Details of a Researcher - Tomita, Masaru

Papers - Tomita, Masaru

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High-quality overlapping paired-end reads for the detection of A-to-I editing on small RNA

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

Methods in Molecular Biology 1823 167 - 183 2018

ISSN 10643745

© 2018, Springer Science+Business Media, LLC, part of Springer Nature. Paired-end RNA sequencing (RNA-seq) is usually applied to the quantification of long transcripts such as messenger or long non-coding RNAs, in which case overlapping pairs are discarded. In contrast, RNA-seq on short RNAs (≤ 200 nt) is typically carried out in single-end mode, as the additional cost associated with paired-end would only translate into redundant sequence information. Here, we exploit paired-end sequencing of short RNAs as a strategy to filter out sequencing errors and apply this method to the identification of adenosine-to-inosine (A-to-I) RNA editing events on human precursor microRNA (pre-miRNA) and mature miRNA. Combined with RNA immunoprecipitation sequencing (RIP-seq) of A-to-I RNA editing enzymes, this method takes full advantage of deep sequencing technology to identify RNA editing sites with unprecedented resolution in terms of editing efficiency.

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Beta-galactosidase-responsive synthetic biomarker for targeted tumor detection

Nishihara T., Kuno S., Nonaka H., Tabata S., Saito N., Fukuda S., Tomita M., Sando S., Soga T.

Chemical Communications (Chemical Communications) 54 ( 83 ) 11745 - 11748 2018

ISSN 13597345

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Accelerated laboratory evolution reveals the influence of replication on the GC skew in Escherichia coli

Kono N., Tomita M., Arakawa K.

Genome Biology and Evolution (Genome Biology and Evolution) 10 ( 11 ) 3110 - 3117 2018

© The Author(s) 2018. Most bacterial genomes display contrasting strand asymmetry in a variety of features, such as nucleotide composition and gene orientation, of the two replichores separated by the replication origin and terminus. The cause for the polarization is often attributed to mutations arising from the asymmetric replication machinery. Notably, a base compositional bias known as a GC skew is focused on as a footprint of the bacterial genome evolution driven by DNA replication. Previously, although a replication driven mutation pattern responsible for the GC skew formation or the related mathematical models have been well reported, an exact impact of the replication-related elements on the genomic structure is yet actively debated, and not confirmed experimentally. However, the GC skew formation is very time consuming and challenging in the laboratory. We, therefore, used cytosine deaminase as a DNA mutator, and by monitoring the mutations during an accelerated laboratory evolution procedure with Illumina sequencing, we enabled the trial and error of the GC skew formation in high resolution. Using this technology, we succeeded in reconfirming the influence of bacterial replication machinery on the genomic structure at high resolution.

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A metabologenomic approach reveals changes in the intestinal environment of mice fed on american diet

Ishii C., Nakanishi Y., Murakami S., Nozu R., Ueno M., Hioki K., Aw W., Hirayama A., Soga T., Ito M., Tomita M., Fukuda S.

International Journal of Molecular Sciences (International Journal of Molecular Sciences) 19 ( 12 ) 2018

ISSN 16616596

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. Intestinal microbiota and their metabolites are strongly associated with host physiology. Developments in DNA sequencing and mass spectrometry technologies have allowed us to obtain additional data that enhance our understanding of the interactions among microbiota, metabolites, and the host. However, the strategies used to analyze these datasets are not yet well developed. Here, we describe an original analytical strategy, metabologenomics, consisting of an integrated analysis of mass spectrometry-based metabolome data and high-throughput-sequencing-based microbiome data. Using this approach, we compared data obtained from C57BL/6J mice fed an American diet (AD), which contained higher amounts of fat and fiber, to those from mice fed control rodent diet. The feces of the AD mice contained higher amounts of butyrate and propionate, and higher relative abundances of Oscillospira and Ruminococcus. The amount of butyrate positively correlated with the abundance of these bacterial genera. Furthermore, integrated analysis of the metabolome data and the predicted metagenomic data from Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) indicated that the abundance of genes associated with butyrate metabolism positively correlated with butyrate amounts. Thus, our metabologenomic approach is expected to provide new insights and understanding of intestinal metabolic dynamics in complex microbial ecosystems.

