Detection of six single-nucleotide polymorphisms associated with rheumatoid arthritis by a loop-mediated isothermal amplification method and an electrochemical DNA chip

An electrochemical DNA chip using an electrochemically active intercalator and DNA probe immobilized on a gold electrode has been developed for genetic analysis. In this study, the six polymorphisms associated with rheumatoid arthritis (RA), N-acetyltransferase2 (NAT2) gene polymorphisms T341C, G590A, and G857A, methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms C677T and A1298C, and serum amyloid A1 (SAA1) gene promoter polymorphism C-13T were simultaneously detected by the electrochemical DNA chip and the loop-mediated isothermal amplification (LAMP) method, which is a novel technique for DNA amplification. Human genomic DNAs were extracted from blood, and the targets containing the six polymorphisms were amplified by the LAMP method. A sample containing the six LAMP products was reacted with the electrochemical DNA chip using a DNA detection system that controls hybridization reaction, washing, electrochemical detection, and data analysis automatically. A total of 31 samples were genotyped by this method, and the results were completely consistent with those determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis or the PCR direct sequence analysis. The time required for this method was only 2 h, and operations were very simple. Therefore, this method is expected to contribute to personalized medicine based on genotype.     

Use of the nuclear reaction for diagnostics of energetic particles in burning plasmas

Application of reaction-produced γ-rays to diagnostics of energetic particles in burning plasmas is analyzed. Particularly, we focus on 0.981 MeV γ-rays emitted in the nuclear reaction solely governed in the plasmas by energetic tritons. It is shown that these γ quanta can serve as a promising tool to diagnose α knock-on tritons and α-particles confined in burning DT plasmas. Key parameters of the α knock-on triton population and the α-particle confinement property can be obtained by comparing the experimental γ-ray yield and spectrum with theoretical slowing-down calculations. Even if the γ-ray spectral shape cannot be acquired, one can monitor densities of these tritons and α-particles at energies of 0.6–1.8 MeV and 2.0–3.5 MeV, respectively, in a nearly steady-state plasma. The 0.981-MeV photons also would help to display time evolution of the α-particle population in experiments of deuterium plasmas with pulsed tritium beam shots.     

Effects of photo-induced graft polymerization of 2-methacryloyloxyethyl phosphorylcholine on physical properties of cross-linked polyethylene in artificial hip joints

Osteolysis caused by wear particles from polyethylene in the artificial hip joints is a serious issue. We have used photo-induced radical graft polymerization to graft 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer onto the surface of cross-linked polyethylene (CLPE-g-MPC) in order to reduce friction and wear at the bearing surface of the joint. The physical and mechanical properties of CLPE and CLPE-g-MPC were not significantly different, expect that the friction coefficient of untreated CLPE cups was 0.0075, compared with 0.0009 for CLPE-g-MPC cup, an 88% reduction. After 3.0 × 10⁶ cycles in the hip joint simulator test, we could not observe any wear of CLPE-g-MPC cups. We concluded that the advantage of photo-induced radical graft polymerization technique was that the grafted MPC polymer gave a high lubricity only on the surface and has no effect on the bulk properties of the CLPE substrate.     

Cytocompatibility of calcium phosphate coatings deposited by an ArF pulsed laser

In the current studies, we deposited ultra-thin hydroxyapatite films on a pure titanium substrate by pulsed laser deposition, and we examined the effects of these surfaces on rat bone marrow (RBM) cells. This method allowed deposition of 500-, 2000-, and 5000-Angstrom-thick hydroxyapatite films. X-ray diffraction showed that the amorphous films recrystallized to a hydroxyapatite crystal structure after annealing. The proliferation of RBM cells was unaffected by the hydroxyapatite films, but osteocalsin and alkaline phosphatase mRNA and protein levels were elevated in cells grown on 2000- and 5000-Angstrom-thick films. These results indicate that ultra-thin hydroxyapatite films generated by pulsed laser deposition are better at promoting osteogenesis than pure titanium surfaces.     

