed with metabolic flux analysis. The upregulation of a malate/lactate dehydrogenase encoding gene could be connected to a flux increase

  17. Flux Balance Analysis Inspired Bioprocess Upgrading for Lycopene Production by a Metabolically Engineered Strain of Yarrowia lipolytica

    Directory of Open Access Journals (Sweden)

    Komi Nambou

    2015-12-01

    Full Text Available Genome-scale metabolic models embody a significant advantage of systems biology since their applications as metabolic flux simulation models enable predictions for the production of industrially-interesting metabolites. The biotechnological production of lycopene from Yarrowia lipolytica is an emerging scope that has not been fully scrutinized, especially for what concerns cultivation conditions of newly generated engineered strains. In this study, by combining flux balance analysis (FBA and Plackett-Burman design, we screened chemicals for lycopene production from a metabolically engineered strain of Y. lipolytica. Lycopene concentrations of 126 and 242 mg/L were achieved correspondingly from the FBA-independent and the FBA-assisted designed media in fed-batch cultivation mode. Transcriptional studies revealed upregulations of heterologous genes in media designed according to FBA, thus implying the efficiency of model predictions. Our study will potentially support upgraded lycopene and other terpenoids production from existing or prospect bioengineered strains of Y. lipolytica and/or closely related yeast species.

  18. Flux Balance Analysis Inspired Bioprocess Upgrading for Lycopene Production by a Metabolically Engineered Strain of Yarrowia lipolytica

    Science.gov (United States)

    Nambou, Komi; Jian, Xingxing; Zhang, Xinkai; Wei, Liujing; Lou, Jiajia; Madzak, Catherine; Hua, Qiang

    2015-01-01

    Genome-scale metabolic models embody a significant advantage of systems biology since their applications as metabolic flux simulation models enable predictions for the production of industrially-interesting metabolites. The biotechnological production of lycopene from Yarrowia lipolytica is an emerging scope that has not been fully scrutinized, especially for what concerns cultivation conditions of newly generated engineered strains. In this study, by combining flux balance analysis (FBA) and Plackett-Burman design, we screened chemicals for lycopene production from a metabolically engineered strain of Y. lipolytica. Lycopene concentrations of 126 and 242 mg/L were achieved correspondingly from the FBA-independent and the FBA-assisted designed media in fed-batch cultivation mode. Transcriptional studies revealed upregulations of heterologous genes in media designed according to FBA, thus implying the efficiency of model predictions. Our study will potentially support upgraded lycopene and other terpenoids production from existing or prospect bioengineered strains of Y. lipolytica and/or closely related yeast species. PMID:26703753

  19. Metabolomic and 13C-Metabolic Flux Analysis of a Xylose-Consuming Saccharomyces cerevisiae Strain Expressing Xylose Isomerase

    Science.gov (United States)

    Wasylenko, Thomas M.; Stephanopoulos, Gregory

    2016-01-01

    Over the past two decades significant progress has been made in the engineering of xylose-consuming Saccharomyces cerevisiae strains for production of lignocellulosic biofuels. However, the ethanol productivities achieved on xylose are still significantly lower than those observed on glucose for reasons that are not well understood. We have undertaken an analysis of central carbon metabolite pool sizes and metabolic fluxes on glucose and on xylose under aerobic and anaerobic conditions in a strain capable of rapid xylose assimilation via xylose isomerase in order to investigate factors that may limit the rate of xylose fermentation. We find that during xylose utilization the flux through the non-oxidative PPP is high but the flux through the oxidative PPP is low, highlighting an advantage of the strain employed in this study. Furthermore, xylose fails to elicit the full carbon catabolite repression response that is characteristic of glucose fermentation in S. cerevisiae. We present indirect evidence that the incomplete activation of the fermentation program on xylose results in a bottleneck in lower glycolysis, leading to inefficient re-oxidation of NADH produced in glycolysis. PMID:25311863

  20. Metabolomic and (13)C-metabolic flux analysis of a xylose-consuming Saccharomyces cerevisiae strain expressing xylose isomerase.

    Science.gov (United States)

    Wasylenko, Thomas M; Stephanopoulos, Gregory

    2015-03-01

    Over the past two decades, significant progress has been made in the engineering of xylose-consuming Saccharomyces cerevisiae strains for production of lignocellulosic biofuels. However, the ethanol productivities achieved on xylose are still significantly lower than those observed on glucose for reasons that are not well understood. We have undertaken an analysis of central carbon metabolite pool sizes and metabolic fluxes on glucose and on xylose under aerobic and anaerobic conditions in a strain capable of rapid xylose assimilation via xylose isomerase in order to investigate factors that may limit the rate of xylose fermentation. We find that during xylose utilization the flux through the non-oxidative Pentose Phosphate Pathway (PPP) is high but the flux through the oxidative PPP is low, highlighting an advantage of the strain employed in this study. Furthermore, xylose fails to elicit the full carbon catabolite repression response that is characteristic of glucose fermentation in S. cerevisiae. We present indirect evidence that the incomplete activation of the fermentation program on xylose results in a bottleneck in lower glycolysis, leading to inefficient re-oxidation of NADH produced in glycolysis. © 2014 Wiley Periodicals, Inc.

  1. Interaction of storage carbohydrates and other cyclic fluxes with central metabolism: A quantitative approach by non-stationary 13C metabolic flux analysis

    Directory of Open Access Journals (Sweden)

    C.A. Suarez-Mendez

    2016-12-01

    Full Text Available 13C labeling experiments in aerobic glucose limited cultures of Saccharomyces cerevisiae at four different growth rates (0.054; 0.101, 0.207, 0.307 h−1 are used for calculating fluxes that include intracellular cycles (e.g., storage carbohydrate cycles, exchange fluxes with amino acids, which are rearranged depending on the growth rate. At low growth rates the impact of the storage carbohydrate recycle is relatively more significant than at high growth rates due to a higher concentration of these materials in the cell (up to 560-fold and higher fluxes relative to the glucose uptake rate (up to 16%. Experimental observations suggest that glucose can be exported to the extracellular space, and that its source is related to storage carbohydrates, most likely via the export and subsequent extracellular breakdown of trehalose. This hypothesis is strongly supported by 13C-labeling experimental data, measured extracellular trehalose, and the corresponding flux estimations. Keywords: Non-stationary 13C labeling, Flux estimation, Trehalose, Glycogen, Amino acids

  2. Metabolic cartography: experimental quantification of metabolic fluxes from isotopic labelling studies

    Energy Technology Data Exchange (ETDEWEB)

    O' Grady J.; Schwender J.; Shachar-Hill, Y.; Morgan, J. A.

    2012-03-01

    For the past decade, flux maps have provided researchers with an in-depth perspective on plant metabolism. As a rapidly developing field, significant headway has been made recently in computation, experimentation, and overall understanding of metabolic flux analysis. These advances are particularly applicable to the study of plant metabolism. New dynamic computational methods such as non-stationary metabolic flux analysis are finding their place in the toolbox of metabolic engineering, allowing more organisms to be studied and decreasing the time necessary for experimentation, thereby opening new avenues by which to explore the vast diversity of plant metabolism. Also, improved methods of metabolite detection and measurement have been developed, enabling increasingly greater resolution of flux measurements and the analysis of a greater number of the multitude of plant metabolic pathways. Methods to deconvolute organelle-specific metabolism are employed with increasing effectiveness, elucidating the compartmental specificity inherent in plant metabolism. Advances in metabolite measurements have also enabled new types of experiments, such as the calculation of metabolic fluxes based on {sup 13}CO{sub 2} dynamic labelling data, and will continue to direct plant metabolic engineering. Newly calculated metabolic flux maps reveal surprising and useful information about plant metabolism, guiding future genetic engineering of crops to higher yields. Due to the significant level of complexity in plants, these methods in combination with other systems biology measurements are necessary to guide plant metabolic engineering in the future.

  3. Metabolic cartography: experimental quantification of metabolic fluxes from isotopic labelling studies

    Energy Technology Data Exchange (ETDEWEB)

    O' Grady, J; Schwender, J; Shachar-Hill, Y; Morgan, JA

    2012-03-26

    For the past decade, flux maps have provided researchers with an in-depth perspective on plant metabolism. As a rapidly developing field, significant headway has been made recently in computation, experimentation, and overall understanding of metabolic flux analysis. These advances are particularly applicable to the study of plant metabolism. New dynamic computational methods such as non-stationary metabolic flux analysis are finding their place in the toolbox of metabolic engineering, allowing more organisms to be studied and decreasing the time necessary for experimentation, thereby opening new avenues by which to explore the vast diversity of plant metabolism. Also, improved methods of metabolite detection and measurement have been developed, enabling increasingly greater resolution of flux measurements and the analysis of a greater number of the multitude of plant metabolic pathways. Methods to deconvolute organelle-specific metabolism are employed with increasing effectiveness, elucidating the compartmental specificity inherent in plant metabolism. Advances in metabolite measurements have also enabled new types of experiments, such as the calculation of metabolic fluxes based on (CO2)-C-13 dynamic labelling data, and will continue to direct plant metabolic engineering. Newly calculated metabolic flux maps reveal surprising and useful information about plant metabolism, guiding future genetic engineering of crops to higher yields. Due to the significant level of complexity in plants, these methods in combination with other systems biology measurements are necessary to guide plant metabolic engineering in the future.

  4. Metabolic cartography: experimental quantification of metabolic fluxes from isotopic labelling studies.

    Science.gov (United States)

    O'Grady, John; Schwender, Jörg; Shachar-Hill, Yair; Morgan, John A

    2012-03-01

    For the past decade, flux maps have provided researchers with an in-depth perspective on plant metabolism. As a rapidly developing field, significant headway has been made recently in computation, experimentation, and overall understanding of metabolic flux analysis. These advances are particularly applicable to the study of plant metabolism. New dynamic computational methods such as non-stationary metabolic flux analysis are finding their place in the toolbox of metabolic engineering, allowing more organisms to be studied and decreasing the time necessary for experimentation, thereby opening new avenues by which to explore the vast diversity of plant metabolism. Also, improved methods of metabolite detection and measurement have been developed, enabling increasingly greater resolution of flux measurements and the analysis of a greater number of the multitude of plant metabolic pathways. Methods to deconvolute organelle-specific metabolism are employed with increasing effectiveness, elucidating the compartmental specificity inherent in plant metabolism. Advances in metabolite measurements have also enabled new types of experiments, such as the calculation of metabolic fluxes based on (13)CO(2) dynamic labelling data, and will continue to direct plant metabolic engineering. Newly calculated metabolic flux maps reveal surprising and useful information about plant metabolism, guiding future genetic engineering of crops to higher yields. Due to the significant level of complexity in plants, these methods in combination with other systems biology measurements are necessary to guide plant metabolic engineering in the future.

  5. Controlling fluxes for microbial metabolic engineering

    OpenAIRE

    Sachdeva, Gairik

    2014-01-01

    This thesis presents novel synthetic biology tools and design principles usable for microbial metabolic engineering. Controlling metabolic fluxes is essential for biological manufacturing of fuels, materials, and high value chemicals. Insulating the flow of metabolites is a successful natural strategy for metabolic flux regulation. Recently, approaches using scaffolds, both in vitro and in vivo, to spatially co-localize enzymes have reported significant gains in product yields. RNA is suitabl...

  6. CeCaFDB: a curated database for the documentation, visualization and comparative analysis of central carbon metabolic flux distributions explored by 13C-fluxomics.

    Science.gov (United States)

    Zhang, Zhengdong; Shen, Tie; Rui, Bin; Zhou, Wenwei; Zhou, Xiangfei; Shang, Chuanyu; Xin, Chenwei; Liu, Xiaoguang; Li, Gang; Jiang, Jiansi; Li, Chao; Li, Ruiyuan; Han, Mengshu; You, Shanping; Yu, Guojun; Yi, Yin; Wen, Han; Liu, Zhijie; Xie, Xiaoyao

    2015-01-01

    The Central Carbon Metabolic Flux Database (CeCaFDB, available at http://www.cecafdb.org) is a manually curated, multipurpose and open-access database for the documentation, visualization and comparative analysis of the quantitative flux results of central carbon metabolism among microbes and animal cells. It encompasses records for more than 500 flux distributions among 36 organisms and includes information regarding the genotype, culture medium, growth conditions and other specific information gathered from hundreds of journal articles. In addition to its comprehensive literature-derived data, the CeCaFDB supports a common text search function among the data and interactive visualization of the curated flux distributions with compartmentation information based on the Cytoscape Web API, which facilitates data interpretation. The CeCaFDB offers four modules to calculate a similarity score or to perform an alignment between the flux distributions. One of the modules was built using an inter programming algorithm for flux distribution alignment that was specifically designed for this study. Based on these modules, the CeCaFDB also supports an extensive flux distribution comparison function among the curated data. The CeCaFDB is strenuously designed to address the broad demands of biochemists, metabolic engineers, systems biologists and members of the -omics community. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Assessment of energetic costs of AhR activation by β-naphthoflavone in rainbow trout (Oncorhynchus mykiss) hepatocytes using metabolic flux analysis

    Energy Technology Data Exchange (ETDEWEB)

    Nault, Rance, E-mail: naultran@msu.edu [Ottawa-Carleton Institute of Biology, Department of Biology and Centre for Advanced Research in Environmental Genomics, University of Ottawa, Ottawa, Ontario, K1N 6N5 (Canada); Abdul-Fattah, Hiba [Ottawa-Carleton Institute of Biology, Department of Biology and Centre for Advanced Research in Environmental Genomics, University of Ottawa, Ottawa, Ontario, K1N 6N5 (Canada); Mironov, Gleb G.; Berezovski, Maxim V. [Ottawa-Carleton Institute of Biology, Department of Biology and Centre for Advanced Research in Environmental Genomics, University of Ottawa, Ottawa, Ontario, K1N 6N5 (Canada); Department of Chemistry, University of Ottawa, Ottawa, Ontario, K1N 6N5 (Canada); Moon, Thomas W. [Ottawa-Carleton Institute of Biology, Department of Biology and Centre for Advanced Research in Environmental Genomics, University of Ottawa, Ottawa, Ontario, K1N 6N5 (Canada)

    2013-08-15

    Exposure to environmental contaminants such as activators of the aryl hydrocarbon receptor (AhR) leads to the induction of defense and detoxification mechanisms. While these mechanisms allow organisms to metabolize and excrete at least some of these environmental contaminants, it has been proposed that these mechanisms lead to significant energetic challenges. This study tests the hypothesis that activation of the AhR by the model agonist β-naphthoflavone (βNF) results in increased energetic costs in rainbow trout (Oncorhynchus mykiss) hepatocytes. To address this hypothesis, we employed traditional biochemical approaches to examine energy allocation and metabolism including the adenylate energy charge (AEC), protein synthesis rates, Na{sup +}/K{sup +}-ATPase activity, and enzyme activities. Moreover, we have used for the first time in a fish cell preparation, metabolic flux analysis (MFA) an in silico approach for the estimation of intracellular metabolic fluxes. Exposure of trout hepatocytes to 1 μM βNF for 48 h did not alter hepatocyte AEC, protein synthesis, or Na{sup +}/K{sup +}-ATPase activity but did lead to sparing of glycogen reserves and changes in activities of alanine aminotransferase and citrate synthase suggesting altered metabolism. Conversely, MFA did not identify altered metabolic fluxes, although we do show that the dynamic metabolism of isolated trout hepatocytes poses a significant challenge for this type of approach which should be considered in future studies. - Highlights: • Energetic costs of AhR activation by βNF was examined in rainbow trout hepatocytes. • Metabolic flux analysis was performed on a fish cell preparation for the first time. • Exposure to βNF led to sparing of glycogen reserves and altered enzyme activities. • Adenylate energy charge was maintained despite temporal changes in metabolism.

  8. Assessment of energetic costs of AhR activation by β-naphthoflavone in rainbow trout (Oncorhynchus mykiss) hepatocytes using metabolic flux analysis

    International Nuclear Information System (INIS)

    Nault, Rance; Abdul-Fattah, Hiba; Mironov, Gleb G.; Berezovski, Maxim V.; Moon, Thomas W.

    2013-01-01

    Exposure to environmental contaminants such as activators of the aryl hydrocarbon receptor (AhR) leads to the induction of defense and detoxification mechanisms. While these mechanisms allow organisms to metabolize and excrete at least some of these environmental contaminants, it has been proposed that these mechanisms lead to significant energetic challenges. This study tests the hypothesis that activation of the AhR by the model agonist β-naphthoflavone (βNF) results in increased energetic costs in rainbow trout (Oncorhynchus mykiss) hepatocytes. To address this hypothesis, we employed traditional biochemical approaches to examine energy allocation and metabolism including the adenylate energy charge (AEC), protein synthesis rates, Na + /K + -ATPase activity, and enzyme activities. Moreover, we have used for the first time in a fish cell preparation, metabolic flux analysis (MFA) an in silico approach for the estimation of intracellular metabolic fluxes. Exposure of trout hepatocytes to 1 μM βNF for 48 h did not alter hepatocyte AEC, protein synthesis, or Na + /K + -ATPase activity but did lead to sparing of glycogen reserves and changes in activities of alanine aminotransferase and citrate synthase suggesting altered metabolism. Conversely, MFA did not identify altered metabolic fluxes, although we do show that the dynamic metabolism of isolated trout hepatocytes poses a significant challenge for this type of approach which should be considered in future studies. - Highlights: • Energetic costs of AhR activation by βNF was examined in rainbow trout hepatocytes. • Metabolic flux analysis was performed on a fish cell preparation for the first time. • Exposure to βNF led to sparing of glycogen reserves and altered enzyme activities. • Adenylate energy charge was maintained despite temporal changes in metabolism

  9. Development of a screening approach for exploring cell factory potential through metabolic flux analysis and physiology

    DEFF Research Database (Denmark)

    Knudsen, Peter Boldsen; Nielsen, Kristian Fog; Thykær, Jette

    2012-01-01

    of the recombinant strains in order to evaluate their potential as fungal cell factories and for guiding further metabolic engineering strategies. To meet the demand for a fast and reliable method for physiological characterisation of fungal strains, a screening approach using a micro titer format was developed...... of the validation, already well described fungal strains were selected and tested using the described method and the developed method was subsequently used to test recombinant fungal strains producing the model polyketide 6-methylsalicylic acid. Diligent application of this strategy significantly reduces the cost...

  10. Regulation of flux through metabolic cycles

    International Nuclear Information System (INIS)

    Walsh, K.

    1984-01-01

    The branchpoint of the tricarboxylic acid and glyoxylate shunt was characterized in the intact organism by a multidimensional approach. Theory and methodology were developed to determine velocities for the net flow of carbon through the major steps of acetate metabolism in E. coli. Rates were assigned based on the 13 C-NMR spectrum of intracellular glutamate, measured rates of substrate incorporation into end products, the constituent composition of E. coli and a series of conservation equations which described the system at steady state. The in vivo fluxes through the branchpoint of the tricarboxylic acid and glyoxylate cycles were compared to rates calculated from the kinetic constants of the branchpoint enzymes and the intracellular concentrations of their substrates. These studies elucidated the role of isocitrate dehydrogenase phosphorylation in the Krebs cycle and led to the development of a generalized mathematical description of the sensitivity of branchpoints to regulatory control. This theoretical analysis was termed the branchpoint effect and it describes conditions which result in large changes in the flux through an enzyme even though that enzyme is not subject to direct regulatory control. The theoretical and experimental characterization of this system provided a framework to study the effects of enzyme overproduction and underproduction on metabolic processes in the cell. An in vivo method was developed to determine the extent to which an enzyme catalyzes a rate-controlling reaction. The enzyme chosen for this study was citrate synthase

  11. 13C Metabolic Flux Analysis for Systematic Metabolic Engineering of S. cerevisiae for Overproduction of Fatty Acids

    DEFF Research Database (Denmark)

    Ghosh, Amit; Ando, David; Gin, Jennifer

    2016-01-01

    Efficient redirection of microbial metabolism into the abundant production of desired bioproducts remains non-trivial. Here, we used flux-based modeling approaches to improve yields of fatty acids in Saccharomyces cerevisiae. We combined 13C labeling data with comprehensive genome-scale models to...

  12. Metabolic Flux Analysis of Lipid Biosynthesis in the Yeast Yarrowia lipolytica Using 13C-Labled Glucose and Gas Chromatography-Mass Spectrometry.

    Science.gov (United States)

    Zhang, Huaiyuan; Wu, Chao; Wu, Qingyu; Dai, Junbiao; Song, Yuanda

    2016-01-01

    The oleaginous yeast Yarrowia lipolytica has considerable potential for producing single cell oil, which can be converted to biodiesel, a sustainable alternative to fossil fuels. However, extensive fundamental and engineering efforts must be carried out before commercialized production become cost-effective. Therefore, in this study, metabolic flux analysis of Y. lipolytica was performed using 13C-labeled glucose as a sole carbon source in nitrogen sufficient and insufficient media. The nitrogen limited medium inhibited cell growth while promoting lipid accumulation (from 8.7% of their biomass to 14.3%). Metabolic flux analysis showed that flux through the pentose phosphate pathway was not significantly regulated by nitrogen concentration, suggesting that NADPH generation is not the limiting factor for lipid accumulation in Y. lipolytica. Furthermore, metabolic flux through malic enzyme was undetectable, confirming its non-regulatory role in lipid accumulation in this yeast. Nitrogen limitation significantly increased flux through ATP:citrate lyase (ACL), implying that ACL plays a key role in providing acetyl-CoA for lipid accumulation in Y. lipolytica.

  13. Metabolic Flux Analysis of Lipid Biosynthesis in the Yeast Yarrowia lipolytica Using 13C-Labled Glucose and Gas Chromatography-Mass Spectrometry.

    Directory of Open Access Journals (Sweden)

    Huaiyuan Zhang

    Full Text Available The oleaginous yeast Yarrowia lipolytica has considerable potential for producing single cell oil, which can be converted to biodiesel, a sustainable alternative to fossil fuels. However, extensive fundamental and engineering efforts must be carried out before commercialized production become cost-effective. Therefore, in this study, metabolic flux analysis of Y. lipolytica was performed using 13C-labeled glucose as a sole carbon source in nitrogen sufficient and insufficient media. The nitrogen limited medium inhibited cell growth while promoting lipid accumulation (from 8.7% of their biomass to 14.3%. Metabolic flux analysis showed that flux through the pentose phosphate pathway was not significantly regulated by nitrogen concentration, suggesting that NADPH generation is not the limiting factor for lipid accumulation in Y. lipolytica. Furthermore, metabolic flux through malic enzyme was undetectable, confirming its non-regulatory role in lipid accumulation in this yeast. Nitrogen limitation significantly increased flux through ATP:citrate lyase (ACL, implying that ACL plays a key role in providing acetyl-CoA for lipid accumulation in Y. lipolytica.

  14. Phototransduction Influences Metabolic Flux and Nucleotide Metabolism in Mouse Retina.

    Science.gov (United States)

    Du, Jianhai; Rountree, Austin; Cleghorn, Whitney M; Contreras, Laura; Lindsay, Ken J; Sadilek, Martin; Gu, Haiwei; Djukovic, Danijel; Raftery, Dan; Satrústegui, Jorgina; Kanow, Mark; Chan, Lawrence; Tsang, Stephen H; Sweet, Ian R; Hurley, James B

    2016-02-26

    Production of energy in a cell must keep pace with demand. Photoreceptors use ATP to maintain ion gradients in darkness, whereas in light they use it to support phototransduction. Matching production with consumption can be accomplished by coupling production directly to consumption. Alternatively, production can be set by a signal that anticipates demand. In this report we investigate the hypothesis that signaling through phototransduction controls production of energy in mouse retinas. We found that respiration in mouse retinas is not coupled tightly to ATP consumption. By analyzing metabolic flux in mouse retinas, we also found that phototransduction slows metabolic flux through glycolysis and through intermediates of the citric acid cycle. We also evaluated the relative contributions of regulation of the activities of α-ketoglutarate dehydrogenase and the aspartate-glutamate carrier 1. In addition, a comprehensive analysis of the retinal metabolome showed that phototransduction also influences steady-state concentrations of 5'-GMP, ribose-5-phosphate, ketone bodies, and purines. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Flux analysis of central metabolic pathways in the Fe(III)-reducing organism Geobacter metallireducens via 13C isotopiclabeling

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Yinjie J.; Chakraborty, Romy; Martin, Hector Garcia; Chu,Jeannie; Hazen, Terry C.; Keasling, Jay D.

    2007-08-13

    We analyzed the carbon fluxes in the central metabolism ofGeobacter metallireducens strain GS-15 using 13C isotopomer modeling.Acetate labeled in the 1st or 2nd position was the sole carbon source,and Fe-NTA was the sole terminal electron acceptor. The measured labeledacetate uptake rate was 21 mmol/gdw/h in the exponential growth phase.The resulting isotope labeling pattern of amino acids allowed an accuratedetermination of the in vivo global metabolic reaction rates (fluxes)through the central metabolic pathways using a computational isotopomermodel. The model indicated that over 90 percent of the acetate wascompletely oxidized to CO2 via a complete tricarboxylic acid (TCA) cyclewhile reducing iron. Pyruvate carboxylase and phosphoenolpyruvatecarboxykinase were present under these conditions, but enzymes in theglyoxylate shunt and malic enzyme were absent. Gluconeogenesis and thepentose phosphate pathway were mainly employed for biosynthesis andaccounted for less than 3 percent of total carbon consumption. The modelalso indicated surprisingly high reversibility in the reaction betweenoxoglutarate and succinate. This step operates close to the thermodynamicequilibrium possibly because succinate is synthesized via a transferasereaction, and its product, acetyl-CoA, inhibits the conversion ofoxoglutarate to succinate. These findings enable a better understandingof the relationship between genome annotation and extant metabolicpathways in G. metallireducens.

  16. Metabolic flux prediction in cancer cells with altered substrate uptake.

    Science.gov (United States)

    Schwartz, Jean-Marc; Barber, Michael; Soons, Zita

    2015-12-01

    Proliferating cells, such as cancer cells, are known to have an unusual metabolism, characterized by an increased rate of glycolysis and amino acid metabolism. Our understanding of this phenomenon is limited but could potentially be used in order to develop new therapies. Computational modelling techniques, such as flux balance analysis (FBA), have been used to predict fluxes in various cell types, but remain of limited use to explain the unusual metabolic shifts and altered substrate uptake in human cancer cells. We implemented a new flux prediction method based on elementary modes (EMs) and structural flux (StruF) analysis and tested them against experimentally measured flux data obtained from (13)C-labelling in a cancer cell line. We assessed the quality of predictions using different objective functions along with different techniques in normalizing a metabolic network with more than one substrate input. Results show a good correlation between predicted and experimental values and indicate that the choice of cellular objective critically affects the quality of predictions. In particular, lactate gives an excellent correlation and correctly predicts the high flux through glycolysis, matching the observed characteristics of cancer cells. In contrast with FBA, which requires a priori definition of all uptake rates, often hard to measure, atomic StruFs (aStruFs) are able to predict uptake rates of multiple substrates. © 2015 Authors; published by Portland Press Limited.

  17. Metabolic profiling and flux analysis of MEL-2 human embryonic stem cells during exponential growth at physiological and atmospheric oxygen concentrations.

    Directory of Open Access Journals (Sweden)

    Jennifer Turner

    Full Text Available As human embryonic stem cells (hESCs steadily progress towards regenerative medicine applications there is an increasing emphasis on the development of bioreactor platforms that enable expansion of these cells to clinically relevant numbers. Surprisingly little is known about the metabolic requirements of hESCs, precluding the rational design and optimisation of such platforms. In this study, we undertook an in-depth characterisation of MEL-2 hESC metabolic behaviour during the exponential growth phase, combining metabolic profiling and flux analysis tools at physiological (hypoxic and atmospheric (normoxic oxygen concentrations. To overcome variability in growth profiles and the problem of closing mass balances in a complex environment, we developed protocols to accurately measure uptake and production rates of metabolites, cell density, growth rate and biomass composition, and designed a metabolic flux analysis model for estimating internal rates. hESCs are commonly considered to be highly glycolytic with inactive or immature mitochondria, however, whilst the results of this study confirmed that glycolysis is indeed highly active, we show that at least in MEL-2 hESC, it is supported by the use of oxidative phosphorylation within the mitochondria utilising carbon sources, such as glutamine to maximise ATP production. Under both conditions, glycolysis was disconnected from the mitochondria with all of the glucose being converted to lactate. No difference in the growth rates of cells cultured under physiological or atmospheric oxygen concentrations was observed nor did this cause differences in fluxes through the majority of the internal metabolic pathways associated with biogenesis. These results suggest that hESCs display the conventional Warburg effect, with high aerobic activity despite high lactate production, challenging the idea of an anaerobic metabolism with low mitochondrial activity. The results of this study provide new insight that

  18. Co-utilization of glucose and xylose by evolved Thermus thermophilus LC113 strain elucidated by 13C metabolic flux analysis and whole genome sequencing

    Science.gov (United States)

    Cordova, Lauren T.; Lu, Jing; Cipolla, Robert M.; Sandoval, Nicholas R.; Long, Christopher P.; Antoniewicz, Maciek R.

    2018-01-01

    We evolved Thermus thermophilus to efficiently co-utilize glucose and xylose, the two most abundant sugars in lignocellulosic biomass, at high temperatures without carbon catabolite repression. To generate the strain, T. thermophilus HB8 was first evolved on glucose to improve its growth characteristics, followed by evolution on xylose. The resulting strain, T. thermophilus LC113, was characterized in growth studies, by whole genome sequencing, and 13C-metabolic flux analysis (13C-MFA) with [1,6-13C]glucose, [5-13C]xylose, and [1,6-13C]glucose + [5-13C]xylose as isotopic tracers. Compared to the starting strain, the evolved strain had an increased growth rate (~ 2-fold), increased biomass yield, increased tolerance to high temperatures up to 90 °C, and gained the ability to grow on xylose in minimal medium. At the optimal growth temperature of 81 °C, the maximum growth rate on glucose and xylose was 0.44 and 0.46 h−1, respectively. In medium containing glucose and xylose the strain efficiently co-utilized the two sugars. 13C-MFA results provided insights into the metabolism of T. thermophilus LC113 that allows efficient co-utilization of glucose and xylose. Specifically, 13C-MFA revealed that metabolic fluxes in the upper part of metabolism adjust flexibly to sugar availability, while fluxes in the lower part of metabolism remain relatively constant. Whole genome sequence analysis revealed two large structural changes that can help explain the physiology of the evolved strain: a duplication of a chromosome region that contains many sugar transporters, and a 5× multiplication of a region on the pVV8 plasmid that contains xylose isomerase and xylulokinase genes, the first two enzymes of xylose catabolism. Taken together, 13C-MFA and genome sequence analysis provided complementary insights into the physiology of the evolved strain. PMID:27164561

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

    Science.gov (United States)

    Zhao, Xinhe; Kasbi, Mayssa; Chen, Jingkui; Peres, Sabine; Jolicoeur, Mario

    2017-12-01

    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. © 2017 Wiley Periodicals, Inc.

  20. ¹³C metabolic flux analysis identifies an unusual route for pyruvate dissimilation in mycobacteria which requires isocitrate lyase and carbon dioxide fixation.

    Directory of Open Access Journals (Sweden)

    Dany J V Beste

    2011-07-01

    Full Text Available Mycobacterium tuberculosis requires the enzyme isocitrate lyase (ICL for growth and virulence in vivo. The demonstration that M. tuberculosis also requires ICL for survival during nutrient starvation and has a role during steady state growth in a glycerol limited chemostat indicates a function for this enzyme which extends beyond fat metabolism. As isocitrate lyase is a potential drug target elucidating the role of this enzyme is of importance; however, the role of isocitrate lyase has never been investigated at the level of in vivo fluxes. Here we show that deletion of one of the two icl genes impairs the replication of Mycobacterium bovis BCG at slow growth rate in a carbon limited chemostat. In order to further understand the role of isocitrate lyase in the central metabolism of mycobacteria the effect of growth rate on the in vivo fluxes was studied for the first time using ¹³C-metabolic flux analysis (MFA. Tracer experiments were performed with steady state chemostat cultures of BCG or M. tuberculosis supplied with ¹³C labeled glycerol or sodium bicarbonate. Through measurements of the ¹³C isotopomer labeling patterns in protein-derived amino acids and enzymatic activity assays we have identified the activity of a novel pathway for pyruvate dissimilation. We named this the GAS pathway because it utilizes the Glyoxylate shunt and Anapleurotic reactions for oxidation of pyruvate, and Succinyl CoA synthetase for the generation of succinyl CoA combined with a very low flux through the succinate--oxaloacetate segment of the tricarboxylic acid cycle. We confirm that M. tuberculosis can fix carbon from CO₂ into biomass. As the human host is abundant in CO₂ this finding requires further investigation in vivo as CO₂ fixation may provide a point of vulnerability that could be targeted with novel drugs. This study also provides a platform for further studies into the metabolism of M. tuberculosis using ¹³C-MFA.

  1. Designing metabolic engineering strategies with genome-scale metabolic flux modeling

    Directory of Open Access Journals (Sweden)

    Yen JY

    2015-01-01

    Full Text Available Jiun Y Yen,1,2 Imen Tanniche,1 Amanda K Fisher,1–3 Glenda E Gillaspy,2 David R Bevan,2,3 Ryan S Senger1 1Department of Biological Systems Engineering, 2Department of Biochemistry, 3Genomics, Bioinformatics, and Computational Biology Interdisciplinary Program, Virginia Tech, Blacksburg, VA, USA Abstract: New in silico tools that make use of genome-scale metabolic flux modeling are improving the design of metabolic engineering strategies. This review highlights the latest developments in this area, explains the interface between these in silico tools and the experimental implementation tools of metabolic engineers, and provides a way forward so that in silico predictions can better mimic reality and more experimental methods can be considered in simulation studies. The several methodologies for solving genome-scale models (eg, flux balance analysis [FBA], parsimonious FBA, flux variability analysis, and minimization of metabolic adjustment all have unique advantages and applications. There are two basic approaches to designing metabolic engineering strategies in silico, and both have demonstrated success in the literature. The first involves: 1 making a genetic manipulation in a model; 2 testing for improved performance through simulation; and 3 iterating the process. The second approach has been used in more recently designed in silico tools and involves: 1 comparing metabolic flux profiles of a wild-type and ideally engineered state and 2 designing engineering strategies based on the differences in these flux profiles. Improvements in genome-scale modeling are anticipated in areas such as the inclusion of all relevant cellular machinery, the ability to understand and anticipate the results of combinatorial enrichment experiments, and constructing dynamic and flexible biomass equations that can respond to environmental and genetic manipulations. Keywords: genome-scale modeling, genome-scale modeling, flux balance analysis, flux variability

  2. Co-utilization of glucose and xylose by evolved Thermus thermophilus LC113 strain elucidated by (13)C metabolic flux analysis and whole genome sequencing.

    Science.gov (United States)

    Cordova, Lauren T; Lu, Jing; Cipolla, Robert M; Sandoval, Nicholas R; Long, Christopher P; Antoniewicz, Maciek R

    2016-09-01

    We evolved Thermus thermophilus to efficiently co-utilize glucose and xylose, the two most abundant sugars in lignocellulosic biomass, at high temperatures without carbon catabolite repression. To generate the strain, T. thermophilus HB8 was first evolved on glucose to improve its growth characteristics, followed by evolution on xylose. The resulting strain, T. thermophilus LC113, was characterized in growth studies, by whole genome sequencing, and (13)C-metabolic flux analysis ((13)C-MFA) with [1,6-(13)C]glucose, [5-(13)C]xylose, and [1,6-(13)C]glucose+[5-(13)C]xylose as isotopic tracers. Compared to the starting strain, the evolved strain had an increased growth rate (~2-fold), increased biomass yield, increased tolerance to high temperatures up to 90°C, and gained the ability to grow on xylose in minimal medium. At the optimal growth temperature of 81°C, the maximum growth rate on glucose and xylose was 0.44 and 0.46h(-1), respectively. In medium containing glucose and xylose the strain efficiently co-utilized the two sugars. (13)C-MFA results provided insights into the metabolism of T. thermophilus LC113 that allows efficient co-utilization of glucose and xylose. Specifically, (13)C-MFA revealed that metabolic fluxes in the upper part of metabolism adjust flexibly to sugar availability, while fluxes in the lower part of metabolism remain relatively constant. Whole genome sequence analysis revealed two large structural changes that can help explain the physiology of the evolved strain: a duplication of a chromosome region that contains many sugar transporters, and a 5x multiplication of a region on the pVV8 plasmid that contains xylose isomerase and xylulokinase genes, the first two enzymes of xylose catabolism. Taken together, (13)C-MFA and genome sequence analysis provided complementary insights into the physiology of the evolved strain. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  3. 13C based proteinogenic amino acid (PAA) and metabolic flux ratio analysis of Lactococcus lactis reveals changes in pentose phosphate (PP) pathway in response to agitation and temperature related stresses

    Science.gov (United States)

    2017-01-01

    Lactococcus lactis subsp. cremoris MG1363 is an important starter culture for dairy fermentation. During industrial fermentations, L. lactis is constantly exposed to stresses that affect the growth and performance of the bacterium. Although the response of L. lactis to several stresses has been described, the adaptation mechanisms at the level of in vivo fluxes have seldom been described. To gain insights into cellular metabolism, 13C metabolic flux analysis and gas chromatography mass spectrometry (GC-MS) were used to measure the flux ratios of active pathways in the central metabolism of L. lactis when subjected to three conditions varying in temperature (30°C, 37°C) and agitation (with and without agitation at 150 rpm). Collectively, the concentrations of proteinogenic amino acids (PAAs) and free fatty acids (FAAs) were compared, and Pearson correlation analysis (r) was calculated to measure the pairwise relationship between PAAs. Branched chain and aromatic amino acids, threonine, serine, lysine and histidine were correlated strongly, suggesting changes in flux regulation in glycolysis, the pentose phosphate (PP) pathway, malic enzyme and anaplerotic reaction catalysed by pyruvate carboxylase (pycA). Flux ratio analysis revealed that glucose was mainly converted by glycolysis, highlighting the stability of L. lactis’ central carbon metabolism despite different conditions. Higher flux ratios through oxaloacetate (OAA) from pyruvate (PYR) reaction in all conditions suggested the activation of pyruvate carboxylate (pycA) in L. lactis, in response to acid stress during exponential phase. Subsequently, more significant flux ratio differences were seen through the oxidative and non-oxidative pentose phosphate (PP) pathways, malic enzyme, and serine and C1 metabolism, suggesting NADPH requirements in response to environmental stimuli. These reactions could play an important role in optimization strategies for metabolic engineering in L. lactis. Overall, the

  4. 13C based proteinogenic amino acid (PAA and metabolic flux ratio analysis of Lactococcus lactis reveals changes in pentose phosphate (PP pathway in response to agitation and temperature related stresses

    Directory of Open Access Journals (Sweden)

    Kamalrul Azlan Azizan

    2017-07-01

    Full Text Available Lactococcus lactis subsp. cremoris MG1363 is an important starter culture for dairy fermentation. During industrial fermentations, L. lactis is constantly exposed to stresses that affect the growth and performance of the bacterium. Although the response of L. lactis to several stresses has been described, the adaptation mechanisms at the level of in vivo fluxes have seldom been described. To gain insights into cellular metabolism, 13C metabolic flux analysis and gas chromatography mass spectrometry (GC-MS were used to measure the flux ratios of active pathways in the central metabolism of L. lactis when subjected to three conditions varying in temperature (30°C, 37°C and agitation (with and without agitation at 150 rpm. Collectively, the concentrations of proteinogenic amino acids (PAAs and free fatty acids (FAAs were compared, and Pearson correlation analysis (r was calculated to measure the pairwise relationship between PAAs. Branched chain and aromatic amino acids, threonine, serine, lysine and histidine were correlated strongly, suggesting changes in flux regulation in glycolysis, the pentose phosphate (PP pathway, malic enzyme and anaplerotic reaction catalysed by pyruvate carboxylase (pycA. Flux ratio analysis revealed that glucose was mainly converted by glycolysis, highlighting the stability of L. lactis’ central carbon metabolism despite different conditions. Higher flux ratios through oxaloacetate (OAA from pyruvate (PYR reaction in all conditions suggested the activation of pyruvate carboxylate (pycA in L. lactis, in response to acid stress during exponential phase. Subsequently, more significant flux ratio differences were seen through the oxidative and non-oxidative pentose phosphate (PP pathways, malic enzyme, and serine and C1 metabolism, suggesting NADPH requirements in response to environmental stimuli. These reactions could play an important role in optimization strategies for metabolic engineering in L. lactis. Overall

  5. Transcript abundance on its own cannot be used to infer fluxes in central metabolism

    Directory of Open Access Journals (Sweden)

    Jörg eSchwender

    2014-11-01

    Full Text Available An attempt has been made to define the extent to which metabolic flux in central plant metabolism is reflected by changes in the transcriptome and metabolome, based on an analysis of in vitro cultured immature embryos of two oilseed rape (Brassica napus accessions which contrast for seed lipid accumulation. Metabolic flux analysis was used to constrain a flux balance metabolic model which included 671 biochemical and transport reactions within the central metabolism. This highly confident flux information was eventually used for comparative analysis of flux versus transcript (metabolite. Metabolite profiling succeeded in identifying 79 intermediates within the central metabolism, some of which differed quantitatively between the two accessions and displayed a significant shift corresponding to flux. An RNA-Seq based transcriptome analysis revealed a large number of genes which were differentially transcribed in the two accessions, including some enzymes/proteins active in major metabolic pathways. With a few exceptions, differential activity in the major pathways (glycolysis, TCA cycle, amino acid and fatty acid synthesis was not reflected in contrasting abundances of the relevant transcripts. The conclusion was that transcript abundance on its own cannot be used to infer metabolic activity/fluxes in central plant metabolism. This limitation needs to be borne in mind in evaluating transcriptome data and designing metabolic engineering experiments.

  6. Pathway confirmation and flux analysis of central metabolic pathways in Desulfovibrio vulgaris Hildenborough using Gas Chromatography-Mass Spectrometry and Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry

    International Nuclear Information System (INIS)

    Tang, Yinjie; Pingitore, Francesco; Mukhopadhyay, Aindrila; Phan, Richard; Hazen, Terry C.; Keasling, Jay D.

    2007-01-01

    Flux distribution in central metabolic pathways of Desulfovibrio vulgaris Hildenborough was examined using 13C tracer experiments. Consistent with the current genome annotation and independent evidence from enzyme activity assays, the isotopomer results from both GC-MS and Fourier Transform-Ion Cyclotron Resonance mass spectrometry (FT-ICR MS) indicate the lack of oxidatively functional TCA cycle and an incomplete pentose phosphate pathway. Results from this study suggest that fluxes through both pathways are limited to biosynthesis. The data also indicate that >80 percent of the lactate was converted to acetate and the reactions involved are the primary route of energy production (NAD(P)H and ATP production). Independent of the TCA cycle, direct cleavage of acetyl-CoA to CO and 5,10-methyl-THF also leads to production of NADH and ATP. Although the genome annotation implicates a ferredoxin-dependent oxoglutarate synthase, isotopic evidence does not support flux through this reaction in either the oxidative or reductive mode; therefore, the TCA cycle is incomplete. FT-ICR MS was used to locate the labeled carbon distribution in aspartate and glutamate and confirmed the presence of an atypical enzyme for citrate formation suggested in previous reports (the citrate synthesized by this enzyme is the isotopic antipode of the citrate synthesized by the (S)-citrate synthase). These findings enable a better understanding of the relation between genome annotation and actual metabolic pathways in D. vulgaris, and also demonstrate FT-ICR MS as a powerful tool for isotopomer analysis, overcoming problems in both GC-MS and NMR spectroscopy

  7. Reliable Metabolic Flux Estimation in Escherichia coli Central Carbon Metabolism Using Intracellular Free Amino Acids

    Directory of Open Access Journals (Sweden)

    Nobuyuki Okahashi

    2014-05-01

    Full Text Available 13C metabolic flux analysis (MFA is a tool of metabolic engineering for investigation of in vivo flux distribution. A direct 13C enrichment analysis of intracellular free amino acids (FAAs is expected to reduce time for labeling experiments of the MFA. Measurable FAAs should, however, vary among the MFA experiments since the pool sizes of intracellular free metabolites depend on cellular metabolic conditions. In this study, minimal 13C enrichment data of FAAs was investigated to perform the FAAs-based MFA. An examination of a continuous culture of Escherichia coli using 13C-labeled glucose showed that the time required to reach an isotopically steady state for FAAs is rather faster than that for conventional method using proteinogenic amino acids (PAAs. Considering 95% confidence intervals, it was found that the metabolic flux distribution estimated using FAAs has a similar reliability to that of the PAAs-based method. The comparative analysis identified glutamate, aspartate, alanine and phenylalanine as the common amino acids observed in E. coli under different culture conditions. The results of MFA also demonstrated that the 13C enrichment data of the four amino acids is required for a reliable analysis of the flux distribution.

  8. Metabolic fuels: regulating fluxes to select mix.

    Science.gov (United States)

    Weber, Jean-Michel

    2011-01-15

    Animals must regulate the fluxes of multiple fuels to support changing metabolic rates that result from variation in physiological circumstances. The aim of fuel selection strategies is to exploit the advantages of individual substrates while minimizing the impact of disadvantages. All exercising mammals share a general pattern of fuel selection: at the same %V(O(2,max)) they oxidize the same ratio of lipids to carbohydrates. However, highly aerobic species rely more on intramuscular fuels because energy supply from the circulation is constrained by trans-sarcolemmal transfer. Fuel selection is performed by recruiting different muscles, different fibers within the same muscles or different pathways within the same fibers. Electromyographic analyses show that shivering humans can modulate carbohydrate oxidation either through the selective recruitment of type II fibers within the same muscles or by regulating pathway recruitment within type I fibers. The selection patterns of shivering and exercise are different: at the same %V(O(2,max)), a muscle producing only heat (shivering) or significant movement (exercise) strikes a different balance between lipid and carbohydrate oxidation. Long-distance migrants provide an excellent model to characterize how to increase maximal substrate fluxes. High lipid fluxes are achieved through the coordinated upregulation of mobilization, transport and oxidation by activating enzymes, lipid-solubilizing proteins and membrane transporters. These endurance athletes support record lipolytic rates in adipocytes, use lipoprotein shuttles to accelerate transport and show increased capacity for lipid oxidation in muscle mitochondria. Some migrant birds use dietary omega-3 fatty acids as performance-enhancing agents to boost their ability to process lipids. These dietary fatty acids become incorporated in membrane phospholipids and bind to peroxisome proliferator-activated receptors to activate membrane proteins and modify gene expression.

  9. Metabolic flux analysis of Escherichia coli in glucose-limited continuous culture. I. Growth-rate-dependent metabolic efficiency at steady state.

    Science.gov (United States)

    Kayser, Anke; Weber, Jan; Hecht, Volker; Rinas, Ursula

    2005-03-01

    The Escherichia coli K-12 strain TG1 was grown at 28 degrees C in aerobic glucose-limited continuous cultures at dilution rates ranging from 0.044 to 0.415 h(-1). The rates of biomass formation, the specific rates of glucose, ammonium and oxygen uptake and the specific carbon dioxide evolution rate increased linearly with the dilution rate up to 0.3 h(-1). At dilution rates between 0.3 h(-1) and 0.4 h(-1), a strong deviation from the linear increase to lower specific oxygen uptake and carbon dioxide evolution rates occurred. The biomass formation rate and the specific glucose and ammonium uptake rates did not deviate that strongly from the linear increase up to dilution rates of 0.4 h(-1). An increasing percentage of glucose carbon flow towards biomass determined by a reactor mass balance and a decreasing specific ATP production rate concomitant with a decreasing adenylate energy charge indicated higher energetic efficiency of carbon substrate utilization at higher dilution rates. Estimation of metabolic fluxes by a stoichiometric model revealed an increasing activity of the pentose phosphate pathway and a decreasing tricarboxylic acid cycle activity with increasing dilution rates, indicative of the increased NADPH and precursor demand for anabolic purposes at the expense of ATP formation through catabolic activities. Thus, increasing growth rates first result in a more energy-efficient use of the carbon substrate for biomass production, i.e. a lower portion of the carbon substrate is channelled into the respiratory, energy-generating pathway. At dilution rates above 0.4 h(-1), close to the wash-out point, respiration rates dropped sharply and accumulation of glucose and acetic acid was observed. Energy generation through acetate formation yields less ATP compared with complete oxidation of the sugar carbon substrate, but is the result of maximized energy generation under conditions of restrictions in the tricarboxylic acid cycle or in respiratory NADH turnover

  10. Metabolic flux distribution and mathematical models for dynamic ...

    African Journals Online (AJOL)

    A simple model was build for the metabolic flux determination based on published articles. A method for metabolic flux determination by carbon labeling experiments was described and developed here in the first part of this study that allows mathematical description relating the measured quantities and the intracellular ...

  11. Metabolic flux distribution and mathematical models for dynamic ...

    African Journals Online (AJOL)

    user

    2011-03-21

    Mar 21, 2011 ... for metabolic flux determination by carbon labeling experiments was described and developed here in the first part of this study that allows mathematical description relating the measured quantities and the intracellular fluxes. The described method was used to investigate the central carbon metabolism of.

  12. Metabolic flux analysis of the phenylpropanoid pathway in wound-healing potato tuber tissue using stable isotope-labeled tracer and LC-MS spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Matsuda, Fumio; Morino, Keiko; Miyashita, Masahiro; Miyagawa, Hisashi [Kyoto Univ. (Japan). Department of Agriculture

    2003-05-01

    The metabolic flux of two phenylpropanoid metabolites, N-p-coumaroyloctopamine (p-CO) and chlorogenic acid (CGA), in the wound-healing potato tuber tissue was quantitatively analyzed by a newly developed method based upon the tracer experiment using stable isotope-labeled compounds and LC-MS. Tuber disks were treated with aqueous solution of L-phenyl-d{sub 5}-alanine, and the change in the ratio of stable isotope-labeled compound to non-labeled (isotope abundance) was monitored for p-CO and CGA in the tissue extract by LC-MS. The time-dependent change in the isotope abundance of each metabolite was fitted to an equation that was derived from the formation and conversion kinetics of each compound. Good correlations were obtained between the observed and calculated isotope abundances for both p-CO and CGA. The rates of p-CO formation and conversion (i.e. fluxes) were 1.15 and 0.96 nmol (g FW){sup -1}h{sup -1}, respectively, and for CGA, the rates 4.63 and 0.42 nmol (g FW){sup -1}h{sup -1}, respectively. This analysis enabled a direct comparison of the biosynthetic activity between these two compounds. (author)

  13. Metabolic control analysis of the penicillin biosynthetic pathway: the influence of the LLD-ACV:bisACV ratio on the flux control.

    Science.gov (United States)

    Theilgaard, H A; Nielsen, J

    1999-01-01

    An extended kinetic model for the first two steps of the penicillin biosynthetic pathway in Penicillium chrysogenum is set up. It includes the formation and reduction of the dimer bis-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (bisACV) from the first pathway intermediate LLD-ACV and their parallel inhibition of the enzyme ACV synthetase (ACVS). The kinetic model is based on Michaelis-Menten type kinetics, with non-competitive inhibition of the ACVS by both LLD-ACV and bisACV, and competitive inhibition of the isopenicillin N synthetase (IPNS) by glutathione. The inhibition constant of LLD-ACV, KACV is determined to be 0.54 mm. With the kinetic model metabolic control analysis is performed to identify the distribution of rate-control in the pathway at all ratios of LLD-ACV:bisACV. It is concluded that the flux control totally resides at the IPNS. This is a result of the regulation of the ACVS by both the LLD-ACV and bisACV demanding a higher flux through the IPNS enzyme to alleviate their inhibition. The measurement of an intracellular ratio of LLD-ACV:bisACV to be in the range of 1-2 moles per moles emphasises the importance of a fast conversion of LLD-ACV to IPN, and accumulation of LLD-ACV above the K(m)-value of the IPNS should therefore be avoided.

  14. Effect of amino acid supplementation on titer and glycosylation distribution in hybridoma cell cultures-Systems biology-based interpretation using genome-scale metabolic flux balance model and multivariate data analysis.

    Science.gov (United States)

    Reimonn, Thomas M; Park, Seo-Young; Agarabi, Cyrus D; Brorson, Kurt A; Yoon, Seongkyu

    2016-09-01

    Genome-scale flux balance analysis (FBA) is a powerful systems biology tool to characterize intracellular reaction fluxes during cell cultures. FBA estimates intracellular reaction rates by optimizing an objective function, subject to the constraints of a metabolic model and media uptake/excretion rates. A dynamic extension to FBA, dynamic flux balance analysis (DFBA), can calculate intracellular reaction fluxes as they change during cell cultures. In a previous study by Read et al. (2013), a series of informed amino acid supplementation experiments were performed on twelve parallel murine hybridoma cell cultures, and this data was leveraged for further analysis (Read et al., Biotechnol Prog. 2013;29:745-753). In order to understand the effects of media changes on the model murine hybridoma cell line, a systems biology approach is applied in the current study. Dynamic flux balance analysis was performed using a genome-scale mouse metabolic model, and multivariate data analysis was used for interpretation. The calculated reaction fluxes were examined using partial least squares and partial least squares discriminant analysis. The results indicate media supplementation increases product yield because it raises nutrient levels extending the growth phase, and the increased cell density allows for greater culture performance. At the same time, the directed supplementation does not change the overall metabolism of the cells. This supports the conclusion that product quality, as measured by glycoform assays, remains unchanged because the metabolism remains in a similar state. Additionally, the DFBA shows that metabolic state varies more at the beginning of the culture but less by the middle of the growth phase, possibly due to stress on the cells during inoculation. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1163-1173, 2016. © 2016 American Institute of Chemical Engineers.

  15. Rational Design of a Corynebacterium glutamicum Pantothenate Production Strain and Ins Characterization by Metabolic Flux Analysis and Genome-Wide Transcriptional Profiling

    Czech Academy of Sciences Publication Activity Database

    Hüser, A.T.; Chassagnole, Ch.; Lindley, N.D.; Merkamm, M.; Guyonvarch, A.; Elišáková, Veronika; Pátek, Miroslav; Kalinowski, J.; Brune, I.; Pühler, A.; Tauch, A.

    2005-01-01

    Roč. 71, č. 6 (2005), s. 3255-3268 ISSN 0099-2240 Institutional research plan: CEZ:AV0Z50200510 Keywords : corynebacterium glutamicum * metabolic flux Subject RIV: EE - Microbiology, Virology Impact factor: 3.818, year: 2005

  16. Computational Flux Balance Analysis Predicts that Stimulation of Energy Metabolism in Astrocytes and their Metabolic Interactions with Neurons Depend on Uptake of K(+) Rather than Glutamate

    DEFF Research Database (Denmark)

    DiNuzzo, Mauro; Giove, Federico; Maraviglia, Bruno

    2017-01-01

    Brain activity involves essential functional and metabolic interactions between neurons and astrocytes. The importance of astrocytic functions to neuronal signaling is supported by many experiments reporting high rates of energy consumption and oxidative metabolism in these glial cells...... utilization. In order to examine the participation of astrocytic energy metabolism in brain ion homeostasis, here we attempted to devise a simple stoichiometric relation linking glutamatergic neurotransmission to Na(+) and K(+) ionic currents. To this end, we took into account ion pumps and voltage....../ligand-gated channels using the stoichiometry derived from available energy budget for neocortical signaling and incorporated this stoichiometric relation into a computational metabolic model of neuron-astrocyte interactions. We aimed at reproducing the experimental observations about rates of metabolic pathways...

  17. MicrobesFlux: a web platform for drafting metabolic models from the KEGG database

    Directory of Open Access Journals (Sweden)

    Feng Xueyang

    2012-08-01

    Full Text Available Abstract Background Concurrent with the efforts currently underway in mapping microbial genomes using high-throughput sequencing methods, systems biologists are building metabolic models to characterize and predict cell metabolisms. One of the key steps in building a metabolic model is using multiple databases to collect and assemble essential information about genome-annotations and the architecture of the metabolic network for a specific organism. To speed up metabolic model development for a large number of microorganisms, we need a user-friendly platform to construct metabolic networks and to perform constraint-based flux balance analysis based on genome databases and experimental results. Results We have developed a semi-automatic, web-based platform (MicrobesFlux for generating and reconstructing metabolic models for annotated microorganisms. MicrobesFlux is able to automatically download the metabolic network (including enzymatic reactions and metabolites of ~1,200 species from the KEGG database (Kyoto Encyclopedia of Genes and Genomes and then convert it to a metabolic model draft. The platform also provides diverse customized tools, such as gene knockouts and the introduction of heterologous pathways, for users to reconstruct the model network. The reconstructed metabolic network can be formulated to a constraint-based flux model to predict and analyze the carbon fluxes in microbial metabolisms. The simulation results can be exported in the SBML format (The Systems Biology Markup Language. Furthermore, we also demonstrated the platform functionalities by developing an FBA model (including 229 reactions for a recent annotated bioethanol producer, Thermoanaerobacter sp. strain X514, to predict its biomass growth and ethanol production. Conclusion MicrobesFlux is an installation-free and open-source platform that enables biologists without prior programming knowledge to develop metabolic models for annotated microorganisms in the KEGG

  18. Software applications for flux balance analysis.

    Science.gov (United States)

    Lakshmanan, Meiyappan; Koh, Geoffrey; Chung, Bevan K S; Lee, Dong-Yup

    2014-01-01

    Flux balance analysis (FBA) is a widely used computational method for characterizing and engineering intrinsic cellular metabolism. The increasing number of its successful applications and growing popularity are possibly attributable to the availability of specific software tools for FBA. Each tool has its unique features and limitations with respect to operational environment, user-interface and supported analysis algorithms. Presented herein is an in-depth evaluation of currently available FBA applications, focusing mainly on usability, functionality, graphical representation and inter-operability. Overall, most of the applications are able to perform basic features of model creation and FBA simulation. COBRA toolbox, OptFlux and FASIMU are versatile to support advanced in silico algorithms to identify environmental and genetic targets for strain design. SurreyFBA, WEbcoli, Acorn, FAME, GEMSiRV and MetaFluxNet are the distinct tools which provide the user friendly interfaces in model handling. In terms of software architecture, FBA-SimVis and OptFlux have the flexible environments as they enable the plug-in/add-on feature to aid prospective functional extensions. Notably, an increasing trend towards the implementation of more tailored e-services such as central model repository and assistance to collaborative efforts was observed among the web-based applications with the help of advanced web-technologies. Furthermore, most recent applications such as the Model SEED, FAME, MetaFlux and MicrobesFlux have even included several routines to facilitate the reconstruction of genome-scale metabolic models. Finally, a brief discussion on the future directions of FBA applications was made for the benefit of potential tool developers.

