Hypothermia increases adenosine monophosphate and xanthosine monophosphate levels in the mouse hippocampus, preventing their reduction by global cerebral ischemia.

Global cerebral ischemia (GCI) caused by clinical conditions such as cardiac arrest leads to delayed neuronal death in the hippocampus, resulting in physical and mental disability. However, the mechanism of delayed neuronal death following GCI remains unclear. To elucidate the mechanism, we performed a metabolome analysis using a mouse model in which hypothermia (HT) during GCI, which was induced by the transient occlusion of the bilateral common carotid arteries, markedly suppressed the development of delayed neuronal death in the hippocampus after reperfusion. Fifteen metabolites whose levels were significantly changed by GCI and 12 metabolites whose levels were significantly changed by HT were identified. Furthermore, the metabolites common for both changes were narrowed down to two, adenosine monophosphate (AMP) and xanthosine monophosphate (XMP). The levels of both AMP and XMP were found to be decreased by GCI, but increased by HT, thereby preventing their decrease. In contrast, the levels of adenosine, inosine, hypoxanthine, xanthine, and guanosine, the downstream metabolites of AMP and XMP, were increased by GCI, but were not affected by HT. Our results may provide a clue to understanding the mechanism by which HT during GCI suppresses the development of delayed neuronal death in the hippocampus.

Exploration of urine metabolic biomarkers for new-onset, untreated pediatric epilepsy: A gas and liquid chromatography mass spectrometry-based metabolomics study.

OBJECTIVE: The discovery of objective indicators for recent epileptic seizures will help confirm the diagnosis of epilepsy and evaluate therapeutic effects. Past studies had shortcomings such as the inclusion of patients under treatment and those with various etiologies that could confound the analysis results significantly. We aimed to minimize such confounding effects and to explore the small molecule biomarkers associated with the recent occurrence of epileptic seizures using urine metabolomics. METHODS: This is a multicenter prospective study. Subjects included pediatric patients aged 2 to 12 years old with new-onset, untreated epilepsy, who had had the last seizure within 1 month before urine collection. Controls included healthy children aged 2 to 12 years old. Those with underlying or chronic diseases, acute illnesses, or recent administration of medications or supplements were excluded. Targeted metabolome analysis of spot urine samples was conducted using gas chromatography (GC)- and liquid chromatography (LC)-tandem mass spectrometry (MS/MS). RESULTS: We enrolled 17 patients and 21 controls. Among 172 metabolites measured by GC/MS/MS and 41 metabolites measured by LC/MS/MS, only taurine was consistently reduced in the epilepsy group. This finding was subsequently confirmed by the absolute quantification of amino acids. No other metabolites were consistently altered between the two groups. CONCLUSIONS: Urine metabolome analysis, which covers a larger number of metabolites than conventional biochemistry analyses, found no consistently altered small molecule metabolites except for reduced taurine in epilepsy patients compared to healthy controls. Further studies with larger samples, subjects with different ages, expanded target metabolites, and the investigation of plasma samples are required.

Reduced glutathione level in the aqueous humor of patients with primary open-angle glaucoma and normal-tension glaucoma.

Glaucoma is a leading cause of blindness worldwide in older people. Profiling the aqueous humor, including the metabolites it contains, is useful to understand physiological and pathological conditions in the eye. In the current study, we used mass spectrometry (MS) to characterize the aqueous humor metabolomic profile and biological features of patients with glaucoma. Aqueous humor samples were collected during trabeculectomy surgery or cataract surgery and analyzed with global metabolomics. We included 40 patients with glaucoma (32 with POAG, 8 with NTG) and 37 control subjects in a discovery study. VIP analysis revealed five metabolites that were elevated and three metabolites that were reduced in the glaucoma patients. The identified metabolomic profile had an area under the receiver operating characteristic curve of 0.953. Among eight selected metabolites, the glutathione level was significantly decreased in association with visual field defects. Moreover, in a validation study to confirm the reproducibility of our findings, the glutathione level was reduced in NTG and POAG patients compared with a cataract control group. Our findings demonstrate that aqueous humor profiling can help to diagnose glaucoma and that various aqueous humor metabolites are correlated with clinical parameters in glaucoma patients. In addition, glutathione is clearly reduced in the aqueous humor of glaucoma patients with both IOP-dependent and IOP-independent disease subtypes. These findings indicate that antioxidant agents in the aqueous humor reflect glaucomatous optic nerve damage and that excessive oxidative stress may be involved in the pathogenesis of glaucoma.

