• International Journal of Stem Cells
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• 제15권4호
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• pp.347-358
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• 2022

Background and Objectives: The search for a suitable alternative for urethral defect is a challenge in the field of urethral tissue engineering. Induced pluripotent stem cells (iPSCs) possess multipotential for differentiation. The in vitro derivation of urothelial cells from mouse-iPSCs (miPSCs) has thus far not been reported. The purpose of this study was to establish an efficient and robust differentiation protocol for the differentiation of miPSCs into urothelial cells. Methods and Results: Our protocol made the visualization of differentiation processes of a 2-step approach possible. We firstly induced miPSCs into posterior definitive endoderm (DE) with glycogen synthase kinase-3𝛽 (GSK3𝛽) inhibitor and Activin A. We investigated the optimal conditions for DE differentiation with GSK3𝛽 inhibitor treatment by varying the treatment time and concentration. Differentiation into urothelial cells, was directed with all-trans retinoic acid (ATRA) and recombinant mouse fibroblast growth factor-10 (FGF-10). Specific markers expressed at each stage of differentiation were validated by flow cytometry, quantitative real-time polymerase chain reaction (qRT-PCR) assay, immunofluorescence staining, and western blotting Assay. The miPSC-derived urothelial cells were successfully in expressed urothelial cell marker genes, proteins, and normal microscopic architecture. Conclusions: We built a model of directed differentiation of miPSCs into urothelial cells, which may provide the evidence for a regenerative potential of miPSCs in preclinical animal studies.

• Journal of Veterinary Science
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• 제25권5호
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• pp.59.1-59.10
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• 2024

Importance: Despite advancements in herd management, feeding, and pharmaceutical interventions, neonatal calf diarrhea (NCD) remains a major global health concern. Bacteria, viruses, and parasites are the major contributors to NCD. Although several pathogens have been identified in the Republic of Korea (ROK), the etiological agents of numerous NCD cases have not been identified. Objective: To identify, for the first time, the prevalence and impact of Boosepivirus (BooV) on calf diarrhea in the ROK. Methods: Here, the unknown cause of calf diarrhea was determined using metagenomics We then explored the prevalence of certain pathogens, including BooV, that cause NCD. Seventy diarrheal fecal samples from Hanwoo (Bos taurus coreanae) calves were analyzed using reverse transcriptase and quantitative real-time polymerase chain reaction for pathogen detection and BooV isolate sequencing. Results: The complete genome of BooV was detected from unknown causes of calf diarrhea. And also, BooV was the most frequently detected pathogen (35.7%) among 8 pathogens in 70 diarrheic feces from Hanwoo calves. Co-infection analyses indicated that most BooV-positive samples were solely infected with BooV, indicating its significance in NCD in the ROK. All isolates were classified as BooV B in phylogenetic analysis. Conclusions and Relevance: This is the first study to determine the prevalence and molecular characteristics of BooV in calf diarrhea in the ROK, highlighting the potential importance of BooV as a causative agent of calf diarrhea and highlighting the need for further research on its epidemiology and pathogenicity.

• 대한화장품학회지
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• 제33권1호
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• pp.17-22
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• 2007

본 연구에서는 돌나물추출물이 각질형성세포에서 hyaluronan synthase (HAS) mRNA 발현과 hyaluronan (HA) 생성에 미치는 영향을 알아보았다. 또한 돌나물추출물이 가지는 인체 피부에서의 보습력 증진 효과를 평가하였다. 돌나물추출물 처리에 따른 각질형성세포에서의 HAS-1, -2, -3 mRNA 발현 변화는 semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR)을 통해 살펴보았고, HA의 생성량 변화는 enzyme-linked immunosorbent assay (ELISA)를 수행하여 확인하였다. 인체 피부에서의 수분함량 및 피부 수분 손실량에 미치는 영향은 Corneometer와 Tewameter를 이용하여 측정하였다. 그 결과, 돌나물추출물은 사람 각질형성세포의 HAS-2, -3 mRNA의 발현을 증가시켰고, HA의 생성량을 농도의존적으로 증가시킴을 확인하였다. 사람 피부에서는 수분 함량을 유지하는 효과가 우수하였으며, 표피 수분 손실량 또한 감소시켜 피부 보습제로서 매우 우수한 효과를 보였다 이상의 결과를 통해 돌나물은 피부에서 HA 생산을 촉진시키며, 피부 보습력을 증진시키는 화장품 소재로 이용될 수 있을 것으로 사료된다.