  19. Flux-Enabled Exploration of the Role of Sip1 in galactose yeast metabolism

    DEFF Research Database (Denmark)

    Shymansky, Christopher M.; Wang, George; Baidoo, Edward E. K.

    2017-01-01

    13C metabolic flux analysis (13C MFA) is an important systems biology technique that has been used to investigate microbial metabolism for decades. The heterotrimer Snf1 kinase complex plays a key role in the preference Saccharomyces cerevisiae exhibits for glucose over galactose, a phenomenon kn...

  20. Metabolic-flux analysis of continuously cultured hybridoma cells using 13CO2 mass spectrometry in combination with 13C-lactate nuclear magnetic resonance spectroscopy and metabolic balancing

    NARCIS (Netherlands)

    Bonarius, H.P.J.; Ozemre, A.; Timmerarends, B.; Skrabal, P.; Tramper, J.; Schmid, G.; Heinzle, E.

    2001-01-01

    Protein production of mammalian-cell culture is limited due to accumulation of waste products such as lactate, CO2, and ammonia. In this study, the intracellular fluxes of hybridoma cells are measured to determine the amount by which various metabolic pathways contribute to the secretion of waste

  1. Invariability of Central Metabolic Flux Distribution in Shewanella oneidensis MR-1 Under Environmental or Genetic Perturbations

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Yinjie; Martin, Hector Garcia; Deutschbauer, Adam; Feng, Xueyang; Huang, Rick; Llora, Xavier; Arkin, Adam; Keasling, Jay D.

    2009-04-21

    An environmentally important bacterium with versatile respiration, Shewanella oneidensis MR-1, displayed significantly different growth rates under three culture conditions: minimal medium (doubling time {approx} 3 hrs), salt stressed minimal medium (doubling time {approx} 6 hrs), and minimal medium with amino acid supplementation (doubling time {approx}1.5 hrs). {sup 13}C-based metabolic flux analysis indicated that fluxes of central metabolic reactions remained relatively constant under the three growth conditions, which is in stark contrast to the reported significant changes in the transcript and metabolite profiles under various growth conditions. Furthermore, ten transposon mutants of S. oneidensis MR-1 were randomly chosen from a transposon library and their flux distributions through central metabolic pathways were revealed to be identical, even though such mutational processes altered the secondary metabolism, for example, glycine and C1 (5,10-Me-THF) metabolism.

  2. OptFlux: an open-source software platform for in silico metabolic engineering

    Directory of Open Access Journals (Sweden)

    Pinto José P

    2010-04-01

    Full Text Available Abstract Background Over the last few years a number of methods have been proposed for the phenotype simulation of microorganisms under different environmental and genetic conditions. These have been used as the basis to support the discovery of successful genetic modifications of the microbial metabolism to address industrial goals. However, the use of these methods has been restricted to bioinformaticians or other expert researchers. The main aim of this work is, therefore, to provide a user-friendly computational tool for Metabolic Engineering applications. Results OptFlux is an open-source and modular software aimed at being the reference computational application in the field. It is the first tool to incorporate strain optimization tasks, i.e., the identification of Metabolic Engineering targets, using Evolutionary Algorithms/Simulated Annealing metaheuristics or the previously proposed OptKnock algorithm. It also allows the use of stoichiometric metabolic models for (i phenotype simulation of both wild-type and mutant organisms, using the methods of Flux Balance Analysis, Minimization of Metabolic Adjustment or Regulatory on/off Minimization of Metabolic flux changes, (ii Metabolic Flux Analysis, computing the admissible flux space given a set of measured fluxes, and (iii pathway analysis through the calculation of Elementary Flux Modes. OptFlux also contemplates several methods for model simplification and other pre-processing operations aimed at reducing the search space for optimization algorithms. The software supports importing/exporting to several flat file formats and it is compatible with the SBML standard. OptFlux has a visualization module that allows the analysis of the model structure that is compatible with the layout information of Cell Designer, allowing the superimposition of simulation results with the model graph. Conclusions The OptFlux software is freely available, together with documentation and other resources, thus

  3. OptFlux: an open-source software platform for in silico metabolic engineering.

    Science.gov (United States)

    Rocha, Isabel; Maia, Paulo; Evangelista, Pedro; Vilaça, Paulo; Soares, Simão; Pinto, José P; Nielsen, Jens; Patil, Kiran R; Ferreira, Eugénio C; Rocha, Miguel

    2010-04-19

    Over the last few years a number of methods have been proposed for the phenotype simulation of microorganisms under different environmental and genetic conditions. These have been used as the basis to support the discovery of successful genetic modifications of the microbial metabolism to address industrial goals. However, the use of these methods has been restricted to bioinformaticians or other expert researchers. The main aim of this work is, therefore, to provide a user-friendly computational tool for Metabolic Engineering applications. OptFlux is an open-source and modular software aimed at being the reference computational application in the field. It is the first tool to incorporate strain optimization tasks, i.e., the identification of Metabolic Engineering targets, using Evolutionary Algorithms/Simulated Annealing metaheuristics or the previously proposed OptKnock algorithm. It also allows the use of stoichiometric metabolic models for (i) phenotype simulation of both wild-type and mutant organisms, using the methods of Flux Balance Analysis, Minimization of Metabolic Adjustment or Regulatory on/off Minimization of Metabolic flux changes, (ii) Metabolic Flux Analysis, computing the admissible flux space given a set of measured fluxes, and (iii) pathway analysis through the calculation of Elementary Flux Modes. OptFlux also contemplates several methods for model simplification and other pre-processing operations aimed at reducing the search space for optimization algorithms. The software supports importing/exporting to several flat file formats and it is compatible with the SBML standard. OptFlux has a visualization module that allows the analysis of the model structure that is compatible with the layout information of Cell Designer, allowing the superimposition of simulation results with the model graph. The OptFlux software is freely available, together with documentation and other resources, thus bridging the gap from research in strain optimization

  4. Modeling the Differences in Biochemical Capabilities of Pseudomonas Species by Flux Balance Analysis: How Good Are Genome-Scale Metabolic Networks at Predicting the Differences?

    Directory of Open Access Journals (Sweden)

    Parizad Babaei

    2014-01-01

    Full Text Available To date, several genome-scale metabolic networks have been reconstructed. These models cover a wide range of organisms, from bacteria to human. Such models have provided us with a framework for systematic analysis of metabolism. However, little effort has been put towards comparing biochemical capabilities of closely related species using their metabolic models. The accuracy of a model is highly dependent on the reconstruction process, as some errors may be included in the model during reconstruction. In this study, we investigated the ability of three Pseudomonas metabolic models to predict the biochemical differences, namely, iMO1086, iJP962, and iSB1139, which are related to P. aeruginosa PAO1, P. putida KT2440, and P. fluorescens SBW25, respectively. We did a comprehensive literature search for previous works containing biochemically distinguishable traits over these species. Amongst more than 1700 articles, we chose a subset of them which included experimental results suitable for in silico simulation. By simulating the conditions provided in the actual biological experiment, we performed case-dependent tests to compare the in silico results to the biological ones. We found out that iMO1086 and iJP962 were able to predict the experimental data and were much more accurate than iSB1139.

  5. Modeling the differences in biochemical capabilities of pseudomonas species by flux balance analysis: how good are genome-scale metabolic networks at predicting the differences?

    Science.gov (United States)

    Babaei, Parizad; Ghasemi-Kahrizsangi, Tahereh; Marashi, Sayed-Amir

    2014-01-01

    To date, several genome-scale metabolic networks have been reconstructed. These models cover a wide range of organisms, from bacteria to human. Such models have provided us with a framework for systematic analysis of metabolism. However, little effort has been put towards comparing biochemical capabilities of closely related species using their metabolic models. The accuracy of a model is highly dependent on the reconstruction process, as some errors may be included in the model during reconstruction. In this study, we investigated the ability of three Pseudomonas metabolic models to predict the biochemical differences, namely, iMO1086, iJP962, and iSB1139, which are related to P. aeruginosa PAO1, P. putida KT2440, and P. fluorescens SBW25, respectively. We did a comprehensive literature search for previous works containing biochemically distinguishable traits over these species. Amongst more than 1700 articles, we chose a subset of them which included experimental results suitable for in silico simulation. By simulating the conditions provided in the actual biological experiment, we performed case-dependent tests to compare the in silico results to the biological ones. We found out that iMO1086 and iJP962 were able to predict the experimental data and were much more accurate than iSB1139.

  6. Maintenance metabolism and carbon fluxes in Bacillus species

    Directory of Open Access Journals (Sweden)

    Decasper Seraina

    2008-06-01

    Full Text Available Abstract Background Selection of an appropriate host organism is crucial for the economic success of biotechnological processes. A generally important selection criterion is a low maintenance energy metabolism to reduce non-productive consumption of substrate. We here investigated, whether various bacilli that are closely related to Bacillus subtilis are potential riboflavin production hosts with low maintenance metabolism. Results While B. subtilis exhibited indeed the highest maintenance energy coefficient, B. licheniformis and B. amyloliquefaciens exhibited only statistically insignificantly reduced maintenance metabolism. Both B. pumilus and B. subtilis (natto exhibited irregular growth patterns under glucose limitation such that the maintenance metabolism could not be determined. The sole exception with significantly reduced maintenance energy requirements was the B. licheniformis strain T380B. The frequently used spo0A mutation significantly increased the maintenance metabolism of B. subtilis. At the level of 13C-detected intracellular fluxes, all investigated bacilli exhibited a significant flux through the pentose phosphate pathway, a prerequisite for efficient riboflavin production. Different from all other species, B. subtilis featured high respiratory tricarboxylic acid cycle fluxes in batch and chemostat cultures. In particular under glucose-limited conditions, this led to significant excess formation of NADPH of B. subtilis, while anabolic consumption was rather balanced with catabolic NADPH formation in the other bacilli. Conclusion Despite its successful commercial production of riboflavin, B. subtilis does not seem to be the optimal cell factory from a bioenergetic point of view. The best choice of the investigated strains is the sporulation-deficient B. licheniformis T380B strain. Beside a low maintenance energy coefficient, this strain grows robustly under different conditions and exhibits only moderate acetate overflow, hence

  7. Physiology and genetics of metabolic flux control in Zymomonas mobilis

    Energy Technology Data Exchange (ETDEWEB)

    Conway, T.

    1992-01-01

    This work seeks to understand the role of gene expression in regulating glycolytic enzyme synthesis in a balance that allows proper glycoltic flux control. The seven genes targeted for study in this laboratory have been cloned and sequenced, and molecular details of regulation have been investigated. Clear that glycolytic enzyme synthesis is coordinated to prevent the build up of toxic metabolic intermediates. The genetic mechanisms responsible for regulating balanced expression of the EntnerDoudoroff and glycolytic genes in Z. mobilis are beginning to be understood. Several layers of genetic control, perhaps in a hierarchal arrangement act in concert to determine the relative abundance of the glycolytic enzymes. These genetic controls involve differential translational efficiency, highly conserved promoter sequences, transcription factors, differential mRNA stabilities, and nucleolytic mRNA processing. The serendipitous cloning of the glucose facilitator, glf, as a result of linkage to several other genes of interest will have a significant impact on the study of Z. mobilis metabolism. The glucose facilitator is being characterized in a genetically reconstituted system in E. coli. Molecular genetic studies indicate that the ratio of glf expression to that of glk, zmf, and edd is carefully regulated, and suggests a critical role in metabolic control. Regulation of glycolytic gene expression is now sufficiently well understood to allow use of the glycolytic genes as tools to manipulate specified enzyme levels for the purpose of analyzing metabolic flux control. The critical genes have been subcloned for stable expression in Z. mobilis and placed under control of a regulated promoter system involving the tac promoter, the lacI repressor, and gene induction in by IPTG. HPLC methods have been developed that allow quantitation of virtually all of the metabolic intermediates in the cell pool.

  8. Metabolic flux profiling of recombinant protein secreting Pichia pastoris growing on glucose:methanol mixtures

    Science.gov (United States)

    2012-01-01

    Background The methylotrophic yeast Pichia pastoris has emerged as one of the most promising yeast hosts for the production of heterologous proteins. Mixed feeds of methanol and a multicarbon source instead of methanol as sole carbon source have been shown to improve product productivities and alleviate metabolic burden derived from protein production. Nevertheless, systematic quantitative studies on the relationships between the central metabolism and recombinant protein production in P. pastoris are still rather limited, particularly when growing this yeast on mixed carbon sources, thus hampering future metabolic network engineering strategies for improved protein production. Results The metabolic flux distribution in the central metabolism of P. pastoris growing on a mixed feed of glucose and methanol was analyzed by Metabolic Flux Analysis (MFA) using 13C-NMR-derived constraints. For this purpose, we defined new flux ratios for methanol assimilation pathways in P. pastoris cells growing on glucose:methanol mixtures. By using this experimental approach, the metabolic burden caused by the overexpression and secretion of a Rhizopus oryzae lipase (Rol) in P. pastoris was further analyzed. This protein has been previously shown to trigger the unfolded protein response in P. pastoris. A series of 13C-tracer experiments were performed on aerobic chemostat cultivations with a control and two different Rol producing strains growing at a dilution rate of 0.09 h−1 using a glucose:methanol 80:20 (w/w) mix as carbon source. The MFA performed in this study reveals a significant redistristribution of carbon fluxes in the central carbon metabolism when comparing the two recombinant strains vs the control strain, reflected in increased glycolytic, TCA cycle and NADH regeneration fluxes, as well as higher methanol dissimilation rates. Conclusions Overall, a further 13C-based MFA development to characterise the central metabolism of methylotrophic yeasts when growing on mixed

  9. Metabolic flux profiling of recombinant protein secreting Pichia pastoris growing on glucose:methanol mixtures

    Directory of Open Access Journals (Sweden)

    Jordà Joel

    2012-05-01

    Full Text Available Abstract Background The methylotrophic yeast Pichia pastoris has emerged as one of the most promising yeast hosts for the production of heterologous proteins. Mixed feeds of methanol and a multicarbon source instead of methanol as sole carbon source have been shown to improve product productivities and alleviate metabolic burden derived from protein production. Nevertheless, systematic quantitative studies on the relationships between the central metabolism and recombinant protein production in P. pastoris are still rather limited, particularly when growing this yeast on mixed carbon sources, thus hampering future metabolic network engineering strategies for improved protein production. Results The metabolic flux distribution in the central metabolism of P. pastoris growing on a mixed feed of glucose and methanol was analyzed by Metabolic Flux Analysis (MFA using 13C-NMR-derived constraints. For this purpose, we defined new flux ratios for methanol assimilation pathways in P. pastoris cells growing on glucose:methanol mixtures. By using this experimental approach, the metabolic burden caused by the overexpression and secretion of a Rhizopus oryzae lipase (Rol in P. pastoris was further analyzed. This protein has been previously shown to trigger the unfolded protein response in P. pastoris. A series of 13C-tracer experiments were performed on aerobic chemostat cultivations with a control and two different Rol producing strains growing at a dilution rate of 0.09 h−1 using a glucose:methanol 80:20 (w/w mix as carbon source. The MFA performed in this study reveals a significant redistristribution of carbon fluxes in the central carbon metabolism when comparing the two recombinant strains vs the control strain, reflected in increased glycolytic, TCA cycle and NADH regeneration fluxes, as well as higher methanol dissimilation rates. Conclusions Overall, a further 13C-based MFA development to characterise the central metabolism of methylotrophic

  10. A procedure for the estimation over time of metabolic fluxes in scenarios where measurements are uncertain and/or insufficient

    Directory of Open Access Journals (Sweden)

    Picó Jesús

    2007-10-01

    Full Text Available Abstract Background An indirect approach is usually used to estimate the metabolic fluxes of an organism: couple the available measurements with known biological constraints (e.g. stoichiometry. Typically this estimation is done under a static point of view. Therefore, the fluxes so obtained are only valid while the environmental conditions and the cell state remain stable. However, estimating the evolution over time of the metabolic fluxes is valuable to investigate the dynamic behaviour of an organism and also to monitor industrial processes. Although Metabolic Flux Analysis can be successively applied with this aim, this approach has two drawbacks: i sometimes it cannot be used because there is a lack of measurable fluxes, and ii the uncertainty of experimental measurements cannot be considered. The Flux Balance Analysis could be used instead, but the assumption of optimal behaviour of the organism brings other difficulties. Results We propose a procedure to estimate the evolution of the metabolic fluxes that is structured as follows: 1 measure the concentrations of extracellular species and biomass, 2 convert this data to measured fluxes and 3 estimate the non-measured fluxes using the Flux Spectrum Approach, a variant of Metabolic Flux Analysis that overcomes the difficulties mentioned above without assuming optimal behaviour. We apply the procedure to a real problem taken from the literature: estimate the metabolic fluxes during a cultivation of CHO cells in batch mode. We show that it provides a reliable and rich estimation of the non-measured fluxes, thanks to considering measurements uncertainty and reversibility constraints. We also demonstrate that this procedure can estimate the non-measured fluxes even when there is a lack of measurable species. In addition, it offers a new method to deal with inconsistency. Conclusion This work introduces a procedure to estimate time-varying metabolic fluxes that copes with the insufficiency of

  11. Flux-Enabled Exploration of the Role of Sip1 in Galactose Yeast Metabolism

    Directory of Open Access Journals (Sweden)

    Christopher M. Shymansky

    2017-05-01

    Full Text Available 13C metabolic flux analysis (13C MFA is an important systems biology technique that has been used to investigate microbial metabolism for decades. The heterotrimer Snf1 kinase complex plays a key role in the preference Saccharomyces cerevisiae exhibits for glucose over galactose, a phenomenon known as glucose repression or carbon catabolite repression. The SIP1 gene, encoding a part of this complex, has received little attention, presumably, because its knockout lacks a growth phenotype. We present a fluxomic investigation of the relative effects of the presence of galactose in classically glucose-repressing media and/or knockout of SIP1 using a multi-scale variant of 13C MFA known as 2-Scale 13C metabolic flux analysis (2S-13C MFA. In this study, all strains have the galactose metabolism deactivated (gal1Δ background so as to be able to separate the metabolic effects purely related to glucose repression from those arising from galactose metabolism. The resulting flux profiles reveal that the presence of galactose in classically glucose-repressing conditions, for a CEN.PK113-7D gal1Δ background, results in a substantial decrease in pentose phosphate pathway (PPP flux and increased flow from cytosolic pyruvate and malate through the mitochondria toward cytosolic branched-chain amino acid biosynthesis. These fluxomic redistributions are accompanied by a higher maximum specific growth rate, both seemingly in violation of glucose repression. Deletion of SIP1 in the CEN.PK113-7D gal1Δ cells grown in mixed glucose/galactose medium results in a further increase. Knockout of this gene in cells grown in glucose-only medium results in no change in growth rate and a corresponding decrease in glucose and ethanol exchange fluxes and flux through pathways involved in aspartate/threonine biosynthesis. Glucose repression appears to be violated at a 1/10 ratio of galactose-to-glucose. Based on the scientific literature, we may have conducted our experiments

  12. Estimation of dynamic flux profiles from metabolic time series data

    Directory of Open Access Journals (Sweden)

    Chou I-Chun

    2012-07-01

    Full Text Available Abstract Background Advances in modern high-throughput techniques of molecular biology have enabled top-down approaches for the estimation of parameter values in metabolic systems, based on time series data. Special among them is the recent method of dynamic flux estimation (DFE, which uses such data not only for parameter estimation but also for the identification of functional forms of the processes governing a metabolic system. DFE furthermore provides diagnostic tools for the evaluation of model validity and of the quality of a model fit beyond residual errors. Unfortunately, DFE works only when the data are more or less complete and the system contains as many independent fluxes as metabolites. These drawbacks may be ameliorated with other types of estimation and information. However, such supplementations incur their own limitations. In particular, assumptions must be made regarding the functional forms of some processes and detailed kinetic information must be available, in addition to the time series data. Results The authors propose here a systematic approach that supplements DFE and overcomes some of its shortcomings. Like DFE, the approach is model-free and requires only minimal assumptions. If sufficient time series data are available, the approach allows the determination of a subset of fluxes that enables the subsequent applicability of DFE to the rest of the flux system. The authors demonstrate the procedure with three artificial pathway systems exhibiting distinct characteristics and with actual data of the trehalose pathway in Saccharomyces cerevisiae. Conclusions The results demonstrate that the proposed method successfully complements DFE under various situations and without a priori assumptions regarding the model representation. The proposed method also permits an examination of whether at all, to what degree, or within what range the available time series data can be validly represented in a particular functional format of

  13. Flux coupling and transcriptional regulation within the metabolic network of the photosynthetic bacterium Synechocystis sp. PCC6803

    DEFF Research Database (Denmark)

    Montagud, Arnau; Zelezniak, Aleksej; Navarro, Emilio

    2011-01-01

    Synechocystis sp. PCC6803 is a model cyanobacterium capable of producing biofuels with CO2 as carbon source and with its metabolism fueled by light, for which it stands as a potential production platform of socio-economic importance. Compilation and characterization of Synechocystis genome...... networks, surrounded by a stable core of pathways leading to biomass building blocks. This analysis identified potential bottlenecks for hydrogen and ethanol production. Integration of transcriptomic data with the Synechocystis flux coupling networks lead to identification of reporter flux coupling pairs...... and reporter flux coupling groups - regulatory hot spots during metabolic shifts triggered by the availability of light. Overall, flux coupling analysis provided insight into the structural organization of Synechocystis sp. PCC6803 metabolic network toward designing of a photosynthesis-based production...

  14. Pathway confirmation and flux analysis of central metabolic pathways in Desulfovibrio vulgaris Hildenborough using gas chromatography-mass spectrometry and fourier transform-ion cyclotron resonance mass spectrometry

    International Nuclear Information System (INIS)

    Tang, Yinjie; Pingitore, Francesco; Mukhopadhyay, Aindrila; Phan, Richard; Hazen, Terry C.; Keasling, Jay D.

    2006-01-01

    It has been proposed that during growth under anaerobic or oxygen-limited conditions Shewanella oneidensis MR-1 uses the serine-isocitrate lyase pathway common to many methylotrophic anaerobes, in which formaldehyde produced from pyruvate is condensed with glycine to form serine. The serine is then transformed through hydroxypyruvate and glycerate to enter central metabolism at phosphoglycerate. To examine its use of the serine-isocitrate lyase pathway under anaerobic conditions, we grew S. oneidensis MR-1 on [1-13C] lactate as the sole carbon source with either trimethylamine N-oxide (TMAO) or fumarate as an electron acceptor. Analysis of cellular metabolites indicates that a large percentage (>75 percent) of lactate was partially oxidized to either acetate or pyruvate. The 13C isotope distributions in amino acids and other key metabolites indicate that, under anaerobic conditions, a complete serine pathway is not present, and lactate is oxidized via a highly reversible serine degradation pathway. The labeling data also suggest significant activity in the anaplerotic (malic enzyme and phosphoenolpyruvatecarboxylase) and glyoxylate shunt (isocitrate lyase and malate synthase) reactions. Although the tricarboxylic acid (TCA) cycle is often observed to be incomplete in many other anaerobes (absence of 2-oxoglutaratede hydrogenase activity), isotopic labeling supports the existence of a complete TCA cycle in S. oneidensis MR-1 under TMAO reduction condition

  15. Tissue-specific metabolic reprogramming drives nutrient flux in diabetic complications

    Science.gov (United States)

    Sas, Kelli M.; Kayampilly, Pradeep; Byun, Jaeman; Nair, Viji; Hinder, Lucy M.; Zhang, Hongyu; Lin, Chengmao; Qi, Nathan R.; Michailidis, George; Groop, Per-Henrik; Nelson, Robert G.; Darshi, Manjula; Sharma, Kumar; Schelling, Jeffrey R.; Sedor, John R.; Pop-Busui, Rodica; Weinberg, Joel M.; Soleimanpour, Scott A.; Abcouwer, Steven F.; Gardner, Thomas W.; Burant, Charles F.; Feldman, Eva L.; Kretzler, Matthias; Brosius, Frank C.

    2016-01-01

    Diabetes is associated with altered cellular metabolism, but how altered metabolism contributes to the development of diabetic complications is unknown. We used the BKS db/db diabetic mouse model to investigate changes in carbohydrate and lipid metabolism in kidney cortex, peripheral nerve, and retina. A systems approach using transcriptomics, metabolomics, and metabolic flux analysis identified tissue-specific differences, with increased glucose and fatty acid metabolism in the kidney, a moderate increase in the retina, and a decrease in the nerve. In the kidney, increased metabolism was associated with enhanced protein acetylation and mitochondrial dysfunction. To confirm these findings in human disease, we analyzed diabetic kidney transcriptomic data and urinary metabolites from a cohort of Southwestern American Indians. The urinary findings were replicated in 2 independent patient cohorts, the Finnish Diabetic Nephropathy and the Family Investigation of Nephropathy and Diabetes studies. Increased concentrations of TCA cycle metabolites in urine, but not in plasma, predicted progression of diabetic kidney disease, and there was an enrichment of pathways involved in glycolysis and fatty acid and amino acid metabolism. Our findings highlight tissue-specific changes in metabolism in complication-prone tissues in diabetes and suggest that urinary TCA cycle intermediates are potential prognostic biomarkers of diabetic kidney disease progression. PMID:27699244

  16. OptFlux: an open-source software platform for in silico metabolic engineering

    DEFF Research Database (Denmark)

    Rocha, I.; Maia, P.; Evangelista, P.

    2010-01-01

    software aimed at being the reference computational application in the field. It is the first tool to incorporate strain optimization tasks, i.e., the identification of Metabolic Engineering targets, using Evolutionary Algorithms/Simulated Annealing metaheuristics or the previously proposed Opt...... to address industrial goals. However, the use of these methods has been restricted to bioinformaticians or other expert researchers. The main aim of this work is, therefore, to provide a user-friendly computational tool for Metabolic Engineering applications. Results: OptFlux is an open-source and modular...... algorithms. The software supports importing/exporting to several flat file formats and it is compatible with the SBML standard. OptFlux has a visualization module that allows the analysis of the model structure that is compatible with the layout information of Cell Designer, allowing the superimposition...

  17. Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

    NARCIS (Netherlands)

    van den Bossche, Jan; Baardman, Jeroen; de Winther, Menno P. J.

    2015-01-01

    Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M-(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of

  18. Stochastic flux analysis of chemical reaction networks.

    Science.gov (United States)

    Kahramanoğulları, Ozan; Lynch, James F

    2013-12-07

    Chemical reaction networks provide an abstraction scheme for a broad range of models in biology and ecology. The two common means for simulating these networks are the deterministic and the stochastic approaches. The traditional deterministic approach, based on differential equations, enjoys a rich set of analysis techniques, including a treatment of reaction fluxes. However, the discrete stochastic simulations, which provide advantages in some cases, lack a quantitative treatment of network fluxes. We describe a method for flux analysis of chemical reaction networks, where flux is given by the flow of species between reactions in stochastic simulations of the network. Extending discrete event simulation algorithms, our method constructs several data structures, and thereby reveals a variety of statistics about resource creation and consumption during the simulation. We use these structures to quantify the causal interdependence and relative importance of the reactions at arbitrary time intervals with respect to the network fluxes. This allows us to construct reduced networks that have the same flux-behavior, and compare these networks, also with respect to their time series. We demonstrate our approach on an extended example based on a published ODE model of the same network, that is, Rho GTP-binding proteins, and on other models from biology and ecology. We provide a fully stochastic treatment of flux analysis. As in deterministic analysis, our method delivers the network behavior in terms of species transformations. Moreover, our stochastic analysis can be applied, not only at steady state, but at arbitrary time intervals, and used to identify the flow of specific species between specific reactions. Our cases study of Rho GTP-binding proteins reveals the role played by the cyclic reverse fluxes in tuning the behavior of this network.

  19. Amino Acid Flux from Metabolic Network Benefits Protein Translation: the Role of Resource Availability.

    Science.gov (United States)

    Hu, Xiao-Pan; Yang, Yi; Ma, Bin-Guang

    2015-06-09

    Protein translation is a central step in gene expression and affected by many factors such as codon usage bias, mRNA folding energy and tRNA abundance. Despite intensive previous studies, how metabolic amino acid supply correlates with protein translation efficiency remains unknown. In this work, we estimated the amino acid flux from metabolic network for each protein in Escherichia coli and Saccharomyces cerevisiae by using Flux Balance Analysis. Integrated with the mRNA expression level, protein abundance and ribosome profiling data, we provided a detailed description of the role of amino acid supply in protein translation. Our results showed that amino acid supply positively correlates with translation efficiency and ribosome density. Moreover, with the rank-based regression model, we found that metabolic amino acid supply facilitates ribosome utilization. Based on the fact that the ribosome density change of well-amino-acid-supplied genes is smaller than poorly-amino-acid-supply genes under amino acid starvation, we reached the conclusion that amino acid supply may buffer ribosome density change against amino acid starvation and benefit maintaining a relatively stable translation environment. Our work provided new insights into the connection between metabolic amino acid supply and protein translation process by revealing a new regulation strategy that is dependent on resource availability.

  20. Effects of drugs in subtoxic concentrations on the metabolic fluxes in human hepatoma cell line Hep G2

    International Nuclear Information System (INIS)

    Niklas, Jens; Noor, Fozia; Heinzle, Elmar

    2009-01-01

    Commonly used cytotoxicity assays assess the toxicity of a compound by measuring certain parameters which directly or indirectly correlate to the viability of the cells. However, the effects of a given compound at concentrations considerably below EC 50 values are usually not evaluated. These subtoxic effects are difficult to identify but may eventually cause severe and costly long term problems such as idiosyncratic hepatotoxicity. We determined the toxicity of three hepatotoxic compounds, namely amiodarone, diclofenac and tacrine on the human hepatoma cell line Hep G2 using an online kinetic respiration assay and analysed the effects of subtoxic concentrations of these drugs on the cellular metabolism by using metabolic flux analysis. Several changes in the metabolism could be detected upon exposure to subtoxic concentrations of the test compounds. Upon exposure to diclofenac and tacrine an increase in the TCA-cycle activity was observed which could be a signature of an uncoupling of the oxidative phosphorylation. The results indicate that metabolic flux analysis could serve as an invaluable novel tool for the investigation of the effects of drugs. The described methodology enables tracking the toxicity of compounds dynamically using the respiration assay in a range of concentrations and the metabolic flux analysis permits interesting insights into the changes in the central metabolism of the cell upon exposure to drugs.

  1. Random sampling of elementary flux modes in large-scale metabolic networks.

    Science.gov (United States)

    Machado, Daniel; Soons, Zita; Patil, Kiran Raosaheb; Ferreira, Eugénio C; Rocha, Isabel

    2012-09-15

    The description of a metabolic network in terms of elementary (flux) modes (EMs) provides an important framework for metabolic pathway analysis. However, their application to large networks has been hampered by the combinatorial explosion in the number of modes. In this work, we develop a method for generating random samples of EMs without computing the whole set. Our algorithm is an adaptation of the canonical basis approach, where we add an additional filtering step which, at each iteration, selects a random subset of the new combinations of modes. In order to obtain an unbiased sample, all candidates are assigned the same probability of getting selected. This approach avoids the exponential growth of the number of modes during computation, thus generating a random sample of the complete set of EMs within reasonable time. We generated samples of different sizes for a metabolic network of Escherichia coli, and observed that they preserve several properties of the full EM set. It is also shown that EM sampling can be used for rational strain design. A well distributed sample, that is representative of the complete set of EMs, should be suitable to most EM-based methods for analysis and optimization of metabolic networks. Source code for a cross-platform implementation in Python is freely available at http://code.google.com/p/emsampler. dmachado@deb.uminho.pt Supplementary data are available at Bioinformatics online.

  2. A robust and efficient method for estimating enzyme complex abundance and metabolic flux from expression data.

    Science.gov (United States)

    Barker, Brandon E; Sadagopan, Narayanan; Wang, Yiping; Smallbone, Kieran; Myers, Christopher R; Xi, Hongwei; Locasale, Jason W; Gu, Zhenglong

    2015-12-01

    A major theme in constraint-based modeling is unifying experimental data, such as biochemical information about the reactions that can occur in a system or the composition and localization of enzyme complexes, with high-throughput data including expression data, metabolomics, or DNA sequencing. The desired result is to increase predictive capability and improve our understanding of metabolism. The approach typically employed when only gene (or protein) intensities are available is the creation of tissue-specific models, which reduces the available reactions in an organism model, and does not provide an objective function for the estimation of fluxes. We develop a method, flux assignment with LAD (least absolute deviation) convex objectives and normalization (FALCON), that employs metabolic network reconstructions along with expression data to estimate fluxes. In order to use such a method, accurate measures of enzyme complex abundance are needed, so we first present an algorithm that addresses quantification of complex abundance. Our extensions to prior techniques include the capability to work with large models and significantly improved run-time performance even for smaller models, an improved analysis of enzyme complex formation, the ability to handle large enzyme complex rules that may incorporate multiple isoforms, and either maintained or significantly improved correlation with experimentally measured fluxes. FALCON has been implemented in MATLAB and ATS, and can be downloaded from: https://github.com/bbarker/FALCON. ATS is not required to compile the software, as intermediate C source code is available. FALCON requires use of the COBRA Toolbox, also implemented in MATLAB. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Predicting growth conditions from internal metabolic fluxes in an in-silico model of E. coli.

    Directory of Open Access Journals (Sweden)

    Viswanadham Sridhara

    Full Text Available A widely studied problem in systems biology is to predict bacterial phenotype from growth conditions, using mechanistic models such as flux balance analysis (FBA. However, the inverse prediction of growth conditions from phenotype is rarely considered. Here we develop a computational framework to carry out this inverse prediction on a computational model of bacterial metabolism. We use FBA to calculate bacterial phenotypes from growth conditions in E. coli, and then we assess how accurately we can predict the original growth conditions from the phenotypes. Prediction is carried out via regularized multinomial regression. Our analysis provides several important physiological and statistical insights. First, we show that by analyzing metabolic end products we can consistently predict growth conditions. Second, prediction is reliable even in the presence of small amounts of impurities. Third, flux through a relatively small number of reactions per growth source (∼10 is sufficient for accurate prediction. Fourth, combining the predictions from two separate models, one trained only on carbon sources and one only on nitrogen sources, performs better than models trained to perform joint prediction. Finally, that separate predictions perform better than a more sophisticated joint prediction scheme suggests that carbon and nitrogen utilization pathways, despite jointly affecting cellular growth, may be fairly decoupled in terms of their dependence on specific assortments of molecular precursors.

  4. Diversity of flux distribution in central carbon metabolism of S. cerevisiae strains from diverse environments.

    Science.gov (United States)

    Nidelet, Thibault; Brial, Pascale; Camarasa, Carole; Dequin, Sylvie

    2016-04-05

    S. cerevisiae has attracted considerable interest in recent years as a model for ecology and evolutionary biology, revealing a substantial genetic and phenotypic diversity. However, there is a lack of knowledge on the diversity of metabolic networks within this species. To identify the metabolic and evolutionary constraints that shape metabolic fluxes in S. cerevisiae, we used a dedicated constraint-based model to predict the central carbon metabolism flux distribution of 43 strains from different ecological origins, grown in wine fermentation conditions. In analyzing these distributions, we observed a highly contrasted situation in flux variability, with quasi-constancy of the glycolysis and ethanol synthesis yield yet high flexibility of other fluxes, such as the pentose phosphate pathway and acetaldehyde production. Furthermore, these fluxes with large variability showed multimodal distributions that could be linked to strain origin, indicating a convergence between genetic origin and flux phenotype. Flux variability is pathway-dependent and, for some flux, a strain origin effect can be found. These data highlight the constraints shaping the yeast operative central carbon network and provide clues for the design of strategies for strain improvement.

  5. Detecting the Significant Flux Backbone of Escherichia coli metabolism.

    Science.gov (United States)

    Güell, Oriol; Sagués, Francesc; Serrano, M Ángeles

    2017-05-01

    The heterogeneity of computationally predicted reaction fluxes in metabolic networks within a single flux state can be exploited to detect their significant flux backbone. Here, we disclose the backbone of Escherichia coli, and compare it with the backbones of other bacteria. We find that, in general, the core of the backbones is mainly composed of reactions in energy metabolism corresponding to ancient pathways. In E. coli, the synthesis of nucleotides and the metabolism of lipids form smaller cores which rely critically on energy metabolism. Moreover, the consideration of different media leads to the identification of pathways sensitive to environmental changes. The metabolic backbone of an organism is thus useful to trace simultaneously both its evolution and adaptation fingerprints. © 2017 The Authors FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

  6. Metabolic network rewiring of propionate flux compensates vitamin B12 deficiency in C. elegans

    Science.gov (United States)

    Watson, Emma; Olin-Sandoval, Viridiana; Hoy, Michael J; Li, Chi-Hua; Louisse, Timo; Yao, Victoria; Mori, Akihiro; Holdorf, Amy D; Troyanskaya, Olga G; Ralser, Markus; Walhout, Albertha JM

    2016-01-01

    Metabolic network rewiring is the rerouting of metabolism through the use of alternate enzymes to adjust pathway flux and accomplish specific anabolic or catabolic objectives. Here, we report the first characterization of two parallel pathways for the breakdown of the short chain fatty acid propionate in Caenorhabditis elegans. Using genetic interaction mapping, gene co-expression analysis, pathway intermediate quantification and carbon tracing, we uncover a vitamin B12-independent propionate breakdown shunt that is transcriptionally activated on vitamin B12 deficient diets, or under genetic conditions mimicking the human diseases propionic- and methylmalonic acidemia, in which the canonical B12-dependent propionate breakdown pathway is blocked. Our study presents the first example of transcriptional vitamin-directed metabolic network rewiring to promote survival under vitamin deficiency. The ability to reroute propionate breakdown according to B12 availability may provide C. elegans with metabolic plasticity and thus a selective advantage on different diets in the wild. DOI: http://dx.doi.org/10.7554/eLife.17670.001 PMID:27383050

  7. Thermodynamic principles governing metabolic operation : inference, analysis, and prediction

    NARCIS (Netherlands)

    Niebel, Bastian

    2015-01-01

    The principles governing metabolic flux are poorly understood. Because diverse organisms show similar metabolic flux patterns, we hypothesized that fundamental thermodynamic constraints might shape cellular metabolism. We developed a constraint-based model for Saccharomyces cerevisiae that included

  8. Modelling central metabolic fluxes by constraint-based optimization reveals metabolic reprogramming of developing Solanum lycopersicum (tomato) fruit.

    Science.gov (United States)

    Colombié, Sophie; Nazaret, Christine; Bénard, Camille; Biais, Benoît; Mengin, Virginie; Solé, Marion; Fouillen, Laëtitia; Dieuaide-Noubhani, Martine; Mazat, Jean-Pierre; Beauvoit, Bertrand; Gibon, Yves

    2015-01-01

    Modelling of metabolic networks is a powerful tool to analyse the behaviour of developing plant organs, including fruits. Guided by our current understanding of heterotrophic metabolism of plant cells, a medium-scale stoichiometric model, including the balance of co-factors and energy, was constructed in order to describe metabolic shifts that occur through the nine sequential stages of Solanum lycopersicum (tomato) fruit development. The measured concentrations of the main biomass components and the accumulated metabolites in the pericarp, determined at each stage, were fitted in order to calculate, by derivation, the corresponding external fluxes. They were used as constraints to solve the model by minimizing the internal fluxes. The distribution of the calculated fluxes of central metabolism were then analysed and compared with known metabolic behaviours. For instance, the partition of the main metabolic pathways (glycolysis, pentose phosphate pathway, etc.) was relevant throughout fruit development. We also predicted a valid import of carbon and nitrogen by the fruit, as well as a consistent CO2 release. Interestingly, the energetic balance indicates that excess ATP is dissipated just before the onset of ripening, supporting the concept of the climacteric crisis. Finally, the apparent contradiction between calculated fluxes with low values compared with measured enzyme capacities suggest a complex reprogramming of the metabolic machinery during fruit development. With a powerful set of experimental data and an accurate definition of the metabolic system, this work provides important insight into the metabolic and physiological requirements of the developing tomato fruits. © 2014 The Authors The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.

  9. Co-regulation of metabolic genes is better explained by flux coupling than by network distance.

    Directory of Open Access Journals (Sweden)

    Richard A Notebaart

    2008-01-01

    Full Text Available To what extent can modes of gene regulation be explained by systems-level properties of metabolic networks? Prior studies on co-regulation of metabolic genes have mainly focused on graph-theoretical features of metabolic networks and demonstrated a decreasing level of co-expression with increasing network distance, a naïve, but widely used, topological index. Others have suggested that static graph representations can poorly capture dynamic functional associations, e.g., in the form of dependence of metabolic fluxes across genes in the network. Here, we systematically tested the relative importance of metabolic flux coupling and network position on gene co-regulation, using a genome-scale metabolic model of Escherichia coli. After validating the computational method with empirical data on flux correlations, we confirm that genes coupled by their enzymatic fluxes not only show similar expression patterns, but also share transcriptional regulators and frequently reside in the same operon. In contrast, we demonstrate that network distance per se has relatively minor influence on gene co-regulation. Moreover, the type of flux coupling can explain refined properties of the regulatory network that are ignored by simple graph-theoretical indices. Our results underline the importance of studying functional states of cellular networks to define physiologically relevant associations between genes and should stimulate future developments of novel functional genomic tools.

  10. In vivo dynamics of galactose metabolism in Saccharomyces cerevisiae: Metabolic fluxes and metabolite levels

    DEFF Research Database (Denmark)

    Østergaard, Simon; Olsson, Lisbeth; Nielsen, Jens

    2001-01-01

    the flux entering the TCA cycle was low and acetate and ethanol started to be excreted to the extracellular medium. During recovery of cell growth the flux entering the TCA cycle increased again, and at t = 30 this flux exceeded the corresponding steady-state flux. During the pulse an enhanced level of Gal...

  11. Carbon-flux distribution within Streptomyces coelicolor metabolism: a comparison between the actinorhodin-producing strain M145 and its non-producing derivative M1146.

    Directory of Open Access Journals (Sweden)

    Fabien Coze

    Full Text Available Metabolic Flux Analysis is now viewed as essential to elucidate the metabolic pattern of cells and to design appropriate genetic engineering strategies to improve strain performance and production processes. Here, we investigated carbon flux distribution in two Streptomyces coelicolor A3 (2 strains: the wild type M145 and its derivative mutant M1146, in which gene clusters encoding the four main antibiotic biosynthetic pathways were deleted. Metabolic Flux Analysis and (13C-labeling allowed us to reconstruct a flux map under steady-state conditions for both strains. The mutant strain M1146 showed a higher growth rate, a higher flux through the pentose phosphate pathway and a higher flux through the anaplerotic phosphoenolpyruvate carboxylase. In that strain, glucose uptake and the flux through the Krebs cycle were lower than in M145. The enhanced flux through the pentose phosphate pathway in M1146 is thought to generate NADPH enough to face higher needs for biomass biosynthesis and other processes. In both strains, the production of NADPH was higher than NADPH needs, suggesting a key role for nicotinamide nucleotide transhydrogenase for redox homeostasis. ATP production is also likely to exceed metabolic ATP needs, indicating that ATP consumption for maintenance is substantial.Our results further suggest a possible competition between actinorhodin and triacylglycerol biosynthetic pathways for their common precursor, acetyl-CoA. These findings may be instrumental in developing new strategies exploiting S. coelicolor as a platform for the production of bio-based products of industrial interest.

  12. Metabolic engineering of Synechococcus elongatus PCC 7942 for improvement of 1,3-propanediol and glycerol production based on in silico simulation of metabolic flux distribution.

    Science.gov (United States)

    Hirokawa, Yasutaka; Matsuo, Shingo; Hamada, Hiroyuki; Matsuda, Fumio; Hanai, Taizo

    2017-11-25

    Production directly from carbon dioxide by engineered cyanobacteria is one of the promising technologies for sustainable future. Previously, we have successfully achieved 1,3-propanediol (1,3-PDO) production using Synechococcus elongatus PCC 7942 with a synthetic metabolic pathway. The strain into which the synthetic metabolic pathway was introduced produced 3.48 mM (0.265 g/L) 1,3-PDO and 14.3 mM (1.32 g/L) glycerol during 20 days of incubation. In this study, the productivities of 1,3-PDO were improved by gene disruption selected by screening with in silico simulation. First, a stoichiometric metabolic model was applied to prediction of cellular metabolic flux distribution in a 1,3-PDO-producing strain of S. elongatus PCC 7942. A genome-scale model of S. elongatus PCC 7942 constructed by Knoop was modified by the addition of a synthetic metabolic pathway for 1,3-PDO production. Next, the metabolic flux distribution predicted by metabolic flux balance analysis (FBA) was used for in silico simulation of gene disruption. As a result of gene disruption simulation, NADPH dehydrogenase 1 (NDH-1) complexes were found by screening to be the most promising candidates for disruption to improve 1,3-PDO production. The effect of disruption of the gene encoding a subunit of the NDH-1 complex was evaluated in the 1,3-PDO-producing strain. During 20 days of incubation, the ndhF1-null 1,3-PDO-producing strain showed the highest titers: 4.44 mM (0.338 g/L) 1,3-PDO and 30.3 mM (2.79 g/L) glycerol. In this study, we successfully improved 1,3-PDO productivity on the basis of in silico simulation of gene disruption.

  13. A holistic view of dietary carbohydrate utilization in lobster: digestion, postprandial nutrient flux, and metabolism.

    Directory of Open Access Journals (Sweden)

    Leandro Rodríguez-Viera

    Full Text Available Crustaceans exhibit a remarkable variation in their feeding habits and food type, but most knowledge on carbohydrate digestion and utilization in this group has come from research on few species. The aim of this study was to make an integrative analysis of dietary carbohydrate utilization in the spiny lobster Panulirus argus. We used complementary methodologies such as different assessments of digestibility, activity measurements of digestive and metabolic enzymes, and post-feeding flux of nutrients and metabolites. Several carbohydrates were well digested by the lobster, but maize starch was less digestible than all other starches studied, and its inclusion in diet affected protein digestibility. Most intense hydrolysis of carbohydrates in the gastric chamber of lobster occurred between 2-6 h after ingestion and afterwards free glucose increased in hemolymph. The inclusion of wheat in diet produced a slow clearance of glucose from the gastric fluid and a gradual increase in hemolymph glucose. More intense hydrolysis of protein in the gastric chamber occurred 6-12 h after ingestion and then amino acids tended to increase in hemolymph. Triglyceride concentration in hemolymph rose earlier in wheat-fed lobsters than in lobsters fed other carbohydrates, but it decreased the most 24 h later. Analyses of metabolite levels and activities of different metabolic enzymes revealed that intermolt lobsters had a low capacity to store and use glycogen, although it was slightly higher in wheat-fed lobsters. Lobsters fed maize and rice diets increased amino acid catabolism, while wheat-fed lobsters exhibited higher utilization of fatty acids. Multivariate analysis confirmed that the type of carbohydrate ingested had a profound effect on overall metabolism. Although we found no evidence of a protein-sparing effect of dietary carbohydrate, differences in the kinetics of their digestion and absorption impacted lobster metabolism determining the fate of other

  14. Bacterial adaptation through distributed sensing of metabolic fluxes

    NARCIS (Netherlands)

    Kotte, Oliver; Zaugg, Judith B.; Heinemann, Matthias

    The recognition of carbon sources and the regulatory adjustments to recognized changes are of particular importance for bacterial survival in fluctuating environments. Despite a thorough knowledge base of Escherichia coli’s central metabolism and its regulation, fundamental aspects of the employed

  15. Detection of Metabolic Fluxes of O and H Atoms into Intracellular Water in Mammalian Cells

    Science.gov (United States)

    Kreuzer, Helen W.; Quaroni, Luca; Podlesak, David W.; Zlateva, Theodora; Bollinger, Nikki; McAllister, Aaron; Lott, Michael J.; Hegg, Eric L.

    2012-01-01

    Metabolic processes result in the release and exchange of H and O atoms from organic material as well as some inorganic salts and gases. These fluxes of H and O atoms into intracellular water result in an isotopic gradient that can be measured experimentally. Using isotope ratio mass spectroscopy, we revealed that slightly over 50% of the H and O atoms in the intracellular water of exponentially-growing cultured Rat-1 fibroblasts were isotopically distinct from growth medium water. We then employed infrared spectromicroscopy to detect in real time the flux of H atoms in these same cells. Importantly, both of these techniques indicate that the H and O fluxes are dependent on metabolic processes; cells that are in lag phase or are quiescent exhibit a much smaller flux. In addition, water extracted from the muscle tissue of rats contained a population of H and O atoms that were isotopically distinct from body water, consistent with the results obtained using the cultured Rat-1 fibroblasts. Together these data demonstrate that metabolic processes produce fluxes of H and O atoms into intracellular water, and that these fluxes can be detected and measured in both cultured mammalian cells and in mammalian tissue. PMID:22848359

  16. Prediction of Metabolic Flux Distribution from Gene Expression Data Based on the Flux Minimization Principle

    Science.gov (United States)

    2014-11-14

    Saccharomyces cerevisiae , and wild-type and mutant Escherichia coli strains, our method achieved high prediction accuracy, as gauged by correlation...the aerobic growth of S. cerevisiae . We used experimental data collected by Lee et al., which included RNA-seq transcriptomic data and exchange flux...measurements of S. cerevisiae aerobically growing in chemostat cultures. This study provides data under two different growth conditions, i.e., glucose

  17. Thermodynamics-based Metabolite Sensitivity Analysis in metabolic networks.

    Science.gov (United States)

    Kiparissides, A; Hatzimanikatis, V

    2017-01-01

    The increasing availability of large metabolomics datasets enhances the need for computational methodologies that can organize the data in a way that can lead to the inference of meaningful relationships. Knowledge of the metabolic state of a cell and how it responds to various stimuli and extracellular conditions can offer significant insight in the regulatory functions and how to manipulate them. Constraint based methods, such as Flux Balance Analysis (FBA) and Thermodynamics-based flux analysis (TFA), are commonly used to estimate the flow of metabolites through genome-wide metabolic networks, making it possible to identify the ranges of flux values that are consistent with the studied physiological and thermodynamic conditions. However, unless key intracellular fluxes and metabolite concentrations are known, constraint-based models lead to underdetermined problem formulations. This lack of information propagates as uncertainty in the estimation of fluxes and basic reaction properties such as the determination of reaction directionalities. Therefore, knowledge of which metabolites, if measured, would contribute the most to reducing this uncertainty can significantly improve our ability to define the internal state of the cell. In the present work we combine constraint based modeling, Design of Experiments (DoE) and Global Sensitivity Analysis (GSA) into the Thermodynamics-based Metabolite Sensitivity Analysis (TMSA) method. TMSA ranks metabolites comprising a metabolic network based on their ability to constrain the gamut of possible solutions to a limited, thermodynamically consistent set of internal states. TMSA is modular and can be applied to a single reaction, a metabolic pathway or an entire metabolic network. This is, to our knowledge, the first attempt to use metabolic modeling in order to provide a significance ranking of metabolites to guide experimental measurements. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier

  18. Framework for network modularization and Bayesian network analysis to investigate the perturbed metabolic network.

    Science.gov (United States)

    Kim, Hyun Uk; Kim, Tae Yong; Lee, Sang Yup

    2011-01-01

    Genome-scale metabolic network models have contributed to elucidating biological phenomena, and predicting gene targets to engineer for biotechnological applications. With their increasing importance, their precise network characterization has also been crucial for better understanding of the cellular physiology. We herein introduce a framework for network modularization and Bayesian network analysis (FMB) to investigate organism's metabolism under perturbation. FMB reveals direction of influences among metabolic modules, in which reactions with similar or positively correlated flux variation patterns are clustered, in response to specific perturbation using metabolic flux data. With metabolic flux data calculated by constraints-based flux analysis under both control and perturbation conditions, FMB, in essence, reveals the effects of specific perturbations on the biological system through network modularization and Bayesian network analysis at metabolic modular level. As a demonstration, this framework was applied to the genetically perturbed Escherichia coli metabolism, which is a lpdA gene knockout mutant, using its genome-scale metabolic network model. After all, it provides alternative scenarios of metabolic flux distributions in response to the perturbation, which are complementary to the data obtained from conventionally available genome-wide high-throughput techniques or metabolic flux analysis.

  19. Quantitative flux analysis reveals folate-dependent NADPH production

    Science.gov (United States)

    Fan, Jing; Ye, Jiangbin; Kamphorst, Jurre J.; Shlomi, Tomer; Thompson, Craig B.; Rabinowitz, Joshua D.

    2014-06-01

    ATP is the dominant energy source in animals for mechanical and electrical work (for example, muscle contraction or neuronal firing). For chemical work, there is an equally important role for NADPH, which powers redox defence and reductive biosynthesis. The most direct route to produce NADPH from glucose is the oxidative pentose phosphate pathway, with malic enzyme sometimes also important. Although the relative contribution of glycolysis and oxidative phosphorylation to ATP production has been extensively analysed, similar analysis of NADPH metabolism has been lacking. Here we demonstrate the ability to directly track, by liquid chromatography-mass spectrometry, the passage of deuterium from labelled substrates into NADPH, and combine this approach with carbon labelling and mathematical modelling to measure NADPH fluxes. In proliferating cells, the largest contributor to cytosolic NADPH is the oxidative pentose phosphate pathway. Surprisingly, a nearly comparable contribution comes from serine-driven one-carbon metabolism, in which oxidation of methylene tetrahydrofolate to 10-formyl-tetrahydrofolate is coupled to reduction of NADP+ to NADPH. Moreover, tracing of mitochondrial one-carbon metabolism revealed complete oxidation of 10-formyl-tetrahydrofolate to make NADPH. As folate metabolism has not previously been considered an NADPH producer, confirmation of its functional significance was undertaken through knockdown of methylenetetrahydrofolate dehydrogenase (MTHFD) genes. Depletion of either the cytosolic or mitochondrial MTHFD isozyme resulted in decreased cellular NADPH/NADP+ and reduced/oxidized glutathione ratios (GSH/GSSG) and increased cell sensitivity to oxidative stress. Thus, although the importance of folate metabolism for proliferating cells has been long recognized and attributed to its function of producing one-carbon units for nucleic acid synthesis, another crucial function of this pathway is generating reducing power.

  20. Dynamic Flux Balance Analysis Using DFBAlab.

    Science.gov (United States)

    Gomez, Jose Alberto; Barton, Paul I

    2018-01-01

    Bioprocesses are of critical importance in several industries such as the food and pharmaceutical industries. Despite their importance and widespread application, bioprocess models remain rather simplistic and based on unstructured models. These simple models have limitations, making it very difficult to model complex bioprocesses. With dynamic flux balance analysis (DFBA) more comprehensive bioprocess models can be obtained. DFBA simulations are difficult to carry out because they result in dynamic systems with linear programs embedded. Therefore, the use of DFBA as a modeling tool has been limited. With DFBAlab, a MATLAB code that performs efficient and reliable DFBA simulations, the use of DFBA as a modeling tool has become more accessible. Here, we illustrate with an example how to implement bioprocess models in DFBAlab.

  1. Simulating Serial-Target Antibacterial Drug Synergies Using Flux Balance Analysis

    DEFF Research Database (Denmark)

    Krueger, Andrew S.; Munck, Christian; Dantas, Gautam

    2016-01-01

    Flux balance analysis (FBA) is an increasingly useful approach for modeling the behavior of metabolic systems. However, standard FBA modeling of genetic knockouts cannot predict drug combination synergies observed between serial metabolic targets, even though such synergies give rise to some...... the possibility for more accurate genome-scale predictions of drug synergies, which can be used to suggest treatments for infections and other diseases....

  2. Regulation of amino-acid metabolism controls flux to lipid accumulation in Yarrowia lipolytica

    DEFF Research Database (Denmark)

    Kerkhoven, Eduard J.; Pomraning, Kyle R.; Baker, Scott E.

    2016-01-01

    accumulation in Y. lipolytica does not involve transcriptional regulation of lipid metabolism but is associated with regulation of amino-acid biosynthesis, resulting in redirection of carbon flux during nitrogen limitation from amino acids to lipids. Lipid accumulation in Y. lipolytica at nitrogen limitation...

  3. Metabolic flux pattern of glucose utilization by Xanthomonas campestris pv. campestris: prevalent role of the Entner-Doudoroff pathway and minor fluxes through the pentose phosphate pathway and glycolysis.

    Science.gov (United States)

    Schatschneider, Sarah; Huber, Claudia; Neuweger, Heiko; Watt, Tony Francis; Pühler, Alfred; Eisenreich, Wolfgang; Wittmann, Christoph; Niehaus, Karsten; Vorhölter, Frank-Jörg

    2014-10-01

    The well-studied plant pathogenic bacterium Xanthomonas campestris pv. campestris (Xcc) synthesizes the biotechnologically important polysaccharide xanthan gum, which is also regarded as a virulence factor in plant interactions. In Xcc, sugars like glucose are utilized as a source to generate energy and biomass for growth and pathogenicity. In this study, we used [1-(13)C]glucose as a tracer to analyze the fluxes in the central metabolism of the bacterium growing in a minimal medium. (13)C-Metabolic flux analysis based on gas chromatography-mass spectrometry (GC-MS) confirmed the prevalent catabolic role of the Entner-Doudoroff pathway. Comparative nuclear magnetic resonance (NMR)-based isotopologue profiling of a mutant deficient in glycolysis gave evidence for a moderate flux via glycolysis in the wild-type. In addition to reconfirming the Entner-Doudoroff pathway as a catabolic main route, this approach affirmed a numerically minor but important flux via the pentose phosphate pathway.

  4. Aerobic glucose metabolism of Saccharomyces kluyveri: Growth, metabolite production, and quantification of metabolic fluxes

    DEFF Research Database (Denmark)

    Møller, Kasper; Christensen, B.; Förster, Jochen

    2002-01-01

    growth in aerobic glucose-limited continuous cultivation. It was found that in S. kluyveri the flux into the pentose phosphate pathway was 18.8 mmole per 100 mmole glucose consumed during respiratory growth in aerobic glucose-limited continuous cultivation. Such a low flux into the pentose phosphate...

  5. A kinetic model describes metabolic response to perturbations and distribution of flux control in the benzenoid network of Petunia hybrida.

