• BMB Reports
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• 제57권2호
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• pp.79-85
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• 2024

Pre-harvest sprouting is a critical phenomenon involving germination of seeds in the mother plant before harvest under relative humid conditions and reduced dormancy. In this paper, we generated HDR mutant lines with one region SNP (C/T) and an insertion of 6 bp (GGT/GGTGGCGGC) in OsERF1 genes for pre-harvest sprouting (PHS) resistance using CRISPR/Cas9 and a geminiviral replicon system. The incidence of HDR was 2.6% in transformed calli. T₁ seeds were harvested from 12 HDR-induced calli and named ERF1-hdr line. Molecular stability, key agronomic properties, physiological properties, and biochemical properties of target genes in the ERF1-hdr line were investigated for three years. The ERF1-hdr line showed significantly enhanced seed dormancy and pre-harvest sprouting resistance. qRT-PCR analysis suggested that enhanced ABA signaling resulted in a stronger phenotype of PHS resistance. These results indicate that efficient HDR can be achieved through SNP/InDel replacement using a single and modular configuration applicable to different rice targets and other crops. This work demonstrates the potential to replace all genes with elite alleles within one generation and greatly expands our ability to improve agriculturally important traits.

• 한국가금학회지
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• 제48권2호
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• pp.81-90
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• 2021

조류 인플루엔자 바이러스는 다양한 숙주 단백질을 이용해야만 증식이 가능하다. 포유류 (사람, 쥐) Fragile X mental retardation protein (FMRP)는 최근 인플루엔자 A 바이러스 viral RNP (vRNP)의 조립을 돕고, 이를 핵에서 세포질로 운반시켜 바이러스 증식에 도움을 준다. 하지만, 조류 인플루엔자 바이러스의 주요 숙주인 닭에서는 FMRP translational regulator 1 (FMR1) 유전자의 기능이 규명되지 않았다. 본 연구는 CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/Cas9) 유전자 가위를 이용해 정확히 닭 FMR1 유전자를 제거하여 닭 FMR1 유전자가 조류 인플루엔자 바이러스 증식에 어떤 영향을 끼치는지 연구하였다. 닭 FMR1 유전자는 닭 배아섬유아세포 (DF1세포)에서 초기 조류 인플루엔자 바이러스의 유전자 발현을 촉진하나, 감염 후 24시간 뒤에는 바이러스 생산 및 바이러스 중합효소 (Polymerase)의 활성에 영향을 끼치지 않았다. 또한, 야생형 닭 FMR1 유전자를 과발현 함에도 불구하고, 조류 인플루엔자 바이러스의 생산량에는 변화가 없었다. 위 결과들은 닭 FMR1은 포유류 FMR1 유전자에 비해 조류 인플루엔자 바이러스의 증식에 큰 영향을 주지 못하는 숙주인자임을 시사한다. 또한, 닭 FMR1처럼 기존에 포유류에서 알려진 숙주 인자를 목표로 하는 조류 인플루엔자 바이러스 저항성 치료제 및 형질전환 동물을 생산할 때, 조류 시스템에서 위 숙주 인자의 기능이 보존돼 있는지 고찰할 필요가 있다고 사료된다.

• 한국결정성장학회지
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• 제29권6호
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• pp.298-307
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• 2019

본 연구에서는 Al₂O₃ 소결체 표면에 두 종류의 CaO-Al₂O₃-SiO₂(CAS) 계 유리 분말을 스프레이 코팅(spray coating) 후 소성 거동에 대하여 연구하였다; (1) Si-rich, SiO₂ 함량이 높은 유리, (2) Ca-rich, CaO 함량이 높은 유리. Ca-rich 유리는 점도가 약 10⁷~10⁹ poise일 때 유리 내부에 잔존하는 기포들의 심한 발포 현상이 일어났고 이에 따라 소결 방해에 따른 수축율이 감소하였으며 조도는 증가하였다. 반면, Si-rich 유리의 경우, 1000℃ 이하에서는 Ca-rich와 같은 심각한 발포현상은 없었으나, 1200℃에서 밀도가 낮은 크리스토발라이트(cristobalite) 결정 발생과 함께 기포의 재발포 현상이 일어나 수축율이 감소하였으며 조도가 증가하는 이상 소성 거동을 보였다. 이는 저밀도 크리스토발라이트 결정 생성으로 인한 유리질의 칼슘 함량 증가에 의한 점도 감소로 생각된다. 따라서 CAS계 유리의 경우 저온 소성에서는 발포 현상에 의한 표면조도 상승과 소결 방해를 고려해야 하며, 특히 SiO₂ 함량이 높을 경우, 고온에서 결정화에 의한 이상 발포 현상도 생각해야 한다.