Identification of predictive biomarkers for endometrial cancer diagnosis and treatment response monitoring using plasma metabolome profiling.

BACKGROUND: Endometrial cancer (EMC) is the most common female genital tract malignancy with an increasing prevalence in many countries including Japan, a fact that renders early detection and treatment necessary to protect health and fertility. Although early detection and treatment are necessary to further improve the prognosis of women with endometrial cancer, biomarkers that accurately reflect the pathophysiology of EMC patients are still unclear. Therefore, it is clinically critical to identify biomarkers to assess diagnosis and treatment efficacy to facilitate appropriate treatment and development of new therapies for EMC. METHODS: In this study, wide-targeted plasma metabolome analysis was performed to identify biomarkers for EMC diagnosis and the prediction of treatment responses. The absolute quantification of 628 metabolites in plasma samples from 142 patients with EMC was performed using ultra-high-performance liquid chromatography with tandem mass spectrometry. RESULTS: The concentrations of 111 metabolites increased significantly, while the concentrations of 148 metabolites decreased significantly in patients with EMC compared to healthy controls. Specifically, LysoPC and TGs, including unsaturated fatty acids, were reduced in patients with stage IA EMC compared to healthy controls, indicating that these metabolic profiles could be used as early diagnostic markers of EMC. In contrast, blood levels of amino acids such as histidine and tryptophan decreased as the risk of recurrence increased and the stages of EMC advanced. Furthermore, a marked increase in total TG and a decrease in specific TGs and free fatty acids including polyunsaturated fatty acids levels were observed in patients with EMC. These results suggest that the polyunsaturated fatty acids in patients with EMC are crucial for disease progression. CONCLUSIONS: Our data identified specific metabolite profiles that reflect the pathogenesis of EMC and showed that these metabolites correlate with the risk of recurrence and disease stage. Analysis of changes in plasma metabolite profiles could be applied for the early diagnosis and monitoring of the course of treatment of EMC patients.

Local Delivery of Nimustine Hydrochloride against Brain Tumors: Basic Characterization Study.

Convection-enhanced delivery (CED) delivers agents directly into tumors and the surrounding parenchyma. Although a promising concept, clinical applications are often hampered by insufficient treatment efficacy. Toward developing an effective CED-based strategy for delivering drugs with proven clinical efficacy, we performed a basic characterization study to explore the locally delivered characteristics of the water soluble nitrosourea nimustine hydrochloride (ACNU). First, ACNU distribution after CED in rodent brain was studied using mass spectrometry imaging. Clearance of 14C-labeled ACNU after CED in striatum was also studied. ACNU was robustly distributed in rodent brain similar to the distribution of the hydrophilic dye Evans blue after CED, and locally delivered ACNU was observed for over 24 h at the delivery site. Subsequently, to investigate the potential of ACNU to induce an immunostimulative microenvironment, Fas and transforming growth factor-ß1 (TGF-ß1) was assessed in vitro. We found that ACNU significantly inhibited TGF-ß1 secretion and reduced Fas expression. Further, after CED of ACNU in 9L-derived intracranial tumors, the infiltration of CD4/CD8 lymphocytes in tumors was evaluated by immunofluorescence.CED of ACNU in xenografted intracranial tumors induced tumor infiltration of CD4/CD8 lymphocytes. ACNU has a robust distribution in rodent brain by CED, and delayed clearance of the drug was observed at the local infusion site. Further, local delivery of ACNU affects the tumor microenvironment and induces immune cell migration in tumor. These characteristics make ACNU a promising agent for CED.

Identification of predictive biomarkers for diagnosis and radiation sensitivity of uterine cervical cancer using wide-targeted metabolomics.