    Science.gov (United States)

    Colón, Amy Marshall; Sengupta, Neelanjan; Rhodes, David; Dudareva, Natalia; Morgan, John

    2010-04-01

    In recent years there has been much interest in the genetic enhancement of plant metabolism; however, attempts at genetic modification are often unsuccessful due to an incomplete understanding of network dynamics and their regulatory properties. Kinetic modeling of plant metabolic networks can provide predictive information on network control and response to genetic perturbations, which allow estimation of flux at any concentration of intermediate or enzyme in the system. In this research, a kinetic model of the benzenoid network was developed to simulate whole network responses to different concentrations of supplied phenylalanine (Phe) in petunia flowers and capture flux redistributions caused by genetic manipulations. Kinetic parameters were obtained by network decomposition and non-linear least squares optimization of data from petunia flowers supplied with either 75 or 150 mm(2)H(5)-Phe. A single set of kinetic parameters simultaneously accommodated labeling and pool size data obtained for all endogenous and emitted volatiles at the two concentrations of supplied (2)H(5)-Phe. The generated kinetic model was validated using flowers from transgenic petunia plants in which benzyl CoA:benzyl alcohol/phenylethanol benzoyltransferase (BPBT) was down-regulated via RNAi. The determined in vivo kinetic parameters were used for metabolic control analysis, in which flux control coefficients were calculated for fluxes around the key branch point at Phe and revealed that phenylacetaldehyde synthase activity is the primary controlling factor for the phenylacetaldehyde branch of the benzenoid network. In contrast, control of flux through the beta-oxidative and non-beta-oxidative pathways is highly distributed.

  6. The Protein Cost of Metabolic Fluxes: Prediction from Enzymatic Rate Laws and Cost Minimization.

    Directory of Open Access Journals (Sweden)

    Elad Noor

    2016-11-01

    Full Text Available Bacterial growth depends crucially on metabolic fluxes, which are limited by the cell's capacity to maintain metabolic enzymes. The necessary enzyme amount per unit flux is a major determinant of metabolic strategies both in evolution and bioengineering. It depends on enzyme parameters (such as kcat and KM constants, but also on metabolite concentrations. Moreover, similar amounts of different enzymes might incur different costs for the cell, depending on enzyme-specific properties such as protein size and half-life. Here, we developed enzyme cost minimization (ECM, a scalable method for computing enzyme amounts that support a given metabolic flux at a minimal protein cost. The complex interplay of enzyme and metabolite concentrations, e.g. through thermodynamic driving forces and enzyme saturation, would make it hard to solve this optimization problem directly. By treating enzyme cost as a function of metabolite levels, we formulated ECM as a numerically tractable, convex optimization problem. Its tiered approach allows for building models at different levels of detail, depending on the amount of available data. Validating our method with measured metabolite and protein levels in E. coli central metabolism, we found typical prediction fold errors of 4.1 and 2.6, respectively, for the two kinds of data. This result from the cost-optimized metabolic state is significantly better than randomly sampled metabolite profiles, supporting the hypothesis that enzyme cost is important for the fitness of E. coli. ECM can be used to predict enzyme levels and protein cost in natural and engineered pathways, and could be a valuable computational tool to assist metabolic engineering projects. Furthermore, it establishes a direct connection between protein cost and thermodynamics, and provides a physically plausible and computationally tractable way to include enzyme kinetics into constraint-based metabolic models, where kinetics have usually been ignored or

  7. Understanding the control of acyl flux through the lipid metabolic network of plant oil biosynthesis.

    Science.gov (United States)

    Bates, Philip D

    2016-09-01

    Plant oil biosynthesis involves a complex metabolic network with multiple subcellular compartments, parallel pathways, cycles, and pathways that have a dual function to produce essential membrane lipids and triacylglycerol. Modern molecular biology techniques provide tools to alter plant oil compositions through bioengineering, however with few exceptions the final composition of triacylglycerol cannot be predicted. One reason for limited success in oilseed bioengineering is the inadequate understanding of how to control the flux of fatty acids through various fatty acid modification, and triacylglycerol assembly pathways of the lipid metabolic network. This review focuses on the mechanisms of acyl flux through the lipid metabolic network, and highlights where uncertainty resides in our understanding of seed oil biosynthesis. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Changes of in vivo fluxes through central metabolic pathways during the production of nystatin by Streptomyces noursei in batch culture

    DEFF Research Database (Denmark)

    Jonsbu, E.; Christensen, Bjarke; Nielsen, Jens

    2001-01-01

    of the amino acids and calculated fluxes of the central metabolism showed that changes in the primary and secondary metabolisms occurred simultaneously. Changes in the profiles for the integrated fluxes showed a decreased flux through the pentose phosphate pathway and an increased flux in the tricarboxylic...... acid cycle relative to the glucose uptake rate when the culture entered a phase with reduced specific growth rate and enhanced nystatin yield. The flux through the pentose phosphate pathway seemed to be adjusted according to the NADPH requirement during the different phases of the batch fermentation....

  9. Revealing differences in metabolic flux distributions between a mutant strain and its parent strain Gluconacetobacter xylinus CGMCC 2955.

    Directory of Open Access Journals (Sweden)

    Cheng Zhong

    Full Text Available A better understanding of metabolic fluxes is important for manipulating microbial metabolism toward desired end products, or away from undesirable by-products. A mutant strain, Gluconacetobacter xylinus AX2-16, was obtained by combined chemical mutation of the parent strain (G. xylinus CGMCC 2955 using DEC (diethyl sulfate and LiCl. The highest bacterial cellulose production for this mutant was obtained at about 11.75 g/L, which was an increase of 62% compared with that by the parent strain. In contrast, gluconic acid (the main byproduct concentration was only 5.71 g/L for mutant strain, which was 55.7% lower than that of parent strain. Metabolic flux analysis indicated that 40.1% of the carbon source was transformed to bacterial cellulose in mutant strain, compared with 24.2% for parent strain. Only 32.7% and 4.0% of the carbon source were converted into gluconic acid and acetic acid in mutant strain, compared with 58.5% and 9.5% of that in parent strain. In addition, a higher flux of tricarboxylic acid (TCA cycle was obtained in mutant strain (57.0% compared with parent strain (17.0%. It was also indicated from the flux analysis that more ATP was produced in mutant strain from pentose phosphate pathway (PPP and TCA cycle. The enzymatic activity of succinate dehydrogenase (SDH, which is one of the key enzymes in TCA cycle, was 1.65-fold higher in mutant strain than that in parent strain at the end of culture. It was further validated by the measurement of ATPase that 3.53-6.41 fold higher enzymatic activity was obtained from mutant strain compared with parent strain.

  10. Development of Computational Tools for Metabolic Model Curation, Flux Elucidation and Strain Design

    Energy Technology Data Exchange (ETDEWEB)

    Maranas, Costas D

    2012-05-21

    An overarching goal of the Department of Energy mission is the efficient deployment and engineering of microbial and plant systems to enable biomass conversion in pursuit of high energy density liquid biofuels. This has spurred the pace at which new organisms are sequenced and annotated. This torrent of genomic information has opened the door to understanding metabolism in not just skeletal pathways and a handful of microorganisms but for truly genome-scale reconstructions derived for hundreds of microbes and plants. Understanding and redirecting metabolism is crucial because metabolic fluxes are unique descriptors of cellular physiology that directly assess the current cellular state and quantify the effect of genetic engineering interventions. At the same time, however, trying to keep pace with the rate of genomic data generation has ushered in a number of modeling and computational challenges related to (i) the automated assembly, testing and correction of genome-scale metabolic models, (ii) metabolic flux elucidation using labeled isotopes, and (iii) comprehensive identification of engineering interventions leading to the desired metabolism redirection.

  11. In vivo metabolic flux profiling with stable isotopes discriminates sites and quantifies effects of mitochondrial dysfunction in C. elegans

    Science.gov (United States)

    Ostrovsky, Julian; Clarke, Colleen; Preston, Judith; Bennett, Michael J.; Yudkoff, Marc; Xiao, Rui; Falk, Marni J.

    2014-01-01

    Mitochondrial respiratory chain (RC) disease diagnosis is complicated both by an absence of biomarkers that sufficiently divulge all cases and limited capacity to quantify adverse effects across intermediary metabolism. We applied high performance liquid chromatography (HPLC) and mass spectrometry (MS) studies of stable-isotope based precursor–product relationships in the nematode, C. elegans, to interrogate in vivo differences in metabolic flux among distinct genetic models of primary RC defects and closely related metabolic disorders. Methods C. elegans strains studied harbor single nuclear gene defects in complex I, II, or III RC subunits (gas-1, mev-1, isp-1); enzymes involved in coenzyme Q biosynthesis (clk-1), the tricarboxylic acid cycle (TCA, idh-1), or pyruvate metabolism (pdha-1); and central nodes of the nutrient-sensing signaling network that involve insulin response (daf-2) or the sirtuin homologue (sir-2.1). Synchronous populations of 2000 early larval stage worms were fed standard Escherichia coli on nematode growth media plates containing 1,6-13C2-glucose throughout their developmental period, with samples extracted on the first day of adult life in 4% perchloric acid with an internal standard. Quantitation of whole animal free amino acid concentrations and isotopic incorporation into amino and organic acids throughout development was performed in all strains by HPLC and isotope ratio MS, respectively. GC/MS analysis was also performed to quantify absolute isotopic incorporation in all molecular species of key TCA cycle intermediates in gas-1 and N2 adult worms. Results Genetic mutations within different metabolic pathways displayed distinct metabolic profiles. RC complex I (gas-1) and III (isp-1) subunit mutants, together with the coenzyme Q biosynthetic mutant (clk-1), shared a similar amino acid profile of elevated alanine and decreased glutamate. The metabolic signature of the complex II mutant (mev-1) was distinct from that of the other RC

  12. Flux Analysis of the Trypanosoma brucei Glycolysis Based on a Multiobjective-Criteria Bioinformatic Approach

    Directory of Open Access Journals (Sweden)

    Amine Ghozlane

    2012-01-01

    Full Text Available Trypanosoma brucei is a protozoan parasite of major of interest in discovering new genes for drug targets. This parasite alternates its life cycle between the mammal host(s (bloodstream form and the insect vector (procyclic form, with two divergent glucose metabolism amenable to in vitro culture. While the metabolic network of the bloodstream forms has been well characterized, the flux distribution between the different branches of the glucose metabolic network in the procyclic form has not been addressed so far. We present a computational analysis (called Metaboflux that exploits the metabolic topology of the procyclic form, and allows the incorporation of multipurpose experimental data to increase the biological relevance of the model. The alternatives resulting from the structural complexity of networks are formulated as an optimization problem solved by a metaheuristic where experimental data are modeled in a multiobjective function. Our results show that the current metabolic model is in agreement with experimental data and confirms the observed high metabolic flexibility of glucose metabolism. In addition, Metaboflux offers a rational explanation for the high flexibility in the ratio between final products from glucose metabolism, thsat is, flux redistribution through the malic enzyme steps.

  13. A flux footprint analysis to understand ecosystem fluxes in an intensively managed landscape

    Science.gov (United States)

    Hernandez Rodriguez, L. C.; Goodwell, A. E.; Kumar, P.

    2017-12-01

    Flux tower studies in agricultural sites have mainly been done at plot scale, where the footprint of the instruments is small such that the data reveals the behaviour of the nearby crop on which the study is focused. In the Midwestern United States, the agricultural ecosystem and its associated drainage, evapotranspiration, and nutrient dynamics are dominant influences on interactions between the soil, land, and atmosphere. In this study, we address large-scale ecohydrologic fluxes and states in an intensively managed landscape based on data from a 25m high eddy covariance flux tower. We show the calculated upwind distance and flux footprint for a flux tower located in Central Illinois as part of the Intensively Managed Landscapes Critical Zone Observatory (IMLCZO). In addition, we calculate the daily energy balance during the summer of 2016 from the flux tower measurements and compare with the modelled energy balance from a representative corn crop located in the flux tower footprint using the Multi-Layer Canopy model, MLCan. The changes in flux footprint over the course of hours, days, and the growing season have significant implications for the measured fluxes of carbon and energy at the flux tower. We use MLCan to simulate these fluxes under land covers of corn and soybeans. Our results demonstrate how the instrument heights impact the footprint of the captured eddy covariance fluxes, and we explore the implication for hydrological analysis. The convective turbulent atmosphere during the daytime shows a wide footprint of more than 10 km2, which reaches 3km length for the 90% contribution, where buoyancy is the dominant mechanism driving turbulence. In contrast, the stable atmosphere during the night-time shows a narrower footprint that goes beyond 8km2 and grows in the direction of the prevalent wind, which exceeds 4 km in length. This study improves our understanding of agricultural ecosystem behaviour in terms of the magnitude and variability of fluxes and

  14. Metabolic control analysis of xylose catabolism in Aspergillus

    DEFF Research Database (Denmark)

    Prathumpai, Wai; Gabelgaard, J.B.; Wanchanthuek, P.

    2003-01-01

    A kinetic model for xylose catabolism in Aspergillus is proposed. From a thermodynamic analysis it was found that the intermediate xylitol will accumulate during xylose catabolism. Use of the kinetic model allowed metabolic control analysis (MCA) of the xylose catabolic pathway to be carried out......, and flux control was shown to be dependent on the metabolite levels. Due to thermodynamic constraints, flux control may reside at the first step in the pathway, i.e., at the xylose reductase, even when the intracellular xylitol concentration is high. On the basis of the kinetic analysis, the general dogma...... specifying that flux control often resides at the step following an intermediate present at high concentrations was, therefore, shown not to hold. The intracellular xylitol concentration was measured in batch cultivations of two different strains of Aspergillus niger and two different strains of Aspergillus...

  15. Tracking the metabolic pulse of plant lipid production with isotopic labeling and flux analyses: Past, present and future.

    Science.gov (United States)

    Allen, Doug K; Bates, Philip D; Tjellström, Henrik

    2015-04-01

    Metabolism is comprised of networks of chemical transformations, organized into integrated biochemical pathways that are the basis of cellular operation, and function to sustain life. Metabolism, and thus life, is not static. The rate of metabolites transitioning through biochemical pathways (i.e., flux) determines cellular phenotypes, and is constantly changing in response to genetic or environmental perturbations. Each change evokes a response in metabolic pathway flow, and the quantification of fluxes under varied conditions helps to elucidate major and minor routes, and regulatory aspects of metabolism. To measure fluxes requires experimental methods that assess the movements and transformations of metabolites without creating artifacts. Isotopic labeling fills this role and is a long-standing experimental approach to identify pathways and quantify their metabolic relevance in different tissues or under different conditions. The application of labeling techniques to plant science is however far from reaching it potential. In light of advances in genetics and molecular biology that provide a means to alter metabolism, and given recent improvements in instrumentation, computational tools and available isotopes, the use of isotopic labeling to probe metabolism is becoming more and more powerful. We review the principal analytical methods for isotopic labeling with a focus on seminal studies of pathways and fluxes in lipid metabolism and carbon partitioning through central metabolism. Central carbon metabolic steps are directly linked to lipid production by serving to generate the precursors for fatty acid biosynthesis and lipid assembly. Additionally some of the ideas for labeling techniques that may be most applicable for lipid metabolism in the future were originally developed to investigate other aspects of central metabolism. We conclude by describing recent advances that will play an important future role in quantifying flux and metabolic operation in plant

  16. Metabolic flux distributions in Corynebacterium glutamicum during growth and lysine overproduction. Reprinted from Biotechnology and Bioengineering, Vol. 41, Pp 633-646 (1993).

    Science.gov (United States)

    Vallino, J J; Stephanopoulos, G

    2000-03-20

    The two main contributions of this article are the solidification of Corynebacterium glutamicum biochemistry guided by bioreaction network analysis, and the determination of basal metabolic flux distributions during growth and lysine synthesis. Employed methodology makes use of stoichiometrically based mass balances to determine flux distributions in the C. glutamicum metabolic network. Presented are a brief description of the methodology, a thorough literature review of glutamic acid bacteria biochemistry, and specific results obtained through a combination of fermentation studies and analysis-directed intracellular assays. The latter include the findings of the lack of activity of glyoxylate shunt, and that phosphoenolpyruvate carboxylase (PPC) is the only anaplerotic reaction expressed in C. glutamicum cultivated on glucose minimal media. Network simplifications afforded by the above findings facilitated the determination of metabolic flux distributions under a variety of culture conditions and led to the following conclusions. Both the pentose phosphate pathway and PPC support significant fluxes during growth and lysine overproduction, and that flux partitioning at the glucosa-6-phosphate branch point does not appear to limit lysine synthesis. Copyright 1993 John Wiley & Sons, Inc.

  17. The nutritional status of Methanosarcina acetivorans regulates glycogen metabolism and gluconeogenesis and glycolysis fluxes.

    Science.gov (United States)

    Santiago-Martínez, Michel Geovanni; Encalada, Rusely; Lira-Silva, Elizabeth; Pineda, Erika; Gallardo-Pérez, Juan Carlos; Reyes-García, Marco Antonio; Saavedra, Emma; Moreno-Sánchez, Rafael; Marín-Hernández, Alvaro; Jasso-Chávez, Ricardo

    2016-05-01

    Gluconeogenesis is an essential pathway in methanogens because they are unable to use exogenous hexoses as carbon source for cell growth. With the aim of understanding the regulatory mechanisms of central carbon metabolism in Methanosarcina acetivorans, the present study investigated gene expression, the activities and metabolic regulation of key enzymes, metabolite contents and fluxes of gluconeogenesis, as well as glycolysis and glycogen synthesis/degradation pathways. Cells were grown with methanol as a carbon source. Key enzymes were kinetically characterized at physiological pH/temperature. Active consumption of methanol during exponential cell growth correlated with significant methanogenesis, gluconeogenic flux and steady glycogen synthesis. After methanol exhaustion, cells reached the stationary growth phase, which correlated with the rise in glycogen consumption and glycolytic flux, decreased methanogenesis, negligible acetate production and an absence of gluconeogenesis. Elevated activities of carbon monoxide dehydrogenase/acetyl-CoA synthetase complex and pyruvate: ferredoxin oxidoreductase suggested the generation of acetyl-CoA and pyruvate for glycogen synthesis. In the early stationary growth phase, the transcript contents and activities of pyruvate phosphate dikinase, fructose 1,6-bisphosphatase and glycogen synthase decreased, whereas those of glycogen phosphorylase, ADP-phosphofructokinase and pyruvate kinase increased. Therefore, glycogen and gluconeogenic metabolites were synthesized when an external carbon source was provided. Once such a carbon source became depleted, glycolysis and methanogenesis fed by glycogen degradation provided the ATP supply. Weak inhibition of key enzymes by metabolites suggested that the pathways evaluated were mainly transcriptionally regulated. Because glycogen metabolism and glycolysis/gluconeogenesis are not present in all methanogens, the overall data suggest that glycogen storage might represent an environmental

  18. Sampling with poling-based flux balance analysis: optimal versus sub-optimal flux space analysis of Actinobacillus succinogenes.

    Science.gov (United States)

    Binns, Michael; de Atauri, Pedro; Vlysidis, Anestis; Cascante, Marta; Theodoropoulos, Constantinos

    2015-02-18

    Flux balance analysis is traditionally implemented to identify the maximum theoretical flux for some specified reaction and a single distribution of flux values for all the reactions present which achieve this maximum value. However it is well known that the uncertainty in reaction networks due to branches, cycles and experimental errors results in a large number of combinations of internal reaction fluxes which can achieve the same optimal flux value. In this work, we have modified the applied linear objective of flux balance analysis to include a poling penalty function, which pushes each new set of reaction fluxes away from previous solutions generated. Repeated poling-based flux balance analysis generates a sample of different solutions (a characteristic set), which represents all the possible functionality of the reaction network. Compared to existing sampling methods, for the purpose of generating a relatively "small" characteristic set, our new method is shown to obtain a higher coverage than competing methods under most conditions. The influence of the linear objective function on the sampling (the linear bias) constrains optimisation results to a subspace of optimal solutions all producing the same maximal fluxes. Visualisation of reaction fluxes plotted against each other in 2 dimensions with and without the linear bias indicates the existence of correlations between fluxes. This method of sampling is applied to the organism Actinobacillus succinogenes for the production of succinic acid from glycerol. A new method of sampling for the generation of different flux distributions (sets of individual fluxes satisfying constraints on the steady-state mass balances of intermediates) has been developed using a relatively simple modification of flux balance analysis to include a poling penalty function inside the resulting optimisation objective function. This new methodology can achieve a high coverage of the possible flux space and can be used with and without

  19. Metabolic control analysis of xylose catabolism in Aspergillus

    DEFF Research Database (Denmark)

    Prathumpai, Wai; Gabelgaard, J.B.; Wanchanthuek, P.

    2003-01-01

    A kinetic model for xylose catabolism in Aspergillus is proposed. From a thermodynamic analysis it was found that the intermediate xylitol will accumulate during xylose catabolism. Use of the kinetic model allowed metabolic control analysis (MCA) of the xylose catabolic pathway to be carried out...... specifying that flux control often resides at the step following an intermediate present at high concentrations was, therefore, shown not to hold. The intracellular xylitol concentration was measured in batch cultivations of two different strains of Aspergillus niger and two different strains of Aspergillus...... nidulans grown on media containing xylose, and a concentration up to 30 mM was found. Applying MCA showed that the first polyol dehydrogenase (XDH) in the catabolic pathway of xylose exerted the main flux control in the two strains of A. nidulans and A. niger NW324, but the flux control was exerted mainly...

  20. Physiology and genetics of metabolic flux control in Zymomonas mobilis. Progress report

    Energy Technology Data Exchange (ETDEWEB)

    Conway, T.

    1992-08-01

    This work seeks to understand the role of gene expression in regulating glycolytic enzyme synthesis in a balance that allows proper glycoltic flux control. The seven genes targeted for study in this laboratory have been cloned and sequenced, and molecular details of regulation have been investigated. Clear that glycolytic enzyme synthesis is coordinated to prevent the build up of toxic metabolic intermediates. The genetic mechanisms responsible for regulating balanced expression of the EntnerDoudoroff and glycolytic genes in Z. mobilis are beginning to be understood. Several layers of genetic control, perhaps in a hierarchal arrangement act in concert to determine the relative abundance of the glycolytic enzymes. These genetic controls involve differential translational efficiency, highly conserved promoter sequences, transcription factors, differential mRNA stabilities, and nucleolytic mRNA processing. The serendipitous cloning of the glucose facilitator, glf, as a result of linkage to several other genes of interest will have a significant impact on the study of Z. mobilis metabolism. The glucose facilitator is being characterized in a genetically reconstituted system in E. coli. Molecular genetic studies indicate that the ratio of glf expression to that of glk, zmf, and edd is carefully regulated, and suggests a critical role in metabolic control. Regulation of glycolytic gene expression is now sufficiently well understood to allow use of the glycolytic genes as tools to manipulate specified enzyme levels for the purpose of analyzing metabolic flux control. The critical genes have been subcloned for stable expression in Z. mobilis and placed under control of a regulated promoter system involving the tac promoter, the lacI repressor, and gene induction in by IPTG. HPLC methods have been developed that allow quantitation of virtually all of the metabolic intermediates in the cell pool.

  1. Aerobic glucose metabolism of Saccharomyces kluyveri: Growth, metabolite production, and quantification of metabolic fluxes

    DEFF Research Database (Denmark)

    Møller, Kasper; Christensen, B.; Förster, Jochen

    2002-01-01

    The growth and product formation of Saccharomyces kluyveri was characterized in aerobic batch cultivation on glucose. At these conditions it was found that ethyl acetate was a major overflow metabolite in S. kluyveri. During the exponential-growth phase on glucose ethyl acetate was produced...... at a constant specific rate of 0.12 g ethyl acetate per g dry weight per hour. The aerobic glucose metabolism in S. kluyveri was found to be less fermentative than in S. cerevisiae, as illustrated by the comparably low yield of ethanol on glucose (0.08 +/- 0.02 g/g), and high yield of biomass on glucose (0...

  2. Flux

    DEFF Research Database (Denmark)

    Ravn, Ib

    . FLUX betegner en flyden eller strømmen, dvs. dynamik. Forstår man livet som proces og udvikling i stedet for som ting og mekanik, får man et andet billede af det gode liv end det, som den velkendte vestlige mekanicisme lægger op til. Dynamisk forstået indebærer det gode liv den bedst mulige...... kanalisering af den flux eller energi, der strømmer igennem os og giver sig til kende i vore daglige aktiviteter. Skal vores tanker, handlinger, arbejde, samvær og politiske liv organiseres efter stramme og faste regelsæt, uden slinger i valsen? Eller skal de tværtimod forløbe ganske uhindret af regler og bånd...

  3. Pathway analysis of Pichia pastoris to elucidate methanol metabolism and its regulation for production of recombinant proteins.

    Science.gov (United States)

    Unrean, Pornkamol

    2014-01-01

    This research rationally analyzes metabolic pathways of Pichia pastoris to study the metabolic flux responses of this yeast under methanol metabolism. A metabolic model of P. pastoris was constructed and analyzed by elementary mode analysis (EMA). EMA was used to comprehensively identify the cell's metabolic flux profiles and its underlying regulation mechanisms for the production of recombinant proteins from methanol. Change in phenotypes and flux profiles during methanol adaptation with varying feed mixture of glycerol and methanol was examined. EMA identified increasing and decreasing fluxes during the glycerol-methanol metabolic shift, which well agreed with experimental observations supporting the validity of the metabolic network model. Analysis of all the identified pathways also led to the determination of the metabolic capacities as well as the optimum metabolic pathways for recombinant protein synthesis during methanol induction. The network sensitivity analysis revealed that the production of proteins can be improved by manipulating the flux ratios at the pyruvate branch point. In addition, EMA suggested that protein synthesis is optimum under hypoxic culture conditions. The metabolic modeling and analysis presented in this study could potentially form a valuable knowledge base for future research on rational design and optimization of P. pastoris by determining target genes, pathways, and culture conditions for enhanced recombinant protein synthesis. The metabolic pathway analysis is also of considerable value for production of therapeutic proteins by P. pastoris in biopharmaceutical applications. © 2013 American Institute of Chemical Engineers.

  4. Cofactor engineering through heterologous expression of an NADH oxidase and its impact on metabolic flux redistribution in Klebsiella pneumoniae

    Directory of Open Access Journals (Sweden)

    Ji Xiao-Jun

    2013-01-01

    Full Text Available Abstract Background Acetoin is an important bio-based platform chemical. However, it is usually existed as a minor byproduct of 2,3-butanediol fermentation in bacteria. Results The present study reports introducing an exogenous NAD+ regeneration sysytem into a 2,3-butanediol producing strain Klebsiella pneumoniae to increse the accumulation of acetoin. Batch fermentation suggested that heterologous expression of the NADH oxidase in K. pneumoniae resulted in large decreases in the intracellular NADH concentration (1.4 fold and NADH/NAD+ ratio (2.0 fold. Metabolic flux analysis revealed that fluxes to acetoin and acetic acid were enhanced, whereas, production of lactic acid and ethanol were decreased, with the accumualation of 2,3-butanediol nearly unaltered. By fed-batch culture of the recombinant, the highest reported acetoin production level (25.9 g/L by Klebsiella species was obtained. Conclusions The present study indicates that microbial production of acetoin could be improved by decreasing the intracellular NADH/NAD+ ratio in K. pneumoniae. It demonstrated that the cofactor engineering method, which is by manipulating the level of intracellular cofactors to redirect cellular metabolism, could be employed to achieve a high efficiency of producing the NAD+-dependent microbial metabolite.

  5. Nutrient fluxes and net metabolism in a coastal lagoon SW peninsula of Baja California, Mexico

    Directory of Open Access Journals (Sweden)

    Cervantes Duarte, R.

    2016-09-01

    Full Text Available Fluxes of nutrients and net metabolism were estimated in coastal lagoon Magdalena Bay using LOICZ biogeochemical model. In situ data were obtained from 14 sites in the lagoon and also from a fixed site in the adjacent ocean area. Intense upwelling (February to July and faint upwelling (August to January were analyzed from monthly time series. The Temperature, nitrite + nitrate, ammonium and phosphate within the lagoon showed significant differences (p<0.05 between the two periods. Salinity (p=0.408 was more homogeneous (no significantly different due to mixing processes. During the intense upwelling period, nutrients increased in and out of the lagoon due to the influence of Transitional Water and Subartic Water transported by the California Current. However, during the faint upwelling, from August to January, the Transition Water and Subtropical Surface Water were predominant. Magdalena Bay showed denitrification processes of throughout the year as it occurred in other semi-arid coastal lagoons. It also showed a net autotrophic metabolism during intense upwelling and heterotrophic metabolism during faint upwelling. Understanding nutrient flows and net metabolism through simple biogeochemical models can provide tools for better management of the coastal zone.

  6. Looking at the metabolic consequences of the colchicine-based in vivo autophagic flux assay

    Science.gov (United States)

    Seiliez, Iban; Belghit, Ikram; Gao, Yujie; Skiba-Cassy, Sandrine; Dias, Karine; Cluzeaud, Marianne; Rémond, Didier; Hafnaoui, Nordine; Salin, Bénédicte; Camougrand, Nadine; Panserat, Stéphane

    2016-01-01

    ABSTRACT Monitoring autophagic flux in vivo or in organs remains limited and the ideal methods relative to the techniques possible with cell culture may not exist. Recently, a few papers have demonstrated the feasibility of measuring autophagic flux in vivo by intraperitoneal (IP) injection of pharmacological agents (chloroquine, leupeptin, vinblastine, and colchicine). However, the metabolic consequences of the administration of these drugs remain largely unknown. Here, we report that 0.8 mg/kg/day IP colchicine increased LC3-II protein levels in the liver of fasted trout, supporting the usefulness of this drug for studying autophagic flux in vivo in our model organism. This effect was accompanied by a decrease of plasma glucose concentration associated with a fall in the mRNA levels of gluconeogenesis-related genes. Concurrently, triglycerides and lipid droplets content in the liver increased. In contrast, transcript levels of β-oxidation-related gene Cpt1a dropped significantly. Together, these results match with the reported role of autophagy in the regulation of glucose homeostasis and intracellular lipid stores, and highlight the importance of considering these effects when using colchicine as an in vivo “autophagometer.” PMID:26902586

  7. Metabolic flux of the oxidative pentose phosphate pathway under low light conditions in Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Ueda, Kentaro; Nakajima, Tsubasa; Yoshikawa, Katsunori; Toya, Yoshihiro; Matsuda, Fumio; Shimizu, Hiroshi

    2018-02-27

    The role of the oxidative pentose phosphate pathway (oxPPP) in Synechocystis sp. PCC 6803 under mixotrophic conditions was investigated by 13 C metabolic flux analysis. Cells were cultured under low (10 μmol m -2  s -1 ) and high light intensities (100 μmol m -2  s -1 ) in the presence of glucose. The flux of CO 2 fixation by ribulose bisphosphate carboxylase/oxygenase under the high light condition was approximately 3-fold higher than that under the low light condition. Although no flux of the oxPPP was observed under the high light condition, flux of 0.08-0.19 mmol gDCW -1  h -1 in the oxPPP was observed under the low light condition. The balance between the consumption and production of NADPH suggested that approximately 10% of the total NADPH production was generated by the oxPPP under the low light condition. The growth phenotype of a mutant with deleted zwf, which encodes glucose-6-phosphate dehydrogenase in the oxPPP, was compared to that of the parental strain under low and high light conditions. Growth of the Δzwf mutant nearly stopped during the late growth phase under the low light condition, whereas the growth rates of the two strains were identical under the high light condition. These results indicate that NADPH production in the oxPPP is essential for anabolism under low light conditions. The oxPPP appears to play an important role in producing NADPH from glucose and ATP to compensate for NADPH shortage under low light conditions. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  8. Linking high-resolution metabolic flux phenotypes and transcriptional regulation in yeast modulated by the global regulator Gcn4p.

    Science.gov (United States)

    Moxley, Joel F; Jewett, Michael C; Antoniewicz, Maciek R; Villas-Boas, Silas G; Alper, Hal; Wheeler, Robert T; Tong, Lily; Hinnebusch, Alan G; Ideker, Trey; Nielsen, Jens; Stephanopoulos, Gregory

    2009-04-21

    Genome sequencing dramatically increased our ability to understand cellular response to perturbation. Integrating system-wide measurements such as gene expression with networks of protein-protein interactions and transcription factor binding revealed critical insights into cellular behavior. However, the potential of systems biology approaches is limited by difficulties in integrating metabolic measurements across the functional levels of the cell despite their being most closely linked to cellular phenotype. To address this limitation, we developed a model-based approach to correlate mRNA and metabolic flux data that combines information from both interaction network models and flux determination models. We started by quantifying 5,764 mRNAs, 54 metabolites, and 83 experimental (13)C-based reaction fluxes in continuous cultures of yeast under stress in the absence or presence of global regulator Gcn4p. Although mRNA expression alone did not directly predict metabolic response, this correlation improved through incorporating a network-based model of amino acid biosynthesis (from r = 0.07 to 0.80 for mRNA-flux agreement). The model provides evidence of general biological principles: rewiring of metabolic flux (i.e., use of different reaction pathways) by transcriptional regulation and metabolite interaction density (i.e., level of pairwise metabolite-protein interactions) as a key biosynthetic control determinant. Furthermore, this model predicted flux rewiring in studies of follow-on transcriptional regulators that were experimentally validated with additional (13)C-based flux measurements. As a first step in linking metabolic control and genetic regulatory networks, this model underscores the importance of integrating diverse data types in large-scale cellular models. We anticipate that an integrated approach focusing on metabolic measurements will facilitate construction of more realistic models of cellular regulation for understanding diseases and constructing

  9. Exhaustive Analysis of a Genotype Space Comprising 10(15 Central Carbon Metabolisms Reveals an Organization Conducive to Metabolic Innovation.

    Directory of Open Access Journals (Sweden)

    Sayed-Rzgar Hosseini

    2015-08-01

    Full Text Available All biological evolution takes place in a space of possible genotypes and their phenotypes. The structure of this space defines the evolutionary potential and limitations of an evolving system. Metabolism is one of the most ancient and fundamental evolving systems, sustaining life by extracting energy from extracellular nutrients. Here we study metabolism's potential for innovation by analyzing an exhaustive genotype-phenotype map for a space of 10(15 metabolisms that encodes all possible subsets of 51 reactions in central carbon metabolism. Using flux balance analysis, we predict the viability of these metabolisms on 10 different carbon sources which give rise to 1024 potential metabolic phenotypes. Although viable metabolisms with any one phenotype comprise a tiny fraction of genotype space, their absolute numbers exceed 10(9 for some phenotypes. Metabolisms with any one phenotype typically form a single network of genotypes that extends far or all the way through metabolic genotype space, where any two genotypes can be reached from each other through a series of single reaction changes. The minimal distance of genotype networks associated with different phenotypes is small, such that one can reach metabolisms with novel phenotypes--viable on new carbon sources--through one or few genotypic changes. Exceptions to these principles exist for those metabolisms whose complexity (number of reactions is close to the minimum needed for viability. Increasing metabolic complexity enhances the potential for both evolutionary conservation and evolutionary innovation.

  10. Modeling the Contribution of Allosteric Regulation for Flux Control in the Central Carbon Metabolism of E. coli

    DEFF Research Database (Denmark)

    Machado, Daniel; Herrgard, Markus; Rocha, Isabel

    2015-01-01

    contribution to the metabolic flux changes. Inspired by these results, we develop a constraint-based method (arFBA) for simulation of metabolic flux distributions that accounts for allosteric interactions. This method can be used for systematic prediction of potential allosteric regulation under the given...... the metabolic flux. Accounting for allosteric interactions in metabolic reconstructions reveals a hidden topology in metabolic networks, improving our understanding of cellular metabolism and fostering the development of novel simulation methods that account for this type of regulation....

  11. BOREAS AFM-11 Aircraft Flux Analysis Reports

    Data.gov (United States)

    National Aeronautics and Space Administration — Reports from the BOREAS AFM-11 team regarding quality control and sampling analysis of data collected by other AFM personnel using the Electra, LongEZ, and Twin...

  12. Study of Stationary Phase Metabolism Via Isotopomer Analysis of Amino Acids from an Isolated Protein

    Energy Technology Data Exchange (ETDEWEB)

    Shaikh, AfshanS.; Tang, YinjieJ.; Mukhopadhyay, Aindrila; Martin, Hector Garcia; Gin, Jennifer; Benke, Peter; Keasling, Jay D.

    2009-09-14

    Microbial production of many commercially important secondary metabolites occurs during stationary phase, and methods to measure metabolic flux during this growth phase would be valuable. Metabolic flux analysis is often based on isotopomer information from proteinogenic amino acids. As such, flux analysis primarily reflects the metabolism pertinent to the growth phase during which most proteins are synthesized. To investigate central metabolism and amino acids synthesis activity during stationary phase, addition of fully 13C-labeled glucose followed by induction of green fluorescent protein (GFP) expression during stationary phase was used. Our results indicate that Escherichia coli was able to produce new proteins (i.e., GFP) in the stationary phase, and the amino acids in GFP were mostly from degraded proteins synthesized during the exponential growth phase. Among amino acid biosynthetic pathways, only those for serine, alanine, glutamate/glutamine, and aspartate/asparagine had significant activity during the stationary phase.

  13. DNA methylation dynamics, metabolic fluxes, gene splicing, and alternative phenotypes in honey bees.

    Science.gov (United States)

    Foret, Sylvain; Kucharski, Robert; Pellegrini, Matteo; Feng, Suhua; Jacobsen, Steven E; Robinson, Gene E; Maleszka, Ryszard

    2012-03-27

    In honey bees (Apis mellifera), the development of a larva into either a queen or worker depends on differential feeding with royal jelly and involves epigenomic modifications by DNA methyltransferases. To understand the role of DNA methylation in this process we sequenced the larval methylomes in both queens and workers. We show that the number of differentially methylated genes (DMGs) in larval head is significantly increased relative to adult brain (2,399 vs. 560) with more than 80% of DMGs up-methylated in worker larvae. Several highly conserved metabolic and signaling pathways are enriched in methylated genes, underscoring the connection between dietary intake and metabolic flux. This includes genes related to juvenile hormone and insulin, two hormones shown previously to regulate caste determination. We also tie methylation data to expressional profiling and describe a distinct role for one of the DMGs encoding anaplastic lymphoma kinase (ALK), an important regulator of metabolism. We show that alk is not only differentially methylated and alternatively spliced in Apis, but also seems to be regulated by a cis-acting, anti-sense non-protein-coding transcript. The unusually complex regulation of ALK in Apis suggests that this protein could represent a previously unknown node in a process that activates downstream signaling according to a nutritional context. The correlation between methylation and alternative splicing of alk is consistent with the recently described mechanism involving RNA polymerase II pausing. Our study offers insights into diet-controlled development in Apis.

  14. Bacterial persistence is an active σS stress response to metabolic flux limitation.

    Science.gov (United States)

    Radzikowski, Jakub Leszek; Vedelaar, Silke; Siegel, David; Ortega, Álvaro Dario; Schmidt, Alexander; Heinemann, Matthias

    2016-09-21

    While persisters are a health threat due to their transient antibiotic tolerance, little is known about their phenotype and what actually causes persistence. Using a new method for persister generation and high-throughput methods, we comprehensively mapped the molecular phenotype of Escherichia coli during the entry and in the state of persistence in nutrient-rich conditions. The persister proteome is characterized by σ(S)-mediated stress response and a shift to catabolism, a proteome that starved cells tried to but could not reach due to absence of a carbon and energy source. Metabolism of persisters is geared toward energy production, with depleted metabolite pools. We developed and experimentally verified a model, in which persistence is established through a system-level feedback: Strong perturbations of metabolic homeostasis cause metabolic fluxes to collapse, prohibiting adjustments toward restoring homeostasis. This vicious cycle is stabilized and modulated by high ppGpp levels, toxin/anti-toxin systems, and the σ(S)-mediated stress response. Our system-level model consistently integrates past findings with our new data, thereby providing an important basis for future research on persisters. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  15. k-OptForce: integrating kinetics with flux balance analysis for strain design.

    Directory of Open Access Journals (Sweden)

    Anupam Chowdhury

    2014-02-01

    Full Text Available Computational strain design protocols aim at the system-wide identification of intervention strategies for the enhanced production of biochemicals in microorganisms. Existing approaches relying solely on stoichiometry and rudimentary constraint-based regulation overlook the effects of metabolite concentrations and substrate-level enzyme regulation while identifying metabolic interventions. In this paper, we introduce k-OptForce, which integrates the available kinetic descriptions of metabolic steps with stoichiometric models to sharpen the prediction of intervention strategies for improving the bio-production of a chemical of interest. It enables identification of a minimal set of interventions comprised of both enzymatic parameter changes (for reactions with available kinetics and reaction flux changes (for reactions with only stoichiometric information. Application of k-OptForce to the overproduction of L-serine in E. coli and triacetic acid lactone (TAL in S. cerevisiae revealed that the identified interventions tend to cause less dramatic rearrangements of the flux distribution so as not to violate concentration bounds. In some cases the incorporation of kinetic information leads to the need for additional interventions as kinetic expressions render stoichiometry-only derived interventions infeasible by violating concentration bounds, whereas in other cases the kinetic expressions impart flux changes that favor the overproduction of the target product thereby requiring fewer direct interventions. A sensitivity analysis on metabolite concentrations shows that the required number of interventions can be significantly affected by changing the imposed bounds on metabolite concentrations. Furthermore, k-OptForce was capable of finding non-intuitive interventions aiming at alleviating the substrate-level inhibition of key enzymes in order to enhance the flux towards the product of interest, which cannot be captured by stoichiometry-alone analysis

  16. Assessment of metabolic flux distribution in the thermophilic hydrogen producer Caloramator celer as affected by external pH and hydrogen partial pressure.

    Science.gov (United States)

    Ciranna, Alessandro; Pawar, Sudhanshu S; Santala, Ville; Karp, Matti; van Niel, Ed W J

    2014-03-28

    Caloramator celer is a strict anaerobic, alkalitolerant, thermophilic bacterium capable of converting glucose to hydrogen (H2), carbon dioxide, acetate, ethanol and formate by a mixed acid fermentation. Depending on the growth conditions C. celer can produce H2 at high yields. For a biotechnological exploitation of this bacterium for H2 production it is crucial to understand the factors that regulate carbon and electron fluxes and therefore the final distribution of metabolites to channel the metabolic flux towards the desired product. Combining experimental results from batch fermentations with genome analysis, reconstruction of central carbon metabolism and metabolic flux analysis (MFA), this study shed light on glucose catabolism of the thermophilic alkalitolerant bacterium C. celer. Two innate factors pertaining to culture conditions have been identified to significantly affect the metabolic flux distribution: culture pH and partial pressures of H2 (PH2). Overall, at alkaline to neutral pH the rate of biomass synthesis was maximized, whereas at acidic pH the lower growth rate and the less efficient biomass formation are accompanied with more efficient energy recovery from the substrate indicating high cell maintenance possibly to sustain intracellular pH homeostasis. Higher H2 yields were associated with fermentation at acidic pH as a consequence of the lower synthesis of other reduced by-products such as formate and ethanol. In contrast, PH2 did not affect the growth of C. celer on glucose. At high PH2 the cellular redox state was balanced by rerouting the flow of carbon and electrons to ethanol and formate production allowing unaltered glycolytic flux and growth rate, but resulting in a decreased H2 synthesis. C. celer possesses a flexible fermentative metabolism that allows redistribution of fluxes at key metabolic nodes to simultaneously control redox state and efficiently harvest energy from substrate even under unfavorable conditions (i.e. low pH and high

  17. An Analysis of Moisture Fluxes into the Gulf of California

    Science.gov (United States)

    Wu, Man-Li C.; Schubert, Siegfried D.; Suarez, Max J.; Huang, Norden E.

    2009-01-01

    This study examines the nature of episodes of enhanced warm-season moisture flux into the Gulf of California. Both spatial structure and primary time scales of the fluxes are examined using the 40-yr ECMWF Re-Analysis data for the period 1980-2001. The analysis approach consists of a compositing technique that is keyed on the low-level moisture fluxes into the Gulf of California. The results show that the fluxes have a rich spectrum of temporal variability, with periods of enhanced transport over the gulf linked to African easterly waves on subweekly (3-8 day) time scales, the Madden-Julian oscillation (MJO) at intraseasonal time scales (20-90 day), and intermediate (10-15 day) time-scale disturbances that appear to originate primarily in the Caribbean Sea-western Atlantic Ocean. In the case of the MJO, enhanced low-level westerlies and large-scale rising motion provide an environment that favors large-scale cyclonic development near the west coast of Central America that, over the course of about 2 weeks, expands northward along the coast eventually reaching the mouth of the Gulf of California where it acts to enhance the southerly moisture flux in that region. On a larger scale, the development includes a northward shift in the eastern Pacific ITCZ, enhanced precipitation over much of Mexico and the southwestern United States, and enhanced southerly/southeasterly fluxes from the Gulf of Mexico into Mexico and the southwestern and central United States. In the case of the easterly waves, the systems that reach Mexico appear to redevelop/reorganize on the Pacific coast and then move rapidly to the northwest to contribute to the moisture flux into the Gulf of California. The most intense fluxes into the gulf on these time scales appear to be synchronized with a midlatitude short-wave trough over the U.S. West Coast and enhanced low-level southerly fluxes over the U.S. Great Plains. The intermediate (10-15 day) time-scale systems have zonal wavelengths roughly twice

  18. Dimensional analysis of membrane distillation flux through fibrous membranes

    Science.gov (United States)

    Mauter, Meagan

    We developed a dimensional-analysis-based empirical modeling method for membrane distillation (MD) flux that is adaptable for novel membrane structures. The method makes fewer simplifying assumptions about membrane pore geometry than existing theoretical (i.e. mechanistic) models, and allows selection of simple, easily-measureable membrane characteristics as structural parameters. Furthermore, the model does not require estimation of membrane surface temperatures; it accounts for convective heat transfer to the membrane surface without iterative fitting of mass and heat transfer equations. The Buckingham-Pi dimensional analysis method is tested for direct contact membrane distillation (DCMD) using non-woven/fibrous structures as the model membrane material. Twelve easily-measured variables to describe DCMD operating conditions, fluid properties, membrane structures, and flux were identified and combined into eight dimensionless parameters. These parameters were regressed using experimentally-collected data for multiple electrospun membrane types and DCMD system conditions, achieving R2 values >95%. We found that vapor flux through isotropic fibrous membranes can be estimated using only membrane thickness, solid fraction, and fiber diameter as structural parameters. Buckingham-Pi model DCMD flux predictions compare favorably with previously-developed empirical and theoretical models, and suggest this simple yet theoretically-grounded empirical modeling method can be used practically for predicting MD vapor flux from membrane structural parameters.

  19. [Metabolic mechanisms underlying reparative action of metal-dependent spectral light flux from a hollow cathode lamp (experimental study)].

    Science.gov (United States)

    Pusyreva, G A; Frolkov, V K; Bobrovnitskiĭ, I P

    2010-01-01

    The objective of the present study was to evaluate the influence of serial irradiation of experimental animals with a visible light flow showing spectral lines of copper and manganese on the rate of reparative processes and the associated metabolic events. The cathode of the lamp that contained both microelements generated the light flux possessed of significant biological activity. Specifically, it accelerated reduction of the wound surface area and optimized regulation of carbohydrate metabolism in the lipid peroxidation system by insulin and cotisol. The light flux emitted by the cathode containing only one of the two elements (either copper or manganese) produced a similar but less pronounced effect.

  20. Pyruvate modifies metabolic flux and nutrient sensing during extracorporeal membrane oxygenation in an immature swine model

    Energy Technology Data Exchange (ETDEWEB)

    Ledee, Dolena R.; Kajimoto, Masaki; O' Kelly-Priddy, Colleen M.; Olson, Aaron; Isern, Nancy G.; Robillard Frayne, Isabelle; Des Rosiers, Christine; Portman, Michael A.

    2015-07-01

    Extracorporeal membrane oxygenation (ECMO) provides mechanical circulatory support for infants and children with postoperative cardiopulmonary failure. Nutritional support is mandatory during ECMO, although specific actions for substrates on the heart have not been delineated. Prior work shows that enhancing pyruvate oxidation promotes successful weaning from ECMO. Accordingly, we closely examined the role of prolonged systemic pyruvate supplementation in modifying metabolic parameters during the unique conditions of ventricular unloading provided by ECMO. Twelve male mixed breed Yorkshire piglets (age 30-49 days) received systemic infusion of either normal saline (Group C) or pyruvate (Group P) during ECMO for 8 hours. Over the final hour piglets received [2-13C] pyruvate, and [13C6]-L-leucine, as an indicator for oxidation and protein synthesis. A significant increase in lactate and pyruvate concentrations occurred, along with an increase in the absolute concentration of all measured CAC intermediates. Group P showed greater anaplerotic flux through pyruvate carboxylation although pyruvate oxidation relative to citrate synthase flux was similar to Group C. The groups demonstrated similar leucine fractional contributions to acetyl-CoA and fractional protein synthesis rates. Pyruvate also promoted an increase in the phosphorylation state of several nutrient sensitive enzymes, such as AMPK and ACC, and promoted O-GlcNAcylation through the hexosamine biosynthetic pathway (HBP). In conclusion, prolonged pyruvate supplementation during ECMO modified anaplerotic pyruvate flux and elicited changes in important nutrient and energy sensitive pathways, while preserving protein synthesis. Therefore, the observed results support the further study of nutritional supplementation and its downstream effects on cardiac adaptation during ventricular unloading.

  1. Analysis of edge stability for models of heat flux width

    Directory of Open Access Journals (Sweden)

    M.A. Makowski

    2017-08-01

    Full Text Available Detailed measurements of the ne, Te, and Ti profiles in the vicinity of the separatrix of ELMing H-mode discharges have been used to examine plasma stability at the extreme edge of the plasma and assess stability dependent models of the heat flux width. The results are strongly contrary to the critical gradient model, which posits that a ballooning instability determines a gradient scale length related to the heat flux width. The results of this analysis are not sensitive to the choice of location to evaluate stability. Significantly, it is also found that the results are completely consistent with the heuristic drift model for the heat flux width. Here the edge pressure gradient scales with plasma density and is proportional to the pressure gradient inferred from the equilibrium in accordance with the predictions of that theory.

  2. Analysis of the low-altitude proton flux asymmetry: methodology

    CERN Document Server

    Kruglanski, M

    1999-01-01

    Existing East-West asymmetry models of the trapped proton fluxes at low altitudes depend on the local magnetic dip angle and a density scale height derived from atmospheric models. We propose an alternative approach which maps the directional flux over a drift shell (B sub m , L) in terms of the local pitch and azimuthal angles alpha and beta, where beta is defined in the local mirror plane as the angle between the proton arrival direction and the surface normal to the drift shell. This approach has the advantage that it only depends on drift shell parameters and does not involve an atmosphere model. A semi-empirical model based on the new methodology is able to reproduce the angular distribution of a set of SAMPEX/PET proton flux measurements. Guidelines are proposed for spacecraft missions and data analysis procedures that are intended to be used for the building of new trapped radiation environment models.

  3. Direct calculation of elementary flux modes satisfying several biological constraints in genome-scale metabolic networks.

    Science.gov (United States)

    Pey, Jon; Planes, Francisco J

    2014-08-01

    The concept of Elementary Flux Mode (EFM) has been widely used for the past 20 years. However, its application to genome-scale metabolic networks (GSMNs) is still under development because of methodological limitations. Therefore, novel approaches are demanded to extend the application of EFMs. A novel family of methods based on optimization is emerging that provides us with a subset of EFMs. Because the calculation of the whole set of EFMs goes beyond our capacity, performing a selective search is a proper strategy. Here, we present a novel mathematical approach calculating EFMs fulfilling additional linear constraints. We validated our approach based on two metabolic networks in which all the EFMs can be obtained. Finally, we analyzed the performance of our methodology in the GSMN of the yeast Saccharomyces cerevisiae by calculating EFMs producing ethanol with a given minimum carbon yield. Overall, this new approach opens new avenues for the calculation of EFMs in GSMNs. Matlab code is provided in the supplementary online materials fplanes@ceit.es. Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  4. Histone Deacetylase AtSRT1 Links Metabolic Flux and Stress Response in Arabidopsis.

    Science.gov (United States)

    Liu, Xiaoyun; Wei, Wei; Zhu, Wenjun; Su, Lufang; Xiong, Zeyang; Zhou, Man; Zheng, Yu; Zhou, Dao-Xiu

    2017-12-04

    How plant metabolic flux alters gene expression to optimize plant growth and response to stress remains largely unclear. Here, we report that Arabidopsis thaliana NAD + -dependent histone deacetylase AtSRT1 negatively regulates plant tolerance to stress and glycolysis but stimulates mitochondrial respiration. We found that AtSRT1 interacts with Arabidopsis cMyc-Binding Protein 1 (AtMBP-1), a transcriptional repressor produced by alternative translation of the cytosolic glycolytic enolase gene LOS2/ENO2. We demonstrated that AtSRT1 could associate with the chromatin of AtMBP-1 targets LOS2/ENO2 and STZ/ZAT10, both of which encode key stress regulators, and reduce the H3K9ac levels at these genes to repress their transcription. Overexpression of both AtSRT1 and AtMBP-1 had synergistic effects on the expression of glycolytic genes, glycolytic enzymatic activities, and mitochondrial respiration. Furthermore, we found that AtMBP-1 is lysine-acetylated and vulnerable to proteasomal protein degradation, while AtSRT1 could remove its lysine acetylation and significantly enhance its stability in vivo. Taken together, these results indicate that AtSRT1 regulates primary metabolism and stress response by both epigenetic regulation and modulation of AtMBP-1 transcriptional activity in Arabidopsis. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

  5. Beaver-mediated lateral hydrologic connectivity, fluvial carbon and nutrient flux, and aquatic ecosystem metabolism

    Science.gov (United States)

    Wegener, Pam; Covino, Tim; Wohl, Ellen

    2017-06-01

    River networks that drain mountain landscapes alternate between narrow and wide valley segments. Within the wide segments, beaver activity can facilitate the development and maintenance of complex, multithread planform. Because the narrow segments have limited ability to retain water, carbon, and nutrients, the wide, multithread segments are likely important locations of retention. We evaluated hydrologic dynamics, nutrient flux, and aquatic ecosystem metabolism along two adjacent segments of a river network in the Rocky Mountains, Colorado: (1) a wide, multithread segment with beaver activity; and, (2) an adjacent (directly upstream) narrow, single-thread segment without beaver activity. We used a mass balance approach to determine the water, carbon, and nutrient source-sink behavior of each river segment across a range of flows. While the single-thread segment was consistently a source of water, carbon, and nitrogen, the beaver impacted multithread segment exhibited variable source-sink dynamics as a function of flow. Specifically, the multithread segment was a sink for water, carbon, and nutrients during high flows, and subsequently became a source as flows decreased. Shifts in river-floodplain hydrologic connectivity across flows related to higher and more variable aquatic ecosystem metabolism rates along the multithread relative to the single-thread segment. Our data suggest that beaver activity in wide valleys can create a physically complex hydrologic environment that can enhance hydrologic and biogeochemical buffering, and promote high rates of aquatic ecosystem metabolism. Given the widespread removal of beaver, determining the cumulative effects of these changes is a critical next step in restoring function in altered river networks.

  6. Oxygen-enriched fermentation of sodium gluconate by Aspergillus niger and its impact on intracellular metabolic flux distributions.

    Science.gov (United States)

    Shen, Yuting; Tian, Xiwei; Zhao, Wei; Hang, Haifeng; Chu, Ju

    2018-01-01

    Different concentrations of oxygen-enriched air were utilized for sodium gluconate (SG) fermentation by Aspergillus niger. The fermentation time shortened from 20 to 15.5 h due to the increase of volumetric oxygen transfer coefficient (K L a) and the formation of more dispersed mycelia when inlet oxygen concentration ascended from 21 to 32%. According to metabolic flux analysis, during the growth phase, extracellular glucose for SG synthesis accounted for 79.0 and 85.3% with air and oxygen-enriched air (25%), respectively, whereas the proportions were 89.4 and 93.0% in the stationary phase. Intracellular glucose consumption decreased in oxygen-enriched fermentation, as cell respiration was more high-efficiently performed. Metabolic profiling indicated that most intermediates in TCA cycle and EMP pathway had smaller pool sizes in oxygen-enriched fermentations. Moreover, the main by-product of citric acid dramatically decreased from 1.36 to 0.34 g L -1 in oxygen-enriched fermentation. And the sodium gluconate yield increased from 0.856 to 0.903 mol mol -1 .

  7. Metabolic flux rearrangement in the amino acid metabolism reduces ammonia stress in the α1-antitrypsin producing human AGE1.HN cell line.

    Science.gov (United States)

    Priesnitz, Christian; Niklas, Jens; Rose, Thomas; Sandig, Volker; Heinzle, Elmar

    2012-03-01

    This study focused on metabolic changes in the neuronal human cell line AGE1.HN upon increased ammonia stress. Batch cultivations of α(1)-antitrypsin (A1AT) producing AGE1.HN cells were carried out in media with initial ammonia concentrations ranging from 0mM to 5mM. Growth, A1AT production, metabolite dynamics and finally metabolic fluxes calculated by metabolite balancing were compared. Growth and A1AT production decreased with increasing ammonia concentration. The maximum A1AT concentration decreased from 0.63g/l to 0.51g/l. Central energy metabolism remained relatively unaffected exhibiting only slightly increased glycolytic flux at high initial ammonia concentration in the medium. However, the amino acid metabolism was significantly changed. Fluxes through transaminases involved in amino acid degradation were reduced concurrently with a reduced uptake of amino acids. On the other hand fluxes through transaminases working in the direction of amino acid synthesis, i.e., alanine and phosphoserine, were increased leading to increased storage of excess nitrogen in extracellular alanine and serine. Glutamate dehydrogenase flux was reversed increasingly fixing free ammonia with increasing ammonia concentration. Urea production additionally observed was associated with arginine uptake by the cells and did not increase at high ammonia stress. It was therefore not used as nitrogen sink to remove excess ammonia. The results indicate that the AGE1.HN cell line can adapt to ammonia concentrations usually present during the cultivation process to a large extent by changing metabolism but with slightly reduced A1AT production and growth. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. Tracking the metabolic pulse of plant lipid production with isotopic labeling and flux analyses: Past, present and future

    Science.gov (United States)

    Metabolic networks are comprised of chemical transformations that are the basis of cellular operation and function to sustain life. The molecular rate of transitioning through biochemical pathways (i.e. flux) establishes cellular phenotypes that can be studied in response to genetic or environmental...

  9. Linking high-resolution metabolic flux phenotypes and transcriptional regulation in yeast modulated by the global regulator Gcn4p

    DEFF Research Database (Denmark)

    Moxley, Joel F.; Jewett, Michael Christopher; Antoniewicz, Maciek R.

    2009-01-01

    Gcn4p. Although mRNA expression alone did not directly predict metabolic response, this correlation improved through incorporating a network-based model of amino acid biosynthesis (from r = 0.07 to 0.80 for mRNA-flux agreement). The model provides evidence of general biological principles: rewiring...

