13C Metabolic Flux Analysis Research Articles

13 C-MFA helps to identify metabolic bottlenecks for improving malic acid production in Myceliophthora thermophila

BackgroundMyceliophthora thermophila has been engineered as a significant cell factory for malic acid production, yet strategies to further enhance production remain unclear and lack rational guidance. 13C-MFA (13C metabolic flux analysis) offers a means to analyze cellular metabolic mechanisms and pinpoint critical nodes for improving product synthesis. Here, we employed 13C-MFA to investigate the metabolic flux distribution of a high-malic acid-producing strain of M. thermophila and attempted to decipher the crucial bottlenecks in the metabolic pathways.ResultsCompared with the wild-type strain, the high-Malic acid-producing strain M. thermophila JG207 exhibited greater glucose uptake and carbon dioxide evolution rates but lower oxygen uptake rates and biomass yields. Consistent with these phenotypes, the 13C-MFA results showed that JG207 displayed elevated flux through the EMP pathway and downstream TCA cycle, along with reduced oxidative phosphorylation flux, thereby providing more precursors and NADH for malic acid synthesis. Furthermore, based on the 13C-MFA results, we conducted oxygen-limited culture and nicotinamide nucleotide transhydrogenase (NNT) gene knockout experiments to increase the cytoplasmic NADH level, both of which were shown to be beneficial for malic acid accumulation.ConclusionsThis work elucidates and validates the key node for achieving high malic acid production in M. thermophila. We propose effective fermentation strategies and genetic modifications for enhancing malic acid production. These findings offer valuable guidance for the rational design of future cell factories aimed at improving malic acid yields.

Fluxomic, Metabolomic, and Transcriptomic Analyses Reveal Metabolic Responses to Phenazine-1-carboxamide Synthesized in Pseudomonas chlororaphis.

Phenazine-1-carboxamide (PCN) has been exploited as a successful biopesticide due to its broad-spectrum antifungal activity. We engineered a PCN-overproducing Pseudomonas chlororaphis strain through overexpressing shikimate pathway genes (aroB, aroQ, aroE, and phzC) and deleting negative regulatory genes (relA, fleQ, and pykF). The optimized strain produced 1.92 g/L PCN with a yield of 0.11 g/g glycerol, the highest titer ever reported by using minimal media. To gain deeper insights into the underlying regulatory network, the final strain and the parental strain were examined using three distinct omic data sets. 13C-metabolic flux analysis revealed a substantial flux reconfiguration in the optimized strain, including the activation of the EDEMP cycle, the PP pathway, the glyoxylate shunt, and the shikimate pathway. Metabolomic results indicated that central carbon was rerouted to the shikimate pathway. Transcriptomic data identified global gene expression changes. This study forms the basis for further engineering of strains to achieve outstanding performance.

Global 13C tracing and metabolic flux analysis of intact human liver tissue ex vivo.

Liver metabolism is central to human physiology and influences the pathogenesis of common metabolic diseases. Yet, our understanding of human liver metabolism remains incomplete, with much of current knowledge based on animal or cell culture models that do not fully recapitulate human physiology. Here, we perform in-depth measurement of metabolism in intact human liver tissue ex vivo using global 13C tracing, non-targeted mass spectrometry and model-based metabolic flux analysis. Isotope tracing allowed qualitative assessment of a wide range of metabolic pathways within a single experiment, confirming well-known features of liver metabolism but also revealing unexpected metabolic activities such as de novo creatine synthesis and branched-chain amino acid transamination, where human liver appears to differ from rodent models. Glucose production ex vivo correlated with donor plasma glucose, suggesting that cultured liver tissue retains individual metabolic phenotypes, and could be suppressed by postprandial levels of nutrients and insulin, and also by pharmacological inhibition of glycogen utilization. Isotope tracing ex vivo allows measuring human liver metabolism with great depth and resolution in an experimentally tractable system.

Metabolic and transcriptomic reprogramming during contact inhibition-induced quiescence is mediated by YAP-dependent and YAP-independent mechanisms.

