Published papers

The aim of this study is to evaluate the effect of duration after meals for saliva collections for oral cancer detection using metabolomics. Saliva samples were collected from oral cancer patients (n = 22) and controls (n = 44). Saliva from cancer patients was collected 12 h after dinner, and 1.5 and 3.5 h after breakfast. Control subjects fasted >1.5 h prior to saliva collection. Hydrophilic metabolites were analyzed using capillary electrophoresis mass spectrometry. Levels of 51 metabolites differed significantly in controls vs. oral cancer patients at the 12-h fasting time point (P < 0.05). Fifteen and ten metabolites differed significantly at the 1.5- and 3.5-h time points, respectively. The area of under receiver operating characteristic curve for discriminating oral cancer patients from controls was greatest at the 12-h fasting time point. The collection time after meals affects levels of salivary metabolites for oral cancer screening. The 12-h fasting after dinner time point is optimal. This study contributes to design of saliva collection protocols for metabolomics-based biomarker discovery.

© 2018, Springer Science+Business Media, LLC, part of Springer Nature. Paired-end RNA sequencing (RNA-seq) is usually applied to the quantification of long transcripts such as messenger or long non-coding RNAs, in which case overlapping pairs are discarded. In contrast, RNA-seq on short RNAs (≤ 200 nt) is typically carried out in single-end mode, as the additional cost associated with paired-end would only translate into redundant sequence information. Here, we exploit paired-end sequencing of short RNAs as a strategy to filter out sequencing errors and apply this method to the identification of adenosine-to-inosine (A-to-I) RNA editing events on human precursor microRNA (pre-miRNA) and mature miRNA. Combined with RNA immunoprecipitation sequencing (RIP-seq) of A-to-I RNA editing enzymes, this method takes full advantage of deep sequencing technology to identify RNA editing sites with unprecedented resolution in terms of editing efficiency.

© 2018, Springer Nature B.V. Metabolomics is often used to comprehensively elucidate the metabolites in organisms like seaweed. Amino acids hydrolysed from proteins and certain targeted metabolites in seaweed have been investigated. However, water-soluble metabolites like free amino acids, organic acids, and sugars have seldom been comprehensively analysed. Metabolomics are valuable tools for these studies, but they require optimisation of pre-treatment methodology. Here, we evaluated various pre-treatment drying and extraction methods for brown seaweed metabolomics. Three edible brown seaweeds (Cladosiphon okamuranus [Mozuku], Saccharina japonica [Kombu], and Undaria pinnatifida [Wakame]) were used. Freeze-drying and oven-drying at both 40 and 80 °C were investigated. Methanol-water extracts with and without chloroform were compared. Metabolites were evaluated and quantified using liquid chromatography-mass spectrometry and capillary electrophoresis-mass spectrometry. The results showed that metabolite profiling was determined mainly by seaweed species identity rather than pre-treatment method. Freeze-drying yielded higher metabolite concentrations than oven-drying at either 40 or 80 °C. The effects of extraction with and without chloroform on metabolite concentration varied with seaweed species.

© 2018 Japanese Society of Phycology In 1960, Butcher isolated a strain of unicellular green alga, ‘Chlamydomonas subehrenbergii’ SAG 18.89, from a brackish water locality in England, facing the North Sea. Although C. subehrenbergii Butcher was previously described by the same author in 1959, the strain SAG 18.89 is in fact morphologically different, possessing shorter flagella, a different-shaped papilla, and different-sized vegetative cells. Light and fluorescence microscopy, molecular phylogenetic analyses, and ITS2 comparisons revealed that this strain represents an undescribed species of the genus Microglena (Volvocales, Chlorophyceae), described here as M. redcarensis sp. nov. The presence of contractile vacuoles, which are osmoregulatory organelles, suggests potential inhabitation in freshwater. Contractile vacuole activity and growth of M. redcarensis were therefore examined in media of different salinity.

