• International Journal of Stem Cells
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• v.16 no.2
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• pp.145-155
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• 2023

Background and Objectives: Embryologically, mesodermal development is closely related to the development of various organs such as muscles, blood vessels, and hearts, which are the main organs that make up the body. However, treatment for mesoderm developmental disorders caused by congenital or acquired factors has so far relied on surgery and drug treatment for symptom relief, and more fundamentally, treatment for mesoderm developmental disorders is needed. Methods and Results: In our study, microRNA (miRNA), which plays an important role in the mesoderm development process, was identified and the developmental function was evaluated. miRNAs consist of small nucleotides, which act as transcription factors that bind to the 3' untranslated region and suppressed target gene expression. We constructed the human embryonic stem cell (hESC) knockout cell line and analyzed the function and characteristics of miR-5739, which plays an important role in mesoderm lineage. miR-5739 acts as a transcription factor targeting SMA, Brachyury T, Hand1, which controls muscle proliferation and differentiation, and KDR gene, which regulates vessel formation in vitro. In vivo results suggest a role in regulating muscle proliferation and differentiation. Gene ontology analysis confirmed that the miR-5739 is closely related to genes that regulate muscle and vessel proliferation and differentiation. Importantly, abnormal expression of miR-5739 was detected in somatic cells derived from patients with congenital muscle disease. Conclusions: Our study demonstrate that miR-5739 gene function significantly affects transcriptional circuits that regulate muscle and vascular differentiation during embryonic development.

• Animal cells and systems
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• v.3 no.3
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• pp.295-301
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• 1999

The nucleotide sequence of a 447 base pair fragment in the mitochondrial cytochrome b gene and the complete sequence of the mitochondrial 12S ribosomal RNA gene, 938 bp, were analyzed to infer inter- and intraspecific genetic relationships of Hyla japonica and H. suweonensis from Korea and H, japonica from Japan. In the mitochondrial cytochrome b gene, genetic differentiation among H. japonica populations were 9.62% and 15.66% between H. japonica and H. suweonensis. Based on the Tamura-Nei distance, the level of sequence divergence ranged from 0.45% to 2.75% within Korean H. japonica, while 8.31%-8.87% between Korean and Japanese H. japonica and 11.51%-12.46% between H. japonica and H. suweonensis. In the neigh-bor-joining tree, Korean populations of H. japonica were clustered first at 2.22% and followed by Japanese H. japonica and H. suweonensis at 8.51% and 12.29%, respectively. In mitochondrial 12S rRNA gene, genetic differentiation between H. japonica and H. suweonensis nras 7.17% (68 bp) including 7 gaps. Based on Tamura-Nei distance, the level of sequence divergence ranged 3.53% between Korean and Japanese H. japonica and from 4.93% to 5.41% between H. japonica and H. suweonensis. Phenogram pattern of the 12S rRNA gene sequence corresponded with that of the mitochondrial cytochrome b gene.

• Korean journal of applied entomology
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• v.39 no.1
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• pp.5-12
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• 2000

The sweetpotato whiteflies, Bemisia tabaci(Gennadius), were found recently in Korea on Glycine max, Euphorbia pulcherrima, and Rosa hybrida. The biotype identity of Bemisia tabaci in Korea was determined by several DNA markers including the random amplified polymorphic DNAs, and restriction fragments length polymorphism of mitochondrial 12S and 16S rRNA genes. The electromorph profiles of DNA fragments from the rose(Jincheon) and poinsettia(Seoul) populations in Korea are both identical to those of B biotypes distributed in Australia, Israel, and Japan. The populations of B. tabaci collected on Glycine max, Ipomea batatas, and Perilla frutescens in different localities retained the same DNA markes with the population from Lonicera japonica and shikoku of Japan. These populations are non-B biotype and considered as an indigenous type in the Far Eastern Asia Region including Korea and Japan, Morphological Characteristics of B. Tabaci were also observed by the scanning electron microscope and described with the comparison to the other important whitefly pest, Trialeurodes vaporariorum (Westwood).

