Acetyl-CoA synthetase 2 contributes to a better prognosis for liver cancer by switching acetate-glucose metabolism.

Acetyl-CoA synthetase 2 (ACSS2)-dependent acetate usage has generally been associated with tumorigenesis and increased malignancy in cancers under nutrient-depleted conditions. However, the nutrient usage and metabolic characteristics of the liver differ from those of other organs; therefore, the mechanism of ACSS2-mediated acetate metabolism may also differ in liver cancer. To elucidate the underlying mechanisms of ACSS2 in liver cancer and acetate metabolism, the relationships between patient acetate uptake and metabolic characteristics and between ACSS2 and tumor malignancies were comprehensively studied in vitro, in vivo and in humans. Clinically, we initially found that ACSS2 expression was decreased in liver cancer patients. Moreover, PET-CT imaging confirmed that lower-grade cancer cells take up more 11C-acetate but less 18F-fluorodeoxyglucose (18F-FDG); however, this trend was reversed in higher-grade cancer. Among liver cancer cells, those with high ACSS2 expression avidly absorbed acetate even in a glucose-sufficient environment, whereas those with low ACSS2 expression did not, thereby showing correlations with their respective ACSS2 expression. Metabolomic isotope tracing in vitro and in vivo revealed greater acetate incorporation, greater lipid anabolic metabolism, and less malignancy in high-ACSS2 tumors. Notably, ACSS2 downregulation in liver cancer cells was associated with increased tumor occurrence in vivo. In human patient cohorts, patients in the low-ACSS2 subgroup exhibited reduced anabolism, increased glycolysis/hypoxia, and poorer prognosis. We demonstrated that acetate uptake by ACSS2 in liver cancer is independent of glucose depletion and contributes to lipid anabolic metabolism and reduced malignancy, thereby leading to a better prognosis for liver cancer patients.

Visualizing Cancer-Originating Acetate Uptake Through MCT1 in Reactive Astrocytes in the Glioblastoma Tumor Microenvironment.

BACKGROUND: Reactive astrogliosis is a hallmark of various brain pathologies, including neurodegenerative diseases and glioblastomas. However, the specific intermediate metabolites contributing to reactive astrogliosis remain unknown. This study investigated how glioblastomas induce reactive astrogliosis in the neighboring microenvironment and explores 11C-acetate PET as an imaging technique for detecting reactive astrogliosis. METHODS: Through in vitro, mouse models, and human tissue experiments, we examined the association between elevated 11C-acetate uptake and reactive astrogliosis in gliomas. We explored acetate from glioblastoma cells, which triggers reactive astrogliosis in neighboring astrocytes by upregulating MAO-B and MCT1 expression. We evaluated the presence of cancer stem cells in the reactive astrogliosis region of glioblastomas and assessed the correlation between the volume of 11C-acetate uptake beyond MRI and prognosis. RESULTS: Elevated 11C-acetate uptake is associated with reactive astrogliosis and astrocytic MCT1 in the periphery of glioblastomas in human tissues and mouse models. Glioblastoma cells exhibit increased acetate production as a result of glucose metabolism, with subsequent secretion of acetate. Acetate derived from glioblastoma cells induces reactive astrogliosis in neighboring astrocytes by increasing the expression of MAO-B and MCT1. We found cancer stem cells within the reactive astrogliosis at the tumor periphery. Consequently, a larger volume of 11C-acetate uptake beyond contrast-enhanced MRI was associated with worse prognosis. CONCLUSION: Our results highlight the role of acetate derived from glioblastoma cells in inducing reactive astrogliosis and underscore the potential value of 11C-acetate PET as an imaging technique for detecting reactive astrogliosis, offering important implications for the diagnosis and treatment of glioblastomas.

Evaluation of Bacterial and Fungal Biomarkers for Differentiation and Prognosis of Patients with Inflammatory Bowel Disease.

