Details of a Researcher - Tomita, Masaru

Papers - Tomita, Masaru

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Metabolomics profiles alterations in cigarette smokers and heated tobacco product users.

Harada S, Ohmomo H, Matsumoto M, Sata M, Iida M, Hirata A, Miyagawa N, Kuwabara K, Kato S, Toki R, Edagawa S, Sugiyama D, Sato A, Hirayama A, Sugimoto M, Soga T, Tomita M, Shimizu A, Okamura T, Takebayashi T

Journal of epidemiology 2023.11

ISSN 0917-5040

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Systematic Analysis of Diverse Polynucleotide Kinase Clp1 Family Proteins in Eukaryotes: Three Unique Clp1 Proteins of Trypanosoma brucei

Saito M., Inose R., Sato A., Tomita M., Suzuki H., Kanai A.

Journal of Molecular Evolution (Journal of Molecular Evolution) 91 ( 5 ) 669 - 686 2023.10

ISSN 00222844

The Clp1 family proteins, consisting of the Clp1 and Nol9/Grc3 groups, have polynucleotide kinase (PNK) activity at the 5′ end of RNA strands and are important enzymes in the processing of some precursor RNAs. However, it remains unclear how this enzyme family diversified in the eukaryotes. We performed a large-scale molecular evolutionary analysis of the full-length genomes of 358 eukaryotic species to classify the diverse Clp1 family proteins. The average number of Clp1 family proteins in eukaryotes was 2.3 ± 1.0, and most representative species had both Clp1 and Nol9/Grc3 proteins, suggesting that the Clp1 and Nol9/Grc3 groups were already formed in the eukaryotic ancestor by gene duplication. We also detected an average of 4.1 ± 0.4 Clp1 family proteins in members of the protist phylum Euglenozoa. For example, in Trypanosoma brucei, there are three genes of the Clp1 group and one gene of the Nol9/Grc3 group. In the Clp1 group proteins encoded by these three genes, the C-terminal domains have been replaced by unique characteristics domains, so we designated these proteins Tb-Clp1-t1, Tb-Clp1-t2, and Tb-Clp1-t3. Experimental validation showed that only Tb-Clp1-t2 has PNK activity against RNA strands. As in this example, N-terminal and C-terminal domain replacement also contributed to the diversification of the Clp1 family proteins in other eukaryotic species. Our analysis also revealed that the Clp1 family proteins in humans and plants diversified through isoforms created by alternative splicing.

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Temporal phosphoproteomic analysis of VEGF-A signaling in HUVECs: an insight into early signaling events associated with angiogenesis

Abhinand C.S., Galipon J., Mori M., Ramesh P., Prasad T.S.K., Raju R., Sudhakaran P.R., Tomita M.

Journal of Cell Communication and Signaling (Journal of Cell Communication and Signaling) 17 ( 3 ) 1067 - 1079 2023.09

ISSN 18739601

Vascular endothelial growth factor-A (VEGF-A) is one of the primary factors promoting angiogenesis in endothelial cells. Although defects in VEGF-A signaling are linked to diverse pathophysiological conditions, the early phosphorylation-dependent signaling events pertinent to VEGF-A signaling remain poorly defined. Hence, a temporal quantitative phosphoproteomic analysis was performed in human umbilical vein endothelial cells (HUVECs) treated with VEGF-A-165 for 1, 5 and 10 min. This led to the identification and quantification of 1971 unique phosphopeptides corresponding to 961 phosphoproteins and 2771 phosphorylation sites in total. Specifically, 69, 153, and 133 phosphopeptides corresponding to 62, 125, and 110 phosphoproteins respectively, were temporally phosphorylated at 1, 5, and 10 min upon addition of VEGF-A. These phosphopeptides included 14 kinases, among others. This study also captured the phosphosignaling events directed through RAC, FAK, PI3K-AKT-MTOR, ERK, and P38 MAPK modules with reference to our previously assembled VEGF-A/VEGFR2 signaling pathway map in HUVECs. Apart from a significant enrichment of biological processes such as cytoskeleton organization and actin filament binding, our results also suggest a role of AAK1-AP2M1 in the regulation of VEGFR endocytosis. Taken together, the temporal quantitative phosphoproteomics analysis of VEGF signaling in HUVECs revealed early signaling events and we believe that this analysis will serve as a starting point for the analysis of differential signaling across VEGF members toward the full elucidation of their role in the angiogenesis processes. Graphical abstract: [Figure not available: see fulltext.]

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Quality Control of Targeted Plasma Lipids in a Large-Scale Cohort Study Using Liquid Chromatography–Tandem Mass Spectrometry

Hirayama A., Ishikawa T., Takahashi H., Yamanaka S., Ikeda S., Hirata A., Harada S., Sugimoto M., Soga T., Tomita M., Takebayashi T.

Metabolites (Metabolites) 13 ( 4 ) 2023.04

ISSN 2218-1989

High-throughput metabolomics has enabled the development of large-scale cohort studies. Long-term studies require multiple batch-based measurements, which require sophisticated quality control (QC) to eliminate unexpected bias to obtain biologically meaningful quantified metabolomic profiles. Liquid chromatography–mass spectrometry was used to analyze 10,833 samples in 279 batch measurements. The quantified profile included 147 lipids including acylcarnitine, fatty acids, glucosylceramide, lactosylceramide, lysophosphatidic acid, and progesterone. Each batch included 40 samples, and 5 QC samples were measured for 10 samples of each. The quantified data from the QC samples were used to normalize the quantified profiles of the sample data. The intra- and inter-batch median coefficients of variation (CV) among the 147 lipids were 44.3% and 20.8%, respectively. After normalization, the CV values decreased by 42.0% and 14.7%, respectively. The effect of this normalization on the subsequent analyses was also evaluated. The demonstrated analyses will contribute to obtaining unbiased, quantified data for large-scale metabolomics.

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iDMET: network-based approach for integrating differential analysis of cancer metabolomics

Matsuta R., Yamamoto H., Tomita M., Saito R.

BMC Bioinformatics (BMC Bioinformatics) 23 ( 1 ) 508 2022.12

Background: Comprehensive metabolomic analyses have been conducted in various institutes and a large amount of metabolomic data are now publicly available. To help fully exploit such data and facilitate their interpretation, metabolomic data obtained from different facilities and different samples should be integrated and compared. However, large-scale integration of such data for biological discovery is challenging given that they are obtained from various types of sample at different facilities and by different measurement techniques, and the target metabolites and sensitivities to detect them also differ from study to study. Results: We developed iDMET, a network-based approach to integrate metabolomic data from different studies based on the differential metabolomic profiles between two groups, instead of the metabolite profiles themselves. As an application, we collected cancer metabolomic data from 27 previously published studies and integrated them using iDMET. A pair of metabolomic changes observed in the same disease from two studies were successfully connected in the network, and a new association between two drugs that may have similar effects on the metabolic reactions was discovered. Conclusions: We believe that iDMET is an efficient tool for integrating heterogeneous metabolomic data and discovering novel relationships between biological phenomena.

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Time-series transcriptomic screening of factors contributing to the cross-tolerance to UV radiation and anhydrobiosis in tardigrades

Yoshida Y., Satoh T., Ota C., Tanaka S., Horikawa D.D., Tomita M., Kato K., Arakawa K.

BMC Genomics (BMC Genomics) 23 ( 1 ) 405 2022.12

Background: Tardigrades are microscopic animals that are capable of tolerating extreme environments by entering a desiccated state of suspended animation known as anhydrobiosis. While antioxidative stress proteins, antiapoptotic pathways and tardigrade-specific intrinsically disordered proteins have been implicated in the anhydrobiotic machinery, conservation of these mechanisms is not universal within the phylum Tardigrada, suggesting the existence of overlooked components. Results: Here, we show that a novel Mn-dependent peroxidase is an important factor in tardigrade anhydrobiosis. Through time-series transcriptome analysis of Ramazzottius varieornatus specimens exposed to ultraviolet light and comparison with anhydrobiosis entry, we first identified several novel gene families without similarity to existing sequences that are induced rapidly after stress exposure. Among these, a single gene family with multiple orthologs that is highly conserved within the phylum Tardigrada and enhances oxidative stress tolerance when expressed in human cells was identified. Crystallographic study of this protein suggested Zn or Mn binding at the active site, and we further confirmed that this protein has Mn-dependent peroxidase activity in vitro. Conclusions: Our results demonstrated novel mechanisms for coping with oxidative stress that may be a fundamental mechanism of anhydrobiosis in tardigrades. Furthermore, localization of these sets of proteins mainly in the Golgi apparatus suggests an indispensable role of the Golgi stress response in desiccation tolerance.

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Identification of Attenuators of Transcriptional Termination: Implications for RNA Regulation in Escherichia coli

Morita T., Majdalani N., Miura M.C., Inose R., Oshima T., Tomita M., Kanai A., Gottesman S.

mBio (mBio) 13 ( 6 ) 2022.12

ISSN 21612129

The regulatory function of many bacterial small RNAs (sRNAs) requires the binding of the RNA chaperone Hfq to the 39 portion of the sRNA intrinsic terminator, and therefore sRNA signaling might be regulated by modulating its terminator. Here, using a multicopy screen developed with the terminator of sRNA SgrS, we identified an sRNA gene (cyaR) and three protein-coding genes (cspD, ygjH, and rof) that attenuate SgrS termination in Escherichia coli. Analyses of CyaR and YgjH, a putative tRNA binding protein, suggested that the CyaR activity was indirect and the effect of YgjH was moderate. Overproduction of the protein attenuators CspD and Rof resulted in more frequent readthrough at terminators of SgrS and two other sRNAs, and regulation by SgrS of target mRNAs was reduced. The effect of Rof, a known inhibitor of Rho, was mimicked by bicyclomycin or by a rho mutant, suggesting an unexpected role for Rho in sRNA termination. CspD, a member of the cold shock protein family, bound both terminated and readthrough transcripts, stabilizing them and attenuating termination. By RNA sequencing analysis of the CspD overexpression strain, we found global effects of CspD on gene expression across some termination sites. We further demonstrated effects of endogenous CspD under slow growth conditions where cspD is highly expressed. These findings provided evidence of changes in the efficiency of intrinsic termination, confirming this as an additional layer of the regulation of sRNA signaling. IMPORTANCE Growing evidence suggests that the modulation of intrinsic termination and readthrough of transcription is more widespread than previously appreciated. For small RNAs, proper termination plays a critical role in their regulatory function. Here, we present a multicopy screen approach to identify factors that attenuate small RNA termination and therefore abrogate signaling dependent on the small RNA. This study highlights a new aspect of regulation of small RNA signaling as well as the modulation of intrinsic termination.

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1000 spider silkomes: Linking sequences to silk physical properties

Arakawa K., Kono N., Malay A.D., Tateishi A., Ifuku N., Masunaga H., Sato R., Tsuchiya K., Ohtoshi R., Pedrazzoli D., Shinohara A., Ito Y., Nakamura H., Tanikawa A., Suzuki Y., Ichikawa T., Fujita S., Fujiwara M., Tomita M., Blamires S.J., Chuah J.A., Craig H., Foong C.P., Greco G., Guan J., Holland C., Kaplan D.L., Sudesh K., Mandal B.B., Norma-Rashid Y., Oktaviani N.A., Preda R.C., Pugno N.M., Rajkhowa R., Wang X., Yazawa K., Zheng Z., Numata K.

Science Advances (Science Advances) 8 ( 41 ) eabo6043 2022.10

Spider silks are among the toughest known materials and thus provide models for renewable, biodegradable, and sustainable biopolymers. However, the entirety of their diversity still remains elusive, and silks that exceed the performance limits of industrial fibers are constantly being found. We obtained transcriptome assemblies from 1098 species of spiders to comprehensively catalog silk gene sequences and measured the mechanical, thermal, structural, and hydration properties of the dragline silks of 446 species. The combination of these silk protein genotype-phenotype data revealed essential contributions of multicomponent structures with major ampullate spidroin 1 to 3 paralogs in high-performance dragline silks and numerous amino acid motifs contributing to each of the measured properties. We hope that our global sampling, comprehensive testing, integrated analysis, and open data will provide a solid starting point for future biomaterial designs.

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Salivary metabolomics with machine learning for colorectal cancer detection

Kuwabara H., Katsumata K., Iwabuchi A., Udo R., Tago T., Kasahara K., Mazaki J., Enomoto M., Ishizaki T., Soya R., Kaneko M., Ota S., Enomoto A., Soga T., Tomita M., Sunamura M., Tsuchida A., Sugimoto M., Nagakawa Y.

Cancer Science (Cancer Science) 113 ( 9 ) 3234 - 3243 2022.09

ISSN 13479032

As the worldwide prevalence of colorectal cancer (CRC) increases, it is vital to reduce its morbidity and mortality through early detection. Saliva-based tests are an ideal noninvasive tool for CRC detection. Here, we explored and validated salivary biomarkers to distinguish patients with CRC from those with adenoma (AD) and healthy controls (HC). Saliva samples were collected from patients with CRC, AD, and HC. Untargeted salivary hydrophilic metabolite profiling was conducted using capillary electrophoresis–mass spectrometry and liquid chromatography–mass spectrometry. An alternative decision tree (ADTree)-based machine learning (ML) method was used to assess the discrimination abilities of the quantified metabolites. A total of 2602 unstimulated saliva samples were collected from subjects with CRC (n = 235), AD (n = 50), and HC (n = 2317). Data were randomly divided into training (n = 1301) and validation datasets (n = 1301). The clustering analysis showed a clear consistency of aberrant metabolites between the two groups. The ADTree model was optimized through cross-validation (CV) using the training dataset, and the developed model was validated using the validation dataset. The model discriminating CRC + AD from HC showed area under the receiver-operating characteristic curves (AUC) of 0.860 (95% confidence interval [CI]: 0.828-0.891) for CV and 0.870 (95% CI: 0.837-0.903) for the validation dataset. The other model discriminating CRC from AD + HC showed an AUC of 0.879 (95% CI: 0.851-0.907) and 0.870 (95% CI: 0.838-0.902), respectively. Salivary metabolomics combined with ML demonstrated high accuracy and versatility in detecting CRC.

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Features of smaller ribosomes in candidate phyla radiation (CPR) bacteria revealed with a molecular evolutionary analysis

Tsurumaki M., Saito M., Tomita M., Kanai A.

RNA (RNA) 28 ( 8 ) 1041 - 1057 2022.08

ISSN 13558382

The candidate phyla radiation (CPR) is a large bacterial group consisting mainly of uncultured lineages. They have small cells and small genomes, and they often lack ribosomal proteins uL1, bL9, and/or uL30, which are basically ubiquitous in non-CPR bacteria. Here, we comprehensively analyzed the genomic information on CPR bacteria and identified their unique properties. The distribution of protein lengths in CPR bacteria peaks at around 100–150 amino acids, whereas the position of the peak varies in the range of 100–300 amino acids in free-living non-CPR bacteria, and at around 100–200 amino acids in most symbiotic non-CPR bacteria. These results show that the proteins of CPR bacteria are smaller, on average, than those of free-living non-CPR bacteria, like those of symbiotic non-CPR bacteria. We found that ribosomal proteins bL28, uL29, bL32, and bL33 have been lost in CPR bacteria in a taxonomic lineage-specific manner. Moreover, the sequences of approximately half of all ribosomal proteins of CPR differ, in part, from those of non-CPR bacteria, with missing regions or specifically added regions. We also found that several regions in the 16S, 23S, and 5S rRNAs of CPR bacteria are lacking, which presumably caused the total predicted lengths of the three rRNAs of CPR bacteria to be smaller than those of non-CPR bacteria. The regions missing in the CPR ribosomal proteins and rRNAs are located near the surface of the ribosome, and some are close to one another. These observations suggest that ribosomes are smaller in CPR bacteria than those in free-living non-CPR bacteria, with simplified surface structures.

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Molecular Evidence of Chemical Disguise by the Socially Parasitic Spiny Ant Polyrhachis lamellidens (Hymenoptera: Formicidae) When Invading a Host Colony

Iwai H., Mori M., Tomita M., Kono N., Arakawa K.

Frontiers in Ecology and Evolution (Frontiers in Ecology and Evolution) 10 2022.06

While most ant species establish a colony independently, some socially parasitic ants build the foundation of their colony by invading other ant (host) colonies and utilizing their labor forces. Many socially parasitic ants disguise their cuticular hydrocarbon (CHC) profile, which is also known as signature mixture for nestmate discrimination, when invading the host colony. Since the strategy of chemical disguise is widespread in socially parasitic ants, elucidating the mechanism of chemical disguise will promote knowledge on the evolutionary history of social parasitism. However, detailed knowledge is still lacking, as the relevant information has only originated from circumstantial evidence, which was obtained from ecological observations. In this study, we investigated the mechanism of chemical disguise in a new queen of a temporary socially parasitic spiny ant (Polyrhachis lamellidens) by measuring its CHC profile, performing a tracing assay with labeled substances, and analyzing gene expression levels. First, after rubbing behavior was observed against the host workers, the CHC profile in P. lamellidens shifted to pronounced peaks that closely resembling that of the host workers. We also observed a reduction in aggressive behaviors by the host ant against P. lamellidens after rubbing behavior was performed. In addition, P. lamellidens acquired artificially-applied labeling substances from host workers through their rubbing behaviors, while gene expression profiling showed the expression of CHC synthesis-related genes did not change during this behavior. These results suggest that P. lamellidens directly obtains host CHCs through rubbing behavior, and these host CHCs enables P. lamellidens to remain disguised during colony invasion.

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Comparative Metabolomics of Small Molecules Specifically Expressed in the Dorsal or Ventral Marginal Zones in Vertebrate Gastrula

Suzuki Y., Hayasaka R., Hasebe M., Ikeda S., Soga T., Tomita M., Hirayama A., Kuroda H.

Metabolites (Metabolites) 12 ( 6 ) 2022.06

ISSN 2218-1989

Many previous studies have reported the various proteins specifically secreted as inducers in the dorsal or ventral regions in vertebrate gastrula. However, little is known about the effect on cell fate of small molecules below 1000 Da. We therefore tried to identify small molecules specifically expressed in the dorsal marginal zone (DMZ) or ventral marginal zone (VMZ) in vertebrate gastrula. Small intracellular and secreted molecules were detected using explants and supernatant samples. Hydrophilic metabolites were analyzed by capillary ion chromatography–mass spectrometry and liquid chromatography–mass spectrometry, and lipids were analyzed by supercritical fluid chromatography–tandem mass spectrometry. In total, 190 hydrophilic metabolites and 396 lipids were identified. The DMZ was found to have high amounts of glycolysis-and glutathione metabolismrelated metabolites in explants, and the VMZ was richer in purine metabolism-related metabolites. We also discovered some hydrophilic metabolites and lipids differentially contained in the DMZ or VMZ. Our research would contribute to a deeper understanding of the cellular physiology that regulates early embryogenesis.

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High quality genome assembly of the anhydrobiotic midge provides insights on a single chromosome-based emergence of extreme desiccation tolerance.

Yoshida Y, Shaikhutdinov N, Kozlova O, Itoh M, Tagami M, Murata M, Nishiyori-Sueki H, Kojima-Ishiyama M, Noma S, Cherkasov A, Gazizova G, Nasibullina A, Deviatiiarov R, Shagimardanova E, Ryabova A, Yamaguchi K, Bino T, Shigenobu S, Tokumoto S, Miyata Y, Cornette R, Yamada TG, Funahashi A, Tomita M, Gusev O, Kikawada T

NAR genomics and bioinformatics (NAR Genomics and Bioinformatics) 4 ( 2 ) lqac029 2022.06

Non-biting midges (Chironomidae) are known to inhabit a wide range of environments, and certain species can tolerate extreme conditions, where the rest of insects cannot survive. In particular, the sleeping chironomid Polypedilum vanderplanki is known for the remarkable ability of its larvae to withstand almost complete desiccation by entering a state called anhydrobiosis. Chromosome numbers in chironomids are higher than in other dipterans and this extra genomic resource might facilitate rapid adaptation to novel environments. We used improved sequencing strategies to assemble a chromosome-level genome sequence for P. vanderplanki for deep comparative analysis of genomic location of genes associated with desiccation tolerance. Using whole genome-based cross-species and intra-species analysis, we provide evidence for the unique functional specialization of Chromosome 4 through extensive acquisition of novel genes. In contrast to other insect genomes, in the sleeping chironomid a uniquely high degree of subfunctionalization in paralogous anhydrobiosis genes occurs in this chromosome, as well as pseudogenization in a highly duplicated gene family. Our findings suggest that the Chromosome 4 in Polypedilum is a site of high genetic turnover, allowing it to act as a 'sandbox' for evolutionary experiments, thus facilitating the rapid adaptation of midges to harsh environments.

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Polarity protein SCRIB interacts with SLC3A2 to regulate proliferation and tamoxifen resistance in ER+ breast cancer.

Saito Y, Matsuda S, Ohnishi N, Endo K, Ashitani S, Ohishi M, Ueno A, Tomita M, Ueda K, Soga T, Muthuswamy SK

Communications biology (Communications Biology) 5 ( 1 ) 403 2022.05

Estrogen receptor (ER) positive breast cancer represents 75% of all breast cancers in women. Although patients with ER+ cancers receive endocrine therapies, more than 30% develop resistance and succumb to the disease, highlighting the need to understand endocrine resistance. Here we show an unexpected role for the cell polarity protein SCRIB as a tumor-promoter and a regulator of endocrine resistance in ER-positive breast cancer cells. SCRIB expression is induced by estrogen signaling in a MYC-dependent manner. SCRIB interacts with SLC3A2, a heteromeric component of leucine amino acid transporter SLC7A5. SLC3A2 binds to the N-terminus of SCRIB to facilitate the formation of SCRIB/SLC3A2/LLGL2/SLC7A5 quaternary complex required for membrane localization of the amino acid transporter complex. Both SCRIB and SLC3A2 are required for cell proliferation and tamoxifen resistance in ER+ cells identifying a new role for the SCRIB/SLC3A2 complex in ER+ breast cancer.

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Cinnamon bark extract suppresses metastatic dissemination of cancer cells through inhibition of glycolytic metabolism.

Nakayama J, Konno Y, Maruyama A, Tomita M, Makinoshima H

Journal of natural medicines (Journal of Natural Medicines) 76 ( 3 ) 686 - 692 2022.04

ISSN 1340-3443

Metastasis is responsible for approximately 90% of cancer-associated mortality and proceeds through multiple steps. Several herbal medicines are reported to inhibit primary tumor growth, but the suppressor effects of the medicines on metastasis progression are still not fully elucidated. Here we report that cinnamon bark extract (CBE) has a suppressor effect on metastatic dissemination of cancer cells. Through a phenotypic screening using zebrafish embryos, CBE was identified to interfere with the gastrulation progression of zebrafish embryos, of which the molecular mechanisms are conserved in metastasis progression. A Boyden chamber assay showed that CBE decreased cell motility and invasion of MDA-MB-231 human breast cancer cells without affecting their cell viability. Furthermore, CBE suppressed metastatic dissemination of the cells in a zebrafish xenotransplantation model. Quantitative metabolome analyses revealed that the productions of glucose-6-phosphate (G6P) and fructose 6-phosphate which are intermediate metabolites of glycolytic metabolism were interrupted in CBE-treated cells. qPCR and western-blotting analyses revealed that CBE-treated cells showed decreased expression of hexokinase 2 (HK2) which yields G6P. Pharmacological inhibition of HK2 with 2-deoxy-D-glucose suppressed cell invasion and migration of the cells without affecting their cell viability. Taken together, CBE suppresses metastatic dissemination of cancer cells through inhibition of glycolysis metabolism.

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Initial parasitic behaviour of the temporary social parasitic ant Polyrhachis lamellidens can be induced by host-like cuticles in laboratory environment.

Kurihara Y, Iwai H, Kono N, Tomita M, Arakawa K

Biology open (Biology Open) 11 ( 3 ) 2022.02

Polyrhachis lamellidens is a temporary social parasitic species. When a newly mated queen encounters a host worker, it opens its jaws and then mounts and rubs the body of the host worker, called rubbing behaviour. This behaviour is different from aggressive behaviour and is considered to be a preparatory action before invasion of the host colony. However, it is unclear what cues trigger rubbing behaviour. Therefore, in this study, we used glass beads that imitated the insect body surfaces and searched for triggers. Although P. lamellidens did not respond to the cuticular compounds only, cuticular compounds and chitin coatings on glass beads elicited responses that were similar to those towards live samples. The rubbing behaviour of P. lamellidens was elicited in response to a cuticle-like surface that mimicked a procuticle by combining the compounds with chitin. These results suggest that host recognition and nest-mate recognition are supported by different mechanisms.

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Hao1 Is Not a Pathogenic Factor for Ectopic Ossifications but Functions to Regulate the TCA Cycle In Vivo.

Kimura A, Hirayama A, Matsumoto T, Sato Y, Kobayashi T, Ikeda S, Maruyama M, Kaneko M, Shigeta M, Ito E, Soma T, Miyamoto K, Soga T, Tomita M, Oya A, Matsumoto M, Nakamura M, Kanaji A, Miyamoto T

Metabolites (Metabolites) 12 ( 1 ) 2022.01

ISSN 2218-1989

Ossification of the posterior longitudinal ligament (OPLL), a disease characterized by the ectopic ossification of a spinal ligament, promotes neurological disorders associated with spinal canal stenosis. While blocking ectopic ossification is mandatory to prevent OPLL development and progression, the mechanisms underlying the condition remain unknown. Here we show that expression of hydroxyacid oxidase 1 (Hao1), a gene identified in a previous genome-wide association study (GWAS) as an OPLL-associated candidate gene, specifically and significantly decreased in fibroblasts during osteoblast differentiation. We then newly established Hao1-deficient mice by generating Hao1-flox mice and crossing them with CAG-Cre mice to yield global Hao1-knockout (CAG-Cre/Hao1flox/flox; Hao1 KO) animals. Hao1 KO mice were born normally and exhibited no obvious phenotypes, including growth retardation. Moreover, Hao1 KO mice did not exhibit ectopic ossification or calcification. However, urinary levels of some metabolites of the tricarboxylic acid (TCA) cycle were significantly lower in Hao1 KO compared to control mice based on comprehensive metabolomic analysis. Our data indicate that Hao1 loss does not promote ectopic ossification, but rather that Hao1 functions to regulate the TCA cycle in vivo.

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Metabolic profiling of charged metabolites in association with menopausal status in Japanese community-dwelling midlife women: Tsuruoka Metabolomic Cohort Study.

Watanabe K, Iida M, Harada S, Kato S, Kuwabara K, Kurihara A, Takeuchi A, Sugiyama D, Okamura T, Suzuki A, Amano K, Hirayama A, Sugimoto M, Soga T, Tomita M, Kobayashi Y, Banno K, Aoki D, Takebayashi T

Maturitas (Maturitas) 155 54 - 62 2022.01

ISSN 0378-5122

Background: Emerging evidence has shown that charged metabolites, such as amino acids, may play an important role in the pathogenesis of various metabolic disorders, many of which women in the postmenopausal period are at high risk of developing. This study examined the metabolic profile of middle-aged Japanese women to investigate alterations in charged metabolites induced by menopausal transition. Methods: The participants were 1193 female residents aged 40–60 at the baseline survey of the Tsuruoka Metabolomics Cohort Study. We investigated the cross-sectional association of menopausal status with 94 metabolomic biomarkers assayed in fasting plasma samples via capillary electrophoresis time-of-flight mass spectrometry using linear regression analysis. Results: Among the participants, 529 were premenopausal, 132 were in menopausal transition (MT), and 532 were postmenopausal. Significant differences were found in age, blood pressure, glucose and lipid levels, and smoking and drinking habits among the three groups. The concentrations of 5 metabolites in the MT group and 15 metabolites in the postmenopausal group were significantly higher than those in the premenopausal group after adjusting for confounding factors. When classified into pathways, these metabolites were related to the tricarboxylic cycle, urea cycle, and homocysteine metabolism, some of which are linked to arteriosclerosis. Conclusion: Multiple charged metabolites were associated with women's menopausal status, showing a gradual increase as women shifted from pre-, to peri-, to postmenopause. These findings might reflect the early changes behind the increased risk of dyslipidemia, diabetes, cardiovascular disease, and osteoporosis in later life.

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Distinct Expansion of Group II Introns During Evolution of Prokaryotes and Possible Factors Involved in Its Regulation.

Miura MC, Nagata S, Tamaki S, Tomita M, Kanai A

Frontiers in microbiology (Frontiers in Microbiology) 13 849080 2022

ISSN 1664-302X

Group II introns (G2Is) are ribozymes that have retroelement characteristics in prokaryotes. Although G2Is are suggested to have been an important evolutionary factor in the prokaryote-to-eukaryote transition, comprehensive analyses of these introns among the tens of thousands of prokaryotic genomes currently available are still limited. Here, we developed a bioinformatic pipeline that systematically collects G2Is and applied it to prokaryotic genomes. We found that in bacteria, 25% (447 of 1,790) of the total representative genomes had an average of 5.3 G2Is, and in archaea, 9% (28 of 296) of the total representative genomes had an average of 3.0 G2Is. The greatest number of G2Is per genome was 101 in Arthrospira platensis (phylum Cyanobacteriota). A comprehensive sequence analysis of the intron-encoded protein (IEP) in each G2I sequence was conducted and resulted in the addition of three new IEP classes (U1–U3) to the previous classification. This analysis suggested that about 30% of all IEPs are non-canonical IEPs. The number of G2Is per genome was defined almost at the phylum level, and at least in the following two phyla, Firmicutes, and Cyanobacteriota, the type of IEP was largely associated as a factor in the G2I increase, i.e., there was an explosive increase in G2Is with bacterial C-type IEPs, mainly in the phylum Firmicutes, and in G2Is with CL-type IEPs, mainly in the phylum Cyanobacteriota. We also systematically analyzed the relationship between genomic signatures and the mechanism of these increases in G2Is. This is the first study to systematically characterize G2Is in the prokaryotic phylogenies.

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Metabolomics of small extracellular vesicles derived from isocitrate dehydrogenase 1-mutant HCT116 cells collected by semi-automated size exclusion chromatography.

Hayasaka R, Tabata S, Hasebe M, Ikeda S, Hikita T, Oneyama C, Yoshitake J, Onoshima D, Takahashi K, Shibata T, Uchida K, Baba Y, Soga T, Tomita M, Hirayama A

Frontiers in molecular biosciences (Frontiers in Molecular Biosciences) 9 1049402 2022

ISSN 2296-889X

Cancer-derived small extracellular vesicles (sEVs) are multifunctional particles with a lipid bilayer structure that are involved in cancer progression, such as malignant proliferation, distant metastasis, and cancer immunity evasion. The separation protocol used to isolate sEVs is an important process and thus, several have been developed, including ultracentrifugation (UC), size exclusion chromatography (SEC), and affinity purification using antibodies against sEV surface antigens. However, the effects of different separation methods on sEV components have not been adequately examined. Here, we developed a semi-automated system for collecting sEVs by combining SEC and preparative high-performance liquid chromatography and applied it to metabolome analysis. The developed SEC system could recover sEVs more efficiently and non-destructively than UC, suggesting that it is an appropriate recovery method for metabolic analysis and reflects biological conditions. Furthermore, using the developed SEC system, we performed metabolome analysis of sEVs from isocitrate dehydrogenase 1 (IDH)-mutated human colon HCT116 cells, which produce the oncogenic metabolite, 2-hydroxyglutaric acid (2-HG). IDH1-mutated HCT116 cells released significantly more sEVs than wild-type (WT) cells. The metabolomic profiles of IDH1 mutant and WT cells showed distinct differences between the cells and their sEVs. Notably, in IDH mutant cells, large amounts of 2-HG were detected not only in cells, but also in sEVs. These results indicate that the SEC system we developed has wide potential applications in sEVs research.

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Meta-analysis of transcriptional regulatory networks for lipid metabolism in neural cells from schizophrenia patients based on an open-source intelligence approach.

Okamoto L, Watanabe S, Deno S, Nie X, Maruyama J, Tomita M, Hatano A, Yugi K

Neuroscience research (Neuroscience Research) 175 82 - 97 2021.12

ISSN 0168-0102

There have been a number of reports about the transcriptional regulatory networks in schizophrenia. However, most of these studies were based on a specific transcription factor or a single dataset, an approach that is inadequate to understand the diverse etiology and underlying common characteristics of schizophrenia. Here we reconstructed and compared the transcriptional regulatory network for lipid metabolism enzymes using 15 public transcriptome datasets of neural cells from schizophrenia patients. Since many of the well-known schizophrenia-related SNPs are in enhancers, we reconstructed a network including enhancer-dependent regulation and found that 53.3 % of the total number of edges (7,577 pairs) involved regulation via enhancers. By examining multiple datasets, we found common and unique transcriptional modes of regulation. Furthermore, enrichment analysis of SNPs that were connected with genes in the transcriptional regulatory networks by eQTL suggested an association with hematological cell counts and some other traits/diseases, whose relationship to schizophrenia was either not or insufficiently reported in previous studies. Based on these results, we suggest that in future studies on schizophrenia, information on genotype, comorbidities and hematological cell counts should be included, along with the transcriptome, for a more detailed genetic stratification and mechanistic exploration of schizophrenia.

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Decoding gut microbiota by imaging analysis of fecal samples.

Furusawa C, Tanabe K, Ishii C, Kagata N, Tomita M, Fukuda S

iScience (iScience) 24 ( 12 ) 103481 2021.12

The gut microbiota plays a crucial role in maintaining health. Monitoring the complex dynamics of its microbial population is, therefore, important. Here, we present a deep convolution network that can characterize the dynamic changes in the gut microbiota using low-resolution images of fecal samples. Further, we demonstrate that the microbial relative abundances, quantified via 16S rRNA amplicon sequencing, can be quantitatively predicted by the neural network. Our approach provides a simple and inexpensive method of gut microbiota analysis.

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

Ishibashi Y., Harada S., Takeuchi A., Iida M., Kurihara A., Kato S., Kuwabara K., Hirata A., Shibuki T., Okamura T., Sugiyama D., Sato A., Amano K., Hirayama A., Sugimoto M., Soga T., Tomita M., Takebayashi T.

Scientific Reports 11 ( 1 ) 7407 2021.12

Currently, large-scale cohort studies for metabolome analysis have been launched globally. However, only a few studies have evaluated the reliability of urinary metabolome analysis. This study aimed to establish the reliability of urinary metabolomic profiling in cohort studies. In the Tsuruoka Metabolomics Cohort Study, 123 charged metabolites were identified and routinely quantified using capillary electrophoresis-mass spectrometry (CE-MS). We evaluated approximately 750 quality control (QC) samples and 6,720 participants’ spot urine samples. We calculated inter- and intra-batch coefficients of variation in the QC and participant samples and technical intraclass correlation coefficients (ICC). A correlation of metabolite concentrations between spot and 24-h urine samples obtained from 32 sub-cohort participants was also evaluated. The coefficient of variation (CV) was less than 20% for 87 metabolites (70.7%) and 20–30% for 19 metabolites (15.4%) in the QC samples. There was less than 20% inter-batch CV for 106 metabolites (86.2%). Most urinary metabolites would have reliability for measurement. The 96 metabolites (78.0%) was above 0.75 for the estimated ICC, and those might be useful for epidemiological analysis. Among individuals, the Pearson correlation coefficient of 24-h and spot urine was more than 70% for 59 of the 99 metabolites. These results show that the profiling of charged metabolites using CE-MS in morning spot human urine is suitable for epidemiological metabolomics studies.

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Reprogramming of glutamine metabolism via glutamine synthetase silencing induces cisplatin resistance in A2780 ovarian cancer cells

Guo J., Satoh K., Tabata S., Mori M., Tomita M., Soga T.

BMC Cancer 21 ( 1 ) 174 2021.12

Background: Cisplatin (CDDP) significantly prolongs survival in various cancers, but many patients also develop resistance that results in treatment failure. Thus, this study aimed to elucidate the underlying mechanisms by which ovarian cancer cells acquire CDDP resistance. Methods: We evaluated the metabolic profiles in CDDP-sensitive ovarian cancer A2780 cells and CDDP-resistant A2780cis cells using capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS). We further examined the expression of glutamine metabolism enzymes using real-time PCR and Western blot analyses. Cell viability was accessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: The results showed that levels of glutamine, glutamate, and glutathione (GSH), a key drug resistance mediator synthesized from glutamate, were significantly elevated in A2780cis cells than those in A2780 cells. Furthermore, glutamine starvation decreased the GSH levels and CDDP resistance in A2780cis cells. Interestingly, the expression of glutamine synthetase (GS/GLUL), which synthesizes glutamine from glutamate and thereby negatively regulates GSH production, was almost completely suppressed in resistant A2780cis cells. In addition, treatment of A2780cis cells with 5-aza-2′-deoxycytidine, a DNA-demethylating agent, restored GS expression and reduced CDDP resistance. In contrast, GS knockdown in CDDP-sensitive A2780 cells induced CDDP resistance. Conclusions: The results indicate that upregulation of GSH synthesis from glutamine via DNA methylation-mediated silencing of GS causes CDDP resistance in A2780cis cells. Therefore, glutamine metabolism could be a novel therapeutic target against CDDP resistance.

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TGF-β-dependent reprogramming of amino acid metabolism induces epithelial–mesenchymal transition in non-small cell lung cancers

Nakasuka F., Tabata S., Sakamoto T., Hirayama A., Ebi H., Yamada T., Umetsu K., Ohishi M., Ueno A., Goto H., Sugimoto M., Nishioka Y., Yamada Y., Tomita M., Sasaki A.T., Yano S., Soga T.

Communications Biology 4 ( 1 ) 782 2021.12

Epithelial–mesenchymal transition (EMT)—a fundamental process in embryogenesis and wound healing—promotes tumor metastasis and resistance to chemotherapy. While studies have identified signaling components and transcriptional factors responsible in the TGF-β-dependent EMT, whether and how intracellular metabolism is integrated with EMT remains to be fully elucidated. Here, we showed that TGF-β induces reprogramming of intracellular amino acid metabolism, which is necessary to promote EMT in non-small cell lung cancer cells. Combined metabolome and transcriptome analysis identified prolyl 4-hydroxylase α3 (P4HA3), an enzyme implicated in cancer metabolism, to be upregulated during TGF-β stimulation. Further, knockdown of P4HA3 diminished TGF-β-dependent changes in amino acids, EMT, and tumor metastasis. Conversely, manipulation of extracellular amino acids induced EMT-like responses without TGF-β stimulation. These results suggest a previously unappreciated requirement for the reprogramming of amino acid metabolism via P4HA3 for TGF-β-dependent EMT and implicate a P4HA3 inhibitor as a potential therapeutic agent for cancer.

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Multiomics study of a heterotardigrade, Echinisicus testudo, suggests the possibility of convergent evolution of abundant heat-soluble proteins in Tardigrada.

Murai Y, Yagi-Utsumi M, Fujiwara M, Tanaka S, Tomita M, Kato K, Arakawa K

BMC genomics (BMC Genomics) 22 ( 1 ) 813 2021.11

Background: Many limno-terrestrial tardigrades can enter an ametabolic state, known as anhydrobiosis, upon desiccation, in which the animals can withstand extreme environments. Through genomics studies, molecular components of anhydrobiosis are beginning to be elucidated, such as the expansion of oxidative stress response genes, loss of stress signaling pathways, and gain of tardigrade-specific heat-soluble protein families designated CAHS and SAHS. However, to date, studies have predominantly investigated the class Eutardigrada, and molecular mechanisms in the remaining class, Heterotardigrada, still remains elusive. To address this gap in the research, we report a multiomics study of the heterotardigrade Echiniscus testudo, one of the most desiccation-tolerant species which is not yet culturable in laboratory conditions. Results: In order to elucidate the molecular basis of anhydrobiosis in E. testudo, we employed a multi-omics strategy encompassing genome sequencing, differential transcriptomics, and proteomics. Using ultra-low input library sequencing protocol from a single specimen, we sequenced and assembled the 153.7 Mbp genome annotated using RNA-Seq data. None of the previously identified tardigrade-specific abundant heat-soluble genes was conserved, while the loss and expansion of existing pathways were partly shared. Furthermore, we identified two families novel abundant heat-soluble proteins, which we named E. testudo Abundant Heat Soluble (EtAHS), that are predicted to contain large stretches of disordered regions. Likewise the AHS families in eutardigrada, EtAHS shows structural changes from random coil to alphahelix as the water content was decreased in vitro. These characteristics of EtAHS proteins are analogous to those of CAHS in eutardigrades, while there is no conservation at the sequence level. Conclusions: Our results suggest that Heterotardigrada have partly shared but distinct anhydrobiosis machinery compared with Eutardigrada, possibly due to convergent evolution within Tardigrada. (276/350).

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The evidence of temporary social parasitism by Polyrhachis lamellidens (Hymenoptera, Formicidae) in a Camponotus obscuripes colony (Hymenoptera, Formicidae)

Iwai H., Kurihara Y., Kono N., Tomita M., Arakawa K.

Insectes Sociaux (Insectes Sociaux) 68 ( 4 ) 375 - 382 2021.11

ISSN 00201812

Polyrhachis lamellidens is a temporary socially parasitic ant whose new queen utilizes other ant species in the early stages of colony foundation. Field observations and rearing experiments suggest that Camponotus japonicus is a host species of P. lamellidens. It is presumed that Camponotus obscuripes is also a host of P. lamellidens by rearing experiments and field observations; however, there are no records of P. lamellidens workers or brood coexisting in C. obscuripes colonies in field observations, and there is no clear evidence that C. obscuripes is a natural host of P. lamellidens. We conducted detailed field observations, behavioral tests, and rearing experiments to show that C. obscuripes is a host of temporary social parasites. We found colonies with P. lamellidens queens, workers, and larvae intermixed with C. obscuripes workers in the field. Behavioral tests showed that workers of both species in mixed colonies did not attack each other and maintained nestmate recognition ability, which suggests a collaborative nestmate relationship. Furthermore, a rearing experiment confirmed social parasitism by P. lamellidens among C. obscuripes by producing a mixed brood-producing colony. These are the first field and laboratory records of temporary social parasitism involving P. lamellidens and C. obscuripes.

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Urinary metabolome analyses of patients with acute kidney injury using capillary electrophoresis-mass spectrometry

Saito R., Hirayama A., Akiba A., Kamei Y., Kato Y., Ikeda S., Kwan B., Pu M., Natarajan L., Shinjo H., Akiyama S., Tomita M., Soga T., Maruyama S.

Metabolites (Metabolites) 11 ( 10 ) 2021.10

ISSN 2218-1989

Acute kidney injury (AKI) is defined as a rapid decline in kidney function. The associated syndromes may lead to increased morbidity and mortality, but its early detection remains difficult. Using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), we analyzed the urinary metabolomic profile of patients admitted to the intensive care unit (ICU) after invasive surgery. Urine samples were collected at six time points: before surgery, at ICU admission and 6, 12, 24 and 48 h after. First, urine samples from 61 initial patients (non-AKI: 23, mild AKI: 24, severe AKI: 14) were measured, followed by the measurement of urine samples from 60 additional patients (non-AKI: 40, mild AKI: 20). Glycine and ethanolamine were decreased in patients with AKI compared with non-AKI patients at 6–24 h in the two groups. The linear statistical model constructed at each time point by machine learning achieved the best performance at 24 h (median AUC: 89%, cross-validated) for the 1st group. When cross-validated between the two groups, the AUC showed the best value of 70% at 12 h. These results identified metabolites and time points that show patterns specific to subjects who develop AKI, paving the way for the development of better biomarkers.

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Multicomponent nature underlies the extraordinary mechanical properties of spider dragline silk

Kono N., Nakamura H., Mori M., Yoshida Y., Ohtoshi R., Malay A.D., Pedrazzoli Moran D.A., Tomita M., Numata K., Arakawa K.

Proceedings of the National Academy of Sciences of the United States of America 118 ( 31 ) 2021.08

ISSN 00278424

Dragline silk of golden orb-weaver spiders (Nephilinae) is noted for its unsurpassed toughness, combining extraordinary extensibility and tensile strength, suggesting industrial application as a sustainable biopolymer material. To pinpoint the molecular composition of dragline silk and the roles of its constituents in achieving its mechanical properties, we report a multiomics approach, combining highquality genome sequencing and assembly, silk gland transcriptomics, and dragline silk proteomics of four Nephilinae spiders.We observed the consistent presence of the MaSp3B spidroin unique to this subfamily as well as several nonspidroin SpiCE proteins. Artificial synthesis and the combination of these components in vitro showed that the multicomponent nature of dragline silk, including MaSp3B and SpiCE, along with MaSp1 and MaSp2, is essential to realize the mechanical properties of spider dragline silk.

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RNA sequencing data for gamma radiation response in the extremotolerant tardigrade Ramazzottius varieornatus.

Yoshida Y, Horikawa DD, Sakashita T, Yokota Y, Kobayashi Y, Tomita M, Arakawa K

Data in brief 36 107111 2021.06

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Quality assessment of untargeted analytical data in a large-scale metabolomic study

Saito R., Sugimoto M., Hirayama A., Soga T., Tomita M., Takebayashi T.

Journal of Clinical Medicine (Journal of Clinical Medicine) 10 ( 9 ) 2021.05

ISSN 2077-0383

Large-scale metabolomic studies have become common, and the reliability of the peak data produced by the various instruments is an important issue. However, less attention has been paid to the large number of uncharacterized peaks in untargeted metabolomics data. In this study, we tested various criteria to assess the reliability of 276 and 202 uncharacterized peaks that were detected in a gathered set of 30 plasma and urine quality control samples, respectively, using capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS). The linear relationship between the amounts of pooled samples and the corresponding peak areas was one of the criteria used to select reliable peaks. We used samples from approximately 3000 participants in the Tsuruoka Metabolome Cohort Study to investigate patterns of the areas of these uncharacterized peaks among the samples and clustered the peaks by combining the patterns and differences in the migration times. Our assessment pipeline removed substantial numbers of unreliable or redundant peaks and detected 35 and 74 reliable uncharacterized peaks in plasma and urine, respectively, some of which may correspond to metabolites involved in important physiological processes such as disease progression. We propose that our assessment pipeline can be used to help establish large-scale untargeted clinical metabolomic studies.

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A metabolomic profile predictive of new osteoporosis or sarcopenia development

Miyamoto K., Hirayama A., Sato Y., Ikeda S., Maruyama M., Soga T., Tomita M., Nakamura M., Matsumoto M., Yoshimura N., Miyamoto T.

Metabolites (Metabolites) 11 ( 5 ) 2021.05

ISSN 2218-1989

The increasing number of patients with osteoporosis and sarcopenia is a global concern among countries with progressively aging societies. The high medical costs of treating those patients suggest that prevention rather than treatment is preferable. We enrolled 729 subjects who attended both the second and third surveys of the Research on Osteoarthritis/Osteoporosis Against Disability (ROAD) study. Blood samples were collected from subjects at the second survey, and then a comprehensive metabolomic analysis was performed. It was found that 35 had newly developed osteoporosis at the third survey performed four years later, and 39 were newly diagnosed with sarcopenia at the third survey. In the second survey, we found that serum Gly levels were significantly higher even after adjustment for age, sex, and BMI in subjects with newly developed osteoporosis relative to those who remained osteoporosis-negative during the four-year follow-up. We also show that serum taurine levels were significantly lower at the second survey, even after adjustment for age, sex, and BMI in subjects with newly developed sarcopenia during the four-year follow-up compared with those not diagnosed with sarcopenia at the second or third surveys. Though our sample size and odds ratios were small, increased Gly and decreased taurine levels were found to be predictive of new development of osteoporosis and sarcopenia, respectively, within four years.

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Metabolomic analysis of small extracellular vesicles derived from pancreatic cancer cells cultured under normoxia and hypoxia

Hayasaka R., Tabata S., Hasebe M., Ikeda S., Ohnuma S., Mori M., Soga T., Tomita M., Hirayama A.

Metabolites (Metabolites) 11 ( 4 ) 2021.04

ISSN 2218-1989

Extracellular vesicles (EVs) released from cancer cells contribute to various malignant phenotypes of cancer, including metastasis, cachexia, and angiogenesis. Although DNA, mRNAs, miRNAs, and proteins contained in EVs have been extensively studied, the function of metabolites in EVs remains unclear. In this study, we performed a comprehensive metabolomic analysis of pancreatic cancer cells, PANC-1, cultured under different oxygen concentrations, and small EVs (sEVs) released from them, considering the fact that hypoxia contributes to the malignant behavior of cells in pancreatic cancer, which is a poorly diagnosed cancer. sEVs were collected by ultracen-trifugation, and hydrophilic metabolites were analyzed using capillary ion chromatography-mass spectrometry and liquid chromatography-mass spectrometry, and lipids were analyzed by supercrit-ical fluid chromatography-tandem mass spectrometry. A total of 140 hydrophilic metabolites and 494 lipids were detected in sEVs, and their profiles were different from those in cells. In addition, the metabolomic profile of sEVs was observed to change under hypoxic stress, and an increase in metabolites involved in angiogenesis was also detected. We reveal the hallmark of the metabolites contained in sEVs and the effect of tumor hypoxia on their profiles, which may help in understanding EV-mediated cancer malignancy.

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Xanthurenic acid is the main pigment of trichonephila clavata gold dragline silk

Fujiwara M., Kono N., Hirayama A., Malay A.D., Nakamura H., Ohtoshi R., Numata K., Tomita M., Arakawa K.

Biomolecules 11 ( 4 ) 2021.04

Spider silk is a natural fiber with remarkable strength, toughness, and elasticity that is attracting attention as a biomaterial of the future. Golden orb-weaving spiders (Trichonephila clavata) construct large, strong webs using golden threads. To characterize the pigment of golden T. clavata dragline silk, we used liquid chromatography and mass spectrometric analysis. We found that the major pigment in the golden dragline silk of T. clavata was xanthurenic acid. To investigate the possible function of the pigment, we tested the effect of xanthurenic acid on bacterial growth using gram-negative Escherichia coli and gram-positive Bacillus subtilis. We found that xanthurenic acid had a slight antibacterial effect. Furthermore, to investigate the UV tolerance of the T. clavata threads bleached of their golden color, we conducted tensile deformation tests and scanning electron microscope observations. However, in these experiments, no significant effect was observed. We therefore speculate that golden orb-weaving spiders use the pigment for other purposes, such as to attract their prey in the sunlight.

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Charged metabolite biomarkers of food intake assessed via plasma metabolomics in a population-based observational study in Japan

Shibutami E., Ishii R., Harada S., Kurihara A., Kuwabara K., Kato S., Iida M., Akiyama M., Sugiyama D., Hirayama A., Sato A., Amano K., Sugimoto M., Soga T., Tomita M., Takebayashi T.

PLoS ONE 16 ( 2 February ) e0246456 2021.02

Food intake biomarkers can be critical tools that can be used to objectively assess dietary exposure for both epidemiological and clinical nutrition studies. While an accurate estimation of food intake is essential to unravel associations between the intake and specific health conditions, random and systematic errors affect self-reported assessments. This study aimed to clarify how habitual food intake influences the circulating plasma metabolome in a free-living Japanese regional population and to identify potential food intake biomarkers. To achieve this aim, we conducted a cross-sectional analysis as part of a large cohort study. From a baseline survey of the Tsuruoka Metabolome Cohort Study, 7, 012 eligible male and female participants aged 40-69 years were chosen for this study. All data on patients' health status and dietary intake were assessed via a food frequency questionnaire, and plasma samples were obtained during an annual physical examination. Ninety-four charged plasma metabolites were measured using capillary electrophoresis mass spectrometry, by a non-targeted approach. Statistical analysis was performed using partial-least-square regression. A total of 21 plasma metabolites were likely to be associated with long-term food intake of nine food groups. In particular, the influential compounds in each food group were hydroxyproline for meat, trimethylamine- N-oxide for fish, choline for eggs, galactarate for dairy, cystine and betaine for soy products, threonate and galactarate for carotenoid-rich vegetables, proline betaine for fruits, quinate and trigonelline for coffee, and pipecolate for alcohol, and these were considered as prominent food intake markers in Japanese eating habits. A set of circulating plasma metabolites was identified as potential food intake biomarkers in the Japanese community-dwelling population. These results will open the way for the application of new reliable dietary assessment tools not by self-reported measurements but through objective quantification of biofluids.

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Quantitative and Molecular Similarity Analyses of the Metabolites of Cold- A nd Hot-Natured Chinese Herbs

Guo J., Wang J., Iino K., Tomita M., Soga T.

Evidence-based Complementary and Alternative Medicine 2021 6646507 2021

ISSN 1741427X

Background. Based on the theory of traditional Chinese medicine, Chinese herbs possess four different medicinal properties: Hot, warm, cold, and cool. These serve as a reference guide for these herbal medicines. However, the molecular mechanisms supporting their relevance remain unclear. Methods. We performed metabolomics based on capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF/MS) and multivariate data analysis for the structural identification of compounds of cold- A nd hot-natured Chinese herbs. Results. To this end, 30 selected herbs were analyzed and a total of 416 metabolites were identified via CE-TOF/MS, of which 193 compounds were detected in most herbs. The observed profiles offered the potential to understand the mechanism of association between the compounds and nature of the Chinese herbs. Comparison of the similarity in terms of chemical and molecular structures and content revealed that hot-natured herbs contained more nucleotides. In contrast, principal component analysis revealed the presence of more amino acid compounds in cold-natured herbs. Conclusion. Comparing the structural similarities between the samples using the Tanimoto coefficient revealed that a general non-specific structure was observed between cold- A nd hot-natured herbs; however, the distribution of the molecular groups seemed to contribute more toward the energy properties.

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Upregulation of Thymidylate Synthase Induces Pemetrexed Resistance in Malignant Pleural Mesothelioma.

Sato Y, Tomita M, Soga T, Ochiai A, Makinoshima H

Frontiers in pharmacology (Frontiers in Pharmacology) 12 718675 2021

ISSN 1663-9812

Malignant pleural mesothelioma (MPM) is an invasive malignancy that develops in the pleural cavity, and antifolates are used as chemotherapeutics for treating. The majority of antifolates, including pemetrexed (PMX), inhibit enzymes involved in purine and pyrimidine synthesis. MPM patients frequently develop drug resistance in clinical practice, however the associated drug-resistance mechanism is not well understood. This study was aimed to elucidate the mechanism underlying resistance to PMX in MPM cell lines. We found that among the differentially expressed genes associated with drug resistance (determined by RNA sequencing), TYMS expression was higher in the established resistant cell lines than in the parental cell lines. Knocking down TYMS expression significantly reduced drug resistance in the resistant cell lines. Conversely, TYMS overexpression significantly increased drug resistance in the parental cells. Metabolomics analysis revealed that the levels of dTMP were higher in the resistant cell lines than in the parental cell lines; however, resistant cells showed no changes in dTTP levels after PMX treatment. We found that the nucleic acid-biosynthetic pathway is important for predicting the efficacy of PMX in MPM cells. The results of chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) assays suggested that H3K27 acetylation in the 5′-UTR of TYMS may promote its expression in drug-resistant cells. Our findings indicate that the intracellular levels of dTMP are potential biomarkers for the effective treatment of patients with MPM and suggest the importance of regulatory mechanisms of TYMS expression in the disease.

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Correlation-centred variable selection of a gene expression signature to predict breast cancer metastasis

Hikichi S., Sugimoto M., Tomita M.

Scientific Reports (Scientific Reports) 10 ( 1 ) 7923 2020.12

Predictions of distant cancer metastasis based on gene signatures are studied intensively to realise precise diagnosis and treatments. Gene selection i.e. feature selection is a cornerstone to both establish accurate predictions and understand underlying pathologies. Here, we developed a simple but robust feature selection method using a correlation-centred approach to select minimal gene sets that have both high predictive and generalisation abilities. A multiple logistic regression model was used to predict 5-year metastases of patients with breast cancer. Gene expression data obtained from tumour samples of lymph node-negative breast cancer patients were randomly split into training and validation data. Our method selected 12 genes using training data and this showed a higher area under the receiver operating characteristic curve of 0.730 compared with 0.579 yielded by previously reported 76 genes. The signature with the predictive model was validated in an independent dataset, and its higher generalization ability was observed. Gene ontology analyses revealed that our method consistently selected genes with identical functions which frequently selected by the 76 genes. Taken together, our method identifies fewer gene sets bearing high predictive abilities, which would be versatile and applicable to predict other factors such as the outcomes of medical treatments and prognoses of other cancer types.

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Spidroin profiling of cribellate spiders provides insight into the evolution of spider prey capture strategies

Kono N., Nakamura H., Mori M., Tomita M., Arakawa K.

Scientific Reports (Scientific Reports) 10 ( 1 ) 15721 2020.12

Orb-weaving spiders have two main methods of prey capture: cribellate spiders use dry, sticky capture threads, and ecribellate spiders use viscid glue droplets. Predation behaviour is a major evolutionary driving force, and it is important on spider phylogeny whether the cribellate and ecribellate spiders each evolved the orb architecture independently or both strategies were derived from an ancient orb web. These hypotheses have been discussed based on behavioural and morphological characteristics, with little discussion on this subject from the perspective of molecular materials of orb web, since there is little information about cribellate spider-associated spidroin genes. Here, we present in detail a spidroin catalogue of six uloborid species of cribellate orb-weaving spiders, including cribellate and pseudoflagelliform spidroins, with transcriptome assembly complemented with long read sequencing, where silk composition is confirmed by proteomics. Comparative analysis across families (Araneidae and Uloboridae) shows that the gene architecture, repetitive domains, and amino acid frequencies of the orb web constituting silk proteins are similar among orb-weaving spiders regardless of the prey capture strategy. Notably, the fact that there is a difference only in the prey capture thread proteins strongly supports the monophyletic origin of the orb web.

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Integrated omics profiling of dextran sodium sulfate-induced colitic mice supplemented with Wolfberry (Lycium barbarum)

Aw W., Jia H., Lyu W., Fukuda S., Tomita M., Otani L., Kato H.

npj Science of Food (npj Science of Food) 4 ( 1 ) 5 2020.12

We used a multi-omics profiling approach to investigate the suppressive effects of 2% Wolfberry (WOL)-enriched diets on dextran sodium sulfate (DSS)-induced colitis in mice. It was observed that in mice fed the WOL diet, the disease activity index, colon shortening, plasma concentrations of matrix metalloproteinase-3 and relative mesenteric fat weight were significantly improved as compared to the DSS group. Results from colon transcriptome and proteome profiles showed that WOL supplementation significantly ameliorated the expression of genes and proteins associated with the integrity of the colonic mucosal wall and colonic inflammation. Based on the hepatic transcriptome, proteome and metabolome data, genes involved in fatty acid metabolism, proteins involved in inflammation and metabolites related to glycolysis were downregulated in WOL mice, leading to lowered inflammation and changes in these molecules may have led to improvement in body weight loss. The integrated nutrigenomic approach thus revealed the molecular mechanisms underlying the ameliorative effect of whole WOL fruit consumption on inflammatory bowel disease.

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Relationship between standard uptake values of positron emission tomography/computed tomography and salivary metabolites in oral cancer: A pilot study

Ishikawa S., Hiraka T., Kirii K., Sugimoto M., Shimamoto H., Sugano A., Kitabatake K., Toyoguchi Y., Kanoto M., Nemoto K., Soga T., Tomita M., Iino M.

Journal of Clinical Medicine (Journal of Clinical Medicine) 9 ( 12 ) 1 - 8 2020.12

ISSN 2077-0383

18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET)/computed tomography (CT) is usually used for staging or evaluation of treatment response rather than for cancer screening. However,18F-FDG PET/CT has also been used in Japan for cancer screening in people with no cancer symptoms, and accumulating evidence supports this application of18F-FDG PET/CT. Previously, we have observed a correlation between the saliva and tumor metabolomic profiles in patients with oral cancer. Hence, if salivary metabolites demonstrate a significant correlation with PET parameters such as the maximum standardized uptake value (SUVmax), they may have the potential to be used as a screening tool before PET/CT to identify patients with high SUVmax. Hence, in this study, we aimed to explore the relationship between salivary metabolites and SUVmax of18F-FDG PET/CT using previously collected data.18F-FDG PET/CT was performed for staging 26 patients with oral cancer. The collected data were integrated and analyzed along with quantified salivary hydrophilic metabolites obtained from the same patients with oral cancer and controls (n = 44). In total, 11 metabolites showed significant correlations with SUVmax in the delayed phases. A multiple logistic regression model of the two metabolites showed the ability to discriminate between patients with oral cancer and controls, with an area under the receiver operating characteristic curve of 0.738 (p = 0.001). This study uniquely confirmed a relationship between salivary metabolites and SUVmax of PET/CT in patients with oral cancer; salivary metabolites were significantly correlated with SUVmax. These salivary metabolites can be used as a screening tool before PET/CT to identify patients with high SUVmax, i.e., to detect the presence of oral cancer.

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Comprehensive dipeptide analysis revealed cancer-specific profile in the liver of patients with hepatocellular carcinoma and hepatitis

Ozawa H., Hirayama A., Shoji F., Maruyama M., Suzuki K., Yamanaka-Okumura H., Tatano H., Morine Y., Soga T., Shimada M., Tomita M.

Metabolites (Metabolites) 10 ( 11 ) 1 - 11 2020.11

ISSN 2218-1989

As the physical properties and functionality of dipeptides differ from those of amino acids, they have attracted attention in metabolomics; however, their functions in vivo have not been clarified in detail. Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, and its major cause is chronic hepatitis. This study was conducted to explore tumor-specific dipeptide characteristics by performing comprehensive dipeptide analysis in the tumor and surrounding nontumor tissue of patients with HCC. Dipeptides were analyzed by liquid chromatography tandem mass spectrometry and capillary electrophoresis tandem mass spectrometry. Principal component analysis using 236 detected dipeptides showed differences in the dipeptide profiles between nontumor and tumor tissues; however, no clear difference was observed in etiological comparison. In addition, the N-and C-terminal amino acid compositions of the detected dipeptides significantly differed, suggesting the substrate specificity of enzyme proteins, such as peptidase. Furthermore, hepatitis-derived HCC may show a characteristic dipeptide profile even before tumor formation. These results provide insight into HCC pathogenesis and may help identify novel biomarkers for diagnosis.

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Effects of feed crops and boiling on chicken egg yolk and white determined by a metabolome analysis

Ogura T., Wakayama M., Ashino Y., Kadowaki R., Sato M., Soga T., Tomita M.

Food Chemistry (Food Chemistry) 327 127077 2020.10

ISSN 03088146

Compositional analyses of eggs have primarily focused on nutritional components, including large molecules, such as proteins. However, few reports have investigated the effects of heating and hen feed crops on taste components, such as free amino acids and sugars. Herein, water-soluble metabolites in raw and boiled eggs produced from chickens raised with corn- or rice-fed were analyzed. Each egg was separated into yolk and white, and freeze-dried samples were analyzed by CE-MS and LC-MS. Abundant metabolites included amino acids in yolks and sugars in whites. Compared to corn-fed, rice-fed resulted in three times higher betaine and uridine monophosphate concentrations in yolks and whites, respectively. Boiled whites contained more than four times higher concentrations of amino acids and fructose than raw whites. Metabolites in yolks exhibited minimal changes after boiling. Our findings support the use of water-soluble metabolomics to evaluate the effects of heating and feed crops on taste components.

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Conjunctival injection reduction in patients with atopic keratoconjunctivitis due to synergic effect of bovine enteric-coated lactoferrin in 0.1% tacrolimus ophthalmic suspension

Fujishima H., Okada N., Matsumoto K., Shimizu E., Fukuda S., Tomita M.

Journal of Clinical Medicine (Journal of Clinical Medicine) 9 ( 10 ) 1 - 9 2020.10

ISSN 2077-0383

Lactoferrin (LF), a multifunctional glycoprotein found in mammalian milk, is reported to have immunoregulatory effects. The present study aimed to evaluate whether enteric-coated LF (eLF) could improve symptoms in patients with atopic keratoconjunctivitis (AKC). This randomized double-blind placebo-controlled single-center trial comprised Japanese patients (n = 20; aged 22–60 years) with AKC. Patients treated with 0.1% tacrolimus ophthalmic suspension (TALYMUS®) were administered eLF (400 mg/d of bovine LF) or placebo tablets for 12 weeks. Conjunctival injection was examined, papillae formation in the palpebral conjunctiva was evaluated, and corneal fluorescein score, itchy sensation in end-point itching scale, and serum allergic parameters were assessed. Conjunctival injection was significantly reduced in the LF group than in the placebo group (p = 0.0017, Mann–Whitney U-test). Papillae formation in the palpebral conjunctiva showed a statistical decrease in the LF group than in the placebo group (p = 0.010, unpaired T-test). LF combined with TALYMUS® could be a promising treatment strategy to mitigate AKC.

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Pre-treatment with D942, a furancarboxylic acid derivative, increases desiccation tolerance in an anhydrobiotic tardigrade Hypsibius exemplaris

Kondo K., Mori M., Tomita M., Arakawa K.

FEBS Open Bio (FEBS Open Bio) 10 ( 9 ) 1774 - 1781 2020.09

The tardigrade Hypsibius exemplaris can undergo anhydrobiosis. Several chemicals that inhibit successful anhydrobiosis in H. exemplaris have been identified, and these chemicals inhibit the activity of signaling molecules. In the present study, we investigated whether upregulation of the activity of these signaling molecules could improve desiccation tolerance of H. exemplaris. Pre-treatment with an indirect activator of AMP-activated protein kinase [AMPK; which directly inhibits mammalian NAD(P)H dehydrogenase [quinone] 1 [NQO1] of mitochondrial complex I (D942)] significantly improved desiccation tolerance of H. exemplaris, whereas a direct activator of AMPK did not. To elucidate the underlying molecular mechanisms, we examined the proteome of tardigrades treated with D942. Two proteins, putative glutathione S-transferase and pirin-like protein, were upregulated by treatment. Both of these proteins are known to be associated with the response to oxidative stress. One of the downregulated proteins was serine/threonine-proteinphosphatase 2A (PP2A) 65-kDa regulatory subunit A alpha isoform, and it is interesting to note that PP2A activity was previously suggested to be required for successful anhydrobiosis in H. exemplaris. Taken together, our results suggest that D942 treatment may partially induce responses common to those of desiccation stress. The identification of a chemical that improves desiccation tolerance of H. exemplaris may facilitate further investigation into desiccation tolerance mechanisms.

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Comprehensive Dipeptide Profiling and Quantitation by Capillary Electrophoresis and Liquid Chromatography Coupled with Tandem Mass Spectrometry

Ozawa H., Hirayama A., Ishikawa T., Kudo R., Maruyama M., Shoji F., Doke T., Ishimoto T., Maruyama S., Soga T., Tomita M.

Analytical Chemistry (Analytical Chemistry) 92 ( 14 ) 9799 - 9806 2020.07

ISSN 00032700

Dipeptides have attracted much attention as post-amino acids with physical properties and functions different from those of amino acids. However, a given dipeptide cannot be distinguished by mass spectrometry from its structural isomer with an opposite amino acid binding order unless these isomers are separated before introduction, which complicates the comprehensive analysis of dipeptides. Herein, a novel analytical platform for dipeptide analysis by capillary electrophoresis tandem mass spectrometry and liquid chromatography tandem mass spectrometry is developed. This method is used to quantitate 335 dipeptides and achieves excellent separation of structural isomers with opposite binding orders, high correlation coefficients, and low instrumental detection limits (0.088-83.1 nM). Moreover, acceptable recoveries (70-135%) are observed for most tested dipeptides in chicken liver samples spiked both before and after preparation. The developed method is also applied to the quantitation of dipeptides in the livers of mice fed different diets to detect 236 dipeptides, and the shift from a normal diet to a high-fat diet is shown to increase/decrease (p < 0.05, fold-change < 0.5) the contents of 0/29 dipeptides, respectively. The developed method is expected to facilitate the search for new dipeptide applications such as novel functional components of foods and biomarkers of diseases.

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Base editors for simultaneous introduction of C-to-T and A-to-G mutations

Sakata R.C., Ishiguro S., Mori H., Tanaka M., Tatsuno K., Ueda H., Yamamoto S., Seki M., Masuyama N., Nishida K., Nishimasu H., Arakawa K., Kondo A., Nureki O., Tomita M., Aburatani H., Yachie N.

Nature Biotechnology (Nature Biotechnology) 38 ( 7 ) 865 - 869 2020.07

ISSN 10870156

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Draft Genome Sequence of Geobacter sp. Strain SVR, Isolated from Antimony Mine Soil

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

Microbiology Resource Announcements (Microbiology Resource Announcements) 9 ( 26 ) 2020.06

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The use of a double coaxial electrospray ionization sprayer improves the peak resolutions of anionic metabolites in capillary ion chromatography-mass spectrometry

Hirayama A., Tabata S., Kudo R., Hasebe M., Suzuki K., Tomita M., Soga T.

Journal of Chromatography A (Journal of Chromatography A) 1619 460914 2020.05

ISSN 00219673

Recently, ion chromatography coupled with mass spectrometry has been used for the determination of anionic metabolites. However, connection with a mass spectrometer in this method is not straightforward because backpressure produced by the addition of a make-up solution often affects the peak resolutions of the target metabolites. To overcome this problem, we developed a capillary ion chromatography-mass spectrometry method utilizing a double coaxial electrospray ionization sprayer. This method was not affected by backpressure and the number of theoretical plates was about three times that of a conventional sprayer. Under optimized conditions, 44 anionic metabolites, including organic acids, sugar phosphates, nucleotides, and cofactors, were successfully separated and selectively detected with a Q Exactive mass spectrometer. The calibration curves of the tested metabolites showed excellent linearity within the range of 1–100,000 nmol/L and the correlation coefficient was greater than 0.991. The detection limits for these metabolites were between 1 and 500 nmol/L (0.4 and 200 fmol). The developed method was applied to the quantitation of anionic metabolites in cultured cancer cell samples with tumor necrosis factor (TNF)-α stimulation. This allowed for the successful determination of 105 metabolites. The levels of tricarboxylic acid cycle intermediates changed significantly after TNF-α stimulation. These results demonstrate that the developed method is a promising new tool for comprehensive analysis of anionic metabolites.

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Effect of blanching on the concentration of metabolites in two parts of Undaria pinnatifida, Wakame (leaf) and Mekabu (sporophyll)

Hamid S.S., Wakayama M., Ashino Y., Kadowaki R., Soga T., Tomita M.

Algal Research 47 2020.05

ISSN 22119264

Undaria pinnatifida is a seaweed native to Japan, where it is cultivated and sold commercially as food. Fresh seaweed commonly blanches before consumption. However, the effects of blanching on metabolites present in different parts of U. pinnatifida, namely, Wakame (leaf) and Mekabu (sporophyll), are not well understood. Here, we investigated changes in metabolites in these two seaweed parts after blanching for different periods. The samples were freeze-dried and extracted using methanol-water, and metabolite concentration was quantitated using liquid chromatography mass spectrometry and capillary electrophoresis mass spectrometry. The concentration of most metabolites was the highest in raw samples, decreasing drastically within 20 s of blanching in Wakame and decreasing gradually in Mekabu. In contrast, the concentration of some fatty acids, such as octanoate, increased in both parts. During blanching, seaweed color changed from brown to green, and blanching did not significantly affect amino acid or the sugar composition. Finally, a relationship was found between metabolite and blanching-related characteristics, such as color and drying percentage.

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Chromatin Remodeling and Immediate Early Gene Activation by SLFN11 in Response to Replication Stress

Murai J., Zhang H., Pongor L., Tang S.W., Jo U., Moribe F., Ma Y., Tomita M., Pommier Y.

Cell Reports (Cell Reports) 30 ( 12 ) 4137 - 4151.e6 2020.03

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Sensory properties and metabolomic profiles of dry-cured ham during the ripening process

Sugimoto M., Sugawara T., Obiya S., Enomoto A., Kaneko M., Ota S., Soga T., Tomita M.

Food Research International (Food Research International) 129 108850 2020.03

ISSN 09639969

Dry-cured ham with a long ripening period is a valuable product worldwide. Ripening time is a key determinant of the endogenous metabolites that characterize the flavor and taste of ham products. While various studies have analyzed the relationship between ripening duration and sensory characteristics, no studies have evaluated ham products produced in Japan. Here, we conducted time-course metabolomic profiling, taste sensor-based analyses, and sensory evaluations by non-trained consumers during ripening. Capillary electrophoresis-mass spectrometry was used to quantify non-volatile metabolites, such as amino acids, organic acids, and nucleotides. In an analysis of eight time-points during 680 days of ripening, the highest score for the after-taste of umami was observed on day 540, despite subtle changes in the scores for other properties. The concentrations of aspartic acid and glutamic acid relative to those of total amino acids were the highest at this point. This approach can contribute to the understanding of the relationship between the metabolite profile and ripening duration.

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Mass Spectrometry-Compatible Subcellular Fractionation for Proteomics

Masuda T., Sugiyama N., Tomita M., Ohtsuki S., Ishihama Y.

Journal of Proteome Research (Journal of Proteome Research) 19 ( 1 ) 75 - 84 2020.01

ISSN 15353893

We found that nuclear envelopes stabilize against surfactants in the presence of ethylene glycol (EG). We, therefore, developed a novel subcellular fractionation approach for proteomics using RIPA buffer containing EG and phase transfer surfactants. This method involves separating the cells into the cytoplasm, organelles, and nucleus, including intermediate filaments without ultracentrifugation. These fractions are directly applicable to sample preparation for shotgun proteomics as they have no mass spectrometry (MS)-incompatible chemicals, whereas those separated by traditional fractionation protocols require desalting. This protocol is successfully applied to subcellular fractionation with only 3.5 × 10 cells. Here, it was combined with phosphoproteomics and proteomics to identify phosphorylation sites regulating protein subcellular localization. In total, 59 phosphorylation sites on 42 phosphopeptides and 32 proteins showing different enrichment patterns between phosphoproteomics and the corresponding proteomics were identified, which are potential candidate sites to regulate the protein subcellular localization, including serine 706 on CD44 and serine 22 on lamin A/C. 5

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Stable mutants of restriction-deficient/modification-proficient Bacillus subtilis 168: Hub strains for giant DNA engineering

Itaya M., Sato M., Watanabe S., Yoshikawa H., Tomita M., Sato R.

Journal of Biochemistry (Journal of Biochemistry) 25 ( 1 ) 231 - 236 2020.01

ISSN 0021924X

Bacillus subtilis 168 has been explored as a platform for the synthesis and transmission of large DNA. Two inherent DNA incorporation systems, natural transformation and pLS20-based conjugation transfer, enable rapid handling of target DNA. Both systems are affected by the Bsu restriction-modification system that recognizes and cleaves unmethylated XhoI sites, limiting the choice of target DNA. We constructed B. subtilis 168 with stable mutation for restriction-deficient and modification-proficient (r m ). It was demonstrated that the r m strains can incorporate and transfer synthesized DNA with multiple XhoI sites. These should be of value as hub strains to integrate and disseminate giant DNA between B. subtilis 168 derivatives. - + - +

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Does the Gut Microbiota Modulate Host Physiology through Polymicrobial Biofilms?

Yang J, Yang Y, Ishii M, Nagata M, Aw W, Obana N, Tomita M, Nomura N, Fukuda S.

Microbes Environ 2020

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Draft Genome Sequences of Bifidobacterium animalis Consecutively Isolated from Healthy Japanese Individuals

Tsukimi T, Watabe T, Tanaka K, Sato MP, Suzuki H, Tomita M, Fukuda S.

J Genomics 8 37 - 42 2020

ISSN 1839-9940

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Comparison of the transcriptomes of two tardigrades with different hatching coordination

Yoshida Y., Sugiura K., Tomita M., Matsumoto M., Arakawa K.

BMC Developmental Biology (BMC Developmental Biology) 19 ( 1 ) 24 2019.12

Background: Tardigrades are microscopic organisms, famous for their tolerance against extreme environments. The establishment of rearing systems of multiple species has allowed for comparison of tardigrade physiology, in particular in embryogenesis. Interestingly, in-lab cultures of limnic species showed smaller variation in hatching timing than terrestrial species, suggesting a hatching regulation mechanism acquired by adaptation to their habitat. Results: To this end, we screened for coordinated gene expression during the development of two species of tardigrades, Hypsibius exemplaris and Ramazzottius varieornatus, and observed induction of the arthropod molting pathway. Exposure of ecdysteroids and juvenile hormone analog affected egg hatching but not embryonic development in only the limnic H. exemplaris. Conclusion: These observations suggest a hatching regulation mechanism by the molting pathway in H. exemplaris.

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Orb-weaving spider Araneus ventricosus genome elucidates the spidroin gene catalogue

Kono N., Nakamura H., Ohtoshi R., Moran D., Shinohara A., Yoshida Y., Fujiwara M., Mori M., Tomita M., Arakawa K.

Scientific Reports (Scientific Reports) 9 ( 1 ) 8380 2019.12

© 2019, The Author(s). Members of the family Araneidae are common orb-weaving spiders, and they produce several types of silks throughout their behaviors and lives, from reproduction to foraging. Egg sac, prey capture thread, or dragline silk possesses characteristic mechanical properties, and its variability makes it a highly attractive material for ecological, evolutional, and industrial fields. However, the complete set of constituents of silks produced by a single species is still unclear, and novel spidroin genes as well as other proteins are still being found. Here, we present the first genome in genus Araneus together with the full set of spidroin genes with unamplified long reads and confirmed with transcriptome of the silk glands and proteome analysis of the dragline silk. The catalogue includes the first full length sequence of a paralog of major ampullate spidroin MaSp3, and several spider silk-constituting elements designated SpiCE. Family-wide phylogenomic analysis of Araneidae suggests the relatively recent acquisition of these genes, and multiple-omics analyses demonstrate that these proteins are critical components in the abdominal spidroin gland and dragline silk, contributing to the outstanding mechanical properties of silk in this group of species.

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AMPK activity is required for the induction of anhydrobiosis in a tardigrade Hypsibius exemplaris, and its potential up-regulator is PP2A

Kondo K., Mori M., Tomita M., Arakawa K.

Genes to Cells (Genes to Cells) 24 ( 12 ) 768 - 780 2019.12

ISSN 13569597

The anhydrobiotic tardigrade, Hypsibius exemplaris, was previously considered to require de novo gene expression and protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) activity for successful anhydrobiosis. These indicate that H. exemplaris has signal transduction systems responding to desiccation stress, with the involvement of phosphorylation events. To this end, we carried out time-series phosphoproteomics of H. exemplaris exposed to mild desiccation stress and detected 48 phosphoproteins with significant differential regulations. Among them, immediate and successive reduction of phosphorylation levels of AMP-activated protein kinase (AMPK) was observed. The subsequent chemical genetic approach showed that AMPK was activated during the preconditioning stage for anhydrobiosis, and inhibition of its activity impaired successful anhydrobiosis. As PP2A is known to dephosphorylate AMPK in other organisms, we suggested that decreased phosphorylation levels of AMPK upon mild desiccation stress were caused by dephosphorylation by PP2A. Accordingly, phosphoproteomics of animals pre-treated with the PP1/PP2A inhibitor cantharidic acid (CA) lacked the decrease in phosphorylation levels of AMPK. These observations suggest that AMPK activity is required for successful anhydrobiosis in H. exemplaris, and its phosphorylation state is possibly regulated by PP2A.

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The bagworm genome reveals a unique fibroin gene that provides high tensile strength

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

Communications Biology (Communications Biology) 2 ( 1 ) 148 2019.12

Arthropod silk is known as a versatile tool, and its variability makes it an attractive biomaterial. Eumeta variegata is a bagworm moth (Lepidoptera, Psychidae) that uses silk throughout all life stages. Notably, the bagworm-specific uses of silk include larval development in a bag coated with silk and plant materials and the use of silk attachments to hang pupae. An understanding at the molecular level of bagworm silk, which enables such unique purposes, is an opportunity to expand the possibilities for artificial biomaterial design. However, very little is known about the bagworm fibroin gene and the mechanical properties of bagworm silk. Here, we report the bagworm genome, including a silk fibroin gene. The genome is approximately 700 Mbp in size, and the newly found fibroin gene has a unique repetitive motif. Furthermore, a mechanical property test demonstrates a phylogenetic relationship between the unique motif and tensile strength of bagworm silk.

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Peptide Synthesis under the Alkaline Hydrothermal Conditions on Enceladus

Takahagi W., Seo K., Shibuya T., Takano Y., Fujishima K., Saitoh M., Shimamura S., Matsui Y., Tomita M., Takai K.

ACS Earth and Space Chemistry 3 ( 11 ) 2559 - 2568 2019.11

Chemical evolution is an abiotic reaction process in which complex organic molecules arise from a combination of simple inorganic and organic chemical compounds. To assess the possible ongoing chemical evolution in the subsurface ocean of Saturn's icy satellite Enceladus, we explored the water-rock aqueous reactions and the peptide formation capability under a hydrothermal environment similar to that on Enceladus. It has been suggested that the core of Enceladus has not experienced high temperatures from the time of satellite formation to the present. The major components of the core are assumed to be carbonaceous chondrites; thus, simple organic substances, including amino acids, are likely present in the alkaline seawater of Enceladus. In this study, we conducted a laboratory-based simulation experiment to describe the chemical alteration of six prebiotically abundant amino acids over 147 days under high pressure with thermal cycling (30 to 100 °C) to simulate the water-rock interface of the ocean on Enceladus. As a result, we detected 28 out of 36 possible dipeptide species during the entire reaction period. We propose that peptide-bond formation is coupled to rock surface chemisorption of amino acids under alkaline condition, which was further supported by the elemental analysis showing carbon and nitrogen signature on the rock surface only when amino acids are added. The above result suggests that ongoing chemical evolution on Enceladus is likely producing short abiotic peptides on the porous core surface.

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Association between dyslipidemia and plasma levels of branched-chain amino acids in the Japanese population without diabetes mellitus

Fukushima K., Harada S., Takeuchi A., Kurihara A., Iida M., Fukai K., Kuwabara K., Kato S., Matsumoto M., Hirata A., Akiyama M., Tomita M., Hirayama A., Sato A., Suzuki C., Sugimoto M., Soga T., Sugiyama D., Okamura T., Takebayashi T.

Journal of Clinical Lipidology (Journal of Clinical Lipidology) 13 ( 6 ) 932 - 939.e2 2019.11

ISSN 19332874

Background: Branched-chain amino acids (BCAAs) play a key role in energy homeostasis. Objective: We aimed to investigate the association between plasma BCAA levels and dyslipidemia in the Japanese population without diabetes mellitus. Methods: This cross-sectional study included 4952 participants without diabetes mellitus, enrolled in the Tsuruoka Metabolomic Cohort Study. Plasma BCAA levels were measured by capillary electrophoresis–mass spectrometry. Correlations between lipid and BCAA profiles were evaluated by sex-stratified multiple linear regression analyses, after adjusting for confounders. Logistic regression was used to identify associations between BCAAs and metabolic dyslipidemia (MD) defined as triglyceride levels ≥150 mg/dL, high-density lipoprotein cholesterol levels ≤40 mg/dL for men and ≤50 mg/dL for women, or low-density lipoprotein cholesterol (LDL-C) levels ≥140 mg/dL. Results: In both sexes, the levels of individual BCAAs and the total BCAA levels correlated positively with triglyceride levels and negatively with high-density lipoprotein cholesterol levels. Valine, leucine, and total BCAA levels were weakly and positively correlated with LDL-C levels. Increased BCAA levels showed positive associations with MD. However, associations between BCAAs and elevated LDL-C levels were unclear. Furthermore, the associations between BCAA levels and MD regardless of fasting blood sugar (FBS) levels (high or low). Although valine, leucine, and total BCAA levels were weakly associated with elevated LDL-C levels in the high-FBS group, no such association was observed in the low-FBS group. Conclusions: BCAAs might be associated with MD independently of the FBS level and might play an important role in lipid metabolism and dyslipidemia.

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Draft Genome Sequences of Enterococcus faecalis Strains Isolated from Healthy Japanese Individuals

Tanaka K., Watabe T., Kato K., Tsukimi T., Sato M.P., Odamaki T., Tomita M., Fukuda S.

Microbiology Resource Announcements (Microbiology Resource Announcements) 8 ( 40 ) 2019.10

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Salivary metabolomics with alternative decision tree-based machine learning methods for breast cancer discrimination

Murata T., Yanagisawa T., Kurihara T., Kaneko M., Ota S., Enomoto A., Tomita M., Sugimoto M., Sunamura M., Hayashida T., Kitagawa Y., Jinno H.

Breast Cancer Research and Treatment (Breast Cancer Research and Treatment) 177 ( 3 ) 591 - 601 2019.10

ISSN 01676806

© 2019, Springer Science+Business Media, LLC, part of Springer Nature. Purpose: The aim of this study is to explore new salivary biomarkers to discriminate breast cancer patients from healthy controls. Methods: Saliva samples were collected after 9 h fasting and were immediately stored at − 80 °C. Capillary electrophoresis and liquid chromatography with mass spectrometry were used to quantify hundreds of hydrophilic metabolites. Conventional statistical analyses and artificial intelligence-based methods were used to assess the discrimination abilities of the quantified metabolites. A multiple logistic regression (MLR) model and an alternative decision tree (ADTree)-based machine learning method were used. The generalization abilities of these mathematical models were validated in various computational tests, such as cross-validation and resampling methods. Results: One hundred sixty-six unstimulated saliva samples were collected from 101 patients with invasive carcinoma of the breast (IC), 23 patients with ductal carcinoma in situ (DCIS), and 42 healthy controls (C). Of the 260 quantified metabolites, polyamines were significantly elevated in the saliva of patients with breast cancer. Spermine showed the highest area under the receiver operating characteristic curves [0.766; 95% confidence interval (CI) 0.671–0.840, P ' 0.0001] to discriminate IC from C. In addition to spermine, polyamines and their acetylated forms were elevated in IC only. Two hundred each of two-fold, five-fold, and ten-fold cross-validation using different random values were conducted and the MLR model had slightly better accuracy. The ADTree with an ensemble approach showed higher accuracy (0.912; 95% CI 0.838–0.961, P ' 0.0001). These prediction models also included spermine as a predictive factor. Conclusions: These data indicated that combinations of salivary metabolomics with the ADTree-based machine learning methods show potential for non-invasive screening of breast cancer.

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Large-Scale Molecular Evolutionary Analysis Uncovers a Variety of Polynucleotide Kinase Clp1 Family Proteins in the Three Domains of Life

Saito M., Sato A., Nagata S., Tamaki S., Tomita M., Suzuki H., Kanai A.

Genome Biology and Evolution (Genome Biology and Evolution) 11 ( 10 ) 2713 - 2726 2019.09

© 2019 The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. Clp1, a polyribonucleotide 5′-hydroxyl kinase in eukaryotes, is involved in pretRNA splicing and mRNA 3′-end formation. Enzymes similar in amino acid sequence to Clp1, Nol9, and Grc3, are present in some eukaryotes and are involved in prerRNA processing. However, our knowledge of how these Clp1 family proteins evolved and diversified is limited. We conducted a large-scale molecular evolutionary analysis of the Clp1 family proteins in all living organisms for which protein sequences are available in public databases. The phylogenetic distribution and frequencies of the Clp1 family proteins were investigated in complete genomes of Bacteria, Archaea and Eukarya. In total, 3,557 Clp1 family proteins were detected in the three domains of life, Bacteria, Archaea, and Eukarya. Many were from Archaea and Eukarya, but a few were found in restricted, phylogenetically diverse bacterial species. The domain structures of the Clp1 family proteins also differed among the three domains of life. Although the proteins were, on average, 555 amino acids long (range, 196-2,728), 122 large proteins with >1,000 amino acids were detected in eukaryotes. These novel proteins contain the conserved Clp1 polynucleotide kinase domain and various other functional domains. Of these proteins, >80% were from Fungi or Protostomia. The polyribonucleotide kinase activity of Thermus scotoductus Clp1 (Ts-Clp1) was characterized experimentally. Ts-Clp1 preferentially phosphorylates single-stranded RNA oligonucleotides (Km value for ATP, 2.5 μM), or single-stranded DNA at higher enzyme concentrations. We propose a comprehensive assessment of the diversification of the Clp1 family proteins and the molecular evolution of their functional domains.

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Chlorococcum turfosum comb. nov. (Volvocales, Chlorophyceae) and visualization of chimeric sequences

Nakada T., Watanabe S., Lewis L., Tomita M.

Phycological Research 67 ( 3 ) 239 - 243 2019.07

ISSN 13220829

© 2019 Japanese Society of Phycology Parachlorococcum turfosum was previously described based on morphological studies of a culture collection strain originally identified as ‘Chlorococcum elkhartiense’ and on an 18S rRNA gene phylogeny of a strain derived from the same original strain but from a different culture collection. However, a subsequently published 18S rRNA gene sequence was inconsistent with the original sequence. Therefore, a new 18S rRNA gene sequence was obtained from the strain from which the holotype was derived. Comparative analyses of the new sequence using the basic local alignment search tool and a newly written Perl code, ChimViewP, which visualizes nucleotide signature shifts characteristic of chimeric sequences, revealed that the sequence used in the original description was of a chimeric nature. Similar analyses also indicated that two published ‘Chlamydomonas acidophila’ sequences are chimeric. Phylogenetic analyses excluding all putative chimeras showed that P. turfosum belongs to Chlorococcum, and thus Chlorococcum turfosum comb. nov. is proposed.

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Genomic analysis of Pseudomonas sp. Strain SCT, an iodate-reducing bacterium isolated from marine sediment, reveals a possible use for bioremediation

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

G3: Genes, Genomes, Genetics (G3: Genes, Genomes, Genetics) 9 ( 5 ) 1321 - 1329 2019.05

Copyright © 2019 Harada et al. Strain SCT is an iodate-reducing bacterium isolated from marine sediment in Kanagawa Prefecture, Japan. In this study, we determined the draft genome sequence of strain SCT and compared it to complete genome sequences of other closely related bacteria, including Pseudomonas stutzeri. A phylogeny inferred from concatenation of core genes revealed that strain SCT was closely related to marine isolates of P. stutzeri. Genes present in the SCT genome but absent from the other analyzed P. stutzeri genomes comprised clusters corresponding to putative prophage regions and possible operons. They included pil genes, which encode type IV pili for natural transformation; the mer operon, which encodes resistance systems for mercury; and the pst operon, which encodes a Pi-specific transport system for phosphate uptake. We found that strain SCT had more prophage-like genes than the other P. stutzeri strains and that the majority (70%) of them were SCT strain-specific. These genes, encoded on distinct prophage regions, may have been acquired after branching from a common ancestor following independent phage transfer events. Thus, the genome sequence of Pseudomonas sp. strain SCT can provide detailed insights into its metabolic potential and the evolution of genetic elements associated with its unique phenotype.

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Stable and efficient delivery of DNA to Bacillus subtilis (natto) using pLS20 conjugational transfer plasmids

Itaya M., Nagasaku M., Shimada T., Ohtani N., Shiwa Y., Yoshikawa H., Kaneko S., Tomita M., Sato M.

FEMS Microbiology Letters (FEMS Microbiology Letters) 366 ( 4 ) 2019.02

ISSN 03781097

© FEMS 2019. Bacillus subtilis (natto) is generally regarded as a safe bacterium and used as a host for the production of several materials. However, genetic engineering of B. subtilis (natto) is not well established because of poor DNA delivery methods and the lack of a standard strain for the aim. Here, we developed a genetic delivery tool in B. subtilis (natto) using the pLS20 conjugational plasmid (65 kbp). Transmission of pLS20 from B. subtilis 168 to wild-type B. subtilis (natto) did not occur via established mating protocols. We isolated B. subtilis (natto) mutants showing dramatically increased recipient activity. Whole-genome sequence analyses revealed three common alterations: mutations in the restriction endonuclease gene and in the methyl-accepting chemotaxis protein gene, and a 43-kbp deletion at the genome replication termination locus. A representative strain named NEST116 was generated as the first B. subtilis (natto) strain suitable for exploring pLS20-based genetic engineering.

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Effects of inter-day and intra-day variation on salivary metabolomic profiles

Kawanishi N., Hoshi N., Masahiro S., Enomoto A., Ota S., Kaneko M., Soga T., Tomita M., Kimoto K.

Clinica Chimica Acta (Clinica Chimica Acta) 489 41 - 48 2019.02

ISSN 00098981

© 2018 Elsevier B.V. Background: Salivary secretion is an important parameter reflecting the health status of an individual and has been used clinically for the diagnosis of various oral diseases, such as xerostomia. Salivary metabolomic profiling is considered an emerging potential tool for the detection of various systemic diseases. To our knowledge, this is the first study to investigate the quantitative relationship between salivary secretion volume and salivary metabolomic profile. Methods: To evaluate inter- and intra-day variations in salivary secretion, 234 saliva samples were collected three times per day for three days from 13 subjects and analyzed. Capillary electrophoresis-mass spectrometry was used for non-targeted quantification of water-soluble metabolites. Results: No significant inter- or intra-day variations were observed in salivary secretion volume. No significant inter-day variations were observed in metabolomic patterns. In contrast, significant intra-day variations were observed in salivary metabolomic profiles. The difference was more obvious for stimulated saliva than for unstimulated saliva. These profile changes were independent of salivary secretion volume. Conclusions: Our results indicated that diurnal change had a greater effect on salivary metabolomic profiles than the other factors. Hence, sampling time should be tightly controlled to minimize unexpected bias in the clinical use of salivary metabolomics.

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Perioperative serum and urine metabolome analyses in patients with hepatocellular carcinoma undergoing partial hepatectomy

Kajiura D., Yamanaka-Okumura H., Hirayama A., Tatano H., Endo K., Honma M., Igarashi K., Shoji F., Ikeda S., Yamaguchi N., Katayama T., Morine Y., Imura S., Utsunomiya T., Soga T., Tomita M., Shimada M.

Nutrition (Nutrition) 58 110 - 119 2019.02

ISSN 08999007

© 2018 Elsevier Ltd Objectives: Perioperative nutritional management is essential for early recovery after liver surgery. The aim of this study was to assess changes in amino acid levels in serum and urine after hepatectomy. Methods: Serum samples were collected from 16 patients with hepatocellular carcinoma before and 1, 3, and 14 d after hepatectomy (S0, S1, S3, and S14, respectively). Spot urine samples were collected before and 3 d after the hepatectomy (U0 and U3). Metabolites in the serum and urine were analyzed. Results: Compared with S0, insulin levels significantly increased in the S1 and S3 samples. Valine levels significantly decreased in S1 and S14, and leucine levels significantly decreased in S14. Phenylalanine levels significantly increased in S1 and S3, and tyrosine levels significantly increased in S1. The Fischer ratio (branched-chain/aromatic amino acids) significantly decreased in S1 and S3. In multiple regression analysis, changes in serum taurine levels were related to the white blood cell count in S1 and S3, and inversely related to alanine aminotransferase levels in S14. Changes in serum glutamine levels were negatively related to C-reactive protein levels in S3. Serum glutamine levels decreased in S3 and S14, and tended to increase in U3, suggesting a deficiency of glutamate resulting from the invasive surgical procedure. Conclusions: These findings highlight the usefulness of metabolome analysis for characterizing perioperative patterns after liver resection. The observed amino acid pattern, including the reduction in Fischer ratio, underscores the need for specialized nutritional support.

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Comparative genomics of Bacteria commonly identified in the built environment

Merino N., Zhang S., Tomita M., Suzuki H.

BMC Genomics (BMC Genomics) 20 ( 1 ) 92 2019.01

© 2019 The Author(s). Background: The microbial community of the built environment (BE) can impact the lives of people and has been studied for a variety of indoor, outdoor, underground, and extreme locations. Thus far, these microorganisms have mainly been investigated by culture-based methods or amplicon sequencing. However, both methods have limitations, complicating multi-study comparisons and limiting the knowledge gained regarding in-situ microbial lifestyles. A greater understanding of BE microorganisms can be achieved through basic information derived from the complete genome. Here, we investigate the level of diversity and genomic features (genome size, GC content, replication strand skew, and codon usage bias) from complete genomes of bacteria commonly identified in the BE, providing a first step towards understanding these bacterial lifestyles. Results: Here, we selected bacterial genera commonly identified in the BE (or "Common BE genomes") and compared them against other prokaryotic genera ("Other genomes"). The "Common BE genomes" were identified in various climates and in indoor, outdoor, underground, or extreme built environments. The diversity level of the 16S rRNA varied greatly between genera. The genome size, GC content and GC skew strength of the "Common BE genomes" were statistically larger than those of the "Other genomes" but were not practically significant. In contrast, the strength of selected codon usage bias (S value) was statistically higher with a large effect size in the "Common BE genomes" compared to the "Other genomes." Conclusion: Of the four genomic features tested, the S value could play a more important role in understanding the lifestyles of bacteria living in the BE. This parameter could be indicative of bacterial growth rates, gene expression, and other factors, potentially affected by BE growth conditions (e.g., temperature, humidity, and nutrients). However, further experimental evidence, species-level BE studies, and classification by BE location is needed to define the relationship between genomic features and the lifestyles of BE bacteria more robustly.

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Fast and global detection of periodic sequence repeats in large genomic resources

Mori H., Evans-Yamamoto D., Ishiguro S., Tomita M., Yachie N.

Nucleic acids research (Nucleic Acids Research) 47 ( 2 ) e8 2019.01

ISSN 03051048

Periodically repeating DNA and protein elements are involved in various important biological events including genomic evolution, gene regulation, protein complex formation, and immunity. Notably, the currently used genome editing tools such as ZFNs, TALENs, and CRISPRs are also all associated with periodically repeating biomolecules of natural organisms. Despite the biological importance of periodically repeating sequences and the expectation that new genome editing modules could be discovered from such periodical repeats, no software that globally detects such structured elements in large genomic resources in a high-throughput and unsupervised manner has been developed. We developed new software, SPADE (Search for Patterned DNA Elements), that exhaustively explores periodic DNA and protein repeats from large-scale genomic datasets based on k-mer periodicity evaluation. With a simple constraint, sequence periodicity, SPADE captured reported genome-editing-associated sequences and other protein families involving repeating domains such as tetratricopeptide, ankyrin and WD40 repeats with better performance than the other software designed for limited sets of repetitive biomolecular sequences, suggesting the high potential of this software to contribute to the discovery of new biological events and new genome editing modules.

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Metabolomics Platform with Capillary Electrophoresis Coupled with High-Resolution Mass Spectrometry for Plasma Analysis

Sasaki K., Sagawa H., Suzuki M., Yamamoto H., Tomita M., Soga T., Ohashi Y.

Analytical Chemistry (Analytical Chemistry) 91 ( 2 ) 1295 - 1301 2019.01

ISSN 00032700

© 2018 American Chemical Society. Metabolome analysis using capillary electrophoresis (CE) coupled with high-resolution mass spectrometry (HRMS) has the potential to improve coverage of metabolite detection because of its high selectivity and sensitivity. Configuration of the interface between CE and HRMS to meet the ground connection is essential for enabling independent regulation of the electrical currents in the CE and electrospray field. In the present study, we applied an electrospray-ionization adapter equipped with a grounded nebulizer to CE-HRMS and tested the analytical performance for 34 charged compounds. The extracted-ion electropherograms, consisting of seven sets of isomers, showed reasonable peak shapes and separation for the annotation of each metabolite. The levels of 34 target analytes in a standard mixture were determined with a dynamic range of at least 10 2 , maintaining linearity with r 2 > 0.9. The repeatability and intermediate precision above the lower limit of quantification showed the relative standard deviation to be lower than 20%. In the spike-recovery experiment, 27 of the 34 metabolites in plasma extract were recovered at a rate of 80 to 120%, suggesting high accuracy. Furthermore, we assessed the feasibility of our platform in metabolome analysis using human-plasma extract. The results showed successful detection of 270 metabolites, indicating the potential of our platform to yield higher coverage of the metabolome. In addition, analysis of dilution integrity demonstrated the quantitative ability of metabolome analysis with CE-HRMS, although the existence of saturation or matrix effects were seen in the case of 33 of the metabolites. This study indicates that our platform has great potential for large-scale metabolome analysis of plasma for biological studies and clinical biomarker screening.

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Reconsidering plasmid maintenance factors for computational plasmid design

Yano H., Shintani M., Tomita M., Suzuki H., Oshima T.

Computational and Structural Biotechnology Journal (Computational and Structural Biotechnology Journal) 17 70 - 81 2019.01

ISSN 2001-0370

© 2018 The Authors Plasmids are genetic parasites of microorganisms. The genomes of naturally occurring plasmids are expected to be polished via natural selection to achieve long-term persistence in the microbial cell population. However, plasmid genomes are extremely diverse, and the rules governing plasmid genomes are not fully understood. Therefore, computationally designing plasmid genomes optimized for model and nonmodel organisms remains challenging. Here, we summarize current knowledge of the plasmid genome organization and the factors that can affect plasmid persistence, with the aim of constructing synthetic plasmids for use in gram-negative bacteria. Then, we introduce publicly available resources, plasmid data, and bioinformatics tools that are useful for computational plasmid design.

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Improved taxon sampling and multigene phylogeny of unicellular chlamydomonads closely related to the colonial volvocalean lineage Tetrabaenaceae-Goniaceae-Volvocaceae (Volvocales, Chlorophyceae)

Nakada T., Tsuchida Y., Tomita M.

Molecular Phylogenetics and Evolution (Molecular Phylogenetics and Evolution) 130 1 - 8 2019.01

ISSN 10557903

© 2018 Elsevier Inc. In the green algal order Volvocales (Chlorophyceae), flagellate colonial forms have evolved at least four times. One of these colonial lineages, Tetrabaenaceae-Goniaceae-Volvocaceae (TGV), which belongs to the clade Reinhardtinia, is closely related to several unicellular chlamydomonads in the genera Chlamydomonas and Vitreochlamys. However, the unicellular sister of TGV has not been specified. Here, the largest ever 18S rRNA phylogenetic tree of Reinhardtinia was constructed including several newly isolated chlamydomonads, and a clade (core-Reinhardtinia) including 32 unicellular lineages and three colonial families were recognized. Interrelationships within core-Reinhardtinia were barely resolved in the tree, and therefore combined 18S-atpB-psaA-psaB-psbC-rbcL gene phylogenetic analyses were performed with selected representatives of 29 of the 32 unicellular lineages and three colonial families. The 29 unicellular lineages were clustered into five metaclades and an unassigned lineage; the metaclade that includes Chlamydomonas pila was resolved, with moderate support, as the sister clade to TGV. To examine possible biases from specific gene(s), long-branch taxa, and the heterogeneous base composition, phylogenetic analyses using several smaller data sets were also performed. Light microscopy of C. pila and its relatives indicated that any early steps towards colony evolution appeared after divergence of TGV from the C. pila lineage.

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Metabolome profiling of various seaweed species discriminates between brown, red, and green algae

Hamid S., Wakayama M., Ichihara K., Sakurai K., Ashino Y., Kadowaki R., Soga T., Tomita M.

Planta (Planta) 249 ( 6 ) 1921 - 1947 2019

ISSN 00320935

© 2019, Springer-Verlag GmbH Germany, part of Springer Nature. Main Conclusion: Among seaweed groups, brown algae had characteristically high concentrations of mannitol, and green algae were characterised by fructose. In red algae, metabolite profiles of individual species should be evaluated. Seaweeds are metabolically different from terrestrial plants. However, general metabolite profiles of the three major seaweed groups, the brown, red, and green algae, and the effect of various extraction methods on metabolite profiling results have not been comprehensively explored. In this study, we evaluated the water-soluble metabolites in four brown, five red, and two green algae species collected from two sites in northern Japan, located in the Sea of Japan and the Pacific Ocean. Freeze-dried seaweed samples were processed by methanol–water extraction with or without chloroform and analysed by capillary electrophoresis- and liquid chromatography-mass spectrometry for metabolite characterisation. The metabolite concentration profiles showed distinctive characteristic depends on species and taxonomic groups, whereas the extraction methods did not have a significant effect. Taxonomic differences between the various seaweed metabolite profiles were well defined using only sugar metabolites but no other major compound types. Mannitol was the main sugar metabolites in brown algae, whereas fructose, sucrose, and glucose were found at high concentrations in green algae. In red algae, individual species had some characteristic metabolites, such as sorbitol in Pyropia pseudolinearis and panose in Dasya sessilis. The metabolite profiles generated in this study will be a resource and provide guidance for nutraceutical research studies because the information about metabolites in seaweeds is still very limited compared to that of terrestrial plants.

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Manufacturing of biomimetic silicone rubber films for experimental fluid mechanics: 3D printed shark skin molds

Yasuda Y., Zhang K., Sasaki O., Tomita M., Rival D., Galipon J.

Journal of the Electrochemical Society 166 ( 9 ) B3302 - B3308 2019

ISSN 00134651

The skin is at the interface of living organisms and their environment, and has evolved interesting structural properties usually over the course of millions of years, providing clues for the design of manmade biomimetic surfaces with favorable fluid mechanics properties. Here, we describe steps to produce silicone rubber films based on microscopy data from shark skin denticles, from data acquisition and manufacturing to attachment to an airfoil for experimental fluid dynamics in large towing tanks. This method is relatively low-cost, may be generalized to other types of patterned micro-structured surfaces, and the manufacturing process may be reproduced by anyone equipped with a 3D printer.

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Cutibacterium acnes (Propionibacterium acnes) 16S rRNA genotyping of microbial samples from possessions contributes to owner identification

Yang J., Tsukimi T., Yoshikawa M., Suzuki K., Takeda T., Tomita M., Fukuda S.

mSystems (mSystems) 4 ( 6 ) 2019

ISSN 2379-5077

The human skin surface harbors huge numbers of microbes. The skin microbiota interacts with its host and forms a skin microbiome profile that is specific for each individual. It has been reported that the skin microbiota that is left on an individual’s possessions can act as a sort of “fingerprint” and be used for owner identification. However, this approach needs to be improved to take into account any long-term instability of skin microbiota and contamination from nonspecific bacteria. Here, we took advantage of single-nucleotide polymorphisms (SNPs) in the 16S-encoding rRNA gene of Cutibacterium acnes, the most common and abundant bacterium on human skin, to perform owner identification. We first developed a high-throughput genotyping method based on next-generation sequencing to characterize the SNPs of the C. acnes 16S rRNA gene and found that the genotype composition of C. acnes 16S rRNA is individual specific. Owner identification accuracy of around 90% based on random forest machine learning was achieved by using a combination of C. acnes 16S rRNA genotype and skin microbiome profile data. Furthermore, our study showed that the C. acnes 16S rRNA genotype remained more stable over time than the skin microbiome profile. This characteristic of C. acnes was further confirmed by the analysis of publicly available human skin metagenome data. Our approach, with its high precision, good reproducibility, and low costs, thus provides new possibilities in the field of microbiome-based owner identification and forensics in general. IMPORTANCE Cutibacterium acnes is the most common and abundant bacterial species on human skin, and the gene that encodes its 16S rRNA has multiple single-nucleotide polymorphisms. In this study, we developed a method to efficiently determine the C. acnes 16S rRNA genotype composition from microbial samples taken from the hands of participants and from their possessions. Using the C. acnes 16S rRNA genotype composition, we could predict the owner of a possession with around 90% accuracy when the 16S rRNA gene-based microbiome profile was included. We also showed that the C. acnes 16S rRNA genotype composition was more stable over time than the skin microbiome profile and thus is more suitable for owner identification.

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Strand-specific Single-stranded DNA Sequencing (4S-seq) of E. coli genomes

Masuda T, Kono N, Tomita M, Arakawa K.

Bio-protocol (Bio-protocol) 9 ( 15 ) e3329 2019

Most bacterial genomes have biased nucleotide composition, and the asymmetry is considered to be caused by a single-stranded DNA (ssDNA) deamination arising from the bacterial replication machinery. In order to evaluate the relationship experimentally, the position and frequency of ssDNA formed during replication must be verified clearly. Although many ssDNA detection technologies exist, almost all methods have been developed for eukaryotic genomes. To apply these to bacterial genomes, which harbor a smaller amount of DNA than those of eukaryotes, more efficient, new methods are required. Therefore, we developed a novel strand-specific ssDNA sequencing method, called 4S-seq, for the bacterial genome. The 4S-seq method enriches ssDNA in the extracted genomic DNA by a dsDNA-specific nuclease and implements a strand-specific library using a biotin label with a customized tag. As a result, the 4S-seq is able to calculate the ssDNA content in each strand (Watson/Crick) at each position of the genome efficiently.

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Complete Genome Sequence of Arthrobacter sp. Strain MN05-02, a UV-Resistant Bacterium from a Manganese Deposit in the Sonoran Desert

Ii KM, Kono N, Paulino-Lima IG, Tomita M, Rothschild LJ, Arakawa K.

Journal of genomics 7 18 - 25 2019

ISSN 1839-9940

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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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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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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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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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Dynamic Organization of Chromatin Domains Revealed by Super-Resolution Live-Cell Imaging

Nozaki T, Imai R, Tanbo M, Nagashima R, Tamura S, Tani T, Joti Y, Tomita M, Hibino K, Kanemaki MT, Wendt KS, Okada Y, Nagai T, Maeshima K.

Molecular Cell (Molecular Cell) 67 ( 2 ) 282-293 - 293.e7 2017

ISSN 10972765

The eukaryotic genome is organized within cells as chromatin. For proper information output, higher-order chromatin structures can be regulated dynamically. How such structures form and behave in various cellular processes remains unclear. Here, by combining super-resolution imaging (photoactivated localization microscopy [PALM]) and single-nucleosome tracking, we developed a nuclear imaging system to visualize the higher-order structures along with their dynamics in live mammalian cells. We demonstrated that nucleosomes form compact domains with a peak diameter of ∼160 nm and move coherently in live cells. The heterochromatin-rich regions showed more domains and less movement. With cell differentiation, the domains became more apparent, with reduced dynamics. Furthermore, various perturbation experiments indicated that they are organized by a combination of factors, including cohesin and nucleosome-nucleosome interactions. Notably, we observed the domains during mitosis, suggesting that they act as building blocks of chromosomes and may serve as information units throughout the cell cycle.

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A serum metabolomics-based profile in low bone mineral density postmenopausal women

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

Bone (Bone) 95 1 - 4 2017

Joint Work, Accepted, ISSN 87563282

Osteoporosis is characterized as a metabolic disorder of bone tissue, and various metabolic markers are now available to support its diagnosis and evaluate treatment effects. Substances produced as end products of metabolomic activities are the correlated factors to the biological or metabolic status, and thus, metabolites are considered highly sensitive markers of particular pathological states, including osteoporosis. Here we undertook comprehensive serum metabolomics analysis in postmenopausal women with or without low bone mineral density (low BMD vs controls) for the first time using capillary electrophoresis/mass spectrometry. Among the metabolites tested, 57 were detected in sera. Levels of hydroxyproline, Gly-Gly and cystine, differed significantly between groups, with Gly-Gly and cystine significantly lower in the low BMD group and hydroxyproline, a reported marker of osteoporosis, significantly higher. Levels of TRACP5b, a bone resorption marker, were significantly higher in the low BMD group, supporting the study's validity. Taken together, our findings represent novel metabolomic profiling in low BMD in postmenopausal women.

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Metabolomic Profiling as a Possible Reverse Engineering Tool for Estimating Processing Conditions of Dry-Cured Hams.

Sugimoto M, Obiya S, Kaneko M, Enomoto A, Honma M, Wakayama M, Soga T, Tomita M.

Journal of Agricultural and Food Chemistry (Journal of Agricultural and Food Chemistry) 65 ( 2 ) 402 - 410 2017

Joint Work, Accepted, ISSN 00218561

Dry-cured hams are popular among consumers. To increase the attractiveness of the product, objective analytical methods and algorithms to evaluate the relationship between observable properties and consumer acceptability are required. In this study, metabolomics, which is used for quantitative profiling of hundreds of small molecules, was applied to 12 kinds of drycured hams from Japan and Europe. In total, 203 charged metabolites, including amino acids, organic acids, nucleotides, and peptides, were successfully identified and quantified. Metabolite profiles were compared for the samples with different countries of origin and processing methods (e.g., smoking or use of a starter culture). Principal component analysis of the metabolite profiles with sensory properties revealed significant correlations for redness, homogeneity, and fat whiteness. This approach could be used to design new ham products by objective evaluation of various features.

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Rearing and observation of immature stages of the hoverfly Microdon katsurai (Diptera, Syrphidae)

Iwai,H., Horikawa, D., Arakawa, K., Tomita, M., Komatsu, T. and Maruyama, M.

Biodiversity Data Journal (Biodiversity Data Journal) 4 ( 1 ) e10185 2017

Joint Work, Accepted, ISSN 1314-2828

Background The hoverfly Microdon (Chymophila) katsurai Maruyama et Hironaga 2004 was speculated to be a myrmecophilous species associated with the ant Polyrhachis lamellidens based on observations of adults near the ant nest. However, there have been no reports regarding the observation of immature stages of this species in association with P. lamellidens. New information For the first time, we found three M. katsurai larvae inside a P. lamellidens nest and conducted rearing experiments on the larval M. katsurai. P. lamellidens workers did not show any inspection or attack behavior against the M. katsurai larvae under rearing conditions, suggesting that M. katsurai larvae can survive inside a P. lamellidens nest. Although no predatory behavior by the M. katsurai larvae was observed, all the M. katsurai larvae pupated and emerged in a rearing environment. The dorsal surface of the larval M. katsurai has a distinct pale green color with a uniform reticular structure. The puparium of M. katsurai shows several morphological features that are characteristic of the subgenus Chymophila. We conclude that M. katsurai is likely a myrmecophilous species that utilizes P. lamellidens as a specific host and that classification of M. katsurai based on puparium morphology is concordant with that based on adult morphology.

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Alteration of fatty acid chain length of Chlamydomonas reinhardtii by simultaneous expression of medium-chain-specific thioesterase and acyl carrier protein

*Inaba, Y., Nakahigashi, K., Ito, T. and Tomita, M.

Phycological Research 65 ( 1 ) 94 - 99 2017

Joint Work, Accepted, ISSN 13220829

Biofuel from fatty acids with chain lengths of 8–15 (C8–C15) have properties similar to those of conventional diesel and jet fuels, thus, can save time and reduce costs for the refurbishment of engines and maintenance of oiling facilities. Most oil-producing algae yield C16–C18 fatty acids; however, the manipulation of algae using genetic engineering is a promising approach to obtain C8–C15 fatty acids. The introduction of a medium-chain-specific thioesterase (TE) is expected to effectively alter algae to produce medium-chain fatty acids (MCFAs). TE is the main determinant of fatty acid chain length as it releases fatty acids from the acyl carrier protein (ACP) in the fatty acid elongation cycle. In a previous study, the introduction of heterologous C8–C12-specific TEs into Chlamydomonas reinhardtii did not increase the yield of MCFAs. This effect was attributed to a low affinity of the heterologous TEs to C. reinhardtii ACP. Therefore, we introduced both the C10–C14-specific TE gene and the ACP gene from the land plant Cuphea lanceolata into C. reinhardtii. We measured free fatty acids (FFAs) and triacylglycerols (TAGs) in the transformants using liquid chromatography–mass spectrometry. The production of C12:0 and C14:0, chain length 12 and 14 without unsaturation, FFAs was not significantly increased in any of the tested strains. However, we found a slight but significant increase in TAG-containing MCFAs in both TE only and TE–ACP transformants. The increased production rate of C14:0-containing TAGs ranged from 1.25- to 1.58-fold, indicating the ability of medium-chain-specific TE to increase MCFAs. These results suggest that the selection of specific TEs is important when modifying eukaryotic algae to produce MCFAs.

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Differential binding of three major human ADAR isoforms to coding and long non-coding transcripts.

Galipon, J., Ishii, R., Suzuki, Y., Tomita, M., and * Ui-Tei, K.

Genes (Genes) 8 ( 2 ) 68 2017

Joint Work, Accepted, ISSN 2073-4425

RNA editing by deamination of adenosine to inosine is an evolutionarily conserved process involved in many cellular pathways, from alternative splicing to miRNA targeting. In humans, it is carried out by no less than three major adenosine deaminases acting on RNA (ADARs): ADAR1-p150, ADAR1-p110, and ADAR2. However, the first two derive from alternative splicing, so that it is currently impossible to delete ADAR1-p110 without also knocking out ADAR1-p150 expression. Furthermore, the expression levels of ADARs varies wildly among cell types, and no study has systematically explored the effect of each of these isoforms on the cell transcriptome. In this study, RNA immunoprecipitation (RIP)-sequencing on overexpressed ADAR isoforms tagged with green fluorescent protein (GFP) shows that each ADAR is associated with a specific set of differentially expressed genes, and that they each bind to distinct set of RNA targets. Our results show a good overlap with known edited transcripts, establishing RIP-seq as a valid method for the investigation of RNA editing biology.

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Developmental changes in the balance of glycolytic ATP production and oxidative phosphorylation in ventricular cells: A simulation study

Sano HI, Toki T, Naito Y, Tomita M.

Journal of Theoretical Biology (Journal of Theoretical Biology) 419 269 - 277 2017

Joint Work, Accepted, ISSN 00225193

The developmental program of the heart requires accurate regulation to ensure continuous circulation and simultaneous cardiac morphogenesis, because any functional abnormalities may progress to congenital heart malformation. Notably, energy metabolism in fetal ventricular cells is regulated in a manner that differs from adult ventricular cells: fetal cardiomyocytes generally have immature mitochondria and fetal ventricular cells show greater dependence on glycolytic ATP production. However, although various characteristics of energy metabolism in fetal ventricular cells have been reported, to our knowledge, a quantitative description of the contributions of these factors to fetal ventricular cell functions has not yet been established. Here, we constructed a mathematical model to integrate various characteristics of fetal ventricular cells and predicted the contribution of each characteristic to the maintenance of intracellular ATP concentration and sarcomere contraction under anoxic conditions. Our simulation results demonstrated that higher glycogen content, higher hexokinase activity, and lower creatine concentration helped prolong the time for which ventricular cell contraction was maintained under anoxic conditions. The integrated model also enabled us to quantitatively assess the contributions of factors related to energy metabolism in ventricular cells. Because fetal cardiomyocytes exhibit similar energy metabolic profiles to stem cell-derived cardiomyocytes and those in the failing heart, an improved understanding of these fetal ventricular cells will contribute to a better comprehension of the processes in stem cell-derived cardiomyocytes or under pathological conditions.

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Evolutionary Analysis of HIV-1 Pol Proteins Reveals Representative Residues for Viral Subtype Differentiation

Nagata S, Imai J, Makino G, Tomita M, Kanai A.

Frontiers in Microbiology (Frontiers in Microbiology) 8 ( NOV ) 2151 2017

ISSN 1664-302X

RNA viruses have been used as model systems to understand the patterns and processes of molecular evolution because they have high mutation rates and are genetically diverse. Human immunodeficiency virus 1 (HIV-1), the etiological agent of acquired immune deficiency syndrome, is highly genetically diverse, and is classified into several groups and subtypes. However, it has been difficult to use its diverse sequences to establish the overall phylogenetic relationships of different strains or the trends in sequence conservation with the construction of phylogenetic trees. Our aims were to systematically characterize HIV-1 subtype evolution and to identify the regions responsible for HIV-1 subtype differentiation at the amino acid level in the Pol protein, which is often used to classify the HIV-1 subtypes. In this study, we systematically characterized the mutation sites in 2,052 Pol proteins from HIV-1 group M (144 subtype A; 1,528 subtype B; 380 subtype C), using sequence similarity networks. We also used spectral clustering to group the sequences based on the network graph structures. A stepwise analysis of the cluster hierarchies allowed us to estimate a possible evolutionary pathway for the Pol proteins. The subtype A sequences also clustered according to when and where the viruses were isolated, whereas both the subtype B and C sequences remained as single clusters. Because the Pol protein has several functional domains, we identified the regions that are discriminative by comparing the structures of the domain-based networks. Our results suggest that sequence changes in the RNase H domain and the reverse transcriptase (RT) connection domain are responsible for the subtype classification. By analyzing the different amino acid compositions at each site in both domain sequences, we found that a few specific amino acid residues (i.e., M357 in the RT connection domain and Q480, Y483, and L491 in the RNase H domain) represent the differences among the subtypes. These residues were located on the surface of the RT structure and in the vicinity of the amino acid sites responsible for RT enzymatic activity or function.

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Thymidine Catabolism as a Metabolic Strategy for Cancer Survival

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, Hanibuchi M, Nishioka Y, Sone S, Esumi H, Tomita M, Soga T, Furukawa T, Akiyama SI.

Cell Reports (Cell Reports) 19 ( 7 ) 1313-1321 - 1321 2017

Thymidine phosphorylase (TP), a rate-limiting enzyme in thymidine catabolism, plays a pivotal role in tumor progression; however, the mechanisms underlying this role are not fully understood. Here, we found that TP-mediated thymidine catabolism could supply the carbon source in the glycolytic pathway and thus contribute to cell survival under conditions of nutrient deprivation. In TP-expressing cells, thymidine was converted to metabolites, including glucose 6-phosphate, lactate, 5-phospho-α-D-ribose 1-diphosphate, and serine, via the glycolytic pathway both in vitro and in vivo. These thymidine-derived metabolites were required for the survival of cells under low-glucose conditions. Furthermore, activation of thymidine catabolism was observed in human gastric cancer. These findings demonstrate that thymidine can serve as a glycolytic pathway substrate in human cancer cells.

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

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

Journal of genomics 5 51 - 53 2017

ISSN 1839-9940

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18S Ribosomal RNA Gene Phylogeny of a Colonial Volvocalean Lineage (Tetrabaenaceae-Goniaceae-Volvocaceae, Volvocales, Chlorophyceae) and Its Close Relatives

Takashi Nakada, Takuro Ito, Masaru Tomita

The Journal of Japanese Botany 345 - 354 2016.12

Serum metabolome profiles characterized by patients with hepatocellular carcinoma associated with hepatitis B and C

Saito T, Sugimoto M, Okumoto K, Haga H, Katsumi T, Mizuno K, Nishina T, Sato S, Igarashi K, Maki H, Tomita M, Ueno Y, Soga T.

World Journal of Gastroenterology (World Journal of Gastroenterology) 22 ( 27 ) 6224 - 6234 2016.07

ISSN 10079327

AIM: To clarify the characteristics of metabolite profiles in virus-related hepatocellular carcinoma (HCC) patients using serum metabolome analysis. METHODS: The serum levels of low-molecular-weight metabolites in 68 patients with HCC were quantified using capillary electrophoresis chromatography and mass spectrometry. Thirty and 38 of the patients suffered from hepatitis B virus-related HCC (HCC-B) and hepatitis C virus-related HCC (HCC-C), respectively. RESULTS: The main metabolites characteristic of HCC were those associated with glutathione metabolism, notably 13 γ-glutamyl peptides, which are by-products of glutathione induction. Two major profiles, i.e. , concentration patterns, of metabolites were identified in HCC patients, and these were classified into two groups: an HCC-B group and an HCC-C group including some of the HCC-B cases. The receiver operating characteristic curve for the multiple logistic regression model discriminating HCC-B from HCC-C incorporating the concentrations of glutamic acid, methionine and γ-glutamyl-glycine-glycine showed a highly significant area under the curve value of 0.94 (95%CI: 0.89-1.0, P < 0.0001). CONCLUSION: The serum levels of γ-glutamyl peptides, as well as their concentration patterns, contribute to the development of potential biomarkers for virus-related HCC. The difference in metabolite profiles between HCC-B and HCC-C may reflect the respective metabolic reactions that underlie the different pathogeneses of these two types of HCC.

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Erratum to: Metabolomic profiling reveals novel biomarkers of alcohol intake and alcohol-induced liver injury in community-dwelling men.

Harada S, Takebayashi T, Kurihara A, Akiyama M, Suzuki A, Hatakeyama Y, Sugiyama D, Kuwabara K, Takeuchi A, Okamura T, Nishiwaki Y, Tanaka T, Hirayama A, Sugimoto M, Soga T, Tomita M

Environmental health and preventive medicine 21 ( 4 ) 283 - 284 2016.07

ISSN 1342-078X

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Smad2/3 proteins are required for immobilization-induced skeletal muscle atrophy

Tando T, Hirayama A, Furukawa M, Sato Y, Kobayashi T, Funayama A, Kanaji A, Hao W, Watanabe R, Morita M, Oike T, Miyamoto K, Soga T, Nomura M, Yoshimura A, Tomita M, Matsumoto M, Nakamura M, Toyama Y, Miyamoto T.

Journal of Biological Chemistry (Journal of Biological Chemistry) 291 ( 23 ) 12184 - 12194 2016.06

ISSN 00219258

Skeletal muscle atrophy promotes muscle weakness, limiting activities of daily living. However, mechanisms underlying atrophy remain unclear. Here, we show that skeletal muscle immobilization elevates Smad2/3 protein but not mRNA levels in muscle, promoting atrophy. Furthermore, we demonstrate that myostatin, which negatively regulates muscle hypertrophy, is dispensable for denervation-induced muscle atrophy and Smad2/3 protein accumulation. Moreover, muscle-specific Smad2/3-deficient mice exhibited significant resistance to denervation-induced muscle atrophy. In addition, expression of the atrogenes Atrogin-1 and MuRF1, which underlie muscle atrophy, did not increase in muscles of Smad2/3-deficient mice following denervation. We also demonstrate that serum starvation promotes Smad2/3 protein accumulation in C2C12 myogenic cells, an in vitro muscle atrophy model, an effect inhibited by IGF1 treatment. In vivo, we observed IGF1 receptor deactivation in immobilized muscle, even in the presence of normal levels of circulating IGF1. Denervation-induced muscle atrophy was accompanied by reduced glucose intake and elevated levels of branched-chain amino acids, effects that were Smad2/3-dependent. Thus, muscle immobilization attenuates IGF1 signals at the receptor rather than the ligand level, leading to Smad2/3 protein accumulation, muscle atrophy, and accompanying metabolic changes.

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Phylogenetic Position and Molecular Chronology of a Colonial Green Flagellate, Stephanosphaera pluvialis (Volvocales, Chlorophyceae), among Unicellular Algae

Munakata H., Nakada T., Nakahigashi K., Nozaki H., Tomita M.

Journal of Eukaryotic Microbiology (Journal of Eukaryotic Microbiology) 63 ( 3 ) 340 - 348 2016.05

ISSN 10665234

The genus Balticola comprises a group of unicellular green flagellate algae and is composed of four species formerly classified in the genus Haematococcus. Balticola is closely related to a colonial green flagellate, Stephanosphaera pluvialis. Although the phylogeny among these genera was previously investigated based on two nuclear gene sequences, the phylogenetic sister of S. pluvialis has yet to be determined. In the present study, the species diversity of Balticola and Stephanosphaera was investigated using 18S rRNA gene sequences, and phylogenetic analyses of combined nuclear and chloroplast gene sequences were performed to understand the evolutionary origin of coloniality in Stephanosphaera. The divergence times of four colonial volvocalean flagellates from their respective unicellular sisters were also estimated. Six Balticola genotypes and a single Stephanosphaera genotype were recognized, and one Balticola genotype was resolved as the sister of S. pluvialis, showing that Balticola is a nonmonophyletic genus. The divergence time of Stephanosphaera from its nearest Balticola relative was estimated to be 4-63 million years ago, and these genera represent the most recently diverged pair of unicellular and colonial flagellates among the Volvocales.

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Combination of quantitative changes in ionic components to enhance the contractile force during T-tubule development

Wakita M., Sano H.I., Naito Y., Tomita M.

Computing in Cardiology 43 1093 - 1096 2016.03

ISSN 23258861

During rodent ventricular cell development, the sarcoplasmic reticulum (SR) is scarce and poorly organized. A Ca2+ transient in embryonic ventricular cells depends mostly on Ca2+ influx through L-type Ca2+ channels. In rat ventricular cells, transverse tubules (t-tubules) begin to form postnatally. As such, Ca2+-induced Ca2+ release (CICR), in which ryanodine receptor channels on the SR are activated via Ca2+ influx through L-type Ca2+ channels on the t-tubules, is not established in embryonic ventricular cells. Here, we modeled developmental changes in Guinea pig ventricular cells and identified the factors that enhance contraction of the cells with an increase in CICR.

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Simulation study on the balance of glycolytic ATP production and oxidative phosphorylation in embryonic and adult ventricular cells

Sano H.I., Naito Y., Tomita M.

Computing in Cardiology 43 249 - 252 2016.03

ISSN 23258861

Here, we integrated various characteristics of fetal ventricular cells based on a mathematical model and predicted the contribution of each characteristic to maintenance of intracellular ATP concentration and sarcomere contraction under anoxic conditions. Our simulation results showed that higher glycogen content, higher hexokinase activity, and lower creatine concentration helped to prolong the time that contraction of ventricular cells was maintained under anoxic conditions. The integrated model enabled us to quantitatively assess the contributions of factors related to energy metabolism in ventricular cells.

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Metabolomic profiling reveals novel biomarkers of alcohol intake and alcohol-induced liver injury in community-dwelling men

Harada S., Takebayashi T., Kurihara A., Akiyama M., Suzuki A., Hatakeyama Y., Sugiyama D., Kuwabara K., Takeuchi A., Okamura T., Nishiwaki Y., Tanaka T., Hirayama A., Sugimoto M., Soga T., Tomita M.

Environmental Health and Preventive Medicine (Environmental Health and Preventive Medicine) 21 ( 1 ) 18 - 26 2016.01

ISSN 1342078X

Objective: Metabolomics is a promising approach to the identification of biomarkers in plasma. Here, we performed a population-based, cross-sectional study to identify potential biomarkers of alcohol intake and alcohol-induced liver injury by metabolomic profiling using capillary electrophoresis-mass spectrometry (CE-MS). Methods: Fasting plasma samples were collected from 896 Japanese men who participated in the baseline survey of the Tsuruoka Metabolomics Cohort Study, and 115 polar metabolites were identified and absolutely quantified by CE-MS. Information on daily ethanol intake was collected through a standardized, self-administered questionnaire. The associations between ethanol intake and plasma concentration of metabolites were examined. Relationships between metabolite concentrations or their ratios and serum liver enzyme levels in the highest ethanol intake group (>46.0 g/day) were then examined by linear regression analysis. Replication analysis was conducted in 193 samples collected from independent population of this cohort. Results: Nineteen metabolites were identified to have an association with daily alcohol consumption both in the original and replication population. Three of these metabolites (threonine, glutamine, and guanidinosuccinate) were found to associate well with elevated levels of serum liver enzymes in the highest ethanol intake group, but not in the non-drinker group. We also found that the glutamate/glutamine ratio had a much stronger relation to serum γ-glutamyltransferase, aspartate transaminase, and alanine transaminase than glutamate or glutamine alone (standardized beta = 0.678, 0.558, 0.498, respectively). Conclusions: We found 19 metabolites associated with alcohol intake, and three biomarker candidates (threonine, guanidinosuccinate and glutamine) of alcohol-induced liver injury. Glutamate/glutamine ratio might also be good biomarker.

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Conversion of graded phosphorylation into switch-like nuclear translocation via autoregulatory mechanisms in ERK signalling

Shindo Y, Iwamoto K, Mouri K, Hibino K, Tomita M, Kosako H, Sako Y, Takahashi K.

Nature Communications (Nature Communications) 7 10485 2016.01

The phosphorylation cascade in the extracellular signal-regulated kinase (ERK) pathway is a versatile reaction network motif that can potentially act as a switch, oscillator or memory. Nevertheless, there is accumulating evidence that the phosphorylation response is mostly linear to extracellular signals in mammalian cells. Here we find that subsequent nuclear translocation gives rise to a switch-like increase in nuclear ERK concentration in response to signal input. The switch-like response disappears in the presence of ERK inhibitor, suggesting the existence of autoregulatory mechanisms for ERK nuclear translocation involved in conversion from a graded to a switch-like response. In vitro reconstruction of ERK nuclear translocation indicates that ERK-mediated phosphorylation of nucleoporins regulates ERK translocation. A mathematical model and knockdown experiments suggest a contribution of nucleoporins to regulation of the ERK nuclear translocation response. Taken together, this study provides evidence that nuclear translocation with autoregulatory mechanisms acts as a switch in ERK signalling.

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Expansion of noncanonical V-Arm-containing tRNAs in eukaryotes

Hamashima K., Tomita M., Kanai A.

Molecular Biology and Evolution (Molecular Biology and Evolution) 33 ( 2 ) 530 - 540 2016

Joint Work, ISSN 07374038

Transfer RNA (tRNA) is essential for the translation of genetic information into proteins, and understanding its molecular evolution is important if we are to understand the genetic code. In general, long variable-arm (V-arm) structures form in tRNALeu, tRNASer, and bacterial and organellar tRNATyr. However, as we have previously reported, noncanonical V-arms occur in nematode tRNAGly and tRNAIle, and potentially affect translational fidelity. Here, we comprehensively analyzed 69 eukaryotic genome sequences and examined the evolutionary divergence of the V-arm-containing tRNAs. In total, 253 V-arm-containing tRNAs, with neither leucine nor serine anticodons, were identified in organisms ranging from nematodes to fungi, plants, and vertebrates. We defined them as "noncanonical V-arm-containing tRNAs" (nov-tRNAs). Moreover, 2,415 nov-tRNA-like sequences lacking some of the conserved features of tRNAs were also identified, largely in vertebrate genomes. These nov-tRNA/nov-tRNA-like sequences can be categorized into three types, based on differences in their possible evolutionary origins. The type A nov-tRNAs in nematodes probably evolved not only from tRNALeu but also from tRNASer and other isotypes on several independent occasions. The type B nov-tRNAs are dispersed abundantly throughout vertebrate genomes, and seem to have originated from retrotransposable elements. The type C nov-tRNAs may have been acquired from plant chloroplasts or from bacteria through horizontal transfer. Our findings provide unexpected insight into the evolution of the tRNA molecule, which was diverse and occurred independently in nematodes, vertebrates, and plants.

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De novo assembly and comparative transcriptome analysis of Euglena gracilis in response to anaerobic conditions

Yoshida Y., Tomiyama T., Maruta T., Tomita M., Ishikawa T., Arakawa K.

BMC Genomics (BMC Genomics) 17 ( 1 ) 182 2016

Joint Work

Background: The phytoflagellated protozoan, Euglena gracilis, has been proposed as an attractive feedstock for the accumulation of valuable compounds such as β-1,3-glucan, also known as paramylon, and wax esters. The production of wax esters proceeds under anaerobic conditions, designated as wax ester fermentation. In spite of the importance and usefulness of Euglena, the genome and transcriptome data are currently unavailable, though another research group has recently published E.gracilis transcriptome study during our submission. We herein performed an RNA-Seq analysis to provide a comprehensive sequence resource and some insights into the regulation of genes including wax ester metabolism by comparative transcriptome analysis of E.gracilis under aerobic and anaerobic conditions. Results: The E.gracilis transcriptome analysis was performed using the Illumina platform and yielded 90.3 million reads after the filtering steps. A total of 49,826 components were assembled and identified as a reference sequence of E.gracilis, of which 26,479 sequences were considered to be potentially expressed (having FPKM value of greater than 1). Approximately half of all components were estimated to be regulated in a trans-splicing manner, with the addition of protruding spliced leader sequences. Nearly 40 % of 26,479 sequences were annotated by similarity to Swiss-Prot database using the BLASTX program. A total of 2080 transcripts were identified as differentially expressed genes (DEGs) in response to anaerobic treatment for 24 h. A comprehensive pathway enrichment analysis using the KEGG pathway revealed that the majority of DEGs were involved in photosynthesis, nucleotide metabolism, oxidative phosphorylation, fatty acid metabolism. We successfully identified a candidate gene set of paramylon and wax esters, including novel β-1,3-glucan and wax ester synthases. A comparative expression analysis of aerobic- and anaerobic-treated E.gracilis cells indicated that gene expression changes in these components were not extensive or dynamic during the anaerobic treatment. Conclusion: The RNA-Seq analysis provided comprehensive transcriptome information on E.gracilis for the first time, and this information will advance our understanding of this unique organism. The comprehensive analysis indicated that paramylon and wax ester metabolic pathways are regulated at post-transcriptional rather than the transcriptional level in response to anaerobic conditions.

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Taxonomic revision of Chlamydomonas subg. Amphichloris (Volvocales, Chlorophyceae), with resurrection of the genus Dangeardinia and descriptions of Ixipapillifera gen. nov. and Rhysamphichloris gen. nov

Nakada, T., Tomita, M., Wu, J. T. and Nozaki, H.

Journal of Phycology (Journal of Phycology) 52 ( 2 ) 208 - 304 2016

Research paper (scientific journal), Joint Work, ISSN 00223646

Chlamydomonas (Cd.) is one of the largest but most polyphyletic genera of freshwater unicellular green algae. It consists of 400-600 morphological species and requires taxonomic revision. Toward reclassification, each morphologically defined classical subgenus (or subgroup) should be examined using culture strains. Chlamydomonas subg. Amphichloris is characterized by a central nucleus between two axial pyrenoids, however, the phylogenetic structure of this subgenus has yet to be examined using molecular data. Here, we examined 12 strains including six newly isolated strains, morphologically identified as Chlamydomonas subg. Amphichloris, using 18S rRNA gene phylogeny, light microscopy, and mitochondria fluorescent microscopy. Molecular phylogenetic analyses revealed three independent lineages of the subgenus, separated from the type species of Chlamydomonas, Cd. reinhardtii. These three lineages were further distinguished from each other by light and fluorescent microscopy-in particular by the morphology of the papillae, chloroplast surface, stigmata, and mitochondria-and are here assigned to three genera: Dangeardinia emend., Ixipapillifera gen. nov., and Rhysamphichloris gen. nov. Based on the molecular and morphological data, two to three species were recognized in each genus, including one new species, I. pauromitos. In addition, Cd. deasonii, which was previously assigned to subgroup "Pleiochloris," was included in the genus Ixipapillifera as I. deasonii comb. nov.

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Evaluation of the impact of RNA preservation methods of spiders for de novo transcriptome assembly

Kono N., Nakamura H., Ito Y., Tomita M., Arakawa K.

Molecular Ecology Resources (Molecular Ecology Resources) 16 ( 3 ) 662 - 672 2016

Research paper (scientific journal), Joint Work, ISSN 1755098X

With advances in high-throughput sequencing technologies, de novo transcriptome sequencing and assembly has become a cost-effective method to obtain comprehensive genetic information of a species of interest, especially in nonmodel species with large genomes such as spiders. However, high-quality RNA is essential for successful sequencing, and sample preservation conditions require careful consideration for the effective storage of field-collected samples. To this end, we report a streamlined feasibility study of various storage conditions and their effects on de novo transcriptome assembly results. The storage parameters considered include temperatures ranging from room temperature to -80°C; preservatives, including ethanol, RNAlater, TRIzol and RNAlater-ICE; and sample submersion states. As a result, intact RNA was extracted and assembly was successful when samples were preserved at low temperatures regardless of the type of preservative used. The assemblies as well as the gene expression profiles were shown to be robust to RNA degradation, when 30 million 150-bp paired-end reads are obtained. The parameters for sample storage, RNA extraction, library preparation, sequencing and in silico assembly considered in this work provide a guideline for the study of field-collected samples of spiders.

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Draft Genome Sequences of 37 Salmonella enterica Strains Isolated from Poultry Sources in Nigeria

Useh, N.,M., Ngbede, E.,O., Akange, N., Thomas, M., Foley, A., Keena, M., C., Nelson, E., Christopher-Hennings, J., Tomita, M., *Suzuki, H. and *Scaria, J.

Genome Announc 4 ( 3 ) e00315 - e00316 2016

Joint Work

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Mice carrying a human GLUD2 gene recapitulate aspects of human transcriptome and metabolome development

Li Q., Guo S., Jiang X., Bryk J., Naumann R., Enard W., Tomita M., Sugimoto M., Khaitovich P., Pääbo S.

Proc Natl Acad Sci U S A (Proceedings of the National Academy of Sciences of the United States of America) 113 ( 19 ) 5358 - 5363 2016

Joint Work, ISSN 00278424

Whereas all mammals have one glutamate dehydrogenase gene (GLUD1), humans and apes carry an additional gene (GLUD2), which encodes an enzyme with distinct biochemical properties. We inserted a bacterial artificial chromosome containing the human GLUD2 gene into mice and analyzed the resulting changes in the transcriptome and metabolome during postnatal brain development. Effects were most pronounced early postnatally, and predominantly genes involved in neuronal development were affected. Remarkably, the effects in the transgenic mice partially parallel the transcriptome and metabolome differences seen between humans and macaques analyzed. Notably, the introduction of GLUD2 did not affect glutamate levels in mice, consistent with observations in the primates. Instead, the metabolic effects of GLUD2 center on the tricarboxylic acid cycle, suggesting that GLUD2 affects carbon flux during early brain development, possibly supporting lipid biosynthesis.

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Simultaneous analysis of consumer variables, acceptability and sensory characteristics of dry-cured ham

Sugimoto, M., Obiya, S., Kaneko, M., Enomoto, A., Honma, M., Wakayama, M., and Tomita, M.

Meat Science (Meat Science) 120 210 - 215 2016

Joint Work, ISSN 03091740

We conducted a consumer acceptability analysis of dry-cured ham based on sensory evaluation. Consumer acceptability data are rendered heterogeneous by the diverse backgrounds and assessment abilities of the participants, requiring versatile analytical methods for their interpretation. Totally, 9 sensory attributes of 12 kinds of dry-cured ham samples collected from Japan (n = 9), Italy (n = 1), Spain (n = 1), and Germany (n = 1) were tasted by 117 Japanese consumers who showed acceptable evaluation abilities during blind sampling. Common techniques, such as hierarchical clustering, principal component analysis, and external preference mapping, were simultaneously utilized to analyze each characteristics scored in modified hedonic scale. These analyses revealed the relationships between the features and preferences of the assessors. For example, consumers aged 20–30 with smoking and drinking habits preferred sweetness and saltiness, and gave high ratings to Spanish Jómon serrano and Italian prosciutto. Our approach could assist ham marketers to identify potential purchasers and the preferred characteristics of their products.

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Genome sequencing of a single tardigrade Hypsibius dujardini individual

Arakawa, K., Yoshida, Y., Tomita, M.

Scientific Data (Scientific Data) 3 160063 2016

Joint Work

Tardigrades are ubiquitous microscopic animals that play an important role in the study of metazoan phylogeny. Most terrestrial tardigrades can withstand extreme environments by entering an ametabolic desiccated state termed anhydrobiosis. Due to their small size and the non-axenic nature of laboratory cultures, molecular studies of tardigrades are prone to contamination. To minimize the possibility of microbial contaminations and to obtain high-quality genomic information, we have developed an ultra-low input library sequencing protocol to enable the genome sequencing of a single tardigrade Hypsibius dujardini individual. Here, we describe the details of our sequencing data and the ultra-low input library preparation methodologies.

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Identification of salivary metabolomic biomarkers for oral cancer screening

Ishikawa S., Sugimoto M., Kitabatake K., Sugano A., Nakamura M., Kaneko M., Ota S., Hiwatari K., Enomoto A., Soga T., Tomita M., Iino M.

Scientific Reports (Scientific Reports) 6 31520 2016

Joint Work

The objective of this study was to explore salivary metabolite biomarkers by profiling both saliva and tumor tissue samples for oral cancer screening. Paired tumor and control tissues were obtained from oral cancer patients and whole unstimulated saliva samples were collected from patients and healthy controls. The comprehensive metabolomic analysis for profiling hydrophilic metabolites was conducted using capillary electrophoresis time-of-flight mass spectrometry. In total, 85 and 45 metabolites showed significant differences between tumor and matched control samples, and between salivary samples from oral cancer and controls, respectively (P < 0.05 correlated by false discovery rate); 17 metabolites showed consistent differences in both saliva and tissue-based comparisons. Of these, a combination of only two biomarkers yielded a high area under receiver operating characteristic curves (0.827; 95% confidence interval, 0.726-0.928, P < 0.0001) for discriminating oral cancers from controls. Various validation tests confirmed its high generalization ability. The demonstrated approach, integrating both saliva and tumor tissue metabolomics, helps eliminate pseudo-molecules that are coincidentally different between oral cancers and controls. These combined salivary metabolites could be the basis of a clinically feasible method of non-invasive oral cancer screening.

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Profiling of plasma metabolites in postmenopausal women with metabolic syndrome

Iida M., Harada S., Kurihara A., Fukai K., Kuwabara K., Sugiyama D., Takeuchi A., Okamura T., Akiyama M., Nishiwaki Y., Suzuki A., Hirayama A., Sugimoto M., Soga T., Tomita M., Banno K., Aoki D., Takebayashi T.

Menopause (Menopause) 23 ( 7 ) 749 - 758 2016

Joint Work, ISSN 10723714

Objective:The aim of the study was to investigate the associations of amino acids and other polar metabolites with metabolic syndrome (MetS) in postmenopausal women in a lean Asian population. Methods:The participants were 1,422 female residents enrolled in a cohort study from April to August 2012. MetS was defined according to the National Cholesterol Education Program Adult Treatment Panel III modified for Japanese women. Associations were examined between MetS and 78 metabolites assayed in fasting plasma samples using capillary electrophoresis-mass spectrometry. Replication analysis was performed to confirm the robustness of the results in a separate population created by random allocation. Results:Analysis was performed for 877 naturally postmenopausal women, including 594 in the original population and 283 in the replication population. The average age, body mass index, and levels of high- and low-density lipoprotein cholesterol of the entire population were 64.6 years, 23.0kg/m2, 72.1mg/dL, and 126.1mg/dL, respectively. There was no significant difference in low-density lipoprotein cholesterol levels between women with and without MetS. Thirteen metabolites were significantly related to MetS: multiple plasma amino acids were elevated in women with MetS, including branched-chain amino acids, alanine, glutamate, and proline; and alpha-aminoadipate, which is generated by lysine degradation, was also significantly increased. Conclusions:Our large-scale metabolomic profiling indicates that Japanese postmenopausal women with MetS have abnormal polar metabolites, suggesting altered catabolic pathways. These results may help to understand metabolic disturbance, including in persons with normal body mass index and relatively high levels of high-density lipoprotein cholesterol, and may have clinical utility based on further studies.

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Metabolic profiling of total physical activity and sedentary behavior in community dwelling men

Fukai, K., Harada, S., Iida, M., Kurihara, A., Takeuchi, A., Kuwabara, K., Sugiyama, D., Okamura, T., Akiyama, M., Nishiwaki, Y., Oguma, Y., Suzuki, A., Suzuki, C., Hirayama, A., Sugimoto, M., Soga, T., Tomita, M. and *Takebayashi, T.

PLoS ONE (PLoS ONE) 11 ( 10 ) e0164877 2016

Joint Work

Objective: Physical activity is known to be preventive against various non-communicable diseases. We investigated the relationship between daily physical activity level and plasma metabolites using a targeted metabolomics approach in a population-based study. Methods: A total of 1,193 participants (male, aged 35 to 74 years) with fasting blood samples were selected from the baseline survey of a cohort study. Information on daily total physical activity, classified into four levels by quartile of metabolic equivalent scores, and sedentary behavior, defined as hours of sitting per day, was collected through a self-administered questionnaire. Plasma metabolite concentrations were quantified by capillary electrophoresis mass spectrometry method. We performed linear regression analysis models with multivariable adjustment and corrected p-values for multiple testing in the original population (n = 808). The robustness of the results was confirmed by replication analysis in a separate population (n = 385) created by random allocation. Results: Higher levels of total physical activity were associated with various metabolite concentrations, including lower concentrations of amino acids and their derivatives, and higher concentrations of pipecolate (FDR p <0.05 in original population). The findings persisted after adjustment for age, body mass index, smoking, alcohol intake, and energy intake. Isoleucine, leucine, valine, 4-methyl-2-oxoisopentanoate, 2-oxoisopentanoate, alanine, and proline concentrations were lower with a shorter sitting time. Conclusions: Physical activity is related to various plasma metabolites, including known biomarkers for future insulin resistance or type 2 diabetes. These metabolites might potentially play a key role in the protective effects of higher physical activity and/or less sedentary behavior on non-communicable diseases.

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Draft genome sequence of Arenibacter sp. strain C-21, an iodine-accumulating bacterium isolated from surface marine sediment

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

Genome Announcements (Genome Announcements) 4 ( 5 ) e01155-16 2016

Joint Work, ISSN 2169-8287

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Morphology and phylogeny of a new wall-less freshwater volvocalean flagellate, Hapalochloris nozakii gen. et sp. nov. (Volvocales, Chlorophyceae)

Nakada, T. and Tomita, M.

Journal of Phycology (Journal of Phycology) 53 ( 1 ) JPY12484 - 117 2016

ISSN 00223646

New strains of a wall-less unicellular volvocalean flagellate were isolated from a freshwater environment in Japan. Observations of the alga, described here as Hapalochloris nozakii Nakada, gen. et sp. nov., were made using light, fluorescence, and electron microscopy. Each vegetative cell had two flagella, four contractile vacuoles, and a spirally furrowed cup-shaped chloroplast with an axial pyrenoid, and mitochondria located in the furrows. Based on the morphology, H. nozakii was distinguished from other known wall-less volvocalean flagellates. Under electron microscopy, fibrous material, instead of a cell wall and dense cortical microtubules, was observed outside and inside the cell membrane, respectively. Based on the phylogenetic analyses of 18S rRNA gene sequences, H. nozakii was found to be closely related to Asterococcus, Oogamochlamys, Rhysamphichloris, and “Dunaliella” lateralis and was separated from other known wall-less flagellate volvocaleans, indicating independent secondary loss of the cell wall in H. nozakii. In the combined 18S rRNA and chloroplast gene tree, H. nozakii was sister to Lobochlamys.

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Systematic characterization of artificial small RNA-mediated inhibition of Escherichia coli growth

Noro, E., Mori, M., Makino, G., Takai, Y., Ohnuma, S., Sato, A., Tomita, M., Nakahigashi, K. and Kanai, A.

RNA Biology (RNA Biology) 16 ( 2 ) 1 - 13 2016

Joint Work, Accepted, ISSN 15476286

A new screening system for artificial small RNAs (sRNAs) that inhibit the growth of Escherichia coli was constructed. In this system, we used a plasmid library to express RNAs of ∼120 nucleotides, each with a random 30-nucleotide sequence that can recognize its target mRNA(s). After approximately 60,000 independent colonies were screened, several plasmids that inhibited bacterial growth were isolated. To understand the inhibitory mechanism, we focused on one sRNA, S-20, that exerted a strong inhibitory effect. A time-course analysis of the proteome of S-20-expressing E. coli and a bioinformatic analysis were used to identify potential S-20 target mRNAs, and suggested that S-20 binds the translation initiation sites of several mRNAs encoding enzymes such as peroxiredoxin (osmC), glycyl-tRNA synthetase α subunit (glyQ), uncharacterized protein ygiM, and tryptophan synthase β chain (trpB). An in vitro translation analysis of chimeric luciferase-encoding mRNAs, each containing a potential S-20 target sequence, indicated that the translation of these mRNAs was inhibited in the presence of S-20. A gel shift analysis combined with the analysis of a series of S-20 mutants suggested that S-20 targets multiple mRNAs that are responsible for inhibiting E. coli growth. These data also suggest that S-20 acts like an endogenous sRNA and that E. coli can utilize artificial sRNAs.

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Bri CA: A modular software platform for whole brain architecture

Itaya K., Takahashi K., Nakamura M., Koizumi M., Arakawa N., Tomita M., Yamakawa H.

Lecture Notes in Computer Science (including subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics) 9947 LNCS 334 - 341 2016

ISSN 03029743

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Curing the megaplasmid pTT27 from Thermus thermophilus HB27 and maintaining exogenous plasmids in the plasmid-free strain

Ohtani N, Tomita M, Itaya M.

Applied and Environmental Microbiology (Applied and Environmental Microbiology) 28;82 ( 5 ) 1537 - 48 2015.12

ISSN 00992240

Stepwise deletions in the only plasmid in Thermus thermophilus HB27, megaplasmid pTT27, showed that two distantly located loci were important for maintenance of the plasmid. One is a minimum replicon including one gene, repT, coding a replication initiator, and the other encodes subunits of class I ribonucleotide reductase (RNR) for deoxynucleoside triphosphate (dNTP) synthesis. Since the initiator protein, RepT, bound to direct repeats downstream from its own gene, it was speculated that a more-downstream A+T-rich region, which was critical for replication ability, could be unwound for replication initiation. On the other hand, the class I RNR is not necessarily essential for cell growth, as evidenced by the generation of the plasmid-free strain by the loss of pTT27. However, the plasmid-free strain culture has fewer viable cells than the wild-type culture, probably due to a dNTP pool imbalance in the cell. This is because of the introduction of the class I RNR genes or the supplementation of 5'-deoxyadenosylcobalamin, which stimulated class II RNR encoded in the chromosome, resolved the decrease in the number of viable cells in the plasmid-free strain. Likewise, these treatments dramatically enhanced the efficiency of transformation by exogenous plasmids and the stability of the plasmids in the strain. Therefore, the class I RNR would enable the stable maintenance of plasmids, including pTT27, as a result of genome replication normalized by reversing the dNTP pool imbalance. The generation of this plasmid-free strain with great natural competence and its analysis in regard to exogenous plasmid maintenance will expand the availability of HB27 for thermophilic cell factories.

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The application of omics technologies in the functional evaluation of inulin and inulin-containing prebiotics dietary supplementation

Tsurumaki M., Kotake M., Iwasaki M., Saito M., Tanaka K., Aw W., Fukuda S., Tomita M.

Nutrition and Diabetes (Nutrition and Diabetes) 5 ( 11 ) e185 2015.11

ISSN 2044-4052

Inulin, a natural renewable polysaccharide resource produced by various plants in nature, has been reported to possess a significant number of diverse pharmaceutical and food applications. Recently, there has been rapid progress in high-throughput technologies and platforms to assay global mRNA, proteins, metabolites and gut microbiota. In this review, we will describe the current status of utilizing omics technologies of elucidating the impact of inulin and inulin-containing prebiotics at the transcriptome, proteome, metabolome and gut microbiome levels. Although many studies in this review have addressed the impact of inulin comprehensively, these omics technologies only enable us to understand physiological information at each different stage of mRNA, protein, metabolite and gut microbe. We believe that a synergistic approach is vital in order to fully illustrate the intricate beauty behind the relatively modest influence of food factors like inulin on host health.

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Taxonomic revision of oil-producing green algae, Chlorococcum oleofaciens (Volvocales, Chlorophyceae), and its relatives

Kawasaki Y, Nakada T, Tomita M.

Journal of Phycology (Journal of Phycology) 51 ( 5 ) 1000 - 16 2015.10

ISSN 00223646

Historically, species in Volvocales were classified based primarily on morphology. Although the taxonomy of Chlamydomonas has been re-examined using a polyphasic approach including molecular phylogeny, that of Chlorococcum (Cc.), the largest coccoid genus in Volvocales, has yet to be reexamined. Six species thought to be synonymous with the oil-producing alga Cc. oleofaciens were previously not confirmed by molecular phylogeny. In this study, seven authentic strains of Cc. oleofaciens and its putative synonyms, along with 11 relatives, were examined based on the phylogeny of the 18S ribosomal RNA (rRNA) gene, comparisons of secondary structures of internal transcribed spacer 1 (ITS1) and ITS2 rDNA, and morphological observations by light microscopy. Seven 18S rRNA types were recognized among these strains and three were distantly related to Cc. oleofaciens. Comparisons of ITS rDNA structures suggested possible separation of the remaining four types into different species. Shapes of vegetative cells, thickness of the cell walls in old cultures, the size of cells in old cultures, and stigma morphology of zoospores also supported the 18S rRNA grouping. Based on these results, the 18 strains examined were reclassified into seven species. Among the putative synonyms, synonymy of Cc. oleofaciens, Cc. croceum, and Cc. granulosum was confirmed, and Cc. microstigmatum, Cc. rugosum, Cc. aquaticum, and Cc. nivale were distinguished from Cc. oleofaciens. Furthermore, another related strain is described as a new species, Macrochloris rubrioleum sp. nov.

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Tracking global gene expression responses in T cell differentiation

Simeoni O., Piras V., Tomita M., Selvarajoo K.

Gene (Gene) 569 ( 2 ) 259 - 266 2015.09

ISSN 03781119

Upon receiving antigens from the innate immune cells, CD4+ T cells differentiate into distinct effector cells. To probe the global responses of distinct effector cells, we analyzed transcriptome-wide expressions of Th1, Th2, Treg and Th17 using Pearson correlation, entropy and principal component analyses, with Th0 as a control. Although the global response of Th0 was quite distinct from Th17, surprisingly, it was highly similar to Th1, Th2 and Treg. Moreover, 8 major temporal groups consisting of 5704 differentially expressed genes were revealed for both Th0 and Th17. Gene functional enrichment analysis showed immune responses and metabolic processes were mainly activated between Th0 and Th17, while genes related to cell cycle and replication were differentially regulated. Moreover, we found the upregulation of several novel genes for Th0 and Th17. Overall, we deduce that Th0 is globally similar to Th1, Th2 and Treg. Our results indicate that Th0 is a differentiated state and, therefore, may not be used as a control cell type.

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Operation of a 130-MHz/9-mm Compact HTS Annulus Magnet With a Micro-NMR Probe.

Hahn S, Kim Y, Song J, Voccio JP, Chu Y, Bascuñán J, Tomita M, Iwasa Y

IEEE transactions on applied superconductivity : a publication of the IEEE Superconductivity Committee 25 ( 3 ) 1 - 5 2015.06

ISSN 1051-8223

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Organization and Evolution of Brain Lipidome Revealed by Large-Scale Analysis of Human, Chimpanzee, Macaque, and Mouse Tissues

Bozek K., Wei Y., Yan Z., Liu X., Xiong J., Sugimoto M., Tomita M., Pääbo S., Sherwood C., Hof P., Ely J., Li Y., Steinhauser D., Willmitzer L., Giavalisco P., Khaitovich P.