  10. Detailed analysis of radon flux studies at Australian uranium projects

    International Nuclear Information System (INIS)

    Mudd, Gavin M.

    2005-01-01

    The release of radon gas and radon progeny from uranium projects is a major issue during operation as well as for the design of rehabilitation works. In Australia, there have been a number of premining radon flux studies as part of the environmental investigation and potential development of recent uranium projects. There is also an increasing amount of operational data on radon fluxes and loads from various aspects of projects, such as tailings, waste rock and mills. Thus there exists much useful measured data which can be used to assess the design radon flux and load targets for rehabilitation. The main projects for which radon data exists includes Ranger, Olympic Dam, Beverley, Honeymoon, Jabiluka, Yeelirrie, Lake Way, Koongarra, Moline, Coronation Hill, Rockhole, Nabarlek, Rum Jungle, Port Pirie and Ben Lomond. To date, much of this data has not been systematically evaluated. The need to compile and assess this data is twofold. Firstly, to assess the loads released from uranium production as an input into life-cycle analyses of the nuclear fuel cycle, such as those undertaken by UNSCEAR and industry groups. Secondly, there is a need to set suitable design standards for radon flux for the rehabilitation of former and current uranium projects. This paper will present such a detailed compilation of radon fluxes and loads which can then be used as the basis for both life-cycle analyses as well as setting appropriate site-specific rehabilitation criteria for radon. The implications for former and current projects is then discussed as well as future data needs. Ultimately, there is a critical need for thorough baseline surveys prior to mining to ensure accurate assessments of changes to radon fluxes and loads. The data and analysis presented is considered applicable to all uranium projects in Australia, as well as being a useful model for considering such issues internationally

  11. Analysis of neutron flux measurement systems using statistical functions

    International Nuclear Information System (INIS)

    Pontes, Eduardo Winston

    1997-01-01

    This work develops an integrated analysis for neutron flux measurement systems using the concepts of cumulants and spectra. Its major contribution is the generalization of Campbell's theorem in the form of spectra in the frequency domain, and its application to the analysis of neutron flux measurement systems. Campbell's theorem, in its generalized form, constitutes an important tool, not only to find the nth-order frequency spectra of the radiation detector, but also in the system analysis. The radiation detector, an ionization chamber for neutrons, is modeled for cylindrical, plane and spherical geometries. The detector current pulses are characterized by a vector of random parameters, and the associated charges, statistical moments and frequency spectra of the resulting current are calculated. A computer program is developed for application of the proposed methodology. In order for the analysis to integrate the associated electronics, the signal processor is studied, considering analog and digital configurations. The analysis is unified by developing the concept of equivalent systems that can be used to describe the cumulants and spectra in analog or digital systems. The noise in the signal processor input stage is analysed in terms of second order spectrum. Mathematical expressions are presented for cumulants and spectra up to fourth order, for important cases of filter positioning relative to detector spectra. Unbiased conventional estimators for cumulants are used, and, to evaluate systems precision and response time, expressions are developed for their variances. Finally, some possibilities for obtaining neutron radiation flux as a function of cumulants are discussed. In summary, this work proposes some analysis tools which make possible important decisions in the design of better neutron flux measurement systems. (author)

  12. Effects of temperature and UVR on organic matter fluxes and the metabolic activity of Acropora muricata

    Directory of Open Access Journals (Sweden)

    Lucile Courtial

    2017-08-01

    Full Text Available Coral bleaching events are predicted to occur more frequently in the coming decades with global warming. The susceptibility of corals to bleaching during thermal stress episodes depends on many factors, including the magnitude of thermal stress and irradiance. The interactions among these two factors, and in particular with ultra-violet radiation (UVR, the most harmful component of light, are more complex than assumed, and are not yet well understood. This paper explores the individual and combined effects of temperature and UVR on the metabolism of Acropora muricata, one of the most abundant coral species worldwide. Particulate and dissolved organic matter (POM/DOM fluxes and organic matter (OM degradation by the mucus-associated bacteria were also monitored in all conditions. The results show that UVR exposure exacerbated the temperature-induced bleaching, but did not affect OM fluxes, which were only altered by seawater warming. Temperature increase induced a shift from POM release and DOM uptake in healthy corals to POM uptake and DOM release in stressed ones. POM uptake was linked to a significant grazing of pico- and nanoplankton particles during the incubation, to fulfil the energetic requirements of A. muricata in the absence of autotrophy. Finally, OM degradation by mucus-associated bacterial activity was unaffected by UVR exposure, but significantly increased under high temperature. Altogether, our results demonstrate that seawater warming and UVR not only affect coral physiology, but also the way corals interact with the surrounding seawater, with potential consequences for coral reef biogeochemical cycles and food webs.

  13. A Diel Flux Balance Model Captures Interactions between Light and Dark Metabolism during Day-Night Cycles in C3 and Crassulacean Acid Metabolism Leaves1[C][W][OPEN

    Science.gov (United States)

    Cheung, C.Y. Maurice; Poolman, Mark G.; Fell, David. A.; Ratcliffe, R. George; Sweetlove, Lee J.

    2014-01-01

    Although leaves have to accommodate markedly different metabolic flux patterns in the light and the dark, models of leaf metabolism based on flux-balance analysis (FBA) have so far been confined to consideration of the network under continuous light. An FBA framework is presented that solves the two phases of the diel cycle as a single optimization problem and, thus, provides a more representative model of leaf metabolism. The requirement to support continued export of sugar and amino acids from the leaf during the night and to meet overnight cellular maintenance costs forces the model to set aside stores of both carbon and nitrogen during the day. With only minimal constraints, the model successfully captures many of the known features of C3 leaf metabolism, including the recently discovered role of citrate synthesis and accumulation in the night as a precursor for the provision of carbon skeletons for amino acid synthesis during the day. The diel FBA model can be applied to other temporal separations, such as that which occurs in Crassulacean acid metabolism (CAM) photosynthesis, allowing a system-level analysis of the energetics of CAM. The diel model predicts that there is no overall energetic advantage to CAM, despite the potential for suppression of photorespiration through CO2 concentration. Moreover, any savings in enzyme machinery costs through suppression of photorespiration are likely to be offset by the higher flux demand of the CAM cycle. It is concluded that energetic or nitrogen use considerations are unlikely to be evolutionary drivers for CAM photosynthesis. PMID:24596328

  14. Dynamic regulation of metabolic flux in engineered bacteria using a pathway-independent quorum-sensing circuit.

    Science.gov (United States)

    Gupta, Apoorv; Reizman, Irene M Brockman; Reisch, Christopher R; Prather, Kristala L J

    2017-03-01

    Metabolic engineering of microorganisms to produce desirable products on an industrial scale can result in unbalanced cellular metabolic networks that reduce productivity and yield. Metabolic fluxes can be rebalanced using dynamic pathway regulation, but few broadly applicable tools are available to achieve this. We present a pathway-independent genetic control module that can be used to dynamically regulate the expression of target genes. We apply our module to identify the optimal point to redirect glycolytic flux into heterologous engineered pathways in Escherichia coli, resulting in titers of myo-inositol increased 5.5-fold and titers of glucaric acid increased from unmeasurable to >0.8 g/L, compared to the parent strains lacking dynamic flux control. Scaled-up production of these strains in benchtop bioreactors resulted in almost ten- and fivefold increases in specific titers of myo-inositol and glucaric acid, respectively. We also used our module to control flux into aromatic amino acid biosynthesis to increase titers of shikimate in E. coli from unmeasurable to >100 mg/L.

  15. Influence of the late winter bloom on migrant zooplankton metabolism and its implications on export fluxes

    Science.gov (United States)

    Putzeys, S.; Yebra, L.; Almeida, C.; Bécognée, P.; Hernández-León, S.

    2011-12-01

    Studies on carbon active fluxes due to diel migrants are scarce and critical for carbon flux models and biogeochemical estimates. We studied the temporal variability and vertical distribution of biomass, indices of feeding and respiration of the zooplanktonic community north off the Canary Islands during the end of the late winter bloom, in order to assess vertical carbon fluxes in this area. Biomass distribution during the day presented two dense layers of organisms at 0-200 m and around 500 m, whereas at night, most of the biomass concentrated in the epipelagic layer. The gut pigment flux (0.05-0.18 mgC·m - 2 ·d - 1 ) represented 0.22% of the estimated passive export flux (POC flux) while potential ingestion represented 3.91% of the POC (1.24-3.40 mgC·m - 2 ·d - 1 ). The active respiratory flux (0.50-1.36 mgC·m - 2 ·d - 1 ) was only 1.57% of the POC flux. The total carbon flux mediated by diel migrants (respiration plus potential ingestion) ranged between 3.37 and 9.22% of the POC flux; which is three-fold higher than calculating ingestion fluxes from gut pigments. Our results suggest that the fluxes by diel migrants play a small role in the downward flux of carbon in the open ocean during the post-bloom period.

  16. Geoneutrinos at Jinping: Flux prediction and oscillation analysis

    Science.gov (United States)

    Wan, Linyan; Hussain, Ghulam; Wang, Zhe; Chen, Shaomin

    2017-03-01

    Geoneutrinos are electron antineutrinos (ν¯e) generated by the beta decays of radionuclides naturally occurring inside the Earth, in particular U 238 , Th 232 , and K 40 . Measurement of these neutrinos provides powerful constraints on the radiogenic heat of the Earth and tests on the Earth models. Since the prediction of ν¯e's in geoneutrino flux is subject to neutrino oscillation effects, we performed a calculation including detailed oscillation analysis in the propagation of geoneutrinos and reactor neutrinos generated around the Earth. The expected geoneutrino signal, the reactor neutrino background rates, and the systematic error budget are provided for a proposed 3 kt neutrino detector at the Jinping underground lab in Sichuan, China. In addition, we evaluated sensitivities for the geoneutrino flux, Th/U ratio, and power of a possible fission reactor in the interior of the Earth.

  17. Consequences of phosphoenolpyruvate:sugar phosphotranferase system and pyruvate kinase isozymes inactivation in central carbon metabolism flux distribution in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Meza Eugenio

    2012-09-01

    Full Text Available Abstract Background In Escherichia coli phosphoenolpyruvate (PEP is a key central metabolism intermediate that participates in glucose transport, as precursor in several biosynthetic pathways and it is involved in allosteric regulation of glycolytic enzymes. In this work we generated W3110 derivative strains that lack the main PEP consumers PEP:sugar phosphotransferase system (PTS- and pyruvate kinase isozymes PykA and PykF (PTS-pykA- and PTS-pykF-. To characterize the effects of these modifications on cell physiology, carbon flux distribution and aromatics production capacity were determined. Results When compared to reference strain W3110, strain VH33 (PTS- displayed lower specific rates for growth, glucose consumption and acetate production as well as a higher biomass yield from glucose. These phenotypic effects were even more pronounced by the additional inactivation of PykA or PykF. Carbon flux analysis revealed that PTS inactivation causes a redirection of metabolic flux towards biomass formation. A cycle involving PEP carboxylase (Ppc and PEP carboxykinase (Pck was detected in all strains. In strains W3110, VH33 (PTS- and VH35 (PTS-, pykF-, the net flux in this cycle was inversely correlated with the specific rate of glucose consumption and inactivation of Pck in these strains caused a reduction in growth rate. In the PTS- background, inactivation of PykA caused a reduction in Ppc and Pck cycling as well as a reduction in flux to TCA, whereas inactivation of PykF caused an increase in anaplerotic flux from PEP to OAA and an increased flux to TCA. The wild-type and mutant strains were modified to overproduce L-phenylalanine. In resting cells experiments, compared to reference strain, a 10, 4 and 7-fold higher aromatics yields from glucose were observed as consequence of PTS, PTS PykA and PTS PykF inactivation. Conclusions Metabolic flux analysis performed on strains lacking the main activities generating pyruvate from PEP revealed the high

  18. Pareto optimality in organelle energy metabolism analysis.

    Science.gov (United States)

    Angione, Claudio; Carapezza, Giovanni; Costanza, Jole; Lió, Pietro; Nicosia, Giuseppe

    2013-01-01

    In low and high eukaryotes, energy is collected or transformed in compartments, the organelles. The rich variety of size, characteristics, and density of the organelles makes it difficult to build a general picture. In this paper, we make use of the Pareto-front analysis to investigate the optimization of energy metabolism in mitochondria and chloroplasts. Using the Pareto optimality principle, we compare models of organelle metabolism on the basis of single- and multiobjective optimization, approximation techniques (the Bayesian Automatic Relevance Determination), robustness, and pathway sensitivity analysis. Finally, we report the first analysis of the metabolic model for the hydrogenosome of Trichomonas vaginalis, which is found in several protozoan parasites. Our analysis has shown the importance of the Pareto optimality for such comparison and for insights into the evolution of the metabolism from cytoplasmic to organelle bound, involving a model order reduction. We report that Pareto fronts represent an asymptotic analysis useful to describe the metabolism of an organism aimed at maximizing concurrently two or more metabolite concentrations.

  19. Computational Functional Analysis of Lipid Metabolic Enzymes.

    Science.gov (United States)

    Bagnato, Carolina; Have, Arjen Ten; Prados, María B; Beligni, María V

    2017-01-01

    The computational analysis of enzymes that participate in lipid metabolism has both common and unique challenges when compared to the whole protein universe. Some of the hurdles that interfere with the functional annotation of lipid metabolic enzymes that are common to other pathways include the definition of proper starting datasets, the construction of reliable multiple sequence alignments, the definition of appropriate evolutionary models, and the reconstruction of phylogenetic trees with high statistical support, particularly for large datasets. Most enzymes that take part in lipid metabolism belong to complex superfamilies with many members that are not involved in lipid metabolism. In addition, some enzymes that do not have sequence similarity catalyze similar or even identical reactions. Some of the challenges that, albeit not unique, are more specific to lipid metabolism refer to the high compartmentalization of the routes, the catalysis in hydrophobic environments and, related to this, the function near or in biological membranes.In this work, we provide guidelines intended to assist in the proper functional annotation of lipid metabolic enzymes, based on previous experiences related to the phospholipase D superfamily and the annotation of the triglyceride synthesis pathway in algae. We describe a pipeline that starts with the definition of an initial set of sequences to be used in similarity-based searches and ends in the reconstruction of phylogenies. We also mention the main issues that have to be taken into consideration when using tools to analyze subcellular localization, hydrophobicity patterns, or presence of transmembrane domains in lipid metabolic enzymes.

  20. ToMI-FBA: A genome-scale metabolic flux based algorithm to select optimum hosts and media formulations for expressing pathways of interest

    Directory of Open Access Journals (Sweden)

    Hadi Nazem-Bokaee

    2015-09-01

    Full Text Available The Total Membrane Influx constrained Flux Balance Analysis (ToMI-FBA algorithm was developed in this research as a new tool to help researchers decide which microbial host and medium formulation are optimal for expressing a new metabolic pathway. ToMI-FBA relies on genome-scale metabolic flux modeling and a novel in silico cell membrane influx constraint that specifies the flux of atoms (not molecules into the cell through all possible membrane transporters. The ToMI constraint is constructed through the addition of an extra row and column to the stoichiometric matrix of a genome-scale metabolic flux model. In this research, the mathematical formulation of the ToMI constraint is given along with four case studies that demonstrate its usefulness. In Case Study 1, ToMI-FBA returned an optimal culture medium formulation for the production of isobutanol from Bacillus subtilis. Significant levels of L-valine were recommended to optimize production, and this result has been observed experimentally. In Case Study 2, it is demonstrated how the carbon to nitrogen uptake ratio can be specified as an additional ToMI-FBA constraint. This was investigated for maximizing medium chain length polyhydroxyalkanoates (mcl-PHA production from Pseudomonas putida KT2440. In Case Study 3, ToMI-FBA revealed a strategy of adding cellobiose as a means to increase ethanol selectivity during the stationary growth phase of Clostridium acetobutylicum ATCC 824. This strategy was also validated experimentally. Finally, in Case Study 4, B. subtilis was identified as a superior host to Escherichia coli, Saccharomyces cerevisiae, and Synechocystis PCC6803 for the production of artemisinate.

  1. Development of thermodynamic optimum searching (TOS) to improve the prediction accuracy of flux balance analysis.

    Science.gov (United States)

    Zhu, Yan; Song, Jiangning; Xu, Zixiang; Sun, Jibin; Zhang, Yanping; Li, Yin; Ma, Yanhe

    2013-03-01

    Flux balance analysis (FBA) has been widely used in calculating steady-state flux distributions that provide important information for metabolic engineering. Several thermodynamics-based methods, for example, quantitative assignment of reaction directionality and energy balance analysis have been developed to improve the prediction accuracy of FBA. However, these methods can only generate a thermodynamically feasible range, rather than the most thermodynamically favorable solution. We therefore developed a novel optimization method termed as thermodynamic optimum searching (TOS) to calculate the thermodynamically optimal solution, based on the second law of thermodynamics, the minimum magnitude of the Gibbs free energy change and the maximum entropy production principle (MEPP). Then, TOS was applied to five physiological conditions of Escherichia coli to evaluate its effectiveness. The resulting prediction accuracy was found significantly improved (10.7-48.5%) by comparing with the (13)C-fluxome data, indicating that TOS can be considered an advanced calculation and prediction tool in metabolic engineering. Copyright © 2012 Wiley Periodicals, Inc.

  2. A metabolic and body-size scaling framework for parasite within-host abundance, biomass, and energy flux.

    Science.gov (United States)

    Hechinger, Ryan F

    2013-08-01

    Energetics may provide a useful currency for studying the ecology of parasite assemblages within individual hosts. Parasite assemblages may also provide powerful models to study general principles of ecological energetics. Yet there has been little ecological research on parasite-host energetics, probably due to methodological difficulties. However, the scaling relationships of individual metabolic rate with body or cell size and temperature may permit us to tackle the energetics of parasite assemblages in hosts. This article offers the foundations and initial testing of a metabolic theory of ecology (MTE) framework for parasites in hosts. I first provide equations to estimate energetic flux through observed parasite assemblages. I then develop metabolic scaling theory for parasite abundance, energetics, and biomass in individual hosts. In contrast to previous efforts, the theory factors in both host and parasite metabolic scaling, how parasites use host space, and whether energy or space dictates carrying capacity. Empirical tests indicate that host energetic flux can set parasite carrying capacity, which decreases as predicted considering the scaling of host and parasite metabolic rates. The theory and results also highlight that the phenomenon of "energetic equivalence" is not an assumption of MTE but a possible outcome contingent on how species partition resources. Hence, applying MTE to parasites can lend mechanistic, quantitative, predictive insight into the nature of parasitism and can inform general ecological theory.

  3. Computational Platform for Flux Analysis Using 13C-Label Tracing- Phase I SBIR Final Report

    Energy Technology Data Exchange (ETDEWEB)

    Van Dien, Stephen J.

    2005-04-12

    Isotopic label tracing is a powerful experimental technique that can be combined with metabolic models to quantify metabolic fluxes in an organism under a particular set of growth conditions. In this work we constructed a genome-scale metabolic model of Methylobacterium extorquens, a facultative methylotroph with potential application in the production of useful chemicals from methanol. A series of labeling experiments were performed using 13C-methanol, and the resulting distribution of labeled carbon in the proteinogenic amino acids was determined by mass spectrometry. Algorithms were developed to analyze this data in context of the metabolic model, yielding flux distributions for wild-type and several engineered strains of M. extorquens. These fluxes were compared to those predicted by model simulation alone, and also integrated with microarray data to give an improved understanding of the metabolic physiology of this organism.

  4. Multidimensional optimality of microbial metabolism

    NARCIS (Netherlands)

    Schuetz, Robert; Zamboni, Nicola; Zampieri, Mattia; Heinemann, Matthias; Sauer, Uwe

    2012-01-01

    Although the network topology of metabolism is well known, understanding the principles that govern the distribution of fluxes through metabolism lags behind. Experimentally, these fluxes can be measured by (13)C-flux analysis, and there has been a long-standing interest in understanding this

  5. Moderate DNA damage promotes metabolic flux into PPP via PKM2 Y-105 phosphorylation: a feature that favours cancer cells.

    Science.gov (United States)

    Kumar, Bhupender; Bamezai, Rameshwar N K

    2015-08-01

    Pyruvate kinase M2, an important metabolic enzyme, promotes aerobic glycolysis (Warburg effect) to facilitate cancer cell proliferation. Unravelling the status of this important glycolytic pathway enzyme under sub-lethal doses of etoposide, a commonly used anti-proliferative genotoxic drug to induce mild/moderate DNA damage in HeLa cells as a model system and discern its effect on: PKM2 expression, phosphorylation, dimer: tetramer ratio, activity and associated effects, was pertinent. Protein expression and phosphorylation of PKM2 from HeLa cells was estimated using Western blotting. Same protein lysate was also used to estimate total pyruvate kinase activity and the total dimer: tetramer content evaluated using glycerol gradient ultra-centrifugation. Intracellular PEP was estimated manually using standard curve; while NADPH was assessed by NADPH estimation kit. Unpaired t test and two-way-ANOVA was used for statistical analysis. A relative decrease in PKM2 expression and a subsequent dose and time dependent increase in Y105-phosphorylation were observed. A concomitant increase in PKM2 dimer content and Y105-phosphorylation responsible for reduced PKM2 activity promoted PEP accumulation and NADPH production, representing increased metabolic flux into PPP, a feature that favours cancer cells. It was apparent that the sub-lethal doses of etoposide induced inadequate damage to DNA in cancer cells in culture promoted pro-survival conditions due to Y105-phosphorylation of PKM2, its stable dimerization and inactivation, a unique association not known earlier, indicating what might happen in tumour revivals or recurrences.

  6. Flux Balance Analysis of Escherichia coli under Temperature and pH Stress Conditions

    KAUST Repository

    Xu, Xiaopeng

    2015-05-12

    An interesting discovery in biology is that most genes in an organism are dispensable. That means these genes have minor effects on survival of the organism in standard laboratory conditions. One explanation of this discovery is that some genes play important roles in specific conditions and are essential genes under those conditions. E. coli is a model organism, which is widely used. It can adapt to many stress conditions, including temperature, pH, osmotic, antibiotic, etc. Underlying mechanisms and associated genes of each stress condition responses are usually different. In our analysis, we combined protein abundance data and mutant conditional fitness data into E. coli constraint-based metabolic models to study conditionally essential metabolic genes under temperature and pH stress conditions. Flux Balance Analysis was employed as the modeling method to analysis these data. We discovered lists of metabolic genes, which are E. coli dispensable genes, but conditionally essential under some stress conditions. Among these conditionally essential genes, atpA in low pH stress and nhaA in high pH stress found experimental evidences from previous studies. Our study provides new conditionally essential gene candidates for biologists to explore stress condition mechanisms.

  7. Ethanol-metabolizing pathways in deermice. Estimation of flux calculated from isotope effects

    International Nuclear Information System (INIS)

    Alderman, J.; Takagi, T.; Lieber, C.S.

    1987-01-01

    The apparent deuterium isotope effects on Vmax/Km (D(V/K] of ethanol oxidation in two deermouse strains (one having and one lacking hepatic alcohol dehydrogenase (ADH] were used to calculate flux through the ADH, microsomal ethanol-oxidizing system (MEOS), and catalase pathways. In vitro, D(V/K) values were 3.22 for ADH, 1.13 for MEOS, and 1.83 for catalase under physiological conditions of pH, temperature, and ionic strength. In vivo, in deermice lacking ADH (ADH-), D(V/K) was 1.20 +/- 0.09 (mean +/- S.E.) at 7.0 +/- 0.5 mM blood ethanol and 1.08 +/- 0.10 at 57.8 +/- 10.2 mM blood ethanol, consistent with ethanol oxidation principally by MEOS. Pretreatment of ADH- animals with the catalase inhibitor 3-amino-1,2,4-triazole did not significantly change D(V/K). ADH+ deermice exhibited D(V/K) values of 1.87 +/- 0.06 (untreated), 1.71 +/- 0.13 (pretreated with 3-amino-1,2,4-triazole), and 1.24 +/- 0.13 (after the ADH inhibitor, 4-methylpyrazole) at 5-7 mM blood ethanol levels. At elevated blood ethanol concentrations (58.1 +/- 2.4 mM), a D(V/K) of 1.37 +/- 0.21 was measured in the ADH+ strain. For measured D(V/K) values to accurately reflect pathway contributions, initial reaction conditions are essential. These were shown to exist by the following criteria: negligible fractional conversion of substrate to product and no measurable back reaction in deermice having a reversible enzyme (ADH). Thus, calculations from D(V/K) indicate that, even when ADH is present, non-ADH pathways (mostly MEOS) participate significantly in ethanol metabolism at all concentrations tested and play a major role at high levels

  8. Pathway analysis and optimization in metabolic engineering

    National Research Council Canada - National Science Library

    Torres, Néstor V; Voit, Eberhard O

    2002-01-01

    ... Engineering introduces researchers and advanced students in biology and engineering to methods of optimizing biochemical systems of biotechnological relevance. It examines the development of strategies for manipulating metabolic pathways, demonstrates the need for effective systems models, and discusses their design and analysis, while placing special emp...

  9. Shigella reroutes host cell central metabolism to obtain high-flux nutrient supply for vigorous intracellular growth

    Science.gov (United States)

    Kentner, David; Martano, Giuseppe; Callon, Morgane; Chiquet, Petra; Brodmann, Maj; Burton, Olga; Wahlander, Asa; Nanni, Paolo; Delmotte, Nathanaël; Grossmann, Jonas; Limenitakis, Julien; Schlapbach, Ralph; Kiefer, Patrick; Vorholt, Julia A.; Hiller, Sebastian; Bumann, Dirk

    2014-01-01

    Shigella flexneri proliferate in infected human epithelial cells at exceptionally high rates. This vigorous growth has important consequences for rapid progression to life-threatening bloody diarrhea, but the underlying metabolic mechanisms remain poorly understood. Here, we used metabolomics, proteomics, and genetic experiments to determine host and Shigella metabolism during infection in a cell culture model. The data suggest that infected host cells maintain largely normal fluxes through glycolytic pathways, but the entire output of these pathways is captured by Shigella, most likely in the form of pyruvate. This striking strategy provides Shigella with an abundant favorable energy source, while preserving host cell ATP generation, energy charge maintenance, and survival, despite ongoing vigorous exploitation. Shigella uses a simple three-step pathway to metabolize pyruvate at high rates with acetate as an excreted waste product. The crucial role of this pathway for Shigella intracellular growth suggests targets for antimicrobial chemotherapy of this devastating disease. PMID:24958876

  10. Shigella reroutes host cell central metabolism to obtain high-flux nutrient supply for vigorous intracellular growth.

    Science.gov (United States)

    Kentner, David; Martano, Giuseppe; Callon, Morgane; Chiquet, Petra; Brodmann, Maj; Burton, Olga; Wahlander, Asa; Nanni, Paolo; Delmotte, Nathanaël; Grossmann, Jonas; Limenitakis, Julien; Schlapbach, Ralph; Kiefer, Patrick; Vorholt, Julia A; Hiller, Sebastian; Bumann, Dirk

    2014-07-08

    Shigella flexneri proliferate in infected human epithelial cells at exceptionally high rates. This vigorous growth has important consequences for rapid progression to life-threatening bloody diarrhea, but the underlying metabolic mechanisms remain poorly understood. Here, we used metabolomics, proteomics, and genetic experiments to determine host and Shigella metabolism during infection in a cell culture model. The data suggest that infected host cells maintain largely normal fluxes through glycolytic pathways, but the entire output of these pathways is captured by Shigella, most likely in the form of pyruvate. This striking strategy provides Shigella with an abundant favorable energy source, while preserving host cell ATP generation, energy charge maintenance, and survival, despite ongoing vigorous exploitation. Shigella uses a simple three-step pathway to metabolize pyruvate at high rates with acetate as an excreted waste product. The crucial role of this pathway for Shigella intracellular growth suggests targets for antimicrobial chemotherapy of this devastating disease.

  11. Annual benthic metabolism and organic carbon fluxes in a semi-enclosed Mediterranean bay dominated by the macroalgae Caulerpa prolifera.

    Directory of Open Access Journals (Sweden)

    Sergio eRuiz-Halpern

    2014-12-01

    Full Text Available Coastal areas play an important role on carbon cycling. Elucidating the dynamics on the production, transport and fate of organic carbon is relevant to gain a better understanding of the role coastal areas play in the global carbon budget. Here, we assess the metabolic status and associated organic carbon fluxes of a semi-enclosed Mediterranean bay supporting a meadow of Caulerpa prolifera. We test whether the EDOC pool is a significant component of the organic carbon pool and associated fluxes in this ecosystem. The Bay of Portocolom was in net metabolic balance on a yearly basis, but heterotrophic during the summer months. Community respiration (CR was positively correlated to C. prolifera biomass, while net community production (NCP had a negative correlation. The benthic compartment represented, on average, 72.6 ± 5.2 % of CR and 86.8 ± 4.5 % of gross primary production (GPP. Dissolved organic carbon (DOC production peaked in summer and was always positive, with the incubations performed in the dark almost doubling the flux of those performed in the light. Exchangeable dissolved organic carbon (EDOC, however, oscillated between production and uptake, being completely recycled within the system and representing around 14% of the DOC flux. The pools of bottom and surface DOC were high for an oligotrophic environment, and were positively correlated to the pool of EDOC. Thus, despite being in metabolic balance, this ecosystem acted as a conduit for organic carbon (OC, as it is able to export OC to adjacent areas derived from allochtonous inputs during heterotrophic conditions. These inputs likely come from groundwater discharge, human activity in the watershed, delivered to the sediments through the high capacity of C. prolifera to remove particles from the water column, and from the air-water exchange of EDOC, demonstrating that these communities are a major contributor to the cycling of OC in coastal embayments.

  12. Continuum analysis of biological systems conserved quantities, fluxes and forces

    CERN Document Server

    Suraishkumar, G K

    2014-01-01

    This book addresses the analysis, in the continuum regime, of biological systems at various scales, from the cellular level to the industrial one. It presents both fundamental conservation principles (mass, charge, momentum and energy) and relevant fluxes resulting from appropriate driving forces, which are important for the analysis, design and operation of biological systems. It includes the concept of charge conservation, an important principle for biological systems that is not explicitly covered in any other book of this kind. The book is organized in five parts: mass conservation; charge conservation; momentum conservation; energy conservation; and multiple conservations simultaneously applied. All mathematical aspects are presented step by step, allowing any reader with a basic mathematical background (calculus, differential equations, linear algebra, etc.) to follow the text with ease. The book promotes an intuitive understanding of all the relevant principles and in so doing facilitates their applica...

  13. Dis-aggregation of airborne flux measurements using footprint analysis

    NARCIS (Netherlands)

    Hutjes, R.W.A.; Vellinga, O.S.; Gioli, B.; Miglietta, F.

    2010-01-01

    Aircraft measurements of turbulent fluxes are generally being made with the objective to obtain an estimate of regional exchanges between land surface and atmosphere, to investigate the spatial variability of these fluxes, but also to learn something about the fluxes from some or all of the land

  14. Systems biology analysis of drivers underlying hallmarks of cancer cell metabolism

    Science.gov (United States)

    Zielinski, Daniel C.; Jamshidi, Neema; Corbett, Austin J.; Bordbar, Aarash; Thomas, Alex; Palsson, Bernhard O.

    2017-01-01

    Malignant transformation is often accompanied by significant metabolic changes. To identify drivers underlying these changes, we calculated metabolic flux states for the NCI60 cell line collection and correlated the variance between metabolic states of these lines with their other properties. The analysis revealed a remarkably consistent structure underlying high flux metabolism. The three primary uptake pathways, glucose, glutamine and serine, are each characterized by three features: (1) metabolite uptake sufficient for the stoichiometric requirement to sustain observed growth, (2) overflow metabolism, which scales with excess nutrient uptake over the basal growth requirement, and (3) redox production, which also scales with nutrient uptake but greatly exceeds the requirement for growth. We discovered that resistance to chemotherapeutic drugs in these lines broadly correlates with the amount of glucose uptake. These results support an interpretation of the Warburg effect and glutamine addiction as features of a growth state that provides resistance to metabolic stress through excess redox and energy production. Furthermore, overflow metabolism observed may indicate that mitochondrial catabolic capacity is a key constraint setting an upper limit on the rate of cofactor production possible. These results provide a greater context within which the metabolic alterations in cancer can be understood.

  15. Determination of key enzymes for threonine synthesis through in vitro metabolic pathway analysis.

    Science.gov (United States)

    Zhang, Yanfei; Meng, Qinglong; Ma, Hongwu; Liu, Yongfei; Cao, Guoqiang; Zhang, Xiaoran; Zheng, Ping; Sun, Jibin; Zhang, Dawei; Jiang, Wenxia; Ma, Yanhe

    2015-06-13

    The overexpression of key enzymes in a metabolic pathway is a frequently used genetic engineering strategy for strain improvement. Metabolic control analysis has been proposed to quantitatively determine key enzymes. However, the lack of quality data often makes it difficult to correctly identify key enzymes through control analysis. Here, we proposed a method combining in vitro metabolic pathway analysis and proteomics measurement to find the key enzymes in threonine synthesis pathway. All enzymes in the threonine synthesis pathway were purified for the reconstruction and perturbation of the in vitro pathway. Label-free proteomics technology combined with APEX (absolute protein expression measurements) data analysis method were employed to determine the absolute enzyme concentrations in the crude enzyme extract obtained from a threonine production strain during the fastest threonine production period. The flux control coefficient of each enzyme in the pathway was then calculated by measuring the flux changes after titration of the corresponding enzyme. The isoenzyme LysC catalyzing the first step in the pathway has the largest flux control coefficient, and thus its concentration change has the biggest impact on pathway flux. To verify that the key enzyme identified through in vitro pathway analysis is also the key enzyme in vivo, we overexpressed LysC in the original threonine production strain. Fermentation results showed that the threonine concentration was increased 30% and the yield was increased 20%. In vitro metabolic pathways simulating in vivo cells can be built based on precise measurement of enzyme concentrations through proteomics technology and used for the determination of key enzymes through metabolic control analysis. This provides a new way to find gene overexpression targets for industrial strain improvement.

  16. Ecological network analysis of China's societal metabolism.

    Science.gov (United States)

    Zhang, Yan; Liu, Hong; Li, Yating; Yang, Zhifeng; Li, Shengsheng; Yang, Naijin

    2012-01-01

    Uncontrolled socioeconomic development has strong negative effects on the ecological environment, including pollution and the depletion and waste of natural resources. These serious consequences result from the high flows of materials and energy through a socioeconomic system produced by exchanges between the system and its surroundings, causing the disturbance of metabolic processes. In this paper, we developed an ecological network model for a societal system, and used China in 2006 as a case study to illustrate application of the model. We analyzed China's basic metabolic processes and used ecological network analysis to study the network relationships within the system. Basic components comprised the internal environment, five sectors (agriculture, exploitation, manufacturing, domestic, and recycling), and the external environment. We defined 21 pairs of ecological relationships in China's societal metabolic system (excluding self-mutualism within a component). Using utility and throughflow analysis, we found that exploitation, mutualism, and competition relationships accounted for 76.2, 14.3, and 9.5% of the total relationships, respectively. In our trophic level analysis, the components were divided into producers, consumers, and decomposers according to their positions in the system. Our analyses revealed ways to optimize the system's structure and adjust its functions, thereby promoting healthier socioeconomic development, and suggested ways to apply ecological network analysis in future socioeconomic research. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. Gene Knockout Identification Using an Extension of Bees Hill Flux Balance Analysis

    Directory of Open Access Journals (Sweden)

    Yee Wen Choon

    2015-01-01

    Full Text Available Microbial strain optimisation for the overproduction of a desired phenotype has been a popular topic in recent years. Gene knockout is a genetic engineering technique that can modify the metabolism of microbial cells to obtain desirable phenotypes. Optimisation algorithms have been developed to identify the effects of gene knockout. However, the complexities of metabolic networks have made the process of identifying the effects of genetic modification on desirable phenotypes challenging. Furthermore, a vast number of reactions in cellular metabolism often lead to a combinatorial problem in obtaining optimal gene knockout. The computational time increases exponentially as the size of the problem increases. This work reports an extension of Bees Hill Flux Balance Analysis (BHFBA to identify optimal gene knockouts to maximise the production yield of desired phenotypes while sustaining the growth rate. This proposed method functions by integrating OptKnock into BHFBA for validating the results automatically. The results show that the extension of BHFBA is suitable, reliable, and applicable in predicting gene knockout. Through several experiments conducted on Escherichia coli, Bacillus subtilis, and Clostridium thermocellum as model organisms, extension of BHFBA has shown better performance in terms of computational time, stability, growth rate, and production yield of desired phenotypes.

  18. Metabolic control analysis of Aspergillus niger L-arabinose catabolism

    DEFF Research Database (Denmark)

    de Groot, M.J.L.; Prathumpai, Wai; Visser, J.

    2005-01-01

    A mathematical model of the L-arabinose/D-xylose catabolic pathway of Aspergillus niger was constructed based on the kinetic properties of the enzymes. For this purpose L-arabinose reductase, L-arabitol dehydrogenase and D-xylose reductase were purified using dye-affinity chromatography, and thei......A mathematical model of the L-arabinose/D-xylose catabolic pathway of Aspergillus niger was constructed based on the kinetic properties of the enzymes. For this purpose L-arabinose reductase, L-arabitol dehydrogenase and D-xylose reductase were purified using dye-affinity chromatography......-arabinose, a level that resulted in realistic intermediate concentrations in the model, flux control coefficients for L-arabinose reductase, L-arabitol dehydrogenase and L-xylulose reductase were 0.68, 0.17 and 0.14, respectively. The analysis can be used as a guide to identify targets for metabolic engineering...

  19. Control of fluxes towards antibiotics and the role of primary metabolism in production of antibiotics

    DEFF Research Database (Denmark)

    Gunnarsson, Nina; Eliasson Lantz, Anna; Nielsen, Jacob

    2004-01-01

    Yield improvements in antibiotic-producing strains have classically been obtained through random mutagenesis and screening. An attractive alternative to this strategy is the rational design of producer strains via metabolic engineering, an approach that offers the possibility to increase yields...... in the metabolic network. Here we describe and discuss available methods for identification of these steps, both in antibiotic biosynthesis pathways and in the primary metabolism, which serves as the supplier of precursors and cofactors for the secondary metabolism. Finally, the importance of precursor...... and cofactor supply from primary metabolism in the biosynthesis of different types of antibiotics is discussed and recent developments in metabolic engineering towards increased product yields in antibiotic producing strains are reviewed....

  20. Control of fluxes towards antibiotics and the role of primary metabolism in production of antibiotics

    DEFF Research Database (Denmark)

    Gunnarsson, Nina; Eliasson Lantz, Anna; Nielsen, Jacob

    2004-01-01

    in the metabolic network. Here we describe and discuss available methods for identification of these steps, both in antibiotic biosynthesis pathways and in the primary metabolism, which serves as the supplier of precursors and cofactors for the secondary metabolism. Finally, the importance of precursor...... and cofactor supply from primary metabolism in the biosynthesis of different types of antibiotics is discussed and recent developments in metabolic engineering towards increased product yields in antibiotic producing strains are reviewed.......Yield improvements in antibiotic-producing strains have classically been obtained through random mutagenesis and screening. An attractive alternative to this strategy is the rational design of producer strains via metabolic engineering, an approach that offers the possibility to increase yields...

  1. Flux Balance Analysis Indicates that Methane Is the Lowest Cost Feedstock for Microbial Cell Factories.

    Science.gov (United States)

    Comer, Austin D; Long, Matthew R; Reed, Jennifer L; Pfleger, Brian F

    2017-12-01

    The low cost of natural gas has driven significant interest in using C 1 carbon sources (e.g. methane, methanol, CO, syngas) as feedstocks for producing liquid transportation fuels and commodity chemicals. Given the large contribution of sugar and lignocellulosic feedstocks to biorefinery operating costs, natural gas and other C1 sources may provide an economic advantage. To assess the relative costs of these feedstocks, we performed flux balance analysis on genome-scale metabolic models to calculate the maximum theoretical yields of chemical products from methane, methanol, acetate, and glucose. Yield calculations were performed for every metabolite (as a proxy for desired products) in the genome-scale metabolic models of three organisms: Escherichia coli (bacterium), Saccharomyces cerevisiae (yeast), and Synechococcus sp. PCC 7002 (cyanobacterium). The calculated theoretical yields and current feedstock prices provided inputs to create comparative feedstock cost surfaces. Our analysis shows that, at current market prices, methane feedstock costs are consistently lower than glucose when used as a carbon and energy source for microbial chemical production. Conversely, methanol is costlier than glucose under almost all price scenarios. Acetate feedstock costs could be less than glucose given efficient acetate production from low-cost syngas using nascent biological gas to liquids (BIO-GTL) technologies. Our analysis suggests that research should focus on overcoming the technical challenges of methane assimilation and/or yield of acetate via BIO-GTL to take advantage of low-cost natural gas rather than using methanol as a feedstock.

  2. Flux balance analysis indicates that methane is the lowest cost feedstock for microbial cell factories

    Directory of Open Access Journals (Sweden)

    Austin D. Comer

    2017-12-01

    Full Text Available The low cost of natural gas has driven significant interest in using C1 carbon sources (e.g. methane, methanol, CO, syngas as feedstocks for producing liquid transportation fuels and commodity chemicals. Given the large contribution of sugar and lignocellulosic feedstocks to biorefinery operating costs, natural gas and other C1 sources may provide an economic advantage. To assess the relative costs of these feedstocks, we performed flux balance analysis on genome-scale metabolic models to calculate the maximum theoretical yields of chemical products from methane, methanol, acetate, and glucose. Yield calculations were performed for every metabolite (as a proxy for desired products in the genome-scale metabolic models of three organisms: Escherichia coli (bacterium, Saccharomyces cerevisiae (yeast, and Synechococcus sp. PCC 7002 (cyanobacterium. The calculated theoretical yields and current feedstock prices provided inputs to create comparative feedstock cost surfaces. Our analysis shows that, at current market prices, methane feedstock costs are consistently lower than glucose when used as a carbon and energy source for microbial chemical production. Conversely, methanol is costlier than glucose under almost all price scenarios. Acetate feedstock costs could be less than glucose given efficient acetate production from low-cost syngas using nascent biological gas to liquids (BIO-GTL technologies. Our analysis suggests that research should focus on overcoming the technical challenges of methane assimilation and/or yield of acetate via BIO-GTL to take advantage of low-cost natural gas rather than using methanol as a feedstock.

  3. Transorgan fluxes in a porcine model reveal a central role for liver in acylcarnitine metabolism

    NARCIS (Netherlands)

    Schooneman, Marieke G.; ten Have, Gabriella A. M.; van Vlies, Naomi; Houten, Sander M.; Deutz, Nicolaas E. P.; Soeters, Maarten R.

    2015-01-01

    Acylcarnitines are derived from mitochondrial acyl-CoA metabolism and have been associated with diet-induced insulin resistance. However, plasma acylcarnitine profiles have been shown to poorly reflect whole body acylcarnitine metabolism. We aimed to clarify the individual role of different organ

  4. Model-driven multi-omic data analysis elucidates metabolic immunomodulators of macrophage activation

    Energy Technology Data Exchange (ETDEWEB)

    Bordbar, Aarash; Mo, Monica L.; Nakayasu, Ernesto S.; Rutledge, Alexandra C.; Kim, Young-Mo; Metz, Thomas O.; Jones, Marcus B.; Frank, Bryan C.; Smith, Richard D.; Peterson, Scott N.; Hyduke, Daniel R.; Adkins, Joshua N.; Palsson, Bernhard O.

    2012-06-26

    Macrophages are central players in the immune response, manifesting divergent phenotypes to control inflammation and innate immunity through the release of cytokines and other regulatory factor-dependent signaling pathways. In recent years, the focus on metabolism has been reemphasized as critical signaling and regulatory pathways of human pathophysiology, ranging from cancer to aging, often converge on metabolic responses. Here, we used genome-scale modeling and multi-omics (transcriptomics, proteomics, and metabolomics) analysis to assess metabolic features critical for macrophage functions. We constructed a genome-scale metabolic network for the RAW 264.7 cell line to determine metabolic modulators of macrophage activation. Metabolites well-known to be associated with immunoactivation (e.g., glucose and arginine) and immunosuppression (e.g., tryptophan and vitamin D3) were amongst the most critical effectors. Intracellular metabolic mechanisms linked to critical suppressive effectors were then assessed, identifying a suppressive role for de novo nucleotide synthesis. Finally, the underlying metabolic mechanisms of macrophage activation are identified by analyzing multi-omic data obtained from LPS-stimulated RAW cells in the context of our flux-based predictions. Our study demonstrates metabolism's role in regulating activation may be greater than previously anticipated and elucidates underlying metabolic connections between activation and metabolic effectors.

  5. Radiopharmaceuticals for Assessment of Altered Metabolism and Biometal Fluxes in Brain Aging and Alzheimer’s Disease with Positron Emission Tomography

    OpenAIRE

    Xie, Fang; Peng, Fangyu

    2017-01-01

    Aging is a risk factor for Alzheimer’s disease (AD). There are changes of brain metabolism and biometal fluxes due to brain aging, which may play a role in pathogenesis of AD. Positron emission tomography (PET) is a versatile tool for tracking alteration of metabolism and biometal fluxes due to brain aging and AD. Age-dependent changes in cerebral glucose metabolism can be tracked with PET using 2-deoxy-2-[18F]-fluoro-D-glucose (18F-FDG), a radiolabeled glucose analogue, as a radiotracer. Bas...

  6. Integrating Kinetic Model of E. coli with Genome Scale Metabolic Fluxes Overcomes Its Open System Problem and Reveals Bistability in Central Metabolism.

    Directory of Open Access Journals (Sweden)

    Ahmad A Mannan

    Full Text Available An understanding of the dynamics of the metabolic profile of a bacterial cell is sought from a dynamical systems analysis of kinetic models. This modelling formalism relies on a deterministic mathematical description of enzyme kinetics and their metabolite regulation. However, it is severely impeded by the lack of available kinetic information, limiting the size of the system that can be modelled. Furthermore, the subsystem of the metabolic network whose dynamics can be modelled is faced with three problems: how to parameterize the model with mostly incomplete steady state data, how to close what is now an inherently open system, and how to account for the impact on growth. In this study we address these challenges of kinetic modelling by capitalizing on multi-'omics' steady state data and a genome-scale metabolic network model. We use these to generate parameters that integrate knowledge embedded in the genome-scale metabolic network model, into the most comprehensive kinetic model of the central carbon metabolism of E. coli realized to date. As an application, we performed a dynamical systems analysis of the resulting enriched model. This revealed bistability of the central carbon metabolism and thus its potential to express two distinct metabolic states. Furthermore, since our model-informing technique ensures both stable states are constrained by the same thermodynamically feasible steady state growth rate, the ensuing bistability represents a temporal coexistence of the two states, and by extension, reveals the emergence of a phenotypically heterogeneous population.

  7. Integrating Kinetic Model of E. coli with Genome Scale Metabolic Fluxes Overcomes Its Open System Problem and Reveals Bistability in Central Metabolism

    Science.gov (United States)

    Mannan, Ahmad A.; Toya, Yoshihiro; Shimizu, Kazuyuki; McFadden, Johnjoe; Kierzek, Andrzej M.; Rocco, Andrea

    2015-01-01

    An understanding of the dynamics of the metabolic profile of a bacterial cell is sought from a dynamical systems analysis of kinetic models. This modelling formalism relies on a deterministic mathematical description of enzyme kinetics and their metabolite regulation. However, it is severely impeded by the lack of available kinetic information, limiting the size of the system that can be modelled. Furthermore, the subsystem of the metabolic network whose dynamics can be modelled is faced with three problems: how to parameterize the model with mostly incomplete steady state data, how to close what is now an inherently open system, and how to account for the impact on growth. In this study we address these challenges of kinetic modelling by capitalizing on multi-‘omics’ steady state data and a genome-scale metabolic network model. We use these to generate parameters that integrate knowledge embedded in the genome-scale metabolic network model, into the most comprehensive kinetic model of the central carbon metabolism of E. coli realized to date. As an application, we performed a dynamical systems analysis of the resulting enriched model. This revealed bistability of the central carbon metabolism and thus its potential to express two distinct metabolic states. Furthermore, since our model-informing technique ensures both stable states are constrained by the same thermodynamically feasible steady state growth rate, the ensuing bistability represents a temporal coexistence of the two states, and by extension, reveals the emergence of a phenotypically heterogeneous population. PMID:26469081

  8. Analysis of flux estimates based on C-13-labelling experiments

    DEFF Research Database (Denmark)

    Christensen, Bjarke; Gombert, Andreas Karoly; Nielsen, Jens

    2002-01-01

    metabolism were estimated. In the various samples, the estimates of the central metabolic pathways, the tricarboxylic acid cycle, the oxidative pentose phosphate pathway and the anaplerotic pathway, showed an unprecedented reproducibility. The high reproducibility was obtained with fractional labellings...

  9. Stability analysis of magnetic flux in thin-film superconductors

    International Nuclear Information System (INIS)

    Denisov, Dmitry

    2007-01-01

    This work presents theoretical results in the physics of superconductivity. The first part of the work is dedicated to the problem of thermomagnetic instabilities and flux avalanches in thin film superconductors. The second part describes the problem of flux trapped in the hole of the superconducting ring (author)

  10. Using PSAMM for the Curation and Analysis of Genome-Scale Metabolic Models.

    Science.gov (United States)

    Dufault-Thompson, Keith; Steffensen, Jon Lund; Zhang, Ying

    2018-01-01

    PSAMM is an open source software package that supports the iterative curation and analysis of genome-scale models (GEMs). It aims to integrate the annotation and consistency checking of metabolic models with the simulation of metabolic fluxes. The model representation in PSAMM is compatible with version tracking systems like Git, which allows for full documentation of model file changes and enables collaborative curations of large, complex models. This chapter provides a protocol for using PSAMM functions and a detailed description of the various aspects in setting up and using PSAMM for the simulation and analysis of metabolic models. The overall PSAMM workflow outlined in this chapter includes the import and export of model files, the documentation of model modifications using the Git version control system, the application of consistency checking functions for model curations, and the numerical simulation of metabolic models.

  11. How to determine control of growth rate in a chemostat. Using metabolic control analysis to resolve the paradox

    DEFF Research Database (Denmark)

    Snoep, Jacky L.; Jensen, Peter Ruhdal; Groeneveld, Philip

    1994-01-01

    how, paradoxically, one can determine control of growth rate, of growth yield and of other fluxes in a chemostat. We develop metabolic control analysis for the chemostat. this analysis does not depend on the particular way in which specific growth rate varies with the concentration of the growth...

  12. Flux balance analysis of ammonia assimilation network in E. coli predicts preferred regulation point.

    Directory of Open Access Journals (Sweden)

    Lu Wang

    Full Text Available Nitrogen assimilation is a critical biological process for the synthesis of biomolecules in Escherichia coli. The central ammonium assimilation network in E. coli converts carbon skeleton α-ketoglutarate and ammonium into glutamate and glutamine, which further serve as nitrogen donors for nitrogen metabolism in the cell. This reaction network involves three enzymes: glutamate dehydrogenase (GDH, glutamine synthetase (GS and glutamate synthase (GOGAT. In minimal media, E. coli tries to maintain an optimal growth rate by regulating the activity of the enzymes to match the availability of the external ammonia. The molecular mechanism and the strategy of the regulation in this network have been the research topics for many investigators. In this paper, we develop a flux balance model for the nitrogen metabolism, taking into account of the cellular composition and biosynthetic requirements for nitrogen. The model agrees well with known experimental results. Specifically, it reproduces all the (15N isotope labeling experiments in the wild type and the two mutant (ΔGDH and ΔGOGAT strains of E. coli. Furthermore, the predicted catalytic activities of GDH, GS and GOGAT in different ammonium concentrations and growth rates for the wild type, ΔGDH and ΔGOGAT strains agree well with the enzyme concentrations obtained from western blots. Based on this flux balance model, we show that GS is the preferred regulation point among the three enzymes in the nitrogen assimilation network. Our analysis reveals the pattern of regulation in this central and highly regulated network, thus providing insights into the regulation strategy adopted by the bacteria. Our model and methods may also be useful in future investigations in this and other networks.

  13. SU-G-TeP3-10: Radiation Induces Prompt Live-Cell Metabolic Fluxes

    Energy Technology Data Exchange (ETDEWEB)

    Campos, D [University of Wisconsin Madison, Madison, WI (United States); Peeters, W; Bussink, J [Radboud University Medical Center, Nijmegen, GA (United States); Nickel, K [University of Wisconsin - Madison, Madison, Wisconsin (United States); Burkel, B; Kimple, R; Kogel, A van der; Eliceiri, K [University of Wisconsin - Madison, Madison, WI (United States); Kissick, M [University of Wisconsin, Madison, WI (United States)

    2016-06-15

    Purpose: To compare metabolic dynamics and HIF-1α expression following radiation between a cancerous cell line (UM-SCC-22B) and a normal, immortalized cell line, NOK (Normal Oral Keratinocyte). HIF-1 is a key factor in metabolism and radiosensitivity. A better understanding of how radiation affects the interplay of metabolism and HIF-1 might give a better understanding of the mechanisms responsible for radiosensitivity. Methods: Changes in cellular metabolism in response to radiation are tracked by fluorescence lifetime of NADH. Expression of HIF-1α was measured by immunofluorescence for both cell lines with and without irradiation. Radiation response is also monitored with additional treatment of a HIF-1α inhibitor (chrysin) as well as a radical scavenger (glutathione). Changes in oxygen consumption and respiratory capacity are also monitored using the Seahorse XF analyzer. Results: An increase in HIF-1α was found to be in response to radiation for the cancer cell line, but not the normal cell line. Radiation was found to shift metabolism toward glycolytic pathways in cancer cells as measured by oxygen consumption and respiratory capacity. Radiation response was found to be muted by addition of glutathione to cell media. HIF-1α inhibition similarly muted radiation response in cancer. Conclusion: The HIF-1 protein complex is a key regulator cellular metabolism through the regulation of glycolysis and glucose transport enzymes. Moreover, HIF-1 has shown radio-protective effects in tumor vascular endothelia, and has been implicated in metastatic aggression. Monitoring interplay between metabolism and the HIF-1 protein complex can give a more fundamental understanding of radiotherapy response.

  14. Substrate cycles in Penicillium chrysogenum quantified by isotopic non-stationary flux analysis

    Directory of Open Access Journals (Sweden)

    Zhao Zheng

    2012-10-01

    Full Text Available Abstract Background Penicillium chrysogenum, the main production strain for penicillin-G, has a high content of intracellular carbohydrates, especially reduced sugars such as mannitol, arabitol, erythritol, as well as trehalose and glycogen. In previous steady state 13C wash-in experiments a delay of labeling enrichments in glycolytic intermediates was observed, which suggests turnover of storage carbohydrates. The turnover of storage pools consumes ATP which is expected to reduce the product yield for energy demanding production pathways like penicillin-G. Results In this study, a 13C labeling wash-in experiment of 1 hour was performed to systematically quantify the intracellular flux distribution including eight substrate cycles. The experiments were performed using a mixed carbon source of 85% CmolGlc/CmolGlc+EtOH labeled glucose (mixture of 90% [1-13C1] and 10% [U-13C6] and 15% ethanol [U-13C2]. It was found, that (1 also several extracellular pools are enriched with 13C labeling rapidly (trehalose, mannitol, and others, (2 the intra- to extracellular metabolite concentration ratios were comparable for a large set of metabolites while for some carbohydrates (mannitol, trehalose, and glucose the measured ratios were much higher. Conclusions The fast enrichment of several extracellular carbohydrates and a concentration ratio higher than the ratio expected from cell lysis (2% indicate active (e.g. ATP consuming transport cycles over the cellular membrane. The flux estimation indicates, that substrate cycles account for about 52% of the gap in the ATP balance based on metabolic flux analysis.

  15. Consequences of gas flux model choice on the interpretation of metabolic balance across 15 lakes

    Science.gov (United States)

    Dugan, Hilary; Woolway, R. Iestyn; Santoso, Arianto; Corman, Jessica; Jaimes, Aline; Nodine, Emily; Patil, Vijay; Zwart, Jacob A.; Brentrup, Jennifer A.; Hetherington, Amy; Oliver, Samantha K.; Read, Jordan S.; Winters, Kirsten; Hanson, Paul; Read, Emily; Winslow, Luke; Weathers, Kathleen

    2016-01-01

    Ecosystem metabolism and the contribution of carbon dioxide from lakes to the atmosphere can be estimated from free-water gas measurements through the use of mass balance models, which rely on a gas transfer coefficient (k) to model gas exchange with the atmosphere. Theoretical and empirically based models of krange in complexity from wind-driven power functions to complex surface renewal models; however, model choice is rarely considered in most studies of lake metabolism. This study used high-frequency data from 15 lakes provided by the Global Lake Ecological Observatory Network (GLEON) to study how model choice of kinfluenced estimates of lake metabolism and gas exchange with the atmosphere. We tested 6 models of k on lakes chosen to span broad gradients in surface area and trophic states; a metabolism model was then fit to all 6 outputs of k data. We found that hourly values for k were substantially different between models and, at an annual scale, resulted in significantly different estimates of lake metabolism and gas exchange with the atmosphere.

  16. An integrative machine learning strategy for improved prediction of essential genes in Escherichia coli metabolism using flux-coupled features.

    Science.gov (United States)

    Nandi, Sutanu; Subramanian, Abhishek; Sarkar, Ram Rup

    2017-07-25

    Prediction of essential genes helps to identify a minimal set of genes that are absolutely required for the appropriate functioning and survival of a cell. The available machine learning techniques for essential gene prediction have inherent problems, like imbalanced provision of training datasets, biased choice of the best model for a given balanced dataset, choice of a complex machine learning algorithm, and data-based automated selection of biologically relevant features for classification. Here, we propose a simple support vector machine-based learning strategy for the prediction of essential genes in Escherichia coli K-12 MG1655 metabolism that integrates a non-conventional combination of an appropriate sample balanced training set, a unique organism-specific genotype, phenotype attributes that characterize essential genes, and optimal parameters of the learning algorithm to generate the best machine learning model (the model with the highest accuracy among all the models trained for different sample training sets). For the first time, we also introduce flux-coupled metabolic subnetwork-based features for enhancing the classification performance. Our strategy proves to be superior as compared to previous SVM-based strategies in obtaining a biologically relevant classification of genes with high sensitivity and specificity. This methodology was also trained with datasets of other recent supervised classification techniques for essential gene classification and tested using reported test datasets. The testing accuracy was always high as compared to the known techniques, proving that our method outperforms known methods. Observations from our study indicate that essential genes are conserved among homologous bacterial species, demonstrate high codon usage bias, GC content and gene expression, and predominantly possess a tendency to form physiological flux modules in metabolism.

