Tricarboxylic Acid Cycle Activity Research Articles

Effects of Hypoxia and Reoxygenation on Metabolic Profiles of Cardiomyocytes.

In vitro cellular models provide valuable insights into the adaptive biochemical mechanisms triggered by cells to cope with the stress situation induced by hypoxia and reoxygenation cycles. The first biological data generated in studies based on this micrometric life-scale has the potential to provide us a global overview about the main biochemical phenomena presented in some reported preconditioning therapies in life-scale of higher dimensions. Thus, in this study, a cell incubator was designed and manufactured to produce a cellular model of heart hypoxia followed by reoxygenation (HfR) through consecutive repetitions of hypoxia-normoxia gas exchange. Samples of cellular extracts and culture media were obtained from non-proliferative cardiomyocytes (CMs) cultivated under challenging HfR (stressed CMs) and regular cultivation (unstressed CMs) in rounds of four days for each case. Metabolomic based on proton magnetic resonance spectroscopy (1H-MRS) was used as an analytical approach to identify and quantify the metabolomes of these samples, the endo- and exo-metabolome. Despite the stressed CMs presented over 90% higher cellular death rate compared to the unstressed CMs, the metabolic profiles indicates that the surviving cells up-regulate their amino acid metabolism either by active protein degradation or by the consumption of culture media components to increase coenzyme A-dependent metabolic pathways. This cell auto-regulation mechanism could be well characterized in the first two days when the difference smears off under once the metabolomes become similar. The metabolic adaptations of stressed CMs identified the relevance of the cyclic oxidation/reduction reactions of nicotinamide adenine dinucleotide phosphate molecules, NADP+/NADPH, and the increased tricarboxylic acid cycle activity in an environment overloaded with such a powerful antioxidant agent to survive an extreme HfR challenge. Thus, the combination of cellular models based on CMs, investigative methods, such as metabolomic and 1H-MRS, and the instrumental development of hypoxia incubator shown in this work were able to provide the first biochemical evidences behind therapies of gaseous exchanges paving the way to future assays.

Effect of radiotherapy on head and neck cancer tissues in patients receiving radiotherapy: a bioinformatics analysis-based study

Radiotherapy is pivotal in treating head and neck cancers including nasopharyngeal, tongue, hypopharyngeal, larynx, maxillary sinus, parotid gland, and oral cancers. It holds the potential for curative effects and finds application in conjunction with chemotherapy, either as a radical method to preserve organ function or as an adjuvant postoperative treatment. We used bioinformatics analysis to investigate the effects of radiotherapy on head and neck cancer tissues in patients who had received radiotherapy. In this study, the expression and mutation profiles of The Cancer Genome Atlas–Head-Neck Squamous Cell Carcinoma were downloaded from the UCSC-Xena database, categorizing patients into two groups—those receiving radiotherapy and those not receiving radiotherapy. Subsequently, differential expression analysis and gene set enrichment analysis (GSEA) were performed. Following this, single-sample GSEA (ssGSEA) scores related to glucose and lipid metabolism were compared between the two groups. Additionally, immune cell infiltration analysis and single-cell verification were performed. Finally, the mutation profiles of the two groups were compared. The analyses revealed that patients receiving radiotherapy exhibited prolonged survival, enhanced apoptosis in head and neck cancer tissue, and diminished keratinocyte proliferation and migration. A comparison of ssGSEA scores related to glucose and lipid metabolism between the two groups indicated a reduction in glycolysis, tricarboxylic acid cycle activity, and fat synthesis in tissues treated with radiotherapy, suggesting that radiotherapy can effectively inhibit tumour cell energy metabolism. Analyses of immune cell infiltration and single-cell verification suggested decreased infiltration of immune cells post-radiotherapy in head and neck cancer tissues. A comparison of mutation profiles revealed a higher frequency of TP53, TTN, and CDKN2A mutations in patients receiving radiotherapy for head and neck cancer. In conclusion, the bioinformatics analyses delved into the effect of radiotherapy on patients with head and neck carcinoma. This study provides a theoretical framework elucidating the molecular mechanisms underlying radiotherapy's efficacy in treating head and neck cancer and presents scientific recommendations for drug therapy following radiotherapy.

Enhanced Bacterial Cuproptosis-Like Death via Reversal of Hypoxia Microenvironment for Biofilm Infection Treatment.