Saccharomyces cerevisiae mutant displaying beta-glucans on cell surface

The deletion of MCD4 leads to an increase in beta-1,6-glucan level and a decrease in glycosylphosphatidylinositol-anchored protein and mannan levels in the cell wall of Saccharomyces cerevisiae, suggesting that mcd4 deletion mutant (mcd4Delta) displays beta-glucans on the cell surface without a mannan cover. An observation of the cell surface of mcd4Delta cells and an examination of the effect of contact between mcd4Delta cells and mouse macrophages indicated that macrophages were activated by contact with mcd4Delta cells displaying beta-glucans on the cell surface. We further examined the effect of intraperitoneal ethanol-fixed mcd4Delta cells on the survival period of mice infected with Candida albicans. mcd4Delta cells prolonged the survival period, implying that mcd4Delta cells may enhance the immune function of mice via macrophage activation. Moreover, we examined the structures of beta-glucans (i.e., alkali- and acetic acid-insoluble beta-glucans) extracted from mcd4Delta with (13)C-NMR and the effect of extracted beta-glucans on TNF-alpha secretion from macrophages. The structures of the beta-glucans from mcd4Delta differed from those of wild type (WT); however, there was no difference in tumor necrosis factor-alpha (TNF-alpha) secretion level between beta-glucans from mcd4Delta and those from WT. The yield of purified beta-glucans obtained from dry cells of mcd4Delta was higher than that obtained from dry cells of WT. mcd4Delta may be a superior strain for the preparation of beta-glucans.     

Production of extracellular bifidogenic growth stimulator by anaerobic and aerobic cultivations of several propionibacterial strains

Production of a bifidogenic growth stimulator (BGS) by propionic acid bacteria was investigated under anaerobic and aerobic culture conditions. To measure the concentration of extracellular BGS produced by propionic acid bacteria, we evaluated the effects of bioassay conditions using Bifidobacterium longum as a test microorganism on the formation of a growth-stimulation zone. The diameter of the growth-stimulation zone was significantly affected by both the component concentrations and the pH of a bioassay medium. The optimum component concentrations and pH of a bioassay medium were one-half of the normal values and 8.5, respectively. Using the bioassay method, we can measure the concentration of BGS produced by propionic acid bacteria ranging in concentrations from 0.1 microg/l to 1 mg/l using 1,4-dihydroxy-2-naphthoic acid (DHNA) and 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ) as standards. Of six dairy propionic acid bacterial strains tested, the four strains (Propionibacterium freudenreichii ET-3, P. shermanii PZ-3, P. acidipropionici JCM 6432, and P. jensenii JCM 6433) produced BGS at a concentration range of 4-23 mg/l under the anaerobic culture conditions. Analysis of high performance liquid chromatography (HPLC) showed that more than 70% of total BGS produced in supernatant samples was DHNA and no ACNQ was produced by the strains. The effect of oxygen supply on BGS production was investigated for the four BGS-producing strains. The aerobic conditions exerted in positive effects on BGS production by only P. acidipropionici JCM 6432. The concentration of BGS obtained in the aerobic cultivation of P. acidipropionici JCM 6432 was 1.3-fold than that in anaerobic cultivation. Different properties (BGS production as well as cell growth and glucose metabolism) occurring in response to the aerobic conditions were observed, depending on the propionic acid bacterial strain used. This paper is the first report on BGS production by propionibacterial strains except for P. freudenreichii.     

