BACKGROUND AND OBJECTIVES:The liver continuously adjusts its metabolic activity to synchronize the nutrient supply with the body's demands. This synchronization involves the complex coordination of acute metabolic needs, nutrient availability, and activity levels, which is orchestrated according to cyclic internal rhythms governed by the circadian clock. This study aimed to decipher the role of circadian rhythms in liver metabolic functions, including mitochondrial activities that are critical for energy production and metabolic adaptation. METHODS:We investigated rhythmic changes in liver metabolism via comprehensive multiomics and kinetic mathematical modeling. The liver proteome of male mice was analyzed and modeled, and complementary serum lipidomic and metabolomic analyses were performed. Mitochondrial proteins were examined to evaluate the role of mitochondria in the oscillating regulation of energy production. RESULTS:Most metabolic functions, particularly those related to carbohydrate and fatty acid metabolism, exhibit rhythmic patterns on a 12-hour rather than a 24-hour cycle. The importance of this rhythmicity is function-dependent and can account for 25% to 50% of the overall variability. Mitochondrial activities also exhibit temporal fluctuations that are closely linked to nutrient availability. The strong correlation between metabolic functions and serum metabolites highlights the precise alignment between physiological demand and metabolic performance. CONCLUSIONS:Hepatic metabolic functions follow a 12-hour cycle rather than a 24-hour cycle, significantly contributing to the liver's ability to meet nutrient demands throughout the day. Mitochondrial dynamics, which are influenced by nutrient availability, play a central role in adapting energy production to the body's metabolic needs.

Targeting ACKR3/CXCR7 regulates enzymatic generation of pro-thrombotic, while favoring anti-thrombotic lipids that inhibit platelets through AC-cAMP-PKA pathway in coordination with prostacyclin-IP receptor. This investigation validated the impact of CXCR7 in modulating non-enzymatic lipid (per)oxidation, platelet response to lipoproteins-(LDL, oxLDL), mitochondrial metabolism and procoagulatory functions. Pharmacological CXCR7-agonist-(VUF11207) preserved mitochondrial membrane integrity-(Δψm), counteracted activation-induced mitochondrial superoxide generation-(MitoSOXRed), and nonenzymatic lipid (per)oxidation. Additionally, CXCR7-agonist regulated lipoprotein-induced platelet adhesion on thrombogenic matrices, degranulation, αIIbβIII-integrin activation, aggregation and thrombotic response, by reducing lipoprotein uptake through scavenger receptors-(CD36, ApoER2). CXCR7-ligation triggered activation of metabolic energy sensor Adenosine MonoPhosphate-dependent Kinase-(AMPKSer-172), prompted AMPK-mediated inhibitory phosphorylation of Acetyl-CoA-Carboxylase-(ACC)Ser-79, to foster lipolysis over lipogenesis. Consequently AMPKSer-172-ACCSer-79 pathway increased anticoagulatory FXa-inhibitory long-chain acylcarnitine-(LC-CARs)-(16:0, 18:1, 18:2) generation in platelets from healthy subjects and CAD patients. Increased intraplatelet LC-CARs was not due to dysregulated mitochondrial respiration; since CXCR7-agonist improved maximal respiration, spare respiratory capacity, and ATP-linked respiration in thrombin-activated platelets, suggesting sustained mitochondrial metabolism. Exerting a two-pronged effect on procoagulant function, CXCR7-agonist downregulated phosphatidylserine exposure on activated platelets, reducing FX/FXa binding, while platelet-derived anticoagulatory-LC-CARs regulated thrombin generation. CXCR7-agonist administration reduced thrombus formation, platelet degranulation, αIIbβIII-integrin activation, procoagulant activity, circulatory platelet-leukocyte aggregates in murine venous thrombosis model; besides, decreased plasma procoagulant lipids-(platelet COX-1, 12-LOX, and leukocyte 5/15-LOX-derived) and thrombo-inflammatory mediators-(IL-1β, IL-6, IFN-γ, TNF-α, MCP-1), and increased plasma LC-CAR levels. Therefore, pharmacological targeting of CXCR7 could regulate (non)enzymatic lipid processing, and promote anticoagulatory LC-CAR generation to check platelet-directed thrombotic propensity, and hypercoagulation, moreover, replenish reduced levels of circulatory anticoagulant-LC-CARs in STEMI and venous thromboembolism-(VTE) patients.