A recently emerging cell death pathway, known as copper-induced cell death, has demonstrated significant potential for treating infections. Existing research suggests that cells utilizing aerobic respiration, as opposed to those reliant on glycolysis, exhibit greater sensitivity to copper-induced death. Herein, a MnO2 -loaded copper metal-organic frameworks platform is developed denoted as MCM, to enhance bacterial cuproptosis-like death via the remodeling of bacterial respiratory metabolism. The reversal of hypoxic microenvironments induced a cascade of responses, encompassing the reactivation of suppressed immune responses and the promotion of osteogenesis and angiogenesis. Initially, MCM catalyzed O2 production, alleviating hypoxia within the biofilm and inducing a transition in bacterial respiration mode from glycolysis to aerobic respiration. Subsequently, the sensitized bacteria, characterized by enhanced tricarboxylic acid cycle activity, underwent cuproptosis-like death owing to increased copper concentrations and aggregated intracellular dihydrolipoamide S-acetyltransferase (DLAT). The disruption of hypoxia also stimulated suppressed dendritic cells and macrophages, thereby strengthening their antimicrobial activity through chemotaxis and phagocytosis. Moreover, the nutritional effects of copper elements, coupled with hypoxia alleviation, synergistically facilitated the regeneration of bones and blood vessels. Overall, reshaping the infection microenvironment to enhance cuproptosis-like cell death presents a promising avenue for eradicating biofilms.

Exploring the Antibacterial Potential and Underlying Mechanisms of Prunella vulgaris L. on Methicillin-Resistant Staphylococcus aureus.

Prunella vulgaris L. (PV) is a widely distributed plant species, known for its versatile applications in both traditional and contemporary medicine, as well as in functional food development. Despite its broad-spectrum antimicrobial utility, the specific mechanism of antibacterial action remains elusive. To fill this knowledge gap, the present study investigated the antibacterial properties of PV extracts against methicillin-resistant Staphylococcus aureus (MRSA) and assessed their mechanistic impact on bacterial cells and cellular functions. The aqueous extract of PV demonstrated greater anti-MRSA activity compared to the ethanolic and methanolic extracts. UPLC-ESI-MS/MS tentatively identified 28 phytochemical components in the aqueous extract of PV. Exposure to an aqueous extract at ½ MIC and MIC for 5 h resulted in a significant release of intracellular nucleic acid (up to 6-fold) and protein (up to 10-fold) into the extracellular environment. Additionally, this treatment caused a notable decline in the activity of several crucial enzymes, including a 41.51% reduction in alkaline phosphatase (AKP), a 45.71% decrease in adenosine triphosphatase (ATPase), and a 48.99% drop in superoxide dismutase (SOD). Furthermore, there was a decrease of 24.17% at ½ MIC and 27.17% at MIC in tricarboxylic acid (TCA) cycle activity and energy transfer. Collectively, these findings indicate that the anti-MRSA properties of PV may stem from its ability to disrupt membrane and cell wall integrity, interfere with enzymatic activity, and impede bacterial cell metabolism and the transmission of information and energy that is essential for bacterial growth, ultimately resulting in bacterial apoptosis. The diverse range of characteristics exhibited by PV positions it as a promising antimicrobial agent with broad applications for enhancing health and improving food safety and quality.

Metabolic alterations in hereditary and sporadic renal cell carcinoma.

Kidney cancer is the seventh leading cause of cancer in the world, and its incidence is on the rise. Renal cell carcinoma (RCC) is the most common form and is a heterogeneous disease comprising three major subtypes that vary in their histology, clinical course and driver mutations. These subtypes include clear cell RCC, papillary RCC and chromophobe RCC. Molecular analyses of hereditary and sporadic forms of RCC have revealed that this complex and deadly disease is characterized by metabolic pathway alterations in cancer cells that lead to deregulated oxygen and nutrient sensing, as well as impaired tricarboxylic acid cycle activity. These metabolic changes facilitate tumour growth and survival. Specifically, studies of the metabolic features of RCC have led to the discovery of oncometabolites - fumarate and succinate - that can promote tumorigenesis, moonlighting functions of enzymes, and substrate auxotrophy owing to the disruption of pathways that enable the production of arginine and cholesterol. These metabolic alterations within RCC can be exploited to identify new therapeutic targets and interventions, in combination with novel approaches that minimize the systemic toxicity of metabolic inhibitors and reduce the risk of drug resistance owing to metabolic plasticity.

