Published papers

Therapy resistance in acute myeloid leukemia (AML) remains a major clinical obstacle, particularly due to the persistence of leukemia stem cells (LSCs) capable of metabolic adaptation. While venetoclax (Ven) inhibits oxidative phosphorylation (OXPHOS), we found that Ven-resistant LSCs undergo glycolytic reprogramming to bypass OXPHOS inhibition. This metabolic shift is supported by enhanced ribosome biogenesis, sustained by upregulated de novo guanine nucleotide biosynthesis. Abundant guanine nucleotides suppress the impaired ribosome biogenesis checkpoint (IRBC), leading to TP53 destabilization and persistent MYC expression. Inhibition of inosine monophosphate dehydrogenases (IMPDH1/2) depletes guanine nucleotides, activates IRBC, stabilizes TP53, represses MYC, and impairs the metabolic shift to glycolysis. This metabolic rewiring disrupts LSC stemness and suppresses the reconstitution of human AML cells in xenotransplantation experiments. Notably, the suppression of LSC stemness was observed regardless of Ven resistance or the TP53 mutational status of AML cells. These findings reveal that mutation-independent TP53 inactivation is involved in resistant AML and suggest that targeting guanine nucleotide biosynthesis may offer a clinically actionable strategy to eradicate therapy-resistant LSCs.

Background: Cancer cells reprogram their metabolism to sustain energy production and biosynthesis for malignant proliferation; however, their metabolic phenotypes vary significantly across different growth environments, creating discrepancies between in vitro and in vivo findings. These inconsistencies pose challenges for translating metabolic research into clinical applications. The emergence of 3D culture models as in vitro systems that accurately mimic the in vivo environment can mitigate these challenges by providing conditions that reflect physiological architecture and metabolic interactions. Therefore, we investigated the impact of the purine metabolism enzyme hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) on the proliferation and metabolism of SCLC cells using 2D and 3D culture models, with the goal of identifying context-specific metabolic regulation not captured in conventional 2D cultures. Methods: We evaluated cell proliferation and performed metabolomic profiling of HPRT1-knockout (KO) SCLC cells grown in 2D cultures, two 3D culture systems, and mouse xenograft models. Metabolomic profiling was performed using CE-TOFMS, followed by PCA and pathway analysis. The expression of β-alanine (β-Ala) metabolism-related genes, including carnosine synthase 1 (CARNS1), was assessed by RNA-seq and RT-PCR. CARNS1 expression was further evaluated in publicly available lung cancer datasets, including both cell line and clinical tumor cohorts, to determine its association with patient prognosis and its correlation with HPRT1 expression. Results: The knockout of HPRT1 significantly reduced cell proliferation in 3D cultures and in vivo but had a minimal impact in 2D cultures. Comprehensive metabolomic analyses of HPRT1-KO cells revealed extensive alterations in amino acid and purine metabolism both in vitro and in vivo. Notably, the effect of HPRT1 KO on β-Ala metabolism differed between 2D and 3D cultures. In 3D cultures, HPRT1 KO led to increased expression of the endogenous antitumor metabolite carnosine and its biosynthetic enzyme CARNS1 within the β-Ala metabolic pathway. Furthermore, analysis of clinical databases showed that high CARNS1 expression correlated with improved prognosis in patients with lung cancer and negatively correlated with HPRT1 expression in tumor tissues. Conclusion: This study highlights the potential of 3D culture systems to elucidate context-specific mechanisms of metabolic regulation, such as the suppressive effect of HPRT1 on carnosine production. Our findings demonstrate that metabolic phenotypes observed in 2D cultures may not fully capture the complexity of in vivo metabolism, whereas 3D models can reveal regulatory pathways that are otherwise overlooked, including context-dependent regulation of carnosine metabolism by HPRT1.

Cellular senescence is deeply involved in physiological homeostasis, development, tissue repair, aging, and diseases. Senescent cells (SnCs) accumulate in aged tissues and exert deleterious effects by secreting proinflammatory molecules that contribute to chronic inflammation and aging-related diseases. We revealed that an aberrant interaction between glycolytic PGAM1 and Chk1 kinase is augmented in SnCs associated with increased glycolysis, whose byproduct, lactate, promotes this binding in a noncell autonomous manner. The pseudo-Warburg effect of SnCs with enhanced PPP (pentose phosphate pathway) activity is maintained by HIF-2α phosphorylation by Chk1 and subsequent upregulation of glycolytic enzymes, creating a vicious cycle reprogramming the glycolytic pathway in SnCs. HIF-2α also activates FoxM1 expression, which transcriptionally suppresses proapoptotic profiles, including BIM, and upregulates DNA repair machineries in SnCs. FoxM1 thus supports the genomic integrity and survival capacity of SnCs during their glycolytic changes. Chemical abrogation of PGAM1-Chk1 binding reverts these phenotypes and eliminates SnCs through senolysis. Inhibition of the PGAM1-Chk1 interaction improves physiological parameters during aging and inhibits lung fibrosis in mouse models. Our study highlights a novel pathway contributing to the metabolic reprogramming of SnCs and how the use of a new senolytic molecule that targets the PGAM-Chk1 interaction creates a specific vulnerability of those cells to potentially fight age-related diseases.

Background: Depression represents a major global disease burden. While the Center for Epidemiologic Studies Depression Scale (CES-D) is widely used, it relies on subjective self-reporting. Saliva enables non-invasive metabolite analysis, but existing metabolomics studies have focused on clinical depression using blood or urine, with limited investigation of salivary metabolites in community populations. This study investigated associations between CES-D scores and salivary metabolite concentrations in Tokyo residents. Methods: This cross-sectional study collected CES-D responses and morning saliva samples from Tokyo residents. Salivary metabolites were analyzed using capillary electrophoresis time-of-flight mass spectrometry. Statistical analyses included permutational multivariate analysis of variance (PERMANOVA) for overall metabolite-CES-D associations, pathway enrichment analysis using fast gene set enrichment analysis (FGSEA), and principal component analysis, adjusted for age and sex. Results: Analysis included 107 participants (40 males, 67 females; mean age 55.3 ± 14.2 years). PERMANOVA revealed significant association between 112 salivary metabolites and CES-D scores (F = 2.870, p = 0.003), with metabolites explaining 7.7% of depressive symptom variance. Pathway enrichment analysis identified significant alterations in amino acid metabolism (normalized enrichment score =-1.750, q = 0.007). Glycolysis and amine/polyamine metabolism showed trend-level enrichment (both q = 0.066). Principal component analysis revealed significant correlations between CES-D scores and pathway activity for amino acid metabolism (r = 0.216, p = 0.027) and amine/polyamine metabolism (r = 0.216, p = 0.027). Conclusion: This study demonstrates significant associations between salivary metabolite profiles and depressive symptoms in a nonclinical community population, with amino acid metabolism emerging as the most robustly altered pathway. These findings provide preliminary evidence for associations between salivary metabolomic profiles and depressive symptoms in a nonclinical community population.

Chronic kidney disease (CKD) is a life-threatening condition, and constipation is a progressive risk factor. We evaluated changes in uremic toxins, renal function, and the safety of lubiprostone, a selective chloride channel activator, in patients with CKD. In this phase 2, randomized, double-blind, placebo-controlled trial across nine centers in Japan, 150 patients with stage IIIb-IV CKD received lubiprostone (8 or 16 micrograms) or placebo for 24 weeks. The primary end point was change in indoxyl sulfate levels. Secondary end points included other uremic toxins and renal function markers. Lubiprostone did not alter uremic toxin levels but improved or preserved estimated glomerular filtration rate and its slope in the 16-microgram group. Mild-to-moderate gastrointestinal events occurred in the placebo and 16-microgram groups. Multiomics analysis revealed that lubiprostone modulated the gut microbial agmatine pathway and increased spermidine levels, thereby improving renal mitochondrial function. Lubiprostone is a previously unknown and safe therapeutic option to mitigate renal decline in CKD.

Background/objective: Ulcerative colitis (UC) is significantly linked with gut microbiota, which is essential for maintaining gut health. Their metabolites mitigate gut inflammation and bolster barrier function. Among these metabolites, we focused on vitamin B3, which has been reported to improve the pathogenesis of UC in mice. This study aimed to compare fecal vitamin B3 and gut microbiota between non-UC and UC patients. Methods: We assessed fecal metabolites and gut microbiota in 71 UC patients (UC group) and 72 non-UC patients (non-UC group) matched by sex and age in 10-year intervals. Fecal samples were collected and metabolites were analyzed using capillary electrophoresis time-of-flight mass spectrometry. Bacterial DNA was extracted for 16S rRNA gene sequencing. We analyzed fecal nicotinamide levels and gut microbiota composition, employing statistical adjustments for confounding factors. Results: We found that the UC group exhibited significantly lower fecal nicotinamide levels and α-diversity (Shannon index) compared to the non-UC group. The relative abundance of bacterial genera such as Treponema, UCG-002, and Fusicatenibacter was decreased, while Sellimonas, Fournierella, and Oscillospira were increased in the UC group. Moreover, a negative correlation was observed between Sellimonas abundance and fecal nicotinamide levels in the UC group. Additionally, the UC group showed higher expression of a bacterial gene encoding nicotinamidase compared to the non-UC group. Conclusions: These findings suggest that gut microbiota dysbiosis contributes to reduced vitamin B3 metabolism in UC patients. The study highlights the potential of replenishing vitamin B3 metabolic pathways as a novel therapeutic approach for UC treatment. Keywords: gut microbiota; inflammatory bowel disease; nicotinamide; ulcerative colitis; vitamin B3.

Tyrosine kinase inhibitors are initially efficacious against anaplastic lymphoma kinase (ALK) fusion gene-positive lung adenocarcinoma, but acquired resistance inevitably occurs. Therefore, alternative treatment strategies are needed for tyrosine kinase inhibitor-resistant cases. Although the use of immune checkpoint inhibitors (ICI) has improved the prognosis of patients with lung cancer, patients with ALK+ lung adenocarcinoma exhibit little or no response to immunotherapy and the underlying resistance mechanisms remain unknown. In this study, we explored the immunologic status of the tumor microenvironment (TME) in ALK+ lung adenocarcinoma tissues. Tumor-infiltrating leukocyte analysis revealed reduced numbers of effector T cells and increased myeloid-derived suppressor cells (MDSC) relative to ALK- lung adenocarcinoma cases, indicating that ALK+ lung adenocarcinoma has a myeloid cell-dominant immunosuppressive TME. Single-cell RNA sequencing analysis identified a subset of macrophages that expressed most T cell-attractant chemokines (CXCL9, CXCL10, and CXCL11), and the macrophages were inactivated in ALK+ lung adenocarcinoma. In contrast, ALK+ lung adenocarcinoma expressed high levels of MDSC-attractant chemokines (CXCL1 and CXCL8). In addition, ALK+ lung adenocarcinoma showed higher levels of IL6, an MDSC-inducing cytokine, than ALK- lung adenocarcinoma. An IL6R inhibitor transformed the TME in a murine ALK+ lung adenocarcinoma model, shifting it from an immunosuppressive to a T cell-dominant status. Although ICI monotherapy lacked antitumor effects, a combination of ICI and the IL6R inhibitor had significant antitumor effects in mice. Our findings illustrate the molecular basis of fusion gene-mediated immunosuppressive TMEs, providing a rationale for a novel combination immunotherapy for ALK+ lung adenocarcinoma. See related Spotlight by Vitale and Bria, p.1326.

It has been reported that accumulation of senescent cells in various tissues contributes to pathological aging and that elimination of senescent cells (senolysis) improves age-associated pathologies. Here, we demonstrate that inhibition of sodium-glucose co-transporter 2 (SGLT2) enhances clearance of senescent cells, thereby ameliorating age-associated phenotypic changes. In a mouse model of dietary obesity, short-term treatment with the SGLT2 inhibitor canagliflozin reduced the senescence load in visceral adipose tissue and improved adipose tissue inflammation and metabolic dysfunction, but normalization of plasma glucose by insulin treatment had no effect on senescent cells. Canagliflozin extended the lifespan of mice with premature aging even when treatment was started in middle age. Metabolomic analyses revealed that short-term treatment with canagliflozin upregulated 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, enhancing immune-mediated clearance of senescent cells by downregulating expression of programmed cell death-ligand 1. These findings suggest that inhibition of SGLT2 has an indirect senolytic effect by enhancing endogenous immunosurveillance of senescent cells.

Oncogenes often promote cell death as well as proliferation. How oncogenes drive these diametrically opposed phenomena remains to be solved. A key question is whether cell death occurs as a response to aberrant proliferation signals or through a proliferation-independent mechanism. Here, we reveal that Src, the first identified oncogene, simultaneously drives cell proliferation and death in an obligatorily coupled manner through parallel MAPK pathways. The two MAPK pathways diverge from a lynchpin protein Slpr. A MAPK p38 drives proliferation whereas another MAPK JNK drives apoptosis independently of proliferation signals. Src-p38-induced proliferation is regulated by methionine-mediated Tor signaling. Reduction of dietary methionine uncouples the obligatory coupling of cell proliferation and death, suppressing tumorigenesis and tumor-induced lethality. Our findings provide an insight into how cells evolved to have a fail-safe mechanism that thwarts tumorigenesis by the oncogene Src. We also exemplify a diet-based approach to circumvent oncogenesis by exploiting the fail-safe mechanism.

Aims: Cardiac ischaemia/reperfusion (I/R) injury remains a critical issue in the therapeutic management of ischaemic heart failure. Although mild hypothermia has a protective effect on cardiac I/R injury, more rapid and safe methods that can obtain similar results to hypothermia therapy are required. 2-Methyl-2-thiazoline (2MT), an innate fear inducer, causes mild hypothermia resulting in resistance to critical hypoxia in cutaneous or cerebral I/R injury. The aim of this study is to demonstrate the protective effect of systemically administered 2MT on cardiac I/R injury and to elucidate the mechanism underlying this effect. Methods and results: A single subcutaneous injection of 2MT (50 mg/kg) was given prior to reperfusion of the I/R injured 10 week-old male mouse heart and its efficacy was evaluated 24 h after the ligation of the left anterior descending coronary artery. 2MT preserved left ventricular systolic function following I/R injury (ejection fraction, %: control 37.9 ± 6.7, 2MT 54.1 ± 6.4, P < 0.01). 2MT also decreased infarct size (infarct size/ischaemic area at risk, %: control 48.3 ± 12.1, 2MT 25.6 ± 4.2, P < 0.05) and serum cardiac troponin levels (ng/mL: control 8.9 ± 1.1, 2MT 1.9 ± 0.1, P < 0.01) after I/R. Moreover, 2MT reduced the oxidative stress-exposed area within the heart (%: control 25.3 ± 4.7, 2MT 10.8 ± 1.4, P < 0.01). These results were supported by microarray analysis of the mouse hearts. 2MT induced a transient, mild decrease in core body temperature (°C: -2.4 ± 1.4), which gradually recovered over several hours. Metabolome analysis of the mouse hearts suggested that 2MT minimized energy metabolism towards suppressing oxidative stress. Furthermore, 18F-fluorodeoxyglucose-positron emission tomography/computed tomography imaging revealed that 2MT reduced the activity of brown adipose tissue (standardized uptake value: control 24.3 ± 6.4, 2MT 18.4 ± 5.8, P < 0.05). 2MT also inhibited mitochondrial respiration and glycolysis in rat cardiomyoblasts. Conclusions: We identified the cardioprotective effect of systemically administered 2MT on cardiac I/R injury by sparing energy metabolism with reversible hypothermia. Our results highlight the potential of drug-induced hypothermia therapy as an adjunct to coronary intervention in severe ischaemic heart disease.