  17. Analysis of high heat flux testing of mock-ups

    International Nuclear Information System (INIS)

    Salavy, J.-F.; Giancarli, L.; Merola, M.; Picard, F.; Roedig, M.

    2003-01-01

    ITER EU Home Team is performing a large R and D effort in support of the development of high heat flux components for ITER. In this framework, this paper describes the thermal analyses, the fatigue lifetime evaluation and the transient VDE with material melting related to the high heat flux thermo-mechanical tests performed in the JUDITH facility. It reports on several mock-ups representative of different proposed component designs based on Be, W and CFC as armour materials

  18. Flux effect analysis in WWER-440 reactor pressure vessel steels

    Science.gov (United States)

    Kryukov, A.; Blagoeva, D.; Debarberis, L.

    2013-11-01

    The results of long term research programme concerning the determination of irradiation embrittlement dependence on fast neutron flux for WWER-440 reactor pressure vessel steels before and after annealing are presented in this paper. The study of flux effect was carried out on commercial WWER-440 steels which differ significantly in phosphorous (0.013-0.036 wt%) and copper (0.08-0.20 wt%) contents. All specimens were irradiated in surveillance channel positions under similar conditions at high ˜4 × 1012 сm-2 s-1 and low ˜6 × 1011 сm-2 s-1 fluxes (E > 0.5 MeV) at a temperature of 270 °С. The radiation embrittlement was evaluated by transition temperature shift on the basis of Charpy specimens test results. In case of low flux, the measured Tk shifts could be 25-50 °C bigger than the Tk shifts obtained from high flux data. A significant flux effect is observed in WWER-440 reactor pressure vessel steels with higher copper content (>0.13 wt%).

  19. Partitioning Water Vapor and Carbon Dioxide Fluxes using Correlation Analysis

    Science.gov (United States)

    Scanlon, T. M.

    2008-12-01

    A variety of methods are currently available to partition water vapor fluxes (into components of transpiration and direct evaporation) and carbon dioxide fluxes (into components of photosynthesis and respiration), using chambers, isotopes, and regression modeling approaches. Here, a methodology is presented that accounts for correlations between high-frequency measurements of water vapor (q) and carbon dioxide (c) concentrations being influenced by their non-identical source-sink distributions and the relative magnitude of their constituent fluxes. Flux-variance similarity assumptions are applied separately to the stomatal and the non-stomatal exchange, and the flux components are identified by considering the q-c correlation. Water use efficiency for the vegetation, and how it varies with respect to vapor pressure deficit, is the only input needed for this approach that uses standard eddy covariance measurements. The method is demonstrated using data collected over a corn field throughout a growing season. In particular, the research focuses on the partitioning of the water flux with the aim of improving how direct evaporation is handled in soil-vegetation- atmosphere transfer models over the course of wetting and dry-down cycles.

  20. Differential RNA-seq, Multi-Network Analysis and Metabolic Regulation Analysis of Kluyveromyces marxianus Reveals a Compartmentalised Response to Xylose.

    Directory of Open Access Journals (Sweden)

    Du Toit W P Schabort

    Full Text Available We investigated the transcriptomic response of a new strain of the yeast Kluyveromyces marxianus, in glucose and xylose media using RNA-seq. The data were explored in a number of innovative ways using a variety of networks types, pathway maps, enrichment statistics, reporter metabolites and a flux simulation model, revealing different aspects of the genome-scale response in an integrative systems biology manner. The importance of the subcellular localisation in the transcriptomic response is emphasised here, revealing new insights. As was previously reported by others using a rich medium, we show that peroxisomal fatty acid catabolism was dramatically up-regulated in a defined xylose mineral medium without fatty acids, along with mechanisms to activate fatty acids and transfer products of β-oxidation to the mitochondria. Notably, we observed a strong up-regulation of the 2-methylcitrate pathway, supporting capacity for odd-chain fatty acid catabolism. Next we asked which pathways would respond to the additional requirement for NADPH for xylose utilisation, and rationalised the unexpected results using simulations with Flux Balance Analysis. On a fundamental level, we investigated the contribution of the hierarchical and metabolic regulation levels to the regulation of metabolic fluxes. Metabolic regulation analysis suggested that genetic level regulation plays a major role in regulating metabolic fluxes in adaptation to xylose, even for the high capacity reactions, which is unexpected. In addition, isozyme switching may play an important role in re-routing of metabolic fluxes in subcellular compartments in K. marxianus.

  1. Radiopharmaceuticals for Assessment of Altered Metabolism and Biometal Fluxes in Brain Aging and Alzheimer's Disease with Positron Emission Tomography.

    Science.gov (United States)

    Xie, Fang; Peng, Fangyu

    2017-01-01

    Aging is a risk factor for Alzheimer's disease (AD). There are changes of brain metabolism and biometal fluxes due to brain aging, which may play a role in pathogenesis of AD. Positron emission tomography (PET) is a versatile tool for tracking alteration of metabolism and biometal fluxes due to brain aging and AD. Age-dependent changes in cerebral glucose metabolism can be tracked with PET using 2-deoxy-2-[18F]-fluoro-D-glucose (18F-FDG), a radiolabeled glucose analogue, as a radiotracer. Based on different patterns of altered cerebral glucose metabolism, 18F-FDG PET was clinically used for differential diagnosis of AD and Frontotemporal dementia (FTD). There are continued efforts to develop additional radiopharmaceuticals or radiotracers for assessment of age-dependent changes of various metabolic pathways and biometal fluxes due to brain aging and AD with PET. Elucidation of age-dependent changes of brain metabolism and altered biometal fluxes is not only significant for a better mechanistic understanding of brain aging and the pathophysiology of AD, but also significant for identification of new targets for the prevention, early diagnosis, and treatment of AD.

  2. The key to acetate: metabolic fluxes of acetic acid bacteria under cocoa pulp fermentation-simulating conditions.

    Science.gov (United States)

    Adler, Philipp; Frey, Lasse Jannis; Berger, Antje; Bolten, Christoph Josef; Hansen, Carl Erik; Wittmann, Christoph

    2014-08-01

    Acetic acid bacteria (AAB) play an important role during cocoa fermentation, as their main product, acetate, is a major driver for the development of the desired cocoa flavors. Here, we investigated the specialized metabolism of these bacteria under cocoa pulp fermentation-simulating conditions. A carefully designed combination of parallel 13C isotope labeling experiments allowed the elucidation of intracellular fluxes in the complex environment of cocoa pulp, when lactate and ethanol were included as primary substrates among undefined ingredients. We demonstrate that AAB exhibit a functionally separated metabolism during coconsumption of two-carbon and three-carbon substrates. Acetate is almost exclusively derived from ethanol, while lactate serves for the formation of acetoin and biomass building blocks. Although this is suboptimal for cellular energetics, this allows maximized growth and conversion rates. The functional separation results from a lack of phosphoenolpyruvate carboxykinase and malic enzymes, typically present in bacteria to interconnect metabolism. In fact, gluconeogenesis is driven by pyruvate phosphate dikinase. Consequently, a balanced ratio of lactate and ethanol is important for the optimum performance of AAB. As lactate and ethanol are individually supplied by lactic acid bacteria and yeasts during the initial phase of cocoa fermentation, respectively, this underlines the importance of a well-balanced microbial consortium for a successful fermentation process. Indeed, AAB performed the best and produced the largest amounts of acetate in mixed culture experiments when lactic acid bacteria and yeasts were both present.

  3. Human taurine metabolism: fluxes and fractional extraction rates of the gut, liver, and kidneys

    NARCIS (Netherlands)

    van Stijn, Mireille F. M.; Vermeulen, Mechteld A. R.; Siroen, Michiel P. C.; Wong, Leanne N.; van den Tol, M. Petrousjka; Ligthart-Melis, Gerdien C.; Houdijk, Alexander P. J.; van Leeuwen, Paul A. M.

    2012-01-01

    Taurine is involved in numerous biological processes. However, taurine plasma level decreases in response to pathological conditions, suggesting an increased need. Knowledge on human taurine metabolism is scarce and only described by arterial-venous differences across a single organ. Here we present

  4. Enzyme allocation problems in kinetic metabolic networks: Optimal solutions are elementary flux modes

    Czech Academy of Sciences Publication Activity Database

    Müller, Stefan; Regensburger, G.; Steuer, Ralf

    2014-01-01

    Roč. 347, APR 2014 (2014), s. 182-190 ISSN 0022-5193 R&D Projects: GA MŠk(CZ) EE2.3.20.0256 Institutional support: RVO:67179843 Keywords : metabolic optimization * enzyme kinetics * oriented matroid * elementary vector * conformal sum Subject RIV: EI - Biotechnology ; Bionics Impact factor: 2.116, year: 2014

  5. {sup 13}C dynamic nuclear polarization for measuring metabolic flux in endothelial progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Nielsen, Nathalie; Laustsen, Christoffer; Bertelsen, Lotte Bonde, E-mail: Lotte@clin.au.dk

    2016-11-15

    Endothelial progenitor cells (EPCs) represent a heterogeneous cell population that is believed to be involved in vasculogenesis. With the purpose of enhancing endothelial repair, EPCs could have a potential for future cell therapies. Due to the low amount of EPCs in the peripheral circulating blood, in vitro expansion is needed before administration to recipients and the effects of in vitro culturing is still an under-evaluated field with little knowledge of how the cells change over time in culture. The aim of this study was to use hyperpolarised carbon-13 magnetic resonance spectroscopy to profile important metabolic pathways in a population of progenitor cells and to show that cell culturing in 3D scaffolds seem to block the metabolic processes that leads to cell senescence. The metabolic breakdown of hyperpolarized [1-{sup 13}C]pyruvate was followed after injection of the substrate to a bioreactor system with EPCs either adhered to 3D printed scaffolds or kept in cell suspension. The pyruvate-to-lactate conversion was elevated in suspension of EPCs compared to the EPCs adhered to scaffolds. Furthermore in the setup with EPCs in suspension, an increase in lactate production was seen over time indicating that the older the cultures of EPCs was before using the cells for cell suspension experiments, the more lactate they produce, compared to a constant lactate level in the cells adhered to scaffolds. It could therefore be stated that cells grown first in 2D culture and subsequent prepared for cell suspension show a metabolism with higher lactate production consistent with cells senescence processes compared to cells grown first at 2D culture and subsequent in the 3D printed scaffolds, where metabolism shows no sign of metabolic shifting during the monitored period. - Highlights: • Hyperpolarized 13C MRS detects EPCs metabolic changes associated with ageing and cultivating conditions. • Increased lactate production in EPC’s correlates positively with aging.

  6. Construction and analysis of a genome-scale metabolic network for Bacillus licheniformis WX-02.

    Science.gov (United States)

    Guo, Jing; Zhang, Hong; Wang, Cheng; Chang, Ji-Wei; Chen, Ling-Ling

    2016-05-01

    We constructed the genome-scale metabolic network of Bacillus licheniformis (B. licheniformis) WX-02 by combining genomic annotation, high-throughput phenotype microarray (PM) experiments and literature-based metabolic information. The accuracy of the metabolic network was assessed by an OmniLog PM experiment. The final metabolic model iWX1009 contains 1009 genes, 1141 metabolites and 1762 reactions, and the predicted metabolic phenotypes showed an agreement rate of 76.8% with experimental PM data. In addition, key metabolic features such as growth yield, utilization of different substrates and essential genes were identified by flux balance analysis. A total of 195 essential genes were predicted from LB medium, among which 149 were verified with the experimental essential gene set of B. subtilis 168. With the removal of 5 reactions from the network, pathways for poly-γ-glutamic acid (γ-PGA) synthesis were optimized and the γ-PGA yield reached 83.8 mmol/h. Furthermore, the important metabolites and pathways related to γ-PGA synthesis and bacterium growth were comprehensively analyzed. The present study provides valuable clues for exploring the metabolisms and metabolic regulation of γ-PGA synthesis in B. licheniformis WX-02. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  7. Quantitative elementary mode analysis of metabolic pathways: the example of yeast glycolysis

    Directory of Open Access Journals (Sweden)

    Kanehisa Minoru

    2006-04-01

    Full Text Available Abstract Background Elementary mode analysis of metabolic pathways has proven to be a valuable tool for assessing the properties and functions of biochemical systems. However, little comprehension of how individual elementary modes are used in real cellular states has been achieved so far. A quantitative measure of fluxes carried by individual elementary modes is of great help to identify dominant metabolic processes, and to understand how these processes are redistributed in biological cells in response to changes in environmental conditions, enzyme kinetics, or chemical concentrations. Results Selecting a valid decomposition of a flux distribution onto a set of elementary modes is not straightforward, since there is usually an infinite number of possible such decompositions. We first show that two recently introduced decompositions are very closely related and assign the same fluxes to reversible elementary modes. Then, we show how such decompositions can be used in combination with kinetic modelling to assess the effects of changes in enzyme kinetics on the usage of individual metabolic routes, and to analyse the range of attainable states in a metabolic system. This approach is illustrated by the example of yeast glycolysis. Our results indicate that only a small subset of the space of stoichiometrically feasible steady states is actually reached by the glycolysis system, even when large variation intervals are allowed for all kinetic parameters of the model. Among eight possible elementary modes, the standard glycolytic route remains dominant in all cases, and only one other elementary mode is able to gain significant flux values in steady state. Conclusion These results indicate that a combination of structural and kinetic modelling significantly constrains the range of possible behaviours of a metabolic system. All elementary modes are not equal contributors to physiological cellular states, and this approach may open a direction toward a

  8. Surface renewal analysis for estimating turbulent surface fluxes

    International Nuclear Information System (INIS)

    Castellvi, F.

    2009-01-01

    A decade ago, the need for a long-term surface monitoring was recognized to better understand the soil-vegetation-atmosphere scalar exchange and interaction processes. the AmeriFlux concept emerged in the IGBP workshop (La Thuile, IT, 1995). Continuous acquisition of surface fluxes for different species such as temperature, water vapour, CO x , halocarbon, ozone, etc.,) and momentum allows determination of the influence of local (canopy) exchanges, fossil fuel emission, large-scale biotic exchange on ambient concentrations which are crucial to take decisions for protecting natural environments and water resources, to develop new perspective for modern agriculture and forest management and to better understand the global climate change. (Author)

  9. Metabolic Flux Distribution during Defatting of Steatotic Human Hepatoma (HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Gabriel Yarmush

    2016-01-01

    Full Text Available Methods that rapidly decrease fat in steatotic hepatocytes may be helpful to recover severely fatty livers for transplantation. Defatting kinetics are highly dependent upon the extracellular medium composition; however, the pathways involved are poorly understood. Steatosis was induced in human hepatoma cells (HepG2 by exposure to high levels of free fatty acids, followed by defatting using plain medium containing no fatty acids, or medium supplemented with a cocktail of defatting agents previously described before. We measured the levels of 28 extracellular metabolites and intracellular triglyceride, and fed the data into a steady-state mass balance model to estimate strictly intracellular fluxes. We found that during defatting, triglyceride content decreased, while beta-oxidation, the tricarboxylic acid cycle, and the urea cycle increased. These fluxes were augmented by defatting agents, and even more so by hyperoxic conditions. In all defatting conditions, the rate of extracellular glucose uptake/release was very small compared to the internal supply from glycogenolysis, and glycolysis remained highly active. Thus, in steatotic HepG2 cells, glycolysis and fatty acid oxidation may co-exist. Together, these pathways generate reducing equivalents that are supplied to mitochondrial oxidative phosphorylation.

  10. Robustness analysis of a constraint-based metabolic model links cell growth and proteomics of Thermoanaerobacter tengcongensis under temperature perturbation.

    Science.gov (United States)

    Tong, Wei; Chen, Zhen; Cao, Zhe; Wang, Quanhui; Zhang, Jiyuan; Bai, Xue; Wang, Rong; Liu, Siqi

    2013-04-05

    The integration of omic data with metabolic networks has been demonstrated to be an effective approach to elucidate the underlying metabolic mechanisms in life. Because the metabolic pathways of Thermoanaerobacter tengcongensis (T. tengcongensis) are incomplete, we used a 1-(13)C-glucose culture to monitor intracellular isotope-labeled metabolites by GC/MS and identified the gap gene in glucose catabolism, Re-citrate synthase. Based on genome annotation and biochemical information, we reconstructed the metabolic network of glucose metabolism and amino acid synthesis in T. tengcongensis, including 253 reactions, 227 metabolites, and 236 genes. Furthermore, we performed constraint based modeling (CBM)-derived robustness analysis on the model to study the dynamic changes of the metabolic network. By perturbing the culture temperature from 75 to 55 °C, we collected the bacterial growth rates and differential proteomes. Assuming that protein abundance changes represent metabolic flux variations, we proposed that the robustness analysis of the CBM model could decipher the effect of proteome change on the bacterial growth under perturbation. For approximately 73% of the reactions, the predicted cell growth changes due to such reaction flux variations matched the observed cell growth data. Our study, therefore, indicates that differential proteome data can be integrated with metabolic network modeling and that robustness analysis is a strong method for representing the dynamic change in cell phenotypes under perturbation.

  11. Characterizing the roles of changing population size and selection on the evolution of flux control in metabolic pathways.

    Science.gov (United States)

    Orlenko, Alena; Chi, Peter B; Liberles, David A

    2017-05-25

    Understanding the genotype-phenotype map is fundamental to our understanding of genomes. Genes do not function independently, but rather as part of networks or pathways. In the case of metabolic pathways, flux through the pathway is an important next layer of biological organization up from the individual gene or protein. Flux control in metabolic pathways, reflecting the importance of mutation to individual enzyme genes, may be evolutionarily variable due to the role of mutation-selection-drift balance. The evolutionary stability of rate limiting steps and the patterns of inter-molecular co-evolution were evaluated in a simulated pathway with a system out of equilibrium due to fluctuating selection, population size, or positive directional selection, to contrast with those under stabilizing selection. Depending upon the underlying population genetic regime, fluctuating population size was found to increase the evolutionary stability of rate limiting steps in some scenarios. This result was linked to patterns of local adaptation of the population. Further, during positive directional selection, as with more complex mutational scenarios, an increase in the observation of inter-molecular co-evolution was observed. Differences in patterns of evolution when systems are in and out of equilibrium, including during positive directional selection may lead to predictable differences in observed patterns for divergent evolutionary scenarios. In particular, this result might be harnessed to detect differences between compensatory processes and directional processes at the pathway level based upon evolutionary observations in individual proteins. Detecting functional shifts in pathways reflects an important milestone in predicting when changes in genotypes result in changes in phenotypes.

  12. Fermentation Process and Metabolic Flux of Ethanol Production from the Detoxified Hydrolyzate of Cassava Residue

    Directory of Open Access Journals (Sweden)

    Xingjiang Li

    2017-08-01

    Full Text Available With the growth of the world population, energy problems are becoming increasingly severe; therefore, sustainable energy sources have gained enormous importance. With respect to ethanol fuel production, biomass is gradually replacing grain as the main raw material. In this study, we explored the fermentation of five- and six-carbon sugars, the main biomass degradation products, into alcohol. We conducted mutagenic screening specifically for Candida tropicalis CICC1779 to obtain a strain that effectively used xylose (Candida tropicalis CICC1779-Dyd. By subsequently studying fermentation conditions under different initial liquid volume oxygen transfer coefficients (kLα, and coupling control of the aeration rate and agitation speed under optimal conditions, the optimal dissolved oxygen change curve was obtained. In addition, we constructed metabolic flow charts and equations to obtain a better understanding of the fermentation mechanism and to improve the ethanol yield. In our experiment, the ethanol production of the wild type stain was 17.58 g·L−1 at a kLα of 120. The highest ethanol yield of the mutagenic strains was 24.85 g·L−1. The ethanol yield increased to 26.56 g·L−1 when the dissolved oxygen content was optimized, and the conversion of sugar into alcohol reached 0.447 g·g−1 glucose (the theoretical titer of yeast-metabolized xylose was 0.46 g ethanol/g xylose and the glucose ethanol fermentation titer was 0.51 g ethanol/g glucose. Finally, the detected activity of xylose reductase and xylose dehydrogenase was higher in the mutant strain than in the original, which indicated that the mutant strain (CICC1779-Dyd could effectively utilize xylose for metabolism.

  13. Application of dynamic flux balance analysis to an industrial Escherichia coli fermentation.

    Science.gov (United States)

    Meadows, Adam L; Karnik, Rahi; Lam, Harry; Forestell, Sean; Snedecor, Brad

    2010-03-01

    We have developed a reactor-scale model of Escherichia coli metabolism and growth in a 1000 L process for the production of a recombinant therapeutic protein. The model consists of two distinct parts: (1) a dynamic, process specific portion that describes the time evolution of 37 process variables of relevance and (2) a flux balance based, 123-reaction metabolic model of E. coli metabolism. This model combines several previously reported modeling approaches including a growth rate-dependent biomass composition, maximum growth rate objective function, and dynamic flux balancing. In addition, we introduce concentration-dependent boundary conditions of transport fluxes, dynamic maintenance demands, and a state-dependent cellular objective. This formulation was able to describe specific runs with high-fidelity over process conditions including rich media, simultaneous acetate and glucose consumption, glucose minimal media, and phosphate depleted media. Furthermore, the model accurately describes the effect of process perturbations--such as glucose overbatching and insufficient aeration--on growth, metabolism, and titer. (c) 2009 Elsevier Inc. All rights reserved.

  14. Automated metabolic gas analysis systems: a review.

    Science.gov (United States)

    Macfarlane, D J

    2001-01-01

    The use of automated metabolic gas analysis systems or metabolic measurement carts (MMC) in exercise studies is common throughout the industrialised world. They have become essential tools for diagnosing many hospital patients, especially those with cardiorespiratory disease. Moreover, the measurement of maximal oxygen uptake (VO2max) is routine for many athletes in fitness laboratories and has become a defacto standard in spite of its limitations. The development of metabolic carts has also facilitated the noninvasive determination of the lactate threshold and cardiac output, respiratory gas exchange kinetics, as well as studies of outdoor activities via small portable systems that often use telemetry. Although the fundamental principles behind the measurement of oxygen uptake (VO2) and carbon dioxide production (VCO2) have not changed, the techniques used have, and indeed, some have almost turned through a full circle. Early scientists often employed a manual Douglas bag method together with separate chemical analyses, but the need for faster and more efficient techniques fuelled the development of semi- and full-automated systems by private and commercial institutions. Yet, recently some scientists are returning back to the traditional Douglas bag or Tissot-spirometer methods, or are using less complex automated systems to not only save capital costs, but also to have greater control over the measurement process. Over the last 40 years, a considerable number of automated systems have been developed, with over a dozen commercial manufacturers producing in excess of 20 different automated systems. The validity and reliability of all these different systems is not well known, with relatively few independent studies having been published in this area. For comparative studies to be possible and to facilitate greater consistency of measurements in test-retest or longitudinal studies of individuals, further knowledge about the performance characteristics of these

  15. Simultaneous investigation of cardiac pyruvate dehydrogenase flux, Krebs cycle metabolism and pH, using hyperpolarized [1,2-(13)C2]pyruvate in vivo.

    Science.gov (United States)

    Chen, Albert P; Hurd, Ralph E; Schroeder, Marie A; Lau, Angus Z; Gu, Yi-ping; Lam, Wilfred W; Barry, Jennifer; Tropp, James; Cunningham, Charles H

    2012-02-01

    (13)C MR spectroscopy studies performed on hearts ex vivo and in vivo following perfusion of prepolarized [1-(13)C]pyruvate have shown that changes in pyruvate dehydrogenase (PDH) flux may be monitored non-invasively. However, to allow investigation of Krebs cycle metabolism, the (13)C label must be placed on the C2 position of pyruvate. Thus, the utilization of either C1 or C2 labeled prepolarized pyruvate as a tracer can only afford a partial view of cardiac pyruvate metabolism in health and disease. If the prepolarized pyruvate molecules were labeled at both C1 and C2 positions, then it would be possible to observe the downstream metabolites that were the results of both PDH flux ((13)CO(2) and H(13)CO(3)(-)) and Krebs cycle flux ([5-(13)C]glutamate) with a single dose of the agent. Cardiac pH could also be monitored in the same experiment, but adequate SNR of the (13)CO(2) resonance may be difficult to obtain in vivo. Using an interleaved selective RF pulse acquisition scheme to improve (13)CO(2) detection, the feasibility of using dual-labeled hyperpolarized [1,2-(13)C(2)]pyruvate as a substrate for dynamic cardiac metabolic MRS studies to allow simultaneous investigation of PDH flux, Krebs cycle flux and pH, was demonstrated in vivo. Copyright © 2011 John Wiley & Sons, Ltd.

  16. Novel use of Tat components to increase metabolic flux in light-driven biosynthesis

    DEFF Research Database (Denmark)

    Henriques de Jesus, Maria Perestrello Ramos

    Oxygenic photosynthesis provides plants, algae and cyanobacteria the ability to convert solar energy and CO2 into complex organic molecules. Thus tapping into photosynthesis for synthetic biology efforts has a huge potential for the industrial production of fuels and high value bioactive compounds...... relocating a biosynthetic pathway into the chloroplast, as well as possible solutions. Paper I compares the potential of chloroplasts and cyanobacteria as sustainable biosynthetic compartments and hosts for metabolic engineering. An estimation of the percentage of photosynthetic electrons that can...... be redirected into the biosynthesis of products is given. The availability of photosynthetic reducing power appears to be non-limiting whereas the redirection of the fixed carbon seems to be main challenge for achieving higher product yields. On paper II a novel strategy where the membrane anchors...

  17. Co-factor engineering in lactobacilli: Effects of uncoupled ATPase activity on metabolic fluxes in Lactobacillus (L.) plantarum and L. sakei

    DEFF Research Database (Denmark)

    Rud, Ida; Solem, Christian; Jensen, Peter Ruhdal

    2008-01-01

    The hydrolytic F-1-part of the F1F0-ATPase was over-expressed in Lactobacillus (L.) plantarum NC8 and L. sakei Lb790x during fermentation of glucose or ribose, in order to study how changes in the intracellular levels of ATP and ADP affect the metabolic fluxes. The uncoupled ATPase activity resul...

  18. Novel Strategy for Non-Targeted Isotope-Assisted Metabolomics by Means of Metabolic Turnover and Multivariate Analysis

    Directory of Open Access Journals (Sweden)

    Yasumune Nakayama

    2014-08-01

    Full Text Available Isotope-labeling is a useful technique for understanding cellular metabolism. Recent advances in metabolomics have extended the capability of isotope-assisted studies to reveal global metabolism. For instance, isotope-assisted metabolomics technology has enabled the mapping of a global metabolic network, estimation of flux at branch points of metabolic pathways, and assignment of elemental formulas to unknown metabolites. Furthermore, some data processing tools have been developed to apply these techniques to a non-targeted approach, which plays an important role in revealing unknown or unexpected metabolism. However, data collection and integration strategies for non-targeted isotope-assisted metabolomics have not been established. Therefore, a systematic approach is proposed to elucidate metabolic dynamics without targeting pathways by means of time-resolved isotope tracking, i.e., “metabolic turnover analysis”, as well as multivariate analysis. We applied this approach to study the metabolic dynamics in amino acid perturbation of Saccharomyces cerevisiae. In metabolic turnover analysis, 69 peaks including 35 unidentified peaks were investigated. Multivariate analysis of metabolic turnover successfully detected a pathway known to be inhibited by amino acid perturbation. In addition, our strategy enabled identification of unknown peaks putatively related to the perturbation.

  19. Dissolved organic matter and lake metabolism: Biogeochemistry and controls of nutrient flux dynamics to fresh waters. Technical progress report, January 1, 1990--December 31, 1991

    Energy Technology Data Exchange (ETDEWEB)

    Wetzel, R.G.

    1992-12-31

    The land-water interface region consists of two major components: the wetland, and the down-gradient adjacent littoral floating-leaved and submersed, macrophyte communities. Because of the importance of very high production and nutrient turnover of attached microbiota, a major emphasis of this investigation was placed upon these biota and their metabolic capacities for assimilation and release of organic compounds and nutrient retention and cycling. Examination of the capacities of wetland littoral communities to regulate fluxes of nutrients and organic compounds often has been limited to input-output analyses. These input-output data are an integral part of these investigations, but most of the research effort concentrated on the biotic and metabolic mechanisms that control fluxes and retention capacities and their effects upon biota in the down-gradient waters. The important regulatory capacities of dissolved organic compounds on enzyme reactivity was examined experimentally and coupled to the wetland-littoral organic carbon flux budgets.

  20. Why does yeast ferment? A flux balance analysis study.

    NARCIS (Netherlands)

    Simeonides, E.; Murabito, E.; Smalbone, K.; Westerhoff, H.V.

    2010-01-01

    Advances in biological techniques have led to the availability of genome-scale metabolic reconstructions for yeast. The size and complexity of such networks impose limits on what types of analyses one can perform. Constraint-based modelling overcomes some of these restrictions by using

  1. Reconstruction and in silico analysis of metabolic network for an oleaginous yeast, Yarrowia lipolytica.

    Directory of Open Access Journals (Sweden)

    Pengcheng Pan

    Full Text Available With the emergence of energy scarcity, the use of renewable energy sources such as biodiesel is becoming increasingly necessary. Recently, many researchers have focused their minds on Yarrowia lipolytica, a model oleaginous yeast, which can be employed to accumulate large amounts of lipids that could be further converted to biodiesel. In order to understand the metabolic characteristics of Y. lipolytica at a systems level and to examine the potential for enhanced lipid production, a genome-scale compartmentalized metabolic network was reconstructed based on a combination of genome annotation and the detailed biochemical knowledge from multiple databases such as KEGG, ENZYME and BIGG. The information about protein and reaction associations of all the organisms in KEGG and Expasy-ENZYME database was arranged into an EXCEL file that can then be regarded as a new useful database to generate other reconstructions. The generated model iYL619_PCP accounts for 619 genes, 843 metabolites and 1,142 reactions including 236 transport reactions, 125 exchange reactions and 13 spontaneous reactions. The in silico model successfully predicted the minimal media and the growing abilities on different substrates. With flux balance analysis, single gene knockouts were also simulated to predict the essential genes and partially essential genes. In addition, flux variability analysis was applied to design new mutant strains that will redirect fluxes through the network and may enhance the production of lipid. This genome-scale metabolic model of Y. lipolytica can facilitate system-level metabolic analysis as well as strain development for improving the production of biodiesels and other valuable products by Y. lipolytica and other closely related oleaginous yeasts.

  2. Unraveling the metabolism of HEK-293 cells using lactate isotopomer analysis.

    Science.gov (United States)

    Henry, Olivier; Jolicoeur, Mario; Kamen, Amine

    2011-03-01

    HEK-293 is the most extensively used human cell line for the production of viral vectors and is gaining increasing attention for the production of recombinant proteins by transient transfection. To further improve the metabolic characterization of this cell line, we have performed cultures using ¹³C-labeled substrates and measured the resulting mass isotopomer distributions in lactate by LC/MS. Simultaneous metabolite and isotopomer balancing allowed improvement and validation of the metabolic model and quantification of key intracellular pathways. We have determined the amounts of glucose carbon channeled through the PPP, incorporated into the TCA cycle for energy production and lipids biosynthesis, as well as the cytosolic and mitochondrial malic enzyme fluxes. Our analysis also revealed that glutamine did not significantly contribute to lactate formation. An improved and quantitative understanding of the central carbon metabolism is greatly needed to pursue the rational development of engineering approaches at both the cellular and process levels.

  3. Analysis of the maximum potential proton flux to CNGS

    CERN Document Server

    Meddahi, M

    2007-01-01

    In this note we investigate the limitations to the proton flux which can be sent to the CNGS facility and estimate the maximum that can be attained. In the first part, the injector chain remains unchanged and the limitations are reviewed for operation up to the so called ultimate CNGS intensity, 7x10^13 protons per CNGS cycle. In the second part, the limitations of the SPS accelerator and CNGS facility are described in the scenario of operating with the new injectors - LINAC4, SPL and PS2, as proposed by the PAF working group (PAF).

  4. Computational systems analysis of dopamine metabolism.

    Directory of Open Access Journals (Sweden)

    Zhen Qi

    2008-06-01

    Full Text Available A prominent feature of Parkinson's disease (PD is the loss of dopamine in the striatum, and many therapeutic interventions for the disease are aimed at restoring dopamine signaling. Dopamine signaling includes the synthesis, storage, release, and recycling of dopamine in the presynaptic terminal and activation of pre- and post-synaptic receptors and various downstream signaling cascades. As an aid that might facilitate our understanding of dopamine dynamics in the pathogenesis and treatment in PD, we have begun to merge currently available information and expert knowledge regarding presynaptic dopamine homeostasis into a computational model, following the guidelines of biochemical systems theory. After subjecting our model to mathematical diagnosis and analysis, we made direct comparisons between model predictions and experimental observations and found that the model exhibited a high degree of predictive capacity with respect to genetic and pharmacological changes in gene expression or function. Our results suggest potential approaches to restoring the dopamine imbalance and the associated generation of oxidative stress. While the proposed model of dopamine metabolism is preliminary, future extensions and refinements may eventually serve as an in silico platform for prescreening potential therapeutics, identifying immediate side effects, screening for biomarkers, and assessing the impact of risk factors of the disease.

  5. Continuous and batch cultures of Escherichia coli KJ134 for succinic acid fermentation: metabolic flux distributions and production characteristics.

    Science.gov (United States)

    van Heerden, Carel D; Nicol, Willie

    2013-09-17

    Succinic acid (SA) has become a prominent biobased platform chemical with global production quantities increasing annually. Numerous genetically modified E. coli strains have been developed with the main aim of increasing the SA yield of the organic carbon source. In this study, a promising SA-producing strain, E. coli KJ134 [Biotechnol. Bioeng. 101:881-893, 2008], from the Department of Microbiology and Cell Science of the University of Florida was evaluated under continuous and batch conditions using D-glucose and CO2 in a mineral salt medium. Production characteristics entailing growth and maintenance rates, growth termination points and metabolic flux distributions under growth and non-growth conditions were determined. The culture remained stable for weeks under continuous conditions. Under growth conditions the redox requirements of the reductive tricarboxylic acid (TCA) cycle was solely balanced by acetic acid (AcA) production via the pyruvate dehydrogenase route resulting in a molar ratio of SA:AcA of two. A maximum growth rate of 0.22 h(-1) was obtained, while complete growth inhibition occurred at a SA concentration of 18 g L(-1). Batch culture revealed that high-yield succinate production (via oxidative TCA or glyoxylate redox balancing) occurred under non-growth conditions where a SA:AcA molar ratio of up to five was attained, with a final SA yield of 0.94 g g(-1). Growth termination of the batch culture was in agreement with that of the continuous culture. The maximum maintenance production rate of SA under batch conditions was found to be 0.6 g g(-1) h(-1). This is twice the maintenance rate observed in the continuous runs. The study revealed that the metabolic flux of E. coli KJ134 differs significantly for growth and non-growth conditions, with non-growth conditions resulting in higher SA:AcA ratios and SA yields. Bioreaction characteristics entailing growth and maintenance rates, as well as growth termination markers will guide future fermentor

  6. Global Bedload Flux Modeling and Analysis in Large Rivers

    Science.gov (United States)

    Islam, M. T.; Cohen, S.; Syvitski, J. P.

    2017-12-01

    Proper sediment transport quantification has long been an area of interest for both scientists and engineers in the fields of geomorphology, and management of rivers and coastal waters. Bedload flux is important for monitoring water quality and for sustainable development of coastal and marine bioservices. Bedload measurements, especially for large rivers, is extremely scarce across time, and many rivers have never been monitored. Bedload measurements in rivers, is particularly acute in developing countries where changes in sediment yields is high. The paucity of bedload measurements is the result of 1) the nature of the problem (large spatial and temporal uncertainties), and 2) field costs including the time-consuming nature of the measurement procedures (repeated bedform migration tracking, bedload samplers). Here we present a first of its kind methodology for calculating bedload in large global rivers (basins are >1,000 km. Evaluation of model skill is based on 113 bedload measurements. The model predictions are compared with an empirical model developed from the observational dataset in an attempt to evaluate the differences between a physically-based numerical model and a lumped relationship between bedload flux and fluvial and basin parameters (e.g., discharge, drainage area, lithology). The initial study success opens up various applications to global fluvial geomorphology (e.g. including the relationship between suspended sediment (wash load) and bedload). Simulated results with known uncertainties offers a new research product as a valuable resource for the whole scientific community.

  7. Measuring and modeling C flux rates through the central metabolic pathways in microbial communities using position-specific 13C-labeled tracers

    Science.gov (United States)

    Dijkstra, P.; van Groenigen, K.; Hagerty, S.; Salpas, E.; Fairbanks, D. E.; Hungate, B. A.; KOCH, G. W.; Schwartz, E.

    2012-12-01

    The production of energy and metabolic precursors occurs in well-known processes such as glycolysis and Krebs cycle. We use position-specific 13C-labeled metabolic tracers, combined with models of microbial metabolic organization, to analyze the response of microbial community energy production, biosynthesis, and C use efficiency (CUE) in soils, decomposing litter, and aquatic communities. The method consists of adding position-specific 13C -labeled metabolic tracers to parallel soil incubations, in this case 1-13C and 2,3-13C pyruvate and 1-13C and U-13C glucose. The measurement of CO2 released from the labeled tracers is used to calculate the C flux rates through the various metabolic pathways. A simplified metabolic model consisting of 23 reactions is solved using results of the metabolic tracer experiments and assumptions of microbial precursor demand. This new method enables direct estimation of fundamental aspects of microbial energy production, CUE, and soil organic matter formation in relatively undisturbed microbial communities. We will present results showing the range of metabolic patterns observed in these communities and discuss results from testing metabolic models.

  8. IDENTIFICATION AND ANALYSIS OF BACTERIAL GENOMIC METABOLIC SIGNATURES.

    Science.gov (United States)

    Bowerman, Nathaniel; Tintle, Nathan; Dejongh, Matthew; Best, Aaron A

    2017-01-01

    With continued rapid growth in the number and quality of fully sequenced and accurately annotated bacterial genomes, we have unprecedented opportunities to understand metabolic diversity. We selected 101 diverse and representative completely sequenced bacteria and implemented a manual curation effort to identify 846 unique metabolic variants present in these bacteria. The presence or absence of these variants act as a metabolic signature for each of the bacteria, which can then be used to understand similarities and differences between and across bacterial groups. We propose a novel and robust method of summarizing metabolic diversity using metabolic signatures and use this method to generate a metabolic tree, clustering metabolically similar organisms. Resulting analysis of the metabolic tree confirms strong associations with well-established biological results along with direct insight into particular metabolic variants which are most predictive of metabolic diversity. The positive results of this manual curation effort and novel method development suggest that future work is needed to further expand the set of bacteria to which this approach is applied and use the resulting tree to test broad questions about metabolic diversity and complexity across the bacterial tree of life.

  9. The reconstruction and analysis of tissue specific human metabolic networks.

    Science.gov (United States)

    Hao, Tong; Ma, Hong-Wu; Zhao, Xue-Ming; Goryanin, Igor

    2012-02-01

    Human tissues have distinct biological functions. Many proteins/enzymes are known to be expressed only in specific tissues and therefore the metabolic networks in various tissues are different. Though high quality global human metabolic networks and metabolic networks for certain tissues such as liver have already been studied, a systematic study of tissue specific metabolic networks for all main tissues is still missing. In this work, we reconstruct the tissue specific metabolic networks for 15 main tissues in human based on the previously reconstructed Edinburgh Human Metabolic Network (EHMN). The tissue information is firstly obtained for enzymes from Human Protein Reference Database (HPRD) and UniprotKB databases and transfers to reactions through the enzyme-reaction relationships in EHMN. As our knowledge of tissue distribution of proteins is still very limited, we replenish the tissue information of the metabolic network based on network connectivity analysis and thorough examination of the literature. Finally, about 80% of proteins and reactions in EHMN are determined to be in at least one of the 15 tissues. To validate the quality of the tissue specific network, the brain specific metabolic network is taken as an example for functional module analysis and the results reveal that the function of the brain metabolic network is closely related with its function as the centre of the human nervous system. The tissue specific human metabolic networks are available at .

  10. Determination of metabolic fluxes during glucose catabolism in Propionibacterium freudenreichii subsp. shermanii.

    Science.gov (United States)

    Meurice, G; Bondon, A; Deborde, C; Boyaval, P

    2001-01-01

    In vivo 13C Nuclear Magnetic Resonance (NMR) spectroscopy was used to investigate the pathways of glucose metabolism, non-invasively, in living cell suspensions of Propionibacterium freudenreichii subsp. shermanii. This species is the main ripening flora of the Swiss-type cheeses and is widely used as propionic acid and vitamin B12 industrial producer. The flow of labelled [1-13C]glucose was monitored in living cell suspensions and enrichment was detected in main products like [1-13C]glycogen, [6-13C]lycogen, [1-13C]trehalose, [6-13C]trehalose, [1-13C]propionate, [2-13C]propionate, [3-13C]propionate, [1-13C]acetate, [2-13C]acetate, [1-13C]succinate, [2-13C]succinate and [1-13C]CO2. alpha and beta glucose consumption could be examined separately and were catabolized at the same rate. Three intermediates were also found out, namely [1-13C]glucose-6-phosphate, [6-13C]glucose-6-phosphate and [1-13C]glucose-1-phosphate. From the formation of intermediates such as [6-3C]glucose-6-phosphate and products like [6-13C]glycogen from [1-13C]glucose we concluded the bidirectionality of reactions in the first part of glycolysis and the isomerization at the triose-phosphate level. Comparison of spectra obtained after addition of [1-13C]glucose or [U-12C]glucose revealed production of [1-13C]CO2 which means that pentose phosphates pathway is active under our experimental conditions. From the isotopic pattern of trehalose, it could be postulated that trehalose biosynthesis occurred either by direct condensation of two glucose molecules or by gluconeogenesis. A chemically defined medium was elaborated for the study and trehalose was the main osmolyte found in the intracellular fraction of P. shermanii grown in this medium.

  11. Metabolic flux in carbohydrate biosynthesis. New methods using stable isotopes, mass spectrometry, and NMR

    Science.gov (United States)

    Structural analysis of carbohydrates involves three parameters: composition, linkage, and conformation, and tends to rely on the various forms of two techniques; mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. These techniques are enhanced and extended by the use of stable...

  12. Modeling and analysis of flux distributions in the two branches of the phosphotransferase system in Pseudomonas putida

    Directory of Open Access Journals (Sweden)

    Kremling Andreas

    2012-12-01

    Full Text Available Abstract Background Signal transduction plays a fundamental role in the understanding of cellular physiology. The bacterial phosphotransferase system (PTS together with the PEP/pyruvate node in central metabolism represents a signaling unit that acts as a sensory element and measures the activity of the central metabolism. Pseudomonas putida possesses two PTS branches, the C-branch (PTSFru and a second branch (PTSNtr, which communicate with each other by phosphate exchange. Recent experimental results showed a cross talk between the two branches. However, the functional role of the crosstalk remains open. Results A mathematical model was set up to describe the available data of the state of phosphorylation of PtsN, one of the PTS proteins, for different environmental conditions and different strain variants. Additionally, data from flux balance analysis was used to determine some of the kinetic parameters of the involved reactions. Based on the calculated and estimated parameters, the flux distribution during growth of the wild type strain on fructose could be determined. Conclusion Our calculations show that during growth of the wild type strain on the PTS substrate fructose, the major part of the phosphoryl groups is provided by the second branch of the PTS. This theoretical finding indicates a new role of the second branch of the PTS and will serve as a basis for further experimental studies.

  13. Hierarchical analysis of dependency in metabolic networks.

    Science.gov (United States)

    Gagneur, Julien; Jackson, David B; Casari, Georg

    2003-05-22

    Elucidation of metabolic networks for an increasing number of organisms reveals that even small networks can contain thousands of reactions and chemical species. The intimate connectivity between components complicates their decomposition into biologically meaningful sub-networks. Moreover, traditional higher-order representations of metabolic networks as metabolic pathways, suffers from the lack of rigorous definition, yielding pathways of disparate content and size. We introduce a hierarchical representation that emphasizes the gross organization of metabolic networks in largely independent pathways and sub-systems at several levels of independence. The approach highlights the coupling of different pathways and the shared compounds responsible for those couplings. By assessing our results on Escherichia coli (E.coli metabolic reactions, Genetic Circuits Research Group, University of California, San Diego, http://gcrg.ucsd.edu/organisms/ecoli.html, 'model v 1.01. reactions') against accepted biochemical annotations, we provide the first systematic synopsis of an organism's metabolism. Comparison with operons of E.coli shows that low-level clusters are reflected in genome organization and gene regulation. Source code, data sets and supplementary information are available at http://www.mas.ecp.fr/labo/equipe/gagneur/hierarchy/hierarchy.html

  14. Validation of the doubly-labeled water (H3H18O) method for measuring water flux and energy metabolism in tenebrionid beetles

    International Nuclear Information System (INIS)

    Cooper, P.D.

    1981-01-01

    Doubly-labeled water (H 3 H 18 O) has been used to determine water flux and energy metabolism in a variety of vertebrates. This study examines the applicability of this technique to arthropods. The theory of the technique depends upon the assumption that doubly-labeled water introduced into the animal's body water equilibrates with water and carbon dioxide by the action of carbonic anhydrase. Tritium ( 3 H) is lost from the animal only with water while oxygen-18 is lost with both water and carbon dioxide. The difference bwtween the rates of loss of the two isotopes is proportional to CO 2 loss rate. Validation of the use of tritiated water for measuring water flux was accomplished by comparing gravimetric measurements of water gain with flux rates determined by loss of tritiated water. At room humidity, an overestimate for influx calculated from labeled water calculations was found, averaging 12 mg H 2 O (g.d) -1 . Comparison of CO 2 loss rate determined isotopically with rates of CO 2 loss determined by standard metabolic rates also yielded overestimates for the isotopic technique, overestimates ranging between 20 and 30%. The relevance of this for studies using labeled water for studying water fluxes and free metabolism of free-ranging arthropods is discussed

  15. Energy Conversion Loops for Flux-Switching PM Machine Analysis

    Directory of Open Access Journals (Sweden)

    E. Ilhan

    2012-10-01

    Full Text Available Induction and synchronous machines have traditionally been the first choice of automotive manufacturers for electric/hybrid vehicles. However, these conventional machines are not able anymore to meet the increasing demands for a higher energy density due to space limitation in cars. Flux-switching PM (FSPM machines with their high energy density are very suitable to answer this demand. In this paper, the energy conversion loop technique is implemented on FSPM for the first time. The energy conversion technique is a powerful tool for the visualization of machine characteristics, both linear and nonlinear. Further, the technique provides insight into the torque production mechanism. A stepwise explanation is given on how to create these loops for FSPM along with the machine operation.

  16. Emergency diesel generator reliability analysis high flux isotope reactor

    International Nuclear Information System (INIS)

    Merryman, L.; Christie, B.

    1993-01-01

    A program to apply some of the techniques of reliability engineering to the High Flux Isotope Reactor (HFIR) was started on August 8, 1992. Part of the program was to track the conditional probabilities of the emergency diesel generators responding to a valid demand. This was done to determine if the performance of the emergency diesel generators (which are more than 25 years old) has deteriorated. The conditional probabilities of the diesel generators were computed and trended for the period from May 1990 to December 1992. The calculations indicate that the performance of the emergency diesel generators has not deteriorated in recent years, i.e., the conditional probabilities of the emergency diesel generators have been fairly stable over the last few years. This information will be one factor than may be considered in the decision to replace the emergency diesel generators

  17. A case study of eddy covariance flux of N2O measured within forest ecosystems: quality control and flux error analysis

    Directory of Open Access Journals (Sweden)

    T. Markkanen

    2010-02-01

    Full Text Available Eddy covariance (EC flux measurements of nitrous oxide (N2O obtained by using a 3-D sonic anemometer and a tunable diode laser gas analyzer for N2O were investigated. Two datasets (Sorø, Denmark and Kalevansuo, Finland from different measurement campaigns including sub-canopy flux measurements of energy and carbon dioxide are discussed with a focus on selected quality control aspects and flux error analysis. Although fast response trace gas analyzers based on spectroscopic techniques are increasingly used in ecosystem research, their suitability for reliable estimates of EC fluxes is still limited, and some assumptions have to be made for filtering and processing data. The N2O concentration signal was frequently dominated by offset drifts (fringe effect, which can give an artificial extra contribution to the fluxes when the resulting concentration fluctuations are correlated with the fluctuations of the vertical wind velocity. Based on Allan variance analysis of the N2O signal, we found that a recursive running mean filter with a time constant equal to 50 s was suitable to damp the influence of the periodic drift. Although the net N2O fluxes over the whole campaign periods were quite small at both sites (~5 μg N m−2 h−1 for Kalevansuo and ~10 μg N m−2 h−1 for Sorø, the calculated sub-canopy EC fluxes were in good agreement with those estimated by automatic soil chambers. However, EC N2O flux measurements show larger random uncertainty than the sensible heat fluxes, and classification according to statistical significance of single flux values indicates that downward N2O fluxes have larger random error.

  18. Gill lipid metabolism and unidirectional Na+ flux in the European eel (Anguilla anguilla) after transfer to dilute media

    DEFF Research Database (Denmark)

    Hansen, H.J.M.; Grosell, M.; Rosenkilde, P.

    1999-01-01

    positive linear correlation of percentage (14C) wax alcohols with log [22Na efflux]. Based on the observed parallel between Naf flux and gill lipid metabolism, it is suggested that the eel reacts at first to a loss of Na+ by synthesizing wax alcohols that can tighten the gill membrane. (C) 1999 Elsevier......+ uptake rate was 12 mu mol kg(-1) h(-1), i.e., the general picture in DW was a net Na+ loss. In another similar experiment, groups of three FW-adapted eels were incubated in vivo for up to 24 h with (C-14) acetate added as lipid precursor to the ambient water. Incubation in FW showed about 20......% of the total C-14-activity incorporated into gill lipids as (C-14) wax alcohols (WA; octadecanol and eicosanol). This percentage went up to 50% shortly after transfer to DW and came down again to about 20% after 2 weeks in DW. Single eels labelled with Na-22 in the plasma showed a statistically significant...

  19. XRD and DTA Analysis of Developed Agglomerated Fluxes for Submerged Arc Welding

    Directory of Open Access Journals (Sweden)

    Ajay Kumar

    2013-01-01

    Full Text Available A unique study of structural and chemical analysis of crystalline phases in developed agglomerated fluxes was carried out. Thirty-two fluxes were developed by using a mixture of oxides, halides, carbonates, silicates, and ferroalloys for submerged arc welding. The present paper focuses on only ten (out of thirty-two fluxes which were analyzed by X-ray diffraction (XRD to know the different types of oxides formed and changed in oxidation number of metallic centers after sintering process at around 850∘C. To know the effect of temperature over phase transformation and melting of different compounds, differential thermal analysis (DTA was carried out from 1000 to 1400∘C. This study aims to know the quantity of ions present (percentage and melting behavior of developed agglomerated fluxes for submerged arc welding process.

  20. Genome-scale reconstruction and analysis of the Pseudomonas putida KT2440 metabolic network facilitates applications in biotechnology.

    Science.gov (United States)

    Puchałka, Jacek; Oberhardt, Matthew A; Godinho, Miguel; Bielecka, Agata; Regenhardt, Daniela; Timmis, Kenneth N; Papin, Jason A; Martins dos Santos, Vítor A P

    2008-10-01

    A cornerstone of biotechnology is the use of microorganisms for the efficient production of chemicals and the elimination of harmful waste. Pseudomonas putida is an archetype of such microbes due to its metabolic versatility, stress resistance, amenability to genetic modifications, and vast potential for environmental and industrial applications. To address both the elucidation of the metabolic wiring in P. putida and its uses in biocatalysis, in particular for the production of non-growth-related biochemicals, we developed and present here a genome-scale constraint-based model of the metabolism of P. putida KT2440. Network reconstruction and flux balance analysis (FBA) enabled definition of the structure of the metabolic network, identification of knowledge gaps, and pin-pointing of essential metabolic functions, facilitating thereby the refinement of gene annotations. FBA and flux variability analysis were used to analyze the properties, potential, and limits of the model. These analyses allowed identification, under various conditions, of key features of metabolism such as growth yield, resource distribution, network robustness, and gene essentiality. The model was validated with data from continuous cell cultures, high-throughput phenotyping data, (13)C-measurement of internal flux distributions, and specifically generated knock-out mutants. Auxotrophy was correctly predicted in 75% of the cases. These systematic analyses revealed that the metabolic network structure is the main factor determining the accuracy of predictions, whereas biomass composition has negligible influence. Finally, we drew on the model to devise metabolic engineering strategies to improve production of polyhydroxyalkanoates, a class of biotechnologically useful compounds whose synthesis is not coupled to cell survival. The solidly validated model yields valuable insights into genotype-phenotype relationships and provides a sound framework to explore this versatile bacterium and to

  1. Genome-scale reconstruction and analysis of the Pseudomonas putida KT2440 metabolic network facilitates applications in biotechnology.

    Directory of Open Access Journals (Sweden)

    Jacek Puchałka

    2008-10-01

    Full Text Available A cornerstone of biotechnology is the use of microorganisms for the efficient production of chemicals and the elimination of harmful waste. Pseudomonas putida is an archetype of such microbes due to its metabolic versatility, stress resistance, amenability to genetic modifications, and vast potential for environmental and industrial applications. To address both the elucidation of the metabolic wiring in P. putida and its uses in biocatalysis, in particular for the production of non-growth-related biochemicals, we developed and present here a genome-scale constraint-based model of the metabolism of P. putida KT2440. Network reconstruction and flux balance analysis (FBA enabled definition of the structure of the metabolic network, identification of knowledge gaps, and pin-pointing of essential metabolic functions, facilitating thereby the refinement of gene annotations. FBA and flux variability analysis were used to analyze the properties, potential, and limits of the model. These analyses allowed identification, under various conditions, of key features of metabolism such as growth yield, resource distribution, network robustness, and gene essentiality. The model was validated with data from continuous cell cultures, high-throughput phenotyping data, (13C-measurement of internal flux distributions, and specifically generated knock-out mutants. Auxotrophy was correctly predicted in 75% of the cases. These systematic analyses revealed that the metabolic network structure is the main factor determining the accuracy of predictions, whereas biomass composition has negligible influence. Finally, we drew on the model to devise metabolic engineering strategies to improve production of polyhydroxyalkanoates, a class of biotechnologically useful compounds whose synthesis is not coupled to cell survival. The solidly validated model yields valuable insights into genotype-phenotype relationships and provides a sound framework to explore this versatile

  2. Use of Principal Components Analysis to Explain Controls on Nutrient Fluxes to the Chesapeake Bay

    Science.gov (United States)

    Rice, K. C.; Mills, A. L.

    2017-12-01

    The Chesapeake Bay watershed, on the east coast of the United States, encompasses about 166,000-square kilometers (km2) of diverse land use, which includes a mixture of forested, agricultural, and developed land. The watershed is now managed under a Total Daily Maximum Load (TMDL), which requires implementation of management actions by 2025 that are sufficient to reduce nitrogen, phosphorus, and suspended-sediment fluxes to the Chesapeake Bay and restore the bay's water quality. We analyzed nutrient and sediment data along with land-use and climatic variables in nine sub watersheds to better understand the drivers of flux within the watershed and to provide relevant management implications. The nine sub watersheds range in area from 300 to 30,000 km2, and the analysis period was 1985-2014. The 31 variables specific to each sub watershed were highly statistically significantly correlated, so Principal Components Analysis was used to reduce the dimensionality of the dataset. The analysis revealed that about 80% of the variability in the whole dataset can be explained by discharge, flux, and concentration of nutrients and sediment. The first two principal components (PCs) explained about 68% of the total variance. PC1 loaded strongly on discharge and flux, and PC2 loaded on concentration. The PC scores of both PC1 and PC2 varied by season. Subsequent analysis of PC1 scores versus PC2 scores, broken out by sub watershed, revealed management implications. Some of the largest sub watersheds are largely driven by discharge, and consequently large fluxes. In contrast, some of the smaller sub watersheds are more variable in nutrient concentrations than discharge and flux. Our results suggest that, given no change in discharge, a reduction in nutrient flux to the streams in the smaller watersheds could result in a proportionately larger decrease in fluxes of nutrients down the river to the bay, than in the larger watersheds.

  3. Bayesian statistics applied to neutron activation data for reactor flux spectrum analysis

    International Nuclear Information System (INIS)

    Chiesa, Davide; Previtali, Ezio; Sisti, Monica

    2014-01-01

    Highlights: • Bayesian statistics to analyze the neutron flux spectrum from activation data. • Rigorous statistical approach for accurate evaluation of the neutron flux groups. • Cross section and activation data uncertainties included for the problem solution. • Flexible methodology applied to analyze different nuclear reactor flux spectra. • The results are in good agreement with the MCNP simulations of neutron fluxes. - Abstract: In this paper, we present a statistical method, based on Bayesian statistics, to analyze the neutron flux spectrum from the activation data of different isotopes. The experimental data were acquired during a neutron activation experiment performed at the TRIGA Mark II reactor of Pavia University (Italy) in four irradiation positions characterized by different neutron spectra. In order to evaluate the neutron flux spectrum, subdivided in energy groups, a system of linear equations, containing the group effective cross sections and the activation rate data, has to be solved. However, since the system’s coefficients are experimental data affected by uncertainties, a rigorous statistical approach is fundamental for an accurate evaluation of the neutron flux groups. For this purpose, we applied the Bayesian statistical analysis, that allows to include the uncertainties of the coefficients and the a priori information about the neutron flux. A program for the analysis of Bayesian hierarchical models, based on Markov Chain Monte Carlo (MCMC) simulations, was used to define the problem statistical model and solve it. The first analysis involved the determination of the thermal, resonance-intermediate and fast flux components and the dependence of the results on the Prior distribution choice was investigated to confirm the reliability of the Bayesian analysis. After that, the main resonances of the activation cross sections were analyzed to implement multi-group models with finer energy subdivisions that would allow to determine the

  4. Metabolic control analysis of biochemical pathways based on a thermokinetic description of reaction rates

    DEFF Research Database (Denmark)

    Nielsen, Jens Bredal

    1997-01-01

    of the thermokinetic description of reaction rates to include the influence of effecters. Here the reaction rate is written as a linear function of the logarithm of the metabolite concentrations. With this type of rate function it is shown that the approach of Delgado and Liao [Biochem. J. (1992) 282, 919-927] can......Metabolic control analysis is a powerful technique for the evaluation of flux control within biochemical pathways. Its foundation is the elasticity coefficients and the flux control coefficients (FCCs). On the basis of a thermokinetic description of reaction rates it is here shown...... that the elasticity coefficients can be calculated directly from the pool levels of metabolites at steady state. The only requirement is that one thermodynamic parameter be known, namely the reaction affinity at the intercept of the tangent in the inflection point of the curve of reaction rate against reaction...

