• Animal Bioscience
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• 제37권3호
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• pp.509-521
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• 2024

Objective: It was shown that microRNAs (miRNAs) play an important role in milk protein synthesis. However, the post-transcriptional regulation of casein expression by exogenous miRNA (xeno-miRNAs) in ruminants remains unclear. This study explores the regulatory roles of alfalfa xeno-miR162 on casein synthesis in bovine mammary epithelial cells (bMECs). Methods: The effects of alfalfa xenomiR-162 and G protein subunit gamma 11 (GNG11) on proliferation and milk protein metabolism of bMECs were detected by 5-Ethynyl-2'-Deoxyuridine (EdU) staining, flow cytometry, cell counting kit-8 (CCK-8), enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot. Dual-luciferase reporter assay was used to verify the targeting relationship between GNG11 and xenomiR-162. Results: Results showed that over-expression of xenomiR-162 inhibited cell proliferation but promoted apoptosis, which also up-regulated the expression of several casein coding genes, including CSN1S1, CSN1S2, and CSN3, while decreasing the expression of CSN2. Furthermore, the targeting relationship between GNG11 and xenomiR-162 was determined, and it was confirmed that GNG11 silencing also inhibited cell proliferation but promoted apoptosis and reduced the expression of casein coding genes and genes related to the mammalian target of rapamycin (mTOR) pathway. Conclusion: Alfalfa xenomiR-162 appears to regulate bMECs proliferation and milk protein synthesis via GNG11 in the mTOR pathway, suggesting that this xeno-miRNA could be harnessed to modulate CSN3 expression in dairy cows, and increase κ-casein contents in milk.

• Animal Bioscience
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• 제37권2호
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• pp.193-202
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• 2024

Objective: Oxidative stress (OS) is a pathological process arising from the excessive production of free radicals in the body. It has the potential to alter animal gene expression and cause damage to the jejunum. However, there have been few reports of changes in the expression of long noncoding RNAs (lncRNAs) in the jejunum in piglets under OS. The purpose of this research was to examine how lncRNAs in piglet jejunum change under OS. Methods: The abdominal cavities of piglets were injected with diquat (DQ) to produce OS. Raw reads were downloaded from the SRA database. RNA-seq was utilized to study the expression of lncRNAs in piglets under OS. Additionally, six randomly selected lncRNAs were verified using quantitative real-time polymerase chain reaction (qRT-PCR) to examine the mechanism of oxidative damage. Results: A total of 79 lncRNAs were differentially expressed (DE) in the treatment group compared to the negative control group. The target genes of DE lncRNAs were enriched in gene ontology (GO) terms and Kyoto encyclopedia of genes and genomes (KEGG) signaling pathways. Chemical carcinogenesis-reactive oxygen species, the Foxo signaling pathway, colorectal cancer, and the AMPK signaling pathway were all linked to OS. Conclusion: Our results demonstrated that DQ-induced OS causes differential expression of lncRNAs, laying the groundwork for future research into the processes involved in the jejunum's response to OS.

• Biomolecules & Therapeutics
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• 제32권1호
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• pp.154-161
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• 2024

