Ether-phosphatidylcholine characterized by consolidated plasma and liver lipidomics is a predictive biomarker for valproic acid-induced hepatic steatosis.

Valproic acid (VPA) is known to induce hepatic steatosis due to mitochondrial toxicity in rodents and humans. In the present study, we administered VPA to SD rats for 3 or 14 days at 250 and 500 mg/kg and then performed lipidomics analysis to reveal VPA-induced alteration of the hepatic lipid profile and its association with the plasma lipid profile. VPA induced hepatic steatosis at the high dose level without any degenerative changes in the liver on day 4 (after 3 days dosing) and at the low dose level on day 15 (after 14 days dosing). We compared the plasma and hepatic lipid profiles obtained on day 4 between the VPA-treated and control rats using a multivariate analysis to determine differences between the two groups. In total, 36 species of plasma lipids and 24 species of hepatic lipids were identified as altered in the VPA-treated group. Of these lipid species, ether-phosphatidylcholines (ePCs), including PC(16:0e/22:4) and PC(16:0e/22:6), were decreased in both the plasma and liver from the low dose level on day 4, however, neither an increase in hepatic TG level nor histopathological hepatic steatosis was observed at either dose level on day 4. Hepatic mRNA levels of glycerone-phosphate O-acyltransferase (Gnpat), which is a key enzyme for biosynthesis of ePC, was also decreased by treatment with VPA along with the decrease in ePCs. In conclusion, the changes in ePCs, (PC[16:0e/22:4] and PC[16:0e/22:6]), have potential utility as predictive biomarkers for VPA-induced hepatic steatosis.

Clinical results of alumina medial pivot total knee arthroplasty at a minimum follow-up of 10years.

BACKGROUND: The medial pivot total knee prosthesis with alumina ceramic femoral components was introduced to reproduce physiological knee kinematics and reduce polyethylene wear. The five-year clinical outcomes of alumina medial pivot total knee arthroplasties (TKA) have previously been reported. The purpose of this study was to provide the longer-term clinical results at a minimum follow-up of 10years. METHODS: The clinical results of 70 alumina medial pivot TKA in 51 consecutive patients, with a minimum follow-up period of 10years, were evaluated. RESULTS: Mean follow-up was 11.8years (range 10 to 13). Alumina medial pivot TKAs improved the patients' Knee Society knee scores, function scores, and postoperative ranges of motion compared with their pre-operative statuses (P<0.05 for each). Revision surgery was required in one knee due to a postoperative fracture of the tibial plateau after a fall that occurred two years postoperatively. Osteolysis and femoral component loosening was identified in one case after the initial five-year time point of analysis. The survival rate was 99.1% at 10years. CONCLUSIONS: This study demonstrated excellent clinical results for patients receiving the alumina medial pivot prosthesis at a minimum follow-up period of 10years.

Arachidonic acid-containing phosphatidylcholine characterized by consolidated plasma and liver lipidomics as an early onset marker for tamoxifen-induced hepatic phospholipidosis.

Lipid profiling has emerged as an effective approach to not only screen disease and drug toxicity biomarkers but also understand their underlying mechanisms of action. Tamoxifen, a widely used antiestrogenic agent for adjuvant therapy against estrogen-positive breast cancer, possesses side effects such as hepatic steatosis and phospholipidosis (PLD). In the present study, we administered tamoxifen to Sprague-Dawley rats and used lipidomics to reveal tamoxifen-induced alteration of the hepatic lipid profile and its association with the plasma lipid profile. Treatment with tamoxifen for 28 days caused hepatic PLD in rats. We compared the plasma and liver lipid profiles in treated vs. untreated rats using a multivariate analysis to determine differences between the two groups. In total, 25 plasma and 45 liver lipids were identified and altered in the tamoxifen-treated group. Of these lipids, arachidonic acid (AA)-containing phosphatidylcholines (PCs), such as PC (17:0/20:4) and PC (18:1/20:4), were commonly reduced in both plasma and liver. Conversely, tamoxifen increased other phosphoglycerolipids in the liver, such as phosphatidylethanolamine (18:1/18:1) and phosphatidylinositol (18:0/18:2). We also examined alteration of AA-containing PCs and some phosphoglycerolipids in the pre-PLD stage and found that these lipid alterations were initiated before pathological alteration in the liver. In addition, changes in plasma and liver levels of AA-containing PCs were linearly associated. Moreover, levels of free AA and mRNA levels of AA-synthesizing enzymes, such as fatty acid desaturase 1 and 2, were decreased by tamoxifen treatment. Therefore, our study demonstrated that AA-containing PCs might have potential utility as novel and predictive biomarkers for tamoxifen-induced PLD. Copyright © 2017 John Wiley & Sons, Ltd.

