Details of a Researcher - Tomita, Masaru

Papers - Tomita, Masaru

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Thymidine catabolism promotes NADPH oxidase-derived reactive oxygen species (ROS) signalling in KB and yumoto cells

Tabata S., Yamamoto M., Goto H., Hirayama A., Ohishi M., Kuramoto T., Mitsuhashi A., Ikeda R., Haraguchi M., Kawahara K., Shinsato Y., Minami K., Saijo A., Toyoda Y., Hanibuchi M., Nishioka Y., Sone S., Esumi H., Tomita M., Soga T., Furukawa T., Akiyama S.

Scientific Reports (Scientific Reports) 8 ( 1 ) 6760 2018.12

© 2018 The Author(s). Thymidine phosphorylase (TP) is a rate-limiting enzyme in the thymidine catabolic pathway. TP is identical to platelet-derived endothelial cell growth factor and contributes to tumour angiogenesis. TP induces the generation of reactive oxygen species (ROS) and enhances the expression of oxidative stress-responsive genes, such as interleukin (IL)-8. However, the mechanism underlying ROS induction by TP remains unclear. In the present study, we demonstrated that TP promotes NADPH oxidase-derived ROS signalling in cancer cells. NADPH oxidase inhibition using apocynin or small interfering RNAs (siRNAs) abrogated the induction of IL-8 and ROS in TP-expressing cancer cells. Meanwhile, thymidine catabolism induced by TP increased the levels of NADPH and intermediates of the pentose phosphate pathway (PPP). Both siRNA knockdown of glucose 6-phosphate dehydrogenase (G6PD), a rate-limiting enzyme in PPP, and a G6PD inhibitor, dihydroepiandrosterone, reduced TP-induced ROS production. siRNA downregulation of 2-deoxy-D-ribose 5-phosphate (DR5P) aldolase, which is needed for DR5P to enter glycolysis, also suppressed the induction of NADPH and IL-8 in TP-expressing cells. These results suggested that TP-mediated thymidine catabolism increases the intracellular NADPH level via the PPP, which enhances the production of ROS by NADPH oxidase and activates its downstream signalling.

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Far rapid synthesis of giant DNA in the Bacillus subtilis genome by a conjugation transfer system

Itaya M., Sato M., Hasegawa M., Kono N., Tomita M., Kaneko S.

Scientific Reports (Scientific Reports) 8 ( 1 ) 8792 2018.12

© 2018 The Author(s). Bacillus subtilis offers a platform for giant DNA synthesis, which is mediated by the connection of overlapping DNA segments called domino DNA, in the cloning locus of the host. The domino method was successfully used to produce DNA fragments as large as 3500 kbp. However, domino DNA is limited to <100 kbp because of size restrictions regarding the transformation (TF) of B. subtilis competent cells. A novel conjugal transfer (CT) method was designed to eliminate the TF size limit. The CT method enables rapid and efficient domino reactions in addition to the transfer of giant DNA molecules of up to 875 kbp to another B. subtilis genome within 4 hours. The combined use of the TF and CT should enable significantly rapid giant DNA production.

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Effect of storage conditions on salivary polyamines quantified via liquid chromatography-mass spectrometry

Tomita A., Mori M., Hiwatari K., Yamaguchi E., Itoi T., Sunamura M., Soga T., Tomita M., Sugimoto M.

Scientific Reports (Scientific Reports) 8 ( 1 ) 12075 2018.12

© 2018, The Author(s). Salivary polyamines are potential non-invasive tools for screening various types of cancers. For clinical use, the reproducibility of these metabolites should be evaluated under various storage conditions, including duration and temperature, to establish standard operating protocols. Polyamines and amino acids in unstimulated whole saliva were quantified via liquid chromatography-mass spectrometry. Concentrations of time course samples were analysed after short-term storage for up to 240 min and long-term storage for up to 8 days under various storage conditions. As expected, storage at the lowest temperature (−18 °C) exerted the least pronounced effects on the quantified values in both tests. At a higher temperature, polyamines were more stable than amino acids, as evident from polyamine profiling. Addition of ethanol significantly stabilized polyamine profiles even at a higher temperature. Comparative processing of saliva revealed a minor effect of the solvent, whereas drying had a more prominent effect on polyamine profiles. Computational analyses evaluated the ability of polyamines to discriminate pancreatic cancer from controls. Repeated noise added tests were designed on the basis of the results of the storage tests; these analyses confirmed that the discriminative abilities were robust. These data contribute to the standardization of salivary storage conditions, thereby highlighting the clinical utility of saliva.

