Elimination of the cryptic plasmid in Leuconostoc citreum by CRISPR/Cas9 system

Jang, Y.J.,Seo, S.O.,Kim, S.A.,Li, L.,Kim, T.J.,Kim, S.C.,Jin, Y.S.,Han, N.S. Elsevier Science Publishers 2017 Journal of biotechnology Vol.251 No.-

<P>Leuconostoc spp. are important lactic acid bacteria for the fermentation of foods. In particular, L. citreum strains isolated from various foods have been used as host strains for genetic and metabolic engineering studies. In order to develop a food-grade genetic engineering system, L. citreum CB2567 was isolated from Kimchi. However, the isolated bacterium contained a cryptic plasmid which was difficult to eliminate. As the existence of the plasmid might hinder strain engineering, we eliminated the plasmid using an RNA-guided DNA endonuclease CRISPR/Cas9 system. We demonstrated that a plasmid-free L. citreum CB2567 host strain could be efficiently constructed through a two-step procedure: 1) transformation of the 'killer' plasmid expressing Cas9 endonuclease and a guide RNA (gRNA) targeting for a specific sequence in the cryptic plasmid, and 2) serial subculture without antibiotics for curing the killer plasmid. When the crude extract of L. citreum expressing Cas9 and the guide RNA was incubated with a PCR fragment containing the specific sequence recognized by the guide RNA, the PCR fragment was cleaved. Also, the cryptic plasmid pCB42 was successfully eliminated from the host strain after transforming the plasmid harboring Cas9 and the guide RNA. The Cas9 and gRNA expression plasmid used in this study can be applied for genome engineering purposes by additionally introducing an editing DNA template to repair the double strand DNA breakage caused by Cas9 in the genome of L. citreum. This study demonstrates the feasibility of developing CRISPR/Cas9-based genetic engineering tools to develop a safe host strain and construct food-grade lactic acid bacteria without residual antibiotic markers.</P>

Phase II study of S-1 combined with oxaliplatin as therapy for patients with metastatic biliary tract cancer: influence of the <i>CYP2A6</i> polymorphism on pharmacokinetics and clinical activity

Kim, K-p,Jang, G,Hong, Y S,Lim, H-S,Bae, K-s,Kim, H-S,Lee, S S,Shin, J-G,Lee, J-L,Ryu, M-H,Chang, H-M,Kang, Y-K,Kim, T W Nature Publishing Group 2011 The British journal of cancer Vol.104 No.4

<P><B>Background:</B></P><P>Advanced biliary cancer is often treated with fluoropyrimidine-based chemotherapy. In this study, we evaluated the efficacy and tolerability of a combination of S-1, an oral fluoropyrimidine prodrug, and oxaliplatin in patients with metastatic biliary cancer.</P><P><B>Methods:</B></P><P>Patients with histologically confirmed metastatic biliary cancer and no history of radiotherapy or chemotherapy were enrolled. Oxaliplatin was administered intravenously (130 mg m<SUP>−2</SUP>), followed by 14-day administration of oral S-1 (40 mg m<SUP>−2</SUP> twice daily) with a subsequent 7-day rest period every 21 days. Pharmacokinetic analysis of S-1 was performed at cycle 1. Patients were genotyped for <I>CYP2A6</I> polymorphisms (<SUP>*</SUP>1, <SUP>*</SUP>4, <SUP>*</SUP>7, <SUP>*</SUP>9 or <SUP>*</SUP>10), and pharmacokinetic and clinical parameters compared according to the <I>CYP2A6</I> genotype.</P><P><B>Results:</B></P><P>In total, 49 patients were evaluated, who received a median of four cycles. The overall response rate was 24.5%. Median progression-free and overall survival was 3.7 and 8.7 months, respectively. The most common haematological grade 3 out of 4 toxicity was neutropenia (14%), while non-hematological grade 3 out of 4 toxicities included anorexia (14%), nausea (12%), asthenia (10%), vomiting (10%), and diarrhoea (4%). Biotransformation of S-1 (AUC<SUB>0−24 h</SUB> of 5-fluorouracil/AUC<SUB>0−24 h</SUB> of tegafur) was 1.85-fold higher for the <I>*1/*1</I> group than for the other groups (90% confidence interval 1.37–2.49). Diarrhoea (<I>P</I>=0.0740), neutropenia (<I>P</I>=0.396), and clinical efficacy (response rate, <I>P</I>=0.583; PFS, <I>P</I>=0.916) were not significantly associated with <I>CYP2A6</I> genotype, despite differences in 5-FU exposure.</P><P><B>Conclusion:</B></P><P>The combination of S-1 and oxaliplatin appears to be active and well tolerated in patients with metastatic biliary cancer, and thus is feasible as a therapeutic modality. <I>CYP2A6</I> genotypes are associated with differences in the biotransformation of S-1. However, the impact of the <I>CYP2A6</I> polymorphism on variations in clinical efficacy or toxicity requires further evaluation.</P>

