Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Yoshihiro Izumi Last modified date:2024.06.27

Associate Professor / Medical Research Center for High Depth Omics / Medical Institute of Bioregulation


Papers
1. Ikeda K, Takahashi M, Bamba T, Izumi Y*, Comparison of amine-modified polymeric stationary phases for polar metabolomic analysis based on unified-hydrophilic interaction/anion exchange liquid chromatography/high-resolution mass spectrometry (unified-HILIC/AEX/HRMS), Mass Spectrometry, 10.5702/massspectrometry.A0143, 13, 1, Article number A0143, 2024.02, In metabolomic analysis, one of the most commonly used techniques to support the detection sensitivity and quantitation of mass spectrometry is combining it with liquid chromatography. Recently, we developed a method that enables comprehensive single-run measurement of hydrophilic metabolites using unified-hydrophilic interaction/anion exchange liquid chromatography/high-resolution mass spectrometry (unified-HILIC/AEX/HRMS) with a polymer-based mixed amines column (Gelpack GL-HilicAex). However, the importance of stationary phase functional groups and mobile phase conditions for the separation mechanisms and sensitive detection in unified-HILIC/AEX/HRMS is not yet fully understood. This study aimed to understand the importance of the mobile and stationary phases in unified-HILIC/AEX/HRMS. Two different alkali-resistant polymer-based amines-modified columns (Gelpack GL-HilicAex, primary, secondary, tertiary, and quaternary amine-modified polyglycerol dimethacrylate gel; Asahipak NH2P-50 2D, secondary amine-modified polyvinyl alcohol gel) and two eluents (acetonitrile and ammonium bicarbonate solution, pH 9.8) were used for comparative validation. A comparison of mobile phase conditions using both columns confirmed that the two-step separation from HILIC to AEX characteristic of unified-HILIC/AEX requires a linear gradient condition from acetonitrile to nearly 50% water and AEX with up to 40 mM bicarbonate ions. We found that when alkali-resistant hydrophilic polymer packing materials are modified with amines, unified-HILIC/AEX separation can be reproduced if at least one secondary amine associated with the amine series is present in the stationary phase. Furthermore, the difference in sensitivity in the HILIC and AEX modes owing to the different columns indicates the need for further improvements in the mobile phase composition and stationary phase..
2. Bai Y, Morita K, Kokaji T, Hatano A, Ohno S, Egami R, Pan Y, Li D, Yugi K, Uematsu S, Inoue H, Inaba Y, Suzuki Y, Matsumoto M, Takahashi M, Izumi Y, Bamba T, Hirayama A, Soga T, Kuroda S*, Trans-omic analysis reveals opposite metabolic dysregulation between feeding and fasting in liver associated with obesity, iScience, 10.1016/j.isci.2024.109121, 27, 3, Article number 109121, 2024.02, Dysregulation of liver metabolism associated with obesity during feeding and fasting leads to the breakdown of metabolic homeostasis. However, the underlying mechanism remains unknown. Here, we measured multi-omics data in the liver of wild-type and leptin-deficient obese (ob/ob) mice at ad libitum feeding and constructed a differential regulatory trans-omic network of metabolic reactions. We compared the trans-omic network at feeding with that at 16 h fasting constructed in our previous study. Intermediate metabolites in glycolytic and nucleotide metabolism decreased in ob/ob mice at feeding but increased at fasting. Allosteric regulation reversely shifted between feeding and fasting, generally showing activation at feeding while inhibition at fasting in ob/ob mice. Transcriptional regulation was similar between feeding and fasting, generally showing inhibiting transcription factor regulations and activating enzyme protein regulations in ob/ob mice. The opposite metabolic dysregulation between feeding and fasting characterizes breakdown of metabolic homeostasis associated with obesity..
3. Ito E, Inuki S, Izumi Y, Takahashi M, Dambayashi Y, Ciacchi L, Awad W, Takeyama A, Shibata K, Mori S, Mak JYW, Fairlie DP, Bamba T, Ishikawa E, Nagae M, Rossjohn J, Yamasaki S*, Sulfated bile acid is a host-derived ligand for MAIT cells, Science Immunology, 10.1126/sciimmunol.ade6924, 9, 91, Article number 100492, 2024.01, Mucosal-associated invariant T (MAIT) cells are innate-like T cells that recognize bacterial riboflavin-based metabolites as activating antigens. Although MAIT cells are found in tissues, it is unknown whether any host tissue-derived antigens exist. Here, we report that a sulfated bile acid, cholic acid 7-sulfate (CA7S), binds the nonclassical MHC class I protein MR1 and is recognized by MAIT cells. CA7S is a host-derived metabolite whose levels were reduced by more than 98% in germ-free mice. Deletion of the sulfotransferase 2a family of enzymes (Sult2a1-8) responsible for CA7S synthesis reduced the number of thymic MAIT cells in mice. Moreover, recognition of CA7S induced MAIT cell survival and the expression of a homeostatic gene signature. By contrast, recognition of a previously described foreign antigen, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU), drove MAIT cell proliferation and the expression of inflammatory genes. Thus, CA7S is an endogenous antigen for MAIT cells, which promotes their development and function..
4. Taihei Torigoe, Masatomo Takahashi*, Omidreza Heravizadeh, Kazuki Ikeda, Kohta Nakatani, Takeshi Bamba, Yoshihiro Izumi*, Predicting retention time in unified-hydrophilic-interaction/anion-exchange liquid chromatography high-resolution tandem mass spectrometry (unified-HILIC/AEX/HRMS/MS) for comprehensive structural annotation of polar metabolome, Analytical Chemistry, 10.1021/acs.analchem.3c04618, 96, 3, 1275-1283, 2024.01, The accuracy of the structural annotation of unidentified peaks obtained in metabolomic analysis using liquid chromatography/tandem mass spectrometry (LC/MS/MS) can be enhanced using retention time (RT) information as well as precursor and product ions. Unified-hydrophilic-interaction/anion-exchange liquid chromatography high-resolution tandem mass spectrometry (unified-HILIC/AEX/HRMS/MS) has been recently developed as an innovative method ideal for nontargeted polar metabolomics. However, the RT prediction for unified-HILIC/AEX has not been developed because of the complex separation mechanism characterized by the continuous transition of the separation modes from HILIC to AEX. In this study, we propose an RT prediction model of unified-HILIC/AEX/HRMS/MS, which enables the comprehensive structural annotation of polar metabolites. With training data for 203 polar metabolites, we ranked the feature importance using a random forest among 12,420 molecular descriptors (MDs) and constructed an RT prediction model with 26 selected MDs. The accuracy of the RT model was evaluated using test data for 51 polar metabolites, and 86.3% of the ΔRTs (difference between measured and predicted RTs) were within ±1.50 min, with a mean absolute error of 0.80 min, indicating high RT prediction accuracy. Nontargeted metabolomic data from the NIST SRM 1950-Metabolites in frozen human plasma were analyzed using the developed RT model and in silico MS/MS prediction, resulting in a successful structural estimation of 216 polar metabolites, in addition to the 62 identified based on standards. The proposed model can help accelerate the structural annotation of unknown hydrophilic metabolites, which is a key issue in metabolomic research..
5. Kohta Nakatani, Yoshihiro Izumi*, Hironobu Umakoshi, Maki Yokomoto-Umakoshi, Tomoko Nakaji, Hiroki Kaneko, Hiroshi Nakao, Yoshihiro Ogawa, Kazutaka Ikeda, Takeshi Bamba*, Wide-scope targeted analysis of bioactive lipids in human plasma by LC/MS/MS, Journal of Lipid Research, 10.1016/j.jlr.2023.100492, 65, 1, Article number 100492, 2024.01, Quantitative information on blood metabolites can be used in developing advanced medical strategies such as early detection and prevention of disease. Monitoring bioactive lipids such as steroids, bile acids, and PUFA metabolites could be a valuable indicator of health status. However, a method for simultaneously measuring these bioactive lipids has not yet been developed. Here, we report a LC/MS/MS method that can simultaneously measure 144 bioactive lipids, including steroids, bile acids, and PUFA metabolites, from human plasma, and a sample preparation method for these targets. Protein removal by methanol precipitation and purification of bioactive lipids by solid-phase extraction improved the recovery of the targeted compounds in human plasma samples, demonstrating the importance of sample preparation methods for a wide range of bioactive lipid analyses. Using the developed method, we studied the plasma from healthy human volunteers and confirmed the presence of bioactive lipid molecules associated with sex differences and circadian rhythms. The developed method of bioactive lipid analysis can be applied to health monitoring and disease biomarker discovery in precision medicine..
6. Noriyuki Tomiyasu, Masatomo Takahashi, Kenji Toyonaga, Sho Yamasaki, Takeshi Bamba, Yoshihiro Izumi*, Efficient lipidomic approach for the discovery of lipid ligands for immune receptors by combining LC-HRMS/MS analysis with fractionation and reporter cell assay, Analytical and Bioanalytical Chemistry, 10.1007/s00216-023-05111-w, in press, 2023.12, C-type lectin receptors (CLRs), which are pattern recognition receptors responsible for triggering innate immune responses, recognize damaged self-components and immunostimulatory lipids from pathogenic bacteria; however, several of their ligands remain unknown. Here, we propose a new analytical platform combining liquid chromatography-high-resolution tandem mass spectrometry with microfractionation capability (LC-FRC-HRMS/MS) and a reporter cell assay for sensitive activity measurements to develop an efficient methodology for searching for lipid ligands of CLR from microbial trace samples (crude cell extracts of approximately 5 mg dry cell/mL). We also developed an in-house lipidomic library containing accurate mass and fragmentation patterns of more than 10,000 lipid molecules predicted in silico for 90 lipid subclasses and 35 acyl side chain fatty acids. Using the developed LC-FRC-HRMS/MS system, the lipid extracts of Helicobacter pylori were separated and fractionated, and HRMS and HRMS/MS spectra were obtained simultaneously. The fractionated lipid extract samples in 96-well plates were thereafter subjected to reporter cell assays using nuclear factor of activated T cells (NFAT)-green fluorescent protein (GFP) reporter cells expressing mouse or human macrophage-inducible C-type lectin (Mincle). A total of 102 lipid molecules from all fractions were annotated using an in-house lipidomic library. Furthermore, a fraction that exhibited significant activity in the NFAT-GFP reporter cell assay contained α-cholesteryl glucoside, a type of glycolipid, which was successfully identified as a lipid ligand molecule for Mincle. Our analytical platform has the potential to be a useful tool for efficient discovery of lipid ligands for immunoreceptors..
7. Masahiko Sugimura, Taisuke Seike, Nobuyuki Okahashi, Yoshihiro Izumi, Takeshi Bamba, Jun Ishii, Fumio Matsuda*, Improved 2,3-Butanediol Production Rate of Metabolically Engineered Saccharomyces cerevisiae by Deletion of RIM15 and Activation of Pyruvate Consumption Pathway, International Journal of Molecular Sciences, 10.3390/ijms242216378, 24, 22, Article number 16378, 2023.11, Saccharomyces cerevisiae is a promising host for the bioproduction of higher alcohols, such as 2,3-butanediol (2,3-BDO). Metabolically engineered S. cerevisiae strains that produce 2,3-BDO via glycolysis have been constructed. However, the specific 2,3-BDO production rates of engineered strains must be improved. To identify approaches to improving the 2,3-BDO production rate, we investigated the factors contributing to higher ethanol production rates in certain industrial strains of S. cerevisiae compared to laboratory strains. Sequence analysis of 11 industrial strains revealed the accumulation of many nonsynonymous substitutions in RIM15, a negative regulator of high fermentation capability. Comparative metabolome analysis suggested a positive correlation between the rate of ethanol production and the activity of the pyruvate-consuming pathway. Based on these findings, RIM15 was deleted, and the pyruvate-consuming pathway was activated in YHI030, a metabolically engineered S. cerevisiae strain that produces 2,3-BDO. The titer, specific production rate, and yield of 2,3-BDO in the test tube-scale culture using the YMS106 strain reached 66.4 ± 4.4 mM, 1.17 ± 0.017 mmol (g dry cell weight h)-1, and 0.70 ± 0.03 mol (mol glucose consumed)-1. These values were 2.14-, 2.92-, and 1.81-fold higher than those of the vector control, respectively. These results suggest that bioalcohol production via glycolysis can be enhanced in a metabolically engineered S. cerevisiae strain by deleting RIM15 and activating the pyruvate-consuming pathway..
8. Kazuki Nishimoto, Nobuyuki Okahashi, Masaharu Maruyama, Yoshihiro Izumi, Kohta Nakatani, Yuki Ito, Junko Iida, Takeshi Bamba, Fumio Matsuda*, Lipidome and metabolome analyses reveal metabolic alterations associated with MCF-7 apoptosis upon 4-hydroxytamoxifen treatment, Scientific Reports, 10.1038/s41598-023-45764-2, 13, 1, Article number 18549, 2023.10, 4-hydroxytamoxifen (OHT) is an anti-cancer drug that induces apoptosis in breast cancer cells. Although changes in lipid levels and mitochondrial respiration have been observed in OHT-treated cells, the overall mechanisms underlying these metabolic alterations are poorly understood. In this study, time-series metabolomics and lipidomics were used to analyze the changes in metabolic profiles induced by OHT treatment in the MCF-7 human breast cancer cell line. Lipidomic and metabolomic analyses revealed increases in ceramide, diacylglycerol and triacylglycerol, and decreases in citrate, respectively. Gene expression analyses revealed increased expression of ATP-dependent citrate lyase (ACLY) and subsequent fatty acid biosynthetic enzymes, suggesting that OHT-treated MCF-7 cells activate citrate-to-lipid metabolism. The significance of the observed metabolic changes was evaluated by co-treating MCF-7 cells with OHT and ACLY or a diacylglycerol O-acyltransferase 1 (DGAT1) inhibitor. Co-treatment ameliorated cell death and reduced mitochondrial membrane potential compared to that in OHT treatment alone. The inhibition of cell death by co-treatment with an ACLY inhibitor has been observed in other breast cancer cell lines. These results suggest that citrate-to-lipid metabolism is critical for OHT-induced cell death in breast cancer cell lines..
9. Yuki Soma, Saki Tominaga, Kanako Tokito, Yuri Imado, Kosuke Naka, Taizo Hanai, Masatomo Takahashi, Yoshihiro Izumi, Takeshi Bamba*, Trace impurities in sodium phosphate influences the physiological activity of Escherichia coli in M9 minimal medium, Scientific Reports, 10.1038/s41598-023-44526-4, 13, 1, Article number 17396, 2023.10, In the field of applied microbiology, reproducibility and experimental variability are important factors that influence both basic research as well as process development for industrial applications. Experimental reproducibility and accuracy depend not only on culture conditions such as temperature and aeration but also on raw materials and procedures used for media preparation. The M9 minimal medium is one of the most common synthetic media for culturing Escherichia coli and other bacteria. This synthetic medium can be used to observe and evaluate the physiological activity of microbes under minimal nutritional requirements and determine the limiting factor for the desired phenotype. Although one of the advantages using the M9 medium is that its composition can be modulated, it is difficult to control presence of trace components and impurities from the reagents for preparing this medium. Herein, we showed that trace ingredients present in the reagents used for M9 media preparation affect the bacterial physiological activities (e.g., cell growth, substrate consumption, and byproduct formation). Additionally, we systematically identified the trace ingredient that influenced phenotypic differences. Our results showed that the selection of reagents and accuracy during reagent preparation is important for experimental reproducibility in the field of bio-engineering and systems biology focused on the systematic and continuous development of biomolecular systems (e.g., biorefinery, metabolic engineering, and synthetic biology)..
10. Shintaro Nakamura, Yasutaka Miyachi*, Akihito Shinjo, Hisashi Yokomizo, Masatomo Takahashi, Kohta Nakatani, Yoshihiro Izumi, Hiroko Otsuka, Naoichi Sato, Ryuichi Sakamoto, Takashi Miyazawa, Takeshi Bamba, Yoshihiro Ogawa*, Improved endurance capacity of diabetic mice during SGLT2 inhibition: Potential role of AICARP, an endogenous AMPK activator in the soleus muscle, Journal of Cachexia, Sarcopenia and Muscle, 10.1016/j.redox.2023.102834, 14, 6, 2866-2881, 2023.12, Background: Diabetes is associated with an increased risk of deleterious changes in muscle mass and function or sarcopenia, leading to physical inactivity and worsening glycaemic control. Given the negative energy balance during sodium-glucose cotransporter-2 (SGLT2) inhibition, whether SGLT2 inhibitors affect skeletal muscle mass and function is a matter of concern. However, how SGLT2 inhibition affects the skeletal muscle function in patients with diabetes remains insufficiently explored. We aimed to explore the effects of canagliflozin (CANA), an SGLT2 inhibitor, on skeletal muscles in genetically diabetic db/db mice focusing on the differential responses of oxidative and glycolytic muscles.

Methods: Db/db mice were treated with CANA for 4 weeks. We measured running distance and handgrip strength to assess skeletal muscle function during CANA treatment. At the end of the experiment, we performed a targeted metabolome analysis of the skeletal muscles.

Results: CANA treatment improved the reduced endurance capacity, as revealed by running distance in db/db mice (414.9 ± 52.8 vs. 88.7 ± 22.7 m, P
Conclusions: This study highlights the potential role of the AICARP/AMPK pathway in oxidative rather than glycolytic skeletal muscles during SGLT2 inhibition, providing novel insights into the mechanism by which SGLT2 inhibitors improve endurance capacity in patients with type 2 diabetes..
11. Haruna Takeda, Shohei Murakami, Zun Liu, Tomohiro Sawa, Masatomo Takahashi, Yoshihiro Izumi, Takeshi Bamba, Hideyo Sato, Takaaki Akaike, Hiroki Sekine, Hozumi Motohashi*, Sulfur metabolic response in macrophage limits excessive inflammatory response by creating a negative feedback loop, Redox Biology, 10.1016/j.redox.2023.102834, 65, Article number 102834, 2023.09, The excessive inflammatory response of macrophages plays a vital role in the pathogenesis of various diseases. The dynamic metabolic alterations in macrophages, including amino acid metabolism, are known to orchestrate their inflammatory phenotype. To explore a new metabolic pathway that regulates the inflammatory response, we examined metabolome changes in mouse peritoneal macrophages (PMs) in response to lipopolysaccharide (LPS) and found a coordinated increase of cysteine and its related metabolites, suggesting an enhanced demand for cysteine during the inflammatory response. Because Slc7a11, which encodes a cystine transporter xCT, was remarkably upregulated upon the pro-inflammatory challenge and found to serve as a major channel of cysteine supply, we examined the inflammatory behavior of Slc7a11 knockout PMs (xCT-KO PMs) to clarify an impact of the increased cysteine demand on inflammation. The xCT-KO PMs exhibited a prolonged upregulation of pro-inflammatory genes, which was recapitulated by cystine depletion in the culture media of wild-type PMs, suggesting that cysteine facilitates the resolution of inflammation. Detailed analysis of the sulfur metabolome revealed that supersulfides, such as cysteine persulfide, were increased in PMs in response to LPS, which was abolished in xCT-KO PMs. Supplementation of N-acetylcysteine tetrasulfide (NAC-S2), a supersulfide donor, attenuated the pro-inflammatory gene expression in xCT-KO PMs. Thus, activated macrophages increase cystine uptake via xCT and produce supersulfides, creating a negative feedback loop to limit excessive inflammation. Our study highlights the finely tuned regulation of macrophage inflammatory response by sulfur metabolism..
12. Shiori Oka, Miyuki Watanabe, Emi Ito, Ami Takeyama, Takuro Matsuoka, Masatomo Takahashi, Yoshihiro Izumi, Norihito Arichi, Hiroaki Ohno*, Sho Yamasaki*, Shinsuke Inuki*, Archaeal Glycerolipids Are Recognized by C-Type Lectin Receptor Mincle, Journal of the American Chemical Society, 10.1021/jacs.3c05473, 145, 33, 18538-18548, 2023.08, Recently, various metabolites derived from host microbes have been reported to modulate the immune system, with potential involvement in health or diseases. Archaea, prokaryotic organisms, are present in the human body, but their connection with the host is largely unknown when compared to other microorganisms such as bacteria. This study focused on unique glycerolipids from symbiotic methanogenic archaea and evaluated their activities toward an innate immune receptor. The results revealed that archaeal lipids were recognized by the C-type lectin receptor Mincle and induced immune responses. A concurrent structure-activity relationship study identified the key structural features of archaeal lipids required for recognition by Mincle. Subsequent gene expression profiling suggested qualitative differences between the symbiotic archaeal lipid and the pathogenic bacteria-derived lipid. These findings have broad implications for understanding the function of symbiotic archaea in host health and diseases..
13. Manabu Kodama, Gouji Toyokawa, Osamu Sugahara, Shigeaki Sugiyama, Naoki Haratake, Yuichi Yamada, Reona Wada, Shinkichi Takamori, Mototsugu Shimokawa, Tomoyoshi Takenaka, Tetsuzo Tagawa, Hiroki Kittaka, Takeshi Tsuruda, Kentaro Tanaka, Yushiro Komatsu, Keisuke Nakata, Yuri Imado, Koji Yamazaki, Isamu Okamoto, Yoshinao Oda, Masatomo Takahashi, Yoshihiro Izumi, Takeshi Bamba, Hideyuki Shimizu, Tomoharu Yoshizumi, Keiichi I Nakayama*, Modulation of host glutamine anabolism enhances the sensitivity of small cell lung cancer to chemotherapy, Cell Reports, 10.1016/j.celrep.2023.112899, 42, 8, Article number 112899, 2023.08, Small cell lung cancer (SCLC) is one of the deadliest human cancers, with a 5-year survival rate of ∼7%. Here, we performed a targeted proteomics analysis of human SCLC samples and thereby identified hypoxanthine phosphoribosyltransferase 1 (HPRT1) in the salvage purine synthesis pathway as a factor that contributes to SCLC malignancy by promoting cell survival in a glutamine-starved environment. Inhibition of HPRT1 by 6-mercaptopurine (6-MP) in combination with methotrexate (MTX), which blocks the de novo purine synthesis pathway, attenuated the growth of SCLC in mouse xenograft models. Moreover, modulation of host glutamine anabolism with the glutamine synthetase inhibitor methionine sulfoximine (MSO) in combination with 6-MP and MTX treatment resulted in marked tumor suppression and prolongation of host survival. Our results thus suggest that modulation of host glutamine anabolism combined with simultaneous inhibition of the de novo and salvage purine synthesis pathways may be of therapeutic benefit for SCLC..
14. Yuki Narukawa, Naoyuki Sugiyama, Jiro Miura, Rentaro Yamashita, Shotaro Tominaga, Yoshihiro Izumi, Takeshi Bamba, Yasushi Ishihama, Yoichiro Kashiwagi*, Shinya Murakami*, Chronic hyperglycemia reduces the expression of intercellular adhesion molecules and increases intercellular hyperpermeability in the periodontal epithelium., Journal of Periodontal Research, doi: 10.1111/jre.13140, 58, 4, 813-826, 2023.08, Background/aims: Hyperglycemia in diabetes is closely associated with periodontal disease progression. This study aimed to investigate the effect of hyperglycemia on the barrier function of gingival epithelial cells as a cause of hyperglycemia-exacerbated periodontitis in diabetes mellitus.
Methods: The abnormal expression of adhesion molecules in gingival epithelium in diabetes was compared between db/db and control mice. To study the effects of hyperglycemia on interepithelial cell permeability, the mRNA and protein expressions of adhesion molecules were investigated using a human gingival epithelial cell line (epi 4 cells) in the presence of either 5.5 mM glucose (NG) or 30 mM glucose (HG). Immunocytochemical and histological analyses were performed. We also studied HG-related intracellular signaling to assess abnormal adhesion molecule expression in the cultured epi 4 cells.
Results: The results of the proteomic analysis implied the abnormal regulation of cell-cell adhesion, and mRNA and protein expression assessments revealed the significant downregulation of Claudin1 expression in the gingival tissues of db/db mice (p Conclusions: High glucose-induced impairment of intercellular adhesion molecule expression in gingival epithelial cells was related to the intercellular permeability of gingival cells, representing a possible link to hyperglycemia-related AGE signaling, oxidative stress, and ERK1/2 activation..
15. Shun Fujinuma, Hirokazu Nakatsumi, Hideyuki Shimizu, Shigeaki Sugiyama, Akihito Harada, Takeshi Goya, Masatake Tanaka, Motoyuki Kohjima, Masatomo Takahashi, Yoshihiro Izumi, Mikako Yagi, Dongchon Kang, Mari Kaneko, Mayo Shigeta, Takeshi Bamba, Yasuyuki Ohkawa, Keiichi I Nakayama*, FOXK1 promotes nonalcoholic fatty liver disease by mediating mTORC1-dependent inhibition of hepatic fatty acid oxidation., Cell Reports, 10.1016/j.celrep.2023.112530, 42, 5, Article number 112530, 2023.05, Nonalcoholic fatty liver disease (NAFLD) is a chronic metabolic disorder caused by overnutrition and can lead to nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). The transcription factor Forkhead box K1 (FOXK1) is implicated in regulation of lipid metabolism downstream of mechanistic target of rapamycin complex 1 (mTORC1), but its role in NAFLD-NASH pathogenesis is understudied. Here, we show that FOXK1 mediates nutrient-dependent suppression of lipid catabolism in the liver. Hepatocyte-specific deletion of Foxk1 in mice fed a NASH-inducing diet ameliorates not only hepatic steatosis but also associated inflammation, fibrosis, and tumorigenesis, resulting in improved survival. Genome-wide transcriptomic and chromatin immunoprecipitation analyses identify several lipid metabolism-related genes, including Ppara, as direct targets of FOXK1 in the liver. Our results suggest that FOXK1 plays a key role in the regulation of hepatic lipid metabolism and that its inhibition is a promising therapeutic strategy for NAFLD-NASH, as well as for HCC..
