• Title/Summary/Keyword: Fragment method

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Involvement of IS26 Element in the Evolution and Dissemination of $bla_{SHV-2a}$ and $bla_{SHV-12}$ ($bla_{SHV-2a}$$bla_{SHV-12}$ 항균제 내성 유전자의 분자적 진화 및 확산에 IS26 Mobile Element의 개입)

  • Kim, Jung-Min;Shin, Haeng-Seop;Cho, Dong-Taek
    • The Journal of the Korean Society for Microbiology
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    • v.35 no.3
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    • pp.263-271
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    • 2000
  • A clinical isolate of Klebsiella pneumoniae K7746 produced the extended-spectrum ${\beta}$-lactamase (ESBL) SHV-12. A 6.6 kb BamHI fragment containing the $bla_{SHV-12}$ gene of K7746 strain was cloned into pCRScriptCAM vector resulting in the recombinant plasmid p7746-Cl. The restriction map of 3.6 kb inserted DNA and sequences immediately surrounding $bla_{SHV-12}$ of p7746-C1 were homologous to plasmid pMPA2a carrying $bla_{SHV-2a}$. In addition, both $bla_{SHV-12}$ and $bla_{SHV-2a}$ were expressed from a common hybrid promoter made of the -35 region derived from the left inverted repeat of IS26 and the -10 region from the $bla_{SHV}$ promoter itself. The results indicate that $bla_{SHV-12}$ and $bla_{SHV-2a}$ may have evolved from a common ancestor in the sequential order of $bla_{SHV-2a}$ first, followed by $bla_{SHV-12}$. Furthermore, by the PCR mapping method using primers corresponding to the IS26 and $bla_{SHV}$, the association between IS26 and $bla_{SHV}$ was studied in 12 clinical isolates carrying $bla_{SHV-2a}$, 27 clinical isolates carrying $bla_{SHV-12}$, and 5 reference strains carrying $bla_{SHV-1}$ to $bla_{SHV-5}$. All 39 strains carrying $bla_{SHV-2a}$ or $bla_{SHV-12}$ were positive by the PCR, providing confirmative evidence that IS26 has been involved in the evolution and dissemination of $bla_{SHV-2a}$ and $bla_{SHV-12}$. But 5 reference strains carrying $bla_{SHV-1}$ to $bla_{SHV-5}$ were negative by the PCR. Therefore, we concluded that the molecular evolutionary pathway of $bla_{SHV-2a}$ and $bla_{SHV-12}$ may be different from that of other $bla_{SHV-ESBL}$, e.g., $bla_{SHV-2}$, $bla_{SHV-3}$, $bla_{SHV-4}$, and $bla_{SHV-5}$.

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Identification of Meat Species Using Species-Specific PCR-RFLP Fingerprint of Mitochondrial 12S rRNA Gene (미토콘드리아 12S rRNA 유전자의 종 특이적 PCR-RFLP Fingerprint를 이용한 식육 원료의 판별)

  • Park, Jong-Keun;Shin, Ki-Hyun;Shin, Sung-Chul;Chung, Ku-Young;Chung, Eui-Ryong
    • Food Science of Animal Resources
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    • v.27 no.2
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    • pp.209-215
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    • 2007
  • In order to develop a sensitive and reliable method for the species-specific molecular markers, PCR-RFLP assay of the mitochondrial DNA(mt DNA) 12S rRNA gene was exploited for the identification of the origin of animal meat species including cattle, pig, sheep, goat, horse, deer, chicken, duck and turkey. A specific primer pairs were designed, based on the nucleotide sequences of mt 12S rRNA gene, for the amplification of the highly conserved region in the gene of the animal species using PCR-RFLP technique. mt DNA was isolated from meat samples followed by DNA amplification using PCR with the specific primers. PCR amplification produced an approximately 455 bp fragment in each of these animal meats. To distinguish pleat species, the PCR amplicons were digested with restriction endonucleases Tsp5091 and MboI, respectively, which generates distinct RFLP profiles. The DNA profiles digested with Tsp5091 allowed the clear discrimination in the mammalian meat species and the DNA profiles digested with MboI in poultry meat species. Therefore, the PCR-RFLP profiles of mt 12S rRNA gene could be very useful to identify the origin of the raw materials in the raw meats as well as the processed meat products.