Neuron (Neuron) 85 ( 4 ) 695 - 702 2015.02

ISSN 08966273

Lipids are prominent components of the nervous system. Here we performed a large-scale mass spectrometry-based analysis of the lipid composition of three brain regions as well as kidney and skeletal muscle of humans, chimpanzees, rhesus macaques, and mice. The human brain shows the most distinct lipid composition: 76% of 5,713 lipid compounds examined in our study are either enriched or depleted in the human brain. Concentration levels of lipids enriched in the brain evolve approximately four times faster among primates compared with lipids characteristic of non-neural tissues and show further acceleration of change in human neocortical regions but not in the cerebellum. Human-specific concentration changes are supported by human-specific expression changes for corresponding enzymes. These results provide the first insights into the role of lipids in human brain evolution.

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Modeling and simulation of developmental changes in contractile apparatus of ventricular cells

Takiguchi M., Toki T., Sano H., Naito Y., Tomita M.

Computing in Cardiology 42 1097 - 1100 2015.02

ISSN 23258861

During development, ventricular cells utilize different isoforms of both myosin heavy chain (MHC) and troponin I. The differences in these isoforms affect Ca2+ sensitivity, ATPase activity, and velocity of contraction. In order to consider the differences in isoforms, we integrated a new contraction model with the Kyoto model. Briefly, the new model considered tropomyosin, which inhibits formation of a cross-bridge between actin and myosin filaments. We varied the level of Ca2+ sensitivity in order to obtain similar traces for contractile force between the original Kyoto model and the modified model. We also modified the new contraction model to consider ATP consumption by myosin-ATPase in order to simulate the changes in ATPase activity caused by the difference in MHC isoforms. The modified model enabled us to compare the contribution of developmental changes in ATP consumption via contraction to excitation-contraction coupling, which is regulated differently in fetal and adult Guinea pigs.

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Sensitivity analysis of ectopic electrical activity in the pulmonary vein myocardium

Sano H., Tanaka Y., Naito Y., Tomita M.

Computing in Cardiology 42 861 - 864 2015.02

ISSN 23258861

The pulmonary vein contains a myocardial layer that is capable of generating spontaneous or triggered action potentials. Although the electrophysiological and pharmacological characteristics of the pulmonary vein myocardium have been compiled in various studies, a comprehensive understanding of the spontaneous action potentials generated in the myocardial layer has yet to be presented. Here we integrated the electrophysiological properties of the pulmonary vein myocardial layer using a Guinea pig ventricular cell model. Based on the preceding research, which reported that approximately half of the isolated pulmonary vein myocardial layer exhibited spontaneous action potentials and the remaining half was quiescent, we constructed various combinations of the pulmonary vein myocardial models to represent variations of the action potential tracings. As a result, we predicted that the spontaneous action potentials, including burst-like action potentials, are observed with varying currents of the relative densities of Na+ (INa), L-type Ca2+ (ICaL), inwardly rectifying K+ (IK1), rapid component of delayed rectifying K+ (IKr), and ACh-activated K+ (IKACh).

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Identification, expression, and molecular evolution of microRNAs in the "living fossil" Triops cancriformis (tadpole shrimp)

Ikeda K., Hirose Y., Hiraoka K., Noro E., Fujishima K., Tomita M., Kanai A.

RNA (RNA) 21 ( 2 ) 230 - 242 2015.02

ISSN 13558382

MicroRNAs have been identified and analyzed in various model species, but an investigation of miRNAs in nonmodel species is required for a more complete understanding of miRNA evolution. In this study, we investigated the miRNAs of the nonmodel species Triops cancriformis (tadpole shrimp), a "living fossil," whose morphological form has not changed in almost 200 million years. Dramatic ontogenetic changes occur during its development. To clarify the evolution of miRNAs, we comparatively analyzed its miRNAs and the components of its RNAi machinery. We used deep sequencing to analyze small RNA libraries from the six different developmental stages of T. cancriformis (egg, first- fourth instars, and adult), and also analyzed its genomic DNA with deep sequencing. We identified 180 miRNAs (87 conserved miRNAs and 93 novel candidate miRNAs), and deduced the components of its RNAi machinery: the DICER1, AGO1-3, PIWI, and AUB proteins. A comparative miRNA analysis of T. cancriformis and Drosophila melanogaster showed inconsistencies in the expression patterns of four conserved miRNAs. This suggests that although the miRNA sequences of the two species are very similar, their roles differ across the species. An miRNA conservation analysis revealed that most of the conserved T. cancriformis miRNAs share sequence similarities with those of arthropods, although T. cancriformis is called a "living fossil." However, we found that let- 7 and DICER1 of T. cancriformis are more similar to those of the vertebrates than to those of the arthropods. These results suggest that miRNA systems of T. cancriformis have evolved in a unique fashion.

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A Generic Software Platform for Brain-inspired Cognitive Computing

Takahashi, K., Itaya, K., Nakamura, M., Koizumi, M., Arakawa, N., Tomita, M. and Yamakawa, H.

Procedia Computer Science 71 31 - 37 2015

Joint Work

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The Consumption of Bicarbonate-Rich Mineral Water Improves Glycemic Control

Murakami S, Goto Y, Ito K, Hayasaka S, Kurihara S, Soga T, Tomita M, Fukuda S.

Evid Based Complement Alternat Med. (Evidence-based Complementary and Alternative Medicine) 2015 10 2015

ISSN 1741427X

Hot spring water and natural mineral water have been therapeutically used to prevent or improve various diseases. Specifically, consumption of bicarbonate-rich mineral water (BMW) has been reported to prevent or improve type 2 diabetes (T2D) in humans. However, the molecular mechanisms of the beneficial effects behind mineral water consumption remain unclear. To elucidate the molecular level effects of BMW consumption on glycemic control, blood metabolome analysis and fecal microbiome analysis were applied to the BMW consumption test. During the study, 19 healthy volunteers drank 500 mL of commercially available tap water (TW) or BMW daily. TW consumption periods and BMW consumption periods lasted for a week each and this cycle was repeated twice. Biochemical tests indicated that serum glycoalbumin levels, one of the indexes of glycemic controls, decreased significantly after BMW consumption. Metabolome analysis of blood samples revealed that 19 metabolites including glycolysis-related metabolites and 3 amino acids were significantly different between TW and BMW consumption periods. Additionally, microbiome analysis demonstrated that composition of lean-inducible bacteria was increased after BMW consumption. Our results suggested that consumption of BMW has the possible potential to prevent and/or improve T2D through the alterations of host metabolism and gut microbiota composition.

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Effects of 3-styrylchromones on metabolic profiles and cell death in oral squamous cell carcinoma cells

Sakagami, H., Shimada, C., Kanda, Y., Amano, O., Sugimoto, M., Ota, S., Soga, T., Tomita, M., Sato, A., Tanuma, S., Takao, K., and Sugita, Y.

Toxicology Reports (Toxicology Reports) 2 1281 - 1290 2015

Joint Work, ISSN 22147500

4H-1-benzopyran-4-ones (chromones) are important naturally-distributing compounds. As compared with flavones, isoflavones and 2-styrylchromones, there are only few papers of 3-styrylchromones that have been published. We have previously reported that among fifteen 3-styrylchromone derivatives, three new synthetic compounds that have OCH3 group at the C-6 position of chromone ring, (E)-3-(4-hydroxystyryl)-6-methoxy-4H-chromen-4-one (compound 11), (E)-6-methoxy-3-(4-methoxystyryl)-4H-chromen-4-one (compound 4), (E)-6-methoxy-3-(3,4,5-trimethoxystyryl)-4H-chromen-4-one (compound 6) showed much higher cytotoxicities against four epithelial human oral squamous cell carcinoma (OSCC) lines than human normal oral mesenchymal cells. In order to further confirm the tumor specificities of these compounds, we compared their cytotoxicities against both human epithelial malignant and non-malignant cells, and then investigated their effects on fine cell structures and metabolic profiles and cell death in human OSCC cell line HSC-2. Cytotoxicities of compounds 4, 6, 11 were assayed with MTT method. Fine cell structures were observed under transmission electron microscope. Cellular metabolites were extracted with methanol and subjected to CE-TOFMS analysis. Compounds 4, 6, 11 showed much weaker cytotoxicity against human oral keratinocyte and primary human gingival epithelial cells, as compared with HSC-2, confirming their tumor-specificity, whereas doxorubicin and 5-FU were highly cytotoxic to these normal epithelial cells, giving unexpectedly lower tumor-specificity. The most cytotoxic compound 11, induced the mitochondrial vacuolization, autophagy suppression followed by apoptosis induction, and changes in the metabolites involved in amino acid and glycerophospholipid metabolisms. Chemical modification of lead compound 11 may be a potential choice for designing new type of anticancer drugs.

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Analysis of genetic code ambiguity arising from nematode-specific misacylated tRNAs

Hamashima, K., Mori, M., Andachi, Y., Yuji Kohara, Y., M. and Kanai, A.

PLoS ONE (PLoS ONE) 10 ( 1 ) e0116981 2015

Joint Work

The faithful translation of the genetic code requires the highly accurate aminoacylation of transfer RNAs (tRNAs). However, it has been shown that nematode-specific V-arm-containing tRNAs (nev-tRNAs) are misacylated with leucine in vitro in a manner that transgresses the genetic code. nev-tRNAGly (CCC) and nev-tRNAIle (UAU), which are the major nev-tRNA isotypes, could theoretically decode the glycine (GGG) codon and isoleucine (AUA) codon as leucine, causing GGG and AUA codon ambiguity in nematode cells. To test this hypothesis, we investigated the functionality of nev-tRNAs and their impact on the proteome of Caenorhabditis elegans. Analysis of the nucleotide sequences in the 3' end regions of the nev-tRNAs showed that they had matured correctly, with the addition of CCA, which is a crucial posttranscriptional modification required for tRNA aminoacylation. The nuclear export of nev-tRNAs was confirmed with an analysis of their subcellular localization. These results show that nev-tRNAs are processed to their mature forms like common tRNAs and are available for translation. However, a whole-cell proteome analysis found no detectable level of nev-tRNA-induced mistranslation in C. elegans cells, suggesting that the genetic code is not ambiguous, at least under normal growth conditions. Our findings indicate that the translational fidelity of the nematode genetic code is strictly maintained, contrary to our expectations, although deviant tRNAs with misacylation properties are highly conserved in the nematode genome.

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Alteration of metabolomic profiles by titanium dioxide nanoparticles in human gingivitis model

Garcia-Contreras, R., Sugimoto, M., Umemura, N., Kaneko, M., Hatakeyama, Y., Soga, T., Tomita, M., Scougall-Vilchis, R., J., Contreras-Bulnes, R., Nakajima, H. and *Sakagami, H.

Biomaterials (Biomaterials) 57 33 - 40 2015

Joint Work, ISSN 01429612

Although nanoparticles (NPs) has afforded considerable benefits in various fields of sciences, several reports have shown their harmful effects, suggesting the necessity of adequate risk assessment. To clarify the mechanism of titanium dioxide nanoparticles (TiO<inf>2</inf> NPs)-enhanced gingival inflammation, we conducted the full-scale metabolomic analyses of human gingival fibroblast cells treated with IL-1β alone or in combination with TiO<inf>2</inf> NPs. Observation with transmission electron microscope demonstrated the incorporation of TiO<inf>2</inf> NPs into vacuoles of the cells. TiO<inf>2</inf> NPs significantly enhanced the IL-1β-induced prostaglandin E<inf>2</inf> production and COX-1 and COX-2 protein expression. IL-1β reduced the intracellular concentrations of overall primary metabolites especially those of amino acid, urea cycle, polyamine, S-adenosylmethione and glutathione synthetic pathways. The addition of TiO<inf>2</inf> NPs further augmented these IL-1β-induced metabolic changes, recommending careful use of dental materials containing TiO<inf>2</inf> NPs towards patients with gingivitis or periodontitis. The impact of the present study is to identify the molecular targets of TiO<inf>2</inf> NPs for the future establishment of new metabolic markers and therapeutic strategy of gingival inflammation.

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Method of preparing an equimolar DNA mixture for one-step DNA assembly of over 50 fragments

Tsuge, K., Sato, Y., Kobayashi, Y., Gondo, M., Hasebe, M., Togashi, T., Tomita, M. and Itaya, M.

Scientific Reports (Scientific Reports) 5 10655 2015

Joint Work

In the era of synthetic biology, techniques for rapidly constructing a designer long DNA from short DNA fragments are desired. To realize this, we attempted to establish a method for one-step DNA assembly of unprecedentedly large numbers of fragments. The basic technology is the Ordered Gene Assembly in Bacillus subtilis (OGAB) method, which uses the plasmid transformation system of B. subtilis. Since this method doesn' t require circular ligation products but needs tandem repeat ligation products, the degree of deviation in the molar concentration of the material DNAs is the only determinant that affects the efficiency of DNA assembly. The strict standardization of the size of plasmids that clone the DNA block and the measurement of the block in the state of intact plasmid improve the reliability of this step, with the coefficient of variation of the molar concentrations becoming 7%. By coupling this method with the OGAB method, one-step assembly of more than 50 DNA fragments becomes feasible.

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Effects of processing and storage conditions on charged metabolomic profiles in blood

Hirayama, A., Sugimoto, M., Suzuki, A., Hatakeyama, Y., Enomoto, A., Harada, S., Soga, T., Tomita, M. and Takebayashi, T.

Electrophoresis (Electrophoresis) 36 ( 18 ) 2148 - 2155 2015

Joint Work, ISSN 01730835

The development of high-throughput metabolite measurement technologies has enabled the use of metabolomics for epidemiologic studies by profiling metabolite concentrations in large cohorts of human blood samples. Standard protocols are necessary to obtain unbiased profiles through multiple runs over long periods of time and to allow reliable statistical analyses. This study assessed the effects of sampling procedures and storage conditions on the stability of metabolomic profiles in plasma and serum. Charged metabolomic profiles were determined by capillary electrophoresis-mass spectrometry (CE-MS) and compared by multivariate analyses. The effects of pre-analytical procedures, including times for clotting and incubation of serum and plasma, respectively; incubation temperatures; and number of freeze-thaw cycles, were assessed. Overall, inter-individual differences in profiles were larger than intra-individual differences, and profiles in plasma showed better stability than those in serum. These quantified datasets of metabolites, along with their stability and variation, may help in interpreting data from long-term cohort studies.

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Metabolomic alterations in human cancer cells by Vitamin C-induced oxidative stress

Uetaki M., Tabata S., Nakasuka F., Soga T., Tomita M.

Scientific Reports (Scientific Reports) 5 13896 2015

Joint Work

Intravenous administration of high-dose vitamin C has recently attracted attention as a cancer therapy. High-dose vitamin C induces pro-oxidant effects and selectively kills cancer cells. However, the anticancer mechanisms of vitamin C are not fully understood. Here, we analyzed metabolic changes induced by vitamin C in MCF7 human breast adenocarcinoma and HT29 human colon cancer cells using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). The metabolomic profiles of both cell lines were dramatically altered after exposure to cytotoxic concentrations of vitamin C. Levels of upstream metabolites in the glycolysis pathway and tricarboxylic acid (TCA) cycle were increased in both cell lines following treatment with vitamin C, while adenosine triphosphate (ATP) levels and adenylate energy charges were decreased concentration-dependently. Treatment with N-acetyl cysteine (NAC) and reduced glutathione (GSH) significantly inhibited vitamin C-induced cytotoxicity in MCF7 cells. NAC also suppressed vitamin C-dependent metabolic changes, and NAD treatment prevented vitamin C-induced cell death. Collectively, our data suggests that vitamin C inhibited energy metabolism through NAD depletion, thereby inducing cancer cell death.

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Comprehensive analysis of microbes and metabolites in human tear fluids

Murakami, S., Baba, F., Fukuda, S., Soga, T., Fujishima, H. and Tomita, M.

KEIO SFC JOURNAL 382 - 400 2015

Joint Work

Particle Simulation of Oxidation Induced Band 3 Clustering in Human Erythrocytes

Shimo H., Arjunan S.N.V., Machiyama H., Nishino T., Suematsu M., Fujita H., Tomita M., Takahashi K.

PLoS Computational Biology (PLoS Computational Biology) 11 ( 6 ) e1004210 2015

Joint Work, ISSN 1553734X

Oxidative stress mediated clustering of membrane protein band 3 plays an essential role in the clearance of damaged and aged red blood cells (RBCs) from the circulation. While a number of previous experimental studies have observed changes in band 3 distribution after oxidative treatment, the details of how these clusters are formed and how their properties change under different conditions have remained poorly understood. To address these issues, a framework that enables the simultaneous monitoring of the temporal and spatial changes following oxidation is needed. In this study, we established a novel simulation strategy that incorporates deterministic and stochastic reactions with particle reaction-diffusion processes, to model band 3 cluster formation at single molecule resolution. By integrating a kinetic model of RBC antioxidant metabolism with a model of band 3 diffusion, we developed a model that reproduces the time-dependent changes of glutathione and clustered band 3 levels, as well as band 3 distribution during oxidative treatment, observed in prior studies. We predicted that cluster formation is largely dependent on fast reverse reaction rates, strong affinity between clustering molecules, and irreversible hemichrome binding. We further predicted that under repeated oxidative perturbations, clusters tended to progressively grow and shift towards an irreversible state. Application of our model to simulate oxidation in RBCs with cytoskeletal deficiency also suggested that oxidation leads to more enhanced clustering compared to healthy RBCs. Taken together, our model enables the prediction of band 3 spatio-temporal profiles under various situations, thus providing valuable insights to potentially aid understanding mechanisms for removing senescent and premature RBCs.

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Precise mapping and dynamics of tRNA-derived fragments (tRFs) in the development of Triops cancriformis (tadpole shrimp)

Hirose Y., Ikeda K., Noro E., Hiraoka K., Tomita M., Kanai A.

BMC Genetics (BMC Genetics) 16 ( 1 ) 83 2015

Joint Work

Background: In a deep sequencing analysis of small RNAs prepared from a living fossil, the tadpole shrimp Triops cancriformis, a 32-nt small RNA was specifically detected in the adult stage. A nucleotide sequence comparison between the 32-nt small RNA and predicted tRNA sequences in the draft nuclear genomic DNA showed that the small RNA was derived from tRNAGly(GCC). To determine the overall features of the tRNA-derived fragments (tRFs) of T. cancriformis, the small RNA sequences in each of the six developmental stages (egg, 1st - 4th instar larvae, and adult) were compared with the mitochondrial and nuclear tRNA sequences. Results: We found that the tRFs were derived from mitochondrial and nuclear tRNAs corresponding to 16 and 39 anticodons, respectively. The total read number of nuclear tRFs was approximately 400 times larger than the number of mitochondrial tRFs. Interestingly, the main regions in each parental tRNA from which these tRFs were derived differed, depending on the parental anticodon. Mitochondrial tRFSer(GCU)s were abundantly produced from the 5' half regions of the parental tRNA, whereas mitochondrial tRFVal(UAC)s were mainly produced from the 3' end regions. Highly abundant nuclear tRFs, tRFGly(GCC)s, tRFGly(CCC)s, tRFGlu(CUC)s, and tRFLys(CUU)s were derived from the 5' half regions of the parental tRNAs. Further analysis of the tRF read counts in the individual developmental stages suggested that the expression of mitochondrial and nuclear tRFs differed during the six stages. Based on these data, we precisely summarized the positions of the tRFs in their parental tRNAs and their expression changes during development. Conclusions: Our results reveal the entire dynamics of the tRFs from both the nuclear and mitochondrial genomes of T. cancriformis and indicate that the majority of tRFs in the cell are derived from nuclear tRNAs. This study provides the first examples of developmentally expressed mitochondrial tRFs.

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Taxonomic reassessment of strains formerly classified as chloromonas insignis (Volvocales, chlorophyceae) and description of Gloeomonas anomalipyrenoides sp. nov.

Nakada T., Matsuzaki R., Krienitz L., Tomita M., Nozaki H.

Acta Phytotaxonomica et Geobotanica 66 ( 1 ) 23 - 33 2015

ISSN 13467565

Two strains previously identified as Chloromonas insignis, NIES-447 and ACOI-3241, and two new strains related to NIES-447 were examined using light microscopy. The molecular phylogeny of their 18S rRNA gene sequences were also examined, and their identifications are reassessed. The strains represent two independent species, both of which can be distinguished from Chloromonas insignis. Strain NIES-447 and the two new strains each have large, irregular atypical pyrenoids without associated starch granules. They are described here as Gloeomonas anomalipyrenoides Nakada et al. sp. nov. Strain ACOI-3241, which has small atypical pyrenoids on the tips of the chloroplast lobes, was identified as C. vernalis (Skuja) Nakada comb. nov. The identity of the three strains of G. anomalipyrenoides was verified by their identical ITS-2 rDNA sequences. Both G. anomalipyrenoides and C. vernalis of the clade Chloromonadinia in the Volvocales (Chlorophyceae) are closely related to other species of Gloeomonas and to C. kasaiae Matsuzaki, Nakada, Y. Hara & Nozaki, respectively.

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Systems biology strategy reveals PKCd is key for sensitizing TRAIL-resistant human fibrosarcoma

Hayashi K., Tabata S., Piras V., Tomita M., Selvarajoo K.

Frontiers in Immunology (Frontiers in Immunology) 6 ( JAN ) 659 2015

ISSN 1664-3224

Cancer cells are highly variable and largely resistant to therapeutic intervention. Recently, the use of the tumor necrosis factor related apoptosis-inducing ligand (TRAIL) induced treatment is gaining momentum due to TRAIL's ability to specifically target cancers with limited effect on normal cells. Nevertheless, several malignant cancer types still remain non-sensitive to TRAIL. Previously, we developed a dynamic computational model, based on perturbation-response differential equations approach, and predicted protein kinase C (PKC) as the most effective target, with over 95% capacity to kill human fibrosarcoma (HT1080) in TRAIL stimulation (1). Here, to validate the model prediction, which has significant implications for cancer treatment, we conducted experiments on two TRAIL-resistant cancer cell lines (HT1080 and HT29). Using PKC inhibitor bisindolylmaleimide I, we demonstrated that cell viability is significantly impaired with over 95% death of both cancer types, in consistency with our previous model. Next, we measured caspase-3, Poly (ADP-ribose) polymerase (PARP), p38, and JNK activations in HT1080, and confirmed cell death occurs through apoptosis with significant increment in caspase-3 and PARP activations. Finally, to identify a crucial PKC isoform, from 10 known members, we analyzed each isoform mRNA expressions in HT1080 cells and shortlisted the highest 4 for further siRNA knock-down (KD) experiments. From these KDs, PKCd produced the most cancer cell death in conjunction with TRAIL. Overall, our approach combining model predictions with experimental validation holds promise for systems biology based cancer therapy.

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G-Links: A gene-centric link acquisition service

Arakawa K., Oshita K., Tomita M.

F1000Research (F1000Research) 3 285 2015

ISSN 20461402

With the availability of numerous curated databases, researchers are now able to efficiently use the multitude of biological data by integrating these resources via hyperlinks and cross-references. A large proportion of bioinformatics research tasks, however, may include labor-intensive tasks such as fetching, parsing, and merging datasets and functional annotations from distributed multi-domain databases. This data integration issue is one of the key challenges in bioinformatics. We aim to provide an identifier conversion and data aggregation system as a part of solution to solve this problem with a service named G-Links, 1) by gathering resource URI information from 130 databases and 30 web services in a gene-centric manner so that users can retrieve all available links about a given gene, 2) by providing RESTful API for easy retrieval of links including facet searching based on keywords and/or predicate types, and 3) by producing a variety of outputs as visual HTML page, tab-delimited text, and in Semantic Web formats such as Notation3 and RDF. G-Links as well as other relevant documentation are available at http://link.g-language.org/.

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Development of quantitative method for determination of γ-glutamyl peptides by capillary electrophoresis tandem mass spectrometry: An efficient approach avoiding matrix effect

Hirayama A., Igarashi K., Tomita M., Soga T.

Journal of Chromatography A (Journal of Chromatography A) 1369 161 - 169 2014.11

ISSN 00219673

Serum γ-glutamyl di- and tripeptides have proven to be useful biomarkers to accurately predict nine different forms of liver disease. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM), serum and liver samples spiked with γ-glutamyl peptide standards were analyzed to estimate accuracy. Unexpectedly, the recovery rates for several γ-glutamyl peptides in the serum samples were quite low, whereas values for some γ-glutamyl peptides in the liver samples were highly elevated. Most of these peptides were barely retained on the reverse-phase column, resulting in significant ion suppression or enhancement. In contrast, a capillary electrophoresis tandem mass spectrometry (CE-MS/MS) method with MRM was minimally affected by matrix effects. Of the 39 tested compounds, most of γ-glutamyl peptides that did not contain a thiol substituent in its structure gave acceptable recoveries (70-120%), and limits of detection for the analytes were between 3.6 and 800. nmol/l with pressure injection at 5. kPa for 10. s (ca. 10. nl). The CE-MS/MS method provided high resolution and proved to be highly selective and sensitive, its utility being demonstrated by the determination of γ-glutamyl di- and tripeptides in serum and liver samples.

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Time resolved DNA occupancy dynamics during the respiratory oscillation uncover a global reset point in the yeast growth program

Amariei C., Machné R., Stolc V., Soga T., Tomita M., Murray D.B.

Microbial Cell (Microbial Cell) 1 ( 9 ) 279 - 288 2014.09

ISSN 2311-2638

The structural dynamics of chromatin have been implicated in the regulation of fundamental eukaryotic processes, such as DNA transcription, replication and repair. Although previous studies have revealed that the chromatin landscape, nucleosome remodeling and histone modification events are intimately tied into cellular energetics and redox state, few studies undertake defined time-resolved measurements of these state variables. Here, we use metabolically synchronous, continuously-grown yeast cultures to measure DNA occupancy and track global patterns with respect to the metabolic state of the culture. Combined with transcriptome analyses and ChIP-qPCR experiments, these paint an intriguing picture where genome-wide nucleosome focusing occurs during the recovery of energy charge, followed by clearance of the promoter regions and global transcriptional slow-down, thus indicating a nucleosome-mediated “reset point” for the cycle. The reset begins at the end of the catabolic and stress-response transcriptional programs and ends prior to the start of the anabolic and cell-growth transcriptional program, and the histones on genes from both the catabolic and anabolic superclusters are deacetylated.

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Temporal Enhancement of Trapped Field in a Compact NMR Magnet Comprising YBCO Annuli.

Hahn S, Kim Y, Voccio JP, Song J, Bascuñán J, Tomita M, Iwasa Y

IEEE transactions on applied superconductivity : a publication of the IEEE Superconductivity Committee 24 ( 3 ) 2014.06

ISSN 1051-8223

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Contribution of developmental changes in energy metabolism to excitation-contraction coupling of a ventricular cell: A simulation study

Sano, I., H., Toki, T., Naito, Y. and Tomita, M.

Computing in Cardiology 41 ( January ) 73 - 76 2014

ISSN 23258861

In fetal guinea pigs, ventricular cells have higher anaerobic glycolytic capacity and lower mitochondrial enzyme activity. Here, we implemented developmental changes in glycolytic enzyme activity, concentrations of glycogen, and total creatine in late embryonic and adult ventricular cell models. We then simulated the effects of hypoxic conditions on dynamic changes in contractile force and ATP concentration. Our model demonstrates that fetal ventricular cells maintain ATP for longer periods of time than adult ventricular cells. This is consistent with reported dynamics of ventricular cells under hypoxic conditions.

Global metabolic network reorganization by adaptive mutations allows fast growth of Escherichia coli on glycerol

Cheng K., Lee B., Masuda T., Ito T., Ikeda K., Hirayama A., Deng L., Dong J., Shimizu K., Soga T., Tomita M., Palsson B., Robert M.

Nature Communications (Nature Communications) 31 3233 2014

Joint Work

Comparative whole-genome sequencing enables the identification of specific mutations during adaptation of bacteria to new environments and allelic replacement can establish their causality. However, the mechanisms of action are hard to decipher and little has been achieved for epistatic mutations, especially at the metabolic level. Here we show that a strain of Escherichia coli carrying mutations in the rpoC and glpK genes, derived from adaptation in glycerol, uses two distinct metabolic strategies to gain growth advantage. A 27-bp deletion in the rpoC gene first increases metabolic efficiency. Then, a point mutation in the glpK gene promotes growth by improving glycerol utilization but results in increased carbon wasting as overflow metabolism. In a strain carrying both mutations, these contrasting carbon/energy saving and wasting mechanisms work together to give an 89% increase in growth rate. This study provides insight into metabolic reprogramming during adaptive laboratory evolution for fast cellular growth.

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Metabolomic profiling of sodium fluoride-induced cytotoxicity in an oral squamous cell carcinoma cell line

Sakagami H., Sugimoto M., Tanaka S., Onuma H., Ota S., Kaneko M., Soga T., Tomita M.

Metabolomics 10 ( 2 ) 270 - 279 2014

Joint Work, ISSN 15733882

Sodium fluoride (NaF) is used in dentistry as a preventive agent for dental caries because of its ability to remineralize the tooth surface and its antibacterial effect. Although one of its target molecules in bacteria is enolase, its site of action in human cells has not been identified. The aim of this study was to identify target metabolites that are coupled to NaF-induced cytotoxicity in the HSC-2 human oral squamous cell carcinoma cell line. Cell viability, membrane integrity and apoptosis induction were analyzed by MTT assay, trypan blue exclusion and caspase-3 activation, respectively. Cells were treated with a minimal cytotoxic concentration of NaF for various times and subjected to comprehensive metabolomics analysis using capillary electrophoresis-mass spectrometry. In the early stages, inhibition of the enolase reaction in glycolysis pathway was observed. This was coupled with rapid inhibition of the progression of TCA cycle. In the later stages, gradual increases in the AMP/ATP ratio (a putative marker of apoptosis) and oxidized products (e.g. GSSH, and methionine sulfoxide), and marginal changes in polyamine levels (putative marker of necrosis) were observed. This manuscript provides the new insight into the global impact of NaF on metabolic pathways in human oral squamous cell carcinoma cells.

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Quantifying periodicity in omics data

Amariei, A., Tomita, M. and Murray, B.D.

Frontiers in Cell and Developmental Biology (Frontiers in Cell and Developmental Biology) 2 ( AUG ) 40 2014

Joint Work, ISSN 2296-634X

Oscillations play a significant role in biological systems, with many examples in the fast, ultradian, circadian, circalunar, and yearly time domains. However, determining periodicity in such data can be problematic. There are a number of computational methods to identify the periodic components in large datasets, such as signal-to-noise based Fourier decomposition, Fisher's g-test and autocorrelation. However, the available methods assume a sinusoidal model and do not attempt to quantify the waveform shape and the presence of multiple periodicities, which provide vital clues in determining the underlying dynamics. Here, we developed a Fourier based measure that generates a de-noised waveform from multiple significant frequencies. This waveform is then correlated with the raw data from the respiratory oscillation found in yeast, to provide oscillation statistics including waveform metrics and multi-periods. The method is compared and contrasted to commonly used statistics. Moreover, we show the utility of the program in the analysis of noisy datasets and other high-throughput analyses, such as metabolomics and flow cytometry, respectively.

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Transcriptome-wide variability in single embryonic development cells

Piras V., Tomita M., Selvarajoo K.

Scientific Reports (Scientific Reports) 4 7137 2014

Joint Work

Molecular heterogeneity of individual molecules within single cells has been recently shown to be crucial for cell fate diversifications. However, on a global scale, the effect of molecular variability for embryonic developmental stages is largely underexplored. Here, to understand the origins of transcriptome-wide variability of oocytes to blastocysts in human and mouse, we examined RNA-Seq datasets. Evaluating Pearson correlation, Shannon entropy and noise patterns (•2 vs.μ), our investigations reveal a phase transition from low to saturating levels of diversity and variability of transcriptome-wide expressions through the development stages. To probe the observed behaviour further, we utilised a stochastic transcriptional model to simulate the global gene expressions pattern for each development stage. From the model, we concur that transcriptome-wide regulation initially begins from 2-cell stage, and becomes strikingly variable from 8-cell stage due to amplification and quantal transcriptional activity.

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Genome Analysis Workshop: a Personal Genomics class at Keio SFC

Arakawa, K. and Tomita, M.

KEIO SFC JOURNAL 14 158 - 197 2014

Joint Work

Undesigned selection for replication termination of bacterial chromosomes

Kono, N., Arakawa, K., Mitsuru Sato; Yoshikawa, H., Masaru Tomita, M. and Itaya, M.

Journal of Molecular Biology (Journal of Molecular Biology) 426 ( 16 ) 2918 - 2927 2014

Joint Work, ISSN 00222836

The oriC DNA replication origin in bacterial chromosomes, the location of which appears to be physically identified, is genetically regulated by relevant molecular machinery. In contrast, the location of the terminus remains obscure for many bacterial replicons, except for terC, the proposed and well-studied chromosome termination site in certain bacteria. The terC locus, which is composed of specific sequences for its binding protein, is located at a site opposite from oriC, exhibiting a symmetric structure around the oriC-terC axis. Here, we investigated Bacillus subtilis 168 strains whose axes were hindered and found that the native terC function was robust. However, eradication of terminus region specific binding protein resulted in the natural terC sites not being used for termination; instead, new termini were selected at a location exactly opposite to oriC. We concluded that replication generally terminates at the loci where the two approaching replisomes meet. This site was automatically selected, and two replisomes moving at the same rate supported symmetrical chromosome structures relative to oriC. The rule, which was even validated by artificial chromosomes irrespective of oriC, should be general for replicons administered by two replisomes. © 2014 The Authors.

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Multi-faceted integrated omics analysis revealed parsley (Petroselinum crispum) as a novel dietary intervention in dextran sodium sulphate induced colitic mice

Jia, H., Aw, W., Hanate, M., Takahashi, S., Kenji, S., Tanaka, H., Tomita, M., and Kato, H.

Journal of Functional Foods 11 ( C ) 438 - 448 2014

Joint Work, ISSN 17564646

Here we describe our unprecedented approach in proposing parsley as a nutraceutical intervention in inflammatory bowel disease (IBD) via multi-omics evaluation using dextran sodium sulphate (DSS)-induced colitis. Seven-week-old male C57BL/6J mice were fed either 2% parsley or basal diet and drank normal-drinking-water for 1 week after which colitis was induced by administering 1.5% (w/v) DSS-drinking-water for 9 days. Parsley supplementation significantly improved colon shortening and increased disease activity index. Colonic transcriptome revealed down-regulation of inflammatory cytokines, hepatic transcriptome and metabolome revealed up-regulation of fatty-acid synthesis genes, thereby improving body weight loss. Down-regulated cancer markers were observed in the hepatic transcriptome and proteome. Plasma metabolite analysis indicated shifts in the citric and urea cycle, implicating improved impaired glycolysis and oxidative stress. Parsley's role in preventing against IBD was highlighted by this pioneering multi-integrated-omics which is expected to be useful and informative, and shape the future of nutraceutical and functional food research.

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Hybridization between Japanese and North American Chlamydomonas reinhardtii (Volvocales, Chlorophyceae)

Nakada T, Tsuchida Y, Arakawa K, Ito T and Tomita M.

Phycological Research 62 ( 3 ) 232 - 266 2014

Joint Work, ISSN 13220829

The microalga Chlamydomonas reinhardtii is a model organism whose whole genome has been sequenced. Although considered a cosmopolitan species, only eastern North American isolates of C. reinhardtii were available before 2010, when new Japanese isolates were reported. In the study describing the new Japanese isolates, zygote formation between Japanese and North American strains was shown, but germination was not demonstrated. In this study, the germination of intercontinental hybrid zygotes was examined using wild-type Japanese strains and mutant American strains that cannot utilize nitrate. Several clonal progeny strains were established, and the progeny strains were screened based on mating type and nitrate utilization to confirm their hybrid nature. The establishment of four intercontinental hybrid strains with different phenotypic combinations was confirmed by sequencing mating type-specific and nitrate reductase-related genes. The potential for hybrid formation between Japanese and North American strains suggests the existence of a worldwide mating population of C. reinhardtii. © 2014 Japanese Society of Phycology.

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Effect of codon adaptation on codon-level and gene-level translation efficiency in vivo

Nakahigashi, K., Takai, Y., Shiwa, Y., Wada, M., Honma, M., Yoshikawa, H., Tomita, M., Kanai, A. and Mori, H

BMC Genomics (BMC Genomics) 15 ( 1 ) 1115 2014

Joint Work

Background: There is a significant difference between synonymous codon usage in many organisms, and it is known that codons used more frequently generally showed efficient decoding rate. At the gene level, however, there are conflicting reports on the existence of a correlation between codon adaptation and translation efficiency, even in the same organism. Results: To resolve this issue, we cultured Escherichia coli under conditions designed to maintain constant levels of mRNA and protein and subjected the cells to ribosome profiling (RP) and mRNA-seq analyses. We showed that the RP results correlated more closely with protein levels generated under similar culture conditions than with the mRNA abundance from the mRNA-seq. Our result indicated that RP/mRNA ratio could be used as a measure of translation efficiency at gene level. On the other hand, the RP data showed that codon-specific ribosome density at the decoding site negatively correlated with codon usage, consistent with the hypothesis that preferred codons display lower ribosome densities due to their faster decoding rate. However, highly codon-adapted genes showed higher ribosome densities at the gene level, indicating that the efficiency of translation initiation, rather than higher elongation efficiency of preferred codons, exerted a greater effect on ribosome density and thus translation efficiency. Conclusions: These findings indicate that evolutionary pressure on highly expressed genes influenced both codon bias and translation initiation efficiency and therefore explains contradictory findings that codon usage bias correlates with translation efficiency of native genes, but not with the artificially created gene pool, which was not subjected to evolution pressure.

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Comparative metabolite profiling of foxglove aphids (Aulacorthum solani Kaltenbach) on leaves of resistant and susceptible soybean strains

Sato D., Sugimoto M., Akashi H., Tomita M., Soga T.

Molecular BioSystems (Molecular BioSystems) 10 ( 4 ) 909 - 915 2014

ISSN 1742206X

Aphid infestations can cause severe decreases in soybean (Glycine max [L.] Merr.) yield. Since planting aphid-resistant soybean strains is a promising approach for pest control, understanding the resistance mechanisms employed by aphids is of considerable importance. We compared aphid resistance in seven soybean strains and found that strain Tohoku149 was the most resistant to the foxglove aphid, Aulacorthum solani Kaltenbach. We subsequently analyzed the metabolite profiles of aphids cultured on the leaves of resistant and susceptible soybean strains using capillary electrophoresis-time-of-flight mass spectrometry. Our findings showed that the metabolite profiles of several amino acids, glucose 6-phosphate, and components of the tricarboxylic acid cycle were similar in aphids reared on Tohoku149 leaves and in aphids maintained under conditions of starvation, suggesting that Tohoku149 is more resistant to aphid feeding. Compared to susceptible strains, we also found that two methylated metabolites, S-methylmethionine and trigonelline, were either not detected or decreased in aphids reared on Tohoku149 plants. Since these metabolites function as important sulfur transporters in phloem sap and osmoprotectants involved in salt and drought stress, respectively, aphid-resistance is considered to be related to sulfur metabolism and methylation. These results contribute to an increase in our understanding of soybean aphid resistance mechanisms at the molecular level. © 2014 The Royal Society of Chemistry.

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Exceptional Evolutionary Divergence of Human Muscle and Brain Metabolomes Parallels Human Cognitive and Physical Uniqueness

Bozek K., Wei Y., Yan Z., Liu X., Xiong J., Sugimoto M., Tomita M., Pääbo S., Pieszek R., Sherwood C.C., Hof P.R., Ely J.J., Steinhauser D., Willmitzer L., Bangsbo J., Hansson O., Call J., Giavalisco P., Khaitovich P.

PLoS Biology (PLoS Biology) 12 ( 5 ) e1001871 2014

ISSN 15449173

Metabolite concentrations reflect the physiological states of tissues and cells. However, the role of metabolic changes in species evolution is currently unknown. Here, we present a study of metabolome evolution conducted in three brain regions and two non-neural tissues from humans, chimpanzees, macaque monkeys, and mice based on over 10,000 hydrophilic compounds. While chimpanzee, macaque, and mouse metabolomes diverge following the genetic distances among species, we detect remarkable acceleration of metabolome evolution in human prefrontal cortex and skeletal muscle affecting neural and energy metabolism pathways. These metabolic changes could not be attributed to environmental conditions and were confirmed against the expression of their corresponding enzymes. We further conducted muscle strength tests in humans, chimpanzees, and macaques. The results suggest that, while humans are characterized by superior cognition, their muscular performance might be markedly inferior to that of chimpanzees and macaque monkeys. © 2014 Bozek et al.

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Light microscopy and phylogenetic analyses of Chlamydomonas species (volvocales, chlorophyceae). II. molecular phylogeny, secondary structure of ITS-2, cell morphology, and nomenclature of Microglena opisthopyren, and m. media, comb. nov.

Nakada T., Tomita M.

Acta Phytotaxonomica et Geobotanica 65 ( 2 ) 67 - 73 2014

ISSN 13467565

Two species of Chlamydomonas Ehrenb. in the clade Monadinia, Chlamydomonas pseudomacrostigma L. Ş. Péterfi ex H. Ettl and C. media G. A. Klebs, were recently reported from Japan. However, several species in the clade were transferred to the genus Microglena Ehrenb. by different authors, and therefore the taxonomic treatment of these two species should be revisited. Here, their identifications were re-examined based on 18S rRNA gene phylogeny, secondary structure comparisons of ITS-2 rDNA, and vegetative cell morphology. Based on these comparisons, C. pseudomacrostigma and C. media were treated as a later synonym of Microglena opisthopyren (Skuja) Demchenko, Mikhailyuk & Pröschold and an earlier synonym of M. skujae Demchenko, Mikhailyuk & Pröschold, respectively. A new combination, Microglena media (G.A. Klebs) Nakada, comb. nov., is proposed for the latter.

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Dynamics of serum metabolites in patients with chronic hepatitis C receiving pegylated interferon plus ribavirin: a metabolomics analysis

Saito T, Sugimoto M, Igarashi K, Saito K, Shao L, Katsumi T, Tomita K, Sato C, Okumoto K, Nishise Y, Watanabe H, Tomita M, Ueno Y, Soga T.

Metabolism (Metabolism: Clinical and Experimental) 62 ( 11 ) 1577 - 1586 2013.11

Joint Work, ISSN 00260495

Objectives Serum samples from patients with chronic hepatitis C were subjected to metabolomics analysis to clarify the pretreatment characteristics of their metabolites and also changes in specific metabolites resulting from antiviral therapy with pegylated interferon plus ribavirin (PegIFN/RBV). Materials/Methods The serum levels of low-molecular-weight metabolites in the twenty patients before and 24 weeks after completion of PegIFN/RBV therapy were analyzed using capillary electrophoresis and liquid chromatography-mass spectrometry. Results Ten patients showed a non-virological response (NVR) and 10 achieved a sustained virological response (SVR) with eradication of viremia. The pretreatment levels of tryptophan were significantly higher in the patients of SVR than in those of NVR (p = 0.010). The area under the curve (AUC) value of tryptophan calculated from the receiver operating characteristic (ROC) curve for discriminating SVR from NVR was 0.84 (95% confidential interval, 0.66-1.02, p = 0.010). The ROC curve of multiple logistic regression model incorporating the pretreatment levels of tryptophan and γ-glutamate-arginine showed that the AUC value was highly significant (AUC = 0.92, 95% confidential interval, 0.79-1.05, p = 0.002). Twenty four weeks after completion of treatment, the levels of γ-glutamyl dipeptides, glutamic acid, 5-oxoproline, glucosamine and methionine sulfoxide were decreased, whereas those of 5-methoxy-3- indoleacetate, glutamine, kynurenine and lysine were increased significantly (p < 0.05) in both the NVR and SVR patients. Conclusions The pretreatment serum levels of certain metabolites including tryptophan are associated with the response to PegIFN/RBV therapy. PegIFN/RBV therapy can ameliorate the oxidative stress responsible for glutathione metabolism. © 2013 Elsevier Inc.

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The dynamics of cellular energetics during continuous yeast culture

Amariei C., Machne R., Sasidharan K., Gottstein W., Tomita M., Soga T., Lloyd D., Murray D.B.

Proceedings of the Annual International Conference of the IEEE Engineering in Medicine and Biology Society, EMBS (Proceedings of the Annual International Conference of the IEEE Engineering in Medicine and Biology Society, EMBS) 2013 2708 - 2711 2013.10

ISSN 1557170X

A plethora of data is accumulating from high throughput methods on metabolites, coenzymes, proteins, and nucleic acids and their interactions as well as the signalling and regulatory functions and pathways of the cellular network. The frozen moment viewed in a single discrete time sample requires frequent repetition and updating before any appreciation of the dynamics of component interaction becomes possible. Even then in a sample derived from a cell population, time-averaging of processes and events that occur in out-of-phase individuals blur the detailed complexity of single cell organization. Continuously-grown cultures of yeast can become spontaneously self-synchronized, thereby enabling resolution of far more detailed temporal structure. Continuous on-line monitoring by rapidly responding sensors (O 2 electrode and membrane-inlet mass spectrometry for O2, CO2 and H2S; direct fluorimetry for NAD(P)H and flavins) gives dynamic information from time-scales of minutes to hours. Supplemented with capillary electophoresis and gas chromatography mass spectrometry and transcriptomics the predominantly oscillatory behaviour of network components becomes evident, with a 40 min cycle between a phase of increased respiration (oxidative phase) and decreased respiration (reductive phase). Highly pervasive, this ultradian clock provides a coordinating function that links mitochondrial energetics and redox balance to transcriptional regulation, mitochondrial structure and organelle remodelling, DNA duplication and cell division events. Ultimately, this leads to a global partitioning of anabolism and catabolism and the enzymes involved, mediated by a relatively simple ATP feedback loop on chromatin architecture. © 2013 IEEE.

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Time-resolved metabolomics of a novel trebouxiophycean alga using 13CO2 feeding

Ito T., Sugimoto M., Toya Y., Ano Y., Kurano N., Soga T., Tomita M.

Journal of Bioscience and Bioengineering (Journal of Bioscience and Bioengineering) 116 ( 3 ) 408 - 415 2013.09

ISSN 13891723

Oil-rich algae are potentially promising as next-generation biofuel feedstock. However, the productivity of oil needs to be improved for industrial use. The biosynthesis of oil and its control mechanism have not been characterized in any algae, and understanding the metabolic network is vital to achieve the precise engineering of algae metabolic pathways. "Pseudochoricystis ellipsoidea" MBIC 11204, a novel microalgal strain, accumulates a large amount of lipids in nitrogen-deficient conditions. In this study, "P.ellipsoidea" was grown in flat flasks with continuous illumination and aeration with 1% CO2 at 25°C. During the exponential growth phase, CO2 was switched to 13C-labeled CO2 and samples were collected for time-course experiments. Seventy-eight pairs of unlabeled and uniformly 13C-labeled metabolites were quantified using a capillary electrophoresis- and liquid chromatography-mass spectrometry for ionic primary metabolites and lipids, respectively. The 13C-exchange indices of the metabolites were calculated from a concentration of unlabeled and uniformly-labeled metabolites. A hierarchical clustering analysis of the dynamics of the indices revealed 4 characteristic clusters, two of which represented rapidly-labeled metabolites, mainly composed of primary metabolites, while the two other clusters represented slowly-labeled metabolites, mainly composed of lipids. Moreover, the labeling order of these clusters was mainly matched to the metabolic process of Chlamydomonas reinhardtii, a model organism of green algae. In TCA cycle, anomalistically different of the labeling order was found. To the author's knowledge, this study for the first time in literature, characterize the features of global metabolism in "P.ellipsoidea.". © 2013 The Society for Biotechnology, Japan.

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Dynamic Simulation and Metabolome Analysis of Long-Term Erythrocyte Storage in Adenine-Guanosine Solution

Nishino T., Yachie-Kinoshita A., Hirayama A., Soga T., Suematsu M., Tomita M.

PLoS ONE (PLoS ONE) 8 ( 8 ) e71060 2013.08

Although intraerythrocytic ATP and 2,3-bisphophoglycerate (2,3-BPG) are known as direct indicators of the viability of preserved red blood cells and the efficiency of post-transfusion oxygen delivery, no current blood storage method in practical use has succeeded in maintaining both these metabolites at high levels for long periods. In this study, we constructed a mathematical kinetic model of comprehensive metabolism in red blood cells stored in a recently developed blood storage solution containing adenine and guanosine, which can maintain both ATP and 2,3-BPG. The predicted dynamics of metabolic intermediates in glycolysis, the pentose phosphate pathway, and purine salvage pathway were consistent with time-series metabolome data measured with capillary electrophoresis time-of-flight mass spectrometry over 5 weeks of storage. From the analysis of the simulation model, the metabolic roles and fates of the 2 major additives were illustrated: (1) adenine could enlarge the adenylate pool, which maintains constant ATP levels throughout the storage period and leads to production of metabolic waste, including hypoxanthine; (2) adenine also induces the consumption of ribose phosphates, which results in 2,3-BPG reduction, while (3) guanosine is converted to ribose phosphates, which can boost the activity of upper glycolysis and result in the efficient production of ATP and 2,3-BPG. This is the first attempt to clarify the underlying metabolic mechanism for maintaining levels of both ATP and 2,3-BPG in stored red blood cells with in silico analysis, as well as to analyze the trade-off and the interlock phenomena between the benefits and possible side effects of the storage-solution additives. © 2013 Yachie-Kinoshita et al.

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GEMBASSY: An EMBOSS associated software package for comprehensive genome analyses

Itaya H, Oshita K, Arakawa K, Tomita M.

Source Code for Biology and Medicine (Source Code for Biology and Medicine) 8 ( 17 ) 17 2013.08

Joint Work, ISSN 1751-0473

The popular European Molecular Biology Open Software Suite (EMBOSS) currently contains over 400 tools used in various bioinformatics researches, equipped with sophisticated development frameworks for interoperability and tool discoverability as well as rich documentations and various user interfaces. In order to further strengthen EMBOSS in the fields of genomics, we here present a novel EMBOSS associated software (EMBASSY) package named GEMBASSY, which adds more than 50 analysis tools from the G-language Genome Analysis Environment and its Representational State Transfer (REST) and SOAP web services. GEMBASSY basically contains wrapper programs of G-language REST/SOAP web services to provide intuitive and easy access to various annotations within complete genome flatfiles, as well as tools for analyzing nucleic composition, calculating codon usage, and visualizing genomic information. For example, analysis methods such as for calculating distance between sequences by genomic signatures and for predicting gene expression levels from codon usage bias are effective in the interpretation of meta-genomic and meta-transcriptomic data. GEMBASSY tools can be used seamlessly with other EMBOSS tools and UNIX command line tools. The source code written in C is available from GitHub (https://github.com/celery-kotone/GEMBASSY/) and the distribution package is freely available from the GEMBASSY web site (http://www.g-language.org/gembassy/). © 2013 Itaya et al.; licensee BioMed Central Ltd.

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Global microRNA elevation by inducible Exportin 5 regulates cell cycle entry

Iwasaki Y.W., Kiga K., Kayo H., Fukuda-Yuzawa Y., Weise J., Inada T., Tomita M., Ishihama Y., Fukao T.

RNA (RNA) 19 ( 4 ) 490 - 497 2013.04

ISSN 13558382

Proper regulation of gene expression during cell cycle entry ensures the successful completion of proliferation, avoiding risks such as carcinogenesis. The microRNA (miRNA) network is an emerging molecular system regulating multiple genetic pathways. We demonstrate here that the global elevation of miRNAs is critical for proper control of gene expression program during cell cycle entry. Strikingly, Exportin 5 (XPO5) is promptly induced during cell cycle entry by a PI3K-dependent post-transcriptional mechanism. Inhibition of XPO5 induction interfered with global miRNA elevation and resulted in a proliferation defect associated with delayed G1/S transition. During cell cycle entry, XPO5 therefore plays a paramount role as a critical molecular hub controlling the gene expression program through global regulation of miRNAs. Our data suggest that XPO5-mediated global miRNA elevation might be involved in a broad range of cellular events associated with cell cycle control. Copyright © 2013 RNA Society.

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Changes of metabolic profiles in an oral squamous cell carcinoma cell line induced by eugenol

Koh T, Murakami Y, Tanaka S, Machino M, Onuma H, Kaneko M, Sugimoto M, Soga T, Tomita M, Sakagami H.

In Vivo (In Vivo) 27 ( 2 ) 233 - 243 2013.03

Joint Work, ISSN 0258851X

Background: We have recently reported that eugenol exerted indiscriminate cytotoxicity towards normal oral cells and oral squamous cell carcinoma (OSCC) cell lines without induction of apoptosis markers. In order to investigate the underlying mechanisms of cytotoxicity induction, we investigated the effect of short-term treatment with eugenol on the metabolic profiles of a human OSCC cell line (HSC-2). Materials and Methods: The viable cell number was determined by direct cell counting with a hemocytometer after trypsinization. After washing with 5% D-mannitol solution (found to retain the highest amounts of intracellular metabolites among several washing conditions), cellular metabolites were extracted with methanol with internal markers and then subjected to metabolomic analysis. Results: Cytotoxic concentrations of eugenol induced the reduction of ATP utilization (assessed by a significant reduction of the AMP/ATP and ADP/ATP ratio), of oxidative stress (assessed by the increase in oxidized form of glutathione, cysteine-glutathione disulfide and methionine sulfoxide), and an increase in the polyamines and glycolytic metabolites. Conclusion: The metabolic changes observed in this study suggest the induction of non-apoptotic cell death by eugenol.

Metabolomic study of Chilean biomining bacteria Acidithiobacillus ferrooxidans strain Wenelen and Acidithiobacillus thiooxidans strain Licanantay

Martínez P., Gálvez S., Ohtsuka N., Budinich M., Cortés M.P., Serpell C., Nakahigashi K., Hirayama A., Tomita M., Soga T., Martínez S., Maass A., Parada P.

Metabolomics (Metabolomics) 9 ( 1 ) 247 - 257 2013.02

ISSN 15733882

In this study, we present the first metabolic profiles for two bioleaching bacteria using capillary electrophoresis coupled with mass spectrometry. The bacteria, Acidithiobacillus ferrooxidans strain Wenelen (DSM 16786) and Acidithiobacillus thiooxidans strain Licanantay (DSM 17318), were sampled at different growth phases and on different substrates: the former was grown with iron and sulfur, and the latter with sulfur and chalcopyrite. Metabolic profiles were scored from planktonic and sessile states. Spermidine was detected in intra- and extracellular samples for both strains, suggesting it has an important role in biofilm formation in the presence of solid substrate. The canonical pathway for spermidine synthesis seems absent as its upstream precursor, putrescine, was not present in samples. Glutathione, a catalytic activator of elemental sulfur, was identified as one of the most abundant metabolites in the intracellular space in A. thiooxidans strain Licanantay, confirming its participation in the sulfur oxidation pathway. Amino acid profiles varied according to the growth conditions and bioleaching species. Glutamic and aspartic acid were highly abundant in intra- and extracellular extracts. Both are constituents of the extracellular matrix, and have a probable role in cell detoxification. This novel metabolomic information validates previous knowledge from in silico metabolic reconstructions based on genomic sequences, and reveals important biomining functions such as biofilm formation, energy management and stress responses. © 2012 The Author(s).

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Identification of a replication initiation protein of the pVV8 plasmid from Thermus thermophilus HB8.

Ohtani, N., Tomita, M. and Itaya, M.

Extremophiles. (Extremophiles) 17 ( 1 ) 15 - 28 2013

Joint Work, Accepted, ISSN 14310651

Recently, the extremely thermophilic bacterium Thermus thermophilus HB8 has been demonstrated to harbor a circular plasmid designated by pVV8 in addition to two well-known plasmids, pTT8 and pTT27, and its entire sequence has been determined. The absence of any obvious replication initiation gene in the 81. 2 kb plasmid prompted us to isolate its minimum replicon. By in vivo replication assays with fragments deleted in a stepwise manner, a minimum replicon containing a single ORF, TTHV001, was identified. A protein encoded by TTHV001 showed no amino acid sequence similarity to other function-known proteins. As the results of in vivo and in vitro experiments strongly suggested that the TTHV001 protein was involved in the replication initiation of pVV8, the protein and the gene were referred to as RepV and repV, respectively. The RepV protein binds to an inverted repeat sequence within its own repV gene and then triggers the unwinding of the DNA duplex in an A + T-rich region located just downstream from the inverted repeat. The in vivo replication assays with minimum replicon mutants in the RepV binding site or the unwinding region demonstrated that the unwinding in the region by the RepV binding was essential for pVV8 replication initiation. © 2012 Springer Japan.

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Metabolic and morphological changes of an oil accumulating trebouxiophycean alga in nitrogen-deficient conditions.

Ito, T., Tanaka, M., Shinkawa, H., Nakada, T., Ano, Y., Kurano, N., Soga, T. and Tomita, M.

Metabolomics (Metabolomics) 9 ( (Suppl 1) ) 178-187 - 187 2013

Research paper (scientific journal), Joint Work, Accepted, ISSN 15733882

Oil-rich algae have promising potential for a next-generation biofuel feedstock. Pseudochoricystis ellipsoidea MBIC 11204, a novel unicellular green algal strain, accumulates a large amount of oil (lipids) in nitrogen-deficient (-N) conditions. Although the oil bodies are easily visualized by lipophilic staining in the cells, little is known about how oil bodies are metabolically synthesized. Clarifying the metabolic profiles in -N conditions is important to understand the physiological mechanisms of lipid accumulations and will be useful to optimize culture conditions efficiently produce industrial oil. Metabolome and lipidome profiles were obtained, respectively, using capillary electrophoresis- and liquid chromatography-mass spectrometry from P. ellipsoidea in both nitrogen-rich (+N; rapid growth) and -N conditions. Relative quantities of more than 300 metabolites were systematically compared between these two conditions. Amino acids in nitrogen assimilation and N-transporting metabolisms were decreased to 1/20 the amount, or less, in -N conditions. In lipid metabolism, the quantities of neutral lipids increased greatly in -N conditions; however, quantities of nearly all the other lipids either decreased or only changed slightly. The morphological changes in +N and -N conditions were also provided by microscopy, and we discuss their relationship to the metabolic changes. This is the first approach to understand the novel algal strain's metabolism using a combination of wide-scale metabolome analysis and morphological analysis. © 2012 The Author(s).

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Metabolomic Profiling of Lung and Prostate Tumor Tissues by Capillary Electrophoresis Time-of-flight Mass Spectrometry

Kami, K., Fujimori, T., Sato, H., Sato, M., Yamamoto, H., Ohashi, Y., Sugiyama, N., Ishihama, Y., Onozuka, H., Kinoshita, T., Saito, N., Ochiai, A., *Esumi, H., Soga, T. and Tomita, M.

Metabolomics (Metabolomics) 9 ( 2 ) 444-453 - 453 2013

Research paper (scientific journal), Joint Work, Accepted, ISSN 15733882

Metabolic microenvironment of tumor cells is influenced by oncogenic signaling and tissue-specific metabolic demands, blood supply, and enzyme expression. To elucidate tumor-specific metabolism, we compared the metabolomics of normal and tumor tissues surgically resected pairwise from nine lung and seven prostate cancer patients, using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Phosphorylation levels of enzymes involved in central carbon metabolism were also quantified. Metabolomic profiles of lung and prostate tissues comprised 114 and 86 metabolites, respectively, and the profiles not only well distinguished tumor from normal tissues, but also squamous cell carcinoma from the other tumor types in lung cancer and poorly differentiated tumors from moderately differentiated tumors in prostate cancer. Concentrations of most amino acids, especially branched-chain amino acids, were significantly higher in tumor tissues, independent of organ type, but of essential amino acids were particularly higher in poorly differentiated than moderately differentiated prostate cancers. Organ-dependent differences were prominent at the levels of glycolytic and tricarboxylic acid cycle intermediates and associated energy status. Significantly high lactate concentrations and elevated activating phosphorylation levels of phosphofructokinase and pyruvate kinase in lung tumors confirmed hyperactive glycolysis. We highlighted the potential of CE-TOFMS-based metabolomics combined with phosphorylated enzyme analysis for understanding tissue-specific tumor microenvironments, which may lead to the development of more effective and specific anticancer therapeutics. © 2012 The Author(s).

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Physiological and environmental parameters associated with mass spectrometry-based salivary metabolomic profiles

Sugimoto, M., Saruta, J., Matsuki, C., To, M., Onuma, H., Kaneko, M., Soga, T., M Tomita, M. and Tsukinoki, K.

Metabolomics 9 ( 2 ) 454-463 - 463 2013

Research paper (scientific journal), Joint Work, Accepted, ISSN 15733882

Mass spectrometry (MS)-based metabolomic methods enable simultaneous profiling of hundreds of salivary metabolites, and may be useful to diagnose a wide range of diseases using saliva. However, few studies have evaluated the effects of physiological or environmental factors on salivary metabolomic profiles. Therefore, we used capillary electrophoresis-MS to analyze saliva metabolite profiles in 155 subjects with reasonable oral hygiene, and examined the effects of physiological and environmental factors on the metabolite profiles. Overall, 257 metabolites were identified and quantified. The global profiles and individual metabolites were evaluated by principle component analysis and univariate tests, respectively. Collection method, collection time, sex, body mass index, and smoking affected the global metabolite profiles. However, age also might contribute to the bias in sex and collection time. The profiles were relatively unaffected by other parameters, such as alcohol consumption and smoking, tooth brushing, or the use of medications or nutritional supplements. Temporomandibular joint disorders had relatively greater effects on salivary metabolites than other dental abnormalities (e. g., stomatitis, tooth alignment, and dental caries). These findings provide further insight into the diversity and stability of salivary metabolomic profiles, as well as the generalizability of disease-specific biomarkers. © 2012 Springer Science+Business Media New York.

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Computational analysis of associations between alternative splicing and histone modifications

Shindo, Y., Nozaki, T., Saito, R., Tomita, M.

FEBS LETTERS (FEBS Letters) 587 ( 5 ) 516-21 - 521 2013

Research paper (scientific journal), Joint Work, Accepted, ISSN 00145793

Pre-mRNA splicing is a complex process involving combinatorial effects of cis- and trans-elements. Here, we focused on histone modifications as typical trans-regulatory elements and performed systematic analyses of associations between splicing patterns and histone modifications by using publicly available ChIP-Seq, mRNA-Seq, and exon-array data obtained in two human cell lines. We found that several types of histone modifications including H3K36me3 were associated with the inclusion or exclusion of alternative exons. Furthermore, we observed that the levels of H3K36me3 and H3K79me1 in the cell lines were well correlated with the differences in alternative splicing patterns between the cell lines. © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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Physical Laws Shape Biology

Selvarajoo, K. and Tomita, M.

Science Feb 8 339 646 2013

Research paper (scientific journal), Single Work, Accepted

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Comprehensive Computational Analysis of Bacterial CRP/FNR Superfamily and its Target Motifs Reveals Stepwise Evolution of Transcriptional Networks

Matsui, M., Tomita, M. and Kanai, A.

Genome Biol Evol (Genome Biology and Evolution) 5 ( 2 ) 267-282 - 282 2013

Research paper (scientific journal), Joint Work

The cAMP receptor protein (CRP)/fumarate and nitrate reduction regulatory protein (FNR)-type transcription factors (TFs) are members of a well-characterized global TF family in bacteria and have two conserved domains: the N-terminal ligand-binding domain for small molecules (e.g., cAMP, NO, or O2) and the C-terminal DNA-binding domain. Although the CRP/FNR-type TFs recognize very similar consensus DNA target sequences, they can regulate different sets of genes in response to environmental signals. To clarify the evolution of the CRP/FNR-type TFs throughout the bacterial kingdom, we undertook a comprehensive computational analysis of a large number of annotated CRP/FNR-type TFs and the corresponding bacterial genomes. Based on theamino acid sequence similarities among 1,455 annotated CRP/FNR-type TFs, spectral clustering classified the TFs into 12 representative groups, and step wise clustering allowed us to propose a possible process of protein evolution. Although each cluster mainly consists of functionally distinct members (e.g., CRP, NTC, FNR-like protein, and Fix K), FNR-related TFs are found in several groups and are distributed in a wide range of bacterial phyla in the sequence similarity network. This result suggests that the CRP/FNR-type TFs originated from an ancestral FNR protein, involved in nitrogen fixation. Furthermore, a phylogenetic profiling analysis showed that combinations of TFs and their target genes have fluctuated dynamically during bacterial evolution. A genome-wide analysis of TF-binding sites also suggested that the diversity of the transcriptional regulatory system was derived by the stepwise adaptation of TF-binding sites to the evolution of TFs. © 2013 The Author(s).

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Quantitative assay for TALEN activity at endogenous genomic loci

Hisano, Y., Ota, S., Arakawa, K., Muraki, M., Kono, N., Oshita, K., Sakuma, T., Tomita, M., Yamamoto, T., Okada, Y. and *Kawahara, A.

Biology Open (Biology Open) 2 ( 4 ) 363-367 - 367 2013

Research paper (scientific journal), Joint Work, Accepted

Artificially designed nucleases such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can induce a targeted DNA double-strand break at the specific target genomic locus, leading to the frameshiftmediated gene disruption. However, the assays for their activity on the endogenous genomic loci remain limited. Herein, we describe a versatile modified lacZ assay to detect frameshifts in the nuclease target site. Short fragments of the genome DNA at the target or putative off-target loci were amplified from the genomic DNA of TALEN-treated or control embryos, and were inserted into the lacZa sequence for the conventional blue-white selection. The frequency of the frameshifts in the fragment can be estimated from the numbers of blue and white colonies. Insertions and/or deletions were easily determined by sequencing the plasmid DNAs recovered from the positive colonies. Our technique should offer broad application to the artificial nucleases for genome editing in various types of model organisms.

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Contribution of quantitative changes in individual ionic current systems to the embryonic development of ventricular myocytes: a simulation study

Okubo, C., Sano, H., Naito, Y. and Tomita, M.

Journal of Physiological Sciences (Journal of Physiological Sciences) 63 ( 5 ) 355-67 - 367 2013

Research paper (scientific journal), Joint Work, Accepted, ISSN 18806546

Early embryonic rodent ventricular cells exhibit spontaneous action potential (AP), which disappears in later developmental stages. Here, we used 3 mathematical models - the Kyoto, Ten Tusscher-Panfilov, and Luo-Rudy models - to present an overview of the functional landscape of developmental changes in embryonic ventricular cells. We switched the relative current densities of 9 ionic components in the Kyoto model, and 160 of 512 representative combinations were predicted to result in regular spontaneous APs, in which the quantitative changes in Na+ current (I Na) and funny current (I f) made large contributions to a wide range of basic cycle lengths. In all three models, the increase in inward rectifier current (I K1) before the disappearance of I f was predicted to result in abnormally high intracellular Ca2+ concentrations. Thus, we demonstrated that the developmental changes in APs were well represented, as I Na increased before the disappearance of I f, followed by a 10-fold increase in I K1. © 2013 The Author(s).

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Capillary electrophoresis-mass spectrometry-based metabolome analysis of serum and saliva from neurodegenerative dementia patients

Tsuruoka, M., Hara, J., Hirayama, A., Sugimoto, M., Soga, T., R. Shankle, R., W., and Tomita, M.

Electrophoresis (Electrophoresis) 34 ( 19 ) 2865-2872 - 2872 2013

Research paper (scientific journal), Joint Work, Accepted, ISSN 01730835

Despite increasing global prevalence, the precise pathogenesis and terms for objective diagnosis of neurodegenerative dementias remain controversial, and comprehensive understanding of the disease remains lacking. Here, we conducted metabolomic analysis of serum and saliva obtained from patients with neurodegenerative dementias (n = 10), including Alzheimer's disease, frontotemporal lobe dementia, and Lewy body disease, as well as from age-matched healthy controls (n = 9). Using CE-TOF-MS, six metabolites in serum (β-alanine, creatinine, hydroxyproline, glutamine, iso-citrate, and cytidine) and two in saliva (arginine and tyrosine) were significantly different between dementias and controls. Using multivariate analysis, serum was confirmed as a more efficient biological fluid for diagnosis compared to saliva; additionally, 45 metabolites in total were identified as candidate markers that could discriminate at least one pair of diagnostic groups from the healthy control group. These metabolites possibly provide an objective method for diagnosing dementia-type by multiphase screening. Moreover, diagnostic-type-dependent differences were observed in several tricarboxylic acid cycle compounds detected in serum, indicating that some pathways in glucose metabolism may be altered in dementia patients. This pilot study revealed novel alterations in metabolomic profiles between various neurodegenerative dementias, which would contribute to etiological investigations. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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Commensal microbe-derived butyrate induces the Differentiation of colonic regulatory T cells

Furusawa, Y., Obata, Y., Fukuda, S., Endo, T., A., Nakato, G., Takahashi, D., Nakanishi, Y., Uetake, C., Kato, K., Kato, T., Takahashi, M., Fukuda, N., N., Murakami, S., Miyauchi, E., Hino, S., Atarashi, K., Onawa, S., Fujimura, Y., Lockett, T., Clarke, J., M., Topping, D., L., Tomita, M., Hori, S., Ohara, O., Morita, T., Koseki, H., Kikuchi, J., Honda, K., Hase, K. and Ohno, H.

Nature (Nature) 504 ( 7480 ) 446 - 450 2013

Joint Work, ISSN 00280836

Gut commensal microbes shape the mucosal immune system by regulating the differentiation and expansion of several types of T cell. Clostridia, a dominant class of commensal microbe, can induce colonic regulatory T (T reg) cells, which have a central role in the suppression of inflammatory and allergic responses. However, the molecular mechanisms by which commensal microbes induce colonic T reg cells have been unclear. Here we show that a large bowel microbial fermentation product, butyrate, induces the differentiation of colonic T reg cells in mice. A comparative NMR-based metabolome analysis suggests that the luminal concentrations of short-chain fatty acids positively correlates with the number of T reg cells in the colon. Among short-chain fatty acids, butyrate induced the differentiation of T reg cells in vitro and in vivo, and ameliorated the development of colitis induced by adoptive transfer of CD4 + CD45RB hi T cells in Rag1 -/- mice. Treatment of naive T cells under the T reg -cell-polarizing conditions with butyrate enhanced histone H3 acetylation in the promoter and conserved non-coding sequence regions of the Foxp3 locus, suggesting a possible mechanism for how microbial-derived butyrate regulates the differentiation of T reg cells. Our findings provide new insight into the mechanisms by which host-microbe interactions establish immunological homeostasis in the gut. © 2013 Macmillan Publishers Limited. All rights reserved.

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A systems biology approach to suppress TNF-induced proinflammatory gene expressions

Hayashi K, Piras V, Tabata S, Tomita M, Selvarajoo K.

Cell Communication and Signaling (Cell Communication and Signaling) 11 ( 1 ) 84 2013

Joint Work

Background: Tumor necrosis factor (TNF) is a widely studied cytokine (ligand) that induces proinflammatory signaling and regulates myriad cellular processes. In major illnesses, such as rheumatoid arthritis and certain cancers, the expression of TNF is elevated. Despite much progress in the field, the targeted regulation of TNF response for therapeutic benefits remains suboptimal. Here, to effectively regulate the proinflammatory response induced by TNF, a systems biology approach was adopted. Results: We developed a computational model to investigate the temporal activations of MAP kinase (p38), nuclear factor (NF)-κ;B, and the kinetics of 3 groups of genes, defined by early, intermediate and late phases, in murine embryonic fibroblast (MEF) and 3T3 cells. To identify a crucial target that suppresses, and not abolishes, proinflammatory genes, the model was tested in several in silico knock out (KO) conditions. Among the candidate molecules tested, in silico RIP1 KO effectively regulated all groups of proinflammatory genes (early, middle and late). To validate this result, we experimentally inhibited TNF signaling in MEF and 3T3 cells with RIP1 inhibitor, Necrostatin-1 (Nec-1), and investigated 10 genes (Il6, Nfkbia, Jun, Tnfaip3, Ccl7, Vcam1, Cxcl10, Mmp3, Mmp13, Enpp2) belonging to the 3 major groups of upregulated genes. As predicted by the model, all measured genes were significantly impaired. Conclusions: Our results demonstrate that Nec-1 modulates TNF-induced proinflammatory response, and may potentially be used as a therapeutic target for inflammatory diseases such as rheumatoid arthritis and osteoarthritis. © 2013 Hayashi et al.; licensee BioMed Central Ltd.

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Metabolomic profiling of the response of susceptible and resistant soybean strains to foxglove aphid, Aulacorthum solani Kaltenbach.

Sato, D., Akashi, H., Sugimoto, M., Tomita, M. and Soga, T.

J Chromatogr B Analyt Technol Biomed Life Sci. (Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences) 925 95 - 103 2013

Joint Work, Accepted, ISSN 15700232

Aphid infection reduces soybean (Glycine max [L.] Merr.) yield. Consequently, cultivation of aphid-resistant strains is a promising approach to pest control, and understanding the resistance mechanism is of importance. Here, we characterized the resistance of soybeans to foxglove aphid, Aulacorthum solani Kaltenbach, at the metabolite level. First, we evaluated aphid mortality and settlement rates on the leaves of two soybean strains, 'Tohoku149' and 'Suzuyutaka', and found that the former had strong resistance soon after introduction of the aphids. The metabolomic response to aphid introduction was analyzed using capillary electrophoresis-time-of-flight mass spectrometry. We found the following three features in the profiles: (1) concentrations of citrate, amino acids, and their intermediates were intrinsically higher for Tohoku149 than Suzuyutaka, (2) concentrations of several metabolites producing secondary metabolites, such as flavonoids and alkaloids, drastically changed 6. h after aphid introduction, and (3) concentrations of TCA cycle metabolites increased in Tohoku149 48. h after aphid introduction. We also profiled free amino acids in aphids reared on both soybean strains and under starvation, and found that the profile of the aphids on Tohoku149 was similar to that of the starved aphids, but different to that of aphids on Suzuyutaka. These tests confirmed that aphids suck phloem sap even from Tohoku149. This study demonstrates the metabolomic profiles of both soybean strains and aphids, which will contribute to the molecular level understanding of mechanisms of soybean resistance to aphids. © 2013 Elsevier B.V.

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Merging multiple omics datasets in silico: statistical analyses and data interpretation.

Arakawa, K. and Tomita, M.

Methods Mol Biol. (Methods in Molecular Biology) 985 459 - 470 2013

Joint Work, Accepted, ISSN 10643745

By the combinations of high-throughput analytical technologies in the fields of transcriptomics, proteomics, and metabolomics, we are now able to gain comprehensive and quantitative snapshots of the intracellular processes. Dynamic intracellular activities and their regulations can be elucidated by systematic observation of these multi-omics data. On the other hand, careful statistical analysis is necessary for such integration, since each of the omics layers as well as the specific analytical methodologies harbor different levels of noise and variations. Moreover, interpretation of such multitude of data requires an intuitive pathway context. Here we describe such statistical methods for the integration and comparison of multiomics data, as well as the computational methods for pathway reconstruction, ID conversion, mapping, and visualization that play key roles for the efficient study of multi-omics information. © Springer Science+Business Media, LLC 2013.

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Role of Na+-Ca2+ exchange in neonatal and adult ventricular cells: A simulation study

Sano H., Naito Y., Tomita M.

Computing in Cardiology 39 877 - 880 2012.12

ISSN 2325887X

The Na+-Ca2+ exchange assumes considerable importance in neonatal cells with a morphologically sparse sarcoplasmic reticulum. Our simulation shows that the neonatal ventricular cell consumed larger amount of ATP than the adult ventricular cell, owing to a long relaxation time. The amount of ATP consumed per beat was reduced to the amount equivalent to the adult ventricular cells, thus increasing the relative current density of Na +-Ca2+ exchange by 4-fold, which is consistent with the observed current density in the neonatal rabbit ventricular cells. © 2012 CCAL.

Prediction of potentially unstable electrical activity during embryonic development of rodent ventricular myocytes

Okubo C., Sano H., Naito Y., Tomita M.

Computing in Cardiology 39 301 - 304 2012.12

ISSN 2325887X

In order to evaluate developmental changes in embryonic ventricular cells at early embryonic (EE) and late embryonic (LE) stage, we aimed to predict potentially unstable action potentials (APs) that could be lethal to developing ventricular cells. Two models of the Kyoto and the Luo-Rudy model were used for simulation of 512 representative combinations by switching the relative activities of 9 ionic components whose activities vary between the EE and LE stages. Out of these 512 combinations in Kyoto model, 144 combinations were predicted potentially unstable resulting from combinations of funny current (If), inward rectifier current (IK1), sustained inward current (Ist), L-type Ca2+ current (ICaL), and Na+ current (INa). Other 208 and 160 combinations were predicted quiescent membrane potentials and regular spontaneous APs. Based on these results, we suggest that sequential switches of the relative activities of INa, If, and IK1 enable cells to avoid unstable patterns. © 2012 CCAL.

Clarification as to why alcoholic beverages have the ability to induce superconductivity in Fe <inf>1+d</inf>Te <inf>1-x</inf>S <inf>x</inf>

Deguchi K., Sato D., Sugimoto M., Hara H., Kawasaki Y., Demura S., Watanabe T., Denholme S., Okazaki H., Ozaki T., Yamaguchi T., Takeya H., Soga T., Tomita M., Takano Y.

Superconductor Science and Technology 25 ( 8 ) 2012.08

ISSN 09532048

To elucidate the mechanism as to why alcoholic beverages can induce superconductivity in Fe 1+dTe 1xS x samples, we performed component analysis and found that a weak acid such as an organic acid has the ability to induce superconductivity. Inductively coupled plasma spectroscopy was performed on weak acid solutions post-annealing. We found that the mechanism of inducement of superconductivity in Fe 1+dTe 1xS x is the deintercalation of excess Fe from the interlayer sites. © 2012 IOP Publishing Ltd.

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The rice proteogenomics database OryzaPG-DB: Development, expansion, and new features

Helmy M., Sugiyama N., Tomita M., Ishihama Y.

Frontiers in Plant Science (Frontiers in Plant Science) 3 ( APR ) 65 2012.04

Our recently developed rice proteogenomics database (OryzaPG-DB) is the first sustainable resource for rice shotgun-based proteogenomics, providing information on peptides identified in rice protein digested peptides measured by means of liquid chromatography-tandem mass spectrometry (LC-MS/MS), and mapping of the peptides to their genomic origins and the genomic novelty of each peptide. The sequences of the peptides, proteins, cDNAs and genes, and the gene annotations are available for download in FASTA and GFF3 formats, respectively. Further, an annotated visualization of the gene models, corresponding peptides, and genomic novelty is available for each gene, and MS/MS spectra are available for each peptide. In this article, we discuss the utilization of OryzaPG-DB and report on its development, recent content expansions, and newly added features in the current version (OryzaPG-DB v1.1). © 2012 Helmy, Sugiyama, Tomita and Ishihama.

