• The Plant Pathology Journal
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• 제38권4호
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• pp.287-295
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• 2022

Citrus tristeza virus (CTV) is efficiently transmitted in a semi-persistent manner by the brown citrus aphid (Toxoptera citricida (Kirkaldy)). Currently, the most sensitive method for detecting plant viruses in insect vectors is reverse-transcription quantitative polymerase chain reaction (RT-qPCR). In this study, the elongation factor-1 alpha (EF-1α) gene and acidic p0 ribosomal protein (RPAP0) gene were confirmed to be suitable reference genes for RT-qPCR normalization in viruliferous T. citricida aphids using the geNorm, NormFinder, and BestKeeper tools. Then the relative CTV titer in aphids (T. citricida) at different post-acquisition feeding times on healthy plants was quantified by RT-qPCR using EF-1α and RPAP0 as reference genes. The relative CTV titer retained in the aphids gradually decreased with increasing feeding time. During the first 0.5 h of feeding time on healthy plants, the remaining CTV titer in aphids showed about 80% rapid loss for the highly transmissible isolate CT11A and 40% loss for the poorly transmissible isolate CTLJ. The relative CTV titer in aphids during more than 12 h post-acquisition times for CT11A was significantly lower than at the other feeding times, which is similar to the trend found for CTLJ. To our knowledge, this is the first report about the relative titer variation of CTV remaining in T. citricida at different post-acquisition feeding times on healthy plants.

• Journal of Veterinary Science
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• 제24권4호
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• pp.53.1-53.12
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• 2023

Background: Mammalian orthoreovirus type 3 (MRV3), which is responsible for gastroenteritis in many mammalian species including pigs, has been isolated from piglets with severe diarrhea. However, the use of pig-derived cells as an infection model for swine-MRV3 has rarely been studied. Objectives: This study aims to establish porcine intestinal organoids (PIOs) and examine their susceptibility as an in vitro model for intestinal MRV3 infection. Methods: PIOs were isolated and established from the jejunum of a miniature pig. Established PIOs were characterized using polymerase chain reaction (PCR) and immunofluorescence assays (IFAs) to confirm the expression of small intestine-specific genes and proteins, such as Lgr5, LYZI, Mucin-2, ChgA, and Villin. The monolayered PIOs and three-dimensional (3D) PIOs, obtained through their distribution to expose the apical surface, were infected with MRV3 for 2 h, washed with Dulbecco's phosphate-buffered saline, and observed. Viral infection was confirmed using PCR and IFA. We performed quantitative real-time reverse transcription-PCR to assess changes in viral copy numbers and gene expressions linked to intestinal epithelial genes and antiviral activity. Results: The established PIOs have molecular characteristics of intestinal organoids. Infected PIOs showed delayed proliferation with disruption of structures. In addition, infection with MRV3 altered the gene expression linked to intestinal epithelial cells and antiviral activity, and these effects were observed in both 2D and 3D models. Furthermore, viral copy numbers in the supernatant of both models increased in a time-dependent manner. Conclusions: We suggest that PIOs can be an in vitro model to study the infection mechanism of MRV3 in detail, facilitating pharmaceutical development.

• Asian Pacific Journal of Cancer Prevention
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• 제14권12호
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• pp.7621-7628
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• 2013

Human epidermal growth factor receptor (HER) status is an important prognostic factor in breast cancer. There is no globally accepted method for determining its status, and which method is most precise is still a matter of debate. We here analyzed HER2 mRNA expression by quantitative reverse transcription-PCR (qRT-PCR) and HER2 DNA amplification using multiplex ligation-dependent probe amplification (MLPA). In parallel, we performed a routine evaluation of HER2 protein by immunohistochemistry (IHC). To assess the accuracy of the RT-PCR and MLPA techniques, a combination of IHC and fluorescence in situ hybridization (FISH) was used, substituting FISH when the results of IHC were ambiguous (2+) and for those IHC results that disagreed with MLPA and qRT-PCR, this approach being termed IHC-FISH. The IHC results for four samples were not compatible with the MLPA and qRT-PCR results; the MLPA and qRT-PCR results for these samples were confirmed by FISH. The correlations between IHC-FISH and qRT-PCR or MLPA were 0.945 and 0.973, respectively. The ASCO/CAP guideline IHC/FISH correlation with MLPA was (0.827) and with RT-PCR was (0.854). The correlations between the IHC results (0, 1+ as negative, and 3+ as positive) and qRT-PCR and MLPA techniques were 0.743 and 0.831, respectively. Given the shortcomings of IHC analysis and greater correlations between MLPA, qRT-PCR, and FISH methods than IHC analysis alone with each of these three methods, we propose that MLPA and real-time PCR are good alternatives to IHC. However a suitable cut-off point for qRTPCR is a prerequisite for determining the exact status of HER2.

• Biomolecules & Therapeutics
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• 제18권2호
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• pp.178-183
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• 2010

Costunolide is an active compound isolated from the stem bark of Magnolia sieboldii, and is considered a potential therapeutic for the treatment of various cancers. In this study, we investigated the underlying mechanism whereby costunolide induces the apoptosis of human leukemia cells. Using apoptosis analysis and quantitative reverse transcription-polymerase chain reaction (RT-PCR) results obtained during this study show that costunolide is a potent inducer of apoptosis and that it is triggered due to the premature activation of Cdc2. $G_1$-synchronized cells, which cannot undergo mitosis, were found to be more sensitive to costunolide, and Cdc2 mRNA levels were increased by costunolide treatment. Furthermore, the Cdk inhibitors, olomucine and butyrolactone I, were found to suppress costunolide-induced apoptosis. In addition, the PKC activator TPA rescued cells from cell death by costunolide, and this was prevented by the PKC inhibitor staurosporin. The present study suggests that costunolide induces the apoptosis of HL-60 leukemic cells by modulating cyclin-dependent kinase Cdc2.

• The Korean Journal of Physiology and Pharmacology
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• 제21권4호
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• pp.449-456
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• 2017

Beauvericin (BEA), a cyclic hexadepsipeptide produced by the fungus Beauveria bassiana, is known to have anti-cancer, anti-inflammatory, and anti-microbial actions. However, how BEA suppresses macrophage-induced inflammatory responses has not been fully elucidated. In this study, we explored the anti-inflammatory properties of BEA and the underlying molecular mechanisms using lipopolysaccharide (LPS)-treated macrophage-like RAW264.7 cells. Levels of nitric oxide (NO), mRNA levels of transcription factors and the inflammatory genes inducible NO synthase (iNOS) and interleukin (IL)-1, and protein levels of activated intracellular signaling molecules were determined by Griess assay, semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), luciferase reporter gene assay, and immunoblotting analysis. BEA dose-dependently blocked the production of NO in LPS-treated RAW264.7 cells without inducing cell cytotoxicity. BEA also prevented LPS-triggered morphological changes. This compound significantly inhibited nuclear translocation of the $NF-{\kappa}B$ subunits p65 and p50. Luciferase reporter gene assays demonstrated that BEA suppresses MyD88-dependent NF-${\kappa}B$ activation. By analyzing upstream signaling events for $NF-{\kappa}B$ activation and overexpressing Src and Syk, these two enzymes were revealed to be targets of BEA. Together, these results suggest that BEA suppresses $NF-{\kappa}B$-dependent inflammatory responses by suppressing both Src and Syk.

• Asian Pacific Journal of Cancer Prevention
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• 제13권7호
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• pp.3259-3264
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• 2012

Curcumin (CM) possesses anti-cancer activity against a variety of tumors. Matrix metalloproteinases (MMPs) play an important role in remodeling the extracellular matrix and their activities are regulated by tissue inhibitor of metalloproteinases (TIMPs) family. Control of MMP and TIMP activity are now of great significance. In this study, the effect of CM is investigated on metastatic MMPs and anti-metastatic TIMPs genes on MDA breast cancer cells cultured in a mixture of DMEM and Ham's F12 medium and treated with different concentrations of CM (10, 20 and $40{\mu}M$ for various lengths of time. Reverse transcription followed by quantitative real time PCR was used to detect the gene expression levels of MMPs and TIMPs in CM-treated versus untreated cases and the data were analyzed by one-way ANOVA. At high concentrations of curcumin, TIMP-1, -2, -3 and -4 genes were up-regulated after 48 hours of treatment, their over-expression being accompanied by down-regulation of MMP-2 and MMP-9 gene expression levels in a concentration- and time-dependent manner. These results suggest that curcumin plays a role in regulating cell metastasis by inhibiting MMP-2 and MMP-9 and up-regulating TIMP1 and TIMP4 gene expression in breast cancer cells.

• Asian Pacific Journal of Cancer Prevention
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• 제15권15호
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• pp.6065-6070
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• 2014

Background: Breast cancer is the most common malignancy in women worldwide, including Thailand, and is a major cause of mortality and morbidity, despite advances in diagnosis and treatment. Novel gene expression in breast cancer is a focus in searches for prognostic biomarkers and new therapeutic targets. Materials and Methods: The mRNA expression of novel B4GALT4, SLC35B2, and WDHD1 genes in breast cancer were examined in invasive ductal breast carcinoma (IDC) patients using quantitative real-time reverse transcription polymerase chain reaction (QRT-PCR). Results: Among these genes, increased expression of SLC35B2 mRNA was significantly associated with TNM stage III + IV of IDC (p<0.001). Hence, up-regulation of SLC35B2 may serve as a prognostic biomarker for poor prognosis, and is also a potential therapeutic target in breast cancer.

• 대한한방내과학회지
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• 제24권1호
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• pp.134-143
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• 2003

Objectives : We performed this study to understand the molecular basis of the antitumor effect of Saussurea lappa, Pharbitis nil, Plantago asiatica and Taraxacum mongolicum, which have been used for cancer treatment in Korean traditional medicine. Design: We analyzed, the effect of these medicinal herbs on proliferation and apoptosis of tumor cells and its association with gene expression, We performed semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR) analysis of cell cycle- and apoptosis-related genes using a gastric cancer cell line AGS. Results : Cell counting assay and $[^3H]thymidine$ uptake analysis showed that Saussurea lappa and Pharbitis nil strongly inhibit cell proliferation of AGS in a dose-dependent manner. Interestingly, gene espression assay revealed that mRNA espression levels of c-Jun, c-Fos, c-Myc, and Cyclin D1 were markedly decreased by Saussurea lappa and Pharbitis nil. Furthermore, Saussurea lappa was identified to activate expression of the p53 tumor suppressor and its downstream effector $p21^{Wafl}$, which leads to $G_1$ cell cycle arrest and apoptosis. These observations suggest that the anticancer effect of Saussurea lappa and Pharbitis nil might be associated with their regulatory capability of tumor-related gene expression.

• Molecules and Cells
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• 제41권8호
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• pp.724-732
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• 2018

Plant defence responses to various biotic stresses via systemic acquired resistance (SAR) are induced by avirulent pathogens and chemical compounds, including certain plant hormones in volatile form, such as methyl salicylate and methyl jasmonate. SAR refers to the observation that, when a local part of a plant is exposed to elicitors, the entire plant exhibits a resistance response. In the natural environment, plants are continuously exposed to avirulent pathogens that induce SAR and volatile emissions affecting neighbouring plants as well as the plant itself. However, the underlying mechanism has not been intensively studied. In this study, we evaluated whether plants "memorise" the previous activation of plant immunity when exposed repeatedly to plant defensive volatiles such as methyl salicylate and methyl jasmonate. We hypothesised that stronger SAR responses would occur in plants treated with repeated applications of the volatile plant defence compound MeSA than in those exposed to a single or no treatment. Nicotiana benthamiana seedlings subjected to repeated applications of MeSA exhibited greater protection against Pseudomonas syringae pv. tabaci and Pectobacterium carotovorum subsp. carotovorum than the control. The increase in SAR capacity in response to repeated MeSA treatment was confirmed by analysing the defence priming of the expression of N. benthamiana Pathogenesis-Related 1a (NbPR1a) and NbPR2 by quantitative reverse-transcription PCR compared with the control. We propose the concept of plant memory of plant defence volatiles and suggest that SAR is strengthened by the repeated perception of volatile compounds in plants.

• 대한의생명과학회지
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• 제25권2호
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• pp.185-189
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• 2019

Breast cancer is the second most common cancer in women with approximately 522,000 deaths annually worldwide. microRNAs have recently been studied as potential biomarkers that regulate gene expression and are involved in tumorigenesis. Here we evaluated circulating miR-221 and miR-222 as potential biomarkers for breast cancer by quantitative reverse transcription PCR using blood plasma of 30 healthy controls and 30 breast cancer patients. The TNM stage on circulating miR-221 and miR-222 was also investigated. Circulating miR-221 and miR-222 were significantly up-regulated in breast cancer patients compared to those in healthy controls (P < 0.0022 and P = 0.0058, respectively). Furthermore, the relative expression level of circulating miR-221 in patients with stage III breast cancer was higher than in those with stage I and II. Taken together, we have shown circulating miR-221 and miR-222 could be useful biomarkers for the screening of breast cancer patients.