• International Journal of Oral Biology
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• v.43 no.4
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• pp.223-230
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• 2018

Exosomes are Nano-sized lipid vesicles secreted from mammalian cells containing diverse cellular materials such as proteins, lipids, and nucleotides. Multiple lines of evidence indicate that in saliva, exosomes and their contents such as microRNAs (miRNAs) mediate numerous cellular responses upon delivery to recipient cells. The objective of this study was to characterize the different expression profile of exosomal miRNAs in saliva samples, periodically isolated from a single periodontitis patient. Unstimulated saliva was collected from a single patient over time periods for managing periodontitis. MicroRNAs extracted from each phase were investigated for the expression of exosomal miRNAs. Salivary exosomal miRNAs were analyzed using Affymetrix miRNA arrays and prediction of target genes and pathways for its different expression performed using DIANA-mirPath, a web-based, computational tool. Following the delivery of miRNA mimics (hsa-miR-4487, -4532, and -7108-5p) into human gingival fibroblasts, the expression of pro-inflammatory cytokines and activation of the MAPK pathway were evaluated through RT-PCR and western blotting. In each phase, 13 and 43 miRNAs were found to be differently expressed $({\mid}FC{\mid}{\geq}2)$. Among these, hsa-miR-4487 $({\mid}FC{\mid}=9.292005)$ and has-miR-4532 $({\mid}FC{\mid}=18.322697)$ were highly up-regulated in the clinically severe phase, whereas hsa-miR-7108-5p $({\mid}FC{\mid}=12.20601)$ was strongly up-regulated in the clinically mild phase. In addition, the overexpression of miRNA mimics in human gingival fibroblasts resulted in a significant induction of IL-6 mRNA expression and p38 phosphorylation. The findings of this study established alterations in salivary exosomal miRNAs which are dependent on the severity of periodontitis and may act as potential candidates for the treatment of oral inflammatory diseases.

• Korean Journal of Microbiology
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• v.55 no.3
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• pp.220-225
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• 2019

Studies based on microbial community analyses have increased in the recent decade since the development of next generation sequencing technology. Associations of gut microbiota with host's health are one of the major outcomes of microbial ecology filed. The major approach for microbial community analysis includes the sequencing of variable regions of 16S rRNA genes, which does not provide the information of bacterial activities. Here, we conducted RNA-based microbial community analysis and compared results obtained from DNA- and its cDNA-based microbial community analyses. Our results indicated that these two approaches differed in the ratio of Firmicutes and Bacteroidetes, known as an obesity indicator, as well as abundance of some key bacteria in gut metabolisms such as butyrate producers and probiotics strains. Therefore, cDNA-based microbial community may provide different insights regarding roles of gut microbiota compared to the previous studies where DNA-based microbial community analyses were performed.

• Journal of Microbiology and Biotechnology
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• v.32 no.2
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• pp.149-159
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• 2022

Cancers of the lung and liver are the top 10 leading causes of cancer death worldwide. Thus, it is essential to identify the genes specifically expressed in these two cancer types to develop new therapeutics. Although many messenger RNA (mRNA) sequencing data related to these cancer cells are available due to the advancement of next-generation sequencing (NGS) technologies, optimized data processing methods need to be developed to identify the novel cancer-specific genes. Here, we conducted an analytical comparison between Bowtie2, a Burrows-Wheeler transform-based alignment tool, and Kallisto, which adopts pseudo alignment based on a transcriptome de Bruijn graph using mRNA sequencing data on normal cells and lung/liver cancer tissues. Before using cancer data, simulated mRNA sequencing reads were generated, and the high Transcripts Per Million (TPM) values were compared. mRNA sequencing reads data on lung/liver cancer cells were also extracted and quantified. While Kallisto could directly give the output in TPM values, Bowtie2 provided the counts. Thus, TPM values were calculated by processing the Sequence Alignment Map (SAM) file in R using package Rsubread and subsequently in python. The analysis of the simulated sequencing data revealed that Kallisto could detect more transcripts and had a higher overlap over Bowtie2. The evaluation of these two data processing methods using the known lung cancer biomarkers concludes that in standard settings without any dedicated quality control, Kallisto is more effective at producing faster and more accurate results than Bowtie2. Such conclusions were also drawn and confirmed with the known biomarkers specific to liver cancer.