Metabolic rewiring during the proliferation-to-quiescence transition is poorly understood. Here, using a model of contact inhibition-induced quiescence, we conducted 13C-metabolic flux analysis in proliferating (P) and quiescent (Q) mouse embryonic fibroblasts (MEFs) to investigate this process. Q cells exhibit reduced glycolysis but increased TCA cycle flux and mitochondrial respiration. Reduced glycolytic flux in Q cells correlates with reduced glycolytic enzyme expression mediated by yes-associated protein (YAP) inhibition. The increased TCA cycle activity and respiration in Q cells is mediated by induced mitochondrial pyruvate carrier (MPC) expression, rendering them vulnerable to MPC inhibition. The malate-to-pyruvate flux, which generates NADPH, is markedly reduced by modulating malic enzyme 1 (ME1) dimerization in Q cells. Conversely, the malate dehydrogenase 1 (MDH1)-mediated oxaloacetate-to-malate flux is reversed and elevated in Q cells, driven by high mitochondrial-derived malate levels, reduced cytosolic oxaloacetate, elevated MDH1 levels, and a high cytoplasmic NAD+/NADH ratio. Transcriptomic analysis revealed large number of genes are induced in Q cells, many of which are associated with the extracellular matrix (ECM), while YAP-dependent and cell cycle-related genes are repressed. The results suggest that high TCA cycle flux and respiration in Q cells are required to generate ATP and amino acids to maintain de-novo ECM protein synthesis and secretion.

Ala-Cys-Cys-Ala dipeptide dimer alleviates problematic cysteine and cystine levels in media formulations and enhances CHO cell growth and metabolism

Cysteine and cystine are essential amino acids present in mammalian cell cultures. While contributing to biomass synthesis, recombinant protein production, and antioxidant defense mechanisms, cysteine poses a major challenge in media formulations owing to its poor stability and oxidation to cystine, a cysteine dimer. Due to its poor solubility, cystine can cause precipitation of feed media, formation of undesired products, and consequently, reduce cysteine bioavailability. In this study, a highly soluble cysteine containing dipeptide dimer, Ala-Cys-Cys-Ala (ACCA), was evaluated as a suitable alternative to cysteine and cystine in CHO cell cultures. Replacing cysteine and cystine in basal medium with ACCA did not sustain cell growth. However, addition of ACCA at 4 mM and 8 mM to basal medium containing cysteine and cystine boosted cell growth up to 15% and 27% in CHO-GS and CHO–K1 batch cell cultures respectively and led to a proportionate increase in IgG titer. 13C-Metabolic flux analysis revealed that supplementation of ACCA reduced glycolytic fluxes by 20% leading to more efficient glucose metabolism in CHO–K1 cells. In fed-batch cultures, ACCA was able to replace cysteine and cystine in feed medium. Furthermore, supplementation of ACCA at high concentrations in basal medium eliminated the need for any cysteine equivalents in feed medium and increased cell densities and viabilities in fed-batch cultures without any significant impact on IgG charge variants. Taken together, this study demonstrates the potential of ACCA to improve CHO cell growth, productivity, and metabolism while also facilitating the formulation of cysteine- and cystine-free feed media. Such alternatives to cysteine and cystine will pave the way for enhanced biomanufacturing by increasing cell densities in culture and extending the storage of highly concentrated feed media as part of achieving intensified bioproduction processes.

Elucidation of triacylglycerol catabolism in Yarrowia lipolytica: How cells balance acetyl-CoA and excess reducing equivalents

Yarrowia lipolytica is an industrial yeast that can convert waste oil to value-added products. However, it is unclear how this yeast metabolizes lipid feedstocks, specifically triacylglycerol (TAG) substrates. This study used 13C-metabolic flux analysis (13C-MFA), genome-scale modeling, and transcriptomics analyses to investigate Y. lipolytica W29 growth with oleic acid, glycerol, and glucose. Transcriptomics data were used to guide 13C-MFA model construction and to validate the 13C-MFA results. The 13C-MFA data were then used to constrain a genome-scale model (GSM), which predicted Y. lipolytica fluxes, cofactor balance, and theoretical yields of terpene products. The three data sources provided new insights into cellular regulation during catabolism of glycerol and fatty acid components of TAG substrates, and how their consumption routes differ from glucose catabolism. We found that (1) over 80% of acetyl-CoA from oleic acid is processed through the glyoxylate shunt, a pathway that generates less CO2 compared to the TCA cycle, (2) the carnitine shuttle is a key regulator of the cytosolic acetyl-CoA pool in oleic acid and glycerol cultures, (3) the oxidative pentose phosphate pathway and mannitol cycle are key routes for NADPH generation, (4) the mannitol cycle and alternative oxidase activity help balance excess NADH generated from β-oxidation of oleic acid, and (5) asymmetrical gene expressions and GSM simulations of enzyme usage suggest an increased metabolic burden for oleic acid catabolism.