16. Mayuko Yoda, Rin Mizuno, Yoshihiro Izumi, Masatomo Takahashi, Takeshi Bamba, Shinpei Kawaoka*, Nicotinamide-N-methyltransferase regulates lipid metabolism via SAM and 1-methylnicotinamide in the AML12 hepatocyte cell line., The Journal of Biochemistry, 10.1093/jb/mvad028, 174, 1, 89-98, 2023.04, Nicotinamide-N-methyltransferase (NNMT) is an enzyme that consumes S-adenosyl-methionine (SAM) and nicotinamide (NAM) to produce S-adenosyl-homocysteine (SAH) and 1-methylnicotinamide (MNAM). How much NNMT contributes to the quantity regulation of these four metabolites depends on whether NNMT is a major consumer or producer of these metabolites, which varies among various cellular contexts. Yet, whether NNMT critically regulates these metabolites in the AML12 hepatocyte cell line has been unexplored. To address this, we knock down Nnmt in AML12 cells and investigate the effects of Nnmt RNAi on metabolism and gene expression. We find that Nnmt RNAi accumulates SAM and SAH, whereas it reduces MNAM with NAM being unaltered. These results indicate that NNMT is a significant consumer of SAM and critical for MNAM production in this cell line. Moreover, transcriptome analyses reveal that altered SAM and MNAM homeostasis is accompanied by various detrimental molecular phenotypes, as exemplified by the down-regulations of lipogenic genes such as Srebf1. Consistent with this, oil-red O-staining experiments demonstrate the decrease of total neutral lipids upon Nnmt RNAi. Treating Nnmt RNAi AML12 cells with cycloleucine, an inhibitor of SAM biogenesis suppresses SAM accumulation and rescues the decrease of neutral lipids. MNAM also shows activity to elevate neutral lipids. These results suggest that NNMT contributes to lipid metabolism by maintaining proper SAM and MNAM homeostasis. This study provides an additional example where NNMT plays a critical role in regulating SAM and MNAM metabolism..
17. Takuya Morikawa, Masatomo Takahashi, Yoshihiro Izumi, Takeshi Bamba, Kosei Moriyama, Gohsuke Hattori, Ryuta Fujioka, Shiroh Miura, Hiroki Shibata*, Oleic acid‑containing phosphatidylinositol is a blood biomarker candidate for SPG28., Biomedicines, 10.3390/biomedicines11041092, 11, 4, Article number 1092, 2023.04, Hereditary spastic paraplegia is a genetic neurological disorder characterized by spasticity of the lower limbs, and spastic paraplegia type 28 is one of its subtypes. Spastic paraplegia type 28 is a hereditary neurogenerative disorder with an autosomal recessive inheritance caused by loss of function of DDHD1. DDHD1 encodes phospholipase A1, which catalyzes phospholipids to lysophospholipids such as phosphatidic acids and phosphatidylinositols to lysophosphatidic acids and lysophoshatidylinositols. Quantitative changes in these phospholipids can be key to the pathogenesis of SPG28, even at subclinical levels. By lipidome analysis using plasma from mice, we globally examined phospholipids to identify molecules showing significant quantitative changes in Ddhd1 knockout mice. We then examined reproducibility of the quantitative changes in human sera including SPG28 patients. We identified nine kinds of phosphatidylinositols that show significant increases in Ddhd1 knockout mice. Of these, four kinds of phosphatidylinositols replicated the highest level in the SPG28 patient serum. All four kinds of phosphatidylinositols contained oleic acid. This observation suggests that the amount of oleic acid-containing PI was affected by loss of function of DDHD1. Our results also propose the possibility of using oleic acid-containing PI as a blood biomarker for SPG28..
18. Kenji Morino, Kazufumi Kunimura*, Yuki Sugiura, Yoshihiro Izumi, Keisuke Matsubara, Sayaka Akiyoshi, Rae Maeda, Kenichiro Hirotani, Daiji Sakata, Seiya Mizuno, Satoru Takahashi, Takeshi Bamba, Takehito Uruno, Yoshinori Fukui*, Cholesterol sulfate limits neutrophil recruitment and gut inflammation during mucosal injury., Frontiers in Immunology, 10.3389/fimmu.2023.1131146, 14, Article number 1131146, 2023.03, During mucosal injury, intestinal immune cells play a crucial role in eliminating invading bacteria. However, as the excessive accumulation of immune cells promotes inflammation and delays tissue repair, it is essential to identify the mechanism that limits the infiltration of immune cells to the mucosal-luminal interface. Cholesterol sulfate (CS) is the lipid product of the sulfotransferase SULT2B1 and suppresses immune reactions by inhibiting DOCK2-mediated Rac activation. In this study, we aimed to elucidate the physiological role of CS in the intestinal tract. We found that, in the small intestine and colon, CS is predominantly produced in the epithelial cells close to the lumen. While dextran sodium sulfate (DSS)-induced colitis was exacerbated in Sult2b1-deficient mice with increased prevalence of neutrophils, the elimination of either neutrophils or intestinal bacteria in Sult2b1-deficient mice attenuated disease development. Similar results were obtained when the Dock2 was genetically deleted in Sult2b1-deficient mice. In addition, we also show that indomethacin-induced ulcer formation in the small intestine was exacerbated in Sult2b1-deficient mice and was ameliorated by CS administration. Thus, our results uncover that CS acts on inflammatory neutrophils, and prevents excessive gut inflammation by inhibiting the Rac activator DOCK2. The administration of CS may be a novel therapeutic strategy for inflammatory bowel disease and non-steroidal anti-inflammatory drug-induced ulcers..
19. Takumi Udo, Yuta Matsuoka, Masatomo Takahashi, Yoshihiro Izumi, Kota Saito, Kaho Tazoe, Moe Tanaka, Hideto Naka, Takeshi Bamba, Ken-Ichi Yamada*, Structural analysis of intracellular lipid radicals by LC/MS/MS using a BODIPY-based profluorescent nitroxide probe., Analytical Chemistry, 10.1021/acs.analchem.2c04950, 95, 10, 4585-4591, 2023.03, Free radical-mediated lipid peroxidation (LPO) induces the formation of numerous lipid radicals, which contribute to the development of several oxidative diseases. To understand the mechanism of LPO in biological systems and the significance of these radicals, identifying the structures of individual lipid radicals is imperative. In this study, we developed an analytical method based on liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) and a profluorescent nitroxide probe, N-(1-oxyl-2,2,6-trimethyl-6-pentylpiperidin-4-yl)-3-(5,5-difluoro-1,3-dimethyl-3H,5H-5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-7-yl)propanamide (BDP-Pen), for the detailed structural analysis of lipid radicals. The MS/MS spectra of BDP-Pen-lipid radical adducts showed product ions and thus allow the prediction of the lipid radical structures and individual detection of isomeric adducts. Using the developed technology, we separately detected the isomers of arachidonic acid (AA)-derived radicals generated in AA-treated HT1080 cells. This analytical system is a powerful tool for elucidating the mechanism of LPO in biological systems..
20. Yuka Fujito, Yoshihiro Izumi, Kohta Nakatani, Masatomo Takahashi, Yoshihiro Hayakawa, Mitsuo Takayama, Takeshi Bamba*, Understanding the mechanism of CO2-assisted electrospray ionization for parameter optimization in supercritical fluid chromatography mass spectrometry, Analytica Chimica Acta, 10.1016/j.aca.2023.340863, 1246, Article number 340863, 2023.03, Supercritical fluid chromatography (SFC) is often coupled with electrospray ionization mass spectrometry (ESI-MS) for analyte detection because of its detection capability to a wide range of chemical properties. However, MS sensitivity is highly dependent on the chromatographic conditions, so that it is important to understand the ionization mechanism to determine the optimal chromatographic conditions. The ionization mechanism in SFC/ESI-MS is different to that of liquid chromatography because of the use of CO2 as a mobile phase. Some studies have suggested that alkoxycarbonic acids are formed in the mixture of CO2 and the alcohol modifier, and these species contribute to ionization in CO2-assisted SFC/ESI-MS. Therefore, in this study, we investigated CO2-assisted ESI to test this hypothesis, and we confirmed that methoxylcarbonic acid is generated in CO2/methanol mixtures and contributed to ion generation and detection because it acts as a proton donor in positive-ion mode. However, methoxylcarbonic acid interfered with ionization in negative-ion mode. Addition of ammonium acetate, which is often added to the modifier for negative ion detection in SFC/MS analysis, did not contribute to the recovery of MS sensitivity, although it tended to suppress the formation of metoxylcarbonic acid. This is likely due to ion suppression and neutralization of the negative sites of the analytes by anions or cations derived from ammonium acetate in the negative ion mode. Thus, additive-free methanol/CO2 was the most suitable mobile phase for obtaining high sensitivity in SFC/MS. To demonstrate the practicality of these findings, we tested our optimal mobile phase selection for pesticide analysis. In addition, we tested the addition of 0, 1, and 5 mM ammonium formate to the modifier and make-up solvent, and found that the addition of 1 mM ammonium formate gave the best results in pesticides analysis. In SFC/MS, salt is often added to improve separation or prevent desorption, but our findings suggest that the concentration of salt must be kept as low as possible to achieve highly sensitive MS detection. The results of this study reveal the best selection of the optimal conditions for the modifier and make-up solvent for CO2-assisted SFC/MS analysis and will be useful for the method development in SFC/MS..
21. Jun Nakamura, Takeshi Yamamoto*, Yoshitsugu Takabatake, Tomoko Namba-Hamano, Satoshi Minami, Atsushi Takahashi, Jun Matsuda, Shinsuke Sakai, Hiroaki Yonishi, Shihomi Maeda, Sho Matsui, Isao Matsui, Takayuki Hamano, Masatomo Takahashi, Maiko Goto, Yoshihiro Izumi, Takeshi Bamba, Miwa Sasai, Masahiro Yamamoto, Taiji Matsusaka, Fumio Niimura, Motoko Yanagita, Shuhei Nakamura, Tamotsu Yoshimori, Andrea Ballabio, Yoshitaka Isaka, TFEB-mediated lysosomal exocytosis alleviates high-fat diet-induced lipotoxicity in the kidney, JCI Insight, 10.1172/jci.insight.162498, 8, 4, Article number e162498, 2023.02, Obesity is a major risk factor for end-stage kidney disease. We previously found that lysosomal dysfunction and impaired autophagic flux contribute to lipotoxicity in obesity-related kidney disease, in both humans and experimental animal models. However, the regulatory factors involved in countering renal lipotoxicity are largely unknown. Here, we found that palmitic acid strongly promoted dephosphorylation and nuclear translocation of transcription factor EB (TFEB) by inhibiting the mechanistic target of rapamycin kinase complex 1 pathway in a Rag GTPase-dependent manner, though these effects gradually diminished after extended treatment. We then investigated the role of TFEB in the pathogenesis of obesity-related kidney disease. Proximal tubular epithelial cell-specific (PTEC-specific) Tfeb-deficient mice fed a high-fat diet (HFD) exhibited greater phospholipid accumulation in enlarged lysosomes, which manifested as multilamellar bodies (MLBs). Activated TFEB mediated lysosomal exocytosis of phospholipids, which helped reduce MLB accumulation in PTECs. Furthermore, HFD-fed, PTEC-specific Tfeb-deficient mice showed autophagic stagnation and exacerbated injury upon renal ischemia/reperfusion. Finally, higher body mass index was associated with increased vacuolation and decreased nuclear TFEB in the proximal tubules of patients with chronic kidney disease. These results indicate a critical role of TFEB-mediated lysosomal exocytosis in counteracting renal lipotoxicity..
22. Takashi Shimizu, Charles R Schutt, Yoshihiro Izumi, Noriyuki Tomiyasu, Zakaria Omahdi, Kuniyuki Kano, Hyota Takamatsu, Junken Aoki, Takeshi Bamba, Atsushi Kumanogoh, Masaki Takao, Sho Yamasaki*, Direct activation of microglia by β-glucosylceramide causes phagocytosis of neurons that exacerbates Gaucher disease, Immunity, 10.1016/j.immuni.2023.01.008, 56, 1-13, 2023.02, Gaucher disease (GD) is the most common lysosomal storage disease caused by recessive mutations in the degrading enzyme of β-glucosylceramide (β-GlcCer). However, it remains unclear how β-GlcCer causes severe neuronopathic symptoms, which are not fully treated by current therapies. We herein found that β-GlcCer accumulating in GD activated microglia through macrophage-inducible C-type lectin (Mincle) to induce phagocytosis of living neurons, which exacerbated Gaucher symptoms. This process was augmented by tumor necrosis factor (TNF) secreted from activated microglia that sensitized neurons for phagocytosis. This characteristic pathology was also observed in human neuronopathic GD. Blockade of these pathways in mice with a combination of FDA-approved drugs, minocycline (microglia activation inhibitor) and etanercept (TNF blocker), effectively protected neurons and ameliorated neuronopathic symptoms. In this study, we propose that limiting unrestrained microglia activation using drug repurposing provides a quickly applicable therapeutic option for fatal neuronopathic GD..
23. Le Si-Hung, Yoshihiro Izumi, Takeshi Bamba*, First proof-of-concept of UC/HILIC for extending the versatility of the current art of supercritical fluid separation, Analytica Chimica Acta, 10.1016/j.aca.2022.340741, 1240, Article number 340741, 2023.02, Supercritical Fluid Chromatography (SFC), a high-throughput separation technique, has been widely applied as a promising routine method in pharmaceutical, pesticides, and metabolome analysis in the same way as conventional liquid chromatography and gas chromatography. Unified chromatography (UC), an advanced version of SFC, which applied gradient elution with mobile phase changing continuously from supercritical to subcritical and to liquid states, can further extend the SFC applications. UC mostly applying the popular mobile phase of 95%:5%/Methanol:Water with additives allows to analyze many hydrophilic compounds. However, many of phosphorylated metabolites or multi carboxylic acids show very poor peak shapes or even can't be eluted under UC conditions, thus hampering the UC's metabolome coverage. In this study, we proposed the first proof-of-concept of UC/HILIC, a novel strategy to extend the current UC metabolome coverage by employing an aqueous gradient right after the UC gradient on a single packed column in a single measurement. The proposed method showed significant improvement regarding the chromatographic performance and metabolome coverage, while still maintaining the precision and high throughput in comparison with conventional UC methods..
24. Naohiro Taya, Naoto Katakami*, Kazuo Omori, Shigero Hosoe, Hirotaka Watanabe, Mitsuyoshi Takahara, Kazuyuki Miyashita, Hitoshi Nishizawa, Yutaka Konya, Sachiko Obara, Ayako Hidaka, Motonao Nakao, Masatomo Takahashi, Yoshihiro Izumi, Iichiro Shimomura, Takeshi Bamba, Change in fatty acids composition of plasma triglyceride caused by a 2-week comprehensive risk management for diabetes: A prospective observational study of type 2 diabetes patients with supercritical fluid chromatography/mass spectrometry-based semi-target lipidomic analysis.

, Journal of Diabetes Investigation, 10.1111/jdi.13924, 14, 1, 102-110, 2023.01, Aims/introduction: Hypertriglyceridemia is common in patients with diabetes. Although the fatty acid (FA) composition of triglycerides (TGs) is suggested to be related to the pathology of diabetes and its complications, changes in the fatty acid composition caused by diabetes treatment remain unclear. This study aimed to identify short-term changes in the fatty acid composition of plasma triglycerides after diabetes treatment.

Materials and methods: This study was a sub-analysis of a prospective observational study of patients with type 2 diabetes aged between 20 and 75 years who were hospitalized to improve glycemic control (n = 31). A lipidomic analysis of plasma samples on the 2nd and 16th hospital days was conducted by supercritical fluid chromatography coupled with mass spectrometry.

Results: In total, 104 types of triglycerides with different compositions were identified. Most of them tended to decrease after treatment. In particular, triglycerides with a lower carbon number and fewer double bonds showed a relatively larger reduction. The inclusion of FA 14:0 (myristic acid), as a constituent of triglyceride, was significantly associated with a more than 50%, and statistically significant, reduction (odds ratio 39.0; P
Conclusions: A 2 week comprehensive risk management for diabetes resulted in decreased levels of plasma triglycerides and a change in the fatty acid composition of triglycerides, characterized by a relatively large reduction in FA 14:0 as a constituent of triglycerides..
25. Naoya Nishimura, Noriyuki Tomiyasu, Shota Torigoe, Satoru Mizuno, Hanako Fukano, Eri Ishikawa, Harutaka Katano, Yoshihiko Hoshino, Kazuhiro Matsuo, Masatomo Takahashi, Yoshihiro Izumi*, Takeshi Bamba, Koichi Akashi, Sho Yamasaki*, Mycobacterial mycolic acids trigger inhibitory receptor Clec12A to suppress host immune responses., Tuberculosis, 10.1016/j.tube.2022.102294, 94, 138, Article number 102294, 2023.01, Mycobacteria often cause chronic infection. To establish persistence in the host, mycobacteria need to evade host immune responses. However, the molecular mechanisms underlying the evasion strategy are not fully understood. Here, we demonstrate that mycobacterial cell wall lipids trigger an inhibitory receptor to suppress host immune responses. Mycolic acids are major cell wall components and are essential for survival of mycobacteria. By screening inhibitory receptors that react with mycobacterial lipids, we found that mycolic acids from various mycobacterial species bind to mouse Clec12A, and more potently to human Clec12A. Clec12A is a conserved inhibitory C-type lectin receptor containing immunoreceptor tyrosine-based inhibitory motif (ITIM). Innate immune responses, such as MCP-1 production, and PPD-specific recall T cell responses were augmented in Clec12A-deficient mice after infection. In contrast, human Clec12A transgenic mice were susceptible to infection with M. tuberculosis. These results suggest that mycobacteria dampen host immune responses by hijacking an inhibitory host receptor through their specific and essential lipids, mycolic acids. The blockade of this interaction might provide a therapeutic option for the treatment or prevention of mycobacterial infection..
26. Kohta Nakatani, Yoshihiro Izumi*, Masatomo Takahashi, Takeshi Bamba*, Unified-hydrophilic-interaction/anion-exchange liquid chromatography mass spectrometry (unified-HILIC/AEX/MS): A single-run method for comprehensive and simultaneous analysis of polar metabolome., Analytical Chemistry, 10.1021/acs.analchem.2c03986, 94, 48, 16877-16866, 2022.12, One of the technical challenges in the field of metabolomics is the development of a single-run method to detect the full complement of polar metabolites in biological samples. However, an ideal method to meet this demand has not yet been developed. Herein, we proposed a simple methodology that enables the comprehensive and simultaneous analysis of polar metabolites using unified-hydrophilic-interaction/anion-exchange liquid chromatography mass spectrometry (unified-HILIC/AEX/MS) with a polymer-based mixed amines column composed of methacrylate-based polymer particles with primary, secondary, tertiary, and quaternary amines as functional groups. The optimized unified-HILIC/AEX/MS method is composed of two consecutive chromatographic separations, HILIC-dominant separation for cationic, uncharged, and zwitterionic polar metabolites [retention times (RTs) = 0-12.8 min] and AEX-dominant separation for polar anionic metabolites (RTs = 12.8-26.5 min), by varying the ratio of acetonitrile to 40 mM ammonium bicarbonate solution (pH 9.8). A total of 400 polar metabolites were analyzed simultaneously through a combination of highly efficient separation using unified-HILIC/AEX and remarkably sensitive detection using multiple reaction monitoring-based triple quadrupole mass spectrometry (unified-HILIC/AEX/MS/MS). A nontargeted metabolomic approach using unified-HILIC/AEX high-resolution mass spectrometry (unified-HILIC/AEX/HRMS) also provided more comprehensive information on polar metabolites (3242 metabolic features) in HeLa cell extracts than the conventional HILIC/HRMS method (2068 metabolic features). Our established unified-HILIC/AEX/MS/MS and unified-HILIC/AEX/HRMS methods have several advantages over conventional techniques, including polar metabolome coverage, throughput, and accurate quantitative performance, and represent potentially useful tools for in-depth studies on metabolism and biomarker discovery..
27. Keiko Morita, Mariko Wada, Kohta Nakatani, Yuki Matsumoto, Nahoki Hayashi, Ikuko Yamahata, Kotone Mitsunari, Nagi Mukae, Masatomo Takahashi, Yoshihiro Izumi, Takeshi Bamba, Michiko Shirane*, PDZD8-deficient mice accumulate cholesteryl esters in the brain as a result of impaired lipophagy., iScience, 10.1016/j.isci.2022.105612, 25, 12, Article number 105612, 2022.11, Dyslipidemia including the accumulation of cholesteryl esters (CEs) in the brain is associated with neurological disorders, although the underlying mechanism has been unclear. PDZD8, a Rab7 effector protein, transfers lipids between endoplasmic reticulum (ER) and Rab7-positive organelles and thereby promotes endolysosome maturation and contributes to the maintenance of neuronal integrity. Here we show that CEs accumulate in the brain of PDZD8-deficient mice as a result of impaired lipophagy. This CE accumulation was not affected by diet, implicating a defect in intracellular lipid metabolism. Whereas cholesterol synthesis appeared normal, degradation of lipid droplets (LDs) was defective, in the brain of PDZD8-deficient mice. PDZD8 may mediate the exchange of cholesterol and phosphatidylserine between ER and Rab7-positive organelles to promote the fusion of CE-containing LDs with lysosomes for their degradation. Our results thus suggest that PDZD8 promotes clearance of CEs from the brain by lipophagy, with this role of PDZD8 likely contributing to brain function..
28. Kensuke Shibata, Chihiro Motozono, Masamichi Nagae, Takashi Shimizu, Eri Ishikawa, Daisuke Motooka, Daisuke Okuzaki, Yoshihiro Izumi, Masatomo Takahashi, Nao Fujimori, James B Wing, Takahide Hayano, Yoshiyuki Asai, Takeshi Bamba, Yoshihiro Ogawa, Makoto Furutani-Seiki, Mutsunori Shirai, Sho Yamasaki* , Symbiotic bacteria-dependent expansion of MR1-reactive T cells causes pancreatitis in the absence of Bcl11b in lymphocytes., Nature communications, 10.1038/s41467-022-34802-8, 13, 1, Article number 6948, 2022.11, MHC class I-related protein 1 (MR1) is a metabolite-presenting molecule that restricts MR1-reactive T cells including mucosal-associated invariant T (MAIT) cells. In contrast to MAIT cells, the function of other MR1-restricted T cell subsets is largely unknown. Here, we report that mice in which a T cell-specific transcription factor, B-cell lymphoma/leukemia 11B (Bcl11b), was ablated in immature thymocytes (Bcl11b∆iThy mice) develop chronic inflammation. Bcl11b∆iThy mice lack conventional T cells and MAIT cells, whereas CD4+IL-18R+ αβ T cells expressing skewed Traj33 (Jα33)+ T cell receptors (TCR) accumulate in the periphery, which are necessary and sufficient for the pathogenesis. The disorders observed in Bcl11b∆iThy mice are ameliorated by MR1-deficiency, transfer of conventional T cells, or germ-free conditions. We further show the crystal structure of the TCR expressed by Traj33+ T cells expanded in Bcl11b∆iThy mice. Overall, we establish that MR1-reactive T cells have pathogenic potential..
29. Naohiro Taya, Naoto Katakami*, Kazuo Omori, Shigero Hosoe, Hirotaka Watanabe, Mitsuyoshi Takahara, Kazuyuki Miyashita, Hitoshi Nishizawa, Yutaka Konya, Sachiko Obara, Ayako Hidaka, Motonao Nakao, Masatomo Takahashi, Yoshihiro Izumi, Iichiro Shimomura, Takeshi Bamba, Change in fatty acids composition of plasma triglyceride caused by a 2-week comprehensive risk management for diabetes: A prospective observational study of type 2 diabetes patients with supercritical fluid chromatography/mass spectrometry-based semi-target lipidomic analysis., Journal of Diabetes Investigation, 10.1111/jdi.13924, in press, 2022.10, Aims/introduction: Hypertriglyceridemia is common in patients with diabetes. Although the fatty acid (FA) composition of triglycerides (TGs) is suggested to be related to the pathology of diabetes and its complications, changes in the fatty acid composition caused by diabetes treatment remain unclear. This study aimed to identify short-term changes in the fatty acid composition of plasma triglycerides after diabetes treatment.
Materials and methods: This study was a sub-analysis of a prospective observational study of patients with type 2 diabetes aged between 20 and 75 years who were hospitalized to improve glycemic control (n = 31). A lipidomic analysis of plasma samples on the 2nd and 16th hospital days was conducted by supercritical fluid chromatography coupled with mass spectrometry.