• Journal of Astronomy and Space Sciences
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• 제17권2호
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• pp.163-172
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• 2000

주성과 반성의 온도차가 큰 $(\mid\bigtriangleup T\mid\geq1,000K)$ EB형 접촉식쌍성에 WD 모웰의 모드 3을 적용 할 경우, 주성과 반성의 표면 반사율 (A), 주변감광계수 (x) 그리고 중력강광계수 (g)가 각각 서로 같은 값을 갖는 일반적인 경우와 각각 서로 다른 값을 갖도록 수정된 모드 3의 경우를 비교 분석하였다. EB형 접촉식쌍성인 DO Cas, GO Cyg 그리고 FS Lup에 적용한 결과, 수정된 모드 3에 의해 얻은 이론적인 광도곡선이 관측된 광도곡선을 더 잘 만족하고 있음을 확인하였다.

• Laboraroty Animal Research
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• 제34권4호
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• pp.257-263
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• 2018

Trefoil factor 1 (TFF1, also known as pS2) is strongly expressed in the gastrointestinal mucosa and plays a critical role in the differentiation of gastric glands. Since approximately 50% of all human gastric cancers are associated with decreased TFF1 expression, it is considered a tumor suppressor gene. Tff1 deficiency in mice results in histological changes in the antral and pyloric gastric mucosa, with severe hyperplasia and dysplasia of epithelial cells, resulting in the development of antropyloric adenoma. Here, we generated Tff1-knockout (KO) mice, without a neomycin resistant ($Neo^R$) cassette, using the clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRSIPR/Cas9) system. Though our Tff1-KO mice showed phenotypes very similar to the previous embryonic stem (ES)-cell-based KO mice, they differed from the previous reports in that a reduction in body weight was observed in males. These results demonstrate that these newly established Tff1-KO mice are useful tools for investigating genetic and environmental factors influencing gastric cancer, without the effects of artificial gene insertion. Furthermore, these findings suggest a novel hypothesis that Tff1 expression influences gender differences.

• 생체재료학회지
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• 제22권4호
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• pp.346-351
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• 2018

Background: Carbonate apatite ($CO_3Ap$) and silica-calcium phosphate composite (SCPC) are bone substitutes with good prospect for dental application. SCPC creates a hydroxyapatite surface layer and stimulate bone cell function while, $CO_3Ap$ induce apatite crystal formation with good adaptation providing good seal between cement and the bone. Together, these materials will add favorable properties as a pulp capping material to stimulate mineral barrier and maintain pulp vitality. The aim of this study is to investigate modification of $CO_3Ap$ cement combined with SCPC, later term as $CO_3Ap-SCPC$ cement (CAS) in means of its chemical (Calcium release) and physical properties (setting time, DTS and pH value). Methods: The study consist of three groups; group 1 (100% calcium hydroxide, group 2 $CO_3Ap$ (60% DCPA: 40% vaterite, and group 3 CAS (60% DCPA: 20% vaterite: 20% SCPC. Distilled water was employed as a solution for group 1, and $0.2mol/L\;Na_3PO_4$ used for group 2 and group 3. Samples were evaluated with respect to important properties for pulp capping application such as pH, setting time, mechanical strength and calcium release evaluation. Results: The fastest setting time was in $CO_3Ap$ cement group without SCPC, while the addition of 20% SCPC slightly increase the pH value but did not improved the cement mechanical strength, however, the mechanical strength of both $CO_3Ap$ groups were significantly higher than calcium hydroxide. All three groups released calcium ions and had alkaline pH. Highest pH level, as well as calcium released level, was in the control group. Conclusion: The CAS cement had good mechanical and acceptable chemical properties for pulp capping application compared to calcium hydroxide as a gold standard. However, improvements and in vivo studies are to be carried out with the further development of this material.

• Membrane and Water Treatment
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• 제8권3호
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• pp.245-257
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• 2017

The separation of nicotine and tobacco-specific N-nitrosamines is a tough problem in tobacco industry. In this study, separation of nicotine from 4-(methylnitrosamino) -1-(3-pyridyl)-1-butanone (NNK) mixtures was investigated using electrodialysis by taking the principle of the protonation status difference between these two components. The results indicated that the solution pH has a dominant impact on the separation process. In a pH range of 5-7, nicotine molecules are existed as mono- and di-protonated ions and can be separated from the uncharged NNK molecules. The acidic electrolyte is conducive to the separation process from the point of flux and energy consumption; while the alkaline electrolyte has negative impact on the separation process. A current density of $10mA/cm^2$ is an appropriate value for the separation process. The lowest energy consumption of the separation process is 0.58 kWh/kg nicotine with the process cost to be estimated at only $0.208 /kg nicotine. Naturally, electrodialysis is a high-efficiency, cost-effective, and environmentally friendly process to separate and purify nicotine from tobacco juice.