Characterization of Lactobacillus acidophilus Isolated from Piglets and Chicken

Ahn, Y.T.,Lim, K.L.,Ryu, J.C.,Kang, D.K.,Ham, J.S.,Jang, Y.H.,Kim, H.U. Asian Australasian Association of Animal Productio 2002 Animal Bioscience Vol.15 No.12

Lactic acid bacteria were isolated from piglets and chicken and characterized. Lactic acid bacteria showing resistance to low pH and bile, adhesion to intestinal epithelium cells, and the inhibition of Escherichia coli and Salmonella spp. were identified as Lactobacillus acidophilus. L. acidophilus PF01 survived for 2 h in MRS broth adjusted to pH 2. L. acidophilus CF07 was less resistant than L. acidophilus PF01 to pH 2, but survived at pH 2.5 for 2 h. Both of isolates were able to grow in MRS broth containing 0.3% (w/v) bile, with L. acidophilus CF07 being more tolerant to bile than L. acidophilus PF01. L. acidophilus PF01 and CF07 adhered specifically to the duodenal and jejunal epithelium cells of piglet, and the cecal and duodenal epithelium cells of chicken, respectively. Both of isolates did not adhere to the epithelium cells of the various animal intestines from which they were isolated. When L. acidophilus was cultured with E. coli and Salmonella spp. in MRS broth, MRS broth containing 2% skim milk powder or modified tryptic soy broth at $37^{\circ}C$, L. acidophilus PF01 and CF07 inhibited the growths of E. coli K88 and K99, and S. enteritidis and S. typhimurium, respectively. Both of isolates were found to possess the essential characteristics of probiotic lactic acid bacteria for piglet and chicken.

여자 고등학생의 성태도와 부모 양육태도가 성폭력 인식수준에 미치는 영향

강수인,김수민,김유진,김지수,박지원,신수경,이지현,장은주,정채린,진애경,차지영,이수연 이화여자대학교 간호과학대학 2019 이화간호학회지 Vol.- No.53

Purpose: The study aimed to investigate the factors influencing consciousness of sexual violence in female high school students, focusing on sexual attitude and parental attitude. ethods: Data were collected from 248 female high school students in South Korea. A structured questionnaire was used for data collection through online survey. The collected data were analyzed by descriptive statistics, t-test, ANOVA, Tukey’s test, Pearson correlation coefficient, and multiple linear regression using the SPSS 25.0 program. Results: The mean score of consciousness of sexual violence in female high school students was 43.54 out of 48. The findings indicate that the mean score of consciousness of sexual violence differed y number of years in high school and academic achievement. Among sexual attitude, parental ttitude, and consciousness of sexual violence, there were meaningful correlations between onsciousness of sexual violence and sexual attitude (r=.132, p=.038,), love-hostility (r=.172, p=.006),and autonomous-control (r=.132, p=.038). Love-hostility aspect of parental attitude (=.159, p=.049) and academic achievement (=.208, p=.001) were found to be the important influencing factors on female high school student’s consciousness of sexual violence (F=5.720, p<.001). Conclusion: The findings of the study suggest that the love-hostility aspect of parental attitude and academic achievement influence consciousness of sexual violence of female high school students. Therefore, in order to improve consciousness of sexual violence in female high school students, sex education program and parent-education program should be developed with further research.