Results: In total, 104 types of triglycerides with different compositions were identified. Most of them tended to decrease after treatment. In particular, triglycerides with a lower carbon number and fewer double bonds showed a relatively larger reduction. The inclusion of FA 14:0 (myristic acid), as a constituent of triglyceride, was significantly associated with a more than 50%, and statistically significant, reduction (odds ratio 39.0; P Conclusions: A 2 week comprehensive risk management for diabetes resulted in decreased levels of plasma triglycerides and a change in the fatty acid composition of triglycerides, characterized by a relatively large reduction in FA 14:0 as a constituent of triglycerides..
30. Yuki Soma, Yoshihiro Izumi, Takehiko Shimohira, Masatomo Takahashi, Yuri Imado, Saki Tominaga, Kanako Tokito, Kosuke Hata, Shoji Shinadama, Mana Oshiro, Yoshihiro Hayakawa, Takeshi Bamba*, In-needle pre-column derivatization for amino acid quantification (iPDAQ) using HPLC., Metabolites, 10.3390/metabo12090807, 12, 9, 807, 2022.08, Pre-column fluorescent derivatization has been used for the fast quantification of amino acids using high-performance liquid chromatography (HPLC) systems. However, it generally requires an offline in-vial derivatization process with multiple derivatization reagents. The offline derivatization requires the same number of reaction vials as the number of sample vials for use as a reaction chamber for the derivatization reaction in an autosampler. Therefore, the number of samples analyzed per batch using the pre-column derivatization method is halved. To benefit from the pre-column derivatization method, we transformed the derivatization process from an offline chamber process to an online in-needle process (in-needle Pre-column Derivatization for Amino acids Quantification; iPDAQ). Fluorescent derivatization in the injection needle obviated the need for vacant vials as reaction chambers. Consequently, the throughput per batch improved up to two times, and the consumption of derivatization reagents was reduced to less than one-tenth of that in the conventional vial method. We demonstrated to separate and quantify the amino acids in various biological samples. Herein, we presented a novel HPLC-based amino acid quantification method that enables the continuous analysis of a large number of samples. The iPDAQ facilitates accurate amino acid quantification due to the automation of derivatization and achieves improvement in the throughput and reduction of analysis labor..
31. Govind Kunduri*, Le Si-Hung, Baena Valentina, Nagampalli Vijaykrishna, Adam Harned, Kunio Nagashima, Daniel Blankenberg, Yoshihiro Izumi, Kedar Narayan, Takeshi Bamba,Usha Acharya*, Jairaj K. Acharya*, Delivery of ceramide phosphoethanolamine lipids to the cleavage furrow through the endocytic pathway is essential for male meiotic cytokinesis., PLoS Biology, 10.1371/journal.pbio.3001599, 20, 9, Article number e3001599, 2022.09, Cell division, wherein 1 cell divides into 2 daughter cells, is fundamental to all living organisms. Cytokinesis, the final step in cell division, begins with the formation of an actomyosin contractile ring, positioned midway between the segregated chromosomes. Constriction of the ring with concomitant membrane deposition in a specified spatiotemporal manner generates a cleavage furrow that physically separates the cytoplasm. Unique lipids with specific biophysical properties have been shown to localize to intercellular bridges (also called midbody) connecting the 2 dividing cells; however, their biological roles and delivery mechanisms remain largely unknown. In this study, we show that ceramide phosphoethanolamine (CPE), the structural analog of sphingomyelin, has unique acyl chain anchors in Drosophila spermatocytes and is essential for meiotic cytokinesis. The head group of CPE is also important for spermatogenesis. We find that aberrant central spindle and contractile ring behavior but not mislocalization of phosphatidylinositol phosphates (PIPs) at the plasma membrane is responsible for the male meiotic cytokinesis defect in CPE-deficient animals. Further, we demonstrate the enrichment of CPE in multivesicular bodies marked by Rab7, which in turn localize to cleavage furrow. Volume electron microscopy analysis using correlative light and focused ion beam scanning electron microscopy shows that CPE-enriched Rab7 positive endosomes are juxtaposed on contractile ring material. Correlative light and transmission electron microscopy reveal Rab7 positive endosomes as a multivesicular body-like organelle that releases its intraluminal vesicles in the vicinity of ingressing furrows. Genetic ablation of Rab7 or Rab35 or expression of dominant negative Rab11 results in significant meiotic cytokinesis defects. Further, we show that Rab11 function is required for localization of CPE positive endosomes to the cleavage furrow. Our results imply that endosomal delivery of CPE to ingressing membranes is crucial for meiotic cytokinesis..
32. Yutaka Konya, Yoshihiro Izumi, Kenji Hamase, Takeshi Bamba*, Ultrafast simultaneous chiral analysis of native amino acid enantiomers using supercritical fluid chromatography/tandem mass spectrometry.

, Journal of Chromatography A, 10.1016/j.chroma.2022.463305, 1677, Article number 463305, 2022.08, In the chiral separation of amino acids, liquid chromatography has been mainly used because of the physicochemical properties of the analytes. To date, only few reports of the use of supercritical fluid chromatography (SFC) for the analysis of chiral amino acids exist, and there is much room for improvement in terms of the number of measurable amino acids, peak shape, and analysis time. In this study, we developed a novel method for the chiral analysis of native amino acids using a system combining SFC and tandem mass spectrometry. Specifically, the separation of amino acid enantiomers was investigated using a CROWNPAK CR-I(+) column with a chiral stationary phase of optically active crown ether. Methanol/water mobile phase with trifluoroacetic acid as a modifier based on supercritical carbon dioxide (CO2) was used. At a low modifier concentration of 30% for the separation of hydrophilic compounds, 18 proteinogenic amino acid enantiomers except glycine and proline were successfully separated with resolution (Rs) = 1.96-33.62 within 6.5 min. In attempt to shorten the analysis time, the flow rate was increased; using a CO2/modifier ratio of 60/40 at a flow rate of 3 mL/min, ultrafast chromatography of 17 amino acid enantiomers, except histidine, was achieved with retention time ≤ 1 min and resolution ≥ 1.5. The developed ultrafast chiral separation method was verified by analyzing a commercially available black vinegar, which detected eight kinds of d-amino acids. The present method has thus confirmed to be successful and practical in terms of both analyte coverage and throughput..
33. Rin Mizuno, Hiroaki Hojo, Masatomo Takahashi, Soshiro Kashio, Sora Enya, Motonao Nakao, Riyo Konishi, Mayuko Yoda, Ayano Harata, Junzo Hamanishi, Hiroshi Kawamoto, Masaki Mandai, Yutaka Suzuki, Masayuki Miura, Takeshi Bamba, Yoshihiro Izumi, Shinpei Kawaoka*, Remote solid cancers rewire hepatic nitrogen metabolism via host nicotinamide-N-methyltransferase., Nature communications, 10.1038/s41467-022-30926-z, 13, Article number 3346, 2022.06, Cancers disrupt host homeostasis in various manners but the identity of host factors underlying such disruption remains largely unknown. Here we show that nicotinamide-N-methyltransferase (NNMT) is a host factor that mediates metabolic dysfunction in the livers of cancer-bearing mice. Multiple solid cancers distantly increase expression of Nnmt and its product 1-methylnicotinamide (MNAM) in the liver. Multi-omics analyses reveal suppression of the urea cycle accompanied by accumulation of amino acids, and enhancement of uracil biogenesis in the livers of cancer-bearing mice. Importantly, genetic deletion of Nnmt leads to alleviation of these metabolic abnormalities, and buffers cancer-dependent weight loss and reduction of the voluntary wheel-running activity. Our data also demonstrate that MNAM is capable of affecting urea cycle metabolites in the liver. These results suggest that cancers up-regulate the hepatic NNMT pathway to rewire liver metabolism towards uracil biogenesis rather than nitrogen disposal via the urea cycle, thereby disrupting host homeostasis..
34. Takaaki Tatsuguchi, Takehito Uruno*, Yuki Sugiura, Kounosuke Oisaki, Daisuke Takaya, Daiji Sakata, Yoshihiro Izumi, Takaya Togo, Yuko Hattori, Kazufumi Kunimura, Tetsuya Sakurai, Teruki Honma, Takeshi Bamba, Masafumi Nakamura, Motomu Kanai, Makoto Suematsu, Yoshinori Fukui*, Pharmacological intervention of cholesterol sulfate-mediated T cell exclusion promotes antitumor immunity., Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2022.04.035, 609, 183-188, 2022.06, Effective cancer immunotherapy requires physical contact of T cells with cancer cells. However, tumors often constitute special microenvironments that exclude T cells and resist immunotherapy. Cholesterol sulfate (CS) is a product of sulfotransferase SULT2B1b and acts as an endogenous inhibitor of DOCK2, a Rac activator essential for migration and activation of lymphocytes. We have recently shown that cancer-derived CS prevents tumor infiltration by effector T cells. Therefore, SULT2B1b may be a therapeutic target to dampen CS-mediated immune evasion. Here, we identified 3β-hydroxy-5-cholenoic acid (3β-OH-5-Chln) as a cell-active inhibitor of SULT2B1b. 3β-OH-5-Chln inhibited the cholesterol sulfotransferase activity of SULT2B1b in vitro and suppressed CS production from cancer cells expressing SULT2B1b. In vivo administration of 3β-OH-5-Chln locally reduced CS level in murine CS-producing tumors and increased infiltration of CD8+ T cells. When combined with immune checkpoint blockade or antigen-specific T cell transfer, 3β-OH-5-Chln suppressed the growth of CS-producing tumors. These results demonstrate that pharmacological inhibition of SULT2B1b can promote antitumor immunity through suppressing CS-mediated T cell exclusion..
35. Shintaro Mise, Akinobu Matsumoto*, Keisuke Shimada, Toshiaki Hosaka, Masatomo Takahashi, Kazuya Ichihara, Hideyuki Shimizu, Chisa Shiraishi, Daisuke Saito, Mikita Suyama, Tomoharu Yasuda, Toru Ide, Yoshihiro Izumi, Takeshi Bamba, Tomomi Kimura-Someya, Mikako Shirouzu, Haruhiko Miyata, Masahito Ikawa, Keiichi I Nakayama*, Kastor and Polluks polypeptides encoded by a single gene locus cooperatively regulate VDAC and spermatogenesis., Nature communications, 10.1038/s41467-022-28677-y, 13, 1, Article number 1071, 2022.02, Although several long noncoding RNAs (lncRNAs) have recently been shown to encode small polypeptides, those in testis remain largely uncharacterized. Here we identify two sperm-specific polypeptides, Kastor and Polluks, encoded by a single mouse locus (Gm9999) previously annotated as encoding a lncRNA. Both Kastor and Polluks are inserted in the outer mitochondrial membrane and directly interact with voltage-dependent anion channel (VDAC), despite their different amino acid sequences. Male VDAC3-deficient mice are infertile as a result of reduced sperm motility due to an abnormal mitochondrial sheath in spermatozoa, and deficiency of both Kastor and Polluks also severely impaired male fertility in association with formation of a similarly abnormal mitochondrial sheath. Spermatozoa lacking either Kastor or Polluks partially recapitulate the phenotype of those lacking both. Cooperative function of Kastor and Polluks in regulation of VDAC3 may thus be essential for mitochondrial sheath formation in spermatozoa and for male fertility..
36. Takaaki Tatsuguchi, Takehito Uruno*, Yuki Sugiura, Daiji Sakata, Yoshihiro Izumi, Tetsuya Sakurai, Yuko Hattori, Eiji Oki, Naoto Kubota, Koshiro Nishimoto, Masafumi Oyama, Kazufumi Kunimura, Takuto Ohki, Takeshi Bamba, Hideaki Tahara, Michiie Sakamoto, Masafumi Nakamura, Makoto Suematsu, Yoshinori Fukui*, Cancer-derived cholesterol sulfate is a key mediator to prevent tumor infiltration by effector T cells., International Immunology, 10.1093/intimm/dxac002, 2022.01, The effective tumor immunotherapy requires physical contact of T cells with cancer cells. However, tumors often constitute a specialized microenvironment that excludes T cells from the vicinity of cancer cells, and its underlying mechanisms are still poorly understood. DOCK2 is a Rac activator critical for migration and activation of lymphocytes. We herein show that cancer-derived cholesterol sulfate (CS), a lipid product of the sulfotransferase SULT2B1b, acts as a DOCK2 inhibitor and prevents tumor infiltration by effector T cells. Using clinical samples, we found that CS was abundantly produced in certain types of human cancers such as colon cancers. Functionally, CS-producing cancer cells exhibited resistance to cancer-specific T cell transfer and immune checkpoint blockade. Although SULT2B1b is known to sulfate oxysterols and inactivate their tumor-promoting activity, the expression levels of cholesterol hydroxylases, which mediate oxysterol production, are low in SULT2B1b-expressing cancers. Therefore, SULT2B1b inhibition could be a therapeutic strategy to disrupt tumor immune evasion in oxysterol-non-producing cancers. Thus, our findings define a previously unknown mechanism for tumor immune evasion and provide a novel insight into the development of effective immunotherapies..
37. Hiroko Otsuka, Hisashi Yokomizo*, Shintaro Nakamura, Yoshihiro Izumi, Masatomo Takahashi, Sachiko Obara, Motonao Nakao, Yosuke Ikeda, Naoichi Sato, Ryuichi Sakamoto, Yasutaka Miyachi, Takashi Miyazawa, Takeshi Bamba, Yoshihiro Ogawa*, Differential effect of canagliflozin, a sodium-glucose cotransporter (SGLT2) inhibitor, on slow and fast skeletal muscles from nondiabetic mice., Biochemical Journal, 10.1042/BCJ20210700, 479, 3, 425-444, 2022.02, There has been a concern that sodium-glucose cotransporter 2 (SGLT2) inhibitors could reduce skeletal muscle mass and function. Here, we examine the effect of canagliflozin (CANA), an SGLT2 inhibitor, on slow and fast muscles from nondiabetic C57BL/6J mice. In this study, mice were fed with or without CANA under ad libitum feeding, and then evaluated for metabolic valuables as well as slow and fast muscle mass and function. We also examined the effect of CANA on gene expressions and metabolites in slow and fast muscles. During SGLT2 inhibition, fast muscle function is increased, as accompanied by increased food intake, whereas slow muscle function is unaffected, although slow and fast muscle mass is maintained. When the amount of food in CANA-treated mice is adjusted to that in vehicle-treated mice, fast muscle mass and function are reduced, but slow muscle was unaffected during SGLT2 inhibition. In metabolome analysis, glycolytic metabolites and ATP are increased in fast muscle, whereas glycolytic metabolites are reduced but ATP is maintained in slow muscle during SGLT2 inhibition. Amino acids and free fatty acids are increased in slow muscle, but unchanged in fast muscle during SGLT2 inhibition. The metabolic effects on slow and fast muscles are exaggerated when food intake is restricted. This study demonstrates the differential effects of an SGLT2 inhibitor on slow and fast muscles independent of impaired glucose metabolism, thereby providing new insights into how they should be used in patients with diabetes, who are at a high risk of sarcopenia..
38. Le Si-Hung, Yoshihiro Izumi, Motonao Nakao, Masatomo Takahashi, Takeshi Bamba*, Investigation of supercritical fluid chromatography retention behaviors using quantitative structure-retention relationships., Analytica Chimica Acta, 10.1016/j.aca.2022.339463, 1197, 339463, 2022.03, Supercritical Fluid Chromatography (SFC), a high-throughput separation technique, has been widely applied as a promising routine method in pharmaceutical, pesticides, and metabolome analysis in the same way as conventional liquid chromatography and gas chromatography. However, the retention behaviors of many compounds in SFC are not fully investigated. In this study, more than 500 pesticides were analyzed on several polar and nonpolar columns using SFC/MS/MS. Then, partial least squares regression (PLS) was used to explore the retention behaviors of pesticides and construct the quantitative structure-retention relationships under practical gradient elution. The optimized relationships between pesticide structures and pesticide retention were established and validated for predicting power using both internal- and external-validations; hence, several important factors affecting retention of the compounds were identified. In the best case, approximately almost all pesticides in the training set and nearly 80% of pesticides in the external validation set could be predicted with the prediction error of less than 0.5 min. Moreover, the proposed workflow successfully established the local interaction profiles, describing the possible interactions in the 8 studied chromatographic systems, and can be further applied for any groups of compounds under any system conditions..
39. Takuji Yamauchi, Kohta Miyawaki, Yuichiro Semba, Masatomo Takahashi, Yoshihiro Izumi, Jumpei Nogami, Fumihiko Nakao, Takeshi Sugio, Kensuke Sasaki, Luca Pinello, Daniel E Bauer, Takeshi Bamba, Koichi Akashi, Takahiro Maeda*, Targeting leukemia-specific dependence on the de novo purine synthesis pathway., Leukemia, 10.1038/s41375-021-01369-0, 36, 2, 383-393, 2022.02, Acute myeloid leukemia (AML) is a devastating disease, and clinical outcomes are still far from satisfactory. Here, to identify novel targets for AML therapy, we performed a genome-wide CRISPR/Cas9 screen using AML cell lines, followed by a second screen in vivo. We show that PAICS, an enzyme involved in de novo purine biosynthesis, is a potential target for AML therapy. AML cells expressing shRNA-PAICS exhibited a proliferative disadvantage, indicating a toxic effect of shRNA-PAICS. Treatment of human AML cells with a PAICS inhibitor suppressed their proliferation by inhibiting DNA synthesis and promoting apoptosis and had anti-leukemic effects in AML PDX models. Furthermore, CRISPR/Cas9 screens using AML cells in the presence of the inhibitor revealed genes mediating resistance or synthetic lethal to PAICS inhibition. Our findings identify PAICS as a novel therapeutic target for AML and further define components of de novo purine synthesis pathway and its downstream effectors essential for AML cell survival..
40. Shin Nishiumi*, Yoshihiro Izumi*, Akiyoshi Hirayama*, Masatomo Takahashi, Motonao Nakao, Kosuke Hata, Daisuke Saigusa, Eiji Hishinuma, Naomi Matsukawa, Suzumi M. Tokuoka, Yoshihiro Kita, Fumie Hamano, Nobuyuki Okahashi, Kazutaka Ikeda, Hiroki Nakanishi, Kosuke Saito, Masami Yokota Hirai, Masaru Yoshida, Yoshiya Oda, Fumio Matsuda, Takeshi Bamba, Comparative evaluation of plasma metabolomic data from multiple laboratories., Metabolites, 10.3390/metabo12020135, 12, 2, 135, 2022.01, In mass spectrometry-based metabolomics, the differences in the analytical results from different laboratories/machines are an issue to be considered because various types of machines are used in each laboratory. Moreover, the analytical methods are unique to each laboratory. It is important to understand the reality of inter-laboratory differences in metabolomics. Therefore, we have evaluated whether the differences in analytical methods, with the exception sample pretreatment and including metabolite extraction, are involved in the inter-laboratory differences or not. In this study, nine facilities are evaluated for inter-laboratory comparisons of metabolomic analysis. Identical dried samples prepared from human and mouse plasma are distributed to each laboratory, and the metabolites are measured without the pretreatment that is unique to each laboratory. In these measurements, hydrophilic and hydrophobic metabolites are analyzed using 11 and 7 analytical methods, respectively. The metabolomic data acquired at each laboratory are integrated, and the differences in the metabolomic data from the laboratories are evaluated. No substantial difference in the relative quantitative data (human/mouse) for a little less than 50% of the detected metabolites is observed, and the hydrophilic metabolites have fewer differences between the laboratories compared with hydrophobic metabolites. From evaluating selected quantitatively guaranteed metabolites, the proportion of metabolites without the inter-laboratory differences is observed to be slightly high. It is difficult to resolve the inter-laboratory differences in metabolomics because all laboratories cannot prepare the same analytical environments. However, the results from this study indicate that the inter-laboratory differences in metabolomic data are due to measurement and data analysis rather than sample preparation, which will facilitate the understanding of the problems in metabolomics studies involving multiple laboratories..
41. Yuki Soma, Masatomo Takahashi, Yuri Fujiwara, Noriyuki Tomiyasu, Maiko Goto, Taizo Hanai, Yoshihiro Izumi, Takeshi Bamba*, Quantitative metabolomics for dynamic metabolic engineering using stable isotope labeled internal standards mixture (SILIS)., Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2021.09.009, 133, 1, 46-55, 2022.01, The production of chemicals and fuels from renewable resources using engineered microbes is an attractive alternative for current fossil-dependent industries. Metabolic engineering has contributed to pathway engineering for the production of chemicals and fuels by various microorganisms. Recently, dynamic metabolic engineering harnessing synthetic biological tools has become a next-generation strategy in this field. The dynamic regulation of metabolic flux during fermentation optimizes metabolic states according to each fermentation stage such as cell growth phase and compound production phase. However, it is necessary to repeat the evaluation and redesign of the dynamic regulation system to achieve the practical use of engineered microbes. In this study, we performed quantitative metabolome analysis to investigate the effects of dynamic metabolic flux regulation on engineered Escherichia coli for γ-amino butyrate (GABA) fermentation. We prepared a stable isotope-labeled internal standard mixture (SILIS) for the stable isotope dilution method (SIDM), a mass spectrometry-based quantitative metabolome analysis method. We found multiple candidate bottlenecks for GABA production. Some metabolic reactions in the GABA production pathway should be engineered for further improvement in the direct GABA fermentation with dynamic metabolic engineering strategy..
42. Hiroko Watanabe, Riku Usami, Shigenobu Kishino, Kengo Osada, Yudai Aoki, Hironobu Morisaka, Masatomo Takahashi, Yoshihiro Izumi, Takeshi Bamba, Wataru Aoki, Hiroyuki Suganuma, Jun Ogawa*, Enzyme systems involved in glucosinolate metabolism in Companilactobacillus farciminis KB1089., Scientific Reports, 10.1038/s41598-021-03064-7, 11, 1, Article number 23715, 2021.12, Cruciferous vegetables are rich sources of glucosinolates (GSLs). GSLs are degraded into isothiocyanates, which are potent anticarcinogens, by human gut bacteria. However, the mechanisms and enzymes involved in gut bacteria-mediated GSL metabolism are currently unclear. This study aimed to elucidate the enzymes involved in GSL metabolism in lactic acid bacteria, a type of gut bacteria. Companilactobacillus farciminis KB1089 was selected as a lactic acid bacteria strain model that metabolizes sinigrin, which is a GSL, into allylisothiocyanate. The sinigrin-metabolizing activity of this strain is induced under glucose-absent and sinigrin-present conditions. A quantitative comparative proteomic analysis was conducted and a total of 20 proteins that were specifically expressed in the induced cells were identified. Three candidate proteins, β-glucoside-specific IIB, IIC, IIA phosphotransferase system (PTS) components (CfPttS), 6-phospho-β-glucosidase (CfPbgS) and a hypothetical protein (CfNukS), were suspected to be involved in sinigrin-metabolism and were thus investigated further. We hypothesize a pathway for sinigrin degradation, wherein sinigrin is taken up and phosphorylated by CfPttS, and subsequently, the phosphorylated entity is degraded by CfPbgS. As expression of both pttS and pbgS genes clearly gave Escherichia coli host strain sinigrin converting activity, these genes were suggested to be responsible for sinigrin degradation. Furthermore, heterologous expression analysis using Lactococcus lactis suggested that CfPttS was important for sinigrin degradation and CfPbgS degraded phosphorylated sinigrin..
43. Kosho Yamauchi, Yuta Matsuoka, Masatomo Takahashi, Yoshihiro Izumi, Hideto Naka, Yosuke Taniguchi, Kazuaki Kawai, Takeshi Bamba, Ken-Ichi Yamada*, Detection and structural analysis of pyrimidine-derived radicals generated on DNA using a profluorescent nitroxide probe., Chemical Communications, 10.1039/d1cc04998d, 58, 1, 56-59, 2021.12, The oxidative damage of DNA is associated with aging and the development of various diseases. Although nucleoside-derived radicals play an important role in DNA oxidation, their analysis methods are limited. Herein, we propose a fluorometric detection and structural analysis of radicals on the surface of oxidatively damaged DNA using a profluorescent nitroxide probe combined with liquid chromatography–fluorometry and high-resolution tandem mass spectrometry..
44. Yuta Matsuoka, Masatomo Takahashi, Yuki Sugiura, Yoshihiro Izumi, Kazuhiro Nishiyama, Motohiro Nishida, Makoto Suematsu, Takeshi Bamba, Ken-Ichi Yamada*, Structural library and visualization of endogenously oxidized phosphatidylcholines using mass spectrometry-based techniques., Nature Communications, 10.1038/s41467-021-26633-w, 12, 1, Article number 6339, 2021.11, Although oxidized phosphatidylcholines (oxPCs) play critical roles in numerous pathological events, the type and production sites of endogenous oxPCs remain unknown because of the lack of structural information and dedicated analytical methods. Herein, a library of 465 oxPCs is constructed using high-resolution mass spectrometry-based non-targeted analytical methods and employed to detect 70 oxPCs in mice with acetaminophen-induced acute liver failure. We show that doubly oxygenated polyunsaturated fatty acid (PUFA)-PCs (PC PUFA;O2), containing epoxy and hydroxide groups, are generated in the early phase of liver injury. Hybridization with in-vivo 18O labeling and matrix-assisted laser desorption/ionization-tandem MS imaging reveals that PC PUFA;O2 are accumulated in cytochrome P450 2E1-expressing and glutathione-depleted hepatocytes, which are the major sites of liver injury. The developed library and visualization methodology should facilitate the characterization of specific lipid peroxidation events and enhance our understanding of their physiological and pathological significance in lipid peroxidation-related diseases..
