Development of an optimized RT-LAMP test for the detection of SARS-CoV-2.

SARS-COV-2 is the causative agent of an acute respiratory syndrome called Coronavirus disease 2019 (COVID-19) with a varying mortality rate from 2019 to 2022. There are several measures for control and prevention of Covid-19 including using mask, vaccine injections, as well as screening the potential cases. We aimed to design and develop a molecular method (RT-LAMP) for detecting coronavirus in biological samples that is cheaper, faster and easier than conventional molecular methods. In this study, various reaction components were explored to make the optimal combination of an RT-LAMP master mix composition. The results revealed the ability of this RT-LAMP test in specifically identifying 100 copies of mixture of N and E genes in just 30-45 min. This study demonstrated the reliable performance of the RT-LAMP method for the detection of SARS-COV-2 in biological samples. Given the significant advantages of this method compared to the gold standard qRT-PCR, it can be employed as a promising tool for the diagnosis of coronavirus as well as other pathogenic viruses.

Maximizing the recovery of the native p28 bacterial peptide with improved activity and maintained solubility and stability in Escherichia coli BL21 (DE3).

p28 is a natural bacterial product, which recently has attracted much attention as an efficient cell penetrating peptide (CPP) and a promising anticancer agent. Considering the interesting biological qualities of p28, maximizing its expression appears to be a prominent priority. The optimization of such bioprocesses might be facilitated by utilizing statistical approaches such as Design of Experiment (DoE). In this study, we aimed to maximize the expression of "biologically active" p28 in Escherichia coli BL21 (DE3) host by harnessing statistical tools and experimental methods. Using Minitab, Plackett-Burman and Box-Behnken Response Surface Methodology (RSM) designs were generated to optimize the conditions for the expression of p28. Each condition was experimentally investigated by assessing the biological activity of the purified p28 in the MCF-7 breast cancer cell line. Seven independent variables were investigated, and three of them including ethanol concentration, OD600 of the culture at the time of induction, and the post-induction temperature were demonstrated to significantly affect the p28 expression in E. coli. The cytotoxicity, penetration efficiency, and total process time were measured as dependent variables. The optimized expression conditions were validated experimentally, and the final products were investigated in terms of expression yield, solubility, and stability in vitro. Following the optimization, an 8-fold increase of the concentration of p28 expression was observed. In this study, we suggest an optimized combination of effective factors to produce soluble p28 in the E. coli host, a protocol that results in the production of a significantly high amount of the biologically active peptide with retained solubility and stability.

CRISPR/Cas9-mediated knockout of MLL5 enhances apoptotic effect of cisplatin in HeLa cells in vitro.

Mixed lineage leukemia 5 (MLL5) transactivates the expression of E6 and E7 oncogenes in cervical cancer cells. In this study, we utilized CRISPR/Cas9 system with the aim to target HPV-E6 and MLL5 to enhance apoptosis efficiency in HPV-18 positive HeLa cells and to improve chemotherapeutic efficacy of Cisplatin as the most common anticancer drug, used for cervical cancer. sgRNAs against MLL5 and E6 were designed and cloned into PX458 plasmid vector. Real-time PCR was used to determine knockout expression of MLL5 and E6 following, transfection with cloned plasmids. Cell viability and apoptosis were evaluated, using Dimethyl-thiazolyl diphenyl tetrazolium bromide (MTT) assay and Annexin V flow cytometry. Cellular p 53 level was measured, using enzyme linked immune sorbent assay (ELISA). Real-time PCR indicated the downregulation of E6 and MLL5 in the transfected cells. A significant increase in the accumulation of P53 was observed due to targeting MLL5 and E6 genes. MTT and apoptosis assays showed a significant decrease in cell viability and enhanced apoptosis rate of transfected cells. Combination therapy showed that targeting E6 and MLL5 enhanced apoptotic effect of Cisplatin in MLL5 knockout cells in a synergistic manner. The results suggest that CRISPR/Cas9 targeting of E6 and MLL5 genes can increase apoptotic effects of Cisplatin and can be considered as a potential combination therapy for the treatment of HPV- related cervical cancer.

The Effect of Bacterial Peptide p28 on Viability and Apoptosis Status of P53-null HeLa Cells.

Purpose: Despite all the efforts for discovery of efficient anti-cancer therapeutics, cancer is still a major health concern worldwide. p28 is a bacterial small peptide which has been widely investigated due to its preferential cell internalization and anti-cancer activities. Intracellularly, p28 offers its anti-cancer traits by impeding the degradation of tumor-suppressor protein "p53". In this study, we investigated the potency of p28 in inducing apoptosis or decreasing cell viability in p53-null "HeLa" cell line. Methods: The coding sequence for p28 peptide was obtained from Pseudomonas aeruginosa by PCR amplification of the p28 gene. The coding gene was cloned in pET-28a vector and transformed into E. coli bacterial host. Subsequently, the expressed peptide was purified using Ni-NTA chromatography system and introduced into the target cells. The anti-proliferative and apoptotic activity of p28 on HeLa and HEK-293 cells were investigated using MTT and PEAnnexin V Flowcytometry assays. Results: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting confirmed the expression of p28 peptide in the bacterial host. Bradford assay revealed a concentration of 0.05 mg/mL for the purified p28. MTT assay of cells treated with p28 at concentrations of 0, 0.5, 1, 2 and 2.5 µM indicated 24h viability values of 97%, 89%, 88%, 87% and 84% for HeLa cells, respectively. Data obtained from flowcytometry analyses revealed 24h apoptosis rate of 7.17%, 8.05%, 8.63% and 8.84% for HeLa cells treated with 0, 0.5, 1, and 2 µM p28, respectively. Conclusion: MTT and flowcytometry apoptosis assays suggest no statistically significant effect of p28 on the viability and apoptosis status of p53-null HeLa cells when results compared to data obtained from HEK-293 cells (P>0.05). These results imply that anti-cancer efficacy of p28 is directly dependent on the presence of p53, suggesting p28 as an inappropriate therapeutic agent for treatment of cancers with negative p53 status.