Exopolysaccharide secreted by Lactiplantibacillus plantarum Y12 showed inhibitory effect on the pathogenicity of Shigella flexneri in vitro and in vivo

Shigella flexneri is a prevalent foodborne and waterborne pathogen that threatens human health. Our previous research indicated that the Lactiplantibacillus plantarum Y12 exopolysaccharide (L-EPS) potentially inhibited the pathogenicity of S. flexneri. The in vitro results of this study demonstrated that L-EPS effectively mitigated the symptoms induced by S. flexneri in HT-29 cells, including inhibited gene expression levels of IL-1β, IL-6, IL-8, TNF-α, TLR 2/4, and NOD1/2; decreased apoptosis ratio; and alleviated damage degree of intestinal barrier function (Zona occludens 1, Occludin, and Claudin-1). The in vivo results demonstrated that S. flexneri treated with L-EPS elicited mild adverse physiological manifestations, an inflammatory response, and tissue damage. The infection of S. flexneri caused significant alterations in the abundance of phylum (Firmicutes, Bacteroidota, Actinobacteriota, and Proteobacteria), family (Lachnospiraceae, Muribaculaceae, Rikenellaceae, Prevotellaceaea, Ruminococcaceae, and Lactobaillaceae), and genus (Escherichia Shigella and Lachnospirillaceae NK4A136 group) within the cecal microbiota. These changes were accompanied by perturbations in taurine and hypotaurine metabolism, tricarboxylic acid (TCA) cycle activity, arginine biosynthesis, and histidine metabolic pathways. However, intervention with L-EPS attenuated the dysbiosis of cecal microbiota and metabolic disturbances. In summary, our research suggested a potential application of L-EPS as a functional food additive for mitigating S. flexneri infection.

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

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

The reprogramming of mitochondrial energy metabolism in Rice: The role of the TCA cycle and GABA shunt under Arsenic exposure

AbstractArsenic (As) pollution is hazardous for all living organisms. Plants growing in As‐contaminated soil are less performant. Recently, seed priming has attracted significant attention for its beneficial effects on plants. Numerous studies have associated priming technique with the induction of stress memory against traumatic conditions, but its relation to crop's energy balance is under‐researched. The present study highlights variations in energy metabolic pathways, particularly TCA (tricarboxylic acid) cycle and GABA (gamma‐aminobutyric acid)‐shunt in multi‐nutrient [silicon (Si), iron (Fe), zinc (Zn), and ascorbic acid (AA)] primed rice seedlings under As stress. Arsenic exposure decreased the TCA cycle enzymes' activity while intensifying GABA accumulation and metabolism. This enhancement in GABA shunt provides an alternative energy source for As‐stressed seedlings. However, priming and further Fe‐supplementation reduced As‐toxicity by accelerating the activity of both energy pathways, namely, TCA and GABA cycle. Improved energy regulation by priming and Fe‐augmentation also enhanced the functionality of anabolic respiratory‐interactive‐pathway, i.e., carbon‐nitrogen metabolism. The As‐tolerance response of priming and Fe‐augmentation was further analyzed from the expression profiling of nitrogen and phosphorous utilization genes. Out of 10 genes, particular enhancement was observed in OsNiR, OsGS, and OsGOGAT expression with improved macro‐micronutrient content of the seedlings. Thus, irrespective of As infestations, multi‐nutrient (Si, Fe, Zn, and AA) seed priming approach along with Fe‐supplementation may emerge as a beneficial strategy to improve plant metabolic capacity in terms of nitrogen, phosphorous, and carbon assimilation to support normal vigor because they balance cellular energy levels.

Nutrient-Sensitising Drug Repurposing Screen Identifies Lomerizine As a Mitochondrial Metabolism Inhibitor in Chronic Myeloid Leukaemia