Novel urushiols with human immunodeficiency virus type 1 reverse transcriptase inhibitory activity from the leaves of Rhus verniciflua.

Two novel urushiols, 1 and 2, and two known urushiols, 3 and 4, were isolated from the leaves of Rhus verniciflua and were examined for their human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) inhibitory activity. The novel urushiols were found to be 1,2-dihydroxyphenyl-3-[7'(E),9'(Z),11'(Z)-pentadecatrienyl]-14'-ol (1) and 1,2-dihydroxyphenyl-3-[8'(Z),10'(E),12'(E)-pentadecatrienyl]-14'-ol (2) by spectroscopic analyses. The absolute configuration at C-14' in 1 and 2 was determined to be a racemic mixture of (R) and (S) isomers by ozonolysis. Compound 2 (IC50: 12.6 µM) showed the highest HIV-1 RT inhibitory activity among the four urushiols, being 2.5-fold more potent than the positive control, adriamycin (IC50: 31.9 µM). Although the known urushiols were isolated from the sap and leaves of R. verniciflua, 1 was exclusively present in the leaves, and higher amounts of 2 were found in the leaves than in the sap. Present findings indicate that the leaves of R. verniciflua represent a new biological resource from which novel and known urushiols may be prepared, and the possible use of novel urushiols as bioactive products.

Mid-term clinical results of alumina medial pivot total knee arthroplasty.

PURPOSE: The medial pivot total knee prosthesis has been designed to reproduce physiological knee kinematics. It has been reported that alumina ceramic femoral components reduce polyethylene wear. Thus, medial pivot total knee prostheses with alumina ceramic femoral components were introduced. The purpose of this study was to evaluate the clinical results of patients who underwent newly introduced alumina medial pivot total knee arthroplasties (TKA). METHODS: We evaluated the clinical results of 107 alumina medial pivot TKAs in 80 consecutive patients with a mean follow-up period of 5 years. RESULTS: Alumina medial pivot TKAs provided significant improvements in the patients' Knee Society knee scores, function scores and post-operative ranges of motion compared with their pre-operative statuses (each, P < 0.05). There was no statistical correlation between the change in maximum knee flexion and the increase in posterior condylar offset. Revision surgery was required in one knee due to a post-operative fracture of the tibial plateau after a fall that occurred 2 years postoperatively. No knees had aseptic loosening, osteolysis, or ceramic fractures. The survival rate was 98.6% at 5 years. CONCLUSIONS: This study demonstrates satisfactory mid-term clinical results for patients receiving the alumina medial pivot prosthesis. LEVEL OF EVIDENCE: Therapeutic study, Level IV.

High-throughput technique for comprehensive analysis of Japanese green tea quality assessment using ultra-performance liquid chromatography with time-of-flight mass spectrometry (UPLC/TOF MS).

