Molecular Dynamics, Simulation of Protein-Protein Complex and their Role in Cytoprotective Process in Ethanol-induced Toxicity of HepG2 Cell Line

Aim: To examine the protein-protein interaction of Wolbachia Surface Protein (WSP of Uzifly) with six proteins involved in Ethanol-induced toxicity and the proteins involved in its cytoprotective process in HepG2 cell line (CYP2E1, Superoxide dismutase, Catalase, Death-associated protein kinase1, Alcohol dehydrogenases (Alpha/beta/gamma) and Cytochrome-C) and to study real time molecular dynamics. