• Laboraroty Animal Research
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• v.34 no.4
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• pp.279-287
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• 2018

Placenta specific 8 (PLAC8, also known as ONZIN) is a multi-functional protein that is highly expressed in the intestine, lung, spleen, and innate immune cells, and is involved in various diseases, including cancers, obesity, and innate immune deficiency. Here, we generated a Plac8 knockout mouse using the CRISPR/Cas9 system. The Cas9 mRNA and two single guide RNAs targeting a region near the translation start codon at Plac8 exon 2 were microinjected into mouse zygotes. This successfully eliminated the conventional translation start site, as confirmed by Sanger sequencing and PCR genotyping analysis. Unlike the previous Plac8 deficient models displaying increased adipose tissue and body weights, our male Plac8 knockout mice showed rather lower body weight than sex-matched littermate controls, though the only difference between these two mouse models is genetic context. Differently from the previously constructed embryonic stem cell-derived Plac8 knockout mouse that contains a neomycin resistance cassette, this knockout mouse model is free from a negative selection marker or other external insertions, which will be useful in future studies aimed at elucidating the multi-functional and physiological roles of PLAC8 in various diseases, without interference from exogenous foreign DNA.

• The Korean Journal of Physiology and Pharmacology
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• v.26 no.6
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• pp.519-530
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• 2022

Recent research indicates that lactate promotes the switching of vascular smooth muscle cells (VSMCs) to a synthetic phenotype, which has been implicated in various vascular diseases. This study aimed to investigate the effects of lactate on the VSMC phenotype switch and the underlying mechanism. The CCK-8 method was used to assess cell viability. The microRNAs and mRNAs levels were evaluated using quantitative PCR. Targets of microRNA were predicted using online tools and confirmed using a luciferase reporter assay. We found that lactate promoted the switch of VSMCs to a synthetic phenotype, as evidenced by an increase in VSMC proliferation, mitochondrial activity, migration, and synthesis but a decrease in VSMC apoptosis. Lactate inhibited miR-23b expression in VSMCs, and miR-23b inhibited VSMC's switch to the synthetic phenotype. Lactate modulated the VSMC phenotype through downregulation of miR-23b expression, suggesting that overexpression of miR-23b using a miR-23b mimic attenuated the effects of lactate on VSMC phenotype modulation. Moreover, we discovered that SMAD family member 3 (SMAD3) was the target of miR-23b in regulating VSMC phenotype. Further findings suggested that lactate promotes VSMC switch to synthetic phenotype by targeting SMAD3 and downregulating miR-23b. These findings suggest that correcting the dysregulation of miR-23b/SMAD3 or lactate metabolism is a potential treatment for vascular diseases.

• Parasites, Hosts and Diseases
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• v.60 no.2
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• pp.127-131
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• 2022

Feline hemotropic mycoplasmosis (hemoplasmosis) is an infection of the red blood cells caused by the Mycoplasma haemofelis (Mhf), Candidatus Mycoplasma haemominutum (CMhm), and Candidatus Mycoplasma turicensis (CMt). The existence of Mhf, CMhm, and CMt has been demonstrated in feral cats in Korea using molecular methods, but no clinical cases have yet been reported. This study reports 2 clinical cases of hemotropic mycoplasmosis caused by CMhm and CMt in 2 anemic cats. The first case was a client-owned intact female domestic shorthair cat that presented with fever, pale mucous membranes, and normocytic normochromic non-regenerative anemia. Prior to referral, an immunosuppressive prednisolone dose was administered at the local veterinary clinic for 1 month. The cat was diagnosed with high-grade alimentary lymphoma. Organisms were found on the surface of the red blood cells on blood smear examination. The second case was of a rescued cat that presented with dehydration and fever. The cat had normocytic normochromic non-regenerative anemia. Necropsy revealed concurrent feline infectious peritonitis. Polymerase chain reaction assay targeting 16S rRNA revealed CMhm infection in case 1 and dual infection of CMhm and CMt in case 2. Normocytic normochromic non-regenerative anemia was observed in both cats before and during the management of the systemic inflammation. This is the first clinical case report in Korea to demonstrate CMhm and CMt infections in symptomatic cats.

