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Isotopically nonstationary metabolic flux analysis (INST-MFA): putting theory into practice.
Cheah YE, Young JD
(2018) Curr Opin Biotechnol 54: 80-87
MeSH Terms: Autotrophic Processes, Carbon Isotopes, Isotope Labeling, Metabolic Flux Analysis, Models, Biological, Software
Show Abstract · Added March 14, 2018
Typically, C flux analysis relies on assumptions of both metabolic and isotopic steady state. If metabolism is steady but isotope labeling is not allowed to fully equilibrate, isotopically nonstationary metabolic flux analysis (INST-MFA) can be used to estimate fluxes. This requires solution of differential equations that describe the time-dependent labeling of network metabolites, while iteratively adjusting the flux and pool size parameters to match the transient labeling measurements. INST-MFA holds a number of unique advantages over approaches that rely solely upon steady-state isotope enrichments. First, INST-MFA can be applied to estimate fluxes in autotrophic systems, which consume only single-carbon substrates. Second, INST-MFA is ideally suited to systems that label slowly due to the presence of large intermediate pools or pathway bottlenecks. Finally, INST-MFA provides increased measurement sensitivity to estimate reversible exchange fluxes and metabolite pool sizes, which represents a potential framework for integrating metabolomic analysis with C flux analysis. This review highlights the unique capabilities of INST-MFA, describes newly available software tools that automate INST-MFA calculations, presents several practical examples of recent INST-MFA applications, and discusses the technical challenges that lie ahead.
Copyright © 2018 Elsevier Ltd. All rights reserved.
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6 MeSH Terms
Isotopically Nonstationary Metabolic Flux Analysis (INST-MFA) of Photosynthesis and Photorespiration in Plants.
Ma F, Jazmin LJ, Young JD, Allen DK
(2017) Methods Mol Biol 1653: 167-194
MeSH Terms: Amino Acids, Arabidopsis, Carbon Dioxide, Carbon Isotopes, Chlorophyll, Chloroplasts, Isotope Labeling, Mass Spectrometry, Metabolic Flux Analysis, Metabolic Networks and Pathways, Oxygen, Oxygen Consumption, Photosynthesis, Plant Leaves, Ribulose-Bisphosphate Carboxylase, Starch, Sucrose
Show Abstract · Added September 11, 2017
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 C metabolic flux analysis (INST-MFA), which is based on transient labeling studies at metabolic steady state, offers a comprehensive platform to quantify plant central metabolism. In this chapter, we describe the application of INST-MFA to investigate metabolism in leaves. Leaves are an autotrophic tissue, assimilating CO over a diurnal period implying that the metabolic steady state is limited to less than 12 h and thus requiring an INST-MFA approach. This strategy results in a comprehensive unified description of photorespiration, Calvin cycle, sucrose and starch synthesis, tricarboxylic acid (TCA) cycle, and amino acid biosynthetic fluxes. We present protocols of the experimental aspects for labeling studies: transient CO labeling of leaf tissue, sample quenching and extraction, mass spectrometry (MS) analysis of isotopic labeling data, measurement of sucrose and amino acids in vascular exudates, and provide details on the computational flux estimation using INST-MFA.
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17 MeSH Terms
High-resolution hyperpolarized in vivo metabolic C spectroscopy at low magnetic field (48.7mT) following murine tail-vein injection.
Coffey AM, Feldman MA, Shchepin RV, Barskiy DA, Truong ML, Pham W, Chekmenev EY
(2017) J Magn Reson 281: 246-252
MeSH Terms: Animals, Carbon Isotopes, Cell Line, Tumor, Contrast Media, Electromagnetic Fields, Female, Injections, Intravenous, Magnetic Resonance Imaging, Magnetic Resonance Spectroscopy, Mice, Mice, Nude, Neoplasms, Experimental, Tail, Veins
Show Abstract · Added March 21, 2018
High-resolution C NMR spectroscopy of hyperpolarized succinate-1-C-2,3-d is reported in vitro and in vivo using a clinical-scale, biplanar (80cm-gap) 48.7mT permanent magnet with a high homogeneity magnetic field. Non-localized C NMR spectra were recorded at 0.52MHz resonance frequency over the torso of a tumor-bearing mouse every 2s. Hyperpolarized C NMR signals with linewidths of ∼3Hz (corresponding to ∼6ppm) were recorded in vitro (2mL in a syringe) and in vivo (over a mouse torso). Comparison of the full width at half maximum (FWHM) for C NMR spectra acquired at 48.7mT and at 4.7T in a small-animal MRI scanner demonstrates a factor of ∼12 improvement for the C resonance linewidth attainable at 48.7mT compared to that at 4.7T in vitro. C hyperpolarized succinate-1-C resonance linewidths in vivo are at least one order of magnitude narrower at 48.7mT compared to those observed in high-field (≥3T) studies employing HP contrast agents. The demonstrated high-resolution C in vivo spectroscopy could be useful for high-sensitivity spectroscopic studies involving monitoring HP agent uptake or detecting metabolism using HP contrast agents with sufficiently large C chemical shift differences.
Copyright © 2017 Elsevier Inc. All rights reserved.
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14 MeSH Terms
Isotopically nonstationary C flux analysis of cyanobacterial isobutyraldehyde production.
Jazmin LJ, Xu Y, Cheah YE, Adebiyi AO, Johnson CH, Young JD
(2017) Metab Eng 42: 9-18
MeSH Terms: Aldehydes, Bacterial Proteins, Carbon Isotopes, Malate Dehydrogenase, Phosphoenolpyruvate Carboxylase, Pyruvate Kinase, Staining and Labeling, Synechococcus
Show Abstract · Added September 11, 2017
We applied isotopically nonstationary C metabolic flux analysis (INST-MFA) to compare the pathway fluxes of wild-type (WT) Synechococcus elongatus PCC 7942 to an engineered strain (SA590) that produces isobutyraldehyde (IBA). The flux maps revealed a potential bottleneck at the pyruvate kinase (PK) reaction step that was associated with diversion of flux into a three-step PK bypass pathway involving the enzymes PEP carboxylase (PEPC), malate dehydrogenase (MDH), and malic enzyme (ME). Overexpression of pk in SA590 led to a significant improvement in IBA specific productivity. Single-gene overexpression of the three enzymes in the proposed PK bypass pathway also led to improvements in IBA production, although to a lesser extent than pk overexpression. Combinatorial overexpression of two of the three genes in the proposed PK bypass pathway (mdh and me) led to improvements in specific productivity that were similar to those achieved by single-gene pk overexpression. Our work demonstrates how C flux analysis can be used to identify potential metabolic bottlenecks and novel metabolic routes, and how these findings can guide rational metabolic engineering of cyanobacteria for increased production of desired molecules.
Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.
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8 MeSH Terms
Application of C flux analysis to identify high-productivity CHO metabolic phenotypes.
Templeton N, Smith KD, McAtee-Pereira AG, Dorai H, Betenbaugh MJ, Lang SE, Young JD
(2017) Metab Eng 43: 218-225
MeSH Terms: Animals, Antibodies, Monoclonal, CHO Cells, Carbon Isotopes, Citric Acid Cycle, Cricetulus, Gene Expression, Immunoglobulin G, Isotope Labeling, Recombinant Proteins
Show Abstract · Added April 27, 2017
Industrial bioprocesses place high demands on the energy metabolism of host cells to meet biosynthetic requirements for maximal protein expression. Identifying metabolic phenotypes that promote high expression is therefore a major goal of the biotech industry. We conducted a series of C flux analysis studies to examine the metabolic response to IgG expression during early stationary phase of CHO cell cultures grown in 3L fed-batch bioreactors. We examined eight clones expressing four different IgGs and compared with three non-expressing host-cell controls. Some clones were genetically manipulated to be apoptosis-resistant by expressing Bcl-2Δ, which correlated with increased IgG production and elevated glucose metabolism. The metabolic phenotypes of the non-expressing, IgG-expressing, and Bcl-2Δ/IgG-expressing clones were fully segregated by hierarchical clustering analysis. Lactate consumption and citric acid cycle fluxes were most strongly associated with specific IgG productivity. These studies indicate that enhanced oxidative metabolism is a characteristic of high-producing CHO cell lines.
Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.
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10 MeSH Terms
Open-Source Automated Parahydrogen Hyperpolarizer for Molecular Imaging Using (13)C Metabolic Contrast Agents.