Efforts to understand microbiome-host interactions in disease development and progression are growing, with short-chain fatty acids (SCFAs) produced by gut bacteria recognized as key metabolic mediators. To facilitate studies on the effect of bacterially secreted metabolites on human cells, we have developed a new LC-MS(/MS) method that allows quantitative analysis of underivatised SCFAs and simultaneous non-targeted screening for additional microbial or endogenous metabolites from cell culture media. Comprehensive method optimization resulted in methanolic gradient elution using a Kinetex XB-C18 column allowing for quantitative QQQ-MS analysis of propionate, butyrate, isobutyrate, valerate, isovalerate, and caproate, using deuterated internal standards. The method was validated in terms of linearity, selectivity, stability as well as inter-and intra-day accuracy and precision. To integrate non-targeted metabolomics, the assay was subsequently transferred to an LC-QTOF-MS platform and cross-validated to ensure accuracy and precision of SCFA analysis. Applying this method to study the effects of the breast cancer drug tamoxifen and 10 human tamoxifen metabolites (desmethyltamoxifen, Z-endoxifen, (E/Z)-4 '-hydroxy-desmethyltamoxifen, (E/Z)-4-hydroxytamoxifen, tamoxifen-Nglucuronide, (E/Z)-tamoxifen-4-glucuronid, (E/Z)-DM-tamoxifen-4-O-glucuronide, (E/Z)-4-hydroxytamoxifen-N-glucuronide, (E/Z)-endoxifen-4-sulfate, (E/Z)-tamoxifen-4-sulfate) on the secretome of faecal bacterial communities revealed a pronounced impact of the parent drug and non-sulfated metabolites. The omission of derivatisation leads to fast and simple sample preparation. Together with the short LC run time (10 min) and the comprehensive information obtained by combining targeted and non-targeted metabolomics in a single run, the new method represents a valuable tool for the in vitro investigation of metabolite-mediated microbiome-host interactions.

Purpose:Brimonidine, a selective α2-adrenergic agonist, is widely used in topical therapy for ocular hypertension or primary open-angle glaucoma. However, effective delivery is hindered by multiple ocular surface barriers. Because brimonidine is a cationic drug, we hypothesized that organic cation transporters (OCTs) and multidrug and toxin extrusion proteins (MATEs) are involved in brimonidine uptake in the human eye. This study aimed to determine if brimonidine is transported by OCT1, OCT2, OCT3, MATE1, and MATE2K and if the identified transporters are localized in anterior eye structures. Methods:Uptake studies were performed using HEK293 cells stably expressing OCT1, OCT2, OCT3, MATE1, and MATE2K. Intracellular brimonidine accumulation was analyzed by mass spectrometry. Immunohistochemistry of glaucomatous human eyes was used to localize relevant transporters in anterior ocular structures. Results:Brimonidine was transported by OCT2 and MATE1 but not by OCT1, OCT3, or MATE2K. Uptake was time- and concentration-dependent. Both OCT2 and MATE1 were expressed in the cornea, the conjunctiva, and the ciliary body. Conclusions:These results imply that OCT2 and MATE1 may play a role in brimonidine uptake into the human eye and may contribute to the interindividual variability of brimonidine concentrations and effects.

Background and purposeThiopurines are used in paediatric inflammatory bowel disease (IBD), but some patients do not respond. Because the gut microbiota influences drug efficacy and IBD-patient microbiota presents increased bacterial abundance, we investigated the impact of candidate Enterobacteriaceae on drug cytotoxicity, metabolism and efficacy.Experimental approachThiopurines were exposed in vitro to bacteria for 4 h at 37 degrees C and drug concentrations measured by UV spectrophotometry. Cytotoxic effects and drug metabolite concentrations on NALM6 and JURKAT cells were determined after treatment with thiopurines exposed or not to bacteria. Drugs were measured in Klebsiella pneumoniae lysates and bacterial conditioned media were used for metabolomic analyses. Shotgun metagenomic sequencing was performed on eight IBD-patient faecal stools.Key resultsIncubation of thiopurines with K. pneumoniae, but not Escherichia coli and Salmonella enterica, reduced thiopurine concentrations and cytotoxicity on NALM6 and JURKAT cells. Thiopurine metabolites were lower in cells treated with drugs previously exposed to K. pneumoniae. Internalisation of drugs was demonstrated by their detection in lysates after bacterial incubation. Untargeted metabolomics revealed biotransformation of thiopurines by K. pneumoniae, as reactions of deconjugation, reduction, glycosylation, acetylation or conjugation with propionic acid. Incubation with thiopurines led to changes in the secretion of endogenous bacterial metabolites. K. pneumoniae faecal abundance was associated with lower thiopurine metabolite concentrations in erythrocytes of paediatric IBD-patients.Conclusions and ImplicationsK. pneumoniae decreases the cytotoxicity of thiopurines through internalisation of MP and TG. We revealed potential bacterial drug biotransformation, as well as negative correlations between bacterial abundance and drug metabolites.

