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BACKGROUND: Gout is a common kind of inflammatory arthritis with metabolic disorders. However, the detailed pathogenesis of gout is complex and not fully clear. We investigated the urine metabolic profiling of gout patients by ultra-performance liquid chromatograph quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). METHOD: Urine metabolites were extracted from 26 acute gout patients, 31 chronic gout patients, and 32 healthy controls. Metabolite extracts were analyzed by UPLC-Q-TOF-MS for untargeted metabolomics. The peak area of creatinine was used to correct the content variations of urine samples for the semi-quantitative analysis. The value of variable importance in the projection (VIP) was obtained through the orthogonal partial least squares-discrimination analysis (OPLS-DA), and several differential metabolites were screened out. RESULTS: The potential metabolic markers of gout in different stages were found based on the t-test. Finally, 18 different metabolites were identified through Human Metabolome Database (HMDB) and Targeted-MS/MS. The receiver operating characteristic (ROC) curve results revealed that all the screened biomarkers exerted high accuracy and diagnostic value. Pathway analysis indicated that the significantly different metabolites were mainly involved in purine metabolism and amino acid metabolism. CONCLUSION: The identified potential biomarkers are mainly involved in purine metabolism and amino acid metabolism, which leads us to further explore the pathogenesis of gout. This will lead us to further explore the pathogenesis of gout and provide the basis and ideas for the prevention and treatment of gout.

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Purine metabolism acts as the core role in human metabolic network. It offers purine metabolites as raw material for building blocks in cell survival and proliferation. Purine metabolites are the most abundant metabolic substrates in organisms. There are few reports to simultaneously quantify canonical purine metabolism in cells. A novel hydrophilic interaction liquid chromatography coupled with mass spectrometry (HILIC-MS/MS) method was developed to simultaneously determine purines profile in biological samples. Chromatographic separation was achieved using a HILIC (Waters Xbridge™ Amide) column. Different optimizing chromatographic conditions and mass spectrometric parameters were tested in order to provide the best separation and the lowest limit of quantification (LLOQ) values for targeted metabolites. The validation was evaluated according to the Food and Drug Administration guidelines. The limit of determination (LOD) and the LOQ values were in the range of 0.02-8.33 ng mL-1 and 0.1-24.5 ng mL-1, respectively. All calibration curves displayed good linear relationship of with excellent correlation coefficient (r) ranging from 0.9943 to 0.9999. Both intra-day and inter-day variability were below 15 %, respectively. Trueness, expressed as relative error, was always within ±15 %. In addition, no derivatization procedure and ion-pair reagents are in need. The innovated approach demonstrates high sensitivity, strong specificity, and good repeatability, making it suitable for absolute quantitative studies of canonical purine metabolism in cultured cells.

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Release of radionuclides to the environment from either nuclear weapon and fuel cycles or from naturally occurring radionuclides (NORM) may cause long term contamination of aquatic ecosystems and chronic exposure of living organisms to ionizing radiation, which in turn could lead to adverse effects compromising the sustainability of populations. To address the effects of chronic ionizing radiation on the development of fish, Atlantic salmon embryos were exposed from fertilization until hatching (88 days, 550 day-degree) to dose rates from 1 to 30 mGy·h-1 gamma radiation (60Co). The lowest adopted dose rate was similar to the highest doses measured in some water bodies right after the Chernobyl accident (1 mGy·h-1), however, well above current environmentally realistic scenarios (20 µGy·h-1), or the threshold assumed for significant effects on fish population (40 µGy·h-1). Dose dependent effects were observed on survival, hatching, morbidity, DNA damage, antioxidant defenses, and metabolic status. Histopathological analysis showed dose rate dependent impairment of eye and brain tissues development and establishment of epidermal mucus cell layers accompanied by increased DNA damage at doses ≥1.3 Gy (dose rates ≥1 mGy·h-1). At ≥32.8 Gy (dose rates ≥20 mGy·h-1) deformities and developmental growth defects resulted in respective 46 and 95 % pre-hatch mortality. The 10 mGy·h-1 exposure (≥ 12 Gy total dose) caused significantly increased DNA damage, impaired eye development, and both premature and delayed hatching, while no deformities or effect on survival were observed. We observed a dose rate dependent reduction from dose rate ≥ 20 mGy·h-1 (≥ 27 Gy total dose) on antioxidant SOD, catalase and glutathione reductase enzyme activities. The reduction of antioxidant enzyme activities was in line with observed developmental delay and disturbance to time of hatching. Metabolomic profiles showed a clear shift at dose rates ≥10 mGy·h-1 (≥ 12 Gy total dose) in pathways related to oxidative stress, detoxification, DNA damage and repair. Due to gamma radiation exposure, a switch of central metabolism from glycolysis, citric acid cycle and lactate production towards pentose phosphate pathway indicated a rewiring mechanism for increased production of reductive equivalents to maintain redox homeostasis at the expense of energy output and thus embryonic development.