AIM: Uterine cervical cancer (UCC) is the fourth most common cancer in women, responsible for more than 300 000 deaths worldwide. Its early detection, by cervical cytology, and prevention, by vaccinating against human papilloma virus, greatly contribute to reducing cervical cancer mortality in women. However, penetration of the effective prevention of UCC in Japan remains low. Plasma metabolome analysis is widely used for biomarker discovery and the identification of cancer-specific metabolic pathways. Here, we aimed to identify predictive biomarkers for the diagnosis and radiation sensitivity of UCC using wide-targeted plasma metabolomics. METHODS: We analyzed 628 metabolites in plasma samples obtained from 45 patients with UCC using ultra-high-performance liquid chromatography with tandem mass spectrometry. RESULTS: The levels of 47 metabolites were significantly increased and those of 75 metabolites were significantly decreased in patients with UCC relative to healthy controls. Increased levels of arginine and ceramides, and decreased levels of tryptophan, ornithine, glycosylceramides, lysophosphatidylcholine, and phosphatidylcholine were characteristic of patients with UCC. Comparison of metabolite profiles in groups susceptible and non-susceptible to radiation therapy, a treatment for UCC, revealed marked variations in polyunsaturated fatty acid, nucleic acid, and arginine metabolism in the group not susceptible to treatment. CONCLUSIONS: Our findings suggest that the metabolite profile of patients with UCC may be an important indicator for distinguishing these patients from healthy cohorts, and may also be useful for predicting sensitivity to radiotherapy.

Prospective exosome-focused translational research for afatinib (EXTRA) study of patients with nonsmall cell lung cancer harboring EGFR mutation: an observational clinical study.

Background: The exosome-focused translational research for afatinib (EXTRA) study is the first trial to identify novel predictive biomarkers for longer treatment efficacy of afatinib in patients with epidermal growth factor receptor (EGFR) mutation-positive nonsmall cell lung cancer (NSCLC) via a comprehensive association study using genomic, proteomic, epigenomic, and metabolomic analyses. Objectives: We report details of the clinical portion prior to omics analyses. Design: A prospective, single-arm, observational study was conducted using afatinib 40 mg/day as an initial dose in untreated patients with EGFR mutation-positive NSCLC. Dose reduction to 20 mg every other day was allowed. Methods: Progression-free survival (PFS), overall survival (OS), and adverse events (AEs) were evaluated. Results: A total of 103 patients (median age 70 years, range 42-88 years) were enrolled from 21 institutions in Japan between February 2017 and March 2018. After a median follow-up of 35.0 months, 21% remained on afatinib treatment, whereas 9% had discontinued treatment because of AEs. The median PFS was 18.4 months, with a 3-year PFS rate of 23.3%. The median afatinib treatment duration in patients with final doses of 40 (n = 27), 30 (n = 23), and 20 mg/day (n = 35), and 20 mg every other day (n = 18) were 13.4, 15.4, 18.8, and 18.3 months, respectively. The median OS was not reached, with a 3-year OS rate of 58.5%. The median OS in patients who did (n = 25) and did not (n = 78) receive osimertinib during the entire course of treatment were 42.4 months and not reached, respectively (p = 0.654). Conclusions: As the largest prospective study in Japan, this study confirmed favorable OS following first-line afatinib in patients with EGFR mutation-positive NSCLC in a real-world setting. Further analysis of the EXTRA study is expected to identify novel predictive biomarkers for afatinib. Trial registration: UMIN-CTR identifier (UMIN000024935, https://center6.umin.ac.jp/cgi-open-bin/ctr/ctr_his_list.cgi?recptno=R000028688.

Nrf2 deficiency deteriorates diabetic kidney disease in Akita model mice.