Clear cell renal cell carcinoma (ccRCC) is characterized by a metabolic shift towards enhanced aerobic glycolysis and increased lactate production. The survival rate for metastatic RCC is still poor. We evaluated the lactate monocarboxylate transporter 4 (MCT4), encoded by SLC16A3, as drug target for metastatic disease. MCT4 protein expression in 209 distant ccRCC metastases, including 40 recurrent metastases, was generally as high as compared to primary tumor tissue and significantly increased compared to non-tumor tissue (P<1E-15). MCT4 expression was irrespective of affected organs and mutations in RCC driver genes. DNA methylation in the SLC16A3 promoter, assessed by MALDI TOF mass spectrometry and correlated with clinicopathological data, were not significantly different in metastases of all investigated organ sites, and between paired tumor and metastases samples. Visualization of expression in single-cell and spatial RNA sequencing datasets reveals main expression of SLC16A3 in cells derived from tumor, tumor-normal interface, metastatic and lymph node tissue. Alone or combined with inhibition of mitochondrial respiration by metformin and phenformin, the MCT4 inhibitor syrosingopine significantly inhibits lactate efflux, induces cell viability reduction in four different RCC cell lines and patient-derived 2D/3D models, and alterations in cellular metabolism and mitochondrial respiration. Six patient-derived RCC air-liquid interface models, mimicking the complex RCC architecture, corroborate these data. Beyond potential prediction of patient outcome using MCT4 expression and DNA methylation at specific CpG sites, drug targeting of MCT4 and inhibiting mitochondrial respiration synergistically is a novel treatment strategy for metastatic ccRCC.

The classical approach of using adjacent pieces of fresh-frozen tissue for various omics analysis from the same sample possesses a risk of biological mismatch between arising from intrinsic tissue heterogeneity. We propose an alternative approach of tissue cryogenic pulverization and lyophilization before distribution for omics studies for a more reliable analysis. Here, we compare individual omics layer readouts from fresh-frozen adjacent tissue pieces and homogenized powder in mouse brain, kidney, and liver. Genomics, transcriptomics, proteomics, and metabolomics analyses showed comparable RNA integrity, DNA methylation, and coverage of transcripts, proteins, and metabolites across both methods. Moreover, the homogenized-lyophilized powder usage led to reduced heterogeneity between biological replicates. We conclude that the cryogenically pulverized-lyophilized tissue approach not only maintains a critical molecular feature coverage and quality but also provides a homogenous basis for various omics analysis enhancing reproducibility, sample transport, storage and enabling multi omics base on one and the same tissue aliquot.

Thiopurines are effective drugs for inflammatory bowel disease, but their use is limited by side effects such as pancreatitis, whose mechanism remains unknown and may be more severe in children. This study investigated in a personalized way thiopurine-induced pancreatitis mechanism using induced pluripotent stem cells from pediatric inflammatory bowel disease patients. Ten pediatric patients, five developing pancreatitis (cases) and five without it (controls), were enrolled. Patient-specific stem cells and their pancreatic differentiated counterparts were used to evaluate thiopurine cytotoxicity, to quantify metabolites levels by liquid chromatography-tandem mass spectrometry, and to assess thiopurine pharmacodynamics by western-blot assay. Statistical analyses were performed applying Student's t-test or two-way ANOVA followed by Bonferroni's post-hoc test for multiple comparisons. Cytotoxicity assays revealed higher thioguanine cytotoxicity in stem and pancreatic cells from cases; pancreatic cells from cases were also more sensitive to mercaptopurine. Moreover, thioguanine treatment on stem cells produced thioguanosine monophosphate and its methylated form, but their concentration did not differ significantly between the groups. In addition, higher TPMT gene expression was observed in stem cells from cases, but no differences were observed in pancreatic cells. No significant differences were detected in HPRT, NUDT15, ITPA, or PACSIN2 expression. Lastly, Rac1 protein concentration was similar in stem cells from cases and controls, but pancreatic cells from cases exhibited significantly higher Rac1 expression. These findings suggest that thiopurine cytotoxicity differences might be linked to pharmacokinetics in stem cells, while altered Rac1 expression in pancreatic cells might contribute to pancreatitis, implicating distinct mechanisms between stem and differentiated cells.

