ATP Metabolism Research Articles

DOP124 Fecal content from IBD patients causes more severe epithelial damage than fecal content from healthy donors, which can be moderated by supplementation of metabolites cocktail

Abstract Background Inflammatory bowel disease (IBD) pathogenesis involves the gut epithelia. However, no specific epithelial interventions exist, and models that capture epithelial and human gut variations for pre-clinical screening of therapies are limited Methods Monolayered organoides, derived from human rectal mucosal biopsies, were incubated with fecal samples from Crohn's disease (CD), ulcerative colitis (UC), and healthy control. Organoid viability was quantified by ATP levels and validated by live microscopy imaging. Fecal metabolites were measured using a semi-targeted library of 550 predefined metabolites. Gut bacteria were characterized using 16Sseq. Results Fecal samples were processed by separating the supernatant, heat-killing (HK) the bacteria, and rejoining the samples (Fig. 1A). We generated three pools per group (CD p#1-3, UC p#1-3, controls p#1-3). Each fecal p#1 (CD, UC, and controls) was a mix of ten subjects, and fecal pools p#2-3 were each a mix of five other independent subjects. An additional saliva/oral pool was processed similarly as an independent bacterial/metabolite source. Microbial characterization shows the overall variations of the original and pooled samples, with separation between IBD and control samples (Fig. 1B). We then inculcated the oral or fecal contents pools with differentiated colonoid and compared epithelial viability to untreated organoids and to those exposed to inflammatory triggers. Fecal content from UC and CD patients significantly reduced colonoid viability and ATP metabolism compared to healthy controls (p<0.001, Fig. 1C-D). This effect was mediated mainly by the fecal supernatant that also includes the metabolites, rather than the HK fraction (Fig. 1E). Live microscopy confirmed cell clumping already after 3 hours with the IBD fecal material (Fig. 1F). Characterizing metabolite levels in the joined and supernatant pools and correlating them with disease state (IBD vs. controls) and the colonoids viability outcomes highlighted several metabolites higher in controls and linked with improved viability (Fig. 2A-B). Notably, supplementing these identified metabolites with the fecal content pools, UC p#3 and CD p#2 improved epithelial viability (Fig. 2C-D). Lower metabolite supplementation mediated the improvement of the CD pool, but only higher concentrations resulted in improvement of the UC pools. Similarly, microscopy showed improvement in cell clumping when the IBD pools were supplemented with the prioritized metabolites (Fig. 2E). Conclusion Fecal content from IBD patients was associated with significantly reduced organoid viability and ATP metabolism compared to fecal content from controls, which improved, in part, with supplementation with prioritized metabolites.

Antagonists of CD39 and CD73 potentiate doxycycline repositioning to induce a potent antitumor immune response.

Studies have reported that cellular metabolism at the tumor-immune microenvironment (TiME) serves as a critical checkpoint and perturbs/supports anti-cancer immunity. Extra cellular ATP (eATP) may mediate anti-cancer immune response; however, its catabolism by ectonucleotidase generates immunosuppressive adenosine. In the presented work, we have tried to repurpose doxycycline with or without an antagonist of ectonucleotidase for mitigating ATP metabolism and immunosuppression. In this methodology eATP and adenosine levels were quantified. Bone marrow-derived M1 and M2 polarized macrophages were maintained in tumor mimicking condition (TMC). Total/CD4+Tcells were co-cultured with macrophages to understand the impact of doxycycline and/or antagonist of ectonucleotidase on T cell/subset differentiation. Preclinical efficacy of doxycycline and/or ectonucleotidase antagonist and their synergy was scored in 4 T1-induced breast carcinoma. We found that Doxycycline manipulated macrophage polarization by decreasing the frequency of CD206+M2 macrophages, which resulted in enhanced CD4+ directed CD8+ T cell response. Doxycycline alleviated the expression of CD39 and CD73, rescuing ATP catabolism. Doxycycline delayed tumor growth by enhancing F4/80+ CD86+ M1 macrophages and subsequently anti-tumor Tbet+ CD4+Tcells, attenuating the frequency of FOXP3+ regulatory T cells, which was cooperatively supported by ARL67156 and AMPCP (CD39 and CD73 antagonist).A synergy was observed with ARL67156 and AMPC Pensuring a possibility of using doxycycline alone or in combination with an antagonist of ectonucleotidase to present adenosine-mediated immunosuppression. Subsequently, our finding indicated that prospective usage of doxycycline as a novel metabolic checkpoint blocker (IMB) against ectonucleotidase and may be modified/delivered appropriately as a monotherapy or in combination with antagonists of ectonucleotidases as an IMB.

Photodynamic therapy photosensitizers and photoactivated chemotherapeutics exhibit distinct bioenergetic profiles to impact ATP metabolism.

Energy is essential for all life, and mammalian cells generate and store energy in the form of ATP by mitochondrial (oxidative phosphorylation) and non-mitochondrial (glycolysis) metabolism. These processes can now be evaluated by extracellular flux analysis (EFA), which has proven to be an indispensable tool in cell biology, providing previously inaccessible information regarding the bioenergetic landscape of cell lines, complex tissues, and in vivo models. Recently, EFA demonstrated its utility as a screening tool in drug development, both by providing insights into small molecule-organelle interactions, and by revealing the peripheral and potentially undesired off-target effects small molecules have within cells. Surprisingly, technologies to quantify cellular bioenergetics have not been systematically applied in phototherapy development, leaving open several questions about how the mechanism of action of a compound can impact essential cellular functions. Here, we utilized the Seahorse analyzer to address this question for photosensitizers (PSs) for photodynamic therapy (PDT) and contrast these systems to molecules that photo-release a ligand and thus act as photocages or photoactivated chemotherapeutics (PACT), intending to understand the influence these two classes of compounds have on cellular bioenergetics. EFA results show that acute treatment of A549 lung adenocarcinoma cells with PDT agents induces a quiescent bioenergetic response as a result of mitochondrial respiration shutdown. The loss of oxidative phosphorylation is followed by disruption of glycolysis, which occurs after an initial increase in glycolytic respiration is unable to compensate for the interruption of the electron transport chain (ETC). In contrast, the PACT agents tested had little impact on cellular respiration, and the minor inhibition of these metabolic processes was not related to the mechanism of action, as reflected by a lack of correlation with photoejection efficiency. Notably, a system capable of both generating 1O2 and photo-releasing a ligand exhibited the dominant profile of a PDT agent and induced the quiescent bioenergetic state, indicating potential implications on cellular bioenergetics for so-called dual-action agents. These findings are presented with the aim to provide the necessary groundwork for expanding the application and utility of EFA to phototherapeutics and to highlight the role of metabolic alterations in PDT.

Chronic unpredictable stress induces anxiety-like behavior and oxidative stress, leading to diminished ovarian reserve

Chronic stress can adversely affect the female reproductive endocrine system, potentially leading to disorders and impairments in ovarian function. However, current research lacks comprehensive understanding regarding the biochemical characteristics and underlying mechanisms of ovarian damage induced by chronic stress. We established a stable chronic unpredictable stress (CUS)-induced diminished ovarian reserve (DOR) animal model. Our findings demonstrated that prolonged CUS treatment over eight weeks resulted in increased atresia follicles in female mice. This atresia was accompanied by decreased AMH and increased FSH levels. Furthermore, we observed elevated levels of corticosterone both in the peripheral blood and within the ovary. Additionally, we detected abnormalities in ATP metabolism within the ovarian tissue. CUS exposure led to oxidative stress in the ovaries, fostering a microenvironment characterized by oxidative damage to mouse ovarian granulosa cells (mGCs) and heightened levels of reactive oxygen species. Furthermore, CUS prompted mGCs to undergo apoptosis via the mitochondrial pathway. These findings indicate a direct association between the fundamental physiological alterations leading to DOR and the oxidative phosphorylation processes within mGCs. The diminished ATP production by mGCs, triggered by CUS, emerges as a pivotal indicator of CUS-induced DOR. Our study establishes an animal model to investigate the impact of chronic stress on ovarian reserve function and sheds light on potential mechanisms underlying this phenomenon.

Circulatory response to exercise relative to oxygen uptake assessed in the follow-up of patients with fatty acid beta-oxidation disorders.

Patients with fatty acid oxidation disorders (FAODs) experience muscle symptoms due to impaired ATP metabolism and the toxicity of accumulated mitochondrial FAO substrates or intermediates, especially during catabolic states. A major issue is the absence of specific and sensible biomarkers to evaluate metabolic equilibrium. The relationship between cardiac output (Q) and oxygen consumption (VO2) during incremental exercise (dQ/dVO2) provides an indirect surrogate of mitochondrial function. A high dQ/dVO2 slope indicates impaired oxidative phosphorylation in skeletal muscle during exercise. Our study aimed to evaluate dQ/dVO2 as a potential marker of the severity of FAODs. We retrospectively collected clinical, laboratory parameters and treatment data for FAOD patients over 6 years old, including a disease severity score, plasma acylcarnitines and cardiopulmonary exercise tests with Q measurement via thoracic bioelectrical impedance. FAO flux was measured in whole blood and in myoblasts when available. We included 27 FAOD patients followed from 2015 to 2022, with deficiencies in LCHAD (n = 10), CPT2 (n = 6), VLCAD (n = 7), or MADD (n = 4). CPT2 deficient patients with severe scores had the highest C18:1-, C16-, C18-acylcarnitines, and dQ/dVO2. In these patients, dQ/dVO2 was positively correlated with C18:1, C16, and C18 acylcarnitines. In a linear multivariate regression model, dQ/dVO2 was significantly associated with the severity score (B = 0.831, p = 0.008) and triheptanoin treatment (B = -0.547, p = 0.025). dQ/dVO2 and plasma long-chain acylcarnitines might be useful to monitor CPT2D, as these parameters associate with our clinical severity score and could reflect altered mitochondrial functions.

Cysteinyl‐tRNA synthetase is involved in damage of renal tubular cells in ischemia–reperfusion‐induced acute kidney injury via pyroptosis

AbstractAcute kidney injury (AKI) is a significant global healthcare burden but lacks specific and effective treatment. Renal tubular cells damage is central to ischemia‐reperfusion injury (IRI) induced AKI. It is critical to clarify the initiation mechanisms of renal IRI and develop early intervention targets of AKI. This study used label‐free quantification proteomic analysis to identify new targets in AKI‐related renal tubular injury and investigate the potential mechanisms. We discovered significant changes in cysteinyl‐tRNA synthetase (CARS) in renal tubular cell during IRI. Considering the involvement of CARS in ATP metabolism and the close correlation between ATP and pyroptosis, we further explored pyroptosis phenotype with and without CARS intervention as well as the expression of CARS during pyroptosis activation and inhibition. Our findings suggest that CARS expression decreased over time and is linked to pyroptosis. Modifying CARS affects ATP metabolism and alters the expression of pyroptosis‐related proteins during H/R and IRI treatments. Regulating pyroptosis may influence CARS expression during IRI treatment. Overall, CARS is associated with renal tubular damage from ischemia‐reperfusion injury, possibly involving pyroptosis, though the regulatory mechanism remains unclear.

Decreases in metabolic ATP open KATP channels and reduce firing in an auditory brainstem neuron: A dynamic mechanism of firing control during intense activity

Cartwheel (CW) neurons are glycinergic interneurons in the dorsal cochlear nucleus (DCN) that exhibit spontaneous firing, resulting in potent tonic inhibition of fusiform neurons. CW neurons expressing open ATP-sensitive potassium (KATP) channels do not fire spontaneously, and activation of KATP channels halts spontaneous firing in these neurons. However, the conditions that regulate KATP channel opening in CW neurons remain unknown. Here, we tested the hypothesis that fluctuations in metabolic ATP levels modulate KATP channels in CW neurons. Using whole-cell patch-clamp recordings in CW neurons from young rat brain slices (p17-22) with an ATP-free internal solution, we observed that the mitochondrial uncoupler CCCP hyperpolarized the membrane potential, reduced spontaneous firing, and generated an outward current, which was inhibited by the KATP channel antagonist tolbutamide. Additionally, a glucose-free external solution quickly activated KATP channels and ceased spontaneous firing. We hypothesized that intense membrane ion ATPase activity during strong depolarization would deplete intracellular ATP, leading to KATP channel opening. Consistent with this, depolarizing CW neurons with a 250 pA DC did not increase spontaneous firing because the depolarization activated KATP channels; however, the same depolarization after tolbutamide administration increased firing, suggesting that ATP depletion triggered KATP channel opening to limit action potential firing. These results indicate that KATP channels in the DCN provide dynamic control over action potential firing, preventing excessive excitation during high-firing activity.

Altered extracellular atp, adp, and amp hydrolysis in blood serum of sedentary individuals after an acute, strength, exercise session

Nucleotidases participate in the regulation of physiological and pathological events, such as inflammation and coagulation. Different types of physical exercise promote changes in purinergic signaling. In the present study, we investigated the activities of soluble nucleotidases in the blood serum of sedentary young male adults before and after a resistance exercise session. In addition, we evaluated how this type of exercise could influence the concentrations of adenine nucleotides in the blood serum. The individuals were submitted to a strength exercise session, consisting of 8 exercises. Blood samples were collected pre- and post-exercise, and serum was separated for analysis. Results showed increases in ATP, ADP, and AMP hydrolysis post-exercise, compared to pre-exercise values. Our results demonstrate that a resistance exercise session modifies ATP metabolism, being one more form of exercise that can positively influence cardiovascular and metabolic health.

Estrogen-mediated inhibition of purine metabolism and cell cycle arrest as a novel therapeutic approach in colorectal cancer

Purine metabolism is upregulated in various cancers including colorectal cancer (CRC). While previous work has elucidated the role of estrogen (E2) in metabolic reprogramming and ATP production, the effect of E2 on purine metabolism remains largely unknown. Herein, the impact of E2 signalling on purine metabolism in CRC cells was investigated using metabolome and transcriptome profiling of cell extracts derived from E2-treated HCT-116 cells with intact or silenced estrogen receptor alpha (ERα). Purine metabolic pathway enrichment analysis showed that 27 genes in the de novo purine synthesis pathway were downregulated in E2-treated CRC cells. Downstream consequences of E2 treatment including the induction of DNA damage, cell cycle arrest, and apoptosis were all shown to be ERα-dependent. These findings demonstrate, for the first time, that E2 exerts a significant anti-growth and survival effect in CRC cells by targeting the purine synthesis pathway in a ERα-dependent manner, meriting further investigation of the therapeutic utility of E2 signalling in CRC.

Digoxin reduces senescence and restores dysfunctional endothelial-subcutaneous adipocyte cross-talk and dysregulated glucose homeostasis in patients with type 2 diabetes mellitus

Abstract Background Normal subcutaneous adipose tissue (SAT) function is dependent on SAT microvascular expansion. Heart failure with reduced ejection fraction (HF) and type 2 diabetes mellitus (DM) have a synergistic deleterious relationship and, of relevance to this, metabolic dysregulation may drive HF progression in DM. Chronic hyperinsulinemia is associated with adipocyte senescence, however any contributing role of SAT microvascular endothelial cell (SATMVEC) senescence on adverse microvascular expansion in type 2 diabetes mellitus (DM) remains unknown. Digoxin has been shown to have senolytic activity in murine cancer models. However, the potential therapeutic role of digoxin on SAT metabolic function is unexplored. Purpose We establish the presence of SATMVEC senescence and its effects on adipocyte function in people with HF and DM (HFDM). Moreover, we establish a therapeutic role for digoxin in targeting SATMVEC senescence, modulating SATMVEC-adipocyte crosstalk and restoring metabolic homeostasis. Methods We took pectoral SAT biopsies from 59 patients undergoing pacemaker implantation of whom 25 (42%) had HF and DM (HFDM). SATMVEC were isolated from SAT and were treated with 1ng/mL digoxin vs control for 24 hours before characterisation (cell function, expression of senescence-associated ß-galactosidase (ß-gal) and senescence associated secretory phenotype, SASP). Crosstalk between SATMVEC and subcutaneous white adipocytes were examined using co-culture techniques (Fig 1A). Paired digoxin vs control treated SATMVEC were seeded co-cultured with adipocytes for 24 hours, before adipocyte health was quantified, through 2-NBD-glucose uptake and expression of key adipogenic genes and proteins. Results Compared to HF alone, isolated SATMVEC from patients with HFDM had a senescent phenotype with increased SASP and ß-gal expression (p<0.05), reduced metabolic activity, ATP production, proliferative capacity, and impaired angiogenesis (p<0.05). In co-culture, SATMVEC from patients with HFDM had deleterious effects on adipocyte function: increasing expression of IL-6 (Fig 1C) and reducing 2-NBD-glucose uptake (p<0.05). Treatment of SATMVEC with digoxin reduced the SATMVEC senescent phenotype (Fig 1B) and increased 2-NBD-glucose adipocyte uptake (Figure 1D) (p<0.001). Conclusions SATMVEC from patients with HFDM have a senescent phenotype, and when in co-culture induce a proinflammatory adipocyte phenotype, resistant to glucose uptake. Digoxin reduces SATMVEC senescence, restores healthy adipocyte glucose homeostasis, and thus has the potential to repair dysfunctional SAT in patients with HFDM.

