Published papers

We report here the draft genome sequence of Geobacter sp. strain SVR, isolated from antimony mine soil in Hyogo Prefecture, Japan. The genome sequence data in this study will provide useful information for understanding bacterial antimonate reduction.

Enterococcus faecalis is a common commensal of the intestines of humans and other mammals but is also a frequent cause of serious ailments. Here, we report 14 draft genome sequences of strains of Enterococcus faecalis, a normal inhabitant and Gram-positive bacterium that was isolated from 7 healthy Japanese volunteers.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

© 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.

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.