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Acquired drug resistance is a major challenge in the management of cancer, which underscores the need for discovery and development of novel therapeutic strategies. We report here the mechanism of the anti-cancer activity of a small coordinate complex composed of the rare earth metal praseodymium (Pr) and mercaptopyridine oxide (MPO; pyrithione). Exposure of cancer cells to relatively low concentrations of the conjugate Pr-MPO (5 µM) significantly impairs cell survival in a p53-independent manner and irrespective of the drug resistant phenotype. Mechanistically, Pr-MPO-induced cell death is caspase-independent, not inhibitable by necrostatin, but associated with the appearance of autophagy markers. However, further analysis revealed incomplete autophagic flux, thus suggesting altered integrity of lysosomal machinery. Supporting the lysosomal targeting activity are data demonstrating increased lysosomal Ca2+ accumulation and alkalinization, which coincides with cytosolic acidification (drop in pHc from 7.75 to 7.00). In parallel, an increase in lysosomal activity of glycosidase alpha acid (GAA), involved in passive glycogen breakdown, correlates with rapid depletion of glucose stores upon Pr-MPO treatment. This is associated with swift cataplerosis of TCA cycle intermediates, loss of NAD+/NADH and increase in pyruvate dehydrogenase (PDH) activity to compensate for pyruvate loss. Addition of exogenous pyruvate rescued cell survival. Notably, lysosomal impairment and metabolic catastrophe triggered by Pr-MPO are suggestive of Zn2+-mediated cytotoxicity, which is confirmed by the ability of Zn2+ chelator TPEN to block Pr-MPO-mediated anti-tumor activity. Together, these results highlight the ability of the small molecule lanthanide conjugate to target the cells' waste clearing machinery as well as mitochondrial metabolism for Zn2+-mediated execution of cancer cells, which could have therapeutic potential against cancers with high metabolic activity. [ABSTRACT FROM AUTHOR]

Cataracts are a disease that reduces vision due to opacity formation of the lens. Diabetic cataracts occur at young age and progress relatively quickly, so the development of effective treatment has been awaited. Several studies have shown that pyruvate inhibits oxidative stress and glycation of lens proteins, which contribute to onset of diabetic cataracts. However, detailed molecular mechanisms have not been revealed. In this study, we attempted to reduce galactose‐induced opacity by pyruvate with rat ex vivo model. Rat lenses were extracted and cultured in galactose‐containing medium to induce lens opacity. After opacity had developed, continued culturing with pyruvate in the medium resulted in a reduction of lens opacity. Subsequently, we conducted microarray analysis to investigate the genes that contribute to the therapeutic effect. We performed quantitative expression measurements using RT‐qPCR for extracted genes that were upregulated in cataract‐induced lenses and downregulated in pyruvate‐treated lenses, resulting in the identification of 34 candidate genes. Functional analysis using the STRING database suggests that metallothionein‐related factors (Mt1a, Mt1m, and Mt2A) and epithelial‐mesenchymal transition‐related factors (Acta2, Anxa1, Cd81, Mki67, Timp1, and Tyms) contribute to the therapeutic effect of cataracts. [ABSTRACT FROM AUTHOR]

Background: The lactate/pyruvate (LP) ratio has been studied as an alternative to serum lactate to determine clinical prognosis. Despite its clinical utility, there is a paucity of evidence evaluating the role of the L/P ratio in patients with sepsis. Methods: We assessed the clinical utility of the L/P ratio in patients with sepsis. The L/P ratio was measured at baseline, 4 and 8 h after admission. Our primary outcome was to determine the prognostic utility of the L/P ratio on the 15-day mortality risk. Our secondary outcomes were to compare the L/P ratio across time and its prognostic utility against standard risk calculators such as APACHE-II and SOFA scores. Results: We had a total of 80 patients, with 18 (22.5%) survivors and 62 (77.5%) non-survivors. While we found that patients having higher L/P ratios at 8 h had an increased 30-mortality risk (OR 1.08, 95% CI 1.02–1.18), the model's performance showed no difference when compared to other measurements of the L/P ratio that showed no association with mortality (p-value: 0.45). For our secondary outcome, we found that the APACHE-II and SOFA scores have better performance and predictability than the L/P ratio (AUC 0.83 and AUC 0.80, respectively), but showed no association with mortality (OR 1.07, 95% CI 1.01–1.17 and OR 1.08, 95% CI 1.02–1.18). Conclusions: Based on our findings, the L/P ratio appears to function more effectively as an early predictor of mortality when used as an adjuvant biomarker with other clinical parameters. [ABSTRACT FROM AUTHOR]

Xanthan gum is a microbial polysaccharide produced by Xanthomonas and widely used in various industries. To produce xanthan gum, the native Xanthomonas citri- 386 was used in a cheese-whey-based culture medium. The culture conditions were investigated in batch experiments based on the response surface methodology to increase xanthan production and viscosity. Three independent variables in this study included feeding times of acetate, pyruvate, and citrate. The maximum xanthan gum production and viscosity within 120 h by X. citri -386 using Box–Behnken design were 25.7 g/l and 65   500 cP, respectively, with a 151% and 394% increase as compared to the control sample. Overall, the findings of this study recommend the use of X. citri -386 in the cheese-whey-based medium as an economical medium with optimal amounts of acetate, pyruvate, and citrate for commercial production of xanthan gum on an industrial scale. The adjustment of the pyruvate and acetate concentrations optimized xanthan gum production in the environment. [ABSTRACT FROM AUTHOR]

The present study aimed to identify the metabolites associated with the physiological activity of kimchi‐derived lactic acid bacteria (LAB). A clear difference was observed between the 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid (ABTS) radical scavenging rates when the pyruvate content was high (273.5 ng/µL; radical removal speed 6.50% per min) and the rates when the pyruvate content had decreased (131.9 ng/µL; radical removal speed 3.63% per min). Additionally, the characteristics of LAB antioxidant activity (increase in ABTS radical scavenging activity with reaction time, low level of 2,2‐diphenyl‐1‐picrylhydrazyl radical scavenging activity) were similar to those of pyruvate‐derived activity. Hydrogen peroxide content (WiKim0124, 2.08 → 0.26; WiKim0121, 0.99 → 0.47; WiKim39, 1.93 → 0.24) and lactate dehydrogenase activity (WiKim0124, 1.53 → 0.00; WiKim0121, 0.73 → 0.01; WiKim39, 1.72 → 0.02) decreased more in heat‐killed LAB than in non‐heat‐killed LAB. Accordingly, this resulted in increased pyruvate content and the inhibitory activity of lipid peroxide production increased by 2–3 times. Our findings indicate that pyruvate is one of the major metabolites regulating LAB physiological activity. Practical Application: The safety of utilizing live probiotics remains a topic of debate. To mitigate associated risks, there is a growing interest in non‐viable microorganisms or microbial cell extracts for use as probiotics. Various methods can be employed for probiotic inactivation. Heat treatment typically emerges as the preferred choice for inactivating probiotic strains in many instances. The present study shows the distinctions between inactivating lactic acid bacteria (LAB) through heat treatment and non‐heat treatment. It may serve as a valuable reference for selecting an appropriate inactivation method for LAB in industrial processes. [ABSTRACT FROM AUTHOR]

The addition of antioxidants to cryopreservation media reportedly improves sperm post‐thaw quality and reproductive performance after artificial insemination. Therefore, the objectives of this study were to evaluate if the addition of L‐carnitine and pyruvate to freezing media, or their addition to samples after thawing, improves the post‐thaw quality of equine spermatozoa. Thus, in Experiment 1, stallion semen samples were cryopreserved in: (1) EDTA–glucose–based extender with 20% egg yolk and 5% dimethylformamide (EDTA control); (2) skim milk–based extender with 20% egg yolk and 5% dimethylformamide (milk control); (3) Extender 1 supplemented with 50 mM L‐carnitine and 10 mM pyruvate (EDTA‐carnitine‐pyruvate); and (4) Extender 2 supplemented with 50 mM L‐carnitine and 10 mM pyruvate (milk‐carnitine‐pyruvate). In Experiment 2, 50 mM L‐carnitine and 10 mM pyruvate were added post‐thaw to samples cryopreserved with extenders 1 and 2 (EDTA control and milk control). Sperm kinematic parameters, DNA fragmentation, membrane lipid peroxidation, acrosome status and viability were evaluated after thawing. No significant differences (p > 0.05) were observed for most of the kinematic parameters, DNA fragmentation, membrane lipid peroxidation, acrosome status and viability of spermatozoa, between the samples frozen in the presence or absence of L‐carnitine and pyruvate, nor between the samples after the post‐thaw addition of these components. A higher (p < 0.05) mean velocity and higher (p < 0.05) amplitude of lateral head displacement were observed in the samples frozen in the milk‐based extender with the addition of L‐carnitine and pyruvate after thawing. The addition of 50 mM L‐carnitine and 10 mM pyruvate, either to the freezing extenders or after thawing, was not deleterious for sperm; however, it did not improve equine sperm motility, viability, acrosome and DNA integrity, nor decrease membrane lipid peroxidation after thawing. [ABSTRACT FROM AUTHOR]

Purpose: We aimed to incorporate a deep learning prior with k‐space data fidelity for accelerating hyperpolarized carbon‐13 MRSI, demonstrated on synthetic cancer datasets. Methods: A two‐site exchange model, derived from the Bloch equation of MR signal evolution, was firstly used in simulating training and testing data, that is, synthetic phantom datasets. Five singular maps generated from each simulated dataset were used to train a deep learning prior, which was then employed with the fidelity term to reconstruct the undersampled MRI k‐space data. The proposed method was assessed on synthetic human brain tumor images (N = 33), prostate cancer images (N = 72), and mouse tumor images (N = 58) for three undersampling factors and 2.5% additive Gaussian noise. Furthermore, varied levels of Gaussian noise with SDs of 2.5%, 5%, and 10% were added on synthetic prostate cancer data, and corresponding reconstruction results were evaluated. Results: For quantitative evaluation, peak SNRs were approximately 32 dB, and the accuracy was generally improved for 5 to 8 dB compared with those from compressed sensing with L1‐norm regularization or total variation regularization. Reasonable normalized RMS error were obtained. Our method also worked robustly against noise, even on a data with noise SD of 10%. Conclusion: The proposed singular value decomposition + iterative deep learning model could be considered as a general framework that extended the application of deep learning MRI reconstruction to metabolic imaging. The morphology of tumors and metabolic images could be measured robustly in six times acceleration using our method. [ABSTRACT FROM AUTHOR]

Obesity has a multifactorial etiology and is known to be a state of chronic low-grade inflammation, known as meta-inflammation. This state is associated with the development of metabolic disorders such as glucose intolerance and nonalcoholic fatty liver disease. Pyruvate is a glycolytic metabolite and a crucial node in various metabolic pathways. However, its role and molecular mechanism in obesity and associated complications are obscure. In this study, we reported that pyruvate substantially inhibited adipogenic differentiation in vitro and its administration significantly prevented HFD-induced weight gain, white adipose tissue inflammation, and metabolic dysregulation. To identify the target proteins of pyruvate, drug affinity responsive target stability was employed with proteomics, cellular thermal shift assay, and isothermal drug response to detect the interactions between pyruvate and its molecular targets. Consequently, we identified cytosolic phospholipase A2 (cPLA2) as a novel molecular target of pyruvate and demonstrated that pyruvate restrained diet-induced obesity, white adipose tissue inflammation, and hepatic steatosis in a cPLA2-dependent manner. Studies with global ablation of cPLA2 in mice showed that the protective effects of pyruvate were largely abrogated, confirming the importance of pyruvate/cPLA2 interaction in pyruvate attenuation of inflammation and obesity. Overall, our study not only establishes pyruvate as an antagonist of cPLA2 signaling and a potential therapeutic option for obesity but it also sheds light on the mechanism of its action. Pyruvate's prior clinical use indicates that it can be considered a safe and viable alternative for obesity, whether consumed as a dietary supplement or as part of a regular diet. [ABSTRACT FROM AUTHOR]

A 3‐year‐old male neutered domestic shorthair cat presented with lethargy, hyporexia, and pyrexia of unknown origin. Biochemical analysis using a Beckman Coulter AU480 demonstrated marked increases in creatine kinase and aspartate aminotransferase, indicative of severe muscle injury, with concurrent presumptive myoglobinuria on urinalysis. A marked, non‐physiologic increase in measured bicarbonate and resultant negative anion gap was documented; however, calculated bicarbonate obtained via a point‐of‐care blood gas analyzer was within normal limits. Laboratory error due to interference by lactate dehydrogenase was suspected and supported by the results of subsequent biochemical testing. Artifactual increases in bicarbonate have been documented in cases of rhabdomyolysis in horses, cows, and a bird. However, to the best of the authors' knowledge, this is the first report to demonstrate this spurious change in a cat. [ABSTRACT FROM AUTHOR]

Abstract Acquired drug resistance is a major challenge in the management of cancer, which underscores the need for discovery and development of novel therapeutic strategies. We report here the mechanism of the anti-cancer activity of a small coordinate complex composed of the rare earth metal praseodymium (Pr) and mercaptopyridine oxide (MPO; pyrithione). Exposure of cancer cells to relatively low concentrations of the conjugate Pr-MPO (5 µM) significantly impairs cell survival in a p53-independent manner and irrespective of the drug resistant phenotype. Mechanistically, Pr-MPO-induced cell death is caspase-independent, not inhibitable by necrostatin, but associated with the appearance of autophagy markers. However, further analysis revealed incomplete autophagic flux, thus suggesting altered integrity of lysosomal machinery. Supporting the lysosomal targeting activity are data demonstrating increased lysosomal Ca2+ accumulation and alkalinization, which coincides with cytosolic acidification (drop in pHc from 7.75 to 7.00). In parallel, an increase in lysosomal activity of glycosidase alpha acid (GAA), involved in passive glycogen breakdown, correlates with rapid depletion of glucose stores upon Pr-MPO treatment. This is associated with swift cataplerosis of TCA cycle intermediates, loss of NAD+/NADH and increase in pyruvate dehydrogenase (PDH) activity to compensate for pyruvate loss. Addition of exogenous pyruvate rescued cell survival. Notably, lysosomal impairment and metabolic catastrophe triggered by Pr-MPO are suggestive of Zn2+-mediated cytotoxicity, which is confirmed by the ability of Zn2+ chelator TPEN to block Pr-MPO-mediated anti-tumor activity. Together, these results highlight the ability of the small molecule lanthanide conjugate to target the cells’ waste clearing machinery as well as mitochondrial metabolism for Zn2+-mediated execution of cancer cells, which could have therapeutic potential against cancers with high metabolic activity.

Background. In diabetes mellitus (DM), analysis of changes in the biochemical profile of erythrocytes is the important stage of complex scientific research to clarify the mechanism of action of medicinal products based on plant raw materials. The fruits of Cornus mas L. are widely known. The biologically active compounds of these fruits show multiple biological effects. However, the effect of the fruit extracts of cornelian cherry on the functional state of erythrocytes in diabetes has not been sufficiently studied. The high glucose concentration in erythrocytes induces various structural and functional changes, which lead to numerous disturbances in their metabolism. Glucose transported into erythrocytes by facilitated diffusion via GLUT2 undergoes catabolic breakdown in anaerobic glycolysis (90 % of all glucose) and pentose phosphate pathway (the rest 10 %). ATP and reduced coenzymes of NADH + H+ and NADPH + H+ formed due to metabolism participate in maintaining the structure of hemoglobin. Enzymes of the antioxidant defense system, which prevent hemoglobin oxidation into methemoglobin, are especially important. Hyperglycemia and the development of oxidative stress in diabetes are the cause of a decrease in the activity of antioxidant enzymes and the accumulation of ligand forms of hemoglobin (HbCO2, MetHb, HbA1c). Therefore, the work aimed to investigate the effect of extracts of red and yellow fruits of Cornus mas L. on the content of end products of the glycolytic breakdown of glucose in erythrocytes and biochemical markers of the antioxidant status of these blood cells in rats with streptozotocin-induced diabetes. Materials and Methods. DM 1 type in animals was induced by intraperitoneal injection of streptozotocin. Experiments were performed on male Wistar rats, who, from the 10th day after diabetes induction, were administered per os extracts of red and yellow fruits of the cornelian cherry and loganic acid obtained from yellow fruits at a dose of 20 mg/kg of body weight for 14 days. On the 24th day of the experiment, the rats were decapitated under ether anesthesia, and blood was taken. The content of pyruvate and lactate (as the end products of anaerobic glycolysis) and L-lactate dehydrogenase activity were determined in plasma and erythrocytes, as well as biochemical markers of the antioxidant status of erythrocytes (activity of superoxide dismutase, catalase and glutathione peroxidase, level of reduced glutathione, TBA-reactive substances, concentration of oxidative modifications of proteins and advanced oxidation protein products). Results. The activity of catalase, superoxide dismutase, glutathione peroxidase and the concentration of reduced glutathione significantly increased against the decrease in the content of oxidative modifications of proteins, advanced oxidation protein products, TBA-reactive substances, pyruvate, L-lactate, and lactate dehydrogenase in rats with DM after administration of the fruit extracts of the cornelian cherry. Noteworthy, these biochemical indicators made it possible to assess the intensity of anaerobic glycolysis and the antioxidant status of blood erythrocytes in streptozotocin diabetes. Conclusions. Extracts of Cornus mas L. fruits might be potential natural drugs for the treatment of metabolic disorders in diabetes, as they have a corrective effect on the catabolic breakdown of glucose and the antioxidant defense system of erythrocytes, preventing the development of oxidative stress. It should be pointed out that the extract of red fruits of cornelian cherry showed the best effect among the studied extracts in normalizing these indicators.

[Display omitted] Population-based studies have highlighted the link between chronic urticaria (CU) and metabolic syndrome, and metabolic alterations have been revealed in CU. However, to our knowledge, a comprehensive metabolomics study on a large cohort of patients with CU has not been reported. We sought to explore the underlying metabolic subtypes and novel metabolite biomarkers for CU diagnosis and therapy. Plasma samples from 80 patients with CU and 82 healthy controls were collected for metabolomics quantification and bioinformatics analysis. Another independent cohort consisting of 144 patients with CU was studied to validate the findings. Bone marrow–derived mast cells and mice with IgE-induced passive cutaneous anaphylaxis were used for in vitro and in vivo experiments, respectively. We observed clear metabolome differences between CU patients and healthy controls. Meanwhile, differential metabolites N6-acetyl- l -lysine, l -aspartate, maleic acid, and pyruvic acid were used to construct random forest classifiers and achieved area under receiver operating characteristic curve values greater than 0.85, suggesting their potential as diagnostic biomarkers of CU. More importantly, by exploring the underlying metabolic subtypes of CU, we found that the low abundance of pyruvic acid and maleic acid was significantly related to the activity of CU, poor efficacy of second-generation H 1 antihistamines, and short relapse-free time. The results were validated in the independent cohort. Moreover, supplementation with pyruvate or maleate could significantly attenuate IgE-mediated mast cell activation in vitro and in vivo. Plasma pyruvic acid and maleic acid may be effective biomarkers for predicting disease activity, therapeutic efficacy, and prognosis for patients with CU. [ABSTRACT FROM AUTHOR]

Background: Pancreatic ductal adenocarcinoma (PDA) is the third leading cause of cancer‐related death in the United States. However, early response assessment using the current approach of measuring changes in tumor size on computed tomography (CT) or MRI is challenging. Purpose: To investigate the feasibility of hyperpolarized (HP) [1‐13C]pyruvate MRI to quantify metabolism in the normal appearing pancreas and PDA, and to assess changes in PDA metabolism following systemic chemotherapy. Study Type: Prospective. Subjects: Six patients (65.0 ± 7.6 years, 2 females) with locally advanced or metastatic PDA enrolled prior to starting a new line of systemic chemotherapy. Field Strength/Sequence: 3‐T, T1‐weighted gradient echo, metabolite‐selective 13C echoplanar imaging. Assessment: Time‐resolved HP [1‐13C]pyruvate data were acquired before (N = 6) and 4‐weeks after (N = 3) treatment initiation. Pyruvate metabolism, as quantified by pharmacokinetic modeling and metabolite area‐under‐the‐curve ratios, was assessed in manually segmented PDA and normal appearing pancreas ROIs (N = 5). The change in tumor metabolism before and 4‐weeks after treatment initiation was assessed in primary PDA (N = 2) and liver metastases (N = 1), and was compared to objective tumor response defined by response evaluation criteria in solid tumors (RECIST) on subsequent CTs. Statistical Tests: Descriptive tests (mean ± standard deviation), model fit error for pharmacokinetic rate constants. Results: Primary PDA showed reduced alanine‐to‐lactate ratios when compared to normal pancreas, due to increased lactate‐to‐pyruvate or reduced alanine‐to‐pyruvate ratios. Of the three patients who received HP [1‐13C]pyruvate MRI before and 4‐weeks after treatment initiation, one patient had a primary tumor with early metabolic response (increase in alanine‐to‐lactate) and subsequent partial response according to RECIST, one patient had a primary tumor with relatively stable metabolism and subsequent stable disease by RECIST, and one patient had metastatic PDA with increase in lactate‐to‐pyruvate of the liver metastases and corresponding progressive disease according to RECIST. Data Conclusion: Altered pyruvate metabolism with increased lactate or reduced alanine was observed in the primary tumor. Early metabolic response assessed at 4‐weeks after treatment initiation correlated with subsequent objective tumor response assessed using RECIST. Level of Evidence: 2 Technical Efficacy: Stage 2 [ABSTRACT FROM AUTHOR]

Purine nucleotide adenosine triphosphate (ATP) is a source of intracellular energy maintained by mitochondrial oxidative phosphorylation. However, when released from ischemic cells into the extracellular space, they act as death-signaling molecules (eATP). Despite there being potential benefit in using pyruvate to enhance mitochondria by inducing a highly oxidative metabolic state, its association with eATP levels is still poorly understood. Therefore, while we hypothesized that pyruvate could beneficially increase intracellular ATP with the enhancement of mitochondrial function after cardiac arrest (CA), our main focus was whether a proportion of the raised intracellular ATP would detrimentally leak out into the extracellular space. As indicated by the increased levels in systemic oxygen consumption, intravenous administrations of bolus (500 mg/kg) and continuous infusion (1000 mg/kg/h) of pyruvate successfully increased oxygen metabolism in post 10-min CA rats. Plasma ATP levels increased significantly from 67 ± 11 nM before CA to 227 ± 103 nM 2 h after the resuscitation; however, pyruvate administration did not affect post-CA ATP levels. Notably, pyruvate improved post-CA cardiac contraction and acidemia (low pH). We also found that pyruvate increased systemic CO2 production post-CA. These data support that pyruvate has therapeutic potential for improving CA outcomes by enhancing oxygen and energy metabolism in the brain and heart and attenuating intracellular hydrogen ion disorders, but does not exacerbate the death-signaling of eATP in the blood. [ABSTRACT FROM AUTHOR]

2-Keto-3-deoxy-galactonate (KDGal) serves as a pivotal metabolic intermediate within both the fungal d-galacturonate pathway, which is integral to pectin catabolism, and the bacterial DeLey-Doudoroff pathway for d-galactose catabolism. The presence of KDGal enantiomers, l-KDGal and d-KDGal, varies across these pathways. Fungal pathways generate l-KDGal through the reduction and dehydration of d-galacturonate, whereas bacterial pathways produce d-KDGal through the oxidation and dehydration of d-galactose. Two distinct catabolic routes further metabolize KDGal: a nonphosphorolytic pathway that employs aldolase and a phosphorolytic pathway involving kinase and aldolase. Recent findings have revealed that l-KDGal, identified in the bacterial catabolism of 3,6-anhydro-l-galactose, a major component of red seaweeds, is also catabolized by Escherichia coli, which is traditionally known to be catabolized by specific fungal species, such as Trichoderma reesei. Furthermore, the potential industrial applications of KDGal and its derivatives, such as pyruvate and d- and l-glyceraldehyde, are underscored by their significant biological functions. This review comprehensively outlines the catabolism of l-KDGal and d-KDGal across different biological systems, highlights stereospecific methods for discriminating between enantiomers, and explores industrial application prospects for producing KDGal enantiomers. Key points: • KDGal is a metabolic intermediate in fungal and bacterial pathways • Stereospecific enzymes can be used to identify the enantiomeric nature of KDGal • KDGal can be used to induce pectin catabolism or produce functional materials [ABSTRACT FROM AUTHOR]