This study aimed to evaluate bacterial and fungal biomarkers to differentiate patients with inflammatory bowel disease (IBD), predict the IBD prognosis, and determine the relationship of these biomarkers with IBD pathogenesis. The composition and function of bacteria and fungi in stool from 100 IBD patients and 97 controls were profiled using next-generation sequencing. We evaluated the cumulative risk of relapse according to bacterial and fungal enterotypes. The microbiome and mycobiome alpha diversity in IBD patients were significantly lower and higher than in the controls, respectively; the micro/mycobiome beta diversity differed significantly between IBD patients and the controls. Ruminococcus gnavus, Cyberlindnera jadinii, and Candida tropicalis increased in IBD patients. Combining functional and species analyses revealed that lower sugar import and higher modified polysaccharide production were associated with IBD pathogenesis. Tricarboxylic acid cycling consuming acetyl CoA was higher in IBD patients than the controls, leading to lower short-chain fatty acid (SCFA) fermentation. Bacterial and fungal enterotypes were not associated with IBD relapse. We found differences in bacterial and fungal species between IBD patients and controls. A working model for the role of gut bacteria in IBD pathogenesis is proposed, wherein bacterial species increase modified N-glycan production and decrease SCFA fermentation.

Lactate as a major epigenetic carbon source for histone acetylation via nuclear LDH metabolism.

Histone acetylation involves the transfer of two-carbon units to the nucleus that are embedded in low-concentration metabolites. We found that lactate, a high-concentration metabolic byproduct, can be a major carbon source for histone acetylation through oxidation-dependent metabolism. Both in cells and in purified nuclei, 13C3-lactate carbons are incorporated into histone H4 (maximum incorporation: ~60%). In the purified nucleus, this process depends on nucleus-localized lactate dehydrogenase (LDHA), knockout (KO) of which abrogates incorporation. Heterologous expression of nucleus-localized LDHA reverses the KO effect. Lactate itself increases histone acetylation, whereas inhibition of LDHA reduces acetylation. In vitro and in vivo settings exhibit different lactate incorporation patterns, suggesting an influence on the microenvironment. Higher nuclear LDHA localization is observed in pancreatic cancer than in normal tissues, showing disease relevance. Overall, lactate and nuclear LDHA can be major structural and regulatory players in the metabolism-epigenetics axis controlled by the cell's own status or the environmental status.

Metabolomic profiling of overnight peritoneal dialysis effluents predicts the peritoneal equilibration test type.

This study primarily aimed to evaluate whether peritoneal equilibration test (PET) results can be predicted through the metabolomic analysis of overnight peritoneal dialysis (PD) effluents. From a total of 125 patients, overnight PD effluents on the day of the first PET after PD initiation were analyzed. A modified 4.25% dextrose PET was performed, and the PET type was categorized according to the dialysate-to-plasma creatinine ratio at the 4-h dwell time during the PET as follows: high, high average, low average, or low transporter. Nuclear magnetic resonance (NMR)-based metabolomics was used to analyze the effluents and identify the metabolites. The predictive performances derived from the orthogonal projection to latent structure discriminant analysis (OPLS-DA) modeling of the NMR spectrum were estimated by calculating the area under the curve (AUC) using receiver operating characteristic curve analysis. The OPLS-DA score plot indicated significant metabolite differences between high and low PET types. The relative concentrations of alanine and creatinine were greater in the high transporter type than in the low transporter type. The relative concentrations of glucose and lactate were greater in the low transporter type than in the high transporter type. The AUC of a composite of four metabolites was 0.975 in distinguish between high and low PET types. Measured PET results correlated well with the total NMR metabolic profile of overnight PD effluents.

Gut Microbial Genes and Metabolism for Methionine and Branched-Chain Amino Acids in Diabetic Nephropathy.