Oral glucose ingestion induces systemic changes of many blood metabolites related not only to glucose, but also other metabolites such as amino acids and lipids through many blood hormones. However, the detailed temporal changes in the concentrations of comprehensive metabolites and hormones over a long time by oral glucose ingestion are uncharacterized. We measured 83 metabolites and 7 hormones in 20 healthy human subjects in response to glucose ingestion. We characterized temporal patterns of blood molecules by four features: (i) the decomposability into "amplitude" and "rate" components, (ii) the similarity of temporal patterns among individuals, (iii) the relation of molecules over time among individuals, and (iv) the similarity of temporal patterns among molecules. Glucose and glucose metabolism-related hormones indicated a rapid increase, and citrulline and lipids, which indicated a rapid decrease, returned to fasting levels faster than amino acids. Compared to glucose metabolism-related molecules and lipids, amino acids showed similar temporal patterns among individuals. The four features of temporal patterns of blood molecules by oral glucose ingestion characterize the differences among individuals and among molecules.

Glucose homeostasis is maintained by modulation of metabolic flux. Enzymes and metabolites regulate the involved metabolic pathways. Dysregulation of glucose homeostasis is a pathological event in obesity. Analyzing metabolic pathways and the mechanisms contributing to obesity-associated dysregulation in vivo is challenging. Here, we introduce OMELET: Omics-Based Metabolic Flux Estimation without Labeling for Extended Trans-omic Analysis. OMELET uses metabolomic, proteomic, and transcriptomic data to identify relative changes in metabolic flux, and to calculate contributions of metabolites, enzymes, and transcripts to the changes in metabolic flux. By evaluating the livers of fasting ob/ob mice, we found that increased metabolic flux through gluconeogenesis resulted primarily from increased transcripts, whereas that through the pyruvate cycle resulted from both increased transcripts and changes in substrates of metabolic enzymes. With OMELET, we identified mechanisms underlying the obesity-associated dysregulation of metabolic flux in the liver.

Accumulating evidence indicates that the gut microbiome and metabolites are associated with colorectal cancer (CRC). However, the influence of surgery for CRC treatment on the gut microbiome and metabolites and how it relates to CRC risk in postoperative CRC patients remain partially understood. Here, we collected 170 fecal samples from 85 CRC patients pre- and approximately 1 year postsurgery and performed shotgun metagenomic sequencing and capillary electrophoresis-time of flight mass spectrometry-based metabolomics analyses to characterize alterations between pre- and postsurgery. We determined that the relative abundance of 114 species was altered postsurgery (P < 0.005). CRC-associated species, such as Fusobacterium nucleatum, were decreased postsurgery. On the other hand, Clostridium scindens, carcinogenesis-associated deoxycholate (DCA)-producing species, and its biotransformed genes (bai operon) were increased postsurgery. The concentration of 60 fecal metabolites was also altered postsurgery (P < 0.005). Two bile acids, cholate and DCA, were increased postsurgery. We developed methods to estimate postoperative CRC risk based on the gut microbiome and metabolomic compositions using a random forest machine-learning algorithm that classifies large adenoma or early-stage CRC and healthy controls from publicly available data sets. We applied methods to preoperative samples and then compared the estimated CRC risk between the two groups according to the presence of large adenoma or tumors 5 years postsurgery (P < 0.05). Overall, our results show that the gut microbiome and metabolites dynamically change from pre- to postsurgery. In postoperative CRC patients, potential CRC risk derived from gut microbiome and metabolites still remains, which indicates the importance of follow-up assessments. IMPORTANCE The gut microbiome and metabolites are associated with CRC progression and carcinogenesis. Postoperative CRC patients are reported to be at an increased CRC risk; however, how gut microbiome and metabolites are related to CRC risk in postoperative patients remains only partially understood. In this study, we investigated the influence of surgical CRC treatment on the gut microbiome and metabolites. We found that the CRC-associated species Fusobacterium nucleatum was decreased postsurgery, whereas carcinogenesis-associated DCA and its producing species and genes were increased postsurgery. We developed methods to estimate postoperative CRC risk based on the gut microbiome and metabolomic compositions. We applied methods to compare the estimated CRC risk between two groups according to the presence of large adenoma or tumors after 5 years postsurgery. To our knowledge, this study is the first report on differences between pre- and postsurgery using metagenomics and metabolomics data analysis. Our methods might be used for CRC risk assessment in postoperative patients.