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The first high frequency of recombination-like conjugal transfer from an integrated origin of transfer sequence in Bacillus subtilis 168

Itaya M., Hasegawa M., Tomita M., Sato M.

Bioscience, Biotechnology and Biochemistry (Bioscience, Biotechnology and Biochemistry) 82 ( 2 ) 356 - 362 2018

ISSN 09168451

© 2018 Japan society for Bioscience, Biotechnology, and agrochemistry. Bacillus subtilis 168 was developed as a genome vector to manipulate large DNA fragments. The system is based on the inherent natural transformation (TF) activity. However, DNA size transferred by TF is limited up to approximately 100 kb. A conjugal transfer system capable of transferring DNA fragments considerably larger than those transferred by TF was developed. A well-defined oriT110 sequence and a cognate relaxase gene from the pUB110 plasmid were inserted into the xkdE gene of the B. subtilis genome. Transfer of antibiotic resistance markers distant from the oriT110 locus to the recipient B. subtilis occurred only in the presence of pLS20, a helper plasmid that provides a type IV secretion system. Marker transmission was consistent with the orientation of oriT110 and required a recA-proficient recipient. The first conjugal transfer system of genomic DNA should provide a valuable alternative genetic tool for editing the B. subtilis genome.

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Morphology and Phylogeny of a Japanese Strain of Chlamydomonas augustae (Volvocales, Chlorophyceae)

Takahashi S, Tomita M, Nakada T.

J. Jpn. Bot 2018

Changes of liver metabolites following hepatectomy with ischemia reperfusion towards liver regeneration

Saito Y, Morine Y, Iwahashi S, Ikemoto T, Imura S, Yamanaka-Okumura H, Hirayama A, Soga T, Tomita M, Shimada M.

Ann Gastroenterol Surg 2 ( 3 ) 204 - 211 2018

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Genomic Analysis of Rhodococcus sp. Br-6, a Bromate Reducing Bacterium Isolated From Soil in Chiba, Japan

Ito K, Harada M, Nakajima N, Yamamura S, Tomita M, Suzuki H, Amachi S.

J Genomics 6 122 - 126 2018

ISSN 1839-9940

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Comparative genomic analysis of Clostridium difficile ribotype 027 strains including the newly sequenced strain NCKUH-21 isolated from a patient in Taiwan

Suzuki H, Tomita M, Tsai PJ, Ko WC, Hung YP, Huang IH, Chen JW.

Gut Pathogens (Gut Pathogens) 9 ( 1 ) 70 2017.10

ISSN 1757-4749

Background: Clostridium difficile is a Gram-positive anaerobe and the leading cause of antibiotic-associated diarrhea worldwide. The emergence of ribotype 027 (RT027) strains is associated with increased incidence of infection and mortality. To further understand the relationship between C. difficile NCKUH-21, a RT027 strain isolated from a patient in Taiwan, and other RT027 strains, we performed whole-genome shotgun sequencing on NCKUH-21 and comparative genomic analyses. Results: The genome size, G+C content, and gene number for the NCKUH-21 strain were determined to be similar to those for other C. difficile strains. The core genome phylogeny indicated that the five RT027 strains R20291, CD196, NCKUH-21, BI1, and 2007855 formed a clade. A pathogenicity locus, tcdR-tcdB-tcdE-orf-tcdA-tcdC, was conserved in the genome. A genomic region highly similar to the Clostridium phage $$\upvarphi$$ φ CD38-2 was present in the NCKUH-21 strain but absent in the other RT027 strains and designated as the prophage $$\upvarphi$$ φ NCKUH-21. The prophage $$\upvarphi$$ φ NCKUH-21 genes were significantly higher in G+C content than the other genes in the NCKUH-21 genome, indicating that the prophage does not match the base composition of the host genome. Conclusions: This is the first whole-genome analysis of a RT027 C. difficile strain isolated from Taiwan. Due to the high identity with $$\upvarphi$$ φ CD38-2, the prophage identified in the NCKUH-21 genome has the potential to regulate toxin production. These results provide important information for understanding the pathogenicity of RT027 C. difficile in Taiwan.