• Asian Pacific Journal of Cancer Prevention
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• 제14권11호
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• pp.6949-6953
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• 2013

Background: Nowadays, the encapsulation of cytotoxic chemotherapeutic agents is attracting interest as a method for drug delivery. We hypothesized that the efficiency of helenalin might be maximized by encapsulation in ${\beta}$-cyclodextrin nanoparticles. Helenalin, with a hydrophobic structure obtained from flowers of Arnica chamissonis and Arnica Montana, has anti-cancer and anti-inflammatory activity but low water solubility and bioavailability. ${\beta}$-Cyclodextrin (${\beta}$-CD) is a cyclic oligosaccharide comprising seven D-glucopyranoside units, linked through 1,4-glycosidic bonds. Materials and Methods: To test our hypothesis, we prepared ${\beta}$-cyclodextrin-helenalin complexes to determine their inhibitory effects on telomerase gene expression by real-time polymerase chain reaction (q-PCR) and cytotoxic effects by colorimetric cell viability (MTT) assay. Results: MTT assay showed that not only ${\beta}$-cyclodextrin has no cytotoxic effect on its own but also it demonstrated that ${\beta}$-cyclodextrin-helenalin complexes inhibited the growth of the T47D breast cancer cell line in a time and dose-dependent manner. Our q-PCR results showed that the expression of telomerase gene was effectively reduced as the concentration of ${\beta}$-cyclodextrin-helenalin complexes increased. Conclusions: ${\beta}$-Cyclodextrin-helenalin complexes exerted cytotoxic effects on T47D cells through down-regulation of telomerase expression and by enhancing Helenalin uptake by cells. Therefore, ${\beta}$-cyclodextrin could be superior carrier for this kind of hydrophobic agent.

• Journal of Veterinary Science
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• 제22권6호
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• pp.87.1-87.12
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• 2021

Background: African swine fever virus (ASFV), classical swine fever virus (CSFV), and porcine reproductive and respiratory syndrome virus (PRRSV) are still prevalent in many regions of China. Co-infections make it difficult to distinguish their clinical symptoms and pathological changes. Therefore, a rapid and specific method is needed for the differential detection of these pathogens. Objectives: The aim of this study was to develop a multiplex real-time quantitative reverse transcription polymerase chain reaction (multiplex qRT-PCR) for the simultaneous differential detection of ASFV, CSFV, and PRRSV. Methods: Three pairs of primers and TaqMan probes targeting the ASFV p72 gene, CSFV 5' untranslated region, and PRRSV ORF7 gene were designed. After optimizing the reaction conditions, including the annealing temperature, primer concentration, and probe concentration, multiplex qRT-PCR for simultaneous and differential detection of ASFV, CSFV, and PRRSV was developed. Subsequently, 1,143 clinical samples were detected to verify the practicality of the assay. Results: The multiplex qRT-PCR assay could specifically and simultaneously detect the ASFV, CSFV, and PRRSV with a detection limit of 1.78 × 10⁰ copies for the ASFV, CSFV, and PRRSV, but could not amplify the other major porcine viruses, such as pseudorabies virus, porcine circovirus type 1 (PCV1), PCV2, PCV3, foot-and-mouth disease virus, porcine parvovirus, atypical porcine pestivirus, and Senecavirus A. The assay had good repeatability with coefficients of variation of intra- and inter-assay of less than 1.2%. Finally, the assay was used to detect 1,143 clinical samples to evaluate its practicality in the field. The positive rates of ASFV, CSFV, and PRRSV were 25.63%, 9.36%, and 17.50%, respectively. The co-infection rates of ASFV+CSFV, ASFV+PRRSV, CSFV+PRRSV, and ASFV+CSFV+PRRSV were 2.45%, 2.36%, 1.57%, and 0.17%, respectively. Conclusions: The multiplex qRT-PCR developed in this study could provide a rapid, sensitive, specific diagnostic tool for the simultaneous and differential detection of ASFV, CSFV, and PRRSV.