  5. Metabolic control analysis of biochemical pathways based on a thermokinetic description of reaction rates

    DEFF Research Database (Denmark)

    Nielsen, Jens Bredal

    1997-01-01

    Metabolic control analysis is a powerful technique for the evaluation of flux control within biochemical pathways. Its foundation is the elasticity coefficients and the flux control coefficients (FCCs). On the basis of a thermokinetic description of reaction rates it is here shown...... that the elasticity coefficients can be calculated directly from the pool levels of metabolites at steady state. The only requirement is that one thermodynamic parameter be known, namely the reaction affinity at the intercept of the tangent in the inflection point of the curve of reaction rate against reaction...... of the thermokinetic description of reaction rates to include the influence of effecters. Here the reaction rate is written as a linear function of the logarithm of the metabolite concentrations. With this type of rate function it is shown that the approach of Delgado and Liao [Biochem. J. (1992) 282, 919-927] can...

  6. Effects of experimental nitrogen fertilization on planktonic metabolism and CO2 flux in a hypereutrophic hardwater lake.

    Directory of Open Access Journals (Sweden)

    Matthew J Bogard

    Full Text Available Hardwater lakes are common in human-dominated regions of the world and often experience pollution due to agricultural and urban effluent inputs of inorganic and organic nitrogen (N. Although these lakes are landscape hotspots for CO2 exchange and food web carbon (C cycling, the effect of N enrichment on hardwater lake food web functioning and C cycling patterns remains unclear. Specifically, it is unknown if different eutrophication scenarios (e.g., modest non point vs. extreme point sources yield consistent effects on auto- and heterotrophic C cycling, or how biotic responses interact with the inorganic C system to shape responses of air-water CO2 exchange. To address this uncertainty, we induced large metabolic gradients in the plankton community of a hypereutrophic hardwater Canadian prairie lake by adding N as urea (the most widely applied agricultural fertilizer at loading rates of 0, 1, 3, 8 or 18 mg N L-1 week-1 to 3240-L, in-situ mesocosms. Over three separate 21-day experiments, all treatments of N dramatically increased phytoplankton biomass and gross primary production (GPP two- to six-fold, but the effects of N on autotrophs plateaued at ~3 mg N L-1. Conversely, heterotrophic metabolism increased linearly with N fertilization over the full treatment range. In nearly all cases, N enhanced net planktonic uptake of dissolved inorganic carbon (DIC, and increased the rate of CO2 influx, while planktonic heterotrophy and CO2 production only occurred in the highest N treatments late in each experiment, and even in these cases, enclosures continued to in-gas CO2. Chemical effects on CO2 through calcite precipitation were also observed, but similarly did not change the direction of net CO2 flux. Taken together, these results demonstrate that atmospheric exchange of CO2 in eutrophic hardwater lakes remains sensitive to increasing N loading and eutrophication, and that even modest levels of N pollution are capable of enhancing autotrophy and CO

  7. Flux control exerted by mitochondrial outer membrane carnitine palmitoyltransferase over beta-oxidation, ketogenesis and tricarboxylic acid cycle activity in hepatocytes isolated from rats in different metabolic states.

    OpenAIRE

    Drynan, L; Quant, P A; Zammit, V A

    1996-01-01

    The Flux Control Coefficients of mitochondrial outer membrane carnitine palmitoyltransferase (CPT I) with respect to the overall rates of beta-oxidation, ketogenesis and tricarboxylic acid cycle activity were measured in hepatocytes isolated from rats in different metabolic states (fed, 24 h-starved, starved-refed and starved/insulin-treated). These conditions were chosen because there is controversy as to whether, when significant control ceases to be exerted by CPT I over the rate of fatty ...

  8. Effects of ocean acidification and hydrodynamic conditions on carbon metabolism and dissolved organic carbon (DOC) fluxes in seagrass populations.

    Science.gov (United States)

    Egea, Luis G; Jiménez-Ramos, Rocío; Hernández, Ignacio; Bouma, Tjeerd J; Brun, Fernando G

    2018-01-01

    Global change has been acknowledged as one of the main threats to the biosphere and its provision of ecosystem services, especially in marine ecosystems. Seagrasses play a critical ecological role in coastal ecosystems, but their responses to ocean acidification (OA) and climate change are not well understood. There have been previous studies focused on the effects of OA, but the outcome of interactions with co-factors predicted to alter during climate change still needs to be addressed. For example, the impact of higher CO2 and different hydrodynamic regimes on seagrass performance remains unknown. We studied the effects of OA under different current velocities on productivity of the seagrass Zostera noltei, using changes in dissolved oxygen as a proxy for the seagrass carbon metabolism, and release of dissolved organic carbon (DOC) in a four-week experiment using an open-water outdoor mesocosm. Under current pH conditions, increasing current velocity had a positive effect on productivity, but this depended on shoot density. However, this positive effect of current velocity disappeared under OA conditions. OA conditions led to a significant increase in gross production rate and respiration, suggesting that Z. noltei is carbon-limited under the current inorganic carbon concentration of seawater. In addition, an increase in non-structural carbohydrates was found, which may lead to better growing conditions and higher resilience in seagrasses subjected to environmental stress. Regarding DOC flux, a direct and positive relationship was found between current velocity and DOC release, both under current pH and OA conditions. We conclude that OA and high current velocity may lead to favourable growth scenarios for Z. noltei populations, increasing their productivity, non-structural carbohydrate concentrations and DOC release. Our results add new dimensions to predictions on how seagrass ecosystems will respond to climate change, with important implications for the

  9. Effects of ocean acidification and hydrodynamic conditions on carbon metabolism and dissolved organic carbon (DOC fluxes in seagrass populations.

    Directory of Open Access Journals (Sweden)

    Luis G Egea

    Full Text Available Global change has been acknowledged as one of the main threats to the biosphere and its provision of ecosystem services, especially in marine ecosystems. Seagrasses play a critical ecological role in coastal ecosystems, but their responses to ocean acidification (OA and climate change are not well understood. There have been previous studies focused on the effects of OA, but the outcome of interactions with co-factors predicted to alter during climate change still needs to be addressed. For example, the impact of higher CO2 and different hydrodynamic regimes on seagrass performance remains unknown. We studied the effects of OA under different current velocities on productivity of the seagrass Zostera noltei, using changes in dissolved oxygen as a proxy for the seagrass carbon metabolism, and release of dissolved organic carbon (DOC in a four-week experiment using an open-water outdoor mesocosm. Under current pH conditions, increasing current velocity had a positive effect on productivity, but this depended on shoot density. However, this positive effect of current velocity disappeared under OA conditions. OA conditions led to a significant increase in gross production rate and respiration, suggesting that Z. noltei is carbon-limited under the current inorganic carbon concentration of seawater. In addition, an increase in non-structural carbohydrates was found, which may lead to better growing conditions and higher resilience in seagrasses subjected to environmental stress. Regarding DOC flux, a direct and positive relationship was found between current velocity and DOC release, both under current pH and OA conditions. We conclude that OA and high current velocity may lead to favourable growth scenarios for Z. noltei populations, increasing their productivity, non-structural carbohydrate concentrations and DOC release. Our results add new dimensions to predictions on how seagrass ecosystems will respond to climate change, with important

  10. Uncertainty analysis of RFSP-IST code flux calculation after cobalt adjuster rod replacement

    International Nuclear Information System (INIS)

    Tang Chuntao; Yang Bo; Bi Guangwen; Wang Jun

    2014-01-01

    In physical analysis of the cobalt adjuster rods design change for Qinshan Phase Ⅲ, the incremental cross section used in RFSP-IST code for the cobalt adjuster rod was calculated by DRAGON, whose methodology was not exactly the same as the MULTICELL used for Qinshan Phase Ⅲ RFSAR (2007), hence it was necessary to do the uncertainty analysis for RFSP-IST code flux calculation after cobalt adjuster rod replacement. Based on the historical data from Unit 1 and Unit 2 of Qinshan Phase Ⅲ, the uncertainty analysis of RFSP-IST code flux calculation was done with 95/95 upper tolerance limit method. Numerical results show that the cobalt adjuster rod design change and different incremental cross section calculation codes do not impact the uncertainty of RFSP-IST code flux calculation. (authors)

  11. BioMet Toolbox: genome-wide analysis of metabolism

    DEFF Research Database (Denmark)

    Cvijovic, M.; Olivares Hernandez, Roberto; Agren, R.

    2010-01-01

    standardized simulations. Model files for various model organisms (fungi and bacteria) are included. Overall, the BioMet Toolbox serves as a valuable resource for exploring the capabilities of these metabolic networks. BioMet Toolbox is freely available at www.sysbio.se/BioMet/....... models. Systematic analysis of biological processes by means of modelling and simulations has made the identification of metabolic networks and prediction of metabolic capabilities under different conditions possible. For facilitating such systemic analysis, we have developed the BioMet Toolbox, a web......-based resource for stoichiometric analysis and for integration of transcriptome and interactome data, thereby exploiting the capabilities of genome-scale metabolic models. The BioMet Toolbox provides an effective user-friendly way to perform linear programming simulations towards maximized or minimized growth...

  12. Applicability of TASS/SMR using drift flux model for SMART LOCA analysis

    International Nuclear Information System (INIS)

    Chung, Young-Jong; Kim, Soo Hyung; Lee, Gyu Hyeung; Lee, Won Jae

    2013-01-01

    Highlights: • SMART plant and TASS/SMR code have been developed by KAERI. • TASS/SMR code adopts a drift flux model to consider relative velocity under two-phase condition. • Drift flux model in TASS/SMR is validated using separate effect test results. • Applicability of TASS/SMR using drift flux model for SMART LOCA analysis is confirmed. -- Abstract: Small reactors can apply to local power demands or remote areas. SMART, which can produce 90 MWe of electricity and 40,000 tons/day of sea-water desalination for a 100,000 population city, is a promising advanced integral type small reactor. The thermal hydraulic analysis code with a drift flux model, TASS/SMR, was developed for a conservative simulation of a small break loss of coolant accident in SMART. Taking into account SMART-specific inherent characteristics, the code adopts the Chexal–Lellouche correlation for a drift flux model. The capability of TASS/SMR code is validated using the results of the experimental data. The code predicts conservatively the void distribution compared with the experimental data. TASS/SMR calculation predicts reasonably major phenomena for the SBLOCA and is more conservative than or nearly the same as the results of the best estimated realistic system code, MARS. TASS/SMR code can be used for a SBLOCA analysis of SMART

  13. [Spectra and thermal analysis of the arc in activating flux plasma arc welding].

    Science.gov (United States)

    Chai, Guo-Ming; Zhu, Yi-Feng

    2010-04-01

    In activating flux plasma arc welding the welding arc was analyzed by spectra analysis technique, and the welding arc temperature field was measured by the infrared sensing and computer image technique. The distribution models of welding arc heat flow density of activating flux PAW welding were developed. The composition of welding arc affected by activated flux was studied, and the welding arc temperature field was studied. The results show that the spectral lines of argon atom and ionized argon atom of primary ionization are the main spectra lines of the conventional plasma welding arc. The spectra lines of weld metal are inappreciable in the spectra lines of the conventional plasma welding arc. The gas particle is the main in the conventional plasma welding arc. The conventional plasma welding arc is gas welding arc. The spectra lines of argon atom and ionized argon atom of primary ionization are intensified in the activating flux plasma welding arc, and the spectra lines of Ti, Cr and Fe elements are found in the activating flux plasma welding arc. The welding arc temperature distribution in activating flux plasma arc welding is compact, the outline of the welding arc temperature field is narrow, the range of the welding arc temperature distribution is concentrated, the welding arc radial temperature gradient is large, and the welding arc radial temperature gradient shows normal Gauss distribution.

  14. Fast flux test facility final safety analysis report amendment 79

    International Nuclear Information System (INIS)

    Dautel, W.A.

    1999-01-01

    This document is provided to replace, remove, or add applicable pages to the chapters on: Heat Transport System; Containment and Structures; Auxiliary Systems; Reactor Refueling System; Conduct of Operations; Safety Analysis; Quality Assurance; FFTF Criticality Specifications; and Appendix H's TRIGA Fuel Storage System

  15. Application of the Principles of Systems Biology and Wiener's Cybernetics for Analysis of Regulation of Energy Fluxes in Muscle Cells in Vivo

    Directory of Open Access Journals (Sweden)

    Rita Guzun

    2010-03-01

    Full Text Available The mechanisms of regulation of respiration and energy fluxes in the cells are analyzed based on the concepts of systems biology, non-equilibrium steady state kinetics and applications of Wiener’s cybernetic principles of feedback regulation. Under physiological conditions cardiac function is governed by the Frank-Starling law and the main metabolic characteristic of cardiac muscle cells is metabolic homeostasis, when both workload and respiration rate can be changed manifold at constant intracellular level of phosphocreatine and ATP in the cells. This is not observed in skeletal muscles. Controversies in theoretical explanations of these observations are analyzed. Experimental studies of permeabilized fibers from human skeletal muscle vastus lateralis and adult rat cardiomyocytes showed that the respiration rate is always an apparent hyperbolic but not a sigmoid function of ADP concentration. It is our conclusion that realistic explanations of regulation of energy fluxes in muscle cells require systemic approaches including application of the feedback theory of Wiener’s cybernetics in combination with detailed experimental research. Such an analysis reveals the importance of limited permeability of mitochondrial outer membrane for ADP due to interactions of mitochondria with cytoskeleton resulting in quasi-linear dependence of respiration rate on amplitude of cyclic changes in cytoplasmic ADP concentrations. The system of compartmentalized creatine kinase (CK isoenzymes functionally coupled to ANT and ATPases, and mitochondrial-cytoskeletal interactions separate energy fluxes (mass and energy transfer from signalling (information transfer within dissipative metabolic structures – intracellular energetic units (ICEU. Due to the non-equilibrium state of CK reactions, intracellular ATP utilization and mitochondrial ATP regeneration are interconnected by the PCr flux from mitochondria. The feedback regulation of respiration occurring via

  16. Design and analysis of a new flux-intensifying permanent magnet brushless motor with multilayer flux barriers

    Directory of Open Access Journals (Sweden)

    Shen Yang

    2017-05-01

    Full Text Available This paper proposes a new flux-intensifying permanent magnet brushless motor for potential application in electric vehicles. The key of the proposed motor is to adopt the concept of flux-intensifying effect, thus the preferable flux-weakening ability and extended speed range can be achieved. The usage of segmented and relatively thinner permanent magnet (PM in the proposed motor contributes to the increase of d-axis inductance Ld. In addition, the multilayer flux barriers along q-axis flux path will effectively decrease q-axis inductance Lq. As a result, the unique feature of Ld>Lq can be obtained, which is beneficial to extending the speed range of the proposed motor. Furthermore, the flux-intensifying effect can reduce the risk of irreversible demagnetization in PMs. The electromagnetic performances of the proposed motor are analyzed and investigated in details by using the finite element methods, which demonstrate the excellent flux-weakening capability and wide speed range can be achieved in the proposed FI-PMBL motor.

  17. Design and analysis of a new flux-intensifying permanent magnet brushless motor with multilayer flux barriers

    Science.gov (United States)

    Yang, Shen; Zhu, Xiaoyong; Xiang, Zixuan; Fan, Deyang; Wu, Wenye; Yin, Jianing

    2017-05-01

    This paper proposes a new flux-intensifying permanent magnet brushless motor for potential application in electric vehicles. The key of the proposed motor is to adopt the concept of flux-intensifying effect, thus the preferable flux-weakening ability and extended speed range can be achieved. The usage of segmented and relatively thinner permanent magnet (PM) in the proposed motor contributes to the increase of d-axis inductance Ld. In addition, the multilayer flux barriers along q-axis flux path will effectively decrease q-axis inductance Lq. As a result, the unique feature of Ld>Lq can be obtained, which is beneficial to extending the speed range of the proposed motor. Furthermore, the flux-intensifying effect can reduce the risk of irreversible demagnetization in PMs. The electromagnetic performances of the proposed motor are analyzed and investigated in details by using the finite element methods, which demonstrate the excellent flux-weakening capability and wide speed range can be achieved in the proposed FI-PMBL motor.

  18. In silico analysis of human metabolism: Reconstruction, contextualization and application of genome-scale models

    DEFF Research Database (Denmark)

    Geng, Jun; Nielsen, Jens

    2017-01-01

    The arising prevalence of metabolic diseases calls for a holistic approach for analysis of the underlying nature of abnormalities in cellular functions. Through mathematic representation and topological analysis of cellular metabolism, GEnome scale metabolic Models (GEMs) provide a promising fram...

  19. A simplified method for power-law modelling of metabolic pathways from time-course data and steady-state flux profiles

    Directory of Open Access Journals (Sweden)

    Sugimoto Masahiro

    2006-07-01

    Full Text Available Abstract Background In order to improve understanding of metabolic systems there have been attempts to construct S-system models from time courses. Conventionally, non-linear curve-fitting algorithms have been used for modelling, because of the non-linear properties of parameter estimation from time series. However, the huge iterative calculations required have hindered the development of large-scale metabolic pathway models. To solve this problem we propose a novel method involving power-law modelling of metabolic pathways from the Jacobian of the targeted system and the steady-state flux profiles by linearization of S-systems. Results The results of two case studies modelling a straight and a branched pathway, respectively, showed that our method reduced the number of unknown parameters needing to be estimated. The time-courses simulated by conventional kinetic models and those described by our method behaved similarly under a wide range of perturbations of metabolite concentrations. Conclusion The proposed method reduces calculation complexity and facilitates the construction of large-scale S-system models of metabolic pathways, realizing a practical application of reverse engineering of dynamic simulation models from the Jacobian of the targeted system and steady-state flux profiles.

  20. Fast flux module detection using matroid theory.

    Science.gov (United States)

    Reimers, Arne C; Bruggeman, Frank J; Olivier, Brett G; Stougie, Leen

    2015-05-01

    Flux balance analysis (FBA) is one of the most often applied methods on genome-scale metabolic networks. Although FBA uniquely determines the optimal yield, the pathway that achieves this is usually not unique. The analysis of the optimal-yield flux space has been an open challenge. Flux variability analysis is only capturing some properties of the flux space, while elementary mode analysis is intractable due to the enormous number of elementary modes. However, it has been found by Kelk et al. (2012) that the space of optimal-yield fluxes decomposes into flux modules. These decompositions allow a much easier but still comprehensive analysis of the optimal-yield flux space. Using the mathematical definition of module introduced by Müller and Bockmayr (2013b), we discovered useful connections to matroid theory, through which efficient algorithms enable us to compute the decomposition into modules in a few seconds for genome-scale networks. Using that every module can be represented by one reaction that represents its function, in this article, we also present a method that uses this decomposition to visualize the interplay of modules. We expect the new method to replace flux variability analysis in the pipelines for metabolic networks.

  1. Use of Germanium as comparator and integral monitor of neutron flux in activation analysis

    International Nuclear Information System (INIS)

    Furnari, Juan C.; Cohen, Isaac M.; Arribere, Maria A.; Kestelman, Abraham J.

    1997-01-01

    The possibility of using germanium as monitor of the thermal and epithermal components of the neutron flux, and comparator in parametric activation analysis, is discussed. The advantages and drawbacks associated to the use of this element are commented on, and the comparison with zirconium, in terms of the determination relative error, is performed. The utilisation of germanium as integral flux monitor, including the fast component of the neutron spectrum, is also discussed. Data corresponding to measurements of k 0 factor for the most relevant gamma transitions from Ge-75 and Be-77 are presented, as well as the results of the reference material analysis, employing germanium as flux monitor and comparator in a simultaneous way. (author). 8 refs., 3 figs., 2 tabs

  2. Development of high flux thermal neutron generator for neutron activation analysis

    International Nuclear Information System (INIS)

    Vainionpaa, Jaakko H.; Chen, Allan X.; Piestrup, Melvin A.; Gary, Charles K.; Jones, Glenn; Pantell, Richard H.

    2015-01-01

    The new model DD110MB neutron generator from Adelphi Technology produces thermal (<0.5 eV) neutron flux that is normally achieved in a nuclear reactor or larger accelerator based systems. Thermal neutron fluxes of 3–5 · 10 7 n/cm 2 /s are measured. This flux is achieved using four ion beams arranged concentrically around a target chamber containing a compact moderator with a central sample cylinder. Fast neutron yield of ∼2 · 10 10 n/s is created at the titanium surface of the target chamber. The thickness and material of the moderator is selected to maximize the thermal neutron flux at the center. The 2.5 MeV neutrons are quickly thermalized to energies below 0.5 eV and concentrated at the sample cylinder. The maximum flux of thermal neutrons at the target is achieved when approximately half of the neutrons at the sample area are thermalized. In this paper we present simulation results used to characterize performance of the neutron generator. The neutron flux can be used for neutron activation analysis (NAA) prompt gamma neutron activation analysis (PGNAA) for determining the concentrations of elements in many materials. Another envisioned use of the generator is production of radioactive isotopes. DD110MB is small enough for modest-sized laboratories and universities. Compared to nuclear reactors the DD110MB produces comparable thermal flux but provides reduced administrative and safety requirements and it can be run in pulsed mode, which is beneficial in many neutron activation techniques

  3. Development of high flux thermal neutron generator for neutron activation analysis

    Energy Technology Data Exchange (ETDEWEB)

    Vainionpaa, Jaakko H., E-mail: hannes@adelphitech.com [Adelphi Technology, 2003 E Bayshore Rd, Redwood City, CA 94063 (United States); Chen, Allan X.; Piestrup, Melvin A.; Gary, Charles K. [Adelphi Technology, 2003 E Bayshore Rd, Redwood City, CA 94063 (United States); Jones, Glenn [G& J Jones Enterprice, 7486 Brighton Ct, Dublin, CA 94568 (United States); Pantell, Richard H. [Department of Electrical Engineering, Stanford University, Stanford, CA (United States)

    2015-05-01

    The new model DD110MB neutron generator from Adelphi Technology produces thermal (<0.5 eV) neutron flux that is normally achieved in a nuclear reactor or larger accelerator based systems. Thermal neutron fluxes of 3–5 · 10{sup 7} n/cm{sup 2}/s are measured. This flux is achieved using four ion beams arranged concentrically around a target chamber containing a compact moderator with a central sample cylinder. Fast neutron yield of ∼2 · 10{sup 10} n/s is created at the titanium surface of the target chamber. The thickness and material of the moderator is selected to maximize the thermal neutron flux at the center. The 2.5 MeV neutrons are quickly thermalized to energies below 0.5 eV and concentrated at the sample cylinder. The maximum flux of thermal neutrons at the target is achieved when approximately half of the neutrons at the sample area are thermalized. In this paper we present simulation results used to characterize performance of the neutron generator. The neutron flux can be used for neutron activation analysis (NAA) prompt gamma neutron activation analysis (PGNAA) for determining the concentrations of elements in many materials. Another envisioned use of the generator is production of radioactive isotopes. DD110MB is small enough for modest-sized laboratories and universities. Compared to nuclear reactors the DD110MB produces comparable thermal flux but provides reduced administrative and safety requirements and it can be run in pulsed mode, which is beneficial in many neutron activation techniques.

  4. Machine Learning Methods for Analysis of Metabolic Data and Metabolic Pathway Modeling.

    Science.gov (United States)

    Cuperlovic-Culf, Miroslava

    2018-01-11

    Machine learning uses experimental data to optimize clustering or classification of samples or features, or to develop, augment or verify models that can be used to predict behavior or properties of systems. It is expected that machine learning will help provide actionable knowledge from a variety of big data including metabolomics data, as well as results of metabolism models. A variety of machine learning methods has been applied in bioinformatics and metabolism analyses including self-organizing maps, support vector machines, the kernel machine, Bayesian networks or fuzzy logic. To a lesser extent, machine learning has also been utilized to take advantage of the increasing availability of genomics and metabolomics data for the optimization of metabolic network models and their analysis. In this context, machine learning has aided the development of metabolic networks, the calculation of parameters for stoichiometric and kinetic models, as well as the analysis of major features in the model for the optimal application of bioreactors. Examples of this very interesting, albeit highly complex, application of machine learning for metabolism modeling will be the primary focus of this review presenting several different types of applications for model optimization, parameter determination or system analysis using models, as well as the utilization of several different types of machine learning technologies.

  5. Machine Learning Methods for Analysis of Metabolic Data and Metabolic Pathway Modeling

    Science.gov (United States)

    Cuperlovic-Culf, Miroslava

    2018-01-01

    Machine learning uses experimental data to optimize clustering or classification of samples or features, or to develop, augment or verify models that can be used to predict behavior or properties of systems. It is expected that machine learning will help provide actionable knowledge from a variety of big data including metabolomics data, as well as results of metabolism models. A variety of machine learning methods has been applied in bioinformatics and metabolism analyses including self-organizing maps, support vector machines, the kernel machine, Bayesian networks or fuzzy logic. To a lesser extent, machine learning has also been utilized to take advantage of the increasing availability of genomics and metabolomics data for the optimization of metabolic network models and their analysis. In this context, machine learning has aided the development of metabolic networks, the calculation of parameters for stoichiometric and kinetic models, as well as the analysis of major features in the model for the optimal application of bioreactors. Examples of this very interesting, albeit highly complex, application of machine learning for metabolism modeling will be the primary focus of this review presenting several different types of applications for model optimization, parameter determination or system analysis using models, as well as the utilization of several different types of machine learning technologies. PMID:29324649

  6. Predicting SPE Fluxes: Coupled Simulations and Analysis Tools

    Science.gov (United States)

    Gorby, M.; Schwadron, N.; Linker, J.; Caplan, R. M.; Wijaya, J.; Downs, C.; Lionello, R.

    2017-12-01

    Presented here is a nuts-and-bolts look at the coupled framework of Predictive Science Inc's Magnetohydrodynamics Around a Sphere (MAS) code and the Energetic Particle Radiation Environment Module (EPREM). MAS simulated coronal mass ejection output from a variety of events can be selected as the MHD input to EPREM and a variety of parameters can be set to run against: bakground seed particle spectra, mean free path, perpendicular diffusion efficiency, etc.. A standard set of visualizations are produced as well as a library of analysis tools for deeper inquiries. All steps will be covered end-to-end as well as the framework's user interface and availability.

  7. Stage-Specific Fatty Acid Fluxes Play a Regulatory Role in Glycerolipid Metabolism during Seed Development in Jatropha curcas L.

    Science.gov (United States)

    Chaitanya, Bharatula Sri Krishna; Kumar, Sumit; Kaki, Shiva Shanker; Balakrishna, Marrapu; Karuna, Mallampalli Sri Lakshmi; Prasad, Rachapudi Badari Narayana; Sastry, Pidaparty Seshadri; Reddy, Attipalli Ramachandra

    2015-12-23

    The present study describes the changes in lipid profile as well as fatty acid fluxes during seed development in Jatropha curcas L. Endosperm from 34, 37, and 40 days after anthesis (DAA), incubated with [(14)C]acetate, showed significant synthesis of phosphatidylcholine (PC) at seed maturation. The fatty acid methyl ester profile showed PC from 34 DAA was rich in palmitic acid (16:0), whereas PC from 37 and 40 DAA was rich in oleic acid (18:1n-9). Molecular species analysis of diacylglycerol (DAG) indicated DAG (16:0/18:2n-6) was in abundance at 34 DAA, whereas DAG (18:1n-9/18:2n-6) was significantly high at 40 DAA. Triacylglycerol (TAG) analysis revealed TAG (16:0/18:2n-6/16:0) was abundant at 34 DAA, whereas TAG (18:1n-9/18:2n-6/18:1n-9) formed the majority at 40 DAA. Expression of two types of diacylglycerol acyltransferases varied with seed maturation. These data demonstrate stage-specific distinct pools of PC and DAG synthesis during storage TAG accumulation in Jatropha seed.

  8. The operating experience and incident analysis for High Flux Engineering Test Reactor

    International Nuclear Information System (INIS)

    Zhao Guang

    1999-01-01

    The paper describes the incidents analysis for High Flux Engineering test reactor (HFETR) and introduces operating experience. Some suggestion have been made to reduce the incidents of HFETR. It is necessary to adopt new improvements which enhance the safety and reliability of operation. (author)

  9. Design, Prototyping, and Analysis of a Novel Modular Permanent Magnet Transverse Flux Disk Generator

    DEFF Research Database (Denmark)

    Hosseini, Seyedmohsen; Moghani, Javad Shokrollahi; Ershad, Nima Farrokhzad

    2011-01-01

    This paper presents the design, prototyping, and analysis of a novel modular transverse flux permanent magnet disk generator. The disk-shaped structure simplifies the construction procedure by using laminated steel sheets. To reduce output harmonics, the excitation of the generator is done by cir...

  10. Thermodynamic and Probabilistic Metabolic Control Analysis of Riboflavin (Vitamin B₂) Biosynthesis in Bacteria.

    Science.gov (United States)

    Birkenmeier, Markus; Mack, Matthias; Röder, Thorsten

    2015-10-01

    In this study, we applied a coupled in silico thermodynamic and probabilistic metabolic control analysis methodology to investigate the control mechanisms of the commercially relevant riboflavin biosynthetic pathway in bacteria. Under the investigated steady-state conditions, we found that several enzyme reactions of the pathway operate far from thermodynamic equilibrium (transformed Gibbs energies of reaction below about -17 kJ mol(-1)). Using the obtained thermodynamic information and applying enzyme elasticity sampling, we calculated the distributions of the scaled concentration control coefficients (CCCs) and scaled flux control coefficients (FCCs). From the statistical analysis of the calculated distributions, we inferred that the control over the riboflavin producing flux is shared among several enzyme activities and mostly resides in the initial reactions of the pathway. More precisely, the guanosine triphosphate (GTP) cyclohydrolase II activity, and therefore the bifunctional RibA protein of Bacillus subtilis because it catalyzes this activity, appears to mainly control the riboflavin producing flux (mean FCCs = 0.45 and 0.55, respectively). The GTP cyclohydrolase II activity and RibA also exert a high positive control over the riboflavin concentration (mean CCCs = 2.43 and 2.91, respectively). This prediction is consistent with previous findings for microbial riboflavin overproducing strains.

  11. Metabolic control analysis of xylose catabolism in Aspergillus

    NARCIS (Netherlands)

    Prathumpai, W.; Gabelgaard, J.B.; Wanchanthuek, P.; Vondervoort, van de P.J.I.; Groot, de M.J.L.; McIntyre, M.; Nielsen, J.

    2003-01-01

    A kinetic model for xylose catabolism in Aspergillus is proposed. From a thermodynamic analysis it was found that the intermediate xylitol will accumulate during xylose catabolism. Use of the kinetic model allowed metabolic control analysis (MCA) of the xylose catabolic pathway to be carried out,

  12. Analysis of neutron flux depression across the pellet radius in CANDU fuel elements

    International Nuclear Information System (INIS)

    Sim, K.S.; Suk, H.C.

    1998-08-01

    The TUBRNP model, originally developed to perform the analysis of the flux depression across the pellet radius in LWR fuel elements, was improved for the application to CANDU fuel elements. The improved model was verified through comparison with existing CANDU model named FLUXDEP in prediction for various fuel conditions. A sensitivity study was also performed to investigate the effects on the flux depression of fuel initial enrichment and burnup, the contents of isotopes U-234 and U-236 and pellet diameter. (author). 9 refs., 8 figs

  13. Design and analysis of a 3D-flux flux-switching permanent magnet machine with SMC cores and ferrite magnets

    Science.gov (United States)

    Liu, Chengcheng; Wang, Youhua; Lei, Gang; Guo, Youguang; Zhu, Jianguo

    2017-05-01

    Since permanent magnets (PM) are stacked between the adjacent stator teeth and there are no windings or PMs on the rotor, flux-switching permanent magnet machine (FSPMM) owns the merits of good flux concentrating and robust rotor structure. Compared with the traditional PM machines, FSPMM can provide higher torque density and better thermal dissipation ability. Combined with the soft magnetic composite (SMC) material and ferrite magnets, this paper proposes a new 3D-flux FSPMM (3DFFSPMM). The topology and operation principle are introduced. It can be found that the designed new 3DFFSPMM has many merits over than the traditional FSPMM for it can utilize the advantages of SMC material. Moreover, the PM flux of this new motor can be regulated by using the mechanical method. 3D finite element method (FEM) is used to calculate the magnetic field and parameters of the motor, such as flux density, inductance, PM flux linkage and efficiency map. The demagnetization analysis of the ferrite magnet is also addressed to ensure the safety operation of the proposed motor.

  14. Design and analysis of a 3D-flux flux-switching permanent magnet machine with SMC cores and ferrite magnets

    Directory of Open Access Journals (Sweden)

    Chengcheng Liu

    2017-05-01

    Full Text Available Since permanent magnets (PM are stacked between the adjacent stator teeth and there are no windings or PMs on the rotor, flux-switching permanent magnet machine (FSPMM owns the merits of good flux concentrating and robust rotor structure. Compared with the traditional PM machines, FSPMM can provide higher torque density and better thermal dissipation ability. Combined with the soft magnetic composite (SMC material and ferrite magnets, this paper proposes a new 3D-flux FSPMM (3DFFSPMM. The topology and operation principle are introduced. It can be found that the designed new 3DFFSPMM has many merits over than the traditional FSPMM for it can utilize the advantages of SMC material. Moreover, the PM flux of this new motor can be regulated by using the mechanical method. 3D finite element method (FEM is used to calculate the magnetic field and parameters of the motor, such as flux density, inductance, PM flux linkage and efficiency map. The demagnetization analysis of the ferrite magnet is also addressed to ensure the safety operation of the proposed motor.

  15. Analysis of the Daya Bay Reactor Antineutrino Flux Changes with Fuel Burnup

    Science.gov (United States)

    Hayes, A. C.; Jungman, Gerard; McCutchan, E. A.; Sonzogni, A. A.; Garvey, G. T.; Wang, X. B.

    2018-01-01

    We investigate the recent Daya Bay results on the changes in the antineutrino flux and spectrum with the burnup of the reactor fuel. We find that the discrepancy between current model predictions and the Daya Bay results can be traced to the original measured U 235 /Pu 239 ratio of the fission β spectra that were used as a base for the expected antineutrino fluxes. An analysis of the antineutrino spectra that is based on a summation over all fission fragment β decays, using nuclear database input, explains all of the features seen in the Daya Bay evolution data. However, this summation method still allows for an anomaly. We conclude that there is currently not enough information to use the antineutrino flux changes to rule out the possible existence of sterile neutrinos.

  16. Analysis of a flux-coupling type superconductor fault current limiter with pancake coils

    Science.gov (United States)

    Liu, Shizhuo; Xia, Dong; Zhang, Zhifeng; Qiu, Qingquan; Zhang, Guomin

    2017-10-01

    The characteristics of a flux-coupling type superconductor fault current limiter (SFCL) with pancake coils are investigated in this paper. The conventional double-wound non-inductive pancake coil used in AC power systems has an inevitable defect in Voltage Sourced Converter Based High Voltage DC (VSC-HVDC) power systems. Due to its special structure, flashover would occur easily during the fault in high voltage environment. Considering the shortcomings of conventional resistive SFCLs with non-inductive coils, a novel flux-coupling type SFCL with pancake coils is carried out. The module connections of pancake coils are performed. The electromagnetic field and force analysis of the module are contrasted under different parameters. To ensure proper operation of the module, the impedance of the module under representative operating conditions is calculated. Finally, the feasibility of the flux-coupling type SFCL in VSC-HVDC power systems is discussed.

  17. Neutron activation analysis: Modelling studies to improve the neutron flux of Americium-Beryllium source

    Energy Technology Data Exchange (ETDEWEB)

    Didi, Abdessamad; Dadouch, Ahmed; Tajmouati, Jaouad; Bekkouri, Hassane [Advanced Technology and Integration System, Dept. of Physics, Faculty of Science Dhar Mehraz, University Sidi Mohamed Ben Abdellah, Fez (Morocco); Jai, Otman [Laboratory of Radiation and Nuclear Systems, Dept. of Physics, Faculty of Sciences, Tetouan (Morocco)

    2017-06-15

    Americium–beryllium (Am-Be; n, γ) is a neutron emitting source used in various research fields such as chemistry, physics, geology, archaeology, medicine, and environmental monitoring, as well as in the forensic sciences. It is a mobile source of neutron activity (20 Ci), yielding a small thermal neutron flux that is water moderated. The aim of this study is to develop a model to increase the neutron thermal flux of a source such as Am-Be. This study achieved multiple advantageous results: primarily, it will help us perform neutron activation analysis. Next, it will give us the opportunity to produce radio-elements with short half-lives. Am-Be single and multisource (5 sources) experiments were performed within an irradiation facility with a paraffin moderator. The resulting models mainly increase the thermal neutron flux compared to the traditional method with water moderator.

  18. Analysis of heat transfer under high heat flux nucleate boiling conditions

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Y.; Dinh, N. [3145 Burlington Laboratories, Raleigh, NC (United States)

    2016-07-15

    Analysis was performed for a heater infrared thermometric imaging temperature data obtained from high heat flux pool boiling and liquid film boiling experiments BETA. With the OpenFOAM solver, heat flux distribution towards the coolant was obtained by solving transient heat conduction of heater substrate given the heater surface temperature data as boundary condition. The so-obtained heat flux data was used to validate them against the state-of-art wall boiling model developed by D. R. Shaver (2015) with the assumption of micro-layer hydrodynamics. Good agreement was found between the model prediction and data for conditions away from the critical heat flux (CHF). However, the data indicate a different heat transfer pattern under CHF, which is not captured by the current model. Experimental data strengthen the notion of burnout caused by the irreversible hot spot due to failure of rewetting. The observation forms a basis for a detailed modeling of micro-layer hydrodynamics under high heat flux.

  19. Theoretical Analysis of Interferometer Wave Front Tilt and Fringe Radiant Flux on a Rectangular Photodetector

    Directory of Open Access Journals (Sweden)

    Franz Konstantin Fuss

    2013-09-01

    Full Text Available This paper is a theoretical analysis of mirror tilt in a Michelson interferometer and its effect on the radiant flux over the active area of a rectangular photodetector or image sensor pixel. It is relevant to sensor applications using homodyne interferometry where these opto-electronic devices are employed for partial fringe counting. Formulas are derived for radiant flux across the detector for variable location within the fringe pattern and with varying wave front angle. The results indicate that the flux is a damped sine function of the wave front angle, with a decay constant of the ratio of wavelength to detector width. The modulation amplitude of the dynamic fringe pattern reduces to zero at wave front angles that are an integer multiple of this ratio and the results show that the polarity of the radiant flux changes exclusively at these multiples. Varying tilt angle causes radiant flux oscillations under an envelope curve, the frequency of which is dependent on the location of the detector with the fringe pattern. It is also shown that a fringe count of zero can be obtained for specific photodetector locations and wave front angles where the combined effect of fringe contraction and fringe tilt can have equal and opposite effects. Fringe tilt as a result of a wave front angle of 0.05° can introduce a phase measurement difference of 16° between a photodetector/pixel located 20 mm and one located 100 mm from the optical origin.

  20. Metabolic Effects of Hypoxia in Colorectal Cancer by 13C NMR Isotopomer Analysis

    Directory of Open Access Journals (Sweden)

    Ana M. Abrantes

    2014-01-01

    Full Text Available 13C NMR isotopomer analysis was used to characterize intermediary metabolism in three colorectal cancer cell lines (WiDr, LS1034, and C2BBe1 and determine the “metabolic remodeling” that occurs under hypoxia. Under normoxia, the three colorectal cancer cell lines present high rates of lactate production and can be seen as “Warburg” like cancer cells independently of substrate availability, since such profile was dominant at both high and low glucose media contents. The LS1034 was the less glycolytic of the three cell lines and was the most affected by the event of hypoxia, raising abruptly glucose consumption and lactate production. The other two colorectal cell lines, WiDr and C2BBe1, adapted better to hypoxia and were able to maintain their oxidative fluxes even at the very low levels of oxygen. These differential metabolic behaviors of the three colorectal cell lines show how important an adequate knowledge of the “metabolic remodeling” that follows a given cancer treatment is towards the correct (redesign of therapeutic strategies against cancer.

  1. Integrated proteomic and metabolic analysis of breast cancer progression.

    Directory of Open Access Journals (Sweden)

    Patrick G Shaw

    Full Text Available One of the most persistent hallmarks of cancer biology is the preference of tumor cells to derive energy through glycolysis as opposed to the more efficient process of oxidative phosphorylation (OXPHOS. However, little is known about the molecular cascades by which oncogenic pathways bring about this metabolic switch. We carried out a quantitative proteomic and metabolic analysis of the MCF10A derived cell line model of breast cancer progression that includes parental cells and derivatives representing three different tumor grades of Ras-driven cancer with a common genetic background. A SILAC (Stable Isotope Labeling by Amino acids in Cell culture labeling strategy was used to quantify protein expression in conjunction with subcellular fractionation to measure dynamic subcellular localization in the nucleus, cytosol and mitochondria. Protein expression and localization across cell lines were compared to cellular metabolic rates as a measure of oxidative phosphorylation (OXPHOS, glycolysis and cellular ATP. Investigation of the metabolic capacity of the four cell lines revealed that cellular OXPHOS decreased with breast cancer progression independently of mitochondrial copy number or electron transport chain protein expression. Furthermore, glycolytic lactate secretion did not increase in accordance with cancer progression and decreasing OXPHOS capacity. However, the relative expression and subcellular enrichment of enzymes critical to lactate and pyruvate metabolism supported the observed extracellular acidification profiles. This analysis of metabolic dysfunction in cancer progression integrated with global protein expression and subcellular localization is a novel and useful technique for determining organelle-specific roles of proteins in disease.

  2. (Im) Perfect robustness and adaptation of metabolic networks subject to metabolic and gene-expression regulation: marrying control engineering with metabolic control analysis

    NARCIS (Netherlands)

    He, F.; Fromion, V.; Westerhoff, H.V.

    2013-01-01

    Background: Metabolic control analysis (MCA) and supply-demand theory have led to appreciable understanding of the systems properties of metabolic networks that are subject exclusively to metabolic regulation. Supply-demand theory has not yet considered gene-expression regulation explicitly whilst a

  3. Reconstruction and analysis of a genome-scale metabolic model for Scheffersomyces stipitis

    Directory of Open Access Journals (Sweden)

    Balagurunathan Balaji

    2012-02-01

    Full Text Available Abstract Background Fermentation of xylose, the major component in hemicellulose, is essential for economic conversion of lignocellulosic biomass to fuels and chemicals. The yeast Scheffersomyces stipitis (formerly known as Pichia stipitis has the highest known native capacity for xylose fermentation and possesses several genes for lignocellulose bioconversion in its genome. Understanding the metabolism of this yeast at a global scale, by reconstructing the genome scale metabolic model, is essential for manipulating its metabolic capabilities and for successful transfer of its capabilities to other industrial microbes. Results We present a genome-scale metabolic model for Scheffersomyces stipitis, a native xylose utilizing yeast. The model was reconstructed based on genome sequence annotation, detailed experimental investigation and known yeast physiology. Macromolecular composition of Scheffersomyces stipitis biomass was estimated experimentally and its ability to grow on different carbon, nitrogen, sulphur and phosphorus sources was determined by phenotype microarrays. The compartmentalized model, developed based on an iterative procedure, accounted for 814 genes, 1371 reactions, and 971 metabolites. In silico computed growth rates were compared with high-throughput phenotyping data and the model could predict the qualitative outcomes in 74% of substrates investigated. Model simulations were used to identify the biosynthetic requirements for anaerobic growth of Scheffersomyces stipitis on glucose and the results were validated with published literature. The bottlenecks in Scheffersomyces stipitis metabolic network for xylose uptake and nucleotide cofactor recycling were identified by in silico flux variability analysis. The scope of the model in enhancing the mechanistic understanding of microbial metabolism is demonstrated by identifying a mechanism for mitochondrial respiration and oxidative phosphorylation. Conclusion The genome

  4. Genome-scale metabolic analysis of Clostridium thermocellum for bioethanol production

    Directory of Open Access Journals (Sweden)

    Brooks J Paul

    2010-03-01

    Full Text Available Abstract Background Microorganisms possess diverse metabolic capabilities that can potentially be leveraged for efficient production of biofuels. Clostridium thermocellum (ATCC 27405 is a thermophilic anaerobe that is both cellulolytic and ethanologenic, meaning that it can directly use the plant sugar, cellulose, and biochemically convert it to ethanol. A major challenge in using microorganisms for chemical production is the need to modify the organism to increase production efficiency. The process of properly engineering an organism is typically arduous. Results Here we present a genome-scale model of C. thermocellum metabolism, iSR432, for the purpose of establishing a computational tool to study the metabolic network of C. thermocellum and facilitate efforts to engineer C. thermocellum for biofuel production. The model consists of 577 reactions involving 525 intracellular metabolites, 432 genes, and a proteomic-based representation of a cellulosome. The process of constructing this metabolic model led to suggested annotation refinements for 27 genes and identification of areas of metabolism requiring further study. The accuracy of the iSR432 model was tested using experimental growth and by-product secretion data for growth on cellobiose and fructose. Analysis using this model captures the relationship between the reduction-oxidation state of the cell and ethanol secretion and allowed for prediction of gene deletions and environmental conditions that would increase ethanol production. Conclusions By incorporating genomic sequence data, network topology, and experimental measurements of enzyme activities and metabolite fluxes, we have generated a model that is reasonably accurate at predicting the cellular phenotype of C. thermocellum and establish a strong foundation for rational strain design. In addition, we are able to draw some important conclusions regarding the underlying metabolic mechanisms for observed behaviors of C. thermocellum

  5. Motor current and leakage flux signature analysis technique for condition monitoring

    International Nuclear Information System (INIS)

    Pillai, M.V.; Moorthy, R.I.K.; Mahajan, S.C.

    1994-01-01

    Till recently analysis of vibration signals was the only means available to predict the state of health of plant equipment. Motor current and leakage magnetic flux signature analysis is acquiring importance as a technique for detection of incipient damages in the electrical machines and as a supplementary technique for diagnostics of driven equipment such as centrifugal and reciprocating pumps. The state of health of the driven equipment is assessed by analysing time signal, frequency spectrum and trend analysis. For example, the pump vane frequency, piston stroke frequency, gear frequency and bearing frequencies are indicated in the current and flux spectra. By maintaining a periodic record of the amplitudes of various frequency lines in the frequency spectra, it is possible to understand the trend of deterioration of parts and components of the pump. All problems arising out of inappropriate mechanical alignment of vertical pumps are easily identified by a combined analysis of current, flux and vibration signals. It is found that current signature analysis technique is a sufficient method in itself for the analysis of state of health of reciprocating pumps and compressors. (author). 10 refs., 4 figs

  6. Metabolic control analysis of L-lactate synthesis pathway in Rhizopus oryzae As 3.2686.

    Science.gov (United States)

    Ke, Wei; Chang, Shu; Chen, Xiaoju; Luo, Shuizhong; Jiang, Shaotong; Yang, Peizhou; Wu, Xuefeng; Zheng, Zhi

    2015-11-01

    The relationship between the metabolic flux and the activities of the pyruvate branching enzymes of Rhizopus oryzae As 3.2686 during L-lactate fermentation was investigated using the perturbation method of aeration. The control coefficients for five enzymes, pyruvate dehydrogenase (PDH), pyruvate carboxylase (PC), pyruvate decarboxylase (PDC), lactate dehydrogenase (LDH), and alcohol dehydrogenase (ADH), were calculated. Our results indicated significant correlations between PDH and PC, PDC and LDH, PDC and ADH, LDH and ADH, and PDC and PC. It also appeared that PDH, PC, and LDH strongly controlled the L-lactate flux; PDH and ADH strongly controlled the ethanol flux; while PDH and PC strongly controlled the acetyl coenzyme A flux and the oxaloacetate flux. Further, the flux control coefficient curves indicated that the control of the system gradually transferred from PDC to PC during the steady state. Therefore, PC was the key rate-limiting enzyme that controls the flux distribution.

  7. Enhanced H2 Production and Redirected Metabolic Flux via Overexpression of fhlA and pncB in Klebsiella HQ-3 Strain.

    Science.gov (United States)

    Jawed, Muhammad; Pi, Jian; Xu, Li; Zhang, Houjin; Hakeem, Abdul; Yan, Yunjun

    2016-03-01

    Genetic modifications are considered as one of the most important technologies for improving fermentative hydrogen yield. Herein, we overexpress fhlA and pncB genes from Klebsiella HQ-3 independently to enhance hydrogen molar yield. HQ-3-fhlA/pncB strain is developed by manipulation of pET28-Pkan/fhlA Kan(r) and pBBR1-MCS5/pncB Gm(r) as expression vectors to examine the synchronous effects of fhlA and pncB. Optimization of anaerobic batch fermentations is achieved and the maximum yield of biohydrogen (1.42 mol H2/mol of glucose) is produced in the range of pH 6.5-7.0 at 33-37 °C. Whole cell H2 yield is increased up to 40 % from HQ-3-fhlA/pncB, as compared with HQ-3-fhlA 20 % and HQ-3-pncB 12 % keeping HQ-3-C as a control. Mechanism of improved H2 yield is studied in combination with metabolic flux analysis by measuring glucose consumption and other metabolites including formate, succinate, 2,3 butanediol, lactate, acetate, ethanol, and hydrogen. The results suggest that under transient conditions, the increase in the total level of NAD by NAPRTase can enhance the rate of NADH-dependent pathways, and therefore, final distribution of metabolites is changed. Combined overexpression of fhlA and pncB eventually modifies the energy and carbon balance leading to enhanced H2 production from FHL as well as by NADH pathway.

  8. A computer program for the algebraic determination of control coefficients in Metabolic Control Analysis.

    Science.gov (United States)

    Thomas, S; Fell, D A

    1993-06-01

    A computer program (MetaCon) is described for the evaluation of flux control, concentration control and branch-point distribution control coefficients of a metabolic pathway. Requiring only the reaction scheme as input, the program produces algebraic expressions for the control coefficients in terms of elasticity coefficients, metabolite concentrations and pathway fluxes. Any of these variables can be substituted by numeric or simple algebraic expressions; the expressions will then be automatically rearranged in terms of the remaining unknown variables. When all variables have been substituted, numeric values will be obtained for the control coefficients. The program is a computerized implementation of the matrix method for the determination of control coefficients. The features of MetaCon are compared with those of other programs available to workers in Metabolic Control Analysis. Potential benefits of, and methods of using, MetaCon are discussed. The mathematical background and validity of the matrix method rules are discussed, and the algorithm used by MetaCon is described. The matrix method is shown to be a specific case of a previously described general formalism for calculating control coefficients.

  9. Controlling the transcription levels of argGH redistributed L-arginine metabolic flux in N-acetylglutamate kinase and ArgR-deregulated Corynebacterium crenatum.

    Science.gov (United States)

    Zhao, Qinqin; Luo, Yuchang; Dou, Wenfang; Zhang, Xian; Zhang, Xiaomei; Zhang, Weiwei; Xu, Meijuan; Geng, Yan; Rao, Zhiming; Xu, Zhenghong

    2016-01-01

    Corynebacterium crenatum SYPA5-5, an L-arginine high-producer obtained through multiple mutation-screening steps, had been deregulated by the repression of ArgR that inhibits L-arginine biosynthesis at genetic level. Further study indicated that feedback inhibition of SYPA5-5 N-acetylglutamate kinase (CcNAGK) by L-arginine, as another rate-limiting step, could be deregulated by introducing point mutations. Here, we introduced two of the positive mutations (H268N or R209A) of CcNAGK into the chromosome of SYPA5-5, however, resulting in accumulation of large amounts of the intermediates (L-citrulline and L-ornithine) and decreased production of L-arginine. Genetic and enzymatic levels analysis involved in L-arginine biosynthetic pathway of recombinants SYPA5-5-NAGKH268N (H-7) and SYPA5-5-NAGKR209A (R-8) showed that the transcription levels of argGH decreased accompanied with the reduction of argininosuccinate synthase and argininosuccinase activities, respectively, which led to the metabolic obstacle from L-citrulline to L-arginine. Co-expression of argGH with exogenous plasmid in H-7 and R-8 removed this bottleneck and increased L-arginine productivity remarkably. Compared with SYPA5-5, fermentation period of H-7/pDXW-10-argGH (H-7-GH) reduced to 16 h; meanwhile, the L-arginine productivity improved about 63.6%. Fed-batch fermentation of H-7-GH in 10 L bioreactor produced 389.9 mM L-arginine with the productivity of 5.42 mM h(-1). These results indicated that controlling the transcription of argGH was a key factor for regulating the metabolic flux toward L-arginine biosynthesis after deregulating the repression of ArgR and feedback inhibition of CcNAGK, and therefore functioned as another regulatory mode for L-arginine production. Thus, deregulating all these three regulatory modes was a powerful strategy to construct L-arginine high-producing C. crenatum.

  10. Predicting the accumulation of storage compounds by Rhodococcus jostii RHA1 in the feast-famine growth cycles using genome-scale flux balance analysis

    Science.gov (United States)

    Tajparast, Mohammad

    2018-01-01

    Feast-famine cycles in biological wastewater resource recovery systems select for bacterial species that accumulate intracellular storage compounds such as poly-β-hydroxybutyrate (PHB), glycogen, and triacylglycerols (TAG). These species survive better the famine phase and resume rapid substrate uptake at the beginning of the feast phase faster than microorganisms unable to accumulate storage. However, ecophysiological conditions favouring the accumulation of either storage compounds remain to be clarified, and predictive capabilities need to be developed to eventually rationally design reactors producing these compounds. Using a genome-scale metabolic modelling approach, the storage metabolism of Rhodococcus jostii RHA1 was investigated for steady-state feast-famine cycles on glucose and acetate as the sole carbon sources. R. jostii RHA1 is capable of accumulating the three storage compounds (PHB, TAG, and glycogen) simultaneously. According to the experimental observations, when glucose was the substrate, feast phase chemical oxygen demand (COD) accumulation was similar for the three storage compounds; when acetate was the substrate, however, PHB accumulation was 3 times higher than TAG accumulation and essentially no glycogen was accumulated. These results were simulated using the genome-scale metabolic model of R. jostii RHA1 (iMT1174) by means of flux balance analysis (FBA) to determine the objective functions capable of predicting these behaviours. Maximization of the growth rate was set as the main objective function, while minimization of total reaction fluxes and minimization of metabolic adjustment (environmental MOMA) were considered as the sub-objective functions. The environmental MOMA sub-objective performed better than the minimization of total reaction fluxes sub-objective function at predicting the mixture of storage compounds accumulated. Additional experiments with 13C-labelled bicarbonate (HCO3−) found that the fluxes through the central

  11. Stoichiometric Correlation Analysis: Principles of Metabolic Functionality from Metabolomics Data

    Directory of Open Access Journals (Sweden)

    Kevin Schwahn

    2017-12-01

    Full Text Available Recent advances in metabolomics technologies have resulted in high-quality (time-resolved metabolic profiles with an increasing coverage of metabolic pathways. These data profiles represent read-outs from often non-linear dynamics of metabolic networks. Yet, metabolic profiles have largely been explored with regression-based approaches that only capture linear relationships, rendering it difficult to determine the extent to which the data reflect the underlying reaction rates and their couplings. Here we propose an approach termed Stoichiometric Correlation Analysis (SCA based on correlation between positive linear combinations of log-transformed metabolic profiles. The log-transformation is due to the evidence that metabolic networks can be modeled by mass action law and kinetics derived from it. Unlike the existing approaches which establish a relation between pairs of metabolites, SCA facilitates the discovery of higher-order dependence between more than two metabolites. By using a paradigmatic model of the tricarboxylic acid cycle we show that the higher-order dependence reflects the coupling of concentration of reactant complexes, capturing the subtle difference between the employed enzyme kinetics. Using time-resolved metabolic profiles from Arabidopsis thaliana and Escherichia coli, we show that SCA can be used to quantify the difference in coupling of reactant complexes, and hence, reaction rates, underlying the stringent response in these model organisms. By using SCA with data from natural variation of wild and domesticated wheat and tomato accession, we demonstrate that the domestication is accompanied by loss of such couplings, in these species. Therefore, application of SCA to metabolomics data from natural variation in wild and domesticated populations provides a mechanistic way to understanding domestication and its relation to metabolic networks.

  12. PSAMM: A Portable System for the Analysis of Metabolic Models.

    Directory of Open Access Journals (Sweden)

    Jon Lund Steffensen

    2016-02-01

    Full Text Available The genome-scale models of metabolic networks have been broadly applied in phenotype prediction, evolutionary reconstruction, community functional analysis, and metabolic engineering. Despite the development of tools that support individual steps along the modeling procedure, it is still difficult to associate mathematical simulation results with the annotation and biological interpretation of metabolic models. In order to solve this problem, here we developed a Portable System for the Analysis of Metabolic Models (PSAMM, a new open-source software package that supports the integration of heterogeneous metadata in model annotations and provides a user-friendly interface for the analysis of metabolic models. PSAMM is independent of paid software environments like MATLAB, and all its dependencies are freely available for academic users. Compared to existing tools, PSAMM significantly reduced the running time of constraint-based analysis and enabled flexible settings of simulation parameters using simple one-line commands. The integration of heterogeneous, model-specific annotation information in PSAMM is achieved with a novel format of YAML-based model representation, which has several advantages, such as providing a modular organization of model components and simulation settings, enabling model version tracking, and permitting the integration of multiple simulation problems. PSAMM also includes a number of quality checking procedures to examine stoichiometric balance and to identify blocked reactions. Applying PSAMM to 57 models collected from current literature, we demonstrated how the software can be used for managing and simulating metabolic models. We identified a number of common inconsistencies in existing models and constructed an updated model repository to document the resolution of these inconsistencies.

  13. Metabolic pathway analysis using a nash equilibrium approach

    NARCIS (Netherlands)

    Lucia, Angelo; DiMaggio, Peter A.; Alonso-Martinez, Diego

    2018-01-01

    A novel approach to metabolic network analysis using a Nash Equilibrium (NE) formulation is proposed in which enzymes are considered players in a multi-player game. Each player has its own payoff function with the objective of minimizing the Gibbs free energy associated with the biochemical

  14. Improved analysis of all-sky meteor radar measurements of gravity wave variances and momentum fluxes

    Directory of Open Access Journals (Sweden)

    V. F. Andrioli

    2013-05-01

    Full Text Available The advantages of using a composite day analysis for all-sky interferometric meteor radars when measuring mean winds and tides are widely known. On the other hand, problems arise if this technique is applied to Hocking's (2005 gravity wave analysis for all-sky meteor radars. In this paper we describe how a simple change in the procedure makes it possible to use a composite day in Hocking's analysis. Also, we explain how a modified composite day can be constructed to test its ability to measure gravity wave momentum fluxes. Test results for specified mean, tidal, and gravity wave fields, including tidal amplitudes and gravity wave momentum fluxes varying strongly with altitude and/or time, suggest that the modified composite day allows characterization of monthly mean profiles of the gravity wave momentum fluxes, with good accuracy at least at the altitudes where the meteor counts are large (from 89 to 92.5 km. In the present work we also show that the variances measured with Hocking's method are often contaminated by the tidal fields and suggest a method of empirical correction derived from a simple simulation model. The results presented here greatly increase our confidence because they show that our technique is able to remove the tide-induced false variances from Hocking's analysis.