Mathematical model developed for environmental samples: prediction of GC/MS dioxin TEQ from XDS-CALUX bioassay data

Remediation of hazardous waste sites requires efficient and cost-effective methods to assess the extent of contamination by toxic substances including dioxin-like chemicals. Traditionally, dioxin-like contamination has been assessed by gas chromatography/high-resolution mass spectrometry (GC/MS) analysis for specific polychlorinated dibenzo-p-dioxins, dibenzofurans, and biphenyl congeners. Toxic equivalency factors for these congeners are then used to estimate the overall dioxin toxic equivalency (TEQ) of complex mixtures found in samples. The XDS-CALUX bioassay estimates contamination by dioxin-like chemicals in a sample extract by measuring expression of a sensitive reporter gene in genetically engineered cells. The output of the XDS-CALUX assay is a CALUX-TEQ value, calibrated based on TCDD standards. Soil samples taken from a variety of hazardous waste sites were measured using the XDS-CALUX bioassay and GC/MS. TEQ and CALUX-TEQ from these methods were compared, and a mathematical model was developed describing the relationship between these two data sets: log(TEQ) = 0.654 x log(CALUX-TEQ) + 0.058-(log(CALUX-TEQ))2. Applying this equation to these samples showed that predicted and GC/MS measured TEQ values strongly correlate (R2 = 0.876) and that TEQ values predicted from CALUX-TEQ were on average nearly identical to the GC/MS-TEQ. The ability of XDS-CALUX bioassay data to predict GC/MS-derived TEQ data should make this procedure useful in risk assessment and management decisions.     

Inductive logic programming for relational knowledge discovery

Inductive logic programming (ILP) is concerned with the induction of logic programs from examples and background knowledge. In ILP, the shift of attention from program synthesis to knowledge discovery resulted in advanced techniques that are practically applicable for discovering knowledge in relational databases. This paper gives a brief introduction to ILP, presents selected ILP techniques for relational knowledge discovery and reviews selected ILP applications. Nada Lavrač, Ph.D.: She is a senior research associate at the Department of Intelligent Systems, J. Stefan Institute, Ljubljana, Slovenia (since 1978) and a visiting professor at the Klagenfurt University, Austria (since 1987). Her main research interest is in machine learning, in particular inductive logic programming and intelligent data analysis in medicine. She received a BSc in Technical Mathematics and MSc in Computer Science from Ljubljana University, and a PhD in Technical Sciences from Maribor University, Slovenia. She is coauthor of KARDIO: A Study in Deep and Qualitative Knowledge for Expert Systems, The MIT Press 1989, and Inductive Logic Programming: Techniques and Applications, Ellis Horwood 1994, and coeditor of Intelligent Data Analysis in Medicine and Pharmacology, Kluwer 1997. She was the coordinator of the European Scientific Network in Inductive Logic Programming ILPNET (1993–1996) and program cochair of the 8th European Machine Learning Conference ECML’95, and 7th International Workshop on Inductive Logic Programming ILP’97. Sašo Džeroski, Ph.D.: He is a research associate at the Department of Intelligent Systems, J. Stefan Institute, Ljubljana, Slovenia (since 1989). He has held visiting researcher positions at the Turing Institute, Glasgow (UK), Katholieke Universiteit Leuven (Belgium), German National Research Center for Computer Science (GMD), Sankt Augustin (Germany) and the Foundation for Research and Technology-Hellas (FORTH), Heraklion (Greece). His research interest is in machine learning and knowledge discovery in databases, in particular inductive logic programming and its applications and knowledge discovery in environmental databases. He is co-author of Inductive Logic Programming: Techniques and Applications, Ellis Horwood 1994. He is the scientific coordinator of ILPnet2, The Network of Excellence in Inductive Logic Programming. He was program co-chair of the 7th International Workshop on Inductive Logic Programming ILP’97 and will be program co-chair of the 16th International Conference on Machine Learning ICML’99. Masayuki Numao, Ph.D.: He is an associate professor at the Department of Computer Science, Tokyo Institute of Technology. He received a bachelor of engineering in electrical and electronics engineering in 1982 and his Ph.D. in computer science in 1987 from Tokyo Institute of Technology. He was a visiting scholar at CSLI, Stanford University from 1989 to 1990. His research interests include Artificial Intelligence, Global Intelligence and Machine Learning. Numao is a member of Information Processing Society of Japan, Japanese Society for Artificial Intelligence, Japanese Cognitive Science Society, Japan Society for Software Science and Technology and AAAI.