Applications of metabolomics techniques along with chemometrics provide an understanding in the relationship between metabolome of green tea and its quality. A coupled of ultra-performance liquid chromatography with time-of-flight mass spectrometry (UPLC/TOF MS) allowed a high-throughput and comprehensive analysis with minimal sample preparation. Using this technique, a wide range of metabolites were investigated. Data analysis was rapid, considering that the fingerprinting technique was performed. A set of green tea samples from 2006 tea contest of the Kansai area was analyzed to prove usefulness of the developed technique. Green tea with different qualities were discriminated through principal component analysis (PCA). Consequently, projection to latent structure by means of partial least-squares (PLS) was performed to create a constructive quality-predictive model by means of metabolic fingerprinting. Beside epigallocatechin, other predominant catechins, including epigallocatechin gallate and epicatechin gallate, detected in green tea were found to be significant biomarkers to the high quality of Japanese green tea (Sencha).

Metabolic profiling of Angelica acutiloba roots utilizing gas chromatography-time-of-flight-mass spectrometry for quality assessment based on cultivation area and cultivar via multivariate pattern recognition.

Gas chromatography time-of-flight mass spectrometry was applied to elucidate the profiling of primary metabolites and to evaluate the differences between quality differences in Angelica acutiloba (or Yamato-toki) roots through the utilization of multivariate pattern recognition-principal component analysis (PCA). Twenty-two metabolites consisting of sugars, amino and organic acids were identified. PCA analysis successfully discriminated the good, the moderate and the bad quality Yamato-toki roots in accordance to their cultivation areas. The results signified two reducing sugars, fructose and glucose being the most accumulated in the bad quality, whereas higher quantity of phosphoric acid, proline, malic acid and citric acid were found in the good and the moderate quality toki roots. PCA was also effective in discriminating samples derive from different cultivars. Yamato-toki roots with the moderate quality were compared by means of PCA, and the results illustrated good discrimination which was influenced most by malic acid. Overall, this study demonstrated that metabolomics technique is accurate and efficient in determining the quality differences in Yamato-toki roots, and has a potential to be a superior and suitable method to assess the quality of this medicinal plant.

High throughput and exhaustive analysis of diverse lipids by using supercritical fluid chromatography-mass spectrometry for metabolomics.

We have developed an analytical system that enables the simultaneous rapid analysis of lipids with varied structures and polarities through the use of supercritical fluid chromatography-mass spectrometry (SFC-MS). The separation conditions for SFC (column, modifier, back pressure, etc.) and the detection conditions for mass spectrometry (ionization method, parameters, etc.) were investigated to develop a simultaneous analytical method for lipid mixtures that included phospholipids, glycolipids, neutral lipids, and sphingolipids. When cyanopropylated silica gel-packed column was used for the separation, all lipids were successfully detected and the analysis time was less than 15 min. The use of an octadecylsilylated column resulted in separation, which was dependent on the differences in the unsaturation of the fatty acid side chains and isomer separation. This system is a powerful tool for studies on lipid metabolomics because it is useful not only as a fingerprinting method for the screening of diverse lipids but also for the detailed profiling of individual components.

Quality prediction of Japanese green tea using pyrolyzer coupled GC/MS based metabolic fingerprinting.

A couple between pyrolyzer and gas chromatography/mass spectrometry (GC/MS) has allowed a fast, simple, and low-cost approach to evaluate a quality of Japanese green tea without any sample preparation or derivatization techniques. Using our method, errors from sample preparation could be avoided since raw samples were directly extracted through the extreme heat of the pyrolyzer. In addition, undesired reactions from expensive derivatizing agents, which are commonly needed to treat the samples before analyzing with GC/MS, could be omitted. In order to illustrate the efficiency of this technique, a set of green tea samples from the Tea contest in 2005 in the Kansai area were used. Projection to latent structure by means of partial least squares (PLS) along with orthogonal signal correction (OSC) was selected to explain the relation between green tea's metabolite profiling and its quality. The quality of the model was validated by testing and comparing the predictive ability to the respective model.

Prediction of Japanese green tea ranking by fourier transform near-infrared reflectance spectroscopy.