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Changes in the charged metabolite and sugar profiles of pasteurized and unpasteurized Japanese sake with storage

Sugimoto M., Kaneko M., Onuma H., Sakaguchi Y., Mori M., Abe S., Soga T., Tomita M.

Journal of Agricultural and Food Chemistry (Journal of Agricultural and Food Chemistry) 60 ( 10 ) 2586 - 2593 2012.03

ISSN 00218561

Japanese sake (rice wine) is commonly heat treated (pasteurized) to maintain its quality. In this study, temporal changes in the metabolite profiles of pasteurized and unpasteurized sake were investigated during storage. Metabolomic analyses were conducted for eight sets of pasteurized and unpasteurized sake obtained from single process batches stored at 8 or 20 °C for 0, 1, 2, or 4 months. Capillary electrophoresis time-of-flight mass spectrometry and liquid chromatography tandem mass spectrometry were used to obtain charged metabolite and sugar profiles, respectively. The total amino acid concentration decreased with storage, and the decrease was faster in pasteurized sake than in unpasteurized. The organic acid concentrations were relatively constant in both types of sake. Peptide and glucose concentrations increased and polysaccharide concentrations decreased in unpasteurized sake, while they were relatively constant in pasteurized sake. Rather than stabilizing the sake metabolite profile during storage, pasteurization results in characteristic changes compared to unpasteurized sake. © 2012 American Chemical Society.

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Genomic heterogeneity in a natural archaeal population suggests a model of tRNA gene disruption

Sugahara J, Fujishima K, Nunoura T, Takaki Y, Takami H, Takai K, Tomita M, Kanai A.

PLoS ONE (PLoS ONE) 7 ( 3 ) 1-8 2012.03

Research paper (scientific journal), Joint Work

Understanding the mechanistic basis of the disruption of tRNA genes, as manifested in the intron-containing and split tRNAs found in Archaea, will provide considerable insight into the evolution of the tRNA molecule. However, the evolutionary processes underlying these disruptions have not yet been identified. Previously, a composite genome of the deep-branching archaeon Caldiarchaeum subterraneum was reconstructed from a community genomic library prepared from a C. subterraneum-dominated microbial mat. Here, exploration of tRNA genes from the library reveals that there are at least three types of heterogeneity at the tRNA Thr(GGU) gene locus in the Caldiarchaeum population. All three involve intronic gain and splitting of the tRNA gene. Of two fosmid clones found that encode tRNA Thr(GGU), one (tRNA Thr-I) contains a single intron, whereas another (tRNA Thr-II) contains two introns. Notably, in the clone possessing tRNA Thr-II, a 5′ fragment of the tRNA Thr-I (tRNA Thr-F) gene was observed 1.8-kb upstream of tRNA Thr-II. The composite genome contains both tRNA Thr-II and tRNA Thr-F, although the loci are >500 kb apart. Given that the 1.8-kb sequence flanked by tRNA Thr-F and tRNA Thr-II is predicted to encode a DNA recombinase and occurs in six regions of the composite genome, it may be a transposable element. Furthermore, its dinucleotide composition is most similar to that of the pNOB8-type plasmid, which is known to integrate into archaeal tRNA genes. Based on these results, we propose that the gain of the tRNA intron and the scattering of the tRNA fragment occurred within a short time frame via the integration and recombination of a mobile genetic element. © 2012 Sugahara et al.

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Bioinformatics Tools for Mass Spectroscopy-Based Metabolomic Data Processing and Analysis

Sugimoto, Masahiro; Kawakami, Masato; Robert, Martin; Soga, Tomoyoshi; Tomita, Masaru

Current Bioinformatics (Current Bioinformatics) 7 ( 1 ) 96-108 - 108 2012.03

Research paper (scientific journal), Joint Work, ISSN 15748936

Biological systems are increasingly being studied in a holistic manner, using omics approaches, to provide quantitative and qualitative descriptions of the diverse collection of cellular components. Among the omics approaches, metabolomics, which deals with the quantitative global profiling of small molecules or metabolites, is being used extensively to explore the dynamic response of living systems, such as organelles, cells, tissues, organs and whole organisms, under diverse physiological and pathological conditions. This technology is now used routinely in a number of applications, including basic and clinical research, agriculture, microbiology, food science, nutrition, pharmaceutical research, environmental science and the development of biofuels. Of the multiple analytical platforms available to perform such analyses, nuclear magnetic resonance and mass spectrometry have come to dominate, owing to the high resolution and large datasets that can be generated with these techniques. The large multidimensional datasets that result from such studies must be processed and analyzed to render this data meaningful. Thus, bioinformatics tools are essential for the efficient processing of huge datasets, the characterization of the detected signals, and to align multiple datasets and their features. This paper provides a state-of-the-art overview of the data processing tools available, and reviews a collection of recent reports on the topic. Data conversion, pre-processing, alignment, normalization and statistical analysis are introduced, with their advantages and disadvantages, and comparisons are made to guide the reader. © 2012 Bentham Science Publishers.

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Measures of Compositional Strand Bias Related to Replication Machinery and its Applications

Arakawa K., Tomita M.

Current Genomics (Current Genomics) 13 ( 1 ) 4 - 15 2012.03

ISSN 13892029

The compositional asymmetry of complementary bases in nucleotide sequences implies the existence of a mutational or selectional bias in the two strands of the DNA duplex, which is commonly shaped by strand-specific mechanisms in transcription or replication. Such strand bias in genomes, frequently visualized by GC skew graphs, is used for the computational prediction of transcription start sites and replication origins, as well as for comparative evolutionary genomics studies. The use of measures of compositional strand bias in order to quantify the degree of strand asymmetry is crucial, as it is the basis for determining the applicability of compositional analysis and comparing the strength of the mutational bias in different biological machineries in various species. Here, we review the measures of strand bias that have been proposed to date, including the ΔGC skew, the B 1 index, the predictability score of linear discriminant analysis for gene orientation, the signal-to-noise ratio of the oligonucleotide bias, and the GC skew index. These measures have been predominantly designed for and applied to the analysis of replication-related mutational processes in prokaryotes, but we also give research examples in eukaryotes. ©2012 Bentham Science Publishers.

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Metatranscriptomic Analysis of Microbes in an Oceanfront Deep-Subsurface Hot Spring Reveals Novel Small RNAs and Type-Specific tRNA Degradation

Murakami S., Fujishima K., Tomita M., Kanai A.

Applied and Environmental Microbiology (Applied and Environmental Microbiology) 78 ( 4 ) 1015-1022 - 1022 2012.02

Research paper (scientific journal), Joint Work, ISSN 00992240

Studies of small noncoding RNAs (sRNAs) have been conducted predominantly using culturable organisms, and the acquisition of further information about sRNAs from global environments containing uncultured organisms now is very important. In this study, hot spring water (57°C, pH 8.1) was collected directly from the underground environment at depths of 250 to 1,000min Yunohama, Japan, and small RNA sequences obtained from the environment were analyzed. A phylogenetic analysis of both archaeal and bacterial 16S rRNA gene sequences was conducted, and the results suggested the presence of unique species in the environment, corresponding to the Archaeal Richmond Mine Acidophilic Nanoorganisms (ARMAN) group and three new Betaproteobacteria. A metatranscriptomic analysis identified 64,194 (20,057 nonredundant) cDNA sequences. Of these cDNAs, 90% were either tRNAs, tRNA fragments, rRNAs, or rRNA fragments, whereas 2,181 reads (10%) were classified as previously uncharacterized putative candidate sRNAs. Among these, 15 were particularly abundant, 14 of which showed no sequence similarity to any known noncoding RNA, and at least six of which form very stable RNA secondary structures. The analysis of a large number of tRNA fragments suggested that unique relationships exist between the anticodons of the tRNAs and the sites of tRNA degradation. Previous bacterial tRNA degradation studies have been limited to specific organisms, such as Escherichia coli and Streptomyces coelicolor, and the current results suggest that specific tRNA decay occurs more frequently than previously expected. © 2012, American Society for Microbiology.

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MMMDB: Mouse multiple tissue metabolome database

Sugimoto M., Ikeda S., Niigata K., Tomita M., Sato H., Soga T.

Nucleic Acids Research (Nucleic Acids Research) 40 ( D1 ) D809 - 14 2012.01

ISSN 03051048

The Mouse Multiple Tissue Metabolome Database (MMMDB) provides comprehensive and quantitative metabolomic information for multiple tissues from single mice. Manually curated databases that integrate literature-based individual metabolite information have been available so far. However, data sets on the absolute concentration of a single metabolite integrated from multiple resources are often difficult to be used when different metabolomic studies are compared because the relative balance of the multiple metabolite concentrations in the metabolic pathways as a snapshot of a dynamic system is more important than the absolute concentration of a single metabolite. We developed MMMDB by performing non-targeted analyses of cerebra, cerebella, thymus, spleen, lung, liver, kidney, heart, pancreas, testis and plasma using capillary electrophoresis time-of-flight mass spectrometry and detected 428 non-redundant features from which 219 metabolites were successfully identified. Quantified concentrations of the individual metabolites and the corresponding processed raw data; for example, the electropherograms and mass spectra with their annotations, such as isotope and fragment information, are stored in the database. MMMDB is designed to normalize users' data, which can be submitted online and used to visualize overlaid electropherograms. Thus, MMMDB allows newly measured data to be compared with the other data in the database. MMMDB is available at: http:// mmmdb.iab.keio.ac.jp. © The Author(s) 2011.

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Time-structure of the yeast metabolism in vivo

Sasidharan K., Tomita M., Aon M., Lloyd D., Murray D.B.

Advances in Experimental Medicine and Biology 2012

ISSN 00652598

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Peptide Identification by Searching Large-Scale Tandem Mass Spectra against Large Databases: Bioinformatics Methods in Proteogenomics

Helmy, M, Tomita M, Ishihama Y

Genes, Genome and Genomics 6 76-85 2012

Research paper (scientific journal), Joint Work, Accepted

Profiling of the charged metabolites of traditional herbal medicines using capillary electrophoresis time-of-flight mass spectrometry

Iino¸ K., Sugimoto¸ M., Soga¸ T. and Tomita, M.

Metabolomics 8 ( 1 ) 99-108 - 108 2012

Research paper (scientific journal), Joint Work, ISSN 15733882

The quantification of a small number of bioactive components in herbal medicines is often inadequate when attempting to elucidate a medicine's biological effects. Despite rapid advances in analytical technologies, obtaining comprehensive metabolomic profiles of herbal medicines remains difficult, due to the complexity of natural product mixtures. Toki-Shakuyaku-San is a Chinese medicine used widely to treat gynecological and obstetric disorders, such as infertility, dysmenorrhea, toxemia during pregnancy and neural dysfunction. It consists of Angelica acutiloba Radix (Toki), Cnidium officinale Rhizoma (Senkyu), Paeonia lactiflora Radix (Shakuyaku), Atractylodes lancea Rhizoma (Sojutsu), Alisma orientale Rhizoma (Takusha) and Poria cocos Hoelen (Bukuryo). To elucidate the composition of these herbal medicines individually, we conducted non-targeted profiling analyses of extracts of these herbs using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), which allows the simultaneous quantification of hundreds of charged metabolites. In total, 737 ± 183. 1 (average ± SD) metabolite-derived features were observed, and of these, 119 metabolites were identified. Score plots of principal component analysis (PCA) showed a clear cluster including Shakuyaku, Bukuryo, and Sojutsu, while the other three herbs were distributed over PCA spaces. Loading plots revealed that amino acids and shikimate-derived alkaloids were the predominant metabolite constituents. Hierarchical clustering analysis revealed that few clusters overlapped in the herbal medicines tested. This report is the first demonstration of the characterization of a herbal medicine using large-scale metabolomic analysis, which is complementary to traditional quality control methods. © 2011 Springer Science+Business Media, LLC.

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Validation of Bacterial Replication Termination Models Using Simulation of Genomic Mutations

Kono, N., Arakawa, K.,and Tomita, M.

PLoS ONE (PLoS ONE) 7 ( 4 ) e34526 2012

Research paper (scientific journal), Joint Work, Accepted

In bacterial circular chromosomes and most plasmids, the replication is known to be terminated when either of the following occurs: the forks progressing in opposite directions meet at the distal end of the chromosome or the replication forks become trapped by Tus proteins bound to Ter sites. Most bacterial genomes have various polarities in their genomic structures. The most notable feature is polar genomic compositional asymmetry of the bases G and C in the leading and lagging strands, called GC skew. This asymmetry is caused by replication-associated mutation bias, and this "footprint" of the replication machinery suggests that, in contrast to the two known mechanisms, replication termination occurs near the chromosome dimer resolution site dif. To understand this difference between the known replication machinery and genomic compositional bias, we undertook a simulation study of genomic mutations, and we report here how different replication termination models contribute to the generation of replication-related genomic compositional asymmetry. Contrary to naive expectations, our results show that a single finite termination site at dif or at the GC skew shift point is not sufficient to reconstruct the genomic compositional bias as observed in published sequences. The results also show that the known replication mechanisms are sufficient to explain the position of the GC skew shift point. © 2012 Kono et al.

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Nematode-specific tRNAs that decode an alternative genetic code for leucine

Hamashima, K., Fujishima, K., Masuda, T., Sugahara, J., Tomita, M. and Kanai, A.

Nucleic Acids Research (Nucleic Acids Research) 40 ( 8 ) 3653-3662 - 3662 2012

Research paper (scientific journal), Joint Work, Accepted, ISSN 03051048

Class II transfer RNAs (tRNAs), including tRNALeu and tRNA Ser, have an additional stem and loop structure, the long variable arm (V-arm). Here, we describe Class II tRNAs with a unique anticodon corresponding to neither leucine nor serine. Because these tRNAs are specifically conserved among the nematodes, we have called them 'nematode-specific V-arm-containing tRNAs' (nev-tRNAs). The expression of nev-tRNA genes in Caenorhabditis elegans was confirmed experimentally. A comparative sequence analysis suggested that the nev-tRNAs derived phylogenetically from tRNALeu. In vitro aminoacylation assays showed that nev-tRNAGly and nev-tRNAIle are only charged with leucine, which is inconsistent with their anticodons. Furthermore, the deletion and mutation of crucial determinants for leucylation in nev-tRNA led to a marked loss of activity. An in vitro translation analysis showed that nev-tRNA Gly decodes GGG as leucine instead of the universal glycine code, indicating that nev-tRNAs can be incorporated into ribosomes and participate in protein biosynthesis. Our findings provide the first example of unexpected tRNAs that do not consistently obey the general translation rules for higher eukaryotes. © 2011 The Author(s).

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The role of Staphylococcal enterotoxin in atopic keratoconjunctivitis and corneal ulceration

Fujishima, H., Okada, N., Dogru., M., Baba, F., Tomita, M., Abe, J., Matsumoto, K. and Saito, H.

Allergy 67 ( 6 ) 799-803 - 803 2012

Research paper (scientific journal), Joint Work, Accepted, ISSN 01054538

Background Patients with atopic eczema frequently experience colonization with Staphylococcus aureus that is directly correlated with the eczema severity. We hypothesized that S. aureus-secreted enterotoxins (SE) are involved in the pathophysiology of atopic keratoconjunctivitis (AKC). Methods A total of 45 subjects (18 with AKC, nine vernal keratoconjunctivitis (VKC), eight seasonal allergic conjunctivitis (SAC), and ten healthy volunteers) were enrolled. Slit lamp examinations, including fluorescein staining, were performed. Scraped samples were collected from the upper tarsal conjunctiva, lower conjunctival sacs, and the skin around the eyelid margins. Superantigen (SAg) genes were detected using polymerase chain reaction (PCR). Results Among 45 cases, S. aureus was detected significantly more in AKC patients than VKC patients (P = 0.026), SAC patients (P = 0.0003), and healthy volunteers (P = 0.0001). SAg genes were detected in 11 patients. SEB (2/11), SEG (8/11), and SEI (8/11) were detected, but no other SE. There was a significant difference in SE detection between AKC and SAC patients (P = 0.03). In severe types of ocular allergic disease such as AKC and VKC (N = 27), SE was detected in six of ten patients with corneal ulcers and two of 17 patients without corneal ulcers. SE was detected in significantly more patients with corneal ulcers (P = 0.025). Conclusions In patients with AKC, S. aureus and SE were detected more frequently compared with other patients and healthy volunteers, especially in association with corneal ulceration suggesting a role of SE. So far, it is unknown whether SE leads to tissue damage of the cornea by initiating an immune response or has direct toxic effects. © 2012 John Wiley & Sons A/S.

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Systems Biology, Metabolomics, and Cancer Metabolism

Tomita, M.,Kami, K.

Science 336 990-991 2012

Research paper (scientific journal), Joint Work, Accepted

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The third plasmid pVV8 from Thermus thermophilus HB8: isolation, characterization, and sequence determination

Ohtani., N., Tomita, M. and Itaya, M.

Extremophiles (Extremophiles) 16 ( 2 ) 237-44 - 244 2012

Research paper (scientific journal), Joint Work, ISSN 14310651

The extremely thermophilic bacterium Thermus thermophilus is a model organism for structural biology and systems biology, and the so-called "Structural and Functional Whole-Cell Project for T. thermophilus HB8" is in progress. The released genomic sequence of the strain HB8 is composed of chromosome, pTT27 megaplasmid, and pTT8 plasmid. In this paper, however, a third plasmid was demonstrated and its sequence was determined. Although this plasmid pVV8 had been reported before, limited information and an unfortunate dropout in the substrain, whose genomic sequence was determined, would have prevented the plasmid from coming to public attention. The intrinsic circular plasmid, which was estimated to be six to ten copies in a cell, is 81151 bp and its G + C content is 68%. Among the identified 91 ORFs, a single gene has been experimentally analyzed before and is known as xylose isomerase. The phnCDEGHIJKLMX operon related to phosphonate metabolism, alkaline phosphatase, putative transcriptional regulators, several sets of toxin-antitoxin system, and transposase-like ORFs are also encoded on the pVV8 plasmid. Although association with cell aggregation was the one phenotypic characteristic of the plasmid that had been reported, it was never confirmed. Comparison of T. thermophilus HB8 strains suggests that the pVV8 is nonessential for growth. © 2011 Springer.

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Prediction of axillary lymph node metastasis in primary breast cancer patients using a decision tree-based model

Takada, M., Sugimoto, M., Naito, Y., Moon, H., G., Han, W., Noh, D., Y., Kondo, M., Kuroi, K., Sasano, H., Inamoto, T., Tomita, M., and Toi, M.

BMC Medical Informatics and Decision Making (BMC Medical Informatics and Decision Making) 12 ( 1 ) 54 2012

Research paper (scientific journal), Joint Work, Accepted

Background: The aim of this study was to develop a new data-mining model to predict axillary lymph node (AxLN) metastasis in primary breast cancer. To achieve this, we used a decision tree-based prediction method-The alternating decision tree (ADTree). Methods: Clinical datasets for primary breast cancer patients who underwent sentinel lymph node biopsy or AxLN dissection without prior treatment were collected from three institutes (institute A, n = 148; institute B, n = 143; institute C, n = 174) and were used for variable selection, model training and external validation, respectively. The models were evaluated using area under the receiver operating characteristics (ROC) curve analysis to discriminate node-positive patients from node-negative patients. Results: The ADTree model selected 15 of 24 clinicopathological variables in the variable selection dataset. The resulting area under the ROC curve values were 0.770 [95% confidence interval (CI), 0.689-0.850] for the model training dataset and 0.772 (95% CI: 0.689-0.856) for the validation dataset, demonstrating high accuracy and generalization ability of the model. The bootstrap value of the validation dataset was 0.768 (95% CI: 0.763-0.774). Conclusions: Our prediction model showed high accuracy for predicting nodal metastasis in patients with breast cancer using commonly recorded clinical variables. Therefore, our model might help oncologists in the decision-making process for primary breast cancer patients before starting treatment. © 2012 Takada et al.; licensee BioMed Central Ltd.

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Rapid DNA, RNA and protein extraction protocols optimized for slow continuously growing yeast cultures

Sasidharan, K., Amariei, C., Tomita, M., and Murray, D, B.

Yeast (Yeast) 29 ( 8 ) 311-22 - 322 2012

Research paper (scientific journal), Joint Work, Accepted, ISSN 0749503X

Conventional extraction protocols for yeast have been developed for relatively rapid-growing low cell density cultures of laboratory strains and often do not have the integrity for frequent sampling of cultures. Therefore, these protocols are usually inefficient for cultures under slow growth conditions or of non-laboratory strains. We have developed a combined mechanical and chemical disruption procedure using vigorous bead-beating that can consistently disrupt yeast cells (> 95%), irrespective of cell cycle and metabolic state. Using this disruption technique coupled with quenching, we have developed DNA, RNA and protein extraction protocols that are optimized for a large number of samples from slow-growing high-density industrial yeast cultures. Additionally, sample volume, the use of expensive reagents/enzymes, handling times and incubations were minimized. We have tested the reproducibility of our methods using triplicate/time-series extractions and compared these with commonly used protocols or commercially available kits. Moreover, we utilized a simple flow-cytometric approach to estimate the mitochondrial DNA copy number. Based on the results, our methods have shown higher reproducibility, yield and quality. © 2012 John Wiley & Sons, Ltd.

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Predictions of the pathological response to neoadjuvant chemotherapy in patients with primary breast cancer using a data mining technique

Takada, M., Sugimoto, M., Ohno, S., Kuroi, K., N. Sato , H., Bando, H., Masuda, N., Iwata, H., Kondo, M., Sasano, H., Chow, L., W., C., Inamoto , T., Naito, Y., Tomita, M. and Toi, M.

Breast Cancer Research and Treatment 134 ( 2 ) 661-70 - 670 2012

Research paper (scientific journal), Joint Work, Accepted, ISSN 01676806

Nomogram, a standard technique that utilizes multiple characteristics to predict efficacy of treatment and likelihood of a specific status of an individual patient, has been used for prediction of response to neoadjuvant chemotherapy (NAC) in breast cancer patients. The aim of this study was to develop a novel computational technique to predict the pathological complete response (pCR) to NAC in primary breast cancer patients. A mathematical model using alternating decision trees, an epigone of decision tree, was developed using 28 clinicopathological variables that were retrospectively collected from patients treated with NAC (n = 150), and validated using an independent dataset from a randomized controlled trial (n = 173). The model selected 15 variables to predict the pCR with yielding area under the receiver operating characteristics curve (AUC) values of 0.766 [95 % confidence interval (≥I)], 0.671-0.861, P value< 0.0001) in cross-validation using training dataset and 0.787 (95 % CI 0.716-0.858, P value < 0.0001) in the validation dataset. Among three subtypes of breast cancer, the luminal subgroup showed the best discrimination (AUC = 0.779, 95 % CI 0.641-0.917, P value = 0.0059). The developed model (AUC = 0.805, 95 % CI 0.716-0.894, P value\0.0001) outperformed multivariate logistic regression (AUC = 0.754, 95 % CI 0.651-0.858, P value = 0.00019) of validation datasets without missing values (n = 127). Several analyses, e.g. bootstrap analysis, revealed that the developed model was insensitive to missing values and also tolerant to distribution bias among the datasets. Our model based on clinicopathological variables showed high predictive ability for pCR. This model might improve the prediction of the response to NAC in primary breast cancer patients. © Springer Science+Business Media, LLC. 2012.

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Non-targeted metabolite profiling in activated macrophage secretion

Sugimoto, M., Sakagami, H., Yokote, Y., Onuma, H., Kaneko, M., Mori, M., Sakaguchi, Y., Soga, T. and Tomita, M.

Metabolomics 8 ( 4 ) 624–633 - 633 2012

Research paper (scientific journal), Joint Work, Accepted, ISSN 15733882

Periodontal diseases are inflammatory infectious diseases that affect the periodontal tissue. Macrophages play a central role in inflammatory conditions, leading to the destruction of tissues. Identifying the signaling molecules secreted by macrophages would be valuable to the study of these diseases. Here, we present non-targeted analysis using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) for the profiling of extracellular metabolites released during macrophage activation. Lipopolysaccharide (LPS)-induced activation of a mouse macrophage-like cell line RAW264. 7 was used as a model system. Cells were treated without (control) or with LPS for 22 h and, after washing, were incubated for 1 h in phosphate-buffered saline. The accumulation of metabolites in the culture supernatant was monitored. LPS treatment significantly enhanced the accumulation of prostaglandins, tumor necrosis factor-α, nitric oxide and citrulline in the culture medium. RAW264. 7 cells produced 46 metabolites and 66% of these showed significant changes (P < 0.05) following cell activation. In particular, the production of leucine, hypoxanthine, choline, putrecine, N 8-acetylspermidine, succinate, itaconate, and 4-methyl-2-oxopentanoate was significantly increased by cell activation (P < 0.001). Significantly elevated production of lactate and glycine was also observed. Here, we present the first catalog of the up and down-regulation of the various metabolites secreted by macrophages following inflammatory activation. © 2011 Springer Science+Business Media, LLC.

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Mass spectrum sequential subtraction speeds up searching large peptide MS/MS spectra datasets against large nucleotide databases for proteogenomics

Helmy, M., Sugiyama, N., Tomita, M. and Ishihama, Y.

Genes to Cells (Genes to Cells) 17 ( 8 ) 633-44 - 644 2012

Research paper (scientific journal), Joint Work, Accepted, ISSN 13569597

We have developed a novel bioinformatics method called mass spectrum sequential subtraction (MSSS) to search large peptide spectra datasets produced by liquid chromatography/mass spectrometry (LC-MS/MS) against protein and large-sized nucleotide sequence databases. The main principle in MSSS is to search the peptide spectra set against the protein database, followed by removal of the spectra corresponding to the identified peptides to create a smaller set of the remaining peptide spectra for searching against the nucleotide sequences database. Therefore, we reduce the number of spectra to be searched to limit the peptide search space. Comparing MSSS and conventional search approach using a dataset of 27 LC-MS/MS runs of rice culture cells indicated that MSSS reduced the search queries to 50% and the search time to 75% on average. In addition, MSSS had no effect on the identification false-positive rate (FPR) or the novel peptide sequences identification ability. We used MSSS to analyze another dataset of 34 LC-MS/MS runs, resulting in identifying additional 74 novel peptides. Proteogenomic analysis with these additional peptides yielded 47 new genomic features in 24 rice genes plus 24 intergenic peptides. These results show that the utility of MSSS in searching large databases with large MS/MS datasets for proteogenomics. © 2012 The Authors Journal compilation © 2012 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

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Investigation of stochasticity in TRAIL signaling cancer model

Piras, V., Hayashi, K., Tomita, M. and Selvarajoo, K.

IEEE/ICME Com. Med. Eng. 609-614. - 614 2012

Research paper (scientific journal), Joint Work, Accepted

In cancer, apoptosis or programmed cell death has been demonstrated through the tumor necrosis factor related apoptosis-inducing ligand (TRAIL) signal transduction. As a result, TRAIL-based therapies have been widely investigated to fight cancers. However, several malignant cancer types still remain resistant to TRAIL. Recently, we developed a dynamic computational model to investigate the resistance mechanisms in TRAIL-stimulated human fibrosarcoma (HT1080) cells. The macroscopic average-cell response model, based on the law of mass action and signaling flux conservation, successfully simulates the semi-quantitative temporal profiles of cell survival (I?B, JNK, p38) and apoptotic (caspase-8 and -3) molecules in wildtype and several mutants (FADD, RIP1 and TRAF2 knockdowns or KD). However, cancer populations are known to be highly heterogeneous, and various studies have demonstrated the importance of stochasticity and variability for phenotypic diversity between identical cells. Here, we extend our original model to investigate the effect of such fluctuations on TRAIL signaling response by adopting probabilities of signaling reactions through the Gillespie algorithm. Notably, when we stimulated the model 1000 times to indicate the variability of 1000 single cell responses in all 4 experimental conditions with different levels of stochasticity, we notice that TRAF2 KD produced the most variable signaling response. This variance subsequently affected the level of cellular apoptosis analysed through the cell-survival metric (CSM). Our work highlights the necessity to understand variable responses of cell signaling reactions to different levels of stochasticity. Thus, prior to the actual development of potential drug targets for killing cancer cells, the effect of stochastic variance could be investigated through dynamic models. © 2012 IEEE.

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A Yeast Metabolite Extraction Protocol Optimised for Time-Series Analyses

Sasidharan, K., Soga, T., Tomita, M. and Murray, D., B.

PLoS ONE (PLoS ONE) 7 ( 8 ) e44283 2012

Research paper (scientific journal), Joint Work, Accepted

There is an increasing call for the absolute quantification of time-resolved metabolite data. However, a number of technical issues exist, such as metabolites being modified/degraded either chemically or enzymatically during the extraction process. Additionally, capillary electrophoresis mass spectrometry (CE-MS) is incompatible with high salt concentrations often used in extraction protocols. In microbial systems, metabolite yield is influenced by the extraction protocol used and the cell disruption rate. Here we present a method that rapidly quenches metabolism using dry-ice ethanol bath and methanol N-ethylmaleimide solution (thus stabilising thiols), disrupts cells efficiently using bead-beating and avoids artefacts created by live-cell pelleting. Rapid sample processing minimised metabolite leaching. Cell weight, number and size distribution was used to calculate metabolites to an attomol/cell level. We apply this method to samples obtained from the respiratory oscillation that occurs when yeast are grown continuously. © 2012 Sasidharan et al.

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Metabolic regulation analysis of wild-type and arcA mutant Escherichia coli under nitrate conditions using different levels of omics data

Toya, Y., Nakahigashi, K., Tomita, M., and Shimizu, K.

Molecular BioSystems (Molecular BioSystems) 8 ( 10 ) 2593-604. - 2604 2012

Research paper (scientific journal), Joint Work, Accepted, ISSN 1742206X

It is of practical interest to investigate the effect of nitrates on bacterial metabolic regulation of both fermentation and energy generation, as compared to aerobic and anaerobic growth without nitrates. Although gene level regulation has previously been studied for nitrate assimilation, it is important to understand this metabolic regulation in terms of global regulators. In the present study, therefore, we measured gene expression using DNA microarrays, intracellular metabolite concentrations using CE-TOFMS, and metabolic fluxes using the 13C-labeling technique for wild-type E. coli and the ΔarcA (a global regulatory gene for anoxic response control, ArcA) mutant to compare the metabolic state under nitrate conditions to that under aerobic and anaerobic conditions without nitrates in continuous culture conditions at a dilution rate of 0.2 h-1. In wild-type, although the measured metabolite concentrations changed very little among the three culture conditions, the TCA cycle and the pentose phosphate pathway fluxes were significantly different under each condition. These results suggested that the ATP production rate was 29% higher under nitrate conditions than that under anaerobic conditions, whereas the ATP production rate was 10% lower than that under aerobic conditions. The flux changes in the TCA cycle were caused by changes in control at the gene expression level. In ΔarcA mutant, the TCA cycle flux was significantly increased (4.4 times higher than that of the wild type) under nitrate conditions. Similarly, the intracellular ATP/ADP ratio increased approximately two-fold compared to that of the wild-type strain. © 2012 The Royal Society of Chemistry.

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Temporal profiling of lapatinib-suppressed phosphorylation signals inEGFR/HER2 pathways

Imami, K., Sugiyama, N., Imamura, H., Wakabayashi, M., Tomita, M., Taniguchi, M., Ueno, T., Toi, M. and Ishihama, Y.

Mol Cell Proteomics (Molecular and Cellular Proteomics) 11 ( 12 ) [Epub ahead of print] - 1757 2012

Research paper (scientific journal), Joint Work, Accepted, ISSN 15359476

Lapatinib is a clinically potent kinase inhibitor for breast cancer patients because of its outstanding selectivity for epidermal growth factor receptor (EGFR) and EGFR2 (also known as HER2). However, there is only limited information about the in vivo effects of lapatinib on EGFR/HER2 and downstream signaling targets. Here, we profiled the lapatinib-induced time- and dose-dependent phosphorylation dynamics in SKBR3 breast cancer cells by means of quantitative phosphoproteomics. Among 4953 identified phosphopeptides from 1548 proteins, a small proportion (5-7%) was regulated at least twofold by 1-10 μM lapatinib. We obtained a comprehensive phosphorylation map of 21 sites on EGFR/HER2, including nine novel sites on HER2. Among them, serine/threonine phosphosites located in a small region of HER2 (amino acid residues 1049-1083) were up-regulated by the drug, whereas all other sites were down-regulated. We show that cAMP-dependent protein kinase is involved in phosphorylation of this particular region of HER2 and regulates HER2 tyrosine kinase activity. Computational analyses of quantitative phosphoproteome data indicated for the first time that protein-protein networks related to cytoskeletal organization and transcriptional/translational regulation, such as RNP complexes (i.e. hnRNP, snRNP, telomerase, ribosome), are linked to EGFR/HER2 signaling networks. To our knowledge, this is the first report to profile the temporal response of phosphorylation dynamics to a kinase inhibitor. The results provide new insights into EGFR/HER2 regulation through region-specific phosphorylation, as well as a global view of the cellular signaling networks associated with the anti-breast cancer action of lapatinib. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

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Sheathless capillary electrophoresis-mass spectrometry with a high- sensitivity porous sprayer for cationic metabolome analysis

Hirayama, A., Tomita, M. and Soga, T.

Analyst (Analyst) 137 ( 21 ) 5026-5033 - 5033 2012

Research paper (scientific journal), Joint Work, Accepted, ISSN 00032654

Sheath-flow capillary electrophoresis-mass spectrometry (CE-MS) has emerged as a new tool for comprehensive analysis of charged metabolites. However, it needs to be more sensitive. Here, we report a sheathless capillary electrophoresis-electrospray ionization-mass spectrometry method for cationic metabolome analysis. This system used a high-sensitivity porous sprayer interface and 10% (v/v) acetic acid as the background electrolyte (BGE). Under optimized conditions, 53 cationic metabolites, including amino acids and their derivatives, amines, nucleic acids and small peptides, were successfully separated and selectively detected with a time-of-flight mass spectrometer. At a signal-to-noise ratio of three, the concentration detection limits for these compounds were between 0.004 and 0.8 μmol L-1 (amount detection limit, 0.01 to 2 fmol) with pressure injection at 20.7 kPa for 5 s (2.6 nL). Compared with conventional sheath-flow CE-MS, the detection limit of the present method was increased more than 5-fold for 21 (40%) of the compounds detected. When the method was applied to the analysis of cationic metabolites obtained from human urine, there was a 10-fold increase in the number of detected peaks compared with conventional methods. More than 180 successive runs could be conducted without any problems, and only the BGE needed to be changed. © 2012 The Royal Society of Chemistry.

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Pilot Study of Changes in Salivary Metabolic Profiles Induced by Template Therapy

Tanaka, S., Taga, H., Maehara, K., Kaneshima, A., Machino, M., Onuma, H., Kaneko, M., Sakagami, H., Sugimoto, M., Soga, T. and Tomita, M.

InVivo. (In Vivo) 26 ( 6 ) 1015-1020 - 1020 2012

Research paper (scientific journal), Joint Work, Accepted, ISSN 0258851X

Background: Occlusal raising method (so-called 'Template therapy') has been reported to alleviate various diseases and symptoms, but the underlying mechanism is not clear. We searched the low-molecular weight metabolite(s) in the saliva, the concentration of which is significantly changed by the template therapy. Materials and Methods: One female patient with headache underwent the template therapy for 12 days, and her total saliva was subjected to non-targeted analysis using capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS). Results: One hundred and thirteen substances were identified in the saliva. Glycine was the most abundant amino acid in the saliva, followed by alanine, serine and proline. After the start of the template therapy, her headache was alleviated, accompanied by a significant (p=0.042) increase of salivary concentration of glycine, as compared with total amino acids whereas that of other amino acids was not significantly changed. In the metabolomics profile, salivary concentration of large number of metabolites as compared with total metabolite concentration decreased, including N-acetylneuraminate (p=0.025) and p-hydroxyphenylacetate (p=0.039). Conclusion: This pilot study demonstrated, to our knowledge for the first time, that only glycine exhibited unique changes among total metabolites, suggesting its significant role in template therapy.

Is central dogma a global property of cellular information flow?

Piras, V., Tomita, M. and Selvarajoo, K

Front. Physiol. (Frontiers in Physiology) 3 439 2012

Research paper (scientific journal), Joint Work, Accepted

The central dogma of molecular biology has come under scrutiny in recent years. Here, we reviewed high-throughput mRNA and protein expression data of Escherichia coli, Saccharomyces cerevisiae, and several mammalian cells. At both single cell and population scales, the statistical comparisons between the entire transcriptomes and proteomes show clear correlation structures. In contrast, the pair-wise correlations of single transcripts to proteins show nullity. These data suggest that the organizing structure guiding cellular processes is observed at omics-wide scale, and not at single molecule level. The central dogma, thus, globally emerges as an average integrated flow of cellular information. © 2012 Piras, Tomita and Selvarajoo.

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Metabolomics: A New Omics Technology for Traditional Herbal Medicine Analysis

Iino, K., Sugimoto, M., Soga, T. and Tomita, M.

Chinese Medicine: Acupuncture, Herbal Medicine and Therapies 45-55 2012

Research paper (scientific journal), Joint Work, Accepted

Metabolic profiling reveals new serum biomarkers for differentiating diabetic nephropathy

Hirayama, A., Nakashima, E., Sugimotoa, M., Akiyama, S., Sato, W., Maruyama, S., Matsuo, S., Tomita, M., Yuzawa, Y. and Soga, T.

Anal Bioanal Chem (Analytical and Bioanalytical Chemistry) 404 ( 10 ) 3101-9 - 3109 2012

Research paper (scientific journal), Joint Work, Accepted, ISSN 16182642

Capillary electrophoresis coupled with time-offlight mass spectrometry was used to explore new serum biomarkers with high sensitivity and specificity for diabetic nephropathy (DN) diagnosis, through comprehensive analysis of serum metabolites with 78 diabetic patients. Multivariate analyses were used for identification of marker candidates and development of discriminative models. Of the 289 profiled metabolites, orthogonal partial leastsquares discriminant analysis identified 19 metabolites that could distinguish between DN with macroalbuminuria and diabetic patients without albuminuria. These identified metabolites included creatinine, aspartic acid, γ-butyrobetaine, citrulline, symmetric dimethylarginine (SDMA), kynurenine, azelaic acid, and galactaric acid. Significant correlations between all these metabolites and urinary albumin-to-creatinine ratios (p<0.009, Spearman's rank test) were observed. When five metabolites (including γ-butyrobetaine, SDMA, azelaic acid and two unknowns) were selected from 19 metabolites and applied for multiple logistic regressionmodel, AUC value for diagnosing DN was 0.927 using the whole dataset, and 0.880 in a cross-validation test. In addition, when four known metabolites (aspartic acid, SDMA, azelaic acid and galactaric acid) were applied, the resulting AUC was still high at 0.844 with the whole dataset and 0.792 with cross-validation. Combination of serum metabolomics with multivariate analyses enabled accurate discrimination of DN patients. The results suggest that capillary electrophoresis-mass spectrometry based metabolome analysis could be used for DN diagnosis. © Springer-Verlag 2012.

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A simple method to provide a shuttling plasmid for delivery to other host ascertained by prolonged stability of extracellular plasmid DNA released from Escherichia coli K12 endA mutant, deficient in major endonuclease

Itaya, M.,　Kawata, Y., Sato, M., Tomita, M. and Nakahigashi, K.

J. Biochem. (Journal of Biochemistry) 152 ( 6 ) 501–504 - 504 2012

Research paper (scientific journal), Joint Work, Accepted, ISSN 0021924X

Escherichia coli lyses by lambda phage propagation. Circular plasmid DNA was present during E. coli lysis as an extracellular plasmid DNA (excpDNA) that was stable enough to transform coexisting competent Bacillus subtilis cells. Detailed investigations unveiled that excpDNA is transient in both quality and quantity, with stability lasting no more than several hours. A survey using E. coli lambda lysogens with various genetic backgrounds demonstrated that the loss of Endonuclease I (ΔendA::kan) conferred extraordinary stability upon excpDNA for as long as 48 h. Studies on endA mutants suggested that excpDNA remained localized in cell debris, in contrast to E. coli genome DNA, which diffused into medium at an early point in lysis. Lambda lysogens constructed on endA recA mutants are presented for potential pipelines in delivery to other competent proficient microbes. © 2012 The Authors.

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A medium-chain fatty acid as an alternative energy source in mouse preimplantation development

Yamada, M., Takanashi, K., Hamatani, T., Hirayama, A., Akutsu, H., Fukunaga, T., Ogawa, S., Sugawara, K., Shinoda, K., Soga, T., Umezawa, A., Kuji, N., Yoshimura, Y. and Tomita, M.

Sci Rep. (Scientific Reports) 2 930 2012

Research paper (scientific journal), Joint Work, Accepted

To further optimize the culturing of preimplantation embryos, we undertook metabolomic analysis of relevant culture media using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). We detected 28 metabolites: 23 embryo-excreted metabolites including 16 amino acids and 5 media-derived metabolites (e.g., octanoate, a medium-chain fatty acid (MCFA)). Due to the lack of information on MCFAs in mammalian preimplantation development, this study examined octanoate as a potential alternative energy source for preimplantation embryo cultures. No embryos survived in culture media lacking FAs, pyruvate, and glucose, but supplementation of octanoate rescued the embryonic development. Immunoblotting showed significant expression of acyl-CoA dehydrogenase and hydroxyacyl-CoA dehydrogenase, important enzymes for β-oxidation of MCFAs, in preimplantation embryo. Furthermore, CE-TOFMS traced [1-13 C 8[ octanoate added to the culture media into intermediate metabolites of the TCA cycle via β-oxidation in mitochondria. These results are the first demonstration that octanoate could provide an efficient alternative energy source throughout preimplantation development.

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Light Microscopy and Phylogenetic Analyses of Chlamydomonas Species (Volvocales, Chlorophyceae). : I. Chlamydomonas pseudomacrostigma and Chlamydomonas media

Nakada, T., Nozaki, H. and Tomita, M.

Acta phytotaxonomica et geobotanica 62:69-78 62 69 - 78 2012

Joint Work

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Serial assembly of Thermus megaplasmid DNA in the genome of Bacillus subtilis 168: a BAC-based domino method applied to DNA with a high GC content

Ohtani, N., Hasegawa, M., Sato, M., Tomita, M., Kaneko, S. and Itaya, M.

Biotechnol J. (Biotechnology Journal) 7 ( 7 ) 867 - 76 2012

Joint Work, Accepted, ISSN 18606768

Bacillus subtilis is the only bacterium-based host able to clone giant DNA above 1000 kbp. DNA previously handled by this host was limited to that with GC content similar to or lower than that of the B. subtilis genome. To expand the target DNA range to higher GC content, we tried to clone a pTT27 megaplasmid (257 kbp, 69% of G+C) from Thermus thermophilus. To facilitate the reconstruction process, we subcloned pTT27 in a bacterial artificial chromosome (BAC) vector of Escherichia coli. Owing to the ability of BAC to carry around 100 kbp DNA, only 4 clones were needed to cover the pTT27 and conduct step-by-step assembly in the B. subtilis genome. The full length of 257 kbp was reconstructed through 3 intermediary lengths (108, 153, and 226 kbp), despite an unexpected difficulty in the maintenance of DNA >200 kbp. Retrieval of these four pTT27 segments from the B. subtilis genome by genetic transfer to a plasmid pLS20 was attempted. A stable plasmid clone was obtained only for the 108 and 153 kbp intermediates. The B. subtilis genome was demonstrated to accommodate large DNA with a high GC content, but may be restricted to less than 200 kbp by unidentified mechanisms. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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Metabolomic profiling rationalized pyruvate efficacy in cybrid cells harboring MELAS mitochondrial DNA mutations.

Kami, K., Fujita, Y., Igarashi, S., Koike, S., Sugawara, S., Ikeda, S., Sato, N., Ito, M., Tanaka, M., Tomita, M. and Soga, T.

Mitochondrion. (Mitochondrion) 12 ( 6 ) 644 - 653 2012

Accepted, ISSN 15677249

Pyruvate treatment was found to alleviate clinical symptoms of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome and is highly promising therapeutic. Using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), we measured time-changes of 161 intracellular and 85 medium metabolites to elucidate metabolic effects of pyruvate treatment on cybrid human 143B osteosarcoma cells harboring normal (2SA) and MELAS mutant (2SD) mitochondria. The results demonstrated dramatic and sustainable effects of pyruvate administration on the energy metabolism of 2SD cells, corroborating pyruvate as a metabolically rational treatment regimen for improving symptoms associated with MELAS and possibly other mitochondrial diseases. © 2012 Elsevier B.V. and Mitochondria Research Society.

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Computational assessment of insulin secretion and insulin sensitivity from 2-h oral glucose tolerance tests for clinical use for type 2 diabetes

Seike, Masayoshi; Saitou, Takeo; Kouchi, Yasuhiro; Ohara, Takeshi; Matsuhisa, Munehide; Sakaguchi, Kazuhiko; Tomita, Koji; Kosugi, Keisuke; Kashiwagi, Atsunori; Kasuga, Masato; Tomita, Masaru; Naito, Yasuhiro; Nakajima, Hiromu

Journal of Physiological Sciences (Journal of Physiological Sciences) 61 ( 4 ) 321-330 - 330 2011.07

Research paper (scientific journal), Joint Work, ISSN 18806546

In type 2 diabetes mellitus, glucose homeostasis is tightly maintained through insulin secretion and insulin sensitivity. Therefore, finding an accurate method to assess insulin secretion and sensitivity using clinically available data would enhance the quality of diabetic medical care. In an effort to find such a method, we developed a computational approach to derive indices of these factors using a 2-h oral glucose tolerance test (OGTT). To evaluate our method, clinical data from subjects who received an OGTT and a glucose clamp test were examined. Our insulin secretion index was significantly correlated with an analogous index obtained from a hyperglycemic clamp test (r = 0.90, n = 46, p < 0.001). Our insulin sensitivity index sensitivity was also significantly correlated with an analogous index obtained from a hyperinsulinemic-euglycemic clamp test (r = 0.56, n = 79, p < 0.001). These results suggest that our method can potentially provide an accurate and convenient tool toward improving the management of diabetes in clinical practice by assessing insulin secretion and insulin sensitivity. © 2011 The Author(s).

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A novel mechanism regulates H2S and SO2 production in Saccharomyces cerevisiae

Yoshida S., Imoto J., Minato T., Oouchi R., Kamada Y., Tomita M., Soga T., Yoshimoto H.

Yeast 28 ( 2 ) 109 - 121 2011.02

ISSN 0749503X

Sulfite (SO2) plays an important role in flavour stability in alcoholic beverages, whereas hydrogen sulfide (H2S) has an undesirable aroma. To discover the cellular processes that control SO2 and H2S production, we screened a library of Saccharomyces cerevisiae deletion mutants. Deletion of 12 genes led to increased H2S productivity. Ten of these genes are known to be involved in sulfur-containing amino acid metabolism, whereas UBI4 functions in the ubiquitin-proteasome system and SKP2 encodes an F-box-containing protein whose function is unknown. We found that the skp2 mutant accumulated H2S and SO2, because the adenosylphophosulfate kinase Met14p is a substrate of SCFSkp2 and more stable in the skp2 mutant than in the wild-type strain. Furthermore, the skp2 mutant grew more slowly than the wild-type strain under nutrient-limited conditions. Metabolome analysis showed that the concentration of intracellular cysteine is lower in the skp2 mutant than in the wild-type strain. The slow growth of the skp2 mutant was due to a lower concentration of intracellular cysteine, because the addition of cysteine suppressed the slow growth. In the skp2 mutant, the cysteine biosynthesis proteins Str2p, Str3p and Str4p are more stable than in the wild-type strain. Moreover, supplementation with methionine, S-adenosylmethionine, S-adenosylhomocysteine and homocysteine also suppressed the slow growth. Overexpression of STR1 or STR4 caused a more severe defect in the skp2 mutant. These results suggest that the balance of methionine and cysteine biosynthesis is important for yeast cell growth. Thus, Skp2p is one of the key components regulating this balance and H2S/SO2 production. © 2010 John Wiley & Sons, Ltd.

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A novel three-unit tRNA splicing endonuclease found in ultrasmall Archaea possesses broad substrate specificity

Fujishima K., Sugahara J., Miller C.S., Baker B.J., Di Giulio M., Takesue K., Sato A., Tomita M., Banfield J.F., Kanai A.

Nucleic Acids Research (Nucleic Acids Research) 39 ( 22 ) 9695-9704 - 9704 2011

Research paper (scientific journal), Joint Work, ISSN 03051048

tRNA splicing endonucleases, essential enzymes found in Archaea and Eukaryotes, are involved in the processing of pre-tRNA molecules. In Archaea, three types of splicing endonuclease [homotetrameric: α4, homodimeric: α2, and heterotetrameric: (αβ)2] have been identified, each representing different substrate specificity during the tRNA intron cleavage. Here, we discovered a fourth type of archaeal tRNA splicing endonuclease (2) in the genome of the acidophilic archaeon Candidatus Micrarchaeum acidiphilum, referred to as ARMAN-2 and its closely related species, ARMAN-1. The enzyme consists of two duplicated catalytic units and one structural unit encoded on a single gene, representing a novel three-unit architecture. Homodimeric formation was confirmed by cross-linking assay, and site-directed mutagenesis determined that the conserved L10-pocket interaction between catalytic and structural unit is necessary for the assembly. A tRNA splicing assay reveal that 2 endonuclease cleaves both canonical and non-canonical bulge-helix-bulge motifs, similar to that of (αβ)2 endonuclease. Unlike other ARMAN and Euryarchaeota, tRNAs found in ARMAN-2 are highly disrupted by introns at various positions, which again resemble the properties of archaeal species with (αβ)2 endonuclease. Thus, the discovery of 2 endonuclease in an archaeon deeply branched within Euryarchaeota represents a new example of the coevolution of tRNA and their processing enzymes. © The Author(s) 2011. Published by Oxford University Press.

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Redox Regulation in Respiring Saccharomyces cerevisiae

Murray, D.B., Haynes, K. and Tomita, M.

Biochim Biophys Acta. (Biochimica et Biophysica Acta - General Subjects) 1810 ( 10 ) 945-58 - 958 2011

Research paper (scientific journal), Joint Work, Accepted, ISSN 03044165

Background: In biological systems, redox reactions are central to most cellular processes and the redox potential of the intracellular compartment dictates whether a particular reaction can or cannot occur. Indeed the widespread use of redox reactions in biological systems makes their detailed description outside the scope of one review. Scope of the Review: Here we will focus on how system-wide redox changes can alter the reaction and transcriptional landscape of Saccharomyces cerevisiae. To understand this we explore the major determinants of cellular redox potential, how these are sensed by the cell and the dynamic responses elicited. Major Conclusions: Redox regulation is a large and complex system that has the potential to rapidly and globally alter both the reaction and transcription landscapes. Although we have a basic understanding of many of the sub-systems and a partial understanding of the transcriptional control, we are far from understanding how these systems integrate to produce coherent responses. We argue that this non-linear system self-organises, and that the output in many cases is temperature-compensated oscillations that may temporally partition incompatible reactions in vivo. General Significance: Redox biochemistry impinges on most of cellular processes and has been shown to underpin ageing and many human diseases. Integrating the complexity of redox signalling and regulation is perhaps one of the most challenging areas of biology. This article is part of a Special Issue entitled Systems Biology of Microorganisms. © 2011 Elsevier B.V. All rights reserved.

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Serum Metabolomics Reveals γ-glutamyl dipeptides as biomarkers for discrimination among different forms of liver disease

Soga, T., Sugimoto, M., Honma, M., Mori, M., Igarashi, K., Kashikura, K., Ikeda, S., Hirayama, A., Yamamoto, T., Yoshida, H., Otsuka, M., Tsuji, S., Yatomi, Y., Sakuragawa, T., Watanabe, H., Nihei, K., Saito, T., Kawata, S., Suzuki

J Hepatology (Journal of Hepatology) 55 ( 4 ) 896-905 - 905 2011

Research paper (scientific journal), Joint Work, Accepted, ISSN 01688278

Background & Aims: We applied a metabolome profiling approach to serum samples obtained from patients with different liver diseases, to discover noninvasive and reliable biomarkers for rapid-screening diagnosis of liver diseases. Methods: Using capillary electrophoresis and liquid chromatography mass spectrometry, we analyzed low molecular weight metabolites in a total of 248 serum samples obtained from patients with nine types of liver disease and healthy controls. Results: We found that γ-glutamyl dipeptides, which were biosynthesized through a reaction with γ-glutamylcysteine synthetase, were indicative of the production of reduced glutathione, and that measurement of their levels could distinguish among different liver diseases. Multiple logistic regression models facilitated the discrimination between specific and other liver diseases and yielded high areas under receiver-operating characteristic curves. The area under the curve values in training and independent validation data were 0.952 and 0.967 in healthy controls, 0.817 and 0.849 in drug-induced liver injury, 0.754 and 0.763 in asymptomatic hepatitis B virus infection, 0.820 and 0.762 in chronic hepatitis B, 0.972 and 0.895 in hepatitis C with persistently normal alanine transaminase, 0.917 and 0.707 in chronic hepatitis C, 0.803 and 0.993 in cirrhosis type C, and 0.762 and 0.803 in hepatocellular carcinoma, respectively. Several γ-glutamyl dipeptides also manifested potential for differentiating between nonalcoholic steatohepatitis and simple steatosis. Conclusions: γ-Glutamyl dipeptides are novel biomarkers for liver diseases, and varying levels of individual or groups of these peptides have the power to discriminate among different forms of hepatic disease. © 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

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Predicting the Kinetic Properties Associated with Redox Imbalance after Oxidative Crisis in G6PD-Deficient Erythrocytes: A Simulation Study.

Shimo, H., Nishino, T. and Tomita, M.

Adv Hematol. (Advances in Hematology) 2011 398945 2011

Research paper (scientific journal), Joint Work, Accepted, ISSN 16879104

It is well known that G6PD-deficient individuals are highly susceptible to oxidative stress. However, the differences in the degree of metabolic alterations among patients during an oxidative crisis have not been extensively studied. In this study, we applied mathematical modeling to assess the metabolic changes in erythrocytes of various G6PD-deficient patients during hydrogen peroxide- (H2O2-) induced perturbation and predict the kinetic properties that elicit redox imbalance after exposure to an oxidative agent. Simulation results showed a discrepancy in the ability to restore regular metabolite levels and redox homeostasis among patients. Two trends were observed in the response of redox status (GSH/GSSG) to oxidative stress, a mild decrease associated with slow recovery and a drastic decline associated with rapid recovery. The former was concluded to apply to patients with severe clinical symptoms. Low Vmax and high KmG6P of G6PD were shown to be kinetic properties that enhance consequent redox imbalance. Copyright © 2011 Hanae Shimo et al.

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Enhancing apoptosis in TRAIL-resistant cancer cells using fundamental response rules

Piras, V., Hayashi, K., Tomita, M. and Selvarajoo, K

Scientific Reports (Scientific Reports) 1 144 2011

Research paper (scientific journal), Joint Work, Accepted

The tumor necrosis factor related apoptosis-inducing ligand (TRAIL) induces apoptosis in malignant cells, while leaving other cells mostly unharmed. However, several carcinomas remain resistant to TRAIL. To investigate the resistance mechanisms in TRAIL-stimulated human fibrosarcoma (HT1080) cells, we developed a computational model to analyze the temporal activation profiles of cell survival (IkB, JNK, p38) and apoptotic (caspase-8 and-3) molecules in wildtype and several (FADD, RIP1, TRAF2 and caspase-8) knock-down conditions. Based on perturbation-response approach utilizing the law of information (signaling flux) conservation, we derived response rules for population-level average cell response. From this approach, i) a FADD-independent pathway to activate p38 and JNK, ii) a crosstalk between RIP1 and p38, and iii) a crosstalk between p62 and JNK are predicted. Notably, subsequent simulations suggest that targeting a novel molecule at p62/sequestosome-1 junction will optimize apoptosis through signaling flux redistribution. This study offers a valuable prospective to sensitive TRAIL-based therapy.

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GTP-dependent RNA 3'-terminal phosphate cyclase from the hyperthermophilic archaeon Pyrococcus furiosus

Sato, A., Soga, T., Igarashi, K., Takesue, K., Tomita, M. and Kanai, A.

Genes to Cells (Genes to Cells) 16 ( 12 ) 1190-1199 - 1199 2011

Research paper (scientific journal), Joint Work, Accepted, ISSN 13569597

We discovered that the PF1549 gene in Pyrococcus furiosus encodes a very heat-stable RNA 3'-terminal phosphate cyclase (Pf-Rtc). Although all previously reported Rtc proteins are ATP-dependent enzymes, we found that Pf-Rtc requires GTP for its cyclase activity at 95°C. Low-level activation of the enzyme was also observed in the presence of dGTP but not other dNTPs, indicating that the guanine base is very important for Pf-Rtc activity. We analyzed a series of GTP analogues and found that the conversion from GTP to GMP is important for Pf-Rtc activity and that an excess of GMP inhibits this activity. Gel-shift analysis clearly showed that the RNA-binding activity of Pf-Rtc is totally dependent on the linear form of the 3'-terminal phosphate, with an apparent K d value of 20nm at 95°C. Furthermore, we found that Pf-Rtc may contribute to GTP-dependent RNA ligation activity through the PF0027 protein (a 2'-5' RNA ligase-like protein in P. furiosus). The possible roles of Pf-Rtc and the importance of terminal phosphate structures in RNA are discussed. © 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

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Microscale phosphoproteome analysis of 10,000 cells from human cancer cell lines

Masuda, T., Sugiyama, N., Tomita, M.. and Ishihama, Y.

Anal. Chem 83 7698-703 2011

Research paper (scientific journal), Joint Work, Accepted

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A screening system for artificial small RNAs that inhibit the growth of Escherichia coli

Komasa M., Fujishima K., Hiraoka K., Shinhara A., Lee B., Tomita M., Kanai A.

Journal of Biochemistry 150 ( 3 ) 289-294 - 294 2011

Research paper (scientific journal), Joint Work, Accepted, ISSN 0021924X

We have developed a screening system for artificial small RNAs (sRNAs) that inhibit the growth of Escherichia coli. In this system, we used a plasmid library to express artificial sRNAs (approximately 200 bases long) containing 60 bases of random nucleotide sequence. The induced expression of the known rydB sRNA in the system reduced the amount of its possible target mRNA, rpoS, supporting the reliability of the method. To isolate clones of sRNAs that inhibited the growth of E. coli, we used two successive screening steps: (i) colony size selection on plates and (ii) monitoring E. coli growth in a 96-well plate format. As a result, 83 artificial sRNAs were identified that showed a range of inhibitory effects on bacterial growth. We also introduced nucleotide replacements into one of the highly inhibitory sRNA clones, H12, which partially abolished the inhibition of bacterial growth, suggesting that bacterial growth was inhibited in a sequence-specific manner. © The Authors 2011. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

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Comprehensive prediction of chromosome dimer resolution sites in bacterial genomes

Kono N., Arakawa K., Tomita M.

BMC Genomics (BMC Genomics) 12 19 2011

Research paper (scientific journal), Joint Work, Accepted

Background: During the replication process of bacteria with circular chromosomes, an odd number of homologous recombination events results in concatenated dimer chromosomes that cannot be partitioned into daughter cells. However, many bacteria harbor a conserved dimer resolution machinery consisting of one or two tyrosine recombinases, XerC and XerD, and their 28-bp target site, dif.Results: To study the evolution of the dif/XerCD system and its relationship with replication termination, we report the comprehensive prediction of dif sequences in silico using a phylogenetic prediction approach based on iterated hidden Markov modeling. Using this method, dif sites were identified in 641 organisms among 16 phyla, with a 97.64% identification rate for single-chromosome strains. The dif sequence positions were shown to be strongly correlated with the GC skew shift-point that is induced by replicational mutation/selection pressures, but the difference in the positions of the predicted dif sites and the GC skew shift-points did not correlate with the degree of replicational mutation/selection pressures.Conclusions: The sequence of dif sites is widely conserved among many bacterial phyla, and they can be computationally identified using our method. The lack of correlation between dif position and the degree of GC skew suggests that replication termination does not occur strictly at dif sites. © 2011 Kono et al; licensee BioMed Central Ltd.

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Integrative features of the yeast phosphoproteome and protein-protein interaction map

Yachie N., Saito R., Sugiyama N., Tomita M., Ishihama Y.

PLoS Computational Biology (PLoS Computational Biology) 7 ( 1 ) e1001064 2011

Research paper (scientific journal), Joint Work, Accepted, ISSN 1553734X

Following recent advances in high-throughput mass spectrometry (MS)-based proteomics, the numbers of identified phosphoproteins and their phosphosites have greatly increased in a wide variety of organisms. Although a critical role of phosphorylation is control of protein signaling, our understanding of the phosphoproteome remains limited. Here, we report unexpected, large-scale connections revealed between the phosphoproteome and protein interactome by integrative data-mining of yeast multi-omics data. First, new phosphoproteome data on yeast cells were obtained by MSbased proteomics and unified with publicly available yeast phosphoproteome data. This revealed that nearly 60% of,6,000 yeast genes encode phosphoproteins. We mapped these unified phosphoproteome data on a yeast protein-protein interaction (PPI) network with other yeast multi-omics datasets containing information about proteome abundance, proteome disorders, literature-derived signaling reactomes, and in vitro substratomes of kinases. In the phospho-PPI, phosphoproteins had more interacting partners than nonphosphoproteins, implying that a large fraction of intracellular protein interaction patterns (including those of protein complex formation) is affected by reversible and alternative phosphorylation reactions. Although highly abundant or unstructured proteins have a high chance of both interacting with other proteins and being phosphorylated within cells, the difference between the number counts of interacting partners of phosphoproteins and nonphosphoproteins was significant independently of protein abundance and disorder level. Moreover, analysis of the phospho-PPI and yeast signaling reactome data suggested that co-phosphorylation of interacting proteins by single kinases is common within cells. These multi-omics analyses illuminate how wide-ranging intracellular phosphorylation events and the diversity of physical protein interactions are largely affected by each other. © 2011 Yachie et al.

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Computational analysis suggests a highly bendable, fragile structure for nucleosomal DNA

Nozaki T., Yachie N., Ogawa R., Saito R., Tomita M.

Gene (Gene) 476 ( 1-2 ) 10-14 - 14 2011

Research paper (scientific journal), Joint Work, Accepted, ISSN 03781119

Eukaryotic chromosomal DNA coils around histones to form nucleosomes. Although histone affinity for DNA depends on DNA sequence patterns, how nucleosome positioning is determined by them remains unknown. Here, we show relationships between nucleosome positioning and two structural characteristics of DNA conferred by DNA sequence. Analysis of bendability and hydroxyl radical cleavage intensity of nucleosomal DNA sequences indicated that nucleosomal DNA is bendable and fragile and that nucleosome positional stability was correlated with characteristics of DNA. This result explains how histone positioning is partially determined by nucleosomal DNA structure, illuminating the optimization of chromosomal DNA packaging that controls cellular dynamics. © 2011 Elsevier B.V.

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Metabolomic anatomy of an animal model revealing homeostatic imbalances in dyslipidaemia

Ooga, Takushi ; Sato, Hajime ; Nagashima, Atsushi ; Sasaki, Kazunori ; Tomita, Masaru ; Soga, Tomoyoshi ; Ohashi, Yoshiaki

Molecular BioSystems (Molecular BioSystems) 7 ( 4 ) 1217-1223 - 1223 2011

Research paper (scientific journal), Joint Work, ISSN 1742-206X

Metabolomics is an emerging technology that reveals homeostatic imbalances in biological systems. Global determination of metabolite concentrations in body fluid and tissues provides novel anatomical aspects of pathological conditions that cannot be obtained from target-specific measurements. Here, we characterised metabolic imbalance in Watanabe heritable hyperlipidaemic rabbits as a model of hypercholesterolaemia. Using a mass spectrometry-based system, we measured a total of 335 metabolites in plasma and tissues (liver, aorta, cardiac muscle, and brain) from WHHL and healthy control rabbits. From the comparison between two metabolomic profiles, pathophysiological features including glutathione and phosphatidylcholine metabolism indicated the occurrence of oxidative stress in several tissues. Especially for the liver, imbalanced purine catabolism shed light on the transcriptional activation of xanthine oxidase, which is thought to act in absorbing or possibly triggering oxidative stress. We also applied this system to assess the therapeutic effects of simvastatin administration. After the treatment, a portion of the metabolomic features in pathological conditions showed alterations suggesting restoration of metabolism to the healthy condition. These changes were considered to be due to the pleiotropic action of statin, including antioxidant effects, rather than its main inhibitory action on cholesterol biosynthesis. © The Royal Society of Chemistry.

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OryzaPG-DB: Rice Proteome Database based on Shotgun Proteogenomics

Helmy, M., Tomita, M. and Ishihama

BMC Plant Biol. (BMC Plant Biology) 11 63 2011

Research paper (scientific journal), Joint Work, Accepted

Background: Proteogenomics aims to utilize experimental proteome information for refinement of genome annotation. Since mass spectrometry-based shotgun proteomics approaches provide large-scale peptide sequencing data with high throughput, a data repository for shotgun proteogenomics would represent a valuable source of gene expression evidence at the translational level for genome re-annotation.Description: Here, we present OryzaPG-DB, a rice proteome database based on shotgun proteogenomics, which incorporates the genomic features of experimental shotgun proteomics data. This version of the database was created from the results of 27 nanoLC-MS/MS runs on a hybrid ion trap-orbitrap mass spectrometer, which offers high accuracy for analyzing tryptic digests from undifferentiated cultured rice cells. Peptides were identified by searching the product ion spectra against the protein, cDNA, transcript and genome databases from Michigan State University, and were mapped to the rice genome. Approximately 3200 genes were covered by these peptides and 40 of them contained novel genomic features. Users can search, download or navigate the database per chromosome, gene, protein, cDNA or transcript and download the updated annotations in standard GFF3 format, with visualization in PNG format. In addition, the database scheme of OryzaPG was designed to be generic and can be reused to host similar proteogenomic information for other species. OryzaPG is the first proteogenomics-based database of the rice proteome, providing peptide-based expression profiles, together with the corresponding genomic origin, including the annotation of novelty for each peptide.Conclusions: The OryzaPG database was constructed and is freely available at http://oryzapg.iab.keio.ac.jp/. © 2011 Helmy et al; licensee BioMed Central Ltd.

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Positive Autoregulation Delays the Expression Phase of Mammalian Clock Gene Per2

Ogawa, Y., Koike, N., Kurosawa, G., Soga, T., Tomita, M. and Tei, H.

PLoS ONE (PLoS ONE) 6 ( 4 ) e18663 2011

Research paper (scientific journal), Joint Work, Accepted

In mammals, cellular circadian rhythms are generated by a transcriptional-translational autoregulatory network that consists of clock genes that encode transcriptional regulators. Of these clock genes, Period1 (Per1) and Period2 (Per2) are essential for sustainable circadian rhythmicity and photic entrainment. Intriguingly, Per1 and Per2 mRNAs exhibit circadian oscillations with a 4-hour phase difference, but they are similarly transactivated by CLOCK-BMAL1. In this study, we investigated the mechanism underlying the phase difference between Per1 and Per2 through a combination of mathematical simulations and molecular experiments. Mathematical analyses of a model for the mammalian circadian oscillator demonstrated that the slow synthesis and fast degradation of mRNA tend to advance the oscillation phase of mRNA expression. However, the phase difference between Per1 and Per2 was not reproduced by the model, which implemented a 1.1-fold difference in degradation rates and a 3-fold difference in CLOCK-BMAL1 mediated inductions of Per1 and Per2 as estimated in cultured mammalian cells. Thus, we hypothesized the existence of a novel transcriptional activation of Per2 by PER1/2 such that the Per2 oscillation phase was delayed. Indeed, only the Per2 promoter, but not Per1, was strongly induced by both PER1 and PER2 in the presence of CLOCK-BMAL1 in a luciferase reporter assay. Moreover, a 3-hour advance was observed in the transcriptional oscillation of the delta-Per2 reporter gene lacking cis-elements required for the induction by PER1/2. These results indicate that the Per2 positive feedback regulation is a significant factor responsible for generating the phase difference between Per1 and Per2 gene expression. © 2011 Ogawa et al.

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Comprehensive expressional analyses of antisense transcripts in colon cancer tissues using artificial antisense probes

Saito, R., Kohno, K., Okada, Y., Osada, Y., Numata, K., Kohama, C., Watanabe, K., Nakaoka, H., Yamamoto, N., Kanai, A., Yasue, H., Murata, S., Abe, K., Tomita, M., Ohkohchi, N. and Kiyosawa, H.

BMC Med. Genomics (BMC Medical Genomics) 4 42 2011

Research paper (scientific journal), Joint Work, Accepted

Background: Recent studies have identified thousands of sense-antisense gene pairs across different genomes by computational mapping of cDNA sequences. These studies have shown that approximately 25% of all transcriptional units in the human and mouse genomes are involved in cis-sense-antisense pairs. However, the number of known sense-antisense pairs remains limited because currently available cDNA sequences represent only a fraction of the total number of transcripts comprising the transcriptome of each cell type. Methods. To discover novel antisense transcripts encoded in the antisense strand of important genes, such as cancer-related genes, we conducted expression analyses of antisense transcripts using our custom microarray platform along with 2376 probes designed specifically to detect the potential antisense transcripts of 501 well-known genes suitable for cancer research. Results: Using colon cancer tissue and normal tissue surrounding the cancer tissue obtained from 6 patients, we found that antisense transcripts without poly(A) tails are expressed from approximately 80% of these well-known genes. This observation is consistent with our previous finding that many antisense transcripts expressed in a cell are poly(A)-. We also identified 101 and 71 antisense probes displaying a high level of expression specifically in normal and cancer tissues respectively. Conclusion: Our microarray analysis identified novel antisense transcripts with expression profiles specific to cancer tissue, some of which might play a role in the regulatory networks underlying oncogenesis and thus are potential targets for further experimental validation. Our microarray data are available at http://www.brc.riken.go.jp/ncrna2007/viewer-Saito-01/index.html. © 2011 Saito et al; licensee BioMed Central Ltd.

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KBWS: an EMBOSS associated package for accessing bioinformatics web services

Oshita, K., Arakawa, K. and Tomita, M.

Source Code Biol Med. (Source Code for Biology and Medicine) 6 8 2011

Research paper (scientific journal), Joint Work, Accepted

The availability of bioinformatics web-based services is rapidly proliferating, for their interoperability and ease of use. The next challenge is in the integration of these services in the form of workflows, and several projects are already underway, standardizing the syntax, semantics, and user interfaces. In order to deploy the advantages of web services with locally installed tools, here we describe a collection of proxy client tools for 42 major bioinformatics web services in the form of European Molecular Biology Open Software Suite (EMBOSS) UNIX command-line tools. EMBOSS provides sophisticated means for discoverability and interoperability for hundreds of tools, and our package, named the Keio Bioinformatics Web Service (KBWS), adds functionalities of local and multiple alignment of sequences, phylogenetic analyses, and prediction of cellular localization of proteins and RNA secondary structures. This software implemented in C is available under GPL from http://www.g-language.org/kbws/ and GitHub repository http://github.com/cory-ko/KBWS. Users can utilize the SOAP services implemented in Perl directly via WSDL file at http://soap.g-language.org/kbws.wsdl (RPC Encoded) and http://soap.g-language.org/kbws_dl.wsdl (Document/literal). © 2011 Oshita et al; licensee BioMed Central Ltd.

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Chlamydomonas neoplanoconvexa nom. nov. and its unique phylogenetic position within Volvocales (Chlorophyceae)

Nakada, T. and Tomita,

Phycol. Res. 59 ( 3 ) 194–199 - 199 2011

Research paper (scientific journal), Joint Work, Accepted, ISSN 13220829

Chlamydomonas Ehrenb. is a unicellular volvocalean genus consisting of 400-600 species, most of which are known solely based on microscopy. In this study, a newly isolated strain of Chlamydomonas neoplanoconvexa (M. O. P. Iyengar) Nakada nom. nov. (≡Chlamydomonas planoconvexa M. O. P. Iyengar non J. W. G. Lund) was examined using light microscopy and combined 18S rRNA, rbcL and psaB gene phylogeny. The C. neoplanoconvexa strain was quite similar to Chlamydomonas perpusilla (Korshikov) Gerloff in terms of its small fusiform vegetative cells (∼10μm in length), parietal chloroplast containing a single pyrenoid, and nucleus posterior to the pyrenoid. However, C. neoplanoconvexa was distinguished from C. perpusilla based on the morphology of the papillae and pyrenoids. The two species belong to the clade Caudivolvoxa in the order Volvocales, but are distantly related to each other. Chlamydomonas perpusilla was shown to be sister to Chlorogonium Ehrenb. (clade Chlorogonia, within Caudivolvoxa), while C. neoplanoconvexa represented a second basal lineage within Caudivolvoxa, next to the clade Characiosiphonia. Although the morphology of C. neoplanoconvexa was not particularly outstanding, its unique phylogenetic position will encourage further investigation of this species and its uncultivated relatives. © 2011 Japanese Society of Phycology.

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Metabolic flux analysis and visualization

Toya, Y., Kono, N., Arakawa, K. and Tomita, M.

J. Proteome Res. (Journal of Proteome Research) 10 ( 8 ) 3313–3323 - 3323 2011

Research paper (scientific journal), Joint Work, Accepted, ISSN 15353893

One of the ultimate goals of systems biology research is to obtain a comprehensive understanding of the control mechanisms of complex cellular metabolisms. Metabolic Flux Analysis (MFA) is a important method for the quantitative estimation of intracellular metabolic flows through metabolic pathways and the elucidation of cellular physiology. The primary challenge in the use of MFA is that many biological networks are underdetermined systems; it is therefore difficult to narrow down the solution space from the stoichiometric constraints alone. In this tutorial, we present an overview of Flux Balance Analysis (FBA) and 13C-Metabolic Flux Analysis ( 13C-MFA), both of which are frequently used to solve such underdetermined systems, and we demonstrate FBA and 13C-MFA using the genome-scale model and the central carbon metabolism model, respectively. Furthermore, because such comprehensive study of intracellular fluxes is inherently complex, we subsequently introduce various pathway mapping and visualization tools to facilitate understanding of these data in the context of the pathways. Specific visualization of MFA results using the BioCyc Omics Viewer and Pathway Projector are shown as illustrative examples. © 2011 American Chemical Society.

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Tight Associations between Transcription Promoter Type and Epigenetic Variation in Histone Positioning and Modification

Nozaki, T., Yachie, N., Ogawa, R., Kratz, A., Saito, R. and Tomita, M.

BMC Genomics (BMC Genomics) 12 416 2011

Research paper (scientific journal), Joint Work, Accepted

Background: Transcription promoters are fundamental genomic cis-elements controlling gene expression. They can be classified into two types by the degree of imprecision of their transcription start sites: peak promoters, which initiate transcription from a narrow genomic region; and broad promoters, which initiate transcription from a wide-ranging region. Eukaryotic transcription initiation is suggested to be associated with the genomic positions and modifications of nucleosomes. For instance, it has been recently shown that histone with H3K9 acetylation (H3K9ac) is more likely to be distributed around broad promoters rather than peak promoters; it can thus be inferred that there is an association between histone H3K9 and promoter architecture.Results: Here, we performed a systematic analysis of transcription promoters and gene expression, as well as of epigenetic histone behaviors, including genomic position, stability within the chromatin, and several modifications. We found that, in humans, broad promoters, but not peak promoters, generally had significant associations with nucleosome positioning and modification. Specifically, around broad promoters histones were highly distributed and aligned in an orderly fashion. This feature was more evident with histones that were methylated or acetylated; moreover, the nucleosome positions around the broad promoters were more stable than those around the peak ones. More strikingly, the overall expression levels of genes associated with broad promoters (but not peak promoters) with modified histones were significantly higher than the levels of genes associated with broad promoters with unmodified histones.Conclusion: These results shed light on how epigenetic regulatory networks of histone modifications are associated with promoter architecture. © 2011 Nozaki et al; licensee BioMed Central Ltd.

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Deep sequencing reveals as-yet-undiscovered small RNAs in Escherichia coli

Shinhara, A., Matsui, M., Hiraoka, K., Nomura, W., Hirano, R., Nakahigashi, K., Tomita, M., Mori, H. and Kanai, A.

BMC Genomics (BMC Genomics) 12 428 2011

Research paper (scientific journal), Joint Work, Accepted

Background: In Escherichia coli, approximately 100 regulatory small RNAs (sRNAs) have been identified experimentally and many more have been predicted by various methods. To provide a comprehensive overview of sRNAs, we analysed the low-molecular-weight RNAs (< 200 nt) of E. coli with deep sequencing, because the regulatory RNAs in bacteria are usually 50-200 nt in length.Results: We discovered 229 novel candidate sRNAs (≥ 50 nt) with computational or experimental evidence of transcription initiation. Among them, the expression of seven intergenic sRNAs and three cis-antisense sRNAs was detected by northern blot analysis. Interestingly, five novel sRNAs are expressed from prophage regions and we note that these sRNAs have several specific characteristics. Furthermore, we conducted an evolutionary conservation analysis of the candidate sRNAs and summarised the data among closely related bacterial strains.Conclusions: This comprehensive screen for E. coli sRNAs using a deep sequencing approach has shown that many as-yet-undiscovered sRNAs are potentially encoded in the E. coli genome. We constructed the Escherichia coli Small RNA Browser (ECSBrowser; http://rna.iab.keio.ac.jp/), which integrates the data for previously identified sRNAs and the novel sRNAs found in this study. © 2011 Shinhara et al; licensee BioMed Central Ltd.

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The Salivary Gland and Systemic Health: Towards the Creation of Salivary Gland and Health Medicine

Tsukinoki K., Saruta J., Yamano S., Tomita M.

Journal of Oral Biosciences 53 ( 4 ) 330 - 337 2011

ISSN 13490079

Salivary glands are intimately connected with the entire body via the nervous system and blood vessels, yet few studies have documented the effects of the salivary glands on the health of the whole body, although saliva has often been reported to possibly contain promising biomarkers of systemic disease states, including cancer. We therefore propose that the investigation of the effects of the salivary gland on the whole body and vice versa could introduce a new field of study, i.e., “salivary gland and health medicine.” This review examines data from novel studies on “salivary gland and health medicine” that were carried out by 3 different research groups. © 2011, Japanese Association for Oral Biology. All rights reserved.

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An Extreme Thermophile, Thermus thermophilus, Is a Polyploid Bacterium

Ohtani N, Tomita M, Itaya M.