Oxidative stress is an essential component in the progression of diabetic kidney disease (DKD), and the transcription factor NF-E2-related factor-2 (Nrf2) plays critical roles in protecting the body against oxidative stress. To clarify the roles of Nrf2 in protecting against DKD, in this study we prepared compound mutant mice with diabetes and loss of antioxidative defense. Specifically, we prepared compound Ins2Akita/+ (Akita) and Nrf2 knockout (Akita::Nrf2-/-) or Akita and Nrf2 induction (Akita::Keap1FA/FA) mutant mice. Eighteen-week-old Akita::Nrf2-/- mice showed more severe diabetic symptoms than Akita mice. In the Akita::Nrf2-/- mouse kidneys, the glomeruli showed distended capillary loops, suggesting enhanced mesangiolysis. Distal tubules showed dilation and an increase in 8-hydroxydeoxyguanosine-positive staining. In the Akita::Nrf2-/- mouse kidneys, the expression of glutathione (GSH) synthesis-related genes was decreased, and the actual GSH level was decreased in matrix-assisted laser desorption/ionization mass spectrometry imaging analysis. Akita::Nrf2-/- mice exhibited severe inflammation and enhancement of infiltrated macrophages in the kidney. To further examine the progression of DKD, we compared forty-week-old Akita mouse kidney compounds with Nrf2-knockout or Nrf2 mildly induced (Akita::Keap1FA/FA) mice. Nrf2-knockout Akita (Akita::Nrf2-/-) mice displayed severe medullary cast formation, but the formation was ameliorated in Akita::Keap1FA/FA mice. Moreover, in Akita::Keap1FA/FA mice, tubule injury and inflammation-related gene expression were significantly suppressed, which was evident in Akita::Nrf2-/- mouse kidneys. These results demonstrate that Nrf2 contributes to the protection of the kidneys against DKD by suppressing oxidative stress and inflammation.

Metabolic and Epigenetic Regulation of SMAD7 by STC1 Ameliorates Lung Fibrosis.

As shown in our previous studies, the intratracheal-administration of STC1 (stanniocalcin-1) ameliorates pulmonary fibrosis by reducing oxidative and endoplasmic reticulum stress through the uncoupling of respiration in a bleomycin-treated mouse model. However, the overall effect of STC1 on metabolism was not examined. Therefore, we first conducted a comprehensive metabolomics analysis to screen the overall metabolic changes induced by STC1 in an alveolar epithelial cell line using capillary electrophoresis time-of-flight mass spectrometry. The results were subsequently validated in multiple alveolar epithelial and fibroblast cell lines by performing precise analyses of each substance. STC1 stimulated glycolysis, acetyl-CoA synthesis, and the methionine and cysteine-glutathione pathways, which are closely related to the uncoupling of respiration, modulation of epigenetics, and reduction in oxidative stress. These results are consistent with our previous study. Subsequently, we focused on the inhibitory factor SMAD7, which exerts an antifibrotic effect and is susceptible to epigenetic regulation. STC1 upregulates SMAD7 in an uncoupling protein 2-dependent manner, induces demethylation of the SMAD7 promoter region and acetylation of the SMAD7 protein in human alveolar epithelial and fibroblast cell lines and a bleomycin-treated mouse model, and subsequently attenuates fibrosis. The antifibrotic effects of STC1 may partially depend on the regulation of SMAD7. In the evaluation using lung tissue from patients with idiopathic pulmonary fibrosis, SMAD7 expression and acetylation were high in the alveolar structure-preserving region and low in the fibrotic region. The intratracheal administration of STC1 may prevent the development of pulmonary fibrosis by regulating the metabolism-mediated epigenetic modification of SMAD7 in patients.

Esterification promotes the intracellular accumulation of roxadustat, an activator of hypoxia-inducible factors, to extend its effective duration.

Kidney injury often causes anemia due to a lack of production of the erythroid growth factor erythropoietin (EPO) in the kidneys. Roxadustat is one of the first oral medicines inducing EPO production in patients with renal anemia by activating hypoxia-inducible factors (HIFs), which are activators of EPO gene expression. In this study, to develop prodrugs of roxadustat with improved permeability through cell membrane, we investigated the effects of 8 types of esterification on the pharmacokinetics and bioactivity of roxadustat using Hep3B hepatoma cells that HIF-dependently produce EPO. Mass spectrometry of cells incubated with the esterified roxadustat derivatives revealed that the designed compounds were deesterified after being taken up by cells and showed low cytotoxicity compared to the original compound. Esterification prolonged the effective duration of roxadustat with respect to EPO gene induction and HIF activation in cells transiently exposed to the compounds. In the kidneys and livers of mice, both of which are unique sites of EPO production, a majority of the methyl-esterified roxadustat was deesterified within 6 h after drug administration. The deesterified roxadustat derivative was continuously detectable in plasma and urine for at least 48 h after administration, while the administered compound became undetectable 24 h after administration. Additionally, we confirmed that methyl-esterified roxadustat activated erythropoiesis in mice by inducing Epo mRNA expression exclusively in renal interstitial cells, which have intrinsic EPO-producing potential. These data suggest that esterification could lead to the development of roxadustat prodrugs with improvements in cell membrane permeability, effective duration and cytotoxicity.