• 한국해양생명과학회지
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• 제5권2호
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• pp.35-42
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• 2020

17β-estradiol (E₂)는 난소로부터 방출되는 호르몬으로 가정 및 축산 오폐수에 포함되어 환경으로 지속적으로 유출된다. E₂는 높은 에스트로겐 활성을 가지고 있어 갑각류의 발달과 생식에 영향을 미치는 내분비계교란물질로 알려져 있다. 갑각류의 발달은 탈피호르몬(ecdysteroid)의 신호 전달 과정에 의해 이루어지지만 E₂가 소형 갑각류의 탈피호르몬 경로 유전자를 어떻게 조절하는지에 대한 연구는 매우 미흡하다. 본 연구에서는 기수산 물벼룩 Diaphanosoma celebensis에서 E₂에 대한 급성 독성 시험을 통해 24-h LC_x 값을 도출하였고, E₂ 노출에 따른 탈피호르몬 경로에 관여하는 7개의 유전자(CYP314a1, EcRA, EcRB, USP, ERR, Vtg, VtgR)의 시간별 발현 변화를 quantitative real time polymerase chain reaction (qRT-PCR)을 이용하여 분석하였다. D. celebensis의 24-h LC₅₀ 값은 9.581 mg/l (95% C.I.: 7.697~11.927 mg/l), 24 h-LC₁₀ 값은 4.842 mg/l(95% C.I.: 3.683~6.366 mg/l)로 나타났다. CYP314a1, EcRA, USP, VtgR 유전자의 발현이 12시간 또는 24시간에 유의하게 증가하는 양상을 보였다. 이러한 결과는 E₂가 D. celebensis의 탈피호르몬 경로에 관련하는 유전자의 발현을 조절함으로써 탈피와 생식에 영향을 미칠 수 있을 것임을 시사한다. 본 연구는 소형 갑각류에서 내분비계교란물질이 탈피 경로에 미치는 영향에 대한 분자 기전을 이해하는데 도움이 될 것이다.

• 한국수정란이식학회지
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• 제24권3호
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• pp.225-230
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• 2009

Sex-sorting of sperm is an assisted reproductive technology (ART) used by the livestock industry for the mass production of animals of a desired sex. The standard method for sorting sperm is the detection of DNA content differences between X and Y chromosome-bearing sperm by flow cytometry. However, this method has variable efficiency and therefore requires verification by a second method. We have developed a sex determination method based on quantitative real-time polymerase chain reaction (qPCR) of the porcine amelogenin (AMEL) gene. The AMEL gene is present on both the X and the Y chromosome, but the length and sequence of its noncoding regions differ between the X and Y chromosomes. By measuring the threshold cycle (Ct) of qPCR, we were able to calculate the relative frequency of X chromosome. Two sets of AMEL primers were used in these studies. One set (AME) targeted AMEL gene sequences present in both X and Y chromosome, but produced PCR products of different lengths for each chromosome. The other set (AXR) bound to AMEL gene sequences present on the X chromosome but absent esholthe Y-chromosome. Relative product levels were calculated by normalizing the AXR fluorescence to the AME fluorescence. The AMEL method accurately predicted the sex ratios of boar sperm, demonstrating that it has potential value as a sex determination method.

• Asian-Australasian Journal of Animal Sciences
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• 제24권11호
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• pp.1552-1559
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• 2011

Real time PCR was used in this study to determine the effect of triticale dried distillers grains with solubles (TDDGS) as a replacement for grain or barley silage in finishing diets on the presence of six classical ruminal bacterial species (Succinivibrio dextrinosolvens, Selenomonas ruminantium, Streptococcus bovis, Megasphaera elsdenii, Prevotella ruminicola and Fibrobacter succinogenes) within the rumen contents of feedlot cattle. This study was divided into a step-wise adaptation experiment (112 days) that examined the effects of adaptation to diets containing increasing levels of TDDGS up to 30% (n = 4), a short-term experiment comparing animals (n = 16) fed control, 20%, 25% or 30% TDDGS diets over 28 days, and a rapid transition experiment (56 days) where animals (n = 4) were rapidly switched from a diet containing 30% TDDGS to a barley-based diet with no TDDGS. It was found that feeding TDDGS as replacement for barley grain (control vs. 20% TDDGS) decreased 16S rRNA copy numbers of starch-fermenting S. ruminantium and S. bovis (p<0.001 and p = 0.04, respectively), but did not alter 16S rRNA copy numbers of the other rumen bacteria. Furthermore, feeding TDDGS as a replacement barley silage (20% vs. 25% and 30% TDDGS) increased 16S rRNA copy numbers of S. ruminantium, M. elsdenii and F. succinogenes (p<0.001; p = 0.03 and p<0.001, respectively), but decreased (p<0.001) the 16S rRNA copy number of P. ruminicola. Upon removal of 30% TDDGS and return to the control diet, 16S rRNA copy numbers of S. ruminantium, M. elsdenii and F. succinogenes decreased (p = 0.01; p = 0.03 and p = 0.01, respectively), but S. dextrinosolvens and S. bovis increased (p = 0.04 and p = 0.009, respectively). The results suggest that replacement of TDDGS for grain reduces 16S rRNA copy numbers of starch-fermenting bacteria, whereas substitution for barley silage increases 16S rRNA copy numbers of bacteria involved in fibre digestion and the metabolism of lactic acid. This outcome supports the contention that the fibre in TDDGS is highly fermentable.