J Bacteriol (Journal of Bacteriology) 192 ( 20 ) 5499-5505 - 5505 2010.10

Research paper (scientific journal), Joint Work, ISSN 0021-9193

An extremely thermophilic bacterium, Thermus thermophilus HB8, is one of the model organisms for systems biology. Its genome consists of a chromosome (1.85 Mb), a megaplasmid (0.26 Mb) designated pTT27, and a plasmid (9.3 kb) designated pTT8, and the complete sequence is available. We show here that T. thermophilus is a polyploid organism, harboring multiple genomic copies in a cell. In the case of the HB8 strain, the copy number of the chromosome was estimated to be four or five, and the copy number of the pTT27 megaplasmid seemed to be equal to that of the chromosome. It has never been discussed whether T. thermophilus is haploid or polyploid. However, the finding that it is polyploid is not surprising, as Deinococcus radiodurans, an extremely radioresistant bacterium closely related to Thermus, is well known to be a polyploid organism. As is the case for D. radiodurans in the radiation environment, the polyploidy of T. thermophilus might allow for genomic DNA protection, maintenance, and repair at elevated growth temperatures. Polyploidy often complicates the recognition of an essential gene in T. thermophilus as a model organism for systems biology. Copyright © 2010, American Society for Microbiology. All Rights Reserved.

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13C-Metabolic flux analysis for batch culture of Escherichia coli and its pyk and pgi gene knockout mutants based on mass isotopomer distribution of intracellular metabolites

Toya Y., Ishii N., Nakahigashi K., Hirasawa T., Soga T., Tomita M., Shimizu K.

Biotechnology Progress 26 ( 4 ) 975 - 992 2010.07

ISSN 87567938

Since most bio-production processes are conducted in a batch or fed-batch manner, the evaluation of metabolism with respect to time is highly desirable. Toward this aim, we applied 13C-metabolic flux analysis to nonstationary conditions by measuring the mass isotopomer distribution of intracellular metabolites. We performed our analysis on batch cultures of wild-type Escherichia coli, as well as on Pyk and Pgi mutants, obtained the fluxes and metabolite concentrations as a function of time. Our results for the wild-type indicated that the TCA cycle flux tended to increase during growth on glucose. Following glucose exhaustion, cells controlled the branch ratio between the glyoxylate pathway and the TCA cycle, depending on the availability of acetate. In the Pyk mutant, the concentrations of glycolytic intermediates changed drastically over time due to the dumping and feedback inhibition caused by PEP accumulation. Nevertheless, the flux distribution and free amino acid concentrations changed little. The growth rate and the fluxes remained constant in the Pgi mutant and the glucose-6-phosphate dehydrogenase reaction was the rate-limiting step. The measured fluxes were compared with those predicted by flux balance analysis using maximization of biomass yield or ATP production. Our findings indicate that the objective function of biosynthesis became less important as time proceeds on glucose in the wild-type, while it remained highly important in the Pyk mutant. Furthermore, ATP production was the primary objective function in the Pgi mutant. This study demonstrates how cells adjust their metabolism in response to environmental changes and/or genetic perturbations in the batch cultivation. © 2010 American Institute of Chemical Engineers.

Predictive value of CD24 and CD44 for neoadjuvant chemotherapy response and prognosis in primary breast cancer patients

Horiguchi K, Toi M, Horiguchi S, Sugimoto M, Naito Y, Hayashi Y, Ueno T, Ohno S, Funata N, Kuroi K, Tomita M, Eishi Y.

J Med Dent Sci (Journal of Medical and Dental Sciences) 57 ( 2 ) 165-175 - 175 2010.06

Research paper (scientific journal), Joint Work, ISSN 1342-8810

Purpose: We investigated the significance of CD24 and CD44 expression for predicting responses to chemotherapy and prognosis in primary breast cancer patients. Patients and Methods: Diagnosis of breast cancer was confirmed by core needle biopsy, and immunohistochemical studies were performed. Preoperatively, patients received anthracyclinecontaining chemotherapy. Expression of CD44 and CD24 was assessed immunohistochemically and the relationship with chemotherapy response and with prognosis was analyzed. Results: Between 2001 and 2004, 139 women were enrolled in this study. In the correlation analysis, CD24 expression was negatively associated with pathological response to chemotherapy (p=0.0003). A machine learning technique with an alternating decision tree (ADTree) showed that four logical rules are involved in predicting the response depending on the combination of CD24, HER2, tumor stage, CD44, progesterone receptor, and patient age. In the survival analysis, patients having CD44 (++) showed a significantly favorable prognosis as compared with others (p=0.0002). A multivariate analysis showed that CD44 expression had an independent prognostic value (p<0.001). Conclusion: We found a significant correlation between CD44 expression and prognosis and between CD24 expression and response to chemotherapy. CD24 and CD44 expressions would be useful predictive markers, although further studies are needed.

Characterization of RNase HII substrate recognition using RNase HII-argonaute chimaeric enzymes from Pyrococcus furiosus

Kitamura S., Fujishima K., Sato A., Tsuchiya D., Tomita M., Kanai A.

Biochemical Journal (Biochemical Journal) 426 ( 3 ) 337 - 344 2010.03

ISSN 02646021

RNase H (ribonuclease H) is an endonuclease that cleaves the RNA strand of RNA-DNA duplexes. It has been reported that the three-dimensional structure of RNase H is similar to that of the PIWI domain of the Pyrococcus furiosus Ago (argonaute) protein, although the two enzymes share almost no similarity in their amino acid sequences. Eukaryotic Ago proteins are key components of the RNA-induced silencing complex and are involved in microRNA or siRNA (small interfering RNA) recognition. In contrast, prokaryotic Ago proteins show greater affinity for RNA-DNA hybrids than for RNA-RNA hybrids. Interestingly, we found that wild-type Pf-RNase HII (P. furiosus, RNase HII) digests RNA-RNA duplexes in the presence of Mn2+ ions. To characterize the substrate specificity of Pf-RNase HII, we aligned the amino acid sequences of Pf-RNase HII and Pf-Ago, based on their protein secondary structures. We found that one of the conserved secondary structural regions (the fourth β-sheet and the fifth α-helix of Pf-RNase HII) contains family-specific amino acid residues. Using a series of Pf-RNase HII-Pf-Ago chimaeric mutants of the region, we discovered that residues Asp110, Arg113 and Phe 114 are responsible for the dsRNA (double-stranded RNA) digestion activity of Pf-RNase HII. On the basis of the reported three-dimensional structure of Ph-RNase HII from Pyrococcus horikoshii, we built a three-dimensional structural model of RNase HII complexed with its substrate, which suggests that these amino acids are located in the region that discriminates DNA from RNA in the non-substrate strand of the duplexes. © The Authors.

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Volvulina compacta ( Volvocaceae, Chlorophyceae ), new to Japan, and its phylogenetic position

Nakada, T., Tomita, M. and Nozaki, H.

J. Jpn. Bot. 85 ( 6 ) 364–369 - 369 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 00222062

Volvulina Playfair is a colonial volvocalean genus with usually 8 or 16 cells forming hollow ellipsoidal colonies. In this study, a strain of Volvulina was isolated from Lake Tsukui, Kanagawa Prefecture, Japan. This strain was morphologically identified as Volvulina compacta Nozaki, which was previously known only from strains isolated from Nepal. Chloroplast multigene analyses using rbcL, atpB, psaA, psaB and psbC genes showed that the Japanese strain of V. compacta is closely related to the Nepalese strain of V. compacta and to an American strain of Volvulina pringsheimii Starr, but only distantly to Volvulina steinii Playfair previously reported from Japan. Although the Japanese strain was more closely related to V. pringsheimii than to Nepalese V. compacta, it was clearly distinguished from V. pringsheimii by its square cell shape in the top view. These results indicate the existence of cryptic species within V. compacta.

Phylogenetic position of a rare loricated green alga, Cephalomonas granulata N. L. Higinb. (Volvocales, Chlorophyceae)

Nakada, T., Soga, T. and Tomita, M.

Phycol. Res. 58 ( 1 ) 62-68 - 68 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 13220829

Cephalomonas (Cp.) granulata N. L. Higinb. (Volvocales, Chlorophyceae), a rare volvocalean phytoflagellate, has recently been isolated from a paddy field in Japan and examined by light and fluorescence microscopy. The vegetative cells of Cp. granulata have granulate loricae and a characteristic mushroom-like shape with a hemispherical to spherical anterior part and narrower posterior part. Cp. granulata has been classified among the Phacotaceae along with other loricated genera. However, its phylogenetic position has not been investigated using molecular phylogeny. To evaluate the phylogenetic position of Cephalomonas, the 18S rRNA gene sequence of the Japanese strain was determined. The phylogenetic analyses revealed that Cp. granulata was not closely related to other loricated taxa with known phylogenetic affinities, such as Phacotaceae sensu stricto (Phacotus, Pteromonas, and Wislouchiella) and Dysmorphococcus globosus. This indicates at least three independent origins of loricae within the Volvocales. © 2010 Japanese Society of Phycology.

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A new multicompartmental reaction-diffusion modeling method links transient membrane attachment of E. coli MinE to E-ring formation

Arjunan, S. N. V. and Tomita, M.

Syst Synth Biol. (Systems and Synthetic Biology) 4 ( 1 ) 35-53 - 53 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 18725325

Many important cellular processes are regulated by reaction-diffusion (RD) of molecules that takes place both in the cytoplasm and on the membrane. To model and analyze such multicompartmental processes, we developed a lattice-based Monte Carlo method, Spatiocyte that supports RD in volume and surface compartments at single molecule resolution. Stochasticity in RD and the excluded volume effect brought by intracellular molecular crowding, both of which can significantly affect RD and thus, cellular processes, are also supported. We verified the method by comparing simulation results of diffusion, irreversible and reversible reactions with the predicted analytical and best available numerical solutions. Moreover, to directly compare the localization patterns of molecules in fluorescence microscopy images with simulation, we devised a visualization method that mimics the microphotography process by showing the trajectory of simulated molecules averaged according to the camera exposure time. In the rod-shaped bacterium Escherichia coli, the division site is suppressed at the cell poles by periodic pole-to-pole oscillations of the Min proteins (MinC, MinD and MinE) arising from carefully orchestrated RD in both cytoplasm and membrane compartments. Using Spatiocyte we could model and reproduce the in vivo MinDE localization dynamics by accounting for the previously reported properties of MinE. Our results suggest that the MinE ring, which is essential in preventing polar septation, is largely composed of MinE that is transiently attached to the membrane independently after recruited by MinD. Overall, Spatiocyte allows simulation and visualization of complex spatial and reaction-diffusion mediated cellular processes in volumes and surfaces. As we showed, it can potentially provide mechanistic insights otherwise difficult to obtain experimentally. © 2009 The Author(s).

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Computational prediction and experimental validation of evolutionarily conserved microRNA target genes in bilaterian animals

Takane, K., Fujishima, K., Watanabe, Y., Sato, A., Saito, N., Tomita, M and Kanai, A.

BMC Genomics (BMC Genomics) 11 ( 1 ) 101 2010

Research paper (scientific journal), Joint Work, Accepted

Background: In many eukaryotes, microRNAs (miRNAs) bind to complementary sites in the 3'-untranslated regions (3'-UTRs) of target messenger RNAs (mRNAs) and regulate their expression at the stage of translation. Recent studies have revealed that many miRNAs are evolutionarily conserved; however, the evolution of their target genes has yet to be systematically characterized. We sought to elucidate a set of conserved miRNA/target-gene pairs and to analyse the mechanism underlying miRNA-mediated gene regulation in the early stage of bilaterian evolution.Results: Initially, we extracted five evolutionarily conserved miRNAs (let-7, miR-1, miR-124, miR-125/lin-4, and miR-34) among five diverse bilaterian animals. Subsequently, we designed a procedure to predict evolutionarily conserved miRNA/target-gene pairs by introducing orthologous gene information. As a result, we extracted 31 orthologous miRNA/target-gene pairs that were conserved among at least four diverse bilaterian animals; the prediction set showed prominent enrichment of orthologous miRNA/target-gene pairs that were verified experimentally. Approximately 84% of the target genes were regulated by three miRNAs (let-7, miR-1, and miR-124) and their function was classified mainly into the following categories: development, muscle formation, cell adhesion, and gene regulation. We used a reporter gene assay to experimentally verify the downregulation of six candidate pairs (out of six tested pairs) in HeLa cells.Conclusions: The application of our new method enables the identification of 31 miRNA/target-gene pairs that were expected to have been regulated from the era of the common bilaterian ancestor. The downregulation of all six candidate pairs suggests that orthologous information contributed to the elucidation of the primordial set of genes that has been regulated by miRNAs; it was also an efficient tool for the elimination of false positives from the predicted candidates. In conclusion, our study identified potentially important miRNA-target pairs that were evolutionarily conserved throughout diverse bilaterian animals and that may provide new insights into early-stage miRNA functions. © 2010 Takane et al; licensee BioMed Central Ltd.

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A Comprehensive Resource of Interacting Protein Regions for Refining Human Transcription Factor Networks

Sato, M. E., Fujimori, S., Ishizaka, M., Hirai1, N., Masuoka1, K., Saito, R., Ozawa, Y., Hino, K., Washio, T., Tomita, M., Yamashita, T., Oshikubo, T., Akasaka, H., Sugiyama, J., Matsumoto, Y. and Yanagawa, H.

PLoS ONE (PLoS ONE) 5 ( 2 ) e9289 2010

Research paper (scientific journal), Joint Work, Accepted

Large-scale data sets of protein-protein interactions (PPIs) are a valuable resource for mapping and analysis of the topological and dynamic features of interactome networks. The currently available large-scale PPI data sets only contain information on interaction partners. The data presented in this study also include the sequences involved in the interactions (i.e., the interacting regions, IRs) suggested to correspond to functional and structural domains. Here we present the first large-scale IR data set obtained using mRNA display for 50 human transcription factors (TFs), including 12 transcription-related proteins. The core data set (966 IRs; 943 PPIs) displays a verification rate of 70%. Analysis of the IR data set revealed the existence of IRs that interact with multiple partners. Furthermore, these IRs were preferentially associated with intrinsic disorder. This finding supports the hypothesis that intrinsically disordered regions play a major role in the dynamics and diversity of TF networks through their ability to structurally adapt to and bind with multiple partners. Accordingly, this domain-based interaction resource represents an important step in refining protein interactions and networks at the domain level and in associating network analysis with biological structure and function. © 2010 Miyamoto-Sato et al.

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Chlorogonium complexum sp. nov. (Volvocales, Chlorophyceae), and morphological evolution of Chlorogonium

Nakada, T., Soga, T., Tomita, M. and Nozaki, H.

Eur. J. Phycol. 45 ( 1 ) 97-107 - 106 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 09670262

We isolated strains of a new species of Chlorogonium (Cg.), Cg. complexum Nakada sp. nov. The genus Chlorogonium was recently emended to have several, irregularly distributed contractile vacuoles, two-to-many pyrenoids associated with fragmented starch plates and tubular thylakoid lamellae penetrating their matrices, and stigma composed of a single layer of globules. Chlorogonium complexum has these diagnostic characters of the emended genus. It is distinguished from other species of Chlorogonium, Cg. euchlorum, Cg. elongatum and Cg. capillatum, on the combination of diagnostic characters, such as the number and stability of pyrenoids and the mode of encystment. Based on a dataset combining 18S rRNA, RuBisCO large subunit (rbcL) and P700 chl a-apoprotein A2 (psaB) gene sequences, the phylogenetic position of Chlorogonium was determined. Chlorogonium complexum represented an independent lineage, sister to Cg. euchlorum and Cg. capillatum. The evolution of diagnostic characters among Chlorogonium species is discussed based on the molecular phylogenetic analyses, and a key to species of Chlorogonium and fusiform chlamydomonads with irregularly distributed contractile vacuoles is presented. © 2010 British Phycological Society.

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Computational prediction of nucleosome positioning by calculating the relative fragment frequency index of nucleosomal sequences

Ogawa, R., Kitagawa, N., Ashida, H., Saito, R. and Tomita, M.

FEBS Letters (FEBS Letters) 584 ( 8 ) 1498-1502 - 1502 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 00145793

We developed an accurate method to predict nucleosome positioning from genome sequences by refining the previously developed method of Peckham et al. (2007) [19]. Here, we used the relative fragment frequency index we developed and a support vector machine to screen for nucleosomal and linker DNA sequences. Our twofold cross-validation revealed that the accuracy of our method based on the area under the receiver operating characteristic curve was 81%, whereas that of Peckham's method was 75% when both of two nucleosomal sequence data obtained from independent experiments were used for validation. We suggest that our method is more effective in predicting nucleosome positioning. © 2010 Federation of European Biochemical Societies.

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One-Dimensional Capillary Liquid Chromatographic Separation Coupled with Tandem Mass Spectrometry Unveils the Escherichia coli Proteome on a Microarray Scale

Iwasaki, M., Miwa, S., Ikegami, T., Tomita, M., Tanaka, N. and Ishihama, Y.

Anal. Chem. (Analytical Chemistry) 82 ( 7 ) 2616-2620 - 2620 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 00032700

We successfully identified the proteome expressed in Escherichia coli (E. coli) cells on a microarray scale using one-dimensional capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a 350 cm long, 100 μm i. d., monolithic silica-C18 capillary column. E. coli tryptic digest (4 μg) was injected onto the column, and a 41 h gradient was applied with a flow rate of 500 nL/min at less than 20 MPa. In total, 22 196 nonredundant tryptic peptides from 2602 proteins, including 830 membrane proteins, were identified from the E. coli cells (triplicate analysis), in which an equivalent number of genes was detected by transcriptome analysis. Approximately a 5-fold larger peak response on average was obtained in this system, compared with that obtained by conventional capillary LC-MS/MS analysis with a 15 cm long, 3 μm diameter C18 silica particle-packed column. The higher response suggests that the influence of ionization suppression was drastically reduced by the high-efficiency separation on the long monolithic silica column coupled with the shallow gradient. Because this high-resolution system does not require any additional separation prior to LC-MS/MS, this "one-shot" proteomics approach can simplify the workflow of shotgun proteomics and minimize the sample amount, as well as reduce the total analysis time, despite the use of prolonged shallow gradient elution. © 2010 American Chemical Society.

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Gene knockout and metabolome analysis of carnitine/organic cation transporter OCTN1

Kato, Y., Kubo, Y., Iwata, D., Kato, S., Sudo, T., Sugiura, T., Kagaya, T., Wakayama, T.,Hirayama, A., Sugimoto, M., Sugihara, K., Kaneko, S., Soga, T., Asano, M., Tomita, M., Matsui, T., Wada, M. and Tsuji A.

Pharm Res 27 ( 5 ) 832-840 - 40 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 0724-8741

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Towards the systematic discovery of signal transduction networks using phosphorylation dynamics data

Imamura, H., Yachie, N., Saito, R., Ishihama, Y. and Tomita, M.

BMC Bioinformatics (BMC Bioinformatics) 11 232 2010

Research paper (scientific journal), Joint Work, Accepted

Background: Phosphorylation is a ubiquitous and fundamental regulatory mechanism that controls signal transduction in living cells. The number of identified phosphoproteins and their phosphosites is rapidly increasing as a result of recent mass spectrometry-based approaches.Results: We analyzed time-course phosphoproteome data obtained previously by liquid chromatography mass spectrometry with the stable isotope labeling using amino acids in cell culture (SILAC) method. This provides the relative phosphorylation activities of digested peptides at each of five time points after stimulating HeLa cells with epidermal growth factor (EGF). We initially calculated the correlations between the phosphorylation dynamics patterns of every pair of peptides and connected the strongly correlated pairs to construct a network. We found that peptides extracted from the same intracellular fraction (nucleus vs. cytoplasm) tended to be close together within this phosphorylation dynamics-based network. The network was then analyzed using graph theory and compared with five known signal-transduction pathways. The dynamics-based network was correlated with known signaling pathways in the NetPath and Phospho.ELM databases, and especially with the EGF receptor (EGFR) signaling pathway. Although the phosphorylation patterns of many proteins were drastically changed by the EGF stimulation, our results suggest that only EGFR signaling transduction was both strongly activated and precisely controlled.Conclusions: The construction of a phosphorylation dynamics-based network provides a useful overview of condition-specific intracellular signal transduction using quantitative time-course phosphoproteome data under specific experimental conditions. Detailed prediction of signal transduction based on phosphoproteome dynamics remains challenging. However, since the phosphorylation profiles of kinase-substrate pairs on the specific pathway were localized in the dynamics-based network, our method will be a complementary strategy to explore new components of protein signaling pathways in combination with previous methods (including software) of predicting direct kinase-substrate relationships. © 2010 Imamura et al; licensee BioMed Central Ltd.

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Transcription-Associated Mutagenesis Increases Protein Sequence Diversity More Effectively than Does Random Mutagenesis in Escherichia coli

Kim, H., Lee, B. S., Tomita, M. and Kanai, A.

PLoS ONE (PLoS ONE) 5 ( 5 ) e10567 2010

Research paper (scientific journal), Joint Work, Accepted

Background: During transcription, the nontranscribed DNA strand becomes single-stranded DNA (ssDNA), which can form secondary structures. Unpaired bases in the ssDNA are less protected from mutagens and hence experience more mutations than do paired bases. These mutations are called transcription-associated mutations. Transcription-associated mutagenesis is increased under stress and depends on the DNA sequence. Therefore, selection might significantly influence protein-coding sequences in terms of the transcription-associated mutability per transcription event under stress to improve the survival of Escherichia coli. Methodology/Principal Findings: The mutability index (MI) was developed by Wright et al. to estimate the relative transcription-associated mutability of bases per transcription event. Using the most stable fold of each ssDNA that have an average length n, MI was defined as (the number of folds in which the base is unpaired)/n ×(highest -ΔG of all n folds in which the base is unpaired), where ΔG is the free energy. The MI values show a significant correlation with mutation data under stress but not with spontaneous mutations in E. coli. Protein sequence diversity is preferred under stress but not under favorable conditions. Therefore, we evaluated the selection pressure on MI in terms of the protein sequence diversity for all the protein-coding sequences in E. coli. The distributions of the MI values were lower at bases that could be substituted with each of the other three bases without affecting the amino acid sequence than at bases that could not be so substituted. Start codons had lower distributions of MI values than did nonstart codons. Conclusions/Significance: Our results suggest that the majority of protein-coding sequences have evolved to promote protein sequence diversity and to reduce gene knockout under stress. Consequently, transcription-associated mutagenesis increases protein sequence diversity more effectively than does random mutagenesis under stress. Nonrandom transcription-associated mutagenesis under stress should improve the survival of E. coli. © 2010 Kim et al.

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Core promoter structure and genomic context reflect histone 3 lysine 9 acetylation patterns

Kratz, A., Arner, E., Saito, R., Kubosaki, A., Kawai, J., Suzuki, H., Carninci, P., Arakawa, T., Tomita, M., Hayashizaki, Y. and Daub, C.O.

BMC Genomics (BMC Genomics) 11 ( 1 ) 257 2010

Research paper (scientific journal), Joint Work, Accepted

Background: Histone modifications play an important role in gene regulation. Acetylation of histone 3 lysine 9 (H3K9ac) is generally associated with transcription initiation and unfolded chromatin, thereby positively influencing gene expression. Deep sequencing of the 5' ends of gene transcripts using DeepCAGE delivers detailed information about the architecture and expression level of gene promoters. The combination of H3K9ac ChIP-chip and DeepCAGE in a myeloid leukemia cell line (THP-1) allowed us to study the spatial distribution of H3K9ac around promoters using a novel clustering approach. The promoter classes were analyzed for association with relevant genomic sequence features.Results: We performed a clustering of 4,481 promoters according to their surrounding H3K9ac signal and analyzed the clustered promoters for association with different sequence features. The clustering revealed three groups with major H3K9ac signal upstream, centered and downstream of the promoter. Narrow single peak promoters tend to have a concentrated activity of H3K9ac in the upstream region, while broad promoters tend to have a concentrated activity of H3K9ac and RNA polymerase II binding in the centered and downstream regions. A subset of promoters with high gene expression level, compared to subsets with low and medium gene expression, shows dramatic increase in H3K9ac activity in the upstream cluster only; this may indicate that promoters in the centered and downstream clusters are predominantly regulated at post-initiation steps. Furthermore, the upstream cluster is depleted in CpG islands and more likely to regulate un-annotated genes.Conclusions: Clustering core promoters according to their surrounding acetylation signal is a promising approach for the study of histone modifications. When examining promoters clustered into groups according to their surrounding H3K9 acetylation signal, we find that the relative localization and intensity of H3K9ac is very specific depending on characteristic sequence features of the promoter. Experimental data from DeepCAGE and ChIP-chip experiments using undifferentiated (monocyte) and differentiated (macrophage) THP-1 cells leads us to the same conclusions. © 2010 Kratz et al; licensee BioMed Central Ltd.

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Unveiling cellular biochemical reactions via metabolomics-driven approaches

Saito, N., Ohashi, Y., Soga, T. and Tomita, M.

Curr Opin Microbiol. (Current Opinion in Microbiology) 13 ( 3 ) 358-362 - 362 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 13695274

Despite the widespread availability of genome sequences and the systematic assignment of individual open reading frames, many of the activities of metabolic enzymes are still veiled. It is difficult to assign biochemical functions to enzymes and uncover the in vivo regulation of their enzymatic activities, but this is crucial to understand cellular functions. The development of metabolomics and metabolite profiling, which can globally analyze intracellular and extracellular metabolites, has provided some valuable tools for systems biology. This report reviews the recent metabolomics approaches to elucidate unidentified metabolic systems while focusing on their enzymatic activities, including their biochemical and biological functions, and their regulation in response to environmental perturbations in microorganisms. © 2010 Elsevier Ltd. All rights reserved.

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Complete genome sequence and comparative analysis of Shewanella violacea, a psychrophilic and piezophilic bacterium from deep sea floor sediments

Aono, E., Baba, T., Ara, T., Nishi, T., Nakamichi, T., Inamoto, E., Toyonaga, H., Hasegawa, M., Takai, Y., Okumura,Y., Baba, M., Tomita, M., Kato, C., Oshima, T., Nakasone, K. and Mori

Mol. BioSyst. (Molecular BioSystems) 6 ( 7 ) 1216-1226 - 1226 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 1742206X

Remineralization of organic matter in deep-sea sediments is important in oceanic biogeochemical cycles, and bacteria play a major role in this process. Shewanella violacea DSS12 is a psychrophilic and piezophilic γ-proteobacterium that was isolated from the surface layer of deep sea sediment at a depth of 5110 m. Here, we report the complete genome sequence of S. violacea and comparative analysis with the genome of S. oneidensis MR-1, isolated from sediments of a freshwater lake. Unlike S. oneidensis, this deep-sea Shewanella possesses very few terminal reductases for anaerobic respiration and no c-type cytochromes or outer membrane proteins involved in respiratory Fe(iii) reduction, which is characteristic of most Shewanella species. Instead, the S. violacea genome contains more terminal oxidases for aerobic respiration and a much greater number of putative secreted proteases and polysaccharases, in particular, for hydrolysis of collagen, cellulose and chitin, than are encoded in S. oneidensis. Transporters and assimilatory reductases for nitrate and nitrite, and nitric oxide-detoxifying mechanisms (flavohemoglobin and flavorubredoxin) are found in S. violacea. Comparative analysis of the S. violacea genome revealed the respiratory adaptation of this bacterium to aerobiosis, leading to predominantly aerobic oxidation of organic matter in surface sediments, as well as its ability to efficiently use diverse organic matter and to assimilate inorganic nitrogen as a survival strategy in the nutrient-poor deep-sea floor. © The Royal Society of Chemistry 2010.

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Differential metabolomics software for capillary electrophoresis-mass spectrometry data analysis

Sugimoto, Masahiro ; Hirayama, Akiyoshi ; Ishikawa, Takamasa ; Robert, Martin ; Baran, Richard ; Uehara, Keizo ; Kawai, Katsuya ; Soga, Tomoyoshi ; Tomita, Masaru

METABOLOMICS 6 ( 1 ) 27-41 - 41 2010

Research paper (scientific journal), Joint Work, ISSN 1573-3882

In metabolomics, the rapid identification of quantitative differences between multiple biological samples remains a major challenge. While capillary electrophoresis-mass spectrometry (CE-MS) is a powerful tool to simultaneously quantify charged metabolites, reliable and easy-to-use software that is well suited to analyze CE-MS metabolic profiles is still lacking. Optimized software tools for CE-MS are needed because of the sometimes large variation in migration time between runs and the wider variety of peak shapes in CE-MS data compared with LC-MS or GC-MS. Therefore, we implemented a stand-alone application named JDAMP (Java application for Differential Analysis of Metabolite Profiles), which allows users to identify the metabolites that vary between two groups. The main features include fast calculation modules and a file converter using an original compact file format, baseline subtraction, dataset normalization and alignment, visualization on 2D plots (m/z and time axis) with matching metabolite standards, and the detection of significant differences between metabolite profiles. Moreover, it features an easy-to-use graphical user interface that requires only a few mouse-actions to complete the analysis. The interface also enables the analyst to evaluate the semiautomatic processes and interactively tune options and parameters depending on the input datasets. The confirmation of findings is available as a list of overlaid electropherograms, which is ranked using a novel difference-evaluation function that accounts for peak size and distortion as well as statistical criteria for accurate difference-detection. Overall, the JDAMP software complements other metabolomics data processing tools and permits easy and rapid detection of significant differences between multiple complex CE-MS profiles. © Springer Science+Business Media, LLC 2009.

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Another Origin of Coloniality in Volvocaleans: The Phylogenetic Position of Pyrobotrys Arnoldi (Spondylomoraceae, Volvocales)

Nakada, T., Nozaki, H. and Tomita, M.

J. Eukaryot. Microbiol. (Journal of Eukaryotic Microbiology) 57 ( 4 ) 379-382 - 382 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 10665234

Colonial volvocaleans (Chlorophyceae) are used as a standard model of multicellular evolution. However, the phylogenetic position of the colonial volvocalean family Spondylomoraceae has yet to be resolved. To examine this, the molecular phylogenies of Pyrobotrys stellata and Pyrobotrys squarrosa were analyzed using combined 18S rRNA, RUBISCO large subunit, and P700 chl a-apoprotein A2 gene sequences. In the phylogenetic trees, Pyrobotrys belonged to the clade Caudivolvoxa and was not closely related to other colonial volvocalean flagellates. The results indicate that colony formation of Spondylomoraceae independently evolved from unicellular volvocaleans. The phylogenetic position of problematic "Pascherina tetras" SAG 159-1 was also analyzed. © 2010 by the International Society of Protistologists.

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Large-scale comparative phosphoproteomics identifies conserved phosphorylation sites in plants

Nakagami, H., Sugiyama, N., Mochida, K., Daudi, A., Yoshida, Y., Toyoda, T., Tomita, M., Ishihama, Y. and Shirasu, K.

Plant Physiology (Plant Physiology) 153 ( 3 ) 1161-1174 - 1174 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 00320889

Knowledge of phosphorylation events and their regulation is crucial to understand the functional biology of plants. Here, we report a large-scale phosphoproteome analysis in the model monocot rice (Oryza sativa japonica 'Nipponbare'), an economically important crop. Using unfractionated whole-cell lysates of rice cells, we identified 6,919 phosphopeptides from 3,393 proteins. To investigate the conservation of phosphoproteomes between plant species, we developed a novel phosphorylation-site evaluation method and performed a comparative analysis of rice and Arabidopsis (Arabidopsis thaliana). The ratio of tyrosine phosphorylation in the phosphoresidues of rice was equivalent to those in Arabidopsis and human. Furthermore, despite the phylogenetic distance and the use of different cell types, more than 50% of the phosphoproteins identified in rice and Arabidopsis, which possessed ortholog(s), had an orthologous phosphoprotein in the other species. Moreover, nearly half of the phosphorylated orthologous pairs were phosphorylated at equivalent sites. Further comparative analyses against the Medicago phosphoproteome also showed similar results. These data provide direct evidence for conserved regulatory mechanisms based on phosphorylation in plants. We also assessed the phosphorylation sites on nucleotide-binding leucine-rich repeat proteins and identified novel conserved phosphorylation sites that may regulate this class of proteins. © 2010 American Society of Plant Biologists.

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Prediction of metabolite identity from accurate mass, migration time prediction and isotopic pattern information in CE-TOFMS data

Sugimoto, M., Hirayama, A., Robert, M., Abe, S., Soga, T. and Tomita, M.

Electrophoresis (Electrophoresis) 31 ( 14 ) 2311–2318 - 2318 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 01730835

CE-TOFMS is a powerful method for profiling charged metabolites. However, the limited availability of metabolite standards hinders the process of identifying compounds from detected features in CE-TOFMS data sets. To overcome this problem, we developed a method to identify unknown peaks based on the predicted migration time (tm) and accurate m/z values. We developed a predictive model using 375 standard cationic metabolites and support vector regression. The model yielded good correlations between the predicted and measured tm (R=0.952 and 0.905 using complete and cross-validation data sets, respectively). Using the trained model, we subsequently predicted the tm for 2938 metabolites available from the public databases and assigned tentative identities to noise-filtered features in human urine samples. While 38.9% of the peaks were assigned metabolite names by matching with the standard library alone, the proportion increased to 52.2%. The proposed methodology increases the value of metabolomic data sets obtained from CE-TOFMS profiling. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.

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Protein complex prediction via verifying and reconstructing the topology of domain-domain interactions

Ozawa, Y., Saito, R., Fujimori, S., Kashima, H., Ishizaka, Hiroshi Yanagawa, H., Miyamoto-Sato, E. and Tomita, M.

BMC Bioinformatics (BMC Bioinformatics) 11 350 2010

Research paper (scientific journal), Joint Work, Accepted

Background: High-throughput methods for detecting protein-protein interactions enable us to obtain large interaction networks, and also allow us to computationally identify the associations of proteins as protein complexes. Although there are methods to extract protein complexes as sets of proteins from interaction networks, the extracted complexes may include false positives because they do not account for the structural limitations of the proteins and thus do not check that the proteins in the extracted complex can simultaneously bind to each other. In addition, there have been few searches for deeper insights into the protein complexes, such as of the topology of the protein-protein interactions or into the domain-domain interactions that mediate the protein interactions.Results: Here, we introduce a combinatorial approach for prediction of protein complexes focusing not only on determining member proteins in complexes but also on the DDI/PPI organization of the complexes. Our method analyzes complex candidates predicted by the existing methods. It searches for optimal combinations of domain-domain interactions in the candidates based on an assumption that the proteins in a candidate can form a true protein complex if each of the domains is used by a single protein interaction. This optimization problem was mathematically formulated and solved using binary integer linear programming. By using publicly available sets of yeast protein-protein interactions and domain-domain interactions, we succeeded in extracting protein complex candidates with an accuracy that is twice the average accuracy of the existing methods, MCL, MCODE, or clustering coefficient. Although the configuring parameters for each algorithm resulted in slightly improved precisions, our method always showed better precision for most values of the parameters.Conclusions: Our combinatorial approach can provide better accuracy for prediction of protein complexes and also enables to identify both direct PPIs and DDIs that mediate them in complexes. © 2010 Ozawa et al; licensee BioMed Central Ltd.

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Chemical dephosphorylation for identification of multiply phosphorylated peptides and phosphorylation site determination

Kyono, Y., Sugiyama, N., Tomita, M. and Ishihama, Y.

Rapid Commun. Mass Spectrom. (Rapid Communications in Mass Spectrometry) 24 ( 15 ) 2277–2282 - 2282 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 09514198

We have developed a novel strategy to improve the efficiency of identification of multiply phosphorylated peptides isolated by hydroxy acid modified metal oxide chromatography (HAMMOC). This strategy consists of alkali-induced chemical dephosphorylation (beta-elimination reaction) of phosphopeptides isolated by HAMMOC prior to analysis by liquid chromatography/mass spectrometry (LC/MS). This approach identified 1.9-fold more multiply phosphorylated peptides than the conventional approach without beta-elimination from a digested mixture of three standard phosphoproteins. In addition, the accuracy of phosphorylation site determination in synthetic phosphopeptides was significantly improved. Finally, we applied this approach to a cell lysate. By combining this dephosphorylation approach with the conventional approach, we successfully identified 1649 unique phosphopeptides, including 325 multiply phosphorylated phosphopeptides, from 200mg of cultured Arabidopsis cells. These results indicate that chemical dephosphorylation prior to LC/MS analysis increases the efficiency of identification of multiply phosphorylated peptides, as well as the accuracy of phosphorylation site determination. © 2010 John Wiley & Sons, Ltd.

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Metabolomic Profiles and Sensory Attributes of Edamame under Various Storage Duration and Temperature Conditions.

Sugimoto, M., Goto, H., Otomo, K., Ito, M., Onuma, H., Suzuki, A., Sugawara, M., Abe, S., Tomita, M. and Soga, T.

J Agric Food Chem. (Journal of Agricultural and Food Chemistry) 58 ( 14 ) 8418–8425 - 8425 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 00218561

Its high nutritional content and sensory characteristics make edamame a popular vegetable bean. However, because of its short shelf-life, it is important to optimize the storage conditions to maintain its quality during distribution to consumers. We focused on storage conditions to investigate the temporal changes in the metabolic profiles and sensory characteristics of edamame during transportation from the site of harvest to the site of purchase/consumption. We conducted metabolomic analysis and sensory evaluation tests of edamame stored for different lengths and at different temperatures. Charged metabolites were profiled by capillary electrophoresis-mass spectrometry, and free sugars were quantified by liquid chromatography-tandem mass spectrometry. In comparison to the gradual decrease in its sensory characteristics over time, the changes in metabolite profiles manifested four different patterns. In particular, changes in amino acid levels were related to sensory attributes. The downstream metabolites of shikimate as well as phospholipids and γ-aminobutyric acid increased with increasing storage temperatures. © 2010 American Chemical Society.

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G-language genome analysis environment with REST and SOAP web service interfaces

Arakawa, K., Kido, N., Oshita, K. and Tomita, M.

Nucleic Acids Res. (Nucleic Acids Research) 38 ( SUPPL. 2 ) W700–W705 - 5 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 03051048

G-language genome analysis environment (G-language GAE) contains more than 100 programs that focus on the analysis of bacterial genomes, including programs for the identification of binding sites by means of information theory, analysis of nucleotide composition bias and the distribution of particular oligonucleotides, calculation of codon bias and prediction of expression levels, and visualization of genomic information. We have provided a collection of web services for these programs by utilizing REST and SOAP technologies. The REST interface, available at http://rest.glanguage. org/, provides access to all 145 functions of the G-language GAE. These functions can be accessed from other online resources. All analysis functions are represented by unique universal resource identifiers. Users can access the functions directly via the corresponding universe resource locators (URLs), and biological web sites can readily embed the functions by simply linking to these URLs. The SOAP services, available at http://www.g-language.org/wiki/soap/, provide language-independent programmatic access to 77 analysis programs. The SOAP service Web Services Definition Language file can be readily loaded into graphical clients such as the Taverna workbench to integrate the programs with other services and workflows. © The Author(s) 2010. Published by Oxford University Press.

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Collective Dynamics of Specific Gene Ensembles Crucial for Neutrophil Differentiation: The Existence of Genome Vehicles Revealed

Tsuchiya, M., Piras, V., Giuliani, A., Tomita, M. and Selvarajoo

PLoS One (PLoS ONE) 5 ( 8 ) e12116 2010

Research paper (scientific journal), Joint Work, Accepted

Cell fate decision remarkably generates specific cell differentiation path among the multiple possibilities that can arise through the complex interplay of high-dimensional genome activities. The coordinated action of thousands of genes to switch cell fate decision has indicated the existence of stable attractors guiding the process. However, origins of the intracellular mechanisms that create "cellular attractor" still remain unknown. Here, we examined the collective behavior of genome-wide expressions for neutrophil differentiation through two different stimuli, dimethyl sulfoxide (DMSO) and alltrans- retinoic acid (atRA). To overcome the difficulties of dealing with single gene expression noises, we grouped genes into ensembles and analyzed their expression dynamics in correlation space defined by Pearson correlation and mutual information. The standard deviation of correlation distributions of gene ensembles reduces when the ensemble size is increased following the inverse square root law, for both ensembles chosen randomly from whole genome and ranked according to expression variances across time. Choosing the ensemble size of 200 genes, we show the two probability distributions of correlations of randomly selected genes for atRA and DMSO responses overlapped after 48 hours, defining the neutrophil attractor. Next, tracking the ranked ensembles' trajectories, we noticed that only certain, not all, fall into the attractor in a fractal-like manner. The removal of these genome elements from the whole genomes, for both atRA and DMSO responses, destroys the attractor providing evidence for the existence of specific genome elements (named "genome vehicle") responsible for the neutrophil attractor. Notably, within the genome vehicles, genes with low or moderate expression changes, which are often considered noisy and insignificant, are essential components for the creation of the neutrophil attractor. Further investigations along with our findings might provide a comprehensive mechanistic view of cell fate decision. © 2010 Tsuchiya et al.

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Large-scale tRNA intron transposition in the archaeal order Thermoproteales represents a novel mechanism of intron gain

Fujishima, K., Sugahara, J., Tomita, M. and Kanai, A.

Mol. Biol. Evol. (Molecular Biology and Evolution) 27 ( 10 ) 2233–2243 - 2243 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 07374038

Recently, diverse arrangements of transfer RNA (tRNA) genes have been found in the domain Archaea, in which the tRNA is interrupted by a maximum of three introns or is even fragmented into two or three genes. Whereas most of the eukaryotic tRNA introns are inserted strictly at the canonical nucleotide position (37/38), archaeal intron-containing tRNAs have a wide diversity of small tRNA introns, which differ in their numbers and locations. This feature is especially pronounced in the archaeal order Thermoproteales. In this study, we performed a comprehensive sequence comparison of 286 tRNA introns and their genes in seven Thermoproteales species to clarify how these introns have emerged and diversified during tRNA gene evolution. We identified 46 intron groups containing sets of highly similar sequences (>70%) and showed that 16 of them contain sequences from evolutionarily distinct tRNA genes. The phylogeny of these 16 intron groups indicates that transposition events have occurred at least seven times throughout the evolution of Thermoproteales. These findings suggest that frequent intron transposition occurs among the tRNA genes of Thermoproteales. Further computational analysis revealed limited insertion positions and corresponding amino acid types of tRNA genes. This has arisen because the bulge-helix-bulge splicing motif is required at the newly transposed position if the pre-tRNA is to be correctly processed. These results clearly demonstrate a newly identified mechanism that facilitates the late gain of short introns at various noncanonical positions in archaeal tRNAs. © 2010 The Author.

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A Metabolic Model of Human Erythrocytes: Practical Application of the E-Cell Simulation Environment

Yachie, A. K., Nishino, T., Shimo, H., Suematsu, M., and Tomita, M.

J. Biomed. Biotechnol. (Journal of Biomedicine and Biotechnology) 2010 642420 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 11107243

The human red blood cell (RBC) has long been used for modeling of complex biological networks, for elucidation of a wide variety of dynamic phenomena, and for understanding the fundamental topology of metabolic pathways. Here, we introduce our recent work on an RBC metabolic model using the E-Cell Simulation Environment. The model is sufficiently detailed to predict the temporal hypoxic response of each metabolite and, at the same time, successfully integrates modulation of metabolism and of the oxygen transporting capacity of hemoglobin. The model includes the mechanisms of RBC maintenance as a single cell system and the functioning of RBCs as components of a higher order system. Modeling of RBC metabolism is now approaching a fully mature stage of realistic predictions at the molecular level and will be useful for predicting conditions in biotechnological applications such as long-term cold storage of RBCs. Copyright © 2010 Ayako Yachie-Kinoshita et al.

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Unbiased quantitation of Escherichia coli membrane proteome using phase-transfer surfactants

Masuda,T., Saito, N., Tomita, M. and Ishihama, M.

Mol Cell Proteomics. (Molecular and Cellular Proteomics) 8 ( 12 ) 2770-2777 - 2777 2009.12

Research paper (scientific journal), Joint Work, Accepted, ISSN 15359476

We developed a sample preparation protocol for rapid and unbiased analysis of the membrane proteome using an alimentary canal-mimicking system in which proteases are activated in the presence of bile salts. In this rapid and unbiased protocol, immobilized trypsin is used in the presence of deoxycholate and lauroylsarcosine to increase digestion efficiency as well as to increase the solubility of the membrane proteins. Using 22.5 μg of Escherichia coli whole cell lysate, we quantitatively demonstrated that membrane proteins were extracted and digested at the same level as soluble proteins without any solubility-related bias. The recovery of membrane proteins was independent of the number of transmembrane domains per protein. In the analysis of the membrane-enriched fraction from 22.5 μg of E. coli cell lysate, the abundance distribution of the membrane proteins was in agreement with that of the membrane protein-coding genes when this protocol, coupled with strong cation exchange prefractionation prior to nano-LC-MS/MS analysis, was used. Because this protocol allows unbiased sample preparation, protein abundance estimation based on the number of observed peptides per protein was applied to both soluble and membrane proteins simultaneously, and the copy numbers per cell for 1,453 E. coli proteins, including 545 membrane proteins, were successfully obtained. Finally, this protocol was applied to quantitative analysis of guanosine tetra- and pentaphosphate-dependent signaling in E. coli wild-type and relA knock-out strains. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Identification of DNA-dependent protein kinase as a cofactor for the forkhead transcription factor FoxA2

Nock A., Ascano J.M., Jones T., Barrero M.J., Sugiyama N., Tomita M., Ishihama Y., Malik S.

Journal of Biological Chemistry (Journal of Biological Chemistry) 284 ( 30 ) 19915 - 19926 2009.07

ISSN 00219258

Forkhead factors are important regulators of animal development and homeostasis. They are among the earliest to bind quiescent genes, which they activate in conjunction with other transcription factors. Many liver-specific genes are under the control of FoxA2, a liver-enriched forkhead protein. Here we confirmed by chromatin immunoprecipitation that FoxA2 is one of the factors bound to the promoter-proximal enhancer of the gene encoding apolipoprotein AI (a component of high density lipoprotein) and that it functions in synergy with the nuclear receptor hepatocyte nuclear. Furthermore, toward identifying additional cofactors that could potentially regulate FoxA2 activity, we identified DNA-dependent protein kinase (DNA-PK) as a FoxA2-associated factor upon affinity purification of epitope-tagged FoxA2.Weshow that FoxA2, found to be a phosphoprotein in vivo, is also an efficient substrate for DNA-PK, which targets serine 283. This residue is contained within a conserved serine-glutamine phosphorylation signal for DNA-PK, located within the C-terminal third of the polypeptide, just distal to its winged-helix DNA binding domain. We establish that this residue is critical for FoxA2 function because FoxA2 bearing a mutation at this site is severely compromised in its ability to activate a reporter gene under the control of its cognate DNA-binding site (apoAI site B). Complementary experiments rule out that this mutation compromises the ability of FoxA2 to either translocate to the nucleus or to bind site B. We therefore conclude that DNA-PK-dependent phosphorylation of FoxA2 plays a critical role in its transcriptional activation function per se. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Degradation of ppGpp by nudix pyrophosphatase modulates the transition of growth phase in the bacterium Thermus thermophilus

Ooga T., Ohashi Y., Kuramitsu S., Koyama Y., Tomita M., Soga T., Masui R.

Journal of Biological Chemistry (Journal of Biological Chemistry) 284 ( 23 ) 15549 - 15556 2009.06

ISSN 00219258

A major bacterial alarmone, guanosine 3′,5′-bispyrophosphate (ppGpp), controls cellular growth under conditions of nutritional starvation. For most bacteria, intracellular ppGpp levels are tightly controlled by the synthesis/degradation cycle of RelA and SpoT activities. This study shows a novel ppGpp regulatory protein governing the cellular growth of Thermus thermophilus, Ndx8, a member of the Nudix pyrophosphatase family that degrades ppGpp to yield guanosine 3′,5′-bisphosphate. The ndx8-null mutant strain exhibited early stage growth arrest accompanied by the stationary phase-specific morphologies and global transcriptional modulation under nutritionally defined conditions. Several possible substrate compounds of Ndx8, which specifically accumulated in the ndx8 mutant cells, were identified by employing a capillary electrophoresis time-of-flight mass spectrometry-based metabolomics approach. Among them, the hydrolytic activity of Ndx8 for ppGpp was significant not only in vitro but also in vivo. Finally, the elimination of ppGpp synthetic activity suppressed the observed phenotype of the ndx8 mutation, suggesting that the function of Ndx8 as a growth regulator is involved in ppGpp accumulation, which is thought to act as a trigger of the growth phase transition. These results suggest a novel mechanism of ppGpp-mediated growth control by the functional relay between Ndx8 and SpoT activity as ppGpp scavengers. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Benzodiazepine and antidepressant use in elderly patients with anxiety disorders: a survey of 796 outpatients in Japan.

Uchida H, Suzuki T, Mamo DC, Mulsant BH, Kikuchi T, Takeuchi H, Tomita M, Watanabe K, Yagi G, Kashima H

Journal of anxiety disorders 23 ( 4 ) 477 - 81 2009.05

ISSN 0887-6185

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Characterization of a heat-stable enzyme possessing GTP-dependent RNA ligase activity from a hyperthermophilic archaeon, Pyrococcus furiosus

Kanai A., Sato A., Fukuda Y., Okada K., Matsuda T., Sakamoto T., Muto Y., Yokoyama S., Kawai G., Tomita M.

RNA 15 ( 3 ) 420 - 431 2009.03

ISSN 13558382

Using an expression protein library of a hyperthermophilic archaeon, Pyrococcus furiosus, we identified a gene (PF0027) that encodes a protein with heat-stable cyclic nucleotide phosphodiesterase (CPDase) activity. The PF0027 gene encoded a 21-kDa protein and an amino acid sequence that showed ∼27% identity to that of the 2′-5′ tRNA ligase protein, ligT (20 kDa), from Escherichia coli. We found that the purified PF0027 protein possessed GTP-dependent RNA ligase activity and that synthetic tRNA halves bearing 2′,3′-cyclic phosphate and 5′-OH termini were substrates for the ligation reaction in vitro. GTP hydrolysis was not required for the reaction, and GTPγS enhanced the tRNA ligation activity of PF0027 protein, suggesting that the ligation step is regulated by a novel mechanism. In comparison to the strong CPDase activity of the PF0027 protein, the RNA ligase activity itself was quite weak, and the ligation product was unstable during in vitro reaction. Finally, we used NMR to determine the solution structure of the PF0027 protein and discuss the implications of our results in understanding the role of the PF0027 protein. Copyright © 2009 RNA Society.

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Six classes of nuclear localization signals specific to different binding grooves of importin α

Kosugi S., Hasebe M., Matsumura N., Takashima H., Miyamoto-Sato E., Tomita M., Yanagawa H.

Journal of Biological Chemistry 284 ( 1 ) 478 - 485 2009.01

ISSN 00219258

The importin α/β pathway mediates nuclear import of proteins containing the classical nuclear localization signals (NLSs). Although the consensus sequences of the classical NLSs have been defined, there are still many NLSs that do not match the consensus rule and many nonfunctional sequences that match the consensus. We report here six different NLS classes that specifically bind to distinct binding pockets of importin α. By screening of random peptide libraries using an mRNA display, we selected peptides bound by importin α and identified six classes of NLSs, including three novel classes. Two noncanonical classes (class 3 and class 4) specifically bound the minor binding pocket of importin α, whereas the classical monopartite NLSs (class 1 and class 2) bound to the major binding pocket. Using a newly developed universal green fluorescent protein expression system, we found that these NLS classes, including plant-specific class 5 NLSs and bipartite NLSs, fundamentally require the regions outside the core basic residues for their activity and have specific residues or patterns that confer the activities differently between yeast, plants, and mammals. Furthermore, amino acid replacement analyses revealed that the consensus basic patterns of the classical NLSs are not essential for activity, thereby generating more unconventional patterns, including redox-sensitive NLSs. These results explain the causes of the NLS diversity. The defined consensus patterns and properties of importin α-dependent NLSs provide useful information for identifying NLSs. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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emPAI Calc -- for the estimation of protein abundance from largescale identification data by liquid chromatography-tandem massspectrometry

Shinoda, K., Tomita, M. and Ishihama,Y.

Bioinformatics (Bioinformatics) 26 ( 4 ) 576-577 - 577 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 13674803

Summary: emPAI Calc is an open-source web application for the estimation of protein abundance. It uses the correlation between the number of identified peptides and protein abundance in mass spectrometry-based proteomic experiments. The program is the first implementation of our previously reported emPAI algorithm; it calculates the emPAI from the protein identification results obtained by database search engines such as Mascot.™ Availability: http://empai.iab.keio.ac.jp/; http://empai.iab.keio.ac.jp/supplement.php Source codes are available under Mozilla Public License. Contact: y-ishi@ttck.keio.ac.jp Supplementary Information: Supplementary data are available at Bioinformatics online. © The Author 2009. Published by Oxford University Press.

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Cooperativity of two active sites in bacterial homodimeric aconitases

Tsuchiya D, Shimizu N, Tomita M

Biochem Biophys Res Commun 379 ( 2 ) 485-488 - 488 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 0006291X

Aconitase catalyzes a reversible isomerization of citrate into isocitrate in the Krebs cycle. Escherichia coli possesses two kinds of aconitases, aconitase A (AcnA) and B (AcnB), whose structural organizations are different. We analyzed the structural state of AcnA by the chemical crosslinking and small-angle X-ray scattering. The protein adopts a homodimer in solution, as AcnB does. The catalytic assay of the two aconitases revealed that the isomerization of isocitrate displayed a negative cooperativity of the two active sites within each homodimer. On the other hand, insignificant cooperativity was observed in the reverse reaction. Therefore, the homodimerization of AcnAB yields a substrate-dependent cooperative effect. In conjunction with the dissociable homodimer of AcnB, the catalytic property could affect the intracellular metabolic process involving the Krebs cycle. © 2008 Elsevier Inc. All rights reserved.

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Predicting novel features of toll-like receptor 3 signaling in macrophages

Helmy, M., Gohda, J, Inoue, J., Tomita, M. Tsuchiya, M. and Selvarajoo, K.

PLoS ONE. 4 ( 11 ) e4661 2009

Research paper (scientific journal), Joint Work, Accepted

The Toll-like receptor (TLR) 3 plays a critical role in mammalian innate immune response against viral attacks by recognizing double-stranded RNA (dsRNA) or its synthetic analog polyinosinic-polycytidylic acid (poly (I:C)). This leads to the activation of MAP kinases and NF-κB which results in the induction of type I interferons and proinflammatory cytokines to combat the viral infection. To understand the complex interplay of the various intracellular signaling molecules in the regulation of NF-κB and MAP kinases, we developed a computational TLR3 model based upon perturbation-response approach. We curated literature and databases to determine the TLR3 signaling topology specifically for murine macrophages. For initial model creation, we used wildtype temporal activation profiles of MAP kinases and NF-κB and, for model testing, used TRAF6 KO and TRADD KO data. From dynamic simulations we predict i) the existence of missing intermediary steps between extracellular poly (I:C) stimulation and intracellular TLR3 binding, and ii) the presence of a novel pathway which is essential for JNK and p38, but not NF-κB, activation. Our work shows activation dynamics of signaling molecules can be used in conjunction with perturbation-response models to decipher novel signaling features of complicated immune pathways. © 2009 Helmy et al.

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Tri-split tRNA is a transfer RNA made from 3 transcripts that provides insight into the evolution of fragmented tRNAs in archaea

Fujishima, K., Sugahara, J., Kikuta, K. Hirano, R.,Sato, A., Tomita, M. and Kanai

Proc Natl Acad Sci U S A. 106 ( 8 ) 2683-7 - 2687 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 00278424

Transfer RNA (tRNA) is essential for decoding the genome sequence into proteins. In Archaea, previous studies have revealed unique multiple intron-containing tRNAs and tRNAs that are encoded on 2 separate genes, so-called split tRNAs. Here, we discovered 10 fragmented tRNA genes in the complete genome of the hyperthermoacidophilic Archaeon Caldivirga maquilingensis that are individually transcribed and further trans-spliced to generate all of the missing tRNAs encoding glycine, alanine, and glutamate. Notably, the 3 mature tRNAGly's with synonymous codons are created from 1 constitutive 3′ half transcript and 4 alternatively switching transcripts, representing tRNA made from a total of 3 transcripts named a "tri-split tRNA." Expression and nucleotide sequences of 10 split tRNA genes and their joined tRNA products were experimentally verified. The intervening sequences of split tRNA have high identity to tRNA intron sequences located at the same positions in ifntron-containing tRNAs in related Thermoproteales species. This suggests that an evolutionary relationship between intron-containing and split tRNAs exists. Our findings demonstrate the first example of split tRNA genes in a free-living organism and a unique tri-split tRNA gene that provides further insight into the evolution of fragmented tRNAs. © 2009 by The National Academy of Sciences of the USA.

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Genome Projector: zoomable genome map with multiple views

Arakawa, K., Tamaki, S., Kono, N., Kido, N., Ikegami, K., Ogawa, R.and Tomita, M

BMC Bioinformatics 10 31 2009

Research paper (scientific journal), Joint Work, Accepted

BACKGROUND: Molecular biology data exist on diverse scales, from the level of molecules to -omics. At the same time, the data at each scale can be categorised into multiple layers, such as the genome, transcriptome, proteome, metabolome, and biochemical pathways. Due to the highly multi-layer and multi-dimensional nature of biological information, software interfaces for database browsing should provide an intuitive interface that allows for rapid migration across different views and scales. The Zoomable User Interface (ZUI) and tabbed browsing have proven successful for this purpose in other areas, especially to navigate the vast information in the World Wide Web. RESULTS: This paper presents Genome Projector, a Web-based gateway for genomics information with a zoomable user interface using Google Maps API, equipped with four seamlessly accessible and searchable views: a circular genome map, a traditional genome map, a biochemical pathways map, and a DNA walk map. The Web application for 320 bacterial genomes is available at http://www.g-language.org/GenomeProjector/. All data and software including the source code, documentations, and development API are freely available under the GNU General Public License. Zoomable maps can be easily created from any image file using the development API, and an online data mapping service for Genome Projector is also available at our Web site. CONCLUSION: Genome Projector is an intuitive Web application for browsing genomics information, implemented with a zoomable user interface and tabbed browsing utilising Google Maps API and Asynchronous JavaScript and XML (AJAX) technology.

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Local and Global responses in complex gene regulation networks

Tsuchiya, M., Selvarajoo, K., Piras, V., Tomita, M. and Giuliani, A.

Physica A 388 ( 8 ) 1738-1746 - 1746 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 03784371

An exacerbated sensitivity to apparently minor stimuli and a general resilience of the entire system stay together side-by-side in biological systems. This apparent paradox can be explained by the consideration of biological systems as very strongly interconnected network systems. Some nodes of these networks, thanks to their peculiar location in the network architecture, are responsible for the sensitivity aspects, while the large degree of interconnection is at the basis of the resilience properties of the system. One relevant feature of the high degree of connectivity of gene regulation networks is the emergence of collective ordered phenomena influencing the entire genome and not only a specific portion of transcripts. The great majority of existing gene regulation models give the impression of purely local 'hard-wired' mechanisms disregarding the emergence of global ordered behavior encompassing thousands of genes while the general, genome wide, aspects are less known. Here we address, on a data analysis perspective, the discrimination between local and global scale regulations, this goal was achieved by means of the examination of two biological systems: innate immune response in macrophages and oscillating growth dynamics in yeast. Our aim was to reconcile the 'hard-wired' local view of gene regulation with a global continuous and scalable one borrowed from statistical physics. This reconciliation is based on the network paradigm in which the local 'hard-wired' activities correspond to the activation of specific crucial nodes in the regulation network, while the scalable continuous responses can be equated to the collective oscillations of the network after a perturbation. © 2008 Elsevier B.V. All rights reserved.

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Can complex cellular processes be governed by simple linear rules?

Selvarajoo, K., Tomita, M. and Tsuchiya, M.

J Bioinform Comput Biol. (Journal of Bioinformatics and Computational Biology) 7 ( 1 ) 243-268 - 268 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 02197200

Complex living systems have shown remarkably well-orchestrated, self-organized, robust, and stable behavior under a wide range of perturbations. However, despite the recent generation of high-throughput experimental datasets, basic cellular processes such as division, differentiation, and apoptosis still remain elusive. One of the key reasons is the lack of understanding of the governing principles of complex living systems. Here, we have reviewed the success of perturbation-response approaches, where without the requirement of detailed in vivo physiological parameters, the analysis of temporal concentration or activation response unravels biological network features such as causal relationships of reactant species, regulatory motifs, etc. Our review shows that simple linear rules govern the response behavior of biological networks in an ensemble of cells. It is daunting to know why such simplicity could hold in a complex heterogeneous environment. Provided physical reasons can be explained for these phenomena, major advancement in the understanding of basic cellular processes could be achieved. © 2009 Imperial College Press.

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In silico analysis of phosphoproteome data suggests a rich-get-richer process of phosphosite accumulation over evolution

Yachie, N., Saito, R., Sugahara, J., Tomita, M. and Ishihama, Y

Mol Cell Proteomics. (Molecular and Cellular Proteomics) 8 ( 5 ) 1061-71 - 1071 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 15359476

Recent phosphoproteome analyses using mass spectrometry-based technologies have provided new insights into the extensive presence of protein phosphorylation in various species and have raised the interesting question of how this protein modification was gained evolutionarily on such a large scale. We investigated this issue by using human and mouse phosphoproteome data. We initially found that phosphoproteins followed a power-law distribution with regard to their number of phosphosites: most of the proteins included only a few phosphosites, but some included dozens of phosphosites. The power-law distribution, unlike more commonly observed distributions such as normal and log-normal distributions, is considered by the field of complex systems science to be produced by a specific rich-get-richer process called preferential attachment growth. Therefore, we explored the factors that may have promoted the rich-get-richer process during phosphosite evolution. We conducted a bioinformatics analysis to evaluate the relationship of amino acid sequences of phosphoproteins with the positions of phosphosites and found an overconcentration of phosphosites in specific regions of protein surfaces and implications that in many phosphoproteins these clusters of phosphosites are activated simultaneously. Multiple phosphosites concentrated in limited spaces on phosphoprotein surfaces may therefore function biologically as cooperative modules that are resistant to selective pressures during phosphoprotein evolution. We therefore proposed a hypothetical model by which the modularization of multiple phosphosites has been resistant to natural selection and has driven the rich-get-richer process of the evolutionary growth of phosphosite numbers. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Chemical Cleavage-Assisted Tryptic Digestion for Membrane Proteome Analysis

Iwasaki, M., Masuda, T., Tomita, M. and Ishihama, Y.

J. Proteome Re (Journal of Proteome Research) 8 ( 6 ) 1061-71 - 3175 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 15353893

We have developed a simple and unbiased method for membrane proteome analysis using cyanocysteine-mediated cleavage in combination with trypsin digestion. In our previous study, application of the trypsin-based phase-transfer surfactants (PTS) protocol for membrane proteome analysis provided a substantial improvement in the identification of the membrane proteome, but the task remains challenging, because trypsin often generates peptides larger than the observable m/z range. Here, we predict computationally that the combination of Cys cleavage with tryptic digestion would be more effective than trypsin digestion alone for membrane proteome analysis. To validate this prediction, we applied a combined Cys cleavage-trypsin approach to 14 μg of Escherichia coli membrane-enriched pellet. By using two-dimensional LC-MS/MS, we identified a total of 1530 proteins, of which 667 were membrane proteins. This represents a 10% increase over the number identified with the PTS protocol using optimized trypsin-based digestion in our previous study [Masuda, T., et al. J. Proteome Res. 2008, 7 (2), 731-40]. The coverage of the E. coli membrane proteome was approximately 40%, ranging from 37% to 42% in various subcategories. Further, the distribution of the number of transmembrane domains per protein was unbiased compared with that in the GenoBase database. These results indicate that the combination Cys chemical cleavage-assisted trypsin digestion protocol will be a powerful tool for membrane proteome analysis. © 2009 American Chemical Society.

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Metabolite profiling reveals YihU as a novel hydroxybutyrate dehydrogenase for alternative succinic semialdehyde metabolism in Escherichia coli

Saito, N., Robert, M., Kochi, H., Matsuo, G., Kakazu, Y., Soga, T. and Tomita, M.

J. Biol. Chem. (Journal of Biological Chemistry) 284 ( 24 ) 16442-51 - 16451 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 00219258

The search for novel enzymes and enzymatic activities is important to map out all metabolic activities and reveal cellular metabolic processes in a more exhaustive manner. Here we present biochemical and physiological evidence for the function of the uncharacterized protein YihU in Escherichia coli using metabolite profiling by capillary electrophoresis time-of-flight mass spectrometry. To detect enzymatic activity and simultaneously identify possible substrates and products of the putative enzyme, we profiled a complex mixture of metabolites in the presence or absence of YihU. In this manner, succinic semialdehyde was identified as a substrate for YihU. The purified YihU Protein catalyzed in vitro the NADH-dependent reduction of succinic semialdehyde to γ-hydroxybutyrate. Moreover, a yihU deletion mutant displayed reduced tolerance to the cytotoxic effects of exogenous addition of succinic semialdehyde. Profiling of intracellular metabolites following treatment of E. coli with succinic semialdehyde supports the existence of a YihU-catalyzed reduction of succinic semialdehyde to γ-hydroxybutyrate in addition to its known oxidation to succinate and through the tricarboxylic acid cycle. These findings suggest that YihU is a novel γ-hydroxybutyrate dehydrogenase involved in the metabolism of succinic semialdehyde, and other potentially toxic intermediates that may accumulate under stress conditions in E. coli. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Emergent genome-wide control in wildtype and genetically mutated lipopolysaccarides-stimulated macrophages

Tsuchiya, M., Piras, V., Choi, S., Akira, S., Tomita, M., Giuliani, A. and Selvarajoo, K.

PLoS One 4 ( 3 ) e4905 2009

Research paper (scientific journal), Joint Work, Accepted

Large-scale gene expression studies have mainly focused on highly expressed and 'discriminatory' genes to decipher key regulatory processes. Biological responses are consequence of the concerted action of gene regulatory network, thus, limiting our attention to genes having the most significant variations is insufficient for a thorough understanding of emergent whole genome response. Here we comprehensively analyzed the temporal oligonucleotide microarray data of lipopolysaccharide (LPS) stimulated macrophages in 4 genotypes; wildtype, Myeloid Differentiation factor 88 (MyD88) knockout (KO), TIR-domain-containing adapter-inducing interferon-b (TRIF) KO and MyD88/ TRIF double KO (DKO). Pearson correlations computed on the whole genome expression between different genotypes are extremely high (>0.98), indicating a strong co-regulation of the entire expression network. Further correlation analyses reveal genome-wide response is biphasic, i) acute-stochastic mode consisting of small number of sharply induced immune-related genes and ii) collective mode consisting of majority of weakly induced genes of diverse cellular processes which collectively adjust their expression level. Notably, temporal correlations of a small number of randomly selected genes from collective mode show scalability. Furthermore, in collective mode, the transition from large scatter in expression distributions for single ORFs to smooth linear lines emerges as an organizing principle when grouping of 50 ORFs and above. With this emergent behavior, the role of MyD88, TRIF and novel MyD88, TRIF-independent processes for gene induction can be linearly superposed to decipher quantitative whole genome differential control of transcriptional and mRNA decay machineries. Our work demonstrates genome-wide co-regulated responses subsequent to specific innate immune stimulus which have been largely neglected. © 2009 Tsuchiya et al.

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Ser/Thr/Tyr phosphoproteome analysis of pathogenic and non-pathogenic Pseudomonas species

Ravichandran, A., Sugiyama, N., Tomita, M., Swarup, S.　and Ishihama, Y.

Proteomics. (Proteomics) 9 ( 10 ) 2764-2775 - 2775 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 16159853

Protein phosphorylation on serine, threonine, and tyrosine is well established as a crucial regulatory posttranslational modification in eukaryotes. With the recent whole-genome sequencing projects reporting the presence of serine/threonine kinases and two-component proteins both in prokaryotes and eukaryotes, the importance of protein phosphorylation in archaea and bacteria is gaining acceptance. While conventional biochemical methods failed to obtain a snapshot of the bacterial phosphoproteomes, advances in MS methods have paved the way for in-depth mapping of phosphorylation sites. Here, we present phosphoproteomes of two ecologically diverse non-enteric Gram-negative bacteria captured by a nanoLC-MS-based approach combined with a novel phosphoenrichment method. While the phosphoproteome data from the two species are not very similar, the results reflect high similarity to the previously published dataset in terms of the pathways the phosphoproteins belong to. This study additionally provides evidence to prior observations that protein phosphorylation is common in bacteria. Notably, phosphoproteins identified in Pseudomonas aeruginosa belong to motility, transport, and pathogenicity pathways that are critical for survival and virulence. We report, for the first time, that motility regulator A, probably acting via the novel secondary messenger cyclic diguanylate monophosphate, significantly affects protein phosphorylation in Pseudomonas putida. ©2009 Wiley-VCH Verlag GmbH & Co. KGaA.

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Network features and pathway analyses of a signal transduction cascade

Yanashima, R, Kitagawa N., Matsubara, Y., Weatheritt, R., Oka, K., Kikuchi, S., Tomita, M,and Ishizaki, S.

Front Neuroinform. 3 ( MAY ) 13 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 16625196

The scale-free and small-world network models reflect the functional units of networks. However, when we investigated the network properties of a signaling pathway using these models, no significant differences were found between the original undirected graphs and the graphs in which inactive proteins were eliminated from the gene expression data. We analyzed signaling networks by focusing on those pathways that best reflected cellular function. Therefore, our analysis of pathways started from the ligands and progressed to transcription factors and cytoskeletal proteins. We employed the Python module to assess the target network. This involved comparing the original and restricted signaling cascades as a directed graph using microarray gene expression profi les of late onset Alzheimer's disease. The most commonly used method of shortest-path analysis neglects to consider the influences of alternative pathways that can affect the activation of transcription factors or cytoskeletal proteins. We therefore introduced included k-shortest paths and k-cycles in our network analysis using the Python modules, which allowed us to attain a reasonable computational time and identify k-shortest paths. This technique reflected results found in vivo and identified pathways not found when shortest path or degree analysis was applied. Our module enabled us to comprehensively analyse the characteristics of biomolecular networks and also enabled analysis of the effects of diseases considering the feedback loop and feedforward loop control structures as an alternative path. © 2009 Yanashima, Kitagawa, Matsubara, Weatheritt, Oka, Kikuchi, Tomita and Ishizaki.

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Quantitative Metabolome Profiling of Colon and Stomach Cancer Microenvironment by Capillary Electrophoresis Time-of-Flight Mass Spectrometry

Hirayama, A., Kami, K., Sugimoto, M., Sugawara, M., Toki, N., Onozuka, H., Kinoshita, T., Saito, N., Ochiai, A., Tomita, M., Esumi, H. and Soga, T.

Cancer Res (Cancer Research) 69 ( 11 ) 4918-25 - 4925 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 00085472

Most cancer cells predominantly produce energy by glycolysis rather than oxidative phosphorylation via the tricarboxylic acid (TCA) cycle, even in the presence of an adequate oxygen supply (Warburg effect). However, little has been reported regarding the direct measurements of global metabolites in clinical tumor tissues. Here, we applied capillary electrophoresis time-of-flight mass spectrometry, which enables comprehensive and quantitative analysis of charged metabolites, to simultaneously measure their levels in tumor and grossly normal tissues obtained from 16 colon and 12 stomach cancer patients. Quantification of 94 metabolites in colon and 95 metabolites in stomach involved in glycolysis, the pentose phosphate pathway, the TCA and urea cycles, and amino acid and nucleotide metabolisms resulted in the identification of several cancer-specific metabolic traits. Extremely low glucose and high lactate and glycolytic intermediate concentrations were found in both colon and stomach tumor tissues, which indicated enhanced glycolysis and thus confirmed the Warburg effect. Significant accumulation of all amino acids except glutamine in the tumors implied autophagic degradation of proteins and active glutamine breakdown for energy production, i.e., glutaminolysis. In addition, significant organ-specific differences were found in the levels of TCA cycle intermediates, which reflected the dependency of each tissue on aerobic respiration according to oxygen availability. The results uncovered unexpectedly poor nutritional conditions in the actual tumor microenvironment and showed that capillary electrophoresis coupled to mass spectrometry-based metabolomics, which is capable of quantifying the levels of energy metabolites in tissues, could be a powerful tool for the development of novel anticancer agents that target cancer-specific metabolism. ©2009 American Association for Cancer Research.

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Measure of synonymous codon usage diversity among genes in bacteria

Suzuki, H., Saito, R. and Tomita, M

BMC Bioinformatics (BMC Bioinformatics) 10 167 2009

Research paper (scientific journal), Joint Work, Accepted

Background: In many bacteria, intragenomic diversity in synonymous codon usage among genes has been reported. However, no quantitative attempt has been made to compare the diversity levels among different genomes. Here, we introduce a mean dissimilarity-based index (Dmean) for quantifying the level of diversity in synonymous codon usage among all genes within a genome. Results: The application of Dmean to 268 bacterial genomes shows that in bacteria with extremely biased genomic G+C compositions there is little diversity in synonymous codon usage among genes. Furthermore, our findings contradict previous reports. For example, a low level of diversity in codon usage among genes has been reported for Helicobacter pylori, but based on Dmean, the diversity level of this species is higher than those of more than half of bacteria tested here. The discrepancies between our findings and previous reports are probably due to differences in the methods used for measuring codon usage diversity. Conclusion: We recommend that Dmean be used to measure the diversity level of codon usage among genes. This measure can be applied to other compositional features such as amino acid usage and dinucleotide relative abundance as a genomic signature. © 2009 Suzuki et al; licensee BioMed Central Ltd.

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Metabolomic Profiling of Anionic Metabolites by Capillary Electrophoresis Mass Spectrometry

Soga, T., Igarashi, K., Itoh, C., Mizobuchi, K., Zimmermann, H. P. and Tomita, M.

Analytical Chemistry (Analytical Chemistry) 81 ( 15 ) 6165-6174 - 6174 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 00032700

We describe a sheath flow capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) method in the negative mode using a platinum electrospray ionization (ESI) spray needle, which allows the comprehensive analysis of anionic metabolites. The material of the spray needle had significant effect on the measurement of anions. A stainless steel spray needle was oxidized and corroded at the anodic electrode due to electrolysis. The precipitation of iron oxides (rust) plugged the capillary outlet, resulting in shortened capillary lifetime. Many anionic metabolites also formed complexes with the iron oxides or migrating nickel ion, which was also generated by electrolysis and moved toward the cathode (the capillary inlet). The metal-anion complex formation significantly reduced detection sensitivity of the anionic compounds. The use of a platinum ESI needle prevented both oxidation of the metals and needle corrosion. Sensitivity using the platinum needle increased from several- to 63-fold, with the largest improvements for anions exhibiting high metal chelating properties such as carboxylic acids, nucleotides, and coenzyme A compounds. The detection limits for most anions were between 0.03 and 0.87 μmol/L (0.8 and 24 fmol) at a signal-to-noise ratio of 3. This method is quantitative, sensitive, and robust, and its utility was demonstrated by the analysis of the metabolites in the central metabolic pathways extracted from mouse liver. © 2009 American Chemical Society.

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Systematic identification of cell cycle-dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs

Kosugi, S., Hasebe, M., Tomita, M. and Yanagawa, H.

Proc Natl Acad Sci U S A. (Proceedings of the National Academy of Sciences of the United States of America) 106 ( 31 ) 13142. - 10176 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 00278424

The cell cycle-dependent nucleocytoplasmic transport of proteins is predominantly regulated by CDK kinase activities; however, it is currently difficult to predict the proteins thus regulated, largely because of the low prediction efficiency of the motifs involved. Here, we report the successful prediction of CDK1-regulated nucleocytoplasmic shuttling proteins using a prediction system for nuclear localization signals (NLSs). By systematic amino acid replacement analyses in budding yeast, we created activity-based profiles for different classes of importin-α-dependent NLSs that represent the functional contributions of different amino acids at each position within an NLS class. We then developed a computer program for prediction of the classical importin-α/β pathway-specific NLSs (cNLS Mapper, available at http//nls-mapper.iab.keio.ac.jp/) that calculates NLS activities by using these profiles and an additivity-based motif scoring algorithm. This calculation method achieved significantly higher prediction accuracy in terms of both sensitivity and specificity than did current methods. The search for NLSs that overlap the consensus CDK1 phosphorylation site by using cNLS Mapper identified all previously reported and 5 previously uncharacterized yeast proteins (Yen1, Psy4, Pds1, Msa1, and Dna2) displaying CDK1- and cell cycle-regulated nuclear transport. CDK1 activated or repressed their nuclear import activity, depending on the position of CDK1-phosphorylation sites within NLSs. The application of this strategy to other functional linear motifs should be useful in systematic studies of protein-protein networks.

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Systematic phenome analysis of Escherichia coli multiple-knockout mutants reveals hidden reactions in central carbon metabolism

Nakahigashi, K., Toya, Y., Ishii, N., Soga, T., Hasegawa, M., Watanabe, H., Takai, Y., Honma, M., Mori, H. and Tomita, M.

Molecular Systems Biology 5 306 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 17444292

Central carbon metabolism is a basic and exhaustively analyzed pathway. However, the intrinsic robustness of the pathway might still conceal uncharacterized reactions. To test this hypothesis, we constructed systematic multiple-knockout mutants involved in central carbon catabolism in Escherichia coli and tested their growth under 12 different nutrient conditions. Differences between in silico predictions and experimental growth indicated that unreported reactions existed within this extensively analyzed metabolic network. These putative reactions were then confirmed by metabolome analysis and in vitro enzymatic assays. Novel reactions regarding the breakdown of sedoheptulose-7-phosphate to erythrose-4-phosphate and dihydroxyacetone phosphate were observed in transaldolase-deficient mutants, without any noticeable changes in gene expression. These reactions, triggered by an accumulation of sedoheptulose-7-phosphate, were catalyzed by the universally conserved glycolytic enzymes ATP-dependent phosphofructokinase and aldolase. The emergence of an alternative pathway not requiring any changes in gene expression, but rather relying on the accumulation of an intermediate metabolite may be a novel mechanism mediating the robustness of these metabolic networks. © 2009 EMBO and Macmillan Publishers Limited.

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A web server for interactive and zoomable Chaos Game Representation images

Arakawa, K., Oshita, K. and Tomita, M.

Source Code for Biology and Medicine (Source Code for Biology and Medicine) 4 6 2009

Research paper (scientific journal), Joint Work, Accepted

Chaos Game Representation (CGR) is a generalized scale-independent Markov transition table, which is useful for the visualization and comparative study of genomic signature, or for the study of characteristic sequence motifs. However, in order to fully utilize the scale-independent properties of CGR, it should be accessible through scale-independent user interface instead of static images. Here we describe a web server and Perl library for generating zoomable CGR images utilizing Google Maps API, which is also easily searchable for specific motifs. The web server is freely accessible at http://www.g-language.org/wiki/cgr/, and the Perl library as well as the source code is distributed with the G-language Genome Analysis Environment under GNU General Public License. © 2009 Arakawa et al; licensee BioMed Central Ltd.

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Modeling reaction-diffusion of molecules on surface and in volume spaces with the E-Cell System

Arjunan, S. N. V., and Tomita, M.

International Journal of Computer Science and Information Security 3 ( 1 ) 211-216 2009

Research paper (scientific journal), Joint Work, Accepted

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In silico modeling and metabolome analysis of long-stored erythrocytes to improve blood storage methods

Nishino, T., Yachie, A. K., Hirayama. A., Soga, T., Suematsu, T., and Tomita, M.