In chronic myeloid leukaemia (CML), the persistence of leukaemic stem cells (LSCs) following first line treatment with tyrosine kinase inhibitors (TKIs) such as imatinib, can lead to disease relapse. We have previously demonstrated that therapy-resistant CML LSCs rely on oxidative phosphorylation (OXPHOS) for survival and that targeting mitochondrial metabolism sensitises CML LSCs to imatinib treatment. However, current OXPHOS inhibitors have demonstrated limited efficacy, or are associated with adverse effects in clinical trials, highlighting that identification of clinically safe oxidative pathway inhibitors is warranted. To identify clinically applicable compounds that target cells dependent on mitochondrial metabolism, we used a nutrient-sensitised repurposing screening strategy. Upon replacement of glucose with galactose as the only sugar source in cell culture media, there is a shift in cellular metabolism towards OXPHOS (as cells are unable to effectively use galactose for glycolysis). Therefore, CML cells grown in galactose-containing media are more sensitive to inhibition of mitochondrial respiration than glucose-grown cells. Using this approach, we screened total of >1,100 unique and structurally diverse FDA-approved drugs in CML cells, cultured in the presence of 11mM glucose or 11mM galactose. This uncovered lomerizine dihydrochloride, a L-type Ca2+ channel blocker used as an anti-migraine drug, as a candidate compound effective against OXPHOS-dependent CML cells. Subsequently, patient derived CML CD34+ cells were shown to have increased ER mass (the main intracellular Ca2+ store) and an increase in mitochondrial Ca2+ levels compared with normal cells. Moreover, transcriptional analysis revealed that Ca2+ channel genes such as voltage-gated CACNA1D and receptor-activated TRPC6 are significantly upregulated in CML LSCs (CD34+CD38-) when compared with normal haematopoietic stem cells (HSCs), which was not reverted following imatinib treatment. Therefore, we hypothesised that the observed increase in mitochondrial metabolism in CML LSCs was dependent on CACNA1D and TRPC6 activity, and the increase in mitochondrial Ca2+ levels, which could be inhibited by lomerizine treatment. Notably, CRISPR-Cas9-mediated CACNA1D or TRPC6 knockout significantly reduced cytoplasmic and mitochondrial Ca2+ levels, and decreased cellular oxygen consumption rate, indicative of reduction in OXPHOS. Similarly, while imatinib treatment did not affect intracellular Ca2+ levels in primary CML CD34+ cells, lomerizine treatment significantly reduced cytoplasmic and mitochondrial Ca2+ levels, and mitochondrial respiration/OXPHOS. Applying liquid chromatography-mass spectrometry and isotope assisted metabolomics, we traced labelled nutrients (glucose and palmitate) in CD34+ cells. This revealed a significant reduction in activity of mitochondria-located Ca2+ dependent dehydrogenases and overall reduction in tricarboxylic acid (TCA) cycle activity in primary CML cells following lomerizine treatment. Of clinical relevance, using combination of cell surface markers, cell division trackers and functional assays, including long-term culture initiating cell (LTC-IC) assay, we demonstrate that lomerizine treatment, alone or in combination with imatinib, targeted CML LSCs when cultured in physiological medium in vitro, without affecting normal HSCs. Notably, combination treatment with imatinib and lomerizine reduced CML tumour burden, targeted CML LSCs and extended survival in xenotransplantation model of human CML. In summary, we reveal for the first time that Ca2+ regulation is disrupted in CML stem cells and influx via CACNA1D and TRPC6 is crucial for maintaining high mitochondrial Ca2+ levels and enhanced mitochondrial OXPHOS. This renders CML stem cells sensitive to lomerizine, an FDA-approved Ca2+ inhibitor identified following metabolism-specific drug-repurposing screening.

Nicotinamide riboside cardioprotection is mediated through glycolysis activation

Abstract Purpose Low NAD+ levels are observed in pathologies such as ageing, diabetes, heart failure and ischemia-reperfusion (IR) injury. Nicotinamide riboside (NR) has emerged as promising therapeutic NAD+ precursor [1,2]. NR was recently reported to protect against in vivo cardiac IR injury [3], but underlying mechanisms of protection were unexplored. Here we used the isolated mouse heart allowing metabolic control to examine NR’s metabolic mechanisms of cardiac protection. Methods Isolated C57BL/6N hearts were Langendorff-perfused with 5.5 mM glucose and 0.2 mM fatty acids (FA), and subjected to 35 min global ischemia and 90 min reperfusion. NR (50 mg/L) or vehicle were administered for 25 min before ischemia, and during the first 25 min of reperfusion. Cardiac functional recovery and cell death parameters (lactate dehydrogenase (LDH) release, % infarct size) were determined. To examine NR’s effects on cardiac metabolism, hearts were perfused with 5.5 mM 13C-glucose and 0.2 mM FA and analyzed by metabolomics and fluxomics using LC-MS techniques before ischemia. The dependency of NR’s protection on glycolysis was tested in hearts perfused with glucose replaced by pyruvate and lactate ("no glycolysis" hearts). Finally, NR’s protection and effects on cardiac metabolism were also examined in "high glycolysis" hearts, by adding insulin to the perfusate. Results NR reduced infarct size, LDH release and improved cardiac functional recovery for glucose/FA perfused hearts, demonstrating NR’s cardioprotection. In these glucose/FA hearts, metabolomics analysis showed NR increased NAD+, glycolytic intermediate phosphoenolpyruvate (PEP), Krebs cycle intermediate succinate and pentose phosphate pathway (PPP) intermediates ribose-5P (R5P) and sedoheptulose-7P (S7P) (Fig. 1). Fluxomics analysis demonstrated that NR’s protection was associated with activated glycolysis, without changes in tricarboxylic acid (TCA) cycle or PPP activities. No 13C-labeling of S7P was observed (Fig. 1). NR protection was lost in the "no glycolysis" hearts. In the "high glycolysis" hearts, NR was unable to protect the heart and increase glycolysis and reduced anaerobic glycolysis and ATP with increases in AMP, indicative of NR-induced energetic stress (Fig. 2). Remarkably, in these insulin perfused hearts, NR still increased PPP intermediates, with high 13C-labeling of S7P (Fig. 2). Conclusions NR’s protection against IR injury is only present in hearts with low glycolysis, associated with activation of pre-ischemic glycolysis. When activation of glycolysis was prevented, through either examining "no glycolysis" hearts or "high glycolysis" hearts, NR protection was abolished. NR treatment increased pre-ischemic PPP intermediates in either low or high glycolysis hearts, whereby insulin was necessary to incorporate 13C-glucose into S7P. The data suggest that NR’s acute cardioprotective effects are mediated through the activation of glycolysis.NR effects in low-glycolysis heartsNR effects in high-glycolysis hearts