Systematic engineering for production of anti-aging sunscreen compound in Pseudomonas putida

Sunscreen has been used for thousands of years to protect skin from ultraviolet radiation. However, the use of modern commercial sunscreen containing oxybenzone, ZnO, and TiO2 has raised concerns due to their negative effects on human health and the environment. In this study, we aim to establish an efficient microbial platform for production of shinorine, a UV light absorbing compound with anti-aging properties. First, we methodically selected an appropriate host for shinorine production by analyzing central carbon flux distribution data from prior studies alongside predictions from genome-scale metabolic models (GEMs). We enhanced shinorine productivity through CRISPRi-mediated downregulation and utilized shotgun proteomics to pinpoint potential competing pathways. Simultaneously, we improved the shinorine biosynthetic pathway by refining its design, optimizing promoter usage, and altering the strength of ribosome binding sites. Finally, we conducted amino acid feeding experiments under various conditions to identify the key limiting factors in shinorine production. The study combines meta-analysis of 13C-metabolic flux analysis, GEMs, synthetic biology, CRISPRi-mediated gene downregulation, and omics analysis to improve shinorine production, demonstrating the potential of Pseudomonas putida KT2440 as platform for shinorine production.

Tanshinone IIA alleviates pulmonary fibrosis by modulating glutamine metabolic reprogramming based on [U-13C5]-glutamine metabolic flux analysis

IntroductionGlutamine metabolic reprogramming, mediated by glutaminase (GLS), is an important signal during pulmonary fibrosis (PF) progression. Tanshinone IIA (Tan IIA) is a naturally lipophilic diterpene with antioxidant and antifibrotic properties. However, the potential mechanisms of Tan IIA for regulating glutamine metabolic reprogramming are not yet clear. ObjectivesThis study aimed was to evaluate the role of Tan IIA in intervening in glutamine metabolic reprogramming to exert anti-PF and to explore the potential new mechanisms of metabolic regulation. MethodsFibrotic characteristics was detected via immunofluorescence and western blotting analysis. Cell proliferation was examined with EdU Assay. Cell metabolites were labeled by using stable isotope [U-13C5]-glutamine. By utilizing 100% 13C glutamine tracers and employing network analysis to investigate the activation of metabolic pathways in fibroblasts, as well as evaluating the impact of Tan IIA on these pathways, we accurately quantified the absolute flux of glutaminolysis, proline synthesis, and the TCA cycle pathway using isotopomer network compartmental analysis (INCA), a user-friendly software tool for 13C metabolic flux analysis (13C-MFA). Molecular docking was used for identifying the binding of Tan IIA with target protein. ResultsTan IIA ameliorate TGF-β1-induced myofibroblast proliferation, reduce collagen I and III and α-SMA protein expression in MRC-5 and NIH-3T3 cells. Furthermore, Tan IIA regulate mitochondrial energy metabolism by modulating TGF-β1-stimulated glutamine metabolic reprogramming in NIH-3T3 cells and inhibiting GLS1 expression, which reduced the metabolic flux of glutamine into mitochondria in myofibroblasts, and also targeted inhibited the expression of Δ1-pyrroline-5-carboxylate synthase (P5CS), P5C reductase 1 (PYCR1), and phosphoserine aminotransferase 1 (PSAT1), and reduced proline hydroxylation and blocked the collagen synthesis pathway. ConclusionTan IIA reverses glutamine metabolic reprogramming, reduces mitochondrial energy expenditure, and inhibits collagen matrix synthesis by modulating potential targets in glutamine metabolism. This novel perspective sheds light on the essential role of glutamine metabolic reprogramming in PF.

Elevated nutrient availability enhances chondrocyte metabolism and biosynthesis in tissue-engineered cartilage