Background: Most commerce of equine seminal doses is carried out using commercial extenders under refrigeration at 5°C. Objectives: To determine if 10 mm pyruvate in a 67 mm glucose extender and storage at 22°C could be the basis of an alternative storage method to cooling to 5°C. Material and methods: Stallion ejaculates were extendedin: INRA96 (67 mm glucose, non‐pyruvate control), modified Tyrode's (67 mm glucose–10 mm pyruvate), supplemented with 0, 10, 50, and 100 μM itaconate. As itaconate was vehiculated in DMSO, a control vehicle was also included. Sperm motility, viability, mitochondrial membrane potential, and production of reactive oxygen species were measured after collection and again after 48 and 96 h of storage at 22°C. To disclose molecular metabolic changes, spermatozoa were incubated up to 3 h in modified Tyrode's 67 mm glucose–10 mm pyruvate and modified Tyrode's 67 mm glucose, and metabolic analysis conducted. Results: After 96 h of storage aliquots stored in the control, INRA96 had a very poor total motility of 5.6% ± 2.3%, while in the 67 mm glucose–10 mm pyruvate/10 μm itaconate extender, total motility was 34.7% ± 3.8% (p = 0.0066). After 96 h, viability was better in most pyruvate‐based media, and the mitochondrial membrane potential in spermatozoa extended in INRA96 was relatively lower (p < 0.0001). Metabolomics revealed that in the spermatozoa incubated in the high pyruvate media, there was an increase in the relative amounts of NAD+, pyruvate, lactate, and ATP. Discussion and conclusions: Aliquots stored in a 67 mm glucose–10 mm pyruvate‐based medium supplemented with 10 μM itaconate, maintained a 35% total motility after 96 h of storage at 22°C, which is considered the minimum acceptable motility for commercialization. Improvements may be related to the conversion of pyruvate to lactate and regeneration of NAD+. [ABSTRACT FROM AUTHOR]

Supplemental Digital Content is Available in the Text. Impairment of mitochondrial pyruvate oxidation in dorsal root ganglia is sufficient to induce long-lasting allodynia and widespread transcriptomic alterations. Metabolism is inextricably linked to every aspect of cellular function. In addition to energy production and biosynthesis, metabolism plays a crucial role in regulating signal transduction and gene expression. Altered metabolic states have been shown to maintain aberrant signaling and transcription, contributing to diseases like cancer, cardiovascular disease, and neurodegeneration. Metabolic gene polymorphisms and defects are also associated with chronic pain conditions, as are increased levels of nerve growth factor (NGF). However, the mechanisms by which NGF may modulate sensory neuron metabolism remain unclear. This study demonstrated that intraplantar NGF injection reprograms sensory neuron metabolism. Nerve growth factor suppressed mitochondrial pyruvate oxidation and enhanced lactate extrusion, requiring 24 hours to increase lactate dehydrogenase A and pyruvate dehydrogenase kinase 1 (PDHK1) expression. Inhibiting these metabolic enzymes reversed NGF-mediated effects. Remarkably, directly disrupting mitochondrial pyruvate oxidation induced severe, persistent allodynia, implicating this metabolic dysfunction in chronic pain. Nanopore long-read sequencing of poly(A) mRNA uncovered extensive transcriptomic changes upon metabolic disruption, including altered gene expression, splicing, and poly(A) tail lengths. By linking metabolic disturbance of dorsal root ganglia to transcriptome reprogramming, this study enhances our understanding of the mechanisms underlying persistent nociceptive sensitization. These findings imply that impaired mitochondrial pyruvate oxidation may drive chronic pain, possibly by impacting transcriptomic regulation. Exploring these metabolite-driven mechanisms further might reveal novel therapeutic targets for intractable pain. [ABSTRACT FROM AUTHOR]

Simple Summary: In cancer cells, gross changes in gene expression help to enhance the uptake of glucose—the blood sugar—for the increased formation of building blocks for the cancer cells' unlimited growth. An issue related to the rewired cancer cell metabolism is that even under sufficient oxygen supply, cancer cells metabolize a large fraction of the glucose to lactic acid (or "lactate"). In non-cancerous cells, lactate would be formed only during oxygen deprivation in order to re-oxidize the cytosolic electron carrier NADH, which forms during the glycolytic glucose breakdown. This phenomenon of lactate secretion from cancer cells under aerobic conditions was named the "Warburg Effect", but the actual reasons for that are often regarded as not yet understood. However, when we acknowledge that the reprogramming of the different metabolic pathways of cancer cells is not neatly fine-tuned, it becomes plausible that lactate formation just serves to dispose of cytosolic electrons that exceed the capacity of the mitochondrial electron transport chain to accept cytosolic electrons. Interestingly, the kinetic properties of the enzymes that metabolize the glycolysis end product pyruvate are sufficient to explain the priorities for metabolite flux at the pyruvate junction in cancer cells: 1. mitochondrial oxidative phosphorylation for efficient ATP production, 2. cytosolic electrons that exceed oxidative phosphorylation capacity need to be disposed of and secreted as lactate, and 3. biosynthesis reactions for cancer cell growth. In other words, a number of cytosolic electrons just take the "emergency exit" from the cell by lactate secretion to maintain the cytosolic redox balance. Cancer cells metabolize a large fraction of glucose to lactate, even under a sufficient oxygen supply. This phenomenon—the "Warburg Effect"—is often regarded as not yet understood. Cancer cells change gene expression to increase the uptake and utilization of glucose for biosynthesis pathways and glycolysis, but they do not adequately up-regulate the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS). Thereby, an increased glycolytic flux causes an increased production of cytosolic NADH. However, since the corresponding gene expression changes are not neatly fine-tuned in the cancer cells, cytosolic NAD+ must often be regenerated by loading excess electrons onto pyruvate and secreting the resulting lactate, even under sufficient oxygen supply. Interestingly, the Michaelis constants (KM values) of the enzymes at the pyruvate junction are sufficient to explain the priorities for pyruvate utilization in cancer cells: 1. mitochondrial OXPHOS for efficient ATP production, 2. electrons that exceed OXPHOS capacity need to be disposed of and secreted as lactate, and 3. biosynthesis reactions for cancer cell growth. In other words, a number of cytosolic electrons need to take the "emergency exit" from the cell by lactate secretion to maintain the cytosolic redox balance. [ABSTRACT FROM AUTHOR]

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Mitochondrial alternative oxidase (AOX) is an important protein that can help in regulating reactive oxygen species and nitric oxide in plants. The role of AOX in regulation of nitro-oxidative stress in chickpea is not known. Using germinating chickpea as a model system, we investigated the role of AOX in nitro-oxidative stress tolerance. NaCl treatment was used as an inducer of nitro-oxidative stress. Treatment of germinating seeds with 150 mM NaCl led to reduced germination and radicle growth. The AOX inhibitor SHAM caused further inhibition of germination, and the AOX inducer pyruvate improved growth of the radicle under NaCl stress. Isolated mitochondria from germinated seeds under salt stress not only increased AOX capacity but also enhanced AOX protein expression. Measurement of superoxide levels revealed that AOX inhibition by SHAM can enhance superoxide levels, whereas the AOX inducer pyruvate reduced superoxide levels. Measurement of NO by gas phase chemiluminescence revealed enhanced NO generation in response to NaCl treatment. Upon NaCl treatment there was enhanced tyrosine nitration, which is an indicator of nitrosative stress response. Taken together, our results revealed that AOX induced under salinity stress in germinating chickpea can help in mitigating nitro-oxidative stress, thereby improving germination. [ABSTRACT FROM AUTHOR]

MRI with hyperpolarized (HP) 13C agents, also known as HP 13CMRI, can measure processes such as localized metabolism that is altered in numerous cancers, liver, heart, kidney diseases, and more. It has been translated into human studies during the past 10 years, with recent rapid growth in studies largely based on increasing availability of HP agent preparation methods suitable for use in humans. This paper aims to capture the current successful practices for HP MRI human studies with [1-13C]pyruvate--by far the most commonly used agent, which sits at a keymetabolic junction in glycolysis. The paper is divided into four major topic areas: (1) HP 13C-pyruvate preparation; (2) MRI system setup and calibrations; (3) data acquisition and image reconstruction; and (4) data analysis and quantification. In each area, we identified the key components for a successful study, summarized both published studies and current practices, and discuss evidence gaps, strengths, and limitations. This paper is the output of the "HP 13C MRI Consensus Group" as well as the ISMRM Hyperpolarized Media MR and Hyperpolarized Methods and Equipment study groups. It further aims to provide a comprehensive reference for future consensus, building as the field continues to advance human studies with this metabolic imaging modality. [ABSTRACT FROM AUTHOR]

