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Rapid Variable-Number Tandem-Repeat Genotyping for Mycobacterium leprae Clinical Specimens
Kimura, M.,Sakamuri, R. M.,Groathouse, N. A.,Rivoire, B. L.,Gingrich, D.,Krueger-Koplin, S.,Cho, S.-N.,Brennan, P. J.,Vissa, V. American Society for Microbiology 2009 Journal of clinical microbiology Vol.47 No.6
<P>Mycobacterium leprae is the noncultivable pathogen of leprosy. Since the genome sequence of an isolate of M. leprae has become available, multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) has been explored as a tool for strain typing and identification of chains of transmission of leprosy. In order to discover VNTRs and develop methods transferable to clinical samples, MLVA was applied to a global collection of M. leprae isolates derived from leprosy patients and propagated in armadillo hosts. PCR amplification, agarose gel electrophoresis, and sequencing methods were applied to DNA extracts from these infected armadillo tissues (n = 21). We identified polymorphisms in 15 out of 25 short-tandem-repeat (STR) loci previously selected by in silico analyses of the M. leprae genome. We then developed multiplex PCR for amplification of these 15 loci in four separate PCRs suitable for fluorescent fragment length analysis and demonstrated STR profiles highly concordant with those from the sequencing methods. Subsequently, we extended this method to DNA extracts from human clinical specimens, such as skin biopsy specimens (n = 30). With these techniques, mapping of multiple loci and differentiation of genotypes have been possible using total DNA extracts from limited amounts of clinical samples at a reduced cost and with less time. These practical methods are therefore available and applicable to answer focused epidemiological questions and to allow monitoring of the transmission of M. leprae in different countries where leprosy is endemic.</P>
Population-Based Molecular Epidemiology of Leprosy in Cebu, Philippines
Sakamuri, R. M.,Kimura, M.,Li, W.,Kim, H.-C.,Lee, H.,Kiran, M. D.,Black, W. C.,Balagon, M.,Gelber, R.,Cho, S.-N.,Brennan, P. J.,Vissa, V. American Society for Microbiology 2009 Journal of clinical microbiology Vol.47 No.9
<P>To address the persisting problem of leprosy in Cebu, Philippines, we compiled a database of more than 200 patients who attend an established referral skin clinic. We described the patient characteristics in conventional demographic parameters and also applied multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) and single nucleotide polymorphism (SNP) typing for Mycobacterium leprae in biopsied skin lesion samples. These combined approaches revealed that transmission is ongoing, with the affected including the young Cebuano population under 40 years of age in both crowded cities and rural areas of the island. The emergence of multicase families (MCF) is indicative of infection unconstrained by standard care measures. For the SNPs, we designed a low-cost PCR-restriction fragment length polymorphism typing method. MLVA in M. leprae was highly discriminatory in this population yet could retain broad groups, as defined by the more stable SNPs, implying temporal marker stability suitable for interpreting population structures and evolution. The majority of isolates belong to an Asian lineage (SNP type 1), and the rest belong to a putative postcolonial lineage (SNP type 3). Specific alleles at two VNTR loci, (GGT)5 and 21-3, were highly associated with SNP type 3 in this population. MLVA identified M. leprae genotype associations for patients with known epidemiological links such as in MCFs and in some villages. These methods provide a molecular database and a rational framework for targeted approaches to search and confirm leprosy transmission in various scenarios.</P>
Nguyen, T.T.H.,Moon, Y.H.,Ryu, Y.B.,Kim, Y.M.,Nam, S.H.,Kim, M.S.,Kimura, A.,Kim, D. IPC Science and Technology Press ; Elsevier Scienc 2013 Enzyme and microbial technology Vol.52 No.1
The human fibroblast collagenase catalytic domain (MMP1ca) that is considered a prototype for all interstitial collagenase and plays an important role in the turnover of collagen fibrils in the matrix was expressed as an inclusion body in the Escherichia coli. The purified enzyme displayed activity with substrate Dnp-Pro-Leu-Ala-Leu-Trp-Ala-Arg-OH with a K<SUB>m</SUB> value of 26.61+/-1.42μM. The inhibition activity of the nine flavonoid compounds and gallic acid against MMP1ca was examined. Among the compounds tested, the IC<SUB>50</SUB> of seven flavonoid compounds were determined and ranged from 14.13 to 339.21μM. Epigallocatechin gallate (EGCG) showed the highest inhibition toward MMP1ca with IC<SUB>50</SUB> values of 14.13+/-0.49μM. EGCG showed a competitive inhibition pattern with a K<SUB>i</SUB> value of 10.47+/-0.51μM. The free binding energy of EGCG against MMP1ca was -13.07kcalmol<SUP>-1</SUP>, which was calculated by using Autodock 3.0.5 software and showed numerous hydrophobic and hydrogen bond interactions. The galloyl group of EGCG, gallocatechin gallate and epicatechin gallate was determined to be important for inhibitory activity against MMP1ca.