45. Daisuke Saigusa*, Eiji Hishinuma, Naomi Matsukawa, Masatomo Takahashi, Jin Inoue, Shu Tadaka, Ikuko N Motoike, Atsushi Hozawa, Yoshihiro Izumi, Takeshi Bamba, Kengo Kinoshita, Kim Ekroos, Seizo Koshiba, Masayuki Yamamoto, Comparison of kit-based metabolomics with other methodologies in a large cohort, towards establishing reference values., Metabolites, 10.3390/metabo11100652, 11, 10, 652, 2021.09, Metabolic profiling is an omics approach that can be used to observe phenotypic changes, making it particularly attractive for biomarker discovery. Although several candidate metabolites biomarkers for disease expression have been identified in recent clinical studies, the reference values of healthy subjects have not been established. In particular, the accuracy of concentrations measured by mass spectrometry (MS) is unclear. Therefore, comprehensive metabolic profiling in large-scale cohorts by MS to create a database with reference ranges is essential for evaluating the quality of the discovered biomarkers. In this study, we tested 8700 plasma samples by commercial kit-based metabolomics and separated them into two groups of 6159 and 2541 analyses based on the different ultra-high-performance tandem mass spectrometry (UHPLC-MS/MS) systems. We evaluated the quality of the quantified values of the detected metabolites from the reference materials in the group of 2541 compared with the quantified values from other platforms, such as nuclear magnetic resonance (NMR), supercritical fluid chromatography tandem mass spectrometry (SFC-MS/MS) and UHPLC-Fourier transform mass spectrometry (FTMS). The values of the amino acids were highly correlated with the NMR results, and lipid species such as phosphatidylcholines and ceramides showed good correlation, while the values of triglycerides and cholesterol esters correlated less to the lipidomics analyses performed using SFC-MS/MS and UHPLC-FTMS. The evaluation of the quantified values by MS-based techniques is essential for metabolic profiling in a large-scale cohort..
46. Yoichiro Kashiwagi, Shunsuke Aburaya, Naoyuki Sugiyama, Yuki Narukawa, Yuta Sakamoto, Masatomo Takahashi, Hayato Uemura, Rentaro Yamashita, Shotaro Tominaga, Satoko Hayashi, Takenori Nozaki, Satoru Yamada, Yoshihiro Izumi, Atsunori Kashiwagi, Takeshi Bamba, Yasushi Ishihama, Shinya Murakami*, Porphyromonas gingivalis induces entero-hepatic metabolic derangements with alteration of gut microbiota in a type 2 diabetes mouse model., Scientific Reports, 10.1038/s41598-021-97868-2, 11, 1, Article number 18398, 2021.09, Periodontal infection induces systemic inflammation; therefore, aggravating diabetes. Orally administered periodontal pathogens may directly alter the gut microbiota. We orally treated obese db/db diabetes mice using Porphyromonas gingivalis (Pg). We screened for Pg-specific peptides in the intestinal fecal specimens and examined whether Pg localization influenced the intestinal microbiota profile, in turn altering the levels of the gut metabolites. We evaluated whether the deterioration in fasting hyperglycemia was related to the changes in the intrahepatic glucose metabolism, using proteome and metabolome analyses. Oral Pg treatment aggravated both fasting and postprandial hyperglycemia (P
47. Kazuki Ikeda, Masatomo Takahashi, Shunsuke Aburaya, Daiki Harada, Maki Ikeda, Yume Kitagawa, Yuki Soma, Yoshihiro Izumi, Takeshi Bamba, Mitsuhiro Furuse*, Produced β-hydroxybutyrate after β-hydroxy-β-methylbutyrate (HMB) administration may contribute HMB function in mice., Biochemistry and Biophysics Reports, 10.1016/j.bbrep.2021.101097, 27, Article number 101097, 2021.08, β-Hydroxy-β-methylbutyrate (HMB) is an intermediate in the metabolism of the branched-chain amino acid leucine. HMB has several demonstrated effects on skeletal muscle function, some of which are contradictory. In addition, the effect of exogenous HMB intake on the levels of intermediate metabolites is not known. Therefore, we investigated changes in HMB metabolites after oral HMB administration in mice. First, ICR mice were treated with either distilled water or HMB (0.215 g/10 mL/kg). Sampling was performed at 0, 1, 6, 12, and 24 h after administration. Next, ICR mice were given distilled water or HMB (0.215 g/10 mL/kg/d) for 10 d. Mice given HMB shown a significant increase in liver β-methylcrotonyl-CoA and increased β-hydroxybutyrate in plasma and the gastrocnemius muscle 1 h after HMB administration. Mice administered HMB for 10 d showed significantly decreased food intake and body weight; however, the relative weight of the gastrocnemius muscle was significantly increased. These results may be attributed to an increase in β-hydroxybutyrate resulting from exogenous HMB, since β-hydroxybutyrate inhibits food intake and suppresses skeletal muscle catabolism. In conclusion, β-hydroxybutyrate, a metabolite of HMB, was found to play an important role in the function of HMB..
48. Amandine Dispas*, Adrian Clarke, Alexandre Grand-Guillaume Perrenoud, Luca Gioacchino Losacco, Jean-Luc Veuthey, Quentin Gros, Jérémy Molineau, Angéline Noireau, Caroline West, Fabio Salafia, Mariosimone Zoccali, Luigi Mondello, Amber Guillen, Jenny Wang, Kelly Zhang, Philipp Jochems, Gesa Schad, Kosuke Nakajima, Shinnosuke Horie, Jan Joseph, Maria Kristina Parr, Pierre Billemont, Antoni Severino, Sonja Schneider, Edgar Naegele, Daniel Kutscher, Rick Wikfors, Regina Black, Lee Ingvaldson, Jimmy Oliveira Da Silva, Raffeal Bennett, Erik L Regalado, Thi Phuong Thuy Hoang, David Touboul, Yana Nikolova, Mariana Kamenova-Nacheva, Vladimir Dimitrov, Blair K Berger, Kevin A Schug, Solène Kerviel-Guillon, Fabien Mauge, Masatomo Takahashi, Yoshihiro Izumi, Takeshi Bamba, Florent Rouvière, Sabine Heinisch, Davy Guillarme, Philippe Hubert, Interlaboratory study of a supercritical fluid chromatography method for the determination of pharmaceutical impurities: evaluation of multi-systems reproducibility., Journal of Pharmaceutical and Biomedical Analysis, 10.1016/j.jpba.2021.114206, 203, Article number 114206, 2021.09, Modern supercritical fluid chromatography (SFC) is now a well-established technique, especially in the field of pharmaceutical analysis. We recently demonstrated the transferability and the reproducibility of a SFC-UV method for pharmaceutical impurities by means of an inter-laboratory study. However, as this study involved only one brand of SFC instrumentation (Waters®), the present study extends the purpose to multi-instrumentation evaluation. Specifically, three instrument types, namely Agilent®, Shimadzu®, and Waters®, were included through 21 laboratories (n = 7 for each instrument). First, method transfer was performed to assess the separation quality and to set up the specific instrument parameters of Agilent® and Shimadzu® instruments. Second, the inter-laboratory study was performed following a protocol defined by the sending lab. Analytical results were examined regarding consistencies within- and between-laboratories criteria. Afterwards, the method reproducibility was estimated taking into account variances in replicates, between-days and between-laboratories. Reproducibility variance was larger than that observed during the first study involving only one single type of instrumentation. Indeed, we clearly observed an 'instrument type' effect. Moreover, the reproducibility variance was larger when considering all instruments than each type separately which can be attributed to the variability induced by the instrument configuration. Nevertheless, repeatability and reproducibility variances were found to be similar than those described for LC methods; i.e. reproducibility as %RSD was around 15 %. These results highlighted the robustness and the power of modern analytical SFC technologies to deliver accurate results for pharmaceutical quality control analysis..
49. Yuki Soma, Masatomo Takahashi, Yuri Fujiwara, Tamaki Shinohara, Yoshihiro Izumi, Taizo Hanai*, Takeshi Bamba*, Design of synthetic quorum sensing achieving induction timing-independent signal stabilization for dynamic metabolic engineering of E. coli., ACS Synthetic Biology, 10.1021/acssynbio.1c00008, 10, 6, 1384-1393, 2021.06, Dynamic metabolic engineering that harnesses synthetic biological tools is a next-generation strategy for microbial chemical and fuel production. We previously reported a synthetic quorum sensing system combined with a metabolic toggle switch (QS-MTS) in E. coli. It autonomously redirected endogenous metabolic flux toward the synthetic metabolic pathway and improved biofuel production. However, its functions and effects on host metabolism were attenuated by induction timing delay. Here, we redesigned the QS-MTS to stabilize QS signaling efficiency and metabolic regulation. We performed a metabolome analysis to clarify the effects of QS-MTS redesign on host metabolism. We compared the contributions of conventional and redesigned QS-MTS to fed-batch fermentation. The redesigned QS-MTS was more conducive than the conventional QS-MTS to long-term processes such as fed-batch fermentation. Here, we present a circuit redesign for metabolic flux control based on dynamic characteristic evaluation and metabolome analysis..
50. Takeshi Hara, Gino V Baron, Kosuke Hata, Yoshihiro Izumi, Takeshi Bamba*, Gert Desme*, Performance of functionalized monolithic silica capillary columns with different mesopore sizes using radical polymerization of octadecyl methacrylate., Journal of Chromatography A, 10.1016/j.chroma.2021.462282, 1651, Article number 462282, 2021.08, We report on the possibility to enhance the phase ratio and retention factor in silica monoliths. According to pioneering work done by Núñez et al. [1], this enhancement is pursued by applying a stationary phase layer via radical polymerization with octadecyl methacrylate (ODM) as an alternative to the customary octadecylsilylation (C18-derivatization). The difference in band broadening, retention factor and separation selectivity between both approaches was compared. Different hydrothermal treatment temperatures for the column preparation were applied to produce monolithic silica structures with three different mesopore sizes (resp. 10, 13, and 16 nm, as determined by argon physisorption) while maintaining similar domain size (sum of through-pore and skeleton size). It has been found that the columns with the poly(octadecyl methacrylate)-phase (ODM columns) provided a 60 to 80% higher retention factor in methanol-water mixture compared to the octadecylsilylated (ODS) columns produced by starting from similar silica backbone structures. In acetonitrile-water mixture, the enhancement is smaller (15 to 30% times higher), yet significant. By adjusting the fabrication conditions (for both the preparation of the monolithic backbones and the surface functionalization), the achieved retention factors (up k = 4.89 for pentylbenzene in 80:20% (v/v) methanol/water) are obviously higher than obtained in the pioneering study on ODM monoliths of Núñez et al. [1], and column clogging could be completely avoided. In addition, also separation efficiencies were significantly higher than shown in Ref. [1], with plate heights as low as 5.8 μm. These plate heights are however inferior to those observed on the ODS-modified sister columns. The difference can be explained by the slower intra-skeleton diffusion displayed by the ODM-modified columns, in turn caused by the larger obstruction to diffusion originating from the thicker stationary phase layer..
51. Takahiro Onoki, Yoshihiro Izumi, Masatomo Takahashi, Shohei Murakami, Daisuke Matsumaru, Nao Ohta, Sisca Meida Wati, Nozomi Hatanaka, Fumiki Katsuoka, Mitsuharu Okutsu, Yutaka Yabe, Yoshihiro Hagiwara, Makoto Kanzaki, Takeshi Bamba, Eiji Itoi, Hozumi Motohashi*, Skeletal muscle-specific Keap1 disruption modulates fatty acid utilization and enhances exercise capacity in female mice, Redox Biology, 10.1016/j.redox.2021.101966, 43, Article number 101966, 2021.07, Skeletal muscle health is important for the prevention of various age-related diseases. The loss of skeletal muscle mass, which is known as sarcopenia, underlies physical disability, poor quality of life and chronic diseases in elderly people. The transcription factor NRF2 plays important roles in the regulation of the cellular defense against oxidative stress, as well as the metabolism and mitochondrial activity. To determine the contribution of skeletal muscle NRF2 to exercise capacity, we conducted skeletal muscle-specific inhibition of KEAP1, which is a negative regulator of NRF2, and examined the cell-autonomous and non-cell-autonomous effects of NRF2 pathway activation in skeletal muscles. We found that NRF2 activation in skeletal muscles increased slow oxidative muscle fiber type and improved exercise endurance capacity in female mice. We also observed that female mice with NRF2 pathway activation in their skeletal muscles exhibited enhanced exercise-induced mobilization and β-oxidation of fatty acids. These results indicate that NRF2 activation in skeletal muscles promotes communication with adipose tissues via humoral and/or neuronal signaling and facilitates the utilization of fatty acids as an energy source, resulting in increased mitochondrial activity and efficient energy production during exercise, which leads to improved exercise endurance..
52. Yan-Yu Chen, Yuki Soma, Masahito Ishikawa, Masatomo Takahashi, Yoshihiro Izumi, Takeshi Bamba, Katsutoshi Hori*, Metabolic alteration of Methylococcus capsulatus str. Bath during a microbial gas-phase reaction, Bioresource Technology, 10.1016/j.biortech.2021.125002, 330, Article number 125002, 2021.06, This study demonstrates the metabolic alteration of Methylococcus capsulatus (Bath), a representative bacterium among methanotrophs, in microbial gas-phase reactions. For comparative metabolome analysis, a bioreactor was designed to be capable of supplying gaseous substrates and liquid nutrients continuously. Methane degradation by M. capsulatus (Bath) was more efficient in a gas-phase reaction operated in the bioreactor than in an aqueous phase reaction operated in a batch reactor. Metabolome analysis revealed remarkable alterations in the metabolism of cells in the gas-phase reaction; in particular, pyruvate, 2-ketoglutarate, some amino acids, xanthine, and hypoxanthine were accumulated, whereas 2,6-diaminopimelate was decreased. Based on the results of metabolome analysis, cells in the gas-phase reaction seemed to alter their metabolism to reduce the excess ATP and NADH generated upon increased availability of methane and oxygen. Our findings will facilitate the development of efficient processes for methane-based bioproduction with low energy consumption..
53. Kosuke Hata, Yuki Soma, Toshiyuki Yamashita, Masatomo Takahashi, Kuniyo Sugitate, Takeshi Serino, Hiromi Miyagawa, Kenichi Suzuki, Kayoko Yamada, Takatomo Kawamukai, Teruhisa Shiota, Yoshihiro Izumi*, Takeshi Bamba*, Calibration-curve-locking database for semi-quantitative metabolomics by gas chromatography/mass spectrometry, Metabolites, 10.3390/metabo11040207, 11, 4, Article number 207, 2021.03, Calibration-Curve-Locking Databases (CCLDs) have been constructed for automatic compound search and semi-quantitative screening by gas chromatography/mass spectrometry (GC/MS) in several fields. CCLD felicitates the semi-quantification of target compounds without calibration curve preparation because it contains the retention time (RT), calibration curves, and electron ionization (EI) mass spectra, which are obtained under stable apparatus conditions. Despite its usefulness, there is no CCLD for metabolomics. Herein, we developed a novel CCLD and semi-quantification framework for GC/MS-based metabolomics. All analytes were subjected to GC/MS after derivatization under stable apparatus conditions using (1) target tuning, (2) RT locking technique, and (3) automatic derivatization and injection by a robotic platform. The RTs and EI mass spectra were obtained from an existing authorized database. A quantifier ion and one or two qualifier ions were selected for each target metabolite. The calibration curves were obtained as plots of the peak area ratio of the target compounds to an internal standard versus the target compound concentration. These data were registered in a database as a novel CCLD. We examined the applicability of CCLD for analyzing human plasma, resulting in time-saving and labor-saving semi-qualitative screening without the need for standard substances..
54. Takuya Morikawa, Hiroaki Ohishi, Kengo Kosaka, Tomofumi Shimojo, Akihiro Nagano, Itsuki Taniguchi, Ryuta Fujioka, Kosei Moriyama, Motoko Unoki, Masatomo Takahashi, Motonao Nakao, Yoshihiro Izumi, Takeshi Bamba, Hiroyuki Sasaki, Shiroh Miura, Hiroki Shibata*, Ddhd1 knockout mouse as a model of locomotive and physiological abnormality in familial spastic paraplegia, Bioscience Reports, 10.1042/BSR20204171, 41, 2, Article number BSR20204171, 2021.02, We have previously reported a novel homozygous 4-bp deletion in DDHD1 as the responsible variant for spastic paraplegia type 28 (SPG28; OMIM#609340). The variant causes a frameshift, resulting in a functionally null allele in the patient. DDHD1 encodes phospholipase A1 (PLA1) catalyzing phosphatidylinositol to lysophosphatidylinositol (LPI). To clarify the pathogenic mechanism of SPG28, we established Ddhd1 knockout mice (Ddhd1[−/−]) carrying a 5-bp deletion in Ddhd1, resulting in a premature termination of translation at a position similar to that of the patient. We observed a significant decrease in foot–base angle (FBA) in aged Ddhd1(−/−) (24 months of age) and a significant decrease in LPI 20:4 (sn-2) in Ddhd1(−/−) cerebra (26 months of age). These changes in FBA were not observed in 14 months of age. We also observed significant changes of expression levels of 22 genes in the Ddhd1(−/−) cerebra (26 months of age). Gene Ontology (GO) terms relating to the nervous system and cell–cell communications were significantly enriched. We conclude that the reduced signaling of LPI 20:4 (sn-2) by PLA1 dysfunction is responsible for the locomotive abnormality in SPG28, further suggesting that the reduction of downstream signaling such as GPR55 which is agonized by LPI is involved in the pathogenesis of SPG28..
55. Kazuhiro Tanaka*, Takashi Sasayama, Hiroaki Nagashima, Yasuhiro Irino, Masatomo Takahashi, Yoshihiro Izumi, Takiko Uno, Naoko Satoh, Akane Kitta, Katsusuke Kyotani, Yuichi Fujita, Mitsuru Hashiguchi, Tomoaki Nakai, Masaaki Kohta, Yoichi Uozumi, Masakazu Shinohara, Kohkichi Hosoda, Takeshi Bamba, Eiji Kohmura, Glioma cells require one-carbon metabolism to survive glutamine starvation, Acta Neuropathologica Communications, 10.1186/s40478-020-01114-1, 9, Article number 16, 2021.01, Cancer cells optimize nutrient utilization to supply energetic and biosynthetic pathways. This metabolic process also includes redox maintenance and epigenetic regulation through nucleic acid and protein methylation, which enhance tumorigenicity and clinical resistance. However, less is known about how cancer cells exhibit metabolic flexibility to sustain cell growth and survival from nutrient starvation. Here, we find that serine and glycine levels were higher in low-nutrient regions of tumors in glioblastoma multiforme (GBM) patients than they were in other regions. Metabolic and functional studies in GBM cells demonstrated that serine availability and one-carbon metabolism support glioma cell survival following glutamine deprivation. Serine synthesis was mediated through autophagy rather than glycolysis. Gene expression analysis identified upregulation of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) to regulate one-carbon metabolism. In clinical samples, MTHFD2 expression was highest in the nutrient-poor areas around “pseudopalisading necrosis.” Genetic suppression of MTHFD2 and autophagy inhibition caused tumor cell death and growth inhibition of glioma cells upon glutamine deprivation. These results highlight a critical role for serine-dependent one-carbon metabolism in surviving glutamine starvation and suggest new therapeutic targets for glioma cells adapting to a low-nutrient microenvironment..
56. Masahiro Nagata, Kenji Toyonaga, Eri Ishikawa, Shojiro Haji, Nobuyuki Okahashi, Masatomo Takahashi, Yoshihiro Izumi, Akihiro Imamura, Koichi Takato, Hideharu Ishida, Shigenori Nagai, Petr Illarionov, Bridget L Stocker, Mattie S M Timmer, Dylan G M Smith, Spencer J Williams, Takeshi Bamba, Tomofumi Miyamoto, Makoto Arita, Ben J Appelmelk, Sho Yamasaki*, Helicobacter metabolites exacerbate gastritis through C-type lectin receptors, Journal of Experimental Medicine, org/10.1084/jem.20200815, 218, 1, e20200815, 2020.10, Helicobacter pylori causes gastritis, which has been attributed to the development of H. pylori–specific T cells during infection. However, the mechanism underlying innate immune detection leading to the priming of T cells is not fully understood, as H. pylori evades TLR detection. Here, we report that H. pylori metabolites modified from host cholesterol exacerbate gastritis through the interaction with C-type lectin receptors. Cholesteryl acyl α-glucoside (αCAG) and cholesteryl phosphatidyl α-glucoside (αCPG) were identified as noncanonical ligands for Mincle (Clec4e) and DCAR (Clec4b1). During chronic infection, H. pylori–specific T cell responses and gastritis were ameliorated in Mincle-deficient mice, although bacterial burdens remained unchanged. Furthermore, a mutant H. pylori strain lacking αCAG and αCPG exhibited an impaired ability to cause gastritis. Thus H. pylori–specific modification of host cholesterol plays a pathophysiological role that exacerbates gastric
inflammation by triggering C-type lectin receptors..
57. Tatsuya Fushimi, Yoshihiro Izumi*, Masatomo Takahashi, Kosuke Hata, Yoshihiro Murano, and Takeshi Bamba, Dynamic metabolome analysis reveals the metabolic fate of medium-chain fatty acid in AML12 cells, Journal of Agricultural and Food Chemistry, org/10.1021/acs.jafc.0c04723, 68, 43, 11997-12010, 2020.10, Several studies in hepatocyte cell lines reported that medium-chain fatty acids (MCFAs) with 6–12 carbons showed different metabolic properties from long-chain fatty acids (LCFAs). However, these studies reported unclear effects of different fatty acid molecules on hepatocyte metabolism. This study is aimed to capture the metabolic kinetics of MCFA assimilation in AML12 cells treated with octanoic acid (FA 8:0), decanoic acid (FA 10:0), or lauric acid (FA12:0) [LCFA; oleic acid (FA 18:1)] via metabolic profiling and dynamic metabolome analysis with 13C-labeling. The concentrations of total ketone bodies in the media of cells treated with FA 8:0 or FA 10:0 were 3.22- or 3.69-fold higher than those obtained with FA 18:1 treatment, respectively. FA 12:0 treatment did not significantly increase ketone body levels compared to DMSO treatment (control), whereas FA 12:0 treatment increased intracellular triacylglycerol (TG) levels 15.4 times compared to the control. Metabolic profiles of FA 12:0-treated samples differed from those of the FA 8:0-treated and FA 10:0-treated samples, suggesting that metabolic assimilation of MCFAs differed significantly depending on the MCFA type. Furthermore, the dynamic metabolome analysis clearly revealed that FA 8:0 was rapidly and quantitatively oxidized to acetyl-CoA and assimilated into ketone bodies, citrate cycle intermediates, and glucogenic amino acids but not readily into TGs..
58. Yutaka Konya, Yoshihiro Izumi, Takeshi Bamba*, Development of a novel method for polar metabolite profiling by supercritical fluid chromatography/tandem mass spectrometry, Journal of Chromatography A, 10.1016/j.chroma.2020.461587, 1632, Article number 461587, 2020.09, Supercritical carbon dioxide (scCO2), the main fluid in the mobile phase for supercritical fluid chromatography (SFC), is non-polar. The majority of polar compounds are little soluble in scCO2, thereby rendering them poor candidates for achieving separation by carbon dioxide-based SFC. There is no reported method for the comprehensive analysis of hydrophilic metabolites by SFC with mobile phases comprising a high CO2 ratio. In this study, we investigated the effect of additives in the modifier for enabling the application of SFC to profile diverse polar compounds for metabolomics. Eleven types of columns were screened by using proteinogenic amino acids as the model compounds. The addition of water and acids (formic acid and trifluoroacetic acid (TFA)) to the modifier was also investigated to improve the solubility of the polar compounds and mitigate the unfavorable interaction between the stationary phase and the polar compounds. A significant improvement in the peak shapes of the amino acids was observed upon addition of TFA. The CO2/modifier ratio and TFA concentration in the mobile phases were investigated using the CROWNPAK CR-I (+) column, which showed the best performance during the column-screening. The CO2/methanol/water/TFA ratio of 70/27/3/0.15 (v/v/v/v) was determined as the optimized mobile phase composition. Furthermore, the applicability of the optimized analytical method to other polar compounds was examined; 100 cationic and amphoteric compounds with predicted logPow values that ranged from –5.9 to 1.7 could be simultaneously analyzed without derivatization. Anionic compounds such as organic acids, phosphates, and sugars were excluded from the target analytes. Most of the previously reported SFC methods for analyzing polar compounds employ a gradient elution and require the use of high modifier ratios at 40% or more. In the proposed method, the use of water and TFA enabled the rapid and simultaneous analysis under isocratic elution within 10 min, even with a high CO2 ratio of 70%. Additionally, a rat serum extract was analyzed using the optimized conditions, and 43 polar metabolites were successfully detected. This result demonstrates the applicability of the SFC/tandem mass spectrometry method to real samples..