• Animal Bioscience
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• v.36 no.4
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• pp.570-583
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• 2023

Objective: Fibroblast growth factors (FGFs) play critical roles in embryo development, and immune responses to infectious diseases. In this study, to investigate the roles of FGFs, we performed genome-wide identification, expression, and functional analyses of FGF family members in chickens. Methods: Chicken FGFs genes were identified and analyzed by using bioinformatics approach. Expression profiles and Hierarchical cluster analysis of the FGFs genes in different chicken tissues were obtained from the genome-wide RNA-seq. Results: A total of 20 FGF genes were identified in the chicken genome, which were classified into seven distinct groups (A-F) in the phylogenetic tree. Gene structure analysis revealed that members of the same clade had the same or similar exon-intron structure. Chromosome mapping suggested that FGF genes were widely dispersed across the chicken genome and were located on chromosomes 1, 4-6, 9-10, 13, 15, 28, and Z. In addition, the interactions among FGF proteins and between FGFs and mitogen-activated protein kinase (MAPK) proteins are limited, indicating that the remaining functions of FGF proteins should be further investigated in chickens. Kyoto encyclopedia of genes and genomes pathway analysis showed that FGF gene interacts with MAPK genes and are involved in stimulating signaling pathway and regulating immune responses. Furthermore, this study identified 15 differentially expressed genes (DEG) in 21 different growth stages during early chicken embryo development. RNA-sequencing data identified the DEG of FGFs on 1- and 3-days post infection in two indigenous Ri chicken lines infected with the highly pathogenic avian influenza virus H5N1 (HPAIV). Finally, all the genes examined through quantitative real-time polymerase chain reaction and RNA-Seq analyses showed similar responses to HPAIV infection in indigenous Ri chicken lines (R² = 0.92-0.95, p<0.01). Conclusion: This study provides significant insights into the potential functions of FGFs in chickens, including the regulation of MAPK signaling pathways and the immune response of chickens to HPAIV infections.

• Animal Bioscience
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• v.36 no.6
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• pp.851-860
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• 2023

Objective: This study aims to evaluate the target genes of gga-miR-20a-5p and the regulated immune responses in the chicken macrophage cell line, HD11, by the exosome-mediated delivery of miR-20a-5p. Methods: Exosomes were purified from the chicken macrophage cell line HD11. Then, mimic gga-miR-20p or negative control miRNA were internalized into HD11 exosomes. HD11 cells were transfected with gga-miR-20a-5p or negative control miRNA containing exosomes. After 44 h of transfection, cells were incubated with or without 5 ㎍/mL poly(I:C) for 4 h. Then, expression of target genes and cytokines was evaluated by quantitative realtime polymerase chain reaction. Results: Using a luciferase reporter assay, we identified that gga-miR-20a-5p directly targeted interferon gamma receptor 2 (IFNGR2), mitogen-activated protein kinase 1 (MAPK1), mitogen-activated protein kinase kinase kinase 5 (MAP3K5), and mitogen-activated protein kinase kinase kinase 14 (MAP3K14). Moreover, the exosome-mediated delivery of gga-miR-20a-5p successfully repressed the expression of IFNGR2, MAPK1, MAP3K5, and MAP3K14 in HD11 cells. The expressions of interferon-stimulated genes (MX dynamin like GTPase 1 [MX1], eukaryotic translation initiation factor 2A [EIF2A], and oligoadenylate synthase-like [OASL]) and proinflammatory cytokines (interferon-gamma [IFNG], interleukin-1 beta [IL1B], and tumor necrosis factor-alpha [TNFA]) were also downregulated by exosomal miR-20a-5p. In addition, the proliferation of HD11 cells was increased by exosomal miR-20a-5p. Conclusion: The exosome-mediated delivery of gga-miR-20a-5p regulated immune responses by controlling the MAPK and apoptotic signaling pathways. Furthermore, we expected that exosomal miR-20a-5p could maintain immune homeostasis against highly pathogenic avian influenza virus H5N1 infection by regulating the expression of proinflammatory cytokines and cell death.