Thiopurines are anticancer agents used for the treatment of leukemia and autoimmune diseases. These purine analogs are characterized by a narrow therapeutic index because of the risk of myelosuppression. With the discovery of NUDIX hydrolase 15 (NUDT15) as a major modulator of thiopurine metabolism and toxicity, we sought to comprehensively examine all members of the NUDIX hydrolase family for their effect on the pharmacologic effects of thiopurine. By performing a NUDIX-targeted CRISPR/Cas9 screen in leukemia cells, we identified NUDT5, whose depletion led to drastic thiopurine resistance. NUDT5 deficiency resulted in a nearly complete depletion of active metabolites of thiopurine and the loss of thioguanine incorporation into DNA. Mechanistically, NUDT5 deletion resulted in substantial alteration in purine nucleotide biosynthesis, as determined by steady-state metabolomics profiling. Stable isotope tracing demonstrated that the loss of NUDT5 was linked to a marked suppression of the purine salvage pathway but with minimal effects on purine de novo synthesis. Finally, we comprehensively identified germline genetic variants in NUDT5 associated with thiopurine-induced myelosuppression in 582 children with acute lymphoblastic leukemia. Collectively, these results pointed to NUDT5 as a key regulator of the thiopurine response primarily through its effects on purine homeostasis, highlighting its potential to inform individualized thiopurine therapy.

Background & Aims: Cholemic nephropathy (CN) is a severe complication of liver diseases associated with cholestasis and represents an unmet medical need. Recently, we identified the molecular mechanism of CN and showed that the systemic apical sodium-dependent bile acid transporter inhibitor (ASBTi) AS0369 prevented CN in mice. However, it is not clear if ASBTi is effective in a therapeutic rather than a preventive setting. Methods: AS0369 was administered daily for 4 weeks to bile duct-ligated (BDL) mice at four CN stages: (1) early stage with proximal tubular epithelial cell (pTEC) death (BDL-day 3); (2) inflammation, leaky peritubular capillaries, and tubular dilatation (BDL-day 21); (3) fibrosis (BDL-day 42); and (4) advanced stage with glomerular cysts (BDL-day 63). Disease progression was evaluated by biochemical, histopathological, and RNA-sequencing analysis. Results: ASBTi increased urinary excretion of bile acids (BAs) and reciprocally reduced BA concentrations in blood and renal tissue at all disease stages. Therapeutic efficacy was highest when ASBTi was given at early disease stages, e.g. urinary BA excretion was increased 9-fold (p <0.001) at the early stage compared to 4-fold (p = 0.021) at the late stage. ASBTi reduced the pTEC injury biomarker KIM-1, tissue damage, replacement proliferation, peritubular capillary damage and renal fibrosis. Additionally, late-stage disease features, such as glomerular cysts, were ameliorated (46% at the late stage, p = 0.005) by the ASBTi. RNA-sequencing revealed that ASBTi attenuated BDL-induced gene deregulation at all stages, with a larger effect size at early stages. Conclusions: Early systemic ASBTi therapy, initiated at the onset of pTEC death, provides the greatest therapeutic benefit. Nonetheless, even at later stages, ASBTi can ameliorate features of advanced CN. Impact and implications: This study demonstrates that systemic inhibition of the apical sodium-dependent bile acid transporter (ASBTi) alleviates cholemic nephropathy across disease stages in a bile duct ligation mouse model. The greatest benefit was achieved when treatment was initiated early, coinciding with proximal tubular epithelial cell death, but even advanced features such as glomerular cysts were partially reversed. These findings highlight ASBTi as a promising therapeutic strategy for cholemic nephropathy, addressing a major unmet need in cholestatic liver disease. By targeting bile acid accumulation and related injury pathways, ASBTi may improve renal outcomes and broaden treatment options in affected patients.

