• Journal of Veterinary Science
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• v.22 no.6
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• pp.87.1-87.12
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• 2021

Background: African swine fever virus (ASFV), classical swine fever virus (CSFV), and porcine reproductive and respiratory syndrome virus (PRRSV) are still prevalent in many regions of China. Co-infections make it difficult to distinguish their clinical symptoms and pathological changes. Therefore, a rapid and specific method is needed for the differential detection of these pathogens. Objectives: The aim of this study was to develop a multiplex real-time quantitative reverse transcription polymerase chain reaction (multiplex qRT-PCR) for the simultaneous differential detection of ASFV, CSFV, and PRRSV. Methods: Three pairs of primers and TaqMan probes targeting the ASFV p72 gene, CSFV 5' untranslated region, and PRRSV ORF7 gene were designed. After optimizing the reaction conditions, including the annealing temperature, primer concentration, and probe concentration, multiplex qRT-PCR for simultaneous and differential detection of ASFV, CSFV, and PRRSV was developed. Subsequently, 1,143 clinical samples were detected to verify the practicality of the assay. Results: The multiplex qRT-PCR assay could specifically and simultaneously detect the ASFV, CSFV, and PRRSV with a detection limit of 1.78 × 10⁰ copies for the ASFV, CSFV, and PRRSV, but could not amplify the other major porcine viruses, such as pseudorabies virus, porcine circovirus type 1 (PCV1), PCV2, PCV3, foot-and-mouth disease virus, porcine parvovirus, atypical porcine pestivirus, and Senecavirus A. The assay had good repeatability with coefficients of variation of intra- and inter-assay of less than 1.2%. Finally, the assay was used to detect 1,143 clinical samples to evaluate its practicality in the field. The positive rates of ASFV, CSFV, and PRRSV were 25.63%, 9.36%, and 17.50%, respectively. The co-infection rates of ASFV+CSFV, ASFV+PRRSV, CSFV+PRRSV, and ASFV+CSFV+PRRSV were 2.45%, 2.36%, 1.57%, and 0.17%, respectively. Conclusions: The multiplex qRT-PCR developed in this study could provide a rapid, sensitive, specific diagnostic tool for the simultaneous and differential detection of ASFV, CSFV, and PRRSV.

• Journal of Marine Life Science
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• v.7 no.2
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• pp.145-153
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• 2022

Mercury (Hg) is a major concern in marine environment because of their bioaccumulation and biomagnification properties, and adverse effects to aquatic organisms at even a trace amount. However, little information on the effects of Hg, compared to other heavy metals, is available in marine small crustaceans. Here, we investigated the transcriptional modulation of metabolism-related genes in the brackish water flea, Diaphanosoma celebensis after exposure to sublethal concentration (0.2, 0.4, 0.8 ㎍/l) of HgCl₂ for 48 h. Relative mRNA expression levels of five detoxification enzyme-coding genes (cytochrome P450; cyp360A1, cyp361A1, cyp4AP3, cyp4C122, and cyp370C5) and six digestive enzyme-coding genes [alpha amylase (AMY), alpha amylase related protein (AMY-like), trypsin (TRYP), chymotrypsin-like protein (CHY), lipase (LIP), pancreatic lipase-related protein (PLRP)] were analyzed using quantitative real time reverse transcription polymerase chain reaction (qRT-PCR). As results, Hg increased the mRNA level of cyp370C5 (clan2) and cyp4AP3 (clan4) in a concentration dependent manner. A significant increase in TRYP mRNA was also concentration-dependently observed after exposure to Hg. These findings suggest that cyp370C5 and cyp4AP3 play a key role in Hg detoxification in D. celebensis, and Hg can affect energy metabolism by modulating the transcription of digestive enzyme. This study will provide better understanding the molecular effects of Hg in marine small crustacean.

• Entomological Research
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• v.48 no.5
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• pp.390-399
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• 2018

In order to precisely assess gene expression levels, the suitable internal reference genes must be served to quantify real-time reverse transcription polymerase chain reaction (RT-qPCR) data. For armyworm, Mythimna separata, which reference genes are suitable for assessing the level of transcriptional expression of target genes have yet to be explored. In this study, eight common reference genes, including ${\beta}$-actin (${\beta}$-ACT), 18 s ribosomal (18S), 28S ribosomal (28S), glyceraldehyde-3-phosphate (GAPDH), elongation fator-alpha ($EF1{\alpha}$), TATA box binding protein (TBP), ribosomal protein L7 (RPL7), and alpha-tubulin (${\alpha}$-TUB) that in different developmental stages, tissues and insecticide treatments of M. separata were evaluated. To further explore whether these genes were suitable to serve as endogenous controls, three software-based approaches (geNorm, BestKeeper, and NormFinder), the delta Ct method, and one web-based comprehensive tool (RefFinder) were employed to analyze and rank the tested genes. The optimal number of reference genes was determined using the geNorm program, and the suitability of particular reference genes was empirically validated according to normalized HSP70, and MsepCYP321A10 gene expression data. We found that the most suitable reference genes for the different experimental conditions. For developmental stages, 28S/RPL7 were the optimal reference genes, both $RPL7/EF1{\alpha}$ were suitable for experiments of different tissues, whereas for insecticide treatments, $28S/{\alpha}-TUB$ were suitable for normalizations of expression data. In addition, $28S/{\alpha}-TUB$ were the suitable reference genes because they have the most stable expression among different developmental stages, tissues and insecticide treatments. Our work is the first report on reference gene selection in M. separata, and might serve as a precedent for future gene expression studies.

• Clinics in Shoulder and Elbow
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• v.25 no.4
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• pp.296-303
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• 2022

Background: A previous study reported that hyperlipidemia increases the incidence of tears in the rotator cuff tendon and affects healing after repair. The aim of our study was to compare the gene and protein expression of torn rotator cuff tendons in patients both with and without hypercholesterolemia. Methods: Thirty patients who provided rotator cuff tendon samples were classified into either a non-hypercholesterolemia group (n=19, serum total cholesterol [TC] <200 mg/dL) and hypercholesterolemia group (n=11, serum TC ≥240 mg/dL) based on their concentrations of serum TC. The expression of various genes of interest, including COL1A1, IGF1, IL-6, MMP2, MMP3, MMP9, MMP13, TNMD, and TP53, was analyzed by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). In addition, Western blot analysis was performed on the proteins encoded by interleukin (IL)-6 and TP53 that showed significantly different expression levels in real-time qRT-PCR. Results: Except for IGF1, the gene expression levels of IL-6, MMP2, MMP9, and TP53 were significantly higher in the hypercholesterolemic group than in the non-hypercholesterolemia group. Western blot analysis confirmed significantly higher protein levels of IL-6 and TP53 in the hypercholesterolemic group (p<0.05). Conclusions: We observed an increase in inflammatory cytokine and matrix metalloproteinase (MMP) levels in hypercholesterolemic patients with rotator cuff tears. Increased levels of IL-6 and TP53 were observed at both the mRNA and protein levels. We suggest that the overexpression of IL-6 and TP53 may be a specific feature in rotator cuff disease patients with hypercholesterolemia.

• BLOOD RESEARCH
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• v.53 no.4
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• pp.320-324
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• 2018

Background Recent studies have devoted much attention to non-protein-coding transcripts in relation to a wide range of malignancies. MALAT1, a long non-coding RNA, has been reported to be associated with cancer progression and prognosis. Thus, we here determined MALAT1 gene expression in chronic lymphocytic leukemia (CLL), a genetically heterogeneous disease, and explored its possible relationships with cytogenetic abnormalities. Methods MALAT1 expression level was evaluated using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) on blood mononuclear cells from 30 non-treated CLL patients and 30 matched healthy controls. Cytogenetic abnormalities were determined in patients by fluorescence in situ hybridization (FISH). Results MALAT1 expression level was up-regulated in the CLL group compared to healthy controls (P=0.008). Del13q14, followed by Del11q22, were the most prevalent cytogenetic abnormalities. We found no association between the FISH results and MALAT1 expression in patients. Conclusion Altered expression of MALAT1 is associated with CLL development. Further investigations are required to assess the relationship between this long non-coding RNA and CLL patient survival and prognosis.

• Parasites, Hosts and Diseases
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• v.56 no.2
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• pp.135-145
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• 2018

Due to the critical location and physiological activities of the retinal pigment epithelial (RPE) cell, it is constantly subjected to contact with various infectious agents and inflammatory mediators. However, little is known about the signaling events in RPE involved in Toxoplasma gondii infection and development. The aim of the study is to screen the host mRNA transcriptional change of 3 inflammation-related gene categories, PI3K/Akt pathway regulatory components, blood vessel development factors and ROS regulators, to prove that PI3K/Akt or mTOR signaling pathway play an essential role in regulating the selected inflammation-related genes. The selected genes include PH domain and leucine- rich-repeat protein phosphatases (PHLPP), casein kinase2 (CK2), vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), glutamate-cysteine ligase (GCL), glutathione S-transferase (GST), and NAD(P)H: quinone oxidoreductase (NQO1). Using reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), we found that T. gondii up-regulates PHLPP2, $CK2{\beta}$, VEGF, GCL, GST and NQO1 gene expression levels, but down-regulates PHLPP1 and PEDF mRNA transcription levels. PI3K inhibition and mTOR inhibition by specific inhibitors showed that most of these host gene expression patterns were due to activation of PI3K/Akt or mTOR pathways with some exceptional cases. Taken together, our results reveal a new molecular mechanism of these gene expression change dependent on PI3K/Akt or mTOR pathways and highlight more systematical insight of how an intracellular T. gondii can manipulate host genes to avoid host defense.

• Journal of Korean Neurosurgical Society
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• v.57 no.5
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• pp.329-334
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• 2015

Objective : To comparatively investigate the expression of several integrins in specimens of human bone metastases and degenerative bone tissue. Methods : Degenerative cancellous tissue was obtained from a sample of human degenerative spine. Thirteen human specimens were obtained from metastatic spine tumors, whose primary cancer was colon cancer (n=3), hepatocellular cancer (n=3), lung cancer (n=4), and breast cancer (n=3). The expression of vimentin and integrins ${\alpha}v$, ${\beta}1$, and ${\beta}3$ was assessed in metastatic and degenerative specimens by immunohistochemistry and real-time reverse transcription-polymerase chain reaction (qRT-PCR). Results : Immunohistochemical staining showed that vimentin and integrin ${\alpha}v$ was broadly expressed in all tissues examined. By contrast, integrin ${\beta}1$ was weakly expressed only in 38.4% (5/13) of tissues. Integrin ${\beta}3$ was consistently negative in all cases examined. qRT-PCR analysis showed that vimentin gene expression was higher in all metastatic specimens, as compared to degenerative bone. The gene expression of integrin ${\alpha}v$ in breast specimen was significantly higher than others (p=0.045). The gene expression of integrin ${\beta}1$ was also higher in all metastatic specimens than in degenerative bone tissue. The gene expression of integrin ${\beta}3$ was variable. Conclusion : Spinal metastatic tumors have mesenchymal characteristics such as increased expression of vimentin. The increased expression of integrin ${\alpha}v$ and ${\beta}1$ in spine metastatic tumors suggests that adhesive molecules such as integrin may have implications for the prevention of spine metastasis.

• Journal of Microbiology and Biotechnology
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• v.31 no.10
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• pp.1358-1365
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• 2021

To clarify the role of long intergenic nonprotein-coding RNA 1232 (LINC01232) in the progression of gastric cancer and the potential mechanism, we analyzed the expression of LINC01232 in TCGA database using the GEPIA online tool, and the LINC01232 level in gastric cancer cell lines was detected by quantitative real time-polymerase chain reaction (qRT-PCR) as well. Cell proliferation assay, colony formation assay, transwell assay and tumor formation experiment in nude mice were conducted to observe the biological behavior changes of gastric cancer cells through the influence of LINC01232 knockdown. LncATLAS database and subcellular isolation assay were used for subcellular distribution of LINC01232 in gastric cancer cells. The interaction among LINC01232, zeste homolog 2 (EZH2) and kruppel-like factor 2 (KLF2) was clarified by RNA-protein interaction prediction (RPISeq), RNA immunoprecipitation (RIP), qRT-PCR and chromatin immunoprecipitation (ChIP) assay. Rescue experiments were further conducted to elucidate the biological function of LINC01232/KLF2 axis in the progression of gastric cancer. LINC01232 was upregulated in stomach adenocarcinoma (STAD) tissues and gastric cancer lines. LINC01232 knockdown inhibited the proliferative capacities of gastric cancer cells in vitro, and impaired in vivo tumorigenicity. LINC01232 was mainly distributed in the cell nucleus where it epigenetically repressed KLF2 expression via binding to the enhancer of EZH2, which was capable of binding to promoter regions of KLF2 to induce histone H3 lysine 27 trimethylation (H3K27me3). LINC01232 exerts oncogenic activities in gastric cancer via inhibition of KLF2, and therefore, the knockdown of KLF2 could reverse the regulatory effect of LINC01232 in the proliferative ability of gastric cancer cells.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.54 no.1
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• pp.16-22
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• 2021

This study investigated the reduction in human norovirus (HNV) GII. 4 count in pacific oyster Crassostrea gigas using electron beam irradiation. Infectious HNV GII. 4 was detected using RT-qPCR (real time reverse transcription-quantitative polymerase chain reaction) with PMA (propidium monoazide)/sarkosyl. At electron beam doses 1, 5, 7, and 10 kGy, the count of HNV GII. 4 was 2.74, 2.37, 2.06, and 1.55 log copies/μL (control, 3.01 log copy/μL), respectively, confirming that as the irradiation dose increased, norovirus count reduced significantly (P<0.05). After PMA/sarkosyl treatment, the counts further reduced at the same irradiation dose, and 10 kGy showed significant differences between the non-treated and PMA/sarkosyl-treated samples (P<0.05). The Ed (decimal reduction dose of electron beam) value based on the first-order kinetic model was 7.33 kGy (R2=0.98). No significant difference was observed in the pH values of the control (6.2) and electron beam-irradiated samples at all doses (6.1). For sensory evaluation, the non-treated sample scored the highest in all categories (5.25-6.17), while the samples treated with 10 kGy showed the lowest score (4.67-5.33), although without statistical significance (P>0.05). Overall, our results suggest that 7 kGy electron beam is sufficient for the non-thermal sterilization of oysters without causing significant changes in quality.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.10
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• pp.1550-1557
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• 2018

Objective: Circular RNAs (circRNAs) are a newfound class of non-coding RNA in animals and plants. Recent studies have revealed that circRNAs play important roles in cell proliferation, differentiation, autophagy and apoptosis during development. However, there are few reports about muscle development-related circRNAs in livestock. Methods: RNA sequencing analysis was employed to identify and annotate circRNAs from longissimus dorsi of sheep. Reverse transcription followed by real-time quantitative (q) polymerase chain reaction (PCR) analysis verified the presence of these circRNAs. Targetscan7.0 and miRanda were used to analyse the interaction of circRNA-microRNA (miRNA). To investigate the function of circRNAs, an experiment was conducted to perform enrichment analysis hosting genes of circRNAs using gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways. Results: About 75.5 million sequences were obtained from RNA libraries of sheep skeletal muscle. These sequences were mapped to 729 genes in the sheep reference genome. We identified 886 circRNAs, including numerous circular intronic RNAs and exonic circRNAs. Reverse transcription PCR (RT-PCR) and DNA sequencing analysis confirmed the presence of several circRNAs. Real-Time RT-PCR analysis exhibited resistance of sheep circRNAs to RNase R digestion. We found that many circRNAs interacted with muscle-specific miRNAs involved in growth and development of muscle, especially circ776. The GO and KEGG enrichment analysis showed that hosting genes of circRNAs was involved in muscle cell development and signaling pathway. Conclusion: The study provides comprehensive expression profiles of circRNAs in sheep skeletal muscle. Our study offers a large number of circRNAs to facilitate a better understanding of their roles in muscle growth. Meanwhile, we suggested that circ776 could be analyzed in future study.