Published papers

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.

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.

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.

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.

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.

© 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.

© 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.

© 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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.