Diabetic mellitus nephropathy (DMN) is a serious complication of diabetes and a major health concern. Although the pathophysiology of diabetes mellitus (DM) leading to DMN is uncertain, recent evidence suggests the involvement of the gut microbiome. This study aimed to determine the relationships among gut microbial species, genes, and metabolites in DMN through an integrated clinical, taxonomic, genomic, and metabolomic analysis. Whole-metagenome shotgun sequencing and nuclear magnetic resonance metabolomic analyses were performed on stool samples from 15 patients with DMN and 22 healthy controls. Six bacterial species were identified to be significantly elevated in the DMN patients after adjusting for age, sex, body mass index, and estimated glomerular filtration rate (eGFR). Multivariate analysis found 216 microbial genes and 6 metabolites (higher valine, isoleucine, methionine, valerate, and phenylacetate levels in the DMN group and higher acetate levels in the control group) that were differentially present between the DMN and control groups. Integrated analysis of all of these parameters and clinical data using the random-forest model showed that methionine and branched-chain amino acids (BCAAs) were among the most significant features, next to the eGFR and proteinuria, in differentiating the DMN group from the control group. Metabolic pathway gene analysis of BCAAs and methionine also revealed that many genes involved in the biosynthesis of these metabolites were elevated in the six species that were more abundant in the DMN group. The suggested correlation among taxonomic, genetic, and metabolic features of the gut microbiome would expand our understanding of gut microbial involvement in the pathogenesis of DMN and may provide potential therapeutic targets for DMN. IMPORTANCE Whole metagenomic sequencing uncovered specific members of the gut microbiota associated with DMN. The gene families derived from the discovered species are involved in the metabolic pathways of methionine and branched-chain amino acids. Metabolomic analysis using stool samples showed increased methionine and branched-chain amino acids in DMN. These integrative omics results provide evidence of the gut microbiota-associated pathophysiology of DMN, which can be further studied for disease-modulating effects via prebiotics or probiotics.

GPX8 regulates clear cell renal cell carcinoma tumorigenesis through promoting lipogenesis by NNMT.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC), with its hallmark phenotype of high cytosolic lipid content, is considered a metabolic cancer. Despite the implication of this lipid-rich phenotype in ccRCC tumorigenesis, the roles and regulators of de novo lipid synthesis (DNL) in ccRCC remain largely unexplained. METHODS: Our bioinformatic screening focused on ccRCC-lipid phenotypes identified glutathione peroxidase 8 (GPX8), as a clinically relevant upstream regulator of DNL. GPX8 genetic silencing was performed with CRISPR-Cas9 or shRNA in ccRCC cell lines to dissect its roles. Untargeted metabolomics, RNA-seq analyses, and other biochemical assays (e.g., lipid droplets staining, fatty acid uptake, cell proliferation, xenograft, etc.) were carried out to investigate the GPX8's involvement in lipid metabolism and tumorigenesis in ccRCC. The lipid metabolic function of GPX8 and its downstream were also measured by isotope-tracing-based DNL flux measurement. RESULTS: GPX8 knockout or downregulation substantially reduced lipid droplet levels (independent of lipid uptake), fatty acid de novo synthesis, triglyceride esterification in vitro, and tumor growth in vivo. The downstream regulator was identified as nicotinamide N-methyltransferase (NNMT): its knockdown phenocopied, and its expression rescued, GPX8 silencing both in vitro and in vivo. Mechanically, GPX8 regulated NNMT via IL6-STAT3 signaling, and blocking this axis suppressed ccRCC survival by activating AMPK. Notably, neither the GPX8-NNMT axis nor the DNL flux was affected by the von Hippel Lindau (VHL) status, the conventional regulator of ccRCC high lipid content. CONCLUSIONS: Taken together, our findings unravel the roles of the VHL-independent GPX8-NNMT axis in ccRCC lipid metabolism as related to the phenotypes and growth of ccRCC, which may be targeted for therapeutic purposes.

TAK-981, a SUMOylation inhibitor, suppresses AML growth immune-independently.