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Rationale: Dysregulated T-cell immune response-mediated inflammation plays critical roles in the pathology of diverse liver diseases, but the underlying mechanism of liver immune homeostasis control and the specific therapies for limiting T-cell overactivation remain unclear. Methods: The metabolic changes in concanavalin A (ConA) mice and autoimmune hepatitis (AIH) patients and their associations with liver injury were analyzed. The expression of purine catabolism nucleases (e.g., CD39 and CD73) on liver cells and immune cells was assessed. The effects of MCregs and their extracellular vesicles (EVs) on CD4+ T-cell overactivation and the underlying mechanism were also explored. Results: Our findings revealed significant alterations in purine metabolism in ConA mice and AIH patients, which correlated with liver injury severity and therapeutic response. CD39 and CD73 were markedly upregulated on CD11b+Gr-1+ MCs under liver injury conditions. The naturally expanded CD39+CD73+Gr-1highCD11b+ MCreg subset during early liver injury effectively suppressed CD4+ T-cell hyperactivation and liver injury both in vitro and in vivo. Mechanistically, MCregs released CD73high EVs, which converted extracellular AMP to immunosuppressive metabolites (e.g., adenosine and inosine), activating the cAMP pathway and inhibiting glycolysis and cytokine secretion in activated CD4+ T cells. Conclusions: This study provides insights into the mechanism controlling immune homeostasis during the early liver injury phase and highlights that MCreg or MCreg-EV therapy may be a specific strategy for preventing diverse liver diseases induced by T-cell overactivation.

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Vesículas Extracelulares , Hepatitis Autoinmune , Ratones Endogámicos C57BL , Purinas , Animales , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/inmunología , Ratones , Purinas/metabolismo , Hepatitis Autoinmune/inmunología , Hepatitis Autoinmune/metabolismo , Hepatitis Autoinmune/patología , Humanos , Apirasa/metabolismo , Hígado/metabolismo , Hígado/inmunología , Hígado/patología , Células Mieloides/metabolismo , Células Mieloides/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Masculino , 5'-Nucleotidasa/metabolismo , Activación de Linfocitos/inmunología , Concanavalina A , Femenino , Modelos Animales de Enfermedad , Inflamación/metabolismo , Inflamación/inmunología , Antígenos CD

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BACKGROUND: Glioblastoma cells preferentially use de-novo purine synthesis pathway, whereas normal brain prefers salvage pathway. Mycophenolate mofetil (MMF), a commonly used oral immunosuppressant that inhibits inosine-5'-monophosphate dehydrogenase (IMPDH), a key enzyme in the de-novo purine pathway. Pre-clinical suggested MMF can improve radiation and temozolomide efficacy in glioblastoma which led to this phase 0/1 trial (NCT04477200) to assess MMF's tolerability with chemoradiation in glioblastoma, mycophenolic acid accumulation, and purine synthesis inhibition in tumor. METHODS: In the phase 0 study, eight recurrent glioblastoma patients received MMF at doses ranging 500-2,000 mg BID for 1-week before surgery. The tissues were analyzed using mass spectrometry for drug accumulation and purine synthesis inhibition. In the phase 1 study, adult patients were given MMF starting at 1,000 mg orally (PO) twice daily (BID), with the possible dose ranging 500-2,000 PO BID. Nineteen recurrent glioblastoma patients (target N=30) received MMF 1-week prior to and concurrently with re-irradiation (40.5 Gy). Thirty newly diagnosed glioblastoma patients received MMF 1-week prior to and concurrently with chemoradiation, followed by MMF 1-day before and during 5 days of each adjuvant temozolomide cycle. RESULTS: Both enhancing and non-enhancing tumors from phase 0 subjects yielded >1 µM active drug metabolite, and the guanosine triphosphate: inosine monophosphate ratio was decreased by 75% in enhancing tumors in MMF-treated patients compared to untreated controls (P=0.009), indicating effective target engagement and inhibition of purine synthesis. In the phase 1 study, no dose-limiting toxicities (DLTs) were observed at the interim analysis at MMF 1,000-1,500 mg BID combined with chemoradiation. At 2,000 mg BID, there was no DLT combined with temozolomide alone, however, there were four DLTs noted (hemiparesis, cognitive disturbance, fatigue, thrombocytopenia) when combined with radiotherapy and temozolomide together, though all were reversible. Interim median overall survival in recurrent phase 1 is 15.6 months, and not reached yet in newly diagnosed phase 1. CONCLUSIONS: MMF with chemoradiation has been reasonably well tolerated and showed promising evidence of brain tumor target engagement and drug accumulation. This study led to a recommended phase 2 dose of MMF 1,500 mg BID and will provide a preliminary efficacy estimate for a randomized phase 2/3 trial through the Alliance for Clinical Trials in Oncology.