• Parasites, Hosts and Diseases
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• v.60 no.2
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• pp.143-147
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• 2022

Acanthamoeba keratitis (AK) is a rare ocular disease, but it is a painful and sight-threatening infectious disease. Early diagnosis and adequate treatment are necessary to prevent serious complications. While AK is frequently diagnosis via several PCR assays or Acanthamoeba-specific antibodies, a more specific and effective diagnostic method is required. This study described the production of a polyclonal peptide antibody against the periplasmic binding protein (PBP) of A. castellanii and investigated its diagnostic potential. Western blot analysis showed that the PBP antibody specifically reacted with the cell lysates of A. castellanii. However, the PBP antibody did not interact with human corneal epithelial (HCE) cells and the other 3 major causative agents of keratitis. Immunocytochemistry (ICC) results revealed the specific detection of A. castellanii trophozoites and cysts by PBP antibodies when A. castellanii were co-cultured with HCE cells. PBP antibody specificity was further confirmed by co-culture of A. castellanii trophozoites with F. solani, S. aureus, and P. aeruginosa via ICC. The PBP antibody specifically reacted with the trophozoites and cysts of A. polyphaga, A. hatchetti, A. culbertsoni, A. royreba, and A. healyi, thus demonstrated its genus-specific nature. These results showed that the PBP polyclonal peptide antibody of A. castellanii could specifically detect several species of Acanthamoeba, contributing to the development of an effective antibody-based AK diagnostics.

• Mycobiology
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• v.50 no.1
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• pp.86-95
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• 2022

Inflammaging in male reproductive organs covers a wide variety of problems, including sexual dysfunction and infertility. In this study, the beneficial effects of cordycepin (COR), isolated from potential medicinal fungi Cordyceps militaris, in aging-associated testicular inflammation and serum biochemical changes in naturally aged rats were investigated. Male Sprague Dawley rats were divided into young control (YC), aged control (AC), and COR (5, 10, and 20 mg/kg) treated aged rat groups. Aging-associated serum biochemical changes and inflammatory parameters were analyzed by biochemical assay kits, Western blotting, and real-time RT-PCR. Results showed a significant (p < 0.05) alteration in the total blood cell count, lipid metabolism, and liver functional parameters in AC group when compared with YC group. However, COR-treated aged rats ameliorated the altered biochemical parameters significantly (p < 0.05 and p < 0.01 at 5, 10, and 20 mg/kg, respectively). Furthermore, the increase in the expression of inflammatory mediators (COX-2, interleukin (IL)-6, IL-1β, and tissue necrosis factor-alpha) in aged rat testis was significant (p < 0.05) when compared with YC group. Treatment with COR at 20 mg/kg to aged rats attenuated the increased expression of inflammatory mediators significantly (p < 0.05). Mechanistic studies revealed that the potential attenuating effects exhibited by COR in aged rats was mediated by regulation of NF-κB activation and MAPKs (c-Jun N-terminal kinase, extracellular signal-regulated kinase 1/2, and p38) signaling. In conclusion, COR restored the altered serum biochemical parameters in aged rats and ameliorated the aging-associated testicular inflammation proving the therapeutic benefits of COR targeting inflammaging-associated male sexual dysfunctions.

• The Korean Journal of Physiology and Pharmacology
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• v.27 no.3
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• pp.197-208
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• 2023

Dysregulation of certain long non-coding RNAs may facilitate tumor initiation and progression. However, numerous carcinogenesis-related long noncoding RNAs have not been characterized. The goal of this study was to elucidate the role of LINC00562 in gastric cancer (GC). The expression of LINC00562 was analyzed using real-time quantitative PCR and Western blotting. The proliferative capacity of GC cells was determined using Cell Counting Kit-8 and colony-formation assays. The migration of GC cells were evaluated using wound-healing assays. The apoptosis of GC cells was assessed by measuring the expression levels of apoptosis-related proteins (Bax and Bcl-2). Xenograft models in nude mice were constructed for in vivo functional analysis of LINC00562. The binding relationship between miR-4636 and LINC00562 or adaptor protein complex 1 sigma 3 (AP1S3), obtained from public databases, was confirmed using dual-luciferase and RNA-binding protein immunoprecipitation experiments. LINC00562 was expressed in GC cells at high levels. Knockdown of LINC00562 repressed GC cell growth and migration, promoted apoptosis in vitro, and inhibited tumor growth in nude mouse models. LINC00562 directly targeted miR-4636, and miR-4636 depletion restored the GC cell behavior inhibited by LINC00562 absence. AP1S3, an oncogene, binds to miR-4636. MiR-4636 downregulation increased AP1S3 level, restoring GC cell malignant behaviors inhibited by AP1S3 downregulation. Thus, LINC00562 exerts carcinogenic effects on GC development by targeting miR-4636-mediated AP1S3 signaling.

• The Plant Pathology Journal
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• v.39 no.5
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• pp.486-493
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• 2023

Cowpea mild mottle virus (CPMMV) is a global plant virus that poses a threat to the production and quality of legume crops. Early and accurate diagnosis is essential for effective managing CPMMV outbreaks. With the advancement in isothermal recombinase polymerase amplification and lateral flow strips technologies, more rapid and sensitive methods have become available for detecting this pathogen. In this study, we have developed a reverse transcription recombinase polymerase amplification combined with lateral flow strips (RT-RPA-LFS) method for the detection of CPMMV, specifically targeting the CPMMV coat protein (CP) gene. The RT-RPA-LFS assay only requires 20 min at 40℃ and demonstrates high specificity. Its detection limit was 10 copies/µl, which is approximately up to 100 times more sensitive than RT-PCR on agarose gel electrophoresis. The developed RT-RPA-LFS method offers a rapid, convenient, and sensitive approach for field detection of CPMMV, which contribute to controlling the spread of the virus.

• The Plant Pathology Journal
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• v.39 no.5
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• pp.494-503
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• 2023

Xanthomonas campestris pv. campestris (Xcc) is a plant pathogen of Brassica crops that causes black rot disease throughout the world. At present, 11 physiological races of Xcc (races 1-11) have been reported. The conventional method of using differential cultivars for Xcc race detection is not accurate and it is laborious and time-consuming. Therefore, the development of specific molecular markers has been used as a substitute tool because it offers an accurate and reliable result, particularly a quick diagnosis of Xcc races. Previously, our laboratory has successfully developed race-specific molecular markers for Xcc races 1-6. In this study, specific molecular markers to identify Xcc race 7 have been developed. In the course of study, whole genome sequences of several Xcc races, X. campestris pv. incanae, X. campestris pv. raphani, and X. campestris pv. vesicatoria were aligned to identify variable regions like sequence-characterized amplified regions and insertions and deletions specific to race 7. Primer pairs were designed targeting these regions and validated against 22 samples. The polymerase chain reaction analysis revealed that three primer pairs specifically amplified the DNA fragment corresponding to race 7. The obtained finding clearly demonstrates the efficiency of the newly developed markers in accurately detecting Xcc race 7 among the other races. These results indicated that the newly developed marker can successfully and rapidly detect Xcc race 7 from other races. This study represents the first report on the successful development of specific molecular markers for Xcc race 7.

• Journal of Animal Reproduction and Biotechnology
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• v.38 no.3
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• pp.99-108
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• 2023

Background: Efficient gene editing technology is needed for successful knock-in. Homologous recombination (HR) is a major double-strand break repair pathway that can be utilized for accurately inserting foreign genes into the genome. HR occurs during the S/G2 phase, and the DNA mismatch repair (MMR) pathway is inextricably linked to HR to maintain HR fidelity. This study was conducted to investigate the effect of inhibiting MMR-related genes using CdCl₂, an MMR-related gene inhibitor, on HR efficiency in HC11 cells. Methods: The mRNA and protein expression levels of MMR-related genes (Msh2, Msh3, Msh6, Mlh1, Pms2), the HR-related gene Rad51, and the NHEJ-related gene DNA Ligase IV were assessed in HC11 cells treated with 10 μM of CdCl₂ for 48 hours. In addition, HC11 cells were transfected with a CRISPR/sgRNA expression vector and a knock-in vector targeting Exon3 of the mouse-beta casein locus, and treated with 10 μM cadmium for 48 hours. The knock-in efficiency was monitored through PCR. Results: The treatment of HC11 cells with a high-dose of CdCl₂ decreased the mRNA expression of the HR-related gene Rad51 in HC11 cells. In addition, the inhibition of MMR-related genes through CdCl₂ treatment did not lead to an increase in knock-in efficiency. Conclusions: The inhibition of MMR-related gene expression through high-dose CdCl₂ treatment reduces the expression of the HR-related gene Rad51, which is active during recombination. Therefore, it was determined that CdCl₂ is an inappropriate compound for improving HR efficiency.

• Journal of Veterinary Science
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• v.25 no.3
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• pp.43.1-43.13
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• 2024

Importance: Haemaphysalis longicornis is an obligate blood-sucking ectoparasite that has gained attention due its role of transmitting medically and veterinary significant pathogens and it is the most common tick species in Republic of Korea. The preferred strategy for controlling ticks is a multi-antigenic vaccination. Testing the efficiency of a combination antigen is a promising method for creating a tick vaccine. Objective: The aim of the current research was to analyze the role of subolesin and enolase in feeding and reproduction of H. longicornis by gene silencing. Methods: In this study, we used RNA interference to silence salivary enolase and subolesin in H. longicornis. Unfed female ticks injected with double-stranded RNA targeting subolesin and enolase were attached and fed normally on the rabbit's ear. Real-time polymerase chain reaction was used to confirm the extent of knockdown. Results: Ticks in the subolesin or enolase dsRNA groups showed knockdown rates of 80% and 60% respectively. Ticks in the combination dsRNA (subolesin and enolase) group showed an 80% knockdown. Knockdown of subolesin and enolase resulted in significant depletion in feeding, blood engorgement weight, attachment rate, and egg laying. Silencing of both resulted in a significant (p < 0.05) reduction in tick engorgement, egg laying, egg hatching (15%), and reproduction. Conclusions and Relevance: Our results suggest that subolesin and enolase are an exciting target for future tick control strategies.