Thiopurine drugs are immunomodulatory antimetabolites relevant for pediatric patients characterized by dose-dependent adverse effects such as myelosuppression and hepatotoxicity, often related to inter-individual differences, involving the activity of important enzymes at the basis of their biotransformation, such as thiopurine S-methyltransferase (TPMT). Surface Enhanced Raman Scattering (SERS) spectroscopy is emerging as a bioanalytical tool and represents a valid alternative in terms of affordable costs, shorter analysis time and easier sample preparation in comparison to the most employed methods for pharmacokinetic analysis of drugs. The aim of this study is to investigate mercaptopurine and thioguanine pharmacokinetics by SERS in cell lysates of a B-lymphoblastoid cell line (NALM-6), that did (TPMT*1) or did not (MOCK) overexpress the wild-type form of TPMT as an in vitro cellular lymphocyte model to discriminate between cells with different levels of TPMT activity on the base of the amount of thioguanosine nucleotides (TGN) metabolites formed. SERS analysis of the cell lysates was carried out using SERS substrates constituted by Ag nanoparticles deposited on paper and parallel samples were used for quantification of thiopurine nucleotides with liquid chromatography-tandem mass spectrometry (LC-MS/MS). A direct SERS detection method has been set up that could be a tool to study thiopurine drug pharmacokinetics in in vitro cellular models to qualitatively discriminate between cells that do and do not overexpress the TPMT enzyme, as an alternative to other more laborious techniques. Results underlined decreased levels of TGN and increased levels of methylated metabolites when TPMT was overexpressed, both after mercaptopurine and thioguanine treatments. A strong positive correlation (Spearman's rank correlation coefficient rho = 0.96) exists between absolute quantification of TGMP (pmol/1 x 106 cells), obtained by LC-MS/MS, and SERS signal (intensity of TGN at 915 cm-1). In future studies, we aim to apply this method to investigate TPMT activity in pediatric patients' leukocytes.

Introduction Pharmacological targeting of the chemokine receptor ACKR3/CXCR7 regulates arterial thrombosis, platelet reactivity and thrombo-inflammatory response following myocardial infarction, those induced by immunothrombotic IgGs, and in coronary artery disease (CAD) patients ex vivo. CXCR7 agonist triggers the generation of anti-platelet lipid 12-HETrE which ligates the prostacyclin receptor and induces the platelet inhibitory cAMP dependent signaling cascade. Current investigation explores the antithrombotic and anticoagulatory implications of targeting CXCR7.

Abstract Background Thiopurines, azathioprine (AZA) and mercaptopurine (MP), are immunomodulators used to maintain remission in patients with inflammatory bowel disease (IBD). Intestinal epithelial cells play a key role in the pathogenesis of IBD, however, their role as target cells for thiopurines has not been elucidated. The aim of the project is to establish an in vitro IBD model using organoids starting from intestinal biopsies of IBD paediatric patients to investigate thiopurine effects on intestinal epithelium. Methods Forty-four IBD patients (mean age 14.4 years, 21 males) were enrolled. Intestinal biopsies were used to isolate crypts and generate adult stem cell-derived organoids. The cytotoxicity of thiopurines (0.2-200 µM for 72 hours) was evaluated by CellTiter-Glo 3D assay. Gene expression was evaluated using TaqMan assays. Thiopurine metabolites were quantified by LC-MS/MS analysis. Proteomic profiles were analyzed by Q-Exactive Plus mass spectrometer, western blot and REACTOME pathway software. Statistical analysis was performed using GraphPad and R software. Results IBD patients-derived organoids were sensitive to thiopurine treatment in a dose-dependent manner with a high interpatient variability (Two-way ANOVA p-value<0.0001; IC50 2.24 µM for AZA (95% CI: 1.28–5.53 µM) and 2.65 µM for MP (95% CI: 1.43–8.27 µM)).A significant negative correlation was observed between the viability after thiopurine exposure and mRNA expression of candidate genes involved in thiopurine pharmacokinetics (ITPA, TPMT, PACSIN2 and NUDT15). The most abundant thiopurine metabolite measured on organoids treated with IC50 of MP was MeTIMP, with a concentration correlated with cell viability after MP exposure (p=0.0056, ρ=-0.99). Proteomic analysis on organoids treated with IC20 of thiopurines (0.2 μM) showed 45 differentially expressed proteins in common between AZA and MP (p<0.05). REACTOME enrichment analysis revealed as the most altered pathways: vesicular traffic, autophagy, protein ubiquitination and interferon signalling, while no proteins related to apoptosis were induced. Among these proteins, TRIM32, a ubiquitin ligase involved in STING activity, a key regulator of both interferon I expression and NF-kB activation, resulted downregulated by the treatment (t-test AZA: p=0.0006; MP: p=0.014) and its basal levels negatively correlated with viability rates after thiopurine exposure (AZA: p=0.006, ρ=-0.85; MP: p=0.002, ρ=-0.88). Organoid exposure to thiopurines also decreased p-STING/STING ratio, particularly after MP treatment (t-test p=0.025), and increased autophagy, assessed as LC3-II levels (t-test AZA: p=0.0006; MP: p=0.014). Conclusion Organoid cultures provide new insights on the mechanism of action of thiopurines in the intestinal epithelium.