Downregulation of CPEB3 exacerbates cardiac injury by inhibiting cardiomyocyte adaptive metabolic reprogramming after myocardial infarction

Abstract Background Adaptive metabolic reprogramming from oxidative phosphorylation (OXPHOS) to glycolysis in hypoxia plays a protective role in cardiomyocyte survival by reducing reactive oxygen species (ROS) and increasing ATP. Cytoplasmic polyadenylation element binding protein 3 (CPEB3) influences protein translation by modulating 3' untranslated region (3'UTR) lengths through alternative polyadenylation (APA). However, the role of CPEB3 in cardiomyocyte after myocardial infarction (MI) remains unknown. Purpose This study aims to explore the function of CPEB3 in cardiomyocyte after MI. Methods Three GEO databases of mice MI (GSE110209, GSE114695 and GSE236374) were analyzed to identify CPEB3 dysregulation. Cardiomyocyte-specific CPEB3 knockout mice and CPEB3-knockdown neonatal mouse cardiomyocytes (NMCMs), RNA sequencing, flow cytometry, TUNEL staining, Seahorse, ROS and ATP measurements were used to investigate the impact of CPEB3. APA events analysis, RIP sequencing, CUT-TAG, 3’RACE, polysome profiling and dual luciferase report were performed to elucidate the mechanisms of CPEB3. Recombinant adeno-associated virus carrying cardiac troponin T promoter was used to evaluate the therapeutic efficacy of CPEB3 in mice with MI. Results CPEB3 expression was markedly reduced in the heart tissue after MI and in NMCMs after hypoxia. Cardiomyocyte-specific CPEB3 deletion aggravated cardiomyocyte apoptosis, enlarged infarct size, and exacerbated cardiac injury after MI. RNA sequencing revealed that CPEB3 deficiency predominantly inhibited glycolysis-related pathway, with a notable decrease in pyruvate dehydrogenase kinase 1 (PDK1) expression. In CPEB3-knockdown NMCMs, adaptive metabolic reprogramming from OXPHOS to glycolysis and ATP level were decreased in hypoxia, whereas ROS production was significantly enhanced, all of which resulted in apoptosis and were regulated by PDK1. Besides, PDK1 overexpression counteracted the effects of CPEB3 knockdown on metabolic pattern, ROS, ATP and apoptosis. Mechanistically, CPEB3 deficiency led to a shortened 3’UTR of the transcription factor forkhead box O3 (FOXO3), and a decline of FOXO3 protein level. CPEB3 protein was confirmed to directly bind to FOXO3 mRNA, and the translation efficiency of FOXO3 was decreased with CPEB3 knockdown. Furthermore, FOXO3 was found to activate the transcription of PDK1, and FOXO3 overexpression alleviated the decline of PDK1 and attenuated CPEB3 knockdown-induced apoptosis in hypoxia. Finally, cardiomyocyte-specific CPEB3 overexpression reduced cardiomyocyte apoptosis, suppressed myocardial fibrosis, and improved cardiac function by upregulating FOXO3 and PDK1 levels after MI. Conclusion CPEB3 could promote FOXO3 protein expression via APA of FOXO3 3’UTR, activate transcription of PDK1 and ultimately protect cardiomyocyte from apoptosis by restoring metabolic balance in hypoxia. CPEB3 may serve as a promising therapeutic target for cardiomyocyte apoptosis after MI.Schematic diagram

BCR, not TCR, repertoire diversity is associated with favorable COVID-19 prognosis.

The SARS-CoV-2 pandemic has had a widespread and severe impact on society, yet there have also been instances of remarkable recovery, even in critically ill patients. In this study, we used single-cell RNA sequencing to analyze the immune responses in recovered and deceased COVID-19 patients during moderate and critical stages. Expanded T cell receptor (TCR) clones were predominantly SARS-CoV-2-specific, but represented only a small fraction of the total repertoire in all patients. In contrast, while deceased patients exhibited monoclonal B cell receptor (BCR) expansions without COVID-19 specificity, survivors demonstrated diverse and specific BCR clones. These findings suggest that neither TCR diversity nor BCR monoclonal expansions are sufficient for viral clearance and subsequent recovery. Differential gene expression analysis revealed that protein biosynthetic processes were enriched in survivors, but that potentially damaging mitochondrial ATP metabolism was activated in the deceased. This study underscores that BCR repertoire diversity, but not TCR diversity, correlates with favorable outcomes in COVID-19.

A regulatory variant rs9379874 in T1D risk region 6p22.2 affects BTN3A1 expression regulating T cell function.