The skin is at the interface of living organisms and their environment, and has evolved interesting structural properties usually over the course of millions of years, providing clues for the design of manmade biomimetic surfaces with favorable fluid mechanics properties. Here, we describe steps to produce silicone rubber films based on microscopy data from shark skin denticles, from data acquisition and manufacturing to attachment to an airfoil for experimental fluid dynamics in large towing tanks. This method is relatively low-cost, may be generalized to other types of patterned micro-structured surfaces, and the manufacturing process may be reproduced by anyone equipped with a 3D printer.

© 2019 Japanese Society of Phycology Parachlorococcum turfosum was previously described based on morphological studies of a culture collection strain originally identified as ‘Chlorococcum elkhartiense’ and on an 18S rRNA gene phylogeny of a strain derived from the same original strain but from a different culture collection. However, a subsequently published 18S rRNA gene sequence was inconsistent with the original sequence. Therefore, a new 18S rRNA gene sequence was obtained from the strain from which the holotype was derived. Comparative analyses of the new sequence using the basic local alignment search tool and a newly written Perl code, ChimViewP, which visualizes nucleotide signature shifts characteristic of chimeric sequences, revealed that the sequence used in the original description was of a chimeric nature. Similar analyses also indicated that two published ‘Chlamydomonas acidophila’ sequences are chimeric. Phylogenetic analyses excluding all putative chimeras showed that P. turfosum belongs to Chlorococcum, and thus Chlorococcum turfosum comb. nov. is proposed.

Chemical evolution is an abiotic reaction process in which complex organic molecules arise from a combination of simple inorganic and organic chemical compounds. To assess the possible ongoing chemical evolution in the subsurface ocean of Saturn's icy satellite Enceladus, we explored the water-rock aqueous reactions and the peptide formation capability under a hydrothermal environment similar to that on Enceladus. It has been suggested that the core of Enceladus has not experienced high temperatures from the time of satellite formation to the present. The major components of the core are assumed to be carbonaceous chondrites; thus, simple organic substances, including amino acids, are likely present in the alkaline seawater of Enceladus. In this study, we conducted a laboratory-based simulation experiment to describe the chemical alteration of six prebiotically abundant amino acids over 147 days under high pressure with thermal cycling (30 to 100 °C) to simulate the water-rock interface of the ocean on Enceladus. As a result, we detected 28 out of 36 possible dipeptide species during the entire reaction period. We propose that peptide-bond formation is coupled to rock surface chemisorption of amino acids under alkaline condition, which was further supported by the elemental analysis showing carbon and nitrogen signature on the rock surface only when amino acids are added. The above result suggests that ongoing chemical evolution on Enceladus is likely producing short abiotic peptides on the porous core surface.

Undaria pinnatifida is a seaweed native to Japan, where it is cultivated and sold commercially as food. Fresh seaweed commonly blanches before consumption. However, the effects of blanching on metabolites present in different parts of U. pinnatifida, namely, Wakame (leaf) and Mekabu (sporophyll), are not well understood. Here, we investigated changes in metabolites in these two seaweed parts after blanching for different periods. The samples were freeze-dried and extracted using methanol-water, and metabolite concentration was quantitated using liquid chromatography mass spectrometry and capillary electrophoresis mass spectrometry. The concentration of most metabolites was the highest in raw samples, decreasing drastically within 20 s of blanching in Wakame and decreasing gradually in Mekabu. In contrast, the concentration of some fatty acids, such as octanoate, increased in both parts. During blanching, seaweed color changed from brown to green, and blanching did not significantly affect amino acid or the sugar composition. Finally, a relationship was found between metabolite and blanching-related characteristics, such as color and drying percentage.

Food intake biomarkers can be critical tools that can be used to objectively assess dietary exposure for both epidemiological and clinical nutrition studies. While an accurate estimation of food intake is essential to unravel associations between the intake and specific health conditions, random and systematic errors affect self-reported assessments. This study aimed to clarify how habitual food intake influences the circulating plasma metabolome in a free-living Japanese regional population and to identify potential food intake biomarkers. To achieve this aim, we conducted a cross-sectional analysis as part of a large cohort study. From a baseline survey of the Tsuruoka Metabolome Cohort Study, 7, 012 eligible male and female participants aged 40-69 years were chosen for this study. All data on patients' health status and dietary intake were assessed via a food frequency questionnaire, and plasma samples were obtained during an annual physical examination. Ninety-four charged plasma metabolites were measured using capillary electrophoresis mass spectrometry, by a non-targeted approach. Statistical analysis was performed using partial-least-square regression. A total of 21 plasma metabolites were likely to be associated with long-term food intake of nine food groups. In particular, the influential compounds in each food group were hydroxyproline for meat, trimethylamine- N-oxide for fish, choline for eggs, galactarate for dairy, cystine and betaine for soy products, threonate and galactarate for carotenoid-rich vegetables, proline betaine for fruits, quinate and trigonelline for coffee, and pipecolate for alcohol, and these were considered as prominent food intake markers in Japanese eating habits. A set of circulating plasma metabolites was identified as potential food intake biomarkers in the Japanese community-dwelling population. These results will open the way for the application of new reliable dietary assessment tools not by self-reported measurements but through objective quantification of biofluids.

Background. Based on the theory of traditional Chinese medicine, Chinese herbs possess four different medicinal properties: Hot, warm, cold, and cool. These serve as a reference guide for these herbal medicines. However, the molecular mechanisms supporting their relevance remain unclear. Methods. We performed metabolomics based on capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF/MS) and multivariate data analysis for the structural identification of compounds of cold- A nd hot-natured Chinese herbs. Results. To this end, 30 selected herbs were analyzed and a total of 416 metabolites were identified via CE-TOF/MS, of which 193 compounds were detected in most herbs. The observed profiles offered the potential to understand the mechanism of association between the compounds and nature of the Chinese herbs. Comparison of the similarity in terms of chemical and molecular structures and content revealed that hot-natured herbs contained more nucleotides. In contrast, principal component analysis revealed the presence of more amino acid compounds in cold-natured herbs. Conclusion. Comparing the structural similarities between the samples using the Tanimoto coefficient revealed that a general non-specific structure was observed between cold- A nd hot-natured herbs; however, the distribution of the molecular groups seemed to contribute more toward the energy properties.

Spider silk is a natural fiber with remarkable strength, toughness, and elasticity that is attracting attention as a biomaterial of the future. Golden orb-weaving spiders (Trichonephila clavata) construct large, strong webs using golden threads. To characterize the pigment of golden T. clavata dragline silk, we used liquid chromatography and mass spectrometric analysis. We found that the major pigment in the golden dragline silk of T. clavata was xanthurenic acid. To investigate the possible function of the pigment, we tested the effect of xanthurenic acid on bacterial growth using gram-negative Escherichia coli and gram-positive Bacillus subtilis. We found that xanthurenic acid had a slight antibacterial effect. Furthermore, to investigate the UV tolerance of the T. clavata threads bleached of their golden color, we conducted tensile deformation tests and scanning electron microscope observations. However, in these experiments, no significant effect was observed. We therefore speculate that golden orb-weaving spiders use the pigment for other purposes, such as to attract their prey in the sunlight.

Currently, large-scale cohort studies for metabolome analysis have been launched globally. However, only a few studies have evaluated the reliability of urinary metabolome analysis. This study aimed to establish the reliability of urinary metabolomic profiling in cohort studies. In the Tsuruoka Metabolomics Cohort Study, 123 charged metabolites were identified and routinely quantified using capillary electrophoresis-mass spectrometry (CE-MS). We evaluated approximately 750 quality control (QC) samples and 6,720 participants’ spot urine samples. We calculated inter- and intra-batch coefficients of variation in the QC and participant samples and technical intraclass correlation coefficients (ICC). A correlation of metabolite concentrations between spot and 24-h urine samples obtained from 32 sub-cohort participants was also evaluated. The coefficient of variation (CV) was less than 20% for 87 metabolites (70.7%) and 20–30% for 19 metabolites (15.4%) in the QC samples. There was less than 20% inter-batch CV for 106 metabolites (86.2%). Most urinary metabolites would have reliability for measurement. The 96 metabolites (78.0%) was above 0.75 for the estimated ICC, and those might be useful for epidemiological analysis. Among individuals, the Pearson correlation coefficient of 24-h and spot urine was more than 70% for 59 of the 99 metabolites. These results show that the profiling of charged metabolites using CE-MS in morning spot human urine is suitable for epidemiological metabolomics studies.

Background: Cisplatin (CDDP) significantly prolongs survival in various cancers, but many patients also develop resistance that results in treatment failure. Thus, this study aimed to elucidate the underlying mechanisms by which ovarian cancer cells acquire CDDP resistance. Methods: We evaluated the metabolic profiles in CDDP-sensitive ovarian cancer A2780 cells and CDDP-resistant A2780cis cells using capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS). We further examined the expression of glutamine metabolism enzymes using real-time PCR and Western blot analyses. Cell viability was accessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: The results showed that levels of glutamine, glutamate, and glutathione (GSH), a key drug resistance mediator synthesized from glutamate, were significantly elevated in A2780cis cells than those in A2780 cells. Furthermore, glutamine starvation decreased the GSH levels and CDDP resistance in A2780cis cells. Interestingly, the expression of glutamine synthetase (GS/GLUL), which synthesizes glutamine from glutamate and thereby negatively regulates GSH production, was almost completely suppressed in resistant A2780cis cells. In addition, treatment of A2780cis cells with 5-aza-2′-deoxycytidine, a DNA-demethylating agent, restored GS expression and reduced CDDP resistance. In contrast, GS knockdown in CDDP-sensitive A2780 cells induced CDDP resistance. Conclusions: The results indicate that upregulation of GSH synthesis from glutamine via DNA methylation-mediated silencing of GS causes CDDP resistance in A2780cis cells. Therefore, glutamine metabolism could be a novel therapeutic target against CDDP resistance.

Epithelial–mesenchymal transition (EMT)—a fundamental process in embryogenesis and wound healing—promotes tumor metastasis and resistance to chemotherapy. While studies have identified signaling components and transcriptional factors responsible in the TGF-β-dependent EMT, whether and how intracellular metabolism is integrated with EMT remains to be fully elucidated. Here, we showed that TGF-β induces reprogramming of intracellular amino acid metabolism, which is necessary to promote EMT in non-small cell lung cancer cells. Combined metabolome and transcriptome analysis identified prolyl 4-hydroxylase α3 (P4HA3), an enzyme implicated in cancer metabolism, to be upregulated during TGF-β stimulation. Further, knockdown of P4HA3 diminished TGF-β-dependent changes in amino acids, EMT, and tumor metastasis. Conversely, manipulation of extracellular amino acids induced EMT-like responses without TGF-β stimulation. These results suggest a previously unappreciated requirement for the reprogramming of amino acid metabolism via P4HA3 for TGF-β-dependent EMT and implicate a P4HA3 inhibitor as a potential therapeutic agent for cancer.

Dragline silk of golden orb-weaver spiders (Nephilinae) is noted for its unsurpassed toughness, combining extraordinary extensibility and tensile strength, suggesting industrial application as a sustainable biopolymer material. To pinpoint the molecular composition of dragline silk and the roles of its constituents in achieving its mechanical properties, we report a multiomics approach, combining highquality genome sequencing and assembly, silk gland transcriptomics, and dragline silk proteomics of four Nephilinae spiders.We observed the consistent presence of the MaSp3B spidroin unique to this subfamily as well as several nonspidroin SpiCE proteins. Artificial synthesis and the combination of these components in vitro showed that the multicomponent nature of dragline silk, including MaSp3B and SpiCE, along with MaSp1 and MaSp2, is essential to realize the mechanical properties of spider dragline silk.

G + C composition at the third codon position (GC3) is widely reported to be correlated with synonymous codon usage bias. However, no quantitative attempt has been made to compare the extent of this correlation among different genomes. Here, we applied Shannon entropy from information theory to measure the degree of GC3 bias and that of synonymous codon usage bias of each gene. The strength of the correlation of GC3 with synonymous codon usage bias, quantified by a correlation coefficient, varied widely among bacterial genomes, ranging from -0.07 to 0.95. Previous analyses suggesting that the relationship between GC3 and synonymous codon usage bias is independent of species are thus inconsistent with the more detailed analyses obtained here for individual species.