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Drying and extraction effects on three edible brown seaweeds for metabolomics

Hamid S., Wakayama M., Soga T., Tomita M.

Journal of Applied Phycology 30 ( 6 ) 3335 - 3350 2018.12

ISSN 09218971

© 2018, Springer Nature B.V. Metabolomics is often used to comprehensively elucidate the metabolites in organisms like seaweed. Amino acids hydrolysed from proteins and certain targeted metabolites in seaweed have been investigated. However, water-soluble metabolites like free amino acids, organic acids, and sugars have seldom been comprehensively analysed. Metabolomics are valuable tools for these studies, but they require optimisation of pre-treatment methodology. Here, we evaluated various pre-treatment drying and extraction methods for brown seaweed metabolomics. Three edible brown seaweeds (Cladosiphon okamuranus [Mozuku], Saccharina japonica [Kombu], and Undaria pinnatifida [Wakame]) were used. Freeze-drying and oven-drying at both 40 and 80 °C were investigated. Methanol-water extracts with and without chloroform were compared. Metabolites were evaluated and quantified using liquid chromatography-mass spectrometry and capillary electrophoresis-mass spectrometry. The results showed that metabolite profiling was determined mainly by seaweed species identity rather than pre-treatment method. Freeze-drying yielded higher metabolite concentrations than oven-drying at either 40 or 80 °C. The effects of extraction with and without chloroform on metabolite concentration varied with seaweed species.

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Metabolomics-based profiles predictive of low bone mass in menopausal women

Miyamoto T., Hirayama A., Sato Y., Koboyashi T., Katsuyama E., Kanagawa H., Fujie A., Morita M., Watanabe R., Tando T., Miyamoto K., Tsuji T., Funayama A., Soga T., Tomita M., Nakamura M., Matsumoto M.

Bone Reports (Bone Reports) 9 11 - 18 2018.12

ISSN 2352-1872

© 2018 The Authors Osteoporosis is a skeletal disorder characterized by compromised bone strength and increased risk of fracture. Low bone mass and/or pre-existing bone fragility fractures serve as diagnostic criteria in deciding when to start medication for osteoporosis. Although osteoporosis is a metabolic disorder, metabolic markers to predict reduced bone mass are unknown. Here, we show serum metabolomics profiles of women grouped as pre-menopausal with normal bone mineral density (BMD) (normal estrogen and normal BMD; NN), post-menopausal with normal BMD (low estrogen and normal BMD; LN) or post-menopausal with low BMD (low estrogen and low BMD; LL) using comprehensive metabolomics analysis. To do so, we enrolled healthy volunteer and osteoporosis patient female subjects, surveyed them with a questionnaire, measured their BMD, and then undertook a comprehensive metabolomics analysis of sera of the three groups named above. We identified 24 metabolites whose levels differed significantly between NN/LN and NN/LL groups, as well as 18 or 10 metabolites whose levels differed significantly between NN/LN and LN/LL, or LN/LL and NN/LN groups, respectively. Our data shows metabolomics changes represent useful markers to predict estrogen deficiency and/or bone loss.

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A metabolomic-based evaluation of the role of commensal microbiota throughout the gastrointestinal tract in mice

Yamamoto Y., Nakanishi Y., Murakami S., Aw W., Tsukimi T., Nozu R., Ueno M., Hioki K., Nakahigashi K., Hirayama A., Sugimoto M., Soga T., Ito M., Tomita M., Fukuda S.