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Quimioradioterapia , Glioblastoma , Purinas , Humanos , Glioblastoma/tratamiento farmacológico , Masculino , Femenino , Persona de Mediana Edad , Adulto , Quimioradioterapia/métodos , Purinas/farmacología , Purinas/uso terapéutico , Anciano , Recurrencia Local de Neoplasia , Neoplasias Encefálicas/tratamiento farmacológico

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Pentachlorophenol (PCP) was used widely as preservative and biocide and has been banned due to with various harmful effects, such as carcinogenicity and teratogenicity. However, the effects of PCP on colitis induced by dextrose sodium sulfate (DSS) remain largely unknown. Serum metabolomics and gut microbiota were investigated to elucidate the underlying mechanisms. Exposure to 20 µg/L PCP aggravated DSS-induced body weight loss, colon shortening, severe histological injuries, and upregulation of TNFα, iNOS, IL-1ß, and IL-6. Serum metabolomics showed that both DSS and PCP could significantly disrupted tryptophan metabolism in normal mice. Interestingly, PCP exposure intensified the disturbance in purine metabolism but not tryptophan metabolism caused by DSS. Quantitative analysis of tryptophan and metabolites further confirmed that PCP exposure significantly increased the serum contents of serotonin, adenine, guanine, guanosine, inosine monophosphate (IMP), inosine, and hypoxanthine in DSS-treated mice. The overall gut microbial community was significantly modified by PCP and DSS treatment alone. Rikenellaceae_RC9_Gut_group, Colidextribacter, and Desulfovibrio were more abundant in colitis mice following PCP exposure. Further integrative analysis of differential bacteria and purine metabolites highlighted a significant correlation between Desulfovibrio and several purine metabolites, including guanine, guanosine, hypoxanthine, IMP, and inosine. Adenosine ribonucleotides de novo biosynthesis, inosine-5'-phosphate biosynthesis I, and urate biosynthesis/inosine 5'-phosphate degradation pathways were depleted in colitis mice upon PCP treatment. Taken together, PCP exposure delayed the recovery of colitis induced by DSS in association with altered gut microbiota and serum metabolites, which were enriched in tryptophan and purine metabolism.

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This study aimed to identify metabolic alterations in the small intestine of newborn rats with intrauterine growth restriction (IUGR), a condition linked to intestinal dysfunction. Pregnant Sprague Dawley rats underwent bilateral uterine artery ligation on gestational day 17 to induce intrauterine growth restriction or sham surgery. Rat pups were delivered spontaneously on gestational day 22. Small intestine tissues were collected on postnatal days 0 and 7 from offspring. Liquid chromatography-mass spectrometry analysis was performed to investigate untargeted metabolomic profiles. Western blot analysis assessed protein expression of key regulators. Newborn rats with intrauterine growth restriction exhibited distinct small intestine metabolic profiles compared to controls on postnatal day 0. Notably, significant alterations were observed in purine metabolism, the pentose phosphate pathway, and related pathways. Western blot analysis revealed a decrease expression in transketolase, a key enzyme of the pentose phosphate pathway, suggesting impaired activity of the pentose phosphate pathway. Additionally, decreased expression of tight junction proteins ZO-1 and occludin indicated compromised intestinal barrier function in rats with intrauterine growth restriction. Similar metabolic disruptions persisted on postnatal day 7, with further reductions in tricarboxylic acid cycle intermediates and folate biosynthesis precursors. Interestingly, lysyl-glycine, a protein synthesis marker, was elevated in rats with intrauterine growth restriction. Our findings reveal a distinct metabolic signature in the small intestine of neonatal rats with intrauterine growth restriction, characterized by disruptions in the pentose phosphate pathway, purine metabolism, and energy production pathways. These novel insights suggest potential mechanisms underlying IUGR-associated intestinal dysfunction and impaired growth.