Changes in Plasma Pyruvate and TCA Cycle Metabolites upon Increased Hepatic Fatty Acid Oxidation and Ketogenesis in Male Wistar Rats.

Altered hepatic mitochondrial fatty acid β-oxidation and associated tricarboxylic acid (TCA) cycle activity contributes to lifestyle-related diseases, and circulating biomarkers reflecting these changes could have disease prognostic value. This study aimed to determine hepatic and systemic changes in TCA-cycle-related metabolites upon the selective pharmacologic enhancement of mitochondrial fatty acid β-oxidation in the liver, and to elucidate the mechanisms and potential markers of hepatic mitochondrial activity. Male Wistar rats were treated with 3-thia fatty acids (e.g., tetradecylthioacetic acid (TTA)), which target mitochondrial biogenesis, mitochondrial fatty acid β-oxidation, and ketogenesis predominantly in the liver. Hepatic and plasma concentrations of TCA cycle intermediates and anaplerotic substrates (LC-MS/MS), plasma ketones (colorimetric assay), and acylcarnitines (HPLC-MS/MS), along with associated TCA-cycle-related gene expression (qPCR) and enzyme activities, were determined. TTA-induced hepatic fatty acid β-oxidation resulted in an increased ratio of plasma ketone bodies/nonesterified fatty acid (NEFA), lower plasma malonyl-CoA levels, and a higher ratio of plasma acetylcarnitine/palmitoylcarnitine (C2/C16). These changes were associated with decreased hepatic and increased plasma pyruvate concentrations, and increased plasma concentrations of succinate, malate, and 2-hydroxyglutarate. Expression of several genes encoding TCA cycle enzymes and the malate-oxoglutarate carrier (Slc25a11), glutamate dehydrogenase (Gdh), and malic enzyme (Mdh1 and Mdh2) were significantly increased. In conclusion, the induction of hepatic mitochondrial fatty acid β-oxidation by 3-thia fatty acids lowered hepatic pyruvate while increasing plasma pyruvate, as well as succinate, malate, and 2-hydroxyglutarate.

Effect of malate on the activity of ciprofloxacin against Pseudomonas aeruginosa in different in vivo and in vivo-like infection models.

The clinical significance of Pseudomonas aeruginosa infections and the tolerance of this opportunistic pathogen to antibiotic therapy makes the development of novel antimicrobial strategies an urgent need. We previously found that D,L-malic acid potentiates the activity of ciprofloxacin against P. aeruginosa biofilms grown in a synthetic cystic fibrosis sputum medium by increasing metabolic activity and tricarboxylic acid cycle activity. This suggested a potential new strategy to improve antibiotic therapy in P. aeruginosa infections. Considering the importance of the microenvironment on microbial antibiotic susceptibility, the present study aims to further investigate the effect of D,L-malate on ciprofloxacin activity against P. aeruginosa in physiologically relevant infection models, aiming to mimic the infection environment more closely. We used Caenorhabditis elegans nematodes, Galleria mellonella larvae, and a 3-D lung epithelial cell model to assess the effect of D,L-malate on ciprofloxacin activity against P. aeruginosa. D,L-malate was able to significantly enhance ciprofloxacin activity against P. aeruginosa in both G. mellonella larvae and the 3-D lung epithelial cell model. In addition, ciprofloxacin combined with D,L-malate significantly improved the survival of infected 3-D cells compared to ciprofloxacin alone. No significant effect of D,L-malate on ciprofloxacin activity against P. aeruginosa in C. elegans nematodes was observed. Overall, these data indicate that the outcome of the experiment is influenced by the model system used which emphasizes the importance of using models that reflect the in vivo environment as closely as possible. Nevertheless, this study confirms the potential of D,L-malate to enhance ciprofloxacin activity against P. aeruginosa-associated infections.