Microorganisms (Microorganisms) 6 ( 4 ) 2018.12

ISSN 2076-2607

Commensal microbiota colonize the surface of our bodies. The inside of the gastrointestinal tract is one such surface that provides a habitat for them. The gastrointestinal tract is a long organ system comprising of various parts, and each part possesses various functions. It has been reported that the composition of intestinal luminal metabolites between the small and large intestine are different; however, comprehensive metabolomic and commensal microbiota profiles specific to each part of the gastrointestinal lumen remain obscure. In this study, by using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS)-based metabolome and 16S rRNA gene-based microbiome analyses of specific pathogen-free (SPF) and germ-free (GF) murine gastrointestinal luminal profiles, we observed the different roles of commensal microbiota in each part of the gastrointestinal tract involved in carbohydrate metabolism and nutrient production. We found that the concentrations of most amino acids in the SPF small intestine were higher than those in the GF small intestine. Furthermore, sugar alcohols such as mannitol and sorbitol accumulated only in the GF large intestine, but not in the SPF large intestine. On the other hand, pentoses, such as arabinose and xylose, gradually accumulated from the cecum to the colon only in SPF mice, but were undetected in GF mice. Correlation network analysis between the gastrointestinal microbes and metabolites showed that niacin metabolism might be correlated to Methylobacteriaceae. Collectively, commensal microbiota partially affects the gastrointestinal luminal metabolite composition based on their metabolic dynamics, in cooperation with host digestion and absorption.

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Metabolic characterization of antifolate responsiveness and nonresponsiveness in malignant pleural mesothelioma cells

Sato Y., Matsuda S., Maruyama A., Nakayama J., Miyashita T., Udagawa H., Umemura S., Yanagihara K., Ochiai A., Tomita M., Soga T., Tsuchihara K., Makinoshima H.

Frontiers in Pharmacology (Frontiers in Pharmacology) 9 ( OCT ) 1129 2018.10

ISSN 1663-9812

© 2007 - 2018 Frontiers Media S.A. All Rights Reserved. Antifolates are a class of drugs effective for treating malignant pleural mesothelioma (MPM). The majority of antifolates inhibit enzymes involved in purine and pyrimidine synthesis such as dihydrofolate reductase (DHFR), thymidylate synthase (TYMS), and glycinamide ribonucleotide formyltransferase (GART). In order to select the most suitable patients for effective therapy with drugs targeting specific metabolic pathways, there is a need for better predictive metabolic biomarkers. Antifolates can alter global metabolic pathways in MPM cells, yet the metabolic profile of treated cells has not yet been clearly elucidated. Here we found that MPM cell lines could be categorized into two groups according to their sensitivity or resistance to pemetrexed treatment. We show that pemetrexed susceptibility could be reversed and DNA synthesis rescued in drug-treated cells by the exogenous addition of the nucleotide precursors hypoxanthine and thymidine (HT). We observed that the expression of pemetrexed-targeted enzymes in resistant MPM cells was quantitatively lower than that seen in pemetrexed-sensitive cells. Metabolomic analysis revealed that glycine and choline, which are involved in one-carbon metabolism, were altered after drug treatment in pemetrexed-sensitive but not resistant MPM cells. The addition of HT upregulated the concentration of inosine monophosphate (IMP) in pemetrexed-sensitive MPM cells, indicating that the nucleic acid biosynthesis pathway is important for predicting the efficacy of pemetrexed in MPM cells. Our data provide evidence that may link therapeutic response to the regulation of metabolism, and points to potential biomarkers for informing clinical decisions regarding the most effective therapies for patients with MPM.

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Microglena redcarensis sp. nov. (Volvocales, Chlorophyceae), a brackish water chlamydomonad with contractile vacuoles

Nakada T., Takahashi S., Tomita M.

