• Journal of Microbiology and Biotechnology
• /
• v.33 no.9
• /
• pp.1228-1237
• /
• 2023

The CRISPR-Cas system has emerged as the most efficient genome editing technique for a wide range of cells. Delivery of the Cas9-sgRNA ribonucleoprotein complex (Cas9 RNP) has gained popularity. The objective of this study was to develop a quantitative polymerase chain reaction (qPCR)-based assay to quantify the double-strand break reaction mediated by Cas9 RNP. To accomplish this, the dextransucrase gene (dsr) from Leuconostoc citreum was selected as the target DNA. The Cas9 protein was produced using recombinant Escherichia coli BL21, and two sgRNAs were synthesized through in vitro transcription to facilitate binding with the dsr gene. Under optimized in vitro conditions, the 2.6 kb dsr DNA was specifically cleaved into 1.1 and 1.5 kb fragments by both Cas9-sgRNA365 and Cas9-sgRNA433. By monitoring changes in dsr concentration using qPCR, the endonuclease activities of the two Cas9 RNPs were measured, and their efficiencies were compared. Specifically, the specific activities of dsr365RNP and dsr433RNP were 28.74 and 34.48 (unit/㎍ RNP), respectively. The versatility of this method was also verified using different target genes, uracil phosphoribosyl transferase (upp) gene, of Bifidobacterium bifidum and specific sgRNAs. The assay method was also utilized to determine the impact of high electrical field on Cas9 RNP activity during an efficient electroporation process. Overall, the results demonstrated that the qPCR-based method is an effective tool for measuring the endonuclease activity of Cas9 RNP.

• The Journal of Korean Academy of Prosthodontics
• /
• v.49 no.4
• /
• pp.324-332
• /
• 2011

Purpose: The purpose of this study was to compare the stress distribution of teeth and jaw on load by differentiating property of materials according to each layer of widely used mouthguard. Materials and methods: A Korean adult having normal cranium and mandible was selected to examine. A customized mouthguard was constructed by use of DRUFOMAT plate and DRUFOMAT-TE/-SQ of Dreve Co. according to Signature Mouthguard system. The cranium was scanned by means of computed tomography with 1mm interval. It was modeled with CANTIBio BIONIX/Body Builder program and simulated and interpreted using Alter HyperMesh program. The mouthguard was classified as follows according to the layers. (1) soft guard (Bioplast)(SG) (2) hard guard (Duran)(HG) (3) medium guard (Drufomat)(MG) (4) soft layer + hard layer (SG + HG) (5) hard layer + soft layer (HG + SG) (6) soft layer + hard layer + soft layer (SG + HG + SG) (7) hard layer + soft layer + hard layer (HG + SG + HG) The impact locations on mandible were gnathion, the center of inferior border, and the anterior edge of gonial angle. And the impact directions were oblique ($45^{\circ}$). The impact load was 800 N for 0.1 sec. The stress distribution was measured at maxillary teeth, TMJ and maxilla. The statistics were conducted using Repeated ANOVA and in case of difference, Duncan test was used as post analysis. Results: In teeth and maxilla, the mouthguard contacting soft layer of mandibular teeth presented lowest stress measure and, in contrast, in condyle, the mouthguard contacting hard layer of mandibular teeth presented lowest stress measure. Conclusion: For all impact directions, soft layer + hard layer + soft layer, the mouthguard with three layers which the hard layer is sandwiched between two soft layers, showed relatively even distribution of stress in impact.