Age-related flexibility of energetic metabolism in the honey bee Apis mellifera.

The mechanisms that underpin aging are still elusive. In this study, we suggest that the ability of mitochondria to oxidize different substrates, which is known as metabolic flexibility, is involved in this process. To verify our hypothesis, we used honey bees (Apis mellifera carnica) at different ages, to assess mitochondrial oxygen consumption and enzymatic activities of key enzymes of the energetic metabolism as well as ATP5A1 content (subunit of ATP synthase) and adenylic energy charge (AEC). We also measured mRNA abundance of genes involved in mitochondrial functions and the antioxidant system. Our results demonstrated that mitochondrial respiration increased with age and favored respiration through complexes I and II of the electron transport system (ETS) while glycerol-3-phosphate (G3P) oxidation was relatively decreased. In addition, glycolytic, tricarboxylic acid cycle and ETS enzymatic activities increased, which was associated with higher ATP5A1 content and AEC. Furthermore, we detected an early decrease in the mRNA abundance of subunits of NADH ubiquinone oxidoreductase subunit B2 (NDUFB2, complex I), mitochondrial cytochrome b (CYTB, complex III) of the ETS as well as superoxide dismutase 1 and a later decrease for vitellogenin, catalase and mitochondrial cytochrome c oxidase subunit 1 (COX1, complex IV). Thus, our study suggests that the energetic metabolism is optimized with aging in honey bees, mainly through quantitative and qualitative mitochondrial changes, rather than showing signs of senescence. Moreover, aging modulated metabolic flexibility, which might reflect an underpinning mechanism that explains lifespan disparities between the different castes of worker bees.

Glucose and redox metabolism in meiotically blocked in vitro grown mouse antral follicles.

Glucose and redox metabolism characterization in mouse antral follicles with meiotically blocked oocytes, after in vitro follicle culture (IFC) from the early secondary stage. Following IFC (10 days), oocytes, corresponding cumulus (CC), and granulosa cells (GC) were collected from antral follicles: (i) on day 9-immature, germinal vesicle (GV) stage; (ii) on day 10, after hCG/EGF stimulation-mature, metaphase II (MII) stage and meiotically blocked (MB) immature GV stage. The metabolic profiles of all samples (GV, MII, and MB) were compared by measuring changes in metabolites involved in glycolysis, tricarboxylic acid (TCA) cycle, pentose phosphate pathway (PPP), and redox activity via enzymatic spectrophotometric assays in each cell type. Within MB follicles, GCs drive higher levels of glycolysis and lactic acid fermentation (LAF) while oocytes exert more PPP activity. MB-oocytes had significantly larger diameters compared to day 9 GVs. MB follicles revealed limited metabolic changes in the somatic compartment compared to their GV counterparts (before stimulation). MB-CCs showed increased aconitase and glucose-6-phosphate dehydrogenase activities with lower malate levels comparted to GV-CCs. MB and MII in vitro grown follicles displayed comparable metabolic profiles, suggesting culture induces metabolic exhaustion regardless of the maturation stage. Current results suggest that in addition to impaired nuclear maturation, metabolic disruption is present in MB follicles. MB follicles either compensate with high levels of TCA cycle and PPP activities in CCs, or are unable to drive proper levels of aerobic metabolism, which might be due to the current culture conditions.

Hyperpolarized [1-13C]Acetyl-l-Carnitine Probes Tricarboxylic Acid Cycle Activity In Vivo.

Mitochondrial oxidative phosphorylation (OXPHOS) is sensitive to a variety of biological factors, and dysregulated OXPHOS is observed during the development of numerous pathological conditions. ATP production via OXPHOS is intrinsically dependent on the availability of acetyl-coenzyme A (CoA), which can enter the tricarboxylic acid (TCA) cycle to drive the oxidative pathway. Acetyl-l-carnitine (ALCAR) is an interchangeable endogenous source of acetyl-CoA, and therefore, ALCAR-derived probes are uniquely positioned for the assessment of OXPHOS. In this report, we develop hyperpolarized (HP) [1-13C]ALCAR as a noninvasive probe to investigate cardiac TCA cycle activity in vivo. We initially synthesized the isotopically labeled substrate and demonstrated that the 13C nucleus maintained a suitable T1 value (50.1 ± 0.8 s at 3 T) and polarization levels (21.3 ± 5.3%) to execute in vivo metabolic measurements. HP [1-13C]ALCAR was employed for cardiac analyses of OXPHOS in rats under fed and fasted conditions. [5-13C]Glutamate was successfully detected, and the metabolite was used to analyze the TCA cycle activity in both nutritional states. These assessments were compared to analogous experiments with the HP [1-13C]pyruvate. Our report represents the first study to demonstrate that HP methods using [1-13C]ALCAR enable direct analyses of mitochondrial function and TCA cycle activity, which are fundamental to cardiac cell homeostasis.