59. Takeshi Yamamoto, Yoshitsugu Takabatake*, Satoshi Minami, Shinsuke Sakai, Ryuta Fujimura, Atsushi Takahashi, Tomoko Namba-Hamano, Jun Matsuda, Tomonori Kimura, Isao Matsui, Jun Ya Kaimori, Hiroaki Takeda, Masatomo Takahashi, Yoshihiro Izumi, Takeshi Bamba, Taiji Matsusaka, Fumio Niimura, Motoko Yanagita, Yoshitaka Isaka, Eicosapentaenoic acid attenuates renal lipotoxicity by restoring autophagic flux, Autophagy, 10.1080/15548627.2020.1782034, 17, 7, 1700-1713, 2020.06, Recently, we identified a novel mechanism of lipotoxicity in the kidney proximal tubular cells (PTECs); lipid overload stimulates macroautophagy/autophagy for the renovation of plasma and organelle membranes to maintain the integrity of the PTECs. However, this autophagic activation places a burden on the lysosomal system, leading to a downstream suppression of autophagy, which manifests as phospholipid accumulation and inadequate acidification in lysosomes. Here, we investigated whether pharmacological correction by eicosapentaenoic acid (EPA) supplementation could restore autophagic flux and alleviate renal lipotoxicity. EPA supplementation to high-fat diet (HFD)-fed mice reduced several hallmarks of lipotoxicity in the PTECs, such as phospholipid accumulation in the lysosome, mitochondrial dysfunction, inflammation, and fibrosis. In addition to improving the metabolic syndrome, EPA alleviated renal lipotoxicity via several mechanisms. EPA supplementation to HFD-fed mice or the isolated PTECs cultured in palmitic acid (PA) restored lysosomal function with significant improvements in the autophagic flux. The PA-induced redistribution of phospholipids from cellular membranes into lysosomes and the HFD-induced accumulation of SQSTM1/p62 (sequestosome 1), an autophagy substrate, during the temporal and genetic ablation of autophagy were significantly reduced by EPA, indicating that EPA attenuated the HFD-mediated increases in autophagy demand. Moreover, a fatty acid pulse-chase assay revealed that EPA promoted lipid droplet (LD) formation and transfer from LDs to the mitochondria for beta-oxidation. Noteworthy, the efficacy of EPA on lipotoxicity is autophagy-dependent and cell-intrinsic. In conclusion, EPA counteracts lipotoxicity in the proximal tubule by alleviating autophagic numbness, making it potentially suitable as a novel treatment for obesity-related kidney diseases. Abbreviations:F: 4-HNE: 4-hydroxy-2-nonenal; ACTB: actin beta; ADGRE1/F4/80: adhesion G protein-coupled receptor E1; ATG: autophagy-related; ATP: adenosine triphosphate; BODIPY: boron-dipyrromethene; BSA: bovine serum albumin; cKO: conditional knockout; CML: N-carboxymethyllysine; COL1A1: collagen type I alpha 1 chain; COX: cytochrome c oxidase; CTRL: control; DGAT: diacylglycerol O-acyltransferase; EPA: eicosapentaenoic acid; FA: fatty acid; FFA: free fatty acid; GFP: green fluorescent protein; HFD: high-fat diet; iKO: inducible knockout; IRI: ischemia-reperfusion injury; LAMP1: lysosomal-associated membrane protein 1; LD: lipid droplet; LRP2: low density lipoprotein receptor-related protein 2; MAP1LC3: microtubule-associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; OA: oleic acid; PAS: periodic-acid Schiff; PPAR: peroxisome proliferator activated receptor; PPARGC1/PGC1: peroxisome proliferator activated receptor, gamma, coactivator 1; PTEC: proximal tubular epithelial cell; ROS: reactive oxygen species; RPS6: ribosomal protein S6; SDH: succinate dehydrogenase complex; SFC/MS/MS: supercritical fluid chromatography triple quadrupole mass spectrometry; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TG: triglyceride; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling..
60. Yuta Matsuoka, Yoshihiro Izumi, Masatomo Takahashi, Takeshi Bamba, Ken Ichi Yamada*, Method for Structural Determination of Lipid-Derived Radicals, Analytical chemistry, 10.1021/acs.analchem.0c00053, 92, 10, 6993-7002, 2020.05, Diversified oxidized-lipid molecules are responsible for inflammation and cell death, including ferroptosis. Lipid radicals are the source of these oxidized lipids, which are the initial key molecules in the lipid peroxidation chain reaction. However, owing to their extremely high reactivity and short half-life, an established detection technique is not available. Here, we propose a high-performance liquid chromatography fluorometry and high-resolution tandem mass spectrometry system combined with a fluorescent probe as a structural analysis method for lipid-derived radicals. We detected 132 lipid-derived radicals, including 111 new species, from five polyunsaturated fatty acids. In addition, a database was constructed for which the initial fatty acid could be determined using the radical structure. Further, 12 endogenous lipid-derived radicals were identified in carcinogen-induced liver cancer mouse models. Therefore, this method and its corresponding database will provide novel insights into mechanisms underlying the lipid peroxidation, including the associated inflammation and ferroptosis..
61. Takeshi Hara, Yoshihiro Izumi, Kosuke Hata, V. Gino Baron, Takeshi Bamba, Gert Desmet*, Performance of small-domain monolithic silica columns in nano-liquid chromatography and comparison with commercial packed bed columns with 2 µm particles, Journal of Chromatography A, 10.1016/j.chroma.2019.460804, 1616, Article number 460804, 2020.04, We report on a direct comparison of the separation performance in capillary nano-LC between commercial packed bed columns and the small-domain silica monoliths in applications. Octadecylsilylated monolithic silica capillary columns with a 50 and 100 µm inner diameter (i.d.) were prepared with a procedure providing domain sizes in the sub-2 µm range. The fabricated monolith columns could provide plate heights (H) of 4.0‒4.2 µm for hexylbenzene (retention factor (k) = 3.6) at an optimal linear velocity range under an isocratic condition, while showing a column permeability (Kv0 = 1.6‒1.8 × 10−14 m2) comparable to that of a column packed with 3‒3.5 µm particles. When the peak capacity (np) for a cytochrome C digest was compared for variable gradient times (tG = 15, 30, 60, and 120 min) and constant gradient steepness (b’), the present monolith columns could show a 30‒40% higher np-value than the packed capillary column with 2 µm particles (e.g. np = 180 versus np = 259 at tG = 30 min). The produced monolith columns showed a high chromatographic repeatability for both isocratic and gradient elution (e.g. relative standard deviation (n = 3, RSD (%)) = 0.5% for H, 2,6% for k, and 5.6% for Kv0 in the isocratic mode using the 100 µm i.d.-columns). The present results show that the domain sizes which can now be achieved for capillary silica monoliths are sufficiently small to result in separation efficiencies that can successfully compete with the commercial packed bed columns available for use in nano-LC applications..
62. Manabu Kodama, Kiyotaka Oshikawa, Hideyuki Shimizu, Susumu Yoshioka, Masatomo Takahashi, Yoshihiro Izumi, Takeshi Bamba, Chisa Tateishi, Takeshi Tomonaga, Masaki Matsumoto*, Keiichi I. Nakayama*, A shift in glutamine nitrogen metabolism contributes to the malignant progression of cancer, Nature communications, 10.1038/s41467-020-15136-9, 11, Article number 1320, 2020.03, Glucose metabolism is remodeled in cancer, but the global pattern of cancer-specific metabolic changes remains unclear. Here we show, using the comprehensive measurement of metabolic enzymes by large-scale targeted proteomics, that the metabolism both carbon and nitrogen is altered during the malignant progression of cancer. The fate of glutamine nitrogen is shifted from the anaplerotic pathway into the TCA cycle to nucleotide biosynthesis, with this shift being controlled by glutaminase (GLS1) and phosphoribosyl pyrophosphate amidotransferase (PPAT). Interventions to reduce the PPAT/GLS1 ratio suppresses tumor growth of many types of cancer. A meta-analysis reveals that PPAT shows the strongest correlation with malignancy among all metabolic enzymes, in particular in neuroendocrine cancer including small cell lung cancer (SCLC). PPAT depletion suppresses the growth of SCLC lines. A shift in glutamine fate may thus be required for malignant progression of cancer, with modulation of nitrogen metabolism being a potential approach to SCLC treatment..
63. Toshiaki Yoshioka, Yoshihiro Izumi*, Masatomo Takahashi, Koji Suzuki, Yasuhisa Miyamoto, Yasushi Nagatomi, Takeshi Bamba*, Identification of Acrylamide Adducts Generated during Storage of Canned Milk Coffee, Journal of Agricultural and Food Chemistry, 10.1021/acs.jafc.9b08139, 68, 12, 3859-3867, 2020.03, Since coffee is a significant contributor to the consumption of acrylamide, its reduction is required. Acrylamide is produced during the roasting of coffee beans, but the roasting process is an essential step in determining the taste of coffee. Acrylamide content in coffee has been suggested to decrease by reacting with proteins and/or other substances during storage, but details are unknown. Investigation of acrylamide adducts may contribute to a strategy for acrylamide reduction in coffee. In this study, a stable isotope labeling technique, combined with high-resolution mass spectrometry, allows the identification of acrylamide adducts (3-hydroxypyridine-acrylamide and pyridine-acrylamide) in canned milk coffee. Other acrylamide adducts derived from milk coffee proteins, Lys-acrylic acid and CysSO2-acrylic acid, were identified. During a 4-month storage period, the formation of these four adducts was found to reduce the total content of acrylamide by 75.3% in canned milk coffee. Therefore, endogenous proteins can be used in acrylamide reduction..
64. Hiroaki Takeda, Yoshihiro Izumi*, Shohei Tamura, Tomonari Koike, Yui Koike, Masashi Shiomi, Takeshi Bamba, Lipid Profiling of Serum and Lipoprotein Fractions in Response to Pitavastatin Using an Animal Model of Familial Hypercholesterolemia, Journal of Proteome Research, 10.1021/acs.jproteome.9b00602, 19, 3, 1100-1108, 2020.03, Statins are widely used for the treatment of atherosclerotic cardiovascular diseases. They inhibit cholesterol biosynthesis in the liver and cause pleiotropic effects, including anti-inflammatory and antioxidant effects. To develop novel therapeutic drugs, the effect of blood-borne lipid molecules on the pleiotropic effects of statins must be elucidated. Myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits, an animal model for hypercholesterolemia, are suitable for the determination of lipid molecules in the blood in response to statins because their lipoprotein metabolism is similar to that of humans. Herein, lipid molecules were investigated by lipidome analysis in response to pitavastatin using WHHLMI rabbits. Various lipid molecules in the blood were measured using a supercritical fluid chromatography triple quadrupole mass spectrometry. Cholesterol and cholesterol ester blood concentrations decreased by reducing the secretion of very low density lipoproteins from the liver. Independent of the inhibition effects of cholesterol biosynthesis, the concentrations of some lipids with anti-inflammation and antioxidant effects (phospholipid molecules with n-6 fatty acid side chains, lysophosphatidylcholines, phosphatidylethanolamine plasmalogens, and ceramide molecules) were significantly altered. These findings may lead to further investigation of the mechanism of statin action..
65. Kosuke Hata, Yoshihiro Izumi*, Takeshi Hara, Masaki Matsumoto*, Takeshi Bamba, In-Line Sample Processing System with an Immobilized Trypsin-Packed Fused-Silica Capillary Tube for the Proteomic Analysis of a Small Number of Mammalian Cells, Analytical chemistry, 10.1021/acs.analchem.9b03993, 92, 4, 2997-3005, 2020.02, Omics analysis at single-cell resolution has helped to demonstrate the shaping of cellular heterogeneity on the basis of the expression of various molecules. However, in-depth proteomic analysis of low-quantity samples has remained challenging because of difficulties associated with the measurement of large numbers of proteins by shotgun proteomics using nanoflow liquid chromatography tandem mass spectrometry (nano-LC/MS/MS). To meet such a demand, we developed a method called in-line sample preparation for efficient cellular proteomics (ISPEC) in which cells were captured, directly lysed, and digested with immobilized trypsin within fused-silica capillaries. ISPEC minimized sample loss during the sample preparation processes with a relatively small number of mammalian cells (
66. Nao Nishida-Aoki, Yoshihiro Izumi*, Hiroaki Takeda, Masatomo Takahashi, Takahiro Ochiya*, Takeshi Bamba, Lipidomic analysis of cells and extracellular vesicles from high-and low-metastatic triple-negative breast cancer, Metabolites, 10.3390/metabo10020067, 10, 2, Article number: 67, 2020.02, Extracellular vesicles (EVs) are lipid bilayer nanovesicles secreted from almost all cells including cancer. Cancer-derived EVs contribute to cancer progression and malignancy via educating the surrounding normal cells. In breast cancer, epidemiological and experimental observations indicated that lipids are associated with cancer malignancy. However, lipid compositions of breast cancer EVs and their contributions to cancer progression are unexplored. In this study, we performed a widely targeted quantitative lipidomic analysis in cells and EVs derived from high-and low-metastatic triple-negative breast cancer cell lines, using supercritical fluid chromatography fast-scanning triple-quadrupole mass spectrometry. We demonstrated the differential lipid compositions between EVs and cells of their origin, and between high-and low-metastatic cell lines. Further, we demonstrated EVs from highly metastatic breast cancer accumulated unsaturated diacylglycerols (DGs) compared with EVs from lower-metastatic cells, without increasing the amount in cells. The EVs enriched with DGs could activate the protein kinase D signaling pathway in endothelial cells, which can lead to stimulated angiogenesis. Our results indicate that lipids are selectively loaded into breast cancer EVs to support tumor progression..
67. Shin Nishiumi*, Yoshihiro Izumi, Takashi Kobayashi, Masaru Yoshida*, Possible involvement of lipids in the effectiveness of kombu in individuals with abnormally high serum triglyceride levels, Journal of Nutritional Science and Vitaminology, 10.3177/jnsv.66.185, 66, 2, 185-190, 2020.02, In Japan, Kombu (Laminaria japonica), which is a type of seaweed, is considered to be a foodstuff with health-promoting benefits, and Japanese people actively incorporate Kombu into their diets. Previously, we reported that the frequent intake of Kombu reduced the serum triglyceride levels of subjects with abnormally high serum triglyceride levels. In the current human study, we performed metabolomic analysis of serum lipids, and then the molecular species profiles of phosphatidylcholines (PC), phosphatidylethanolamines (PE), lysophosphatidylcholines (LPC), lysophosphatidylethanolamines (LPE), and free fatty acids (FFA) were evaluated. As a result, it was found that there were no marked differences between the lipid profiles obtained before and after the intake of Kombu for 4 wk in all subjects. In the subjects with abnormal serum triglyceride levels, the intake of Kombu improved the subjects’ molecular species profiles in terms of their serum levels of the diacyl and acyl forms of PC, PE, LPC, and LPE, and FFA. Furthermore, the intake of Kombu also tended to increase the serum levels of both the plasmanyl and plasmenyl forms of PC and PE in these subjects. The lipid alterations observed in our study might be related to the functionality of Kombu. Furthermore, it is important to evaluate the quality of lipids as well as the quantity of lipids in various types of research, including food functionality studies..
68. Kohta Nakatani, Yoshihiro Izumi*, Kosuke Hata, Takeshi Bamba, An analytical system for single-cell metabolomics of typical mammalian cells based on highly sensitive nano-liquid chromatography tandem mass spectrometry, Mass Spectrometry, 10.5702/massspectrometry.A0080, 9, 1, 2020.01, The rapid development of next-generation sequencing techniques has enabled single-cell genomic and transcriptomic analyses, which have revealed the importance of heterogeneity in biological systems. However, analytical methods to accurately identify and quantify comprehensive metabolites from single mammalian cells with a typical diameter of 10–20 µm are still in the process of development. The aim of this study was to develop a single-cell metabolomic analytical system based on highly sensitive nanoliquid chromatography tandem mass spectrometry (nano-LC-MS/MS) with multiple reaction monitoring. A packed nano-LC column (3-µm particle-size pentafluorophenylpropyl Discovery HSF5 of dimensions 100 µm i.d.×180 mm) was prepared using a slurry technique. The optimized nano-LC-MS/MS method showed 3–132-fold (average value, 26-fold) greater sensitivity than semimicro-LC-MS/MS, and the detection limits for several hydrophilic metabolites, including amino acids and nucleic acid related metabolites were in the sub-fmol range. By combining live single-cell sampling and nano-LC-MS/MS, we successfully detected 18 relatively abundant hydrophilic metabolites (16 amino acids and 2 nucleic acid related metabo-lites) from single HeLa cells (n=22). Based on single-cell metabolic profiles, the 22 HeLa cells were classified into three distinct subclasses, suggesting differences in metabolic function in cultured HeLa cell populations. Our single-cell metabolomic analytical system represents a potentially useful tool for in-depth studies focused on cell metabolism and heterogeneity..
69. Jun Matsuda, Atsushi Takahashi, Yoshitsugu Takabatake*, Shinsuke Sakai, Satoshi Minami, Takeshi Yamamoto, Ryuta Fujimura, Tomoko Namba-Hamano, Hiroaki Yonishi, Jun Nakamura, Tomonori Kimura, Jun Ya Kaimori, Isao Matsui, Masatomo Takahashi, Motonao Nakao, Yoshihiro Izumi, Takeshi Bamba, Taiji Matsusaka, Fumio Niimura, Motoko Yanagita, Tamotsu Yoshimori, Yoshitaka Isaka, Metabolic effects of RUBCN/Rubicon deficiency in kidney proximal tubular epithelial cells, Autophagy, 10.1080/15548627.2020.1712107, 2020.01, Macroautophagy/autophagy is a lysosomal degradation system which plays a protective role against kidney injury. RUBCN/Rubicon (RUN domain and cysteine-rich domain containing, Beclin 1-interacting protein) inhibits the fusion of autophagosomes and lysosomes. However, its physiological role in kidney proximal tubular epithelial cells (PTECs) remains uncertain. In the current study, we analyzed the phenotype of newly generated PTEC-specific rubcn-deficient (KO) mice. Additionally, we investigated the role of RUBCN in lipid metabolism using isolated rubcn-deficient PTECs. Although KO mice exhibited sustained high autophagic flux in PTECs, they were not protected from acute ischemic kidney injury. Unexpectedly, KO mice exhibited hallmark features of metabolic syndrome accompanied by expanded lysosomes containing multi-layered phospholipids in PTECs. RUBCN deficiency in cultured PTECs promoted the mobilization of phospholipids from cellular membranes to lysosomes via enhanced autophagy. Treatment of KO PTECs with oleic acid accelerated fatty acids transfer to mitochondria. Furthermore, KO PTECs promoted massive triglyceride accumulation in hepatocytes (BNL-CL2 cells) co-cultured in transwell, suggesting accelerated fatty acids efflux from the PTECs contributes to the metabolic syndrome in KO mice. This study shows that sustained high autophagic flux by RUBCN deficiency in PTECs leads to metabolic syndrome concomitantly with an accelerated mobilization of phospholipids from cellular membranes to lysosomes. Abbreviations: ABC: ATP binding cassette; ACADM: acyl-CoA dehydrogenase medium chain; ACTB: actin, beta; ATG: autophagy related; AUC: area under the curve; Baf: bafilomycin A1; BAT: brown adipose tissue; BODIPY: boron-dipyrromethene; BSA: bovine serum albumin; BW: body weight; CAT: chloramphenicol acetyltransferase; CM: complete medium; CPT1A: carnitine palmitoyltransferase 1a, liver; CQ: chloroquine; CTRL: control; EGFP: enhanced green fluorescent protein; CTSD: cathepsin D; EAT: epididymal adipose tissue; EGFR: epidermal growth factor receptor; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; FA: fatty acid; FBS: fetal bovine serum; GTT: glucose tolerance test; HE: hematoxylin and eosin; HFD: high-fat diet; I/R: ischemia-reperfusion; ITT: insulin tolerance test; KAP: kidney androgen regulated protein; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LD: lipid droplet; LRP2: low density lipoprotein receptor related protein 2; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MAT: mesenteric adipose tissue; MS: mass spectrometry; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; NDRG1: N-myc downstream regulated 1; NDUFB5: NADH:ubiquinone oxidoreductase subunit B5; NEFA: non-esterified fatty acid; OA: oleic acid; OCT: optimal cutting temperature; ORO: Oil Red O; PAS: Periodic-acid Schiff; PFA: paraformaldehyde; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PPARA: peroxisome proliferator activated receptor alpha; PPARGC1A: PPARG coactivator 1 alpha; PTEC: proximal tubular epithelial cell; RAB7A: RAB7A, member RAS oncogene family; RPS6: ribosomal protein S6; RPS6KB1: ribosomal protein S6 kinase B1; RT: reverse transcription; RUBCN: rubicon autophagy regulator; SAT: subcutaneous adipose tissue; SFC: supercritical fluid chromatography; SQSTM1: sequestosome 1; SREBF1: sterol regulatory element binding transcription factor 1; SV-40: simian virus-40; TFEB: transcription factor EB; TG: triglyceride; TS: tissue specific; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; UN: urea nitrogen; UQCRB: ubiquinol-cytochrome c reductase binding protein; UVRAG: UV radiation resistance associated; VPS: vacuolar protein sorting; WAT: white adipose tissue..
70. Shin Nishiumi*, Yoshihiro Izumi, Takashi Kobayashi, Masaru Yoshida*, A pilot study Effects of kombu intake on lifestyle-related diseases -possibility that kombu intake is effective in individuals with abnormally high serum triglyceride levels, Food Science and Technology Research, 10.3136/fstr.25.827, 25, 6, 827-834, 2019.12, In Japan, kombu (Laminaria japonica), a type of seaweed, has been consumed for centuries. It contains a variety of active compounds such as minerals, vitamins, and dietary fiber. The aim of this human pilot study is to investigate the effects of kombu on lifestyle-related diseases. The study had a randomized crossover design, and the subjects (N=48) freely took 6 g of roasted kombu a day for 4 weeks. The subjects' responses to the Gastrointestinal Symptom Rating Scale questionnaire suggested that the frequent intake of kombu may lead to the relief of constipation, diarrhea, and hard stools. In addition, blood tests indicated the possibility that the frequent intake of kombu can decrease the serum triglyceride levels of subjects with abnormally high serum triglyceride levels. Kombu intake might lead to relief from intestinal ailments and improvements in hypertriglyceridemia..
71. Yoshihiro Izumi, Fumio Matsuda*, Akiyoshi Hirayama, Kazutaka Ikeda, Yoshihiro Kita, Kanta Horie, Daisuke Saigusa, Kosuke Saito, Yuji Sawada, Hiroki Nakanishi, Nobuyuki Okahashi, Masatomo Takahashi, Motonao Nakao, Kosuke Hata, Yutaro Hoshi, Motohiko Morihara, Kazuhiro Tanabe, Takeshi Bamba*, Yoshiya Oda, Inter-laboratory comparison of metabolite measurements for metabolomics data integration, Metabolites, 10.3390/metabo9110257, 9, 11, Article number: 257, 2019.11, Background: One of the current problems in the field of metabolomics is the difficulty in integrating data collected using different equipment at different facilities, because many metabolomic methods have been developed independently and are unique to each laboratory. Methods: In this study, we examined whether different analytical methods among 12 different laboratories provided comparable relative quantification data for certain metabolites. Identical samples extracted from two cell lines (HT-29 and AsPc-1) were distributed to each facility, and hydrophilic and hydrophobic metabolite analyses were performed using the daily routine protocols of each laboratory. Results: The results indicate that there was no difference in the relative quantitative data (HT-29/AsPc-1) for about half of the measured metabolites among the laboratories and assay methods. Data review also revealed that errors in relative quantification were derived from issues such as erroneous peak identification, insufficient peak separation, a difference in detection sensitivity, derivatization reactions, and extraction solvent interference. Conclusion: The results indicated that relative quantification data obtained at different facilities and at different times would be integrated and compared by using a reference materials shared for data normalization..
72. Toshiaki Yoshioka, Yoshihiro Izumi, Yasushi Nagatomi, Yasuhisa Miyamoto, Koji Suzuki, Takeshi Bamba*, A highly sensitive determination method for acrylamide in beverages, grains, and confectioneries by supercritical fluid chromatography tandem mass spectrometry, Food Chemistry, 10.1016/j.foodchem.2019.05.033, 294, 486-492, 2019.10, Acrylamide (AA) analysis is an important topic in food safety. However, it is difficult to rapidly and accurately analyze low concentrations of AA with currently available methods. In the present study, we introduce a highly sensitive method that enables the determination of AA in beverages, grains, and confectioneries by supercritical fluid chromatography tandem mass spectrometry (SFC/MS/MS). The sensitivity of the SFC/MS/MS technique is 11-times higher than that obtained by ultra-high performance liquid chromatography tandem mass spectrometry. We demonstrated that the highly sensitive SFC/MS/MS method was able to quantify low concentrations of AA in beverages (i.e., roasted barley tea and coffee) extracts at less than 10 µg kg −1 level without solid-phase purification. Furthermore, the simplification of the sample preparation procedure provided an improvement in data acquisition time (60 samples per 12 h). In conclusion, the developed analytical system is a potentially useful tool for practical AA determination..