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Glioblastoma (GB) is a lethal brain tumor that rapidly adapts to the dynamic changes of the tumor microenvironment (TME). Mesenchymal stem/stromal cells (MSCs) are one of the stromal components of the TME playing multiple roles in tumor progression. GB progression is prompted by the immunosuppressive microenvironment characterized by high concentrations of the nucleoside adenosine (ADO). ADO acts as a signaling molecule through adenosine receptors (ARs) but also as a genetic and metabolic regulator. Herein, the effects of high extracellular ADO concentrations were investigated in a human glioblastoma cellular model (U343MG) and MSCs. The modulation of the purinome machinery, i.e., the ADO production (CD39, CD73, and adenosine kinase [ADK]), transport (equilibrative nucleoside transporters 1 (ENT1) and 2 (ENT2)), and degradation (adenosine deaminase [ADA]) were investigated in both cell lines to evaluate if ADO could affect its cell management in a positive or negative feed-back loop. Results evidenced a different behavior of GB and MSC cells upon exposure to high extracellular ADO levels: U343MG were less sensitive to the ADO concentration and only a slight increase in ADK and ENT1 was evidenced. Conversely, in MSCs, the high extracellular ADO levels reduced the ADK, ENT1, and ENT2 expression, which further sustained the increase of extracellular ADO. Of note, MSCs primed with the GB-conditioned medium or co-cultured with U343MG cells were not affected by the increase of extracellular ADO. These results evidenced how long exposure to ADO could produce different effects on cancer cells with respect to MSCs, revealing a negative feedback loop that can support the GB immunosuppressive microenvironment. These results improve the knowledge of the ADO role in the maintenance of TME, which should be considered in the development of therapeutic strategies targeting adenosine pathways as well as cell-based strategies using MSCs.

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Selenium is a trace element that plays critical roles in redox biology; it is typically incorporated into "selenoproteins" as the 21st amino acid selenocysteine. Additionally, selenium exists as a labile non-selenocysteine cofactor in a small subset of selenoproteins known as selenium-dependent molybdenum hydroxylases (SDMHs). In purinolytic clostridia, SDMHs are implicated in the degradation of hypoxanthine, xanthine, and uric acid for carbon and nitrogen. While SDMHs have been biochemically analyzed, the genes responsible for the insertion and maturation of the selenium cofactor lack characterization. In this study, we utilized the nosocomial pathogen Clostridioides difficile as a genetic model to begin characterizing this poorly understood selenium utilization pathway and its role in the catabolism of host-derived purines. We first observed that C. difficile could utilize hypoxanthine, xanthine, or uric acid to overcome a growth defect in a minimal medium devoid of glycine and threonine. However, strains lacking selenophosphate synthetase (selD mutants) still grew poorly in the presence of xanthine and uric acid, suggesting a selenium-dependent purinolytic process. Previous computational studies have identified yqeB and yqeC as potential candidates for cofactor maturation, so we subsequently deleted each gene using CRISPR-Cas9 technology. We surprisingly found that the growth of the ΔyqeB mutant in response to each purine was similar to the behavior of the selD mutants, while the ΔyqeC mutant exhibited no obvious phenotype. Our results suggest an important role for YqeB in selenium-dependent purine catabolism and also showcase C. difficile as an appropriate model organism to study the biological use of selenium.IMPORTANCEThe apparent modification of bacterial molybdenum hydroxylases with a catalytically essential selenium cofactor is the least understood mechanism of selenium incorporation. Selenium-dependent molybdenum hydroxylases play an important role in scavenging carbon and nitrogen from purines for purinolytic clostridia. Here, we used Clostridioides difficile as a genetic platform to begin dissecting the selenium cofactor trait and found genetic evidence for a selenium-dependent purinolytic pathway. The absence of selD or yqeB-a predicted genetic marker for the selenium cofactor trait-resulted in impaired growth on xanthine and uric acid, known substrates for selenium-dependent molybdenum hydroxylases. Our findings provide a genetic foundation for future research of this pathway and suggest a novel metabolic strategy for C. difficile to scavenge host-derived purines from the gut.

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Heat stress is a prevalent factor that significantly damages crops, especially with the ongoing global warming and increasing frequency of extreme weather events. Tobacco is particularly sensitive to temperature fluctuations, experiencing reduced yield and quality under high temperatures. However, the underlying molecular mechanisms of heat resistance in tobacco remain poorly understood. This study comprehensively analyzed biochemical, transcriptomic, and metabolomic responses to heat stress on the root and shoot of the tobacco cultivar K326 compared to control conditions. Heat stress significantly increased the activities of antioxidant enzymes (CAT, POD, and SOD) and levels of osmotic mediators (soluble sugars, sucrose, and proline) in the shoot. Furthermore, transcriptome analysis identified 13,176 differentially expressed genes (DEGs) in the root (6,129 up-regulated and 7,047 down-regulated) and 12,283 DEGs (6,621 up-regulated and 5,662 down-regulated) in the shoot. The root had 24 enriched KEGG pathways, including phenylpropanoid metabolism, while the shoot had 32 significant pathways, such as galactose metabolism and MAPK signaling. The metabolomic data identified 647 metabolites in the root and 932 in the shoot, with carbohydrates and amino acids being the main categories. The root had 116 differentially abundant metabolites (DAMs) (107 up-regulated and 9 down-regulated), and the shoot contained 256 DAMs (251 up-regulated and 5 down-regulated). Joint transcriptome and metabolome analysis showed that galactose metabolism and starch and sucrose metabolism were co-enriched in both tissues. In contrast, amino sugar and nucleotide sugar metabolism was enriched in the root, and purine metabolism in the shoot. The purine metabolic pathway in the shoot can modulate the expression of MYB transcription factors by influencing ABA synthesis and signaling, thereby controlling the accumulation of HSPs, raffinose, sucrose, and trehalose to enhance heat tolerance. Furthermore, NtMYB78, an MYB transcription factor, enhances tolerance for heat stress in tobacco. This research offers a foundational framework for investigating and implementing heat-resistant genes and metabolic pathways in the root and shoot of tobacco seedlings.

