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.

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.

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.

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.

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.

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.

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.

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.

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.
CONCLUSIONS: 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.

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.