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder that causes progressive paralysis. L-Citrulline is a nonessential neutral amino acid produced by L-arginine via nitric oxide synthase (NOS). According to previous studies, the pathogenesis of ALS entails glutamate toxicity, oxidative stress, protein misfolding, and neurofilament disruption. In addition, L-citrulline prevents neuronal cell death in brain ischemia; therefore, we investigated the change in the transport of L-citrulline under various pathological conditions in a cell line model of ALS. We examined the uptake of [¹⁴C]L-citrulline in wild-type (hSOD1wt/WT) and mutant NSC-34/ SOD1^G93A (MT) cell lines. The cell viability was determined via MTT assay. A transport study was performed to determine the uptake of [¹⁴C]L-citrulline. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed to determine the expression levels of rat large neutral amino acid transported 1 (rLAT1) in ALS cell lines. Nitric oxide (NO) assay was performed using Griess reagent. L-Citrulline had a restorative effect on glutamate induced cell death, and increased [¹⁴C]L-citrulline uptake and mRNA levels of the large neutral amino acid transporter (LAT1) in the glutamate-treated ALS disease model (MT). NO levels increased significantly when MT cells were pretreated with glutamate for 24 h and restored by co-treatment with L-citrulline. Co-treatment of MT cells with L-arginine, an NO donor, increased NO levels. NSC-34 cells exposed to high glucose conditions showed a significant increase in [¹⁴C]L-citrulline uptake and LAT1 mRNA expression levels, which were restored to normal levels upon co-treatment with unlabeled L-citrulline. In contrast, exposure of the MT cell line to tumor necrosis factor alpha, lipopolysaccharides, and hypertonic condition decreased the uptake significantly which was restored to the normal level by co-treating with unlabeled L-citrulline. L-Citrulline can restore NO levels and cellular uptake in ALS-affected cells with glutamate cytotoxicity, pro-inflammatory cytokines, or other pathological states, suggesting that L-citrulline supplementation in ALS may play a key role in providing neuroprotection.

• 식물병연구
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• 제30권1호
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• pp.60-65
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• 2024

2020년 7월, 전라남도 해남에서 퇴록병반과 엽맥녹대 등 바이러스 병징이 보이는 패션프루트(Passiflora edulis) 잎으로부터 total RNA를 추출했으며, RT-PCR 검정과 염기서열 결정을 통해 CMV-HN2를 동정하였다. 패션프루트에 감염하는 CMV의 생물학적 특성을 확인하기 위해 10개 지표식물에 CMV-HN2를 접종한 결과, CMV-Fny와 비교하여 전형적인 CMV의 감염 패턴을 나타냈다. CMV 패션프루트 분리주들의 외피단백질에 대한 계통발생학적 분석한 결과, CMV의 패션프루트 분리주들은 subgroup I에 속하는 것으로 나타났으며, 그중 CMV-HN2는 subgroup IA에 속하는 것으로 나타났다. 또한 국내 패션프루트 CMV 분리주들 사이의 subgroup 차이가 확인되었다. 패션프루트 분리주 간의 외피단백질 아미노산 서열을 비교하였을때, CMV-HN2는 다른 분리주들과 특이적으로 4-8개 아미노산 차이가 존재하였다. 이 결과를 통해, 패션프루트 CMV 분리주들의 외피단백질에서 유전적 다양성이 있음을 확인하였다.

• 한국식품위생안전성학회지
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• 제39권3호
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• pp.260-265
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• 2024

하수기반역학을 이용한 코로나19 감시 결과, 연구기간(2022년 8월-2023년 8월)동안 울산지역 4곳 하수처리장의 전체 174건 모든 시료에서 코로나바이러스-19가 검출되었다. 확진자 수와 하수 내 코로나바이러스 농도와의 상관분석 결과, 높은 상관성이 나타났으며 특히 하수감시가 임상감시보다 2-3주 앞서 농도가 증가함으로써 조기 인지의 가능성도 볼 수 있었다. 또한 코로나19 변이 분석 결과 역시 유행 시기별 우세종화된 변이와 비교적 유사하여 변이 예측도 가능하였다. 하수감시가 전국적, 전세계적으로 적용되고 있으며 많은 연구가 국가적 사업으로 진행되고 있다. 이에 따라, 하수 분석방법 및 분석기기 발전 등의 지속적 연구 업데이트가 필요하다. 또한 코로나19를 통해 감염병의 선제적 모니터링 및 유행 예측의 가능성을 확인하였으므로 다양한 병원체 및 식품·의약품 등에 확대 적용이 진행 중이다. 따라서 본 연구는 감염병 검출분야에서 더 나아가 하수 내 식품 성분, 활성물질 및 미생물 등의 분석을 통해 지역사회의 식품안전 및 전반적인 위생환경 감시를 위해 활용될 수 있을 것으로 기대된다.