Wide-Targeted Metabolome Analysis Identifies Potential Biomarkers for Prognosis Prediction of Epithelial Ovarian Cancer.

Epithelial ovarian cancer (EOC) is a fatal gynecologic cancer, and its poor prognosis is mainly due to delayed diagnosis. Therefore, biomarker identification and prognosis prediction are crucial in EOC. Altered cell metabolism is a characteristic feature of cancers, and metabolomics reflects an individual's current phenotype. In particular, plasma metabolome analyses can be useful for biomarker identification. In this study, we analyzed 624 metabolites, including uremic toxins (UTx) in plasma derived from 80 patients with EOC using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Compared with the healthy control, we detected 77 significantly increased metabolites and 114 significantly decreased metabolites in EOC patients. Especially, decreased concentrations of lysophosphatidylcholines and phosphatidylcholines and increased concentrations of triglycerides were observed, indicating a metabolic profile characteristic of EOC patients. After calculating the parameters of each metabolic index, we found that higher ratios of kynurenine to tryptophan correlates with worse prognosis in EOC patients. Kynurenine, one of the UTx, can affect the prognosis of EOC. Our results demonstrated that plasma metabolome analysis is useful not only for the diagnosis of EOC, but also for predicting prognosis with the variation of UTx and evaluating response to chemotherapy.

Filopodium-derived vesicles produced by MIM enhance the migration of recipient cells.

Extracellular vesicles (EVs) are classified as large EVs (l-EVs, or microvesicles) and small EVs (s-EVs, or exosomes). S-EVs are thought to be generated from endosomes through a process that mainly depends on the ESCRT protein complex, including ALG-2 interacting protein X (ALIX). However, the mechanisms of l-EV generation from the plasma membrane have not been identified. Membrane curvatures are generated by the bin-amphiphysin-rvs (BAR) family proteins, among which the inverse BAR (I-BAR) proteins are involved in filopodial protrusions. Here, we show that the I-BAR proteins, including missing in metastasis (MIM), generate l-EVs by scission of filopodia. Interestingly, MIM-containing l-EV production was promoted by in vivo equivalent external forces and by the suppression of ALIX, suggesting an alternative mechanism of vesicle formation to s-EVs. The MIM-dependent l-EVs contained lysophospholipids and proteins, including IRS4 and Rac1, which stimulated the migration of recipient cells through lamellipodia formation. Thus, these filopodia-dependent l-EVs, which we named as filopodia-derived vesicles (FDVs), modify cellular behavior.

Development of a highly sensitive method for the quantitative analysis of modified nucleosides using UHPLC-UniSpray-MS/MS.

There are more than 150 types of naturally occurring modified nucleosides, which are believed to be involved in various biological processes. Recently, an ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) technique has been developed to measure low levels of modified nucleosides. A comprehensive analysis of modified nucleosides will lead to a better understanding of intracellular ribonucleic acid modification, but this analysis requires high-sensitivity measurements. In this perspective, we established a highly sensitive and quantitative method using the newly developed ion source, UniSpray. A mass spectrometer was used with a UniSpray source in positive ion mode. Our UHPLC-UniSpray-MS/MS methodology separated and detected the four major nucleosides, 42 modified nucleosides, and dG15N5 (internal standard) in 15 min. The UniSpray method provided good correlation coefficients (>0.99) for all analyzed nucleosides, and a wide range of linearity for 35 of the 46 nucleosides. Additionally, the accuracy and precision values satisfied the criteria of <15% for higher concentrations and <20% for the lowest concentrations of all nucleosides. We also investigated whether this method could measure nucleosides in biological samples using mouse tissues and non-small cell lung cancer clinical specimens. We were able to detect 43 and 31 different modified nucleosides from mouse and clinical tissues, respectively. We also found significant differences in the levels of N6-methyl-N6-threonylcarbamoyladenosine (m6t6A), 1-methylinosine (m1I), 2'-O-methylcytidine (Cm), 5-carbamoylmethyluridine (ncm5U), 5-methoxycarbonylmethyl-2-thiouridine (mcm5S2U), and 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um) between cancerous and noncancerous tissues. In conclusion, we developed a highly sensitive methodology using UHPLC-UniSpray-MS/MS to simultaneously detect and quantify modified nucleosides, which can be used for analysis of biological samples.