• Journal of Plant Biotechnology
• /
• v.44 no.1
• /
• pp.89-96
• /
• 2017

The CRISPR/Cas9 is a core technology that can result in a paradigm for breeding new varieties. This study describes in detail the sgRNA design, vector construction, and the development of a transgenic plant and its molecular analysis, and demonstrates how gene editing technology through the CRISPR/Cas9 system can be applied easily and accurately. CRISPR/Cas9 facilitates targeted gene editing through RNA-guided DNA cleavage, followed by cellular DNA repair mechanisms that introduce sequence changes at the site of cleavage. It also allows the generation of heritable-targeted gene mutations and corrections. Here, we present detailed procedures involved in the CRISPR/Cas9 system to acquire faster, easier and more cost-efficient gene edited transgenic rice. The protocol described here establishes the strategies and steps for the selection of targets, design of sgRNA, vector construction, and analysis of the transgenic lines. The same principles can be used to customize the versatile CRISPR/Cas9 system, for application to other plant species.

• Journal of Physiology & Pathology in Korean Medicine
• /
• v.21 no.5
• /
• pp.1210-1218
• /
• 2007

Recently Atopic Dermatitis(AD) is increasing along with allergic disease. At present, there is no infallible cure for AD. Then AD patients undergo great suffering. This study is carried out to see whether or not the administering Danggwieumja(DG) along with Samhwangseje-gamibang(SG} as a medicine for external aplication, is effective in treating atopic dermatitis. To examine the effectiveness of the above prescription, the author made an observation of diverse immune responses. through the model of NC/Nga atopic mice. Results provided evidence that the DG administration along with SG can be used as a treatment means to atopic dermatitis. The results are as follows: The extent of Clinical skin severities in 13 and 16 week old NC/Nga mice treated with DG and SG, were reduced by 50.9%, 53.9% respectively, compared to the control NC/Nga mice with no drug treatment. IgE, IL-4, IL-5, IL-6, IgM and IgG1 levels in the serum of the NC/Nga mice treated with DG and SG were significantly decreased compared to those of the untreated control mice. In contrary, to the $IFN-{\gamma}$ level, significantly increased. The spleen weight of the NC/Nga mice treated with DG and SG significantly decreased compared to those of the untreated control mice. CCR3 gene expression in the skin tissue of NC/Nga mice treated with DG and SG were highly decreased, and the IL-6 expression significantly decreased, and the $IFN-{\gamma}$ gene expression increased compared to those of the untreated control mice. Histological observation of the ear and dorsal skin tissue of the NC/Nga mice treated with DG and SG, showed that the extents of inflammation and infiltrated immune cells in the epidermal tissue and dermis, were highly reduced compared to those of the untreated control mice. In the model inducing COX-2 activity in RAW 264.7 cell, the denser DG became, the more COX-2 activity was inhibited, compared to those of the untreated control group. $IL-1{\beta}$, and $TNF-{\alpha}$, IL-6 gene expression in RAW 264.7 cell with DG, significantly decreased, compared to those of the untreated control group. According to the assessment of cell toxicity in L929 cell, the rate of cell multiplication increased by 3% in consistency to 100ppm of DG compared to the untreated control group and in more than the 200 ppm consistency, cell toxicity was occurred.

• Molecules and Cells
• /
• v.24 no.1
• /
• pp.83-94
• /
• 2007

During monocarpic senescence in higher plants, functional stay-green delays leaf yellowing, maintaining photosynthetic competence, whereas nonfunctional stay-green retains leaf greenness without sustaining photosynthetic activity. Thus, functional stay-green is considered a beneficial trait that can increase grain yield in cereal crops. A stay-green japonica rice 'SNU-SG1' had a good seed-setting rate and grain yield, indicating the presence of a functional stay-green genotype. SNU-SG1 was crossed with two regular cultivars to determine the inheritance mode and identify major QTLs conferring stay-green in SNU-SG1. For QTL analysis, linkage maps with 100 and 116 DNA marker loci were constructed using selective genotyping with $F_2$ and RIL (recombinant inbred line) populations, respectively. Molecular marker-based QTL analyses with both populations revealed that the functional stay-green phenotype of SNU-SG1 is regulated by several major QTLs accounting for a large portion of the genetic variation. Three main-effect QTLs located on chromosomes 7 and 9 were detected in both populations and a number of epistatic-effect QTLs were also found. The amount of variation explained by several digenic interactions was larger than that explained by main-effect QTLs. Two main-effect QTLs on chromosome 9 can be considered the target loci that most influence the functional stay-green in SNU-SG1. The functional stay-green QTLs may help develop low-input high-yielding rice cultivars by QTL-marker-assisted breeding with SNU-SG1.