• Journal of Animal Science and Technology
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• 제66권4호
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• pp.717-725
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• 2024

The Hanwoo traceability system currently utilizes 11 dinucleotide repeat microsatellite (MS) markers. However, dinucleotide repeat markers are known to have a high incidence of polymerase chain reaction (PCR) artifacts, such as stutter bands, which can complicate the accurate reading of alleles. In this study, we examined the polymorphisms of the 11 dinucleotide repeat MS markers currently employed in traceability systems. Additionally, we explored four trinucleotide repeat MS markers and one tetranucleotide repeat MS marker in a sample of 1,106 Hanwoo cattle. We also assessed the potential utility of the tri- and tetranucleotide repeat MS markers. The polymorphic information content (PIC) of the five tri- and tetranucleotide repeat markers ranged from 0.663 to 0.767 (mean: 0.722), sufficiently polymorphic and slightly higher than the mean (0.716) of the current 11 dinucleotide repeat markers. Using all 16 markers, the mean PIC was 0.718. The estimated probability of identity (PI) was 3.13 × 10^-12 using the 11 dinucleotide repeat markers, 7.03 × 10^-6 using the five tri- and tetranucleotide repeat markers, and 2.39 × 10^-17 using all 16 markers; the respective PI_half-sibs values were 2.69 × 10^-9, 1.29 × 10^-4, and 3.42 × 10^-13; and the respective PIsibs values were 3.89 × 10^-5, 9.6 × 10^-3, and 3.69 × 10^-7. The probability of exclusion1 (PE₁) was 0.999864 for the 11 dinucleotide repeat markers, 0.981141 for five of the tri- and tetranucleotide repeat markers, and > 0.99 for all 16 markers; the respective PE₂ values were 0.994632, 0.901369, and > 0.99; and the respective PE₃ values were 0.998702, > 0.99, and > 0.99. The five investigated triand tetranucleotide repeat MS markers can be used in combination with the 11 existing MS markers to improve the accuracy of individual identification and paternity testing in Hanwoo.

• Journal of Animal Science and Technology
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• 제66권4호
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• pp.663-681
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• 2024

Co-infection with porcine reproductive and respiratory syndrome virus (PRRSV) and Haemophilus parasuis (HPS) has severely restricted the healthy development of pig breeding. Exploring disease resistance of non-coding RNAs in pigs co-infected with PRRSV and HPS is therefore critical to complement and elucidate the molecular mechanisms of disease resistance in Kele piglets and to innovate the use of local pig germplasm resources in China. RNA-seq of lungs from Kele piglets with single-infection of PRRSV or HPS and co-infection of both pathogens was performed. Two hundred and twenty-five differentially expressed long non-coding RNAs (DElncRNAs) and 30 DEmicroRNAs (DEmiRNAs) were identified and characterized in the PRRSV and HPS co-infection (PRRSV-HPS) group. Compared with the single-infection groups, 146 unique DElncRNAs, 17 unique DEmiRNAs, and 206 target differentially expressed genes (DEGs) were identified in the PRRSV-HPS group. The expression patterns of 20 DEmiRNAs and DElncRNAs confirmed by real-time quantitative polymerase chain reaction (RT-qPCR) were consistent with those determined by high-throughput sequencing. In the PRRSV-HPS group, the target DEGs were enriched in eight immune Gene Ontology terms relating to two unique DEmiRNAs and 16 DElncRNAs, and the unique target DEGs participated the host immune response to pathogens infection by affecting 15 immune-related Kyoto Encyclopedia of Genes and Genomes enrichment pathways. Notably, competitive endogenous RNA (ceRNA) networks of different groups were constructed, and the ssc-miR-671-5p miRNA was validated as a potential regulatory factor to regulate DTX4 and AEBP1 genes to achieve innate antiviral effects and inhibit pulmonary fibrosis by dual-luciferase reporter assays. These results provided insight into further study on the molecular mechanisms of resistance to PRRSV and HPS co-infection in Kele piglets.