Effects of Corn Distiller's Dried Grains with Solubles on Production and Egg Quality in Laying Hens

Cheon, Y.J.,Lee, H.L.,Shin, M.H.,Jang, A.,Lee, S.K.,Lee, J.H.,Lee, B.D.,Son, C.K. Asian Australasian Association of Animal Productio 2008 Animal Bioscience Vol.21 No.9

Corn distiller's dried grains with solubles (DDGS) is a completely new feed ingredient in the Korean feed market. There is an ever increasing need for the Korean feed industry to import and make the best of it as a high protein and high energy feed ingredient. A layer feeding trial was conducted for 10 weeks to investigate the effects of addition of light-colored DDGS to layer diets on laying performance, egg qualities and yolk fatty acid composition. Also, the economics of using DDGS in the Korean situation was analyzed. Nine hundred Hy-line Brown layers, 24 weeks of age, were employed in a feeding trial consisting of four dietary treatments (0, 10, 15, and 20% DDGS), and five replicates per treatment. All experimental diets were prepared as iso-protein (17%) and iso-calorie (TMEn 2,780 kcal/kg). The use of DDGS up to 20% in layer diets did not exert any influence on feed intake, laying rate, total egg mass, mean egg weight and feed conversion ratio (p>0.05). The color and breaking strength of eggshell, as well as the albumin height and Haugh unit were not affected by the addition of DDGS up to 20% in the diet. The yolk color was significantly increased by DDGS supplementation (p<0.05). As the DDGS level increased, the oleic acid content decreased, and the linoleic acid increased (p<0.05) in egg yolk. The degree of saturation of yolk fatty acids was not affected by DDGS supplementation. The inclusion of light-colored DDGS up to 20% in layer diets resulted in a decrease of feed cost per kg without any undesirable effect on laying performance. In conclusion, the light-colored DDGS (L* 56.65) could be used up to 20% in layer diets without any harmful effect on laying performance, and possibly provide economic benefits to the Korean poultry industry.

OsWRKY51, a rice transcription factor, functions as a positive regulator in defense response against Xanthomonas oryzae pv. oryzae

Hwang, S. H.,Kwon, S. I.,Jang, J. Y.,Fang, I. L.,Lee, H.,Choi, C.,Park, S.,Ahn, I.,Bae, S. c.,Hwang, D. J. Springer International 2016 Plant cell reports Vol. No.

<P>OsWRKY51 functions as a positive transcriptional regulator in defense signaling against Xanthomonas oryzae pv. oryzae by direct DNA binding to the promoter of defense related gene, OsPR10a. OsWRKY51 in rice (Oryza sativa L.) is induced by exogenous salicylic acid (SA) and inoculation with Xanthomonas oryzae pv. oryzae (Xoo). To examine the role of OsWRKY51 in the defense response of rice, we generated OsWRKY51 overexpressing and underexpressing transgenic rice plants. OsWRKY51-overexpressing transgenic rice lines were more resistant to Xoo and showed greater expression of defense-related genes than wild-type (WT) plants, while OsWRKY51-underexpressing lines were more susceptible to Xoo and showed less expression of defense-associated genes than WT plants. Transgenic lines overexpressing OsWRKY51 showed growth retardation compared to WT plants. In contrast, transgenic lines underexpressing OsWRKY51 by RNA interference showed similar plant height with WT plants. Transient expression of OsWRKY51-green fluorescent protein fusion protein in rice protoplasts revealed that OsWRKY51 was localized in the nucleus. OsWRKY51 bound to the W-box and WLE1 elements of the OsPR10a promoter. Based on these results, we suggest that OsWRKY51 is a positive transcriptional regulator of defense signaling and has direct DNA binding ability to the promoter of OsPR10a, although it is reported to be a negative regulator in GA signaling.</P>