• Food Science and Preservation
• /
• v.10 no.3
• /
• pp.354-359
• /
• 2003

This study was conducted to investigate the fermentation characteristics of kimchi treated with different methods of water extracts of powdered green tea(GT). Four different kimchis, the SB-kimchi(control) which was not treatment of the extracts, SG-kimchi prepared with Chinese cabbage(CC) salted in 1％ GT containing 10％ brine, DG-kimchi prepared with CC dipped in 1％ GT for 30 min after salting, MS-kimchi prepared with the salted CC mixed with 1％ GT containing seasonings. All kimchis was fermented at 10$^{\circ}C$. The pH of treated-kimchis were maintained higher than those of control products during fermentation, but it showed no big difference between each treated groups. Total microbe of SB-kimchi(6.27-9.37 cfu/mL) was higher than those of GT-treated kimchi(5.17-9.20) during fermentation. The ratio of lactic acid bacteria against total microbe was higher than the treated kimchis. Total polyphenol content of kimchi was 52.75 mg％ in DG-kimchi, 47.71 mg％ in MS-kimchi, 44.89 mg％ in SG-kimchi, 30.70 mg％ in SB-kimchi on the 5th days of fermentation. Scores of crispy taste of SG- and DG-kimchi on the 5th days of fermentation was 4.03 and 4.01 points, respectively which was higher than control products. Scores of fishy and hot taste of GT-treated kimchi were lower than those of control products during all fermentation periods.

• Food Science and Preservation
• /
• v.10 no.3
• /
• pp.345-359
• /
• 2003

This study was conducted to investigate the fermentation characteristics of kimchi treated with different methods of water extracts of powdered green tea(GT). Four different kimchis, the SB-kimchi(control) which was not treatment of the extracts, SG-kimchi prepared with Chinese cabbage(CC) salted in 1％ GT containing 10％ brine, DG-kimchi prepared with CC dipped in 1％ GT for 30 min after salting, MS-kimchi prepared with the salted CC mixed with 1％ GT containing seasonings. All kimchis was fermented at 10$^{\circ}C$. The pH of treated-kimchis were maintained higher than those of control products during fermentation, but it showed no big difference between each treated groups. Total microbe of SB-kimchi(6.27-9.37 cfu/mL) was higher than those of GT-treated kimchi(5.17-9.20) during fermentation. The ratio of lactic acid bacteria against total microbe was higher than the treated kimchis. Total polyphenol content of kimchi was 52.75 mg％ in DG-kimchi, 47.71 mg％ in MS-kimchi, 44.89 mg％ in SG-kimchi, 30.70 mg％ in SB-kimchi on the 5th days of fermentation. Scores of crispy taste of SG- and DG-kimchi on the 5th days of fermentation was 4.03 and 4.01 points, respectively which was higher than control products. Scores of fishy and hot taste of GT-treated kimchi were lower than those of control products during all fermentation periods.

• Korean Journal of Food Science and Technology
• /
• v.30 no.6
• /
• pp.1404-1408
• /
• 1998