Acute myeloid leukemia (AML) generally has an unsatisfactory prognosis despite the recent introduction of new regimens, including targeted agents and antibodies. To find a new druggable pathway, we performed integrated bioinformatic pathway screening on large OHSU and MILE AML databases, discovered the SUMOylation pathway, and validated it independently with an external data set (totaling 2959 AML and 642 normal sample data). The clinical relevance of SUMOylation in AML was supported by its core gene expression which is correlated with patient survival, European LeukemiaNet 2017 risk classification, and AML-relevant mutations. TAK-981, a first-in-class SUMOylation inhibitor currently under clinical trials for solid tumors, showed antileukemic effects with apoptosis induction, cell-cycle arrest, and induction of differentiation marker expression in leukemic cells. It exhibited potent nanomolar activity, often stronger than that of cytarabine, which is part of the standard of care. TAK-981's utility was further demonstrated in in vivo mouse and human leukemia models as well as patient-derived primary AML cells. Our results also indicate direct and cancer cell-inherent anti-AML effects by TAK-981, different from the type 1 interferon and immune-dependent mechanism in a previous solid tumor study. Overall, we provide a proof-of-concept for SUMOylation as a new targetable pathway in AML and propose TAK-981 as a promising direct anti-AML agent. Our data should prompt studies on optimal combination strategies and transitions to clinical trials in AML.

Non-bioenergetic roles of mitochondrial GPD2 promote tumor progression.

Rationale: Despite growing evidence for mitochondria's involvement in cancer, the roles of specific metabolic components outside the respiratory complex have been little explored. We conducted metabolomic studies on mitochondrial DNA (mtDNA)-deficient (ρ0) cancer cells with lower proliferation rates to clarify the undefined roles of mitochondria in cancer growth. Methods and results: Despite extensive metabolic downregulation, ρ0 cells exhibited high glycerol-3-phosphate (G3P) level, due to low activity of mitochondrial glycerol-3-phosphate dehydrogenase (GPD2). Knockout (KO) of GPD2 resulted in cell growth suppression as well as inhibition of tumor progression in vivo. Surprisingly, this was unrelated to the conventional bioenergetic function of GPD2. Instead, multi-omics results suggested major changes in ether lipid metabolism, for which GPD2 provides dihydroxyacetone phosphate (DHAP) in ether lipid biosynthesis. GPD2 KO cells exhibited significantly lower ether lipid level, and their slower growth was rescued by supplementation of a DHAP precursor or ether lipids. Mechanistically, ether lipid metabolism was associated with Akt pathway, and the downregulation of Akt/mTORC1 pathway due to GPD2 KO was rescued by DHAP supplementation. Conclusion: Overall, the GPD2-ether lipid-Akt axis is newly described for the control of cancer growth. DHAP supply, a non-bioenergetic process, may constitute an important role of mitochondria in cancer.

Chemical Biopsy for GNMT as Noninvasive and Tumorigenesis-Relevant Diagnosis of Liver Cancer.

Early diagnosis of hepatocellular carcinoma (HCC) is difficult; the lack of convenient biomarker-based diagnostic modalities renders high-risk HCC patients burdened by life-long periodical examinations. Here, a new chemical biopsy approach was developed for noninvasive diagnosis of HCC using urine samples. Bioinformatic screening for tumor suppressors yielded glycine N-methyltransferase (GNMT) as a biomarker with clinical relevance to HCC tumorigenesis. A liquid chromatography-mass spectrometry (LC-MS)-based chemical biopsy detecting nonradioactive 13C-sarcosine from 13C-glycine was designed to noninvasively assess liver GNMT activity extrahepatically. 13C-Sarcosine showed a strong correlation with GNMT in normal and cancerous liver cells. In an autochthonous animal model developing visible cancer nodules at 17 weeks, the urinary 13C-sarcosine chemical biopsy exhibited notable changes as early as 8 weeks, showing significant correlations with liver GNMT and molecular pathological changes. Our chemical biopsy approach should facilitate early and noninvasive diagnosis of HCC, with direct relevance to tumorigenesis, which can be straightforwardly applied to other diseases.

Involvement of cell shape and lipid metabolism in glioblastoma resistance to temozolomide.