Genome-wide association studies (GWAS) have identified that 6p22.2 region is associated with type 1 diabetes (T1D) risk in the Chinese Han population. This study aims to reveal associations between this risk region and T1D subgroups and related clinical features, and further identify causal variant(s) and target gene(s) in this region. 2608 T1D and 4814 healthy controls were recruited from East, Central, and South China. Baseline data and genotyping for rs4320356 were collected. The most likely causal variant and gene were identified by bioinformatics analysis, dual-luciferase reporter assays, expression quantitative trait loci (eQTL), and functional annotation of the non-coding region within the 6p22.2 region. The leading variant rs4320356 in the 6p22.2 region was associated with T1D risk in the Chinese and Europeans. However, this variant was not significantly associated with islet function or autoimmunity. In silico analysis suggested rs9379874 was the most potential causal variant for T1D risk among thymus, spleen, and T cells, overlapping with the enhancer-related histone mark in multiple T cell subsets. Dual luciferase reporter assay and eQTL showed that the T allele of rs9379874 increased BTN3A1 expression by binding to FOXA1. Public single-cell RNA sequencing analysis indicated that BTN3A1 was related to T-cell activation, ATP metabolism, and cytokine metabolism pathways, which might contribute to T1D development. This study indicates that a functional variant rs9379874 regulates BTN3A1 expression, expanding the genomic landscape of T1D risk and offering a potential target for developing novel therapies.

SH3 domain‑binding glutamic acid‑rich protein‑like 3 is associated with hyperglycemia and a poor outcome in Epstein‑Barr virus‑negative gastric carcinoma.

SH3 domain-binding glutamic acid-rich protein-like 3 (SH3BGRL3) is involved in several human cancers. However, its relationship with gastric cancer (GC) remains elusive. Multiple online bioinformatic tools were used to evaluate the messenger (m)RNA expression levels of SH3BGRL3 in GC using data from The Cancer Genome Atlas and Gene Expression Omnibus databases. Reverse transcription-quantitative PCR and tissue microarray-based immunohistochemistry were performed to assess SH3BGRL3 expression in relation to clinicopathological parameters and outcomes in patients with GC. Significant differentially expressed genes (DEGs) of SH3BGRL3 were enriched and visualized. Furthermore, associations between the expression of SH3BGRL3 and the infiltration of immune cells were explored. SH3BGRL3 exhibited aberrant expression in tumor tissues compared with adjacent normal tissues at the mRNA and protein expression levels, especially in Epstein-Barr virus-negative GC (EBVnGC). Higher SH3BGRL3 expression was significantly associated with increasing tumor-node-metastasis staging, tumor budding, perineural invasion, EGFR expression, and a notably higher preoperative blood glucose concentration in clinical specimens. Multivariate analysis revealed that higher SH3BGRL3 expression was an independent adverse prognostic factor for the overall survival of patients with EBVnGC (hazard ratio, 1.666; P=0.018). Furthermore, the stratified analysis revealed that the SH3BGRL3 phenotype could help to refine prognosis in patients. The C-index of the nomogram was 0.740 when combining SH3BGRL3 with other clinicopathological parameters, which indicated a good model for clinical follow-up decisions. Gene functional enrichment analysis also revealed that the DEGs of SH3BGRL3 were mainly enriched in regulating ATP metabolism, ATP synthesis, oxidative phosphorylation and the electron transport chain in GC. Moreover, a higher SH3BGRL3 expression was significantly positively correlated with the infiltrating macrophages in GC. In conclusion, SH3BGRL3 is upregulated in GC, particularly in EBVnGC. Higher SH3BGRL3 expression is closely associated with hyperglycemia and poor outcomes in patients with EBVnGC, suggesting its potential as a biomarker and prognostic predictor.

The effect of m6A methyltransferase METTL3 mediated TMEM30A regulation on tumor energy metabolism and cisplatin anti-tumor activity in oral squamous cell carcinoma