Aberrant metabolite trafficking and fuel sensitivity in human pluripotent stem cell-derived islets

Pancreatic islets regulate blood glucose homeostasis through the controlled release of insulin; however, current metabolic models of glucose-sensitive insulin secretion are incomplete. A comprehensive understanding of islet metabolism is integral to studies of endocrine cell development as well as diabetic islet dysfunction. Human pluripotent stem cell-derived islets (SC-islets) are a developmentally relevant model of human islet function that have great potential in providing a cure for type 1 diabetes. Using multiple 13C-labeled metabolic fuels, we demonstrate that SC-islets show numerous divergent patterns of metabolite trafficking in proposed insulin release pathways compared with primary human islets but are still reliant on mitochondrial aerobic metabolism to derive function. Furthermore, reductive tricarboxylic acid cycle activity and glycolytic metabolite cycling occur in SC-islets, suggesting that non-canonical coupling factors are also present. In aggregate, we show that many facets of SC-islet metabolism overlap with those of primary islets, albeit with a retained immature signature.

Designing glucose utilization "highway" for recombinant biosynthesis

cAMP receptor protein (CRP) is known as a global regulatory factor mainly mediating carbon source catabolism. Herein, we successfully engineered CRP to develop microbial chassis cells with improved recombinant biosynthetic capability in minimal medium with glucose as single carbon source. The obtained best-performing cAMP-independent CRPmu9 mutant conferred both faster cell growth and a 133-fold improvement in expression level of lac promoter in presence of 2% glucose, compared with strain under regulation of CRPwild-type. Promoters free from “glucose repression” are advantageous for recombinant expression, as glucose is a frequently used inexpensive carbon source in high-cell-density fermentations. Transcriptome analysis demonstrated that the CRP mutant globally rewired cell metabolism, displaying elevated tricarboxylic acid cycle activity; reduced acetate formation; increased nucleotide biosynthesis; and improved ATP synthesis, tolerance, and stress-resistance activity. Metabolites analysis confirmed the enhancement of glucose utilization with the upregulation of glycolysis and glyoxylate-tricarboxylic acid cycle. As expected, an elevated biosynthetic capability was demonstrated with vanillin, naringenin and caffeic acid biosynthesis in strains regulated by CRPmu9. This study has expanded the significance of CRP optimization into glucose utilization and recombinant biosynthesis, beyond the conventionally designated carbon source utilization other than glucose. The Escherichiacoli cell regulated by CRPmu9 can be potentially used as a beneficial chassis for recombinant biosynthesis.

Gp05, a Prophage-Encoded Virulence Factor, Contributes to Persistent Methicillin-Resistant Staphylococcus aureus Endovascular Infection.

Persistent methicillin-resistant Staphylococcus aureus (MRSA) endovascular infections represent a serious public health threat. We recently demonstrated that the presence of a novel prophage ϕSA169 was associated with vancomycin (VAN) treatment failure in experimental MRSA endocarditis. In this study, we assessed the role of a ϕSA169 gene, ϕ80α_gp05 (gp05), in VAN-persistent outcome using gp05 isogenic MRSA strain sets. Of note, Gp05 significantly influences the intersection of MRSA virulence factors, host immune responses, and antibiotic treatment efficacy, including the following: (i) activity of the significant energy-yielding metabolic pathway (e.g., tricarboxylic acid cycle); (ii) carotenoid pigment production; (iii) (p)ppGpp (guanosine tetra- and pentaphosphate) production, which activates the stringent response and subsequent downstream functional factors (e.g., phenol-soluble modulins and polymorphonuclear neutrophil bactericidal activity); and (iv) persistence to VAN treatment in an experimental infective endocarditis model. These data suggest that Gp05 is a significant virulence factor which contributes to the persistent outcomes in MRSA endovascular infection by multiple pathways. IMPORTANCE Persistent endovascular infections are often caused by MRSA strains that are susceptible to anti-MRSA antibiotics in vitro by CLSI breakpoints. Thus, the persistent outcome represents a unique variant of traditional antibiotic resistance mechanisms and a significant therapeutic challenge. Prophage, a critical mobile genetic element carried by most MRSA isolates, provides their bacterial host with metabolic advantages and resistance mechanisms. However, how prophage-encoded virulence factors interact with the host defense system and antibiotics, driving the persistent outcome, is not well known. In the current study, we demonstrated that a novel prophage gene, gp05, significantly impacts tricarboxylic acid cycle activity, stringent response, and pigmentation, as well as vancomycin treatment outcome in an experimental endocarditis model using isogenic gp05 overexpression and chromosomal deletion mutant MRSA strain sets. The findings significantly advance our understanding of the role of Gp05 in persistent MRSA endovascular infection and provide a potential target for development of novel drugs against these life-threatening infections.