Phycological Research 66 ( 4 ) 310 - 317 2018.10

ISSN 13220829

© 2018 Japanese Society of Phycology In 1960, Butcher isolated a strain of unicellular green alga, ‘Chlamydomonas subehrenbergii’ SAG 18.89, from a brackish water locality in England, facing the North Sea. Although C. subehrenbergii Butcher was previously described by the same author in 1959, the strain SAG 18.89 is in fact morphologically different, possessing shorter flagella, a different-shaped papilla, and different-sized vegetative cells. Light and fluorescence microscopy, molecular phylogenetic analyses, and ITS2 comparisons revealed that this strain represents an undescribed species of the genus Microglena (Volvocales, Chlorophyceae), described here as M. redcarensis sp. nov. The presence of contractile vacuoles, which are osmoregulatory organelles, suggests potential inhabitation in freshwater. Contractile vacuole activity and growth of M. redcarensis were therefore examined in media of different salinity.

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Ultralow input genome sequencing library preparation from a single tardigrade specimen

Yoshida Y., Konno S., Nishino R., Murai Y., Tomita M., Arakawa K.

Journal of Visualized Experiments (Journal of Visualized Experiments) 2018 ( 137 ) 2018.07

ISSN 1940087X

© 2018 Journal of Visualized Experiments. Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state when water is supplied. The genomic sequencing of microscopic animals such as tardigrades risks bacterial contamination that sometimes leads to erroneous interpretations, for example, regarding the extent of horizontal gene transfer in these animals. Here, we provide an ultralow input method to sequence the genome of the tardigrade, Hypsibius dujardini, from a single specimen. By employing rigorous washing and contaminant exclusion along with an efficient extraction of the 50 ~ 200 pg genomic DNA from a single individual, we constructed a library sequenced with a DNA sequencing instrument. These libraries were highly reproducible and unbiased, and an informatics analysis of the sequenced reads with other H. dujardini genomes showed a minimal amount of contamination. This method can be applied to unculturable tardigrades that could not be sequenced using previous methods.

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Base-pairing probability in the microRNA stem region affects the binding and editing specificity of human A-to-I editing enzymes ADAR1-p110 and ADAR2

Ishiguro S., Galipon J., Ishii R., Suzuki Y., Kondo S., Okada-Hatakeyama M., Tomita M., Ui-Tei K.

RNA Biology (RNA Biology) 15 ( 7 ) 976 - 989 2018.07

ISSN 15476286

© 2018, © 2018 Taylor & Francis Group, LLC. Adenosine deaminases acting on RNA (ADARs) catalyze the deamination of adenosine (A) to inosine (I). A-to-I RNA editing targets double-stranded RNA (dsRNA), and increases the complexity of gene regulation by modulating base pairing-dependent processes such as splicing, translation, and microRNA (miRNA)-mediated gene silencing. This study investigates the genome-wide binding preferences of the nuclear constitutive isoforms ADAR1-p110 and ADAR2 on human miRNA species by RNA immunoprecipitation of ADAR-bound small RNAs (RIP-seq). Our results suggest that secondary structure predicted by base-pairing probability in the mainly double-stranded region of a pre-miRNA or mature miRNA duplex may determine ADAR isoform preference for binding distinct subpopulations of miRNAs. Furthermore, we identify 31 unique editing sites with statistical significance, 19 sites of which are novel editing sites. Editing sites are enriched in the seed region responsible for target recognition by miRNAs, and isoform-specific nucleotide motifs in the immediate vicinity and opposite of editing sites are consistent with previous studies, and further reveal that ADAR2 may edit A/C bulges more frequently than ADAR1-p110 in the context of miRNA.

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The complete mitochondrial genome of Eumeta variegata (Lepidoptera: Psychidae)

Arakawa K., Kono N., Ohtoshi R., Nakamura H., Tomita M.