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eRP arrangement: a strategy for assembled genomic contig rearrangement based on replication profiling in bacteria

Kono N, Tomita M, Arakawa K.

BMC Genomics (BMC Genomics) 18 ( 1 ) 784 2017.09

Background: The reduced cost of sequencing has made de novo sequencing and the assembly of draft microbial genomes feasible in any ordinary biology lab. However, the process of finishing and completing the genome remains labor-intensive and computationally challenging in some cases, such as in the study of complete genome sequences, genomic rearrangements, long-range syntenic relationships, and structural variations. Methods: Here, we show a contig reordering strategy based on experimental replication profiling (eRP) to recapitulate the bacterial genome structure within draft genomes. During the exponential growth phase, the majority of bacteria show a global genomic copy number gradient that is enriched near the replication origin and gradually declines toward the terminus. Therefore, if genome sequencing is performed with appropriate timing, the short-read coverage reflects this copy number gradient, providing information about the contig positions relative to the replication origin and terminus. Results: We therefore investigated the appropriate timing for genomic DNA sampling and developed an algorithm for the reordering of the contigs based on eRP. As a result, this strategy successfully recapitulates the genomic structure of various structural mutants with draft genome sequencing. Conclusions: Our strategy was successful for contig rearrangement with intracellular DNA replication behavior mechanisms and can be applied to almost all bacteria because the DNA replication system is highly conserved. Therefore, eRP makes it possible to understand genomic structural information and long-range syntenic relationships using a draft genome that is based on short reads.

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Global metabolic reprogramming of colorectal cancer occurs at adenoma stage and is induced by MYC

Satoh K, Yachida S, Sugimoto M, Oshima M, Nakagawa T, Akamoto S, Tabata S, Saitoh K, Kato K, Sato S, Igarashi K, Aizawa Y, Kajino-Sakamoto R, Kojima Y, Fujishita T, Enomoto A, Hirayama A, Ishikawa T, Taketo MM, Kushida Y, Haba R, Okano K, Tomita M, Suzuki Y, Fukuda S, Aoki M, Soga T.

Proc Natl Acad Sci U S A. (Proceedings of the National Academy of Sciences of the United States of America) 114 ( 37 ) E7697-E7706 - E7706 2017.09

ISSN 00278424

Cancer cells alter their metabolism for the production of precursors of macromolecules. However, the control mechanisms underlying this reprogramming are poorly understood. Here we show that metabolic reprogramming of colorectal cancer is caused chiefly by aberrant MYC expression. Multiomics-based analyses of paired normal and tumor tissues from 275 patients with colorectal cancer revealed that metabolic alterations occur at the adenoma stage of carcinogenesis, in a manner not associated with specific gene mutations involved in colorectal carcinogenesis. MYC expression induced at least 215 metabolic reactions by changing the expression levels of 121 metabolic genes and 39 transporter genes. Further, MYC negatively regulated the expression of genes involved in mitochondrial biogenesis and maintenance but positively regulated genes involved in DNA and histone methylation. Knockdown of MYC in colorectal cancer cells reset the altered metabolism and suppressed cell growth. Moreover, inhibition of MYC target pyrimidine synthesis genes such as CAD, UMPS, and CTPS blocked cell growth, and thus are potential targets for colorectal cancer therapy.

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An archaeal RNA binding protein, FAU-1, is a novel ribonuclease related to rRNA stability in Pyrococcus and Thermococcus

Ikeda Y, Okada Y, Sato A, Kanai T, Tomita M, Atomi H, Kanai A.

