• Molecules and Cells
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• v.41 no.10
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• pp.917-922
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• 2018

The CRISPR-Cas system is a well-established RNA-guided DNA editing technique widely used to modify genomic DNA sequences. I used the CRISPR-Cas9 system to change the second and third nucleotides of the triplet $T{\underline{CT}}$ of human TNSFSF9 in HepG2 cells to $T{\underline{AG}}$ to create an amber stop codon. The $T{\underline{CT}}$ triplet is the codon for Ser at the $172^{nd}$ position of TNSFSF9. The two substituted nucleotides, AG, were confirmed by DNA sequencing of the PCR product followed by PCR amplification of the genomic TNFSF9 gene. Interestingly, sequencing of the cDNA of transcripts of the edited TNFSF9 gene revealed that the $T{\underline{AG}}$ had been re-edited to the wild type triplet $T{\underline{CT}}$, and 1 or 2 bases just before the triplet had been deleted. These observations indicate that CRISPR-Cas9-mediated editing of bases in target genomic DNA can be followed by spontaneous re-editing (correcting) of the bases during transcription.

• 한국균학회소식:학술대회논문집
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• 2016.05a
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• pp.17-17
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• 2016

Calcineurin governs stress survival, sexual differentiation, and virulence of the human fungal pathogen Cryptococcus neoformans. Herein, we identified and characterized calcineurin substrates in C. neoformans by employing phosphoproteomic $TiO_2$ enrichment and quantitative mass spectrometry. The identified targets include the zinc finger transcription factor Crz1 and proteins whose functions are linked to P-bodies/stress granules (PBs/SGs) and mRNA translation and decay, such as Pbp1 and Puf4. We show that Crz1 is a bona fide calcineurin substrate, and localization and transcriptional activity of Crz1 are controlled by calcineurin. Several of the calcineurin targets localized to PBs/SGs, including Puf4 and Pbp1, and are required for survival at high temperature and for virulence. Genetic epistasis analysis revealed that Crz1 and the novel targets Lhp1, Puf4, and Pbp1 function in a branched calcineurin pathway that orchestrates stress survival and virulence. These findings propose that calcineurin controls thermal stress and virulence at the transcriptional level via Crz1 and post-transcriptionally by regulating target factors involved in mRNA metabolism.

• Development and Reproduction
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• v.27 no.1
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• pp.39-46
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• 2023

Pumilio (Pum) is an RNA-binding protein and translational repressor important to diverse biological processes. In the Drosophila ovary, Pum is expressed in female germline stem cells (GSCs), wherein it acts as an intrinsic stem cell maintenance factor via repressing target mRNAs that are as yet mostly unknown. Pum recognizes the Pum binding sequence (PBS) in the mRNA 3'UTR through its C-terminus Puf domain. Translational repression is mediated through its N-terminal domain, but the molecular mechanism remains largely unknown. We previously showed that Bag-of-marbles, a critical differentiation-promoting factor of female GSCs, physically interacts with the N-terminus of Pum. We further showed that this interaction is critical to Bam inhibition of Pum repressive action in cultured cells, but the physiological relevance was not addressed. Here we design an in vivo GFP translational reporter bearing the PBS in its 3'UTR and show that GFP expression is reduced in cells wherein Pum is known to be active. Furthermore, we demonstrate in pum mutant ovary that this GFP repression requires Pum, and also that the sensor faithfully monitors Pum activity. Finally, we show that forced expression of Bam inhibits Pum-mediated repression, validating that Bam inhibits Pum activity in vivo.

• Asian-Australasian Journal of Animal Sciences
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• v.33 no.11
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• pp.1824-1836
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• 2020

Objective: On the hypothesis that grazing of cattle prompts organs to secrete or internalize circulating microRNAs (c-miRNAs) in parallel with changes in energy metabolism, we aimed to clarify biological events in adipose, skeletal muscle, and liver tissues in grazing Japanese Shorthorn (JSH) steers by a transcriptomic approach. Methods: The subcutaneous fat (SCF), biceps femoris muscle (BFM), and liver in JSH steers after three months of grazing or housing were analyzed using microarray and quantitative polymerase chain reaction (qPCR), followed by gene ontology (GO) and functional annotation analyses. Results: The results of transcriptomics indicated that SCF was highly responsive to grazing compared to BFM and liver tissues. The 'Exosome', 'Carbohydrate metabolism' and 'Lipid metabolism' were extracted as the relevant GO terms in SCF and BFM, and/or liver from the >1.5-fold-altered mRNAs in grazing steers. The qPCR analyses showed a trend of upregulated gene expression related to exosome secretion and internalization (charged multivesicular body protein 4A, vacuolar protein sorting-associated protein 4B, vesicle associated membrane protein 7, caveolin 1) in the BFM and SCF, as well as upregulation of lipolysis-associated mRNAs (carnitine palmitoyltransferase 1A, hormone-sensitive lipase, perilipin 1, adipose triglyceride lipase, fatty acid binding protein 4) and most of the microRNAs (miRNAs) in SCF. Moreover, gene expression related to fatty acid uptake and inter-organ signaling (solute carrier family 27 member 4 and angiopoietin-like 4) was upregulated in BFM, suggesting activation of SCF-BFM organ crosstalk for energy metabolism. Meanwhile, expression of plasma exosomal miR-16a, miR-19b, miR-21-5p, and miR-142-5p was reduced. According to bioinformatic analyses, the c-miRNA target genes are associated with the terms 'Endosome', 'Caveola', 'Endocytosis', 'Carbohydrate metabolism', and with pathways related to environmental information processing and the endocrine system. Conclusion: Exosome and fatty acid metabolism-related gene expression was altered in SCF of grazing cattle, which could be regulated by miRNA such as miR-142-5p. These changes occurred coordinately in both the SCF and BFM, suggesting involvement of exosome in the SCF-BFM organ crosstalk to modulate energy metabolism.

• The Journal of Korean Medicine Ophthalmology and Otolaryngology and Dermatology
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• v.36 no.4
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• pp.30-50
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• 2023

Objectives : To investigate the active compounds and therapeutic mechanisms of Atractylodes Lancea(Thunb.) D.C. and Magnolia Officinalis Rehder et Wilson in the treatment of dermatitis accompanied by pruritus, as well as their potential to complement or replace standard drugs. Methods : We conducted the network pharmacological analysis. We selected effective ingredients among the active compounds of research target herbs. Then we explore pathway/terms of the common target proteins among research target herbs, fexofenadine and disease. Results : We selected 9 active compounds are selected from Atractylodes lancea and identified 231 target proteins. Among them, 74 proteins are associated with inflammatory skin diseases that cause pruritus. These proteins are involved in various pathways including, 'Nitric-oxide synthase regulator activity', 'Hydroperoxy icosatetraenoate dehydratase activity, Aromatase activity', 'RNA-directed DNA polymerase activity', 'Arachidonic acid metabolism', 'Peptide hormone processing', 'Chemokine binding' and 'Sterol biosynthetic process'. Additionally, coregenes are involved in 'IL-17 signaling pathway'. Similarly, we selected 2 active compounds from Magnolia officinalis and identified 133 target proteins. Among them, 33 proteins are related to inflammatory skin diseases that cause pruritus. These proteins are primarily involved in 'Vascular associated smooth muscle cell proliferation' and 'Arachidonic acid metabolism'. There is no significant difference between the pathways in which coregenes are involved. Conclusions : It is expected that Atractylodes Lancea will be able to show direct or indirect anti-pruritus and anti-inflammatory effects on skin inflammation accompanied pruritus through suppressing inflammation and protecting skin barrier. Meanwhile, it is expected that Magnolia Officinalis will only be able to show indirect anti-inflammation effects. Therefore, Atractylodes Lancea and fexofenadine are believed to complement each other, whereas Magnolia Officialinalis is expected to provide supplementary support on skin disease.

• BMB Reports
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• v.55 no.2
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• pp.104-109
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• 2022

Skeletal myogenesis is essential to keep muscle mass and integrity, and impaired myogenesis is closely related to the etiology of muscle wasting. Recently, miR-141-3p has been shown to be induced under various conditions associated with muscle wasting, such as aging, oxidative stress, and mitochondrial dysfunction. However, the functional significance and mechanism of miR-141-3p in myogenic differentiation have not been explored to date. In this study, we investigated the roles of miR-141-3p on CFL2 expression, proliferation, and myogenic differentiation in C2C12 myoblasts. MiR-141-3p appeared to target the 3'UTR of CFL2 directly and suppressed the expression of CFL2, an essential factor for actin filament (F-actin) dynamics. Transfection of miR-141-3p mimic in myoblasts increased F-actin formation and augmented nuclear Yes-associated protein (YAP), a key component of mechanotransduction. Furthermore, miR-141-3p mimic increased myoblast proliferation and promoted cell cycle progression throughout the S and G2/M phases. Consequently, miR-141-3p mimic led to significant suppressions of myogenic factors expression, such as MyoD, MyoG, and MyHC, and hindered the myogenic differentiation of myoblasts. Thus, this study reveals the crucial role of miR-141-3p in myogenic differentiation via CFL2-YAP-mediated mechanotransduction and provides implications of miRNA-mediated myogenic regulation in skeletal muscle homeostasis.