Mitochondrial DNA Part B: Resources (Mitochondrial DNA Part B: Resources) 3 ( 2 ) 812 - 813 2018.07

© 2018, © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. The complete mitochondrial genome of Eumeta variegate, largest bagworm moth in Japan, has been sequenced using a nanopore sequencer as a single long read. The genome has a total length of 16,601 bp, consisting of 13 protein-coding genes, 20 tRNA, 2 rRNA genes, and an AT-rich control region. The nucleotide composition was extremely AT-rich, with 42.4% A, 40.4% T, 6.67% G, and 10.6% C. This is the second report of a complete mitochondrial genome of Psychidae, and the sequence information together with a phylogenetic analysis would provide a reference data in the future studies of Lepidoptera and Psychidae.

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Efficient delivery of large DNA from Escherichia coli to Synechococcus elongatus PCC7942 by broad-host-range conjugal plasmid pUB307

Itaya M., Kusakabe H., Sato M., Tomita M., Sato R.

Journal of Biochemistry (Journal of Biochemistry) 164 ( 1 ) 15 - 20 2018.07

ISSN 0021924X

© The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved. Synechococcus elongatus PCC7942, a cyanobacterium that uses light and carbon dioxide to grow, has a high ability to incorporate DNA by transformation. To assess the effective delivery of large DNA in plasmid form, we cloned the endogenous plasmid pANL (46.4 kbp) into a BAC vector of Escherichia coli. The plasmid p38ANL (54.3 kbp) replaced the native plasmid. To assess the delivery of larger DNA into PCC7942, p38ANL was fused to the broad-host-range conjugal transfer plasmid pUB307IP (53.5 kbp). The resulting plasmid pUB307IP501 (107.9 kbp) was transmitted from E. coli to PCC7942 by simple mixing of donor and recipient cultures. PCC7942 transcipients possessed only pUB307IP501, replacing the preexisting pANL. In contrast, the pUB307IP501 plasmid was unable to transform PCC7942, indicating that natural transformation of DNA may be restricted by size limitations. The ability to deliver large DNA by conjugation may lead to genetic engineering in PCC7942.

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MICOP: Maximal information coefficient-based oscillation prediction to detect biological rhythms in proteomics data

Iuchi H., Sugimoto M., Tomita M.

BMC Bioinformatics (BMC Bioinformatics) 19 ( 1 ) 249 2018.06

© 2018 The Author(s). Background: Circadian rhythms comprise oscillating molecular interactions, the disruption of the homeostasis of which would cause various disorders. To understand this phenomenon systematically, an accurate technique to identify oscillating molecules among omics datasets must be developed; however, this is still impeded by many difficulties, such as experimental noise and attenuated amplitude. Results: To address these issues, we developed a new algorithm named Maximal Information Coefficient-based Oscillation Prediction (MICOP), a sine curve-matching method. The performance of MICOP in labeling oscillation or non-oscillation was compared with four reported methods using Mathews correlation coefficient (MCC) values. The numerical experiments were performed with time-series data with (1) mimicking of molecular oscillation decay, (2) high noise and low sampling frequency and (3) one-cycle data. The first experiment revealed that MICOP could accurately identify the rhythmicity of decaying molecular oscillation (MCC > 0.7). The second experiment revealed that MICOP was robust against high-level noise (MCC > 0.8) even upon the use of low-sampling-frequency data. The third experiment revealed that MICOP could accurately identify the rhythmicity of noisy one-cycle data (MCC > 0.8). As an application, we utilized MICOP to analyze time-series proteome data of mouse liver. MICOP identified that novel oscillating candidates numbered 14 and 30 for C57BL/6 and C57BL/6 J, respectively. Conclusions: In this paper, we presented MICOP, which is an MIC-based algorithm, for predicting periodic patterns in large-scale time-resolved protein expression profiles. The performance test using artificially generated simulation data revealed that the performance of MICOP for decaying data was superior to that of the existing widely used methods. It can reveal novel findings from time-series data and may contribute to biologically significant results. This study suggests that MICOP is an ideal approach for detecting and characterizing oscillations in time-resolved omics data sets.

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Tuning Gene Activity by Inducible and Targeted Regulation of Gene Expression in Minimal Bacterial Cells

Mariscal A., Kakizawa S., Hsu J., Tanaka K., González-González L., Broto A., Querol E., Lluch-Senar M., Piñero-Lambea C., Sun L., Weyman P., Wise K., Merryman C., Tse G., Moore A., Hutchison C., Smith H., Tomita M., Venter J., Glass J., Piñol J., Suzuki Y.

