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In vivo multiplex gene targeting with Streptococcus pyogens and Campylobacter jejuni Cas9 for pancreatic cancer modeling in wild-type animal

  • Chang, Yoo Jin;Bae, Jihyeon;Zhao, Yang;Lee, Geonseong;Han, Jeongpil;Lee, Yoon Hoo;Koo, Ok Jae;Seo, Sunmin;Choi, Yang-Kyu;Yeom, Su Cheong
    • Journal of Veterinary Science
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    • v.21 no.2
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    • pp.26.1-26.14
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    • 2020
  • Pancreatic ductal adenocarcinoma is a lethal cancer type that is associated with multiple gene mutations in somatic cells. Genetically engineered mouse is hardly applicable for developing a pancreatic cancer model, and the xenograft model poses a limitation in the reflection of early stage pancreatic cancer. Thus, in vivo somatic cell gene engineering with clustered regularly interspaced short palindromic repeats is drawing increasing attention for generating an animal model of pancreatic cancer. In this study, we selected Kras, Trp53, Ink4a, Smad4, and Brca2 as target genes, and applied Campylobacter jejuni Cas9 (CjCas9) and Streptococcus pyogens Cas9 (SpCas9) for developing pancreatic cancer using adeno associated virus (AAV) transduction. After confirming multifocal and diffuse transduction of AAV2, we generated SpCas9 overexpression mice, which exhibited high double-strand DNA breakage (DSB) in target genes and pancreatic intraepithelial neoplasia (PanIN) lesions with two AAV transductions; however, wild-type (WT) mice with three AAV transductions did not develop PanIN. Furthermore, small-sized Cjcas9 was applied to WT mice with two AAV system, which, in addition, developed high extensive DSB and PanIN lesions. Histological changes and expression of cancer markers such as Ki67, cytokeratin, Mucin5a, alpha smooth muscle actin in duct and islet cells were observed. In addition, the study revealed several findings such as 1) multiple DSB potential of AAV-CjCas9, 2) peri-ductal lymphocyte infiltration, 3) multi-focal cancer marker expression, and 4) requirement of > 12 months for initiation of PanIN in AAV mediated targeting. In this study, we present a useful tool for in vivo cancer modeling that would be applicable for other disease models as well.

Lectin histochemistry of the olfactory mucosa of Korean native cattle, Bos taurus coreanae

  • Sungwoong, Jang;Bohye, Kim;Jeongmin, Lee;Sohi, Kang;Joong-Sun, Kim;Jong-Choon, Kim;Sung-Ho, Kim;Taekyun, Shin;Changjong, Moon
    • Journal of Veterinary Science
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    • v.23 no.6
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    • pp.88.01-88.14
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    • 2022
  • Background: The olfactory mucosa (OM) is crucial for odorant perception in the main olfactory system. The terminal carbohydrates of glycoconjugates influence chemoreception in the olfactory epithelium (OE). Objectives: The histological characteristics and glycoconjugate composition of the OM of Korean native cattle (Hanwoo, Bos taurus coreae) were examined to characterize their morphology and possible functions during postnatal development. Methods: The OM of neonate and adult Korean native cattle was evaluated using histological, immunohistochemical, and lectin histochemical methods. Results: Histologically, the OM in both neonates and adults consists of the olfactory epithelium and the lamina propria. Additionally, using periodic acid Schiff and Alcian blue (pH 2.5), the mucus specificity of the Bowman's gland duct and acini in the lamina propria was determined. Immunohistochemistry demonstrated that mature and immature olfactory sensory neurons of OEs express the olfactory marker protein and growth associated protein-43, respectively. Lectin histochemistry indicated that numerous glycoconjugates, including as N-acetylglucosamine, mannose, galactose, N-acetylgalactosamine, complex type N-glycan, and fucose groups, were expressed at varied levels in the different cell types in the OMs of neonates and adults at varying levels. According to our observations, the cattle possessed a well-developed olfactory system, and the expression patterns of glycoconjugates in neonatal and adult OMs varied considerably. Conclusions: This is the first study to describe the morphological assessment of the OM of Korean native cattle with a focus on lectin histochemistry. The findings suggest that glycoconjugates may play a role in olfactory chemoreception, and that their labeling properties may be closely related to OM development and maturity.