Scientific Reports (Scientific Reports) 7 ( 1 ) 12674 2017.09

Ribosome biogenesis and turnover are processes necessary for cell viability and proliferation, and many kinds of proteins are known to regulate these processes. However, many questions still remain, especially in the Archaea. Generally, several ribonucleases are required to process precursor rRNAs to their mature forms, and to degrade rRNAs for quality control. Here, we found that FAU-1, which is known to be an RNA binding protein, possesses an RNase activity against precursor 5S rRNA derived from P. furiosus and T. kodakarensis in the order Thermococcales in vitro. An in vitro analysis revealed that UA sequences in the upstream of 5S rRNA were preferentially degraded by addition of FAU-1. Moreover, a fau-1 gene deletion mutant of T. kodakarensis showed a delay of exponential phase, reduction of maximum cell number and significant changes in the nucleotide sequence lengths of its 5S, 16S, and 23S rRNAs in early exponential phase. Our results suggest that FAU-1 is a potential RNase involved in rRNA stability through maturation and/or degradation processes.

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Effect of masticatory stimulation on the quantity and quality of saliva and the salivary metabolomic profile

Okuma N, Saita M, Hoshi N, Soga T, Tomita M, Sugimoto M, Kimoto K.

PLoS ONE (PLoS ONE) 12 ( 8 ) e0183109 2017.08

Background: This study characterized the changes in quality and quantity of saliva, and changes in the salivary metabolomic profile, to understand the effects of masticatory stimulation. Methods: Stimulated and unstimulated saliva samples were collected from 55 subjects and salivary hydrophilic metabolites were comprehensively quantified using capillary electrophoresis-time-of-flight mass spectrometry. Results: In total, 137 metabolites were identified and quantified. The concentrations of 44 metabolites in stimulated saliva were significantly higher than those in unstimulated saliva. Pathway analysis identified the upregulation of the urea cycle and synthesis and degradation pathways of glycine, serine, cysteine and threonine in stimulated saliva. A principal component analysis revealed that the effect of masticatory stimulation on salivary metabolomic profiles was less dependent on sample population sex, age, and smoking. The concentrations of only 1 metabolite in unstimulated saliva, and of 3 metabolites stimulated saliva, showed significant correlation with salivary secretion volume, indicating that the salivary metabolomic profile and salivary secretion volume were independent factors. Conclusions: Masticatory stimulation affected not only salivary secretion volume, but also metabolite concentration patterns. A low correlation between the secretion volume and these patterns supports the conclusion that the salivary metabolomic profile may be a new indicator to characterize masticatory stimulation.

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Comparative genomics of the tardigrades Hypsibius dujardini and Ramazzottius varieornatus

Yoshida Y, Koutsovoulos G, Laetsch DR, Stevens L, Kumar S, Horikawa DD, Ishino K, Komine S, Kunieda T, Tomita M, Blaxter M, Arakawa K.

PLoS Biology (PLoS Biology) 15 ( 7 ) e2002266 2017.07

ISSN 15449173

Tardigrada, a phylum of meiofaunal organisms, have been at the center of discussions of the evolution of Metazoa, the biology of survival in extreme environments, and the role of horizontal gene transfer in animal evolution. Tardigrada are placed as sisters to Arthropoda and Onychophora (velvet worms) in the superphylum Panarthropoda by morphological analyses, but many molecular phylogenies fail to recover this relationship. This tension between molecular and morphological understanding may be very revealing of the mode and patterns of evolution of major groups. Limnoterrestrial tardigrades display extreme cryptobiotic abilities, including anhydrobiosis and cryobiosis, as do bdelloid rotifers, nematodes, and other animals of the water film. These extremophile behaviors challenge understanding of normal, aqueous physiology: how does a multicellular organism avoid lethal cellular collapse in the absence of liquid water? Meiofaunal species have been reported to have elevated levels of horizontal gene transfer (HGT) events, but how important this is in evolution, and particularly in the evolution of extremophile physiology, is unclear. To address these questions, we resequenced and reassembled the genome of H. dujardini, a limnoterrestrial tardigrade that can undergo anhydrobiosis only after extensive pre-exposure to drying conditions, and compared it to the genome of R. varieornatus, a related species with tolerance to rapid desiccation. The 2 species had contrasting gene expression responses to anhydrobiosis, with major transcriptional change in H. dujardini but limited regulation in R. varieornatus. We identified few horizontally transferred genes, but some of these were shown to be involved in entry into anhydrobiosis. Whole-genome molecular phylogenies supported a Tardigrada+Nematoda relationship over Tardigrada+Arthropoda, but rare genomic changes tended to support Tardigrada+Arthropoda.