Colorectal Cancer-Derived CAT1-Positive Extracellular Vesicles Alter Nitric Oxide Metabolism in Endothelial Cells and Promote Angiogenesis.

Accumulating scientific evidences strongly support the importance of cancer-derived extracellular vesicles (EV) in organization of tumor microenvironment and metastatic niches, which are also considered as ideal tools for cancer liquid biopsy. To uncover the full scope of proteomic information packaged within EVs secreted directly from human colorectal cancer, we cultured surgically resected viable tissues and obtained tissue-exudative EVs (Te-EV). Our quantitative profiling of 6,307 Te-EV proteins and 8,565 tissue proteins from primary colorectal cancer and adjacent normal mucosa (n = 17) allowed identification of a specific cargo in colorectal cancer-derived Te-EVs, high-affinity cationic amino acid transporter 1 (CAT1, P = 5.0 × 10-3, fold change = 6.2), in addition to discovery of a new class of EV markers, VPS family proteins. The EV sandwich ELISA confirmed escalation of the EV-CAT1 level in plasma from patients with colorectal cancer compared with healthy donors (n = 119, P = 3.8 × 10-7). Further metabolomic analysis revealed that CAT1-overexpressed EVs drastically enhanced vascular endothelial cell growth and tubule formation via upregulation of arginine transport and downstream NO metabolic pathway. These findings demonstrate the potency of CAT1 as an EV-based biomarker for colorectal cancer and its functional significance on tumor angiogenesis. IMPLICATIONS: This study provides a proteome-wide compositional dataset for viable colorectal cancer tissue-derived EVs and especially emphasizes importance of EV-CAT1 as a key regulator of angiogenesis.

Geldanamycin-Derived HSP90 Inhibitors Are Synthetic Lethal with NRF2.

Activating mutations in KEAP1-NRF2 are frequently found in tumors of the lung, esophagus, and liver, where they are associated with aggressive growth, resistance to cancer therapies, and low overall survival. Despite the fact that NRF2 is a validated driver of tumorigenesis and chemotherapeutic resistance, there are currently no approved drugs which can inhibit its activity. Therefore, there is an urgent clinical need to identify NRF2-selective cancer therapies. To this end, we developed a novel synthetic lethal assay, based on fluorescently labeled isogenic wild-type and Keap1 knockout cell lines, in order to screen for compounds which selectively kill cells in an NRF2-dependent manner. Through this approach, we identified three compounds based on the geldanamycin scaffold which display synthetic lethality with NRF2. Mechanistically, we show that products of NRF2 target genes metabolize the quinone-containing geldanamycin compounds into more potent HSP90 inhibitors, which enhances their cytotoxicity while simultaneously restricting the synthetic lethal effect to cells with aberrant NRF2 activity. As all three of the geldanamycin-derived compounds have been used in clinical trials, they represent ideal candidates for drug repositioning to target the currently untreatable NRF2 activity in cancer.

Metabolomics of a mouse model of preeclampsia induced by overexpressing soluble fms-like tyrosine kinase 1.

Preeclampsia (PE) is a leading cause of maternal morbidity and mortality. Nicotinamide has beneficial effects on PE. In this study, we evaluated the effect of nicotinamide on placental development using a PE mouse model. To generate the PE model, a recombinant adenovirus to overproduce soluble fms-like tyrosine kinase 1 (sFlt-1) was administered to mice (Jcl:ICR) at 8.5 day post-coitum (dpc). Plasma and placenta samples were harvested at 12.5 dpc. Fetal and placental weight was significantly decreased at 12.5 dpc in PE mice. Plasma and placental acylcarnitine levels were significantly higher in PE mice than those in control mice. Glycolysis was accelerated and glucose metabolic flow was altered with hypoxia, leading to ATP shortage in the labyrinth of PE mice. In PE mice, ATP production was diminished, and fatty acid oxidation was accelerated in the placenta, consequently, blood carnitine and acylcarnitine levels were increased. The mitochondrial morphology in BeWo cells was impaired under hypoxia. Nicotinamide treatment reversed fetal growth restriction, placental development, and altered metabolic flow in the early stage in PE. In addition, nicotinamide normalized impaired mitochondrial morphology. Hence, targeting this metabolic alteration in the placenta using nicotinamide may serve as a potential therapeutic approach for PE treatment.