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Exploring the landscape of protein phosphorylation, this investigation focuses on skin samples from LCG (Liaoning Cashmere Goats), characterized by different levels of cashmere fineness. Employing LC-MS/MS technology, we meticulously scrutinized FT-LCG (fine-type Liaoning Cashmere Goats) and CT-LCG (coarse-type Liaoning Cashmere Goats). Identifying 512 modified proteins, encompassing 1368 phosphorylated peptide segments and 1376 quantifiable phosphorylation sites, our exploration further revealed consistent phosphorylation sites in both groups. Analysis of phosphorylated peptides unveiled kinase substrates, prominently featuring Protein Kinase C, Protein Kinase B and MAPK3-MAPK1-MAPK7-NLK-group. Differential analysis spotlighted 28 disparate proteins, comprising six upregulated and twenty-two downregulated. Cluster analysis showcased the robust clustering efficacy of the two sample groups. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses underscored the significance of the purine metabolism pathway, suggesting its pivotal role in modulating cashmere fineness in LCG. Notably, through differential protein analysis, two crucial proteins were identified: HSL-X (hormone-sensitive lipase isoform X1) and KPRP (keratinocyte proline-rich protein). Further evidence supports LIPE and KPRP as key genes regulating cashmere fineness, paving the way for promising avenues in further research. These findings not only contribute to a nuanced understanding of protein-level dynamics in cashmere but also provide a theoretical foundation for the selective breeding of superior Liaoning Cashmere Goat strands.

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Salmonella enterica serovar Typhimurium (S. Typhimurium) infection triggers an inflammatory response that changes the concentration of metabolites in the gut impacting the luminal environment. Some of these environmental adjustments are conducive to S. Typhimurium growth, such as the increased concentrations of nitrate and tetrathionate or the reduced levels of Clostridia-produced butyrate. We recently demonstrated that S. Typhimurium can form biofilms within the host environment and respond to nitrate as a signaling molecule, enabling it to transition between sessile and planktonic states. To investigate whether S. Typhimurium utilizes additional metabolites to regulate its behavior, our study delved into the impact of inflammatory metabolites on biofilm formation. The results revealed that lactate, the most prevalent metabolite in the inflammatory environment, impedes biofilm development by reducing intracellular c-di-GMP levels, suppressing the expression of curli and cellulose, and increasing the expression of flagellar genes. A transcriptomic analysis determined that the expression of the de novo purine pathway increases during high lactate conditions, and a transposon mutagenesis genetic screen identified that PurA and PurG, in particular, play a significant role in the inhibition of curli expression and biofilm formation. Lactate also increases the transcription of the type III secretion system genes involved in tissue invasion. Finally, we show that the pyruvate-modulated two-component system BtsSR is activated in the presence of high lactate, which suggests that lactate-derived pyruvate activates BtsSR system after being exported from the cytosol. All these findings propose that lactate is an important inflammatory metabolite used by S. Typhimurium to transition from a biofilm to a motile state and fine-tune its virulence.IMPORTANCEWhen colonizing the gut, Salmonella enterica serovar Typhimurium (S. Typhimurium) adopts a dynamic lifestyle that alternates between a virulent planktonic state and a multicellular biofilm state. The coexistence of biofilm formers and planktonic S. Typhimurium in the gut suggests the presence of regulatory mechanisms that control planktonic-to-sessile transition. The signals triggering the transition of S. Typhimurium between these two lifestyles are not fully explored. In this work, we demonstrated that in the presence of lactate, the most dominant host-derived metabolite in the inflamed gut, there is a reduction of c-di-GMP in S. Typhimurium, which subsequently inhibits biofilm formation and induces the expression of its invasion machinery, motility genes, and de novo purine metabolic pathway genes. Furthermore, high levels of lactate activate the BtsSR two-component system. Collectively, this work presents new insights toward the comprehension of host metabolism and gut microenvironment roles in the regulation of S. Typhimurium biology during infection.