The Study of Anti-inflammatory Effect of Hwanggeumjakyak-tang Extract in RAW 264.7 Macrophage (황금작약탕(黃芩芍藥湯)의 RAW 264.7 대식 세포에서의 항염증 효과에 관한 연구)

  • Kim, Ma-Ryong;Kang, Ok-Hwa;Kim, Sung-Bae;Kang, Hee-Jung;Kim, Ji-Eun;Hwang, Hyeong-Chil;Kim, In-Won;Kwon, Dong-Yeul
    • The Korea Journal of Herbology
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    • v.28 no.1
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    • pp.43-50
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    • 2013
  • Objectives : Hwanggeumjakyak-tang (huangqin shaoyao tang, HJT) has been used to treat acute enteritis in traditional oriental medicine. However, there has been a lack of studies regarding the effects of HJT on the inflammatory activities and effector inflammatory disease mechanism about macrophage before is not known. So we examined the effect of HJT water extract on pro-inflammatory mediators in lipopolysaccharide (LPS) - stimulated mouse macrophage, RAW 264.7 cells. Methods : Cells were treated with 2 ug/mL of LPS 1 h prior to the addition of HJT. Cell viability was measured by MTS assay. The production of nitric oxide (NO) was determined by reacting cultured medium with Griess reagent. The expression of cyclooxygenase-2 (COX-2), inducible NO synthase (iNOS) and mitogen-activated protein kinases (MAPKs) was investigated by Western blot, RT-PCR. The content of level of cytokines (prostaglandin (PG) $E_2$, interleukin (IL)-6, IL-12, Tumor necrosis factor-alpha (TNF-${\alpha}$) and monocyte chemoattractant protein-1 (MCP-1)) in media from LPS-stimulated Raw 264.7 cells was analyed by ELISA kit. Results : HJT inhibited the production of NO, $PGE_2$, IL-6 as well as the expressions of iNOS, COX-2 but did not inhibit the production of IL-12, TNF-${\alpha}$, MCP-1 in the murine macrophage, RAW 264.7 cells. HJT also had suppression effects of LPS-induced MAPKs activation Conclusion : These results suggest that HJT has an anti-inflammatory therapeutic potential, which may result from inhibition of MAPK phosphorylation, thereby decreasing the expression of pro-inflammatory genes.

Studies on the regulation of Hematopoietic enhancement of Brassica campestris var narinosa., Canavalia gladiata DC semen and their combinational prescription via Jak2/STAT5/GATA1 Pathway in Sca-1+ hematopoietic stem cells (Sca-1+골수조혈세포에서 JAK2/STAT5/GATA-1 신호전달 경로를 통한 다채, 도두 그리고 두 조합물에 의한 조혈증진 조절에 관한 연구)

  • Kim, Kunhoae;Kim, Seung-Hyung;Cho, In-Sik;Kim, Han-Young;Kim, Dong-Seon;Lee, Young-Cheol
    • The Korea Journal of Herbology
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    • v.28 no.4
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    • pp.7-16
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    • 2013
  • Objectives : Brassica campestris var narinosa (BCN), Canavalia gladiata DC semen (CGD) and their combinational prescription (BCN+CGD) have been use to demonstrate to regulate hematopoiesis. In the current study, we investigated whether Brassica campestris var narinosa, Canavalia gladiata DC semen and their combinational prescription is related to hemato-potentiating function using Sca-$1^+$ hematopoietic stem cells (Sca-$1^+HSCs$) as a testing system. Methods : Sca-$1^+HSCs$ isolated from femur in C57bl/6 mice with leukopenia and thrombocytopenia induced by cyclophosphamide (CTX). Then, Real-time PCR was performed to measure the mRNA expression, ELISA and haematopoiesis-related gene (EPO, TPO, IL-3, SCF, c-kit, GM-CSF), the phosphorylation of JAK2, GATA-1 and STAT-5a/b were observed by western blot, and the numbers of $CD117^+/Sca-1^+$ cell and the number of granulocyte erythrocyte monocyte macrophage colony-forming units (CFU-GEMM) and erythroid burst forming units (BFU-E), semisolid clonogenic assay was performed. Result : When Sca-$1^+HSCs$ were treated with Brassica campestris var narinosa, Canavalia gladiata DC semen and their combinational prescription with rIL-3/rSCF, the expression of haematopoiesis-related (EPO, TPO, IL-3, SCF, c-kit, and GM-CSF) were significantly increased at the levels of mRNA as well as production in Sca-$1^+HSCs$. Additionally, CGS enhanced phosphorylation of JAK2, GATA-1, and signal transducer and activator of transcription-5a/b (STAT-5a/b) in Sca-$1^+HSCs$. Furthermore, their combinational prescription (BCN+CGD) significantly enhanced the growth rate of granulocyte erythrocyte monocyte macrophage colony-forming units (CFU-GEMM) and erythroid burst forming units (BFU-E) in vitro. Conclusion : These result suggest that Brassica campestris var narinosa (BCN) and Canavalia gladiata DC have hematopoietic enhancement via hematopoietic cytokine-mediated JAK2/GATA-1/STAT-5a/b pathway, and their combinational prescription (BCN+CGD) has superior hematopoietic enhancement to those of individual extracts.

The study of anti-inflammatory effect of Hyeonto-dan extract in RAW 264.7 macrophage (현토단(玄兎丹)의 RAW 264.7 대식 세포에서의 항염증 효과에 관한 연구)

  • Kim, Ma-Ryong;Kang, Ok-Hua;Kong, Ryong;Seo, Yun-Soo;Zhou, Tian;Kim, Sang-A;Kim, Eun-Su;Sin, Min-A;Lee, Young-Seob;Kwon, Dong-Yeul
    • The Korea Journal of Herbology
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    • v.32 no.2
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    • pp.77-85
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    • 2017
  • Objectives : This study aimed to investigate the unknown mechanisms behind the anti- inflammatory activity of Hyeonto-dan(HT) 70% ethanol extract on LPS-stimulated RAW 264.7 cells. Methods : Cells were treated with Hyeonto-dan 1 h prior to addition of 200 ng/mL of LPS. Cell viability was measured by the MTS assay. Nitric oxide levels were determined by the Griess assay. $PGE_2$ were measured using EIA kit. Pro-inflammatory cytokine production was measured by the enzyme-linked immunosorbent assay (ELISA). The expression of COX-2, iNOS, and MAPKs was investigated by Western blot, qRT-PCR. $NF-{\kappa}B$/p65 localization and interaction of the TLR-4 receptor with LPS was examined by immunofluorescence assays. Results : Hyeonto-dan had no cytotoxicity at the measured concentration. Hyeonto-dan inhibited NO production and pro-inflammatory cytokines such as IL-6, $TNF-{\alpha}$, and PGE2 as well as the protein and mRNA expression of iNOS and COX-2. Moreover, Hyeonto-dan inhibited the interaction between LPS and TLR-4 in murine macrophages. It suppressed phosphorylation of extracellular signal-regulated kinase (ERK 1/2), c-jun N-terminal kinase (JNK 1/2) and p38. Finally, it inhibited translocation of $NF-{\kappa}B$ in response to competitive LPS. Conclusions : Based on the results of this study, Hyeonto-dan inhibited the binding of TLR-4 receptor to LPS and inhibited the phosphorylation of extracellular signaling pathway MAPKs. These inhibitory effects are thought that the amount of $NF-{\kappa}B$ delivered to the nucleus was decreased and the inflammatory reaction was prevented by decreasing the production of LPS-induced $PGE_2$, NO, IL-6 and $TNF-{\alpha}$.