SURGICAL EXTRUSION OF THE CROWN-ROOT FRACTURED INCISORS: CASE REPORTS (외과적 정출술을 이용한 치관-치근 파절된 미성숙 영구치의 치험례)

  • Lee, Eun-Mi;Kim, Tae-Wan;Kim, Hyun-Jung;Kim, Young-Jin;Nam, Sun-Hyun
    • Journal of the korean academy of Pediatric Dentistry
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    • v.35 no.2
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    • pp.305-312
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    • 2008
  • Crown-root fractures occur throughout both crown and root, and are defined as fractures involving enamel, dentin and cementum. The fractures may be grouped according to pulpal involvement into complicated and uncomplicated one. Crown-root fractures often occur on maxillary anterior teeth and comprise 5% of injuries affecting the permanent dentition and 2% in the primary dentition. To restore crown-root fractured tooth, biologic width must be maintained. For maintaining biologic width, such methods as gingivectomy following osteoplasty or orthodontic extrusion or surgical extrusion are available. Surgical extrusion is a method that extracts the tooth and replants the fractured tooth supragingivally. It is indicated when the length of the crown fragment is less than half the length of the clinical root. In these cases, root canal treatment and crown restoration using light-cured composite resin were performed after surgical extrusion. In following periodic examinations, favorable outcome was observed.

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CYP2C19 Genotype Could be a Predictive Factor for Aggressive Manifestations of Hepatocellular Carcinoma Related with Chronic Hepatitis B Infection in Thailand

  • Nun-anan, Pongjarat;Chonprasertsuk, Soonthorn;Siramolpiwat, Sith;Tangaroonsanti, Anupong;Bhanthumkomol, Patommatat;Pornthisarn, Bubpha;Vilaichone, Ratha-korn
    • Asian Pacific Journal of Cancer Prevention
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    • v.16 no.8
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    • pp.3253-3256
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    • 2015
  • Background: Chronic hepatitis B virus (HBV) infection related hepatocellular carcinoma (HCC) is a major health problem in the Asia-Pacific region including Thailand. Several factors have been proposed as contributing to hepatocarcinogenesis. This study was aimed to investigate the impact of CYP2C19 genotypic polymorphism in HCC related to chronic HBV infection in Thailand. Materials and Methods: A cross-sectional study was performed between April 2014 and January 2015. Chronic HBV patients with HCC (n=50) and without HCC (n=50) were included. Clinical information and blood samples of all patients were collected. The CYP2C19 genotype was determined by polymerase chain reaction-restriction fragment length polymorphism method, and was classified as rapid metabolizer (RM), intermediate metabolizer (IM) or poor metabolizer (PM). Results: The CYP2C19 genotype frequencies of RM, IM and PM in HBV patients were found to be 19/50 (38%), 25/50 (50%) and 6/50 (12%), respectively. The CYP2C19 genotype frequencies of RM, IM and PM in HBV with HCC patients were 21/50 (42%), 25/50 (50%) and 4/50 (8%), respectively. The distribution of CYP2C19 genotype was not different between patients with and without HCC. Interestingly, among HBV with HCC patients, the RM genotype of CYP2C19 tended to increase risk of aggressive manifestation (OR=2.89, 95%CI=0.76-11.25, P-value=0.07), compared with non RM genotype carriers. Conclusions: CYP2C19 genotype IM was the most common genotype in Thai patients with chronic HBV infection. In addition, genotype RM could be an associated factor for aggressive presentation in HCC related to chronic HBV infection.

Association of leptin, toll-like receptor 4, and chemokine receptor of interleukin 8 C-X-C motif single nucleotide polymorphisms with fertility traits in Czech Fleckvieh cattle

  • Jecminkova, Katerina;Muller, Uwe;Kyselova, Jitka;Sztankoova, Zuzana;Zavadilova, Ludmila;Stipkova, Miloslava;Majzlik, Ivan
    • Asian-Australasian Journal of Animal Sciences
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    • v.31 no.11
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    • pp.1721-1728
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    • 2018
  • Objective: The use of genetic markers can help to enhance reproduction in cattle, which is a very important trait for profitability in dairy production systems. This study evaluated the association between genotypes of leptin (LEP), toll-like receptor 4 (TLR4), and chemokine receptor of interleukin 8 C-X-C motif (CXCR1) genes and fertility traits in Czech Fleckvieh cattle. Methods: Phenotypic data from 786 Czech Fleckvieh cows raised on 5 farms in the Czech Republic were used, along with information from the 1st three parities. To determine genotype, the polymerase chain reaction-restriction fragment length polymorphism method was used. Results: Except for LEP g.-963C>T, all studied genotype frequencies of single nucleotide polymorphisms (SNPs) were distributed according to the Hardy-Weinberg equilibrium. Two LEP SNPs (g.-963C>T and c.357C>T) were associated with the age at the 1st calving, days open (DO), pregnancy rate after 1st service (PR), and calving interval (CLI). In LEP g.-963C>T the TT genotype heifers firstly calved 24 days earlier than CC genotype and the CT genotype cow showed a tendency for shorter DO and higher PR. In LEP c.357C>T we observed longer CLI and DO period in TT cows. In general, we can propose the TT genotype of g.-963C>T as favorable and the TT genotype of c.357C>T as unfavorable for a cow's fertility. Heterozygotes in TLR4 c.-226C>G were significantly associated with shorter CLI, and presented a nonsignificant tendency to be associated with higher PR. In CXCR1 c.777 C>G, we did not observe any relationship of this SNP with reproduction. Conclusion: Overall, the results showed that LEP could be an effective marker for improving reproduction in Czech Fleckvieh cattle. This study also provides novel insights into the relationship between TLR4 and CXCR1 SNPs and reproduction in dual-purpose cattle.