These studies were carried out to investigate the effect of mixed medicinal herbs, such as Curcuma longa and Lithospermum erythrorhozon (CL), Lithospermum erythrorhozon and Sophrora flavescens AITON (LS), Sophrora flavescens AITON and Glycyrrhiza uralensis (SG), and Glycyrrhiza uralensis and Schizandra chinensis (GS) on shelf-life of kimchi. The pH of kimchi containg 1% of medicinal herb extracts such as CL, LS, SG and GS was higher than that of control during fermentation for 25 days at $10^{\circ}C$ Titratable acidity, viable cells count of total bacteria and lactic acid bacteria in the kimchi were changed more slowly than in control. Shelf-life of kimchi was extended by addition of 1% mixed medicinal extracts such as CL, LS, SG and GS (1:1), respectively. The sensory quality (taste, flavor, crispness and overall acceptability) of CL, LS, SG and GS added kimchi was similar to that of control at 10 days of fermentation. But the sourness of LS, SG and GS (1%) added kimchi was more slowly developed than control after 15 days of fermentation, respectively. The effect of LS, SG and GS mixture on shelf-life in kimchi was significant. But the medicinal herb extracts added kimchi decreased its sensory quality with increasing concentration of the extracts.

• Korean Chemical Engineering Research
• /
• v.57 no.4
• /
• pp.475-483
• /
• 2019

Special chemical warfare agents are lethal gases that attack the human respiratory system. One of such gases are blood agents that react with the irons present in the electron transfer system of the human body. This reaction stops internal respiration and eventually causes death. The molecular sizes of these agents are smaller than the pores of an activated carbon, making chemical adsorption the only alternative method for removing them. In this study, we carried out a Computational Fluid Dynamics simulation by passing a blood agent: cyanogen chloride gas through an SG-1 gas mask canister developed by SG Safety Corporation. The adsorption bed consisted of a Silver-Zinc-Molybdenum-Triethylenediamine activated carbon impregnated with copper, silver, zinc and molybdenum ions. The kinetic analysis of the chemical adsorption was performed in accordance with the test procedure for the gas mask canister and was validated by the kinetic data obtained from experimental results. We predicted the dynamic behaviors of the main variables such as the pressure drop inside the canister and the amount of gas adsorbed by chemisorption. By using a granular packed bed instead of the Ergun equation that is used to model porous materials in Computational Fluid Dynamics, applicable results of the activated carbon were obtained. Dynamic simulations and flow analyses of the chemical adsorption with varying gas flow rates were also executed.

• Journal of the Korean Society of Food Culture
• /
• v.22 no.1
• /
• pp.157-165
• /
• 2007

This study was designed to evaluate the effects of fructose(F) or sucrose(S) and guar gum intake on carbohydrate and lipid metabolism in 15-week-old male Goto-Kakizaki(GK) rats. Fifty rats were randomly assigned to 5 groups which were different in carbohydrate(25% of carbohydrate) and fiber(5% w/w) sources. The carbohydrate(CHO) sources of each group were comstarch(control group, 100% of CHO), fructose with cellulose(F), fructose with guar gum(FG), sucrose with cellulose(S), and sucrose with guar gum(SG). Each group was fed exterimental diet for 4 weeks. We measured food intake, body weight gain, adipose tissues weight and organs weight. We conducted oral glucose tolerance test(OGTT) and measured plasma insulin concentration to examine carbohydrate metabolism. To evaluate lipid metabolism, we measured the lipid profile of plasma, liver and feces. Food intake and weight gain of FG or SG groups tended to be less than those of F or S groups. Perirenal and epididymal fat pad weights of SG group were significantly lower than those of S group and those of FG group tended to be lower than those of F group. In OGTT, blood glucose values of F or S groups were significantly higher than those of C group, and FG or SG groups tended to be lower than those of F or S groups during the experimental time. The area under the curve(AUC) of C group was significantly highest among the groups, AUC and plasma insulin concentration of FG or SG groups tended to be lower than those of F or S groups. Plasma and hepatic triglyceride (TG) of FG and SG groups were significantly lower than those of F and S groups, plasma and hepatic total lipid(TL) and total cholesterol(TC) of FG and SG groups tended to be lower than those of F and S groups. Fecal TL, TG and TC of FG or SG groups tended to be higher than those of F and S groups. In conclusion, intake of guar gum should improve carbohydrate and lipid metabolism in partial substitution of fructose or sucrose for cornstarch in GK rats.