ObjectiveChondrocytes, which typically rely on anaerobic metabolism, exhibit upregulated biosynthetic activity when subjected to conditions that elicit mixed aerobic-anaerobic metabolism. Previously, we observed that increasing media volume resulted in the transition from anaerobic to mixed aerobic-anaerobic metabolism. Maximal extracellular matrix (ECM) accumulation occurred at this transition as a result of changes in HIF-1α signaling and associated hypoxic gene expression. This study aimed to explore the effect of further increases in media availability on ECM synthesis and chondrocyte metabolism. MethodsPrimary bovine chondrocytes were grown in 3D high-density tissue culture under varying levels of media availability (4 – 16 mL/106 cells). Changes in ECM accumulation and metabolism were determined through biochemical assays and 13C-metabolic flux analysis (13C-MFA). ResultsIncreasing media volumes resulted in higher accumulation of cartilaginous extracellular matrix (collagen and proteoglycans) and cellularity. Extracellular metabolite measurements revealed that elevated media availability led to increased glucose and glutamine metabolism, along with increased anaerobic activity. 13C-MFA utilizing [U-13C] glucose demonstrated that increased media availability significantly impacted central carbon metabolism, upregulating all glucose-related metabolic pathways (glycolysis, lactate fermentation, the TCA cycle, hexosamine biosynthetic pathway, and the malate-aspartate shuttle). Furthermore, 13C-MFA indicated that glutamine was donating carbons to the TCA cycle, and additional studies involving [U-13C] glutamine tracing supported this notion. ConclusionsElevated media availability upregulates extracellular matrix synthesis and leads to significant changes in metabolic phenotype. Glutamine plays an important role in chondrocyte metabolism and increases in glutamine metabolism correlate with increases in extracellular matrix accumulation.

Context-dependent modification of PFKFB3 in hematopoietic stem cells promotes anaerobic glycolysis and ensures stress hematopoiesis

Metabolic pathways are plastic and rapidly change in response to stress or perturbation. Current metabolic profiling techniques require lysis of many cells, complicating the tracking of metabolic changes over time after stress in rare cells such as hematopoietic stem cells (HSCs). Here, we aimed to identify the key metabolic enzymes that define differences in glycolytic metabolism between steady-state and stress conditions in murine HSCs and elucidate their regulatory mechanisms. Through quantitative 13C metabolic flux analysis of glucose metabolism using high-sensitivity glucose tracing and mathematical modeling, we found that HSCs activate the glycolytic rate-limiting enzyme phosphofructokinase (PFK) during proliferation and oxidative phosphorylation (OXPHOS) inhibition. Real-time measurement of ATP levels in single HSCs demonstrated that proliferative stress or OXPHOS inhibition led to accelerated glycolysis via increased activity of PFKFB3, the enzyme regulating an allosteric PFK activator, within seconds to meet ATP requirements. Furthermore, varying stresses differentially activated PFKFB3 via PRMT1-dependent methylation during proliferative stress and via AMPK-dependent phosphorylation during OXPHOS inhibition. Overexpression of Pfkfb3 induced HSC proliferation and promoted differentiated cell production, whereas inhibition or loss of Pfkfb3 suppressed them. This study reveals the flexible and multilayered regulation of HSC glycolytic metabolism to sustain hematopoiesis under stress and provides techniques to better understand the physiological metabolism of rare hematopoietic cells.

Context-dependent modification of PFKFB3 in hematopoietic stem cells promotes anaerobic glycolysis and ensures stress hematopoiesis.

Metabolic pathways are plastic and rapidly change in response to stress or perturbation. Current metabolic profiling techniques require lysis of many cells, complicating the tracking of metabolic changes over time after stress in rare cells such as hematopoietic stem cells (HSCs). Here, we aimed to identify the key metabolic enzymes that define differences in glycolytic metabolism between steady-state and stress conditions in murine HSCs and elucidate their regulatory mechanisms. Through quantitative 13C metabolic flux analysis of glucose metabolism using high-sensitivity glucose tracing and mathematical modeling, we found that HSCs activate the glycolytic rate-limiting enzyme phosphofructokinase (PFK) during proliferation and oxidative phosphorylation (OXPHOS) inhibition. Real-time measurement of ATP levels in single HSCs demonstrated that proliferative stress or OXPHOS inhibition led to accelerated glycolysis via increased activity of PFKFB3, the enzyme regulating an allosteric PFK activator, within seconds to meet ATP requirements. Furthermore, varying stresses differentially activated PFKFB3 via PRMT1-dependent methylation during proliferative stress and via AMPK-dependent phosphorylation during OXPHOS inhibition. Overexpression of Pfkfb3 induced HSC proliferation and promoted differentiated cell production, whereas inhibition or loss of Pfkfb3 suppressed them. This study reveals the flexible and multilayered regulation of HSC glycolytic metabolism to sustain hematopoiesis under stress and provides techniques to better understand the physiological metabolism of rare hematopoietic cells.

13C Metabolic Flux Analysis in Chondrocytes Reveals a Novel Switch in Metabolic Phenotype.