Identification of the Polymorphisms in IFITM2 and IFITM5 Genes and their Association with Ulcerative Colitis (IFITM2 및 IFITM5 유전자다형성의 발굴과 궤양성대장염의 감수성과의 연관성)

  • Kim, Hun-Soo;Mo, Ji-Su;Alam, Khondoker Jahengir;Park, Won-Cheol;Kim, Keun Young;Chae, Soo-Cheon
    • Journal of Life Science
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    • v.25 no.1
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    • pp.84-92
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    • 2015
  • Interferon inducible transmembrane protein (IFITM) family genes have been implicated in various cellular processes such as the homotypic cell adhesion functions of IFNs and cellular anti-proliferative activities. The present study aimed to investigate whether the polymorphisms of the IFITM2 and IFITM5 genes are associated with susceptibility to UC. We identified a total of thirteen polymorphisms (eleven SNPs and two variations) in the IFITM2 gene and twelve polymorphisms (eleven SNPs and one variation) in the IFITM5 gene, by the direct sequencing method. Genotype analysis in the IFITM2 and IFITM5 SNPs was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and Taq-Man probe analysis, and the haplotype frequencies of IFITM2 and IFITM5 SNPs for multiple loci were estimated using the expectation maximization (EM) algorithm. The genotype and allele frequencies of IFITM2 SNPs, as well as IFITM5 SNPs, in UC patients were not significantly different from those of the healthy controls. We also analyzed the combined frequencies of rs77537847 of IFITM1, rs909097 of IFITM2, and rs56069858 of IFITM5 in the UC patients and the healthy controls. Although the distribution of the major combined genotype frequency did not differ significantly between the healthy controls and the UC patients, the GGT combined frequency in the healthy controls was significantly different from that in the UC patients (P=0.002). This result suggests that the combined genotype of the IFITMs polymorphisms may be associated with a susceptibility to UC and could be a useful genetic marker for UC.

Acute Oral Toxicity of dsRNA to Honey Bee, Apis mellifera (꿀벌에 대한 dsRNA의 급성섭식독성 평가)

  • Lim, Hye Song;Jung, Young Jun;Kim, Il Ryong;Kim, Jin;Ryu, Sungmin;Kim, Banni;Lee, Jung Ro;Choi, Wonkyun
    • Korean Journal of Environmental Agriculture
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    • v.36 no.4
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    • pp.241-248
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    • 2017
  • BACKGROUND: RNA interference (RNAi) eliminates or decreases gene expression by disrupting the target mRNA or by interfering with translation. Recently, RNAi technique was applied to generate new crop traits which provide protection against pests. To establish the environmental risk assessment protocol of RNAi LMO in lab scale, we developed dsRNA expression system using E. coli and tested acute oral toxicity assay to honey. METHOD AND RESULTS: The dsRNA expression vector, L4440, was chosen and cloned 240 bp of Snf7 and GFP gene fragment. To develop the maximum dsRNA induction condition in E. coli, we tested induction time, temperature and IPTG concentration in media. To estimate the risk assessment of dsRNA to honey bee, it has been selected and cultured with dsRNA supplement for 48 hours according to OECD guideline. As a result, the optimum condition of dsRNA induction was $37^{\circ}C$, 4 hours and 0.4 mM IPTG concentration and the difference between Snf7 and GFP dsRNA molecules from E. coli was not significant in survival and behavior to honey bee. Furthermore, blast search results indicated that effective match of predicted dsRNA fragments were not existed in honey bee genome. CONCLUSION: In this study, we developed and tested the acute oral toxicity of dsRNA using E. coli expression system to honey bee.