73. Masashi Shiomi*, Hiroaki Takeda, Yasuhiro Irino, Norie Kimura, Satoshi Yamada, Nobue Kuniyoshi, Akio Kikumori, Yu Koike, Tomonari Koike, Masaru Yoshida, Yoshihiro Izumi, Masakazu Shinohara, Takeshi Bamba, Tatsuro Ishida, Identification of novel serum markers for the progression of coronary atherosclerosis in WHHLMI rabbits, an animal model of familial hypercholesterolemia, Atherosclerosis, 10.1016/j.atherosclerosis.2019.02.020, 284, 18-23, 2019.05, Background and aims: The development of serum markers specific for coronary lesions is important to prevent coronary events. However, analyses of serum markers in humans are affected by environmental factors and non-target diseases. Using an appropriate model animal can reduce these effects. To identify specific markers for coronary atherosclerosis, we comprehensively analyzed the serum of WHHLMI rabbits, which spontaneously develop coronary atherosclerosis. Methods: Female WHHLMI rabbits were fed standard chow. Serum and plasma were collected under fasting at intervals of 4 months from 4 months old, and a total of 313 lipid molecules, 59 metabolites, lipoprotein lipid levels, and various plasma biochemical parameters were analyzed. The severity of coronary lesions was evaluated with cross-sectional narrowing (CSN) corrected with a frequency of 75%–89% CSN and CSN> 90%. Results: There was a large variation in the severity of coronary lesions in WHHLMI rabbits despite almost no differences in plasma biochemical parameters and aortic lesion area between rabbits with severe and mild coronary lesions. The metabolites and lipid molecules selected as serum markers for coronary atherosclerosis were lysophosphatidylcholine (LPC) 22:4 and diacylglycerol 18:0–18:0 at 4 months old, LPC 20:4 (sn-2), ceramide d18:1–18:2, citric acid plus isocitric acid, and pyroglutamic acid at 8 months old, and phosphatidylethanolamine plasminogen 16:1p-22:2 at 16 months old. Conclusions: These serum markers were coronary lesion-specific markers independent of cholesterol levels and aortic lesions and may be useful to detect patients who develop cardiovascular disease..
74. Tsunehisa Hirose*, Daniel Keck, Yoshihiro Izumi, Takeshi Bamba, Comparison of retention behavior between supercritical fluid chromatography and normal-phase high-performance liquid chromatography with various stationary phases, Molecules, 10.3390/molecules24132425, 24, 13, 2019.01, The retention behavior of a wide variety of stationary phases was compared in supercritical fluid chromatography (SFC) and normal-phase high-performance liquid chromatography (NP-HPLC). We also attempted to elucidate the retention behavior in SFC by investigating the selectivity of the different stationary phases. SFC separation conditions with polar stationary phases, such as silica gel (SL) and diol (Diol) phases, operate via adsorptions that include hydrophilic and ionic interactions similar to those in NP-HPLC. Moreover, non-polar stationary phases, such as pentabromophenyl (PBr), pyrenylethyl (PYE), and octadecyl (C18), could be used despite the non-polar mobile phase conditions, because the dispersion and p-p interactions were stronger in SFC than in HPLC. These results reflect the selectivity of the stationary phase and its retention factor, thus providing useful information for the selection of appropriate stationary phases for particular analytes..
75. Hiroaki Takeda, Masatomo Takahashi, Takeshi Hara, Yoshihiro Izumi, Takeshi Bamba*, Improved quantitation of lipid classes using supercritical fluid chromatography with a charged aerosol detector, Journal of Lipid Research, 10.1194/jlr.D094516, 60, 8, 1465-1474, 2019.01, Quantitatively and rapidly analyzing lipids is necessary to elucidate their biological functions. Herein, we developed a quantitative method for various lipid classes using supercritical fluid chromatography (SFC) coupled with a charged aerosol detector (CAD), providing high-throughput data analysis to detect a large number of molecules in each lipid class as one peak. Applying the CAD was useful for analyzing lipid molecules in the same lipid class with a constant response under the same mobile phase composition. First, we optimized the washing method for the diethylamine column, achieving baseline separation of lipid classes while maintaining good peak shapes. In addition, the CAD conditions (organic solvent evaporation and numerical correction of the CAD data) were optimized to improve the signal-to-noise ratio. We used an internal standard (ceramide phosphoethanolamine d17:1-12:0), which did not coelute with the lipid classes and showed high extraction efficiency. Based on a quantitative analysis of HepG2 cells, the concentration of lipid classes detected by CAD was adequate compared with that obtained by triple-quadrupole MS (QqQMS) in a previous study because the deviations of the concentrations were 0.6- to 2.3-fold. These results also supported the quantitative performance of SFC-QqQMS developed in our previous report.-Takeda, H., M. Takahashi, T. Hara, Y. Izumi, and T. Bamba. Improved quantitation of lipid classes using supercritical fluid chromatography with a charged aerosol detector. J. Lipid Res. 2019. 60: 1465-1474..
76. Masuko Kobori*, Yumiko Takahashi, Hiroaki Takeda, Masatomo Takahashi, Yoshihiro Izumi, Yukari Akimoto, Mutsumi Sakurai, Hideaki Oike, Toshiyuki Nakagawa, Masanori Itoh, Takeshi Bamba, Toshiyuki Kimura, Dietary Intake of Curcumin Improves eIF2 Signaling and Reduces Lipid Levels in the White Adipose Tissue of Obese Mice, Scientific reports, 10.1038/s41598-018-27105-w, 8, 1, 2018.12, White adipose tissue (eWAT) plays a crucial role in preventing metabolic syndrome. We aimed to investigate WAT distribution and gene expression and lipidomic profiles in epididymal WAT (eWAT) in diet-induced obese mice, reflecting a Western-style diet of humans to elucidate the bioactive properties of the dietary antioxidant curcumin in preventing lifestyle-related diseases. For 16 weeks, we fed C57BL/6J mice with a control diet, a high-fat, high-sucrose and high-cholesterol Western diet or Western diet supplemented with 0.1% (w/w) curcumin. Although the dietary intake of curcumin did not affect eWAT weight or plasma lipid levels, it reduced lipid peroxidation markers' levels in eWAT. Curcumin accumulated in eWAT and changed gene expressions related to eukaryotic translation initiation factor 2 (eIF2) signalling. Curcumin suppressed eIF2α phosphorylation, which is induced by endoplasmic reticulum (ER) stress, macrophage accumulation and nuclear factor-κB (NF-κB) p65 and leptin expression, whereas it's anti-inflammatory effect was inadequate to decrease TNF-α and IFN-γ levels. Lipidomic and gene expression analysis revealed that curcumin decreased some diacylglycerols (DAGs) and DAG-derived glycerophospholipids levels by suppressing the glycerol-3-phosphate acyltransferase 1 and adipose triglyceride lipase expression, which are associated with lipogenesis and lipolysis, respectively. Presumably, these intertwined effects contribute to metabolic syndrome prevention by dietary modification..
77. Takeshi Hara, Yoshihiro Izumi, Motonao Nakao, Kosuke Hata, Gino V. Baron, Takeshi Bamba, Gert Desmet*, Silica-based hybrid porous layers to enhance the retention and efficiency of open tubular capillary columns with a 5 μm inner diame9ter, Journal of Chromatography A, 10.1016/j.chroma.2018.10.023, 1580, 63-71, 2018.12, We report on possibility to enhance the hydrophobicity of octadecylsilylated silica-based porous layered open tubular (PLOT) columns with an inner diameter (i.d.) of 5 μm by applying hybrid tetramethoxysilane (TMOS)/methyltrimethoxysilane (MTMS) layers with inserted methyl groups. Due to this higher hydrophobicity, thinner porous layers suffice to achieve similar retention factor (k) as in octadecylsilylated silica-based PLOT columns synthesized using TMOS only. Since thinner layers have a lower intra-layer mass transfer resistance, this in turn allows to obtain superior column efficiencies in comparison with separations carried out with TMOS-based PLOT columns at the same retention. Since layer thickness contributes to the C-term type of band broadening, this is most pronounced at high velocities. Typical gains in column efficiency at a reduced velocity of νi = 30 are on the order of 15%. Preparing the hybrid PLOT columns in 5 μm i.d.-capillaries with a length of 0.4 m using different TMOS/MTMS preparation mixtures leads to different layer thickness in the capillaries. It is shown that column efficiencies for the most retained compound (k = 0.9–1.5) went from N = 101,000 for PLOT columns with a layer thickness (df) of 250 nm, over N = 95,000 for df = 320 nm to N = 89,000 for df = 400 nm, corresponding to plate heights (H) in the order of 3.5–3.9 μm (reduced plate heights (h = 0.8–1.0)). By applying the same preparation mixtures for much longer capillaries of 1.3 m, a high repeatability of the volumetric phase ratio (m) (difference
78. Govind Kunduri, Daniel Turner-Evans, Yutaka Konya, Yoshihiro Izumi, Kunio Nagashima, Stephen Lockett, Joost Holthuis, Takeshi Bamba, Usha Acharya, Jairaj K. Acharya*, Defective cortex glia plasma membrane structure underlies light-induced epilepsy in cpes mutants, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.1808463115, 115, 38, E8919-E8928, 2018.09, Seizures induced by visual stimulation (photosensitive epilepsy; PSE) represent a common type of epilepsy in humans, but the molecular mechanisms and genetic drivers underlying PSE remain unknown, and no good genetic animal models have been identified as yet. Here, we show an animal model of PSE, in Drosophila, owing to defective cortex glia. The cortex glial membranes are severely compromised in ceramide phosphoethanolamine synthase (cpes)-null mutants and fail to encapsulate the neuronal cell bodies in the Drosophila neuronal cortex. Expression of human sphingomyelin synthase 1, which synthesizes the closely related ceramide phosphocholine (sphingomyelin), rescues the cortex glial abnormalities and PSE, underscoring the evolutionarily conserved role of these lipids in glialmembranes. Further,we show the compromise in plasma membrane structure that underlies the glial cell membrane collapse in cpes mutants and leads to the PSE phenotype..
79. Masatomo Takahashi, Yoshihiro Izumi*, Fukumatsu Iwahashi, Yasumune Nakayama, Mitsuhiko Iwakoshi, Motonao Nakao, Seiji Yamato, Eiichiro Fukusaki, Takeshi Bamba*, Highly Accurate Detection and Identification Methodology of Xenobiotic Metabolites Using Stable Isotope Labeling, Data Mining Techniques, and Time-Dependent Profiling Based on LC/HRMS/MS, Analytical Chemistry, 10.1021/acs.analchem.8b01388, 90, 15, 9068-9076, 2018.08, A generally applicable method to discover xenobiotic metabolites is important to safely and effectively develop xenobiotics. We propose an advanced method to detect and identify comprehensive xenobiotic metabolites using stable isotope labeling, liquid chromatography coupled with benchtop quadrupole Orbitrap high-resolution tandem mass spectrometry (LC/HRMS/MS), data mining techniques (alignment, peak picking, and paired-peaks filtering), in silico metabolism prediction, and time-dependent profiling. The LC/HRMS analysis was carried out using Arabidopsis T87 cultured cells treated with unlabeled, or 13C- or 2H-labeled 2,4-dichlorophenoxyacetic acid (2,4-D). Paired-peak filtering enabled accurate detection of 83 candidates for 2,4-D metabolites without any false positive peaks derived from solvents or the biological matrix. We confirmed 10 previously reported 2,4-D metabolites and identified 16 novel 2,4-D metabolites. Our method provides accurate detection and identification of comprehensive xenobiotic metabolites and represents a potentially useful tool to elucidate xenobiotic metabolism..
80. Shin Nishiumi*, Yoshihiro Izumi, Masaru Yoshida*, Alterations in Docosahexaenoic Acid-Related Lipid Cascades in Inflammatory Bowel Disease Model Mice, Digestive Diseases and Sciences, 10.1007/s10620-018-5025-4, 63, 6, 1485-1496, 2018.06, Background: Inflammatory bowel disease (IBD) is an intestinal disorder, involving chronic and relapsing inflammation of the digestive tract. Dysregulation of the immune system based on genetic, environmental, and other factors seems to be involved in the onset of IBD, but its exact pathogenesis remains unclear. Therefore, radical treatments for ulcerative colitis and Crohn’s disease remain to be found, and IBD is considered to be a refractory disease. Aims: The aim of this study is to obtain novel insights into IBD via metabolite profiling of interleukin (IL)-10 knockout mice (an IBD animal model that exhibits a dysregulated immune system). Methods: In this study, the metabolites in the large intestine and plasma of IL-10 knockout mice were analyzed. In our analytical system, two kinds of analysis (gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry) were used to detect a broader range of metabolites, including both hydrophilic and hydrophobic metabolites. In addition, an analysis of lipid mediators in the large intestine and ascites of IL-10 knockout mice was carried out. Results: The levels of a variety of metabolites, including lipid mediators, were altered in IL-10 knockout mice. For example, high large intestinal and plasma levels of docosahexaenoic acid (DHA) were observed. In addition, arachidonic acid- and DHA-related lipid cascades were upregulated in the ascites of the IL-10 knockout mice. Conclusions: Our findings based on metabolite profiles including lipid mediators must contribute to development of researches about IBD..
81. Yoshinari Obata, Shunbun Kita*, Yoshihisa Koyama, Shiro Fukuda, Hiroaki Takeda, Masatomo Takahashi, Yuya Fujishima, Hirofumi Nagao, Shigeki Masuda, Yoshimitsu Tanaka, Yuto Nakamura, Hitoshi Nishizawa, Tohru Funahashi, Barbara Ranscht, Yoshihiro Izumi, Takeshi Bamba, Eiichiro Fukusaki, Rikinari Hanayama, Shoichi Shimada, Norikazu Maeda, Iichiro Shimomura, Adiponectin/T-cadherin system enhances exosome biogenesis and decreases cellular ceramides by exosomal release, JCI insight, 10.1172/jci.insight.99680, 3, 8, 2018.04, Adiponectin, an adipocyte-derived circulating protein, accumulates in vasculature, heart, and skeletal muscles through interaction with a unique glycosylphosphatidylinositol-anchored cadherin, T-cadherin. Recent studies have demonstrated that such accumulation is essential for adiponectin-mediated cardiovascular protection. Here, we demonstrate that the adiponectin/T-cadherin system enhances exosome biogenesis and secretion, leading to the decrease of cellular ceramides. Adiponectin accumulated inside multivesicular bodies, the site of exosome generation, in cultured cells and in vivo aorta, and also in exosomes in conditioned media and in blood, together with T-cadherin. The systemic level of exosomes in blood was significantly affected by adiponectin or T-cadherin in vivo. Adiponectin increased exosome biogenesis from the cells, dependently on T-cadherin, but not on AdipoR1 or AdipoR2. Such enhancement of exosome release accompanied the reduction of cellular ceramides through ceramide efflux in exosomes. Consistently, the ceramide reduction by adiponectin was found in aortas of WT mice treated with angiotensin II, but not in T-cadherin-knockout mice. Our findings provide insights into adiponectin/T-cadherin-mediated organ protection through exosome biogenesis and secretion..
82. Shohei Tamura, Yui Koike, Hiroaki Takeda, Tomonari Koike, Yoshihiro Izumi, Ryosuke Nagasaka, Tetsuto Tsunoda, Motoo Tori, Kazuo Ogawa, Takeshi Bamba, Masashi Shiomi*, Ameliorating effects of D-47, a newly developed compound, on lipid metabolism in an animal model of familial hypercholesterolemia (WHHLMI rabbits), European Journal of Pharmacology, 10.1016/j.ejphar.2018.01.013, 822, 147-153, 2018.03, Improvements induced in lipid metabolism in the liver by D-47, a newly developed compound, were examined herein. WHHLMI rabbits, an animal model of hypercholesterolemia and coronary atherosclerosis, was fed D-47-supplemented chow for 5 weeks at a dose of 30 mg/kg. Lipid concentration were assayed using enzymatic methods. Plasma lipoproteins were fractionated with an ultracentrifuge. mRNA expression was analyzed with real-time PCR. Lipidome analyses of lipoproteins were performed using supercritical fluid chromatography mass spectrometry. In the D-47-treated group, serum lipid levels decreased by 23% for total cholesterol and by 40% for triglycerides. These reductions were mainly attributed to decreases in the VLDL fraction. Compared with the control, in the D-47 group, lipid contents in the liver were decreased by 22% in cholesterol and by 69% in triglycerides, and fat accumulation was decreased by 57% in pericardial fat and by 17% in mesenteric fat. In lipidome analyses of VLDL fraction, lysophosphatidylcholine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylethanolamine plasmalogen, sphingomyelin, and ceramide were decreased by the D-47 treatment. mRNA expression in the liver was 51% lower for FAS and 24% lower for MTP, but 5.9- and 5.1-fold higher for CYP7A1 and CPT-1, respectively, in the D-47 group than in the control. mRNA expression was 72%, 64%, and 36% higher for LPL, CTP-1, and PPARγ, respectively, in mesenteric fat in the D-47 group. D-47 is a potent lipid-lowering compound that uses a different mechanism of action from that of statins. It has potential as a compound in the treatment of steatohepatitis and metabolic syndrome..
83. Hiroaki Takeda, Yoshihiro Izumi, Masatomo Takahashi, Thanai Paxton, Shohei Tamura, Tomonari Koike, Ying Yu, Noriko Kato, Katsutoshi Nagase, Masashi Shiomi, Takeshi Bamba*, Widely-targeted quantitative lipidomics method by supercritical fluid chromatography triple quadrupole mass spectrometry, Journal of Lipid Research, 10.1194/jlr.D083014, 59, 7, 1283-1293, 2018.01, Lipidomics, the mass spectrometry-based comprehensive analysis of lipids, has attracted attention as an analytical approach to provide novel insight into lipid metabolism and to search for biomarkers. However, an ideal method for both comprehensive and quantitative analysis of lipids has not been fully developed. Here, we have proposed a practical methodology for widely targeted quantitative lipidome analysis using supercritical fluid chromatography fast-scanning triple-quadrupole mass spectrometry (SFC/ QqQMS) and theoretically calculated a comprehensive lipid multiple reaction monitoring (MRM) library. Lipid classes can be separated by SFC with a normal-phase diethylaminebonded silica column with high resolution, high throughput, and good repeatability. Structural isomers of phospholipids can be monitored by mass spectrometric separation with fatty acyl-based MRM transitions. SFC/QqQMS analysis with an internal standard-dilution method offers quantitative information for both lipid class and individual lipid molecular species in the same lipid class. Additionally, data acquired using this method has advantages, including reduction of misidentification and acceleration of data analysis. Using the SFC/QqQMS system, alteration of plasma lipid levels in myocardial infarction-prone rabbits to the supplementation of EPA was first observed. Our developed SFC/QqQMS method represents a potentially useful tool for in-depth studies focused on complex lipid metabolism and biomarker discovery..
84. Takaiku Sakamoto, Eiji Sakuradani*, Tomoyo Okuda, Hiroshi Kikukawa, Akinori Ando, Shigenobu Kishino, Yoshihiro Izumi, Takeshi Bamba, Jun Shima, Jun Ogawa, Metabolic engineering of oleaginous fungus Mortierella alpina for high production of oleic and linoleic acids, Bioresource Technology, 10.1016/j.biortech.2017.06.089, 245, 1610-1615, 2017.12, The aim of this work was to study the molecular breeding of oleaginous filamentous Mortierella alpina for high production of linoleic (LA) or oleic acid (OA). Heterologous expression of the Δ12-desaturase (DS) gene derived from Coprinopsis cinerea in the Δ6DS activity-defective mutant of M. alpina increased the LA production rate as to total fatty acid to 5 times that in the wild strain. By suppressing the endogenous Δ6I gene expression by RNAi in the Δ12DS activity-defective mutant of M. alpina, the OA accumulation rate as to total fatty acid reached 68.0%. The production of LA and OA in these transformants reached 1.44 and 2.76 g/L, respectively, on the 5th day. The Δ6I transcriptional levels of the RNAi-treated strains were suppressed to 1/10th that in the parent strain. The amount of Δ6II RNA in the Δ6I RNAi-treated strain increased to 8 times that in the wild strain..
85. Satoshi Minami, Takeshi Yamamoto, Yoshitsugu Takabatake*, Atsushi Takahashi, Tomoko Namba, Jun Matsuda, Tomonori Kimura, Jun ya Kaimori, Isao Matsui, Takayuki Hamano, Hiroaki Takeda, Masatomo Takahashi, Yoshihiro Izumi, Takeshi Bamba, Taiji Matsusaka, Fumio Niimura, Yoshitaka Isaka, Lipophagy maintains energy homeostasis in the kidney proximal tubule during prolonged starvation, Autophagy, 10.1080/15548627.2017.1341464, 13, 10, 1629-1647, 2017.10, Macroautophagy/autophagy is a self-degradation process that combats starvation. Lipids are the main energy source in kidney proximal tubular cells (PTCs). During starvation, PTCs increase fatty acid (FA) uptake, form intracellular lipid droplets (LDs), and hydrolyze them for use. The involvement of autophagy in lipid metabolism in the kidney remains largely unknown. Here, we investigated the autophagy-mediated regulation of renal lipid metabolism during prolonged starvation using PTC-specific Atg5-deficient (atg5-TSKO) mice and an in vitro serum starvation model. Twenty-four h of starvation comparably induced LD formation in the PTCs of control and atg5-TSKO mice; however, additional 24 h of starvation reduced the number of LDs in control mice, whereas increases were observed in atg5-TSKO mice. Autophagic degradation of LDs (lipophagy) in PTCs was demonstrated by electron microscopic observation and biochemical analysis. In vitro pulse-chase assays demonstrated that lipophagy mobilizes FAs from LDs to mitochondria during starvation, whereas impaired LD degradation in autophagy-deficient PTCs led to decreased ATP production and subsequent cell death. In contrast to the in vitro assay, despite impaired LD degradation, kidney ATP content was preserved in 48-h starved atg5-TSKO mice, probably due to increased utilization of ketone bodies. This compensatory mechanism was accompanied by a higher plasma FGF21 (fibroblast growth factor 21) level and its expression in the PTCs; however, this was not essential for the production of ketone bodies in the liver during prolonged starvation. In conclusion, lipophagy combats prolonged starvation in PTCs to avoid cellular energy depletion..
86. Yuka Fujito, Yoshihiro Hayakawa, Yoshihiro Izumi, Takeshi Bamba*, Importance of optimizing chromatographic conditions and mass spectrometric parameters for supercritical fluid chromatography/mass spectrometry, Journal of Chromatography A, 10.1016/j.chroma.2017.05.071, 1508, 138-147, 2017.07, Supercritical fluid chromatography/mass spectrometry (SFC/MS) has great potential in high-throughput and the simultaneous analysis of a wide variety of compounds, and it has been widely used in recent years. The use of MS for detection provides the advantages of high sensitivity and high selectivity. However, the sensitivity of MS detection depends on the chromatographic conditions and MS parameters. Thus, optimization of MS parameters corresponding to the SFC condition is mandatory for maximizing performance when connecting SFC to MS. The aim of this study was to reveal a way to decide the optimum composition of the mobile phase and the flow rate of the make-up solvent for MS detection in a wide range of compounds. Additionally, we also showed the basic concept for determination of the optimum values of the MS parameters focusing on the MS detection sensitivity in SFC/MS analysis. To verify the versatility of these findings, a total of 441 pesticides with a wide polarity range (logPow from −4.21 to 7.70) and pKa (acidic, neutral and basic). In this study, a new SFC-MS interface was used, which can transfer the entire volume of eluate into the MS by directly coupling the SFC with the MS. This enabled us to compare the sensitivity or optimum MS parameters for MS detection between LC/MS and SFC/MS for the same sample volume introduced into the MS. As a result, it was found that the optimum values of some MS parameters were completely different from those of LC/MS, and that SFC/MS-specific optimization of the analytical conditions is required. Lastly, we evaluated the sensitivity of SFC/MS using fully optimized analytical conditions. As a result, we confirmed that SFC/MS showed much higher sensitivity than LC/MS when the analytical conditions were fully optimized for SFC/MS; and the high sensitivity also increase the number of the compounds that can be detected with good repeatability in real sample analysis. This result indicates that SFC/MS has potential for practical use in the multiresidue analysis of a wide range of compounds that requires high sensitivity..
