Published papers

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

A major bacterial alarmone, guanosine 3′,5′-bispyrophosphate (ppGpp), controls cellular growth under conditions of nutritional starvation. For most bacteria, intracellular ppGpp levels are tightly controlled by the synthesis/degradation cycle of RelA and SpoT activities. This study shows a novel ppGpp regulatory protein governing the cellular growth of Thermus thermophilus, Ndx8, a member of the Nudix pyrophosphatase family that degrades ppGpp to yield guanosine 3′,5′-bisphosphate. The ndx8-null mutant strain exhibited early stage growth arrest accompanied by the stationary phase-specific morphologies and global transcriptional modulation under nutritionally defined conditions. Several possible substrate compounds of Ndx8, which specifically accumulated in the ndx8 mutant cells, were identified by employing a capillary electrophoresis time-of-flight mass spectrometry-based metabolomics approach. Among them, the hydrolytic activity of Ndx8 for ppGpp was significant not only in vitro but also in vivo. Finally, the elimination of ppGpp synthetic activity suppressed the observed phenotype of the ndx8 mutation, suggesting that the function of Ndx8 as a growth regulator is involved in ppGpp accumulation, which is thought to act as a trigger of the growth phase transition. These results suggest a novel mechanism of ppGpp-mediated growth control by the functional relay between Ndx8 and SpoT activity as ppGpp scavengers. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

Despite the current availability of selective inhibitors for the classical nuclear export pathway, no inhibitor for the classical nuclear import pathway has been developed. Here we describe the development of specific inhibitors for the importin α/β pathway using a novel method of peptide inhibitor design. An activity-based profile was created via systematic mutational analysis of a peptide template of a nuclear localization signal. An additivity-based design using the activity-based profile generated two peptides with affinities for importin α that were approximately 5 million times higher than that of the starting template sequence. The high affinity of these peptides resulted in specific inhibition of the importin α/β pathway. These peptide inhibitors provide a useful tool for studying nuclear import events. Moreover, our inhibitor design method should enable the development of potent inhibitors from a peptide seed. © 2008 Elsevier Ltd. All rights reserved.

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

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

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

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

Cell fate decision remarkably generates specific cell differentiation path among the multiple possibilities that can arise through the complex interplay of high-dimensional genome activities. The coordinated action of thousands of genes to switch cell fate decision has indicated the existence of stable attractors guiding the process. However, origins of the intracellular mechanisms that create "cellular attractor" still remain unknown. Here, we examined the collective behavior of genome-wide expressions for neutrophil differentiation through two different stimuli, dimethyl sulfoxide (DMSO) and alltrans- retinoic acid (atRA). To overcome the difficulties of dealing with single gene expression noises, we grouped genes into ensembles and analyzed their expression dynamics in correlation space defined by Pearson correlation and mutual information. The standard deviation of correlation distributions of gene ensembles reduces when the ensemble size is increased following the inverse square root law, for both ensembles chosen randomly from whole genome and ranked according to expression variances across time. Choosing the ensemble size of 200 genes, we show the two probability distributions of correlations of randomly selected genes for atRA and DMSO responses overlapped after 48 hours, defining the neutrophil attractor. Next, tracking the ranked ensembles' trajectories, we noticed that only certain, not all, fall into the attractor in a fractal-like manner. The removal of these genome elements from the whole genomes, for both atRA and DMSO responses, destroys the attractor providing evidence for the existence of specific genome elements (named "genome vehicle") responsible for the neutrophil attractor. Notably, within the genome vehicles, genes with low or moderate expression changes, which are often considered noisy and insignificant, are essential components for the creation of the neutrophil attractor. Further investigations along with our findings might provide a comprehensive mechanistic view of cell fate decision. © 2010 Tsuchiya et al.

We have developed a novel strategy to improve the efficiency of identification of multiply phosphorylated peptides isolated by hydroxy acid modified metal oxide chromatography (HAMMOC). This strategy consists of alkali-induced chemical dephosphorylation (beta-elimination reaction) of phosphopeptides isolated by HAMMOC prior to analysis by liquid chromatography/mass spectrometry (LC/MS). This approach identified 1.9-fold more multiply phosphorylated peptides than the conventional approach without beta-elimination from a digested mixture of three standard phosphoproteins. In addition, the accuracy of phosphorylation site determination in synthetic phosphopeptides was significantly improved. Finally, we applied this approach to a cell lysate. By combining this dephosphorylation approach with the conventional approach, we successfully identified 1649 unique phosphopeptides, including 325 multiply phosphorylated phosphopeptides, from 200mg of cultured Arabidopsis cells. These results indicate that chemical dephosphorylation prior to LC/MS analysis increases the efficiency of identification of multiply phosphorylated peptides, as well as the accuracy of phosphorylation site determination. © 2010 John Wiley & Sons, Ltd.

Tardigrada, a phylum of meiofaunal organisms, have been at the center of discussions of the evolution of Metazoa, the biology of survival in extreme environments, and the role of horizontal gene transfer in animal evolution. Tardigrada are placed as sisters to Arthropoda and Onychophora (velvet worms) in the superphylum Panarthropoda by morphological analyses, but many molecular phylogenies fail to recover this relationship. This tension between molecular and morphological understanding may be very revealing of the mode and patterns of evolution of major groups. Limnoterrestrial tardigrades display extreme cryptobiotic abilities, including anhydrobiosis and cryobiosis, as do bdelloid rotifers, nematodes, and other animals of the water film. These extremophile behaviors challenge understanding of normal, aqueous physiology: how does a multicellular organism avoid lethal cellular collapse in the absence of liquid water? Meiofaunal species have been reported to have elevated levels of horizontal gene transfer (HGT) events, but how important this is in evolution, and particularly in the evolution of extremophile physiology, is unclear. To address these questions, we resequenced and reassembled the genome of H. dujardini, a limnoterrestrial tardigrade that can undergo anhydrobiosis only after extensive pre-exposure to drying conditions, and compared it to the genome of R. varieornatus, a related species with tolerance to rapid desiccation. The 2 species had contrasting gene expression responses to anhydrobiosis, with major transcriptional change in H. dujardini but limited regulation in R. varieornatus. We identified few horizontally transferred genes, but some of these were shown to be involved in entry into anhydrobiosis. Whole-genome molecular phylogenies supported a Tardigrada+Nematoda relationship over Tardigrada+Arthropoda, but rare genomic changes tended to support Tardigrada+Arthropoda.

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

Schlafen 11 (SLFN11), a promising therapeutic biomarker, binds chromatin and sensitizes cancer cells to DNA-targeting agents by blocking DNA replication. Murai et al. show that, in response to replication stress, SLFN11 selectively increases chromatin accessibility at promoters and activates a subset of genes known as the immediate early genes (IEGs).

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

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

RNase H (ribonuclease H) is an endonuclease that cleaves the RNA strand of RNA-DNA duplexes. It has been reported that the three-dimensional structure of RNase H is similar to that of the PIWI domain of the Pyrococcus furiosus Ago (argonaute) protein, although the two enzymes share almost no similarity in their amino acid sequences. Eukaryotic Ago proteins are key components of the RNA-induced silencing complex and are involved in microRNA or siRNA (small interfering RNA) recognition. In contrast, prokaryotic Ago proteins show greater affinity for RNA-DNA hybrids than for RNA-RNA hybrids. Interestingly, we found that wild-type Pf-RNase HII (P. furiosus, RNase HII) digests RNA-RNA duplexes in the presence of Mn2+ ions. To characterize the substrate specificity of Pf-RNase HII, we aligned the amino acid sequences of Pf-RNase HII and Pf-Ago, based on their protein secondary structures. We found that one of the conserved secondary structural regions (the fourth β-sheet and the fifth α-helix of Pf-RNase HII) contains family-specific amino acid residues. Using a series of Pf-RNase HII-Pf-Ago chimaeric mutants of the region, we discovered that residues Asp110, Arg113 and Phe 114 are responsible for the dsRNA (double-stranded RNA) digestion activity of Pf-RNase HII. On the basis of the reported three-dimensional structure of Ph-RNase HII from Pyrococcus horikoshii, we built a three-dimensional structural model of RNase HII complexed with its substrate, which suggests that these amino acids are located in the region that discriminates DNA from RNA in the non-substrate strand of the duplexes. © The Authors.

The analysis of archaeal tRNA genes is becoming more important to evaluate the origin and evolution of tRNA molecule. Even with the recent accumulation of complete genomes of numerous archaeal species, several tRNA genes are still required for a full complement of the codon table. We conducted comprehensive screening of tRNA genes from 47 archaeal genomes by using a combination of different types of tRNA prediction programs and extracted a total of 2,143 reliable tRNA gene candidates including 437 intron-containing tRNA genes, which covered more than 99.9% of the codon tables in Archaea. Previously, the content of intron-containing tRNA genes in Archaea was estimated to be approximately 15% of the whole tRNA genes, and most of the introns were known to be located at canonical positions (nucleotide position between 37 and 38) of precursor tRNA (pre-tRNA). Surprisingly, we observed marked enrichment of tRNA introns in five species of the archaeal order Thermoproteales; about 70% of tRNA gene candidates were found to be intron-containing tRNA genes, half of which contained multiple introns, and the introns were located at various noncanonical positions. Sequence similarity analysis revealed that approximately half of the tRNA introns found at Thermoproteales-specific intron locations were highly conserved among several tRNA genes. Intriguingly, identical tRNA intron sequences were found within different types of tRNA genes that completely lacked exon sequence similarity, suggesting that the tRNA introns in Thermoproteales could have been gained via intron insertion events at a later stage of tRNA evolution. Moreover, although the CCA sequence at the 3′ terminal of pre-tRNA is added by a CCA-adding enzyme after gene transcription in Archaea, most of the tRNA genes containing highly conserved introns already encode the CCA sequence at their 3′ terminal. Based on these results, we propose possible models explaining the rapid increase of tRNA introns as a result of intron insertion events via retrotransposition of pre-tRNAs. The sequences and secondary structures of the tRNA genes and their bulge-helix-bulge motifs were registered in SPLITSdb (http://splits.iab.keio.ac.jp/splitsdb/), a novel and comprehensive database for archaeal tRNA genes. © The Author 2008. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved.

Using an expression protein library of a hyperthermophilic archaeon, Pyrococcus furiosus, we identified a gene (PF0027) that encodes a protein with heat-stable cyclic nucleotide phosphodiesterase (CPDase) activity. The PF0027 gene encoded a 21-kDa protein and an amino acid sequence that showed ∼27% identity to that of the 2′-5′ tRNA ligase protein, ligT (20 kDa), from Escherichia coli. We found that the purified PF0027 protein possessed GTP-dependent RNA ligase activity and that synthetic tRNA halves bearing 2′,3′-cyclic phosphate and 5′-OH termini were substrates for the ligation reaction in vitro. GTP hydrolysis was not required for the reaction, and GTPγS enhanced the tRNA ligation activity of PF0027 protein, suggesting that the ligation step is regulated by a novel mechanism. In comparison to the strong CPDase activity of the PF0027 protein, the RNA ligase activity itself was quite weak, and the ligation product was unstable during in vitro reaction. Finally, we used NMR to determine the solution structure of the PF0027 protein and discuss the implications of our results in understanding the role of the PF0027 protein. Copyright © 2009 RNA Society.

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

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