Published papers

There is an increasing call for the absolute quantification of time-resolved metabolite data. However, a number of technical issues exist, such as metabolites being modified/degraded either chemically or enzymatically during the extraction process. Additionally, capillary electrophoresis mass spectrometry (CE-MS) is incompatible with high salt concentrations often used in extraction protocols. In microbial systems, metabolite yield is influenced by the extraction protocol used and the cell disruption rate. Here we present a method that rapidly quenches metabolism using dry-ice ethanol bath and methanol N-ethylmaleimide solution (thus stabilising thiols), disrupts cells efficiently using bead-beating and avoids artefacts created by live-cell pelleting. Rapid sample processing minimised metabolite leaching. Cell weight, number and size distribution was used to calculate metabolites to an attomol/cell level. We apply this method to samples obtained from the respiratory oscillation that occurs when yeast are grown continuously. © 2012 Sasidharan et al.

Colonial volvocaleans (Chlorophyceae) are used as a standard model of multicellular evolution. However, the phylogenetic position of the colonial volvocalean family Spondylomoraceae has yet to be resolved. To examine this, the molecular phylogenies of Pyrobotrys stellata and Pyrobotrys squarrosa were analyzed using combined 18S rRNA, RUBISCO large subunit, and P700 chl a-apoprotein A2 gene sequences. In the phylogenetic trees, Pyrobotrys belonged to the clade Caudivolvoxa and was not closely related to other colonial volvocalean flagellates. The results indicate that colony formation of Spondylomoraceae independently evolved from unicellular volvocaleans. The phylogenetic position of problematic "Pascherina tetras" SAG 159-1 was also analyzed. © 2010 by the International Society of Protistologists.

An extremely thermophilic bacterium, Thermus thermophilus HB8, is one of the model organisms for systems biology. Its genome consists of a chromosome (1.85 Mb), a megaplasmid (0.26 Mb) designated pTT27, and a plasmid (9.3 kb) designated pTT8, and the complete sequence is available. We show here that T. thermophilus is a polyploid organism, harboring multiple genomic copies in a cell. In the case of the HB8 strain, the copy number of the chromosome was estimated to be four or five, and the copy number of the pTT27 megaplasmid seemed to be equal to that of the chromosome. It has never been discussed whether T. thermophilus is haploid or polyploid. However, the finding that it is polyploid is not surprising, as Deinococcus radiodurans, an extremely radioresistant bacterium closely related to Thermus, is well known to be a polyploid organism. As is the case for D. radiodurans in the radiation environment, the polyploidy of T. thermophilus might allow for genomic DNA protection, maintenance, and repair at elevated growth temperatures. Polyploidy often complicates the recognition of an essential gene in T. thermophilus as a model organism for systems biology. Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Osteoporosis is characterized as a metabolic disorder of bone tissue, and various metabolic markers are now available to support its diagnosis and evaluate treatment effects. Substances produced as end products of metabolomic activities are the correlated factors to the biological or metabolic status, and thus, metabolites are considered highly sensitive markers of particular pathological states, including osteoporosis. Here we undertook comprehensive serum metabolomics analysis in postmenopausal women with or without low bone mineral density (low BMD vs controls) for the first time using capillary electrophoresis/mass spectrometry. Among the metabolites tested, 57 were detected in sera. Levels of hydroxyproline, Gly-Gly and cystine, differed significantly between groups, with Gly-Gly and cystine significantly lower in the low BMD group and hydroxyproline, a reported marker of osteoporosis, significantly higher. Levels of TRACP5b, a bone resorption marker, were significantly higher in the low BMD group, supporting the study's validity. Taken together, our findings represent novel metabolomic profiling in low BMD in postmenopausal women.

Ribosome biogenesis and turnover are processes necessary for cell viability and proliferation, and many kinds of proteins are known to regulate these processes. However, many questions still remain, especially in the Archaea. Generally, several ribonucleases are required to process precursor rRNAs to their mature forms, and to degrade rRNAs for quality control. Here, we found that FAU-1, which is known to be an RNA binding protein, possesses an RNase activity against precursor 5S rRNA derived from P. furiosus and T. kodakarensis in the order Thermococcales in vitro. An in vitro analysis revealed that UA sequences in the upstream of 5S rRNA were preferentially degraded by addition of FAU-1. Moreover, a fau-1 gene deletion mutant of T. kodakarensis showed a delay of exponential phase, reduction of maximum cell number and significant changes in the nucleotide sequence lengths of its 5S, 16S, and 23S rRNAs in early exponential phase. Our results suggest that FAU-1 is a potential RNase involved in rRNA stability through maturation and/or degradation processes.

Although nanoparticles (NPs) has afforded considerable benefits in various fields of sciences, several reports have shown their harmful effects, suggesting the necessity of adequate risk assessment. To clarify the mechanism of titanium dioxide nanoparticles (TiO2 NPs)-enhanced gingival inflammation, we conducted the full-scale metabolomic analyses of human gingival fibroblast cells treated with IL-1β alone or in combination with TiO2 NPs. Observation with transmission electron microscope demonstrated the incorporation of TiO2 NPs into vacuoles of the cells. TiO2 NPs significantly enhanced the IL-1β-induced prostaglandin E2 production and COX-1 and COX-2 protein expression. IL-1β reduced the intracellular concentrations of overall primary metabolites especially those of amino acid, urea cycle, polyamine, S-adenosylmethione and glutathione synthetic pathways. The addition of TiO2 NPs further augmented these IL-1β-induced metabolic changes, recommending careful use of dental materials containing TiO2 NPs towards patients with gingivitis or periodontitis. The impact of the present study is to identify the molecular targets of TiO2 NPs for the future establishment of new metabolic markers and therapeutic strategy of gingival inflammation.

The faithful translation of the genetic code requires the highly accurate aminoacylation of transfer RNAs (tRNAs). However, it has been shown that nematode-specific V-arm-containing tRNAs (nev-tRNAs) are misacylated with leucine in vitro in a manner that transgresses the genetic code. nev-tRNAGly (CCC) and nev-tRNAIle (UAU), which are the major nev-tRNA isotypes, could theoretically decode the glycine (GGG) codon and isoleucine (AUA) codon as leucine, causing GGG and AUA codon ambiguity in nematode cells. To test this hypothesis, we investigated the functionality of nev-tRNAs and their impact on the proteome of Caenorhabditis elegans. Analysis of the nucleotide sequences in the 3' end regions of the nev-tRNAs showed that they had matured correctly, with the addition of CCA, which is a crucial posttranscriptional modification required for tRNA aminoacylation. The nuclear export of nev-tRNAs was confirmed with an analysis of their subcellular localization. These results show that nev-tRNAs are processed to their mature forms like common tRNAs and are available for translation. However, a whole-cell proteome analysis found no detectable level of nev-tRNA-induced mistranslation in C. elegans cells, suggesting that the genetic code is not ambiguous, at least under normal growth conditions. Our findings indicate that the translational fidelity of the nematode genetic code is strictly maintained, contrary to our expectations, although deviant tRNAs with misacylation properties are highly conserved in the nematode genome.

© The Author(s) 2018. Most bacterial genomes display contrasting strand asymmetry in a variety of features, such as nucleotide composition and gene orientation, of the two replichores separated by the replication origin and terminus. The cause for the polarization is often attributed to mutations arising from the asymmetric replication machinery. Notably, a base compositional bias known as a GC skew is focused on as a footprint of the bacterial genome evolution driven by DNA replication. Previously, although a replication driven mutation pattern responsible for the GC skew formation or the related mathematical models have been well reported, an exact impact of the replication-related elements on the genomic structure is yet actively debated, and not confirmed experimentally. However, the GC skew formation is very time consuming and challenging in the laboratory. We, therefore, used cytosine deaminase as a DNA mutator, and by monitoring the mutations during an accelerated laboratory evolution procedure with Illumina sequencing, we enabled the trial and error of the GC skew formation in high resolution. Using this technology, we succeeded in reconfirming the influence of bacterial replication machinery on the genomic structure at high resolution.

© 2018, © 2018 Taylor & Francis Group, LLC. Adenosine deaminases acting on RNA (ADARs) catalyze the deamination of adenosine (A) to inosine (I). A-to-I RNA editing targets double-stranded RNA (dsRNA), and increases the complexity of gene regulation by modulating base pairing-dependent processes such as splicing, translation, and microRNA (miRNA)-mediated gene silencing. This study investigates the genome-wide binding preferences of the nuclear constitutive isoforms ADAR1-p110 and ADAR2 on human miRNA species by RNA immunoprecipitation of ADAR-bound small RNAs (RIP-seq). Our results suggest that secondary structure predicted by base-pairing probability in the mainly double-stranded region of a pre-miRNA or mature miRNA duplex may determine ADAR isoform preference for binding distinct subpopulations of miRNAs. Furthermore, we identify 31 unique editing sites with statistical significance, 19 sites of which are novel editing sites. Editing sites are enriched in the seed region responsible for target recognition by miRNAs, and isoform-specific nucleotide motifs in the immediate vicinity and opposite of editing sites are consistent with previous studies, and further reveal that ADAR2 may edit A/C bulges more frequently than ADAR1-p110 in the context of miRNA.

© 2018 The Royal Society of Chemistry. Tumor biomarkers are highly desirable for the screening of patients with a risk of tumor development and progression. Here, we report a beta-galactosidase (β-gal)-responsive acetaminophen (β-GR-APAP) as a synthetic plasma biomarker for targeted tumor detection. Tumor β-gal labeling via the recognition of tumor-related antigen enabled the detection of a tumor using β-GR-APAP.

The anhydrobiotic tardigrade, Hypsibius exemplaris, was previously considered to require de novo gene expression and protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) activity for successful anhydrobiosis. These indicate that H. exemplaris has signal transduction systems responding to desiccation stress, with the involvement of phosphorylation events. To this end, we carried out time-series phosphoproteomics of H. exemplaris exposed to mild desiccation stress and detected 48 phosphoproteins with significant differential regulations. Among them, immediate and successive reduction of phosphorylation levels of AMP-activated protein kinase (AMPK) was observed. The subsequent chemical genetic approach showed that AMPK was activated during the preconditioning stage for anhydrobiosis, and inhibition of its activity impaired successful anhydrobiosis. As PP2A is known to dephosphorylate AMPK in other organisms, we suggested that decreased phosphorylation levels of AMPK upon mild desiccation stress were caused by dephosphorylation by PP2A. Accordingly, phosphoproteomics of animals pre-treated with the PP1/PP2A inhibitor cantharidic acid (CA) lacked the decrease in phosphorylation levels of AMPK. These observations suggest that AMPK activity is required for successful anhydrobiosis in H. exemplaris, and its phosphorylation state is possibly regulated by PP2A.

Background: Branched-chain amino acids (BCAAs) play a key role in energy homeostasis. Objective: We aimed to investigate the association between plasma BCAA levels and dyslipidemia in the Japanese population without diabetes mellitus. Methods: This cross-sectional study included 4952 participants without diabetes mellitus, enrolled in the Tsuruoka Metabolomic Cohort Study. Plasma BCAA levels were measured by capillary electrophoresis–mass spectrometry. Correlations between lipid and BCAA profiles were evaluated by sex-stratified multiple linear regression analyses, after adjusting for confounders. Logistic regression was used to identify associations between BCAAs and metabolic dyslipidemia (MD) defined as triglyceride levels ≥150 mg/dL, high-density lipoprotein cholesterol levels ≤40 mg/dL for men and ≤50 mg/dL for women, or low-density lipoprotein cholesterol (LDL-C) levels ≥140 mg/dL. Results: In both sexes, the levels of individual BCAAs and the total BCAA levels correlated positively with triglyceride levels and negatively with high-density lipoprotein cholesterol levels. Valine, leucine, and total BCAA levels were weakly and positively correlated with LDL-C levels. Increased BCAA levels showed positive associations with MD. However, associations between BCAAs and elevated LDL-C levels were unclear. Furthermore, the associations between BCAA levels and MD regardless of fasting blood sugar (FBS) levels (high or low). Although valine, leucine, and total BCAA levels were weakly associated with elevated LDL-C levels in the high-FBS group, no such association was observed in the low-FBS group. Conclusions: BCAAs might be associated with MD independently of the FBS level and might play an important role in lipid metabolism and dyslipidemia.

We describe base editors that combine both cytosine and adenine base-editing functions. A codon-optimized fusion of the cytosine deaminase PmCDA1, the adenosine deaminase TadA and a Cas9 nickase (Target-ACEmax) showed a high median simultaneous C-to-T and A-to-G editing activity at 47 genomic targets. On-target as well as DNA and RNA off-target activities of Target-ACEmax were similar to those of existing single-function base editors.

We have developed a screening system for artificial small RNAs (sRNAs) that inhibit the growth of Escherichia coli. In this system, we used a plasmid library to express artificial sRNAs (approximately 200 bases long) containing 60 bases of random nucleotide sequence. The induced expression of the known rydB sRNA in the system reduced the amount of its possible target mRNA, rpoS, supporting the reliability of the method. To isolate clones of sRNAs that inhibited the growth of E. coli, we used two successive screening steps: (i) colony size selection on plates and (ii) monitoring E. coli growth in a 96-well plate format. As a result, 83 artificial sRNAs were identified that showed a range of inhibitory effects on bacterial growth. We also introduced nucleotide replacements into one of the highly inhibitory sRNA clones, H12, which partially abolished the inhibition of bacterial growth, suggesting that bacterial growth was inhibited in a sequence-specific manner. © The Authors 2011. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

tRNA splicing endonucleases, essential enzymes found in Archaea and Eukaryotes, are involved in the processing of pre-tRNA molecules. In Archaea, three types of splicing endonuclease [homotetrameric: α4, homodimeric: α2, and heterotetrameric: (αβ)2] have been identified, each representing different substrate specificity during the tRNA intron cleavage. Here, we discovered a fourth type of archaeal tRNA splicing endonuclease (2) in the genome of the acidophilic archaeon Candidatus Micrarchaeum acidiphilum, referred to as ARMAN-2 and its closely related species, ARMAN-1. The enzyme consists of two duplicated catalytic units and one structural unit encoded on a single gene, representing a novel three-unit architecture. Homodimeric formation was confirmed by cross-linking assay, and site-directed mutagenesis determined that the conserved L10-pocket interaction between catalytic and structural unit is necessary for the assembly. A tRNA splicing assay reveal that 2 endonuclease cleaves both canonical and non-canonical bulge-helix-bulge motifs, similar to that of (αβ)2 endonuclease. Unlike other ARMAN and Euryarchaeota, tRNAs found in ARMAN-2 are highly disrupted by introns at various positions, which again resemble the properties of archaeal species with (αβ)2 endonuclease. Thus, the discovery of 2 endonuclease in an archaeon deeply branched within Euryarchaeota represents a new example of the coevolution of tRNA and their processing enzymes. © The Author(s) 2011. Published by Oxford University Press.

To further optimize the culturing of preimplantation embryos, we undertook metabolomic analysis of relevant culture media using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). We detected 28 metabolites: 23 embryo-excreted metabolites including 16 amino acids and 5 media-derived metabolites (e.g., octanoate, a medium-chain fatty acid (MCFA)). Due to the lack of information on MCFAs in mammalian preimplantation development, this study examined octanoate as a potential alternative energy source for preimplantation embryo cultures. No embryos survived in culture media lacking FAs, pyruvate, and glucose, but supplementation of octanoate rescued the embryonic development. Immunoblotting showed significant expression of acyl-CoA dehydrogenase and hydroxyacyl-CoA dehydrogenase, important enzymes for β-oxidation of MCFAs, in preimplantation embryo. Furthermore, CE-TOFMS traced [1-13 C 8[ octanoate added to the culture media into intermediate metabolites of the TCA cycle via β-oxidation in mitochondria. These results are the first demonstration that octanoate could provide an efficient alternative energy source throughout preimplantation development.

The human red blood cell (RBC) has long been used for modeling of complex biological networks, for elucidation of a wide variety of dynamic phenomena, and for understanding the fundamental topology of metabolic pathways. Here, we introduce our recent work on an RBC metabolic model using the E-Cell Simulation Environment. The model is sufficiently detailed to predict the temporal hypoxic response of each metabolite and, at the same time, successfully integrates modulation of metabolism and of the oxygen transporting capacity of hemoglobin. The model includes the mechanisms of RBC maintenance as a single cell system and the functioning of RBCs as components of a higher order system. Modeling of RBC metabolism is now approaching a fully mature stage of realistic predictions at the molecular level and will be useful for predicting conditions in biotechnological applications such as long-term cold storage of RBCs. Copyright © 2010 Ayako Yachie-Kinoshita et al.

Large-scale data sets of protein-protein interactions (PPIs) are a valuable resource for mapping and analysis of the topological and dynamic features of interactome networks. The currently available large-scale PPI data sets only contain information on interaction partners. The data presented in this study also include the sequences involved in the interactions (i.e., the interacting regions, IRs) suggested to correspond to functional and structural domains. Here we present the first large-scale IR data set obtained using mRNA display for 50 human transcription factors (TFs), including 12 transcription-related proteins. The core data set (966 IRs; 943 PPIs) displays a verification rate of 70%. Analysis of the IR data set revealed the existence of IRs that interact with multiple partners. Furthermore, these IRs were preferentially associated with intrinsic disorder. This finding supports the hypothesis that intrinsically disordered regions play a major role in the dynamics and diversity of TF networks through their ability to structurally adapt to and bind with multiple partners. Accordingly, this domain-based interaction resource represents an important step in refining protein interactions and networks at the domain level and in associating network analysis with biological structure and function. © 2010 Miyamoto-Sato et al.

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. Intestinal microbiota and their metabolites are strongly associated with host physiology. Developments in DNA sequencing and mass spectrometry technologies have allowed us to obtain additional data that enhance our understanding of the interactions among microbiota, metabolites, and the host. However, the strategies used to analyze these datasets are not yet well developed. Here, we describe an original analytical strategy, metabologenomics, consisting of an integrated analysis of mass spectrometry-based metabolome data and high-throughput-sequencing-based microbiome data. Using this approach, we compared data obtained from C57BL/6J mice fed an American diet (AD), which contained higher amounts of fat and fiber, to those from mice fed control rodent diet. The feces of the AD mice contained higher amounts of butyrate and propionate, and higher relative abundances of Oscillospira and Ruminococcus. The amount of butyrate positively correlated with the abundance of these bacterial genera. Furthermore, integrated analysis of the metabolome data and the predicted metagenomic data from Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) indicated that the abundance of genes associated with butyrate metabolism positively correlated with butyrate amounts. Thus, our metabologenomic approach is expected to provide new insights and understanding of intestinal metabolic dynamics in complex microbial ecosystems.

Background: Many limno-terrestrial tardigrades can enter an ametabolic state, known as anhydrobiosis, upon desiccation, in which the animals can withstand extreme environments. Through genomics studies, molecular components of anhydrobiosis are beginning to be elucidated, such as the expansion of oxidative stress response genes, loss of stress signaling pathways, and gain of tardigrade-specific heat-soluble protein families designated CAHS and SAHS. However, to date, studies have predominantly investigated the class Eutardigrada, and molecular mechanisms in the remaining class, Heterotardigrada, still remains elusive. To address this gap in the research, we report a multiomics study of the heterotardigrade Echiniscus testudo, one of the most desiccation-tolerant species which is not yet culturable in laboratory conditions. Results: In order to elucidate the molecular basis of anhydrobiosis in E. testudo, we employed a multi-omics strategy encompassing genome sequencing, differential transcriptomics, and proteomics. Using ultra-low input library sequencing protocol from a single specimen, we sequenced and assembled the 153.7 Mbp genome annotated using RNA-Seq data. None of the previously identified tardigrade-specific abundant heat-soluble genes was conserved, while the loss and expansion of existing pathways were partly shared. Furthermore, we identified two families novel abundant heat-soluble proteins, which we named E. testudo Abundant Heat Soluble (EtAHS), that are predicted to contain large stretches of disordered regions. Likewise the AHS families in eutardigrada, EtAHS shows structural changes from random coil to alphahelix as the water content was decreased in vitro. These characteristics of EtAHS proteins are analogous to those of CAHS in eutardigrades, while there is no conservation at the sequence level. Conclusions: Our results suggest that Heterotardigrada have partly shared but distinct anhydrobiosis machinery compared with Eutardigrada, possibly due to convergent evolution within Tardigrada. (276/350).