87. Takahiro Ogawa, Yoshihiro Izumi, Kenichi Kusumoto, Eiichiro Fukusaki, Takeshi Bamba*, Wide target analysis of acylglycerols in miso (Japanese fermented soybean paste) by supercritical fluid chromatography coupled with triple quadrupole mass spectrometry and the analysis of the correlation between taste and both acylglycerols and free fatty acids, Rapid Communications in Mass Spectrometry, 10.1002/rcm.7862, 31, 11, 928-936, 2017.06, Rationale: The acylglycerols in miso have not been studied although it is known that they are important to the taste. In order to determine the fatty acid constituents in the acylglycerols and analyze them individually, multiple reaction monitoring (MRM) was performed utilizing a single platform, typically using both gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry. Methods: Acylglycerols and fatty acids (FAs) in miso were extracted using the Bligh-Dyer method. Supercritical fluid chromatography (SFC) with a C30 column was conducted for separation, and mass spectrometric (MS) analysis was performed with electrospray ionization using a triple quadrupole mass spectrometer (QqQMS) in the MRM mode. Results: The detection of FAs from the hydrolysis of acylglycerols and individual acylglycerols was achieved using only an SFC/MS platform. From the quality control (QC) sample of miso, we determined the main FA constituents, and then performed wide target analysis using MRM. In total, 23 triacylglycerols, 10 diacylglycerols, two monoacylglycerols, and five FAs were annotated effectively. Furthermore, the important compounds related to taste were determined through the analysis using both the relative quantitative data of acylglycerols and FAs and the quantitative descriptive analysis data of miso. Conclusions: A method for the determination of the FA constituents in acylglycerols after hydrolysis and the comprehensive analysis of acylglycerols and FAs using MRM with SFC/QqQMS was developed. Using the data from the comprehensive analysis of acylglycerols and quantitative descriptive data, the key compounds related to taste were investigated. This type of research on lipids and the taste of food is expected to progress hereafter..
88. Makoto Suzuki, Shin Nishiumi, Takashi Kobayashi, Arata Sakai, Yosuke Iwata, Takato Uchikata, Yoshihiro Izumi, Takeshi Azuma, Takeshi Bamba, Masaru Yoshida*, Use of on-line supercritical fluid extraction-supercritical fluid chromatography/tandem mass spectrometry to analyze disease biomarkers in dried serum spots compared with serum analysis using liquid chromatography/tandem mass spectrometry, Rapid Communications in Mass Spectrometry, 10.1002/rcm.7857, 31, 10, 886-894, 2017.05, Rationale: The analytical stability and throughput of biomarker assays based on dried serum spots (DSS) are strongly dependent on the extraction process and determination method. In the present study, an on-line system based on supercritical fluid extraction-supercritical fluid chromatography coupled with tandem mass spectrometry (SFE-SFC/MS/MS) was established for analyzing the levels of disease biomarkers in DSS. Methods: The chromatographic conditions were investigated using the ODS-EP, diol, and SIL-100A columns. Then, we optimized the SFE-SFC/MS/MS method using the diol column, focusing on candidate biomarkers of oral, colorectal, and pancreatic cancer that were identified using liquid chromatography (LC)/MS/MS. Results: By using this system, four hydrophilic metabolites and 17 hydrophobic metabolites were simultaneously detected within 15 min. In an experiment involving clinical samples, PC 16:0-18:2/16:1-18:1 exhibited 93.8% sensitivity and 64.3% specificity, whereas PC 17:1-18:1/17:0-18:2 showed 81.3% sensitivity and 92.9% specificity for detecting oral cancer. In addition, assessments of the creatine levels demonstrated 92.3% sensitivity and 78.6% specificity for detecting colorectal cancer. Conclusions: The results of this study indicate that our method has great potential for clinical diagnosis and would be suitable for large-scale screening..
89. Masahiro Nagata, Yoshihiro Izumi, Eri Ishikawa, Ryoko Kiyotake, Rieko Doi, Satoru Iwai, Zakaria Omahdi, Toshiyuki Yamaji, Tomofumi Miyamoto, Takeshi Bamba, Shou Yamasaki*, Intracellular metabolite β-glucosylceramide is an endogenous Mincle ligand possessing immunostimulatory activity, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.1618133114, 114, 16, E3285-E3294, 2017.04, Sensing and reacting to tissue damage is a fundamental function of immune systems. Macrophage inducible C-type lectin (Mincle) is an activating C-type lectin receptor that senses damaged cells. Notably, Mincle also recognizes glycolipid ligands on pathogens. To elucidate endogenous glycolipids ligands derived from damaged cells, we fractionated supernatants from damaged cells and identified a lipophilic component that activates reporter cells expressing Mincle. Mass spectrometry and NMR spectroscopy identified the component structure as β-glucosylceramide (GlcCer), which is a ubiquitous intracellularmetabolite. Synthetic β-GlcCer activated myeloid cells and induced production of inflammatory cytokines; this production was abrogated inMincle-deficient cells. Sterile inflammation induced by excessive cell death in the thymus was exacerbated by hematopoieticspecific deletion of degrading enzyme of β-GlcCer (β-glucosylceramidase, GBA1). However, this enhanced inflammation was ameliorated in a Mincle-deficient background. GBA1-deficient dendritic cells (DCs) in which β-GlcCer accumulates triggered antigen-specific T-cell responses more efficiently than WT DCs, whereas these responses were compromised in DCs from GBA1 × Mincle double-deficient mice. These results suggest that β-GlcCer is an endogenous ligand for Mincle and possesses immunostimulatory activity..
90. John A. Bowden*, Alan Heckert, Candice Z. Ulmer, Christina M. Jones, Jeremy P. Koelmel, Laila Abdullah, Linda Ahonen, Yazen Alnouti, Aaron M. Armando, John M. Asara, Takeshi Bamba, John R. Barr, Jonas Bergquist, Christoph H. Borchers, Joost Brandsma, Susanne B. Breitkopf, Tomas Cajka, Amaury Cazenave-Gassiot, Antonio Checa, Michelle A. Cinel, Romain A. Colas, Serge Cremers, Edward A. Dennis, James E. Evans, Alexander Fauland, Oliver Fiehn, Michael S. Gardner, Timothy J. Garrett, Katherine H. Gotlinger, Jun Han, Yingying Huang, Aveline Huipeng Neo, Tuulia Hyötyläinen, Yoshihiro Izumi, Hongfeng Jiang, Houli Jiang, Jiang Jiang, Maureen Kachman, Reiko Kiyonami, Kristaps Klavins, Christian Klose, Harald C. Köfeler, Johan Kolmert, Therese Koal, Grielof Koster, Zsuzsanna Kuklenyik, Irwin J. Kurland, Michael Leadley, Karen Lin, Krishna Rao Maddipati, Danielle McDougall, Peter J. Meikle, Natalie A. Mellett, Cian Monnin, M. Arthur Moseley, Renu Nandakumar, Matej Oresic, Rainey Patterson, David Peake, Jason S. Pierce, Martin Post, Anthony D. Postle, Rebecca Pugh, Yunping Qiu, Oswald Quehenberger, Parsram Ramrup, Jon Rees, Barbara Rembiesa, Denis Reynaud, Mary R. Roth, Susanne Sales, Kai Schuhmann, Michal Laniado Schwartzman, Charles N. Serhan, Andrej Shevchenko, Stephen E. Somerville, Lisa St John-Williams, Michal A. Surma, Hiroaki Takeda, Rhishikesh Thakare, J. Will Thompson, Federico Torta, Alexander Triebl, Martin Trötzmüller, S. J.Kumari Ubhayasekera, Dajana Vuckovic, Jacquelyn M. Weir, Ruth Welti, Markus R. Wenk, Craig E. Wheelock, Libin Yao, Min Yuan, Xueqing Heather Zhao, Senlin Zhou, Harmonizing lipidomics
NIST interlaboratory comparison exercise for lipidomics using SRM 1950-Metabolites in frozen human plasma, Journal of Lipid Research, 10.1194/jlr.M079012, 58, 12, 2275-2288, 2017.01, As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950- Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each laboratory using a different lipidomics workflow. A total of 1,527 unique lipids were measured across all laboratories and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra-And interlaboratory quality control and method validation. These analyses were performed using nonstandardized laboratory-independent workflows. The consensus locations were also compared with a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement..
91. Hiroaki Takeda, Yoshihiro Izumi, Atsumi Tomita, Tomonari Koike, Masashi Shiomi, Eiichiro Fukusaki, Fumio Matsuda, Takeshi Bamba*, Lipoprotein profiling methodology based on determination of apolipoprotein concentration, Bioanalysis, 10.4155/bio-2016-0234, 9, 1, 9-19, 2017.01, Aim: Abnormal lipid metabolism results in the alteration of lipid compositions in lipoproteins; therefore an accurate and quantitative analytical approach is required for the detailed structural characterization of lipoproteins. However, the specific lipid composition of each lipoprotein particle is poorly understood. Materials & methods: Lipid composition of very-low-density lipoprotein and low-density lipoprotein particles derived from myocardial infarction-prone rabbits was determined by normalization of lipidomics data using apoB-100 levels. Results: The ratio of lipid levels between very-low-density lipoprotein and low-density lipoprotein particles was different according to not only lipid classes, but also phosphatidylethanolamine subclasses by applying our developed methodology to myocardial infarction-prone rabbits. Conclusion: Our novel analytical approach represents to be a potentially useful tool to obtain particle-specific lipid components of lipoproteins..
92. Hiroaki Takeda, Tomonari Koike, Yoshihiro Izumi, Takayuki Yamada, Masaru Yoshida, Masashi Shiomi, Eiichiro Fukusaki, Takeshi Bamba*, Lipidomic analysis of plasma lipoprotein fractions in myocardial infarction-prone rabbits, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2015.02.015, 120, 4, 476-482, 2015.10, Lipids play important roles in the body and are transported to various tissues via lipoproteins. It is commonly assumed that alteration of lipid levels in lipoproteins leads to dyslipidemia and serious diseases such as coronary artery disease (CAD). However, lipid compositions in each lipoprotein fraction induced by lipoprotein metabolism are poorly understood. Lipidomics, which involves the comprehensive and quantitative analysis of lipids, is expected to provide valuable information regarding the pathogenic mechanism of CAD. Here, we performed a lipidomic analysis of plasma and its lipoprotein fractions in myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits. In total, 172 lipids in plasma obtained from normal and WHHLMI rabbits were quantified with high throughput and accuracy using supercritical fluid chromatography hybrid quadrupole-Orbitrap mass spectrometry (SFC/Q-Orbitrap-MS). Plasma levels of each lipid class (i.e., phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, lysophosphatidylcholine, lysophosphatidylethanolamine, sphingomyelin, ceramide, triacylglycerol, diacylglycerol, and cholesterol ester, except for free fatty acids) in 21-month-old WHHLMI rabbits were significantly higher than those in normal rabbits. High levels of functional lipids, such as alkyl-phosphatidylcholines, phospholipids including ω-6 fatty acids, and plasmalogens, were also observed in WHHLMI rabbit plasma. In addition, high-resolution lipidomic analysis using very low density lipoprotein (VLDL) and low density lipoprotein (LDL) provided information on the specific molecular species of lipids in each lipoprotein fraction. In particular, higher levels of phosphatidylethanolamine plasmalogens were detected in LDL than in VLDL. Our lipidomics approach for plasma lipoprotein fractions will be useful for in-depth studies on the pathogenesis of CAD..
93. Megumi Ishibashi, Yoshihiro Izumi, Miho Sakai, Takashi Ando, Eiichiro Fukusaki, Takeshi Bamba*, High-throughput simultaneous analysis of pesticides by supercritical fluid chromatography coupled with high-resolution mass spectrometry, Journal of Agricultural and Food Chemistry, 10.1021/jf5056248, 63, 18, 4457-4463, 2015.05, Recently, a generally applicable screening method for multiresidue pesticide analysis, which is simple, quick, and accurate and has a reliable performance, is becoming increasingly important for food safety and international trade. This paper proposes a high-throughput screening methodology that enables the detection of multiresidue pesticides using supercritical fluid chromatography coupled to a high-performance benchtop quadrupole Orbitrap mass spectrometry (SFC/Q Exactive) and an automated library-based detection. A total of 444 chemicals covering a wide polarity range (logPow from -4.2 to 7.7) and a wide molecular weight range (from 99.0 to 872.5) were analyzed simultaneously through a combination of high mass resolution (a value of m/Δm = 70000), high mass accuracy (-1 level (provisional maximum residue limits in Japan). In conclusion, the developed analytical system is a potentially useful tool for practical multiresidue pesticide screening with high throughput (time for data acquisition, 72 samples per day; and time for data processing of 72 samples, approximately 45 min)..
94. Hiroshi Tsugawa*, Erika Ohta, Yoshihiro Izumi, Atsushi Ogiwara, Daichi Yukihira, Takeshi Bamba, Eiichiro Fukusaki, Masanori Arita, MRM-DIFF
Data processing strategy for differential analysis in large scale MRM-based lipidomics studies, Frontiers in Genetics, 10.3389/fgene.2014.00471, 5, JAN, 2015.01, Based on theoretically calculated comprehensive lipid libraries, in lipidomics as many as 1000 multiple reaction monitoring (MRM) transitions can be monitored for each single run. On the other hand, lipid analysis from each MRM chromatogram requires tremendous manual efforts to identify and quantify lipid species. Isotopic peaks differing by up to a few atomic masses further complicate analysis. To accelerate the identification and quantification process we developed novel software, MRM-DIFF, for the differential analysis of large-scale MRM assays. It supports a correlation optimized warping (COW) algorithm to align MRM chromatograms and utilizes quality control (QC) sample datasets to automatically adjust the alignment parameters. Moreover, user-defined reference libraries that include the molecular formula, retention time, and MRM transition can be used to identify target lipids and to correct peak abundances by considering isotopic peaks. Here, we demonstrate the software pipeline and introduce key points for MRM-based lipidomics research to reduce the mis-identification and overestimation of lipid profiles. The MRM-DIFF program, example data set and the tutorials are downloadable at the "Standalone software" section of the PRIMe..
95. Atsuki Matsubara, Yoshihiro Izumi, Shin Nishiumi, Makoto Suzuki, Takeshi Azuma, Eiichiro Fukusaki, Takeshi Bamba*, Masaru Yoshida*, Supercritical fluid extraction as a preparation method for mass spectrometry of dried blood spots, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 10.1016/j.jchromb.2014.08.013, 969, 199-204, 2014.10, The potential of supercritical fluid extraction (SFE) as a preparation method for mass spectrometry of dried blood spots (DBS) was examined. SFE is generally used for the extraction of hydrophobic compounds, but hydrophilic metabolites such as amino acids, amines, and nucleic-acid-related metabolites could be extracted by adding a low level of methanol as a modifier. Under the optimized conditions, over 200 metabolites were detected from a dried serum spot, of which over 160 metabolites could be analyzed stably (RSD
96. Yoshihiro Izumi, Kosuke Aritake, Yoshihiro Urade, Eiichiro Fukusaki*, Practical evaluation of liquid chromatography/tandem mass spectrometry and enzyme immunoassay method for the accurate quantitative analysis of prostaglandins, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2013.12.022, 118, 1, 116-118, 2014.01, The accurate and robust measurement of prostaglandins (PG) concentration could help to understand the many physiological functions. The present study revealed that liquid chromatography/tandem mass spectrometry method for the PGs analysis can satisfy the requirements for both qualitative and quantitative performance as compared to competitive enzyme immunoassays..
97. Yoshihiro Izumi, Shimpei Aikawa, Fumio Matsuda, Tomohisa Hasunuma, Akihiko Kondo*, Aqueous size-exclusion chromatographic method for the quantification of cyanobacterial native glycogen, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 10.1016/j.jchromb.2013.04.037, 930, 90-97, 2013.07, Cyanobacterial glycogen has gained interest as a valuable biomass feedstock for biofuel production. However, an ideal method for native glycogen quantification has not been developed. Here, we have proposed a simple methodology that enables the quantitative determination of cyanobacterial glycogen concentration with high repeatability using aqueous size-exclusion chromatography with a differential refractive index detector (SEC/RID). Our SEC/RID system also allows size distributions for native glycogen based on hydrodynamic volumes (Vh), which is proportional to the product of the molecular mass (M) and intrinsic viscosity [η], obtained by universal calibration using linear homopolymers of known M with Mark-Houwink-Sakurada parameters. The universal calibration curve achieved a broad linear range (Vh parameter [η]M=2×102-8×108mLg-1) with a high correlation coefficient (R2=0.9942), because the developed system is equipped with an OHpak SB-806M HQ aqueous column containing four types of polyhydroxy methacrylate-based particles with different particle and pore sizes. Based on the SEC/RID system, response of molecular size distribution of glycogen in microalgae to the cultivation condition was first observed. Our established SEC/RID method has several advantages over conventional techniques, including the simultaneous quantitative and size distribution analyses of glycogen, and represents a potentially useful tool to elucidate the relationship between structural properties and the roles of glycogen in metabolism..
98. Tomohisa Hasunuma*, Fumi Kikuyama, Mami Matsuda, Shimpei Aikawa, Yoshihiro Izumi, Akihiko Kondo, Dynamic metabolic profiling of cyanobacterial glycogen biosynthesis under conditions of nitrate depletion, Journal of Experimental Botany, 10.1093/jxb/ert134, 64, 10, 2943-2954, 2013.07, Cyanobacteria represent a globally important biomass because they are responsible for a substantial proportion of primary production in the hydrosphere. Arthrospira platensis is a fast-growing halophilic cyanobacterium capable of accumulating glycogen and has the potential to serve as a feedstock in the fermentative production of third-generation biofuels. Accordingly, enhancing cyanobacterial glycogen production is a promising biofuel production strategy. However, the regulatory mechanism of glycogen metabolism in cyanobacteria is poorly understood. The aim of the present study was to determine the metabolic flux of glycogen biosynthesis using a dynamic metabolomic approach. Time-course profiling of widely targeted cyanobacterial metabolic intermediates demonstrated a global metabolic reprogramming that involves transient increases in the levels of some amino acids during the glycogen production phase induced by nitrate depletion. Also, in vivo labelling with NaH13CO3 enabled direct measurement of metabolic intermediate turnover in A. platensis, revealing that under conditions of nitrate depletion glycogen is biosynthesized with carbon derived from amino acids released from proteins via gluconeogenesis. This dynamic metabolic profiling approach provided conclusive evidence of temporal alterations in the metabolic profile in cyanobacterial cells..
99. Hideo Ohira*, Yoshio Fujioka, Chikae Katagiri, Rie Mamoto, Michiko Aoyama-Ishikawa, Katsumi Amako, Yoshihiro Izumi, Shin Nishiumi, Masaru Yoshida, Makoto Usami, Masamichi Ikeda, Butyrate attenuates inflammation and lipolysis generated by the interaction of adipocytes and macrophages, Journal of atherosclerosis and thrombosis, 10.5551/jat.15065, 20, 5, 425-442, 2013.06, Aim: Paracrine interaction between macrophages and adipocytes in obese visceral fat tissues is thought to be a trigger of chronic inflammation. The immunomodulatory effect of the short chain fatty acid, butyric acid, has been demonstrated. We hypothesize that sodium butyrate (butyrate) attenuates inflammatory responses and lipolysis generated by the interaction of macrophages and adipocytes. Methods: Using contact or transwell co-culture methods with differentiated 3T3-L1 adipocytes and RAW264.7 macrophages, we investigated the effects of butyrate on the production of tumor necrosis factor alpha (TNF-α), monocyte chemoattractant protein 1 (MCP-1), interleukin 6 (IL-6), and the release of free glycerol, free fatty acids (FFAs) into the medium. We also examined the activity of nuclear factor-kappaB (NF-κB) and the phosphorylation of mitogen-activated protein kinases (MAPKs) in co-cultured macrophages, as well as lipase activity and expression in co-cultured adipocytes. Results: We found increased production of TNF-α, MCP-1, IL-6, and free glycerol, FFAs in the coculture medium, and butyrate significantly reduced them. Butyrate inhibited the phosphorylation of MAPKs, the activity of NF-κB in co-cultured macrophages, and suppressed lipase activity in co-cultured adipocytes. Lipase inhibitors significantly attenuated the production of TNF-α, MCP-1 and IL-6 in the co-culture medium as effectively as butyrate. Butyrate suppressed the protein production of adipose triglyceride lipase, hormone sensitive lipase, and fatty acid-binding protein 4 in co-cultured adipocytes. Pertussis toxin, which is known to block GPR41 completely, inhibited the antilipolysis effect of butyrate. Conclusion: Butyrate suppresses inflammatory responses generated by the interaction of adipocytes and macrophages through reduced lipolysis and inhibition of inflammatory signaling..
100. Shimpei Aikawa, Ancy Joseph, Ryosuke Yamada, Yoshihiro Izumi, Takahiro Yamagishi, Fumio Matsuda, Hiroshi Kawai, Jo Shu Chang, Tomohisa Hasunuma, Akihiko Kondo*, Direct conversion of Spirulina to ethanol without pretreatment or enzymatic hydrolysis processes, Energy and Environmental Science, 10.1039/c3ee40305j, 6, 6, 1844-1849, 2013.06, Oxygenic photosynthetic microorganisms such as cyanobacteria and microalgae have attracted attention as feedstocks for next-generation biofuels. To date, however, there have been no reports on efficient bioethanol production from cyanobacterial glycogen by yeast fermentation. Additionally, multiple pretreatment and enzymatic hydrolysis steps of polysaccharides are required for conventional ethanol production from agricultural crops and microalgae. Here, we investigate direct ethanol production from Arthrospira (Spirulina) platensis, a fast-growing halophilic cyanobacterium that accumulates large amounts of glycogen, using lysozyme and a recombinant amylase-expressing yeast strain to eliminate the need for biomass pretreatment and amylase hydrolysis. In the direct conversion process from A. platensis to ethanol, 6.5 g L-1 (ethanol productivity of 1.08 g per L per day) of ethanol was produced. The total ethanol yield based on glycogen consumption was 86% of theoretical yield, which to our knowledge, is the highest yield of bioethanol from an oxygenic photosynthetic microorganism. The present findings indicate that A. platensis is a remarkable carbohydrate feedstock in the form of glycogen, which is a promising material for the production of bioethanol and various other commercially valuable chemicals..
101. Toshihiko Okada, Shinji Fukuda, Koji Hase, Shin Nishiumi, Yoshihiro Izumi, Masaru Yoshida, Teruki Hagiwara, Rei Kawashima, Motomi Yamazaki, Tomoyuki Oshio, Takeshi Otsubo, Kyoko Inagaki-Ohara, Kazuki Kakimoto, Kazuhide Higuchi, Yuki I. Kawamura, Hiroshi Ohno, Taeko Dohi*, Microbiota-derived lactate accelerates colon epithelial cell turnover in starvation-refed mice, Nature communications, 10.1038/ncomms2668, 4, Article number 1654, 2013.05, Oral food intake influences the morphology and function of intestinal epithelial cells and maintains gastrointestinal cell turnover. However, how exactly these processes are regulated, particularly in the large intestine, remains unclear. Here we identify microbiota-derived lactate as a major factor inducing enterocyte hyperproliferation in starvation-refed mice. Using bromodeoxyuridine staining, we show that colonic epithelial cell turnover arrests during a 12- to 36-h period of starvation and increases 12-24 h after refeeding. Enhanced epithelial cell proliferation depends on the increase in live Lactobacillus murinus, lactate production and dietary fibre content. In the model of colon tumorigenesis, mice exposed to a carcinogen during refeeding develop more aberrant crypt foci than mice fed ad libitum. Furthermore, starvation after carcinogen exposure greatly reduced the incidence of aberrant crypt foci. Our results indicate that the content of food used for refeeding as well as the timing of carcinogen exposure influence the incidence of colon tumorigenesis in mice..
102. Takashi Kobayashi, Shin Nishiumi, Atsuki Ikeda, Tomoo Yoshie, Aya Sakai, Atsuki Matsubara, Yoshihiro Izumi, Hidetaka Tsumura, Masahiro Tsuda, Hogara Nishisaki, Nobuhide Hayashi, Seiji Kawano, Yutaka Fujiwara, Hironobu Minami, Tadaomi Takenawa, Takeshi Azuma, Masaru Yoshida*, Anovel serum metabolomics-based diagnostic approach to pancreatic cancer, Cancer Epidemiology Biomarkers and Prevention, 10.1158/1055-9965.EPI-12-1033, 22, 4, 571-579, 2013.04, Background: To improve the prognosis of patients with pancreatic cancer, more accurate serum diagnostic methods are required. We used serum metabolomics as a diagnostic method for pancreatic cancer. Methods: Sera from patients with pancreatic cancer, healthy volunteers, and chronic pancreatitis were collected at multiple institutions. The pancreatic cancer and healthy volunteers were randomly allocated to the training or the validation set. All of the chronic pancreatitis cases were included in the validation set. In each study, the subjects' serum metabolites were analyzed by gas chromatography mass spectrometry (GC/MS) and a data processing system using an in-house library. The diagnostic model constructed via multiple logistic regression analysis in the training set study was evaluated on the basis of its sensitivity and specificity, and the results were confirmed by the validation set study. Results: In the training set study, which included 43 patients with pancreatic cancer and 42 healthy volunteers, the model possessed high sensitivity (86.0%) and specificity (88.1%) for pancreatic cancer. The use of the model was confirmed in the validation set study, which included 42 pancreatic cancer, 41 healthy volunteers, and 23 chronic pancreatitis; that is, it displayed high sensitivity (71.4%) and specificity (78.1%); and furthermore, it displayed higher sensitivity (77.8%) in resectable pancreatic cancer and lower false-positive rate (17.4%) in chronic pancreatitis than conventional markers. Conclusions: Our model possessed higher accuracy than conventional tumor markers at detecting the resectable patients with pancreatic cancer in cohort including patients with chronic pancreatitis. Impact: It is a promising method for improving the prognosis of pancreatic cancer via its early detection and accurate discrimination from chronic pancreatitis..