• International Journal of Stem Cells
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• 제15권3호
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• pp.324-333
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• 2022

Background and Objectives: This study was to investigate the role of microRNA-29a-3p (miR-29a-3p) in human bone marrow mesenchymal stem cells (hBMSCs), and its relationship with steroid-associated osteonecrosis. Methods and Results: The online tool GEO2R was used to screen out the differentially expressed genes (DEGs) in GSE123568 dataset. Quantitative real time-polymerase chain reaction (qRT-PCR) was performed to detect the expression of miR-29a-3p, forkhead box O3 (FOXO3), alkaline phosphatase (ALP), bone gamma-carboxyglutamate protein (OCN) and RUNX family transcription factor 2 (Runx2) in the hBMSCs isolated from the patients with steroid-associated osteonecrosis. CCK-8 assay was executed to measure cell viability; western blot assay was utilized to detect FOXO3, ALP, Runx2, OCN and β-catenin expression. Cell apoptosis and cell cycle were detected by flow cytometry. Immunofluorescence assay was used to detect the sub-cellular localization of β-catenin. Bioinformatics analysis and luciferase reporter gene assay were performed to confirm whether miR-29a-3p can combine with FOXO3 3'UTR. MiR-29a-3p was markedly up-regulated in the hBMSCs of patients with steroid-associated osteonecrosis, while FOXO3 mRNA was significantly down-regulated. Transfection of miR-29a-3p mimics significantly inhibited the hBMSCs' proliferation, osteogenic differentiation markers' expressions, including ALP, Runx2, OCN, and repressed the ALP activity, as well as promoted cell apoptosis and cell-cycle arrest. FOXO3 was identified as a target gene of miR-29a-3p, and miR-29a-3p can inhibit the expression of FOXO3 and β-catenin, and inhibition of miR-29a-3p promoted translocation of β-catenin to the nucleus. Conclusions: MiR-29a-3p can modulate FOXO3 expression and Wnt/β-catenin signaling to inhibit viability and osteogenic differentiation of hBMSCs, thereby promoting the development of steroid-associated osteonecrosis.

• 보건의료생명과학 논문지
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• 제9권2호
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• pp.303-308
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• 2021

최근 MRSA와 같은 항생제 내성 균주에 감염되는 사례가 점점 증가하는 추세이며, 특히 원내에서 감염되는 경우도 상당수 찾아볼 수 있다. 본 연구에서는 강원에 소재한 대학에 재학생들을 대상으로 병원 실습 경험 여부를 조사하고, 각각 비강 검체를 채취하여 황색포도알균인 Staphylococcus aureus 균주 및 MRSA (methicillin resistant Staphylococcus aureus)의 존재 여부를 동정하여 임상 실습 경험이 있는 학생과 MRSA 검출에 대한 관계를 확인하고자 하였다. 실험에 참여한 인원은 64명 학생으로 남학생 22명, 여학생 42명이다. 멸균된 면봉으로 비강 검체를 채취하여 수송 배지인 Thioglycollate broth로 수송한 뒤, 선택배지인 MSA(mannitol salt agar)에 접종했다. 균을 배양하여 다양한 생화학적 test 및 상용화 키트인 API Kit를 사용하여 분리 및 동정을 하였고, 최종으로 분자생물학적 방법을 사용하여 MRSA 검출 양상을 확인하였다. 64명 학생 중 Staphylococcus aureus 균주가 46명에서 검출되었으며, 46명의 검체 중에 22명에서 MRSA가 검출되었다, 22명의 학생 중에 남학생은 5명, 여학생은 17명이었으며, 15명의 학생은 최근 1년 이내에 입원, 수술력 또는 임상현장실습 등의 의료기관 관련 MRSA(HR-MRSA)로 확인되었고, 나머지 7명(%)은 CA-MRSA로 확인되었다. MRSA가 검출된 학생들의 감염경로를 확인한 결과, 평상시 의료기관 방문 여부와 임상 실습경험 등으로 비추어 볼 때 원내에서 감염되었다고 추정할 수 있으며, 임상실습에 참가하는 학생들을 대상으로 원내 감염 예방을 위한 추가적인 교육과 학생들의 적극적인 참여가 필요하다고 여겨진다.