Coffey AM, Shchepin RV, Truong ML, Wilkens K, Pham W, Chekmenev EY
(2016) Anal Chem 88: 8279-88
MeSH Terms: Animals, Automation, Carbon Isotopes, Catalysis, Contrast Media, Hydrogen, Lactic Acid, Magnetic Resonance Imaging, Magnetic Resonance Spectroscopy, Mice, Mice, Nude, Software, Succinates, Water
Show Abstract · Added March 21, 2018
An open-source hyperpolarizer producing (13)C hyperpolarized contrast agents using parahydrogen induced polarization (PHIP) for biomedical and other applications is presented. This PHIP hyperpolarizer utilizes an Arduino microcontroller in conjunction with a readily modified graphical user interface written in the open-source processing software environment to completely control the PHIP hyperpolarization process including remotely triggering an NMR spectrometer for efficient production of payloads of hyperpolarized contrast agent and in situ quality assurance of the produced hyperpolarization. Key advantages of this hyperpolarizer include: (i) use of open-source software and hardware seamlessly allowing for replication and further improvement as well as readily customizable integration with other NMR spectrometers or MRI scanners (i.e., this is a multiplatform design), (ii) relatively low cost and robustness, and (iii) in situ detection capability and complete automation. The device performance is demonstrated by production of a dose (∼2-3 mL) of hyperpolarized (13)C-succinate with %P13C ∼ 28% and 30 mM concentration and (13)C-phospholactate at %P13C ∼ 15% and 25 mM concentration in aqueous medium. These contrast agents are used for ultrafast molecular imaging and spectroscopy at 4.7 and 0.0475 T. In particular, the conversion of hyperpolarized (13)C-phospholactate to (13)C-lactate in vivo is used here to demonstrate the feasibility of ultrafast multislice (13)C MRI after tail vein injection of hyperpolarized (13)C-phospholactate in mice.
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14 MeSH Terms
Modeling Method for Increased Precision and Scope of Directly Measurable Fluxes at a Genome-Scale.
McCloskey D, Young JD, Xu S, Palsson BO, Feist AM
(2016) Anal Chem 88: 3844-52
MeSH Terms: Carbon Isotopes, Escherichia coli, Genome, Bacterial, Metabolic Flux Analysis, Models, Biological
Show Abstract · Added March 31, 2016
Metabolic flux analysis (MFA) is considered to be the gold standard for determining the intracellular flux distribution of biological systems. The majority of work using MFA has been limited to core models of metabolism due to challenges in implementing genome-scale MFA and the undesirable trade-off between increased scope and decreased precision in flux estimations. This work presents a tunable workflow for expanding the scope of MFA to the genome-scale without trade-offs in flux precision. The genome-scale MFA model presented here, iDM2014, accounts for 537 net reactions, which includes the core pathways of traditional MFA models and also covers the additional pathways of purine, pyrimidine, isoprenoid, methionine, riboflavin, coenzyme A, and folate, as well as other biosynthetic pathways. When evaluating the iDM2014 using a set of measured intracellular intermediate and cofactor mass isotopomer distributions (MIDs),1 it was found that a total of 232 net fluxes of central and peripheral metabolism could be resolved in the E. coli network. The increase in scope was shown to cover the full biosynthetic route to an expanded set of bioproduction pathways, which should facilitate applications such as the design of more complex bioprocessing strains and aid in identifying new antimicrobials. Importantly, it was found that there was no loss in precision of core fluxes when compared to a traditional core model, and additionally there was an overall increase in precision when considering all observable reactions.
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5 MeSH Terms
Application of isotope labeling experiments and (13)C flux analysis to enable rational pathway engineering.
McAtee AG, Jazmin LJ, Young JD
(2015) Curr Opin Biotechnol 36: 50-6
MeSH Terms: Bioengineering, Carbon Isotopes, Isotope Labeling, Models, Biological
Show Abstract · Added March 31, 2016
Isotope labeling experiments (ILEs) and (13)C flux analysis provide actionable information for metabolic engineers to identify knockout, overexpression, and/or media optimization targets. ILEs have been used in both academic and industrial labs to increase product formation, discover novel metabolic functions in previously uncharacterized organisms, and enhance the metabolic efficiency of host cell factories. This review highlights specific examples of how ILEs have been used in conjunction with enzyme or metabolic engineering to elucidate host cell metabolism and improve product titer, rate, or yield in a directed manner. We discuss recent progress and future opportunities involving the use of ILEs and (13)C flux analysis to characterize non-model host organisms and to identify and subsequently eliminate wasteful byproduct pathways or metabolic bottlenecks.
Copyright © 2015 Elsevier Ltd. All rights reserved.
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4 MeSH Terms
Mass spectrometry-based microassay of (2)H and (13)C plasma glucose labeling to quantify liver metabolic fluxes in vivo.