• BMB Reports
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• 제57권2호
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• pp.86-91
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• 2024

The fatty acids content of castor (Ricinus communis L.) seed oil is 80-90% ricinoleic acid, which is a hydroxy fatty acid (HFA). The structures and functional groups of HFAs are different from those of common fatty acids and are useful for various industrial applications. However, castor seeds contain the toxin ricin and an allergenic protein, which limit their cultivation. Accordingly, many researchers are conducting studies to enhance the production of HFAs in Arabidopsis thaliana, a model plant for oil crops. Oleate 12-hydroxylase from castor (RcFAH12), which synthesizes HFA (18:1-OH), was transformed into an Arabidopsis fae1 mutant, resulting in the CL37 line producing a maximum of 17% HFA content. In addition, castor phospholipid:diacylglycerol acyltransferase 1-2 (RcPDAT1-2), which catalyzes the production of triacylglycerol by transferring HFA from phosphatidylcholine to diacylglycerol, was transformed into the CL37 line to develop a P327 line that produces 25% HFA. In this study, we investigated changes in HFA content when endogenous Arabidopsis PDAT1 (AtPDAT1) of the P327 line was edited using the CRISPR/Cas9 technique. The successful mutation resulted in three independent lines with different mutation patterns, which were transmitted until the T4 generation. Fatty acid analysis of the seeds showed that HFA content decreased in all three mutant lines. These findings indicate that AtPDAT1 as well as RcPDAT1-2 in the P327 line are involved in transferring and increasing HFAs to triacylglycerol.

• Animal Bioscience
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• 제37권3호
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• pp.437-450
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• 2024

Objective: Vanin-1 (VNN1) is a pantetheinase that catalyses the hydrolysis of pantetheine to produce pantothenic acid and cysteamine. Our previous studies have shown that the VNN1 is specifically expressed in chicken liver which negatively regulated by microRNA-122. However, the functions of the VNN1 in lipid metabolism in chicken liver haven't been elucidated. Methods: First, we detected the VNN1 mRNA expression in 4-week chickens which were fasted 24 hours. Next, knocked out VNN1 via CRISPR/Cas9 system in the chicken Leghorn Male Hepatoma cell line. Detected the lipid deposition via oil red staining and analysis the content of triglycerides (TG), low-density lipoprotein-C (LDL-C), and high-density lipoprotein-C (HDL-C) after VNN1 knockout in Leghorn Male Hepatoma cell line. Then we captured various differentially expressed genes (DEGs) between VNN1-modified LMH cells and original LMH cells by RNA-seq. Results: Firstly, fasting-induced expression of VNN1. Meanwhile, we successfully used the CRISPR/Cas9 system to achieve targeted mutations of the VNN1 in the chicken LMH cell line. Moreover, the expression level of VNN1 mRNA in LMH-KO-VNN1 cells decreased compared with that in the wild-type LMH cells (p<0.0001). Compared with control, lipid deposition was decreased after knockout VNN1 via oil red staining, meanwhile, the contents of TG and LDL-C were significantly reduced, and the content of HDL-C was increased in LMH-KO-VNN1 cells. Transcriptome sequencing showed that there were 1,335 DEGs between LMH-KO-VNN1 cells and original LMH cells. Of these DEGs, 431 were upregulated, and 904 were downregulated. Gene ontology analyses of all DEGs showed that the lipid metabolism-related pathways, such as fatty acid biosynthesis and long-chain fatty acid biosynthesis, were enriched. KEGG pathway analyses showed that "lipid metabolism pathway", "energy metabolism", and "carbohydrate metabolism" were enriched. A total of 76 DEGs were involved in these pathways, of which 29 genes were upregulated (such as cytochrome P450 family 7 subfamily A member 1, ELOVL fatty acid elongase 2, and apolipoprotein A4) and 47 genes were downregulated (such as phosphoenolpyruvate carboxykinase 1) by VNN1 knockout in the LMH cells. Conclusion: These results suggest that VNN1 plays an important role in coordinating lipid metabolism in the chicken liver.

• IMMUNE NETWORK
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• 제23권6호
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• pp.44.1-44.16
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• 2023

Mesenchymal stem cells (MSCs) are effective in treating autoimmune diseases and managing various conditions, such as engraftment of allogeneic islets. Additionally, autologous and HLA-matched allogeneic MSCs can aid in the engraftment of human allogeneic kidneys with or without low doses of tacrolimus, respectively. However, HLA alloantigens are problematic because cell therapy uses more HLA-mismatched allogeneic cells than autologous for convenience and standardization. In particular, HLA-mismatched MSCs showed increased Ag-specific T/B cells and reduced viability faster than HLA-matched MSCs. In CRISPR/Cas9-based cell therapy, Cas9 induce T cell activation in the recipient's immune system. Interestingly, despite their immunogenicity being limited to the cells with foreign Ags, the accumulation of HLA alloantigen-sensitized T/B cells may lead to allograft rejection, suggesting that alloantigens may have a greater scope of adverse effects than foreign Ags. To avoid alloantigen recognition, the β2-microglobulin knockout (B2MKO) system, eliminating class-I MHC, was able to avoid rejection by alloreactive CD8 T cells compared to controls. Moreover, universal donor cells in which both B2M and Class II MHC transactivator (CIITA) were knocked out was more effective in avoiding immune rejection than single KO. However, B2MKO and CIITA KO system remain to be controlled and validated for adverse effects such as the development of tumorigenicity due to deficient Ag recognition by CD8 T and CD4 T cells, respectively. Overall, better HLA-matching or depletion of HLA alloantigens prior to cell therapy can reduce repetitive transplantation through the long-term survival of allogeneic cell therapy, which may be especially important for patients seeking allogeneic transplantation.