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Wound rehabilitation is invariably time-consuming, scar formation further weakens therapeutic efficacy, and detailed mechanisms at the molecular level remain unclear. In this work, a Mo4/3B2-x nanoscaffold was fabricated and utilized for wound healing and scar removing in a mice model, while metabolomics was used to study the metabolic reprogramming of metabolome during therapy at the molecular level. The results showed that transition metal borides, called Mo4/3B2-x nanoscaffolds, could mimic superoxide dismutase and glutathione peroxidase to eliminate excess reactive oxygen species (ROS) in the wound microenvironment. During the therapeutic process, the Mo4/3B2-x nanoscaffold could facilitate the regeneration of wounds and removal of scars by regulating the biosynthesis of collagen, fibers, and blood vessels at the pathological, imaging, and molecular levels. Subsequent metabolomics study revealed that the Mo4/3B2-x nanoscaffold effectively ameliorated metabolic disorders in both wound and scar microenvironments through regulating ROS-related pathways including the amino acid metabolic process (including glycine and serine metabolism and glutamate metabolism) and the purine metabolic process. This study is anticipated to illuminate the potential clinical application of the Mo4/3B2-x nanoscaffold as an effective therapeutic agent in traumatic diseases and provide insights into the development of analytical methodology for interrogating wound healing and scar removal-related metabolic mechanisms.

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Aminoácidos , Cicatriz , Purinas , Cicatrización de Heridas , Animales , Cicatrización de Heridas/efectos de los fármacos , Cicatriz/metabolismo , Cicatriz/patología , Cicatriz/tratamiento farmacológico , Ratones , Aminoácidos/química , Aminoácidos/metabolismo , Purinas/química , Purinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Masculino

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Previous studies suggest that uric acid or reactive oxygen species, products of xanthine oxidoreductase (XOR), may associate with neurodegenerative diseases. However, neither relationship has ever been firmly established. Here, we analyzed human brain samples, obtained under protocols approved by research ethics committees, and found no expression of XOR and only low levels of uric acid in various regions of the brain. In the absence of XOR, hypoxanthine will be preserved and available for incorporation into the purine salvage pathway. To clarify the importance of salvage in the brain, we tested using human-induced pluripotent stem cell-derived neuronal cells. Stable isotope analyses showed that the purine salvage pathway was more effective for ATP synthesis than purine de novo synthesis. Blood uric acid levels were related to the intracellular adenylate pool (ATP + ADP + AMP), and reduced levels of this pool result in lower uric acid levels. XOR inhibitors are related to extracellular hypoxanthine levels available for uptake into the purine salvage pathway by inhibiting the oxidation of hypoxanthine to xanthine and uric acid in various organs where XOR is present and can prevent further decreases in the intracellular adenylate pool under stress. Furthermore, adding precursors of the pentose phosphate pathway enhanced hypoxanthine uptake, indicating that purine salvage is activated by phosphoribosyl pyrophosphate replenishment. These findings resolve previous contradictions regarding XOR products and provide new insights into clinical studies. It is suggested that therapeutic strategies maximizing maintenance of intracellular adenylate levels may effectively treat pathological conditions associated with ischemia and energy depletion.

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Encéfalo , Purinas , Ácido Úrico , Xantina Deshidrogenasa , Humanos , Purinas/metabolismo , Encéfalo/metabolismo , Xantina Deshidrogenasa/metabolismo , Ácido Úrico/metabolismo , Hipoxantina/metabolismo , Masculino , Neuronas/metabolismo , Femenino , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Vía de Pentosa Fosfato , Persona de Mediana Edad , Adenosina Trifosfato/metabolismo , Anciano , Adulto

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Introduction: Colistin (CMS) is used for the curation of infections caused by multidrug-resistant bacteria. CMS is constrained by toxicity, particularly in kidney and neuronal cells. The recommended human doses are 2.5-5 mg/kg/day, and the toxicity is linked to higher doses. So far, the in vivo toxicity studies have used doses even 10-fold higher than human doses. It is essential to investigate the impact of metabolic response of doses, that are comparable to human doses, to identify biomarkers of latent toxicity. The innovation of the current study is the in vivo stimulation of CMS's impact using a range of CMS doses that have never been investigated before, i.e., 1 and 1.5 mg/kg. The 1 and 1.5 mg/kg, administered in mice, correspond to the therapeutic and toxic human doses, based on previous expertise of our team, regarding the human exposure. The study mainly focused on the biochemical impact of CMS on the metabolome, and on the alterations provoked by 50%-fold of dose increase. The main objectives were i) the comprehension of the biochemical changes resulting after CMS administration and ii) from its dose increase; and iii) the determination of dose-related metabolites that could be considered as toxicity monitoring biomarkers. Methods: The in vivo experiment employed two doses of CMS versus a control group treated with normal saline, and samples of plasma, kidney, and liver were analysed with a UPLC-MS-based metabolomics protocol. Both univariate and multivariate statistical approaches (PCA, OPLS-DA, PLS regression, ROC) and pathway analysis were combined for the data interpretation. Results: The results pointed out six dose-responding metabolites (PAA, DA4S, 2,8-DHA, etc.), dysregulation of renal dopamine, and extended perturbations in renal purine metabolism. Also, the study determined altered levels of liver suberylglycine, a metabolite linked to hepatic steatosis. One of the most intriguing findings was the detection of elevated levels of renal xanthine and uric acid, that act as AChE activators, leading to the rapid degradation of acetylcholine. This evidence provides a naïve hypothesis, for the potential association between the CMS induced nephrotoxicity and CMS induced 39 neurotoxicity, that should be further investigated.