Analysis of Off-flavor Generated from a Polyethylene Terephthalate Water Bottles and Caps by Using an Electronic Nose (MS-전자코를 사용한 페트(polyethylene terephthalate) 생수병과 뚜껑의 이취 이행 분석)

  • Han, Hyun Jung;Park, Su Won;Jung, Hyo Yeon;Kim, Jung Sun;Dong, Hyemin;Noh, Bong Soo
    • Korean Journal of Food Science and Technology
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    • v.47 no.4
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    • pp.425-430
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    • 2015
  • The objective of the study was to investigate the off-flavor generated from PET water bottles and their caps by using a mass spectrometry-based electronic nose. The ion fragment data obtained from the electronic nose were used for discriminant function analysis (DFA). In the case of increased concentrations of the contamination of water, the off-flavor pattern depended on the discriminant function second score instead of the discriminant function first score. To identify the cause of off-flavor in polyethylene terephthalate (PET) bottled water, the PET bottle and its cap were analyzed by DFA. The results showed that the cap generated more volatile compounds than the bottle or mineral water did. The substances causing the off-flavor were predicted to be 2,4-di-tert-butylphenol (2,4-DTBP), nonanal, and decanal when the main peak of the mass spectrum was compared with the major ion fragments of the electronic nose. Thus, using this method, we could determine whether the PET water bottle was contaminated and whether the off-flavor resulted from contamination of the bottle cap.

Comparison of Partial Least Squares and Support Vector Machine for the Flash Point Prediction of Organic Compounds (유기물의 인화점 예측을 위한 부분최소자승법과 SVM의 비교)

  • Lee, Chang Jun;Ko, Jae Wook;Lee, Gibaek
    • Korean Chemical Engineering Research
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    • v.48 no.6
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    • pp.717-724
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    • 2010
  • The flash point is one of the most important physical properties used to determine the potential for fire and explosion hazards of flammable liquids. Despite the needs of the experimental flash point data for the design and construction of chemical plants, there is often a significant gap between the demands for the data and their availability. This study have built and compared two models of partial least squares(PLS) and support vector machine(SVM) to predict the experimental flash points of 893 organic compounds out of DIPPR 801. As the independent variables of the models, 65 functional groups were chosen based on the group contribution method that was oriented from the assumption that each fragment of a molecule contributes a certain amount to the value of its physical property, and the logarithm of molecular weight was added. The prediction errors calculated from cross-validation were employed to determine the optimal parameters of two models. And, an optimization technique should be used to get three parameters of SVM model. This work adopted particle swarm optimization that is one of heuristic optimization methods. As the selection of training data can affect the prediction performance, 100 data sets of randomly selected data were generated and tested. The PLS and SVM results of the average absolute errors for the whole data range from 13.86 K to 14.55 K and 7.44 K to 10.26 K, respectively, indicating that the predictive ability of the SVM is much superior than PLS.

DNA Markers Applicable for Identification of Two Internal Apple Feeders, Grapholita molesta and Carposina sasakii (두 종의 사과 심식나방류 [복숭아순나방 (Grapholita molesta), 복숭아심식나방 (Carposina sasakii)] 동정용 DNA 분자지표)

  • Song, Seung-Baeck;Choi, Kyeung-Hee;Lee, Soon-Won;Kim, Yong-Gyun
    • Korean journal of applied entomology
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    • v.46 no.2
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    • pp.175-182
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    • 2007
  • Two fruit moths of the oriental fruit moth, Grapholita molesta (Busck), and the peach fruit moth, Carposina sasakii (Matsumura), infest apples in Korea by internally feeding behavior. C. sasakii is a quarantine insect pest from some other countries importing Korean apples. G. molesta is not a quarantine insect pest, but can be incorrectly identified as C. sasakii especially when it is found inside apple fruits at its larval stages because it is not easy to identify the two species by morphological characters alone. This incomplete identification results in massive economical loss by fruits needlessly destroyed or turned away at border inspection stations of the importing nations. This difficulty can be overcome by molecular DNA markers. Several polymorphic regions of mitochondrial DNA of both species were sequenced and used for developing specific striction sites and polymerase chain reaction (PCR) primers. Based on these sequences, three diagnostic PCR-restriction fragment length polymorphism (RFLP) sites were detected and validated for their practical uses. Also, species-specific PCR primers were devised to develop diagnostic PCR method for identifying the internal feeders.