  15. DFBAlab: a fast and reliable MATLAB code for dynamic flux balance analysis.

    Science.gov (United States)

    Gomez, Jose A; Höffner, Kai; Barton, Paul I

    2014-12-18

    Dynamic Flux Balance Analysis (DFBA) is a dynamic simulation framework for biochemical processes. DFBA can be performed using different approaches such as static optimization (SOA), dynamic optimization (DOA), and direct approaches (DA). Few existing simulators address the theoretical and practical challenges of nonunique exchange fluxes or infeasible linear programs (LPs). Both are common sources of failure and inefficiencies for these simulators. DFBAlab, a MATLAB-based simulator that uses the LP feasibility problem to obtain an extended system and lexicographic optimization to yield unique exchange fluxes, is presented. DFBAlab is able to simulate complex dynamic cultures with multiple species rapidly and reliably, including differential-algebraic equation (DAE) systems. In addition, DFBAlab's running time scales linearly with the number of species models. Three examples are presented where the performance of COBRA, DyMMM and DFBAlab are compared. Lexicographic optimization is used to determine unique exchange fluxes which are necessary for a well-defined dynamic system. DFBAlab does not fail during numerical integration due to infeasible LPs. The extended system obtained through the LP feasibility problem in DFBAlab provides a penalty function that can be used in optimization algorithms.

  16. Design and analysis of a flux intensifying permanent magnet embedded salient pole wind generator

    Science.gov (United States)

    Guo, Yujing; Jin, Ping; Lin, Heyun; Yang, Hui; Lyu, Shukang

    2018-05-01

    This paper presents an improved flux intensifying permanent magnet embedded salient pole wind generator (FI-PMESPWG) with mirror symmetrical magnetizing directions permanent magnet (PM) for improving generator's performances. The air-gap flux densities, the output voltage, the cogging torque and the d- and q-axis inductances of FI-PMESPWG are all calculated and analyzed by using the finite element method (FEM). To highlight the advantages of the proposed FI-PMESPWG, an original permanent magnet embedded salient pole wind generator (PMESPWG) model is adopted for comparison under the same operating conditions. The calculating results show that the air-gap flux densities of FI-PMESPWG are intensified with the same magnet amounts because the PMs are set in a form of V shape in each pole. The difference between d-axis inductance and q-axis inductance of the proposed FI-PMESPWG is reduced. Thus, the output power of the proposed FI-PMESPWG reaches a higher value than that of the original PMESPWG at the same current phase angle. The cogging torque is diminished because the flux path is changed. All the analysis results indicate that the electromagnetic characteristics of the proposed FI-PMESPWG are significantly better than that of the original PMESPWG.

  17. Enzyme oscillation can enhance the thermodynamic efficiency of cellular metabolism: consequence of anti-phase coupling between reaction flux and affinity

    Science.gov (United States)

    Himeoka, Yusuke; Kaneko, Kunihiko

    2016-04-01

    Cells generally convert nutrient resources to products via energy transduction. Accordingly, the thermodynamic efficiency of this conversion process is one of the most essential characteristics of living organisms. However, although these processes occur under conditions of dynamic metabolism, most studies of cellular thermodynamic efficiency have been restricted to examining steady states; thus, the relevance of dynamics to this efficiency has not yet been elucidated. Here, we develop a simple model of metabolic reactions with anabolism-catabolism coupling catalyzed by enzymes. Through application of external oscillation in the enzyme abundances, the thermodynamic efficiency of metabolism was found to be improved. This result is in strong contrast with that observed in the oscillatory input, in which the efficiency always decreased with oscillation. This improvement was effectively achieved by separating the anabolic and catabolic reactions, which tend to disequilibrate each other, and taking advantage of the temporal oscillations so that each of the antagonistic reactions could progress near equilibrium. In this case, anti-phase oscillation between the reaction flux and chemical affinity through oscillation of enzyme abundances is essential. This improvement was also confirmed in a model capable of generating autonomous oscillations in enzyme abundances. Finally, the possible relevance of the improvement in thermodynamic efficiency is discussed with respect to the potential for manipulation of metabolic oscillations in microorganisms.

  18. Temperature and void reactivity coefficient calculations for the high flux isotope reactor safety analysis report

    International Nuclear Information System (INIS)

    Engle, W.W. Jr.; Williams, L.R.

    1994-07-01

    This report provides documentation of a series of calculations performed in 1991 in order to provide input for the High Flux Isotope Reactor Safety Analysis Report. In particular, temperature and void reactivity coefficients were calculated for beginning-of-life, end-of-life, and xenon equilibrium (29 h) conditions. Much of the data used to prepare the computer models for these calculations was derived from the original HFIR nuclear design study

  19. Analysis of traces at ORNL's new high-flux neutron activation laboratory

    International Nuclear Information System (INIS)

    Ricci, E.; Handley, T.H.; Dyer, F.F.

    1974-01-01

    The investigations are outlined, which are carried out in order to develop (preferably instrumental) methods for multielement analysis of various trace elements. For this reason a new High-Flux NAA Laboratory was constructed at ORNL's. A general review is given on the Laboratory, further some methods and applications are shown. In the field of comparator activation analysis comparative data are given on mercury determinations in various matrices, and on arsenic determination in grasshoppers. This later method was used to trace the transport of arsenic containing pesticides. Some data are given on absolute activation analysis of Na, Ci, Mn, Br, and Au, too. (K.A.)

  20. Hybrid elementary flux analysis/nonparametric modeling: application for bioprocess control

    Directory of Open Access Journals (Sweden)

    Alves Paula M

    2007-01-01

    Full Text Available Abstract Background The progress in the "-omic" sciences has allowed a deeper knowledge on many biological systems with industrial interest. This knowledge is still rarely used for advanced bioprocess monitoring and control at the bioreactor level. In this work, a bioprocess control method is presented, which is designed on the basis of the metabolic network of the organism under consideration. The bioprocess dynamics are formulated using hybrid rigorous/data driven systems and its inherent structure is defined by the metabolism elementary modes. Results The metabolic network of the system under study is decomposed into elementary modes (EMs, which are the simplest paths able to operate coherently in steady-state. A reduced reaction mechanism in the form of simplified reactions connecting substrates with end-products is obtained. A dynamical hybrid system integrating material balance equations, EMs reactions stoichiometry and kinetics was formulated. EMs kinetics were defined as the product of two terms: a mechanistic/empirical known term and an unknown term that must be identified from data, in a process optimisation perspective. This approach allows the quantification of fluxes carried by individual elementary modes which is of great help to identify dominant pathways as a function of environmental conditions. The methodology was employed to analyse experimental data of recombinant Baby Hamster Kidney (BHK-21A cultures producing a recombinant fusion glycoprotein. The identified EMs kinetics demonstrated typical glucose and glutamine metabolic responses during cell growth and IgG1-IL2 synthesis. Finally, an online optimisation study was conducted in which the optimal feeding strategies of glucose and glutamine were calculated after re-estimation of model parameters at each sampling time. An improvement in the final product concentration was obtained as a result of this online optimisation. Conclusion The main contribution of this work is a

  1. Performance Analysis and Modeling of a Tubular Staggered-Tooth Transverse-Flux PM Linear Machine

    Directory of Open Access Journals (Sweden)

    Shaohong Zhu

    2016-03-01

    Full Text Available This paper investigates the performance analysis and mathematical modeling of a staggered-tooth transverse-flux permanent magnet linear synchronous machine (STTF-PMLSM, which is characterized by simple structure and low flux leakage. Firstly, the structure advantages and operation principle of the STTF-PMLSM are introduced, and a simplified one phase model is established to investigate the performance of the machine in order to save the computation time. Then, the electromagnetic characteristics, including no-load flux linkage, electromotive force (EMF, inductance, detent force and thrust force, are simulated and analyzed in detail. After that, the theoretical analysis of the detent force, thrust force, and power factor are carried out. And the theoretical analysis results are validated with 3-D finite-element method (FEM. Finally, an improved mathematical model of the machine based on d-q rotating coordinate system is proposed, in which inductance harmonics and coupling between d- and q-axis inductance is considered. The results from the proposed mathematical model are in accordance with the results from 3-D FEM, which proves the validity and effectiveness of the proposed mathematical model. This provides a powerful foundation for the control of the machine.

  2. Uncertainty analysis of the Operational Simplified Surface Energy Balance (SSEBop) model at multiple flux tower sites

    Science.gov (United States)

    Chen, Mingshi; Senay, Gabriel B.; Singh, Ramesh K.; Verdin, James P.

    2016-01-01

    Evapotranspiration (ET) is an important component of the water cycle – ET from the land surface returns approximately 60% of the global precipitation back to the atmosphere. ET also plays an important role in energy transport among the biosphere, atmosphere, and hydrosphere. Current regional to global and daily to annual ET estimation relies mainly on surface energy balance (SEB) ET models or statistical and empirical methods driven by remote sensing data and various climatological databases. These models have uncertainties due to inevitable input errors, poorly defined parameters, and inadequate model structures. The eddy covariance measurements on water, energy, and carbon fluxes at the AmeriFlux tower sites provide an opportunity to assess the ET modeling uncertainties. In this study, we focused on uncertainty analysis of the Operational Simplified Surface Energy Balance (SSEBop) model for ET estimation at multiple AmeriFlux tower sites with diverse land cover characteristics and climatic conditions. The 8-day composite 1-km MODerate resolution Imaging Spectroradiometer (MODIS) land surface temperature (LST) was used as input land surface temperature for the SSEBop algorithms. The other input data were taken from the AmeriFlux database. Results of statistical analysis indicated that the SSEBop model performed well in estimating ET with an R2 of 0.86 between estimated ET and eddy covariance measurements at 42 AmeriFlux tower sites during 2001–2007. It was encouraging to see that the best performance was observed for croplands, where R2 was 0.92 with a root mean square error of 13 mm/month. The uncertainties or random errors from input variables and parameters of the SSEBop model led to monthly ET estimates with relative errors less than 20% across multiple flux tower sites distributed across different biomes. This uncertainty of the SSEBop model lies within the error range of other SEB models, suggesting systematic error or bias of the SSEBop model is within

  3. Squeezing Flux Out of Fat

    DEFF Research Database (Denmark)

    Gonzalez-Franquesa, Alba; Patti, Mary-Elizabeth

    2018-01-01

    Merging transcriptomics or metabolomics data remains insufficient for metabolic flux estimation. Ramirez et al. integrate a genome-scale metabolic model with extracellular flux data to predict and validate metabolic differences between white and brown adipose tissue. This method allows both metab...

  4. Use of CITATION code for flux calculation in neutron activation analysis with voluminous sample using an Am-Be source

    International Nuclear Information System (INIS)

    Khelifi, R.; Idiri, Z.; Bode, P.

    2002-01-01

    The CITATION code based on neutron diffusion theory was used for flux calculations inside voluminous samples in prompt gamma activation analysis with an isotopic neutron source (Am-Be). The code uses specific parameters related to the energy spectrum source and irradiation system materials (shielding, reflector). The flux distribution (thermal and fast) was calculated in the three-dimensional geometry for the system: air, polyethylene and water cuboidal sample (50x50x50 cm). Thermal flux was calculated in a series of points inside the sample. The results agreed reasonably well with observed values. The maximum thermal flux was observed at a distance of 3.2 cm while CITATION gave 3.7 cm. Beyond a depth of 7.2 cm, the thermal flux to fast flux ratio increases up to twice and allows us to optimise the detection system position in the scope of in-situ PGAA

  5. Modeling and Robustness Analysis of Biochemical Networks of Glycerol Metabolism by Klebsiella Pneumoniae

    Science.gov (United States)

    Ye, Jianxiong; Feng, Enmin; Wang, Lei; Xiu, Zhilong; Sun, Yaqin

    Glycerol bioconversion to 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae (K. pneumoniae) can be characterized by an intricate network of interactions among biochemical fluxes, metabolic compounds, key enzymes and genetic regulatory. To date, there still exist some uncertain factors in this complex network because of the limitation in bio-techniques, especially in measuring techniques for intracellular substances. In this paper, among these uncertain factors, we aim to infer the transport mechanisms of glycerol and 1,3-PD across the cell membrane, which have received intensive interest in recent years. On the basis of different inferences of the transport mechanisms, we reconstruct various metabolic networks correspondingly and subsequently develop their dynamical systems (S-systems). To determine the most reasonable metabolic network from all possible ones, we establish a quantitative definition of biological robustness and undertake parameter identification and robustness analysis for each system. Numerical results show that it is most possible that both glycerol and 1,3-PD pass the cell membrane by active transport and passive diffusion.

  6. A comparative meta-analysis of maximal aerobic metabolism of vertebrates: implications for respiratory and cardiovascular limits to gas exchange.

    Science.gov (United States)

    Hillman, Stanley S; Hancock, Thomas V; Hedrick, Michael S

    2013-02-01

    Maximal aerobic metabolic rates (MMR) in vertebrates are supported by increased conductive and diffusive fluxes of O(2) from the environment to the mitochondria necessitating concomitant increases in CO(2) efflux. A question that has received much attention has been which step, respiratory or cardiovascular, provides the principal rate limitation to gas flux at MMR? Limitation analyses have principally focused on O(2) fluxes, though the excess capacity of the lung for O(2) ventilation and diffusion remains unexplained except as a safety factor. Analyses of MMR normally rely upon allometry and temperature to define these factors, but cannot account for much of the variation and often have narrow phylogenetic breadth. The unique aspect of our comparative approach was to use an interclass meta-analysis to examine cardio-respiratory variables during the increase from resting metabolic rate to MMR among vertebrates from fish to mammals, independent of allometry and phylogeny. Common patterns at MMR indicate universal principles governing O(2) and CO(2) transport in vertebrate cardiovascular and respiratory systems, despite the varied modes of activities (swimming, running, flying), different cardio-respiratory architecture, and vastly different rates of metabolism (endothermy vs. ectothermy). Our meta-analysis supports previous studies indicating a cardiovascular limit to maximal O(2) transport and also implicates a respiratory system limit to maximal CO(2) efflux, especially in ectotherms. Thus, natural selection would operate on the respiratory system to enhance maximal CO(2) excretion and the cardiovascular system to enhance maximal O(2) uptake. This provides a possible evolutionary explanation for the conundrum of why the respiratory system appears functionally over-designed from an O(2) perspective, a unique insight from previous work focused solely on O(2) fluxes. The results suggest a common gas transport blueprint, or Bauplan, in the vertebrate clade.

  7. Transcriptomics, Targeted Metabolomics and Gene Expression of Blackberry Leaves and Fruits Indicate Flavonoid Metabolic Flux from Leaf to Red Fruit.

    Science.gov (United States)

    Gutierrez, Enrique; García-Villaraco, Ana; Lucas, José A; Gradillas, Ana; Gutierrez-Mañero, F Javier; Ramos-Solano, Beatriz

    2017-01-01

    Blackberries ( Rubus spp.) are among the high added value food products relevant for human health due to the increasing evidence of the beneficial effects of polyphenols, which are very abundant in these fruits. Interestingly, these compounds also play a role on plant physiology, being especially relevant their role in plant defense against biotic and abiotic stress. Hence, we hypothesize that since blackberry fruits have high amounts of flavonols and anthocyanins, leaves would also have high amounts of these compounds, and can be studied as a source of active molecules; furthermore, leaf synthesis would support their high contents in fruits. To explore this hypothesis, the present study reports a de novo transcriptome analysis on field grown blackberry leaves and fruits at the same time point, to establish the metabolic relationship of these compounds in both organs. Transcripts were aligned against Fragaria vesca genome, and genes were identified and annotated in different databases; tissue expression pattern showed 20,463 genes common to leaves and fruits, while 6,604 genes were significantly overexpressed only in fruits, while another 6,599 genes were significantly overexpressed in leaves, among which flavonol-anthocyanin transporter genes were present. Bioactives characterization indicated that total phenolics in leaves were three-fold, and flavonols were six-fold than in fruits, while concentration of anthocyanins was higher in fruits; HPLC-MS analysis indicated different composition in leaves and fruits, with cyanidin-3-glucoside as the only common compound identified. Next, RT-qPCR of the core genes in the flavonol anthocyanin pathway and regulatory MYB genes were carried out. Interestingly, genes in the flavonol-anthocyanin pathway and flavonol-transport families were overexpressed in leaves, consistent with the higher bioactive levels. On the other hand, transcription factors were overexpressed in fruits anticipating an active anthocyanin biosynthesis

  8. Thermal-hydraulic analysis of a 600 MW supercritical CFB boiler with low mass flux

    International Nuclear Information System (INIS)

    Pan Jie; Yang Dong; Chen Gongming; Zhou Xu; Bi Qincheng

    2012-01-01

    Supercritical Circulating Fluidized Bed (CFB) boiler becomes an important development trend for coal-fired power plant and thermal-hydraulic analysis is a key factor for the design and operation of water wall. According to the boiler structure and furnace-sided heat flux, the water wall system of a 600 MW supercritical CFB boiler is treated in this paper as a flow network consisting of series-parallel loops, pressure grids and connecting tubes. A mathematical model for predicting the thermal-hydraulic characteristics in boiler heating surface is based on the mass, momentum and energy conservation equations of these components, which introduces numerous empirical correlations available for heat transfer and hydraulic resistance calculation. Mass flux distribution and pressure drop data in the water wall at 30%, 75% and 100% of the boiler maximum continuous rating (BMCR) are obtained by iteratively solving the model. Simultaneity, outlet vapor temperatures and metal temperatures in water wall tubes are estimated. The results show good heat transfer performance and low flow resistance, which implies that the water wall design of supercritical CFB boiler is applicable. - Highlights: → We proposed a model for thermal-hydraulic analysis of boiler heating surface. → The model is applied in a 600 MW supercritical CFB boiler. → We explore the pressure drop, mass flux and temperature distribution in water wall. → The operating safety of boiler is estimated. → The results show good heat transfer performance and low flow resistance.

  9. Quantitative analysis of bidirectional electron fluxes within coronal mass ejections at 1 AU

    Science.gov (United States)

    Phillips, J. L.; Gosling, J. T.; Mccomas, D. J.; Bame, S. J.; Feldman, W. C.

    1992-01-01

    The solar wind electron heat flux is carried primarily by suprathermal electrons beamed antisunward along the interplanetary magnetic field. However, analysis of electron observations at 1 AU has shown that counterstreaming electron beams, suggesting closed magnetic structures, prevail within coronal mass ejections (CMEs). These structures might be magnetic 'tongues', magnetically detached plasmoids, or complex flux ropes. Here we show results of analysis of ISEE-3 observations within 39 CMEs, including the asymmetry between the two beams, its control by magnetic field orientation, and the variation of the electron distributions as CMEs convect past the spacecraft. We find that some CMEs are strongly asymmetric, with the antisunward beam generally dominant, while others contain nearly symmetric beams. The beam asymmetries, and the magnetic field orientations, exhibit characteristic trends as CMEs pass over the spacecraft. We present an example of a distinctive 'strahl-on-strahl' distribution, suggesting continued magnetic connection to the corona, in which a narrow antisunward beam is superimposed on a broader beam. Our results favor continuing magnetic connection to the Sun in a tongue or flux rope geometry rather than a fully detached plasmoid.

  10. #FluxFlow: Visual Analysis of Anomalous Information Spreading on Social Media.

    Science.gov (United States)

    Zhao, Jian; Cao, Nan; Wen, Zhen; Song, Yale; Lin, Yu-Ru; Collins, Christopher

    2014-12-01

    We present FluxFlow, an interactive visual analysis system for revealing and analyzing anomalous information spreading in social media. Everyday, millions of messages are created, commented, and shared by people on social media websites, such as Twitter and Facebook. This provides valuable data for researchers and practitioners in many application domains, such as marketing, to inform decision-making. Distilling valuable social signals from the huge crowd's messages, however, is challenging, due to the heterogeneous and dynamic crowd behaviors. The challenge is rooted in data analysts' capability of discerning the anomalous information behaviors, such as the spreading of rumors or misinformation, from the rest that are more conventional patterns, such as popular topics and newsworthy events, in a timely fashion. FluxFlow incorporates advanced machine learning algorithms to detect anomalies, and offers a set of novel visualization designs for presenting the detected threads for deeper analysis. We evaluated FluxFlow with real datasets containing the Twitter feeds captured during significant events such as Hurricane Sandy. Through quantitative measurements of the algorithmic performance and qualitative interviews with domain experts, the results show that the back-end anomaly detection model is effective in identifying anomalous retweeting threads, and its front-end interactive visualizations are intuitive and useful for analysts to discover insights in data and comprehend the underlying analytical model.

  11. Evolution of amino acid metabolism inferred through cladistic analysis.

    Science.gov (United States)

    Cunchillos, Chomin; Lecointre, Guillaume

    2003-11-28

    Because free amino acids were most probably available in primitive abiotic environments, their metabolism is likely to have provided some of the very first metabolic pathways of life. What were the first enzymatic reactions to emerge? A cladistic analysis of metabolic pathways of the 16 aliphatic amino acids and 2 portions of the Krebs cycle was performed using four criteria of homology. The analysis is not based on sequence comparisons but, rather, on coding similarities in enzyme properties. The properties used are shared specific enzymatic activity, shared enzymatic function without substrate specificity, shared coenzymes, and shared functional family. The tree shows that the earliest pathways to emerge are not portions of the Krebs cycle but metabolisms of aspartate, asparagine, glutamate, and glutamine. The views of Horowitz (Horowitz, N. H. (1945) Proc. Natl. Acad. Sci. U. S. A. 31, 153-157) and Cordón (Cordón, F. (1990) Tratado Evolucionista de Biologia, Aguilar, Madrid, Spain), according to which the upstream reactions in the catabolic pathways and the downstream reactions in the anabolic pathways are the earliest in evolution, are globally corroborated; however, with some exceptions. These are due to later opportunistic connections of pathways (actually already suggested by these authors). Earliest enzymatic functions are mostly catabolic; they were deaminations, transaminations, and decarboxylations. From the consensus tree we extracted four time spans for amino acid metabolism development. For some amino acids catabolism and biosynthesis occurred at the same time (Asp, Glu, Lys, Leu, Ala, Val, Ile, Pro, Arg). For others ultimate reactions that use amino acids as a substrate or as a product are distinct in time, with catabolism preceding anabolism for Asn, Gln, and Cys and anabolism preceding catabolism for Ser, Met, and Thr. Cladistic analysis of the structure of biochemical pathways makes hypotheses in biochemical evolution explicit and parsimonious.

  12. Accounting for the thermal neutron flux depression in voluminous samples for instrumental neutron activation analysis

    International Nuclear Information System (INIS)

    Overwater, R.M.W.; Hoogenboom, J.E.

    1994-01-01

    At the Delft University of Technology Interfaculty Reactor Institute, a facility has been installed to irradiate cylindrical samples with diameters up to 15 cm and weights up to 50 kg for instrumental neutron activation analysis (INAA) purposes. To be able to do quantitative INAA on voluminous samples, it is necessary to correct for gamma-ray absorption, gamma-ray scattering, neutron absorption, and neutron scattering in the sample. The neutron absorption and the neutron scattering are discussed. An analytical solution is obtained for the diffusion equation in the geometry of the irradiation facility. For samples with known composition, the neutron flux--as a function of position in the sample--can be calculated directly. Those of unknown composition require additional flux measurements on which least-squares fitting must be done to obtain both the thermal neutron diffusion coefficient D s and the diffusion length L s of the sample. Experiments are performed to test the theory

  13. Optimization of a blueprint for in vitro glycolysis by metabolic real-time analysis.

    Science.gov (United States)

    Bujara, Matthias; Schümperli, Michael; Pellaux, René; Heinemann, Matthias; Panke, Sven

    2011-05-01

    Recruiting complex metabolic reaction networks for chemical synthesis has attracted considerable attention but frequently requires optimization of network composition and dynamics to reach sufficient productivity. As a design framework to predict optimal levels for all enzymes in the network is currently not available, state-of-the-art pathway optimization relies on high-throughput phenotype screening. We present here the development and application of a new in vitro real-time analysis method for the comprehensive investigation and rational programming of enzyme networks for synthetic tasks. We used this first to rationally and rapidly derive an optimal blueprint for the production of the fine chemical building block dihydroxyacetone phosphate (DHAP) via Escherichia coli's highly evolved glycolysis. Second, the method guided the three-step genetic implementation of the blueprint, yielding a synthetic operon with the predicted 2.5-fold-increased glycolytic flux toward DHAP. The new analytical setup drastically accelerates rational optimization of synthetic multienzyme networks.

  14. Genome-scale metabolic modeling and in silico analysis of lipid accumulating yeast Candida tropicalis for dicarboxylic acid production.

    Science.gov (United States)

    Mishra, Pranjul; Park, Gyu-Yeon; Lakshmanan, Meiyappan; Lee, Hee-Seok; Lee, Hongweon; Chang, Matthew Wook; Ching, Chi Bun; Ahn, Jungoh; Lee, Dong-Yup

    2016-09-01

    Recently, the bio-production of α,ω-dicarboxylic acids (DCAs) has gained significant attention, which potentially leads to the replacement of the conventional petroleum-based products. In this regard, the lipid accumulating yeast Candida tropicalis, has been recognized as a promising microbial host for DCA biosynthesis: it possess the unique ω-oxidation pathway where the terminal carbon of α-fatty acids is oxidized to form DCAs with varying chain lengths. However, despite such industrial importance, its cellular physiology and lipid accumulation capability remain largely uncharacterized. Thus, it is imperative to better understand the metabolic behavior of this lipogenic yeast, which could be achieved by a systems biological approach. To this end, herein, we reconstructed the genome-scale metabolic model of C. tropicalis, iCT646, accounting for 646 unique genes, 945 metabolic reactions, and 712 metabolites. Initially, the comparative network analysis of iCT646 with other yeasts revealed several distinctive metabolic reactions, mainly within the amino acid and lipid metabolism including the ω-oxidation pathway. Constraints-based flux analysis was, then, employed to predict the in silico growth rates of C. tropicalis which are highly consistent with the cellular phenotype observed in glucose and xylose minimal media chemostat cultures. Subsequently, the lipid accumulation capability of C. tropicalis was explored in comparison with Saccharomyces cerevisiae, indicating that the formation of "citrate pyruvate cycle" is essential to the lipid accumulation in oleaginous yeasts. The in silico flux analysis also highlighted the enhanced ability of pentose phosphate pathway as NADPH source rather than malic enzyme during lipogenesis. Finally, iCT646 was successfully utilized to highlight the key directions of C. tropicalis strain design for the whole cell biotransformation application to produce long-chain DCAs from alkanes. Biotechnol. Bioeng. 2016;113: 1993-2004.

  15. Analysis of Subclinical Hyperthyroidism Influence on Parameters of Bone Metabolism

    Directory of Open Access Journals (Sweden)

    I.V. Pankiv

    2016-03-01

    Full Text Available State of subclinical hypothyroidism can be considered as the optimal model for assessing the significance of thyroid stimulating hormone (TSH for bone tissue in clinical practice. Objective: to make a comparative analysis of the impact of subclinical hyperthyroidism of various origins on the performance of bone mineral density (BMD and bone metabolism parameters. Materials and methods. The study in an outpatient setting included 112 women with a diagnosis of subclinical hyperthyroidism and duration of menopause for at least 5 years. Among the examinees, endogenous subclinical hyperthyroidism has been detected in 78 women (group I, exogenous subclinical hyperthyroidism on the background of suppressive levothyroxine therapy (group II — in 34. The control group (group III included 20 women without thyroid dysfunction. Results. The study first conducted a comparative analysis of bone metabolism, BMD indicators, as well as parameters of phosphorus and calcium, blood lipids in women with subclinical hyperthyroidism of various origins. A positive correlation between markers of bone metabolism and free triiodothyronine (fT3 as hormones necessary for the development of the skeleton and to maintain its homeostasis indicates a physiological effect of parathyroid hormone and fT3 on bone tissue. It is shown that the bone metabolism and BMD depend not only on the content of TSH, but also on the causes of subclinical hyperthyroidism.Conclusions. In postmenopausal women with endogenous subclinical hyperthyroidism, there is a significant decline in BMD indices, more pronounced in the bones with the cortical structure. A negative correlation between markers of bone metabolism and TSH has been observed among all patients included in the study.

  16. A computer program for the algebraic determination of control coefficients in Metabolic Control Analysis.

    OpenAIRE

    Thomas, S; Fell, D A

    1993-01-01

    A computer program (MetaCon) is described for the evaluation of flux control, concentration control and branch-point distribution control coefficients of a metabolic pathway. Requiring only the reaction scheme as input, the program produces algebraic expressions for the control coefficients in terms of elasticity coefficients, metabolite concentrations and pathway fluxes. Any of these variables can be substituted by numeric or simple algebraic expressions; the expressions will then be automat...

  17. Core Fueling and Edge Particle Flux Analysis in Ohmically and Auxiliary Heated NSTX Plasmas

    Energy Technology Data Exchange (ETDEWEB)

    V.A. Soukhanovskii; R. Maingi; R. Raman; H.W. Kugel; B.P. LeBlanc; L. Roquemore; C.H. Skinner; NSTX Research Team

    2002-06-12

    The Boundary Physics program of the National Spherical Torus Experiment (NSTX) is focusing on optimization of the edge power and particle flows in b * 25% L- and H-mode plasmas of t {approx} 0.8 s duration heated by up to 6 MW of high harmonic fast wave and up to 5 MW of neutral beam injection. Particle balance and core fueling efficiencies of low and high field side gas fueling of L-mode homic and NBI heated plasmas have been compared using an analytical zero dimensional particle balance model and measured ion and neutral fluxes. Gas fueling efficiencies are in the range of 0.05-0.20 and do not depend on discharge magnetic configuration, density or poloidal location of the injector. The particle balance modeling indicates that the addition of HFS fueling results in a reversal of the wall loading rate and higher wall inventories. Initial particle source estimates obtained from neutral pressure and spectroscopic measurements indicate that ion flux into the divertor greatly exceeds midplane ion flux from the main plasma, suggesting that the scrape-off cross-field transport plays a minor role in diverted plasmas. Present analysis provides the basis for detailed fluid modeling of core and edge particle flows and particle confinement properties of NSTX plasmas. This research was supported by the U.S. Department of Energy under contracts No. DE-AC02-76CH03073, DE-AC05-00OR22725, and W-7405-ENG-36.

  18. Thermodynamic Analysis for the Refining Ability of Salt Flux for Aluminum Recycling

    Directory of Open Access Journals (Sweden)

    Takehito Hiraki

    2014-07-01

    Full Text Available The removability of impurities during the aluminum remelting process by oxidation was previously investigated by our research group. In the present work, alternative impurity removal with chlorination has been evaluated by thermodynamic analysis. For 43 different elements, equilibrium distribution ratios among metal, chloride flux and oxide slag phases in the aluminum remelting process were calculated by assuming the binary systems of aluminum and an impurity element. It was found that the removability of impurities isn’t significantly affected by process parameters such as chloride partial pressure, temperature and flux composition. It was shown that Ho, Dy, Li, La, Mg, Gd, Ce, Yb, Ca and Sr can be potentially eliminated into flux by chlorination from the remelted aluminum. Chlorination and oxidation are not effective to remove other impurities from the melting aluminum, due to the limited parameters which can be controlled during the remelting process. It follows that a proper management of aluminum scrap such as sorting based on the composition of the products is important for sustainable aluminum recycling.

  19. Thermodynamic Analysis for the Refining Ability of Salt Flux for Aluminum Recycling.

    Science.gov (United States)

    Hiraki, Takehito; Miki, Takahiro; Nakajima, Kenichi; Matsubae, Kazuyo; Nakamura, Shinichiro; Nagasaka, Tetsuya

    2014-07-30

    The removability of impurities during the aluminum remelting process by oxidation was previously investigated by our research group. In the present work, alternative impurity removal with chlorination has been evaluated by thermodynamic analysis. For 43 different elements, equilibrium distribution ratios among metal, chloride flux and oxide slag phases in the aluminum remelting process were calculated by assuming the binary systems of aluminum and an impurity element. It was found that the removability of impurities isn't significantly affected by process parameters such as chloride partial pressure, temperature and flux composition. It was shown that Ho, Dy, Li, La, Mg, Gd, Ce, Yb, Ca and Sr can be potentially eliminated into flux by chlorination from the remelted aluminum. Chlorination and oxidation are not effective to remove other impurities from the melting aluminum, due to the limited parameters which can be controlled during the remelting process. It follows that a proper management of aluminum scrap such as sorting based on the composition of the products is important for sustainable aluminum recycling.

  20. Thermodynamic Analysis for the Refining Ability of Salt Flux for Aluminum Recycling

    Science.gov (United States)

    Hiraki, Takehito; Miki, Takahiro; Nakajima, Kenichi; Matsubae, Kazuyo; Nakamura, Shinichiro; Nagasaka, Tetsuya

    2014-01-01

    The removability of impurities during the aluminum remelting process by oxidation was previously investigated by our research group. In the present work, alternative impurity removal with chlorination has been evaluated by thermodynamic analysis. For 43 different elements, equilibrium distribution ratios among metal, chloride flux and oxide slag phases in the aluminum remelting process were calculated by assuming the binary systems of aluminum and an impurity element. It was found that the removability of impurities isn’t significantly affected by process parameters such as chloride partial pressure, temperature and flux composition. It was shown that Ho, Dy, Li, La, Mg, Gd, Ce, Yb, Ca and Sr can be potentially eliminated into flux by chlorination from the remelted aluminum. Chlorination and oxidation are not effective to remove other impurities from the melting aluminum, due to the limited parameters which can be controlled during the remelting process. It follows that a proper management of aluminum scrap such as sorting based on the composition of the products is important for sustainable aluminum recycling. PMID:28788144

  1. Stability analysis of feedforward anticipation optimal flux difference in traffic lattice hydrodynamic theory

    Science.gov (United States)

    Sun, Di-Hua; Zhang, Geng; Zhao, Min; Cheng, Sen-Lin; Cao, Jian-Dong

    2018-03-01

    Recently, the influence of driver's individual behaviors on traffic stability is research hotspot with the fasting developing transportation cyber-physical systems. In this paper, a new traffic lattice hydrodynamic model is proposed with consideration of driver's feedforward anticipation optimal flux difference. The neutral stability condition of the new model is obtained through linear stability analysis theory. The results show that the stable region will be enlarged on the phase diagram when the feedforward anticipation optimal flux difference effect is taken into account. In order to depict traffic jamming transition properties theoretically, the mKdV equation near the critical point is derived via nonlinear reductive perturbation method. The propagation behavior of traffic density waves can be described by the kink-antikink solution of the mKdV equation. Numerical simulations are conducted to verify the analytical results and all the results confirms that traffic stability can be enhanced significantly by considering the feedforward anticipation optimal flux difference in traffic lattice hydrodynamic theory.

  2. Flux and energy analysis of species in hollow cathode magnetron ionized physical vapor deposition of copper

    International Nuclear Information System (INIS)

    Wu, L.; Ko, E.; Dulkin, A.; Park, K. J.; Fields, S.; Leeser, K.; Meng, L.; Ruzic, D. N.

    2010-01-01

    To meet the stringent requirements of interconnect metallization for sub-32 nm technologies, an unprecedented level of flux and energy control of film forming species has become necessary to further advance ionized physical vapor deposition technology. Such technology development mandates improvements in methods to quantify the metal ion fraction, the gas/metal ion ratio, and the associated ion energies in the total ion flux to the substrate. In this work, a novel method combining planar Langmuir probes, quartz crystal microbalance (QCM), and gridded energy analyzer (GEA) custom instrumentation is developed to estimate the plasma density and temperature as well as to measure the metal ion fraction and ion energy. The measurements were conducted in a Novellus Systems, Inc. Hollow Cathode Magnetron (HCM TM ) physical vapor deposition source used for deposition of Cu seed layer for 65-130 nm technology nodes. The gridded energy analyzer was employed to measure ion flux and ion energy, which was compared to the collocated planar Langmuir probe data. The total ion-to-metal neutral ratio was determined by the QCM combined with GEA. The data collection technique and the corresponding analysis are discussed. The effect of concurrent resputtering during the deposition process on film thickness profile is also discussed.

  3. Analysis of the influence of environmental parameters on methane flux from floodplains and lakes in the Abobral River, Pantanal, Brazil

    Directory of Open Access Journals (Sweden)

    Marcelo Gomes da Silva

    2016-04-01

    Full Text Available This study evaluates the influence of physical and chemical characteristics of the water column in the processes that result in methane emissions in the Pantanal. Two surveys were conducted in 2009 and 2010, corresponding to the drought and flood seasons, respectively. The CH4 fluxes were determined using the static chamber technique. Environmental variables, such as depth, water temperature, pH, redox potential, dissolved oxygen and total dissolved solids were also measured. Diffusive and bubble flux presented an average value of 11.1 ± 13.5 mg CH4 m-2 d-1 e 275.9 ± 348.5 mg CH4 m-2 d-1, which is a value near those observed in other tropical flooded regions. Statistical t-tests have shown significant differences between drought and flood seasons (p ≤ 0.05. Diffusive fluxes represented about 87% of the total fluxes measured in drought. During the flood season there were more instances of bubble fluxes (77%. Statistical analysis (correlation and Principal Components Analysis indicate that physical and chemical characteristics could affect methane fluxes. Diffusive fluxes correlated mainly with pH, redox potential and dissolved oxygen, which is in accordance with the increase of organic matter during flooding. Bubble fluxes measured during the drought season correlated mainly with water depth and temperature, pH and redox potential, which is an indication of an environment to suitable to the formation and liberation of the bubbles from sediment.

  4. Lidar and Hyperspectral Remote Sensing for the Analysis of Coniferous Biomass Stocks and Fluxes

    Science.gov (United States)

    Halligan, K. Q.; Roberts, D. A.

    2006-12-01

    Airborne lidar and hyperspectral data can improve estimates of aboveground carbon stocks and fluxes through their complimentary responses to vegetation structure and biochemistry. While strong relationships have been demonstrated between lidar-estimated vegetation structural parameters and field data, research is needed to explore the portability of these methods across a range of topographic conditions, disturbance histories, vegetation type and climate. Additionally, research is needed to evaluate contributions of hyperspectral data in refining biomass estimates and determination of fluxes. To address these questions we are a conducting study of lidar and hyperspectral remote sensing data across sites including coniferous forests, broadleaf deciduous forests and a tropical rainforest. Here we focus on a single study site, Yellowstone National Park, where tree heights, stem locations, above ground biomass and basal area were mapped using first-return small-footprint lidar data. A new method using lidar intensity data was developed for separating the terrain and vegetation components in lidar data using a two-scale iterative local minima filter. Resulting Digital Terrain Models (DTM) and Digital Canopy Models (DCM) were then processed to retrieve a diversity of vertical and horizontal structure metrics. Univariate linear models were used to estimate individual tree heights while stepwise linear regression was used to estimate aboveground biomass and basal area. Three small-area field datasets were compared for their utility in model building and validation of vegetation structure parameters. All structural parameters were linearly correlated with lidar-derived metrics, with higher accuracies obtained where field and imagery data were precisely collocated . Initial analysis of hyperspectral data suggests that vegetation health metrics including measures of live and dead vegetation and stress indices may provide good indicators of carbon flux by mapping vegetation

  5. Metabolic network analysis of Bacillus clausii on minimal and semirich medium using C-13-Labeled glucose

    DEFF Research Database (Denmark)

    Christiansen, Torben; Christensen, Bjarke; Nielsen, Jens

    2002-01-01

    to increase with increasing specific growth rate but at a much lower level than previously reported for Bacillus subtilis. Two futile cycles in the pyruvate metabolism were included in the metabolic network. A substantial flux in the futile cycle involving malic enzyme was estimated, whereas only a very small...... or zero flux through PEP carboxykinase was estimated, indicating that the latter enzyme was not active during growth on glucose. The uptake of the amino acids in a semirich medium containing 15 of the 20 amino acids normally present in proteins was estimated using fully labeled glucose in batch...

  6. Metabolic Flux Between Unsaturated and Saturated Fatty Acids is Controlled by the FabA:FabB Ratio in the Fully Reconstituted Fatty Acid Biosynthetic Pathway of E. coli#

    Science.gov (United States)

    Xiao, Xirui; Yu, Xingye; Khosla, Chaitan

    2013-01-01

    The entire fatty acid biosynthetic pathway from Escherichia coli, starting from the acetyl-CoA carboxylase, has been reconstituted in vitro from fourteen purified protein components. Radiotracer analysis verified stoichiometric conversion of acetyl-CoA and NAD(P)H into the free fatty acid product, allowing implementation of a facile spectrophotometric assay for kinetic analysis of this multi-enzyme system. At steady state, a maximum turnover rate of 0.5 s−1 was achieved. Under optimal turnover conditions, the predominant products were C16 and C18 saturated as well as monounsaturated fatty acids. The reconstituted system allowed us to quantitatively interrogate the factors that influence metabolic flux toward unsaturated versus saturated fatty acids. In particular, the concentrations of the dehydratase FabA and the β-ketoacyl synthase FabB were found to be crucial for controlling this property. By altering these variables, the percentage of unsaturated fatty acid produced could be adjusted between 10 and 50% without significantly affecting the maximum turnover rate of the pathway. Our reconstituted system provides a powerful tool to understand and engineer rate-limiting and regulatory steps in this complex and practically significant metabolic pathway. PMID:24147979

  7. Metabolic flux between unsaturated and saturated fatty acids is controlled by the FabA:FabB ratio in the fully reconstituted fatty acid biosynthetic pathway of Escherichia coli.

    Science.gov (United States)

    Xiao, Xirui; Yu, Xingye; Khosla, Chaitan

    2013-11-19

    The entire fatty acid biosynthetic pathway of Escherichia coli, starting from the acetyl-CoA carboxylase, has been reconstituted in vitro from 14 purified protein components. Radiotracer analysis verified stoichiometric conversion of acetyl-CoA and NAD(P)H to the free fatty acid product, allowing implementation of a facile spectrophotometric assay for kinetic analysis of this multienzyme system. At steady state, a maximal turnover rate of 0.5 s(-1) was achieved. Under optimal turnover conditions, the predominant products were C16 and C18 saturated as well as monounsaturated fatty acids. The reconstituted system allowed us to quantitatively interrogate the factors that influence metabolic flux toward unsaturated versus saturated fatty acids. In particular, the concentrations of the dehydratase FabA and the β-ketoacyl synthase FabB were found to be crucial for controlling this property. Via changes in these variables, the percentage of unsaturated fatty acid produced could be adjusted between 10 and 50% without significantly affecting the maximal turnover rate of the pathway. Our reconstituted system provides a powerful tool for understanding and engineering rate-limiting and regulatory steps in this complex and practically significant metabolic pathway.

  8. Uncertainties in measuring trace amounts of cobalt and europium with low-flux neutron activation analysis

    Directory of Open Access Journals (Sweden)

    Burnham Steven

    2017-01-01

    Full Text Available Neutron activation analysis is widely used for identification of elements and their quantities even in trace amounts in the samples of almost any type. The challenges in detecting trace amounts of particular elements are often associated with the neutron flux produced at the research reactors. Low-flux neutron activation analysis usually presents the biggest challenge when analyzing trace quantities of elements with lower magnitude of radiative capture cross-sections. In this paper, we present the methodology and the quantified uncertainties associated with the detection of trace amounts of cobalt and europium, using as an example concrete aggregates. Recent growing interest is in improving structural concrete (increasing its strength but reducing its activation in nuclear power plant environments. Aside from buildings, structural concrete is also used as a biological shield in nuclear power plant that become radioactive after exposure to neutron flux. Due to radiative capture interactions, artificial radionuclides are generated to high enough concentrations that classify concrete as low-level radioactive waste at the time of the plant's decommissioning. Disposal of this concrete adds to the expense of nuclear power plant financing and its construction. Three radionuclides, 60Co, 152Eu, and 154Eu, account for 99 % of total residual radioactivity of nuclear power plant decommissioned concrete. IAEA document RS-G-1.7, Application of the Concepts of Exclusion, Exemption, and Clearance, specifies clearance levels of radionuclides specific activities: a specific activity lower than 0.1 Bqg-1 for 60Co and 152Eu, and 154Eu allows for a concrete to be recycled after decommissioning of the nuclear power plant. Therefore, low-flux neutron activation analysis is used to test the detection limits of trace elements in samples of cement, coarse, and fine concrete aggregates. These samples are irradiated at the University of Utah's 100 kW TRIGA Reactor at

  9. Permanent Magnet Flux-Switching Machine, Optimal Design and Performance Analysis

    Directory of Open Access Journals (Sweden)

    Liviu Emilian Somesan

    2013-01-01

    Full Text Available In this paper an analytical sizing-design procedure for a typical permanent magnet flux-switching machine (PMFSM with 12 stator and respectively 10 rotor poles is presented. An optimal design, based on Hooke-Jeeves method with the objective functions of maximum torque density, is performed. The results were validated via two dimensions finite element analysis (2D-FEA applied on the optimized structure. The influence of the permanent magnet (PM dimensions and type, respectively of the rotor poles' shape on the machine performance were also studied via 2D-FEA.

  10. Metabolism

    Science.gov (United States)

    ... functions: Anabolism (uh-NAB-uh-liz-um), or constructive metabolism, is all about building and storing. It ... in infants and young children. Hypothyroidism slows body processes and causes fatigue (tiredness), slow heart rate, excessive ...

  11. Metabolism

    Science.gov (United States)

    ... a particular food provides to the body. A chocolate bar has more calories than an apple, so ... acid phenylalanine, needed for normal growth and protein production). Inborn errors of metabolism can sometimes lead to ...

  12. Effects of Citrus aurantium (bitter orange) fruit extracts and p-synephrine on metabolic fluxes in the rat liver.

    Science.gov (United States)

    Peixoto, Jéssica Sereno; Comar, Jurandir Fernando; Moreira, Caroline Tessaro; Soares, Andréia Assunção; de Oliveira, Andrea Luiza; Bracht, Adelar; Peralta, Rosane Marina

    2012-05-16

    The fruit extracts of Citrus aurantium (bitter orange) are traditionally used as weight-loss products and as appetite supressants. An important fruit component is p-synephrine, which is structurally similar to the adrenergic agents. Weight-loss and adrenergic actions are always related to metabolic changes and this work was designed to investigate a possible action of the C. aurantium extract on liver metabolism. The isolated perfused rat liver was used to measure catabolic and anabolic pathways, including oxygen uptake and perfusion pressure. The C. aurantium extract and p-synephrine increased glycogenolysis, glycolysis, oxygen uptake and perfusion pressure. These changes were partly sensitive to α- and β-adrenergic antagonists. p-Synephrine (200 μM) produced an increase in glucose output that was only 15% smaller than the increment caused by the extract containing 196 μM p-synephrine. At low concentrations the C. aurantium extract tended to increase gluconeogenesis, but at high concentrations it was inhibitory, opposite to what happened with p-synephrine. The action of the C. aurantium extract on liver metabolism is similar to the well known actions of adrenergic agents and can be partly attributed to its content in p-synephrine. Many of these actions are catabolic and compatible with the weight-loss effects usually attributed to C. aurantium.

  13. Assessment of the W7-X high heat flux divertor with thermo-mechanical analysis

    International Nuclear Information System (INIS)

    Qian, Xinyuan; Peng, Xuebing; Fellinger, Joris; Boscary, Jean; Bykov, Victor; Wang, Zhongwei; Ye, Minyou; Song, Yuntao

    2016-01-01

    Highlights: • Thermo-mechanical analysis of HHF divertor module, TM2H. • Temperature of all parts is acceptable for long pulse operation. • Stress in different parts is mainly caused by different load. • Radial displacement need to be improved based on FE calculations. - Abstract: The Wendelstein 7-X is an experimental device designed with a stellarator magnetic confinement for stationary plasma operation (up to 30 min). At the first stage, it is scheduled to start with an inertially cooled test divertor unit and a shorter plasma pulse operation up to 10 s. After the completion of this stage, a water-cooled high heat flux (HHF) divertor will be installed for the steady-state operation phase. The divertor consists of individual target modules, which are sets of target elements armored with CFC tiles supported by a stainless steel structure and fed in parallel with manifolds. Detailed thermo-mechanical analysis of the target modules using the finite element method has been performed to validate and/or improve the elected design of the HHF divertor under operation. Different operating conditions have been studied and the effect of the variation of the convective heat flux pattern with localized heating loads as high as 10 MW/m 2 onto the target elements has been computed. The analysis of the thermal response, stress distribution and deformation allowed a better understanding of the behavior of the divertor modules under operation and confirmed the suitability of the design.

  14. Assessment of the W7-X high heat flux divertor with thermo-mechanical analysis

    Energy Technology Data Exchange (ETDEWEB)

    Qian, Xinyuan [School of Nuclear Science and Technology, University of Science and Technology of China, Jinzhai Road 96, 230026 Hefei,Anhui (China); Institute of Plasma Physics, Chinese Academy of Sciences, Shushanhu Road 350, 230031 Hefei, Anhui (China); Peng, Xuebing, E-mail: pengxb@ipp.ac.cn [Institute of Plasma Physics, Chinese Academy of Sciences, Shushanhu Road 350, 230031 Hefei, Anhui (China); Fellinger, Joris [Max Planck Institute for Plasma Physics, Wendelsteinstr. 1, 17491 Greifswald (Germany); Boscary, Jean [Max Planck Institute for Plasma Physics, Boltzmannstr. 2, 85748 Garching (Germany); Bykov, Victor [Max Planck Institute for Plasma Physics, Wendelsteinstr. 1, 17491 Greifswald (Germany); Wang, Zhongwei [Institute of Plasma Physics, Chinese Academy of Sciences, Shushanhu Road 350, 230031 Hefei, Anhui (China); Ye, Minyou; Song, Yuntao [School of Nuclear Science and Technology, University of Science and Technology of China, Jinzhai Road 96, 230026 Hefei,Anhui (China); Institute of Plasma Physics, Chinese Academy of Sciences, Shushanhu Road 350, 230031 Hefei, Anhui (China)

    2016-11-01

    Highlights: • Thermo-mechanical analysis of HHF divertor module, TM2H. • Temperature of all parts is acceptable for long pulse operation. • Stress in different parts is mainly caused by different load. • Radial displacement need to be improved based on FE calculations. - Abstract: The Wendelstein 7-X is an experimental device designed with a stellarator magnetic confinement for stationary plasma operation (up to 30 min). At the first stage, it is scheduled to start with an inertially cooled test divertor unit and a shorter plasma pulse operation up to 10 s. After the completion of this stage, a water-cooled high heat flux (HHF) divertor will be installed for the steady-state operation phase. The divertor consists of individual target modules, which are sets of target elements armored with CFC tiles supported by a stainless steel structure and fed in parallel with manifolds. Detailed thermo-mechanical analysis of the target modules using the finite element method has been performed to validate and/or improve the elected design of the HHF divertor under operation. Different operating conditions have been studied and the effect of the variation of the convective heat flux pattern with localized heating loads as high as 10 MW/m{sup 2} onto the target elements has been computed. The analysis of the thermal response, stress distribution and deformation allowed a better understanding of the behavior of the divertor modules under operation and confirmed the suitability of the design.

  15. redGEM: Systematic reduction and analysis of genome-scale metabolic reconstructions for development of consistent core metabolic models.

    Directory of Open Access Journals (Sweden)

    Meric Ataman

    2017-07-01

    Full Text Available 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.

  16. Some advance on the comprehension of SR analysis for estimating the flux of a scalar

    Science.gov (United States)

    Castellví, Dr

    2009-04-01

    In agronomy, the eddy covariance, EC, method likely is the preferred for measuring surface scalar fluxes. For latent heat flux, however, weighing lysimeters maybe preferred in agriculture, but they are rarely affordable and not portable. The dissipation method, DM, is considered the most reliable technique for measuring scalar fluxes over open water because instrument motion contaminates the EC measurements. The main advantage of DM over EC is that it is less sensitive to low frequency instrument platform motions (such as ship and buoys), sensor alignment, precise orientation and stringent steadiness in the mean meteorological conditions (Fairall and Larsen, 1986; Kader, 1992; Edson and Fairall, 1998). Over land, keeping in mind that the EC and DM methods require the same measurements for scalar flux measurement, the DM has several disadvantages versus the EC. Direct measurement of the scalar variance dissipation rate, VDR, requires to capture eddies in the Kolmogorov's microscale (thus scalar time series measured at frequencies in the order of kHz are needed). Therefore, it is not practical. Indirect methods to estimate VDR (such as spectral analysis and second or third order structure functions) requires implementing iterative methods involving similarity relationships that are not well established (Hsieh and Katul, 1997; Castellvi and Snyder, 2008). Currently, there is ample evidence that the DM as explained in traditional micrometeorological books (such as, Panofsky and Dutton, 1984; Brutsaert, 1988; Kaimal and Finnigan; 1994) is, in general, not correct. Accordingly, it likely explains why DM is typically omitted in revisits of micrometeorological methods for estimating scalar fluxes in agronomy. Within the last decade, over some agricultural surfaces, evidence has been shown on the advantages over other micrometeorological methods and the reliability (i.e., close performance to the EC method) of Surface Renewal, SR, theory in conjunction with the Analysis of

  17. Flight muscle enzymes and metabolic flux rates during hovering flight of the nectar bat, Glossophaga soricina: further evidence of convergence with hummingbirds.

    Science.gov (United States)

    Suarez, R K; Welch, K C; Hanna, S K; Herrera M, L G

    2009-06-01

    Given their high metabolic rates, nectarivorous diet, and ability to directly fuel their energetically-expensive flight using recently-ingested sugar, we tested the hypothesis that Pallas long tongued nectar bats (Glossophaga soricina) possess flight muscles similar to those of hummingbirds with respect to enzymatic flux capacities in bioenergetic pathways. In addition, we compared these biochemical capacities with flux rates achieved in vivo during hovering flight. Rates of oxygen consumption (V(O(2))) were measured during hover-feeding and used to estimate rates of ATP turnover, glucose and long-chain fatty acid oxidation per unit mass of flight muscle. Enzyme V(max) values at key steps in glucose and fatty acid oxidation obtained in vitro from pectoralis muscle samples exceed those found in the locomotory muscles of other species of small mammals and resemble data obtained from hummingbird flight muscles. The ability of nectar bats and hummingbirds to hover in fed and fasted states, fueled almost exclusively by carbohydrate or fat, respectively, allowed the estimation of fractional velocities (v/V(max)) at both the hexokinase and carnitine palmitoyltransferase-2 steps in glucose and fatty acid oxidation, respectively. The results further support the hypothesis of convergent evolution in biochemical and physiological traits in nectar bats and hummingbirds.

  18. Analysis of the influence of rainfall variables on urban effluents concentrations and fluxes in wet weather

    Science.gov (United States)

    Gooré Bi, Eustache; Monette, Frédéric; Gasperi, Johnny

    2015-04-01

    Urban rainfall runoff has been a topic of increasing importance over the past years, a result of both the increase in impervious land area arising from constant urban growth and the effects of climate change on urban drainage. The main goal of the present study is to assess and analyze the correlations between rainfall variables and common indicators of urban water quality, namely event mean concentrations (EMCs) and event fluxes (EFs), in order to identify and explain the impacts of each of the main rainfall variables on the generation process of urban pollutants during wet periods. To perform this analysis, runoff from eight summer rainfall events that resulted in combined sewer overflow (CSO) was sampled simultaneously from two distinct catchment areas in order to quantify discharges at the respective outfalls. Pearson statistical analysis of total suspended solids (TSS), chemical oxygen demand (COD), carbonaceous biochemical oxygen demand at 5 days (CBOD5), total phosphorus (Ptot) and total kedjal nitrogen (N-TKN) showed significant correlations (ρ = 0.05) between dry antecedent time (DAT) and EMCs on one hand, and between total rainfall (TR) and the volume discharged (VD) during EFs, on the other. These results show that individual rainfall variables strongly affect either EMCs or EFs and are good predictors to consider when selecting variables for statistical modeling of urban runoff quality. The results also show that in a combined sewer network, there is a linear relationship between TSS event fluxes and COD, CBOD5, Ptot, and N-TKN event fluxes; this explains 97% of the variability of these pollutants which adsorb onto TSS during wet weather, which therefore act as tracers. Consequently, the technological solution selected for TSS removal will also lead to a reduction of these pollutants. Given the huge volumes involved, urban runoffs contribute substantially to pollutant levels in receiving water bodies, a situation which, in a climate change context, may

  19. Quantitative Multilevel Analysis of Central Metabolism in Developing Oilseeds of Oilseed Rape during in Vitro Culture.

    Science.gov (United States)

    Schwender, Jörg; Hebbelmann, Inga; Heinzel, Nicolas; Hildebrandt, Tatjana; Rogers, Alistair; Naik, Dhiraj; Klapperstück, Matthias; Braun, Hans-Peter; Schreiber, Falk; Denolf, Peter; Borisjuk, Ljudmilla; Rolletschek, Hardy

    2015-07-01

    Seeds provide the basis for many food, feed, and fuel products. Continued increases in seed yield, composition, and quality require an improved understanding of how the developing seed converts carbon and nitrogen supplies into storage. Current knowledge of this process is often based on the premise that transcriptional regulation directly translates via enzyme concentration into flux. In an attempt to highlight metabolic control, we explore genotypic differences in carbon partitioning for in vitro cultured developing embryos of oilseed rape (Brassica napus). We determined biomass composition as well as 79 net fluxes, the levels of 77 metabolites, and 26 enzyme activities with specific focus on central metabolism in nine selected germplasm accessions. Overall, we observed a tradeoff between the biomass component fractions of lipid and starch. With increasing lipid content over the spectrum of genotypes, plastidic fatty acid synthesis and glycolytic flux increased concomitantly, while glycolytic intermediates decreased. The lipid/starch tradeoff was not reflected at the proteome level, pointing to the significance of (posttranslational) metabolic control. Enzyme activity/flux and metabolite/flux correlations suggest that plastidic pyruvate kinase exerts flux control and that the lipid/starch tradeoff is most likely mediated by allosteric feedback regulation of phosphofructokinase and ADP-glucose pyrophosphorylase. Quantitative data were also used to calculate in vivo mass action ratios, reaction equilibria, and metabolite turnover times. Compounds like cyclic 3',5'-AMP and sucrose-6-phosphate were identified to potentially be involved in so far unknown mechanisms of metabolic control. This study provides a rich source of quantitative data for those studying central metabolism. © 2015 American Society of Plant Biologists. All Rights Reserved.