ABSTRACT Dental caries is associated with microbial dysbiosis caused by the excessive proliferation of Streptococcus mutans in dental biofilms, where oxidative stress serves as the major stressor to microbial communities. The adaptability of S. mutans to oxidative stress is a prerequisite for its proliferation and even for exerting its virulence. Protein acetylation is a reversible and conserved regulatory mechanism enabling bacteria to rapidly respond to external environmental stressors. However, the functions of protein acetylation in regulating oxidative stress adaptability of S. mutans are still unknown. Here, we unveil the impact of acetyltransferase ActA-mediated acetylation on regulating the oxidative stress response of S. mutans. actA overexpression increased the sensitivity of S. mutans to hydrogen peroxide and diminished its competitive ability against Streptococcus sanguinis. In contrast, actA deletion enhanced oxidative stress tolerance and competitiveness of S. mutans. The mass spectrometric analysis identified pyruvate kinase (PykF) as a substrate of ActA, with its acetylation impairing its enzymatic activity and reducing pyruvate production. Supplementation with exogenous pyruvate mitigated oxidative stress sensitivity and restored competitiveness in multi-species biofilms. In vitro acetylation analysis further confirmed that ActA directly acetylates PykF, negatively affecting its enzymatic activity. Moreover, 18 potential lysine-acetylated sites on PykF were identified in vitro, which account for 75% of lysine-acetylated sites detected in vivo. Taken together, our study elucidates a novel regulatory mechanism of ActA-mediated acetylation of PykF in modulating oxidative stress adaptability of S. mutans by influencing pyruvate production, providing insights into the importance of protein acetylation in microbial environmental adaptability and interspecies interactions within dental biofilms.IMPORTANCEDental caries poses a significant challenge to global oral health, driven by microbial dysbiosis within dental biofilms. The pathogenicity of Streptococcus mutans, a major cariogenic bacterium, is closely linked to its ability to adapt to changing environments and cellular stresses. Our investigation into the protein acetylation mechanisms, particularly through the acetyltransferase ActA, reveals a critical pathway by which S. mutans modulates its adaptability to oxidative stress, the dominant stressor within dental biofilms. By elucidating how ActA affects the oxidative stress adaptability and competitiveness of S. mutans through the regulatory axis of ActA-PykF-pyruvate, our findings provide insights into the dynamic interplay between cariogenic and commensal bacteria within dental biofilms. This work emphasizes the significance of protein acetylation in bacterial stress response and competitiveness, opening avenues for the development of novel strategies to maintain oral microbial balance within dental biofilms.

Myofibroblasts are key cellular effectors of corneal wound healing from trauma, surgery, or infection. However, their persistent deposition of disorganized extracellular matrix can also cause corneal fibrosis and visual impairment. Recent work showed that the PPARγ agonist Troglitazone can mitigate established corneal fibrosis, and parallel in vitro data suggested this occurred through inhibition of the mitochondrial pyruvate carrier (MPC) rather than PPARγ. In addition to oxidative phosphorylation (Ox-Phos), pyruvate and other mitochondrial metabolites provide carbon for the synthesis of biological macromolecules. However, it is currently unclear how these roles selectively impact fibrosis. Here, we performed bioenergetic, metabolomic, and epigenetic analyses of corneal fibroblasts treated with TGF-β1 to stimulate myofibroblast trans-differentiation, with further addition of Troglitazone or the MPC inhibitor UK5099, to identify MPC-dependencies that may facilitate remodeling and loss of the myofibroblast phenotype. Our results show that a shift in energy metabolism is associated with, but not sufficient to drive cellular remodeling. Metabolites whose abundances were sensitive to MPC inhibition suggest that sustained carbon influx into the Krebs’ cycle is prioritized over proline synthesis to fuel collagen deposition. Furthermore, increased abundance of acetyl-CoA and increased histone H3 acetylation suggest that epigenetic mechanisms downstream of metabolic remodeling may reinforce cellular phenotypes. Overall, our results highlight a novel molecular target and metabolic vulnerability that affects myofibroblast persistence in the context of corneal wounding.

Cardiovascular disease shows, or may even be caused by, changes in metabolism. Hyperpolarized magnetic resonance spectroscopy and imaging is a technique that could assess the role of different aspects of metabolism in heart disease, allowing real-time metabolic flux assessment in vivo. In this review, we introduce the main hyperpolarization techniques. Then, we summarize the use of dedicated radiofrequency 13C coils, and report a state of the art of 13C data acquisition. Finally, this review provides an overview of the pre-clinical and clinical studies on cardiac metabolism in the healthy and diseased heart. We furthermore show what advances have been made to translate this technique into the clinic in the near future and what technical challenges still remain, such as exploring other metabolic substrates. [ABSTRACT FROM AUTHOR]

Astrocyte-derived pyruvate is considered to have neuroprotective functions. In order to investigate the processes that are involved in astrocytic pyruvate release, we used primary rat astrocyte cultures as model system. Depending on the incubation conditions and medium composition, astrocyte cultures established extracellular steady state pyruvate concentrations in the range between 150 µM and 300 µM. During incubations for up to 2 weeks in DMEM culture medium, the extracellular pyruvate concentration remained almost constant for days, while the extracellular lactate concentration increased continuously during the incubation into the millimolar concentration range as long as glucose was present. In an amino acid-free incubation buffer, glucose-fed astrocytes released pyruvate with an initial rate of around 60 nmol/(h × mg) and after around 5 h an almost constant extracellular pyruvate concentration was established that was maintained for several hours. Extracellular pyruvate accumulation was also observed, if glucose had been replaced by mannose, fructose, lactate or alanine. Glucose-fed astrocyte cultures established similar extracellular steady state concentrations of pyruvate by releasing pyruvate into pyruvate-free media or by consuming excess of extracellular pyruvate. Inhibition of the monocarboxylate transporter MCT1 by AR-C155858 lowered extracellular pyruvate accumulation, while inhibition of mitochondrial pyruvate uptake by UK5099 increased the extracellular pyruvate concentration. Finally, the presence of the uncoupler BAM15 or of the respiratory chain inhibitor antimycin A almost completely abolished extracellular pyruvate accumulation. The data presented demonstrate that cultured astrocytes establish a transient extracellular steady state concentration of pyruvate which is strongly affected by modulation of the mitochondrial pyruvate metabolism. [ABSTRACT FROM AUTHOR]

Lung branching morphogenesis relies on intricate epithelial–mesenchymal interactions and signaling networks. Still, the interplay between signaling and energy metabolism in shaping embryonic lung development remains unexplored. Retinoic acid (RA) signaling influences lung proximal–distal patterning and branching morphogenesis, but its role as a metabolic modulator is unknown. Hence, this study investigates how RA signaling affects the metabolic profile of lung branching. We performed ex vivo lung explant culture of embryonic chicken lungs treated with DMSO, 1 µM RA, or 10 µM BMS493. Extracellular metabolite consumption/production was evaluated by using 1H-NMR spectroscopy. Mitochondrial respiration and biogenesis were also analyzed. Proliferation was assessed using an EdU-based assay. The expression of crucial metabolic/signaling components was examined through Western blot, qPCR, and in situ hybridization. RA signaling stimulation redirects glucose towards pyruvate and succinate production rather than to alanine or lactate. Inhibition of RA signaling reduces lung branching, resulting in a cystic-like phenotype while promoting mitochondrial function. Here, RA signaling emerges as a regulator of tissue proliferation and lactate dehydrogenase expression. Furthermore, RA governs fatty acid metabolism through an AMPK-dependent mechanism. These findings underscore RA's pivotal role in shaping lung metabolism during branching morphogenesis, contributing to our understanding of lung development and cystic-related lung disorders. [ABSTRACT FROM AUTHOR]

Purpose: Improving the quality and maintaining the fidelity of large coverage abdominal hyperpolarized (HP) 13C MRI studies with a patch based global–local higher‐order singular value decomposition (GL‐HOVSD) spatiotemporal denoising approach. Methods: Denoising performance was first evaluated using the simulated [1‐13C]pyruvate dynamics at different noise levels to determine optimal kglobal and klocal parameters. The GL‐HOSVD spatiotemporal denoising method with the optimized parameters was then applied to two HP [1‐13C]pyruvate EPI abdominal human cohorts (n = 7 healthy volunteers and n = 8 pancreatic cancer patients). Results: The parameterization of kglobal = 0.2 and klocal = 0.9 denoises abdominal HP data while retaining image fidelity when evaluated by RMSE. The kPX (conversion rate of pyruvate‐to‐metabolite, X = lactate or alanine) difference was shown to be <20% with respect to ground‐truth metabolic conversion rates when there is adequate SNR (SNRAUC > 5) for downstream metabolites. In both human cohorts, there was a greater than nine‐fold gain in peak [1‐13C]pyruvate, [1‐13C]lactate, and [1‐13C]alanine apparent SNRAUC. The improvement in metabolite SNR enabled a more robust quantification of kPL and kPA. After denoising, we observed a 2.1 ± 0.4 and 4.8 ± 2.5‐fold increase in the number of voxels reliably fit across abdominal FOVs for kPL and kPA quantification maps. Conclusion: Spatiotemporal denoising greatly improves visualization of low SNR metabolites particularly [1‐13C]alanine and quantification of [1‐13C]pyruvate metabolism in large FOV HP 13C MRI studies of the human abdomen. [ABSTRACT FROM AUTHOR]