A rapid and easy determination method of green tea's quality was developed by using Fourier transform near-infrared (FT-NIR) reflectance spectroscopy and metabolomics techniques. The method is applied to an online measurement and an online prediction of green tea's quality. FT-NIR was employed to measure green tea metabolites' alteration affected by green tea varieties and manufacturing processes. A set of ranked green tea samples from a Japanese commercial tea contest was analyzed to create a reliable quality-prediction model. As multivariate analyses, principal component analysis (PCA) and partial least-squares projections to latent structures (PLS) were used. It was indicated that the wavenumber region from 5500 to 5200 cm(-1) had high correlation with the quality of the tea. In this study, a reliable quality-prediction model of green tea has been achieved.

1H NMR based metabolic profiling in the evaluation of Japanese green tea quality.

Classification of tea quality is now mainly performed according to the sensory results by professional tea tasters. However, this evaluation method is inconsistent in differentiating their qualities. A combination of a (1)H NMR technique and a multivariate analysis was introduced to the quality evaluation of green tea by means of a metabolomic technique. A broad range of metabolites were detected by (1)H NMR spectrometry. The principal component analysis (PCA) was used to reduce the complexity of the (1)H NMR spectra data set and provided the quality discrimination result. It offered an extensive clue for classification and quality assessment without any prepurification method. A set of green teas from a Japanese tea contest were analyzed by (1)H NMR to classify the quality with respect to that judged by tea tasters and to conceive a quality prediction model. Metabolic profiling and fingerprinting of (1)H NMR spectra of green teas with different quality were studied. PCA showed a separation between the high- and the low-quality green teas. The taste marker compounds contributing to the discrimination of tea quality were identified. Reliable prediction models were obtained by the partial least-squares projection to latent structure (PLS) analysis together with a preprocessing filter of both orthogonal signal correction (OSC) and a combination between OSC and wavelet transform algorithms.

Prediction of Japanese green tea ranking by gas chromatography/mass spectrometry-based hydrophilic metabolite fingerprinting.

An innovative technique for green tea's quality determination was developed by means of metabolomics. Gas-chromatography coupled with time-of-flight mass spectrometry and multivariate data analysis was employed to evaluate the quality of green tea. Alteration of green tea varieties and manufacturing processes effects a variation in green tea metabolites, which leads to a classification of the green tea's grade. Therefore, metabolic fingerprinting of green tea samples of different qualities was studied. A set of ranked green tea samples from a Japanese commercial tea contest was analyzed with the aim of creating a reliable quality-prediction model. Several multivariate algorithms were performed. Among those, the partial least-squares projections to latent structures (PLS) analysis with the spectral filtering technique, orthogonal signal correction (OCS), was found to be the most practical approach. In addition, metabolites that play an important role in green tea's grade classification were identified.

Historical aspects for the control of soil-transmitted helminthiases.

Japan has been one of the very few countries that operated the nationwide control programme against soil-transmitted helminths (STH) infections and gained a great success. On the basis of researches and operational studies, periodical mass-examination using cellophane thick smear (Kato technique) and selective mass-treatment targeted at school children were employed as the most useful control measure. In later years, Japan's experience and the strategy for STH control programme were transferred to Asian countries through Asian Parasite Control Organization (APCO) and also to countries of Asia, Africa and Latin-America through seminar at a modified form of the integration programme (IP) of STH control with family planning and nutritional improvement. The parasitologists group conducted studies in all aspects to seek effective STH control measures for respective countries and obtained many successful results.

A novel microsurgery method for intact plant tissue at the single cell level using ArF excimer laser microprojection.