Chondrocytes are typically known for their anaerobic metabolism both in vivo and under culture conditions in vitro. However, chondrocytes have been shown to display greater biosynthetic activity when subjected to conditions that elicit aerobic metabolism. We have previously shown that tissue formation by chondrocytes can be upregulated by controlling nutrient availability and that this response arises from changes in glucose metabolism. The aim of the present study was to further characterize these changes through 13C-metabolic flux analysis (13C-MFA), as well as to determine the most optimal response. Primary bovine chondrocytes were grown in scaffold-free high-density tissue culture. [U-13C] glucose labeling experiments were combined with a tissue-specific metabolic network model to carry out 13C-MFA under varying levels of nutrient availability. 13C-MFA results demonstrated that when subjected to increasing nutrient availability, chondrocytes switch from a predominately anaerobic to a mixed aerobic-anaerobic phenotype. This metabolic switch was attributed to the saturation of the lactate fermentation pathway and metabolite overflow toward the tricarboxylic acid cycle. This effect appears to be similar to, but the inverse of, the Crabtree effect ("inverse Crabtree effect"). The relationships between metabolic flux and nutrient availability were then utilized to identify culture conditions that promote enhanced tissue formation. This novel metabolic effect presents a simple but effective approach for enhancing the biosynthetic response of chondrocytes-a key requirement to develop functional engineered cartilaginous tissue for joint resurfacing.

Abstract 6612: Therapy-resistant endocycling cancer cells alter mitochondrial structure and metabolism to survive therapeutic stress

Abstract Therapy resistance is responsible for 90% of cancer-related deaths. We identified a novel mechanism of therapy resistance in which cancer cells enter an endocycling cell state, undergoing repeated S and G phases without dividing. As a result, resistant endocycling cells are 40 times larger than mitotic cancer cell controls. Interestingly, we observed that these non-proliferative endocycling cells can eventually give rise to proliferative progeny weeks after chemotherapy is removed, modeling cancer recurrence. We hypothesize that this endocycling cancer cell state is a critical driver of therapy resistance and cancer lethality. We evaluated oxidative stress in endocycling cells that survived the days and weeks following chemotherapy treatment using DCF-DA staining. We found increased levels of reactive oxygen species (ROS) in endocycling prostate cancer cells compared to untreated mitotic cells when normalized to cell size. Given that mitochondria are major producers and regulators of ROS, we hypothesize that cells surviving in this resistant state alter mitochondrial structure and function in response to increased oxidative stress. We performed immunofluorescence and confocal microscopy on mitochondria in untreated cells and cells following chemotherapy treatment to observe their morphology. Surviving endocycling cells had increased mitochondrial fragmentation when compared to the untreated group, a result that was further supported by reduced expression of phospho-DRP1 Ser637 via western blot. To assess changes in glucose metabolism, we then utilized isotope tracing-based metabolomics with [U-13C]glucose on untreated and surviving endocycling cells following chemotherapy. Endocycling cells had decreased labeling of 13C in TCA cycle metabolites, suggesting a decrease in mitochondrial function when compared to untreated cells. Ongoing work involves further studying metabolic reprogramming as cells enter this resistant state with parallel labeling experiments using [1,2-13C]glucose and [U-13C]glutamine to perform mathematical modeling-based 13C-metabolic flux analysis. In addition, we engineered a cell line to track mitochondria in real-time to quantify mitochondrial dynamics underlying the morphology observed in untreated and surviving cells. Our goal is to characterize how this therapy-resistant cell state alters its mitochondrial structure and function to survive therapeutic stress. Identifying vulnerabilities of this phenotype will enable new approaches for eliminating drug resistance in cancer. Citation Format: Melvin Li, Sarah R. Amend, Kenneth J. Pienta. Therapy-resistant endocycling cancer cells alter mitochondrial structure and metabolism to survive therapeutic stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6612.

Ex Vivo 13C-Metabolic Flux Analysis of Porcine Circulating Immune Cells Reveals Cell Type-Specific Metabolic Patterns and Sex Differences in the Pentose Phosphate Pathway.