• Parasites, Hosts and Diseases
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• v.54 no.1
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• pp.39-46
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• 2016

Theileria annulata is a tick-borne intracellular protozoan parasite that causes tropical theileriosis, a fatal bovine lymphoproliferative disease. The parasite predominantly invades bovine B lymphocytes and macrophages and induces host cell transformation by a mechanism that is not fully comprehended. Analysis of signaling pathways by quantitative real-time PCR (qPCR) could be a highly efficient means to understand this transformation mechanism. However, accurate analysis of qPCR data relies on selection of appropriate reference genes for normalization, yet few papers on T. annulata contain evidence of reference gene validation. We therefore used the geNorm and NormFinder programs to evaluate the stability of 5 candidate reference genes; 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ACTB (${\beta}-actin$), PRKG1 (protein kinase cGMP-dependent, type I) and TATA box binding protein (TBP). The results showed that 18S rRNA was the reference gene most stably expressed in bovine PBMCs transformed and non-transformed with T. annulata, followed by GAPDH and TBP. While 18S rRNA and GAPDH were the best combination, these 2 genes were chosen as references to study signaling pathways involved in the transformation mechanism of T. annulata.

• Journal of Microbiology and Biotechnology
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• v.18 no.7
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• pp.1290-1297
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• 2008

To investigate the diversities of Accumulibacter phosphatis and its polyhydroxyalkanoate (PHA) synthase gene (phaC) in enhanced biological phosphorus removal (EBPR) sludge, an acetate-fed sequencing batch reactor was operated. Analysis of microbial communities using fluorescence in situ hybridization and 16S rRNA gene clone libraries showed that the population of Accumulibacter phosphatis in the EBPR sludge comprised more than 50% of total bacteria, and was clearly divided into two subgroups with about 97.5% sequence identity of the 16S rRNA genes. PAO phaC primers targeting the phaC genes of Accumulibacter phosphatis were designed and applied to retrieve fragments of putative phaC homologs of Accumulibacter phosphatis from EBPR sludge. PAO phaC primers targeting $G_{1PAO},\;G_{2PAO},\;and\;G_{3PAO}$ groups produced PCR amplicons successfully; the resulting sequences of the phaC gene homologs were diverse, and were distantly related to metagenomic phaC sequences of Accumulibacter phosphatis with 75-98% DNA sequence identities. Degenerate NPAO (non-PAO) phaC primers targeting phaC genes of non-Accumulibacter phosphatis bacteria were also designed and applied to the EBPR sludge. Twenty-four phaC homologs retrieved from NPAO phaC primers were different from the phaC gene homologs derived from Accumulibacter phosphatis, which suggests that the PAO phaC primers were specific for the amplification of phaC gene homologs of Accumulibacter phosphatis, and the putative phaC gene homologs by PAO phaC primers were derived from Accumulibacter phosphatis in the EBPR sludge. Among 24 phaC homologs, a phaC homolog (GINPAO-2), which was dominant in the NPAO phaC clone library, showed the strongest signal in slot hybridization and shared approximately 60% nucleotide identity with the $G_{4PAO}$ group of Accumulibacter phosphatis, which suggests that GINPAO-2 might be derived from Accumulibacter phosphatis. In conclusion, analyses of the 16S rRNA and phaC genes showed that Accumulibacter phosphatis might be phylogenetically and metabolically diverse.