Journal of Biotechnology (Journal of Biotechnology) 144 ( 3 ) 212-223 - 223 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 01681656

There is currently no effective method for preventing ATP and 2,3-bisphosphoglycerate (2,3-BPG) depletion during long-term erythrocyte storage in the cold, although these metabolites are strongly associated with cell viability and oxygen delivery after transfusion. Metabolite reduction is caused by whole metabolic networks in the cell, which are regulated by various physical or chemical factors. Mathematical modeling is a powerful tool for integrating such complex and dynamic systems. Here, we developed a mathematical model to predict metabolism in erythrocytes preserved with a mannitol-adenine-phosphate solution (MAP) at 4 °C, by modifying a published model of large-scale erythrocyte metabolism. Our model successfully reproduced the reported decreases in ATP and 2,3-BPG during storage. Analysis of our model identified several enzymatic reactions and factors related to ATP and 2,3-BPG depletions, which may serve as possible targets for improving blood storage methods. We also performed metabolome analysis of laboratory-made MAP-stored erythrocytes using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), which provided a comprehensive view of the metabolism dynamics. Alterations in the metabolic intermediate concentrations after long storage were qualitatively predicted by the model. Finally, through further systematic analysis, we also discuss the usability of our model. © 2009 Elsevier B.V. All rights reserved.

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Pathway Projector: Web-based Zoomable Pathway Browser using KEGG Atlas and Google Maps API

Kono, N., Arakawa, K., Ogawa, R., Kido, N., Oshita, K., Ikegami, K., Tamaki, S. and Tomita, M.

PLoS ONE (PLoS ONE) 4 ( 11 ) e7710 2009

Research paper (scientific journal), Joint Work, Accepted

Background: Biochemical pathways provide an essential context for understanding comprehensive experimental data and the systematic workings of a cell. Therefore, the availability of online pathway browsers will facilitate post-genomic research, just as genome browsers have contributed to genomics. Many pathway maps have been provided online as part of public pathway databases. Most of these maps, however, function as the gateway interface to a specific database, and the comprehensiveness of their represented entities, data mapping capabilities, and user interfaces are not always sufficient for generic usage. Methodology/Principal Findings: We have identified five central requirements for a pathway browser: (1) availability of large integrated maps showing genes, enzymes, and metabolites; (2) comprehensive search features and data access; (3) data mapping for transcriptomic, proteomic, and metabolomic experiments, as well as the ability to edit and annotate pathway maps; (4) easy exchange of pathway data; and (5) intuitive user experience without the requirement for installation and regular maintenance. According to these requirements, we have evaluated existing pathway databases and tools and implemented a web-based pathway browser named Pathway Projector as a solution. Conclusions/Significance: Pathway Projector provides integrated pathway maps that are based upon the KEGG Atlas, with the addition of nodes for genes and enzymes, and is implemented as a scalable, zoomable map utilizing the Google Maps API. Users can search pathway-related data using keywords, molecular weights, nucleotide sequences, and amino acid sequences, or as possible routes between compounds. In addition, experimental data from transcriptomic, proteomic, and metabolomic analyses can be readily mapped. Pathway Projector is freely available for academic users at http://www.g-language.org/PathwayProjector/. © 2009 Kono et al.

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Disrupted tRNA Gene Diversity and Possible Evolutionary Scenarios

Sugahara, J., Fujishima, K., Morita, K., Tomita, M. and Kanai , A.

Journal of Molecular Evolution (Journal of Molecular Evolution) 69 ( 5 ) 497-504 - 504 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 00222844

The following unusual tRNAs have recently been discovered in the genomes of Archaea and primitive Eukaryota: multiple-intron-containing tRNAs, which have more than one intron; split tRNAs, which are produced from two pieces of RNA transcribed from separate genes; tri-split tRNAs, which are produced from three separate genes; and permuted tRNA, in which the 5′ and 3′ halves are encoded with permuted orientations within a single gene. All these disrupted tRNA genes can form mature contiguous tRNA, which is aminoacylated after processing by cis or trans splicing. The discovery of such tRNA disruptions has raised the question of when and why these complex tRNA processing pathways emerged during the evolution of life. Many previous reports have noted that tRNA genes contain a single intron in the anticodon loop region, a feature common throughout all three domains of life, suggesting an ancient trait of the last universal common ancestor. In this context, these unique tRNA disruptions recently found only in Archaea and primitive Eukaryota provide new insight into the origin and evolution of tRNA genes, encouraging further research in this field. In this paper, we summarize the phylogeny, structure, and processing machinery of all known types of disrupted tRNAs and discuss possible evolutionary scenarios for these tRNA genes. © 2009 Springer Science+Business Media, LLC.

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Update on the Keio collection of Escherichia coli single-gene deletion mutants

Yamamoto, N., Nakahigashi, K., Nakamichi, T., Yoshino, M., Takai, Y., Touda, Y., Furubayashi, A., Kinjyo, S., Dose, H., Hasegawa, M., Datsenko, K. A., Toru Nakayashiki, T., Tomita, M., Barry L Wanner, L. B. and Hirotada Mori, H.

Molecular Systems Biology 5 335 2009

Research paper (scientific journal), Joint Work, Accepted

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Recharacterization of Chlamydomonas reinhardtii and its relatives with new isolates from Japan

Nakada, T., Shinkawa, H., Ito, T., and Tomita, M.

J Plant Res (Journal of Plant Research) 123 ( 1 ) 67–78 - 78 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 09189440

Chlamydomonas reinhardtii P. A. Dang. (Volvocales, Chlorophyceae) is one of the most intensely studied algae, and its whole genome was sequenced. Although this species was originally described based on materials from France and is often referred to as a cosmopolitan species, all culture strains available today have been isolated from eastern North America. The distinctions with similar and/or closely related species, such as Chlamydomonas globosa J. Snow and Chlamydomonas orbicularis E. G. Pringsh., are also contentious. In this study, new strains of C. reinhardtii and C. globosa were isolated from Japan and compared with several strains similar to C. reinhardtii. Based on the morphological, genealogical, phylogenetical, and mating studies including the new Japanese strains, the circumscription of C. reinhardtii was clarified. C. reinhardtii was most closely related to C. globosa, and they were shown to be different species. Although C. reinhardtii was similar to C. orbicularis, the authentic strain of C. orbicularis was morphologically distinguishable and phylogenetically distant from C. reinhardtii. Discovery of the Japanese strains of C. reinhardtii supports the cosmopolitan distribution of this species. Based on Japanese strains and/or strains from other countries, emended descriptions of C. reinhardtii, C. globosa, and C. orbicularis are given. © The Botanical Society of Japan and Springer 2009.

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Quantitative analysis of replication-related mutation and selection pressures in bacterial chromosomes and plasmids using generalised GC skew index

Arakawa, K., Suzuki, H. and Tomita, M.

BMC Genomics (BMC Genomics) 10 640 2009

Research paper (scientific journal), Joint Work, Accepted

Background: Due to their bi-directional replication machinery starting from a single finite origin, bacterial genomes show characteristic nucleotide compositional bias between the two replichores, which can be visualised through GC skew or (C-G)/(C+G). Although this polarisation is used for computational prediction of replication origins in many bacterial genomes, the degree of GC skew visibility varies widely among different species, necessitating a quantitative measurement of GC skew strength in order to provide confidence measures for GC skew-based predictions of replication origins.Results: Here we discuss a quantitative index for the measurement of GC skew strength, named the generalised GC skew index (gGCSI), which is applicable to genomes of any length, including bacterial chromosomes and plasmids. We demonstrate that gGCSI is independent of the window size and can thus be used to compare genomes with different sizes, such as bacterial chromosomes and plasmids. It can suggest the existence of different replication mechanisms in archaea and of rolling-circle replication in plasmids. Correlation of gGCSI values between plasmids and their corresponding host chromosomes suggests that within the same strain, these replicons have reproduced using the same replication machinery and thus exhibit similar strengths of replication strand skew.Conclusions: gGCSI can be applied to genomes of any length and thus allows comparative study of replication-related mutation and selection pressures in genomes of different lengths such as bacterial chromosomes and plasmids. Using gGCSI, we showed that replication-related mutation or selection pressure is similar for replicons with similar machinery. © 2009 Arakawa et al; licensee BioMed Central Ltd.

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Correlation between Sensory Evaluation Scores of Japanese Sakeand Metabolome Profiles

Sugimoto, M., Koseki ,T., Hirayama, A., Abe, S., Sano, T., Tomita, M., and Soga, T.

J. Agric. FoodChem 2010 (Journal of Agricultural and Food Chemistry) 58 ( 1 ) 374–383 - 383 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 00218561

The aim of this study was to explore the association between taste and metabolite profiles of Japanese refined sake. Nontarget metabolome analysis was conducted using capillary electrophoresis mass spectrometry. Zatsumi, an unpleasant not clear flavor, and sweetness, bitterness, and sourness were graded by four experienced panelists. Regression models based on support vector regression (SVR) were used to estimate the relationships among sensory evaluation scores and quantified metabolites and visualized as a nonlinear relationship between sensory scores and metabolite components. The SVR model was highly accurate and versatile: the correlation coefficients for whole training data, cross-validation, and separated validation data were 0.86, 0.73, and 0.73, respectively, for zatsumi. Other sensory scores were also analyzed and modeled by SVR. The methodology demonstrated here carries great potential for predicting the relevant parameters and quantitative relationships between charged metabolites and sensory evaluation in Japanese refined sake. © 2009 American Chemical Society.

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Capillary electrophoresis mass spectrometry-based saliva metabolomics identified oral, breast and pancreatic cancer-specific profiles

Sugimoto, M., Wong, D. T., Hirayama, A., Soga, T. and Tomita

Metabolomics (Metabolomics) 6 ( 1 ) 78-95 - 95 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 15733882

Saliva is a readily accessible and informative biofluid, making it ideal for the early detection of a wide range of diseases including cardiovascular, renal, and autoimmune diseases, viral and bacterial infections and, importantly, cancers. Saliva-based diagnostics, particularly those based on metabolomics technology, are emerging and offer a promising clinical strategy, characterizing the association between salivary analytes and a particular disease. Here, we conducted a comprehensive metabolite analysis of saliva samples obtained from 215 individuals (69 oral, 18 pancreatic and 30 breast cancer patients, 11 periodontal disease patients and 87 healthy controls) using capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS). We identified 57 principal metabolites that can be used to accurately predict the probability of being affected by each individual disease. Although small but significant correlations were found between the known patient characteristics and the quantified metabolites, the profiles manifested relatively higher concentrations of most of the metabolites detected in all three cancers in comparison with those in people with periodontal disease and control subjects. This suggests that cancer-specific signatures are embedded in saliva metabolites. Multiple logistic regression models yielded high area under the receiver-operating characteristic curves (AUCs) to discriminate healthy controls from each disease. The AUCs were 0.865 for oral cancer, 0.973 for breast cancer, 0.993 for pancreatic cancer, and 0.969 for periodontal diseases. The accuracy of the models was also high, with cross-validation AUCs of 0.810, 0.881, 0.994, and 0.954, respectively. Quantitative information for these 57 metabolites and their combinations enable us to predict disease susceptibility. These metabolites are promising biomarkers for medical screening. © The Author(s) 2009.

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Quantitative Proteome and Phosphoproteome Analyses of Cultured Cells Based on SILAC Labeling without Requirement of Serum Dialysis

Imami, K., Sugiyama, N., Tomita, M. and Ishihama, Y.

Mol. BioSyst. (Molecular BioSystems) 6 ( 3 ) 594-602 - 602 2009

Research paper (scientific journal), Joint Work, ISSN 1742206X

The use of dialyzed serum is essential in the application of the conventional stable isotope labeling by amino acids in cell culture (SILAC) approach to achieve complete labeling of proteins for quantitative proteomics. Here, we first evaluated the impact of dialyzed serum on the proteome and phosphoproteome of hormone-sensitive breast cancer MCF-7 cells and found that dialyzed serum influenced the expression of proteins related to signaling systems via hormone receptors, inducing a marked change of the phosphoproteome compared with the use of non-dialyzed serum. We also evaluated 9 other cell lines, including HeLa, HEK293 and Panc1, and found that the influence of serum dialysis on the expression profiles of the proteome and phosphoproteome varied, depending on the cell type. To avoid these problems, we established a SILAC-based quantification approach without the requirement of serum dialysis. Our simple approach is based on dual labeling of two populations of cells with two kinds of heavy amino acids of different mass, using non-dialyzed serum. Using our SILAC approach with non-dialyzed serum, we successfully quantified the phosphoproteome of MCF-7 cells induced by lapatinib, an EGFR1/Her2 dual kinase inhibitor. Because of the dual labeling approach, our method is widely applicable to cultured cells in which protein labeling is incomplete for any reason, e.g., owing to the use of non-dialyzed serum or a low growth rate. © 2010 The Royal Society of Chemistry.

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Comprehensive analysis of archaeal tRNA genes reveals rapid increase of tRNA introns in the order thermoproteales

Sugahara J., Kikuta K., Fujishima K., Yachie N., Tomita M., Kanai A.

Molecular Biology and Evolution 25 ( 12 ) 2709 - 2716 2008.12

ISSN 07374038

The analysis of archaeal tRNA genes is becoming more important to evaluate the origin and evolution of tRNA molecule. Even with the recent accumulation of complete genomes of numerous archaeal species, several tRNA genes are still required for a full complement of the codon table. We conducted comprehensive screening of tRNA genes from 47 archaeal genomes by using a combination of different types of tRNA prediction programs and extracted a total of 2,143 reliable tRNA gene candidates including 437 intron-containing tRNA genes, which covered more than 99.9% of the codon tables in Archaea. Previously, the content of intron-containing tRNA genes in Archaea was estimated to be approximately 15% of the whole tRNA genes, and most of the introns were known to be located at canonical positions (nucleotide position between 37 and 38) of precursor tRNA (pre-tRNA). Surprisingly, we observed marked enrichment of tRNA introns in five species of the archaeal order Thermoproteales; about 70% of tRNA gene candidates were found to be intron-containing tRNA genes, half of which contained multiple introns, and the introns were located at various noncanonical positions. Sequence similarity analysis revealed that approximately half of the tRNA introns found at Thermoproteales-specific intron locations were highly conserved among several tRNA genes. Intriguingly, identical tRNA intron sequences were found within different types of tRNA genes that completely lacked exon sequence similarity, suggesting that the tRNA introns in Thermoproteales could have been gained via intron insertion events at a later stage of tRNA evolution. Moreover, although the CCA sequence at the 3′ terminal of pre-tRNA is added by a CCA-adding enzyme after gene transcription in Archaea, most of the tRNA genes containing highly conserved introns already encode the CCA sequence at their 3′ terminal. Based on these results, we propose possible models explaining the rapid increase of tRNA introns as a result of intron insertion events via retrotransposition of pre-tRNAs. The sequences and secondary structures of the tRNA genes and their bulge-helix-bulge motifs were registered in SPLITSdb (http://splits.iab.keio.ac.jp/splitsdb/), a novel and comprehensive database for archaeal tRNA genes. © The Author 2008. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved.

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Cancer metabolomics: Toward the development of novel anticancer therapeutics targeting the energy metabolism in tumor microenvironment

Kami K., Tomitsuka E., Kita K., Tomita M., Soga T., Esumi H.

Japanese Journal of Clinical Chemistry 37 ( 4 ) 361 - 367 2008.12

ISSN 03705633

Role of Na+-Ca2+ exchange in neonatal and adult ventricular cells

Sano H., Naito Y., Tomita M.

WMSCI 2008 - The 12th World Multi-Conference on Systemics, Cybernetics and Informatics, Jointly with the 14th International Conference on Information Systems Analysis and Synthesis, ISAS 2008 - Proc. 2 142 - 144 2008.12

The present paper expanded an integrated mathematical model, which successfully simulated developmental changes in electrophysiological activities of rodent ventricular cells, to contrive models that reflect one of the hypothesis on the role of Na+-Ca"+ exchange in neonatal ventricular cell: the Na+-Ca+ exchange assumes a relatively greater role in neonatal cells with a morphologically sparse SR. Our simulation with the expanded model showed that neonatal ventricular cell consumed larger amount of adenosine triphosphate (ATP) than adult ventricular cell did, owing to long relaxation time. The amount of ATP consumed per beat was reduced to the amount equivalent to adult ventricular cell, as increasing the relative current density of Na+-Ca2+ exchange (F NCx) by 4-fold, which is consistent with the observed current density in neonatal rabbit ventricular cell. To further validate the hypothesis, the relative functional activity of SR (FSR) was incrementally increased from neonatal to adult levels and subsequently increased FNCX, and found that increasing FNcx had negative impact on sarcomere relaxation velocity in ventricular cell model with large FSR. All the results thus theoretically supported the hypothesis that the Na+-Ca2+ exchange assumes a relatively greater role in neonatal cells with a morphologically sparse SR.

Signaling flux redistribution at toll-like receptor pathway junctions

Selvarajoo K., Takada Y., Gohda J., Helmy M., Akira S., Tomita M., Tsuchiya M., Inoue J.I., Matsuo K.

PLoS ONE 3 ( 10 ) e3430 2008.10

Various receptors on cell surface recognize specific extracellular molecules and trigger signal transduction altering gene expression in the nucleus, Gain or loss-of-function mutations of one molecule have shown to affect alternative signaling pathways with a poorly understood mechanism. In Toll-like receptor (TLR) 4 signaling, which branches into MyD88- and TRAM-dependent pathways upon lipopolysaccharide (LPS) stimulation, we investigated the gain or loss-of-function mutations of MyD88. We predict, using a computational model built on the perturbation-response approach and the law of mass conservation, that removal and addition of MyD88 in TLR4 activation, enhances and impairs, respectively, the alternative TRAM-dependent pathway through signaling flux redistribution (SFR) at pathway branches. To verify SFR, we treated MyD88-deficient macrophages with LPS and observed enhancement of TRAM-dependent pathway based on increased IRF3 phosphorylation and induction of Cxcl10 and lfit2. Furthermore, increasing the amount of MyD88 in cultured cells showed decreased TRAM binding to TLR4. Investigating another TLR4 pathway junction, from TRIF to TRAF6, RIP1 and TBK1, the removal of MyD88-dependent TRAF6 increased expression of TRAM-dependent Cxl10 and lfit2. Thus, we demonstrate that SFR is a novel mechanism for enhanced activation of alternative pathways when molecules at pathway junctions are removed. Our data suggest that SFR may enlighten hitherto unexplainable intracellular signaling alterations in genetic diseases where gain or loss-of-function mutations are observed. © 2008 Selvarajoo et al.

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Design of Peptide Inhibitors for the Importin α/β Nuclear Import Pathway by Activity-Based Profiling

Kosugi S., Hasebe M., Entani T., Takayama S., Tomita M., Yanagawa H.

Chemistry and Biology 15 ( 9 ) 940 - 949 2008.09

ISSN 10745521

Despite the current availability of selective inhibitors for the classical nuclear export pathway, no inhibitor for the classical nuclear import pathway has been developed. Here we describe the development of specific inhibitors for the importin α/β pathway using a novel method of peptide inhibitor design. An activity-based profile was created via systematic mutational analysis of a peptide template of a nuclear localization signal. An additivity-based design using the activity-based profile generated two peptides with affinities for importin α that were approximately 5 million times higher than that of the starting template sequence. The high affinity of these peptides resulted in specific inhibition of the importin α/β pathway. These peptide inhibitors provide a useful tool for studying nuclear import events. Moreover, our inhibitor design method should enable the development of potent inhibitors from a peptide seed. © 2008 Elsevier Ltd. All rights reserved.

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Stabilizing synthetic data in the DNA of living organisms

Yachie N, Ohashi Y, Tomita M

Syst Synth Biol. 2 ( 1-2 ) 19-25 - 25 2008.06

Research paper (scientific journal), Joint Work, ISSN 18725325

Data-encoding synthetic DNA, inserted into the genome of a living organism, is thought to be more robust than the current media. Because the living genome is duplicated and copied into new generations, one of the merits of using DNA material is long-term data storage within heritable media. A disadvantage of this approach is that encoded data can be unexpectedly broken by mutation, deletion, and insertion of DNA, which occurs naturally during evolution and prolongation, or laboratory experiments. For this reason, several information theory-based approaches have been developed as an error check of broken DNA data in order to achieve data durability. These approaches cannot efficiently recover badly damaged data- encoding DNA. We recently developed a DNA data-storage approach based on the multiple sequence alignment method to achieve a high level of data durability. In this paper, we overview this technology and discuss strategies for optimal application of this approach.

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GANDivAWeb: A web server for detecting early folding units ("foldons") from protein 3D structures

Laborde T., Tomita M., Krishnan A.

BMC Structural Biology 8 2008.05

Background. It has long been known that small regions of proteins tend to fold independently and are then stabilized by interactions between these distinct subunits or modules. Such units, also known as autonomous folding units (AFUs) or"foldons" play a key role in protein folding. A knowledge of such early folding units has diverse applications in protein engineering as well as in developing an understanding of the protein folding process. Such AFUs can also be used as model systems in order to study the structural organization of proteins. Results. In an earlier work, we had utilized a global network partitioning algorithm to identify modules in proteins. We had shown that these modules correlate well with AFUs. In this work, we have developed a webserver, GANDivAWeb, to identify early folding units or "foldons" in networks using the algorithm described earlier. The website has three functionalities: (a) It is able to display information on the modularity of a database of 1420 proteins used in the original work, (b) It can take as input an uploaded PDB file, identify the modules using the GANDivA algorithm and email the results back to the user and (c) It can take as input an uploaded PDB file and a results file (obtained from functionality (b)) and display the results using the embedded viewer. The results include the module decomposition of the protein, plots of cartoon representations of the protein colored by module identity and connectivity as well as contour plots of the hydrophobicity and relative accessible surface area (RASA) distributions. Conclusion. We believe that the GANDivAWeb server, will be a useful tool for scientists interested in the phenomena of protein folding as well as in protein engineering. Our tool not only provides a knowledge of the AFUs through a natural graph partitioning approach but is also able to identify residues that are critical during folding. It is our intention to use this tool to study the topological determinants of protein folding by analyzing the topological changes in proteins over the unfolding/folding pathways. © 2008 Laborde et al; licensee BioMed Central Ltd.

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Large-scale phosphorylation mapping reveals the extent of tyrosine phosphorylation in Arabidopsis

Sugiyama,N., Nakagami,H., Mochida,K., Daudi,A., Tomita,M., Shirasu,K., and Ishihama,Y.

Mol Syst Biol. 4 193 2008.05

Research paper (scientific journal), Joint Work, ISSN 17444292

Protein phosphorylation regulates a wide range of cellular processes. Here, we report the proteome-wide mapping of in vivo phosphorylation sites in Arabidopsis by using complementary phosphopeptide enrichment techniques coupled with high-accuracy mass spectrometry. Using unfractionated whole cell lysates of Arabidopsis, we identified 2597 phosphopeptides with 2172 high-confidence, unique phosphorylation sites from 1346 proteins. The distribution of phosphoserine, phosphothreonine, and phosphotyrosine sites was 85.0, 10.7, and 4.3%. Although typical tyrosine-specific protein kinases are absent in Arabidopsis, the proportion of phosphotyrosines among the phospho-residues in Arabidopsis is similar to that in humans, where over 90 tyrosine-specific protein kinases have been identified. In addition, the tyrosine phosphoproteome shows features distinct from those of the serine and threonine phosphoproteomes. Taken together, we highlight the extent and contribution of tyrosine phosphorylation in plants. © 2008 EMBO and Nature Publishing Group.

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Developing Bottom-fermenting Yeast Strains That Produce High SO2 Levels by Integrated Metabolome and Transcriptome Analysis

Yoshida S., Imoto J., Minato T., Oouchi R., Sugihara M., Imai T., Ishiguro T., Mizutani S., Tomita M., Soga T., Yoshimoto H.

Applied and Environmental Microbiology 74 ( 9 ) 2787 - 2796 2008.05

ISSN 00992240

Sulfite plays an important role in beer flavor stability. Although breeding of bottom-fermenting Saccharomyces strains that produce high levels of SO 2 is desirable, it is complicated by the fact that undesirable H 2S is produced as an intermediate in the same pathway. Here, we report the development of a high-level SO2-producing bottom-fermenting yeast strain by integrated metabolome and transcriptome analysis. This analysis revealed that O-acetylhomoserine (OAH) is the rate-limiting factor for the production of SO2 and H2S. Appropriate genetic modifications were then introduced into a prototype strain to increase metabolic fluxes from aspartate to OAH and from sulfate to SO 2, resulting in high SO2 and low H2S production. Spontaneous mutants of an industrial strain that were resistant to both methionine and threonine analogs were then analyzed for similar metabolic fluxes. One promising mutant produced much higher levels of SO2 than the parent but produced parental levels of H2S. Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis

Masuda T., Tomita M., Ishihama Y.

Journal of Proteome Research 7 ( 2 ) 731 - 740 2008.02

ISSN 15353893

We have developed a new protocol for digesting hydrophobic proteins using trypsin with the aid of phase-transfer surfactants (PTS), such as sodium deoxycholate (SDC). SDC increases the solubility of hydrophobic proteins, enhances the activity of trypsin, and improves the accessibility to trypsin of proteins denatured during the extraction process. After digestion, SDC was successfully removed from the acidified solution containing tryptic peptides by adding a water-immiscible organic solvent, into which SDC was predominantly transferred, while the digested peptides remained in the aqueous phase. Compared with a protocol using an acid-labile surfactant, this PTS protocol increased the number of identified proteins and the recovery of hydrophobic peptides in the analysis of 400 ng of a membrane-enriched fraction of Escherichia coli. Application of the PTS protocol to 9.0 μg of a membrane-enriched pellet from human cervical cancer HeLa cells resulted in identification of a total of 1450 proteins, of which 764 (53%) were membrane proteins, by two-dimensional strong cation exchange (SCX)-C18 LC-MSMS with 5 SCX fractions. The distribution of the number of transmembrane domains in proteins identified in this study was in agreement with that in the IPI human database, suggesting that the PTS protocol can provide unbiased digestion of the membrane proteome. © 2008 American Chemical Society.

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Perspective in the evolution of human microRNAs: Copy number expansion and acquisition of target gene specialization

Watanabe Y., Tomita M., Kanai A.

Progress of Theoretical Physics Supplement ( 173 ) 219 - 228 2008

ISSN 03759687

A novel class of small, noncoding RNAs called microRNAs (miRNAs) was recently identified, and up to 20%-30% of human genes are said to be regulated by the members of this class of RNA. miRNAs regulate their target genes by imperfect binding to the 3′ untranslated region (UTR) of the gene transcript, and computational predictions of the binding between miRNAs and target gene transcripts have been performed in order to determine which genes are regulated by these RNAs. Phylogenetic analysis has also been used as a powerful tool to predict miRNA target genes. Much emphasis has therefore been placed on studying the phylogenetic conservation and evolution of this novel type of gene regulator, although there still is much that is not known. Here, we propose a hypothesis of how human miRNAs optimized their gene regulation by adjusting their transcript levels, and how they evolved through specific selection of their target genes. We analyzed the correlation between the conservation of miRNAs among species and three features: the number of transcripts, the formation of duplicates, and the number of target genes. The number of miRNA transcripts and the formation of duplicates increased as the conservation rate increased. In contrast, the number of target genes decreased as the conservation rate increased. Therefore, we propose that miRNAs gradually gain an ability to regulate specific target genes when such regulation has a positive effect on the organism. As its pool of target genes is refined, the ability of an miRNA to regulate the genes may be stabilized by an increase in the miRNA transcript number and the formation of duplicates.

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Nuclear export signal consensus sequences defined using a localization-based yeast selection system

Kosugi, S., Hasebe, M., Tomita, M. and Yanagawa, H.

Traffic 9 ( 12 ) 2053 - 2062 2008

Joint Work, ISSN 13989219

Proteins bearing nuclear export signals (NESs) are translocated to the cytoplasm from the nucleus mainly through the CRM1-dependent pathway. However, the NES consensus sequence remains poorly defined, and there are currently no high-throughput methods for identifying NESs. In this study, we report the development of an efficient yeast selection system for detecting nuclear export activity as well as several reliable NES consensus sequences identified using this method. Our selection system is based on the nuclear export-dependent rescue of Tys1p, an essential cytoplasmic protein that has been artificially localized to the nucleus in a haploid Δtys1 knockout strain. A screen of a random peptide library revealed 101 distinct CRM1-dependent NESs, which were classified into six patterns according to the conserved hydrophobic spacing. By combining mutational analyses, we have defined new NES consensus sequences with more specific and redundant residues than the traditional consensus sequence, which are consistent with most experimentally confirmed NESs. These NES consensus sequences should help identify functional NESs, and our selection system can be used to identify other targeting signals or proteins imported to specific subcellular compartments. © 2008 The Authors Journal compilation © 2008 Blackwell Munksgaard.

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Computational Genome Analysis Using The G-language System

Arakawa,K., Suzuki,H. and Tomita,M.

Genges,Genomes and Genomics 2 1-13 2008

Research paper (scientific journal), Joint Work

Visualizing Conplex Omics Information-Scientific Visualization for Genomics and Systems Biology

Arakawa, K., Yachie, N. and Tomita M.

BIOforum Europe 6 27-29 2008

Research paper (scientific journal), Joint Work

Junction ribonuclease activity specified in RNases HII/2

Ohtani, N., Tomita, M. and Itaya, M.

FEBS J. 275 ( 21 ) 5444-55 - 5455 2008

Research paper (scientific journal), Joint Work, ISSN 1742464X

Junction ribonuclease (JRNase) recognizes the transition from RNA to DNA of an RNA-DNA/DNA hybrid, such as an Okazaki fragment, and cleaves it, leaving a mono-ribonucleotide at the 5′ terminus of the RNA-DNA junction. Although this JRNase activity was originally reported in calf RNase H2, some other RNases H have recently been suggested to possess it. This paper shows that these enzymes can also cleave an RNA-DNA/RNA heteroduplex in a manner similar to the RNA-DNA/DNA substrate. The cleavage site of the RNA-DNA/RNA substrate corresponds to the RNA//RNA duplex region, indicating that the cleavage activity cannot be categorized as RNase H activity, which specifically cleaves an RNA strand of an RNA/DNA hybrid. Examination of several RNases H with respect to JRNase activity suggested that the activity is only found in RNase HII orthologs. Therefore, RNases HIII, which are RNase HII paralogs, are distinguished from RNases HII by the absence of JRNase activity. Whether a substrate can be targeted by JRNase activity would depend only on whether or not an RNA-DNA junction consisting of one ribonucleotide and one deoxyribonucleotide is included in the duplex. In addition, although the activity has been reported not to occur on completely single-stranded RNA-DNA, it can recognize a single-stranded RNA-DNA junction if a double-stranded region is located adjacent to the junction. © 2008 The Authors.

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Versatile architecture of a bacterial aconitase B and its catalytic performance in the sequential reaction coupled with isocitrate dehydrogenase

Tsuchiya D., Shimizu N., and Tomita M.

BBA - Proteins and Proteomics. 1784 ( 11 ) 1847-56. - 1856 2008

Research paper (scientific journal), Joint Work, ISSN 15709639

Aconitase B (AcnB) and isocitrate dehydrogenase (ICDH) catalyze the sequential reaction in the Krebs cycle. Since each enzyme was characterized as an independent protein in a diluted condition, the catalytic performance within the cellular metabolism remains unclear. In particular, high macromolecular concentration in the cytosol promotes weak interactions, which affects structure and function of the proteins. We found that the two bacterial enzymes exhibit variable catalytic performance of the sequential reaction, depending on the oligomerization state. The small-angle solution X-ray scattering and the chemical crosslinking analyses revealed that not only the two enzymes but also the fusion protein of the two enzymes assume homodimers in solution. Interestingly, the fusion protein maintains the homodimeric architecture of ICDH, but not AcnB. Instead, one of the two monomeric AcnB regions associates with the homodimeric ICDH region. The fusion protein displayed different catalytic performance of the sequential reaction from that observed in the mixture of the AcnB and ICDH proteins in an equimolar ratio. Connecting the two proteins by a flexible linker yielded a locally high concentration to promote the weak protein-protein interaction. The versatile architecture of AcnB may alter the metabolic process involving the Krebs cycle. © 2008 Elsevier B.V. All rights reserved.

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Aligning LC Peaks by Converting Gradient Retention Times to Retention Index of Peptides in Proteomic Experiments

Shinoda, K., Tomita, M., Ishihama, Y.

Bioinformatics. 24 ( 14 ) 1590-5 - 1595 2008

Research paper (scientific journal), Joint Work, ISSN 13674803

Motivation: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful tool in proteomics studies, but when peptide retention information is used for identification purposes, it remains challenging to compare multiple LC-MS/MS runs or to match observed and predicted retention times, because small changes of LC conditions unavoidably lead to variability in retention times. In addition, non-contiguous retention data obtained with different LC-MS instruments or in different laboratories must be aligned to confirm and utilize rapidly accumulating published proteomics data. Results: We have developed a new alignment method for peptide retention times based on linear solvent strength (LSS) theory. We found that log k0 (logarithm of retention factor for a given organic solvent) in the LSS theory can be utilized as a 'universal' retention index of peptides (RIP) that is independent of LC gradients, and depends solely on the constituents of the mobile phase and the stationary phases. We introduced a machine learning-based scheme to optimize the conversion function of gradient retention times (tg) to log k0. Using the optimized function, tg values obtained with different LC-MS systems can be directly compared with each other on the RIP scale. In an examination of Arabidopsis proteomic data, the vast majority of retention time variability was removed, and five datasets obtained with various LC-MS systems were successfully aligned on the RIP scale. © The Author 2008. Published by Oxford University Press. All rights reserved.

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Restriction on conjugational transfer of pLS20 in Bacillus subtilis 168

Ohtani, N., Sato, M., Tomita, M. and Itaya, M ,

Biosci. Biotechnol. Biochem. , 72 ( 9 ) 2472-5 - 2475 2008

Research paper (scientific journal), Joint Work, ISSN 09168451

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Junction ribonuclease: a ribonuclease HII orthologue from Thermus thermophilus HB8 prefers the RNA-DNA junction to the RNA/DNA heteroduplex

Ohtani, N., Tomita, M. and Itaya, M.

Biochem. J. 412 ( 3 ) 517-26 - 526 2008

Research paper (scientific journal), Joint Work, ISSN 02646021

The genome of an extremely thermophilic bacterium, Thermus thermophilus HB8, contains a single ORF (open reading frame) encoding an RNase-HII-like sequence. Despite the presence of significant amino acid sequence identities with RNase (ribonuclease) HII enzymes, the ORF TTHA0198 could not suppress the temperature-sensitive growth defect of an RNase-H-deficient Escherichia coli mutant and the purified recombinant protein could not cleave an RNA strand of an RNA/DNA heteroduplex, suggesting that the TTHA0198 exhibited no RNase H activity both in vivo and in vitro. When oligomeric RNA-DNA/DNAs were used as a mimic substrate for Okazaki fragments, however, the protein cleaved them only at the 5′ side of the last ribonucleotide at the RNA-DNA junction. In fact, the TTHA0198 protein prefers the RNA-DNA junction to the RNA/DNA hybrid. We have referred to this activity as JRNase (junction RNase) activity, which recognizes an RNA-DNA junction of the RNA-DNA/DNA heteroduplex and cleaves it leaving a mono-ribonucleotide at the 5′ terminus of the RNA-DNA junction. E. coli and Deinococcus radiodurans RNases HII also cleaved the RNA-DNA/DNA substrates at the same site with a different metal-ion preference from that for RNase H activity, implying that the enzymes have JRNase activity as well as RNase H activity. The specialization in the JRNase activity of the RNase HII orthologue from T. thermophilus HB8 (Tth-JRNase) suggests that the JRNase activity of RNase HII enzymes might be independent of the RNase H activity. © The Authors.

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GeNESiS: Gene Network Evolution SImulation Software

Anton Kratz, Masaru Tomita and Arun Krishnan

BMC Bioinformatics 9 541 2008

Research paper (scientific journal), Joint Work

Background: There has been a lot of interest in recent years focusing on the modeling and simulation of Gene Regulatory Networks (GRNs). However, the evolutionary mechanisms that give rise to GRNs in the first place are still largely unknown. In an earlier work, we developed a framework to analyze the effect of objective functions, input types and starting populations on the evolution of GRNs with a specific emphasis on the robustness of evolved GRNs. Results: In this work, we present a parallel software package, GeNESiS for the modeling and simulation of the evolution of gene regulatory networks (GRNs). The software models the process of gene regulation through a combination of finite-state and stochastic models. The evolution of GRNs is then simulated by means of a genetic algorithm with the network connections represented as binary strings. The software allows users to simulate the evolution under varying selective pressures and starting conditions. We believe that the software provides a way for researchers to understand the evolutionary behavior of populations of GRNs. Conclusion: We believe that GeNESiS will serve as a useful tool for scientists interested in understanding the evolution of gene regulatory networks under a range of different conditions and selective pressures. Such modeling efforts can lead to a greater understanding of the network characteristics of GRNs. © 2008 Kratz et al; licensee BioMed Central Ltd.

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Successive and Selective Release of Phosphorylated Peptides Captured by hydroxy Acid-Modified Metal Oxide Chromatography

Kyono, Y.,Sugiyama, N., Imami, K., Tomita, M. and Ishihama, Y.

Journal of Proteome Research 7 ( 10 ) 4585-4593 - 4593 2008

Research paper (scientific journal), Joint Work, ISSN 15353893

We developed a novel approach to enlarge phosphoproteome coverage using successive elution of phosphopeptides with various buffers in series from a single microcolumn packed with hydroxy acid-modified metal oxides, such as titania and zirconia. Elution conditions were investigated to maximize the recovery of phosphopeptides from three standard phosphoproteins. Secondary amines, such as piperidine and pyrrolidine, provided better efficiency than the conventional conditions using ammonium hydroxide and phosphate buffers. Furthermore, elution with these secondary amines provided unique phosphopeptides that were not eluted under the conventional conditions in the analysis of HeLa cell lysates. On the basis of these results, we fractionated phosphopeptides captured by a single metal oxide microcolumn using successive elution with 5% ammonium hydroxide solution, 5% piperidine solution and 5% pyrrolidine solution in series. We identified 1803 nonredun- dant phosphopeptides from 100 μg of HeLa cells, which represented a 1.6-fold increase in phosphopeptide number and a 1.9-fold increase in total peak area of phosphopeptides in comparison with the results obtained under the conventional conditions. Since this approach is applicable to any single tip-based protocol without coupling with other enrichment methods, this simple strategy can be easily incorporated as an option into existing protocols for phosphopeptide enrichment, and would be suitable for high-throughput analysis in a parallel format. © 2008 American Chemical Society.

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Comparison and characterization of proteomes in the three domains of life using 2D correlation analysis

Fujishima K., Komasa M., Kitamura S., Tomita M., Kanai A.

Progress of Theoretical Physics Supplement ( 173 ) 206 - 218 2008

ISSN 03759687

Proteins are a major regulatory component in complex biological systems. Among them, DNA/RNA-binding proteins, the key components of the central dogma of molecular biology, and membrane proteins, which are necessary for both signal transduction and metabolite transport, are suggested to be the most important protein families that arose in the early stage of life. In this study, we computationally analyzed the whole proteome data of six model species to overview the protein diversity in the three domains of life (Bacteria, Archaea and Eukaryota), especially focusing on the above two protein families. To compare the protein distribution among the six model species, we calculated various protein profiles: hydropathy, molecular weight, amino acid composition and periodicity for each protein. We found a domain-specific distribution of the proteome based on 2D correlation analysis of hydropathy and molecular weight. Further, the merged protein distribution of Archaea and other domains revealed many membrane proteins localized in Bacteria-specific regions with a high ratio of hydropathy and many DNA/RNA-binding proteins localized in Eukaryota-specific regions with a low ratio of hydropathy. Since about half of the proteins encoded in the genome are still functionally unknown, we further conducted Support Vector Machine (SVM)-based functional prediction using amino acid composition (CO score) and periodicity (PD score) as feature vectors to predict the overall number of DNA/RNA-binding proteins and membrane proteins in the proteome. Our estimation indicated that two functional categories occupy approximately 60% to 80% of the proteome, and further, the proportion of the two categories varied among the three domains of life, suggesting that the proteome has gone through different selective pressure during evolution.

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Construction of a Biological Tissue Model Based on a Single-Cell Model: A Computer Simulation of Metabolic Heterogeneity in the Liver Lobule

Ohno, H., Naito, Y., Nakajima, H. and Tomita, M.

Artificial Life 14 ( 1 ) 3-28 - 28 2008

Research paper (scientific journal), Joint Work, ISSN 10645462

An enormous body of information has been obtained by molecular and cellular biology in the last half century, However, even these powerful approaches are not adequate when it comes to higher-level biological structures, such as tissues, organs, and individual organisms, because of the complexities involved. Thus, accumulation of data at the higher levels supports and broadens the context for that obtained on the molecular and cellular levels. Under such auspices, an attempt to clucidate mesoscopic and macroscopic subjects based on plentiful nanoscopic and microscopic data is ot great potential value. On the other hand, fully realistic simulation is impracticable because of the extensive cost entailed and enormous amount of data required. Abstraction and modeling that balance the dual requirements of prediction accuracy and manageable calculation cost are of great importance for systems biology. We have constructed an ammonia metabolism model of the hepatic lobule, a histological component of the liver, based on a single-hepatocyte model that consists of the biochemical kinetics of enzymes and transporters. To bring the calculation cost within reason, the porto-central axis, which is an elemental structure of the lobule, is defined as the systems biological unit of the liver, and is accordingly modeled. A model including both histological structure and position-specific gene expression of major enzymes largely represents the physiological dynamics of the hepatic lobule in nature. In addition, heterogeneous gene expression is suggested to have evolved to optimize the energy efficiency of ammonia detoxificattion at the macroscopic level, implying that approaches like this may elucidate how properties at the molecular and cellular levels, such as regulated gene expression, modify higher-level phenomena of multicellular tissue, organs, and organisms. © 2008 Massachusetts Institute of Technology.

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Comparative Study of Circadian Oscillatory Network Models of Drosophila

Ogawa, Y., Arakawa, K., Kaizu, K., Miyoshi, F., Nakayama, Y. and Tomita, M.

Artificial Life 14 ( 1 ) 29-48 - 48 2008

Research paper (scientific journal), Joint Work, ISSN 10645462

The circadian clock of Drosophila is a model pathway for research in biological dock mechanisms, both with traditional experimental approaches and with emerging systems biology approaches utilizing mathematical modeling and in silico computer simulation. Dynamic diurnal oscillations are achieved by the complex interaction of components as a system, and mathematical reconstruction his proven to be an invaluable means of understanding such systematic behavior. In this study, we implemented eight published models of the Drosqpbila circadian clock in Systems Biology Markup Language (SBML) for comparative systems biology studies using E-Cell Simulation Environment version 3, to examine the system-level requirements for the clock mechanism to be robust, by calculating the period and amplitude sensitivity coefficients with simulation experiments. While all models were generally robust as determined by the network topology of the oscillatory feedback loop structure, existing models place relatively strong emphasis on transcription regulation, although this is a limitation on robustness. We suggest that more comprehensive modeling including protein phosphorylation, polymerization, and nuclear transport with regard to amplitude sensitivity will be necessary for understanding the light entrainment and temperature compensation of circadian clocks. © 2008 Massachusetts Institute of Technology.

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Algebraic Method for the Analysis of Signaling Crosstalk

Matsubara, Y., Kikuchi, S., Sugimoto, M., Oka, K. and Tomita, M.

Artificial Life 14 ( 1 ) 81-94 - 94 2008

Research paper (scientific journal), Joint Work, ISSN 10645462

A unified mathematical description that expresses the characteristics of whole systems is necessary for an understanding of signal transduction cascades. In this study we explore an algebraic method, named extreme signaling flow, enhanced from the concept of extreme pathway, to analyze signal transduction systems. This method enables us to represent the long-term potentiation (LTP) and the long-term depression (LTD) of hippocampal neuronal plasticity in an integrated simulation model. The model is validated by comparing the results of redundancy, reaction participation, and in silico knockout analysis with biological knowledge available from the literature. The following properties are assumed in these computational analyses: (1) LTP is fault-tolerant under network modification, (2) protein kinase C and MAPK have numerous routes to LTP induction, (3) calcium-calmodulin kinase II has a few routes to LTP induction, and (4) calcineurin has many routes to LTD induction. These results demonstrate that our approach produces an integrated framework for analyzing properties of large-scale systems with complicated signal transduction. © 2008 Massachusetts Institute of Technology.

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Automated Phosphoproteome Analysis for Cultured Cancer Cells by Two-Dimensional NanoLC-MS Using a Calcined Titania/C18 Biphasic Column

Imami, K., Sugiyama, N., Kyono, Y., Tomita, M. and Ishihama, Y.

Innovations in Analytical Sciences 24 ( 1 ) 161 - 166 2008

Research paper (scientific journal), Joint Work, ISSN 09106340

We have developed an on-line automated system for phosphoproteome analysis using titania-based phosphopeptide enrichment followed by nanoLC-MS/MS. Titania beads were prepared by calcination of commercial chromatographic titania beads at 800°C to convert the crystalline structure. The obtained rutile-form titania exhibited higher selectivity in phosphopeptide enrichment than commercial titania, even in the absence of a competitive chelating reagent for non-phosphopeptides. For phosphoproteome analysis of human cervical cancer HeLa cells, tryptic digests of the cell extracts were directly injected into this on-line system, and 696 non-redundant phosphopeptides with 671 unambiguously determined phosphorylation sites, derived from 512 phosphoproteins, were successfully identified. This is the first successful application of an on-line automated phosphoproteome analysis system to complex biological samples. 2008 © The Japan Society for Analytical Chemistry.

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Depiction of metabolome changes in histidine-starved Escherichia coli by CE-TOFMS

Ohashi,Y.,Hirayama,A.,Ishikawa,T.,Nakamura,S.,Shimizu,K.,Ueno,Y.,Tomita,M.and Soga,T.

Mol.BioSyst 4 ( 2 ) 135-147 - 147 2008

Research paper (scientific journal), Joint Work, ISSN 1742206X

Metabolic changes in response to histidine starvation were observed in histidine-auxotrophic Escherichia coli using a capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS)-based metabolomics technique. Prior to the analysis, we prepared an E. coli metabolome list of 727 metabolites reported in the literature. An improved method for metabolite extraction was developed, which resulted in higher extraction efficiency in phosphate-rich metabolites, e.g., ATP and GTP. Based on the results, 375 charged, hydrophilic intermediates in primary metabolisms were analysed simultaneously, providing quantitative data of 198 metabolites. We confirmed that the intracellular levels of intermediates in histidine biosynthesis are rapidly accumulated in response to a drop in histidine level under histidine-starved conditions. Simultaneously, disciplined responses were observed in the glycolysis, tricarboxylic acid cycle, and amino acid and nucleotide biosynthesis pathways as regulated by amino acid starvation. © The Royal Society of Chemistry.

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Evolution of gene regulatory networks:Robustness as an emergent property of evolution

Krishnan,A.,Tomita,M.and Giuliani,A.

Physica A:Statistical Mechanics nad its Applications 387 ( 8-9 ) 2170-2186 - 2186 2008

Research paper (scientific journal), Joint Work, ISSN 03784371

Gene Regulatory Networks (GRNs) have become a major focus of interest in recent years. Although much work has been done in elucidating the transcriptional regulatory network, the underlying mechanisms that have possibly influenced the evolution of these GRNs are still debatable. We have developed a framework to analyze the effect of objective functions, input types and starting populations on the evolution of GRNs with a specific emphasis on the robustness of evolved GRNs. We observed that robustness evolves along with the networks as an emergent property even in the absence of specific selective pressure towards more robust systems. In addition, robustness was independent of the selective pressure, input types or the initial starting populations. We also observed the existence of multiple genotypes giving rise to the same phenotype in accordance with the theoretical view that natural selection operates on phenotypes thereby accommodating variation in the genotype by fixing those changes that are phenotype-neutral. This study gives a proof-of-concept of the fact that robustness is an emergent property of GRNs as well as of the degeneracy of the network topology/function relationship analogous to the sequence/structure problem in proteins. © 2007 Elsevier Ltd. All rights reserved.

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Reshuffling of the Bacillus subtilis 168 genome by multifold inversion

Kuroki,A.,TOda,T.,Matsui,K.,Uotsu-Tomita,R.,Tomita,M.and Itaya,M.

Journal of Biochemistry 143 ( 1 ) 97-105 - 105 2008

Research paper (scientific journal), Joint Work, ISSN 0021924X

The genome of Bacillus subtilis 168 was modified to yield a genome vector for the cloning of DNA several Mb in size. Unlike contemporary plasmid-based vectors, this 4.2 Mb genome vector requires specific in vivo handling protocols because of its large size. Inversion mutagenesis, a method to modify local genome structure without gain or loss of genes, was applied intensively to the B. subtilis genome; this technique made possible both exchange and translocation of designated regions of the genome. This method not only reshuffles the genome of B. subtilis, but can provide insight into the biologic principles underlying genome plasticity. © 2007 The Japanese Biochemical Society.

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Informatics for Peptide Retention Properties in Proteomic LC-MS

Shinoda, K., Sugimoto, M., Tomita, M. and Ishihama, Y.

Proteomics 8 ( 4 ) 787-98 - 798 2008

Research paper (scientific journal), Joint Work, ISSN 16159853

Retention times in HPLC yield valuable information for the identification of various analytes and the prediction of peptide retention is useful for the identification of peptides/proteins in LC-MS-based proteomics. Informatics methods such as artificial neural networks and support vector machines capable of solving nonlinear problems made possible the accurate modeling of quantitative structure-retention relationships of peptides (including large polymers) up to 5 kDa to which classical linear models cannot be applied, as well as the proteome-wide prediction of peptide retention. Proteome-wide retention prediction and accurate mass-information facilitate the identification of peptides in complex proteomic samples. In this review, we address recent developments in solid informatics methods and their application to peptide-retention properties in 'bottom-up' shotgun proteomics. We also describe future prospects for the standardization and application of retention times. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA.

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Proteins as Networks: Usefulness of Graph Theory in Protein Science

Krishnan, A., Zbilut, P.J., Tomita, M. and Giuliani, A.

Currents in Protein and Peptide Science 9 ( 1 ) 28-38 - 38 2008

Research paper (scientific journal), Joint Work, ISSN 13892037

The network paradigm is based on the derivation of emerging properties of studied systems by their representation as oriented graphs: any system is traced back to a set of nodes (its constituent elements) linked by edges (arcs) correspondent to the relations existing between the nodes. This allows for a straightforward quantitative formalization of systems by means of the computation of mathematical descriptors of such graphs (graph theory). The network paradigm is particularly useful when it is clear which elements of the modelled system must play the role of nodes and arcs respectively, and when topological constraints have a major role with respect to kinetic ones. In this review we demonstrate how nodes and arcs of protein topology are characterized at different levels of definition: 1. Recurrence matrix of hydrophobicity patterns along the sequence 2. Contact matrix of alpha carbons of 3D structures 3. Correlation matrix of motions of different portion of the molecule in molecular dynamics. These three conditions represent different but potentially correlated reticular systems that can be profitably analysed by means of network analysis tools. © 2008 Bentham Science Publishers Ltd.

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Sequence Evidence in the Archaeal Genomes that tRNAs Emerged Through the Combination of Ancestral Genes as 5′ and 3′ tRNA Halves

Fujishima, K., Sugahara, J., Tomita, M. and Kanai, A.

PLoS ONE journal 3 ( 2 ) e1622 2008

Research paper (scientific journal), Joint Work

The discovery of separate 5′ and 3′ halves of transfer RNA (tRNA) molecules - so called split tRNA - in the archaeal parasite Nanoarchaeum equitans made us wonder whether ancestral tRNA was encoded on 1 or 2 genes. We performed a comprehensive phylogenetic analysis of tRNAs in 45 archaal species to explore the relationship between the three types of tRNAs (nonintronic, intronic and split). We classified 1953 mature tRNA sequences into 22 clusters. All split tRNAs have shown phylogenetic relationships with other tRNAs possessing the same anticodon. We also mimicked split tRNA by artificially separating the tRNA sequences of 7 primitive archaeal species at the anticodon and analyzed the sequence similarity and diversity of the 5′ and 3′ tRNA halves. Network analysis revealed specific characteristics of and topological differences between the 5′ and 3′ tRNA halves: the 5′ half sequences were categorized into 6 distinct groups with a sequence similarity of > 80%, while the 3′ half sequences were categorized into 9 groups with a higher sequence similarity of > 88%, suggesting different evolutionary backgrounds of the 2 halves. Furthermore, the combinations of 5′ and 3′ halves coresponded with the variation of amino acids in the codon table. We found not only universally conserved combinations of 5′-3′ tRNA halves in tRNS Imet, tRNA Thr, tRNAIle, tRNAGly, tRNAGin, tRNAGlu, tRNAAsp, tRNALys, tRNA Arg and tRNA but also phylum-specific combinations in tRNAPro, tRNAAla, and tRNATrp. Our results support the idea that tRNA emerged through the combination of separate genes and explain the sequence diversity that arose during archaeal tRNA evolution. © 2008 Fujishima et al.

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Comparative expression analysis uncovers novel features of endogenous antisense transcription

Yuki Okada, Chiaki Tashiro, Koji Numata, Kazufumi Watanabe, Hajime Nakaoka, Naoyuki Yamamoto, Kazue Okubo, Rieko Ikeda, Rintaro Saito, Akio Kanai, Kuniya Abe, Masaru Tomita, and Hidenori Kiyosawa

Hum. Mol. Genet. 17 ( 11 ) 1631-1640 - 1640 2008

Research paper (scientific journal), Joint Work, ISSN 09646906

Increasing numbers of sense - antisense transcripts (SATs), which are transcribed from the same chromosomal location but in opposite directions, have been identified in various eukaryotic species, but the biological meanings of most SATs remain unclear. To improve understanding of natural sense - antisense transcription, we performed comparative expression profiling of SATs conserved among humans and mice. Using custom oligo-arrays loaded with probes that represented SATs with both protein-coding and non-protein-coding transcripts, we showed that 33% of the 291 conserved SATs displayed identical expression patterns in the two species. Among these SATs, expressional balance inversion of sense - antisense genes was mostly observed in testis at a tissue-specific manner. Northern analyses of the individual conserved SAT loci revealed that: (i) a smeary hybridization pattern was present in mice, but not in humans, and (2) small RNAs (about 60 to 80 nt) were detected from the exon-overlapping regions of SAT loci. In addition, further analyses showed marked alteration of sense - antisense expression balance throughout spermatogenesis in testis. These results suggest that conserved SAT loci are rich in potential regulatory roles that will help us understand this new class of transcripts underlying the mammalian genome. © The Author 2008. Published by Oxford University Press. All rights reserved.

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Implications from a Network-Based Topological Analysis of Ubiquitin Unfolding Simulations

Arun Krishnan, Alessandro Giuliani, Joseph P. Zbilut, Masaru Tomita

PLoS ONE 3 ( 5 ) e2149 2008

Research paper (scientific journal), Joint Work

Background: The architectural organization of protein structures has been the focus of intense research since it can hopefully lead to an understanding of how proteins fold. In earlier works we had attempted to identify the inherent structural organization in proteins through a study of protein topology. We obtained a modular partitioning of protein structures with the modules correlating well with experimental evidence of early folding units or "foldons". Residues that connect different modules were shown to be those that were protected during the transition phase of folding. Methodology/Principal Findings: In this work, we follow the topological path of ubiquitin through molecular dynamics unfolding simulations. We observed that the use of recurrence quantification analysis (RQA) could lead to the identification of the transition state during unfolding. Additionally, our earlier contention that the modules uncovered through our graph partitioning approach correlated well with early folding units was vindicated through our simulations. Moreover, residues identified from native structure as connector hubs and which had been shown to be those that were protected during the transition phase of folding were indeed more stable (less flexible) well beyond the transition state. Further analysis of the topological pathway suggests that the all pairs shortest path in a protein is minimized during folding. Conclusions: We observed that treating a protein native structure as a network by having amino acid residues as nodes and the non-covalent interactions among them as links allows for the rationalization of many aspects of the folding process. The possibility to derive this information directly from 3D structure opens the way to the prediction of important residues in proteins, while the confirmation of the minimization of APSP for folding allows for the establishment of a potentially useful proxy for kinetic optimality in the validation of sequence-structure predictions. © 2008 Krishnan et al.

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Time-resolved metabolomics reveals metabolic modulation in rice foliage

Shigeru Sato , Masanori Arita , Tomoyoshi Soga , Takaaki Nishioka and Masaru Tomita

BMC Systems Biology 2 51 2008

Research paper (scientific journal), Joint Work

Background: To elucidate the interaction of dynamics among modules that constitute biological systems, comprehensive datasets obtained from "omics" technologies have been used. In recent plant metabolomics approaches, the reconstruction of metabolic correlation networks has been attempted using statistical techniques. However, the results were unsatisfactory and effective data-mining techniques that apply appropriate comprehensive datasets are needed. Results: Using capillary electrophoresis mass spectrometry (CE-MS) and capillary electrophoresis diode-array detection (CE-DAD), we analyzed the dynamic changes in the level of 56 basic metabolites in plant foliage (Oryza sativa L. ssp. japonica) at hourly intervals over a 24-hr period. Unsupervised clustering of comprehensive metabolic profiles using Kohonen's self-organizing map (SOM) allowed classification of the biochemical pathways activated by the light and dark cycle. The carbon and nitrogen (C/N) metabolism in both periods was also visualized as a phenotypic linkage map that connects network modules on the basis of traditional metabolic pathways rather than pairwise correlations among metabolites. The regulatory networks of C/N assimilation/dissimilation at each time point were consistent with previous works on plant metabolism. In response to environmental stress, glutathione and spermidine fluctuated synchronously with their regulatory targets. Adenine nucleosides and nicotinamide coenzymes were regulated by phosphorylation and dephosphorylation. We also demonstrated that SOM analysis was applicable to the estimation of unidentifiable metabolites in metabolome analysis. Hierarchical clustering of a correlation coefficient matrix could help identify the bottleneck enzymes that regulate metabolic networks. Conclusion: Our results showed that our SOM analysis with appropriate metabolic time-courses effectively revealed the synchronous dynamics among metabolic modules and elucidated the underlying biochemical functions. The application of discrimination of unidentified metabolites and the identification of bottleneck enzymatic steps even to non-targeted comprehensive analysis promise to facilitate an understanding of large-scale interactions among components in biological systems. © 2008 Sato et al; licensee BioMed Central Ltd.

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E. coli metabolomics: Capturing the complexity of a "simple" model

Robert M., Soga T., Tomita M.

Topics in Current Genetics 18 189 - 234 2007.09

ISSN 16106970

As the workhorse of early studies on metabolism, the metabolic pathways of E. coli are arguably the best characterized. The richness of information available about its pathways is broader than for any other model. However, in spite of decades of descriptive work, only recently can a significant number of E. coli metabolic network constituents be analyzed simultaneously. The advent of metabolomic methods that allow to capture qualitative as well as quantitative information about the intracellular and extracellular metabolite profiles is starting to shed light on the remaining complexity of this simpler model. Here we describe important findings about the physiology of E. coli resulting from emerging metabolomic studies. While a vast number of intracellular metabolites in E. coli still remain to be characterized, the information obtained from those studies can provide an unprecedented amount of information about metabolic pathways including their functional elucidation, enzyme activity, metabolic fluxes, network robustness, or even the discovery of completely novel reactions or pathways. These results are also being used to populate rich databases and to develop computational models of E. coli metabolism that have already proven effective to predict cellular states and will shed light on complex and until now still elusive regulatory principles. © 2007 Springer-Verlag Berlin Heidelberg.

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Bioinformatic analysis of post-transcriptional regulation by uORF in human and mouse

Matsui M., Yachie N., Okada Y., Saito R., Tomita M.

FEBS Letters 581 ( 22 ) 4184 - 4188 2007.09

ISSN 00145793

RNA decay is thought to exert an important influence on gene expression by maintaining a steady-state level of transcripts and/or by eliminating aberrant transcripts. However, the sequence elements which control such processes have not been determined. Upstream open reading frames (uORFs) in the transcripts of several genes are reported to control translational initiation by stalling ribosomes and thereby promote RNA decay. We therefore performed bioinformatic analysis of the tissue-wide expression profiles and mRNA half-life of transcripts containing uORFs in humans and mice to assess the relationship between RNA decay and the presence of uORFs in transcripts. The expression levels of transcripts containing uORF were markedly lower than those not containing uORF. Moreover, the half-life of the uORF-containing transcripts was also shorter. These results suggest that uORFs are sequence elements that down-regulate RNA transcripts via RNA decay mechanisms. © 2007 Federation of European Biochemical Societies.

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Sequential logic model deciphers dynamic transciptional control of gene expressions

Yeo Z., Wong S., Vel Arjunan S., Piras V., Tomita M., Selvarajoo K., Giuliani A., Tsuchiya M.

PLoS ONE 2 ( 8 ) 2007.08

Background. Cellular signaling involves a sequence of events from ligand binding to membrane receptors through transcription factors activation and the induction of mRNA expression. The transcriptional-regulatory system plays a pivotal role in the control of gene expression. A novel computational approach to the study of gene regulation circuits is presented, here. Methodology. Based on the concept of finite state machine, which provides a discrete view of gene regulation, a novel sequential logic model (SLM) is developed to decipher control mechanisms of-dynamic transcriptional regulation of gene expressions. The SLM technique is also used to systematically analyze the dynamic function of transcriptional inputs, the dependency and cooperativity, such as synergy effect, among the binding sites with respect to when, how much and how fast the gene of interest is expressed. Principal Findings. SLM is verified by a set of well studied expression data on endo 16 of Strongylocentrotus purpuratus (sea urchin) during the embryonic midgut development. A dynamic regulatory mechanism for endo 16 expression controlled by three binding sites, UI, R and Otx is identified and demonstrated to be consistent with experimental findings. Furthermore, we show that during transition from specification to differentiation in wild type endo16 expression profile, SLM reveals three binary activities are not sufficient to explain the transcriptional regulation of endo16 expression and additional activities of binding sites are required. Further analyses suggest detailed mechanism of R switch activity where indirect dependency occurs in between UI activity and R switch during specification to differentiation stage. Conclusions/Significatwe. The sequential logic formalism allows for a simplification of regulation network dynamics going from a continuous to a discrete representation of gene activation in time. In effect our SLM is non-parametric and model-independent, yet providing rich biological insight. The demonstration of the efficacy of this approach in endo 16 is a promising step for further application of the proposed method. © 2007 Yeo et al.

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P-BOSS: A new filtering method for treasure hunting in metabolomics

Morohashi M., Shimizu K., Ohashi Y., Abe J., Mori H., Tomita M., Soga T.

Journal of Chromatography A 1159 ( 1-2 ) 142 - 148 2007.08

ISSN 00219673

Metabolomics is expected to boost data driven research. In biomarker discovery, powerful filtering methods to remove noise and outliers are essential for screening significant candidates from the huge volume of omic data. Here we propose a post-measurement peak filtering method (named P-BOSS) for CE electrospray ionization-time-of-flight MS (CE-TOFMS) data. Combining outlier detection method functions in parallel, we applied P-BOSS to the data using Escherichia coli knockout mutants of the tryptophan and purine biosynthesis pathways. As the result, P-BOSS showed remarkably superior performance, reducing 65% of all peaks, while leaving significant peaks. © 2007 Elsevier B.V. All rights reserved.

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Distinguishing enzymes using metabolome data for the hybrid dynamic/static method

Ishii N., Nakayama Y., Tomita M.

Theoretical Biology and Medical Modelling 4 2007.06

Background. In the process of constructing a dynamic model of a metabolic pathway, a large number of parameters such as kinetic constants and initial metabolite concentrations are required. However, in many cases, experimental determination of these parameters is time-consuming. Therefore, for large-scale modelling, it is essential to develop a method that requires few experimental parameters. The hybrid dynamic/static (HDS) method is a combination of the conventional kinetic representation and metabolic flux analysis (MFA). Since no kinetic information is required in the static module, which consists of MFA, the HDS method may dramatically reduce the number of required parameters. However, no adequate method for developing a hybrid model from experimental data has been proposed. Results. In this study, we develop a method for constructing hybrid models based on metabolome data. The method discriminates enzymes into static modules and dynamic modules using metabolite concentration time series data. Enzyme reaction rate time series were estimated from the metabolite concentration time series data and used to distinguish enzymes optimally for the dynamic and static modules. The method was applied to build hybrid models of two microbial central-carbon metabolism systems using simulation results from their dynamic models. Conclusion. A protocol to build a hybrid model using metabolome data and a minimal number of kinetic parameters has been developed. The proposed method was successfully applied to the strictly regulated central-carbon metabolism system, demonstrating the practical use of the HDS method, which is designed for computer modelling of metabolic systems. © 2007 Ishii et al; licensee BioMed Central Ltd.

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Gene expression waves: Cell cycle independent collective dynamics in cultured cells

Tsuchyia M., Wong S., Yeo Z., Colosimo A., Palumbo M., Farina L., Crescenzi M., Mazzola A., Negri R., Bianchi M., Selvarajoo K., Tomita M., Giuliani A.

FEBS Journal 274 ( 11 ) 2878 - 2886 2007.06

ISSN 1742464X

The ergodic hypothesis, which assumes the independence of each cell of the ensemble from all the others, is a necessary prerequisite to attach single cell based explanations to the grand averages taken from population data. This was the prevailing view about the interpretation of cellular biology experiments that typically are performed on colonies of billions of cells. By analysing gene expression data of different cells going from yeast to mammalian cell cultures, we demonstrate that cell cultures display a sort of 'ecology-in-a-plate' giving rise to a rich dynamics of gene expression that are independent from reproductive cycles, hence contradicting simple ergodic assumptions The aspecific character of the observed coordinated gene expression activity inhibits any simple mechanistic hypothesis and highlights the need to consider population effects in the interpretation of data coming from cell cultures. © 2007 The Authors.

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Validating the significance of genomic properties of Chi sites from the distribution of all octamers in Escherichia coli

Arakawa K., Uno R., Nakayama Y., Tomita M.

Gene 392 ( 1-2 ) 239 - 246 2007.05

ISSN 03781119

Chi sites (5′-GCTGGTGG-3′) are homologous recombinational hotspot octamer sequences, which attenuate the exonuclease activity of RecBCD in Escherichia coli. They are overrepresented in the genome (1008 occurrences), preferentially located within coding regions (98%), oriented in the direction of replication (75%), and occur most commonly on the mRNA-synonymous sense strand of the double helix (79%). Previous statistical studies of the genome sequence suggested that these genomic properties of Chi sites appear to be related to their role in recombinational repair and therefore to replication and transcription. In this study, we employ three mathematical models to predict the properties of Chi sites from single nucleotide and multi-nucleotide compositions, and validate them statistically using the distribution of all octamer sequences in the entire genome, or exclusively within ORFs. The model based on the overall distribution of all octamers provided better predictions than the single nucleotide composition model, and the ORF and sense strand preference of Chi sites were shown to be within the standard deviation of all octamers. In contrast, the orientation bias of the Chi sites in the direction of replication was significant, although the bias was not as pronounced as with the single nucleotide composition model, suggesting a selective pressure related to the role of RecBCD in replication. © 2007 Elsevier B.V. All rights reserved.

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Roles of hemoglobin allostery in hypoxia-induced metabolic alterations in erythrocytes: Simulation and its verification by metabolome analysis

Kinoshita A., Tsukada K., Soga T., Hishiki T., Ueno Y., Nakayama Y., Tomita M., Suematsu M.

Journal of Biological Chemistry 282 ( 14 ) 10731 - 10741 2007.04

ISSN 00219258

When erythrocytes are exposed to hypoxia, hemoglobin (Hb) stabilizes in theT-state by capturing 2,3-bisphosphoglycerate. This process could reduce the intracellular pool of glycolytic substrates, jeopardizing cellular energetics. Recent observations suggest that hypoxia-induced activation of glycolytic enzymes is correlated with their release from Band III (BIII) on the cell membrane. Based on these data, we developed a mathematical model of erythrocyte metabolism and compared hypoxia-induced differences in predicted activities of the enzymes, their products, and cellular energetics between models with and without the interaction of Hb with BIII. The models predicted that the allostery-dependent Hb interaction with BIII accelerates consumption of upstream glycolytic substrates such as glucose 6-phosphate and increases downstream products such as phosphoenolpyruvate. This prediction was consistent with metabolomic data from capillary electrophoresis mass spectrometry. The hypoxia-induced alterations in the metabolites resulted from acceleration of glycolysis, as judged by increased conversion of [13C]glucose to [13C]lactate. The allostery-dependent interaction of Hb with BIII appeared to contribute not only to maintenance of energy charge but also to further synthesis of 2,3-bisphosphoglycerate, which could help sustain stabilization of T-state Hb during hypoxia. Furthermore, such an activation of glycolysis was not observed when Hb was stabilized in R-state by treating the cells with CO. These results suggest that Hb allostery in erythrocytes serves as an O2-sensing trigger that drives glycolytic acceleration to stabilize intracellular energetics and promote the ability to release O 2 from the cells. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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Simulation study of methemoglobin reduction in erythrocytes: Differential contributions of two pathways to tolerance to oxidative stress

Kinoshita A., Nakayama Y., Kitayama T., Tomita M.

FEBS Journal 274 ( 6 ) 1449 - 1458 2007.03

ISSN 1742464X

Methemoglobin (metHb), an oxidized form of hemoglobin, is unable to bind and carry oxygen. Erythrocytes are continuously subjected to oxidative stress and nitrite exposure, which results in the spontaneous formation of metHb. To avoid the accumulation of metHb, reductive pathways mediated by cytochrome b5 or flavin, coupled with NADH-dependent or NADPH-dependent metHb reductases, respectively, keep the level of metHb in erythrocytes at less than 1% of the total hemoglobin under normal conditions. In this work, a mathematical model has been developed to quantitatively assess the relative contributions of the two major metHb-reducing pathways, taking into consideration the supply of NADH and NADPH from central energy metabolism. The results of the simulation experiments suggest that these pathways have different roles in the reduction of metHb; one has a high response rate to hemoglobin oxidation with a limited reducing flux, and the other has a low response rate with a high capacity flux. On the basis of the results of our model, under normal oxidative conditions, the NADPH-dependent system, the physiological role of which to date has been unclear, is predicted to be responsible for most of the reduction of metHb. In contrast, the cytochrome b5-NADH pathway becomes dominant under conditions of excess metHb accumulation, only after the capacity of the flavin-NADPH pathway has reached its limit. We discuss the potential implications of a system designed with two metHb-reducing pathways in human erythrocytes. © 2007 The Authors.

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A mathematical model for the Kai-protein-based chemical oscillator and clock gene expression rhythms in cyanobacteria

Miyoshi, Fumihiko ; Nakayama, Yoichi ; Kaizu, Kazunari ; Iwasaki, Hideo ; Tomita, Masaru

JOURNAL OF BIOLOGICAL RHYTHMS 22 ( 1 ) 69-80 - 80 2007.02

Research paper (scientific journal), Joint Work, ISSN 0748-7304

In the cyanobacterium, Synechococcus elongatus, most promoters are regulated by a circadian clock under continuous light (LL) conditions. Nevertheless, the basic circadian oscillation is primarily generated by alternating KaiC phosphorylation/dephosphorylation reactions at the posttranslational level. Indeed, the KaiC phosphorylation cycle was recently reconstituted in vitro by incubating KaiA, KaiB, and KaiC proteins with ATP. However, the molecular dynamics of this chemical oscillation and the mechanism that drives the circadian transcription/translation rhythms remain unknown. In this report, the KaiC phosphorylation cycle and the gene regulatory network in the cyanobacterial circadian system have been modeled. The model reproduces the robust KaiC phosphorylation cycle in the absence of de novo gene expression as is observed in vitro, as well as its coupling to transcriptional/translational feedback in LL conditions in vivo. Moreover, the model is consistent with most previous experiments, including various combinations of genetic knockout or overexpression of kai genes. It also predicts that multiple KaiC phosphorylation states and dynamic Kai protein interactions may be required for the cyanobacterial circadian system. © 2007 Sage Publications.

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Noise-reduction filtering for accurate detection of replication termini in bacterial genomes

Arakawa, Kazuharu ; Saito, Rintaro ; Tomita, Masaru

FEBS LETTERS 581 ( 2 ) 253-258 - 258 2007.01

Research paper (scientific journal), Joint Work, ISSN 0014-5793

Bacterial chromosomes are highly polarized in their nucleotide composition through mutational selection related to replication. Using compositional skews such as the GC skew, replication origin and terminus can be predicted in silico by observing the shift points. However, the genome sequence is affected by myriad functional requirements and selection on numerous subgenomic features, and elimination of this "noise" should lead to better predictions. Here, we present a noise-reduction approach that uses low-pass filtering through Fast Fourier transform coupled with cumulative skew graphs. It increases the prediction accuracy of the replication termini compared with previously documented methods based on genomic base composition. © 2006 Federation of European Biochemical Societies.

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eXpanda: an Integrated Platform for Network Analysis and Visualization

Negishi, Y., Nakamura, H., Yachie, N., Saito, R. and Tomita, M.

In silico Biology 7 ( 2 ) 141 - 144 2007

ISSN 13866338

Analysis and visualization of biological networks, such as protein-protein and protein-DNA interactions, are crucially important toward obtaining a thorough understanding of living systems. Here, we present an integrative software platform, eXpanda, which enables an analysis of a very broad range of biological networks, with a special focus on the extraction of characteristic topologies which potentially function as units in the networks. eXpanda is provided as a Perl library which gives full-automatic connections to various biological databases via a Perl programmable interface and can perform topological analysis based on graph theory. The results of these analyses are visualizable by vector graphics. eXpanda is under GNU General Public License. Software package, detailed documentations, source codes, and some sample scripts are downloadable at http://medcd.iab.keio.ac.jp/expanda/. © 2007 IOS Press. All rights reserved.

Direct measurement of isotopomer of intracellular metabolites using capillary electrophoresis time-of-flight mass spectrometry for efficient metabolic flux analysis

Toya, Y., Ishii, N., Hirasawa, T., Tomita, M. and Soga, T.

Journal of Chromatography A 1159 ( 1-2 ) 134–141 - 141 2007

Research paper (scientific journal), Joint Work, ISSN 00219673

We have developed a metabolic flux analysis method that is based on 13C-labeling patterns of the intracellular metabolites directly measured by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). The flux distribution of the central carbon metabolism in Escherichia coli was determined by this new approach and the results were compared with findings obtained by conventional GC-MS analysis based on isotopomer of the proteinogenic amino acids. There were some differences in estimation results between new approach using CE-TOFMS and conventional approach using GC-MS. These were thought to be attributable to variations in measured mass distributions between amino acids and the corresponding precursors and to differences in the sensitivity of the exchange coefficients to mass distributions. However, our CE-TOFMS method facilitates high-throughput flux analysis without requiring complicated sample preparation such as hydrolysis of proteins and derivatization of amino acids. © 2007 Elsevier B.V. All rights reserved.

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Analysis of nucleotides by pressure-assisted capillary electrophoresis　mass spectrometry using silanol mask technique

Soga, T., Ishikawa, T., Igarashi, S., Sugawara, K., Kakazu, Y. and Tomita, M.

Journal of Chromatography A 1159 ( 1-2 ) 125–133 - 133 2007

Research paper (scientific journal), Joint Work, ISSN 00219673

A method for the determination of nucleotides based on pressure-assisted capillary electrophoresis-electrospray ionization mass spectrometry (PACE-MS) is described. To prevent multi-phosphorylated species from adsorbing onto the fused-silica capillary, silanol groups were masked with phosphate ions by preconditioning the capillary with the background electrolyte containing phosphate. During preconditioning, nebulizer gas was turned off to avoid contamination of MS detector with phosphate ions. To detect nucleotides using the CE positive mode at a pH 7.5, it was necessary to apply air pressure to the inlet capillary during electrophoresis to supplement the electroosmotic flow (EOF) toward the cathode. Moreover, we exchanged the running electrolyte every analysis using the buffer replenishment system to obtain the required reproducibility. Under the optimized conditions, 14 phosphorylated species such as nucleotides, nicotinamide-adenine dinucleotides and coenzyme A (CoA) compounds were well determined in less than 20 min. The relative standard deviations (n = 6) of the method were better than 0.9% for migration times and between 1.7% and 8.1% for peak areas. The detection limits for these species were between 0.5 and 1.7 μmol/L with pressure injection of 50 mbar for 30 s (30 nL) at a signal-to-noise ratio of 3. This approach is robust and quantitative compared to the previous method, and its utility is demonstrated by the analysis of intracellular nucleotides and CoA compounds extracted from Escherichia coli wild type, pfkA and pfkB knockout mutants. The methodology was used to suggest that pfkA is the main functional enzyme. © 2007 Elsevier B.V. All rights reserved.

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Conjugational transfer system to shuttle giant DNA cloned by Bacillus subtilis genome (BGM) vector

Kuroki, A., Ohtani, N., Tsuge, K., Tomita, M. and Itaya, M.

Gene 399 ( 1 ) 72-80 - 80 2007

Research paper (scientific journal), Joint Work, ISSN 03781119

The Bacillus subtilis GenoMe (BGM) vector was designed as a versatile vector for the cloning of giant DNA segments. Cloned DNA in the BGM can be retrieved to a plasmid using our Bacillus recombinational transfer (BReT) method that takes advantage of competent cell transformation. However, delivery of the plasmid to a different B. subtilis strain by the normal transformation method is hampered by DNA size-related inefficiency. Therefore, we designed a novel method, conjugational plasmid-mediated DNA retrieval and transfer (CReT) from the BGM vector, and investigated conjugational transmission to traverse DNA between cells to circumvent the transformation-induced size limitation. pLS20, a 65-kb plasmid capable of conjugational transfer between B. subtilis strains, was modified to retrieve DNA cloned in the BGM vector by homologous recombination during normal culture. As the plasmid copy number was estimated to be 3, the retrieval plasmid was selected using increased numbers of marker genes derived from the retrieved DNA. We applied this method to retrieve Synechocystis genome segments up to 90 kb in length. We observed retrieved plasmid transfers between B. subtilis strains by conjugation in the absence of structural alterations in the DNA fragment. Our observations extend DNA transfer protocols over previously exploited size ranges. © 2007 Elsevier B.V. All rights reserved.

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Proteome-Wide Prediction of Novel DNA/RNA-Binding Proteins Using Amino Acid Composition and Periodicity in the Hyperthermophilic Archaeon Pyrococcus furiosus

Fujishima, K., Komasa, M., Kitamura, S., Suzuki, H., Tomita, M. and Kanai, A.