Temozolomide (TMZ) has been used as standard-of-care for glioblastoma multiforme (GBM), but the resistance to TMZ develops quickly and frequently. Thus, more studies are needed to elucidate the resistance mechanisms. In the current study, we investigated the relationship among the three important phenotypes, namely TMZ-resistance, cell shape and lipid metabolism, in GBM cells. We first observed the distinct difference in cell shapes between TMZ-sensitive (U87) and resistant (U87R) GBM cells. We then conducted NMR-based lipid metabolomics, which revealed a significant increase in cholesterol and fatty acid synthesis as well as lower lipid unsaturation in U87R cells. Consistent with the lipid changes, U87R cells exhibited significantly lower membrane fluidity. The transcriptomic analysis demonstrated that lipid synthesis pathways through SREBP were upregulated in U87R cells, which was confirmed at the protein level. Fatostatin, an SREBP inhibitor, and other lipid pathway inhibitors (C75, TOFA) exhibited similar or more potent inhibition on U87R cells compared to sensitive U87 cells. The lower lipid unsaturation ratio, membrane fluidity and higher fatostatin sensitivity were all recapitulated in patient-derived TMZ-resistant primary cells. The observed ternary relationship among cell shape, lipid composition, and TMZ-resistance may be applicable to other drug-resistance cases. SREBP and fatostatin are suggested as a promising target-therapeutic agent pair for drug-resistant glioblastoma.

Ex vivo NMR metabolomics approach using cerebrospinal fluid for the diagnosis of primary CNS lymphoma: Correlation with MR imaging characteristics.

PURPOSE: Primary central nervous system lymphoma (PCNSL) is an uncommon extranodal non-Hodgkin's lymphoma. Here, the feasibility of nuclear magnetic resonance (NMR) metabolomics for the diagnosis and prognosis prediction of PCNSL, as well as its correlation with magnetic resonance imaging (MRI) characteristics, was assessed. PATIENTS AND METHODS: Cerebrospinal fluid (CSF) samples from PCNSL and normal groups (n = 41 for each) were obtained along with MRI data including pre- and postcontrast as well as T1-, T2-, and diffusion-weighted imaging for the treatment-naïve PCNSL patients (n = 24). The CSF samples were analyzed using nuclear magnetic resonance (NMR). RESULTS: The CSF NMR metabolomic exhibited clear differences with a diagnostic sensitivity of 100% and a specificity of 97.6%. The citrate level of the leptomeningeal enhancement (LE) (+) group was significantly lower than that of the LE (-) group (p = 0.018). In addition, the MRI apparent diffusion coefficient (ADC) value of the tumor was positively correlated with the glucose level (p = 0.025). However, none of the marker metabolites were significant prognosis predictors in univariate analysis. CONCLUSIONS: In conclusion, the NMR metabolomics could be helpful to diagnose PCNSL, but not for the prognosis, and MRI features (LE or ADC) can reflect the metabolic profiles of PCNSL.

Differential Background Music as Attentional Resources Interacting with Cognitive Control.

We examined the effects of background music on cognitive task performances using different musical arrangements from an excerpt of Mozart's Piano Sonata K.448. The participants were 126 university students: 70 music majors and 56 nonmusic majors. Three types of musical arrangements were used as background conditions: rhythm-only, melody, and original music conditions. Participants were asked to perform cognitive tasks in the presence of each music condition. The participants' percentage of completed items and accuracy on these tasks were compared for music and nonmusic majors, controlling for the effect of perceived level of arousal and their performance during no background music. Whether a participant's perceptions of background music predicted their cognitive performance was also analyzed. We found that music majors demonstrated decreased task performance for the original background condition, while nonmusic majors demonstrated no significant differences in performance across the arrangements. When pitch or rhythm information was modified, emotional valence and arousal were perceived differently. Perception of the complexity of the background music depending on the arrangement type differed between music majors and nonmusic majors. While the perceived complexity significantly predicted nonmusic majors' cognitive performance, its predictive effect was not found in music majors. The findings imply that perceptions of musical arrangements in terms of expectancy and complexity can be critical factors in determining how arrangements affect concurrent cognitive activity, while suggesting that music itself is not a facilitating or detrimental factor for cognitive performance.

Omics profiling identifies the regulatory functions of the MAPK/ERK pathway in nephron progenitor metabolism.