Lipidome-based rapid diagnosis with machine learning for detection of TGF-ß signalling activated area in head and neck cancer.

BACKGROUND: Several pro-oncogenic signals, including transforming growth factor beta (TGF-ß) signalling from tumour microenvironment, generate intratumoural phenotypic heterogeneity and result in tumour progression and treatment failure. However, the precise diagnosis for tumour areas containing subclones with cytokine-induced malignant properties remains clinically challenging. METHODS: We established a rapid diagnostic system based on the combination of probe electrospray ionisation-mass spectrometry (PESI-MS) and machine learning without the aid of immunohistological and biochemical procedures to identify tumour areas with heterogeneous TGF-ß signalling status in head and neck squamous cell carcinoma (HNSCC). A total of 240 and 90 mass spectra were obtained from TGF-ß-unstimulated and -stimulated HNSCC cells, respectively, by PESI-MS and were used for the construction of a diagnostic system based on lipidome. RESULTS: This discriminant algorithm achieved 98.79% accuracy in discrimination of TGF-ß1-stimulated cells from untreated cells. In clinical human HNSCC tissues, this approach achieved determination of tumour areas with activated TGF-ß signalling as efficiently as a conventional histopathological assessment using phosphorylated-SMAD2 staining. Furthermore, several altered peaks on mass spectra were identified as phosphatidylcholine species in TGF-ß-stimulated HNSCC cells. CONCLUSIONS: This diagnostic system combined with PESI-MS and machine learning encourages us to clinically diagnose intratumoural phenotypic heterogeneity induced by TGF-ß.

Nrf2 Suppresses Oxidative Stress and Inflammation in App Knock-In Alzheimer's Disease Model Mice.

Nrf2 (NF-E2-related-factor 2) is a stress-responsive transcription factor that protects cells against oxidative stresses. To clarify whether Nrf2 prevents Alzheimer's disease (AD), AD model AppNL-G-F/NL-G-F knock-in (AppNLGF ) mice were studied in combination with genetic Nrf2 induction model Keap1FA/FA mice. While AppNLGF mice displayed shorter latency to escape than wild-type mice in the passive-avoidance task, the impairment was improved in AppNLGF ::Keap1FA/FA mice. Matrix-assisted laser desorption ionization-mass spectrometry imaging revealed that reduced glutathione levels were elevated by Nrf2 induction in AppNLGF ::Keap1FA/FA mouse brains compared to AppNLGF mouse brains. Genetic Nrf2 induction in AppNLGF mice markedly suppressed the elevation of the oxidative stress marker 8-OHdG and Iba1-positive microglial cell number. We also determined the plasmalogen-phosphatidylethanolamine (PlsPE) level as an AD biomarker. PlsPE containing polyunsaturated fatty acids was decreased in the AppNLGF mouse brain, but Nrf2 induction attenuated this decline. To evaluate whether pharmacological induction of Nrf2 elicits beneficial effects for AD treatment, we tested the natural compound 6-MSITC [6-(methylsulfinyl)hexyl isothiocyanate]. Administration of 6-MSITC improved the impaired cognition of AppNLGF mice in the passive-avoidance task. These results demonstrate that the induction of Nrf2 ameliorates cognitive impairment in the AD model mouse by suppressing oxidative stress and neuroinflammation, suggesting that Nrf2 is an important therapeutic target of AD.

The guanylate cyclase C agonist linaclotide ameliorates the gut-cardio-renal axis in an adenine-induced mouse model of chronic kidney disease.