In general, females present with stronger immune responses than males, but scarce data are available on sex-specific differences in immunometabolism. In this study, we characterized porcine peripheral blood mononuclear cell (PBMC) and granulocyte energy metabolism using a Bayesian 13C-metabolic flux analysis, which allowed precise determination of the glycolytic, pentose phosphate pathway (PPP), and tricarboxylic acid cycle (TCA) fluxes, together with an assessment of the superoxide anion radical (O2•-) production and mitochondrial O2 consumption. A principal component analysis allowed for identifying the cell type-specific patterns of metabolic plasticity. PBMCs displayed higher TCA cycle activity, especially glutamine-derived aspartate biosynthesis, which was directly related to mitochondrial respiratory activity and inversely related to O2•- production. In contrast, the granulocytes mainly utilized glucose via glycolysis, which was coupled to oxidative PPP utilization and O2•- production rates. The granulocytes of the males had higher oxidative PPP fluxes compared to the females, while the PBMCs of the females displayed higher non-oxidative PPP fluxes compared to the males associated with the T helper cell (CD3+CD4+) subpopulation of PBMCs. The observed sex-specific differences were not directly attributable to sex steroid plasma levels, but we detected an inverse correlation between testosterone and aldosterone plasma levels and showed that aldosterone levels were related with non-oxidative PPP fluxes of both cell types.

13C-Metabolic flux analysis detected a hyperoxemia-induced reduction of tricarboxylic acid cycle metabolism in granulocytes during two models of porcine acute subdural hematoma and hemorrhagic shock.

Supplementation with increased inspired oxygen fractions has been suggested to alleviate the harmful effects of tissue hypoxia during hemorrhagic shock (HS) and traumatic brain injury. However, the utility of therapeutic hyperoxia in critical care is disputed to this day as controversial evidence is available regarding its efficacy. Furthermore, in contrast to its hypoxic counterpart, the effect of hyperoxia on the metabolism of circulating immune cells remains ambiguous. Both stimulating and detrimental effects are possible; the former by providing necessary oxygen supply, the latter by generation of excessive amounts of reactive oxygen species (ROS). To uncover the potential impact of increased oxygen fractions on circulating immune cells during intensive care, we have performed a 13C-metabolic flux analysis (MFA) on PBMCs and granulocytes isolated from two long-term, resuscitated models of combined acute subdural hematoma (ASDH) and HS in pigs with and without cardiovascular comorbidity. Swine underwent resuscitation after 2 h of ASDH and HS up to a maximum of 48 h after HS. Animals received normoxemia (PaO2 = 80 - 120 mmHg) or targeted hyperoxemia (PaO2 = 200 - 250 mmHg for 24 h after treatment initiation, thereafter PaO2 as in the control group). Blood was drawn at time points T1 = after instrumentation, T2 = 24 h post ASDH and HS, and T3 = 48h post ASDH and HS. PBMCs and granulocytes were isolated from whole blood to perform electron spin resonance spectroscopy, high resolution respirometry and 13C-MFA. For the latter, we utilized a parallel tracer approach with 1,2-13C2 glucose, U-13C glucose, and U-13C glutamine, which covered essential pathways of glucose and glutamine metabolism and supplied redundant data for robust Bayesian estimation. Gas chromatography-mass spectrometry further provided multiple fragments of metabolites which yielded additional labeling information. We obtained precise estimations of the fluxes, their joint credibility intervals, and their relations, and characterized common metabolic patterns with principal component analysis (PCA). 13C-MFA indicated a hyperoxia-mediated reduction in tricarboxylic acid (TCA) cycle activity in circulating granulocytes which encompassed fluxes of glutamine uptake, TCA cycle, and oxaloacetate/aspartate supply for biosynthetic processes. We further detected elevated superoxide levels in the swine strain characterized by a hypercholesterolemic phenotype. PCA revealed cell type-specific behavioral patterns of metabolic adaptation in response to ASDH and HS that acted irrespective of swine strains or treatment group. In a model of resuscitated porcine ASDH and HS, we saw that ventilation with increased inspiratory O2 concentrations (PaO2 = 200 - 250 mmHg for 24 h after treatment initiation) did not impact mitochondrial respiration of PBMCs or granulocytes. However, Bayesian 13C-MFA results indicated a reduction in TCA cycle activity in granulocytes compared to cells exposed to normoxemia in the same time period. This change in metabolism did not seem to affect granulocytes' ability to perform phagocytosis or produce superoxide radicals.

Application of cold-adapted microbial agents in soil contaminate remediation: biodegradation mechanisms, case studies, and safety assessments.