The hydrogen isotope ratios (d2HAA values) of amino acids in all organisms are substantially fractionated relative to growth water. In addition, they exhibit large variations within microbial biomass, animals, and human tissues, hinting at rich biochemical information encoded in such signals. In lipids, such d2H variations are thought to primarily reflect NADPH metabolism. Analogous biochemical controls for amino acids remain largely unknown, but must be elucidated to inform the interpretation of these measurements. Here, we measured the d2H values of amino acids from five aerobic, heterotrophic microbes grown on different carbon substrates, as well as five Escherichia coli mutant organisms with perturbed NADPH metabolisms. We observed similar d2HAA patterns across all organisms and growth conditions, which-consistent with previous hypotheses-suggests a first-order control by biosynthetic pathways. Moreover, d2HAA values varied systematically with the catabolic pathways activated for substrate degradation, with variations explainable by the isotopic compositions of important cellular metabolites, including pyruvate and NADPH, during growth on each substrate. As such, amino acid d2H valuesmay be useful for interrogating organismal physiology and metabolism in the environment, provided we can further elucidate the mechanisms underpinning these signals. [ABSTRACT FROM AUTHOR]

Despite the significant role of pyruvic acid in biotransformations, the utilization of pyruvates in the field of catalytic asymmetric synthesis has been unexpectedly limited. This statement also applies to enamine catalysis, in which pyruvates seem to be challenging substrates. Recently, the first illustration of the asymmetric addition of pyruvic acid to β‐nitrostyrenes, catalyzed by a NahE enzyme using the enamine mechanism, has appeared in the literature. Now, we demonstrate pyrrolidine‐based catalyst can mimick enamine mechanism used by this enzyme to activate pyruvates in the asymmetric addition to structurally diverse nitroalkenes to produce highly enantioenriched Michael adducts without the limitations in substrate structures associated with biocatalysts. In addition to confirming that optically pure pyrrolidines can serve as ultimately small organic catalysts in pyruvate activation, we present an exceedingly simple and versatile method for the synthesis of optically pure γ‐nitro acids, which are precursors for well‐known active pharmaceutical ingredients namely GABA derivatives [ABSTRACT FROM AUTHOR]

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The neuroblastoma cell lines SH-SY5Y and Neuro2A are commonly utilized models in neurobiological research. DMEM supplemented with different nutrients and 5–10% Fetal Calf Serum (FCS) is typically used for culturing these cell lines. During special treatments, a reduced FCS content is often deployed to reduce cellular proliferation or the content of bioactive compounds. The impact of the reduction of FCS in culture media on the metabolic profile of SH-SY5Y and Neuro2A cells is currently unknown. Using an Amplex Red Assay, this study showed that the consumption of L-glutamine decreased after FCS reduction. Glucose and pyruvate consumption increased in both cell lines after the reduction of FCS. Thus, lactate production also increased with reduced FCS concentration. The reduction of FCS in the cell culture medium resulted in a reduced aerobic ATP production for SH-SY5Y cells and a complete shut down of aerobic ATP production for Neuro2A cells, measured using the Seahorse XF Real-Time ATP Rate Assay. Utilizing the Seahorse XF Glutamine Oxidation Stress Test, Neuro2A cells showed an increased utilization of L-glutamine oxidation after reduction of FCS. These results indicate that changes in FCS concentration in culture media have an impact on the different energy production strategies of SH-SY5Y and Neuro2A cells which must be considered when planning special treatments. [ABSTRACT FROM AUTHOR]

Onion is an important vegetable crop because it adds nutritional value and diversity to food preparation. Understanding recent advancements in onion molecular genetics is essential to improve production, quality, and disease resistance. Cutting-edge genomic technologies like genetic mapping and RNA sequencing reveal important genes and pathways. The review examines the progress in utilizing various molecular markers to study genetic divergence. The exploration extends to understanding the genes and pathways responsible for bulb color and chemical composition and the genetic factors influencing bulbing, flowering, and vernalization. Additionally, the article explores quantitative trait loci associated with resistance to major damaging diseases and delves into the role of different loci in male sterility and hybrid development. The recent publication of the whole genome sequence of onions will lead to further identification of genes and understanding their roles and functions in metabolic pathways. [ABSTRACT FROM AUTHOR]

Introduction: The healthy heart has remarkable metabolic flexibility that permits rapid switching between mitochondrial glucose oxidation and fatty acid oxidation to generate ATP. Loss of metabolic flexibility has been implicated in the genesis of contractile dysfunction seen in cardiomyopathy. Metabolic flexibility has been imaged in experimental models, using hyperpolarized (HP) [2‐13C]pyruvate MRI, which enables interrogation of metabolites that reflect tricarboxylic acid (TCA) cycle flux in cardiac myocytes. This study aimed to develop methods, demonstrate feasibility for [2‐13C]pyruvate MRI in the human heart for the first time, and assess cardiac metabolic flexibility. Methods: Good manufacturing practice [2‐13C]pyruvic acid was polarized in a 5 T polarizer for 2.5–3 h. Following dissolution, quality control parameters of HP pyruvate met all safety and sterility criteria for pharmacy release, prior to administration to study subjects. Three healthy subjects each received two HP injections and MR scans, first under fasting conditions, followed by oral glucose load. A 5 cm axial slab‐selective spectroscopy approach was prescribed over the left ventricle and acquired at 3 s intervals on a 3 T clinical MRI scanner. Results: The study protocol, which included HP substrate injection, MR scanning, and oral glucose load, was performed safely without adverse events. Key downstream metabolites of [2‐13C]pyruvate metabolism in cardiac myocytes include the glycolytic derivative [2‐13C]lactate, TCA‐associated metabolite [5‐13C]glutamate, and [1‐13C]acetylcarnitine, catalyzed by carnitine acetyltransferase (CAT). After glucose load, 13C‐labeling of lactate, glutamate, and acetylcarnitine from 13C‐pyruvate increased by an average of 39.3%, 29.5%, and 114% respectively in the three subjects, which could result from increases in lactate dehydrogenase, pyruvate dehydrogenase, and CAT enzyme activity as well as TCA cycle flux (glucose oxidation). Conclusions: HP [2‐13C]pyruvate imaging is safe and permits noninvasive assessment of TCA cycle intermediates and the acetyl buffer, acetylcarnitine, which is not possible using HP [1‐13C]pyruvate. Cardiac metabolite measurement in the fasting/fed states provides information on cardiac metabolic flexibility and the acetylcarnitine pool. [ABSTRACT FROM AUTHOR]

The goal of this study was to investigate the origin of brain lactate (Lac) signal in the healthy anesthetized rat after injection of hyperpolarized (HP) [1‐13C]pyruvate (Pyr). Dynamic two‐dimensional spiral chemical shift imaging with flow‐sensitizing gradients revealed reduction in both vascular and brain Pyr, while no significant dependence on the level of flow suppression was detected for Lac. These results support the hypothesis that the HP metabolites predominantly reside in different compartments in the brain (i.e., Pyr in the blood and Lac in the parenchyma). Data from high‐resolution metabolic imaging of [1‐13C]Pyr further demonstrated that Lac detected in the brain was not from contributions of vascular signal attributable to partial volume effects. Additionally, metabolite distributions and kinetics measured with dynamic imaging after injection of HP [1‐13C]Lac were similar to Pyr data when Pyr was used as the substrate. These data do not support the hypothesis that Lac observed in the brain after Pyr injection was generated in other organs and then transported across the blood–brain barrier (BBB). Together, the presented results provide further evidence that even in healthy anesthetized rats, the transport of HP Pyr across the BBB is sufficiently fast to permit detection of its metabolic conversion to Lac within the brain. [ABSTRACT FROM AUTHOR]

Hyperpolarized [1–13C]pyruvate is the leading metabolite used in the emerging field of hyperpolarization-enhanced MRI. Signal amplification by reversible exchange (SABRE) is a straight forward hyperpolarization method that has recently been shown to hyperpolarize [1–13C]pyruvate at low (microtesla and below) magnetic fields. Here, we show that commercial optical magnetometers with Rb-vapor media can be used to readily monitor the build-up and decay of the hyperpolarized MR signal. In addition, we measure ZULF-NMR spectra in various conditions, ranging from a J-coupling-dominated regime transitioning into a Zeeman-dominated regime when going from a sub-nT field to a µT field. The experimentally acquired spectra are matched well by numerical simulations.

