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Showing 2 results for Inhibition Assay
Volume 10, Issue 4 (12-2019)
Abstract
Aims: Aequorin as a bioluminescence protein due to ease of use, non-toxic, and high capability of detecting has long been the interest of researchers. The aim of this study was to design a method for accurate and simple detection of important therapeutic agents using a bioluminescence inhibition based assay by using aequorin.
Materials & Methods: In this study, important drugs in therapeutic monitoring with structural similarity to Coelenterazine, were selected and their interaction with aequorin was investigated. Further, the conditions of the bioluminescence assay were optimized to achieve the lowest detection limit.
Findings: Among the drugs whose effects have been tested on aequorin, the only benserazide resulted in inhibition of the bioluminescence activity. This analyte can significantly reduce the bioluminescence of aequorin in a concentration-dependent manner. The best dose-response curve was obtained and IC50 of 0.26µM was calculated. The linear calibration curve was obtained in a range of about 100 to 1500nM with LOD and LOQ of 79 and 260nM, respectively. Furthermore, we demonstrated the application of the approach in human serum samples with a recovery of 97%. Guddem-Schild graph was plotted to determine the mechanism of inhibition which indicated that the IC50 of benserazide changed in the presence of different concentrations of Coelenterazine.
Conclusion: The proposed method can be used for measuring benserazide which can easily be applicable for real samples. Also, the results show that benserazide inhibits the bioluminescence activity of aequorin by competitive inhibition.
S. Ansari, A. Mousavi, M. R. Safarnejad, N. Farrokhi, S. M. Alavi,
Volume 25, Issue 5 (9-2023)
Abstract
Aspergillus flavus is a major fungal phytopathogen and an opportunistic pathogen to humans and livestock. The fungus produces immunosuppressive and carcinogenic aflatoxins that acts as a burden in food and feed industries. Phylogenetic analysis indicated that the cell wall protein, A. flavus mannoprotein 1 (AFLMP1), is exclusively present in Aspergillus section Flavi such as A. flavus and A. parasiticus. This makes AFLMP1 an excellent candidate for siRNA-based control of aflatoxigenic fungi in farms and processing units, and fungal therapy in hospitals. Here and for the first time, mode of action of a chemically synthesized RNA interference (siRNA) was investigated for the control of AFLMP1 synthesis. The efficacy of direct uptake of different concentration of siRNA on spore germination of A. flavus was monitored via Opera High Content Screening confocal microscope. siRNA caused growth inhibition at lower concentrations (0.65 nM) and germination failure (more than 90%) at higher concentrations (5 nM), most likely by interfering in mannoprotein biosynthesis. It is assumed that siRNA technology can be implemented as a promising suppressive agent in treatment of target genes for inactivation. It can be considered as an intervention in food/feed industries to control the development and reproduction of fungi to keep the fungal population below hazard critical points.
