Metabolic adaptation in epithelial ovarian cancer metastasis.

Epithelial ovarian cancer (EOC) is highly lethal due to its unique metastatic characteristics. EOC spheroids enter a non-proliferative state, with hypoxic cores and reduced oncogenic signaling, all of which contribute to tumour dormancy during metastasis. We investigated the metabolomic states of EOC cells progressing through the three steps to metastasis. Metabolomes of adherent, spheroid, and re-adherent cells were validated by isotopic metabolic flux analysis and mitochondrial functional assays to identify metabolic pathways that were previously unknown to promote EOC metastasis. Although spheroids were thought to exist in a dormant state, metabolomic analysis revealed an unexpected upregulation of energy production pathways in spheroids, accompanied by increased abundance of tricarboxylic acid (TCA) cycle and electron transport chain proteins. Tracing of 13C-labelled glucose and glutamine showed increased pyruvate carboxylation and decreased glutamine anaplerosis in spheroids. Increased reductive carboxylation suggests spheroids adjust redox homeostasis by shuttling cytosolic NADPH into mitochondria via isocitrate dehydrogenase. Indeed, we observed spheroids have increased respiratory capacity and mitochondrial ATP production. Relative to adherent cells, spheroids reduced serine consumption and metabolism, processes which were reversed upon spheroid re-adherence. The data reveal a distinct metabolism in EOC spheroids that enhances energy production by the mitochondria while maintaining a dormant state with respect to growth and proliferation. The findings advance our understanding of EOC metastasis and identify the TCA cycle and mitochondrional activity as novel targets to disrupt EOC metastasis, providing new approaches to treat advanced disease.

Differential cell type-specific function of the aryl hydrocarbon receptor and its repressor in diet-induced obesity and fibrosis.

OBJECTIVE: The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor regulating xenobiotic responses as well as physiological metabolism. Dietary AhR ligands activate the AhR signaling axis, whereas AhR activation is negatively regulated by the AhR repressor (AhRR). While AhR-deficient mice are known to be resistant to diet-induced obesity (DIO), the influence of the AhRR on DIO has not been assessed so far. METHODS: In this study, we analyzed AhRR-/- mice and mice with a conditional deletion of either AhRR or AhR in myeloid cells under conditions of DIO and after supplementation of dietary AhR ligands. Moreover, macrophage metabolism was assessed using Seahorse Mito Stress Test and ROS assays as well as transcriptomic analysis. RESULTS: We demonstrate that global AhRR deficiency leads to a robust, but not as profound protection from DIO and hepatosteatosis as AhR deficiency. Under conditions of DIO, AhRR-/- mice did not accumulate TCA cycle intermediates in the circulation in contrast to wild-type (WT) mice, indicating protection from metabolic dysfunction. This effect could be mimicked by dietary supplementation of AhR ligands in WT mice. Because of the predominant expression of the AhRR in myeloid cells, AhRR-deficient macrophages were analyzed for changes in metabolism and showed major metabolic alterations regarding oxidative phosphorylation and mitochondrial activity. Unbiased transcriptomic analysis revealed increased expression of genes involved in de novo lipogenesis and mitochondrial biogenesis. Mice with a genetic deficiency of the AhRR in myeloid cells did not show alterations in weight gain after high fat diet (HFD) but demonstrated ameliorated liver damage compared to control mice. Further, deficiency of the AhR in myeloid cells also did not affect weight gain but led to enhanced liver damage and adipose tissue fibrosis compared to controls. CONCLUSIONS: AhRR-deficient mice are resistant to diet-induced metabolic syndrome. Although conditional ablation of either the AhR or AhRR in myeloid cells did not recapitulate the phenotype of the global knockout, our findings suggest that enhanced AhR signaling in myeloid cells deficient for AhRR protects from diet-induced liver damage and fibrosis, whereas myeloid cell-specific AhR deficiency is detrimental.

From Planning Stage Towards FAIR Data: A Practical Metadatasheet For Biomedical Scientists.

Datasets consist of measurement data and metadata. Metadata provides context, essential for understanding and (re-)using data. Various metadata standards exist for different methods, systems and contexts. However, relevant information resides at differing stages across the data-lifecycle. Often, this information is defined and standardized only at publication stage, which can lead to data loss and workload increase. In this study, we developed Metadatasheet, a metadata standard based on interviews with members of two biomedical consortia and systematic screening of data repositories. It aligns with the data-lifecycle allowing synchronous metadata recording within Microsoft Excel, a widespread data recording software. Additionally, we provide an implementation, the Metadata Workbook, that offers user-friendly features like automation, dynamic adaption, metadata integrity checks, and export options for various metadata standards. By design and due to its extensive documentation, the proposed metadata standard simplifies recording and structuring of metadata for biomedical scientists, promoting practicality and convenience in data management. This framework can accelerate scientific progress by enhancing collaboration and knowledge transfer throughout the intermediate steps of data creation.

