• BMB Reports
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• 제54권12호
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• pp.608-613
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• 2021

Melanoma, the most serious type of skin cancer, exhibits a high risk of metastasis. Although chemotherapeutic treatment for metastatic melanoma improves disease outcome and patient survival, some patients exhibit resistance or toxicity to the drug treatment regime. OTUB1 is a deubiquitinating enzyme overexpressed in several cancers. In this study, we investigated the effects of inhibiting OTUB1 expression on melanoma-cell proliferation and viability and identified the underlying molecular mechanism of action of OTUB1. We did endogenous OTUB1 knockdown in melanoma cells using short interfering RNA, and assessed the resulting phenotypes via MTT assays, Western blotting, and cell-cycle analysis. We identified differentially expressed genes between OTUB1-knockdown cells and control cells using RNA sequencing and confirmed them via Western blotting and reverse transcription polymerase chain reaction. Furthermore, we investigated the involvement of apoptotic and cell survival signaling pathways upon OTUB1 depletion. OTUB1 depletion in melanoma cells decreased cell viability and caused simultaneous accumulation of cells in the sub-G1 phase, indicating an increase in the apoptotic-cell population. RNA sequencing of OTUB1-knockdown cells revealed an increase in the levels of the apoptosis-inducing protein TRAIL. Additionally, OTUB1-knockdown cells exhibited increased sensitivity to PLX4032, a BRAF inhibitor, implying that OTUB1 and BRAF act collectively in regulating apoptosis. Taken together, our findings show that OTUB1 induces apoptosis of melanoma cells in vitro, likely by upregulating TRAIL, and suggest that approaches targeting OTUB1 can be developed to provide novel therapeutic strategies for treating melanoma.

• Asian-Australasian Journal of Animal Sciences
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• 제31권2호
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• pp.189-197
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• 2018

Objective: Hemicastration is a unilateral orchiectomy to remove an injured testis, which can induce hormonal changes and compensatory hypertrophy of the remaining testis, and may influence spermatogenesis. However, the underlying molecular mechanisms are poorly understood. Here, we investigated the impact of hemicastration on remaining testicular function. Methods: Prepubertal mice (age 24 days) were hemicastrated, and their growth was monitored until they reached physical maturity (age 72 days). Subsequently, we determined testis DNA methylation patterns using reduced representation bisulfite sequencing of normal and hemicastrated mice. Moreover, we profiled the testicular gene expression patterns by RNA sequencing (RNA-seq) to examine whether methylation changes affected gene expression in hemicastrated mice. Results: Hemicastration did not significantly affect growth or testosterone (p>0.05) compared with control. The genome-wide DNA methylation pattern of remaining testis suggested that substantial genes harbored differentially methylated regions (1,139) in gene bodies, which were enriched in process of protein binding and cell adhesion. Moreover, RNA-seq results indicated that 46 differentially expressed genes (DEGs) involved in meiotic cell cycle, synaptonemal complex assembly and spermatogenesis were upregulated in the hemicastration group, while 197 DEGs were downregulated, which were related to arachidonic acid metabolism. Integrative analysis revealed that proteasome 26S subunit ATPase 3 interacting protein gene, which encodes a protein crucial for homologous recombination in spermatocytes, exhibited promoter hypomethylation and higher expression level in hemicastrated mice. Conclusion: Global profiling of DNA methylation and gene expression demonstrated that hemicastration-induced compensatory response maintained normal growth and testicular morphological structure in mice.

• Journal of Veterinary Science
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• 제23권1호
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• pp.2.1-2.18
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• 2022