Antibacterial and Antiviral Activities of Multi-coating Polyester Textiles (다중 코팅 폴리에스터 섬유 여재의 항균 및 항바이러스 특성)

  • Ko, Sangwon;Lee, Jae-Young;Park, Duckshin
    • Applied Chemistry for Engineering
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    • v.33 no.4
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    • pp.444-450
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    • 2022
  • The effect of coated polyester (PET) textiles with metal oxide, chitosan, and copper ion on the antibacterial and antiviral activities was evaluated to investigate the applicability of multi-coated PET textiles as antiviral materials. Compared to coated PETs with a single agent, multi-coated PETs reduced the loading amount of coating materials as well as the contact time with bacteria for a bacterial cell number of < 10 CFU/mL, which was not detectable with the naked eyes. Metal oxides generate reactive oxygen species (ROS) such as free radicals by a catalytic reaction, and copper ions can promote contact killing by the generation of ROS. Chitosan not only enhanced antibacterial activities due to amine groups, but enabled it to be a template to load copper ions. We observed that multi-coated PET textiles have both antibacterial activities for E. coli and S. aureus and antiviral efficiency of more than 99.9% for influenza A (H1N1) and SARS-CoV-2. The multi-coated PET textiles could also be prepared via a roll-to-roll coating process, which showed high antiviral efficacy, demonstrating its potential use in air filtration and antiviral products such as masks and personal protective equipment.

Upstream Risks in Domestic Battery Raw Material Supply Chain and Countermeasures in the Mineral Resource Exploration Sector in Korea (국내 배터리원료광종 공급망 업스트림 리스크와 광물자원탐사부문에서의 대응방안)

  • Oh, Il-Hwan;Heo, Chul-Ho;Kim, Seong-Yong
    • Economic and Environmental Geology
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    • v.55 no.4
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    • pp.399-406
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    • 2022
  • In line with the megatrend of 2050 carbon neutrality, the amount of critical minerals used in clean-energy technology is expected to increase fourfold and sixfold, respectively, according to the Paris Agreement-based scenario as well as the 2050 carbon-neutrality scenario. And, in the case of Korea, in terms of the battery supply chain used for secondary batteries, the midstream that manufactures battery materials and battery cell packs shows strength, but the upstream that provides and processes raw materials is experiencing difficulties. The Korea Institute of Geoscience and Mineral Resources has established a strategy to secure lithium, nickel, and cobalt and is conducting surveys to respond to the upstream risk of these types of battery raw materials. In the case of lithium, exploration has been carried out in Uljin, Gyeongsangbuk-do since 2020, and by the end of 2021, the survey area was selected for precision exploration by synthesizing all exploration data and building a 3D model. Potential resources will be assessed in 2022. In the case of nickel, the prospective site will be selected by the end of 2022 through a preliminary survey targeting 10 nickel sulfide deposits that have been prospected in the past. In the case of cobalt, Boguk cobalt is known only in South Korea, but there is only a record that cobalt was produced as a minor constituent of hydrothermal deposit. According to the literature, a cobalt ore body was found in the contact area between serpentinite and granite, and a protocol for cobalt exploration in Korea will be established.

Dietary crude protein levels during growth phase affects reproductive characteristics but not reproductive efficiency of adult male Japanese quails

  • Retes, Pamela Lacombe;Neves, Danusa Gebin das;Bernardes, Laryssa Fernanda;Alves, Victoria Veiga;Goncalves, Natalia de Castro;Lima, Diego de Rezende;Alvarenga, Renata Ribeiro;Pereira, Barbara Azevedo;Seidavi, Alireza;Zangeronimo, Marcio Gilberto
    • Animal Bioscience
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    • v.35 no.3
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    • pp.385-398
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    • 2022
  • Objective: The objective was to evaluate the influence of different dietary crude protein (CP) levels during the growth phase on reproductive characteristics and reproductive efficiency as well as the body development of adult male Japanese quail. Methods: Three hundred one-day-old male quails were distributed into five treatments with diets containing different CP levels (18%, 20%, 22%, 24%, and 26%) in a completely randomized design, with six replicates of ten birds each. The CP diets were applied only during the growth phase (1 to 35 days). At 36 days of age, the birds were transferred to 30 laying cages with three males and nine females each, and all birds received the same diet formulated to meet production-phase requirements until 96 days of age. Results: The growth rate of the birds increased linearly (p<0.01) with increasing dietary CP, but the age of maximum growth decreased (p<0.05). At growth maturity, all birds had the same body weight (p>0.05). At 35 days of age, higher weight gain was obtained (p<0.05) with diets containing 22% CP or higher. No effects on feed conversion were observed in this phase. The increase in dietary CP enhanced (p<0.01) nitrogen intake and nitrogen excretion but did not affect (p>0.05) nitrogen retention. Testis size, seminiferous tubular area, number of spermatogonia, and germinal epithelial height at 35 days of age increased linearly (p<0.05) with dietary CP, while the number of Leydig cells decreased (p<0.01). The Sertoli cell number at 60 days of age increased linearly (p<0.01) with dietary CP. Dietary CP levels did not affect cloacal gland size, foam weight, foam protein concentration, semen volume, or flock fertility at 90 days of age. Conclusion: Dietary CP concentration affected body and testicular development in male Japanese quails but did not affect reproductive efficiency.