Metabolomics analysis reveals novel serum metabolite alterations in cancer cachexia.

Background: Cachexia is a body wasting syndrome that significantly affects well-being and prognosis of cancer patients, without effective treatment. Serum metabolites take part in pathophysiological processes of cancer cachexia, but apart from altered levels of select serum metabolites, little is known on the global changes of the overall serum metabolome, which represents a functional readout of the whole-body metabolic state. Here, we aimed to comprehensively characterize serum metabolite alterations and analyze associated pathways in cachectic cancer patients to gain new insights that could help instruct strategies for novel interventions of greater clinical benefit. Methods: Serum was sampled from 120 metastatic cancer patients (stage UICC IV). Patients were grouped as cachectic or non-cachectic according to the criteria for cancer cachexia agreed upon international consensus (main criterium: weight loss adjusted to body mass index). Samples were pooled by cachexia phenotype and assayed using non-targeted gas chromatography-mass spectrometry (GC-MS). Normalized metabolite levels were compared using t-test (p < 0.05, adjusted for false discovery rate) and partial least squares discriminant analysis (PLS-DA). Machine-learning models were applied to identify metabolite signatures for separating cachexia states. Significant metabolites underwent MetaboAnalyst 5.0 pathway analysis. Results: Comparative analyses included 78 cachectic and 42 non-cachectic patients. Cachectic patients exhibited 19 annotable, significantly elevated (including glucose and fructose) or decreased (mostly amino acids) metabolites associating with aminoacyl-tRNA, glutathione and amino acid metabolism pathways. PLS-DA showed distinct clusters (accuracy: 85.6%), and machine-learning models identified metabolic signatures for separating cachectic states (accuracy: 83.2%; area under ROC: 88.0%). We newly identified altered blood levels of erythronic acid and glucuronic acid in human cancer cachexia, potentially linked to pentose-phosphate and detoxification pathways. Conclusion: We found both known and yet unknown serum metabolite and metabolic pathway alterations in cachectic cancer patients that collectively support a whole-body metabolic state with impaired detoxification capability, altered glucose and fructose metabolism, and substrate supply for increased and/or distinct metabolic needs of cachexia-associated tumors. These findings together imply vulnerabilities, dependencies and targets for novel interventions that have potential to make a significant impact on future research in an important field of cancer patient care.

Cutting Edge: STING Induces ACLY Activation and Metabolic Adaptations in Human Macrophages through TBK1.

The 2'3'-cyclic GMP-AMP (cGAMP) synthase (cGAS)-stimulator of IFN genes (STING) pathway can sense infection and cellular stress by detecting cytosolic DNA. Upon ligand binding, cGAS produces the cyclic dinucleotide messenger cGAMP, which triggers its receptor STING. Active STING initiates gene transcription through the transcription factors IFN regulatory factor 3 (IRF3) and NF-κB and induces autophagy, but whether STING can cause changes in the metabolism of macrophages is unknown. In this study, we report that STING signaling activates ATP-citrate lyase (ACLY) by phosphorylation in human macrophages. Using genetic and pharmacologic perturbation, we show that STING targets ACLY via its prime downstream signaling effector TANK (TRAF family member-associated NF-κB activator)-binding kinase 1 (TBK1). We further identify that TBK1 alters cellular metabolism upon cGAMP treatment. Our results suggest that STING-mediated metabolic reprogramming adjusts the cellular response to DNA sensing in addition to transcription factor activation and autophagy induction.

A reversible state of hypometabolism in a human cellular model of sporadic Parkinson's disease.

Sporadic Parkinson's Disease (sPD) is a progressive neurodegenerative disorder caused by multiple genetic and environmental factors. Mitochondrial dysfunction is one contributing factor, but its role at different stages of disease progression is not fully understood. Here, we showed that neural precursor cells and dopaminergic neurons derived from induced pluripotent stem cells (hiPSCs) from sPD patients exhibited a hypometabolism. Further analysis based on transcriptomics, proteomics, and metabolomics identified the citric acid cycle, specifically the α-ketoglutarate dehydrogenase complex (OGDHC), as bottleneck in sPD metabolism. A follow-up study of the patients approximately 10 years after initial biopsy demonstrated a correlation between OGDHC activity in our cellular model and the disease progression. In addition, the alterations in cellular metabolism observed in our cellular model were restored by interfering with the enhanced SHH signal transduction in sPD. Thus, inhibiting overactive SHH signaling may have potential as neuroprotective therapy during early stages of sPD.