A novel microsurgery technique for the partial removal of rigid cell-walls in intact plant tissue is established. Using a size-variable slit, an ArF excimer laser was microprojected on the surface of the targeted cell, and this method enabled the area- and depth-controllable processing of the cortical structure of plant cells including the cuticle and cell wall layer. In epidermal cells of all tested plants, viabilities of more than 90% were retained 24 h after irradiation. Scanning electron microscope (SEM) observation revealed that the cuticle layer of the irradiated region was completely ablated, and the cellulose microfibrils of the secondary cell wall were partially removed; furthermore, 4 days after laser treatment, the regeneration of cell wall fibrils was observed. As a model experiment, the transient expression of synthetic green fluorescent protein (sGFP) was performed by the microinjection of cauliflower mosaic virus (CMV) 35S promoter-derived sGFP gene through an "aperture" in the treated cell surface. Moreover, micron-sized fluorescent beads were successfully introduced by the same method into the onion cells indicating that this method can be used to introduce foreign materials as large as organelles.

A novel crude oil emulsifier excreted in the culture supernatant of a marine bacterium, Myroides sp. strain SM1.

A marine bacterium, Myroides sp. SM1, can grow on weathered crude oil and show emulsification of it. The biosurfactant able to emulsify crude oil was excreted in culture supernatant of Myroides sp. SM1 grown on marine broth, which was extracted with chloroform/methanol (1:1) at pH 7 and purified by normal and reverse phase silica gel column chromatographies. The compound was ninhydrin-positive, and the chemical structure was elucidated by nuclear magnetic resonance (NMR), infrared spectroscopy (IR), fast atom bombardment mass spectrometry, and gas chromatography-mass spectrometry (GC-MS) to be a mixture of L: -ornithine lipids, which were composed of L: -ornithine and a different couple of iso-3-hydroxyfatty acid (C(15)-C(17)) and iso-fatty acid (C(15) or C(16)) in a ratio of 1:1:1. The critical micelle concentration for a mixture of ornithine lipids was measured to be approximately 40 mg/l. A mixture of ornithine lipids exhibited emulsifying activity for crude oil in a broad range of pH, temperature, and salinity and showed higher surface activity for oil displacement test than other several artificial surfactants and a biosurfactant, surfactin.

Polyethylene wear particle generation in vivo in an alumina medial pivot total knee prosthesis.

Polyethylene wear particle generation is one of the most important factors affecting mid- to long-term results of total knee arthroplasties. It has been reported that the medial pivot total knee prosthesis (MP) design and alumina ceramic femoral component reduce polyethylene wear. The aim of this study is to evaluate in vivo polyethylene wear particle generation in the newly introduced alumina MP, in comparison with a metal MP. Synovial fluid was obtained from 11 knees with alumina MP and 15 knees with metal MP at nine months after the operation. Polyethylene particles were isolated, and examined using scanning electron microscope and image analyzer. Total number of particles in each knee was 7.10+/-2.86x10(6) in alumina (mean+/-standard error), and 5.70+/-2.82x10(7) in metal MP (p=0.048). Particle size (equivalent circle diameter) was 0.78+/-0.04 microm in alumina, and 0.66+/-0.06 microm in metal MP (p=0.120). Particle shape (aspect ratio) was 1.52+/-0.05 in alumina, and 1.88+/-0.11 in metal MP (p=0.014). Apart from the femoral component, the material and manufacturing method of polyethylene insert differed between the two groups, although the sterilization method was the same. Alumina MP generated fewer and rounder polyethylene wear particles than metal MP in early clinical stage, and could potentially reduce prevalence of osteolysis and aseptic loosening.

Functional analysis of two solanesyl diphosphate synthases from Arabidopsis thaliana.

