Anti-poliovirus activity ofBaccharis dracunculifoliaand propolis by cell viability determination and real-time PCR

Objective The primary purpose of this paper is to investigate the relationship between the microRNA 146a (miR-146a) and the proliferation of cells occurring in glioblastoma multiforme. The secondary purpose of the paper is to investigate abnormalities of expression in miR-146a. Methods A real-time PCR assay was used to investigate the abnormal expression of miR-146a in glioma and adjacent tissue. Lipofection was used to transfect a mimic of miR-146a and induce the upregulation of miR-146a. Real-time PCR was used to observe the expression level of miR-146a. A cell viability analysis was conducted using MTT. A luciferase report vector was used to identify potential targets for miR-146a. Results The miR-146a component was found to be downregulated in glioma tissue compared with adjacent nontumor tissue (p<0.05). The upregulation of miR-146a in glioma cells through miR-146a mimic transfection led to reduction of cell viability and to an increase in the percentage of apoptosis. Notch1 was the name of the potential targeted gene for miR-146a in glioma. Conclusions The study found that the presence of miR-146a potentially affected the proliferation of glioma cells by regulating the rate of Notch1 expression.

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Environmental Monitoring of Mycobacterium bovis in Badger Feces and Badger Sett Soil by Real-Time PCR, as Confirmed by Immunofluorescence, Immunocapture, and Cultivation

Applied and Environmental Microbiology ◽

10.1128/aem.00978-07 ◽

2007 ◽

Vol 73 (22) ◽

pp. 7471-7473 ◽

Cited By ~ 34

Author(s):

F. P. Sweeney ◽

O. Courtenay ◽

V. Hibberd ◽

R. G. Hewinson ◽

L. A. Reilly ◽

...

Keyword(s):

Environmental Monitoring ◽

Cell Viability ◽

Real Time ◽

Species Identification ◽

Mycobacterium Bovis ◽

Real Time Pcr ◽

Immunomagnetic Capture

ABSTRACT Real-time PCR was used to detect and quantify Mycobacterium bovis cells in naturally infected soil and badger feces. Immunomagnetic capture, immunofluorescence, and selective culture confirmed species identification and cell viability. These techniques will prove useful for monitoring M. bovis in the environment and for elucidating transmission routes between wildlife and cattle.

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Evaluation of Kohlrabi (Brassica oleracea Var. Gongylodes) Extract Effect on Mesenchymal Stem Cells Viability and Apoptosis

Research in Molecular Medicine ◽

10.32598/rmm.8.2.1 ◽

2020 ◽

Vol 8 (2) ◽

pp. 93-102

Author(s):

Naser Kalhor Qom ◽

◽

Mohsen Sheykhhasan ◽

Ali Kowsari ◽

◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Mesenchymal Stem Cells ◽

Cell Viability ◽

Brassica Oleracea ◽

Real Time ◽

Real Time Pcr ◽

Surface Markers ◽

Expression Levels ◽

Significant Difference

Background: Cell viability and apoptosis are two crucial factors that may determine cell fate. There are several factors, such as hypoxia, which may be effective in cell processes. Because of its unique features, such as its antioxidant, anti-inflammatory, and anti-apoptosis mechanisms, kohlrabi (Brassica oleracea var. gongylodes) extract may be used in the amelioration of cell viability and a decrease in cell apoptosis. In this study, we evaluate the effect of kohlrabi extract on the viability and apoptosis of adipose-derived mesenchymal stem cells (AD-MSCs). Materials and Methods: In this study, extract from kohlrabi and mesenchymal stem cells from adipose tissue were isolated in a laboratory under sterile conditions. Expression of mesenchymal stem cell (MSC) surface markers, including CD44, CD90, and CD105 was evaluated by flow cytometry method. Besides, CD34 was used as a negative marker. MTT assay was carried out to determine the cell viability. Evaluation of BCL2 and BAX expression levels was performed by real-time PCR. Results: MSC surface markers were verified by flow cytometry. The obtained results demonstrated a significant difference between the cell viability of the kohlrabi-extract treated and control group over time (P=0.03). In addition, the real-time PCR analysis showed that expression levels of BCL2 significantly increased in hypoxic condition after treatment with leaf extract (P=0.019). However, there was no significant expression change in the BAX gene. Conclusion: Our study illustrates that kohlrabi extract may have positive effects on cell survival while having inhibitory effects on apoptosis.