Multi-omics analysis reveals the molecular response to heat stress in a "red tide" dinoflagellate.

BACKGROUND: "Red tides" are harmful algal blooms caused by dinoflagellate microalgae that accumulate toxins lethal to other organisms, including humans via consumption of contaminated seafood. These algal blooms are driven by a combination of environmental factors including nutrient enrichment, particularly in warm waters, and are increasingly frequent. The molecular, regulatory, and evolutionary mechanisms that underlie the heat stress response in these harmful bloom-forming algal species remain little understood, due in part to the limited genomic resources from dinoflagellates, complicated by the large sizes of genomes, exhibiting features atypical of eukaryotes. RESULTS: We present the de novo assembled genome (~ 4.75 Gbp with 85,849 protein-coding genes), transcriptome, proteome, and metabolome from Prorocentrum cordatum, a globally abundant, bloom-forming dinoflagellate. Using axenic algal cultures, we study the molecular mechanisms that underpin the algal response to heat stress, which is relevant to current ocean warming trends. We present the first evidence of a complementary interplay between RNA editing and exon usage that regulates the expression and functional diversity of biomolecules, reflected by reduction in photosynthesis, central metabolism, and protein synthesis. These results reveal genomic signatures and post-transcriptional regulation for the first time in a pelagic dinoflagellate. CONCLUSIONS: Our multi-omics analyses uncover the molecular response to heat stress in an important bloom-forming algal species, which is driven by complex gene structures in a large, high-G+C genome, combined with multi-level transcriptional regulation. The dynamics and interplay of molecular regulatory mechanisms may explain in part how dinoflagellates diversified to become some of the most ecologically successful organisms on Earth.

Stable Isotope-Assisted Untargeted Metabolomics Identifies ALDH1A1-Driven Erythronate Accumulation in Lung Cancer Cells.

Using an untargeted stable isotope-assisted metabolomics approach, we identify erythronate as a metabolite that accumulates in several human cancer cell lines. Erythronate has been reported to be a detoxification product derived from off-target glycolytic metabolism. We use chemical inhibitors and genetic silencing to define the pentose phosphate pathway intermediate erythrose 4-phosphate (E4P) as the starting substrate for erythronate production. However, following enzyme assay-coupled protein fractionation and subsequent proteomics analysis, we identify aldehyde dehydrogenase 1A1 (ALDH1A1) as the predominant contributor to erythrose oxidation to erythronate in cell extracts. Through modulating ALDH1A1 expression in cancer cell lines, we provide additional support. We hence describe a possible alternative route to erythronate production involving the dephosphorylation of E4P to form erythrose, followed by its oxidation by ALDH1A1. Finally, we measure increased erythronate concentrations in tumors relative to adjacent normal tissues from lung cancer patients. These findings suggest the accumulation of erythronate to be an example of metabolic reprogramming in cancer cells, raising the possibility that elevated levels of erythronate may serve as a biomarker of certain types of cancer.

Systems toxicology of complex wood combustion aerosol reveals gaseous carbonyl compounds as critical constituents.

Epidemiological studies identified air pollution as one of the prime causes for human morbidity and mortality, due to harmful effects mainly on the cardiovascular and respiratory systems. Damage to the lung leads to several severe diseases such as fibrosis, chronic obstructive pulmonary disease and cancer. Noxious environmental aerosols are comprised of a gas and particulate phase representing highly complex chemical mixtures composed of myriads of compounds. Although some critical pollutants, foremost particulate matter (PM), could be linked to adverse health effects, a comprehensive understanding of relevant biological mechanisms and detrimental aerosol constituents is still lacking. Here, we employed a systems toxicology approach focusing on wood combustion, an important source for air pollution, and demonstrate a key role of the gas phase, specifically carbonyls, in driving adverse effects. Transcriptional profiling and biochemical analysis of human lung cells exposed at the air-liquid-interface determined DNA damage and stress response, as well as perturbation of cellular metabolism, as major key events. Connectivity mapping revealed a high similarity of gene expression signatures induced by wood smoke and agents prompting DNA-protein crosslinks (DPCs). Indeed, various gaseous aldehydes were detected in wood smoke, which promote DPCs, initiate similar genomic responses and are responsible for DNA damage provoked by wood smoke. Hence, systems toxicology enables the discovery of critical constituents of complex mixtures i.e. aerosols and highlights the role of carbonyls on top of particulate matter as an important health hazard.