Nephron endowment is defined by fetal kidney growth and crucially dictates renal health in adults. Defects in the molecular regulation of nephron progenitors contribute to only a fraction of reduced nephron mass cases, suggesting alternative causative mechanisms. The importance of MAPK/ERK activation in nephron progenitor maintenance has been previously demonstrated, and here, we characterized the metabolic consequences of MAPK/ERK deficiency. Liquid chromatography/mass spectrometry-based metabolomics profiling identified 42 reduced metabolites, of which 26 were supported by in vivo transcriptional changes in MAPK/ERK-deficient nephron progenitors. Among these, mitochondria, ribosome and amino acid metabolism, together with diminished pyruvate and proline metabolism, were the most affected pathways. In vitro cultures of mouse kidneys demonstrated a dosage-specific function for pyruvate in controlling the shape of the ureteric bud tip, a regulatory niche for nephron progenitors. In vivo disruption of proline metabolism caused premature nephron progenitor exhaustion through their accelerated differentiation in pyrroline-5-carboxylate reductases 1 (Pycr1) and 2 (Pycr2) double-knockout kidneys. Pycr1/Pycr2-deficient progenitors showed normal cell survival, indicating no changes in cellular stress. Our results suggest that MAPK/ERK-dependent metabolism functionally participates in nephron progenitor maintenance by monitoring pyruvate and proline biogenesis in developing kidneys.

Extracellular Citrate Treatment Induces HIF1α Degradation and Inhibits the Growth of Low-Glycolytic Hepatocellular Carcinoma under Hypoxia.

HCC is well known for low glycolysis in the tumors, whereas hypoxia induces glycolytic phenotype and tumor progression. This study was conducted to evaluate the expression of SLCs in human HCCs and investigated whether extracellular nutrient administration related to SLCs in low-glycolytic HCC can prevent hypoxic tumor progression. SLCs expression was screened according to the level of glycolysis in HCCs. Then, whether extracellular nutrient treatment can affect hypoxic tumor progression, as well as the mechanisms, were evaluated in an in vitro cell line and an in vivo animal model. Low-glycolytic HCCs showed high SLC13A5/NaCT and SLC16A1/MCT1 but low SLC2A1/GLUT1 and HIF1α/HIF1α expression. Especially, high SLC13A5 expression was significantly associated with good overall survival in the Cancer Genome Atlas (TCGA) database. In HepG2 cells with the highest NaCT expression, extracellular citrate treatment upon hypoxia induced HIF1α degradation, which led to reduced glycolysis and cellular proliferation. Finally, in HepG2-animal models, the citrate-treated group showed smaller tumor with less hypoxic areas than the vehicle-treated group. In patients with HCC, SLC13A5/NaCT is an important SLC, which is associated with low glycolysis and good prognosis. Extracellular citrate treatment induced the failure of metabolic adaptation to hypoxia and tumor growth inhibition, which can be a potential therapeutic strategy in HCCs.

Comparative whole-genome transcriptome analysis in renal cell populations reveals high tissue specificity of MAPK/ERK targets in embryonic kidney.