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GLI1 Directly Regulates the Transcription of AKT Genes in Diffuse Large B-Cell Lymphoma.

Blood ◽

10.1182/blood.v120.21.2399.2399 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2399-2399

Author(s):

Nitin K Agarwal ◽

Changju Qu ◽

Kranthi Kunkalla ◽

Yadong Liu ◽

Francisco Vega

Keyword(s):

Cell Viability ◽

Cell Survival ◽

Mrna Expression ◽

Real Time ◽

Cell Lines ◽

Real Time Pcr ◽

Luciferase Reporter ◽

Knock Down ◽

Hh Signaling

Abstract Abstract 2399 Activation of the Hedgehog (Hh)/glioma-associated oncogene (GLI) pathway has been found in a growing number of malignancies. We have provided evidence that canonical Hh signaling is required for cell survival and proliferation of DLBCL cell lines. To confirm the pathogenic role of GLI1 in DLBCL, we established GLI1 knock down DLBCL cell lines (OCI-Ly19, HBL-1 and BJAB) using a lentiviral shRNA system and performed cell viability and apoptosis assays. Cell viability assays demonstrated that GLI1 knockdown DLBCL cells experienced a statistically significantly decrease in the number of viable cells in comparison with control cells harboring scramble shRNA. To examine whether decreases number of cell viability in GLI1 knock down cells were due to apoptosis, we performed annexin V and PI assays. We observed marked increase of apoptosis in GLI1 knock down DLBCL cells versus controls (2.5 fold increase for OCI-Ly10, and 5 fold for HBL1 and BJAB). To investigate the mechanism by which GLI1 regulates tumorigenesis and cell survival, we searched for whole genome GLI1-target genes in DLBCL cells using CHIP sequencing technique and identified AKT genes as potential targets of GLI1. Using pharmacological and silencing approaches, we observed that Hh signaling modulates the expression of AKT genes in DLBCL cells. We further identified two putative binding sites for GLI1 in the AKT1 promoter region and confirmed their functionality using chromatin immunoprecipitation, luciferase reporter and site-directed mutagenesis assays. To investigate whether there is any correlation between AKT1 and GLI1 mRNA expression in human DLBCL tumors, we performed quantitative real-time PCR analyses in 17 frozen DLBCL specimens including apharesis samples from pleural effusions. The real time PCR analysis revealed a strong Spearmen correlation coefficient (R2=0.9) between GLI1 and AKT1 mRNA expression. In summary, we provide evidence of the role of GLI1 in the pathobiology of DLBCL and demonstrated a cross talk, at the transcriptional level, between Hh signaling and AKT in DLBCL. A link between these 2 pathways at the trasncriptional level was not previoulsy documented. This finding is of clinical interest as AKT has a key role in lymphoma cell survival and constitutive activation of AKT has been described in DLBCL. Disclosures: No relevant conflicts of interest to declare.

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Use of a Real Time PCR Assay to Assess the Effect of Pulsed Light Inactivation on Bacterial Cell Membranes and Associated Cell Viability

Water Environment Research ◽

10.2175/106143016x14504669767210 ◽

2016 ◽

Vol 88 (2) ◽

pp. 168-174 ◽

Cited By ~ 5

Author(s):

Mary Garvey ◽

Alessia Stocca ◽

Neil Rowan

Keyword(s):

Cell Viability ◽

Real Time ◽

Real Time Pcr ◽

Bacterial Cell ◽

Cell Membranes ◽

Pulsed Light ◽

Pcr Assay

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AI-2/LuxS Is Involved in Increased Biofilm Formation by Streptococcus intermedius in the Presence of Antibiotics

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00546-09 ◽

2009 ◽

Vol 53 (10) ◽

pp. 4258-4263 ◽

Cited By ~ 62

Author(s):

Nibras A. Ahmed ◽

Fernanda C. Petersen ◽

Anne A. Scheie

Keyword(s):