Abstract The bioconversion of 4-hydroxy-2-keto acid derivatives via aldol condensation of formaldehyde and pyruvate has received substantial attention as potential source of chemicals for production of amino acids, hydroxy carboxylic acids, and chiral aldehydes. We developed an environmentally friendly biocatalyst consisting of a novel thermostable class II pyruvate aldolase from Deinococcus radiodurans with maltose-binding protein (MBP-DrADL), which has specific activity of 46.3 µmol min–1 mg–1. Surprisingly, MBP-DrADL maintained over 60% of enzyme activity for 4 days at 50 to 65 °C, we used MBP-DrADL as the best candidate enzyme to produce 2-keto-4-hydroxybutyrate (2-KHB) from formaldehyde and pyruvate via aldol condensation. The optimum reaction conditions for 2-KHB production were 50 °C, pH 8.0, 5 mM Mg2+, 100 mM formaldehyde, and 200 mM pyruvate. Under these optimized conditions, MBP-DrADL produced 76.5 mM (8.94 g L–1) 2-KHB over 60 min with a volumetric productivity of 8.94 g L–1 h–1 and a specific productivity of 357.6 mg mg-enzyme–1 h–1. Furthermore, 2-KHB production was improved by continuous addition of substrates, which produced approximately 124.8 mM (14.6 g L–1) of 2-KHB over 60 min with a volumetric productivity and specific productivity of 14.6 g L–1 h–1 and 583.4 mg mg-enzyme–1 h–1, respectively. MBP-DrADL showed the highest specific productivity for 2-KHB production yet reported. Our study provides a highly efficient biocatalyst for the synthesis of 2-KHB and lays the foundation for large-scale production and application of high-value compounds from formaldehyde. Graphical Abstract

If a mechanism of more efficient glycolysis depending on pyruvate is present in stallion spermatozoa, detrimental effects of higher glucose concentrations that are common in current commercial extenders could be counteracted. To test this hypothesis, spermatozoa were incubated in a 67 mM Glucose modified Tyrode's media in the presence of 1- or 10-mM pyruvate and in the Tyrode's basal media which contains 5 mM glucose. Spermatozoa incubated for 3 h at 37 °C in 67 mM Tyrode's media with 10 mM pyruvate showed increased motility in comparison with aliquots incubated in Tyrode's 5 mM glucose and Tyrode's 67 mM glucose (57.1 ± 3.5 and 58.1 ± 1.9 to 73.0 ± 1.1 %; P < 0.01). Spermatozoa incubated in Tyrode's with 67 mM glucose 10 mM pyruvate maintained the viability along the incubation (64.03 ± 15.4 vs 61.3 ± 10.2), while spermatozoa incubated in 67 mM Glucose-Tyrode's showed a decrease in viability (38.01 ± 11.2, P < 0.01). 40 mM oxamate, an inhibitor of the lactate dehydrogenase LDH, reduced sperm viability (P < 0.05, from 76 ± 5 in 67 mM Glucose/10 mM pyruvate to 68.0 ± 4.3 %, P < 0.05). Apoptotic markers increased in the presence of oxamate. (P < 0.01). UHPLC/MS/MS showed that 10 mM pyruvate increased pyruvate, lactate, ATP and NAD+ while phosphoenolpyruvate decreased. The mechanisms that explain the improvement of in presence of 10 mM pyruvate involve the conversion of lactate to pyruvate and increased NAD+ enhancing the efficiency of the glycolysis. • 10 mM pyruvate in a 67 mM Glucose-Tyrode's media maintains sperm functionality in comparison with a 67 mM glucose- Tyrode's. • Motility, linear motility, velocities membrane integrity, mitochondrial membrane potential and glutathione content were higher in the 10 mM pyruvate, 67 mM glucose media. • LACTATE and NAD+ content were higher in the spermatozoa incubated in the 10 mM pyruvate in a 67 mM Glucose-Tyrode's media. • The mechanism explaining the improvements may involve conversion of pyruvate to lactate and regeneration of NAD+. • The stallion spermatozoa may produce significant amounts of lactate under high Oxygen (Warburg effect). [ABSTRACT FROM AUTHOR]

The division of glycolysis into two separate pathways, aerobic and anaerobic, depending on the presence or absence of oxygen, respectively, was formulated over eight decades ago. The former ends with pyruvate, while the latter ends with lactate. Today, this division is confusing and misleading as research over the past 35 years clearly has demonstrated that glycolysis ends with lactate not only in cancerous cells but also in healthy tissues and cells. The present essay offers a review of the history of said division and the more recent knowledge that has been gained about glycolysis and its end-product, lactate. Then, it presents arguments in an attempt to explain why separating glycolysis into aerobic and anaerobic pathways persists among scientists, clinicians and teachers alike, despite convincing evidence that such division is not only wrong scientifically but also hinders progress in the field of energy metabolism. [ABSTRACT FROM AUTHOR]

d‐Serine plays vital physiological roles in the functional regulation of the mammalian brain, where it is produced from l‐serine by serine racemase and degraded by d‐amino acid oxidase. In the present study, we identified a new d‐serine metabolizing activity of serine hydroxymethyltransferase (SHMT) in bacteria as well as mammals. SHMT is known to catalyze the conversion of l‐serine and tetrahydrofolate (THF) to glycine and 5,10‐methylenetetrahydrofolate, respectively. In addition, we found that human and Escherichia coli SHMTs have d‐serine dehydratase activity, which degrades d‐serine to pyruvate and ammonia. We characterized this enzymatic activity along with canonical SHMT activity. Intriguingly, SHMT required THF to catalyze d‐serine dehydration and did not exhibit dehydratase activity toward l‐serine. Furthermore, SHMT did not use d‐serine as a substrate in the canonical hydroxymethyltransferase reaction. The d‐serine dehydratase activities of two isozymes of human SHMT were inhibited in the presence of a high concentration of THF, whereas that of E. coli SHMT was increased. The pH and temperature profiles of d‐serine dehydratase and serine hydroxymethyltransferase activities of these three SHMTs were partially distinct. The catalytic efficiency (kcat/Km) of dehydratase activity was lower than that of hydroxymethyltransferase activity. Nevertheless, the d‐serine dehydratase activity of SHMT was physiologically important because d‐serine inhibited the growth of an SHMT deletion mutant of E. coli, ∆glyA, more than that of the wild‐type strain. Collectively, these results suggest that SHMT is involved not only in l‐ but also in d‐serine metabolism through the degradation of d‐serine. [ABSTRACT FROM AUTHOR]

The bioconversion of 4-hydroxy-2-keto acid derivatives via aldol condensation of formaldehyde and pyruvate has received substantial attention as potential source of chemicals for production of amino acids, hydroxy carboxylic acids, and chiral aldehydes. We developed an environmentally friendly biocatalyst consisting of a novel thermostable class II pyruvate aldolase from Deinococcus radiodurans with maltose-binding protein (MBP-DrADL), which has specific activity of 46.3 µmol min–1 mg–1. Surprisingly, MBP-DrADL maintained over 60% of enzyme activity for 4 days at 50 to 65 °C, we used MBP-DrADL as the best candidate enzyme to produce 2-keto-4-hydroxybutyrate (2-KHB) from formaldehyde and pyruvate via aldol condensation. The optimum reaction conditions for 2-KHB production were 50 °C, pH 8.0, 5 mM Mg2+, 100 mM formaldehyde, and 200 mM pyruvate. Under these optimized conditions, MBP-DrADL produced 76.5 mM (8.94 g L–1) 2-KHB over 60 min with a volumetric productivity of 8.94 g L–1 h–1 and a specific productivity of 357.6 mg mg-enzyme–1 h–1. Furthermore, 2-KHB production was improved by continuous addition of substrates, which produced approximately 124.8 mM (14.6 g L–1) of 2-KHB over 60 min with a volumetric productivity and specific productivity of 14.6 g L–1 h–1 and 583.4 mg mg-enzyme–1 h–1, respectively. MBP-DrADL showed the highest specific productivity for 2-KHB production yet reported. Our study provides a highly efficient biocatalyst for the synthesis of 2-KHB and lays the foundation for large-scale production and application of high-value compounds from formaldehyde. [ABSTRACT FROM AUTHOR]