  20. Quantitative Multilevel Analysis of Central Metabolism in Developing Oilseeds of Oilseed Rape During In Vitro Culture

    Energy Technology Data Exchange (ETDEWEB)

    Schwender, Jorg [Brookhaven National Lab. (BNL), Upton, NY (United States); Hebbelmann, Inga [Brookhaven National Lab. (BNL), Upton, NY (United States); Heinzel, Nicholas [Leibniz Inst. of Plant Genetics and Crop Plant Research, Gatersleben (Germany); Hildebrandt, Tatjana [Univ. of Hannover (Germany); Rogers, Alistair [Brookhaven National Lab. (BNL), Upton, NY (United States); Naik, Dhiraj [Brookhaven National Lab. (BNL), Upton, NY (United States); Indian Inst. of Advanced Research Koba, Gujarat (India); Klapperstuck, Matthias [Monash Univ., Melbourne, VIC (Australia); Braun, Hans -Peter [Univ. of Hannover (Germany); Schreiber, Falk [Monash Univ., Melbourne, VIC (Australia); Univ. Halle-Wittenberg, Melbourne (Australia); Denolf, Peter [Bayer CropScience (Belgium); Borisjuk, Ljudmilla [Leibniz Inst. of Plant Genetics and Crop Plant Research, Gatersleben (Germany); Rolletschek, Hardy [Leibniz Inst. of Plant Genetics and Crop Plant Research, Gatersleben (Germany)

    2015-07-01

    Seeds provide the basis for many food, feed, and fuel products. Continued increases in seed yield, composition, and quality require an improved understanding of how the developing seed converts carbon and nitrogen supplies into storage. Current knowledge of this process is often based on the premise that transcriptional regulation directly translates via enzyme concentration into flux. In an attempt to highlight metabolic control, we explore genotypic differences in carbon partitioning for in vitro cultured developing embryos of oilseed rape (Brassica napus). We determined biomass composition as well as 79 net fluxes, the levels of 77 metabolites, and 26 enzyme activities with specific focus on central metabolism in nine selected germplasm accessions. We observed a tradeoff between the biomass component fractions of lipid and starch. With increasing lipid content over the spectrum of genotypes, plastidic fatty acid synthesis and glycolytic flux increased concomitantly, while glycolytic intermediates decreased. The lipid/starch tradeoff was not reflected at the proteome level, pointing to the significance of (posttranslational) metabolic control. Enzyme activity/flux and metabolite/flux correlations suggest that plastidic pyruvate kinase exerts flux control and that the lipid/starch tradeoff is most likely mediated by allosteric feedback regulation of phosphofructokinase and ADP-glucose pyrophosphorylase. Also, quantitative data were used to calculate in vivo mass action ratios, reaction equilibria, and metabolite turnover times. Compounds like cyclic 3',5'-AMP and sucrose-6-phosphate were identified to potentially be involved in so far unknown mechanisms of metabolic control. This study provides a rich source of quantitative data for those studying central metabolism..

  1. Whole-system metabolism and CO2 fluxes in a Mediterranean Bay dominated by seagrass beds (Palma Bay, NW Mediterranean

    Directory of Open Access Journals (Sweden)

    F. Gazeau

    2005-01-01

    Full Text Available Planktonic and benthic incubations (bare and Posidonia oceanica vegetated sediments were performed at monthly intervals from March 2001 to October 2002 in a seagrass vegetated area of the Bay of Palma (Mallorca, Spain. Results showed a contrast between the planktonic compartment, which was on average near metabolic balance (−4.6±5.9 mmol O2 m-2 d-1 and the benthic compartment, which was autotrophic (17.6±8.5 mmol O2 m-2 d-1. During two cruises in March and June 2002, planktonic and benthic incubations were performed at several stations in the bay to estimate the whole-system metabolism and to examine its relationship with partial pressure of CO2 (pCO2 and apparent oxygen utilisation (AOU spatial patterns. Moreover, during the second cruise, when the residence time of water was long enough, net ecosystem production (NEP estimates based on incubations were compared, over the Posidonia oceanica meadow, to rates derived from dissolved inorganic carbon (DIC and oxygen (O2 mass balance budgets. These budgets provided NEP estimates in fair agreement with those derived from direct metabolic estimates based on incubated samples over the Posidonia oceanica meadow. Whereas the seagrass community was autotrophic, the excess organic carbon production therein could only balance the planktonic heterotrophy in shallow waters relative to the maximum depth of the bay (55 m. This generated a horizontal gradient from autotrophic or balanced communities in the shallow seagrass-covered areas, to strongly heterotrophic communities in deeper areas of the bay. It seems therefore that, on an annual scale in the whole bay, the organic matter production by the Posidonia oceanica may not be sufficient to fully compensate the heterotrophy of the planktonic compartment, which may require external organic carbon inputs, most likely from land.

  2. Bioremediation in marine ecosystems: a computational study combining ecological modelling and flux balance analysis

    Directory of Open Access Journals (Sweden)

    Marianna eTaffi

    2014-09-01

    Full Text Available The pressure to search effective bioremediation methodologies for contaminated ecosystems has led to the large-scale identification of microbial species and metabolic degradation pathways. However, minor attention has been paid to the study of bioremediation in marine food webs and to the definition of integrated strategies for reducing bioaccumulation in species. We propose a novel computational framework for analysing the multiscale effects of bioremediation at the ecosystem level, based on coupling food web bioaccumulation models and metabolic models of degrading bacteria. The combination of techniques from synthetic biology and ecological network analysis allows the specification of arbitrary scenarios of contaminant removal and the evaluation of strategies based on natural or synthetic microbial strains.In this study, we derive a bioaccumulation model of polychlorinated biphenyls (PCBs in the Adriatic food web, and we extend a metabolic reconstruction of Pseudomonas putida KT2440 (iJN746 with the aerobic pathway of PCBs degradation. We assess the effectiveness of different bioremediation scenarios in reducing PCBs concentration in species and we study indices of species centrality to measure their importance in the contaminant diffusion via feeding links.The analysis of the Adriatic sea case study suggests that our framework could represent a practical tool in the design of effective remediation strategies, providing at the same time insights into the ecological role of microbial communities within food webs.

  3. Bioremediation in marine ecosystems: a computational study combining ecological modeling and flux balance analysis.

    Science.gov (United States)

    Taffi, Marianna; Paoletti, Nicola; Angione, Claudio; Pucciarelli, Sandra; Marini, Mauro; Liò, Pietro

    2014-01-01

    The pressure to search effective bioremediation methodologies for contaminated ecosystems has led to the large-scale identification of microbial species and metabolic degradation pathways. However, minor attention has been paid to the study of bioremediation in marine food webs and to the definition of integrated strategies for reducing bioaccumulation in species. We propose a novel computational framework for analysing the multiscale effects of bioremediation at the ecosystem level, based on coupling food web bioaccumulation models and metabolic models of degrading bacteria. The combination of techniques from synthetic biology and ecological network analysis allows the specification of arbitrary scenarios of contaminant removal and the evaluation of strategies based on natural or synthetic microbial strains. In this study, we derive a bioaccumulation model of polychlorinated biphenyls (PCBs) in the Adriatic food web, and we extend a metabolic reconstruction of Pseudomonas putida KT2440 (iJN746) with the aerobic pathway of PCBs degradation. We assess the effectiveness of different bioremediation scenarios in reducing PCBs concentration in species and we study indices of species centrality to measure their importance in the contaminant diffusion via feeding links. The analysis of the Adriatic sea case study suggests that our framework could represent a practical tool in the design of effective remediation strategies, providing at the same time insights into the ecological role of microbial communities within food webs.

  4. Discrimination of cardiac subcellular creatine kinase fluxes by NMR spectroscopy: a new method of analysis.

    Science.gov (United States)

    Joubert, F; Hoerter, J A; Mazet, J L

    2001-12-01

    A challenge in the understanding of creatine kinase (CK) fluxes reflected by NMR magnetization transfer in the perfused rat heart is the choice of a kinetic model of analysis. The complexity of the energetic pathways, due to the presence of adenosine triphosphate (ATP)-inorganic phosphate (Pi) exchange, of metabolite compartmentation and of subcellular localization of CK isozymes cannot be resolve from the sole information obtained from a single NMR protocol. To analyze multicompartment exchanges, we propose a new strategy based on the simultaneous analysis of four inversion transfer protocols. The time course of ATP and Phosphocreatine (PCr) magnetizations computed from the McConnell equations were adjusted to their experimental value for exchange networks of increasing complexity (up to six metabolite pools). Exchange schemes were selected by the quality of their fit and their consistency with data from other sources: the size of mitochondrial pools and the ATP synthesis flux. The consideration of ATP-Pi exchange and of ATP compartmentation were insufficient to describe the data. The most appropriate exchange scheme in our normoxic heart involved the discrimination of three specific CK activities (cytosolic, mitochondrial, and close to ATPases). At the present level of heart contractility, the energy is transferred from mitochondria to myofibrils mainly by PCr.

  5. Job/task analysis for I ampersand C [Instrumentation and Controls] instrument technicians at the High Flux Isotope Reactor

    International Nuclear Information System (INIS)

    Duke, L.L.

    1989-09-01

    To comply with Department of Energy Order 5480.XX (Draft), a job/task analysis was initiated by the Maintenance Management Department at Oak Ridge National Laboratory (ORNL). The analysis was applicable to instrument technicians working at the ORNL High Flux Isotope Reactor (HFIR). This document presents the procedures and results of that analysis. 2 refs., 2 figs

  6. Primary metabolism in Lactobacillus sakei food isolates by proteomic analysis

    Directory of Open Access Journals (Sweden)

    Champomier-Vergès Marie-Christine

    2010-04-01

    Full Text Available Abstract Background Lactobacillus sakei is an important food-associated lactic acid bacterium commonly used as starter culture for industrial meat fermentation, and with great potential as a biopreservative in meat and fish products. Understanding the metabolic mechanisms underlying the growth performance of a strain to be used for food fermentations is important for obtaining high-quality and safe products. Proteomic analysis was used to study the primary metabolism in ten food isolates after growth on glucose and ribose, the main sugars available for L. sakei in meat and fish. Results Proteins, the expression of which varied depending on the carbon source were identified, such as a ribokinase and a D-ribose pyranase directly involved in ribose catabolism, and enzymes involved in the phosphoketolase and glycolytic pathways. Expression of enzymes involved in pyruvate and glycerol/glycerolipid metabolism were also affected by the change of carbon source. Interestingly, a commercial starter culture and a protective culture strain down-regulated the glycolytic pathway more efficiently than the rest of the strains when grown on ribose. The overall two-dimensional gel electrophoresis (2-DE protein expression pattern was similar for the different strains, though distinct differences were seen between the two subspecies (sakei and carnosus, and a variation of about 20% in the number of spots in the 2-DE gels was observed between strains. A strain isolated from fermented fish showed a higher expression of stress related proteins growing on both carbon sources. Conclusions It is obvious from the data obtained in this study that the proteomic approach efficiently identifies differentially expressed proteins caused by the change of carbon source. Despite the basic similarity in the strains metabolic routes when they ferment glucose and ribose, there were also interesting differences. From the application point of view, an understanding of regulatory

  7. Flux balance analysis predicts Warburg-like effects of mouse hepatocyte deficient in miR-122a.

    Directory of Open Access Journals (Sweden)

    Hua-Qing Wu

    2017-07-01

    Full Text Available The liver is a vital organ involving in various major metabolic functions in human body. MicroRNA-122 (miR-122 plays an important role in the regulation of liver metabolism, but its intrinsic physiological functions require further clarification. This study integrated the genome-scale metabolic model of hepatocytes and mouse experimental data with germline deletion of Mir122a (Mir122a-/- to infer Warburg-like effects. Elevated expression of MiR-122a target genes in Mir122a-/-mice, especially those encoding for metabolic enzymes, was applied to analyze the flux distributions of the genome-scale metabolic model in normal and deficient states. By definition of the similarity ratio, we compared the flux fold change of the genome-scale metabolic model computational results and metabolomic profiling data measured through a liquid-chromatography with mass spectrometer, respectively, for hepatocytes of 2-month-old mice in normal and deficient states. The Ddc gene demonstrated the highest similarity ratio of 95% to the biological hypothesis of the Warburg effect, and similarity of 75% to the experimental observation. We also used 2, 6, and 11 months of mir-122 knockout mice liver cell to examined the expression pattern of DDC in the knockout mice livers to show upregulated profiles of DDC from the data. Furthermore, through a bioinformatics (LINCS program prediction, BTK inhibitors and withaferin A could downregulate DDC expression, suggesting that such drugs could potentially alter the early events of metabolomics of liver cancer cells.

  8. Metabolic process engineering of Clostridium tyrobutyricum Δack-adhE2 for enhanced n-butanol production from glucose: effects of methyl viologen on NADH availability, flux distribution, and fermentation kinetics.

    Science.gov (United States)

    Du, Yinming; Jiang, Wenyan; Yu, Mingrui; Tang, I-Ching; Yang, Shang-Tian

    2015-04-01

    Butanol biosynthesis through aldehyde/alcohol dehydrogenase (adhE2) is usually limited by NADH availability, resulting in low butanol titer, yield, and productivity. To alleviate this limitation and improve n-butanol production by Clostridium tyrobutyricum Δack-adhE2 overexpressing adhE2, the NADH availability was increased by using methyl viologen (MV) as an artificial electron carrier to divert electrons from ferredoxin normally used for H2 production. In the batch fermentation with the addition of 500 μM MV, H2 , acetate, and butyrate production was reduced by more than 80-90%, while butanol production increased more than 40% to 14.5 g/L. Metabolic flux analysis revealed that butanol production increased in the fermentation with MV because of increased NADH availability as a result of reduced H2 production. Furthermore, continuous butanol production of ∼55 g/L with a high yield of ∼0.33 g/g glucose and extremely low ethanol, acetate, and butyrate production was obtained in fed-batch fermentation with gas stripping for in situ butanol recovery. This study demonstrated a stable and reliable process for high-yield and high-titer n-butanol production by metabolically engineered C. tyrobutyricum by applying MV as an electron carrier to increase butanol biosynthesis. © 2014 Wiley Periodicals, Inc.

  9. Photoneutron Flux Measurement via Neutron Activation Analysis in a Radiotherapy Bunker with an 18 MV Linear Accelerator

    Directory of Open Access Journals (Sweden)

    Çeçen Yiğit

    2017-01-01

    Full Text Available In cancer treatment, high energy X-rays are used which are produced by linear accelerators (LINACs. If the energy of these beams is over 8 MeV, photonuclear reactions occur between the bremsstrahlung photons and the metallic parts of the LINAC. As a result of these interactions, neutrons are also produced as secondary radiation products (γ,n which are called photoneutrons. The study aims to map the photoneutron flux distribution within the LINAC bunker via neutron activation analysis (NAA using indium-cadmium foils. Irradiations made at different gantry angles (0°, 90°, 180° and 270° with a total of 91 positions in the Philips SLI-25 linear accelerator treatment room and location-based distribution of thermal neutron flux was obtained. Gamma spectrum analysis was carried out with high purity germanium (HPGe detector. Results of the analysis showed that the maximum neutron flux in the room occurred at just above of the LINAC head (1.2x105 neutrons/cm2.s which is compatible with an americium-beryllium (Am-Be neutron source. There was a 90% decrease of flux at the walls and at the start of the maze with respect to the maximum neutron flux. And, just in front of the LINAC door, inside the room, neutron flux was measured less than 1% of the maximum.

  10. Analysis of carbon dioxide, water vapour and energy fluxes over an ...

    Indian Academy of Sciences (India)

    to measure ecosystem carbon exchange (Running et al. 1999; Canadell et al. 2000; Geider ... sense CO2 flux at the rate of 10 Hz are being operationally used from mid 1980s till .... (a) Diurnal variations (monthly mean) in CO2 flux with ±1 standard deviation and (b) CO2 flux variations. (weekly mean) from November 2011 ...

  11. Ratiometric analysis of fura red by flow cytometry: a technique for monitoring intracellular calcium flux in primary cell subsets.

    Directory of Open Access Journals (Sweden)

    Emily R Wendt

    Full Text Available Calcium flux is a rapid and sensitive measure of cell activation whose utility could be enhanced with better techniques for data extraction. We describe a technique to monitor calcium flux by flow cytometry, measuring Fura Red calcium dye by ratiometric analysis. This technique has several advantages: 1 using a single calcium dye provides an additional channel for surface marker characterization, 2 allows robust detection of calcium flux by minority cell populations within a heterogeneous population of primary T cells and monocytes 3 can measure total calcium flux and additionally, the proportion of responding cells, 4 can be applied to studying the effects of drug treatment, simultaneously stimulating and monitoring untreated and drug treated cells. Using chemokine receptor activation as an example, we highlight the utility of this assay, demonstrating that only cells expressing a specific chemokine receptor are activated by cognate chemokine ligand. Furthermore, we describe a technique for simultaneously stimulating and monitoring calcium flux in vehicle and drug treated cells, demonstrating the effects of the Gαi inhibitor, pertussis toxin (PTX, on chemokine stimulated calcium flux. The described real time calcium flux assay provides a robust platform for characterizing cell activation within primary cells, and offers a more accurate technique for studying the effect of drug treatment on receptor activation in a heterogeneous population of primary cells.

  12. Central Russia agroecosystem monitoring with CO2 fluxes analysis by eddy covariance method

    Directory of Open Access Journals (Sweden)

    Joulia Meshalkina

    2015-07-01

    Full Text Available The eddy covariance (EC technique as a powerful statistics-based method of measurement and calculation the vertical turbulent fluxes of greenhouses gases within atmospheric boundary layers provides the continuous, long-term flux information integrated at the ecosystem scale. An attractive way to compare the agricultural practices influences on GHG fluxes is to divide a crop area into subplots managed in different ways. The research has been carried out in the Precision Farming Experimental Field of the Russian Timiryazev State Agricultural University (RTSAU, Moscow in 2013 under the support of RF Government grant # 11.G34.31.0079, EU grant # 603542 LUС4С (7FP and RF Ministry of education and science grant # 14-120-14-4266-ScSh. Arable Umbric Albeluvisols have around 1% of SOC, 5.4 pH (KCl and NPK medium-enhanced contents in sandy loam topsoil. The CO2 flux seasonal monitoring has been done by two eddy covariance stations located at the distance of 108 m. The LI-COR instrumental equipment was the same for the both stations. The stations differ only by current crop version: barley or vetch and oats. At both sites, diurnal patterns of NEE among different months were very similar in shape but varied slightly in amplitude. NEE values were about zero during spring time. CO2 fluxes have been intensified after crop emerging from values of 3 to 7 µmol/s∙m2 for emission, and from 5 to 20 µmol/s∙m2 for sink. Stabilization of the fluxes has come at achieving plants height of 10-12 cm. Average NEE was negative only in June and July. Maximum uptake was observed in June with average values about 8 µmol CO2 m−2 s−1. Although different kind of crops were planted on the fields A and B, GPP dynamics was quite similar for both sites: after reaching the peak values at the mid of June, GPP decreased from 4 to 0.5 g C CO2 m-2 d-1 at the end of July. The difference in crops harvesting time that was equal two weeks did not significantly influence the daily

  13. Analysis of Neutron Flux Distribution in Rsg-Gas Reactor With U-Mo Fuels

    Directory of Open Access Journals (Sweden)

    Taswanda Taryo

    2004-01-01

    Full Text Available The use of U-Mo fuels in research reactors seems to be promising and, recently, world researchers have carried out these such activities actively. The National Nuclear Energy Agency (BATAN which owns RSG-GAS reactor available in Serpong Research Center for Atomic Energy should anticipate this trend. It is, therefore, this research work on the use of U-Mo fuels in RSG-GAS reactor should be carried out. The work was focused on the analysis of neutron flux distribution in the RSG-GAS reactor using different content of molybdenum in U-Mo fuels. To begin with, RSG-GAS reactor core model was developed and simulated into X, Y and Z dimensions. Cross section of materials based on the developed cells of standard and control fuels was then generated using WIMS-D5-B. The criticality calculations were finally carried out applying BATAN-2DIFF code. The results showed that the neutron flux distribution obtained in U-Mo-fuel-based RSG-GAS core is very similar to those achieved in the 300-gram sillicide-fuel-based RSG-GAS reactor core. Indeed, the utilization of the U-Mo RSG-GAS core can be very similar to that of the high-density sillicide reactor core and even could be better in the future.

  14. Torque Characteristic Analysis of a Transverse Flux Motor Using a Combined-Type Stator Core

    Directory of Open Access Journals (Sweden)

    Xiaobao Yang

    2016-11-01

    Full Text Available An external rotor transverse flux motor using a combined-type stator core is proposed for a direct drive application in this paper. The stator core is combined by two kinds of components that can both be manufactured conveniently by generic laminated silicon steel used in traditional motors. The motor benefits from the predominance of low manufacturing cost and low iron loss by using a silicon-steel sheet. Firstly, the basic structure and operation principles of the proposed motor are introduced. Secondly, the expressions of the electromagnetic torque and the cogging torque are deduced by theoretical analysis. Thirdly, the basic characteristics such as permanent magnet flux linkage, no-load back electromotive force, cogging torque and electromagnetic torque are analyzed by a three-dimensional finite element method (3D FEM. Then, the influence of structure parameters on the torque density is investigated, which provides a useful foundation for optimum design of the novel motor. Finally, the torque density of the proposed motor is calculated and discussed, and the result shows that the proposed motor in this paper can provide considerable torque density by using few permanent magnets.

  15. Bibliometric analysis of Indian Journal of Endocrinology and Metabolism

    Directory of Open Access Journals (Sweden)

    Garima Bhutani

    2013-01-01

    Full Text Available Background: Bibliometric analysis of the journal is a method to assess the research impact or research influence of that journal. This information can also be used to evaluate the influence/performance of a researcher and to provide a comparison between researchers. This work was aimed at performing bibliometric analysis of Indian Journal of Endocrinology and Metabolism (IJEM. Materials and Methods: The publications of year 2011-12 of IJEM were analyzed. Total number of articles published, type of articles, their authorship, and the coverage of various subspecialties was studied. The publications were also classified as Indian or foreign, from endocrine or nonendocrine departments and from academic or nonacademic institutions according to the institution of first author. Results and Conclusions: A total of 10 main issues and 7 supplementary issues were published in IJEM in year 2011 and 2012. These included a total of 605 publications, which depict a dramatic increase in the number of publications in last 2 years as compared to the previous years. Taking collectively, review articles were published in majority. Maximum number of articles was dealing with pancreas and metabolic disorders followed by thyroid. Other endocrine organs were given almost similar importance. Publications were largely originating from endocrine departments and from academic institutions. Although maximum number of articles were from India, but the publications from other countries are also on an increase. Thus, the widespread coverage of this journal suggests that IJEM has begun to represent global face of Indian endocrinology.

  16. Fermentation of Xylose Causes Inefficient Metabolic State Due to Carbon/Energy Starvation and Reduced Glycolytic Flux in Recombinant Industrial Saccharomyces cerevisiae

    Science.gov (United States)

    Matsushika, Akinori; Nagashima, Atsushi; Goshima, Tetsuya; Hoshino, Tamotsu

    2013-01-01

    In the present study, comprehensive, quantitative metabolome analysis was carried out on the recombinant glucose/xylose-cofermenting S. cerevisiae strain MA-R4 during fermentation with different carbon sources, including glucose, xylose, or glucose/xylose mixtures. Capillary electrophoresis time-of-flight mass spectrometry was used to determine the intracellular pools of metabolites from the central carbon pathways, energy metabolism pathways, and the levels of twenty amino acids. When xylose instead of glucose was metabolized by MA-R4, glycolytic metabolites including 3- phosphoglycerate, 2- phosphoglycerate, phosphoenolpyruvate, and pyruvate were dramatically reduced, while conversely, most pentose phosphate pathway metabolites such as sedoheptulose 7- phosphate and ribulose 5-phosphate were greatly increased. These results suggest that the low metabolic activity of glycolysis and the pool of pentose phosphate pathway intermediates are potential limiting factors in xylose utilization. It was further demonstrated that during xylose fermentation, about half of the twenty amino acids declined, and the adenylate/guanylate energy charge was impacted due to markedly decreased adenosine triphosphate/adenosine monophosphate and guanosine triphosphate/guanosine monophosphate ratios, implying that the fermentation of xylose leads to an inefficient metabolic state where the biosynthetic capabilities and energy balance are severely impaired. In addition, fermentation with xylose alone drastically increased the level of citrate in the tricarboxylic acid cycle and increased the aromatic amino acids tryptophan and tyrosine, strongly supporting the view that carbon starvation was induced. Interestingly, fermentation with xylose alone also increased the synthesis of the polyamine spermidine and its precursor S-adenosylmethionine. Thus, differences in carbon substrates, including glucose and xylose in the fermentation medium, strongly influenced the dynamic metabolism of MA-R4

  17. Advances in the quantification of mitochondrial function in primary human immune cells through extracellular flux analysis.

    Directory of Open Access Journals (Sweden)

    Dequina Nicholas

    Full Text Available Numerous studies show that mitochondrial energy generation determines the effectiveness of immune responses. Furthermore, changes in mitochondrial function may regulate lymphocyte function in inflammatory diseases like type 2 diabetes. Analysis of lymphocyte mitochondrial function has been facilitated by introduction of 96-well format extracellular flux (XF96 analyzers, but the technology remains imperfect for analysis of human lymphocytes. Limitations in XF technology include the lack of practical protocols for analysis of archived human cells, and inadequate data analysis tools that require manual quality checks. Current analysis tools for XF outcomes are also unable to automatically assess data quality and delete untenable data from the relatively high number of biological replicates needed to power complex human cell studies. The objectives of work presented herein are to test the impact of common cellular manipulations on XF outcomes, and to develop and validate a new automated tool that objectively analyzes a virtually unlimited number of samples to quantitate mitochondrial function in immune cells. We present significant improvements on previous XF analyses of primary human cells that will be absolutely essential to test the prediction that changes in immune cell mitochondrial function and fuel sources support immune dysfunction in chronic inflammatory diseases like type 2 diabetes.

  18. A study of influence of material properties on magnetic flux density induced in magneto rheological damper through finite element analysis

    Directory of Open Access Journals (Sweden)

    Gurubasavaraju T. M.

    2018-01-01

    Full Text Available Magnetorheological fluids are smart materials, which are responsive to the external stimulus and changes their rheological properties. The damper performance (damping force is dependent on the magnetic flux density induced at the annular gap. Magnetic flux density developed at fluid flow gap of MR damper due to external applied current is also dependent on materials properties of components of MR damper (such as piston head, outer cylinder and piston rod. The present paper discus about the influence of different materials selected for components of the MR damper on magnetic effect using magnetostatic analysis. Different materials such as magnetic and low carbon steels are considered for piston head of the MR damper and magnetic flux density induced at fluid flow gap (filled with MR fluid is computed for different DC current applied to the electromagnetic coil. Developed magnetic flux is used for calculating the damper force using analytical method for each case. The low carbon steel has higher magnetic permeability hence maximum magnetic flux could pass through the piston head, which leads to higher value of magnetic effect induction at the annular gap. From the analysis results it is observed that the magnetic steel and low carbon steel piston head provided maximum magnetic flux density. Eventually the higher damping force can be observed for same case.

  19. Decoupling analysis of a novel bearingless flux-switching permanent magnet motor

    Science.gov (United States)

    Zhao, Chenyin; Zhu, Huangqiu; Qin, Yuemei

    2017-05-01

    In this paper, a novel 12/10 stator/rotor pole bearingless flux-switching permanent magnet (BFSPM) motor is proposed to overcome the drawbacks of rotor permanent magnet type bearingless motors. The basic motor configuration, including motor configuration and winding configuration, is introduced firstly. Then, based on the principle of reverse direction magnetization for symmetrical rotor teeth, the radial suspension forces generation principle is analyzed in detail. Finally, decoupling performances between suspension force windings and torque windings are investigated. The results show that the proposed BFSPM not only ensures the merits of high torque output capability compared with conventional 12/10 stator/rotor pole FSPM motor, but also achieves stable radial suspension forces which have little mutual effect with torque. The validity of the proposed structure has been verified by finite element analysis (FEA).

  20. Design of next step tokamak: Consistent analysis of plasma performance flux composition and poloidal field system

    International Nuclear Information System (INIS)

    Ane, J.M.; Grandgirard, V.; Albajar, F.; Johner, J.

    2001-01-01

    A consistent and simple approach to derive plasma scenarios for next step tokamak design is presented. It is based on successive plasma equilibria snapshots from plasma breakdown to end of ramp-down. Temperature and density profiles for each equilibrium are derived from a 2D plasma model. The time interval between two successive equilibria is then computed from the toroidal field magnetic energy balance, the resistive term of which depends on n, T profiles. This approach provides a consistent analysis of plasma performance, flux consumption and PF system, including average voltages waveforms across the PF coils. The plasma model and the Poynting theorem for the toroidal magnetic energy are presented. Application to ITER-FEAT and to M2, a Q=5 machine designed at CEA, are shown. (author)

  1. 3-D FEM Analysis, Prototyping and Tests of an Axial Flux Permanent-Magnet Wind Generator

    Directory of Open Access Journals (Sweden)

    Joya C. Kappatou

    2017-08-01

    Full Text Available This paper contributes to the research and development of Axial Flux Permanent Magnet Synchronous Machines (AFPMSM; and in particular the design, the construction stages and measurements of a double rotor single internal non-ferromagnetic stator with a trapezoidal-concentrated winding machine for wind power generation applications. The initial dimensions of the machine were calculated using analytical formulas and a model was created and analyzed using the 3D Finite Element Method (FEM. The shape of the magnets of the machine was optimized and presented in a previous paper and a prototype was constructed and tested in the laboratory. In addition, a temperature test of the stator was performed experimentally. Finally, the effect of the different axial widths of the two air gaps on the electrical magnitudes and the field of the machine were investigated using both FEM analysis and experiments.

  2. Genome-scale metabolic network validation of Shewanella oneidensis using transposon insertion frequency analysis.

    Directory of Open Access Journals (Sweden)

    Hong Yang

    2014-09-01

    Full Text Available Transposon mutagenesis, in combination with parallel sequencing, is becoming a powerful tool for en-masse mutant analysis. A probability generating function was used to explain observed miniHimar transposon insertion patterns, and gene essentiality calls were made by transposon insertion frequency analysis (TIFA. TIFA incorporated the observed genome and sequence motif bias of the miniHimar transposon. The gene essentiality calls were compared to: 1 previous genome-wide direct gene-essentiality assignments; and, 2 flux balance analysis (FBA predictions from an existing genome-scale metabolic model of Shewanella oneidensis MR-1. A three-way comparison between FBA, TIFA, and the direct essentiality calls was made to validate the TIFA approach. The refinement in the interpretation of observed transposon insertions demonstrated that genes without insertions are not necessarily essential, and that genes that contain insertions are not always nonessential. The TIFA calls were in reasonable agreement with direct essentiality calls for S. oneidensis, but agreed more closely with E. coli essentiality calls for orthologs. The TIFA gene essentiality calls were in good agreement with the MR-1 FBA essentiality predictions, and the agreement between TIFA and FBA predictions was substantially better than between the FBA and the direct gene essentiality predictions.

  3. Analysis of factors controlling sediment phosphorus flux potential of wetlands in Hulun Buir grassland by principal component and path analysis method.

    Science.gov (United States)

    He, Jing; Su, Derong; Lv, Shihai; Diao, Zhaoyan; Ye, Shengxing; Zheng, Zhirong

    2017-11-08

    Phosphorus (P) flux potential can predict the trend of phosphorus release from wetland sediments to water and provide scientific parameters for further monitoring and management for phosphorus flux from wetland sediments to overlying water. Many studies have focused on factors affecting sediment P flux potential in sediment-water interface, but rarely on the relationship among these factors. In the present study, experiment on sediment P flux potential in sediment-water interface was conducted in six wetlands in Hulun Buir grassland, China and the relationships among sediment P flux potential in sediment-water interface, sediment physical properties, and sediment chemical characteristics were examined. Principal component analysis and path analysis were used to discuss these data in correlation coefficient, direct, and indirect effects on sediment P flux potential in sediment-water interface. Results indicated that the major factors affecting sediment P flux potential in sediment-water interface were amount of organophosphate-degradation bacterium in sediment, Ca-P content, and total phosphorus concentrations. The factors of direct influence sediment P flux potential were sediment Ca-P content, Olsen-P content, SOC content, and sediment Al-P content. The indirect influence sediment P flux potential in sediment-water interface was sediment Olsen-P content, sediment SOC content, sediment Ca-P content, and sediment Al-P content. And the standard multiple regression describing the relationship between sediment P flux potential in sediment-water interface and its major effect factors was Y = 5.849 - 1.025X 1  - 1.995X 2  + 0.188X 3  - 0.282X 4 (r = 0.9298, p properties to analyze the interrelation among sediment properties factors, main vegetable factors, and environment factors which influence the sediment P flux potential in sediment-water interface.

  4. Prevalence of metabolic syndrome and metabolic syndrome components in young adults: A pooled analysis

    Directory of Open Access Journals (Sweden)

    Paul B. Nolan

    2017-09-01

    Full Text Available Metabolic syndrome (MetSyn represents a clustering of different metabolic abnormalities. MetSyn prevalence is present in approximately 25% of all adults with increased prevalence in advanced ages. The presence of one component of MetSyn increases the risk of developing MetSyn later in life and likely represents a high lifetime burden of cardiovascular disease risk. Therefore we pooled data from multiple studies to establish the prevalence of MetSyn and MetSyn component prevalence across a broad range of ethnicities. PubMed, SCOPUS and Medline databases were searched to find papers presenting MetSyn and MetSyn component data for 18–30 year olds who were apparently healthy, free of disease, and MetSyn was assessed using either the harmonized, National Cholesterol Education Program Adult Treatment Panel III (NCEP-ATPIII, American Heart Association/National Heart, Blood and Lung Institute (AHA/NHBLI, or International Diabetes Federation (IDF definitions of MetSyn. After reviewing returned articles, 26,609 participants' data from 34 studies were included in the analysis and the data were pooled. MetSyn was present in 4.8–7% of young adults. Atherogenic dyslipidaemia defined as low high density lipoprotein (HDL cholesterol was the most prevalent MetSyn component (26.9–41.2%, followed by elevated blood pressure (16.6–26.6%, abdominal obesity (6.8–23.6%, atherogenic dyslipidaemia defined as raised triglycerides (8.6–15.6%, and raised fasting glucose (2.8–15.4%. These findings highlight that MetSyn is prevalent in young adults. Establishing the reason why low HDL is the most prevalent component may represent an important step in promoting primary prevention of MetSyn and reducing the incidence of subsequent clinical disease.

  5. Construction and analysis of the model of energy metabolism in E. coli.

    Directory of Open Access Journals (Sweden)

    Zixiang Xu

    Full Text Available Genome-scale models of metabolism have only been analyzed with the constraint-based modelling philosophy and there have been several genome-scale gene-protein-reaction models. But research on the modelling for energy metabolism of organisms just began in recent years and research on metabolic weighted complex network are rare in literature. We have made three research based on the complete model of E. coli's energy metabolism. We first constructed a metabolic weighted network using the rates of free energy consumption within metabolic reactions as the weights. We then analyzed some structural characters of the metabolic weighted network that we constructed. We found that the distribution of the weight values was uneven, that most of the weight values were zero while reactions with abstract large weight values were rare and that the relationship between w (weight values and v (flux values was not of linear correlation. At last, we have done some research on the equilibrium of free energy for the energy metabolism system of E. coli. We found that E(out (free energy rate input from the environment can meet the demand of E(ch(in (free energy rate dissipated by chemical process and that chemical process plays a great role in the dissipation of free energy in cells. By these research and to a certain extend, we can understand more about the energy metabolism of E. coli.

  6. Flux analysis of the Lactobacillus reuteri propanediol-utilization pathway for production of 3-hydroxypropionaldehyde, 3-hydroxypropionic acid and 1,3-propanediol from glycerol.

    Science.gov (United States)

    Dishisha, Tarek; Pereyra, Luciana P; Pyo, Sang-Hyun; Britton, Robert A; Hatti-Kaul, Rajni

    2014-05-27

    Lactobacillus reuteri converts glycerol to 3-hydroxypropionic acid (3HP) and 1,3-propanediol (1,3PDO) via 3-hydroxypropionaldehyde (3HPA) as an intermediate using enzymes encoded in its propanediol-utilization (pdu) operon. Since 3HP, 1,3PDO and 3HPA are important building blocks for the bio-based chemical industry, L. reuteri can be an attractive candidate for their production. However, little is known about the kinetics of glycerol utilization in the Pdu pathway in L. reuteri. In this study, the metabolic fluxes through the Pdu pathway were determined as a first step towards optimizing the production of 3HPA, and co-production of 3HP and 1,3PDO from glycerol. Resting cells of wild-type (DSM 20016) and recombinant (RPRB3007, with overexpressed pdu operon) strains were used as biocatalysts. The conversion rate of glycerol to 3HPA by the resting cells of L. reuteri was evaluated by in situ complexation of the aldehyde with carbohydrazide to avoid the aldehyde-mediated inactivation of glycerol dehydratase. Under operational conditions, the specific 3HPA production rate of the RPRB3007 strain was 1.9 times higher than that of the wild-type strain (1718.2 versus 889.0 mg/gCDW.h, respectively). Flux analysis of glycerol conversion to 1,3PDO and 3HP in the cells using multi-step variable-volume fed-batch operation showed that the maximum specific production rates of 3HP and 1,3PDO were 110.8 and 93.7 mg/gCDW.h, respectively, for the wild-type strain, and 179.2 and 151.4 mg/gCDW.h, respectively, for the RPRB3007 strain. The cumulative molar yield of the two compounds was ~1 mol/mol glycerol and their molar ratio was ~1 mol3HP/mol1,3PDO. A balance of redox equivalents between the glycerol oxidative and reductive pathway branches led to equimolar amounts of the two products. Metabolic flux analysis was a useful approach for finding conditions for maximal conversion of glycerol to 3HPA, 3HP and 1,3PDO. Improved specific production rates were obtained with resting cells of

  7. Flux balance analysis of genome-scale metabolic model of rice ...

    Indian Academy of Sciences (India)

    Depending on the physiological conditions of a cell, the transport capacities of intracellular transporters (ICTs) can vary. To mimic this physiological state, we randomly vary the ICTs' transport capacities and investigate their effects. The results show that the rice leaf has the potential to increase glycine and starch in a wide ...

  8. Modeling and Simulation of Optimal Resource Management during the Diurnal Cycle in Emiliania huxleyi by Genome-Scale Reconstruction and an Extended Flux Balance Analysis Approach.

    Science.gov (United States)

    Knies, David; Wittmüß, Philipp; Appel, Sebastian; Sawodny, Oliver; Ederer, Michael; Feuer, Ronny

    2015-10-28

    The coccolithophorid unicellular alga Emiliania huxleyi is known to form large blooms, which have a strong effect on the marine carbon cycle. As a photosynthetic organism, it is subjected to a circadian rhythm due to the changing light conditions throughout the day. For a better understanding of the metabolic processes under these periodically-changing environmental conditions, a genome-scale model based on a genome reconstruction of the E. huxleyi strain CCMP 1516 was created. It comprises 410 reactions and 363 metabolites. Biomass composition is variable based on the differentiation into functional biomass components and storage metabolites. The model is analyzed with a flux balance analysis approach called diurnal flux balance analysis (diuFBA) that was designed for organisms with a circadian rhythm. It allows storage metabolites to accumulate or be consumed over the diurnal cycle, while keeping the structure of a classical FBA problem. A feature of this approach is that the production and consumption of storage metabolites is not defined externally via the biomass composition, but the result of optimal resource management adapted to the diurnally-changing environmental conditions. The model in combination with this approach is able to simulate the variable biomass composition during the diurnal cycle in proximity to literature data.

  9. Analysis of the neutron flux in an annular pulsed reactor by using finite volume method

    Energy Technology Data Exchange (ETDEWEB)

    Silva, Mário A.B. da; Narain, Rajendra; Bezerra, Jair de L., E-mail: mabs500@gmail.com, E-mail: narain@ufpe.br, E-mail: jairbezerra@gmail.com [Universidade Federal de Pernambuco (UFPE), Recife, PE (Brazil). Centro de Tecnologia e Geociências. Departamento de Energia Nuclear

    2017-07-01

    Production of very intense neutron sources is important for basic nuclear physics and for material testing and isotope production. Nuclear reactors have been used as sources of intense neutron fluxes, although the achievement of such levels is limited by the inability to remove fission heat. Periodic pulsed reactors provide very intense fluxes by a rotating modulator near a subcritical core. A concept for the production of very intense neutron fluxes that combines features of periodic pulsed reactors and steady state reactors was proposed by Narain (1997). Such a concept is known as Very Intense Continuous High Flux Pulsed Reactor (VICHFPR) and was analyzed by using diffusion equation with moving boundary conditions and Finite Difference Method with Crank-Nicolson formalism. This research aims to analyze the flux distribution in the Very Intense Continuous Flux High Pulsed Reactor (VICHFPR) by using the Finite Volume Method and compares its results with those obtained by the previous computational method. (author)

  10. Climate-driven uncertainties in modeling terrestrial energy and water fluxes: a site-level to global-scale analysis.

    Science.gov (United States)

    Barman, Rahul; Jain, Atul K; Liang, Miaoling

    2014-06-01

    We used a land surface model constrained using data from flux tower sites, to analyze the biases in ecosystem energy and water fluxes arising due to the use of meteorological reanalysis datasets. Following site-level model calibration encompassing major vegetation types from the tropics to the northern high-latitudes, we repeated the site and global simulations using two reanalysis datasets: the NCEP/NCAR and the CRUNCEP. In comparison with the model simulations using observed meteorology from sites, the reanalysis-driven simulations produced several systematic biases in net radiation (Rn ), latent heat (LE), and sensible heat (H) fluxes. These include: (i) persistently positive tropical/subtropical biases in Rn using the NCEP/NCAR, and gradually transitioning to negative Rn biases in the higher latitudes; (ii) large positive H biases in the tropics/subtropics using the NCEP/NCAR; (iii) negative LE biases using the NCEP/NCAR above 40°N; (iv) high tropical LE using the CRUNCEP in comparison with observationally derived global estimates; and (v) flux-partitioning biases from canopy and ground components. Across vegetation types, we investigated the role of the meteorological drivers (shortwave and longwave radiation, atmospheric humidity, temperature, precipitation) and their seasonal biases in controlling these reanalysis-driven uncertainties. At the global scale, our site-level analysis explains several model-data differences in the LE and H fluxes when compared with observationally derived global estimates of these fluxes. Using our results, we discuss the implications of site-level model calibration on subsequent regional/global applications to study energy and hydrological processes. The flux-partitioning biases presented in this study have potential implications on the couplings among terrestrial carbon, energy, and water fluxes, and for the calibration of land-atmosphere parameterizations that are dependent on LE/H partitioning. © 2013 John Wiley & Sons Ltd.

  11. Modelling and analysis of central metabolism operating regulatory interactions in salt stress conditions in a L-carnitine overproducing E. coli strain.

    Science.gov (United States)

    Santos, Guido; Hormiga, José A; Arense, Paula; Cánovas, Manuel; Torres, Néstor V

    2012-01-01

    Based on experimental data from E. coli cultures, we have devised a mathematical model in the GMA-power law formalism that describes the central and L-carnitine metabolism in and between two steady states, non-osmotic and hyperosmotic (0.3 M NaCl). A key feature of this model is the introduction of type of kinetic order, the osmotic stress kinetic orders (g(OSn)), derived from the power law general formalism, which represent the effect of osmotic stress in each metabolic process of the model.By considering the values of the g(OSn) linked to each metabolic process we found that osmotic stress has a positive and determinant influence on the increase in flux in energetic metabolism (glycolysis); L-carnitine biosynthesis production; the transformation/excretion of Acetyl-CoA into acetate and ethanol; the input flux of peptone into the cell; the anabolic use of pyruvate and biomass decomposition. In contrast, we found that although the osmotic stress has an inhibitory effect on the transformation flux from the glycolytic end products (pyruvate) to Acetyl-CoA, this inhibition is counteracted by other effects (the increase in pyruvate concentration) to the extent that the whole flux increases. In the same vein, the down regulation exerted by osmotic stress on fumarate uptake and its oxidation and the production and export of lactate and pyruvate are reversed by other factors up to the point that the first increased and the second remained unchanged.The model analysis shows that in osmotic conditions the energy and fermentation pathways undergo substantial rearrangement. This is illustrated by the observation that the increase in the fermentation fluxes is not connected with fluxes towards the tricaboxylic acid intermediates and the synthesis of biomass. The osmotic stress associated with these fluxes reflects these changes. All these observations support that the responses to salt stress observed in E. coli might be conserved in halophiles.Flux evolution during osmotic

  12. Modelling and analysis of central metabolism operating regulatory interactions in salt stress conditions in a L-carnitine overproducing E. coli strain.

    Directory of Open Access Journals (Sweden)

    Guido Santos

    Full Text Available Based on experimental data from E. coli cultures, we have devised a mathematical model in the GMA-power law formalism that describes the central and L-carnitine metabolism in and between two steady states, non-osmotic and hyperosmotic (0.3 M NaCl. A key feature of this model is the introduction of type of kinetic order, the osmotic stress kinetic orders (g(OSn, derived from the power law general formalism, which represent the effect of osmotic stress in each metabolic process of the model.By considering the values of the g(OSn linked to each metabolic process we found that osmotic stress has a positive and determinant influence on the increase in flux in energetic metabolism (glycolysis; L-carnitine biosynthesis production; the transformation/excretion of Acetyl-CoA into acetate and ethanol; the input flux of peptone into the cell; the anabolic use of pyruvate and biomass decomposition. In contrast, we found that although the osmotic stress has an inhibitory effect on the transformation flux from the glycolytic end products (pyruvate to Acetyl-CoA, this inhibition is counteracted by other effects (the increase in pyruvate concentration to the extent that the whole flux increases. In the same vein, the down regulation exerted by osmotic stress on fumarate uptake and its oxidation and the production and export of lactate and pyruvate are reversed by other factors up to the point that the first increased and the second remained unchanged.The model analysis shows that in osmotic conditions the energy and fermentation pathways undergo substantial rearrangement. This is illustrated by the observation that the increase in the fermentation fluxes is not connected with fluxes towards the tricaboxylic acid intermediates and the synthesis of biomass. The osmotic stress associated with these fluxes reflects these changes. All these observations support that the responses to salt stress observed in E. coli might be conserved in halophiles.Flux evolution

  13. Design and Finite Element Analysis of a Novel Transverse Flux Permanent Magnet Disk Generator

    DEFF Research Database (Denmark)

    Hosseini, Seyedmohsen; Moghani, Javad Shokrollahi; Ershad, Nima Farrokhzad

    2011-01-01

    This paper presents a novel structure of a transverse flux permanent magnet disk generator. The proposed disk shape structure simplifies prototyping by using simple laminated steel sheets in comparison with previous transverse flux structures that employ bent laminations and soft magnetic...... composites. Also the proposed structure has a higher power factor than what has been reported previously for transverse flux permanent magnet structures. After introducing the transverse flux permanent magnet disk generator, the design process is explained and a small power generator is designed...

  14. In-depth performance analysis of the HyperFlux spectrometer

    Science.gov (United States)

    Meade, Jeffrey T.; Behr, Bradford B.; Bismilla, Yusuf; Cenko, Andrew T.; Hajian, Arsen R.

    2013-03-01

    Tornado Spectral Systems introduced the HyperFluxTM spectrometer to the market in early 2012. The Hyper- Flux is the world's first HTVS (high-throughput virtual slit) enabled spectrometer and is able to achieve much greater system flux compared to slit-based spectrometers. Since the HyperFlux's debut extensive studies into the manufacturability, stability, and detector electronic performance have been performed and are presented in this paper. A generalized quantitative approach to spectrometer comparison by using a clearly-defined Quality Factor is presented at the end of the paper.

  15. Activation analysis of stainless steel flux monitors using 252Cf neutron sources

    International Nuclear Information System (INIS)

    Williams, J.G.; Newton, T.H. Jr.; Cogburn, C.O.

    1984-01-01

    Activation analysis was performed on stainless steel beads from a chain which is used in reactor pressure vessel surveillance experiments at the Arkansas Power and Light Company reactors. The beads allow monitoring of two fast and three thermal neutron induced reactions: 58 Ni(n,p) 58 Co, 54 Fe(n,p) 54 Mn, 58 Fe(n,γ) 59 Fe, 59 Co(n,γ) 60 Co and 50 Cr(n,γ) 51 Cr. The analysis was performed using 12 beads from various positions along 5 different batches of chain and standard materials in an H 2 O moderator tank using two intense californium sources which had a total neutron emission rate of 3.97 x 10 10 /s. Semiconductor gamma spectrometers were used to count the products of the above reactions in the specimens. The percentage by weight of the iron, chromium and cobalt in the beads were found to be 62.1%, 20.2% and 0.120%, respectively. The excellent uniformity found in the bead compositions demonstrates the reproducibility of the experimental techniques and enhances considerably the value of the beads as neutron flux montitors

  16. On-site testing of advective flux probes for enlarging the range of soil hydrocarbon analysis

    International Nuclear Information System (INIS)

    Kerfoot, W.B.

    1991-01-01

    The success of soil vapor as a means of assessing subsurface conditions depends upon the volatility of the compounds and the ability of the vapor to migrate through soil pores. Normally, soil gas techniques are not considered valid for poorly volatile compounds or tight soils. Both of these factors can be overcome by simultaneous application of heat and vacuum, combined with a means of creating an artificially porous substrate out of compacted poorly permeable soils. A special rock bit was devised to fit on a miniature hollow stem shaft. The bit receives a continuous flow of heated air or liquid which removes organics from the pulverized soil as the bit penetrates. The recirculation of hot air then transports the gas or liquid to the surface for chromatographic analysis. A comparison of vapor pressures of different organics versus absolute temperatures can be used to extrapolate the extension of soil gas detection. Heavy oils, petroleum, coal, creosotes, naphthalenes, turpenes and quinolines, all compounds whose boiling points exist in excess of 200 degree C, can be analyzed by soil vapor techniques. Samples of the volatile organic can be obtained by either: direct injection onto a heated column, bubbling the vapor through a solvent or adsorption-desorption tubes. The use of solvents allows the technique to extend to fluorometric or IR analysis. The paper compares current vacuum procedures with those obtained from the advective flux procedure for selected sites on Cape Cod

  17. Heat flux of driven granular mixtures at low density: Stability analysis of the homogeneous steady state

    Science.gov (United States)

    Khalil, Nagi; Garzó, Vicente

    2018-02-01

    The Navier-Stokes order hydrodynamic equations for a low-density driven granular mixture obtained previously [Khalil and Garzó, Phys. Rev. E 88, 052201 (2013), 10.1103/PhysRevE.88.052201] from the Chapman-Enskog solution to the Boltzmann equation are considered further. The four transport coefficients associated with the heat flux are obtained in terms of the mass ratio, the size ratio, composition, coefficients of restitution, and the driven parameters of the model. Their quantitative variation on the control parameters of the system is demonstrated by considering the leading terms in a Sonine polynomial expansion to solve the exact integral equations. As an application of these results, the stability of the homogeneous steady state is studied. In contrast to the results obtained in undriven granular mixtures, the stability analysis of the linearized Navier-Stokes hydrodynamic equations shows that the transversal and longitudinal modes are (linearly) stable with respect to long enough wavelength excitations. This conclusion agrees with a previous analysis made for single granular gases.

  18. Entropy Analysis of Kinetic Flux Vector Splitting Schemes for the Compressible Euler Equations

    Science.gov (United States)

    Shiuhong, Lui; Xu, Jun

    1999-01-01

    Flux Vector Splitting (FVS) scheme is one group of approximate Riemann solvers for the compressible Euler equations. In this paper, the discretized entropy condition of the Kinetic Flux Vector Splitting (KFVS) scheme based on the gas-kinetic theory is proved. The proof of the entropy condition involves the entropy definition difference between the distinguishable and indistinguishable particles.

  19. Analysis of carbon dioxide, water vapour and energy fluxes over an ...

    Indian Academy of Sciences (India)

    Su H B, Scott S L, Offerle B, Randolph J C and. Ehman J 2004 Heat storage and energy balance fluxes for a temperate deciduous forest; Agric. For. Meteor. 126. 185–201. Running S W, Baldocchi D D and Turner D 1999 A global terrestrial monitoring network, scaling tower fluxes with ecosystem modeling and EOS satellite ...

  20. Impact of water use efficiency on eddy covariance flux partitioning using correlation structure analysis

    Science.gov (United States)

    Partitioned land surfaces fluxes (e.g. evaporation, transpiration, photosynthesis, and ecosystem respiration) are needed as input, calibration, and validation data for numerous hydrological and land surface models. However, one of the most commonly used techniques for measuring land surface fluxes,...

  1. Analysis of flux-flow curves in superconductors as a function of temperature

    International Nuclear Information System (INIS)

    Boboshko, N.

    1978-01-01

    A phenomenological method to analyse the experimental data of flux-flow resistivity in superconductors as a function of temperature is proposed and successfully applied to existing data for t = kappa >= 0.07. An empirical curve relating the flux-flow resistivity with the Ginsburg-Landau parameter kappa was obtained. (author)

  2. Analysis of the microclimate and CO2 flux characteristics in arid ...

    Indian Academy of Sciences (India)

    Xihu desert wetland is an important and unusual environment in China or even in the world. However, until now, little research has been focused on the microclimate and CO2 flux characteristics in this area. This paper reports the characteristics of daily variations of microclimate and CO2 flux in the Dunhuang. Xihu desert ...

  3. Central carbon metabolism influences cellulase production in Bacillus licheniformis.

    Science.gov (United States)

    Wang, J; Liu, S; Li, Y; Wang, H; Xiao, S; Li, C; Liu, B

    2018-01-01

    Bacillus licheniformis that can produce cellulase including endo glucanase and glucosidase is an important industrial microbe for cellulose degradation. The purpose of this research was to assess the effect of endo glucanase gene bglC and glucosidase gene bglH on the central metabolic flux in B. licheniformis. bglC and bglH were knocked out using homologous recombination method, respectively, and the corresponding knockout strains were obtained for 13 C metabolic flux analysis. A significant change was observed in metabolic fluxes after 13 C metabolic flux ratio analysis. In both of the knockout strains, the increased fluxes of the pentose phosphate pathway and malic enzyme reaction enabled an elevated supply of NADPH which provided enough reducing power for the in vivo synthesis reactions. The fluxes through tricarboxylic acid cycle and anaplerotic reactions increased fast in the two knockout strains, which meant more energy generated. The changed fluxes in central carbon metabolism provided a holistic view of the physiological status in B. licheniformis and possible targets for further strain engineering. Cellulase is very important in the field of agriculture and bioenergy because of its degrading effect on cellulosic biomass. This study presented the effect of central carbon metabolism on cellulase production in Bacillus licheniformis. The study also provided a holistic view of the physiological status in B. licheniformis. The shifted metabolism provided a quantitative evaluation of the biosynthesis of cellulase and a priority ranked target list for further strain engineering. © 2017 The Society for Applied Microbiology.

  4. Partitioning carbon fluxes from a Midwestern corn and soybean rotation system using footprint analysis

    Science.gov (United States)

    Dold, C.; Hatfield, J.; Prueger, J. H.; Wacha, K.

    2017-12-01

    Midwestern US agriculture is dominated by corn and soybean production. Corn has typically higher Net Ecosystem Exchange (NEE) than soybean due to increased carboxylation efficiency and different crop management. The conjoined NEE may be measured with eddy covariance (EC) stations covering both crops, however, it is often unclear what the contribution of each crop is, as the CO2 source area remains unknown. The aim of this study was to quantify the contribution of CO2 fluxes from each crop for a conventional corn-soybean rotation system from 2007 - 2015. Therefore, the combined CO2 flux of three adjacent fields with annual corn-soybean rotation was measured with a 9.1 m EC tower (Flux 30). In the center of two of these fields, additional EC towers (Flux 10 and Flux 11) were positioned above the corn and soybean canopy to validate Flux 30 NEE. For each EC system the annual 90% NEE footprint area was calculated, footprints were partitioned among fields, and NEE separated accordingly. The average annual 90% footprint area of Flux 30, and Flux 10/11 corn and soybean was estimated to 206, 11 and 7 ha, respectively. The annual average (±SE) NEE of Flux 30 was -693 ± 47 g CO2 m-2 yr-1, of which 83% out of 90% originated from the three adjacent fields. Corn and soybean NEE measured at Flux 10 and 11 was -1124 ± 95 and 173 ± 73 g CO2 m-2 yr-1, respectively, and 89% and 90% originated from these fields. That demonstrates, that Flux 30 represents the combined NEE of a corn-soybean rotation, and Flux 10 and 11 measured NEE from a single crop. However, the share of Flux 30 NEE originating from corn and soybean grown on the Flux 10/11 fields was -192 ± 16 and -205 ± 18 g CO2 m-2 yr-1, indicating a substantial difference to single crop NEE. While it was possible to measure the NEE of a corn-soybean rotation with a tall EC tower, footprint partitioning could not retrieve NEE for each crop, probably due to differences in measurement height and footprint source area.

  5. INAA analysis of rocks: A routine method using Fe as an internal flux monitor

    International Nuclear Information System (INIS)

    Kay, R.W.; Kay, S. Mahlburg

    1992-01-01

    Over the past decade at Cornell, trace elements in over 2500 rocks have been analyzed by INAA. The samples, mainly volcanic rocks, have known concentrations of major elements (e.g. Si, Ti, Al, Mg, Ca, K, Fe, Na) and the last two of these (Fe and Na) are also determined by activation, using rock standards (e.g. USGS standards BCRl, BHVO, etc.). Differences between Fe determined by INAA and that determined as a part of the major element analysis are mainly attributed to volatile (H 2 O, CO 2 ) loss (especially when major element analyses were done by electron microprobe on fused powders, whereas the INAA analyses were done on the powders), and to flux variability during irradiation. Instead of reporting two values for Fe we use Fe as an internal flux monitor, with Na and the trace elements being reported relative to the given Fe value. The ratio Na/Fe is used as a sensitive check on the identity of the sample and as a monitor of alkali loss affecting the major element analysis. Other than this modification (Kay et aL 1987, also reported in Chappell and Hergt, 1989) w