ACS Synthetic Biology (ACS Synthetic Biology) 7 ( 6 ) 1538 - 1552 2018.06

© 2018 American Chemical Society. Functional genomics studies in minimal mycoplasma cells enable unobstructed access to some of the most fundamental processes in biology. Conventional transposon bombardment and gene knockout approaches often fail to reveal functions of genes that are essential for viability, where lethality precludes phenotypic characterization. Conditional inactivation of genes is effective for characterizing functions central to cell growth and division, but tools are limited for this purpose in mycoplasmas. Here we demonstrate systems for inducible repression of gene expression based on clustered regularly interspaced short palindromic repeats-mediated interference (CRISPRi) in Mycoplasma pneumoniae and synthetic Mycoplasma mycoides, two organisms with reduced genomes actively used in systems biology studies. In the synthetic cell, we also demonstrate inducible gene expression for the first time. Time-course data suggest rapid kinetics and reversible engagement of CRISPRi. Targeting of six selected endogenous genes with this system results in lowered transcript levels or reduced growth rates that agree with lack or shortage of data in previous transposon bombardment studies, and now produces actual cells to analyze. The ksgA gene encodes a methylase that modifies 16S rRNA, rendering it vulnerable to inhibition by the antibiotic kasugamycin. Targeting the ksgA gene with CRISPRi removes the lethal effect of kasugamycin and enables cell growth, thereby establishing specific and effective gene modulation with our system. The facile methods for conditional gene activation and inactivation in mycoplasmas open the door to systematic dissection of genetic programs at the core of cellular life.

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Development of a sheathless CE-ESI-MS interface

Hirayama A., Abe H., Yamaguchi N., Tabata S., Tomita M., Soga T.

Electrophoresis (Electrophoresis) 39 ( 11 ) 1382 - 1389 2018.06

ISSN 01730835

© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim A sheath-flow interface is the most common ionization technique in CE-ESI-MS. However, this interface dilutes the analytes with the sheath liquid and decreases the sensitivity. In this study, we developed a sheathless CE-MS interface to improve sensitivity. The interface was fabricated by making a small crack approximately 2 cm from the end of a capillary column fixed on a plastic plate, and then covering the crack with a dialysis membrane to prevent metabolite loss during separation. A voltage for CE separation was applied between the capillary inlet and the buffer reservoir. Under optimum conditions, 52 cationic metabolite standards were separated and selectively detected using MS. With a pressure injection of 5 kPa for 15 s (ca. 1.4 nL), the detection limits for the tested compounds were between 0.06 and 1.7 μmol/L (S/N = 3). The method was applied to analysis of cationic metabolites extracted from a small number (12 000) of cancer cells, and the number of peaks detected was about 2.5 times higher than when using conventional sheath-flow CE-MS. Because the interface is easy to construct, it is cost-effective and can be adapted to any commercially available capillaries. This method is a powerful new tool for highly sensitive CE-MS-based metabolomic analysis.

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Urinary polyamine biomarker panels with machine-learning differentiated colorectal cancers, benign disease, and healthy controls

Nakajima T., Katsumata K., Kuwabara H., Soya R., Enomoto M., Ishizaki T., Tsuchida A., Mori M., Hiwatari K., Soga T., Tomita M., Sugimoto M.