Progress in R&D of coated conductor in M-PACC project
Izumi, T.,Ibi, A.,Nakaoka, K.,Taneda, T.,Yoshida, T.,Takagi, Y.,Nakamura, T.,Machi, T.,Katayama, K.,Sakai, N.,Yoshizumi, M.,Koizumi, T.,Kimura, K.,Kato, T.,Kiss, T.,Shiohara, Y. The Korea Institute of Applied Superconductivity a 2014 한국초전도저온공학회논문지 Vol.16 No.2
The five-year national project in Japan for R&D of coated conductors and applications, named as the Materials and Power Applications of Coated Conductors (M-PACC) project, was finished at the end of FY2013. The project consists of four sub-themes as cable, transformer, SMES and coated conductors. In the theme of coated conductors, the fabrication process had been developed to satisfy the requirements from the applications such as in-field $I_c$ performance, low AC loss in the long tapes etc. Through the project, the remarkable progress was achieved as follows; a high in-field minimum $I_c$ value over 54A/cm-width under 3T at 77K was realized in a 200m long EuBCO tape with artificial pinning centers of $BaHfO_3$ by the pulsed laser deposition (PLD) technique on the IBAD template. On the other hand, the AC loss reduction was confirmed in the tapes fabricated by both PLD and the metal organic deposition (MOD) techniques by scribing 100m tapes into 10-filamments. Additionally, the mechanism of the delamination phenomenon was systematically investigated and the strength was improved by eliminating the origins of the weak points in the films. Through the development, all targeted goals were accomplished and the several results were appreciated as a world champion data.
Woo, H.J.,Kang, H.K.,Nguyen, T.T.H.,Kim, G.E.,Kim, Y.M.,Park, J.S.,Kim, D.,Cha, J.,Moon, Y.H.,Nam, S.H.,Xia, Y.m.,Kimura, A.,Kim, D. IPC Science and Technology Press ; Elsevier Scienc 2012 Enzyme and microbial technology Vol.51 No.6
Novel ampelopsin glucosides (AMPLS-Gs) were enzymatically synthesized and purified using a Sephadex LH-20 column. Each structure of the purified AMPLS-Gs was determined by nuclear magnetic resonance, and the ionic product of AMPLS-G1 was observed at m/z 505 (C<SUB>21</SUB>H<SUB>22</SUB>O<SUB>13</SUB>.Na)<SUP>+</SUP> using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. AMPLS-G1 was identified as ampelopsin-4'-O-α-d-glucopyranoside. The optimum condition for AMPLS-G1, determined using response surface methodology, was 70mM ampelopsin, 150mM sucrose, and 1U/mL dextransucrase, which resulted in an AMPLS-G1 yield of 34g/L. The purified AMPLS-G1 displayed 89-fold increased water solubility and 14.5-fold browning resistance compared to those of AMPLS and competitive inhibition against tyrosinase with a K<SUB>i</SUB> value of 40.16μM. This value was smaller than that of AMPLS (K<SUB>i</SUB>=62.56μM) and much smaller than that of β-arbutin (K<SUB>i</SUB>=514.84μM), a commercial active ingredient of whitening cosmetics. These results indicate the potential of AMPLS and AMPLS-G1 as superior ingredients for functional cosmetics.
Yahaya, M.S.,Kimura, A.,Harai, J.,Nguyen, H.V.,Kawai, M.,Takahashi, J.,Matsuoka, S. Asian Australasian Association of Animal Productio 2001 Animal Bioscience Vol.14 No.12
The evaluation of structural carbohydrate losses and its effect on silages digestibility in alfalfa (Medicago sativa L.) and orchardgrass (Dactylis glomerata L.) was studied during 5, 21 and 56 days ensiling. About 70 and 60 kg fresh matter of the two forages were ensiled in 9 silos of 120 L capacity. The digestion trials were conducted in two phases using the two grasses in two $4{\times}4$ Latin square design according to the four treatments being the grass and the three silages. There were no differences in the DM and CP contents resulting from 5 to 56 days ensiling in both forages. The water-soluble carbohydrates (WSC), hemicelllose, pectin, and energy were slightly reduced and appeared lower in 56 days silage. The ether extract and cellulose contents slightly increased as the ensiling process advanced in the two species. Hemicellulose losses of 29 and 41 g/kg DM were obtained in alfalfa and orchardgrass, respectively, 56 days after ensiling. While the cellulose losses in both species were very little, compared to that for hemicellulose, the pectin losses, 56 days after ensiling were 15 and 12 g/kg DM in alfalfa and orchardgras respectively. The total structural carbohydrates lost (ie., hemicellulose + cellulose + pectin) in g/kg DM of fresh material forage ensiled, is about four fifths the amount lost by WSC, in alfalfa and about two thirds, in orchardgrass, by 21 days ensiling after the activity of microorganism terminated, indicating that appreciable amount was used as substrate for silage fermentation. Ensiling alfalfa and orchardgrass for 0, 5, 21 and 56 days maintained a decreasing trend of 83.8, 82.5, 79.3 and 78.9% digestibility in alfalfa and 80.5, 77.0, 77.1 and 76.4% digestibility in orchardgrass. While the digestibility of cellulose and ether extract increased in silage in both species, the digestible energy values in silage were reduced from 2.6 to 2.3 and 2.9 to 2.7 Mcal/kg DM respectively in alfalfa and orchard during 5-56 days ensiling.