Details of a Researcher - Tomita, Masaru

Papers - Tomita, Masaru

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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 <sup>13</sup>C-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<sup>-1</sup>. 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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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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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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