Published papers

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Arenibacter sp. strain C-21, isolated from surface marine sediment of Japan, accumulates iodine in the presence of glucose and iodide (I-). We report here the draft genome sequence of this strain to provide insight into the molecular mechanism underlying its iodine-accumulating ability.

© 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.

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.

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.

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.