DNA Research 14 ( 3 ) 91-102 - 102 2007

Research paper (scientific journal), Joint Work, ISSN 13402838

Proteins play a critical role in complex biological systems, yet about half of the proteins in publicly available databases are annotated as functionally unknown. Proteome-wide functional classification using bioinformatics approaches thus is becoming an important method for revealing unknown protein functions. Using the hyperthermophilic archaeon Pyrococcus furiosus as a model species, we used the support vector machine (SVM) method to discriminate DNA/RNA-binding proteins from proteins with other functions, using amino acid composition and periodicities as feature vectors. We defined this value as the composition score (CO) and periodicity score (PD). The P. furiosus proteins were classified into three classes (I-III) on the basis of the two-dimensional correlation analysis of CO score and PD score. As a result, approximately 87 of the functionally known proteins categorized as class I proteins (CO score + PD score > 0.6) were found to be DNA/RNA-binding proteins. Applying the two-dimensional correlation analysis to the 994 hypothetical proteins in P. furiosus, a total of 151 proteins were predicted to be novel DNA/RNA-binding protein candidates. DNA/RNA-binding activities of randomly chosen hypothetical proteins were experimentally verified. Six out of seven candidate proteins in class I possessed DNA/RNA-binding activities, supporting the efficacy of our method. © The Author 2007. Kazusa DNA Research Institute.

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Evolution of ribonuclease H genes in prokaryotes to avoid inheritance of redundant genes

Kochiwa, H., Tomita, M. and Kanai, A.

BMC Evolutionary Biology 7 128 2007

Research paper (scientific journal), Joint Work

Background. A theoretical model of genetic redundancy has proposed that the fates of redundant genes depend on the degree of functional redundancy, and that functionally redundant genes will not be inherited together. However, no example of actual gene evolution has been reported that can be used to test this model. Here, we analyzed the molecular evolution of the ribonuclease H (RNase H) family in prokaryotes and used the results to examine the implications of functional redundancy for gene evolution. Results. In prokaryotes, RNase H has been classified into RNase HI, HII, and HIII on the basis of amino acid sequences. Using 353 prokaryotic genomes, we identified the genes encoding the RNase H group and examined combinations of these genes in individual genomes. We found that the RNase H group may have evolved in such a way that the RNase HI and HIII genes will not coexist within a single genome - in other words, these genes are inherited in a mutually exclusive manner. Avoiding the simultaneous inheritance of the RNase HI and HIII genes is remarkable when RNase HI contains an additional non-RNase H domain, double-stranded RNA, and an RNA-DNA hybrid-binding domain, which is often observed in eukaryotic RNase H1. This evolutionary process may have resulted from functional redundancy of these genes, because the substrate preferences of RNase HI and RNase HIII are similar. Conclusion. We provide two possible evolutionary models for RNase H genes in which functional redundancy contributes to the exclusion of redundant genes from the genome of a species. This is the first empirical study to show the effect of functional redundancy on changes in gene constitution during the course of evolution. © 2007 Kochiwa et al; licensee BioMed Central Ltd.

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Indeterminacy of Reverse Engineering of Gene Regulatory Networks: The Curse of Gene Elasticity

Krishnan, A., Giuliani, A. and Tomita, M

PLoS ONE 2 ( 6 ) e562 2007

Research paper (scientific journal), Joint Work

Background. Gene Regulatory Networks (GRNs) have become a major focus in interest in recent years. A number of reverse engineering approaches have been developed to help uncover the regulatory networks giving rise to the observed gene expression profiles. However, this is an overspecified problem due to the fact that more than one genotype (network wiring) can give rise to the same phenotype. We refer to this phenomenon as "gene elasticity." In this work, we study the effect of this particular problem on the pure, data-driven inference of gene regulatory networks. Methodology. We simulated a four-gene network in order to produce "data" (protein levels) that we use in lieu of real experimental data. We then optimized the network connections between the four genes with a view to obtain the original network that gave rise to the data. We did this for two different cases: one in which only the network connections were optimized and the other in which both the network connections as well as the kinetic parameters (given as reaction probabilities in our case) were estimated. We observed that multiple genotypes gave rise to very similar protein levels. Statistical experimentation indicates that it is impossible to differentiate between the different networks on the basis of both equilibrium as well as dynamic data. Conclusions. We show explicitly that reverse engineering of GRNs from pure expression data is an indeterminate problem. Our results suggest the unsuitability of an inferential, purely data-driven approach for the reverse engineering transcriptional networks in the case of gene regulatory networks displaying a certain level of complexity. © 2007 Krishnan et al.

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Restauro-G: A Rapid Genome Re-Annotation System for Comparative Genomics

Tamaki, S., Arakawa, K., Kono, N. and Tomita, M.

Genomics Proteomics Bioinformatics 5; 53-58(2007) 5 ( 1 ) 53-58 - 58 2007

Research paper (scientific journal), Joint Work, ISSN 16720229

Annotations of complete genome sequences submitted directly from sequencing projects are diverse in terms of annotation strategies and update frequencies. These inconsistencies make comparative studies difficult. To allow rapid data preparation of a large number of complete genomes, automation and speed are important for genome re-annotation. Here we introduce an open-source rapid genome re-annotation software system, Restauro-G, specialized for bacterial genomes. Restauro-G re-annotates a genome by similarity searches utilizing the BLAST-Like Alignment Tool, referring to protein databases such as UniProt KB, NCBI nr, NCBI COGs, Pfam, and PSORTb. Re-annotation by Restauro-G achieved over 98% accuracy for most bacterial chromosomes in comparison with the original manually curated annotation of EMBL releases. Restauro-G was developed in the generic bioinformatics workbench G-language Genome Analysis Environment and is distributed at http://restauro-g.iab.keio.ac.jp/under the GNU General Public License. © 2007 Beijing Genomics Institute.

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Computational analysis of microRNA-mediated antiviral defense in humans

Watanabe, Y., Kishi, A., Yachie, N., Kanai, A. and Tomita, M.

FEBS Letters 581 ( 24 ) 4603-4610 - 4610 2007

Research paper (scientific journal), Joint Work, ISSN 00145793

Recent studies have proposed the interesting perspective that viral gene expression is downregulated by host microRNAs (miRNAs), small non-coding RNAs well known as post-transcriptional gene regulators. We computationally predicted human miRNA target sites within 228 human-infecting and 348 invertebrate-infecting virus genomes, and we observed that human-infecting viruses were more likely than invertebrate-infecting ones to be targeted by human miRNAs. We listed 62 possible human miRNA-targeted viruses from 6 families, most of which consisted of single-stranded RNA viruses. These results suggest that miRNAs extensively mediate antiviral defenses in humans. © 2007 Federation of European Biochemical Societies.

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Polymer Entrapment in Polymerized Silicate for Preparing Highly Stable Capillary Coatings for CE and CE-MS

Monton, M., Tomita, M., Soga, T. and Ishihama, Y.

Analytical Chemistry 79 ( 20 ) 7838-7844 - 7844 2007

Research paper (scientific journal), Joint Work, ISSN 00032700

An easy-to-implement capillary coating strategy based on polymer entrapment in the network of polymerized silicate is described. In this manner, cationic polymers are tightiy fixed onto the inner wall of the capillary for electroosmotic flow control without necessitating complex surface modification chemistries. The resulting coated capillary exhibited good stability over a wide range of pH, good reproducibility, strong endurance in more than 300 electrophoretic runs, and tolerance of commonly employed organic solvent additives in CE. Applications in CE-MS analysis of biologically important anions as well as sample enrichment are shown. Additionally, it was used as a durable base for attachment of multiple layers of charged polymers on the wall, via electrostatic interaction with the preceding layer. Thus, two novel types of highly stable coated capillaries, one with anodic EOF and the other cathodic, were developed. © 2007 American Chemical Society.

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Phosphopeptide Enrichment by Aliphatic Hydroxy Acid-modified Metal Oxide Chromatography for Nano-LC-MS/MS in Proteomics Applications

Sugiyama, N., Masuda, T., Shinoda, K., Nakamura, A., Tomita, M. and Ishihama, Y.

Mol Cell Proteomics 6 ( 6 ) 1103-1109 - 1109 2007

Research paper (scientific journal), Joint Work, ISSN 15359476

We developed novel methods for phosphopeptide enrichment using aliphatic hydroxy acid-modified metal oxide chromatography (MOC). Titania and zirconia were successfully applied to enrich phosphopeptides with the aid of aliphatic hydroxy acids, such as lactic acid and β-hydroxypropanoic acid, to reduce the interaction between acidic non-phosphopeptides and the metal oxides. These methods removed the vast majority of non-phosphopeptides from phosphoprotein standard digests, and large numbers of phosphopeptides could be readily identified. The methods were coupled with nano-LC-MS/MS systems without difficulty. Recovery of phosphopeptides in MOC varied greatly from peptide to peptide, ranging from a few percent to 100%, and the average was almost 50%. Repeatability and linearity were satisfactory. In an examination of the cytoplasmic fraction of HeLa cells, more than 1000 phosphopeptides were identified using lactic acid-modified titania MOC and β-hydroxypropanoic acid-modified zirconia MOC, respectively. The overlap between phosphopeptides enriched by these two methods was 40%, and the combined results provided 1646 unique phosphopeptides. To our knowledge, this is the first successful application of a single MOC-based approach to phosphopeptide enrichment from complex biological samples such as cell lysates. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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Statistical Analysis of Gene Expression in Innate Immune Responses: Dynamic Interactions between MicroRNA and Signaling Molecules

Piras, V., Selvarajoo, K., Fujikawa, N., Choi, S., Tomita, M., Giuliani, A. and Tsuchiya, M.

Genomics and Informatics 5 107-112 2007

Research paper (scientific journal), Joint Work

Multiple high throughput analyses monitor the response of E. coli to perturbations

Ishii, N., Nakahigashi, K., Baba, T., Robert, M., Soga, T., Kanai, A., Hirasawa, T., Naba, M., Hirai, K., Hoque, A., Yee, Ho, Pei., Kakazu, Y., Sugawara, K., Igarashi, S., Harada, S., Masuda, T., Sugiyama, N., Togashi, T., Hasegawa, M., Takai, T., Yugi, K., Arakawa, K., Iwata, N., Toya, Y., Nakayama, Y., Nishioka, T., Shimizu, K., Mori, H. and Tomita, M.

Science 316 ( 5824 ) 593-597 - 597 2007

Research paper (scientific journal), Joint Work, ISSN 00368075

Analysis of cellular components at multiple levels of biological information can provide valuable functional insights. We performed multiple high-throughput measurements to study the response of Escherichia coli cells to genetic and environmental perturbations. Analysis of metabolic enzyme gene disruptants revealed unexpectedly small changes in messenger RNA and proteins for most disruptants. Overall, metabolite levels were also stable, reflecting the rerouting of fluxes in the metabolic network. In contrast, E. coli actively regulated enzyme levels to maintain a stable metabolic state in response to changes in growth rate. E. coli thus seems to use complementary strategies that result in a metabolic network robust against perturbations.

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Network scaling invariants help to elucidate basic topological principles of proteins

Krishnan, A., Giuliani, A., Zbilut, J.P. and Tomita, M.

Journal of Proteome Research 6 ( 10 ) 3924-3934 - 3934 2007

Research paper (scientific journal), Joint Work, ISSN 15353893

The structural architecture of proteins continues to be an area of active research. Despite the difference in models dealing with the way proteins fold into their tertiary structures, it is recognized that small regions of proteins tend to fold independently and are then stabilized by interactions between these distinct subunits. However, there are a number of different definitions of what comprises an independent subunit. In the belief that an unequivocal definition of a domain must be based on the most fundamental property of protein 3D structure, namely, the adjacency matrix of inter-residues contact, we adopt a network representation of the protein. In this work, we used a well-established, global method for identifying modules in networks, without any specific reference to the kind of network being analyzed. The algorithm converges toward the maximization of the modularity of the given protein network and, in doing so, allows the representation of the residues of the protein in terms of their intramodule degree, z, and participation coefficient, P. We demonstrate that the labeling of residues in terms of these invariants allows for information-rich representations of the studied proteins as well as to sketch a new way to link sequence, structure, and the dynamical properties of proteins. We discovered a strong invariant character of protein molecules in terms of P/z characterization, pointing to a common topological design of all protein structures. This invariant representation, applied to different protein systems, enabled us to identify the possible functional role of high P/z residues during the folding process. Additionally, we observe a hierarchical behavior of protein structural organization that provides a sequence-secondary-tertiary structure link. The discovery of similar and repeatable scaling laws at different level of definitions going from hydrophobicity patterning along the sequence up to the size of an autonomous folding unit (AFU) and general contact distribution of the entire molecule suggest a hierarchical-like behavior of protein architecture. This implies the possibility to select different privileged scales of observation for deriving useful information on protein systems. © 2007 American Chemical Society.

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Inferring rules of Escherichia coli translational efficiency using an artificial neural network

Mori, K., Saito, R., Kikuchi, S. and Tomita, M.

BioSystems 90 ( 2 ) 414-420 - 420 2007

Research paper (scientific journal), Joint Work, ISSN 03032647

Although the machinery for translation initiation in Escherichia coli is very complicated, the translational efficiency has been reported to be predictable from upstream oligonucleotide sequences. Conventional models have difficulties in their generalization ability and prediction nonlinearity and in their ability to deal with a variety of input attributions. To address these issues, we employed structural learning by artificial neural networks to infer general rules for translational efficiency. The correlation between translational activities measured by biological experiments and those predicted by our method in the test data was significant (r = 0.78), and our method uncovered underlying rules of translational activities and sequence patterns from the obtained skeleton structure. The significant rules for predicting translational efficiency were (1) G- and A-rich oligonucleotide sequences, resembling the Shine-Dalgarno sequence, at positions -10 to -7; (2) first base A in the initiation codon; (3) transport/binding or amino acid metabolism gene function; (4) high binding energy between mRNA and 16S rRNA at positions -15 to -5. An additional inferred novel rule was that C at position -1 increases translational efficiency. When our model was applied to the entire genomic sequence of E. coli, translational activities of genes for metabolism and translational were significantly high. © 2006 Elsevier Ireland Ltd. All rights reserved.

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Computational Methods for MicroRNA Target Prediction

Watanabe, Y., Tomita, M. and Kanai, A.

Methods in Enzymology 427 65-86 - 86 2007

Research paper (scientific journal), Joint Work, ISSN 00766879

The discovery of microRNAs (miRNAs) has introduced a new paradigm into gene regulatory systems. Large numbers of miRNAs have been identified in a wide range of species, and most of them are known to downregulate translation of messenger RNAs (mRNAs) via imperfect binding of the miRNA to a specific site or sites in the 3′ untranslated region (UTR) of the mRNA. Identification of genes targeted by miRNAs is widely believed to be an important step toward understanding the role of miRNAs in gene regulatory networks. As part of the effort to understand interactions between miRNAs and their targets, computational algorithms have been developed based on observed rules for features such as the degree of hybridization between the two RNA molecules. These in silico approaches provide important tools for miRNA target detection, and together with experimental validation, help to reveal regulated targets of miRNAs. Here, we summarize the knowledge that has been accumulated about the principles of target recognition by miRNAs and the currently available computational methodologies for prediction of miRNA target genes. © 2007 Elsevier Inc. All rights reserved.

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Selection effects on the positioning of genes and gene structures from the interplay of replication and transcription in bacterial genomes

Arakawa, K. and Tomita, M.

Evolutionary Bioinformatics 3 279-286 - 286 2007

Research paper (scientific journal), Joint Work

Bacterial chromosomes are partly shaped by the functional requirements for effi cient replication, which lead to strand bias as commonly characterized by the excess of guanines over cytosines in the leading strand. Gene structures are also highly organized within bacterial genomes as a result of such functional constraints, displaying characteristic positioning and structuring along the genome. Here we analyze the gene structures in completely sequenced bacterial chromosomes to observe the positional constraints on gene orientation, length, and codon usage with regard to the positions of replication origin and terminus. Selection on these gene features is different in regions surrounding the terminus of replication from the rest of the genome, but the selection could be either positive or negative depending on the species, and these positional effects are partly attributed to the A-T enrichment near the terminus. Characteristic gene structuring relative to the position of replication origin and terminus is commonly observed among most bacterial species with circular chromosomes, and therefore we argue that the highly organized gene positioning as well as the strand bias should be considered for genomics studies of bacteria.

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In silico screening of archaeal tRNA-encoding genes having multiple introns with bulge-helix-bulge splicing motifs

Sugahara, J., Yachie, N., Arakawa, K. and Tomita, M.

RNA 13 ( 5 ) 671-681 - 681 2007

Research paper (scientific journal), Joint Work, ISSN 13558382

In archaeal species, several transfer RNA genes have been reported to contain endogenous introns. Although most of the introns are located at anticodon loop regions between nucleotide positions 37 and 38, a number of introns at noncanonical sites and six cases of tRNA genes containing two introns have also been documented. However, these tRNA genes are often missed by tRNAscan-SE, the software most widely used for the annotation of tRNA genes. We previously developed SPLITS, a computational tool to identify tRNA genes containing one intron at a noncanonical position on the basis of its discriminative splicing motif, but the software was limited in the detection of tRNA genes with multiple introns at noncanonical sites. In this study, we initially updated the system as SPLITSX in order to correctly predict known tRNA genes as well as novel ones with multiple introns. By a comprehensive search for tRNA genes in 29 archaeal genomes using SPLITSX, we listed 43 novel candidates that contain introns at noncanonical sites. As a result, 15 contained two introns and three contained three introns within the respective putative tRNA genes. Moreover, the candidates completely complemented all the codons of two archaeal species of uncultured methanogenic archaeon, RC-I and Thermofilum pendens Hrk 5, with novel candidates that were not detectable by tRNAscan-SE alone. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 RNA Society.

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Inhibitory effect of phosphoenolpyruvate on glycolytic enzymes in Escherichia coli

Ogawa, T., Mori, H., Tomita, M. and Yoshino, M.

Research in Microbiology 158 ( 2 ) 159-163 - 163 2007

Research paper (scientific journal), Joint Work, ISSN 09232508

For analyzing the control of energy metabolism in Escherichia coli, we carried out kinetic analyses of glycolytic enzymes purified from the overexpressing clones of E. coli K12 W3110 that were constructed with the vector pCA24N. Phosphoenolpyruvate (PEP) acted as an effective inhibitor of enzymes of the preparatory phase in glycolysis. Glucokinase was potently inhibited by PEP in a competitive manner with respect to ATP: the Ki value for PEP was 0.1 mM. PEP further inhibited phosphoglucoisomerase to a lesser extent, and phosphofructokinase A and aldolase A with 10-fold the Ki values of glucokinase and phosphoglucoisomerase. Glucose is incorporated into E. coli through two pathways: the PTS (PEP-dependent phosphotransferase system) and the glucokinase reaction. PEP, a potent inhibitor of E. coli glucokinase, unlike most eukaryotic hexokinases, can act as a signal molecule controlling glucose uptake and glycolytic flux in cells. © 2006 Elsevier Masson SAS. All rights reserved.

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Simulation of developmental changes in action potentials with ventricular cell models

Itoh, H., Naito, Y. and Tomita, M.

Systems and Synthetic Biology 1 ( 1 ) 11-23 - 23 2007

Research paper (scientific journal), Joint Work, ISSN 18725325

During cardiomyocyte development, early embryonic ventricular cells show spontaneous activity that disappears at a later stage. Dramatic changes in action potential are mediated by developmental changes in individual ionic currents. Hence, reconstruction of the individual ionic currents into an integrated mathematical model would lead to a better understanding of cardiomyocyte development. To simulate the action potential of the rodent ventricular cell at three representative developmental stages, quantitative changes in the ionic currents, pumps, exchangers, and sarcoplasmic reticulum (SR) Ca2+ kinetics were represented as relative activities, which were multiplied by conductance or conversion factors for individual ionic systems. The simulated action potential of the early embryonic ventricular cell model exhibited spontaneous activity, which ceased in the simulated action potential of the late embryonic and neonatal ventricular cell models. The simulations with our models were able to reproduce action potentials that were consistent with the reported characteristics of the cells in vitro. The action potential of rodent ventricular cells at different developmental stages can be reproduced with common sets of mathematical equations by multiplying conductance or conversion factors for ionic currents, pumps, exchangers, and SR Ca 2+ kinetics by relative activities. © Springer Science + Business Media B.V. 2007.

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Visualization of three-way comparisons of omics data

Baran, R., Robert, M., Suematsu, M., Soga, T. and Tomita, M.

BMC Bioinformatics 8 72 2007

Research paper (scientific journal), Joint Work

Background: Density plot visualizations (also referred to as heat maps or color maps) are widely used in different fields including large-scale omics studies in biological sciences. However, the current color-codings limit the visualizations to single datasets or pairwise comparisons. Results: We propose a color-coding approach for the representation of three-way comparisons. The approach is based on the HSB (hue, saturation, brightness) color model. The three compared values are assigned specific hue values from the circular hue range (e.g. red, green, and blue). The hue value representing the three-way comparison is calculated according to the distribution of three compared values. If two of the values are identical and one is different, the resulting hue is set to the characteristic hue of the differing value. If all three compared values are different, the resulting hue is selected from a color gradient running between the hues of the two most distant values (as measured by the absolute value of their difference) according to the relative position of the third value between the two. The saturation of the color representing the three-way comparison reflects the amplitude (or extent) of the numerical difference between the two most distant values according to a scale of interest. The brightness is set to a maximum value by default but can be used to encode additional information about the three-way comparison. Conclusion: We propose a novel color-coding approach for intuitive visualization of three-way comparisons of omics data. © 2007 Baran et al; licensee BioMed Central Ltd.

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Alignment-Based Approach for Durable Data Storage into Living Organisms

Yachie, N., Sekiyama, K., Sugahara, J., Ohashi, Y. and Tomita, M.

Biotechnology Progress 23 ( 2 ) 501-505 - 505 2007

Research paper (scientific journal), Joint Work, ISSN 87567938

The practical realization of DNA data storage is a major scientific goal. Here we introduce a simple, flexible, and robust data storage and retrieval method based on sequence alignment of the genomic DNA of living organisms. Duplicated data encoded by different oligonucleotide sequences was inserted redundantly into multiple loci of the Bacillus subtilis genome. Multiple alignment of the bit data sequences decoded by B. subtilis genome sequences enabled the retrieval of stable and compact data without the need for template DNA, parity checks, or error-correcting algorithms. Combined with the computational simulation of data retrieval from mutated message DNA, a practical use of this alignment-based method is discussed. © 2007 American Chemical Society and American Institute of Chemical Engineers.

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Dynamic simulation of an in vitro multi-enzyme system

Ishii, N., Suga, Y., Hagiya, A., Watanabe, H., Mori, H., Yoshino, M, and Tomita, M.

FEBS Letters 581 ( 3 ) 413-420 - 420 2007

Research paper (scientific journal), Joint Work, ISSN 00145793

Parameters often are tuned with metabolite concentration time series data to build a dynamic model of metabolism. However, such tuning may reduce the extrapolation ability (generalization capability) of the model. In this study, we determined detailed kinetic parameters of three purified Escherichia coli glycolytic enzymes using the initial velocity method for individual enzymes; i.e., the parameters were determined independently from metabolite concentration time series data. The metabolite concentration time series calculated by the model using the parameters matched the experimental data obtained in an actual multi-enzyme system consisting of the three purified E. coli glycolytic enzymes. Thus, the results indicate that kinetic parameters can be determined without using an undesirable tuning process. © 2007 Federation of European Biochemical Societies.

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Comparative analysis of cis-encoded antisense RNAs in eukaryotes

Numata, K., Okada, Y., Saito, R., Kiyosawa, H., Kanai, A. and Tomita, M.

Gene　392 392 ( 1-2 ) 134-141 - 141 2007

Research paper (scientific journal), Joint Work, ISSN 03781119

Recent large-scale transcriptomic analyses have identified numerous endogenously encoded cis-antisense RNAs that are thought to play important roles in diverse cellular processes although comprehensive comparative studies among multiple species have yet to be performed. To investigate conserved genomic features across various species that may be related to sense-antisense regulation, we performed comparative analysis of approximately 1000-2000 cis-encoded antisense RNA pairs from five model eukaryotes (Homo sapiens, Mus musculus, Drosophila melanogaster, Arabidopsis thaliana, and Oryza sativa). Analysis of overlapping patterns relative to the exon-intron structure revealed that the number of pairs sharing the 3′ part of the transcripts was larger than that of the 5′-sharing pairs except in rice. Moreover, most of the well-conserved sense-antisense pairs between human and mouse exhibited 3′-overlaps, suggesting that regulatory mechanisms involving these regions may be important in sense-antisense transcription. Functional classification using Gene Ontology revealed that genes related to catalytic activity, nucleotide binding, DNA metabolism, and mitochondria were preferentially distributed within the set of exon-overlapping sense-antisense genes compared to the non-exon-overlapping group in animals. Despite the numerous sense-antisense pairs identified in human and mouse individually, the number of conserved pairs was extremely small (6.6% of the entire set). Whereas both genes of most of the conserved sense-antisense pairs had protein-coding potential, nearly half of the non-conserved pairs included a non-coding RNA, suggesting that non-coding sense-antisense RNAs may function in species-specific regulatory pathways. © 2006 Elsevier B.V. All rights reserved.

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The Hsp70 chaperone system maintains high concentrations of active proteins and suppresses ATP consumption during heat shock

Hu, B. and Tomita, M.

Systems and Synthetic Biology 1 ( 1 ) 47-58 - 58 2007

Research paper (scientific journal), Joint Work, ISSN 18725325

Hsp70 chaperones assist protein folding by cycling between the ATP-bound T state with low affinity for substrates and the ADP-bound R state with high affinity for substrates. The transition from the T to R state is catalyzed by the synergistic action of the substrate and DnaJ cochaperones. The reverse transition from the R state to the T state is accelerated by the nucleotide exchange factor GrpE. These two processes, T-to-R and R-to-T conversion, are affected differently by temperature change. Here we modeled Hsp70-mediated protein folding under permanent and transient heat shock based on published experimental data. Our simulation results were in agreement with in vitro wild-type Escherichia coli chaperone experimental data at 25°C and reflected R-to-T ratio dynamics in response to temperature effects. Our simulation results suggested that the chaperone system evolved naturally to maintain the concentration of active protein as high as possible during heat shock, even at the cost of recovered activity after return to optimal growth conditions. They also revealed that the chaperone system evolved to suppress ATP consumption at non-optimal high growing temperatures. © Springer Science + Business Media B.V. 2007.

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The GC skew index: a measure of genomic compositional asymmetry and the degree of replicational selection

Arakawa, K. and Tomita, M.

Evolutionary Bioinformatics 3 159 - 168 2007

Research paper (scientific journal), Joint Work

Circular bacterial chromosomes have highly polarized nucleotide composition in the two replichores, and this genomic strand asymmetry can be visualized using GC skew graphs. Here we propose and discuss the GC skew index (GCSI) for the quantifi cation of genomic compositional skew, which combines a normalized measure of fast Fourier transform to capture the shape of the skew graph and Euclidean distance between the two vertices in a cumulative skew graph to represent the degree of skew. We calculated GCSI for all available bacterial genomes, and GCSI correlated well with the visibility of GC skew. This novel index is useful for estimating confi dence levels for the prediction of replication origin and terminus by methods based on GC skew and for measuring the strength of replicational selection in a genome.

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Robust effects of Tsr-CheBp and CheA-CheYp affinity in bacterial chemotaxis

Matsuzaki, Y., Kikuchi, S. and Tomita, M.

Artificial intelligence in medicine 41 ( 2 ) 145-150 - 150 2007

Research paper (scientific journal), Joint Work, ISSN 09333657

Objective: Cell motility and chemotaxis play a role in the virulence of pathogenic bacteria, such as escape from host immune responses. Escherichia coli chemotaxis provides a well-characterized model system for the bacterial chemotaxis network. Two features of E. coli chemotaxis include signal amplification and robustly accurate adaptation. Recent simulation studies with models considering the effects of other receptors have suggested possible mechanisms for signal amplification. Although precise adaptation to aspartate has been explained by conventional kinetic models, the adaptation behavior of models incorporating the effects of other receptors remains unclear. Methods: We concentrated on how receptor crosstalk affects minimization of adaptation error and compared models in which the contribution of other receptors varied. Results: We demonstrated that the model is adaptable to attractant concentrations ranging from 0.1μ M to 10 mM with a decreased error rate (from 8% to 2%) when the kinetic constant of CheA and phosphorylated CheY dissociation is increased. Conclusion: The results suggest that accurate adaptation is maintained through control of both the interaction of cytoplasmic Che proteins and the activity of the receptor complex. © 2007 Elsevier B.V. All rights reserved.

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Microelectrospray interface with coaxial sheath flow for high-resolution capillary electrophoresis/mass spectrometry separation

Hashimoto, M., Ishihama, Y., Tomita, M. and Soga, T.

Rapid Communications in Mass Spectrometry 21 ( 22 ) 3579 - 3584 - 3584 2007

Research paper (scientific journal), Joint Work, ISSN 09514198

We have fabricated a coaxial sheath liquid flow microelectrospray ionization (μESI) interface for capillary electrophoresis coupled with mass spectrometry (CE/MS). The ESI interface, which features a reduced probe diameter (130 μm i.d. x 174 μm o.d.) with a nebulizer-free format, can relatively easily electrospray a large amount of make-up sheath liquid (5-10 μL/min) over the long term (more than 80 runs) with a high degree of stability. The interface also provides higher separation qualities and improved detection sensitivities compared with a conventional ion spray (IS) interface. Copyright © 2007 John Wiley & Sons, Ltd.

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Permuted tRNA Genes Expressed via a Circular RNA Intermediate in Cyanidioschyzon merolae

Soma, A., Onodera, A., Sugahara, J., Kanai, A., Yachie, N., Tomita, M., Kawamura, F., Sekine, Y.

Science 318 ( 5849 ) 450-453 - 453 2007

Research paper (scientific journal), Joint Work, ISSN 00368075

A computational analysis of the nuclear genome of a red alga, Cyanidioschyzon merolae, identified 11 transfer RNA (tRNA) genes in which the 3′ half of the tRNA lies upstream of the 5′ half in the genome. We verified that these genes are expressed and produce mature tRNAs that are aminoacylated. Analysis of tRNA-processing intermediates for these genes indicates an unusual processing pathway in which the termini of the tRNA precursor are ligated, resulting in formation of a characteristic circular RNA intermediate that is then processed at the acceptor stem to generate the correct termini.

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Model based definition of population heterogeneity and its effects on metabolism in sporulating Bacillus subtilis

Morohashi, M., Ohashi, Y., Tani, S., Ishii, K., Itaya, M., Nanamiya, H., Kawamura, F., Tomita, M. and Soga, T.

Journal of Biochemistry 142 ( 2 ) 183-191 - 191 2007

Research paper (scientific journal), Joint Work, ISSN 0021924X

The soil bacterium Bacillus subtilis forms dormant, robust spores as a tactic to ensure survival under conditions of starvation. However, the sporulating culture includes sporulating and non-sporulating cells, because a portion of the cell population initiates sporulation in wild-type strain. We anticipated that the population effect must be considered carefully to analyse samples yielding population heterogeneity. We first built a mathematical model and simulated for signal transduction of the sporulation cue to see what mechanisms are responsible for generating the heterogeneity. The simulated results were confirmed experimentally, where heterogeneity is primarily modulated by negative feedback circuits, resulting in generation of a bistable response within the sporulating culture. We also confirmed that mutants relevant to negative feedback yield either sporulating or non-sporulating subpopulations. To see the effect of molecular mechanism between sporulating and non-sporulating cells in distinct manner, metabolome analysis was conducted using the above mutants. The metabolic profiles exhibited distinct characteristics with time regardless of whether sporulation was initiated or not. In addition, several distinct characteristics of metabolites were observed between strains, which was inconsistent with previously reported data. The results imply that careful consideration must be made in the interpretation of data obtained from cells yielding population heterogeneity. © 2007 The Japanese Biochemical Society.

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Variation in the Correlation of G + C Composition with Synonymous Codon Usage Bias among Bacteria

Suzuki H, Saito R, Tomita M.

EURASIP J Bioinform Syst Biol. 2007 ( 61374 ) 2007

Research paper (scientific journal), Joint Work, ISSN 16874145

G + C composition at the third codon position (GC3) is widely reported to be correlated with synonymous codon usage bias. However, no quantitative attempt has been made to compare the extent of this correlation among different genomes. Here, we applied Shannon entropy from information theory to measure the degree of GC3 bias and that of synonymous codon usage bias of each gene. The strength of the correlation of GC3 with synonymous codon usage bias, quantified by a correlation coefficient, varied widely among bacterial genomes, ranging from -0.07 to 0.95. Previous analyses suggesting that the relationship between GC3 and synonymous codon usage bias is independent of species are thus inconsistent with the more detailed analyses obtained here for individual species.

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Multiresolution analysis uncovers hidden conservation of properties in structurally and functionally similar protiens

Chua G.H., Krishnan A., Li K.B., Tomita M.

Journal of Bioinformatics and Computational Biology 4 ( 6 ) 1245 - 1267 2006.12

ISSN 02197200

Physicochemcial properties of amino acids are important factors in determining protein structure and function. Most approaches make use of averaged properties over entire domains or even proteins to analyze their structure or function. This level of coarseness tends to hide the richness of the variability in the different properties across functional domains. This paper studies the conservation of physicochemical properties in a functionally similar family of proteins using a novel wavelet-based technique known as multiresolution analysis. Such an analysis can help uncover characteristics that can otherwise remain hidden. We have studied the protein kinase family of sequences and our findings are as follows: (a) a number of different properties are conserved over the functional catalytic domain irrespective of the sequence identities; (b) conservation of properties can be observed at different frequency levels and they agree well with the known structural/ functional properties of the subdomains for the protein kinase family; (c) structural differences between the different kinase family members are reflected in the waveforms; and (d) functionally important mutations show distortions in the waveforms of conserved properties. The potential usefulness of the above findings in identifying functionally similar sequences in the twilight and midnight zones is demonstrated through a simple prediction model for the protein kinase family which achieved a recall of 93.7% and a precision of 96.75% in cross-validation tests. © Imperial College Press.

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Prediction of liquid chromatographic retention times of peptides generated by protease digestion of the Escherichia coli proteome usina artificial neural networks

Shinoda K., Sugimoto M., Yachie N., Sugiyama N., Masuda T., Robert M., Soga T., Tomita M.

Journal of Proteome Research 5 ( 12 ) 3312 - 3317 2006.12

ISSN 15353893

We developed a computational method to predict the retention times of peptides in HPLC using artificial neural networks (ANN). We performed stepwise multiple linear regressions and selected for ANN input amino acids that significantly affected the LC retention time. Unlike conventional linear models, the trained ANN accurately predicted the retention time of peptides containing up to 50 amino acid residues. In 834 peptides, there was a strong correlation (R2= 0.928) between measured and predicted retention times. We demonstrated the utility of our method by the prediction of the retention time of 121 273 peptides resulting from LysC-digestion of the Escherichia coli proteome. Our approach is useful for the proteome-wide characterization of peptides and the identification of unknown peptide peaks obtained in proteome analysis. © 2006 American Chemical Society.

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HybGFS: a hybrid method for genome-fingerprint scanning

Shinoda, Kosaku ; Yachie, Nozomu ; Masuda, Takeshi ; Sugiyama, Naoyuki ; Sugimoto, Masahiro ; Soga, Tomoyoshi ; Tomita, Masaru

BMC Bioinformatics 7 8 2006.10

Research paper (scientific journal), Joint Work, ISSN 1471-2105

Background: Protein identification based on mass spectrometry (MS) has previously been performed using peptide mass fingerprinting (PMF) or tandem MS (MS/MS) database searching. However, these methods cannot identify proteins that are not already listed in existing databases. Moreover, the alternative approach of de novo sequencing requires costly equipment and the interpretation of complex MS/MS spectra. Thus, there is a need for novel high-throughput protein-identification methods that are independent of existing predefined protein databases. Results: Here, we present a hybrid method for genome-fingerprint scanning, known as HybGFS. This technique combines genome sequence-based peptide MS/MS ion searching with liquid-chromatography elution-time (LC-ET) prediction, to improve the reliability of identification. The hybrid method allows the simultaneous identification and mapping of proteins without a priori information about their coding sequences. The current study used standard LC-MS/MS data to query an in silico-generated six-reading-frame translation and the enzymatic digest of an entire genome. Used in conjunction with precursor/product ion-mass searching, the LC-ETs increased confidence in the peptide-identification process and reduced the number of false-positive matches. The power of this method was demonstrated using recombinant proteins from the Escherichia coli K12 strain. Conclusion: The novel hybrid method described in this study will be useful for the large-scale experimental confirmation of genome coding sequences, without the need for transcriptome-level expression analysis or costly MS database searching. © 2006 Shinoda et al; licensee BioMed Central Ltd.

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Evaluation of metabolic alteration in transgenic rice overexpressing dihydroflavonol-4-reductase

Takahashi, Hideyuki ; Hayashi, Mitsunori ; Goto, Fumiyuki ; Sato, Shigeru ; Soga, Tomoyoshi ; Nishioka, Takaaki ; Tomita, Masaru ; Kawai-Yamada, Maki ; Uchimiya, Hirofumi

Annals of Botany 98 ( 4 ) 819-825 - 825 2006.10

Research paper (scientific journal), Joint Work, ISSN 0305-7364

Background and Aims: Previous studies have shown that transgenic rice plants overexpressing YK1, which possesses dihydroflavonol-4-reductase (DFR) activity, showed biotic and abiotic stress tolerance. High throughput profiles of metabolites have also been shown in such transgenic plants by Fourier transform ion cyclotron mass spectrometry. In this study, capillary electrophoresis mass spectrometry analysis (CE/MS) was employed to identify precise metabolites such as organic acids, amino acids and sugars. Methods: Using CE/MS, we analysed several metabolites of glycolysis, the tricarboxylic acid (TCA) cycle and the pentose phosphate pathway. In addition, the concentrations of sugars and ion were quantified. Key Results: In YK1 (DFR)-overexpressing plants, the concentrations of cis-aconitate, isocitrate and 2-oxoglutarate were higher in leaves, whereas those of fructose-1,6- bisphosphate and glyceraldehyde-3-phosphate were lower in roots. In seeds, the amounts of free amino acids and metals were altered, whereas sugars in seeds were kept constant. In YK1 calli, an approx. 3-fold increase in glutathione was observed, whereas the activities of glutathione peroxidase and glutathione reductase were concomitantly increased. Conclusions: The overexpression of YK1 (DFR) was associated with slight changes in the amounts of several metabolites analysed in whole plants, whilst glutathione derivatives were substantially increased in suspension-cultured cells. © The Author 2006. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.

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Over-representation of Chi sequences caused by di-codon increase in Escherichia coli K-12

Uno, Reina ; Nakayama, Yoichi ; Tomita, Masaru

Gene 380 ( 1 ) 30-37 - 37 2006.09

Research paper (scientific journal), Joint Work, ISSN 0378-1119

Chi sequences (5′-GCTGGTGG-3′) are cis-acting 8 bp sequence elements that enhance homologous recombination promoted by the RecBCD pathway in Escherichia coli. The genome of E. coli K-12 MG1655 contains 1009 Chi sequences and this frequency far exceeds the expected value for occurrence of an 8 bp sequence in a genome of this size. It is generally thought that the over-representation of Chi sequences indicates that they have been selected for during evolution because of their function in recombination. The genes from three E. coli strains (K-12, O157 and CFT) were classified into three categories (island, match to other E. coli, and backbone). Island genes have a different base composition and codon usage in comparison with those in the backbone genes, therefore they were relatively new and not yet adapted to the base composition patterns and codon usage typical of the recipient genome. The over-representation of Chi sequences was examined by comparing Chi frequencies and codon frequencies between island and backbone genes. The difference in the CTGGTG di-codon frequency between the backbone and island genes was correlated with the frequency of Chi sequences which were translated in the Leu-Val (-G|CTG|GTG|G-) reading frame in the K-12 strain. These results suggest that the main reading frame of Chi sequences increased as a result of the di-codon CTG-GTG increasing under a genome-wide pressure for adapting to the codon usage and base composition of the E. coli K-12 strain, and that the RecBCD recombinase might adjust its recognition sequence to a frequently occurring oligomer such as G-CTG-GTG-G. © 2006 Elsevier B.V. All rights reserved.

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Metabolomics approach for enzyme discovery

Saito, Natsumi ; Robert, Martin ; Kitamura, Sayaka ; Baran, Richard ; Soga, Tomoyoshi ; Mori, Hirotada ; Nishioka, Takaaki ; Tomita, Masaru

Journal of Proteome Research 5 ( 8 ) 1979-1987 - 1987 2006.08

Research paper (scientific journal), Joint Work, ISSN 1535-3893

The search for novel enzymes is an important but difficult task in functional genomics. Here, we present a systematic method based on in vitro assays in combination with metabolite profiling to discover novel enzymatic activities. A complex mixture of metabolites is incubated with purified candidate proteins and the reaction mixture is subsequently profiled by capillary electrophoresis electrospray ionization mass spectrometry (CE-MS). Specific changes in the metabolite composition can directly suggest the presence of an enzymatic activity while subsequent identification of the compounds whose level changed specifically can pinpoint the actual substrate(s) and product(s) of the reaction. We first evaluated the method using several Escherichia coli metabolic enzymes and then applied it to the functional screening of uncharacterized proteins. In this manner, YbhA and YbiV proteins were found to display both phosphotransferase and phosphatase activity toward different sugars/sugar phosphates. Our approach should be broadly applicable and useful for enzyme discovery in any system. © 2006 American Chemical Society.

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A simplified method for power-law modelling of metabolic pathways from time-course data and steady-state flux profiles

Kitayama, Tomoya ; Kinoshita, Ayako ; Sugimoto, Masahiro ; Nakayama, Yoichi ; Tomita, Masaru

Theor Biol Med Model 3 24 2006.07

Research paper (scientific journal), Joint Work, ISSN 1742-4682

Background: In order to improve understanding of metabolic systems there have been attempts to construct S-system models from time courses. Conventionally, non-linear curve-fitting algorithms have been used for modelling, because of the non-linear properties of parameter estimation from time series. However, the huge iterative calculations required have hindered the development of large-scale metabolic pathway models. To solve this problem we propose a novel method involving power-law modelling of metabolic pathways from the Jacobian of the targeted system and the steady-state flux profiles by linearization of S-systems. Results: The results of two case studies modelling a straight and a branched pathway, respectively, showed that our method reduced the number of unknown parameters needing to be estimated. The time-courses simulated by conventional kinetic models and those described by our method behaved similarly under a wide range of perturbations of metabolite concentrations. Conclusion: The proposed method reduces calculation complexity and facilitates the construction of large-scale S-system models of metabolic pathways, realizing a practical application of reverse engineering of dynamic simulation models from the Jacobian of the targeted system and steady-state flux profiles. © 2006 Kitayama et al; licensee BioMed Central Ltd.

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Modeling Hsp70-mediated Protein Folding

Hu B, Mayer MP, Tomita M.

Biophysical Journal 91 ( 2 ) 496-507 - 507 2006.07

Research paper (scientific journal), Joint Work, ISSN 00063495

The Hsp70 chaperone system is the major molecular chaperone system that assists protein-folding processes in all cells. To understand these processes, we analyzed the kinetic characteristics of the Escherichia coli homologs of this chaperone system during folding of a denatured protein using computer simulations and compared the results with in vitro refolding experiments. Rate constants used for the model were derived from recent literature or were determined and scrutinized for their applicability to the refolding reaction. Our simulation results are consistent with reported laboratory experiments, not only simulating the refolding reaction of wild-type proteins but also the behavior of mutant variants. Variation of kinetic parameters and concentrations of components of the Hsp70 system demonstrate the robustness of the chaperone system in assisting protein folding. Furthermore, the importance of the synergistic stimulation of the ATPase activity of Hsp70 is demonstrated. The limitations of our kinetic model indicate sore spots in our understanding of this chaperone system. Our model provides a platform for further research on chaperone action and the mechanism of chaperone-assisted refolding of denatured proteins. © 2006 by the Biophysical Society.

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Comparative analysis of base correlations in 5′ untranslated regions of various species

Osada Y., Saito R., Tomita M.

Gene 375 ( 1-2 ) 80 - 86 2006.06

ISSN 03781119

Translational initiation signals, such as Shine-Dalgarno (SD) sequences in bacteria and Kozak consensus sequences in vertebrates, direct ribosomes to initiate protein synthesis from mRNAs. Investigating sequence characteristics of these signals is important, particularly to infer translational initiation mechanisms. Although various statistical analyses of translational initiation signals have been done, few have focused on base correlations that assess base dependencies in the signal sequences. We used relative entropy and mutual information to analyze base conservation and correlation, respectively, in the 5′ UTRs of various species. In eukaryotes, we found peaks of relative entropy at - 3 from the translational start site but no peak of mutual information at that position, indicating that the base at that position (known as the core base of the Kozak sequence) is well conserved but not correlated with neighboring bases and thus functions as a single base. We observed unexpected peaks of mutual information between positions - 2 and - 1 in most eukaryotes. Surprisingly these base correlation also occurred in some bacteria and archaea, although there were no base preferences at neither position. Various dinucleotide patterns existed at these positions, and the correlation between bases at - 2 and - 1 may be relevant to the context of translational initiation. Because dinucleotide patterns of correlated pairs of nucleotides at - 2 and - 1 were not unique within respective organisms, the correlation could not be found when analyzing single-nucleotide conservation. Therefore, mutual information allowed us to discover signals that were not found by simply analyzing base conservation. © 2006 Elsevier B.V. All rights reserved.

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Differential metabolomics reveals ophthalmic acid as an oxidative stress biomarker indicating hepatic glutathione consumption

Soga, Tomoyoshi; Baran, Richard; Suematsu, Makoto; Ueno, Yuki; Ikeda, Satsuki; Sakurakawa, Tadayuki; Kakazu, Yuji; Ishikawa, Takamasa; Robert, Martin; Nishioka, Takaaki; Tomita, Masaru

Journal of Biological Chemistry 281 ( 24 ) 16768-16776 - 16776 2006.06

Research paper (scientific journal), Joint Work, ISSN 0021-9258

Metabolomics is an emerging tool that can be used to gain insights into cellular and physiological responses. Here we present a metabolome differential display method based on capillary electrophoresis time-of-flight mass spectrometry to profile liver metabolites following acetaminophen-induced hepatotoxicity. We globally detected 1,859 peaks in mouse liver extracts and highlighted multiple changes in metabolite levels, including an activation of the ophthalmate biosynthesis pathway. We confirmed that ophthalmate was synthesized from 2-aminobutyrate through consecutive reactions with γ-glutamylcysteine and glutathione synthetase. Changes in ophthalmate level in mouse serum and liver extracts were closely correlated and ophthalmate levels increased significantly in conjunction with glutathione consumption. Overall, our results provide a broad picture of hepatic metabolite changes following acetaminophen treatment. In addition, we specifically found that serum ophthalmate is a sensitive indicator of hepatic GSH depletion, and may be a new biomarker for oxidative stress. Ourmethod can thus pinpoint specific metabolite changes and provide insights into the perturbation of metabolic pathways on a large scale and serve as a powerful new tool for discovering low molecular weight biomarkers. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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Prediction of non-coding and antisense RNA genes in Escherichia coli with Gapped Markov Model

Yachie, Nozomu ; Numata, Koji ; Saito, Rintaro ; Kanai, Akio ; Tomita, Masaru

Gene 372 ( 1-2 ) 171-181 - 181 2006.05

Research paper (scientific journal), Joint Work, ISSN 0378-1119

A new mathematical index was developed to identify and characterize non-coding RNA (ncRNA) genes encoded within the Escherichia coli (E. coli) genome. It was designated the GMMI (Gapped Markov Model Index) and used to evaluate sequence patterns located at the separate positions of consensus sequences, codon biases and/or possible RNA structures on the basis of the Markov model. The GMMI was able to separate a set of known mRNA sequences from a mixture of ncRNAs including tRNAs and rRNAs. Consequently, the GMMI was employed to predict novel ncRNA candidates. At the beginning, possible transcription units were extracted from the E. coli genome using consensus sequences for the sigma70 promoter and the rho-independent terminator. Then, these units were evaluated by using the GMMI. This identified 133 candidate ncRNAs, which contain 29 previously annotated small RNA genes and 46 possible antisense ncRNAs. Furthermore 12 transcripts (including five antisense RNAs) were confirmed according to the expression analysis. These data suggests that the expression of small antisense RNAs might be more common than previously thought in the E. coli genome. © 2006 Elsevier B.V. All rights reserved.

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Large-scale identification of protein-protein interaction of Escherichia coli K-12

Arifuzzaman, M. ; Maeda, M. ; Itoh, A. ; Nishikata, K. ; Takita, C. ; Saito, R. ; Ara, T. ; Nakahigashi, K. ; Huang, H. C. ; Hirai, A. ; Tsuzuki, K. ; Nakamura, S. ; Altaf-Ul-Amin, M. ; Oshima, T. ; Baba, T. ; Yamamoto, N. ; Kawamura, T. ; Ioka-Nakamichi, T. ; Kitagawa, M. ; Tomita, M. ; Kanaya, S. ; Wada, C. ; Mori, H. ; Tomita, M

Genome Research 16 ( 5 ) 686-691 - 691 2006.05

Research paper (scientific journal), Joint Work, ISSN 1088-9051

Protein-protein interactions play key roles in protein function and the structural organization of a cell. A thorough description of these interactions should facilitate elucidation of cellular activities, targeted-drug design, and whole cell engineering. A large-scale comprehensive pull-down assay was performed using a His-tagged Escherichia coli ORF clone library. Of 4339 bait proteins tested, partners were found for 2667, including 779 of unknown function. Proteins copurifying with hexahistidine-tagged baits on a Ni 2+-NTA column were identified by MALDI-TOF MS (matrix-assisted laser desorption ionization time of flight mass spectrometry). An extended analysis of these interacting networks by bioinformatics and experimentation should provide new insights and novel strategies for E. coli systems biology. ©2006 by Cold Spring Harbor Laboratory Press.

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Parameter estimation for stiff equations of biosystems using radial basis function networks

Matsubara, Yoshiya ; Kikuchi, Shinichi ; Sugimoto, Masahiro ; Tomita, Masaru

BMC Bioinformatics 7 11 2006.04

Research paper (scientific journal), Joint Work, ISSN 1471-2105

Background: The modeling of dynamic systems requires estimating kinetic parameters from experimentally measured time-courses. Conventional global optimization methods used for parameter estimation, e.g. genetic algorithms (GA), consume enormous computational time because they require iterative numerical integrations for differential equations. When the target model is stiff, the computational time for reaching a solution increases further. Results: In an attempt to solve this problem, we explored a learning technique that uses radial basis function networks (RBFN) to achieve a parameter estimation for biochemical models. RBFN reduce the number of numerical integrations by replacing derivatives with slopes derived from the distribution of searching points. To introduce a slight search bias, we implemented additional data selection using a GA that searches data-sparse areas at low computational cost. In addition, we adopted logarithmic transformation that smoothes the fitness surface to obtain a solution simply. We conducted numerical experiments to validate our methods and compared the results with those obtained by GA. We found that the calculation time decreased by more than 50% and the convergence rate increased from 60% to 90%. Conclusion: In this work, our RBFN technique was effective for parameter optimization of stiff biochemical models. © 2006 Matsubara et al; licensee BioMed Central Ltd.

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GEM System: automatic prototyping of cell-wide metabolic pathway models from genomes

Arakawa, Kazuharu ; Yamada, Yohei ; Shinoda, Kosaku ; Nakayama, Yoichi ; Tomita, Masaru

BMC Bioinformatics 7 11 2006.03

Research paper (scientific journal), Joint Work, ISSN 1471-2105

Background: Successful realization of a "systems biology" approach to analyzing cells is a grand challenge for our understanding of life. However, current modeling approaches to cell simulation are labor-intensive, manual affairs, and therefore constitute a major bottleneck in the evolution of computational cell biology. Results: We developed the Genome-based Modeling (GEM) System for the purpose of automatically prototyping simulation models of cell-wide metabolic pathways from genome sequences and other public biological information. Models generated by the GEM System include an entire Escherichia coli metabolism model comprising 968 reactions of 1195 metabolites, achieving 100% coverage when compared with the KEGG database, 92.38% with the EcoCyc database, and 95.06% with iJR904 genome-scale model. Conclusion: The GEM System prototypes qualitative models to reduce the labor-intensive tasks required for systems biology research. Models of over 90 bacterial genomes are available at our web site. © 2006 Arakawa et al; licensee BioMed Central Ltd.

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Characteristics and clustering of human ribosomal protein genes

Ishii, Kyota, Washio, Takanori, Uechi, Tamayo, Yoshihama, Maki, Kenmochi, Naoya, Tomita, Masaru

Bmc Genomics 7 37 2006.02

Research paper (scientific journal), Joint Work, ISSN 1471-2164

Background: The ribosome is a central player in the translation system, which in mammals consists of four RNA species and 79 ribosomal proteins (RPs). The control mechanisms of gene expression and the functions of RPs are believed to be identical. Most RP genes have common promoters and were therefore assumed to have a unified gene expression control mechanism. Results: We systematically analyzed the homogeneity and heterogeneity of RP genes on the basis of their expression profiles, promoter structures, encoded amino acid compositions, and codon compositions. The results revealed that (1) most RP genes are coordinately expressed at the mRNA level, with higher signals in the spleen, lymph node dissection (LND), and fetal brain. However, 17 genes, including the P protein genes (RPLP0, RPLP1, RPLP2), are expressed in a tissue-specific manner. (2) Most promoters have GC boxes and possible binding sites for nuclear respiratory factor 2, Yin and Yang 1, and/or activator protein 1. However, they do not have canonical TATA boxes. (3) Analysis of the amino acid composition of the encoded proteins indicated a high lysine and arginine content. (4) The major RP genes exhibit a characteristic synonymous codon composition with high rates of G or C in the third-codon position and a high content of AAG, CAG, ATC, GAG, CAC, and CTG. Conclusion: Eleven of the RP genes are still identified as being unique and did not exhibit at least some of the above characteristics, indicating that they may have unknown functions not present in other RP genes. Furthermore, we found sequences conserved between human and mouse genes around the transcription start sites and in the intronic regions. This study suggests certain overall trends and characteristic features of human RP genes. © 2006 Ishii et al; licensee BioMed Central Ltd.

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Archaeal Pyrococcus furiosus thymidylate synthase 1 is an RNA-binding protein

Kanai A., Sato A., Imoto J., Tomita M.

Biochemical Journal 393 ( 1 ) 373 - 379 2006.01

ISSN 02646021

Using a stem-loop RNA oligonucleotide (19-mer) containing an AUG sequence in the loop region as a probe, we screened the protein library from a hyperthermophilic archaeon, Pyrococcus furiosus, and found that a flavin-dependent thymidylate synthase, Pf-Thy1 (Pyrococcus furiosus thymidylate synthase 1), possessed RNA-binding activity. Recombinant Pf-Thy1 was able to bind to the stem-loop structure at a high temperature (75°C) with an apparent dissociation constant of 0.6 μM. A similar stem-loop RNA structure was located around the translation start AUG codon of Pf-Thy1 RNA, and gel-shift analysis revealed that Pf-Thy1 could also bind to this stem-loop structure. In vitro translation analysis using chimaeric constructs containing the stem-loop sequence in their Pf-Thy1 RNA and a luciferase reporter gene indicated that the stem-loop structure acted as an inhibitory regulator of translation by preventing the binding of its Shine-Dalgarno-like sequence by positioning it in the stem region. Addition of Pf-Thy1 into the in vitro translation system also inhibited translation. These results suggested that this class of thymidylate synthases may autoregulate their own translation in a manner analogous to that of the well characterized thymidylate synthase A proteins, although there is no significant amino acid sequence similarity between them. © 2006 Biochemical Society.

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Stage-specific expression of Caenorhabditis elegans ribonuclease H1 enzymes with different substrate specificities and bivalent cation requirements

Kochiwa H., Itaya M., Tomita M., Kanai A.

FEBS Journal 273 ( 2 ) 420 - 429 2006.01

ISSN 1742464X

Ribonuclease H1 (RNase H1) is a widespread enzyme found in a range of organisms from viruses to humans. It is capable of degrading the RNA moiety of DNA-RNA hybrids and requires a bivalent ion for activity. In contrast with most eukaryotes, which have one gene encoding RNase H1, the activity of which depends on Mg2+ ions, Caenorhabditis elegans has four RNase H1-related genes, and one of them has an isoform produced by alternative splicing. However, little is known about the enzymatic features of the proteins encoded by these genes. To determine the differences between these enzymes, we compared the expression patterns of each RNase H1-related gene throughout the development of the nematode and the RNase H activities of their recombinant proteins. We found gene-specific expression patterns and different enzymatic features. In particular, besides the enzyme that displays the highest activity in the presence of Mg2+ ions, C. elegans has another enzyme that shows preference for Mn2+ ion as a cofactor. We characterized this Mn 2+-dependent RNase H1 for the first time in eukaryotes. These results suggest that there are at least two types of RNase H1 in C. elegans depending on the developmental stage of the organism. © 2005 FEBS.

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Computational analysis of microRNA targets in Caenorhabditis elegans

Watanabe, Yuka, Yachie, Nozomu, Numata, Koji, Saito, Rintaro, Kanai, Akio, Tomita, Masaru

Gene 365 ( 1-2 SPEC. ISS. ) 2-10 - 10 2006.01

Research paper (scientific journal), Joint Work, ISSN 0378-1119

MicroRNAs (miRNAs) are endogenous ∼ 22-nucleotide (nt) non-coding RNAs that post-transcriptionally regulate the expression of target genes via hybridization to target mRNA. Using known pairs of miRNA and target mRNA in Caenorhabditis elegans, we first performed computational analysis for specific hybridization patterns between these two RNAs. We counted the numbers of perfectly complementary dinucleotide sequences and calculated the free energy within complementary base pairs of each dinucleotide, observed by sliding a 2-nt window along all nucleotides of the miRNA-mRNA duplex. We confirmed not only strong base pairing within the 5′ region of miRNAs (nts 1-8) in C. elegans, but also the required mismatch within the central region (nt 9 or nt 10), and we found weak binding within the 3′ region (nts 13-14). We also predicted 687 possible miRNA target transcripts, many of which are thought to be involved in C. elegans development, by combining the abovementioned hybridization tendency with the following analyses: (1) prediction of the miRNA-mRNA duplex with free-energy minimization; (2) identification of the complementary pattern within the miRNA-mRNA duplex; (3) conservation of target sites between C. elegans and C. briggsae, a related soil nematode; and (4) extraction of mRNA candidates with multiple target sites. Rigorous tests using shuffled miRNA controls supported these predictions. Our results suggest that miRNAs recognize their target mRNAs by their hybridization pattern and that many target mRNAs may be regulated through a combination of several specific miRNA target sites in C. elegans. © 2005 Elsevier B.V. All rights reserved.

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SPLITS: a new program for predicting split and intron-containing tRNA genes at the genome level

Sugahara, Junichi ; Yachie, Nozomu ; Sekine, Yasuhiko ; Soma, Akiko ; Matsui, Motomu ; Tomita, Masaru ; Kanai, Akio

In Silico Biology 6 ( 5 ) 411-418 - 418 2006

Research paper (scientific journal), Joint Work, ISSN 1386-6338

In the archaea, some tRNA precursors contain intron(s) not only in the anticodon loop region but also in diverse sites of the gene (intron-containing tRNA or cis-spliced tRNA). The parasite Nanoarchaeum equitans, a member of the Nanoarchaeota kingdom, creates functional tRNA from separate genes, one encoding the 5'-half and the other the 3'-half (split tRNA or trans-spliced tRNA). Although recent genome projects have revealed a huge amount of nucleotide sequence data in the archaea, a comprehensive methodology for intron-containing and split tRNA searching is yet to be established. We therefore developed SPLITS, which is aimed at searching for any type of tRNA gene and is especially focused on intron-containing tRNAs or split tRNAs at the genome level. SPLITS initially predicts the bulge-helix-bulge splicing motif (a well-known, required structure in archaeal pre-tRNA introns) to determine and remove the intronic regions of tRNA genes. The intron-removed DNA sequences are automatically queried to tRNAscan-SE. SPLITS can predict known tRNAs with single introns located at unconventional sites on the genes (100%, tRNAs with double introns (85.7%), and known split tRNAs (100%). Our program will be very useful for identifying novel tRNA genes after completion of genome projects. The SPLITS source code is freely downloadable at http://splits.iab.keio.ac.jp/. © 2006 IOS Press. All rights reserved.

Genome-wide screening of dioxin-responsive genes in fetal brain: bioinformatic and experimental approaches

Fujita, Hideki ; Samejima, Hazuki ; Kitagawa, Noriyuki ; Mitsuhashi, Takayuki ; Washio, Takanori ; Yonemoto, Junzo ; Tomita, Masaru ; Takahashi, Takao ; Kosaki, Kenjiro

Congenital Anomalies 46 ( 3 ) 135-143 - 143 2006

Research paper (scientific journal), Joint Work, ISSN 0914-3505

Many of the effects of dioxins, which are potent environmental pollutants and teratogens, are mediated through the aryl hydrocarbon receptor, also known as the dioxin receptor. The purpose of the present study was to characterize dioxin-responsive genes in a comprehensive manner using two complementary approaches: bioinformatic analysis and microarray analysis. First, we characterized the overall distribution of the cis-regulatory element for the dioxin-responsive element sequence (DRE) 'gcgtg' within putative promoter regions. We assembled the upstream sequences 10 kb from the transcription start site and evaluated their location and frequency in the human and mouse genomes. Second, we characterized the expression profile of mouse embryonic day 12 fetal brain exposed to 2,3,7,8-tetrarchlorodibenzo-p-dioxin. The distributions of 26 680 DREs among 2843 human genes and 98 711 DREs among 18 541 mouse genes were examined. In both species, the DREs tended to be located close to the transcription start site. Forty genes exhibited significant induction or repression following dioxin exposure in fetal mice. The set of genes exhibited a strong functional coherence, with statistically significant enrichment in organogenesis and the DNA-dependent regulation of transcription, according to Gene Ontology annotations. In both humans and mice, DREs were preferentially distributed close to transcription start sites. Evolutionary conservation of this unique DRE distribution pattern suggests that DREs may be involved in transcriptional regulation. In mice, prenatal dioxin exposure altered the expression of 10 transcription factors, many of which have been documented to play a role in organogenesis. These genes may represent potential mediators of dioxin's effects in fetal tissues. © 2006 The Authors.

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Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection

Baba, Tomoya ; Ara, Takeshi ; Hasegawa, Miki ; Takai, Yuki ; Okumura, Yoshiko ; Baba, Miki ; Datsenko, Kirill A. ; Tomita, Masaru ; Wanner, Barry L. ; Mori, Hirotada

Molecular Systems Biology 2 2006.0008. 2006

Research paper (scientific journal), Joint Work, ISSN 1744-4292

We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high-throughput studies, two independent mutants were saved for every deleted gene. These mutants - the 'Keio collection' - provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome-wide testing of mutational effects in a common strain background, E. coli K-12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (http://ecoli.aist-nara.ac.jp/). © 2006 EMBO and Nature Publishing Group.

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GPAC: benchmarking the sensitivity of genome informatics analysis to genome annotation completeness

Arakawa, Kazuharu ; Nakayama, Yoichi ; Tomita, Masaru

In Silico Biology 6 ( 1-2 ) 49-60 - 60 2006

Research paper (scientific journal), Joint Work, ISSN 1386-6338

In view of the recent explosion in genome sequence data, and the 200 or more complete genome sequences currently available, the importance of genome-scale bioinformatics analysis is increasing rapidly. However, computational genome informatics analyses often lack a statistical assessment of their sensitivity to the completeness of the functional annotation. Therefore, a pre-analysis method to automatically validate the sensitivity of computational genome analyses with regard to genome annotation completeness is useful for this purpose. In this report we developed the Gene Prediction Accuracy Classification (GPAC) test, which provides statistical evidence of sensitivity by repeating the same analysis for five different gene groups (classified according to annotation accuracy level), and for randomly sampled gene groups, with the same number of genes as each of the five classified groups. Variability in these results is then assessed, and if the results vary significantly with different data subsets, the analysis is considered "sensitive" to annotation completeness, and careful selection of data is advised prior to the actual in silico analysis. The GPAC test has been applied to the analyses of Sakai et al., 2001, and Ohno et al., 2001, and it revealed that the analysis of Ohno et al. was more sensitive to annotation completeness. It showed that GPAC could be employed to ascertain the sensitivity of an analysis. The GPAC benchmarking software is freely available in the latest G-language Genome Analysis Environment package, at http://www.g-language.org/. © 2006 IOS Press. All rights reserved.

MEGU: pathway mapping Web-service based on KEGG and SVG

Kono, Nobuaki ; Arakawa, Kazuharu ; Tomita, Masaru

In Silico Biology 6 ( 6 ) 621-625 - 625 2006

Research paper (scientific journal), Joint Work, ISSN 1386-6338

Scientific visualization is a key technology to enhance our understanding of huge masses of omics data, especially for cell-wide, high-throughput measurements of the physiological networks. We have developed a web application that visualizes complex omics data of multiple layers simultaneously, including transcriptome, proteome, and metabolome, onto an integrated pathway diagram derived by connecting the individual KEGG pathway maps; the mapped images are generated in Scalable Vector Graphics for easy editing by hand or with computer programs or drawing software. The web application is available at http://megu.iab.keio.ac.jp/. © 2006 IOS Press. All rights reserved.

Identification and expression analysis of putative mRNA-like non-coding RNA in Drosophila

Inagaki S., Numata K., Kondo T., Tomita M., Yasuda K., Kanai A., Kageyama Y.

Genes to Cells 10 ( 12 ) 1163 - 1173 2005.12

ISSN 13569597

One of the most surprising results to emerge from mammalian cDNA sequencing projects is that thousands of mRNA-like non-coding RNAs (ncRNAs) are expressed and constitute at least 10% of poly(A)+ RNAs. In most cases, however, the functions of these RNA molecules remain unclear. To clarify the biological significance of mRNA-like ncRNAs, we computationally screened 11 691 Drosophila melanogaster full-length cDNAs. After eliminating presumable protein-coding transcripts, 136 were identified as strong candidates for mRNA-like ncRNAs. Although most of these putative ncRNAs are found throughout the Drosophila genus, predicted amino acid sequences are not conserved even in related species, suggesting that these transcripts are actually non-coding RNAs. In situ hybridization analyses revealed that 35 of the transcripts are expressed during embryogenesis, of which 27 were detected only in specific tissues including the tracheal system, midgut primordial cells, visceral mesoderm, germ cells and the central and peripheral nervous system. These highly regulated expression patterns suggest that many mRNA-like ncRNAs play important roles in multiple steps of organogenesis and cell differentiation in Drosophila. This is the first report that the majority of mRNA-like ncRNAs in a model organism are expressed in specific tissues and cell types. © 2005 Blackwell Publishing Ltd.

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A problem in multivariate analysis of codon usage data and a possible solution

Suzuki H., Saito R., Tomita M.

FEBS Letters 579 ( 28 ) 6499 - 6504 2005.11

ISSN 00145793

Multivariate analyses are often used to identify major trends of variation in synonymous codon usage among genes. These analyses need to be performed on properly normalized codon usage data to avoid biases masking this synonymous variation, i.e., gene length, amino acid usage, and codon degeneracy; however, previous studies have failed to do so. In this paper, we demonstrate that the use of alternative normalized data (called 'relative adaptiveness' in the literature) can avoid all these biases and furthermore, can identify more trends of variation among genes, including GC-ending codon usage, GT-ending codon usage, and gene expression level. © 2005 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

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Dynamic responses of the intracellular metabolite concentrations of the wild type and pykA mutant Escherichia coli against pulse addition of glucose or NH<inf>3</inf> under those limiting continuous cultures

Hoque M., Ushiyama H., Tomita M., Shimizu K.

Biochemical Engineering Journal 26 ( 1 ) 38 - 49 2005.11

ISSN 1369703X

The dynamics of the intracellular metabolite concentrations were investigated for the wild type and pykA gene knockout mutant Escherichia coli in responses to the glucose pulse addition during glucose-limited continuous culture and in responses to the ammonia pulse addition during ammonia-limited continuous culture. For this, we developed a new automated rapid sampling device, which enables us to take samples rapidly within a second. The intracellular concentrations of G6P, F6P, 2PG, PEP, OAA, 6PG, Ribu5P, E4P and NADPH were higher for pykA mutant as compared with the wild type under both limited continuous cultures, and the concentrations of PYR, ATP and acetate were much lower for pykA mutant than those of the wild type. These phenomena reflected the fact that the accumulation of PEP caused the increased flux from PEP to OAA and that the accumulated PEP inhibited pfk, which caused the accumulation of G6P and F6P, which in turn increased the flux toward pentose phosphate (PP) pathway and increased the PP pathway metabolite concentrations. Oxygen uptake rate (OUR) was a little higher for pykA mutant as compared with that of its wild type, while CO2 production rate (CER) shows the reverse trend. OUR and CER were much less for NH3-limited condition than that of NH3-rich condition. The intracellular concentrations of PEP, ATP and PYR decreased rapidly within several seconds, whereas the concentrations of G6P, F6P FBP, 6PG, ADP, NADH and NADPH increased after glucose pulse addition during glucose-limited condition for both wild type and pykA knockout mutant. Initial decrease in PEP concentration was considered to be due to PTS system. The intracellular concentration of NADPH decreased after NH 3 pulse addition under NH3-limited condition for both strains, which is due to the fact that NADPH is utilized through glutamate production under NH3 addition. © 2005 Elsevier B.V. All rights reserved.

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A comprehensive software suite for the analysis of cDNAs

Arakawa K., Suzuki H., Fujishima K., Fujimoto K., Ueda S., Matsui M., Tomita M.

Genomics, Proteomics and Bioinformatics 3 ( 3 ) 179 - 188 2005.08

ISSN 16720229

We have developed a comprehensive software suite for bioinformatics research of cDNAs; it is aimed at rapid characterization of the features of genes and the proteins they code. Methods implemented include the detection of translation initiation and termination signals, statistical analysis of codon usage, comparative study of amino acid composition, comparative modeling of the structures of product proteins, prediction of alternative splice forms, and metabolic pathway reconstruction.

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All systems go: launching cell simulation fueled by integrated experimental biology data

Arita, M; Robert, M; Tomita, M

Current Opinion in Biotechnology 16 ( 3 ) 344-349 - 349 2005.06

Research paper (scientific journal), Joint Work, ISSN 09581669

Biological simulation serves to unify the basic elements of systems biology, namely, model selection, experimentation and model refinement. To select biochemical models for simulation, metabolome analysis can be performed using capillary electrophoresis or liquid chromatography coupled with mass spectrometry. In this manner, selected models can be elaborated with temporal/spatial gene and protein expression data obtained from model organisms such as Escherichia coli. The E. coli single gene deletion mutant library (KO collection) and His-tag/GFP-fusion single open reading frame clone expression library (ASKA) are powerful resources for this task. The integration of parallel experimental datasets into dynamic simulation tools forms the remaining challenge for the systematic analysis and elucidation of biological networks and holds promise for biotechnological applications. © 2005 Elsevier Ltd. All rights reserved.

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TheSCO2299gene fromStreptomyces coelicolorA3(2) encodes a bifunctional enzyme consisting of an RNase H domain and an acid phosphatase domain

Ohtani, Naoto Saito, Natsumi Tomita, Masaru Itaya, Mitsuhiro Itoh, Aya

FEBS Journal 272 ( 11 ) 2828-2837 - 2837 2005.06

Research paper (scientific journal), Joint Work, ISSN 1742464X

The SCO2299 gene from Streptomyces coelicolor encodes a single peptide consisting of 497 amino acid residues. Its N-terminal region shows high amino acid sequence similarity to RNase HI, whereas its C-terminal region bears similarity to the CobC protein, which is involved in the synthesis of cobalamin. The SCO2299 gene suppressed a temperature-sensitive growth defect of an Escherichia coli RNase H-deficient strain, and the recombinant SCO2299 protein cleaved an RNA strand of RNA-DNA hybrid in vitro. The N-terminal domain of the SCO2299 protein, when overproduced independently, exhibited RNase H activity at a similar level to the full length protein. On the other hand, the C-terminal domain showed no CobC-like activity but an acid phosphatase activity. The full length protein also exhibited acid phosphatase activity at almost the same level as the C-terminal domain alone. These results indicate that RNase H and acid phosphatase activities of the full length SCO2299 protein depend on its N-terminal and C-terminal domains, respectively. The physiological functions of the SCO2299 gene and the relation between RNase H and acid phosphatase remain to be determined. However, the bifunctional enzyme examined here is a novel style in the Type 1 RNase H family. Additionally, S. coelicolor is the first example of an organism whose genome contains three active RNase H genes. © 2005 FEBS.

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Space in systems biology of signaling pathways--towards intracellular molecular crowding in silico

Takahashi, Kouichi Arjunan, Satya Nanda Vel Tomita, Masaru

Febs Letters 579 ( 8 ) 1783-1788 2005.03

Research paper (scientific journal), Joint Work, ISSN 00145793

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GC-compositional strand bias around transcription start sites in plants and fungi

Fujimori S., Washio T., Tomita M.

BMC Genomics 6 2005.02

ISSN 14712164

Background: A GC-compositional strand bias or GC-skew (=(C-G)/(C+G)), where C and G denote the numbers of cytosine and guanine residues, was recently reported near the transcription start sites (TSS) of Arabidopsis genes. However, it is unclear whether other eukaryotic species have equally prominent GC-skews, and the biological meaning of this trait remains unknown. Results: Our study confirmed a significant GC-skew (C > G) in the TSS of Oryza sativa (rice) genes. The full-length cDNAs and genomic sequences from Arabidopsis and rice were compared using statistical analyses. Despite marked differences in the G+C content around the TSS in the two plants, the degrees of bias were almost identical. Although slight GC-skew peaks, including opposite skews (C < G), were detected around the TSS of genes in human and Drosophila, they were qualitatively and quantitatively different from those identified in plants. However, plant-like GC-skew in regions upstream of the translation initiation sites (TIS) in some fungi was identified following analyses of the expressed sequence tags and/or genomic sequences from other species. On the basis of our dataset, we estimated that >70 and 68% of Arabidopsis and rice genes, respectively, had a strong GC-skew (>0.33) in a 100-bp window (that is, the number of C residues was more than double the number of G residues in a +/-100-bp window around the TSS). The mean GC-skew value in the TSS of highly-expressed genes in Arabidopsis was significantly greater than that of genes with low expression levels. Many of the GC-skew peaks were preferentially located near the TSS, so we examined the potential value of GC-skew as an index for TSS identification. Our results confirm that the GC-skew can be used to assist the TSS prediction in plant genomes. Conclusion: The GC-skew (C > G) around the TSS is strictly conserved between monocot and eudicot plants (ie. angiosperms in general), and a similar skew has been observed in some fungi. Highly-expressed Arabidopsis genes had overall a more marked GC-skew in the TSS compared to genes with low expression levels. We therefore propose that the GC-skew around the TSS in some plants and fungi is related to transcription. It might be caused by mutations during transcription initiation or the frequent use of transcription factor-biding sites having a strand preference. In addition, GC-skew is a good candidate index for TSS prediction in plant genomes, where there is a lack of correlation among CpG islands and genes. © 2005 Fujimori et al; licensee BioMed Central Ltd.

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Large-scale prediction of cationic metabolite identity and migration time in capillary electrophoresis mass spectrometry using artificial neural networks

Sugimoto M., Kikuchi S., Arita M., Soga T., Nishioka T., Tomita M.

Analytical Chemistry 77 ( 1 ) 78 - 84 2005.01

ISSN 00032700

We developed a computational technique to assist in the huge-scale identification of charged metabolites. The electrophoretic mobility of metabolites in capillary electrophoresis-mass spectrometry (CE-MS) was predicted from their structure, using an ensemble of artificial neural networks (ANNs). Comparison between relative migration times of 241 various cations measured by CE-MS and predicted by a trained ANN ensemble produced a correlation coefficient of 0.931. When we used our technique to characterize all metabolites listed in the KEGG ligand database, the correct compounds among the top three candidates were predicted in 78.0% of cases. We suggest that this approach can be used for the prediction of the migration time of any cation and that it represents a powerful method for the identification of un characterized CE-MS peaks in metabolome analysis.

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A microarray data-based semi-kinetic method for predicting quantitative dynamics of genetic networks

Yugi K, Nakayama Y, Kojima S, Kitayama T, Tomita M.

BMC Bioinformatics 6 ( 299 ) 2005

Research paper (scientific journal), Joint Work, ISSN 14712105

Background: Elucidating the dynamic behaviour of genetic regulatory networks is one of the most significant challenges in systems biology. However, conventional quantitative predictions have been limited to small networks because publicly available transcriptome data has not been extensively applied to dynamic simulation. Results: We present a microarray data-based semi-kinetic (MASK) method which facilitates the prediction of regulatory dynamics of genetic networks composed of recurrently appearing network motifs with reasonable accuracy. The MASK method allows the determination of model parameters representing the contribution of regulators to transcription rate from time-series microarray data. Using a virtual regulatory network and a Saccharomyces cerevisiae ribosomal protein gene module, we confirmed that a MASK model can predict expression profiles for various conditions as accurately as a conventional kinetic model. Conclusions: We have demonstrated the MASK method for the construction of dynamic simulation models of genetic networks from time-series microarray data, initial mRNA copy number and first-order degradation constants of mRNA. The quantitative accuracy of the MASK models has been confirmed, and the results indicated that this method enables the prediction of quantitative dynamics in genetic networks composed of commonly used network motifs, which cover considerable fraction of the whole network. © 2005 Yugi et al., licensee BioMed Central Ltd.

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Reverse engineering of biochemical equations from time-course data by means of genetic programming

Sugimoto M., Kikuchi S., Tomita M.

Biosystems 80 ( 2 ) 155 - 164 2005

Research paper (scientific journal), Joint Work, ISSN 03032647

Increased research aimed at simulating biological systems requires sophisticated parameter estimation methods. All current approaches, including genetic algorithms, need pre-existing equations to be functional. A generalized approach to predict not only parameters but also biochemical equations from only observable time-course information must be developed and a computational method to generate arbitrary equations without knowledge of biochemical reaction mechanisms must be developed. We present a technique to predict an equation using genetic programming. Our technique can search topology and numerical parameters of mathematical expression simultaneously. To improve the search ability of numeric constants, we added numeric mutation to the conventional procedure. As case studies, we predicted two equations of enzyme-catalyzed reactions regarding adenylate kinase and phosphofructokinase. Our numerical experimental results showed that our approach could obtain correct topology and parameters that were close to the originals. The mean errors between given and simulation-predicted time-courses were 1.6 × 10-5% and 2.0 × 10-3%, respectively. Our equation prediction approach can be applied to identify metabolic reactions from observable time-courses. © 2004 Elsevier Ireland Ltd. All rights reserved.

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In silico diagnosis of inherently inhibited gene expression focusing on initial codon combinations

Ohashi Y., Yamashiro A., Washio T., Ishii N., Ohshima H., Michishita T., Tomita M., Itaya M.