EZH2 mediated metabolic rewiring promotes tumor growth independently of histone methyltransferase activity in ovarian cancer

BackgroundEnhancer of zeste homolog 2 (EZH2), the key catalytic subunit of polycomb repressive complex 2 (PRC2), is overexpressed and plays an oncogenic role in various cancers through catalysis-dependent or catalysis-independent pathways. However, the related mechanisms contributing to ovarian cancer (OC) are not well understood.MethodsThe levels of EZH2 and H3K27me3 were evaluated in 105 OC patients by immunohistochemistry (IHC) staining, and these patients were stratified based on these levels. Canonical and noncanonical binding sites of EZH2 were defined by chromatin immunoprecipitation sequencing (ChIP-Seq). The EZH2 solo targets were obtained by integrative analysis of ChIP-Seq and RNA sequencing data. In vitro and in vivo experiments were performed to determine the role of EZH2 in OC growth.ResultsWe showed that a subgroup of OC patients with high EZH2 expression but low H3K27me3 exhibited the worst prognosis, with limited therapeutic options. We demonstrated that induction of EZH2 degradation but not catalytic inhibition profoundly blocked OC cell proliferation and tumorigenicity in vitro and in vivo. Integrative analysis of genome-wide chromatin and transcriptome profiles revealed extensive EZH2 occupancy not only at genomic loci marked by H3K27me3 but also at promoters independent of PRC2, indicating a noncanonical role of EZH2 in OC. Mechanistically, EZH2 transcriptionally upregulated IDH2 to potentiate metabolic rewiring by enhancing tricarboxylic acid cycle (TCA cycle) activity, which contributed to the growth of OC.ConclusionsThese data reveal a novel oncogenic role of EZH2 in OC and identify potential therapeutic strategies for OC by targeting the noncatalytic activity of EZH2.

Heat-treated Pediococcus acidilactici LM1013-mediated inhibition of biofilm formation by Cutibacterium acnes and its application in acne vulgaris: A single-arm clinical trial.

Acne vulgaris is a common skin disease accompanied by chronic inflammation in the pilosebaceous follicles, resulting from excessive Cutibacterium acnes. This study aimed to investigate the inhibition of biofilm formation by C. acnes ATCC 6919 using heat-treated Pediococcus acidilactici LM1013 (HT-LM1013), previously isolated from the Korean traditional fermented alcoholic beverage-makgeolli, and its application as a leave-on-type product for patients with acne vulgaris. HT-LM1013 was prepared by Lactomason and homogenized using a high-pressure homogenizer. The minimum inhibitory concentration (MIC), tricarboxylic acid (TCA) cycle, and lipase activity were evaluated for C. acnes inhibition. Inhibition of biofilm formation was demonstrated using a crystal violet solution. Damaged C. acnes was observed using field-emission scanning electron microscopy (FE-SEM). Clinical trials were performed using a leave-on-type product containing HT-LM1013. HT-LM1013 inhibited the TCA cycle (36.80%) and lipase activity using palmitate (31.89%), stearate (36.91%), and oleate (30.86%) as substrates at 1 × MIC (p < 0.01). After treatment with HT-LM1013, concave and elongated shapes of C. acnes were observed by FE-SEM. In addition, HT-LM1013 inhibited biofilm formation by 71.75% at 1 × MIC (p < 0.001) and removed 73.35% of mature biofilms (p < 0.01). In the clinical trial, the leave-on-type product decreased the number of closed comedones from 14.04 to 10.22, open comedones from 7.22 to 4.39%, and sebum content to 76.23% at week 4 (p < 0.01). The satisfaction score of the participants was recorded 3.83 on a five-point scale. HT-LM1013 is potent for the treatment of acne vulgaris.