International Journal of Molecular Sciences (International Journal of Molecular Sciences) 19 ( 3 ) 2018.03

ISSN 16616596

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. Colorectal cancer (CRC) is one of the most daunting diseases due to its increasing worldwide prevalence, which requires imperative development of minimally or non-invasive screening tests. Urinary polyamines have been reported as potential markers to detect CRC, and an accurate pattern recognition to differentiate CRC with early stage cases from healthy controls are needed. Here, we utilized liquid chromatography triple quadrupole mass spectrometry to profile seven kinds of polyamines, such as spermine and spermidine with their acetylated forms. Urinary samples from 201 CRCs and 31 non-CRCs revealed the N1,N12-diacetylspermine showing the highest area under the receiver operating characteristic curve (AUC), 0.794 (the 95% confidence interval (CI): 0.704–0.885, p < 0.0001), to differentiate CRC from the benign and healthy controls. Overall, 59 samples were analyzed to evaluate the reproducibility of quantified concentrations, acquired by collecting three times on three days each from each healthy control. We confirmed the stability of the observed quantified values. A machine learning method using combinations of polyamines showed a higher AUC value of 0.961 (95% CI: 0.937–0.984, p < 0.0001). Computational validations confirmed the generalization ability of the models. Taken together, polyamines and a machine-learning method showed potential as a screening tool of CRC.

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Human AK2 links intracellular bioenergetic redistribution to the fate of hematopoietic progenitors

Oshima K., Saiki N., Tanaka M., Imamura H., Niwa A., Tanimura A., Nagahashi A., Hirayama A., Okita K., Hotta A., Kitayama S., Osawa M., Kaneko S., Watanabe A., Asaka I., Fujibuchi W., Imai K., Yabe H., Kamachi Y., Hara J., Kojima S., Tomita M., Soga T., Noma T., Nonoyama S., Nakahata T., Saito M.

Biochemical and Biophysical Research Communications (Biochemical and Biophysical Research Communications) 497 ( 2 ) 719 - 725 2018.03

ISSN 0006291X

© 2018 The Authors AK2 is an adenylate phosphotransferase that localizes at the intermembrane spaces of the mitochondria, and its mutations cause a severe combined immunodeficiency with neutrophil maturation arrest named reticular dysgenesis (RD). Although the dysfunction of hematopoietic stem cells (HSCs) has been implicated, earlier developmental events that affect the fate of HSCs and/or hematopoietic progenitors have not been reported. Here, we used RD-patient-derived induced pluripotent stem cells (iPSCs) as a model of AK2-deficient human cells. Hematopoietic differentiation from RD-iPSCs was profoundly impaired. RD-iPSC-derived hemoangiogenic progenitor cells (HAPCs) showed decreased ATP distribution in the nucleus and altered global transcriptional profiles. Thus, AK2 has a stage-specific role in maintaining the ATP supply to the nucleus during hematopoietic differentiation, which affects the transcriptional profiles necessary for controlling the fate of multipotential HAPCs. Our data suggest that maintaining the appropriate energy level of each organelle by the intracellular redistribution of ATP is important for controlling the fate of progenitor cells.

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Elevated polyamines in saliva of pancreatic cancer

Asai Y., Itoi T., Sugimoto M., Sofuni A., Tsuchiya T., Tanaka R., Tonozuka R., Honjo M., Mukai S., Fujita M., Yamamoto K., Matsunami Y., Kurosawa T., Nagakawa Y., Kaneko M., Ota S., Kawachi S., Shimazu M., Soga T., Tomita M., Sunamura M.

Cancers (Cancers) 10 ( 2 ) 2018.02

ISSN 2072-6694

© 2018 by the authors. Detection of pancreatic cancer (PC) at a resectable stage is still difficult because of the lack of accurate detection tests. The development of accurate biomarkers in low or non-invasive biofluids is essential to enable frequent tests, which would help increase the opportunity of PC detection in early stages. Polyamines have been reported as possible biomarkers in urine and saliva samples in various cancers. Here, we analyzed salivary metabolites, including polyamines, using capillary electrophoresis-mass spectrometry. Salivary samples were collected from patients with PC (n = 39), those with chronic pancreatitis (CP, n = 14), and controls (C, n = 26). Polyamines, such as spermine, N1-acetylspermidine, and N1-acetylspermine, showed a significant difference between patients with PC and those with C, and the combination of four metabolites including N1-acetylspermidine showed high accuracy in discriminating PC from the other two groups. These data show the potential of saliva as a source for tests screening for PC.