• Tuberculosis and Respiratory Diseases
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• 제41권4호
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• pp.339-353
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• 1994

연구배경 : p53 유전자는 염색체 17p에 존재하는 종양억제유전자인데, 돌연변이가 있는 경우의 mutant protein은 그 반감기가 4~8시간으로 wild type protein의 반감기 6~20분에 비하여 현저한 증가를 보이게 된다. 결과적으로 돌연변이가 있는 경우 p53 단백질이 과축적되어 과발현이 유발되는데, 이러한 기전을 이용하여 p53 면역조직화학염색은 p53유전자의 돌연변이의 검색 방법으로 많이 이용되고 있다. 유방암에서는 p53 핵단백질의 과발현이 있는 경우에 없을 때보다 무병생존기간 및 전체 생존기간 모두에서 유의하게 나쁜 임상 경과를 취함이 보고되고 있지만 폐암에서는 p53 유전자의 돌연변이 유무, 또는 p53단백의 과발현 유무가 예후에 미치는 영향은 아직까지 확실하지 않다. 저자 등은 한국인의 폐암에서 p53 종양억제 유전자의 돌연변이 빈도를 확인하고 이들이 폐암 환자의 임상 경과에 미치는 영향을 분석하며, 면역조직화학염색 및 PCR-SSCP 분석의 감수성과 특이성을 염기서열분석과 비교하여 확인하고자 본 연구를 시행하였다. 방법 : 근치적 폐절제술 후 임상경과를 추적중인 원발성폐암환자 75예를 대상으로 하였으며, 면역조직화학검사 및 분자생물학적 연구를 위하여는 이들의 종양조직(paraffin block)을 이용하였다. p53 단백질의 면역조직화학검사를 위한 일차항체로는 DO7(Novocastra, U.K.)을 사용하였고, PCR-SSCP 및 염기서열분석 등을 위하여는 파라핀 포매조직에서 암조직을 선택적으로 박절하여 DNA를 추출하여 이용하였다. 결과: 1) 전체 75예 폐암환자의 면역조직화학염색 결과 27예(36%)에서 p53단백질의 과발현을 보였다(Table 2.3, Fig 1). 2) 전체 환자의 p53 핵단백질 과발현 여부에 따른 중앙전체생존기간은 p53 음성군과 양성군 모두 25개월, 무병생존기간은 두군 모두 13개월로 동일하였다(Fig. 2). 3) PCR-SSCP 분석에 의한 p53 유전자의 mobility shift는 시험한 58예중 16예(27.6%)에서 확인되었다(Fig 3). Mobility shift 유무에 따른 중앙전체생존기간은 shift가 있는 군과 없는 군에서 각각 27개월과 20개월, 무병생존기간은 각각 8개월, 10개월로 유의한 차이가 없었다. 4) 염기서열분석을 통한 p53 유전자의 돌연변이는 시험한 29예중 10예(34.5%)에서 확인되었다(Fig 4). 염기서열분석 결과 돌연변이 유무에 따른 중앙전체생존기간은 돌연변이가 있었던 군과 없었던 군에서 각각 27개월, 22개월이었으며, 무병생존기간은 각각 20개월과 10개월로 유의한 차이는 없었으나 돌연변이가 있는 경우 조기재발을 하는 경향을 보였다(Fig 5, 6). 5) 면역조직화학염색은 PCR-SSCP 결과를 기준으로 할 때 민감도 67.0%, 특이도 74.0%, 그리고 일치도는 62.5% 이었다. PCR-SSCP의 결과는 염기서열분석 결과를 기준으로 할 때 민감도 91.8%, 특이도 96.2%, 일치도는 95.3% 이었다. 결론 : p53 핵단백질의 과발현 정도, PCR-SSCP 및 염기서열분석상 돌연변이 유무는 비소세포폐암환자의 근치적수술후 예후의 예측지표로서는 그 효용성이 적었으나, 돌연변이가 있는 경우 조기재발을 하는 경향을 보였다. 또한 p53유전자의 돌연변이 검색법으로는 면역조직화학염색보다는 PCR-SSCP법이 우수하였다.