Solanesyl diphosphate (SPP) is regarded as the precursor of the side-chains of both plastoquinone and ubiquinone in Arabidopsis thaliana. We previously analyzed A. thaliana SPP synthase (At-SPS1) (Hirooka et al., Biochem. J., 370, 679-686 (2003)). In this study, we cloned a second SPP synthase (At-SPS2) gene from A. thaliana and characterized the recombinant protein. Kinetic analysis indicated that At-SPS2 prefers geranylgeranyl diphosphate to farnesyl diphosphate as the allylic substrate. Several of its features, including the substrate preference, were similar to those of At-SPS1. These data indicate that At-SPS1 and At-SPS2 share their basic catalytic machinery. Moreover, analysis of the subcellular localization by the transient expression of green fluorescent protein-fusion proteins showed that At-SPS2 is transported into chloroplasts, whereas At-SPS1 is likely to be localized in the endoplasmic reticulum in the A. thaliana cells. It is known that the ubiquinone side-chain originates from isopentenyl diphosphate derived from the cytosolic mevalonate pathway, while the plastoquinone side-chain is synthesized from isopentenyl diphosphate derived from the plastidial methylerythritol phosphate pathway. Based on this information, we propose that At-SPS1 contributes to the biosynthesis of the ubiquinone side-chain and that At-SPS2 supplies the precursor of the plastoquinone side-chain in A. thaliana.

Expression of fungal pectin methylesterase in transgenic tobacco leads to alteration in cell wall metabolism and a dwarf phenotype.

A transgenic tobacco plant (Nicotiana tabacum L.) expressing a fungal pectin methylesterase (PME; EC 3.1.1.11) gene derived from a black filamentous fungus, Aspergillus niger was created. Fungal PME should have a wider range of adaptability to substrate pectin compared with plant PME. As expected, the proportion of methyl esters in pectin was reduced in the transgenic tobacco. Consequently, the transgenic plant showed short internodes, small leaves and a dwarf phenotype. At a cellular level, the longitudinal lengths of stem epidermal cells were shorter than those of control plants. This is the first report that fungal PME promotes dwarfism in plants. It is worth noting that in the PME-expressing dwarf plant, the expression levels of cell wall metabolism related genes that included endo-1,4-beta-glucanase, cellulose synthase, endo-xyloglucan transferase and expansin gene were decreased. These results suggest that the expression of fungal PME in plants affects the cell wall metabolism.

Real-time quantification of methanol in plants using a hybrid alcohol oxidase-peroxidase biosensor.

An amperometric biosensor immobilizing two enzymes and an electron mediator in an identical plane has been fabricated by the self-assembly technique for determination of methanol in crude plant samples. A self-assembled mixed monolayer of 4,4'-dithiodibutyric acid covalently attached two enzymes (Hansenula sp. alcohol oxidase and horseradish peroxidase) and 11-ferrocenyl-1-undecanethiol as an electron mediator on an Au electrode is exploited to produce a two-dimensional reaction matrix. The composition of the two enzymes and electron mediator molecules was optimized for detection of methanol in 0.1 M sodium phosphate buffer (pH 6.0). We successfully quantified methanol in low-purity tobacco (Nicotiana tabacum) plant extracts with the biosensor, which showed sensitivity comparable to that of gas chromatography/mass spectrometry. The redox-relay biosensor is quite simple and stable due to its covalent attachment to the Au surface, making it possible to downsize the construction. We fabricated a miniature methanol biosensor that fitted a well of a 96-well micro assay plate available for high-throughput assay. The biosensor is advantageous for the sensitive, continuous, and convenient determination of methanol.

Phytotoxicity of the tetramic acid metabolite trichosetin.

Trichosetin, a tetramic acid-containing metabolite produced in the dual culture of Trichoderma harzianum and Catharanthus roseus (L.) G. Don callus, was subjected to phytotoxicity assays. In seedling growth assays, trichosetin inhibited root and shoot growth of all five plant species tested by damaging the cell membrane, as evidenced by the dose-dependent increase in electrolyte leakage and lipid peroxidation. Vital staining of trichosetin-treated Nicotiana tabacum BY-2 cells, with rhodamine 123, showed a weaker green fluorescence compared to controls indicating damaging effects on mitochondria. FDA-PI staining, to determine cell viability, indicated that cells of the trichosetin-treated roots were mostly dead.