AimsCisplatin (CDDP) is still one of the most commonly used first-line treatments for advanced and recurrent oral squamous cell carcinoma (OSCC) patients in clinical practice. However, the decrease in tumor sensitivity to CDDP weakens its therapeutic effect. There is still limited research on the effect of METTL3-mediated methylation of m6A on CDDP sensitivity in OSCC. TMEM30A widely exists in biomembranes and regulates the lipid asymmetry of the membrane, but there is no report on its function in OSCC. This study aims to explore the specific mechanism by which METTL3 regulates m6A methylation of TMEM30A and affects the occurrence and development of OSCC, and further investigate the effects of METTL3 and TMEM30A on the anti-tumor activity of CDDP. Key findingsIn OSCC, METTL3 plays a pro-cancer role and weakens the anti-tumor efficacy of CDDP; METTL3 positively regulates the expression of TMEM30A by m6A methylation modification and binding to TMEM30A; The abnormally high expression of TMEM30A in OSCC not only weakens CDDP sensitivity, but also enhances the malignant evolution of cancer cells, regulates the metabolic balance of ATP and lactate in cells, and is a potential oncogenic gene. SignificanceTMEM30A promotes malignant progression of tumors through METTL3 mediated m6A methylation modification, participates in maintaining the balance of tumor ATP and lactate metabolism, and reduces the anti-tumor activity of CDDP. TMEM30A is a potential gene target for CDDP anti-tumor activity in OSCC.

Effect of Mesenchymal Stem Cell–Derived Extracellular Vesicles Induced by Advanced Glycation End Products on Energy Metabolism in Vascular Endothelial Cells

IntroductionAdvanced glycation end products (AGEs) play a critical role in the development of vascular diseases in diabetes. While stem cell therapies often involve exposure to AGEs, the impact of this environment on extracellular vesicles (EVs) and endothelial cell metabolism remains unclear. MethodsHuman umbilical cord mesenchymal stem cells (MSC) were treated with either 0 ng/mL or 100 ng/mL AGEs in a serum-free medium for 48 hours, after which MSC-EVs were isolated. The EVs were characterized for morphology, particle size, and protein markers of MSC-EVs, and performed miRNA sequencing to identify differentially expressed miRNAs. MSC-EVs were co-cultured with HUVEC cells to assess effects on cell viability, metabolic activity, oxidative stress, and antioxidant capacity. Tube formation and glucose transporter protein analyses were conducted to evaluate the angiogenic ability and glucose metabolism capacity. ResultsMSC-EVs ranged from 30 to 150 nm, consistenting with exosomal properties. AGEs treatment reduced MSC viability but had minimal effect on EV morphology and protein markers. miRNAs sequencing showed downregulation of hsa-miR-223-3p, hsa-miR-126-3p_R-1, with upregulation of hsa-miR-574-5p, implicating changes in glycolytic and oxidative phosphorylation pathways. MSC-EVs treated with AGEs decreased HUVEC viability (P<0.05), pH (P<0.05), ATP metabolism (P<0.05), glucose metabolism (P<0.05), while enhancing glycolysis processes, including glycolytic activity, capacity, and reserve (P<0.05). This likely resulted from impaired mitochondrial function, including reduced ATP production, maximal respiration, basal respiration, and spare respiratory capacity (P<0.05), or increased ROS (P<0.05) and G6PD activity (P<0.05). Additionally, AGEs reduced GLUT1, GLUT3, GLUT4, and SCO2 expression (P<0.05), along with angiogenic capacity (P<0.05) in HUVEC cells. ConclusionExposure to AGEs diminishes the therapeutic potential of MSC-derived EVs by disrupting energy metabolism and promoting metabolic reprogramming in endothelial cells. These findings suggest that adjusting the dosage or frequency of MSC-EVs may enhance their efficacy for treating diabetes-related vascular conditions. Further research is warranted to evaluate AGEs' broader impact on various cell types and metabolic pathways for improved exosome-based therapies.

Proteomic changes in Alzheimer’s disease associated with progressive Aβ plaque and tau tangle pathologies

Proteomics can shed light on the dynamic and multifaceted alterations in neurodegenerative disorders like Alzheimer’s disease (AD). Combining radioligands measuring β-amyloid (Aβ) plaques and tau tangles with cerebrospinal fluid proteomics, we uncover molecular events mirroring different stages of AD pathology in living humans. We found 127 differentially abundant proteins (DAPs) across the AD spectrum. The strongest Aβ-related proteins were mainly expressed in glial cells and included SMOC1 and ITGAM. A dozen proteins linked to ATP metabolism and preferentially expressed in neurons were independently associated with tau tangle load and tau accumulation. Only 20% of the DAPs were also altered in other neurodegenerative diseases, underscoring AD’s distinct proteome. Two co-expression modules related, respectively, to protein metabolism and microglial immune response encompassed most DAPs, with opposing, staggered trajectories along the AD continuum. We unveil protein signatures associated with Aβ and tau proteinopathy in vivo, offering insights into complex neural responses and potential biomarkers and therapeutics targeting different disease stages.