Resistance to immune checkpoint blockade remains challenging in patients with non-small cell lung cancer (NSCLC). Tumor-infiltrating leukocyte (TIL) quantity, composition, and activation status profoundly influence responsiveness to cancer immunotherapy. This study examined the immune landscape in the NSCLC tumor microenvironment by analyzing TIL profiles of 281 fresh resected NSCLC tissues. Unsupervised clustering based on numbers and percentages of 30 TIL types classified adenocarcinoma (LUAD) and squamous cell carcinoma (LUSQ) into the cold, myeloid cell-dominant, and CD8+ T cell-dominant subtypes. These were significantly correlated with patient prognosis; the myeloid cell subtype had worse outcomes than the others. Integrated genomic and transcriptomic analyses, including RNA sequencing, whole-exome sequencing, T-cell receptor repertoire, and metabolomics of tumor tissue, revealed that immune reaction-related signaling pathways were inactivated, while the glycolysis and K-ras signaling pathways activated in LUAD and LUSQ myeloid cell subtypes. Cases with ALK and ROS1 fusion genes were enriched in the LUAD myeloid subtype, and the frequency of TERT copy-number variations was higher in LUSQ myeloid subtype than in the others. These classifications of NSCLC based on TIL status may be useful for developing personalized immune therapies for NSCLC. Significance: The precise TIL profiling classified NSCLC into novel three immune subtypes that correlates with patient outcome, identifying subtype-specific molecular pathways and genomic alterations that should play important roles in constructing subtype-specific immune tumor microenvironments. These classifications of NSCLC based on TIL status are useful for developing personalized immune therapies for NSCLC.

Cancer-specific metabolic activities play a crucial role in the pathogenesis of human malignancies. To investigate human acute leukemia-specific metabolic properties, we comprehensively measured the cellular metabolites within the CD34+ fraction of normal hematopoietic stem progenitor cells (HSPCs), primary human acute myelogenous leukemia (AML), and acute lymphoblastic leukemia (ALL) cells. Here, we show that human leukemia cells are addicted to the branched-chain amino acid (BCAA) metabolism to maintain their stemness, irrespective of myeloid or lymphoid types. Human primary acute leukemias had BCAA transporters for BCAA uptake, cellular BCAA, α-ketoglutarate (α-KG), and cytoplasmic BCAA transaminase-1 (BCAT1) at significantly higher levels than control HSPCs. Isotope-tracing experiments showed that in primary leukemia cells, BCAT1 actively catabolizes BCAA using α-KG into branched-chain α-ketoacids, whose metabolic processes provide leukemia cells with critical substrates for the trichloroacetic acid cycle and the synthesis of nonessential amino acids, both of which reproduce α-KG to maintain its cellular level. In xenogeneic transplantation experiments, deprivation of BCAA from daily diet strongly inhibited expansion, engraftment and self-renewal of human acute leukemia cells. Inhibition of BCAA catabolism in primary AML or ALL cells specifically inactivates the function of the polycomb repressive complex 2, an epigenetic regulator for stem cell signatures, by inhibiting the transcription of PRC components, such as zeste homolog 2 and embryonic ectoderm development. Accordingly, BCAA catabolism plays an important role in the maintenance of stemness in primary human AML and ALL, and molecules related to the BCAA metabolism pathway should be critical targets for acute leukemia treatment.

Growth regulation by estrogen in breast cancer 1 (GREB1) is involved in hormone-dependent and -independent tumor development (e.g., hepatoblastoma). In this study, we found that a GREB1 splicing variant, isoform 4 (Is4), which encodes C-terminal half of full-length GREB1, is specifically expressed via microphthalmia-associated transcription factor (MITF) in melanocytic melanoma, and that two MITF-binding E-box CANNTG motifs at the 5'-upstream region of GREB1 exon 19 are necessary for GREB1 Is4 transcription. MITF and GREB1 Is4 were strongly co-expressed in approximately 20% of the melanoma specimens evaluated (17/89 cases) and their expression was associated with tumor thickness. GREB1 Is4 silencing reduced melanoma cell proliferation in association with altered expression of cell proliferation-related genes in vitro. In addition, GREB1 Is4 targeting by antisense oligonucleotide (ASO) decreased melanoma xenograft tumor formation and GREB1 Is4 expression in a BRAFV600E; PTENflox melanoma mouse model promoted melanoma formation, demonstrating the crucial role of GREB1 Is4 for melanoma proliferation in vivo. GREB1 Is4 bound to CAD, the rate-limiting enzyme of pyrimidine metabolism, and metabolic flux analysis revealed that GREBI Is4 is necessary for pyrimidine synthesis. These results suggest that MITF-dependent GREB1 Is4 expression leads to melanoma proliferation and GREB1 Is4 represents a new molecular target in melanoma.