The microbial agent technology has made significant progress in remediating nitro-aromatic compounds (NACs), such as p-nitrophenol, 2,4-dinitrophenol, and 2,4,6-Trinitrotoluene, in farmland soil over the past decade. However, there are still gaps in our understanding of the bioavailability and degradation mechanisms of these compounds in low-temperature environments. In this review, we provide a comprehensive summary of the strategies employed by cold-adapted microorganisms and elucidate the degradation pathways of NACs pollutants. To further analyze their metabolic mechanisms, we propose using mass balance to improve our understanding of biochemical processes and refine the degradation pathways through stoichiometry analysis. Additionally, we suggest employing 13C-metabolic flux analysis to track enzyme activity and intermediate products during bio-degradation processes with the aim of accelerating the remediation of nitro-aromatic compounds, particularly in cold regions. Through a comprehensive analysis of pollutant metabolic activities and a commitment to the 'One Health' approach, with an emphasis on selecting non-pathogenic strains, the environmental management strategies for soil remediation could be positioned to develop and implement safe and effective measure.

Bayesian 13C-Metabolic Flux Analysis of Parallel Tracer Experiments in Granulocytes: A Directional Shift within the Non-Oxidative Pentose Phosphate Pathway Supports Phagocytosis.

The pentose phosphate pathway (PPP) plays a key role in the cellular regulation of immune function; however, little is known about the interplay of metabolic adjustments in granulocytes, especially regarding the non-oxidative PPP. For the determination of metabolic mechanisms within glucose metabolism, we propose a novel set of measures for 13C-metabolic flux analysis based on ex vivo parallel tracer experiments ([1,2-13C]glucose, [U-13C]glucose, [4,5,6-13C]glucose) and gas chromatography-mass spectrometry labeling measurements of intracellular metabolites, such as sugar phosphates and their fragments. A detailed constraint analysis showed that the permission range for net and irreversible fluxes was limited to a three-dimensional space. The overall workflow, including its Bayesian flux estimation, resulted in precise flux distributions and pairwise confidence intervals, some of which could be represented as a line due to the strength of their correlation. The principal component analysis that was enabled by these behaviors comprised three components that explained 99.6% of the data variance. It showed that phagocytic stimulation reversed the direction of non-oxidative PPP net fluxes from ribose-5-phosphate biosynthesis toward glycolytic pathways. This process was closely associated with the up-regulation of the oxidative PPP to promote the oxidative burst.

Metabolic Signatures and Pathway Rewiring in TET2-Mutated Myeloid Neoplasia

Background. TET2 mutations ( TET2 MT) are the most frequently observed somatic lesions in myeloid neoplasms (MN). TET2 belongs to an Fe2+ and α-ketoglutarate (αKG) dependent DNA-dioxygenase family that progressively oxidizes 5-methyldeoxycytidine (5mC), leading to promoter and enhancer mCpG demethylation essential for efficient transcription. TET2 accounts for the majority of DNA dioxygenase activity in hematopoietic stem and progenitor cells (HSPCs), and following its loss, the resulting accumulation of 5mC leads to a loss of lineage plasticity and transcriptional repression of tumor suppressor genes, thereby establishing a volatile pre-neoplastic state. TET2 is one of the major consumers of αKG, a major cofactor produced by the tricarboxylic acid (TCA) cycle. Therefore, loss of TET2 creates a metabolic perturbation in HSPCs. However, the impact of TET2 loss on the metabolism of leukemic cells and its subsequent effects on malignant evolution remains unclear. Methods. We generated isogenic TET2 -/- ( TET2 ko) leukemic cell models in THP1 and K562 using CRISPR-Cas9 knockout. We then utilized targeted and untargeted metabolic analyses by ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry in these models. We aimed to investigate the intricate metabolic changes associated with TET2 and TET2 MT. To validate our findings, we extended our studies to primary cells derived from MN patients with and without TET2 lesions. We performed extensive cellular metabolism studies using 13C isotope labelled glucose and glutamine. Additionally, levels of 5mC and its oxidation products, namely 5-hydroxymethyldeoxycytidine, 5-formyldeoxycytidine, and 5-carboxydeoxycytidine were documented for correlating epigenetic modifications with TET2 activities. Results. Targeted metabolic analysis uncovered profound impacts of TET2 MT on key metabolic pathways including the TCA cycle, glycolysis, glutamine metabolism, and nucleoside biosynthesis (Figure A). Distinct changes were observed in cells from different lineage backgrounds, suggesting diverse metabolic pathway rewiring specific to cell origin. Despite the divergence among lineages, loss of TET2 created common and unique metabolic signatures, highlighting their crucial role in sustaining the survival and clonal advantages specific to TET2 MT. Representative signature compounds and their relative quantities in TET2 wildtype (WT) and TET2 MT are depicted in Figure A. The targeted metabolic analysis revealed a significantly lower level of glutamine in TET2 MTcells, suggesting a potential preference for exhausting glutamine as a source of energy production. For validation, we conducted 13C metabolic flux analysis (13C-MFA) using glucose (U-13C, 99%) and L-glutamine (U-13C, 99%). 13C-MFA revealed distinct enrichment patterns (Figure B). In the TCA cycle, the dominant isotopologues receiving [U-13C]-glucose were M+2 for citrate and isocitrate. However, for aKG, succinate (SA) and fumarate (FA), the M+2s were significantly lower in TET2 -/- compared to WT, suggesting that the major source for these metabolites were rewired from glycolysis to glutamine metabolism in TET2 -/-. This was confirmed by [U-13C]-glutamine labeling where dominant isotopologues, M+5 for aKG and M+4 for SA, FA and malate, were significantly higher at 24-hour time point in in TET2 -/- compared to WT. Untargeted metabolic analyses from isogenic cells were used to identify metabolic features associated with TET2 loss. These metabolic features were also assayed in primary AML cells with TET2 MT (n=12) and without: RUNX1 (n=20), SF3B1 (n=11), IDH1 (n=10), IDH2 (n=13), FH (n=3), as well as healthy donors as controls (n=6). Based on the molecular features identified in the TET2 ko isogenic cells, MN patients with TET2, IDH1/2, and germline FH mutations clustered more closely, suggesting shared metabolic signatures among these groups. In contrast, patients with RUNX1 and SF3B1 mutations formed separate clusters, demonstrating distinct metabolic profiles associated with these genetic alterations. Conclusion. Our findings provide comprehensive and novel insights into the metabolic consequences of TET2 inactivation in MN. The identified metabolic signatures and rewiring of key pathways may hold promise as potential therapeutic targets and offer opportunities for tailored treatment approaches in TET2MT MN.