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Synthetic Biomarker Design by Using Analyte-Responsive Acetaminophen

Nishihara T, Inoue J, Tabata S, Murakami S, Ishikawa T, Saito N, Fukuda S, Tomita M, Soga T.

ChemBioChem (ChemBioChem) 18 ( 10 ) 910-913 - 913 2017.05

ISSN 14394227

The use of synthetic biomarkers is an emerging technique to improve disease diagnosis. Here, we report a novel design strategy that uses analyte-responsive acetaminophen (APAP) to expand the catalogue of analytes available for synthetic biomarker development. As proof-of-concept, we designed hydrogen peroxide (H2O2)-responsive APAP (HR-APAP) and succeeded in H2O2 detection with cellular and animal experiments. In fact, for blood samples following HR-APAP injection, we demonstrated that the plasma concentration ratio [APAP+APAP conjugates]/[HR-APAP] accurately reflects in vivo differences in H2O2 levels. We anticipate that our practical methodology will be broadly useful for the preparation of various synthetic biomarkers.

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Draft Genome Sequence of Roseovarius sp. A-2, an Iodide-Oxidizing Bacterium Isolated from Natural Gas

Yuliana T, Nakajima N, Yamamura S, Tomita M, Suzuki H, Amachi S.

J Genomics. 6 ( 5 ) 51-53 2017.04

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Effect of timing of collection of salivary metabolomic biomarkers on oral cancer detection

Ishikawa S., Sugimoto M., Kitabatake K., Tu M., Sugano A., Yamamori I., Iba A., Yusa K., Kaneko M., Ota S., Hiwatari K., Enomoto A., Masaru T., Iino M.

Amino Acids 49 ( 4 ) 761 - 770 2017.04

ISSN 09394451

The aim of this study is to evaluate the effect of duration after meals for saliva collections for oral cancer detection using metabolomics. Saliva samples were collected from oral cancer patients (n = 22) and controls (n = 44). Saliva from cancer patients was collected 12 h after dinner, and 1.5 and 3.5 h after breakfast. Control subjects fasted >1.5 h prior to saliva collection. Hydrophilic metabolites were analyzed using capillary electrophoresis mass spectrometry. Levels of 51 metabolites differed significantly in controls vs. oral cancer patients at the 12-h fasting time point (P < 0.05). Fifteen and ten metabolites differed significantly at the 1.5- and 3.5-h time points, respectively. The area of under receiver operating characteristic curve for discriminating oral cancer patients from controls was greatest at the 12-h fasting time point. The collection time after meals affects levels of salivary metabolites for oral cancer screening. The 12-h fasting after dinner time point is optimal. This study contributes to design of saliva collection protocols for metabolomics-based biomarker discovery.

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Robotic crowd biology with Maholo LabDroids

Yachie, N., Robotic Biology Consortium and Natsume, T. < Collabolators> Takahashi, K., Katayama, T., Sakurada, T., Kanda, G. N., Takagi, E., Hirose, T., Katsura, T., Moriya, T., Kitano, H., Tsujii, J., Shiraki, T., Kariyazaki, H., Kamei, M., Abe, N., Fukuda, T., Sawada, Y., Hashiguchi, Y., Matsukuma, K., Murai, S., Sasaki, N., Ipposhi, T., Urabe, H., Kudo, T., Umeno, M., Ono, S., Miyauchi, K., Nakamura, M., Kizaki, T., Suyama, T., Hatta, T., Natsume, T., Ohta, T., Ozawa, Y., Ihara, S., Tamaki, S., Antezana, E., Garcia-Castro, A., Perret, J., L., Ishiguro, S., Mori, H., Evans-Yamamoto, D., Masuyama N, Tomita M, Katayama T, Matsumoto M, Nakayama H, Shirasawa A, Shimbo, K., Yamada, N., Nakayama, K., I., Shimizu, T., Saya, H., Yamashita, S., Matsushima, T., Asahara, H., Eguchi, H., Mikamori, M. and Mori, M.