Guanosine triphosphate (GTP) and adenosine triphosphate (ATP) are essential nucleic acid building blocks and serve as energy molecules for a wide range of cellular reactions. Cellular GTP concentration fluctuates independently of ATP and is significantly elevated in numerous cancers, contributing to malignancy. Quantitative measurement of ATP and GTP has become increasingly important to elucidate how concentration changes regulate cell function. Liquid chromatography-coupled mass spectrometry (LC-MS) and capillary electrophoresis-coupled MS (CE-MS) are powerful methods widely used for the identification and quantification of biological metabolites. However, these methods have limitations related to specialized instrumentation and expertise, low throughput, and high costs. Here, we introduce a novel quantitative method for GTP concentration monitoring (GTP-quenching resonance energy transfer (QRET)) in homogenous cellular extracts. CE-MS analysis along with pharmacological control of cellular GTP levels shows that GTP-QRET possesses high dynamic range and accuracy. Furthermore, we combined GTP-QRET with luciferase-based ATP detection, leading to a new technology, termed QT-LucGTP&ATP, enabling high-throughput compatible dual monitoring of cellular GTP and ATP in a homogenous fashion. Collectively, GTP-QRET and QT-LucGTP&ATP offer a unique, high-throughput opportunity to explore cellular energy metabolism, serving as a powerful platform for the development of novel therapeutics and extending its usability across a range of disciplines.

The gut microbiota is closely associated with human health and is greatly impacted by the host diet. Bacteria such as Escherichia coli live in the gut all throughout the life of a human host and adapt to the intestinal environment. Adaptive mutations in E. coli are reported to enhance fitness in the mammalian intestine, but to what extent is still poorly known. It is also unknown whether the host diet affects what genes are mutated and to what extent fitness is affected. This study suggests that genetic mutations in the E. coli K-12 strain are selected in response to the intestinal environment and facilitate efficient utilization of abundant nutrients in the germ-free mouse intestine. Our study provides a better understanding of these intestinal adaptation mechanisms of gut microbes.

In autosomal dominant polycystic kidney disease (ADPKD), renal cyst lesions predominantly arise from collecting ducts (CDs). However, relevant CD cyst models using human cells are lacking. Although previous reports have generated in vitro renal tubule cyst models from human induced pluripotent stem cells (hiPSCs), therapeutic drug candidates for ADPKD have not been identified. Here, by establishing expansion cultures of hiPSC-derived ureteric bud tip cells, an embryonic precursor that gives rise to CDs, we succeed in advancing the developmental stage of CD organoids and show that all CD organoids derived from PKD1-/- hiPSCs spontaneously develop multiple cysts, clarifying the initiation mechanisms of cystogenesis. Moreover, we identify retinoic acid receptor (RAR) agonists as candidate drugs that suppress in vitro cystogenesis and confirm the therapeutic effects on an ADPKD mouse model in vivo. Therefore, our in vitro CD cyst model contributes to understanding disease mechanisms and drug discovery for ADPKD.

Capillary electrophoresis mass spectrometry (CE-MS) is now regarded as a powerful tool for the comprehensive analysis of low-molecular-weight charged metabolites. Over the past decades, several CE-MS methods have been established and made significant contributions to metabolomics research in various areas. This review describes the general strategies and principles of the CE-MS-based metabolomics analyses, including sample preparation, CE separation modes, interfacing techniques, and mass spectrometers. Furthermore, new techniques to increase throughput ability in CE-MS for metabolomics are highlighted. Finally, general conclusions and perspectives are provided.