Regulation of HSVtk gene by endogenous microRNA-122a in liver cell lines as suicide gene therapy.

AIM: Here, we use miR-122a that exhibits liver-specific expression for developing a post-transcriptional regulative system mediated by microRNAs. BACKGROUND: Gene therapy with adenovirus (Ad) vectors that express herpes simplex virus thymidine kinase (HSVtk) and ganciclovir (GCV) have been suggested as a therapeutic strategy to cancer. However, Ad vectors injected into tumors are dispersed into the systemic circulation and transduce liver cells, resulting in severe hepatotoxicity. To be effective, the delivery and expression of suicide genes to cancer treatment ought to be specific to tumor cells, and avoid death of healthy cells. Researchers have demonstrated that expression of transgene could be suppressed in healthy cells with use of vectors that are reactive to microRNA regulation. METHODS: We constructed an Ad vector carrying four tandem copies of target sequences of miR-122a that were incorporated into 3'-UTR of HSVtk gene. The expression level of miR-122a was quantified in HepG2 and Huh7 cell lines. RESULTS: Quantitative RT- PCR analysis demonstrated that Huh7 cells express large amounts of miR-122a compared to HepG2 cells. The viability of Huh7 cells and HepG2 cells after infection by Ad-tk-122aT vector was 83% and 23.5%, respectively. The viability of Huh7 cells was not reduced in the presence of GCV after infection by Ad-tk-122a vector. In contrast, cytotoxicity of HSV-tk/GCV was similar in Huh7 cells and HepG2 cells by Ad-tk vector, with 35.3% and 27% viability, respectively. CONCLUSION: Inclusion of the miR-122a target sequences in the HSVtk expression cassette yielded a feasible strategy for reducing cytotoxicity of suicide gene in a liver cell line with high miR-122a expression.

Molecular Analysis of Ganciclovir-Resistant Cytomegalovirus in Renal Transplant Recipients with High Viral Load.

BACKGROUND: Gancyclovir-resistant (GanR) cytomegalovirus (CMV) remains an issue, especially in solid organ transplant (SOT) recipients. Some mutations in UL54 and UL97 confer this resistance. Long-lasting high-dose drug exposure, high viral load, together with lack of sufficient compliance with treatment may account for these mutations. The aim of this study was to detect UL97 and UL54 putative mutations conferring ganciclovir-resistance in renal organ transplant recipients with high CMV load. METHODS: In this cross-sectional study, 58 serum samples were collected from renal transplant recipients who had referred to three hospitals in Tehran from January 2014 to June 2015. Specific criteria such as CMV syndrome, presence of CMV in blood and organ dysfunction were considered. Then, they were tested for viral load in their early fourth month of intravenous ganciclovir treatment. Fifty cases revealing more than 200 copies/mL were analyzed for mutations. Two fragments of UL54 and Ul97 genes were amplified and sequenced bidirectionally. Sequence alignment and statistical analysis were performed by Mutation Surveyor software and t-test respectively. RESULTS: A significant difference was observed in viral load between seronegative and seropositive recipients (P = 0.036). The most frequent mutation was related to D605E in UL97 gene with the rate of 25%. Regardless of viral load, neither putative mutation nor simultaneous mutation was detected in either UL97 and UL54 regions. CONCLUSION: In spite of high viral load and persistence of symptoms, our population study did not reveal putative mutations. Hence, the direct relationship between the presence of high quantity of CMV and the occurrence of putative mutation cannot be considered. Non-putative gancyclovir resistant mutations and prolonged drug exposure may have a role in these manifestations.

Use of TaqMan® real-time PCR for rapid detection of Salmonella enterica serovar Typhi.

We evaluated the performances of a newly designed real-time polymerase chain reaction (PCR) assay using TaqMan® probes to detect Salmonella Typhi. TaqMan® real-time PCR assays were performed by designed primers and probe based on the staG gene for detecting S. Typhi. The specificity of the assay was evaluated on 15 Salmonella serovars. The analytical specificity was evaluated on 20 non-Salmonella microorganisms. The analytical sensitivity was assessed using decreasing DNA quantities of S. Typhi ATCC 19430. Finally the detection capability of the TaqMan® real-time PCR assay on isolates recovered from patients with Salmonella infections was compared to the conventional PCR assay. Only S. Typhi strain had positive results when subjected to the assay using Typhi-specific real-time PCR. No amplification products were observed in real-time PCR with any of the non-Salmonella microorganisms tested. The TaqMan® real-time PCR was more sensitive than the conventional PCR. In conclusion, we found that the easy-to-use real-time PCR assays were faster than conventional PCR systems. The staG-based TaqMan® real-time PCR assay showed to be specific and sensitive method for the safe and rapid detection of the S. Typhi.