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Neural stem/progenitor cells (NSPCs) in specific brain regions require precisely regulated metabolite production during critical development periods. Purines-vital components of DNA, RNA, and energy carriers like ATP and GTP-are crucial metabolites in brain development. Purine levels are tightly controlled through two pathways: de novo synthesis and salvage synthesis. Enzymes driving de novo pathway are assembled into a large multienzyme complex termed the "purinosome." Here, we review purine metabolism and purinosomes as spatiotemporal regulators of neural development. Notably, around postnatal day 0 (P0) during mouse cortical development, purine synthesis transitions from the de novo pathway to the salvage pathway. Inhibiting the de novo pathway affects mTORC1 pathway and leads to specific forebrain malformations. In this review, we also explore the importance of protein-protein interactions of a newly identified NSPC protein-NACHT and WD repeat domain-containing 1 (Nwd1)-in purinosome formation. Reduced Nwd1 expression disrupts purinosome formation, impacting NSPC proliferation and neuronal migration, resulting in periventricular heterotopia. Nwd1 interacts directly with phosphoribosylaminoimidazole-succinocarboxamide synthetase (PAICS), an enzyme involved in de novo purine synthesis. We anticipate this review will be valuable for researchers investigating neural development, purine metabolism, and protein-protein interactions.

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BACKGROUND: The effects of Isopsoralen (ISO) in promoting osteoblast differentiation and inhibiting osteoclast formation are well-established, but the mechanism underlying ISO's improvement of Glucocorticoid- Induced Osteoporosis (GIOP) by regulating metabolism remains unclear. METHODS: This study aims to elucidate the mechanism of ISO treatment for GIOP through non-targeted metabolomics based on ISO's efficacy in GIOP. Initially, we established a GIOP female mouse model and assessed ISO's therapeutic effects using micro-CT detection, biomechanical testing, serum calcium (Ca), and phosphorus (P) level detection, along with histological analyses using hematoxylin and eosin (HE), Masson, and tartrate-resistant acidic phosphatase (TRAP) staining. Subsequently, non-targeted metabolomics was employed to investigate ISO's impact on serum metabolites in GIOP mice. RT-qPCR and Western blot analyses were conducted to measure the levels of enzymes associated with these metabolites. Building on the metabolomic results, we explored the effects of ISO on the cyclic Guanosine Monophosphate (cGMP)/Protein Kinase G (PKG) pathway and its role in mediating osteoblast differentiation. RESULTS: Our findings demonstrate that ISO intervention effectively enhances the bone microarchitecture and strength of GIOP mice. It mitigates pathological damage, such as structural damage in bone trabeculae, reduced collagen fibers, and increased osteoclasts, while improving serum Ca and P levels in GIOP mice. Non-- targeted metabolomics revealed purine metabolism as a common pathway between the Control and GIOP groups, as well as between the ISO high-dose (ISOH) group and the GIOP group. ISO intervention upregulated inosine and adenosine levels, downregulated guanosine monophosphate levels, increased Adenosine Deaminase (ADA) expression, and decreased cGMP-specific 3',5'-cyclic phosphodiesterase (PDE5) expression. Additionally, ISO intervention elevated serum cGMP levels, upregulated PKGI and PKGII expression in bone tissues, as well as the expression of Runt-related transcription factor 2 (Runx2) and Osterix, and increased serum Alkaline Phosphatase (ALP) activity. CONCLUSION: In summary, ISO was able to enhance the bone microstructure and bone strength of GIOP mice and improve their Ca, P, and ALP levels, which may be related to ISO's regulation of purine metabolism and promotion of osteoblast differentiation mediated by the cGMP/PKG pathway. This suggests that ISO is a potential drug for treating GIOP. However, further research is still needed to explore the specific targets and clinical applications of ISO.