Nat Biotechnol 35 ( 4 ) 310-312 2017.04

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Serum Metabolomic Profiles for Human Pancreatic Cancer Discrimination

Itoi T, Sugimoto M, Umeda J, Sofuni A, Tsuchiya T, Tsuji S, Tanaka R, Tonozuka R, Honjo M, Moriyasu F, Kasuya K, Nagakawa Y, Abe Y, Takano K, Kawachi S, Shimazu M, Soga T, Tomita M, Sunamura M.

International Journal of Molecular Sciences (International Journal of Molecular Sciences) 18 ( 4 ) 767 2017.04

ISSN 16616596

This study evaluated the clinical use of serum metabolomics to discriminate malignant cancers including pancreatic cancer (PC) from malignant diseases, such as biliary tract cancer (BTC), intraductal papillary mucinous carcinoma (IPMC), and various benign pancreaticobiliary diseases. Capillary electrophoresis–mass spectrometry was used to analyze charged metabolites. We repeatedly analyzed serum samples (n = 41) of different storage durations to identify metabolites showing high quantitative reproducibility, and subsequently analyzed all samples (n = 140). Overall, 189 metabolites were quantified and 66 metabolites had a 20% coefficient of variation and, of these, 24 metabolites showed significant differences among control, benign, and malignant groups (p < 0.05; Steel–Dwass test). Four multiple logistic regression models (MLR) were developed and one MLR model clearly discriminated all disease patients from healthy controls with an area under receiver operating characteristic curve (AUC) of 0.970 (95% confidential interval (CI), 0.946–0.994, p < 0.0001). Another model to discriminate PC from BTC and IPMC yielded AUC = 0.831 (95% CI, 0.650–1.01, p = 0.0020) with higher accuracy compared with tumor markers including carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), pancreatic cancer-associated antigen (DUPAN2) and s-pancreas-1 antigen (SPAN1). Changes in metabolomic profiles might be used to screen for malignant cancers as well as to differentiate between PC and other malignant diseases.

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Eggshell membrane powder ameliorates intestinal inflammation by facilitating the restitution of epithelial injury and alleviating microbial dysbiosis

Jia H, Hanate M, Aw W, Itoh H, Saito K, Kobayashi S, Hachimura S, Fukuda S, Tomita M, Hasebe Y, Kato H.

Scientific Reports (Scientific Reports) 7 43993 2017.03

Gut microbiota is an essential factor in the shaping of intestinal immune system development and driving inflammation in inflammatory bowel disease (IBD). We report the effects and microbe-host interactions underlying an intervention using fine powder of eggshell membrane (ESM) against IBD. ESM attenuated lipopolysaccharide-induced inflammatory cytokine production and promoted the Caco-2 cell proliferation by up-regulating growth factors in vitro. In a murine model of dextran sodium sulphate-induced colitis, ESM significantly suppressed the disease activity index and colon shortening. These effects were associated with significant ameliorations of gene expressions of inflammatory mediators, intestinal epithelial cell proliferation, restitution-related factors and antimicrobial peptides. Multifaceted integrated omics analyses revealed improved levels of energy metabolism-related genes, proteins and metabolites. Concomitantly, cecal metagenomic information established an essential role of ESM in improving dysbiosis characterized by increasing the diversity of bacteria and decreasing absolute numbers of pathogenic bacteria such as Enterobacteriaceae and E. coli, as well as in the regulation of the expansion of Th17 cells by suppressing the overgrowth of segmented filamentous bacteria. Such modulations have functional effects on the host; i.e., repairing the epithelium, regulating energy requirements and eventually alleviating mucosal inflammation. These findings are first insights into ESM's modulation of microbiota and IBD suppression, providing new perspectives on the prevention/treatment of IBD.

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