Cell Viability ◽

Real Time ◽

Real Time Pcr ◽

Antibiotic Susceptibility ◽

Biofilm Formation ◽

Planktonic Cell ◽

Chronic Infections ◽

Streptococcus Intermedius ◽

Microtiter Plates ◽

Communication Signal

ABSTRACT Bacteria utilize quorum-sensing communication to organize their behavior by monitoring the concentration of bacterial signals, referred to as autoinducers (AIs). The widespread detection of AI-2 signals and its enzymatic synthase (LuxS) in bacteria suggests that AI-2 is an inter- and intraspecies communication signal. We have previously shown that antibiotic susceptibility is affected by AI-2 signaling in Streptococcus anginosus. Since chronic infections involve persistent biofilms resilient to antibiotic treatment, we explored the role of AI-2/LuxS in Streptococcus intermedius biofilm formation and cell viability when the organism was exposed to sub-MICs of ampicillin, ciprofloxacin, or tetracycline. The S. intermedius wild type (WT) and its isogenic luxS mutant, strain SI006, were exposed to sub-MICs of ampicillin, ciprofloxacin, or tetracycline. Biofilms were formed on polystyrene discs in microtiter plates. To assess planktonic cell viability, the ATP microbial viability assay was performed and the numbers of CFU were determined. For complementation assays, the AI-2 precursor dihydroxy pentanedione (DPD) was used as a supplement for SI006. Relative luxS expression was quantified by real-time PCR. The sub-MICs of all three antibiotics increased biofilm formation in S. intermedius WT. However, biofilm formation by SI006 was either unaffected or reduced (P ≤ 0.05). Bacterial viability tests of biofilm and planktonic cell cultures indicated that SI006 was more susceptible to antibiotics than the WT. DPD complemented the luxS mutant phenotype. Real-time PCR revealed modest yet significant changes in luxS expression in the presence of antibiotic concentrations that increased biofilm formation. In conclusion, in S. intermedius, AI-2/LuxS was involved in antibiotic susceptibility and increased biofilm formation at sub-MICs of antibiotic.

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126 POTENTIAL EFFECTS OF ENGINEERED STEM CELLS TRANSDUCED WITH THERAPEUTIC GENES AGAINST HeLa HUMAN CERVICAL CANCER CELLS VIA A SELECTIVE TUMOR TROPISM CAUSED BY VASCULAR ENDOTHELIAL GROWTH FACTOR

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab126 ◽

2014 ◽

Vol 26 (1) ◽

pp. 177

Author(s):

B.-R. Yi ◽

S. U. Kim ◽

K.-C. Choi

Keyword(s):

Cervical Cancer ◽

Stem Cells ◽

Cell Viability ◽

Cancer Cells ◽

Real Time ◽

Hela Cells ◽

Real Time Pcr ◽

Migration Assay ◽

Cervical Cancer Cells ◽

Human Cervical Cancer

According to the World Health Organization, cancer of cervix uteri is the second most common cancer among women worldwide. Recently, cervical cancer still remains a significant public health problem for women despite the development of the human papilloma virus vaccine. The aim of the present study was to investigate the therapeutic efficacy of genetically engineered stem cells (GESTEC) expressing bacterial cytosine deaminase (CD), human interferon-β (IFN-b) gene, or both against HeLa human cervical cancer and the migration factors of the GESTEC toward the cancer cells. A continuously dividing immortalized cell line of neural stem cells (NSC) from a single clone of human fetal brain, HB1.F3, was developed by introducing v-myc. The further introduction of these NSC with bacterial CD and human IFN-b resulted in the generation of HB1.F3.CD and HB1.F3.CD.IFN-b cells. The anticancer effect of these GESTEC was examined in a co-culture with HeLa cells using MTT assay to measure cell viability. A transwell migration assay was performed to assess the migration capability of the stem cells to cervical cancer cells. Next, several chemoattractant ligands and their receptors related to a selective migration of the stem cells towards HeLa cells were determined by real-time PCR. The cell viability of HeLa cells was decreased in response to 5-fluorocytosine (5-FC), a prodrug, indicating that 5-fluorouracil (5-FU), a toxic metabolite, was converted from 5-FC by the CD gene and it caused the cell death in a co-culture system. When IFN-b was additionally expressed with the CD gene by these GESTEC, the anticancer activity was significantly increased. In the migration assay, the GESTEC selectively migrated to HeLa cells. As results of real-time PCR, chemoattractant ligands such as MCP-1, SCF, and VEGF were expressed in HeLa cells, and several receptors such as uPAR, VEGFR2, and c-kit were produced by the GESTEC. These GESTEC transduced with the CD gene and IFN-b may provide the potential of a novel gene therapy for anti-cervical cancer treatments via their selective tumour tropism derived from VEGF and VEGFR2 expressions between HeLa cells and the GESTEC. This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ009599), Rural Development Administration, Republic of Korea.

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LncRNA UCA1 Promotes Mitochondrial Function of Bladder Cancer via the MiR-195/ARL2 Signaling Pathway

Cellular Physiology and Biochemistry ◽

10.1159/000484507 ◽

2017 ◽

Vol 43 (6) ◽

pp. 2548-2561 ◽

Cited By ~ 53

Author(s):

Hui-Jin Li ◽

Xiao-Min Sun ◽

Zheng-Kun Li ◽

Qian-Wen Yin ◽

Huan Pang ◽

...

Keyword(s):

Bladder Cancer ◽

Cell Viability ◽

Real Time ◽

Signaling Pathway ◽

Mitochondrial Function ◽

Real Time Pcr ◽

Ectopic Expression ◽

Metabolic Reprogramming ◽

Cell Counting

Background/Aims: This study aims to identify whether Urothelial Cancer Associated 1 (UCA1) regulates mitochondrial metabolic reprogramming in bladder cancer, and to explore how UCA1 participates in mitochondrial metabolism by the UCA1/miR-195/ARL2 signaling pathway; these findings may be aid in the development of tumor diagnostic and therapeutic strategies. Methods: Bladder tissues were obtained from patients. Stable cell lines were constructed, with ectopic expression of UCA1 in UMUC2 cells and knockdown of UCA1 in 5637 cells. The expression levels of UCA1, miR-195, and ARL2 were detected by real-time PCR, western blotting, and immunohistochemistry Cell viability was detected by Cell Counting Kit-8 (CCK8) assay; mitochondrial DNA copy numbers were tested by realtime PCR; ATP level was evaluated by ATP assay kit; mitochondrial membrane potential was analyzed by 5,5’,6,6’-tetrachloro-1,1’,3,3’- tetraethylbenzimidazolylcarbocyanine iodide (JC-1) fluorescent probe. miRNAs between UCA1 and ARL2 were predicted by TargetScan and RNAHybrid, and then determined by real-time PCR. Dual-luciferase activity assay and RNA immunoprecipitation (RIP) assay were used to verify the relationship between UCA1 and miR-195. The expression level of ARL2 was silenced by small interfering RNA(siRNA). For in vivo experiments, UCA1-silencing 5637 cells were subcutaneously injected into BALB/C nude mice to evaluate the effects of UCA1 on tumor progression by the regulation of miR-195 and ARL2. Results: We demonstrate here that UCA1 enhances mitochondrial function in bladder cancer cells. UCA1 contributes to ARL2-induced mitochondrial activity, which plays an important role in mitochondrial function. UCA1, as a competing endogenous RNA (ceRNA), regulates mitochondrial function through upregulating ARL2. In this way, it inhibited the miR-195 signaling pathway to enhance mitochondrial function in bladder cancer. Additionally, ARL2 is a direct target of miR-195 and can be repressed by either miR-195 overexpression or UCA1 inhibition. Knockdown of ARL2 was analogous to the inhibition of UCA1 and the upregulation of miR-195. Animal experiments further indicated that UCA1 promoted bladder tumor growth by regulating miR-195 /ARL2. Conclusion: These data suggest that UCA1 enhanced mitochondrial function and cell viability through the UCA1/miR-195/ARL2 axis in vitro and in vivo. The elucidation of this signaling network provides a more adequate theoretical basis for understanding the molecular pathology of bladder cancer, and also UCA1 as a potential diagnosis and treatment target for bladder cancer.

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