Sulforaphane rewires central metabolism to support antioxidant response and achieve glucose homeostasis.

Cruciferous-rich diets, particularly broccoli, have been associated with reduced risk of developing cancers of various sites, cardiovascular disease and type-2 diabetes. Sulforaphane (SF), a sulfur-containing broccoli-derived metabolite, has been identified as the major bioactive compound mediating these health benefits. Sulforaphane is a potent dietary activator of the transcription factor Nuclear factor erythroid-like 2 (NRF2), the master regulator of antioxidant cell capacity responsible for inducing cytoprotective genes, but its role in glucose homeostasis remains unclear. In this study, we set to test the hypothesis that SF regulates glucose metabolism and ameliorates glucose overload and its resulting oxidative stress by inducing NRF2 in human hepatoma HepG2 cells. HepG2 cells were exposed to varying glucose concentrations: basal (5.5 mM) and high glucose (25 mM), in the presence of physiological concentrations of SF (10 µM). SF upregulated the expression of glutathione (GSH) biosynthetic genes and significantly increased levels of reduced GSH. Labelled glucose and glutamine experiments to measure metabolic fluxes identified that SF increased intracellular utilisation of glycine and glutamate by redirecting the latter away from the TCA cycle and increased the import of cysteine from the media, likely to support glutathione synthesis. Furthermore, SF altered pathways generating NADPH, the necessary cofactor for oxidoreductase reactions, namely pentose phosphate pathway and 1C-metabolism, leading to the redirection of glucose away from glycolysis and towards PPP and of methionine towards methylation substrates. Finally, transcriptomic and targeted metabolomics LC-MS analysis of NRF2-KD HepG2 cells generated using CRISPR-Cas9 genome editing revealed that the above metabolic effects are mediated through NRF2. These results suggest that the antioxidant properties of cruciferous diets are intricately connected to their metabolic benefits.

The Virulence Factor Macrophage Infectivity Potentiator (Mip) Influences Branched-Chain Amino Acid Metabolism and Pathogenicity of Legionella pneumophila.

Legionella pneumophila (Lp) is a common etiological agent of bacterial pneumonia that causes Legionnaires' disease (LD). The bacterial membrane-associated virulence factor macrophage infectivity potentiator (Mip) exhibits peptidyl-prolyl-cis/trans-isomerase (PPIase) activity and contributes to the intra- and extracellular pathogenicity of Lp. Though Mip influences disease outcome, little is known about the metabolic consequences of altered Mip activity during infections. Here, we established a metabolic workflow and applied mass spectrometry approaches to decipher how Mip activity influences metabolism and pathogenicity. Impaired Mip activity in genetically engineered Lp strains decreases intracellular replication in cellular infection assays, confirming the contribution of Mip for Lp pathogenicity. We observed that genetic and chemical alteration of Mip using the PPIase inhibitors rapamycin and FK506 induces metabolic reprogramming in Lp, specifically branched-chain amino acid (BCAA) metabolism. Rapamycin also inhibits PPIase activity of mammalian FK506 binding proteins, and we observed that rapamycin induces a distinct metabolic signature in human macrophages compared to bacteria, suggesting potential involvement of Mip in normal bacteria and in infection. Our metabolic studies link Mip to alterations in BCAA metabolism and may help to decipher novel disease mechanisms associated with LD.

Branched-chain amino acid synthesis is coupled to TOR activation early in the cell cycle in yeast.

How cells coordinate their metabolism with division determines the rate of cell proliferation. Dynamic patterns of metabolite synthesis during the cell cycle are unexplored. We report the first isotope tracing analysis in synchronous, growing budding yeast cells. Synthesis of leucine, a branched-chain amino acid (BCAA), increases through the G1 phase of the cell cycle, peaking later during DNA replication. Cells lacking Bat1, a mitochondrial aminotransferase that synthesizes BCAAs, grow slower, are smaller, and are delayed in the G1 phase, phenocopying cells in which the growth-promoting kinase complex TORC1 is moderately inhibited. Loss of Bat1 lowers the levels of BCAAs and reduces TORC1 activity. Exogenous provision of valine and, to a lesser extent, leucine to cells lacking Bat1 promotes cell division. Valine addition also increases TORC1 activity. In wild-type cells, TORC1 activity is dynamic in the cell cycle, starting low in early G1 but increasing later in the cell cycle. These results suggest a link between BCAA synthesis from glucose to TORC1 activation in the G1 phase of the cell cycle.