Anti-inflammatory and Anti-allergic Effects of Lentinula edodes Extract by UVIrradiation (UV-B 조사에 따른 버섯 추출물의 항염증 및 항알레르기 활성)

  • Hwang, Mi Sun;Pyo, Jaesung;Kim, Hyun Jin;Do, Sun Gil;Song, Il Dae;Kim, Kang Min
    • Journal of Life Science
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    • v.32 no.5
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    • pp.368-374
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    • 2022
  • In this study, the effects of UV irradiation-enhanced ergocalciferol (vitamin D2) content containing Lentinula edodes extract on inflammation and allergic responses were investigated in vitro. The anti-inflammatory and anti-allergic effects of the mushroom extract were tested by estimating the cytokine secretions, such as TNF-α, IL-6, and IL-1β in LPS-activated macrophages (RAW 264.7), or histamine release in PMA and A23187-activated mast cells (RBL-2H3). Under the condition of macrophage activation with LPS, mushroom extract significantly reduced the secretions of pro-inflammatory cytokines, TNF-α and IL-6, and their mRNA expression also matched the observation. The current mushroom extract also significantly reduced the amount of mast cell degranulation-induced histamine secretion from PMA- and A23187-treated mast cells as well as the reduced secretion of IL-4. These results suggest that mushroom extract, which has increased ergocalciferol content by UV irradiation, inhibits the expression of cytokines in inflammation and allergic reactions; therefore, it can be used effectively for the prevention and treatment of inflammatory and allergic diseases.

A Study on Improved Open-Circuit Voltage Characteristics Through Bi-Layer Structure in Heterojunction Solar Cells (이종접합 태양전지에서의 Bi-Layer 구조를 통한 향상된 개방전압특성에 대한 고찰)

  • Kim, Hongrae;Jeong, Sungjin;Cho, Jaewoong;Kim, Sungheon;Han, Seungyong;Dhungel, Suresh Kumar;Yi, Junsin
    • Journal of the Korean Institute of Electrical and Electronic Material Engineers
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    • v.35 no.6
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    • pp.603-609
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    • 2022
  • Passivation quality is mainly governed by epitaxial growth of crystalline silicon wafer surface. Void-rich intrinsic a-Si:H interfacial layer could offer higher resistivity of the c-Si surface and hence a better device efficiency as well. To reduce the resistivity of the contact area, a modification of void-rich intrinsic layer of a-Si:H towards more ordered state with a higher density is adopted by adapting its thickness and reducing its series resistance significantly, but it slightly decreases passivation quality. Higher resistance is not dominated by asymmetric effects like different band offsets for electrons or holes. In this study, multilayer of intrinsic a-Si:H layers were used. The first one with a void-rich was a-Si:H(I1) and the next one a-SiOx:H(I2) were used, where a-SiOx:H(I2) had relatively larger band gap of ~2.07 eV than that of a-Si:H (I1). Using a-SiOx:H as I2 layer was expected to increase transparency, which could lead to an easy carrier transport. Also, higher implied voltage than the conventional structure was expected. This means that the a-SiOx:H could be a promising material for a high-quality passivation of c-Si. In addition, the i-a-SiOx:H microstructure can help the carrier transportation through tunneling and thermal emission.