Background: Co-infections of the porcine reproductive and respiratory syndrome virus (PRRSV) and the Haemophilus parasuis (HPS) are severe in Chinese pigs, but the immune response genes against co-infected with 2 pathogens in the lungs have not been reported. Objectives: To understand the effect of PRRSV and/or HPS infection on the genes expression associated with lung immune function. Methods: The expression of the immune-related genes was analyzed using RNA-sequencing and bioinformatics. Differentially expressed genes (DEGs) were detected and identified by quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemistry (IHC) and western blotting assays. Results: All experimental pigs showed clinical symptoms and lung lesions. RNA-seq analysis showed that 922 DEGs in co-challenged pigs were more than in the HPS group (709 DEGs) and the PRRSV group (676 DEGs). Eleven DEGs validated by qRT-PCR were consistent with the RNA sequencing results. Eleven common Kyoto Encyclopedia of Genes and Genomes pathways related to infection and immune were found in single-infected and co-challenged pigs, including autophagy, cytokine-cytokine receptor interaction, and antigen processing and presentation, involving different DEGs. A model of immune response to infection with PRRSV and HPS was predicted among the DEGs in the co-challenged pigs. Dual oxidase 1 (DUOX1) and interleukin-21 (IL21) were detected by IHC and western blot and showed significant differences between the co-challenged pigs and the controls. Conclusions: These findings elucidated the transcriptome changes in the lungs after PRRSV and/or HPS infections, providing ideas for further study to inhibit ROS production and promote pulmonary fibrosis caused by co-challenging with PRRSV and HPS.

• 한국동물학회지
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• 제35권1호
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• pp.80-86
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• 1992

The nucleotide sequences of 185 rRNAs of the five Korean decapods were partially determined by the direct sequencing method using the reverse transcriptase. ne average GC content of five species was 51.1% which is higher than that of yeast(45.0%) and lower than those of frog (53.0%) and rat (55.6%). This result follows the general patterns of the GC content in the nucleotides of the nucleic acid shown among the various phylogenetic groups. The average ratio of transrional/transversional nucleotide substitution of pairwise comparison among six species (including Anemia salina) was 1.200 $\pm$ 0.310 when whole region alas examined. However, the ratio showed some differences when the conservative regions and variable regions frere separatelv examined. The molecular phylogenies of the five species were constructed by using two different tree making methods. In general the results support the previously reported molecular phylogeny of the decapod crustaceans. However, our results indicate thats in the analysis of the sequence dat3, the UPGMA clustering method of the distance matrix method should be carefully employed after considering the rate of nucneotide substitution in the different regions of the molecule.

• Parasites, Hosts and Diseases
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• 제53권2호
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• pp.237-242
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• 2015

Analysis of ancient DNA (aDNA) extracted from Ascaris is very important for understanding the phylogenetic lineage of the parasite species. When aDNAs obtained from a Joseon tomb (SN2-19-1) coprolite in which Ascaris eggs were identified were amplified with primers for cytochrome b (cyt b) and 18S small subunit ribosomal RNA (18S rRNA) gene, the outcome exhibited Ascaris specific amplicon bands. By cloning, sequencing, and analysis of the amplified DNA, we obtained information valuable for comprehending genetic lineage of Ascaris prevalent among pre-modern Joseon peoples.

• Quantitative Bio-Science
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• 제37권2호
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• pp.125-132
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• 2018

In this study, noble strains of lactic acid bacteria were isolated and identified by genetic analysis of 16s rRNA. Also, pH-dependent growth curve, cholesterol assimilation ability and sugar production efficiency were measured. Lactic acid bacteria were identified to inhabit in the milks from various animals. Results of sequence analysis showed that there were differences in 16S rRNA sequence among strains and part of gene deletion was also recognized. Growth rates were varied, too, depending on the pH of the medium. Lactobacillus rhamnosus LOCK908 isolated from cow milk showed the highest growth rate and high cholesterol assimilation ability. Results of sugar fermentation tests were relatively consistent with the sequencing results. So, we propose newly isolated Lactobacillus rhamnosus LOCK908 as useful candidate for a starter of fermented beverage and probiotics. Results of this study will contribute to the isolation and identification of noble Lactic acid bacteria and to the public health.

• Clinical and Experimental Reproductive Medicine
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• 제49권4호
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• pp.225-238
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• 2022

The ultimate goal of human assisted reproductive technology is to achieve a healthy pregnancy and birth, ideally from the selection and transfer of a single competent embryo. Recently, techniques for efficiently evaluating the state and quality of preimplantation embryos using time-lapse imaging systems have been applied. Artificial intelligence programs based on deep learning technology and big data analysis of time-lapse monitoring system during in vitro culture of preimplantation embryos have also been rapidly developed. In addition, several molecular markers of the secretome have been successfully analyzed in spent embryo culture media, which could easily be obtained during in vitro embryo culture. It is also possible to analyze small amounts of cell-free nucleic acids, mitochondrial nucleic acids, miRNA, and long non-coding RNA derived from embryos using real-time polymerase chain reaction (PCR) or digital PCR, as well as next-generation sequencing. Various efforts are being made to use non-invasive evaluation of embryo quality (NiEEQ) to select the embryo with the best developmental competence. However, each NiEEQ method has some limitations that should be evaluated case by case. Therefore, an integrated analysis strategy fusing several NiEEQ methods should be urgently developed and confirmed by proper clinical trials.