BACKGROUND: Cardiorenal syndrome is a major cause of mortality in patients with chronic kidney disease (CKD). However, the involvement of detrimental humoral mediators in the pathogenesis of cardiorenal syndrome is still controversial. Trimethylamine-N-oxide (TMAO), a hepatic metabolic product of trimethylamine generated from dietary phosphatidylcholine or carnitine derived by the gut microbiota, has been linked directly with progression of cardiovascular disease and renal dysfunction. Thus, targeting TMAO may be a novel strategy for the prevention of cardiovascular disease and chronic kidney disease. METHODS: Linaclotide, a guanylate cyclase C agonist, was administered to adenine-induced renal failure (RF) mice and changes in renal function and levels of gut-derived uremic toxins, as well as the gut microbiota community, were analyzed using metabolomic and metagenomic methods to reveal its cardiorenal effect. RESULTS: Linaclotide decreased the plasma levels of TMAO at a clinically used low dose of 10 µg/kg in the adenine-induced RF mouse model. At a high concentration of 100 µg/kg, linaclotide clearly improved renal function and reduced the levels of various uremic toxins. A reduction in TMAO levels following linaclotide treatment was also observed in a choline-fed pro-atherosclerotic model. Linaclotide ameliorated renal inflammation and fibrosis and cardiac fibrosis, as well as decreased the expression of collagen I, transforming growth factor-ß, galectin-3 (Gal-3) and ST2 genes. Plasma levels of Gal-3 and ST2 were also reduced. Because exposure of cardiomyocytes to TMAO increased fibronectin expression, these data suggest that linaclotide reduced the levels of TMAO and various uremic toxins and may result in not only renal, but also cardiac, fibrosis. F4/80-positive macrophages were abundant in small intestinal crypts in RF mice, and this increased expression was decreased by linaclotide. Reduced colonic claudin-1 levels were also restored by linaclotide, suggesting that linaclotide ameliorated the 'leaky gut' in RF mice. Metagenomic analysis revealed that the microbial order Clostridiales could be responsible for the change in TMAO levels. CONCLUSION: Linaclotide reduced TMAO and uremic toxin levels and could be a powerful tool for the prevention and control of the cardiorenal syndrome by modification of the gut-cardio-renal axis.

Identification of novel biomarkers of hepatocellular carcinoma by high-definition mass spectrometry: Ultrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry and desorption electrospray ionization mass spectrometry imaging.

RATIONALE: Hepatocellular carcinoma (HCC) is a highly malignant disease for which the development of prospective or prognostic biomarkers is urgently required. Although metabolomics is widely used for biomarker discovery, there are some bottlenecks regarding the comprehensiveness of detected features, reproducibility of methods, and identification of metabolites. In addition, information on localization of metabolites in tumor tissue is needed for functional analysis. Here, we developed a wide-polarity global metabolomics (G-Met) method, identified HCC biomarkers in human liver samples by high-definition mass spectrometry (HDMS), and demonstrated localization in cryosections using desorption electrospray ionization MS imaging (DESI-MSI) analysis. METHODS: Metabolic profiling of tumor (n = 38) and nontumor (n = 72) regions in human livers of HCC was performed by an ultrahigh-performance liquid chromatography quadrupole time-of-flight MS (UHPLC/QTOFMS) instrument equipped with a mixed-mode column. The HCC biomarker candidates were extracted by multivariate analyses and identified by matching values of the collision cross section and their fragment ions on the mass spectra obtained by HDMS. Cryosections of HCC livers, which included both tumor and nontumor regions, were analyzed by DESI-MSI. RESULTS: From the multivariate analysis, m/z 904.83 and m/z 874.79 were significantly high and low, respectively, in tumor samples and were identified as triglyceride (TG) 16:0/18:1(9Z)/20:1(11Z) and TG 16:0/18:1(9Z)/18:2(9Z,12Z) using the synthetic compounds. The TGs were clearly localized in the tumor or nontumor areas of the cryosection. CONCLUSIONS: Novel biomarkers for HCC were identified by a comprehensive and reproducible G-Met method with HDMS using a mixed-mode column. The combination analysis of UHPLC/QTOFMS and DESI-MSI revealed that the different molecular species of TGs were associated with tumor distribution and were useful for characterizing the progression of tumor cells and discovering prospective biomarkers.