Gene 347 ( 1 ) 11 - 19 2005

Research paper (scientific journal), Joint Work, ISSN 03781119

The translation start site, immediately downstream from the start codon, is a dominant factor for gene expression in Escherichia coli. At present, no method exists to improve the expression level of cloned genes, since it remains difficult to find the best codon combination within the region. We determined the expression parameters that correspond to all sense codons within the first four codons using GFPuv which encodes a derivative of green fluorescent protein. Using a genetic algorithm (GA)-based computer program, these parameters were incorporated in a simple, static model for the prediction of translation efficiency, and optimized to the expression level for 137 randomly isolated GFPuv genes. The calculated initial translation index (ITI), also proven for the DsRed2 gene that encodes a red fluorescent protein, should provide a solution to overcome the gene expression problem in cloned genes whose expression is often inherently blocked at the translation process. The proposed method facilitates heterologous protein production in E. coli, the most commonly used host in biological and industrial fields. © 2004 Published by Elsevier B.V.

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Computational analysis suggests that alternative first exons are involved in tissue-specific transcription in rice ( Oryza sativa)

Kitagawa, Noriyuki ; Washio, Takanori ; Kosugi, Shunichi ; Yamashita, Tomoya ; Higashi, Kenji ; Yanagawa, Hiroshi ; Higo, Kenichi ; Satoh, Koji ; Ohtomo, Yasuhiro ; Sunako, Tomomi ; Murakami, Kazuo ; Matsubara, Kenichi ; Kawai, Jun ; Carninci, Piero ; Hayashizaki, Yoshihide ; Kikuchi, Shoshi ; Tomita, Masaru

Bioinformatics 21 ( 9 ) 1758-1763 - 1763 2005

Research paper (scientific journal), Joint Work, ISSN 13674803

Motivation: Transcription start site selection and alternative splicing greatly contribute to diversifying gene expression. Recent studies have revealed the existence of alternative first exons, but most have involved mammalian genes, and as yet the regulation of usage of alternative first exons has not been clarified, especially in plants. Results: We systematically identified putative alternative first exon transcripts in rice, verified the candidates using RT-PCR, and searched for the promoter elements that might regulate the alternative first exons. As a result, we detected a number of unreported alternative first exons, some of which are regulated in a tissue-specific manner. © The Author 2005. Published by Oxford University Press. All rights reserved.

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Hybrid dynamic/static method for large-scale simulation of metabolism

Yugi, K., Nakayama, Y., Kinoshita, A., and Tomita, M.

Theoretical Biology and Medical Modeling 2 ( 42 ) 2005

Research paper (scientific journal), Joint Work, ISSN 17424682

Background: Many computer studies have employed either dynamic simulation or metabolic flux analysis (MFA) to predict the behaviour of biochemical pathways. Dynamic simulation determines the time evolution of pathway properties in response to environmental changes, whereas MFA provides only a snapshot of pathway properties within a particular set of environmental conditions. However, owing to the large amount of kinetic data required for dynamic simulation, MFA, which requires less information, has been used to manipulate large-scale pathways to determine metabolic outcomes. Results: Here we describe a simulation method based on cooperation between kinetics-based dynamic models and MFA-based static models. This hybrid method enables quasi-dynamic simulations of large-scale metabolic pathways, while drastically reducing the number of kinetics assays needed for dynamic simulations. The dynamic behaviour of metabolic pathways predicted by our method is almost identical to that determined by dynamic kinetic simulation. Conclusion: The discrepancies between the dynamic and the hybrid models were sufficiently small to prove that an MFA-based static module is capable of performing dynamic simulations as accurately as kinetic models. Our hybrid method reduces the number of biochemical experiments required for dynamic models of large-scale metabolic pathways by replacing suitable enzyme reactions with a static module. © 2005 Yugi et al; licensee BioMed Central Ltd.

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KEGG-based pathway visualization tool for complex omics data

Arakawa, K., Kono, N., Yamada, Y. and Tomita, M.

In Silico Biology 5 ( 0039 ) 419 - 423 2005

Research paper (scientific journal), Joint Work, ISSN 13866338

Pathway-level visualization of omics data provides an essential means for systems biology, to capture the systematic properties of the inner activities of cells. Here we describe a web-based resource consisting of a web-application for the visualization of complex omics data onto KEGG pathways to overview all entities in the context of cellular pathways, and databases created with the software to visualize a series of microarray data. The web-application accepts transcriptome, proteome, metabolome, or the combination of these data as input, and because of this scalability it is advantageous for the visualization of cell simulation results. The web server can be accessed at http://www.g-language.org/data/marray/. © 2005 IOS Press. All rights reserved.

Metabolome Analysis and Metabolic Simulation

Ishii N., Soga T., Nishioka T. and Tomita M.

Metabolomics 1 ( 1 ) 29-37 - 37 2005

Research paper (scientific journal), Joint Work, ISSN 15733882

For many decades microorganisms have been used for industrial purposes; traditional fermentations such as brewing and production of food additives, aroma molecules, organic acids and pharmaceutical-like antibiotics or recombinant proteins are instances of the industrial microorganism utilization. Therefore, microorganism modeling and simulation have been required for engineering purposes, because of demands for design, optimization and quality control of large-scale fermentation plants. Modeling has recently become more highly developed, aided by the deciphering of microorganism genomes, the completion of metabolic databases, the development of analytical methodologies and improvements in the performance of computers. This paper reviews past and recent metabolic simulation of microorganisms, and also discusses the metabolome analytical techniques and the construction of large-scale microorganism models which are now being developed in our group. © 2005 Springer Science+Business Media, Inc.

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Dynamic simulation of red blood cell metabolism and its application to the analysis of a pathological condition

Nakayama Y., Kinoshita A., Tomita M.

Theoretical Biology and Medical Modeling 2 ( 18 ) 2005

Research paper (scientific journal), Joint Work, ISSN 17424682

Background: Cell simulation, which aims to predict the complex and dynamic behavior of living cells, is becoming a valuable tool. In silico models of human red blood cell (RBC) metabolism have been developed by several laboratories. An RBC model using the E-Cell simulation system has been developed. This prototype model consists of three major metabolic pathways, namely, the glycolytic pathway, the pentose phosphate pathway and the nucleotide metabolic pathway. Like the previous model by Joshi and Palsson, it also models physical effects such as osmotic balance. This model was used here to reconstruct the pathology arising from hereditary glucose-6-phosphate dehydrogenase (G6PD) deficiency, which is the most common deficiency in human RBC. Results: Since the prototype model could not reproduce the state of G6PD deficiency, the model was modified to include a pathway for de novo glutathione synthesis and a glutathione disulfide (GSSG) export system. The de novo glutathione (GSH) synthesis pathway was found to compensate partially for the lowered GSH concentrations resulting from G6PD deficiency, with the result that GSSG could be maintained at a very low concentration due to the active export system. Conclusion: The results of the simulation were consistent with the estimated situation of real G6PD-deficient cells. These results suggest that the de novo glutathione synthesis pathway and the GSSG export system play an important role in alleviating the consequences of G6PD deficiency. © 2005 Nakayama et al; licensee BioMed Central Ltd.

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Cleavage of double-stranded RNA by RNase HI from a thermoacidophilic archaeon, Sulfolobus tokodaii 7

Ohtani N., Yanagawa H., Tomita M., Itaya M.

Nucleic Acids Research 32 ( 19 ) 5809 - 5819 2004.12

ISSN 03051048

ST0753, the orthologous gene of Type 1 RNase H found in a thermoacidophilic archaeon, Sulfolobus tokodaii, was analyzed. The recombinant ST0753 protein exhibited RNase H activity in both in vivo and in vitro assays. The protein expressed in an RNase H-deficient mutant Escherichia coli strain functioned to suppress the temperature-sensitive phenotype associated with the lack of RNase H. The in vitro characteristics of the gene's RNase H activity were similar to those of Halobacterium RNase HI, the first archaeal Type 1 RNase H to be characterized. Surprisingly, the S.tokodaii RNase HI cleaved not only the RNA strand of an RNA/DNA hybrid but also an RNA strand of an RNA/RNA duplex in the presence of Mn2+ or Co2+. The result of gel filtration column chromatography showed this double-stranded RNA-dependent RNase (dsRNase) activity was coincident with S.tokodaii RNase HI. A site-directed mutagenesis study of essential amino acids for RNase H activity indicated that this activity also affected dsRNase activity. A single amino acid replacement of Asp-125 by Asn resulted in loss of dsRNase activity but not RNase H activity, suggesting that amino acid residues required for dsRNase activity seemed slightly different from those of RNase H activity. Some reverse transcriptases from retroelements can cleave double-stranded RNA, and this activity requires the RNase H domain. Similarities in primary structure and biochemical characteristics between S.tokodaii RNase HI and reverse transcriptases imply that the S.tokodaii enzyme might be derived from the RNase H domain of reverse transcriptase. © Oxford University Press 2004; all rights reserved.

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Comparative Analysis of Physical Maps of Four Bacillus subtilis (natto) Genomes

Qiu, Dongru Fujita, Kyoko Sakuma, Yuko Tanaka, Teruo Ohashi, Yoshiaki Ohshima, Hideyuki Tomita, Masaru Itaya, Mitsuhiro

Applied & Environmental Microbiology 70 ( 10 ) 6247-6256 - 6256 2004.10

Research paper (scientific journal), Joint Work, ISSN 00992240

The complete SfiI and I-CeuI physical maps of four Bacillus subtilis (natto) strains, which were previously isolated as natto (fermented soybean) starters, were constructed to elucidate the genome structure. Not only the similarity in genome size and organization but also the microheterogeneity of the gene context was revealed. No large-scale genome rearrangements among the four strains were indicated by mapping of the genes, including 10 rRNA operons (rrn) and relevant genes required for natto production, to the loci corresponding to those of the B. subtilis strain Marburg 168. However, restriction fragment length polymorphism and the presence or absence of strain-specific DNA sequences, such as the prophages SPβ, skin element, and PBSX, as well as the insertion element IS4Bsu1, could be used to identify one of these strains as a Marburg type and the other three strains as natto types. The genome structure and gene heterogeneity were also consistent with the type of indigenous plasmids harbored by the strains.

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Computational comparative analyses of alternative splicing regulation using full-length cDNA of various eukaryotes.

Itoh H, Washio T, Tomita M.

RNA 10 ( 7 ) 1005-1018 - 1018 2004.07

Research paper (scientific journal), Joint Work, ISSN 13558382

We previously reported a computational approach to infer alternative splicing patterns from Mus musculus full-length cDNA clones and microarray data. Although we predicted a large number of unreported splice variants, the general mechanisms regulating alternative splicing were yet unknown. In the present study, we compared alternative exons and constitutive exons in terms of splice-site strength and frequency of potential regulatory sequences. These regulatory features were further compared among five different species: Homo sapiens, M. musculus, Arabidopsis thaliana, Oryza sativa, and Drosophila melanogaster. Solid statistical validations of our comparative analyses indicated that alternative exons have (1) weaker splice sites and (2) more potential regulatory sequences than constitutive exons. Based on our observations, we propose a combinatorial model of alternative splicing mechanisms, which suggests that alternative exons contain weak splice sites regulated alternatively by potential regulatory sequences on the exons.

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A new role for expressed pseudogenes as ncRNA: Regulation of mRNA stability of is homologous coding gene

Yano Y., Saito R., Yoshida N., Yoshiki A., Wynshaw-Boris A., Tomita M., Hirotsune S.

Journal of Molecular Medicine 82 ( 7 ) 414 - 422 2004.07

ISSN 09462716

We have earlier generated a mutant mouse in a course of making a transgenic line that exhibited interesting heterozygote phenotypes, which exhibited failure to thrive, severe bone deformities, and polycystic kidneys. This mutant mouse provided a clue to uncover a unique role of expressed pseudogenes. In this mutant the transgene was integrated into the vicinity of the expressing pseudogene of Makorin1 called Makorin1-p1. This insertion reduced transcription of the Makorin1-p1, resulting in destabilization of the Makorin1 mRNA in trans via a cis-acting RNA decay element within the 5′ region of Makorin1 that is homologous between Makorin1 and Makorin1-p1. These findings demonstrate a novel and specific regulatory role of an expressed pseudogene as well as functional significance for noncoding RNAs. Next, we developed an original algorithm to determine how many pseudogenes are expressed. Based on our examination 2-3% of human processed pseudogenes are expressed using the most strict criteria. Interestingly, the mouse has a much smaller proportion of expressed pseudogenes (0.5-1%). Pseudogenes are functionally less constrained, and have accumulated more mutations than translated genes. If they have some functions in gene regulation, this property would allow more rapid functional diversification than protein-coding genes. In addition, some genetic phenomena that exhibit incomplete penetrance might be attributed to "mutation" or "variation" of pseudogenes. © Springer-Verlag 2004.

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Sustained MAPK activation is dependent on continual NGF receptor regeneration

Qiu, D., Mao, L., Kikuchi, S. and Tomita, M.

Development, Growth & Differentiation 46 ( 5 ) 393 - 403 2004.06

Research paper (conference, symposium, etc.), Joint Work, Accepted, ISSN 00121592

It still remains intriguing how signal specificity is achieved when different signals are relayed by the common intracellular signal transduction pathways. A well documented example for signal specificity determination is found in rat phaeochromocytoma PC12 cells where epidermal growth factor (EGF) stimulation produces a transient mitogen-activated protein kinase (MAPK) activation and leads to cell proliferation while nerve growth factor (NGF) initiates a sustained MAPK activation and induces cell differentiation. In this simulation, we demonstrated that NGF-induced sustained MARK activation may mainly depend on continual regeneration of NGF receptors and that the presence of a small pool of surface receptors is enough to maintain a sustained MAPK activation. On the other hand, MAPK activation is not significantly sensitive to the half-life of internalized receptors and the levels of NGF-specific MAPK phosphatase MAP kinase phosphatase-3 (MKP-3), though cytoplasmic persistence of internalized NGF-bound receptors and the MKP-3 dependent feedback control also contribute to the sustaining of MAPK activation. These results are consistent with the recent experimental evidence that persistent tyrosine receptor kinase A (TrkA) activity is necessary to maintain transcription in the differentiating PC12 cells (Chang et al. 2003) and a sustained Src kinase activity is detected in response to NGF stimulation (Gatti 2003). It is suggested that sustained or transient MAPK activation induced by different growth factor and neurotrophins, which is crucial to their signaling specificity, could be satisfactorily accounted for by their specific receptor turnover kinetics rather than by the activation of specific downstream signaling cascades.

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Characterization of the splice sites in GT-AG and GC-AG introns in higher eukaryotes using full-length cDNAs

Kitamura-Abe S., Itoh H., Washio T., Tsutsumi A., Tomita M.

Journal of Bioinformatics and Computational Biology 2 ( 2 ) 309 - 331 2004.06

ISSN 02197200

For the purpose of analyzing the relation between the splice sites and the order of introns, we conducted the following analysis for the GT-AG and GC-AG splice site groups. First, the pre-mRNAs of H. sapiens, M. musculus, D. melanogaster, A. thaliana and O. sativa were sampled by mapping the full-length cDNA to the genomes. Next, the consensus sequences at different regions of pre-mRNAs were analyzed in the five species. We also investigated the mononucleotide and dinucleotide frequencies in the extensive regions around the 5′ splice sites (5′ss) and 3′ splice sites (3′ss). As a result, differential frequencies of nucleotides at the first 5′ss in both the GT-AG and GC-AG splice site groups were observed in A. thaliana and O. sativa pre-mRNAs. The trend, which indicates that GC 5′ss possess strong consensus sequences, was observed not only in mammalian pre-mRNAs but also in the pre-mRNAs of D. melanogaster, A. thaliana and O. sativa. Furthermore, we examined the consensus sequences of the constitutive and alternative splice sites. It was suggested that in the case of the alternative GC-AG introns, the tendency to have a weak consensus sequence at 5′ss is different between H. sapiens and M. musculus pre-mRNAs. © Imperial College Press.

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Application of capillary electrophoresis-mass spectrometry to syntheitc in vitro glycolysis studies

Itoh A., Ohashi Y., Soga T., Mori H., Nishioka T. and Tomita M.

Electrophoresis 25 1996-2002 2004

Research paper (scientific journal), Joint Work

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Simultaneous determination of the main metabolites in rice leaves using capillary electrophoresis mass spectrometry and capillary electrophoresis diode array detection

Sato, S., Soga, T., Nishioka, T. and Tomita, M.

Plant Journal 40 ( 1 ) 151-163 2004

Research paper (scientific journal), Joint Work, Accepted

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A general computational model of mitochondrial metabolism in a whole organelle scale

Yugi K., Tomita M.

Bioinformatics 20 ( 20 ) 1795-1796 - 1796 2004

Research paper (scientific journal), Joint Work, Accepted, ISSN 13674803

Summary: A computational tool for mitochondrial systems biology has been developed as a simulation model of E-Cell2, a publicly available simulation system. The general model consists of 58 enzymatic reactions and 117 metabolites, representing the respiratory chain, the TCA cycle, the fatty acid β-oxidation and the inner-membrane transport system. It is based on previously published enzyme kinetics studies in the literature; we have successfully integrated and packaged them into a single large model. The model can be easily extended and modified so that mitochondrial biologists/physiologists can integrate their own models and evaluate them in the context of the whole organelle metabolism. © Oxford University Press 2004; all rights reserved.

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Position-specific expression of Hox genes along the gastrointestinal tract

Yahagi N, Kosaki R, Ito T, Mitsuhashi T, Shimada H, Tomita M, Takahashi T, and Kosaki K

Congenital Anomalies 44 ( 1 ) 18-26 - 26 2004

Research paper (scientific journal), Joint Work, Accepted, ISSN 09143505

Hox genes play a critical role in morphogenesis of the early embryo along the anteroposterior axis. In mammals, 39 Hox genes with extensive homology are organized into 13 paralogous groups, forming four clusters on four separate chromosomes. The genes within each cluster are arranged in a 3' to 5' direction and expressed in a temporally and spatially coordinated manner along the anteroposterior axis in the vertebrae, limbs and viscera, including the gastrointestinal tract, but little is known about their spatial expression in the adult gastrointestinal tract. We used the quantitative polymerase chain reaction (PCR) intercalater method with SYBR Green to quantify human Hox gene expression in the adult gastrointestinal tract tissue: esophagus, stomach, duodenum, jejunum, ileum, ileocecum, cecum, ascending colon, transverse colon, descending colon and rectum. Hox gene expression was normalized to glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) gene expression. The spatial expression pattern was analyzed by the multivariate method. The expression level of all 39 Hox genes could be measured in a reproducible manner. Genes with higher expression in the foregut-derived segments tended to have lower expression in hindgut-derived segments, whereas those with low expression in the former tended to have higher in the latter. Principal components analysis and permax analysis revealed a position-specific expression pattern of Hox genes along the anteroposterior axis of the adult gastrointestinal tract. The pattern recapitulates the expression pattern in the embryonic gastrointestinal tract. We suggest that Hox genes may play a pivotal role in the position-specific regenerative process of intestinal epithelial cells.

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A multi-algorithm, multi-timescale method for cell simulation

Takahashi, K., Kaizu, K., Bin, H., Tomita, M

Bioinformatics 20 ( 4 ) 538-546 - 546 2004

Research paper (scientific journal), Joint Work, Accepted, ISSN 13674803

Motivation: Many important problems in cell biology require the dense nonlinear interactions between functional modules to be considered. The importance of computer simulation in understanding cellular processes is now widely accepted, and a variety of simulation algorithms useful for studying certain subsystems have been designed. Many of these are already widely used, and a large number of models constructed on these existing formalisms are available. A significant computational challenge is how we can integrate such sub-cellular models running on different types of algorithms to construct higher order models. Results: A modular, object-oriented simulation meta-algorithm based on a discrete-event scheduler and Hermite polynomial interpolation has been developed and implemented. It is shown that this new method can efficiently handle many components driven by different algorithms and different timescales. The utility of this simulation framework is demonstrated further with a 'composite' heat-shock response model that combines the Gillespie - Gibson stochastic algorithm and deterministic-differential equations. Dramatic improvements in performance were obtained without significant accuracy drawbacks. A multi-timescale demonstration of coupled harmonic oscillators is also shown. © Oxford University Press 2004; all rights reserved.

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Identification of the first archaeal Type 1 RNase H gene from Halobacterium sp. NRC-1: archaeal RNase HI can cleave an RNA-DNA junction

Otani, N., Yanagawa, H., Tomita, M. and Itaya, M

Biochemical Journal 381 ( 3 ) 795-802 - 802 2004

Research paper (scientific journal), Joint Work, Accepted, ISSN 02646021

All the archaeal genomes sequenced to date contain a single Type 2 RNase H gene. We found that the genome of a halophilic archaeon, Halobacterium sp. NRC-1, contains an open reading frame with similarity to Type 1 RNase H. The protein encoded by the Vng0255c gene, possessed amino acid sequence identities of 33 % with Escherichia coli RNase HI and 34 % with a Bacillus subtilis RNase HI homologue. The B. subtilis RNase HI homologue, however, lacks amino acid sequences corresponding to a basic protrusion region of the E. coli RNase HI, and the Vng0255c has the similar deletion. As this deletion apparently conferred a complete loss of RNase H activity on the B. subtilis RNase HI homologue protein, the Vng0255c product was expected to exhibit no RNase H activity. However, the purified recombinant Vng0255c protein specifically cleaved an RNA strand of the RNA/DNA hybrid in vitro, and when the Vng0255c gene was expressed in an E. coli strain MIC2067 it could suppress the temperature-sensitive growth defect associated with the loss of RNase H enzymes of this strain. These results in vitro and in vivo strongly indicate that the Halobacterium Vng0255c is the first archaeal Type 1 RNase H. This enzyme, unlike other Type 1 RNases H, was able to cleave an Okazaki fragment-like substrate at the junction between the 3′-side of ribonucleotide and 5′-side of deoxyribonucleotide. It is likely that the archaeal Type 1 RNase H plays a role in the removal of the last ribonucleotide of the RNA primer from the Okazaki fragment during DNA replication.

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Toward large-scale modeling of the microbial cell for computer simulation

Ishii, N., Robert, M. Nakayama, Y., Kanai, A. and Tomita, M

Journal of Biotechnology 113 ( 1-3 ) 281-294 - 294 2004

Research paper (scientific journal), Joint Work, Accepted, ISSN 01681656

In the post-genomic era, the large-scale, systematic, and functional analysis of all cellular components using transcriptomics, proteomics, and metabolomics, together with bioinformatics for the analysis of the massive amount of data generated by these "omics" methods are the focus of intensive research activities. As a consequence of these developments, systems biology, whose goal is to comprehend the organism as a complex system arising from interactions between its multiple elements, becomes a more tangible objective. Mathematical modeling of microorganisms and subsequent computer simulations are effective tools for systems biology, which will lead to a better understanding of the microbial cell and will have immense ramifications for biological, medical, environmental sciences, and the pharmaceutical industry.In this review, we describe various types of mathematical models (structured, unstructured, static, dynamic, etc.), of microorganisms that have been in use for a while, and others that are emerging. Several biochemical/cellular simulation platforms to manipulate such models are summarized and the E-Cell system 1 developed in our laboratory is introduced. Finally, our strategy for building a "whole cell metabolism model", including the experimental approach, is presented. © 2004 Elsevier B.V. All rights reserved.

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The 'weighted sum of relative entropy': a new index for synonymous codon usage bias

Suzuki, H., Saito, R. and Tomita, M

GENE 335 ( 335 ) 19-23 - 23 2004

Research paper (scientific journal), Joint Work, Accepted, ISSN 03781119

Shannon entropy from information theory has been applied to estimate the degree of deviation from equal usage of synonymous codons; however, previous attempts have failed to take into account all three aspects of amino acid usage, i.e. (i) the number of distinct amino acids, (ii) their relative frequencies, and (iii) their degree of codon degeneracy. A new index taking into account all of these aspects is proposed. The index, designated as the 'weighted sum of relative entropy' (Ew), is defined as the sum of the relative entropy of each amino acid weighted by its relative frequency in the sequence. In this paper, we demonstrate that Ew allows us to avoid some amino acid usage biases and can yield results contradictory to those obtained by previous methods. © 2004 Elsevier B.V. All rights reserved.

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Qualitative and quantitative analysis of amino acids by capillary electrophresis Electrospray ionization tandem mass spectrometry

Soga, T., Kakazu Yuji, Robert, M., Tomita, M. and Nishioka, T

Electrophoresis ( 25 ) 1964-1972 2004

Research paper (scientific journal), Joint Work, Accepted

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On dynamics of overlapping genes in bacterial genomes

Fukuda Y., Nakayama Y., Tomita M.

Gene 323 ( 1-2 ) 181 - 187 2003.12

ISSN 03781119

A genome-wide computational analysis of overlapping genes in various prokaryotic genomes was conducted to study the evolutionary rates of the formation and degradation of overlapping genes. Overlapping genes are adjacent genes that overlap partially or entirely. Overlapping gene pairs extracted from complete bacterial genome sequences were classified into three directional patterns, namely, 'convergent' (→←), 'unidirectional' (→→), and 'divergent' (←→). Divergent overlapping genes were rare compared to the other two patterns. We also compared the structures of overlapping genes that are not conserved in the homologous genes of different species. We found that overlapping genes are mainly generated from gene pairs with short intergenic regions. Overlapping genes caused by mutations at the 3′-end of genes occur more frequently than overlapping genes generated by mutations at the 5′-end of genes in closely related species. In contrast, gene overlaps due to mutations at the 5′-ends accumulate in distant species. These differences presumably correlate to the genomic divergence among species. The evolution of these overlapping gene structures is probably related to the evolutionary time scale, which suggests that the evolution of overlapping genes occurs at a universal mutation rate across species. © 2003 Elsevier B.V. All rights reserved.

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A novel feature of microsatellites in plants: a distribution gradient along the direction of transcription

Fujimori S, Washio T, Higo K, Ohtomo Y, Murakami K, Matsubara K, Kawai J, Carninci P, Hayashizaki Y, Kikuchi S, Tomita M

FEBS LETTERS 554 ( 1-2 ) 17 - 22 2003.11

Research paper (scientific journal), Single Work, ISSN 0014-5793

A computer-based analysis was conducted to assess the characteristics of microsatellites in transcribed regions of rice and Arabidopsis. In addition, two mammals were simultaneously analyzed for a comparative analysis. Our analyses confirmed a novel plant-specific feature in which there is a gradient in microsatellite density along the direction of transcription. It was also confirmed that pyrimidine-rich microsatellites are found intensively near the transcription start sites, specifically in the two plants, but not in the mammals. Our results suggest that microsatellites located at high frequency in the 5′-flanking regions of plant genes can potentially act as factors in regulating gene expression. © 2003 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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Kinetic simulation of signal transduction system in hippocampal long-term potentiation with dynamic modeling of protein phosphatase 2A

Kikuchi S., Fujimoto K., Kitagawa N., Fuchikawa T., Abe M., Oka K., Takei K., Tomita M.

Neural Networks 16 ( 9 ) 1389 - 1398 2003.11

ISSN 08936080

We modeled and analyzed a signal transduction system of long-term potentiation (LTP) in hippocampal post-synapse. Bhalla and Iyengar [Science 283(1999) 381] have developed a hippocampal LTP model. In the conventional model, the concentration of protein phosphatase 2A (PP2A) was fixed. However, it was reported that dynamic inactivation of PP2A was essential for LTP [J. Neurochem. 74 (2000) 807]. We introduced a dynamic modeling of PP2A; inactivation (phosphorylation) of PP2A by calcium/calmodulin-dependent protein kinase II (CaMKII) in the presence of calcium/calmodulin, self-activation (autodephosphorylation) of PP2A, and inactivation (dephosphorylation) of CaMKII by PP2A. This model includes complex feedback loops; both CaMKII and PP2A are autoactivated, while they inactivate each other. Moreover, we proposed an analysis strategy for model validation by applying the results of sensitivity analysis. In our system, calcineurin (CaN) played an essential role, rather than the activation of protein kinase C (PKC) as documented in the conventional model. From results of the analysis of our model, we found the following robustness as characteristics of bistability in our model: (1) PP2A reactions against calcium ion (Ca2+) perturbation; (2) PP2A inactivation against PP2A increase; (3) protein phosphatase 1 (PP1) activation against PF2A increase; and (4) PP2A reactions against PP2A initial concentration. These properties facilitated LTP induction in our system. We showed that another mechanism could introduce bistable behavior by adding dynamic reactions of PP2A. © 2003 Elsevier Ltd. All rights reserved.

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E-cell 2: Multi-platform E-Cell simulation system

Takahashi K, Ishikawa N, Sadamoto Y, Sasamoto H, Ohta S, Shiozawa A, Miyoshi F, Naito Y, Nakayama Y, Tomita M

BIOINFORMATICS 19 ( 13 ) 1727 - 1729 2003.09

Research paper (scientific journal), Single Work, ISSN 1367-4803

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Comparative study of translation termination sites and release factors (RF1 and RF2) in procaryotes

Ozawa Y., Saito R., Washio T., Tomita M.

Journal of Molecular Evolution 56 ( 6 ) 665 - 672 2003.06

ISSN 00222844

Translation termination is catalyzed by release factors that recognize stop codons. However, previous works have shown that in some bacteria, the termination process also involves bases around stop codons. Recently, Ito et al, analyzed release factors and identified the amino acids therein that recognize stop codons. However, the amino acids that recognize bases around stop codons remain unclear. To identify the candidate amino acids that recognize the bases around stop codons, we aligned the protein sequences of the release factors of various bacteria and searched for amino acids that were conserved specifically in the sequence of bacteria that seemed to regulate translation termination by bases around stop codons. As a result, species having several highly conserved residues in RF1 and RF2 showed positive correlations between their codon usage bias and conservation of the bases around the stop codons. In addition, some of the residues were located very close to the SPF motif, which deciphers stop codons. These results suggest that these conserved amino acids enable the release factors to recognize the bases around the stop codons.

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Dynamics modeling of genetic networks using genetic algorithm and S-system

Kikuchi S., Tominaga D., Arita M., Takahashi K., Tomita M.

Bioinformatics 19 ( 5 ) 643 - 650 2003.03

ISSN 13674803

Motivation: The modeling of system dynamics of genetic networks, metabolic networks or signal transduction cascades from time-course data is formulated as a reverse-problem. Previous studies focused on the estimation of only network structures, and they were ineffective in inferring a network structure with feedback loops. We previously proposed a method to predict not only the network structure but also its dynamics using a Genetic Algorithm (GA) and an S-system formalism. However, it could predict only a small number of parameters and could rarely obtain essential structures. In this work, we propose a unified extension of the basic method. Notable improvements are as follows: (1) an additional term in its evaluation function that aims at eliminating futile parameters; (2) a crossover method called Simplex Crossover (SPX) to improve its optimization ability; and (3) a gradual optimization strategy to increase the number of predictable parameters. Results: The proposed method is implemented as a C program called PEACE1 (Predictor by Evolutionary Algorithms and Canonical Equations 1). Its performance was compared with the basic method. The comparison showed that: (1) the convergence rate increased about 5-fold; (2) the optimization speed was raised about 1.5-fold; and (3) the number of predictable parameters was increased about 5-fold. Moreover, we successfully inferred the dynamics of a small genetic network constructed with 60 parameters for 5 network variables and feedback loops using only time-course data of gene expression.

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E-cell simulation system and its application to the modeling of circadian rhythm

Miyoshi F, Nakayama Y, Tomita M

Seikagaku 75 ( 1 ) 5-16 - 16 2003.01

Research paper (scientific journal), Single Work, ISSN 0037-1017

G-language Genome Analysis Environment: a workbench for nucleotide sequence data mining

Arakawa, K., Mori, K., Ikeda, K., Matsuzaki, T., Kobayashi, Y., and Tomita, M.

Bioinformatics 19 ( 2 ) 305 - 306 2003

Research paper (scientific journal), Joint Work, ISSN 13674803

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The Systems Biology Markup Language (SBML): A Medium for Representation and Exchange of Biochemical Network Models

M. Hucka, A. Finney, H. M. Sauro, H. Bolouri, J. C. Doyle, H. Kitano, A. P. Arkin, B. J. Bornstein, D. Bray, A. Cornish-Bowden, A. A. Cuellar, S. Dronov, E. D. Gilles, M. Ginkel, V. Gor, I. I. Goryanin, W. J. Hedley, T. C. Hodgman, J.-H. Hofmeyr, P. J. Hunter, N. S. Juty, J. L. Kasberger, A.Kremling, U. Kummer, N. Le Novere, L. M. Loew, D. Lucio, P. Mendes, E. D.Mjolsness, Y. Nakayama, M. R. Nelson, P. F. Nielsen, T. Sakurada, J. C.Schaff, B. E. Shapiro, T. S. Shimizu, H. D. Spence, J. Stelling, K.Takahashi, M. Tomita, J. Wagner, J. Wang

Bioinformatics 19 ( 4 ) 524 - 531 2003

Research paper (scientific journal), Joint Work, Accepted, ISSN 13674803

Motivation: Molecular biotechnology now makes it possible to build elaborate systems models, but the systems biology community needs information standards if models are to be shared, evaluated and developed cooperatively. Results: We summarize the Systems Biology Markup Language (SBML) Level 1, a free, open, XML-based format for representing biochemical reaction networks. SBML is a software-independent language for describing models common to research in many areas of computational biology, including cell signaling pathways, metabolic pathways, gene regulation, and others.

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Cooperative regulation for Okazaki fragment processing by RNase HII and FEN-1 purified from a hyperthermophilic archaeon, Pyrococcus furiosus.

Sato, A., Kanai, A., Itaya, M. and Tomita, M

Biochemical and Biophysical Research Communication 309 ( 1 ) 247 - 252 2003

Research paper (scientific journal), Joint Work, ISSN 0006291X

A reconstitution system that recapitulates the processing of Okazaki-primer RNA was established by the heat-stable recombinant enzymes RNase HII and FEN-1 (termed Pf-RNase HII and Pf-FEN-1, respectively) prepared from a hyperthermophilic archaeon, Pyrococcus furiosus. A 35-mer RNA-DNA/DNA hybrid substrate mimicking an Okazaki fragment was used to investigate the properties of the processing reaction in vitro at 50°C. Pf-RNase HII endonucleolytically cleaves the RNA primer region, but does not cut the junction between RNA and DNA. Removal of the RNA of the RNA-DNA junction was brought about by Pf-FEN-1 after Pf-RNase HII digestion. In the presence of 0.25-5mM MnCl2, Pf-FEN-1 alone weakly cleaved the junction. The addition of Pf-RNase HII to the reaction mixture increased removal efficiency and optimal Pf-FEN-1 activity was achieved at an equal amount of the two enzymes. These results indicate that there are at least two steps in the degradation of primer RNA requiring a step-specific enzyme. It is likely that Pf-RNase HII and Pf-FEN-1 cooperatively process Okazaki fragment during lagging-strand DNA replication. © 2003 Elsevier Inc. All rights reserved.

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Computational analysis of stop codon readthrough in D.melanogaster

Sato, M., Umeki, H., Saito, R., Kanai, A. and Tomita, M.

Bioinformatics 19 ( 11 ) 1371 - 1380 2003

Research paper (scientific journal), Joint Work, ISSN 13674803

Motivation: Readthrough is an unusual process in which a stop codon is misread or skipped. Recently it has been shown that some translation is regulated by the readthrough reactions although the complete mechanism is not clear. Therefore, the discovery of 'readthrough genes' is important for further investigation of their cellular roles, which may provide additional insights into the mechanism of translational regulation. Results: We constructed a system that lists candidates of readthrough genes based on the existence of a 'protein motif' at the 3′ untranslated region (UTR). Using this system, we extracted 85 candidates from 4082 nucleic acid sequences of Drosophila melanogaster in GenBank database. The sequences of these candidates had a slightly more stable secondary structure and different base preferences compared to the non-candidates. As these features are known to have an effect on readthrough events, we would like to suggest that these candidates contain actual readthrough genes.

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Identification of putative noncoding RNAs among the RIKEN mouse full-length cDNA collection.

Numata K., Kanai, A., Saito, R., Kondo, S., Adachi, J., Wilming, L.G., Hume, D.A., Hayashizaki, Y. and Tomita, M. ;RIKEN GER Group; GSL Members. Hayashizaki, Y. and Tomita, M.

Genome Research 13 ( 6 B ) 1301 - 1306 2003

Research paper (scientific journal), Joint Work, ISSN 10889051

With the sequencing and annotation of genomes and transcriptomes of several eukaryotes, the importance of noncoding RNA (ncRNA)-RNA molecules that are not translated to protein products - has become more evident. A subclass of ncRNA transcripts are encoded by highly regulated, multi-exon, transcriptional units, are processed like typical protein-coding mRNAs and are increasingly implicated in regulation of many cellular functions in eukaryotes. This study describes the identification of candidate functional ncRNAs from among the RIKEN mouse full-length cDNA collection, which contains 60,770 sequences, by using a systematic computational filtering approach. We initially searched for previously reported ncRNAs and found nine murine ncRNAs and homologs of several previously described nonmouse ncRNAs. Through our computational approach to filter artifact-free clones that lack protein coding potential, we extracted 4280 transcripts as the largest-candidate set. Many clones in the set had EST hits, potential CpG islands surrounding the transcription start sites, and homologies with the human genome. This implies that many candidates are indeed transcribed in a regulated manner. Our results demonstrate that ncRNAs are a major functional subclass of processed transcripts in mammals.

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Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs

Okazaki Y, Furuno M, Kasukawa T, Adachi J, Bono H, Kondo S, Nikaido I, Osato N, Saito R, Suzuki H, Yamanaka I, Kiyosawa H, Yagi K, Tomaru Y, Hasegawa Y, Nogami A, Schonbach C, Gojobori T, Baldarelli R, Hill DP, Bult C, Hume DA, Quackenbush J, Schriml LM, Kanapin A, Matsuda H, Batalov S, Beisel KW, Blake JA, Bradt D, Brusic V, Chothia C, Corbani LE, Cousins S, Dalla E, Dragani TA, Fletcher CF, Forrest A, Frazer KS, Gaasterland T, Gariboldi M, Gissi C, Godzik A, Gough J, Grimmond S, Gustincich S, Hirokawa N, Jackson IJ, Jarvis ED, Kanai A, Kawaji H, Kawasawa Y, Kedzierski RM, King BL, Konagaya A, Kurochkin IV, Lee Y, Lenhard B, Lyons PA, Maglott DR, Maltais L, Marchionni L, McKenzie L, Miki H, Nagashima T, Numata K, Okido T, Pavan WJ, Pertea G, Pesole G, Petrovsky N, Pillai R, Pontius JU, Qi D, Ramachandran S, Ravasi T, Reed JC, Reed DJ, Reid J, Ring BZ, Ringwald M, Sandelin A, Schneider C, Semple CAM, Setou M, Shimada K, Sultana R, Takenaka Y, Taylor MS, Teasdale RD, Tomita M, Verardo R

Nature 420 ( 6915 ) 563-573 - 573 2002.12

Research paper (scientific journal), Joint Work, ISSN 0028-0836

Only a small proportion of the mouse genome is transcribed into mature messenger RNA transcripts. There is an international collaborative effort to identify all full-length mRNA transcripts from the mouse, and to ensure that each is represented in a physical collection of clones. Here we report the manual annotation of 60,770 full-length mouse complementary DNA sequences. These are clustered into 33,409 'transcriptional units', contributing 90.1% of a newly established mouse transcriptome database. Of these transcriptional units, 4,258 are new protein-coding and 11,665 are new non-coding messages, indicating that non-coding RNA is a major component of the transcriptome. 41% of all transcriptional units showed evidence of alternative splicing. In protein-coding transcripts, 79% of splice variations altered the protein product. Whole-transcriptome analyses resulted in the identification of 2,431 sense-antisense pairs. The present work, completely supported by physical clones, provides the most comprehensive survey of a mammalian transcriptome so far, and is a valuable resource for functional genomics.

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Comprehensive sequence analysis of translation termination sites in various eukaryotes

Ozawa Y, Hanaoka S, Saito R, Washio T, Nakano S, Shinagawa A, Itoh M, Shibata K, Carninci P, Konno H, Kawai J, Hayashizaki Y, Tomita M

Gene 300 ( 1-2 ) 79-87 - 87 2002.10

Research paper (scientific journal), Joint Work, ISSN 0378-1119

Recent investigations into the translation termination sites of various organisms have revealed that not only stop codons but also sequences around stop codons have an effect on translation termination. To investigate the relationship between these sequence patterns and translation as well as its termination efficiency, we analysed the correlation between strength of consensus and translation efficiency, as predicted according to Codon Adaptation Index (CAI) value. We used RIKEN full-length mouse cDNA sequences and ten other eukaryotic UniGene datasets from NCBI for the analyses. First, we conducted sequence profile analyses following translation termination sites. We found base G and A at position +1 as a strong consensus for mouse cDNA. A similar consensus was found for other mammals, such as Homo sapiens, Rattus norvegicus and Bos taurus. However, some plants had different consensus sequences. We then analysed the correlation between the strength of consensus at each position and the codon biases of whole coding regions, using information content and CAI value. The results showed that in mouse cDNA, CAI value had a positive correlation with information content at positions +1. We also found that, for positions with strong consensus, the strength of the consensus is likely to have a positive correlation with CAI value in some other eukaryotes. Along with these observations, biological insights into the relationship between gene expression level, codon biases and consensus sequence around stop codons will be discussed. © 2002 Published by Elsevier Science B.V.

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Towards Computer Aided Design (CAD) of Useful Microorganisms

Tomita, M.

Bioinformatics 17 1091-1092 2002

Research paper (scientific journal), Single Work

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Characterization of the aryl hydrocarbon receptor repressor gene and association of its Pro185Ala polymorphism with micropenis

Fujita H, Hasegawa T, Takahashi T, Matsuo N, and Kosaki K, Tomita M.

Teratology 65 ( 1 ) 10 - 18 2002

Joint Work, ISSN 00403709

Background: Genetic background of a fetus contributes to the abnormal development after teratogen exposure. In rodents, in utero exposure to dioxins affects male external genital development. The effects of dioxins are mediated via the aryl hydrocarbon receptor (AHR) and its binding protein, aryl hydrocarbon receptor nuclear translocator (ARNT). In mice, aryl hydrocarbon receptor repressor (AHRR), which binds to ARNT in competition with AHR, plays a critical negative regulatory role in AHR signaling. We attempt to characterize the human AHRR gene and investigate the relationship between AHRR polymorphisms and the incidence of micropenis, a phenotype of undermasculinization. Methods: We identified and characterized the human homolog of mouse AHRR, taking advantage of the publicly available draft version of the human genome sequence. After detecting an AHRR protein polymorphism by the direct sequencing of pooled human genomic DNA, we evaluated the association between the polymorphism and the presence or absence of micropenis (<-2.5 SD) in patients with micropenis and control subjects. Results: The deduced sequence for human AHRR (715 residues) and the mouse AHRR protein exhibited 81% sequence homology to each other. The Pro185Ala polymorphism was identified between the PAS-A region and the highly conserved arginine/cysteine-rich RCFRCRL/VRC region. Forty-six percent (27/59) of patients with micropenis and 27% (22/80) of the controls were homozygous for 185Pro; this difference in frequencies was significant (P = 0.03). Conclusions: Homozygosity for the 185Pro allele of AHRR may increase the susceptibility of a fetus to the undermasculinizing effects of dioxin exposure in utero, presumably through the diminished inhibition of AHR-mediated signaling. © 2002 Wiley-Liss, Inc.

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Causal analysis of CpG suppression in the Mycoplasma genome

Goto, M., Washio, T. Tomita M.

Microbial and Comparative Genomics 5 51-58 2002

Joint Work

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Simultaneous Determination of Anionic Intermediates for Bacillus Subtilis Metabolic Pathways by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry

Soga, T. Ueno, Y., Naraoka, H., Ohashi, O., Nishioka, T. Tomita M.

Analytical Chemistry 74 ( 10 ) 2233-2239 - 2239 2002

Research paper (scientific journal), Joint Work, Accepted, ISSN 00032700

A method for simultaneous determination of anionic metabolites based on capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry is described. To prevent current drop by the system, electroosmotic flow (EOF) reversal by using a cationic polymer-coated capillary was indispensable. A mixture containing 32 standards including carboxylic acids, phosphorylated carboxylic acids, phosphorylated saccharides, nucleotides, and nicotinamide and flavin adenine coenzymes of glycolysis and the tricarboxylic acid cycle pathways were separated by CE and selectively detected by a quadrupole mass spectrometer with a sheath-flow electrospray ionization interface. Key to the analysis was EOF reversal using a cationic polymer-coated capillary and an electrolyte system consisting of 50 mM ammonium acetate, pH 9.0. The relative standard deviations of the method were better than 0.4% for migration times and between 0.9% and 5.4% for peak areas. The concentration detection limits for these metabolites were between 0.3 and 6.7 μmol/L with pressure injection of 50 mbar for 30 s (30 nL); i.e., mass detection limits ranged from 9 to 200 fmol, at a signal-to-noise ratio of 3. This method was applied to the comprehensive analysis of metabolic intermediates extracted from Bacillus subtilis, and 27 anionic metabolites could be directly detected and quantified.

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Integrative modeling of gene expression and metabolism with E-CELL simulation system

Hashimoto, K., Seno, S., Dhar, P.K. and Tomita, M

Artificial Life and Robotics 6 99-107 2002

Research paper (scientific journal), Joint Work, Accepted

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Proposition of cortical connectivity between 30 cytoarchitectural areas of human cerebral cortex

Fukuda, R., Hara, J., Shankle, W. R., Tomita M.

Neurocomputing 32-33 ( 33 ) 749-755 - 755 2002

Research paper (scientific journal), Joint Work, ISSN 09252312

Connectivity between 30 cytoarchitectural areas covering almost the whole region of neocortex has been proposed. The data source provided by J.L. Conel was used for the analyses. The analyses consisted of computing correlation coefficients between each cortical layer of one cortical area to each cortical layer of another cortical area over the eight age-points and comparison of the time of the first appearance of myelinated axons per layer per area. Derived connectivity supports some interesting findings reported recently by others and is in good agreement with neural network model deduced from neuroimaging and psychological experiments. (C) 2000 Elsevier Science B.V. All rights reserved.

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Pressure-assisted capillary electrophoresis electrospray ionization mass spectrometry for analysis of multivalent anions

Soga, T., Ueno, Y., Naraoka, H., Matsuda, K., Tomita, M., and Nishioka, T

Analytical Chemistry 74 ( 24 ) 6224-6229 - 6229 2002

Research paper (scientific journal), Joint Work, ISSN 00032700

We describe a method, based on pressure-assisted capillary electrophoresis coupled to electrospray ionization mass spectrometry (PACE/ESI-MS), that allows the simultaneous and quantitative analysis of multivalent anions, such as citrate isomers, nucleotides, nicotinamideadenine dinucleotides, and flavin adenine dinucleotide, and coenzyme A (CoA) compounds. Key to the analysis was using a noncharged polymer, poly(dimethylsiloxane), coated to the inner surface of the capillary to prevent anionic species from adsorbing onto the capillary wall. It was also necessary to drive a constant liquid flow toward the MS by applying air pressure to the inlet capillary during electrophoresis to maintain a conductive liquid junction between the capillary and the electrospray needle. Although theoretical plates were inferior to those obtained by CE/ESI-MS using a cationic polymer-coated capillary, the PACE/ESI-MS method improved reproducibility and sensitivity of these anions. Eighteen anions were separated by PACE and selectively detected by a quadrupole mass spectrometer with a sheath-flow electrospray ionization interface. The relative standard deviations (n = 6) of the method were better than 0.6% for migration times and between 1.4% and 6.2% for peak areas. The detection limits for these species were between 0.4 and 3.7 μmol/L with pressure injection of 50 mbar for 30 s (30 nL), that is, mass detection limits calculated in the range from 12 to 110 fmol at a signal-to-noise ratio of 3. The utility of the method was demonstrated by analysis of Citrate isomers, nucleotides, dinucleotides, and CoA compounds extracted from Bacillus subtilis cells.

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On biased distribution of introns in various eukaryotes

Sakurai, A., Fujimori, S., Kochiwa, H., Kitamura-Abe, S., Washio, T., Saito, R., The RIKEN Genome Exploration Research Group Phase II Team, Carninci, P., Hayashizaki, Y., and Tomita, M.

Gene, 300 ( 1-2 ) 89-95 - 95 2002

Research paper (scientific journal), Joint Work, ISSN 03781119

We conducted comprehensive analyses on intron positions in the Mus musculus genome by comparing genomic sequences in the GenBank database and cDNA sequences in the mouse cDNA library recently developed by Riken Genomic Sciences Center. Our results confirm that introns have a tendency to be located toward the 5′ end of the gene. The same type of analysis was conducted in the coding region of seven eukaryotes (Saccharomyces cerevisiae, Plasmodium falciparum, Caenorhabditis elegans, Drosophila melanogaster, M. musculus, Homo sapiens, Arabidopsis thaliana). Introns in genes with a single intron have a locational bias toward the 5′ end in all species except A. thaliana. We also measured the distance from the start codon to the position of the intron, and found that single introns prefer the location immediately after the start codon in S. cerevisiae and P. falciparum. We discuss three possible explanations for these findings: (1) they are the consequence of intron loss by reverse-transcriptase; (2) they are necessary to accommodate the function; and (3) they are concerned with the mechanism of pre-mRNA splicing. © 2002 Elsevier Science B.V. All rights reserved.

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Inferring alternative splicing patterns in mouse from a full-length cDNA library and micro-array data

Kochiwa, H., Suzuki, R., Washio, T., Saito, R., The RIKEN Genome Exploration Research Group Phase II Team, Bono, H., Carninci, P., Okazaki, Y., Miki, R., Hayashizaki, Y., and Tomita, M.

Genome Research 12 ( 8 ) 1286-1293 - 1293 2002

Research paper (scientific journal), Joint Work, ISSN 10889051

Although many studies on alternative splicing of specific genes have been reported in the literature, the general mechanism that regulates alternative splicing has not been clearly understood. In this study, we systematically aligned each pair of the 21,076 cDNA sequences of Mus musculus, searched for putative alternative splicing patterns, and constructed a list of potential alternative splicing sites. Two cDNAs are suspected to be alternatively spliced and originating from a common gene if they share most of their region with a high degree of sequence homology, but parts of the sequences are very distinctive or deleted in either cDNA. The list contains the following information: (1) tissue, (2) developmental stage, (3) sequences around splice sites, (4) the length of each gapped region, and (5) other comments. The list is available at http://www.bioinfo.sfc.keio.ac.jp/ intron. Our results have predicted a number of unreported alternatively spliced genes, some of which are expressed only in a specific tissue or at a specific developmental stage. ©2002 by Cold Spring Harbor Laboratory Press.

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Computational challenges in cell simulation: A software engineering approach

Takahashi, K., Yugi, K., Hashimoto, K., Yamada, Y., Pickett, C., and Tomita, M

IEEE Intelligent Systems 17 ( 5 ) 64-71 2002

Research paper (scientific journal), Joint Work

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Complete mutation analysis panel of the 39 human HOX genes

Kosaki K, Takahashi T, Sasaki K, Tomita M, McGinnis W, and Matsuo N.

Teratology 65 ( 2 ) 50-62 - 62 2002

Research paper (scientific journal), Joint Work, ISSN 00403709

Background: The HOX gene family consists of highly conserved transcription factors that specify the identity of the body segments along the anteroposterior axis of the embryo. Because the phenotypes of mice with targeted disruptions of Hox genes resemble some patterns of human malformations, mutations in HOX genes have been expected to be associated with a significant number of human malformations. Thus far, however, mutations have been documented in only three of the 39 human HOX genes (HOXD13, HOXA13, and HOXA11) partly because current knowledge on the complete coding sequence and genome structure is limited to only 20 of the 39 human HOX genes. Methods: Taking advantage of the human and mouse draft genome sequences, we attempted to characterize the remaining 19 human HOX genes by bioinformatic analysis including phylogenetic footprinting, the probabilistic prediction method, and comparison of genomic sequences with the complete set of the human anonymous cDNA sequences. Results: We were able to determine the full coding sequences of 19 HOX genes and their genome structure and successfully designed a complete set of PCR primers to amplify the entire coding region of each of the 39 HOX genes from genomic DNA. Conclusions: Our results indicate the usefulness of bioinformatic analysis of the draft genome sequences for clinically oriented research projects. It is hoped that the mutation panel provided here will serve as a launchpad for a new discourse on the genetic basis of human malformations. © 2002 Wiley-Liss, Inc.

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Computer analysis of potential stem structures of rRNA operons in various procaryote genomes

Saito, R., Ozawa, Y., Kuzuno, N., Tomita M.

Gene 259 ( 1-2 ) 217-222 - 222 2001.12

Research paper (scientific journal), Joint Work, ISSN 03781119

The processing of 16S rRNA and 23S rRNA by RNase III in E.coli is known to involve stem structures formed by both ends of the rRNA. Indeed, complementary nucleotide sequences are usually found at both ends of 16S rRNA and 23S rRNA. However, whether or not this phenomenon exists in various other bacteria has not yet been adequately studied. We have conducted computer analyses of potential stem structures of rRNA operons in 12 bacterial and 3 archaeal genomes, and compared characteristics of the stem structures among these species. We systematically computed free energy values by exhaustively 'annealing' sequences around the 5′ end and sequences around the 3′ end of both 16S rRNA and 23S rRNA genes, in order to predict potential stem structures. The results suggest that rRNAs in most species form stem structures at both ends. Some species, such as A.aeolicus, seem to form unusually stable stem structures. On the other hand, some rRNAs, such as rRNAs of D.radiodurans, seem not to form solid stem structures. This suggests that rRNA processing in those species must employ a reliable targeting mechanism other than recognizing stem structures by RNase III. © 2000 Elsevier Science B.V.

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Gene: Foreword

Bernardi G., Macaya G., Tomita M.

Gene 276 ( 1-2 ) 2001.10

ISSN 03781119

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Correlation between sequence conservation of the 5′ untranslated region and codon usage bias in Mus musculus genes

Sakai H., Washio T., Saito R., Shinagawa A., Itoh M., Shibata K., Carninci P., Konno H., Kawai J., Hayashizaki Y., Tomita M.

Gene 276 ( 1-2 ) 101 - 105 2001.10

ISSN 03781119

The codon adaptation index (CAI) values of all protein-coding sequences of the full-length cDNA libraries of Mus musculus were computed based on the RIKEN mouse full-length cDNA library. We have also computed the extent of consensus in flanking sequences of the initiator ATG codon based on the 'relative entropy' values of respective nucleotide positions (from -20 to +12 bp relative to the initiator ATG codon) for each group of genes classified by CAI values. With regard to the two nucleotides positions (-3 and +4) known to be highly conserved in Kozak's consensus sequence, a clear correlation between CAI values and relative entropy values was observed at position -3 but this was not significant at position +4, although a significant correlation was found at position -1 of the consensus sequence. Further, although no correlation was observed at any additional positions, relative entropy values were very high at positions -4, -6, and -8 in genes with high CAI values. These findings suggest that the extent of conservation in the flanking sequence of the initiator ATG codon including Kozak's consensus sequence was an important factor in modulation of the translation efficiency as well as synonymous codon usage bias particularly in highly expressed genes. © 2001 Published by Elsevier Science B.V.

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Functional annotation of a full-length mouse cDNA collection

Kawai J, Shinagawa A, Shibata K, Yoshino M, Itoh M, Ishii Y, Arakawa T, Hara A, Fukunishi Y, Konno H, Adachi J, Fukuda S, Aizawa K, Izawa M, Nishi K, Kiyosawa H, Kondo S, Yamanaka I, Saito T, Okazaki Y, Gojobori T, Bono H, Kasukawa T, Saito R, Kadota K, Matsuda H, Ashburner M, Batalov S, Casavant T, Fleischmann W, Gaasterland T, Gissi C, King B, Kochiwa H, Kuehl P, Lewis S, Matsuo Y, Nikaido I, Pesole G, Quackenbush J, Schriml LM, Staubli F, Suzuki R, Tomita M, Wagner L, Washio T, Sakai K, Okido T, Furuno M, Aono H, Baldarelli R, Barsh G, Blake J, Boffelli D, Bojunga N, Carninci P, de Bonaldo MF, Brownstein MJ, Bult C, Fletcher C, Fujita M, Gariboldi M, Gustincich S, Hill D, Hofmann M, Hume DA, Kamiya M, Lee NH, Lyons P, Marchionni L, Mashima J, Mazzarelli J, Mombaerts P, Nordone P, Ring B, Ringwald M, Rodriguez I, Sakamoto N, Sasaki H, Sato K, Schonbach C, Seya T, Shibata Y, Storch KF, Suzuki H, Toyo-oka K, Wang KH, Weitz C, Whittaker C, Wilming L, Wynshaw-Boris A, Yoshida K, Hasegawa

Nature 409 ( 6821 ) 685-690 - 689 2001.02

Research paper (scientific journal), Joint Work, ISSN 0028-0836

The RIKEN Mouse Gene Encyclopaedia Project, a systematic approach to determining the full coding potential of the mouse genome, involves collection and sequencing of full-length complementary DNAs and physical mapping of the corresponding genes to the mouse genome. We organized an international functional annotation meeting (FANTOM) to annotate the first 21,076 cDNAs to be analysed in this project. Here we describe the first RIKEN clone collection, which is one of the largest described for any organism. Analysis of these cDNAs extends known gene families and identifies new ones.

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Correlation between Shine-Dalgarno sequence conservation and codon usage of bacterial genes

Sakai, H., Imamura, C., Osada, Y., Saito, R., Washio, T. and Tomita, M.

Journal of Molecular Evolution 52 ( 2 ) 164-170 - 170 2001

Research paper (scientific journal), Joint Work, ISSN 00222844

In this study, we analyzed the correlation between codon usage bias and Shine-Dalgarno (SD) sequence conservation, using complete genome sequences of nine prokaryotes. For codon usage bias, we adopted the codon adaptation index (CAI), which is based on the codon usage preference of genes encoding ribosomal proteins, elongation factors, heat shock proteins, outer membrane proteins, and RNA polymerase subunit proteins. To compute SD sequence conservation, we used SD motif sequences predicted by Tompa and systematically aligned them with 5′UTR sequences. We found that there exists a clear correlation between the CAI values and SD sequence conservation in the genomes of Escherichia coli, Bacillus subtilis, Haemophilus influenzae, Archaeoglobus fulgidus, Methanobacterium thermoautotrophicum, and Methanococcus jannaschii, and no relationship is found in M. genitalium, M. pneumoniae, and Synechocystis. That is, genes with higher CAI values tend to have more conserved SD sequences than do genes with lower CAI values in these organisms. Some organisms, such as M. thermoautotrophicum, do not clearly show the correlation. The biological significance of these results is discussed in the context of the translation initiation process and translation efficiency.

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The E-CELL Project: Towards Integrative Simulation of Cellular Processes

Hashimoto, K., Takahashi, K., Matsuzaki, Y., Matsushima, R., Saito, K., Yugi, K., Miyoshi, F., Nakano, H., Tanida, S., Saito, Y., Kawase, A., Watanabe, N., Shimizu, T. and Nakayama, Y., Tomita M.

New Generation Computing 18 ( 1 ) 1-12 2001

Joint Work

Preferential usage of some minor codons in bacteria

Ohno, H., Sakai, H., Washio, T. and Tomita, M.

Gene 276 ( 1-2 ) 107-15 - 115 2001

Research paper (scientific journal), Joint Work, ISSN 03781119

In many bacterial species, such as Deinococcus radiodurans, Haemophilus influenzae, and Methanobacterium thermoautotrophicum, some minor codons are preferentially used near the initiation codon. Among these codons, there are some minor codons that have strong preference for the initiation site in the high codon adaptation index (CAI) group (comprising of highly expressed genes) rather than in the low CAI genes group (comprising of low expressing genes). In the present study, codon usage in the initiation site and in the rest of the gene was systematically compared in the 27 complete bacterial genomes and Saccharomyces cerevisiae genome. Furthermore, we classified genes into two groups according to the CAI values and conducted the same analysis for each of the two groups. Our results suggest a role for some minor codons in the initiation site of the regulating translation system in many bacteria. We have summarized codons that are preferentially used in the initiation site and probably play a role in regulating genes expression in these organisms. © 2001 Elsevier Science B.V. All rights reserved.

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Whole cell simulation: A grand challenge of the 21st century

Tomita, M

Trends in Biotechnology 19 ( 6 ) 205-210 - 210 2001

Research paper (scientific journal), Single Work, ISSN 01677799

Study of the cell will never be complete unless its dynamic behavior is understood. The complex behavior of the cell cannot be determined or predicted unless a computer model of the cell is constructed and computer simulation is undertaken. Rapid accumulation of biological data from genome, proteome, transcriptome and metabolome projects can bring us to the point where it is no longer purely speculative to discuss how to construct virtual cells in silico. This article describes attempts to construct whole cell models. The E-CELL project has completed a couple of virtual cell models, and computer simulations have revealed some biological surprises.

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E-cell project: towards computer modeling of the whole cell

Tomita M.

Annals of Biomedical Engineering (Annals of Biomedical Engineering) 28 ( SUPPL. 1 ) 2000.12

ISSN 00906964

The E-CELL System (http://www.e-cell.org) is a genetic software package developed, to model not only metabolic pathways but also other higher-order cellular processes such as protein synthesis and signal transduction. Using the system, we have successfully constructed a virtual cell with 127 genes sufficient for 'self-support'. The gene set was selected from the genome of Mycoplasma genitalium, and the metabolisms include transcription, translation, membrane transport, the glycolysis pathway for energy production, and the phospholipid biosynthesis pathway or membrane structure.

Characteristic Sequence Pattern in the 5-to 20-bp Upstream Region of Primate Alu Elements

Toda, Y., Saito, R. and Tomita, M.

Journal of Molecular Evolution 50 ( 3 ) 232-237 - 237 2000

Research paper (scientific journal), Joint Work, ISSN 00222844

We conducted comprehensive sequence analysis of 5' flanking regions of primate Alu elements. Information contents were computed and frequencies of 1024 pentanucleotides were measured to approximate the location of a characteristic sequence and to specify its pattern(s), which may be involved in the integration of Alu elements into their host genomes. A large number of samples was used, the wide region of the 5' end of Alu elements was analyzed, and comparisons were made among different subfamilies. Through our analyses, 'TTTTAAAAA' or '(T)(ms)(A)(n)' can be stated as a candidate for the characteristic sequence pattern, which resides around the region 5 to 20 base pairs upstream of the 5' end of Alu elements. This characteristic sequence pattern was more prominent in the sequences of younger Alus, which is a strong indication that the sequence pattern has a role at the time of Alu integration.

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The orientation bias of Chi sequences is a general tendency of G-rich oligomers

Uno R., Nakayama Y., Arakawa K., Tomita M.

Gene 36 ( 1-2 ) 207-215 - 215 2000

Research paper (scientific journal), Joint Work, ISSN 03781119

The Chi sequences are specific oligomers that stimulate DNA repair by homologous recombination, and are different sequences in each organism. Approximately 75% of the copies of the Chi sequence (5′-GCTGGTGG-3′) of Escherichia coli reside on the leading strand, and this orientation bias is often believed to be a consequence of the biological role of Chi sequences as the signal sequence of RecBCD pathway in DNA replication. However, our computer analysis found that many G-rich oligomers also show this asymmetric orientation pattern. The shift in the Chi orientation bias appears around the replication origin and terminus, but these locations are also coincident with the shift points in GC content or GC skew. We conducted the same analysis with the genome of Bacillus subtilis, and found that in addition to Chi, other G-rich oligomers show similar asymmetric orientation patterns, whose shift points were coincident with those of the GC skew. However, the genome of Haemophilus influenzae Rd, whose GC skew is not so pronounced, does not clearly show asymmetric orientation patterns of Chi or other G-rich oligomers. These results lead us to suggest that the uneven distribution of the Chi orientation between the two strands of the double helix is mostly due to the uneven distribution of G content (GC skew) and that the replication-related function of Chi sequences is not the primary factor responsible for the evolutionary pressure causing the orientation bias. © 2000 Elsevier Science B.V.

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Computer Simulation of Human erythrocyte Using the E-CELL Simulation System

Yoichi Nakayama, Ryo Matsushima, Hisako Nakano, Akiko Kawase, Kouichi Takahashi and Masaru Tomita

BRIDGING THE GAP BETWEEN WEQUENCE & FUNCTION 1999.09

Joint Work

ApA dinucleotide periodicity in prokaryote, eukaryote, and organelle genomes

Tomita M., Wada M., Kawashima Y.

Journal of Molecular Evolution 49 ( 2 ) 182 - 192 1999.08

ISSN 00222844

Computer analyses of various genome sequences revealed the existence of certain periodical patterns of adenine-adenine dinucleotides (ApA). For each genome sequence of 13 eubacteria, 3 archaebacteria, 10 eukaryotes, 60 mitochondria, and 9 chloroplasts, we counted frequencies of ApA dinucleotides at each downstream position within 50 bp from every ApA. We found that the complete genomes of all three archaebacteria have clear ApA periodicities of about 10 bps. On the other hand, all of the 13 eubacteria we analyzed were found to have an ApA periodicity of about 11 bp. Similar periodicities exist in the 10 eukaryotes, although higher organisms such as primates tend to have weaker periodic patterns. None of the mitochondria and chroloplasts we analyzed showed an evident periodic pattern.

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Analysis of base-pairing potentials between 16S rRNA and 5' UTR for translation initiation in various prokaryotes

Osada, Y., Saito, R. and Tomita, M.

Bioinformatics 15 ( 7-8 ) 578-581 - 581 1999

Research paper (scientific journal), Joint Work, ISSN 13674803

Motivation: It is well accepted that the 3' end of 16S rRNA is directly involved in prokaryotic translation initiation by pairing with the Shine-Dalgarno (SD) sequence, which is located in the ribosome-binding site of mRNA. According to Shine and Dalgarno, Escherichia coli's 5' UTR has the pattern of 'AGGAGG' (SD sequence), which is complementary to the 3' end sequence of 16S rRNA. In this work, we systematically calculated free-energy values of the base pairing between the 3' end of 16S rRNA and the 5' UTR of mRNA, in order to analyze the base-pairing potentials in various prokaryotes. The free-energy values were then plotted over distances from the start codon to visualize the free-energy pattern of 5' UTRs. Results: The average free-energy values fell sharply before the start codon in E. coli, which is consistent with the model that the 3' end of 16S rRNA base pairs with the SD sequence. Haemophilus influenzae, Bacillus subtilis and Helicobacter pylori show a similar pattern, suggesting that the organisms have basically the same mechanism of translation initiation as E. coli. Other eubacteria, such as Synechocystis PCC6803, Mycoplasma genitalium, Mycoplasma pneumoniae and Borrelia burgdorferi also show decreases in their free-energy values, although they are less evident. We also did the same analysis with a eukaryote genome as a control; no fall in free-energy values was observed between the 3' end of 18S rRNA and 5' UTRs of Saccharomyces cerevisiae, suggesting that this organism does not base pair in translation initiation. The three archaebacteria A. fulgidus, M. jannaschii and M. thermoautotrophicum show patterns similar to eubacteria, but not to S. cerevisiae, indicating that archaebacteria are closer to eubacteria than to eukaryotes with respect to the mechanism of translation initiation. From these observations, it appears that the shape of the curve produced by the algorithm can be used to predict the mechanism of translation initiation. Availability: The C programs used in our analysis are available upon request.

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Computer analyses of complete genomes suggest that some archaebacteria employ both eukaryotic and eubacterial mechanisms in translation initiation.

Saito, R. and Tomita, M.

Gene 238 ( 1 ) 79-83 - 83 1999

Research paper (scientific journal), Joint Work, ISSN 03781119

The translation initiation mechanism of archaebacteria is still not clearly understood. Our previous work showed that ATG triplets before start codons have been strongly depleted in eukaryotic genomes, presumably because ribosome of eukaryotes scans mRNA from the 5' to 3' direction to find proper start codons. Extra ATG triplets before start codons would confuse the process and thus they have been negatively selected in eukaryotic genomes. In eubacterial genomes, on the other hand, ribosome binds to the Shine-Dalgarno (SD) sequence at once without mRNA scanning, and the characteristic patterns of ATG triplet depletion were not observed (Saito, R., Tomita, M., 1999. On negative selection against ATG triplets near start codons in eukaryotic and procaryotic genomes. J. Mol. Evol. 48, 213-217). The ATG triplet analysis on archaebacterial genomes revealed that Methanococcus jannaschii and Pyrococcus horikoshii show patterns similar to eukaryotes, implying that these species employ scanning of mRNA from the 5' to 3' direction in the process of translation initiation. On the other hand, our earlier study found that these archaea have SD-like sequences, which are complementary to the 3' end sequence of 16S rRNA, as in eubacterial translation initiation (Osada, Y., Saito, R., Tomita, M. Analysis of base-pairing potentials between 16S rRNA and 5' UTR for translation initiation in various procaryotes. Bioinformatics, in press). These two results combined lead us to conclude that these archaea probably use a hybrid mechanism; their ribosome scans mRNAs from the 5' to 3' direction and then 16S rRNA binds to the SD-like sequence of the 5' UTR. (C) 1999 Elsevier Science B.V. All rights reserved.

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E-CELL: Software environment for whole cell simulation

Tomita, M., Hashimoto, K., Takahashi, K., Shimizu, T., Matsuzaki, Y., Miyoshi, F., Saito, K., Tanida, S., Yugi, K., Venter, J.,C., Hutchison, C.,A .,3rd.

Bioinformatics 15 ( 1 ) 72 - 84 1999

Research paper (scientific journal), Joint Work, ISSN 13674803

Motivation: Genome sequencing projects and further systematic functional analyses of complete gene sets are producing an unprecedented mass of molecular information for a wide range of model organisms. This provides us with a detailed account of the cell with which we may begin to build models for simulating intracellular molecular processes to predict the dynamic behaviour of living cells. Previous work in biochemical and genetic simulation has isolated well-characterized pathways for detailed analysis, but methods for building integrative models of the cell that incorporate gene regulation, metabolism and signaling have not been established. We, therefore, were motivated to develop a software environment for building such integrative models based on gene sets, and running simulations to conduct experiments in silico. Results: E-CELL, a modeling and simulation environment for biochemical and genetic processes, has been developed. The E-CELL system allows a user to define functions of proteins, protein-protein interactions, protein-DNA interactions, regulation of gene expression and other features of cellular metabolism, as a set of reaction rules. E-CELL simulates cell behavior by numerically integrating the differential equations described implicitly in these reaction rules. The user can observe, through a computer display, dynamic changes in concentrations of proteins, protein complexes and other chemical compounds in the cell. Using this software, we constructed a model of a hypothetical cell with only 127 genes sufficient for transcription, translation, energy production and phospholipid synthesis. Most of the genes are taken from Mycoplasma genitalium, the organism having the smallest known chromosome, whose complete 580 kb genome sequence was determined at TIGR in 1995. We discuss future applications of the E-CELL system with special respect to genome engineering. Availability: The E-CELL software is available upon request. Supplementary information: The complete list of rules of the developed cell model with kinetic parameters can be obtained via our web site at: http://e-cell.org/. Contact: mt@@@sfc.keio.ac.jp.

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Comparative study of overlapping genes in the genomes of Mycoplasma genitalium and Mycoplasma pneumoniae

Fukuda, Y., Washio, T. and Tomita, M.

Nucleic Acids Research 27 ( 8 ) 1847-1853 - 1853 1999

Research paper (scientific journal), Joint Work, ISSN 03051048

Overlapping genes are defined, in this paper, as a pair of adjacent genes whose coding regions are partly overlapping. We systematically analyzed all overlapping genes in the genomes of two closely related species: Mycoplasma genitalium and Mycoplasma pneumoniae. Careful comparisons were made for homologous genes that are overlapped in one species but not in the other. This comparative analysis allows us to propose a model of how overlapping genes emerged in the course of evolution. It was found that overlapping genes were generated primarily due to the loss of a stop codon in either gene, in many cases, the absence of which resulted in elongation of the 3' end of the gene's coding region. More specifically, the loss of the stop codon took place as a result of the following events: deletion of the stop codon (64.4%), point mutation at the stop codon (4.4%), and frame shift at the end of the coding region (6.7%). Overlapping genes, in a sense, can be thought of as the results of evolutionary pressure to minimize genome size. However, our analysis indicates that many overlapping genes, at least in the genomes of M. genitalium and M. pneumoniae, are due to incidental elongation of the coding regions.

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Sequence patterns observed in 5' flanking regions of primate Alu elements

Toda, Y., Saito, R. and Tomita, M.

Annals of the New York Academy of Sciences 18 369-374 - 374 1999

Research paper (scientific journal), Joint Work, ISSN 00778923

Retrotransposons have generally been known to integrate randomly into host genomes. Jurka, however, showed some consensus sequence patterns at insertion sites of some mammalian retrotransposons and proposed enzymatic involvement that mediates integration. Jurka used about 400 complete human Alu and rodent ID sequences which retain full length with identical flanking sequences at both ends. In our study, more than 25,000 Alu sequences longer than 250 bp were used for comprehensive analysis to identify any consensus sequence(s) preceding the 5' end of Alu elements. 'Entropy' at each nucleotide position within 500 bases of the 5' Alu end was computed. Significant drop of entropy was observed between position -20 and -10 of the 5' end of Alu, suggesting the existence of certain consensus sequence(s) in the region. Frequencies of all possible triplets (total of 64) were measured in the same region. Observation that frequencies of triplets 'aaa,' 'taa,' and 'tta' in the 5' flanking sequences were high is consistent with Jurka. However, frequencies of triplets 'att' and 'aca' were also significantly high, which are not the primary candidates for nick site in Jurka.

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On negative selection against ATG triplets near start codons in eukaryotic and prokaryotic genomes

Saito R, Tomita M

Journal of Molecular Evolution 26 ( 23 ) 5456-5463 - 217 1998.12

Research paper (scientific journal), Joint Work, ISSN 0022-2844

The frequencies of ATG triplets in the genomes of various species were systematically analyzed, and the frequency of ATG triplets was significantly low around start codons in both prokaryotic and eukaryotic genomes. In eukaryotes, however, the frequency decrease before the start codon is much more evident than that after the start codon. In prokaryotes, on the other hand, the ATG frequency pattern around the start codon is less evident, and - more importantly - symmetric. We also computed average distances between a start codon and its nearest upstream-located ATG triplet and found a general tendency for the average distances to be longer in higher organisms.

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Analysis of complete genomes suggests that many prokaryotes do not rely on hairpin formation in transcription termination

Washio T., Sasayama J., Tomita M.

Nucleic Acids Research 26 ( 23 ) 5456 - 5463 1998.12

ISSN 03051048

Free energy values of mRNA tertiary structures around stop codons were systematically calculated to surmise the hairpin-forming potential for all genes in each of the 16 complete prokaryote genomes. Instead of trying to detect each individual hairpin, we averaged the free energy values around the stop codons over the entire genome to predict how extensively the organism relies on hairpin formation in the process of transcription termination. The free energy values of Escherichia coli K-12 shows a sharp drop, as expected, at 30 bp downstream of the stop codon, presumably due to hairpin-forming sequences. Similar drops are observed for Haemophilus influenzae Rd, Bacillus subtilis and Chlamydia trachomatis, suggesting that these organisms also form hairpins at their transcription termination sites. On the other hand, 12 other prokaryotes - Mycoplasma genitalium, Mycoplasma pneumoniae, Synechocystis PCC6803, Helicobacter pylori, Borrelia burgdorferi, Methanococcus jannaschii, Archaeoglobus fulgidus, Methanobacterium thermoautotrophicum, Aquifex aeolicus, Pyrococcus horikoshii, Mycobacterium tuberculosis and Treponema pallidum - show no apparent decrease in free energy value at the corresponding regions. This result suggests that these prokaryotes, or at least some of them, may never form hairpins at their transcription termination sites.

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Alu elements as an aid in deciphering genome rearrangements

Toda, Y. and Tomita, M.

Gene 205 ( 1-2 ) 173 - 176 1997

Research paper (scientific journal), Joint Work, ISSN 03781119

Genomic rearrangements result in genomic duplications that lead to the generation of more complex genomes. Some attempts have been made to trace duplication histories of different loci using Alu elements because of their large population in primate genomes. In this short report, using the human growth hormone locus as an example, we demonstrate the usefulness of Alu repetitive elements in computer sequence analyses when tracing duplication histories. Information on subfamily classification, direction, arrangements, Poly(A) tails and direct repeats can aid our understanding of genome rearrangements.

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CpG distribution patterns in methylated and nonmethylated species

Shimizu, T., Takahashi, K. and Tomita, M.

Gene 205 ( 1-2 ) 103-107 - 107 1997

Research paper (scientific journal), Joint Work, ISSN 03781119

To characterize the extent of DNA methylation and its possible biological roles in a wide variety of organisms, we have analyzed gene sequences extracted from the GenBank database. Sequences of both methylated and non-methylated species were used for comparative analysis. The local CpG dinucleotide distribution near the 5' ends of genes as well as the degree of overall CpG suppression/depletion in the entire gene region were examined in all complete gene sequences for each species. We show that the distribution patterns of CpG near the 5' region of genes differ among vertebrates, invertebrates, plants and bacteria. CpG island-like peaks in CpG O/E (observed/expected ratio) were observed not only in methylated species, but also in non-methylated species. In methylated non-vertebrates, overall CpG O/E values were lower, and peaks in the CpG profile of 5' regions were larger than in non-methylated species. We discuss the implications of such biases with respect to DNA methylation.

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Introns and reading frames: Correlation between splicing sites and their codon positions

Tomita, M., Shimizu, N. and Brutlag, D.L.

Molecular Biology and Evolution 13 ( 9 ) 1219-1223 - 1223 1996

Research paper (scientific journal), Joint Work, ISSN 07374038

Computer analyses of the entire GenBank database were conducted to examine correlation between splicing sites and codon positions in reading frames. Intron insertion patterns (i.e., splicing site locations with respect to codon positions) have been analyzed for all of the 64 codons of all the eukaryote taxonomic groups: primates, rodents, mammals, vertebrates, invertebrates, and plants. We found that reading frames are interrupted by an intron at a codon boundary (as opposed to the middle of a codon) significantly more often than expected. This observation is consistent with the exon shuffling hypothesis, because exons that end at codon boundaries can be concatenated without causing a frame shift and thus are evolutionarily advantageous. On the other hand, when introns interrupt at the middles of codons, they exist in between the first and second bases much more frequently than between the second and third bases, despite the fact that boundaries between the first and second bases of codons are generally far more important than those between the second and third bases. The reason for this is not clear and yet to be explained. We also show that the length of an exon is a multiple of 3 more frequently than expected. Furthermore, the total length of two consecutive exons is also more frequently a multiple of 3. All the observations above are consistent with results recently published by Long, Rosenberg, and Gilbert (1995).

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CMU Project

72-82 1989

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