Hasenour CM, Wall ML, Ridley DE, Hughey CC, James FD, Wasserman DH, Young JD
(2015) Am J Physiol Endocrinol Metab 309: E191-203
MeSH Terms: Animals, Biological Transport, Blood Glucose, Carbon Isotopes, Citric Acid Cycle, Deuterium, Gas Chromatography-Mass Spectrometry, Glucose, Isotope Labeling, Liver, Liver Glycogen, Male, Mice, Mice, Inbred C57BL
Show Abstract · Added May 27, 2015
Mouse models designed to examine hepatic metabolism are critical to diabetes and obesity research. Thus, a microscale method to quantitatively assess hepatic glucose and intermediary metabolism in conscious, unrestrained mice was developed. [(13)C3]propionate, [(2)H2]water, and [6,6-(2)H2]glucose isotopes were delivered intravenously in short- (9 h) and long-term-fasted (19 h) C57BL/6J mice. GC-MS and mass isotopomer distribution (MID) analysis were performed on three 40-μl arterial plasma glucose samples obtained during the euglycemic isotopic steady state. Model-based regression of hepatic glucose and citric acid cycle (CAC)-related fluxes was performed using a comprehensive isotopomer model to track carbon and hydrogen atom transitions through the network and thereby simulate the MIDs of measured fragment ions. Glucose-6-phosphate production from glycogen diminished, and endogenous glucose production was exclusively gluconeogenic with prolonged fasting. Gluconeogenic flux from phosphoenolpyruvate (PEP) remained stable, whereas that from glycerol modestly increased from short- to long-term fasting. CAC flux [i.e., citrate synthase (VCS)] was reduced with long-term fasting. Interestingly, anaplerosis and cataplerosis increased with fast duration; accordingly, pyruvate carboxylation and the conversion of oxaloacetate to PEP were severalfold higher than VCS in long-term fasted mice. This method utilizes state-of-the-art in vivo methodology and comprehensive isotopomer modeling to quantify hepatic glucose and intermediary fluxes during physiological stress in mice. The small plasma requirements permit serial sampling without stress and the affirmation of steady-state glucose kinetics. Furthermore, the approach can accommodate a broad range of modeling assumptions, isotope tracers, and measurement inputs without the need to introduce ad hoc mathematical approximations.
Copyright © 2015 the American Physiological Society.
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14 MeSH Terms
Voxelwise Relationships Between Distribution Volume Ratio and Cerebral Blood Flow: Implications for Analysis of β-Amyloid Images.
Sojkova J, Goh J, Bilgel M, Landman B, Yang X, Zhou Y, An Y, Beason-Held LL, Kraut MA, Wong DF, Resnick SM
(2015) J Nucl Med 56: 1042-7
MeSH Terms: Aged, Aged, 80 and over, Amyloid beta-Peptides, Benzothiazoles, Carbon Isotopes, Cerebrovascular Circulation, False Positive Reactions, Female, Humans, Image Processing, Computer-Assisted, Linear Models, Longitudinal Studies, Male, Middle Aged, Oxygen Isotopes, Positron-Emission Tomography, Reference Values, Reproducibility of Results, Water
Show Abstract · Added November 2, 2016
UNLABELLED - Quantification of β-amyloid (Aβ) in vivo is often accomplished using the distribution volume ratio (DVR), based on a simplified reference tissue model. We investigated the local relationships between DVR and cerebral blood flow (CBF), as well as relative CBF (R1), in nondemented older adults.
METHODS - Fifty-five nondemented participants (mean age, 78.5 y) in the Baltimore Longitudinal Study of Aging underwent (15)O-H2O PET CBF and dynamic (11)C-PiB PET. (15)O-H2O PET images were normalized and smoothed using SPM. A simplified reference tissue model with linear regression and spatial constraints was used to generate parametric DVR images. The DVR images were regressed on CBF images on a voxel-by-voxel basis using robust biologic parametric mapping, adjusting for age and sex (false discovery rate, P = 0.05; spatial extent, 50 voxels). DVR images were also regressed on R1 images, a measure of the transport rate constant from vascular space to tissue. All analyses were performed on the entire sample, and on high and low tertiles of mean cortical DVR.
RESULTS - Voxel-based analyses showed that increased DVR is associated with increased CBF in the frontal, parietal, temporal, and occipital cortices. However, this association appears to spare regions that typically show early Aβ deposition. A more robust relationship between DVR and CBF was observed in the lower tertile of DVR, that is, negligible cortical Aβ load, compared with the upper tertile of cortical DVR and Aβ load. The spatial distributions of the DVR-CBF and DVR-R1 correlations showed similar patterns. No reliable negative voxelwise relationships between DVR and CBF or R1 were observed.
CONCLUSION - Robust associations between DVR and CBF at negligible Aβ levels, together with similar spatial distributions of DVR-CBF and DVR-R1 correlations, suggest that regional distribution of DVR reflects blood flow and tracer influx rather than pattern of Aβ deposition in those with minimal Aβ load. DVR-CBF associations in individuals with a higher DVR are more likely to reflect true associations between patterns of Aβ deposition and CBF or neural activity. These findings have important implications for analysis and interpretation of voxelwise correlations with external variables in individuals with varying amounts of Aβ load.
© 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.
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19 MeSH Terms