왕겨에 의한 신령버섯균사체 액체배양액의 생쥐 항복수암성 증가

Young S. Kim(김영숙),Wook J. Jang(장욱진),Md. A. Rakib(라키브),Jung M. Kwon(권정민),Chae R. Ahn(안채린),So Y. Kim(김소영),Yong U. Cho(조용운),Young K. Ha(하영권),Jeong O. Kim(김정옥),Yeong L. Ha(하영래) 한국생명과학회 2010 생명과학회지 Vol.20 No.9

왕겨가 Agaricus blazei Murill (AB: 신령버섯)균사체 액체배양 추출물의 항암성을 증가시키는지에 관한 연구를 수행하였다. AB균사체를 대두박을 기본으로 한 액체배지에 다양한 조건으로 배양하여, β-glucan 함량, Brix, 균사체를 측정하여 적정 생육조건을 선정하고, 이들의 열수추출물의 S-180 cell로 유도한 mouse 복수암에 대한 항암성을 조사하였다. AB균사체는 25℃에서 5일간 배양하였을 때 최적 생육을 나타내었고, 이 배양물이 다른 조건에서 배양한 배양물보다 우수한 항암성을 나타내었다. AB균사체의 생육 및 항암성은 변온배양에 따른 효과는 없었다. 따라서 이 최적배양조건(25℃, 5일 배양)에서 AB균사체를 1% 왕겨분말이 함유된 액체배지에 배양하고, 이의 열수 추출물의 항암성을 검증하였다. 1% 왕겨가 함유된 액체배지에서 배양한 열수추출물의 항암성은 왕겨가 함유되지 않은 배지의 열수추출물보다 항암성이 유의성 있게 증가되었다(p<0.05). 왕겨의 첨가는 AB균사체의 생육을 오히려 촉진시켰다. 이 결과는 왕겨가 AB균사체 뿐 만 아니라 다른 버섯균사체 액체배양물의 항암성 증진을 위한 원료로 활용될 수 있을 것임을 의미한다. The effects of rice hull (RH) powder on the anticarcinogenic activity of submerged-liquid cultures of Agaricus blazei Murill (AB) were assessed for mouse ascites cancers induced by mouse Sarcoma S-180 (S-180) cancer cells. Optimal growth of AB mycelia in the basal liquid culture medium, containing soybean meal, was achieved by culturing at 25℃ for 5 days, when evaluated by β-glucan content, Brix, and mycelial weight, relative to other culture conditions. Hot-water extract (HWE) of the submergedliquid culture of AB mycelia grown at 25℃ for 5 days exhibited a stronger anticarcinogenic activity, relative to HWE from other culture conditions. No such effects were obtained from AB mycelial cultures by alternative temperature-controlling cultures. Both cytotoxicity for S-180 cells and anticarcinogenic potentials for mouse ascites cancer of the HWE from AB mycelia grown in the basal medium containing 1% RH powder for 5 days at 25℃ were significantly (p<0.05) enhanced, relative to HWE from the AB mycelia culture of the basal medium without RH powder. These results indicate that HWE of submerged-liquid culture of AB mycelia, incubated in media containing 1% RH powder at 25℃ for 5 days, enhanced anticarcinogenic activity against S-180 cell-induced mouse ascites cancer, and suggest that RH powder is an excellent ingredient for the improvement of the anticarcinogenic potentials of the submerged-liquid culture of mushroom mycelia.

Expression and characterization of an M cell-specific ligand-fused dengue virus tetravalent epitope using Saccharomyces cerevisiae

Nguyen, N.L.,So, K.K.,Kim, J.M.,Kim, S.H.,Jang, Y.S.,Yang, M.S.,Kim, D.H. Society for Bioscience and Bioengineering, Japan ; 2015 Journal of bioscience and bioengineering Vol.119 No.1