103. Kosuke Takebayashi, Kenji Hirose, Yoshihiro Izumi, Takeshi Bamba, Eiichiro Fukusaki*, Application of ion mobility-mass spectrometry to microRNA analysis, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2012.10.006, 115, 3, 332-338, 2013.03, Liquid chromatography/mass spectrometry is widely used for studying sequence determination and modification analysis of small RNAs. However, the efficiency of liquid chromatography-based separation of intact small RNA species is insufficient, since the physiochemical properties among small RNAs are very similar. In this study, we focused on ion mobility-mass spectrometry (IM-MS), which is a gas-phase separation technique coupled with mass spectrometry; we have evaluated the utility of IM-MS for microRNA (miRNA) analysis. A multiply charged deprotonated ion derived from an 18-24-nt-long miRNA was formed by electrospray ionization, and then the time, called the " drift time" , taken by each ion to migrate through a buffer gas was measured. Each multivalent ion was temporally separated on the basis of the charge state and structural formation; 3 types of unique mass-mobility correlation patterns (i.e., chainlike-form, hairpin-form, and dimer-form) were present on the two-dimensional mobility-mass spectrum. Moreover, we found that the ion size (sequence length) and the secondary structures of the small RNAs strongly contributed to the IM-MS-based separation, although solvent conditions such as pH had no effect. Therefore, sequence isomers could also be discerned by the selection of each specific charged ion, i.e., the 6- charged ion reflected a majority among chainlike-, hairpin-, and other structures. We concluded that the IM-MS provides additional capability for separation; thus, this analytical method will be a powerful tool for comprehensive small RNA analysis..
104. Ping Lai, Atsushi Okazawa*, Yoshihiro Izumi, Takeshi Bamba, Eiichiro Fukusaki, Masaaki Yoshikawa, Akio Kobayashi, Effect of gallic acid on peptides released by trypsin digestion of bovine α-casein, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2012.10.003, 115, 3, 259-267, 2013.03, In this study, the effects of gallic acid (GA) on trypsin digestion of commercial α-casein (α-CN), which contains αs1-CN and αs2-CN, and the peptides released during digestion were investigated. Gallic acid showed no effect on the initial rate of digestion. However, the apparent degree of hydrolysis achieved its maximum value after 1 h, then decreased in the presence of GA, suggesting the cross-linking between peptides once released from α-CN during digestion. In the presence of GA, three peaks derived from αs1-CN disappeared and three new peaks appeared in high-performance liquid chromatography (HPLC) analysis. In these peptides, two Met residues corresponding to the Met135 and Met196 in αs1-CN were oxidized to Met sulfoxide residues. The oxidation of Met196 was quicker than that of Met135. The inhibitory activity of TTMPLW (αs1-CN 193-199) against angiotensin I-converting enzyme was reduced slightly by the oxidation of its Met residue..
105. Ping Lai, Atsushi Okazawa*, Yoshihiro Izumi, Takeshi Bamba, Eiichiro Fukusaki, Masaaki Yoshikawa, Akio Kobayashi, Gallic acid oxidizes Met residues in peptides released from bovine β-lactoglobulin by in vitro digestion, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2012.04.019, 114, 3, 297-305, 2012.09, Phenolic compounds (PCs) are frequently present in foods. However, little is known about the effect of PCs on enzymatic digestion process of food proteins and their products. In this study, the effect of gallic acid (GA) on in vitro digestion of β-lactoglobulin (β-LG) was investigated as a model system for analysis of the interaction between PCs and food proteins. GA showed no effect on the initial rate of β-LG digestion. However, after 1.5 h of digestion, the observed degree of hydrolysis of β-LG was lower in the presence than in the absence of GA. The peptides released from β-LG were characterized by LC/IT-TOF-MS and thirty peptides were identified. In particular, four new peaks were obtained following in vitro digestion of β-LG in the presence of GA. Met7, Met24 and Met145 in the peptides corresponding to these peaks were oxidized to methionine sulfoxide residues..
106. Shin Nishiumi, Takashi Kobayashi, Atsuki Ikeda, Tomoo Yoshie, Megumi Kibi, Yoshihiro Izumi, Tatsuya Okuno, Nobuhide Hayashi, Seiji Kawano, Tadaomi Takenawa, Takeshi Azuma, Masaru Yoshida*, A novel serum metabolomics-based diagnostic approach for colorectal cancer, PloS one, 10.1371/journal.pone.0040459, 7, 7, 2012.07, Background: To improve the quality of life of colorectal cancer patients, it is important to establish new screening methods for early diagnosis of colorectal cancer. Methodology/Principal Findings: We performed serum metabolome analysis using gas-chromatography/mass-spectrometry (GC/MS). First, the accuracy of our GC/MS-based serum metabolomic analytical method was evaluated by calculating the RSD% values of serum levels of various metabolites. Second, the intra-day (morning, daytime, and night) and inter-day (among 3 days) variances of serum metabolite levels were examined. Then, serum metabolite levels were compared between colorectal cancer patients (N = 60; N = 12 for each stage from 0 to 4) and age- and sex-matched healthy volunteers (N = 60) as a training set. The metabolites whose levels displayed significant changes were subjected to multiple logistic regression analysis using the stepwise variable selection method, and a colorectal cancer prediction model was established. The prediction model was composed of 2-hydroxybutyrate, aspartic acid, kynurenine, and cystamine, and its AUC, sensitivity, specificity, and accuracy were 0.9097, 85.0%, 85.0%, and 85.0%, respectively, according to the training set data. In contrast, the sensitivity, specificity, and accuracy of CEA were 35.0%, 96.7%, and 65.8%, respectively, and those of CA19-9 were 16.7%, 100%, and 58.3%, respectively. The validity of the prediction model was confirmed using colorectal cancer patients (N = 59) and healthy volunteers (N = 63) as a validation set. At the validation set, the sensitivity, specificity, and accuracy of the prediction model were 83.1%, 81.0%, and 82.0%, respectively, and these values were almost the same as those obtained with the training set. In addition, the model displayed high sensitivity for detecting stage 0-2 colorectal cancer (82.8%). Conclusions/Significance: Our prediction model established via GC/MS-based serum metabolomic analysis is valuable for early detection of colorectal cancer and has the potential to become a novel screening test for colorectal cancer..
107. Aya Sakai, Shin Nishiumi, Yuuki Shiomi, Takashi Kobayashi, Yoshihiro Izumi, Hiromu Kutsumi, Takanobu Hayakumo, Takeshi Azuma, Masaru Yoshida*, Metabolomic analysis to discover candidate therapeutic agents against acute pancreatitis, Archives of Biochemistry and Biophysics, 10.1016/j.abb.2012.03.025, 522, 2, 107-120, 2012.06, Novel and effective drugs against acute pancreatitis are required. Therefore, we examined the changes in the metabolite levels in the serum and pancreatic tissue of mice with cerulein- and arginine-induced pancreatitis using gas-chromatography/mass-spectrometry (GC/MS) and investigated whether these alterations affected the severity of acute pancreatitis. In the cerulein-induced pancreatitis model, 93 and 129 metabolites were detected in the serum and pancreatic tissue, respectively. In the l-arginine-induced acute pancreatitis model, 120 and 133 metabolites were detected in the serum and pancreatic tissue, respectively. Among the metabolites, the concentrations of tricarboxylic acid (TCA) cycle intermediates and amino acids were altered in pancreatitis, and in pancreatic tissue, the levels of the intermediates involved in the initial part of the TCA cycle were increased and those of the intermediates involved in the latter part of the TCA cycle were decreased. Some metabolites exhibited similar changes in both pancreatitis mouse models, e.g., the levels of glutamic acid and O-phosphoethanolamine were significantly decreased in the pancreatic tissue. Supplementation with glutamic acid and O-phosphoethanolamine attenuated the severity of cerulein-induced acute pancreatitis. Our results suggest that GC/MS-based metabolomics is capable of accurately representing the status of acute pancreatitis, leading to the discovery of therapeutic agents for pancreatitis..
108. Tomoo Yoshie, Shin Nishiumi, Yoshihiro Izumi, Aya Sakai, Jun Inoue, Takeshi Azuma, Masaru Yoshida*, Regulation of the metabolite profile by an APC gene mutation in colorectal cancer, Cancer Science, 10.1111/j.1349-7006.2012.02262.x, 103, 6, 1010-1021, 2012.06, Mutation of the APC gene occurs during the early stages of colorectal cancer development. To obtain new insights into the mechanisms underlying the aberrant activation of the Wnt pathway that accompanies APC mutation, we carried out a gas chromatography-mass spectrometry-based semiquantitative metabolome analysis. In vitro experiments comparing SW480 cells expressing normal APC and truncated APC indicated that the levels of metabolites involved in the latter stages of the intracellular tricarboxylic acid cycle, including succinic acid, fumaric acid, and malic acid, were significantly higher in the SW480 cells expressing the truncated APC. In an in vivo study, we found that the levels of most amino acids were higher in the non-polyp tissues of APC
min/+
mice than in the normal tissues of the control mice and the polyp tissues of APC
min/+
mice. Ribitol, the levels of which were decreased in the polyp lesions of the APC
min/+
mice and the SW480 cells expressing the truncated APC, reduced the growth of SW480 cells with the APC mutation, but did not affect the growth of SW480 transfectants expressing full-length APC. The level of sarcosine was found to be significantly higher in the polyp tissues of APC
min/+
mice than in their non-polyp tissues and the normal tissues of the control mice, and the treatment of SW480 cells with 50 μM sarcosine resulted in a significant increase in their growth rate. These findings suggest that APC mutation causes changes in energetic metabolite pathways and that these alterations might be involved in the development of colorectal cancer..
109. Hiroko Kato, Yoshihiro Izumi, Tomohisa Hasunuma, Fumio Matsuda, Akihiko Kondo*, Widely targeted metabolic profiling analysis of yeast central metabolites, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2011.12.013, 113, 5, 665-673, 2012.05, A method for a widely targeted analysis was developed for the metabolic profiling of yeast central metabolism. The widely targeted method consists of 2 analyses, namely, gas chromatography-quadrupole-mass spectrometry (GC-Q-MS) operated in selected ion monitoring mode with 25m/z channels, and liquid chromatography triple-stage quadrupole (LC-QqQ)-MS operated in multiple reaction monitoring mode. This platform was set up to identify and quantify preselected 99 compounds, including sugars, sugar phosphates, organic acids, amino acids, and cofactors. The method showed good sensitivity and a wide dynamic range. For example, limits of detection for lactate and l-phenylalanine were 1.4fmol and 2.0fmol, respectively. The dynamic ranges for GC-Q-MS analysis and LC-QqQ-MS analysis were approximately 102-105 and 103-104, respectively. The metabolite profiles of 2 yeast strains, YPH499 and BY4741, under glucose-fermenting conditions were compared using the developed method. Although YPH499 and BY4741 were derived from an identical experimental strain, the profiling analysis successfully revealed a variation in metabolic phenotypes among experimental yeast strains demonstrating that the widely targeted method could be a robust and useful method for the investigation of metabolic phenotypes of Saccharomyces cerevisiae..
110. Yoshihiro Izumi, Shin Takimura, Shinichi Yamaguchi, Junko Iida, Takeshi Bamba, Eiichiro Fukusaki*, Application of electrospray ionization ion trap/time-of-flight mass spectrometry for chemically-synthesized small RNAs, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2011.11.007, 113, 3, 412-419, 2012.03, In this study, we have demonstrated an accurate and rapid small RNA analytical method with both sequence determination and detailed modification analysis by electrospray ionization-ion trap/time-of-flight mass spectrometry (ESI-IT/TOFMS). To develop this ideal method, we have examined the performance of ESI-IT/TOFMS using various chemically-synthesized model sequences of modified or unmodified microRNAs (miRNAs). The deconvoluted mass of a 22-nucleotide (nt) miRNA was obtained from a multiply charged precursor ion (MS
1
). The ion exhibited high mass accuracy ( 2
method using ion trap collision-induced dissociation, as well as automatic annotation analysis of product ions based on the accurate mass information, enabled the precise sequencing determination of intact miRNAs. Further, the detailed structural analysis of 3'-terminal modified nucleic acid in intact methylated miRNA was carried out using the MS
3
capability of the hybrid IT/TOFMS. The direct infusion method also provided a high throughput and good sensitivity because the analytical time and sample concentration needed in a series of experiments with reliable data were only 3min and 100nM, respectively. This study provides a novel approach for characterizing the intact chemically-synthesized small RNA without chemical and enzymatic digestions and would be widely applicable for the structural analysis of complicated modified small RNAs..
111. Shimpei Aikawa, Yoshihiro Izumi, Fumio Matsuda, Tomohisa Hasunuma, Jo Shu Chang, Akihiko Kondo*, Synergistic enhancement of glycogen production in Arthrospira platensis by optimization of light intensity and nitrate supply, Bioresource Technology, 10.1016/j.biortech.2012.01.004, 108, 211-215, 2012.03, Arthrospira (Spirulina) platensis, a fast-growing halophilic cyanobacterium able to accumulate glycogen, was investigated for its feasibility to serve as feedstock for fermentative production of biofuels and chemicals. The culture conditions most appropriate for glycogen production were identified. Glycogen production was maximized by the depleting nitrate source under a high light intensity of 700μmol photons m -2s -1. With optimal control of both light intensity and nitrate supply, glycogen production of A. platensis reached nearly 1.03gL -1 (a glycogen productivity of 0.29gL -1d -1), which is, to the best of our knowledge, the highest α-polyglucan (glycogen or starch) production performance ever reported in microalgae. The outcome of this work supports A. platensis as a promising carbohydrate source for biorefinery..
112. Masaru Yoshida*, Naoya Hatano, Shin Nishiumi, Yasuhiro Irino, Yoshihiro Izumi, Tadaomi Takenawa, Takeshi Azuma, Diagnosis of gastroenterological diseases by metabolome analysis using gas chromatography-mass spectrometry, Journal of Gastroenterology, 10.1007/s00535-011-0493-8, 47, 1, 9-20, 2012.01, Recently, metabolome analysis has been increasingly applied to biomarker detection and disease diagnosis in medical studies. Metabolome analysis is a strategy for studying the characteristics and interactions of low molecular weight metabolites under a specific set of conditions and is performed using mass spectrometry and nuclear magnetic resonance spectroscopy. There is a strong possibility that changes in metabolite levels reflect the functional status of a cell because alterations in their levels occur downstream of DNA, RNA, and protein. Therefore, the metabolite profile of a cell is more likely to represent the current status of a cell than DNA, RNA, or protein. Thus, owing to the rapid development of mass spectrometry analytical techniques metabolome analysis is becoming an important experimental method in life sciences including the medical field. Here, we describe metabolome analysis using liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry (GC-MS), capillary electrophoresis-mass spectrometry, and matrix assisted laser desorption ionization-mass spectrometry. Then, the findings of studies about GC-MS-based metabolome analysis of gastroenterological diseases are summarized, and our research results are also introduced. Finally, we discuss the realization of disease diagnosis by metabolome analysis. The development of metabolome analysis using mass spectrometry will aid the discovery of novel biomarkers, hopefully leading to the early detection of various diseases..
113. Takanori Sugimoto, Takeshi Bamba, Yoshihiro Izumi, Hironari Nomura, Takashi Shiina, Eiichiro Fukusaki*, Use of ultra-performance liquid chromatography/time-of-flight mass spectrometry with nozzle-skimmer fragmentation for comprehensive quantitative analysis of secondary metabolites in Arabidopsis thaliana, Journal of Separation Science, 10.1002/jssc.201100552, 34, 24, 3587-3596, 2011.12, This study sought to develop techniques for LC/MS-based metabolomics and to verify that an MS/MS spectral tag (MS2T) could be used in practical secondary metabolite profiling. The retention time (RT), precursor ions, and fragment ions generated by nozzle-skimmer fragmentation were determined using ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC/TOF-MS) and compared with the MS2T. A standard mix was analyzed with UPLC/TOF-MS under the same conditions as were used to construct the MS2T. The difference in RT for the standards was less than 0.15 min and the average RSD was about 2.8%, suggesting that the analysis was highly repeatable. Both precursor ions and fragment ions were observed when the cone voltage was 75 V. Experimental data and fragmentation pattern in the MS2T annotation list were highly similar. Wild-type and cas-1 mutant Arabidopsis thaliana samples treated with an elicitor were analyzed using UPLC/TOF-MS. Sixty-five peaks were successfully annotated. Fragment ions were observed with nozzle-skimmer fragmentation in 50 of 65 (77%) peaks. The reliability of annotation may have increased as a result of fragment ions. Results of multivariate analysis suggested that cas-1 was related to induction of the biosynthesis of these flavonoids. The devised method facilitated practical secondary metabolite profiling..
114. Atsushi Okazawa*, Katsuhito Hori, Yohei Okumura, Yoshihiro Izumi, Naoki Hata, Takeshi Bamba, Eiichiro Fukusaki, Eiichiro Ono, Honoo Satake, Akio Kobayashi, Simultaneous quantification of lignans in Arabidopsis thaliana by highly sensitive capillary liquid chromatography-electrospray ionization-ion trap mass spectrometry, Plant Biotechnology, 10.5511/plantbiotechnology.11.0221a, 28, 3, 287-293, 2011.08, Lignans are phenylpropanoid dimers in which the monomers are linked by the central carbon (C8) atoms. Because many lignans have physiological effects, including antioxidant activity, they are now in demand as components in health foods. However, the lignan biosynthetic pathways in plants are only now being understood. Recently, lariciresinol was detected in Arabidopsis thaliana. This observation indicated the existence of common lignan biosynthetic pathways in A. thaliana, despite a low amount of lignans other than lariciresinol glycosides. In this study, we established a highly sensitive analytical method that enables quantification of both glycoside and aglycone forms of lignans in A. thaliana simultaneously using capillary liquid chromatography-electrospray ionization-ion trap mass spectrometry. Some lignans not previously detected in A. thaliana were quantified in extracts of both roots and shoots. Our method can be used for the comprehensive analysis of lignans in small samples from mutants and transformants. This method will be utilized to elucidate the metabolic pathways and physiological roles of lignans as well as the regulation of their biosynthesis in plants..
115. Yoshihiro Izumi, Atsushi Okazawa, Takeshi Bamba, Akio Kobayashi, Eiichiro Fukusaki*, Development of a method for comprehensive and quantitative analysis of plant hormones by highly sensitive nanoflow liquid chromatography-electrospray ionization-ion trap mass spectrometry, Analytica Chimica Acta, 10.1016/j.aca.2009.07.001, 648, 2, 215-225, 2009.08, In recent plant hormone research, there is an increased demand for a highly sensitive and comprehensive analytical approach to elucidate the hormonal signaling networks, functions, and dynamics. We have demonstrated the high sensitivity of a comprehensive and quantitative analytical method developed with nanoflow liquid chromatography-electrospray ionization-ion trap mass spectrometry (LC-ESI-IT-MS/MS) under multiple-reaction monitoring (MRM) in plant hormone profiling. Unlabeled and deuterium-labeled isotopomers of four classes of plant hormones and their derivatives, auxins, cytokinins (CK), abscisic acid (ABA), and gibberellins (GA), were analyzed by this method. The optimized nanoflow-LC-ESI-IT-MS/MS method showed ca. 5-10-fold greater sensitivity than capillary-LC-ESI-IT-MS/MS, and the detection limits (S/N = 3) of several plant hormones were in the sub-fmol range. The results showed excellent linearity (R2 values of 0.9937-1.0000) and reproducibility of elution times (relative standard deviations, RSDs,
116. Yoshihiro Izumi, Shin'Ichiro Kajiyama*, Ryosuke Nakamura, Atsushi Ishihara, Atsushi Okazawa, Eiichiro Fukusaki, Yasuo Kanematsu, Akio Kobayashi, High-resolution spatial and temporal analysis of phytoalexin production in oats, Planta, 10.1007/s00425-008-0887-x, 229, 4, 931-943, 2009.03, The production of oat (Avena sativa L.) phytoalexins, avenanthramides, occurs in response to elicitor treatment with oligo-N- acetylchitooligosaccharides. In this study, avenanthramides production was investigated by techniques that provide high spatial and temporal resolution in order to clarify the process of phytoalexin production at the cellular level. The amount of avenanthramides accumulation in a single mesophyll cell was quantified by a combination of laser micro-sampling and low-diffuse nanoflow liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) techniques. Avenanthramides, NAD(P)H and chlorophyll were also visualized in elicitor-treated mesophyll cells using line-scanning fluorescence microscopy. We found that elicitor-treated mesophyll cells could be categorized into three characteristic cell phases, which occurred serially over time. Phase 0 indicated the normal cell state before metabolic or morphological change in response to elicitor, in which the cells contained abundant NAD(P)H. In phase 1, rapid NAD(P)H oxidation and marked movement of chloroplasts occurred, and this phase was the early stage of avenanthramides biosynthesis. In phase 2, avenanthramides accumulation was maximized, and chloroplasts were degraded. Avenanthramides appear to be synthesized in the chloroplast, because a fluorescence signal originating from avenanthramides was localized to the chloroplasts. Moreover, our results indicated that avenanthramides biosynthesis and the hypersensitive response (HR) occurred in identical cells. Thus, the avenanthramides production may be one of sequential events programmed in HR leading to cell death. Furthermore, the phase of the defense response was different among mesophyll cells simultaneously treated with elicitor. These results suggest that individual cells may have different susceptibility to the elicitor..
117. Daisuke Igarashi*, Yoshihiro Izumi, Yuko Dokiya, Kazuhiko Totsuka, Eiichiro Fukusaki, Chieko Ohsumi, Reproductive organs regulate leaf nitrogen metabolism mediated by cytokinin signal, Planta, 10.1007/s00425-008-0858-2, 229, 3, 633-644, 2009.02, The metabolism of vegetative organs in plants changes during the development of the reproductive organs. The regulation of this metabolism is important in the control of crop productivity. However, the complexity of the regulatory systems makes it difficult to elucidate their mechanisms. To examine these mechanisms, we constructed model experiments using Arabidopsis to analyze metabolic and gene expression changes during leaf-stage progression and after removal of the reproductive organs. Leaf gene expression levels and content of major amino acids, both of which decreased during leaf-stage progression, increased after removal of the reproductive organs. In particular, the levels of expression of cytokinin biosynthesis genes and cytokinin-responsive genes and the cytokinin content increased after removal of the reproductive organs. Analysis of plants with knockout of a cytokinin-biosynthetic gene (AtIPT3) and a cytokinin receptor gene (AHK3) indicated that glutamate dehydrogenase genes (GDH3) were regulated by cytokinin signaling. These data suggest that cytokinins regulate communication between reproductive and vegetative organs, and that GDH3 is one target of the cytokinin-mediated regulation of nitrogen metabolism..
118. Ryosuke Nakamura, Yoshihiro Izumi, Shin'Ichiro Kajiyama, Akio Kobayashi, Yasuo Kanematsu*, Line-scanning microscopy for time-gated and spectrally resolved fluorescence imaging, Journal of Biological Physics, 10.1007/s10867-008-9113-0, 34, 1-2 SPEC. ISS., 51-62, 2008.04, Laser-scanning fluorescence microscopy for efficient acquisition of time-gated and spectrally resolved fluorescence images was developed based on line illumination of the laser beam and detection of the fluorescence image through a slit. In this optical arrangement, the fluorescence image was obtained by scanning only one axis perpendicular to the excitation line, and the acquisition time was significantly reduced compared with conventional laser-scanning confocal microscopy. A multidimensional fluorescence dataset consisting of fluorescence intensities as a function of x-position, y-position, fluorescence wavelength, and delay time after photoexcitation was analyzed and decomposed based on the parallel factor analysis model. The performance of the line-scanning microscopy was examined by applying it to the analysis of one of the plant defense responses, accumulation of antimicrobial compounds of phytoalexin in oat (Avena sativa), induced by the elicitor treatment..
119. Shin'ichiro Kajiyama*, Takeshi Shoji, Shinya Okuda, Yoshihiro Izumi, Ei Ichiro Fukusaki, Akio Kobayashi, A novel microsurgery method for intact plant tissue at the single cell level using ArF excimer laser microprojection, Biotechnology and Bioengineering, 10.1002/bit.20709, 93, 2, 325-331, 2006.02, A novel microsurgery technique for the partial removal of rigid cell-walls in intact plant tissue is established. Using a size-variable slit, an ArF excimer laser was microprojected on the surface of the targeted cell, and this method enabled the area- and depth-controllable processing of the cortical structure of plant cells including the cuticle and cell wall layer. In epidermal cells of all tested plants, viabilities of more than 90% were retained 24 h after irradiation. Scanning electron microscope (SEM) observation revealed that the cuticle layer of the irradiated region was completely ablated, and the cellulose microfibrils of the secondary cell wall were partially removed; furthermore, 4 days after laser treatment, the regeneration of cell wall fibrils was observed. As a model experiment, the transient expression of synthetic green fluorescent protein (sGFP) was performed by the microinjection of cauliflower mosaic virus (CMV) 35S promoter-derived sGFP gene through an "aperture" in the treated cell surface. Moreover, micron-sized fluorescent beads were successfully introduced by the same method into the onion cells indicating that this method can be used to introduce foreign materials as large as organelles..