• The Plant Pathology Journal
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• 제38권6호
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• pp.656-664
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• 2022

Pectobacterium odoriferum is the primary causative agent in Kimchi cabbage soft-rot diseases. The pathogenic bacteria Pectobacterium genera are responsible for significant yield losses in crops. However, P. odoriferum shares a vast range of hosts with P. carotovorum, P. versatile, and P. brasiliense, and has similar biochemical, phenotypic, and genetic characteristics to these species. Therefore, it is essential to develop a P. odoriferumspecific diagnostic method for soft-rot disease because of the complicated diagnostic process and management as described above. Therefore, in this study, to select P. odoriferum-specific genes, species-specific genes were selected using the data of the P. odoriferum JK2.1 whole genome and similar bacterial species registered with NCBI. Thereafter, the specificity of the selected gene was tested through blast analysis. We identified novel species-specific genes to detect and quantify targeted P. odoriferum and designed specific primer sets targeting HAD family hydrolases. It was confirmed that the selected primer set formed a specific amplicon of 360 bp only in the DNA of P. odoriferum using 29 Pectobacterium species and related species. Furthermore, the population density of P. odoriferum can be estimated without genomic DNA extraction through SYBR Green-based real-time quantitative PCR using a primer set in plants. As a result, the newly developed diagnostic method enables rapid and accurate diagnosis and continuous monitoring of soft-rot disease in Kimchi cabbage without additional procedures from the plant tissue.

• Molecules and Cells
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• 제45권8호
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• pp.550-563
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• 2022

Hepatocellular carcinoma (HCC) is an aggressive and incurable cancer. Although understanding of the molecular pathogenesis of HCC has greatly advanced, therapeutic options for the disease remain limited. In this study, we demonstrated that SETD5 expression is positively associated with poor prognosis of HCC and that SETD5 depletion decreased HCC cell proliferation and invasion while inducing cell death. Transcriptome analysis revealed that SETD5 loss downregulated the interferon-mediated inflammatory response in HCC cells. In addition, SETD5 depletion downregulated the expression of a critical glycolysis gene, PKM (pyruvate kinase M1/2), and decreased glycolysis activity in HCC cells. Finally, SETD5 knockdown inhibited tumor growth in xenograft mouse models. These results collectively suggest that SETD5 is involved in the tumorigenic features of HCC cells and that targeting SETD5 may suppress HCC progression.

• The Plant Pathology Journal
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• 제39권5호
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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.

• Journal of Microbiology and Biotechnology
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• 제33권3호
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• pp.329-338
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• 2023

Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen that produces attaching and effacing lesions on the large intestine and causes hemorrhagic colitis. It is primarily transmitted through the consumption of contaminated meat or fresh produce. Similar to other bacterial pathogens, antibiotic resistance is of concern for EHEC. Furthermore, since the production of Shiga toxin by this pathogen is enhanced after antibiotic treatment, alternative agents that control EHEC are necessary. This study aimed to discover alternative treatments that target virulence factors and reduce EHEC toxicity. The locus of enterocyte effacement (LEE) is essential for EHEC attachment to host cells and virulence, and most of the LEE genes are positively regulated by the transcriptional regulator, Ler. GrlA protein, a transcriptional activator of ler, is thus a potential target for virulence inhibitors of EHEC. To identify the GrlA inhibitors, an in vivo high-throughput screening (HTS) system consisting of a GrlA-expressing plasmid and a reporter plasmid was constructed. Since the reporter luminescence gene was fused to the ler promoter, the bioluminescence would decrease if inhibitors affected the GrlA. By screening 8,201 compounds from the Korea Chemical Bank, we identified a novel GrlA inhibitor named Grlactin [3-[(2,4-dichlorophenoxy)methyl]-4-(3-methylbut-2-en-1-yl)-4,5-dihydro-1,2,4-oxadiazol-5-one], which suppresses the expression of LEE genes. Grlactin significantly diminished the adhesion of EHEC strain EDL933 to human epithelial cells without inhibiting bacterial growth. These findings suggest that the developed screening system was effective at identifying GrlA inhibitors, and Grlactin has potential for use as a novel anti-adhesion agent for EHEC while reducing the incidence of resistance.

• Journal of Veterinary Science
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• 제24권3호
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• pp.38.1-38.12
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• 2023

Background: Poor disease management and irregular vector control could predispose sheltered animals to disease such as feline Bartonella infection, a vector-borne zoonotic disease primarily caused by Bartonella henselae. Objectives: This study investigated the status of Bartonella infection in cats from eight (n = 8) shelters by molecular and serological approaches, profiling the CD4:CD8 ratio and the risk factors associated with Bartonella infection in shelter cats. Methods: Bartonella deoxyribonucleic acid (DNA) was detected through polymerase chain reaction (PCR) targeting 16S-23S rRNA internal transcribed spacer gene, followed by DNA sequencing. Bartonella IgM and IgG antibody titre, CD4 and CD8 profiles were detected using indirect immunofluorescence assay and flow cytometric analysis, respectively. Results: B. henselae was detected through PCR and sequencing in 1.0% (1/101) oral swab and 2.0% (1/50) cat fleas, while another 3/50 cat fleas carried B. clarridgeiae. Only 18/101 cats were seronegative against B. henselae, whereas 30.7% (31/101) cats were positive for both IgM and IgG, 8% (18/101) cats had IgM, and 33.7% (34/101) cats had IgG antibody only. None of the eight shelters sampled had Bartonella antibody-free cats. Although abnormal CD4:CD8 ratio was observed in 48/83 seropositive cats, flea infestation was the only significant risk factor observed in this study. Conclusions: The present study provides the first comparison on the Bartonella spp. antigen, antibody status and CD4:CD8 ratio among shelter cats. The high B. henselae seropositivity among shelter cats presumably due to significant flea infestation triggers an alarm of whether the infection could go undetectable and its potential transmission to humans.

• BMB Reports
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• 제56권11호
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• pp.600-605
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• 2023

Intrahepatic cholangiocarcinoma (ICC) is a bile duct cancer and a rare malignant tumor with a poor prognosis owing to the lack of an early diagnosis and resistance to conventional chemotherapy. A combination of gemcitabine and cisplatin is the typically attempted first-line treatment approach. However, the underlying mechanism of resistance to chemotherapy is poorly understood. We addressed this by studying dynamics in the human ICC SCK cell line. Here, we report that the regulation of glucose and glutamine metabolism was a key factor in overcoming cisplatin resistance in SCK cells. RNA sequencing analysis revealed a high enrichment cell cycle-related gene set score in cisplatin-resistant SCK (SCK-R) cells compared to parental SCK (SCK WT) cells. Cell cycle progression correlates with increased nutrient requirement and cancer proliferation or metastasis. Commonly, cancer cells are dependent upon glucose and glutamine availability for survival and proliferation. Indeed, we observed the increased expression of GLUT (glucose transporter), ASCT2 (glutamine transporter), and cancer progression markers in SCK-R cells. Thus, we inhibited enhanced metabolic reprogramming in SCK-R cells through nutrient starvation. SCK-R cells were sensitized to cisplatin, especially under glucose starvation. Glutaminase-1 (GLS1), which is a mitochondrial enzyme involved in tumorigenesis and progression in cancer cells, was upregulated in SCK-R cells. Targeting GLS1 with the GLS1 inhibitor CB-839 (telaglenastat) effectively reduced the expression of cancer progression markers. Taken together, our study results suggest that a combination of GLUT inhibition, which mimics glucose starvation, and GLS1 inhibition could be a therapeutic strategy to increase the chemosensitivity of ICC.

• IMMUNE NETWORK
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• 제22권6호
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• pp.51.1-51.20
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• 2022

Trypanosoma cruzi, the etiological agent of Chagas disease, is an intracellular protozoan parasite, which is now present in most industrialized countries. About 40% of T. cruzi infected individuals will develop severe, incurable cardiovascular, gastrointestinal, or neurological disorders. The molecular mechanisms by which T. cruzi induces cardiopathogenesis remain to be determined. Previous studies showed that increased IL-6 expression in T. cruzi patients was associated with disease severity. IL-6 signaling was suggested to induce pro-inflammatory and pro-fibrotic responses, however, the role of this pathway during early infection remains to be elucidated. We reported that T. cruzi can dysregulate the expression of host PIWI-interacting RNAs (piRNAs) during early infection. Here, we aim to evaluate the dysregulation of IL-6 signaling and the piRNAs computationally predicted to target IL-6 molecules during early T. cruzi infection of primary human cardiac fibroblasts (PHCF). Using in silico analysis, we predict that piR_004506, piR_001356, and piR_017716 target IL6 and SOCS3 genes, respectively. We validated the piRNAs and target gene expression in T. cruzi challenged PHCF. Secreted IL-6, soluble gp-130, and sIL-6R in condition media were measured using a cytokine array and western blot analysis was used to measure pathway activation. We created a network of piRNAs, target genes, and genes within one degree of biological interaction. Our analysis revealed an inverse relationship between piRNA expression and the target transcripts during early infection, denoting the IL-6 pathway targeting piRNAs can be developed as potential therapeutics to mitigate T. cruzi cardiomyopathies.

• 한국작물학회지
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• 제68권4호
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• pp.294-303
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• 2023

Lipoxygenase gives soybeans their grassy flavor, which can disrupt food processing efficiency. This study aimed to identify soybean genotypes with lipoxygenase deficiency among 1,001 soybean accessions and to develop kompetitive allele specific PCR (KASP) markers that can detect lipoxygenase mutations. Three lipoxygenase isozymes (Lox1, Lox2, and Lox3) were analyzed using a colorimetric assay based on a substrate-enzyme reaction. Among the 1,001 accessions examined, two (IT160160 and IT276392) exhibited a deficiency solely in Lox1, and one (IT269984) lacked both Lox1 and Lox2. IT160160 had a 74-bp deletion in exon 8 of Lox1 (Glyma13g347600), whereas IT276392 displayed a missense mutation involving the change of C to A at position 2,880 of Lox1. Moreover, we successfully developed four KASP markers that specifically target Lox1, Lox2, and Lox3 mutations. To validate the Lox1 KASP markers, we used two F_2:3 populations generated through a cross between Daepung 2 (lipoxygenase wild type, maternal parent), IT160160, and IT276392 (null Lox1, paternal parent). The results revealed that the Daepung 2 × IT160160 group followed the expected 3:1 ratio according to Mendel's law, whereas the Daepung 2 × IT276392 group did not. Furthermore, a comparison between the colorimetric and KASP marker analyses results revealed a high agreement rate of 96%. KASP markers offer a distinct advantage by allowing the distinction of heterozygous types independent of other variables. As a result, we present an opportunity to expedite the lipoxygenase-deficient cultivar development.

• Korean Circulation Journal
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• 제52권9호
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• pp.680-696
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• 2022

Background and Objectives: Circular RNAs were known to play vital role in myocardial ischemia reperfusion injury (MIRI), while the role of CircZNF609 in MIRI remains unclear. This study was aimed to investigate the function of CircZNF609 in MIRI. Methods: Hypoxia/reoxygenation (H/R) model was established to mimic MIRI in vitro. Quantitative polymerase chain reaction was performed to evaluate gene transcripts. Cellular localization of CircZNF609 and miR-214-3p were visualized by fluorescence in situ hybridization. Cell proliferation was determined by CCK-8. TUNEL assay and flow cytometry were applied to detect apoptosis. Lactate dehydrogenase was determined by commercial kit. ROS was detected by DCFH-DA probe. Direct interaction of indicated molecules was determined by RIP and dual luciferase assays. Western blot was used to quantify protein levels. In vivo model was established to further test the function of CircZNF609 in MIRI. Results: CircZNF609 was upregulated in H/R model. Inhibition of CircZNF609 alleviated H/R induced apoptosis, ROS generation, restored cell proliferation in cardiomyocytes and human umbilical vein endothelial cells. Mechanically, CircZNF609 directly sponged miR-214-3p to release PTGS2 expression. Functional rescue experiments showed that miR-214-3p/PTGS2 axis was involved in the function of circZNG609 in H/R model. Furthermore, data in mouse model revealed that knockdown of CircZNF609 significantly reduced the area of myocardial infarction and decreased myocardial cell apoptosis. Conclusions: CircZNF609 aggravated the progression of MIRI via targeting miR-214-3p/PTGS2 axis, which suggested CircZNF609 might act as a vital modulator in MIRI.

• 한국발생생물학회지:발생과생식
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• 제6권1호
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• pp.55-66
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• 2002

배아줄기세포(embryonic stem cell, ESC)는 미분화상태로 지속적인 계대가 가능하며, 정상 핵형과 전 분화능(pluripotency)을 가져 생체내-외에서 분화 유도시 삼배엽성의 모든 세포로 분화 가능하다. ESC를 feeder 세포 없이 부유배양하면 배아체(embryoid body, EB)를 형성하고, 초기 배아 발생과 유사한 분화 양상을 갖는다. ESC의 분화 유도가 초기배아 발생처럼 생식호르몬(GTH: FSH, LH; steroids)의 영향을 받는지는 불명하다. 본 연구는 ESC가 분화과정중 생식호르몬처리에 의해 그들 수용체가 발현되는가를 알아보고자 하였다. 순계혈통 생쥐인 C57BL/6J에서 과배란 유도후 포배를 수획하고, 유사분열적으로 불활성화된 feeder 세포와 공배양하여, 계대배양 하는 중 배아줄기세포주(JHYl)를 확립하였다. JHY1의 alkaline phosphatase 활성과 SSEA-1, 3, 4 발현을 통해 ESC임을 확인하였다. Feeder 세포 없이 ESC를 계대배양 후 호르몬처리(FSH LH E$_2$, P$_4$, T)하에서 5일 동안 부유배양하여 배아체를 형성시키고, 이후 7일 동안 부착배양하여 분화를 유도하였다. GTH와 스테로이드의 수용체 발현 실험에서 ESC에 E$_2$ 처리에 의한 LHR의 발현 증가를 제외한 나머지 호르몬 처리군에서 ESC보다 낮은 생식호르몬의 수용체 발현이 관찰되었다. 생식호르몬을 농도별 수용체 발현 정도는 증감되지 않았다. 미분화 ESC 표지유전자인 Oct-4는 호르몬 처리군에서도 발현되었다. 각 배엽의 표지유전자들(영양세포, handl; 외배엽성, keratin와fgf-5; 중배엽성, enolase와 $\alpha$ -globin; 내배엽성, gata-4와 $\alpha$ -fetoprotein) 등의 발현 양상을 조사한 결과 호르몬 처리후 내배엽성 표지유전자외에는 발현 증가가 관찰되지 않았다. 즉 생식호르몬에 의해 gata-4, $\alpha$-fetoprotein의 발현이 증가되는 것으로 보아 내배엽성 계열로의 분화 유도가 이루어진 것으로 사료된다.