Instrumental Drift in Untargeted Metabolomics: Optimizing Data Quality with Intrastudy QC Samples.

Untargeted metabolomics is an important tool in studying health and disease and is employed in fields such as biomarker discovery and drug development, as well as precision medicine. Although significant technical advances were made in the field of mass-spectrometry driven metabolomics, instrumental drifts, such as fluctuations in retention time and signal intensity, remain a challenge, particularly in large untargeted metabolomics studies. Therefore, it is crucial to consider these variations during data processing to ensure high-quality data. Here, we will provide recommendations for an optimal data processing workflow using intrastudy quality control (QC) samples that identifies errors resulting from instrumental drifts, such as shifts in retention time and metabolite intensities. Furthermore, we provide an in-depth comparison of the performance of three popular batch-effect correction methods of different complexity. By using different evaluation metrics based on QC samples and a machine learning approach based on biological samples, the performance of the batch-effect correction methods were evaluated. Here, the method TIGER demonstrated the overall best performance by reducing the relative standard deviation of the QCs and dispersion-ratio the most, as well as demonstrating the highest area under the receiver operating characteristic with three different probabilistic classifiers (Logistic regression, Random Forest, and Support Vector Machine). In summary, our recommendations will help to generate high-quality data that are suitable for further downstream processing, leading to more accurate and meaningful insights into the underlying biological processes.

Pyruvate dehydrogenase fuels a critical citrate pool that is essential for Th17 cell effector functions.

Pyruvate dehydrogenase (PDH) is the central enzyme connecting glycolysis and the tricarboxylic acid (TCA) cycle. The importance of PDH function in T helper 17 (Th17) cells still remains to be studied. Here, we show that PDH is essential for the generation of a glucose-derived citrate pool needed for Th17 cell proliferation, survival, and effector function. In vivo, mice harboring a T cell-specific deletion of PDH are less susceptible to developing experimental autoimmune encephalomyelitis. Mechanistically, the absence of PDH in Th17 cells increases glutaminolysis, glycolysis, and lipid uptake in a mammalian target of rapamycin (mTOR)-dependent manner. However, cellular citrate remains critically low in mutant Th17 cells, which interferes with oxidative phosphorylation (OXPHOS), lipid synthesis, and histone acetylation, crucial for transcription of Th17 signature genes. Increasing cellular citrate in PDH-deficient Th17 cells restores their metabolism and function, identifying a metabolic feedback loop within the central carbon metabolism that may offer possibilities for therapeutically targeting Th17 cell-driven autoimmunity.

In Vivo Investigation of Glucose Metabolism in Idiopathic and PRKN-Related Parkinson's Disease.

BACKGROUND: Alterations in mitochondrial dysfunction have been implicated in the pathogenesis of Parkinson's disease (PD). Mitochondrial energy production is linked to glucose metabolism, and diabetes is associated with PD. However, studies investigating glucose metabolism in vivo in genetically stratified PD patients and controls have yet to be performed. OBJECTIVES: The objectives of this study were to explore glucose production, gluconeogenesis, and the contribution of gluconeogenesis to glucose production in idiopathic and PRKN PD compared with healthy controls with state-of-the-art biochemical methods. METHODS: We applied a dried-blood sampling/gas chromatography/mass spectrometry approach to monitor fluxes in the Cori cycle in vivo. RESULTS: The contribution of gluconeogenesis to total glucose production is increased in idiopathic PD patients (n = 33), but not in biallelic PRKN mutation carriers (n = 5) compared with healthy controls (n = 13). CONCLUSIONS: We provide first-time in vivo evidence for alterations in glucose metabolism in idiopathic PD, in keeping with the epidemiological evidence for an association between PD and diabetes. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Plasma Metabolome Alterations Discriminate between COVID-19 and Non-COVID-19 Pneumonia.