Downregulating miR-432-5p exacerbates adriamycin-induced cardiotoxicity via activating the RTN3 signaling pathway.

Adriamycin (ADR) is a widely used chemotherapy drug in clinical practice and it causes toxicity in the myocardium affecting its clinical use. miR-432-5p is a miRNA primarily expressed in myocardial cells and has a protective effect in the myocardium. We aim to explore the protective effect of miR-432-5p on ADR-caused impaired mitochondrial ATP metabolism and endoplasmic reticulum stress (ERs). The primary cardiomyocytes were obtained from neonatal mice and the ADR was added to cells, meanwhile, a mice model was constructed through intravenous ADR challenge, and expression levels of miR-432-5p were examined. Subsequently, the miR-432-5p was introduced in vitro and in vivo to explore its effect on the activity of mitochondrial ATP synthesis, autophagy, and ER stress. The bioinformatics analysis was performed to explore the target of miR-432-5p. ADR decreased the expression of miR-432-5p in cardiomyocytes. It also decreases mitochondrial ATP production and activates the ER stress pathway by increasing the expression of LC3B, Beclin 1, cleaved caspase 3, and induces cardiac toxicity. miR-432-5p exogenous supplementation can reduce the cardiotoxicity caused by ADR, and its protective effect on cardiomyocytes depends on the down-regulation of the RTN3 signaling pathway in ER. ADR can induce the low expression of miR-432-5p, and activate the RTN3 pathway in ER, increase the expression of LC3B, Beclin 1, cleaved caspase 3, CHOP, and RTN3, and induce cardiac toxicity.

A Simplified Method for Comprehensive Capture of the Staphylococcus aureus Proteome.

Staphylococcus aureus is a major human pathogen causing myriad infections in both community and healthcare settings. Although well studied, a comprehensive exploration of its dynamic and adaptive proteome is still somewhat lacking. Herein, we employed streamlined liquid- and gas-phase fractionation with PASEF analysis on a TIMS-TOF instrument to expand coverage and explore the S. aureus dark proteome. In so doing, we captured the most comprehensive S. aureus proteome to date, totaling 2,231 proteins (85.6% coverage), using a significantly simplified process that demonstrated high reproducibility with minimal input material. We then showcase application of this library for differential expression profiling by investigating temporal dynamics of the S. aureus proteome. This revealed alterations in metabolic processes, ATP production, RNA processing, and stress-response proteins as cultures progressed to stationary growth. Notably, a significant portion of the library (94%) and proteome (80.5%) was identified by this single-shot, DIA-based analysis. Overall, our study shines new light on the hidden S. aureus proteome, generating a valuable new resource to facilitate further study of this dangerous pathogen.

Assessment of ATP metabolism to adenosine by ecto-nucleotidases carried by tumor-derived small extracellular vesicles.

Immunosuppression is a hallmark of cancer progression. Tumor-derived small extracellular vesicles (sEV), also known as TEX, produce adenosine (ADO) and can mediate tumor-induced immunosuppression.Here, the ATP pathway of ADO production (ATP ADP AMP ADO) by ecto-nucleotidases carried on the sEV surface was evaluated by a method using N6-etheno-ATP (eATP) and N6-etheno-AMP (eAMP) as substrates for enzymatic activity. The "downstream" N6-etheno-purines (ePurines) were measured by high performance liquid chromatography with fluorescence detection (HPLC-FL).Human melanoma cell-derived TEX (MTEX) metabolized eATP to N6-etheno-ADP (eADP), eAMP and N6-etheno-Adenosine (eADO) more robustly than control keratinocyte cell-derived sEV (CEX); due to accelerated conversion of eATP to eADP and eADP to eAMP. MTEX and CEX similarly metabolized eAMP to eADO. Blocking of the ATP pathway with the selective CD39 inhibitor ARL67156 or pan ecto-nucleotidase inhibitor POM-1 normalized the ATP pathway but neither inhibitor completely abolished it. In contrast, inhibition of CD73 by PSB12379 or AMPCP abolished eADO formation by both MTEX and CEX, suggesting that targeting CD73 is the preferred approach to eliminating ADO produced by ecto-nucleotidases located on the sEV surface.The noninvasive, sensitive, and specific assay assessing ePurine metabolism ± ecto-nucleotidase inhibitors in TEX enables the personalized identification of ecto-nucleotidase activity primarily involved in ADO production in patients with cancer. The assay could guide precision medicine by determining which purine is the preferred target for inhibitory therapeutic interventions.