Metabolic flux reprogramming in Mycobacterium tuberculosis-infected human macrophages.

Metabolic fluxes are at the heart of metabolism and growth in any living system. During tuberculosis (TB) infection, the pathogenic Mycobacterium tuberculosis (Mtb) adapts its nutritional behaviour and metabolic fluxes to survive in human macrophages and cause infection. The infected host cells also undergo metabolic changes. However, our knowledge of the infected host metabolism and identification of the reprogrammed metabolic flux nodes remains limited. In this study, we applied systems-based 13C-metabolic flux analysis (MFA) to measure intracellular carbon metabolic fluxes in Mtb-infected human THP-1 macrophages. We provide a flux map for infected macrophages that quantified significantly increased fluxes through glycolytic fluxes towards pyruvate synthesis and reduced pentose phosphate pathway fluxes when compared to uninfected macrophages. The tri carboxylic acid (TCA) cycle fluxes were relatively low, and amino acid fluxes were reprogrammed upon Mtb infection. The knowledge of host metabolic flux profiles derived from our work expands on how the host cell adapts its carbon metabolism in response to Mtb infection and highlights important nodes that may provide targets for developing new therapeutics to improve TB treatment.

BayFlux: A Bayesian method to quantify metabolic Fluxes and their uncertainty at the genome scale.

Metabolic fluxes, the number of metabolites traversing each biochemical reaction in a cell per unit time, are crucial for assessing and understanding cell function. 13C Metabolic Flux Analysis (13C MFA) is considered to be the gold standard for measuring metabolic fluxes. 13C MFA typically works by leveraging extracellular exchange fluxes as well as data from 13C labeling experiments to calculate the flux profile which best fit the data for a small, central carbon, metabolic model. However, the nonlinear nature of the 13C MFA fitting procedure means that several flux profiles fit the experimental data within the experimental error, and traditional optimization methods offer only a partial or skewed picture, especially in "non-gaussian" situations where multiple very distinct flux regions fit the data equally well. Here, we present a method for flux space sampling through Bayesian inference (BayFlux), that identifies the full distribution of fluxes compatible with experimental data for a comprehensive genome-scale model. This Bayesian approach allows us to accurately quantify uncertainty in calculated fluxes. We also find that, surprisingly, the genome-scale model of metabolism produces narrower flux distributions (reduced uncertainty) than the small core metabolic models traditionally used in 13C MFA. The different results for some reactions when using genome-scale models vs core metabolic models advise caution in assuming strong inferences from 13C MFA since the results may depend significantly on the completeness of the model used. Based on BayFlux, we developed and evaluated novel methods (P-13C MOMA and P-13C ROOM) to predict the biological results of a gene knockout, that improve on the traditional MOMA and ROOM methods by quantifying prediction uncertainty.