• Biomedical Science Letters
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• v.10 no.2
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• pp.65-73
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• 2004

I report that single-stranded antisense as a part of large circular (LC-) genomic DNA of recombinant M13 phage exhibits enhanced stability, sequence specific antisense activity, and no need for target site search. A cDNA fragment (708 bp) of rat TNF-$\alpha$ was inserted into a phagemid vector, and TNF-$\alpha$ antisense molecules (TNF$\alpha$-LCAS) were produced as single-stranded circular DNA. When introduced into a rat monocyte/macrophage cell line, WRT7/P2, TNF$\alpha$-LCAS was able to ablate LPS-induced TNF-$\alpha$ mRNA to completion. The antisense effect of TNF$\alpha$-LCAS was shown to be sequence-specific because expressions of three control genes ($\beta$-actin, GAPDH and IL-1$\beta$) were not significantly altered by the antisense treatment. Further, TNF$\alpha$-LCAS was found to be highly efficacious as only 0.1 $\mu$g (0.24 nM) of TNF$\alpha$-LCAS was sufficient to block TNF-$\alpha$ expression in 1$\times10^5$ WRT7/P2 cells. I have also observed specific antisense activity in reduction of NF-$\kappa$B gene expression. The results suggest that an antisense sequence as a part of single-stranded circular genomic DNA has a specific antisense activity.

• Biomedical Science Letters
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• v.14 no.3
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• pp.139-146
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• 2008

The peroxisome proliferator-activated receptor ${\gamma}$ $(PPAR{\gamma})$ plays a central role in adipogenesis and lipid storage. The $(PPAR{\gamma})$ ligands, thiazolidinediones (TZDs), enhance in vitro adipogenesis in several cell types, but the role of the TZDs on in vivo adipogenesis is still poorly understood. To investigate how $PPAR{\gamma}$ ligand troglitazone regulates adipogenesis in female mice, we examined the effects of the troglitazone on adipose tissue mass, morphological changes of adipocytes, and the expression of $PPAR{\gamma}$ target and adipocyte-specific genes in low fat diet-fed female C57BL/6 mice. Administration of troglitazone for 13 weeks did not change body and total white adipose tissue weights compared with control mice. Troglitazone treatment also did not cause a significant decrease in the average size of adipocytes in parametrial adipose tissue although it is reported to increase the number of small adipocytes in male animals. Troglitazone did not affect the mRNA expression of $PPAR{\gamma}$ and its target genes as well as adipocyte-specific genes in parametrial adipose tissue. These results suggest that $PPAR{\gamma}$ does not seem to be associated with adipogenesis in females with functioning ovaries and that its inability to induce adipogenesis may be due to sex-related factors.

• The Plant Pathology Journal
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• v.34 no.6
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• pp.575-579
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• 2018

Apple stem grooving virus (ASGV) is considered to cause the most economically important viral disease in pears in Korea. The current PCR-based methods used to diagnose ASGV are time-consuming in terms of target detection. In this study, a novel assay for specific ASGV detection that is based on reverse transcription-recombinase polymerase amplification is described. This assay has been shown to be reproducible and able to detect as little as $4.7ng/{\mu}l$ of purified RNA obtained from an ASGV-infected plant. The major advantage of this assay is that the reaction for the target virus is completed in 1 min, and amplification only requires an incubation temperature of $42^{\circ}C$. This assay is a promising alternative method for pear breeding programs or virus-free certification laboratories.

• Journal of Microbiology and Biotechnology
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• v.22 no.7
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• pp.1021-1028
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• 2012

In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid, sensitive, and inexpensive detection of nervous necrosis virus (NNV) in olive flounder, Paralichthys olivaceus, in Korea. A set of six specific primers was designed to target the RNA 2 gene encoding the coat protein of Korean NNV strains. The RT-LAMP reaction successfully detected NNV after 30 min at $65^{\circ}C$. When the sensitivities among RT-LAMP, RT-PCR, and nested RTPCR were compared, the RT-LAMP was shown to be able to detect the RNA template at $2.58{\times}10^{-2}\;TCID_{50}/ml$, whereas the RT-PCR and nested RT-PCR were only able to detect the RNA template at $2.58{\times}10^2\;TCID_{50}/ml$ and $2.58TCID_{50}/ml$, respectively. Thus, the sensitivity of the RT-LAMP assay was higher than those of the RT-PCR assays. In the specificity test of the RT-LAMP, 2 genotypes of NNVs (SJNNV and RGNNV) were positive; however, no other fish viruses were positive with the primers, indicating that the RT-LAMP assay is only specific to NNV. A total of 102 olive flounder were collected from hatcheries between 2009 and 2011. The occurrence of NNV in olive flounder was determined to be 53.9% (55/102) by the RT-LAMP. On the other hand, the prevalence based on the nested RT-PCR and RT-PCR results was 33.8% (34/102) and 20.6% (21/102), respectively. This result indicates that the RT-LAMP assay developed in this study is suitable for early field diagnosis of NNV with high sensitivity.