• 한국버섯학회지
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• 제8권2호
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• pp.41-50
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• 2010

Mycoviruses have been found in many fungal species including mushrooms. Double-stranded (ds) RNA genomes were common type in mycoviruses, but single-stranded (ss) RNA mycoviruses were also reported in some fungal species. Sequencing analysis using cDNA cloning experiments revealed that mycoviruses can be classified into several different virus families such as Totiviridae, Hypoviridae, Partitiviridae and Barnaviridae etc. Because the nucleotide sequence data that are available in these days are very limited in a number of mycoviruses, the existence of more diverse viral groups in fungi are currently expected. In this review, we selected four different fungal groups, which were considered as the model systems for mycovirus related studies in both plant pathogenic fungi and edible mushroom species, and discussed about their molecular characteristics of diverse mycoviruses. The plant pathogenic fungi introduced here were Cryphonectria parasitica and Helminthosporium victoriae and the edible mushroom species were Agaricus bisporus and Pleurotus ostreatus.

• Journal of Species Research
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• 제9권4호
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• pp.339-345
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• 2020

The Earth contains billions of microbial species, although the vast majority cannot be cultured in laboratories and are thus considered unidentified and uncharacterized. Extremophiles are microorganisms that thrive in extreme conditions, including temperature, salinity, and pH. Extremophilic microorganisms have provided important insights for biological, metabolic, and evolutionary studies. Between 2017 and 2019, as part of a comprehensive investigation to identify bacterial species in Korea, eight bacterial strains were isolated from marine and non-marine environments in Jeju Island. These strains were cultured under extreme salinity or pH conditions. Phylogenetic analysis using 16S ribosomal RNA(rRNA) gene sequencing indicated that all eight strains belonged to the phyla Gammaproteobacteria, Bacilli, and Alphaproteobacteria. Based on their high 16S rRNA gene sequence similarities(>98.7%) and the formation of strong monophyletic clades with their closest related species, all isolated strains were considered as an unrecorded strain, previously unidentified species. Gram stain reaction, culture conditions, colony and cell morphology, biochemical characteristics, isolation source, and National Institute of Biological Resources(NIBR) IDs are described in this article. The characterization of these unrecorded strains provides information on microorganisms living in Korea.

• 한국식물병리학회지
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• 제10권3호
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• pp.228-234
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• 1994

Genomic RNA was extracted from Cy strain of odontoglossum ringspot tobamovirus (ORSV-Cy) isolated from infected leaves of tobacco cv. Samsun. Size of the genomic RNA was about 6.6 kb in length. The genomic RNA was fractionated using Sephadex G-50 column chromatography into 2 fractions. They were polyadenylated at their 3'-end using E. coli poly(A) polymerase. Polyadenylated viral RNA was recovered by oligo (dT) primer adapter containing NotI restriction site and Moloney murine leukemia virus SuperScript reverse transcriptase (RNase H-). Second-strand cDNA was synthesized by using E. coli DNA ligase, E. coli DNA polymerase I and E. coli RNase H. Recombinant plasmids containing cDNAs for ORSV-Cy RNA ranged from about 800 bp to 3,000 bp. Among the selected 238 recombinants, pORCY-124 clone was the largest one covering 3'-terminal half of the viral RNA. This clone contained two restriction sites for EcoRI and XbaI and one site for AccI, AvaI, BglII, BstXI, HindIII, PstI, and TthIII 1. respectively. The clone contained partial viral replicase, a full-length movement protein and a complete coat protein genes followed by a 3' untranslated region of 414 nucleotides based on restriction mapping and nucleotide sequencing analyses. Clones pORCY-028, -068, -072, -187 and -224 were overlapped with the pORCY-124. Clones pORCY-014 and -095 covered 5' half upstream from the middle region of the viral RNA, which was estimated based on restriction mapping and partial sequence analysis. Constructed cDNA library covered more than 90% of the viral genome.