Pneumonia is a common cause of morbidity and mortality and is most often caused by bacterial pathogens. COVID-19 is characterized by lung infection with potential progressive organ failure. The systemic consequences of both disease on the systemic blood metabolome are not fully understood. The aim of this study was to compare the blood metabolome of both diseases and we hypothesize that plasma metabolomics may help to identify the systemic effects of these diseases. Therefore, we profiled the plasma metabolome of 43 cases of COVID-19 pneumonia, 23 cases of non-COVID-19 pneumonia, and 26 controls using a non-targeted approach. Metabolic alterations differentiating the three groups were detected, with specific metabolic changes distinguishing the two types of pneumonia groups. A comparison of venous and arterial blood plasma samples from the same subjects revealed the distinct metabolic effects of pulmonary pneumonia. In addition, a machine learning signature of four metabolites was predictive of the disease outcome of COVID-19 subjects with an area under the curve (AUC) of 86 ± 10 %. Overall, the results of this study uncover systemic metabolic changes that could be linked to the etiology of COVID-19 pneumonia and non-COVID-19 pneumonia.

Transcriptional licensing is required for Pyrin inflammasome activation in human macrophages and bypassed by mutations causing familial Mediterranean fever.

Pyrin is a cytosolic immune sensor that nucleates an inflammasome in response to inhibition of RhoA by bacterial virulence factors, triggering the release of inflammatory cytokines, including IL-1ß. Gain-of-function mutations in the MEFV gene encoding Pyrin cause autoinflammatory disorders, such as familial Mediterranean fever (FMF) and Pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND). To precisely define the role of Pyrin in pathogen detection in human immune cells, we compared initiation and regulation of the Pyrin inflammasome response in monocyte-derived macrophages (hMDM). Unlike human monocytes and murine macrophages, we determined that hMDM failed to activate Pyrin in response to known Pyrin activators Clostridioides difficile (C. difficile) toxins A or B (TcdA or TcdB), as well as the bile acid analogue BAA-473. The Pyrin inflammasome response was enabled in hMDM by prolonged priming with either LPS or type I or II interferons and required an increase in Pyrin expression. Notably, FMF mutations lifted the requirement for prolonged priming for Pyrin activation in hMDM, enabling Pyrin activation in the absence of additional inflammatory signals. Unexpectedly, in the absence of a Pyrin response, we found that TcdB activated the NLRP3 inflammasome in hMDM. These data demonstrate that regulation of Pyrin activation in hMDM diverges from monocytes and highlights its dysregulation in FMF.

Erythritol synthesis is elevated in response to oxidative stress and regulated by the non-oxidative pentose phosphate pathway in A549 cells.

Background: Erythritol is a predictive biomarker of cardiometabolic diseases and is produced from glucose metabolism through the pentose phosphate pathway (PPP). Little is known regarding the regulation of endogenous erythritol synthesis in humans. Objective: In the present study, we investigated the stimuli that promote erythritol synthesis in human lung carcinoma cells and characterized potential points of regulation along the PPP. Methods: Human A549 lung carcinoma cells were chosen for their known ability to synthesize erythritol. A549 cells were treated with potential substrates for erythritol production, including glucose, fructose, and glycerol. Using siRNA knockdown, we assessed the necessity of enzymes G6PD, TKT, TALDO, and SORD for erythritol synthesis. We also used position-specific 13C-glucose tracers to determine whether the carbons for erythritol synthesis are derived directly from glycolysis or through the oxidative PPP. Finally, we assessed if erythritol synthesis responds to oxidative stress using chemical and genetic models. Results: Intracellular erythritol was directly associated with media glucose concentration. In addition, siRNA knockdown of TKT or SORD inhibited erythritol synthesis, whereas siG6PD did not. Both chemically induced oxidative stress and constitutive activation of the antioxidant response transcription factor NRF2 elevated intracellular erythritol. Conclusion: Our findings indicate that in A549 cells, erythritol synthesis is proportional to flux through the PPP and is regulated by non-oxidative PPP enzymes.

BCAT1 redox function maintains mitotic fidelity.

The metabolic enzyme branched-chain amino acid transaminase 1 (BCAT1) drives cell proliferation in aggressive cancers such as glioblastoma. Here, we show that BCAT1 localizes to mitotic structures and has a non-metabolic function as a mitotic regulator. Furthermore, BCAT1 is required for chromosome segregation in cancer and induced pluripotent stem cells and tumor growth in human cerebral organoid and mouse syngraft models. Applying gene knockout and rescue strategies, we show that the BCAT1 CXXC redox motif is crucial for controlling cysteine sulfenylation specifically in mitotic cells, promoting Aurora kinase B localization to centromeres, and securing accurate chromosome segregation. These findings offer an explanation for the well-established role of BCAT1 in promoting cancer cell proliferation. In summary, our data establish BCAT1 as a component of the mitotic apparatus that safeguards mitotic fidelity through a moonlighting redox functionality.