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Tumor cells reprogram their metabolism to meet the increased demand for nucleotides and other molecules necessary for growth and proliferation. In fact, cancer cells are characterized by an increased "de novo" synthesis of purine nucleotides. Therefore, it is not surprising that specific enzymes of purine metabolism are the targets of drugs as antineoplastic agents, and a better knowledge of the mechanisms underlying their regulation would be of great help in finding new therapeutic approaches. The mammalian target of the rapamycin (mTOR) signaling pathway, which is often activated in cancer cells, promotes anabolic processes and is a major regulator of cell growth and division. Among the numerous effects exerted by mTOR, noteworthy is its empowerment of the "de novo" synthesis of nucleotides, accomplished by supporting the formation of purinosomes, and by increasing the availability of necessary precursors, such as one-carbon formyl group, bicarbonate and 5-phosphoribosyl-1-pyrophosphate. In this review, we highlight the connection between purine and mitochondrial metabolism, and the bidirectional relation between mTOR signaling and purine synthesis pathways.

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Neoplasias , Purinas , Transducción de Señal , Serina-Treonina Quinasas TOR , Humanos , Neoplasias/metabolismo , Neoplasias/patología , Serina-Treonina Quinasas TOR/metabolismo , Purinas/metabolismo , Animales , Mitocondrias/metabolismo

RESUMEN

Mitochondria house many metabolic pathways required for homeostasis and growth. To explore how human cells respond to mitochondrial dysfunction, we performed metabolomics in fibroblasts from patients with various mitochondrial disorders and cancer cells with electron transport chain (ETC) blockade. These analyses revealed extensive perturbations in purine metabolism, and stable isotope tracing demonstrated that ETC defects suppress de novo purine synthesis while enhancing purine salvage. In human lung cancer, tumors with markers of low oxidative mitochondrial metabolism exhibit enhanced expression of the salvage enzyme hypoxanthine phosphoribosyl transferase 1 (HPRT1) and high levels of the HPRT1 product inosine monophosphate. Mechanistically, ETC blockade activates the pentose phosphate pathway, providing phosphoribosyl diphosphate to drive purine salvage supplied by uptake of extracellular bases. Blocking HPRT1 sensitizes cancer cells to ETC inhibition. These findings demonstrate how cells remodel purine metabolism upon ETC blockade and uncover a new metabolic vulnerability in tumors with low respiration.

Asunto(s)

Mitocondrias , Purinas , Humanos , Purinas/metabolismo , Purinas/farmacología , Mitocondrias/metabolismo , Transporte de Electrón , Hipoxantina Fosforribosiltransferasa/metabolismo , Hipoxantina Fosforribosiltransferasa/genética , Vía de Pentosa Fosfato , Fibroblastos/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/tratamiento farmacológico , Línea Celular Tumoral , Animales , Transporte Biológico

RESUMEN

The global surge in multidrug-resistant bacteria owing to antibiotic misuse and overuse poses considerable risks to human and animal health. With existing antibiotics losing their effectiveness and the protracted process of developing new antibiotics, urgent alternatives are imperative to curb disease spread. Notably, improving the bactericidal effect of antibiotics by using non-antibiotic substances has emerged as a viable strategy. Although reduced nicotinamide adenine dinucleotide (NADH) may play a crucial role in regulating bacterial resistance, studies examining how the change of metabolic profile and bacterial resistance following by exogenous administration are scarce. Therefore, this study aimed to elucidate the metabolic changes that occur in Edwardsiella tarda (E. tarda), which exhibits resistance to various antibiotics, following the exogenous addition of NADH using metabolomics. The effects of these alterations on the bactericidal activity of neomycin were investigated. NADH enhanced the effectiveness of aminoglycoside antibiotics against E. tarda ATCC15947, achieving bacterial eradication at low doses. Metabolomic analysis revealed that NADH reprogrammed the ATCC15947 metabolic profile by promoting purine metabolism and energy metabolism, yielding increased adenosine triphosphate (ATP) levels. Increased ATP levels played a crucial role in enhancing the bactericidal effects of neomycin. Moreover, exogenous NADH promoted the bactericidal efficacy of tetracyclines and chloramphenicols. NADH in combination with neomycin was effective against other clinically resistant bacteria, including Aeromonas hydrophila, Vibrio parahaemolyticus, methicillin-resistant Staphylococcus aureus, and Listeria monocytogenes. These results may facilitate the development of effective approaches for preventing and managing E. tarda-induced infections and multidrug resistance in aquaculture and clinical settings.

Asunto(s)

Aminoglicósidos , Antibacterianos , Edwardsiella tarda , NAD , Edwardsiella tarda/efectos de los fármacos , Antibacterianos/farmacología , NAD/metabolismo , Aminoglicósidos/farmacología , Animales , Enfermedades de los Peces/microbiología , Enfermedades de los Peces/tratamiento farmacológico , Pruebas de Sensibilidad Microbiana , Infecciones por Enterobacteriaceae/microbiología , Infecciones por Enterobacteriaceae/tratamiento farmacológico , Adenosina Trifosfato/metabolismo , Neomicina/farmacología , Sinergismo Farmacológico , Metabolómica , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos