Published papers

Periodontal diseases are inflammatory infectious diseases that affect the periodontal tissue. Macrophages play a central role in inflammatory conditions, leading to the destruction of tissues. Identifying the signaling molecules secreted by macrophages would be valuable to the study of these diseases. Here, we present non-targeted analysis using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) for the profiling of extracellular metabolites released during macrophage activation. Lipopolysaccharide (LPS)-induced activation of a mouse macrophage-like cell line RAW264. 7 was used as a model system. Cells were treated without (control) or with LPS for 22 h and, after washing, were incubated for 1 h in phosphate-buffered saline. The accumulation of metabolites in the culture supernatant was monitored. LPS treatment significantly enhanced the accumulation of prostaglandins, tumor necrosis factor-α, nitric oxide and citrulline in the culture medium. RAW264. 7 cells produced 46 metabolites and 66% of these showed significant changes (P < 0.05) following cell activation. In particular, the production of leucine, hypoxanthine, choline, putrecine, N 8-acetylspermidine, succinate, itaconate, and 4-methyl-2-oxopentanoate was significantly increased by cell activation (P < 0.001). Significantly elevated production of lactate and glycine was also observed. Here, we present the first catalog of the up and down-regulation of the various metabolites secreted by macrophages following inflammatory activation. © 2011 Springer Science+Business Media, LLC.

Nomogram, a standard technique that utilizes multiple characteristics to predict efficacy of treatment and likelihood of a specific status of an individual patient, has been used for prediction of response to neoadjuvant chemotherapy (NAC) in breast cancer patients. The aim of this study was to develop a novel computational technique to predict the pathological complete response (pCR) to NAC in primary breast cancer patients. A mathematical model using alternating decision trees, an epigone of decision tree, was developed using 28 clinicopathological variables that were retrospectively collected from patients treated with NAC (n = 150), and validated using an independent dataset from a randomized controlled trial (n = 173). The model selected 15 variables to predict the pCR with yielding area under the receiver operating characteristics curve (AUC) values of 0.766 [95 % confidence interval (≥I)], 0.671-0.861, P value< 0.0001) in cross-validation using training dataset and 0.787 (95 % CI 0.716-0.858, P value < 0.0001) in the validation dataset. Among three subtypes of breast cancer, the luminal subgroup showed the best discrimination (AUC = 0.779, 95 % CI 0.641-0.917, P value = 0.0059). The developed model (AUC = 0.805, 95 % CI 0.716-0.894, P value\0.0001) outperformed multivariate logistic regression (AUC = 0.754, 95 % CI 0.651-0.858, P value = 0.00019) of validation datasets without missing values (n = 127). Several analyses, e.g. bootstrap analysis, revealed that the developed model was insensitive to missing values and also tolerant to distribution bias among the datasets. Our model based on clinicopathological variables showed high predictive ability for pCR. This model might improve the prediction of the response to NAC in primary breast cancer patients. © Springer Science+Business Media, LLC. 2012.

Background Patients with atopic eczema frequently experience colonization with Staphylococcus aureus that is directly correlated with the eczema severity. We hypothesized that S. aureus-secreted enterotoxins (SE) are involved in the pathophysiology of atopic keratoconjunctivitis (AKC). Methods A total of 45 subjects (18 with AKC, nine vernal keratoconjunctivitis (VKC), eight seasonal allergic conjunctivitis (SAC), and ten healthy volunteers) were enrolled. Slit lamp examinations, including fluorescein staining, were performed. Scraped samples were collected from the upper tarsal conjunctiva, lower conjunctival sacs, and the skin around the eyelid margins. Superantigen (SAg) genes were detected using polymerase chain reaction (PCR). Results Among 45 cases, S. aureus was detected significantly more in AKC patients than VKC patients (P = 0.026), SAC patients (P = 0.0003), and healthy volunteers (P = 0.0001). SAg genes were detected in 11 patients. SEB (2/11), SEG (8/11), and SEI (8/11) were detected, but no other SE. There was a significant difference in SE detection between AKC and SAC patients (P = 0.03). In severe types of ocular allergic disease such as AKC and VKC (N = 27), SE was detected in six of ten patients with corneal ulcers and two of 17 patients without corneal ulcers. SE was detected in significantly more patients with corneal ulcers (P = 0.025). Conclusions In patients with AKC, S. aureus and SE were detected more frequently compared with other patients and healthy volunteers, especially in association with corneal ulceration suggesting a role of SE. So far, it is unknown whether SE leads to tissue damage of the cornea by initiating an immune response or has direct toxic effects. © 2012 John Wiley & Sons A/S.

The quantification of a small number of bioactive components in herbal medicines is often inadequate when attempting to elucidate a medicine's biological effects. Despite rapid advances in analytical technologies, obtaining comprehensive metabolomic profiles of herbal medicines remains difficult, due to the complexity of natural product mixtures. Toki-Shakuyaku-San is a Chinese medicine used widely to treat gynecological and obstetric disorders, such as infertility, dysmenorrhea, toxemia during pregnancy and neural dysfunction. It consists of Angelica acutiloba Radix (Toki), Cnidium officinale Rhizoma (Senkyu), Paeonia lactiflora Radix (Shakuyaku), Atractylodes lancea Rhizoma (Sojutsu), Alisma orientale Rhizoma (Takusha) and Poria cocos Hoelen (Bukuryo). To elucidate the composition of these herbal medicines individually, we conducted non-targeted profiling analyses of extracts of these herbs using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), which allows the simultaneous quantification of hundreds of charged metabolites. In total, 737 ± 183. 1 (average ± SD) metabolite-derived features were observed, and of these, 119 metabolites were identified. Score plots of principal component analysis (PCA) showed a clear cluster including Shakuyaku, Bukuryo, and Sojutsu, while the other three herbs were distributed over PCA spaces. Loading plots revealed that amino acids and shikimate-derived alkaloids were the predominant metabolite constituents. Hierarchical clustering analysis revealed that few clusters overlapped in the herbal medicines tested. This report is the first demonstration of the characterization of a herbal medicine using large-scale metabolomic analysis, which is complementary to traditional quality control methods. © 2011 Springer Science+Business Media, LLC.

To elucidate the mechanism as to why alcoholic beverages can induce superconductivity in Fe 1+dTe 1xS x samples, we performed component analysis and found that a weak acid such as an organic acid has the ability to induce superconductivity. Inductively coupled plasma spectroscopy was performed on weak acid solutions post-annealing. We found that the mechanism of inducement of superconductivity in Fe 1+dTe 1xS x is the deintercalation of excess Fe from the interlayer sites. © 2012 IOP Publishing Ltd.

Mass spectrometry (MS)-based metabolomic methods enable simultaneous profiling of hundreds of salivary metabolites, and may be useful to diagnose a wide range of diseases using saliva. However, few studies have evaluated the effects of physiological or environmental factors on salivary metabolomic profiles. Therefore, we used capillary electrophoresis-MS to analyze saliva metabolite profiles in 155 subjects with reasonable oral hygiene, and examined the effects of physiological and environmental factors on the metabolite profiles. Overall, 257 metabolites were identified and quantified. The global profiles and individual metabolites were evaluated by principle component analysis and univariate tests, respectively. Collection method, collection time, sex, body mass index, and smoking affected the global metabolite profiles. However, age also might contribute to the bias in sex and collection time. The profiles were relatively unaffected by other parameters, such as alcohol consumption and smoking, tooth brushing, or the use of medications or nutritional supplements. Temporomandibular joint disorders had relatively greater effects on salivary metabolites than other dental abnormalities (e. g., stomatitis, tooth alignment, and dental caries). These findings provide further insight into the diversity and stability of salivary metabolomic profiles, as well as the generalizability of disease-specific biomarkers. © 2012 Springer Science+Business Media New York.

The microalga Chlamydomonas reinhardtii is a model organism whose whole genome has been sequenced. Although considered a cosmopolitan species, only eastern North American isolates of C. reinhardtii were available before 2010, when new Japanese isolates were reported. In the study describing the new Japanese isolates, zygote formation between Japanese and North American strains was shown, but germination was not demonstrated. In this study, the germination of intercontinental hybrid zygotes was examined using wild-type Japanese strains and mutant American strains that cannot utilize nitrate. Several clonal progeny strains were established, and the progeny strains were screened based on mating type and nitrate utilization to confirm their hybrid nature. The establishment of four intercontinental hybrid strains with different phenotypic combinations was confirmed by sequencing mating type-specific and nitrate reductase-related genes. The potential for hybrid formation between Japanese and North American strains suggests the existence of a worldwide mating population of C. reinhardtii. © 2014 Japanese Society of Phycology.

Here we describe our unprecedented approach in proposing parsley as a nutraceutical intervention in inflammatory bowel disease (IBD) via multi-omics evaluation using dextran sodium sulphate (DSS)-induced colitis. Seven-week-old male C57BL/6J mice were fed either 2% parsley or basal diet and drank normal-drinking-water for 1 week after which colitis was induced by administering 1.5% (w/v) DSS-drinking-water for 9 days. Parsley supplementation significantly improved colon shortening and increased disease activity index. Colonic transcriptome revealed down-regulation of inflammatory cytokines, hepatic transcriptome and metabolome revealed up-regulation of fatty-acid synthesis genes, thereby improving body weight loss. Down-regulated cancer markers were observed in the hepatic transcriptome and proteome. Plasma metabolite analysis indicated shifts in the citric and urea cycle, implicating improved impaired glycolysis and oxidative stress. Parsley's role in preventing against IBD was highlighted by this pioneering multi-integrated-omics which is expected to be useful and informative, and shape the future of nutraceutical and functional food research.

Sodium fluoride (NaF) is used in dentistry as a preventive agent for dental caries because of its ability to remineralize the tooth surface and its antibacterial effect. Although one of its target molecules in bacteria is enolase, its site of action in human cells has not been identified. The aim of this study was to identify target metabolites that are coupled to NaF-induced cytotoxicity in the HSC-2 human oral squamous cell carcinoma cell line. Cell viability, membrane integrity and apoptosis induction were analyzed by MTT assay, trypan blue exclusion and caspase-3 activation, respectively. Cells were treated with a minimal cytotoxic concentration of NaF for various times and subjected to comprehensive metabolomics analysis using capillary electrophoresis-mass spectrometry. In the early stages, inhibition of the enolase reaction in glycolysis pathway was observed. This was coupled with rapid inhibition of the progression of TCA cycle. In the later stages, gradual increases in the AMP/ATP ratio (a putative marker of apoptosis) and oxidized products (e.g. GSSH, and methionine sulfoxide), and marginal changes in polyamine levels (putative marker of necrosis) were observed. This manuscript provides the new insight into the global impact of NaF on metabolic pathways in human oral squamous cell carcinoma cells.

Two species of Chlamydomonas Ehrenb. in the clade Monadinia, Chlamydomonas pseudomacrostigma L. Ş. Péterfi ex H. Ettl and C. media G. A. Klebs, were recently reported from Japan. However, several species in the clade were transferred to the genus Microglena Ehrenb. by different authors, and therefore the taxonomic treatment of these two species should be revisited. Here, their identifications were re-examined based on 18S rRNA gene phylogeny, secondary structure comparisons of ITS-2 rDNA, and vegetative cell morphology. Based on these comparisons, C. pseudomacrostigma and C. media were treated as a later synonym of Microglena opisthopyren (Skuja) Demchenko, Mikhailyuk & Pröschold and an earlier synonym of M. skujae Demchenko, Mikhailyuk & Pröschold, respectively. A new combination, Microglena media (G.A. Klebs) Nakada, comb. nov., is proposed for the latter.

In fetal guinea pigs, ventricular cells have higher anaerobic glycolytic capacity and lower mitochondrial enzyme activity. Here, we implemented developmental changes in glycolytic enzyme activity, concentrations of glycogen, and total creatine in late embryonic and adult ventricular cell models. We then simulated the effects of hypoxic conditions on dynamic changes in contractile force and ATP concentration. Our model demonstrates that fetal ventricular cells maintain ATP for longer periods of time than adult ventricular cells. This is consistent with reported dynamics of ventricular cells under hypoxic conditions.

We have been developing BriCA (Brain-inspired Computing Architecture), the generic software platform that can combine an arbitrary number of machine learning modules to construct higher structures such as cognitive architectures inspired by the brain. We discuss requirements analysis and design principles of this cognitive computing platform, report its implementation, and describe plans for further development.

Two strains previously identified as Chloromonas insignis, NIES-447 and ACOI-3241, and two new strains related to NIES-447 were examined using light microscopy. The molecular phylogeny of their 18S rRNA gene sequences were also examined, and their identifications are reassessed. The strains represent two independent species, both of which can be distinguished from Chloromonas insignis. Strain NIES-447 and the two new strains each have large, irregular atypical pyrenoids without associated starch granules. They are described here as Gloeomonas anomalipyrenoides Nakada et al. sp. nov. Strain ACOI-3241, which has small atypical pyrenoids on the tips of the chloroplast lobes, was identified as C. vernalis (Skuja) Nakada comb. nov. The identity of the three strains of G. anomalipyrenoides was verified by their identical ITS-2 rDNA sequences. Both G. anomalipyrenoides and C. vernalis of the clade Chloromonadinia in the Volvocales (Chlorophyceae) are closely related to other species of Gloeomonas and to C. kasaiae Matsuzaki, Nakada, Y. Hara & Nozaki, respectively.

Biofuel from fatty acids with chain lengths of 8–15 (C8–C15) have properties similar to those of conventional diesel and jet fuels, thus, can save time and reduce costs for the refurbishment of engines and maintenance of oiling facilities. Most oil-producing algae yield C16–C18 fatty acids; however, the manipulation of algae using genetic engineering is a promising approach to obtain C8–C15 fatty acids. The introduction of a medium-chain-specific thioesterase (TE) is expected to effectively alter algae to produce medium-chain fatty acids (MCFAs). TE is the main determinant of fatty acid chain length as it releases fatty acids from the acyl carrier protein (ACP) in the fatty acid elongation cycle. In a previous study, the introduction of heterologous C8–C12-specific TEs into Chlamydomonas reinhardtii did not increase the yield of MCFAs. This effect was attributed to a low affinity of the heterologous TEs to C. reinhardtii ACP. Therefore, we introduced both the C10–C14-specific TE gene and the ACP gene from the land plant Cuphea lanceolata into C. reinhardtii. We measured free fatty acids (FFAs) and triacylglycerols (TAGs) in the transformants using liquid chromatography–mass spectrometry. The production of C12:0 and C14:0, chain length 12 and 14 without unsaturation, FFAs was not significantly increased in any of the tested strains. However, we found a slight but significant increase in TAG-containing MCFAs in both TE only and TE–ACP transformants. The increased production rate of C14:0-containing TAGs ranged from 1.25- to 1.58-fold, indicating the ability of medium-chain-specific TE to increase MCFAs. These results suggest that the selection of specific TEs is important when modifying eukaryotic algae to produce MCFAs.