• Asian Pacific Journal of Cancer Prevention
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• 제16권9호
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• pp.4065-4069
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• 2015

Background: Epidermal growth factor-like domain multiple 7 (EGFL7), a secreted protein specifically expressed by endothelial cells during embryogenesis, recently was identified as a critical gene in tumor metastasis. Epithelial-mesenchymal transition (EMT) was found to be closely related with tumor progression. Accordingly, it is important to investigate the migration and EMT change after knock-down of EGFL7 gene expression in human pancreatic cancer cells. Materials and Methods: EGFL7 expression was firstly testified in 4 pancreatic cancer cell lines by real-time polymerase chain reaction (Real-time PCR) and western blot, and the highest expression of EGFL7 was found in PANC-1 cell line. Then, PANC-1 cells transfected with small interference RNA (siRNA) of EGFL7 using plasmid vector were named si-PANC-1, while transfected with negative control plasmid vector were called NC-PANC-1. Transwell assay was used to analyze the migration of PANC-1 cells. Real-time PCR and western blotting were used to detect the expression change of EGFL7 gene, EMT markers like E-Cadherin, N-Cadherin, Vimentin, Fibronectin and transcription factors like snail, slug in PANC-1, NCPANC-1, and si-PANC-1 cells, respectively. Results: After successful plasmid transfection, EGFL7 gene were dramatically knock-down by RNA interference in si-PANC-1 group. Meanwhile, migration ability decreased significantly, compared with PANC-1 and NC-PANC-1 group. Meanwhile, the expression of epithelial phenotype marker E-Cadherin increased and that of mesenchymal phenotype markers N-Cadherin, Vimentin, Fibronectin dramatically decreased in si-PANC-1 group, indicating a reversion of EMT. Also, transcription factors snail and slug decreased significantly after RNA interference. Conclusions: Current study suggested that highly-expressed EGFL7 promotes migration of PANC-1 cells and acts through transcription factors snail and slug to induce EMT, and further study is needed to confirm this issue.

• Asian-Australasian Journal of Animal Sciences
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• 제30권7호
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• pp.1037-1047
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• 2017

Objective: Despite an increasing number of investigations into the pathophysiology of necrotic enteritis (NE) disease, etiology of NE-associated diseases, and gene expression profiling of NE-affected tissues, the microRNA (miRNA) profiles of NE-affected poultry have been poorly studied. The aim of this study was to induce NE disease in the genetically disparate Fayoumi chicken lines, and to perform non-coding RNA sequencing in the intestinal mucosal layer. Methods: NE disease was induced in the Fayoumi chicken lines (M5.1 and M15.2), and non-coding RNA sequencing was performed in the intestinal mucosal layer of both NE-affected and uninfected chickens to examine the differential expression of miRNAs. Next, quantitative real-time polymerase chain reaction (real-time qPCR) was performed to further examine four miRNAs that showed the highest fold differences. Finally, bioinformatics analyses were performed to examine the four miRNAs target genes involvement in the signaling pathways, and to examine their interaction. Results: According to non-coding RNA sequencing, total 50 upregulated miRNAs and 26 downregulated miRNAs were detected in the NE-induced M5.1 chickens. While 32 upregulated miRNAs and 11 downregulated miRNAs were detected in the NE-induced M15.2 chickens. Results of real-time qPCR analysis on the four miRNAs (gga-miR-9-5p, gga-miR-20b-5p, ggamiR-196-5p, and gga-let-7d) were mostly correlated with the results of RNAseq. Overall, ggamiR-20b-5p was significantly downregulated in the NE-induced M5.1 chickens and this was associated with the upregulation of its top-ranking target gene, mitogen-activated protein kinase, kinase 2. Further bioinformatics analyses revealed that 45 of the gene targets of gga-miR-20b-5p were involved in signal transduction and immune system-related pathways, and 35 of these targets were predicted to interact with each other. Conclusion: Our study is a novel report of miRNA expression in Fayoumi chickens, and could be very useful in understanding the role of differentially expressed miRNAs in a NE disease model.