BACKGROUND: MAPK/ERK signaling is a well-known mediator of extracellular stimuli controlling intracellular responses to growth factors and mechanical cues. The critical requirement of MAPK/ERK signaling for embryonic stem cell maintenance is demonstrated, but specific functions in progenitor regulation during embryonic development, and in particular kidney development remain largely unexplored. We previously demonstrated MAPK/ERK signaling as a key regulator of kidney growth through branching morphogenesis and normal nephrogenesis where it also regulates progenitor expansion. Here, we performed RNA sequencing-based whole-genome expression analysis to identify transcriptional MAPK/ERK targets in two distinct renal populations: the ureteric bud epithelium and the nephron progenitors. RESULTS: Our analysis revealed a large number (5053) of differentially expressed genes (DEGs) in nephron progenitors and significantly less (1004) in ureteric bud epithelium, reflecting likely heterogenicity of cell types. The data analysis identified high tissue-specificity, as only a fraction (362) of MAPK/ERK targets are shared between the two tissues. Tissue-specific MAPK/ERK targets participate in the regulation of mitochondrial energy metabolism in nephron progenitors, which fail to maintain normal mitochondria numbers in the MAPK/ERK-deficient tissue. In the ureteric bud epithelium, a dramatic decline in progenitor-specific gene expression was detected with a simultaneous increase in differentiation-associated genes, which was not observed in nephron progenitors. Our experiments in the genetic model of MAPK/ERK deficiency provide evidence that MAPK/ERK signaling in the ureteric bud maintains epithelial cells in an undifferentiated state. Interestingly, the transcriptional targets shared between the two tissues studied are over-represented by histone genes, suggesting that MAPK/ERK signaling regulates cell cycle progression and stem cell maintenance through chromosome condensation and nucleosome assembly. CONCLUSIONS: Using tissue-specific MAPK/ERK inactivation and RNA sequencing in combination with experimentation in embryonic kidneys, we demonstrate here that MAPK/ERK signaling maintains ureteric bud tip cells, suggesting a regulatory role in collecting duct progenitors. We additionally deliver new mechanistic information on how MAPK/ERK signaling regulates progenitor maintenance through its effects on chromatin accessibility and energy metabolism.

Real-Time Monitoring of Host-Gut Microbial Interspecies Interaction in Anticancer Drug Metabolism.

Gut microbiome can affect drug metabolism considerably, leading to modified drug response. However, quantitative estimation of host vs. microbial contributions in a living host-gut microbiome system has been challenging. Using the interspecies system of Caenorhabditis elegans and gut bacteria, we developed a real-time approach for monitoring their metabolic interaction in vivo during anticancer drug 5-fluorouracil (5-FU) metabolism. The fluorine NMR-based approach yielded the quantitative contributions to the host 5-FU metabolism made by human gut-microbial species of variable genetic backgrounds. It also experimentally confirmed a bacterial gene-metabolism relationship. Differential 5-FU catabolism among bacterial substrains and the contributions to the host metabolism, unobservable by conventional 16S rRNA metagenomic sequencing, were also found. The metabolic contributions could be correlated with phenotypic developmental toxicity of 5-FU to the host fed with different substrains. Our convenient platform should help to reveal heterogeneity in host-gut microbiome interactions for many drugs in a living symbiotic system.

Highly conserved protein Rv1211 in Mycobacterium tuberculosis is a natively unfolded protein that binds to a calmodulin antagonist, trifluoperazine.

Rv1211 is a conserved hypothetical protein in Mycobacterium tuberculosis and is required for the growth and pathogenesis of the bacteria. The protein has been suggested as a calmodulin-like calcium-binding protein with an EF-hand motif and as a target of trifluoperazine, a calmodulin antagonist in eukaryotes that inhibits mycobacterial growth. Here, we expressed the recombinant protein of Rv1211 and performed structural and biochemical studies of Rv1211 and its interaction with Ca2+ or trifluoperazine. Surprisingly, Rv1211 exhibited an elution property typical of a natively unfolded protein. Subsequent circular dichroism experiments with temperature elevation and trifluoroethanol treatment showed that Rv1211 has unfolded structure. Additional NMR experiment confirmed the unfolded state of the protein and further showed that it does not bind to Ca2+. Still, Rv1211 did bind to trifluoperazine, as evidenced by the two-dimensional NMR spectra of 15N-labeled Rv1211. However, there were no peak shifts upon binding, showing that Rv1211 retained its unfolded state even after the trifluoperazine binding. The residues involved in the binding were clustered in the C-terminal region, as identified by the sequence assignment. Isothermal titration calorimetry showed that the Kd of trifluoperazine-Rv1211 binding is 41 µM and that the stoichiometry is 1 : 2 (Rv1211: trifluoperazine). Our results argue against the suggestion of Rv1211 as a Ca2+-binding calmodulin-like protein, and show that Rv1211 is a natively unfolded protein that binds to trifluoperazine. In addition, our results suggest the evidence of the "Fuzziness" in the Rv1211-trifluoperazine interaction that differs from the conventional binding-induced folding of natively unfolded proteins.