A fusion construct (Tet-EDIII-Co1) consisting of an M cell-specific peptide ligand (Co1) at the C-terminus of a recombinant tetravalent gene encoding the amino acid sequences of dengue envelope domain III (Tet-EDIII) from four serotypes was expressed and tested for binding activity to the mucosal immune inductive site M cells for the development of an oral vaccine. The yeast episomal expression vector, pYEGPD-TER, which was designed to direct gene expression using the glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, a functional signal peptide of the amylase 1A protein from rice, and the GAL7 terminator, was used to clone the Tet-EDIII-Co1 gene and resultant plasmids were then used to transform Saccharomyces cerevisiae. PCR and back-transformation into Escherichia coli confirmed the presence of the Tet-EDIII-Co1 gene-containing plasmid in transformants. Northern blot analysis of transformed S. cerevisiae identified the presence of the Tet-EDIII-Co1-specific transcript. Western blot analysis indicated that the produced Tet-EDIII-Co1 protein with the expected molecular weight was successfully secreted into the culture medium. Quantitative Western blot analysis and ELISA revealed that the recombinant Tet-EDIII-Co1 protein comprised approximately 0.1-0.2% of cell-free extracts (CFEs). In addition, 0.1-0.2 mg of Tet-EDIII-Co1 protein per liter of culture filtrate was detected on day 1, and this quantity peaked on day 3 after cultivation. In vivo binding assays showed that the Tet-EDIII-Co1 protein was delivered specifically to M cells in Peyer's patches (PPs) while the Tet-EDIII protein lacking the Co1 ligand did not, which demonstrated the efficient targeting of this antigenic protein through the mucosal-specific ligand.

Polyphenol-enriched Vaccinium uliginosum L. fractions reduce retinal damage induced by blue light in A2E-laden ARPE19 cell cultures and mice

Lee, B.L.,Kang, J.H.,Kim, H.M.,Jeong, S.H.,Jang, D.S.,Jang, Y.P.,Choung, S.Y. Pergamon Press 2016 Nutrition research Vol.36 No.12

<P>Polyphenols exert beneficial effects on vision. We hypothesized that polyphenol components of Vaccinium uliginosum L. (V.U.) extract protect retinal pigment epithelial (RPE) cells against blue light-induced damage. Our aim was to test extracts containing polyphenol components to ascertain effects to reduce damage against blue light in RPEs. We measured the activity in fractions eluted from water, ethanol, and HP20 resin (FH), and found that the FH fraction had the highest beneficial activity. We isolated the individual active compounds from the FH fraction using chromatographic techniques, and found that FH contained flavonoids, anthocyanins, phenyl propanoids, and iridoids. Cell cultures of A2E-laden ARPE-19 exposed to blue light after treatment with V.U. extract fractions and their individual constituents indicated improvement. V uliginosum L extract fractions and constituent compounds significantly reduced A2E photo-oxidation-induced RPE cell death and inhibited intracellular A2E accumulation. Furthermore, Balb/c male mice were exposed to blue light at 10000 lux for 1 h/d for 2 weeks to induce retinal damage. One week after the final blue light exposure, retinal damage evaluated revealed that the outer nuclear layer thickness and nuclei count were improved. Histologic examination of murine photoreceptor cells demonstrated that FH, rich in polyphenols, inhibited the loss of outer nuclear layer thickness and nuclei. Our findings suggest that V.U. extract and eluted fractions are a potential source of bioactive compounds that potentially serve a therapeutic approach for age-related macular degeneration. (C) 2016 Elsevier Inc. All rights reserved.</P>

PCB 조립공정의 성능향상을 위한 자동계수기 개발

장국렬,오성훈,김상미,박순영,방만원 木浦大學校 工業技術硏究所 1996 工業技術硏究誌 Vol.6 No.-

In this paper, we present an automatic PCB counting system which can automatically count the PCBs in assembling process and store the results in computer to be used as the data base of production managerment. The system is composed of optical sensors, a PC interface board, a PC and DB program. The optical sensors which are located at upside and downside of the PCB assembling line send the signal to the computer when the PCB is passing through the sensor position. The DB stored in the computer can be searched according to the production date or the PCB models. The experimental results show that the developed counting system can lead to the performance improvement of PCB assembling process by reduction of employes and automation of production management.