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Systematic analysis of the binding surfaces between tRNAs and their respective aminoacyl tRNA synthetase based on structural and evolutionary data

Tamaki S., Tomita M., Suzuki H., Kanai A.

Frontiers in Genetics (Frontiers in Genetics) 8 ( JAN ) 227 2018.01

ISSN 1664-8021

To determine the mechanism underlying the flow of genetic information, it is important to understand the relationship between a tRNA and its binding enzyme, a member of the aminoacyl-tRNA synthetase (aaRS) family. We have developed a novel method to project the interacting regions of tRNA-aaRS complexes, obtained from their three-dimensional structures, onto two-dimensional space. The interacting surface between each tRNA and its aaRS was successfully identified by determining these interactions with an atomic distance threshold of 3.3 Å. We analyzed their interactions, using 60 mainly bacterial and eukaryotic tRNA-aaRS complexes, and showed that the tRNA sequence regions that interacted most strongly with each aaRS are the anticodon loop and the CCA terminal region, followed by the D-stem. A sequence conservation analysis of the canonical tRNAs was conducted in 83 bacterial, 182 archaeal, and 150 eukaryotic species. Our results show that the three tRNA regions that interact with the aaRS and two additional loop regions (D-loop and TψC-loop) known to be important for formation of the tRNA L-shaped structure are broadly conserved. We also found sequence conservations near the tRNA discriminator in the Bacteria and Archaea, and an enormous number of noncanonical tRNAs in the Eukaryotes. This is the first global view of tRNA evolution based on its structure and an unprecedented number of sequence data.

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Reliability of plasma polar metabolite concentrations in a large-scale cohort study using capillary electrophoresis-mass spectrometry

Harada S., Hirayama A., Chan Q., Kurihara A., Fukai K., Iida M., Kato S., Sugiyama D., Kuwabara K., Takeuchi A., Akiyama M., Okamura T., Ebbels T., Elliott P., Tomita M., Sato A., Suzuki C., Sugimoto M., Soga T., Takebayashi T.

PLoS ONE (PLoS ONE) 13 ( 1 ) e0191230 2018.01

© 2018 Harada et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Background Cohort studies with metabolomics data are becoming more widespread, however, large-scale studies involving 10,000s of participants are still limited, especially in Asian populations. Therefore, we started the Tsuruoka Metabolomics Cohort Study enrolling 11,002 community-dwelling adults in Japan, and using capillary electrophoresis-mass spectrometry (CE-MS) and liquid chromatography–mass spectrometry. The CE-MS method is highly amenable to absolute quantification of polar metabolites, however, its reliability for large-scale measurement is unclear. The aim of this study is to examine reproducibility and validity of large-scale CE-MS measurements. In addition, the study presents absolute concentrations of polar metabolites in human plasma, which can be used in future as reference ranges in a Japanese population. Methods Metabolomic profiling of 8,413 fasting plasma samples were completed using CE-MS, and 94 polar metabolites were structurally identified and quantified. Quality control (QC) samples were injected every ten samples and assessed throughout the analysis. Inter- and intra-batch coefficients of variation of QC and participant samples, and technical intraclass correlation coefficients were estimated. Passing-Bablok regression of plasma concentrations by CE-MS on serum concentrations by standard clinical chemistry assays was conducted for creatinine and uric acid. Results and conclusions In QC samples, coefficient of variation was less than 20% for 64 metabolites, and less than 30% for 80 metabolites out of the 94 metabolites. Inter-batch coefficient of variation was less than 20% for 81 metabolites. Estimated technical intraclass correlation coefficient was above 0.75 for 67 metabolites. The slope of Passing-Bablok regression was estimated as 0.97 (95% confidence interval: 0.95, 0.98) for creatinine and 0.95 (0.92, 0.96) for uric acid. Compared to published data from other large cohort measurement platforms, reproducibility of metabolites common to the platforms was similar to or better than in the other studies. These results show that our CE-MS platform is suitable for conducting large-scale epidemiological studies.

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