In the present study, cryo‐electron tomography was used to investigate the localization of 2‐oxoacid dehydrogenase complexes (OADCs) in cardiac mitochondria and mitochondrial inner membrane samples. Two classes of ordered OADC inner cores with different symmetries were distinguished and their quaternary structures modeled. One class corresponds to pyruvate dehydrogenase complexes and the other to dehydrogenase complexes of α‐ketoglutarate and branched‐chain α‐ketoacids. OADCs were shown to be localized in close proximity to membrane‐embedded respirasomes, as observed both in densely packed lamellar cristae of cardiac mitochondria and in ruptured mitochondrial samples where the dense packing is absent. This suggests the specificity of the OADC–respirasome interaction, which allows localized NADH/NAD+ exchange between OADCs and complex I of the respiratory chain. The importance of this local coupling is based on OADCs being the link between respiration, glycolysis and amino acid metabolism. The coupling of these basic metabolic processes can vary in different tissues and conditions and may be involved in the development of various pathologies. The present study shows that this important and previously missing parameter of mitochondrial complex coupling can be successfully assessed using cryo‐electron tomography. [ABSTRACT FROM AUTHOR]

Embryo quality evaluation during in vitro development is a crucial factor for the success of assisted reproductive technologies (ARTs). However, the subjectivity inherent in the morphological evaluation by embryologists can introduce inconsistencies that impact the optimal embryo choice for transfer. To provide a more comprehensive evaluation of embryo quality, we undertook the integration of embryo metabolomics alongside standardized morphokinetic classification. The culture medium of 55 embryos (derived from 21 couples undergoing ICSI) was collected at two timepoints (days 3 and 5). Samples were split into Good (n = 29), Lagging (n = 19), and Bad (n = 10) according to embryo morphokinetic evaluation. Embryo metabolic performance was assessed by monitoring the variation in specific metabolites (pyruvate, lactate, alanine, glutamine, acetate, formate) using 1H-NMR. Adjusted metabolite differentials were observed during the first 3 days of culture and found to be discriminative of embryo quality at the end of day 5. Pyruvate, alanine, glutamine, and acetate were major contributors to this discrimination. Good and Lagging embryos were found to export and accumulate pyruvate and glutamine in the first 3 days of culture, while Bad embryos consumed them. This suggests that Bad embryos have less active metabolic activity than Good and Lagging embryos, and these two metabolites are putative biomarkers for embryo quality. This study provides a more comprehensive evaluation of embryo quality and can lead to improvements in ARTs by enabling the selection of the best embryos. By combining morphological assessment and metabolomics, the selection of high-quality embryos with the potential to result in successful pregnancies may become more accurate and consistent. [ABSTRACT FROM AUTHOR]

3-hydroxypropionic acid (3-HP) is a valuable platform chemical with high demand in the global market. Owing to its sustainability and environmental affability, the biosynthesis of 3-HP has gained a great deal of attention. The major challenges in biological production of 3-HP is by-product accumulation which significantly affects the titre and yield of 3-HP. In this work, efforts have been made to investigate the metabolic engineering of Escherichia coli to circumvent these constraints. From the mRNA expression studies, it was found that yqhD, poxB, and pta-ackA were the primary targets. The deletion of genes showed significant changes in the synthesis of 3-HP and other major metabolites such as propanediol and acetate. The deletion of yqhD significantly decreased the synthesis of propanediol and diverted the carbon flux towards 3-HP. Furthermore, the combined deletion of poxB and pta-ackA significantly showed the reduction of acetate concentration. The enhancement of 3-HP production has been attained by the combinatorial deletion of yqhD, poxB, and pta-ackA resulted the titre (440 mM vs. 556 mM) and yield (0.31 vs. 0.40). The present work could be an effective tool for the enhanced production of 3-HP with the reduced level of by-product accumulation. [ABSTRACT FROM AUTHOR]

The hydrogen isotope ratios (δ2HAA values) of amino acids in all organisms are substantially fractionated relative to growth water. In addition, they exhibit large variations within microbial biomass, animals, and human tissues, hinting at rich biochemical information encoded in such signals. In lipids, such δ2H variations are thought to primarily reflect NADPH metabolism. Analogous biochemical controls for amino acids remain largely unknown, but must be elucidated to inform the interpretation of these measurements. Here, we measured the δ2H values of amino acids from five aerobic, heterotrophic microbes grown on different carbon substrates, as well as five Escherichia coli mutant organisms with perturbed NADPH metabolisms. We observed similar δ2HAA patterns across all organisms and growth conditions, which–consistent with previous hypotheses–suggests a first-order control by biosynthetic pathways. Moreover, δ2HAA values varied systematically with the catabolic pathways activated for substrate degradation, with variations explainable by the isotopic compositions of important cellular metabolites, including pyruvate and NADPH, during growth on each substrate. As such, amino acid δ2H values may be useful for interrogating organismal physiology and metabolism in the environment, provided we can further elucidate the mechanisms underpinning these signals.

ABSTRACTEngineering microbial hosts to synthesize pyruvate derivatives depends on blocking pyruvate oxidation, thereby causing severe growth defects in aerobic glucose-based bioprocesses. To decouple pyruvate metabolism from cell growth to improve pyruvate availability, a genome-scale metabolic model combined with constraint-based flux balance analysis, geometric flux balance analysis, and flux variable analysis was used to identify genetic targets for strain design. Using translation elements from a ~3,000 cistronic library to modulate fxpK expression in a bicistronic cassette, a bifido shunt pathway was introduced to generate three molecules of non-pyruvate-derived acetyl-CoA from one molecule of glucose, bypassing pyruvate oxidation and carbon dioxide generation. The dynamic control of flux distribution by T7 RNAP-mediated synthetic small RNA decoupled pyruvate catabolism from cell growth. Adaptive laboratory evolution and multi-omics analysis revealed that a mutated isocitrate dehydrogenase functioned as a metabolic switch to activate the glyoxylate shunt as the only C4 anaplerotic pathway to generate malate from two molecules of acetyl-CoA input and bypass two decarboxylation reactions in the tricarboxylic acid cycle. A chassis strain for pyruvate derivative synthesis was constructed to reduce carbon loss by using the glyoxylate shunt as the only C4 anaplerotic pathway and the bifido shunt as a non-pyruvate-derived acetyl-CoA synthetic pathway and produced 22.46, 27.62, and 6.28 g/L of l-leucine, l-alanine, and l-valine by a controlled small RNA switch, respectively. Our study establishes a novel metabolic pattern of glucose-grown bacteria to minimize carbon loss under aerobic conditions and provides valuable insights into cell design for manufacturing pyruvate-derived products.IMPORTANCEBio-manufacturing from biomass-derived carbon sources using microbes as a cell factory provides an eco-friendly alternative to petrochemical-based processes. Pyruvate serves as a crucial building block for the biosynthesis of industrial chemicals; however, it is different to improve pyruvate availability in vivo due to the coupling of pyruvate-derived acetyl-CoA with microbial growth and energy metabolism via the oxidative tricarboxylic acid cycle. A genome-scale metabolic model combined with three algorithm analyses was used for strain design. Carbon metabolism was reprogrammed using two genetic control tools to fine-tune gene expression. Adaptive laboratory evolution and multi-omics analysis screened the growth-related regulatory targets beyond rational design. A novel metabolic pattern of glucose-grown bacteria is established to maintain growth fitness and minimize carbon loss under aerobic conditions for the synthesis of pyruvate-derived products. This study provides valuable insights into the design of a microbial cell factory for synthetic biology to produce industrial bio-products of interest.

Introduction: Multiple system atrophy (MSA) is a rapidly progressing neurodegenerative disorder. Although diverse biomarkers have been established for Parkinson's disease (PD), no widely accepted markers have been identified in MSA. Pyruvate and lactate are the end-product of glycolysis and crucial for brain metabolism. However, their correlation with MSA remains unclear. Moreover, it is elusive how lifestyles modify these metabolites. Methods: To investigate the correlation and diagnostic value of plasma pyruvate and lactate levels in MSA and PD. Moreover, we explored how lifestyle-related metabolites interact with these metabolites in determining the disease risk. We assayed the 3 metabolites in pyruvate/lactate and 6 in the tea/coffee metabolic pathways by targeted mass spectrometry and evaluate their interactions and performance in diagnosis and differentiation between MSA and PD. Results: We found that 7 metabolites were significantly different between MSA, PD and healthy controls (HCs). Particularly, pyruvate was increased in PD while significantly decreased in MSA patients. Moreover, the tea/coffee metabolites were negatively associated with the pyruvate level in HCs, but not in MSA and PD patients. Using machine-learning models, we showed that the combination of pyruvate and tea/coffee metabolites diagnosed MSA (AUC = 0.878) and PD (AUC = 0.833) with good performance. Additionally, pyruvate had good performance in distinguishing MSA from PD (AUC = 0.860), and the differentiation increased (AUC = 0.922) when combined with theanine and 1,3-dimethyluric acid. Conclusions: This study demonstrates that pyruvate correlates reversely with MSA and PD, and may play distinct roles in their pathogenesis, which can be modified by lifestyle-related tea/coffee metabolites.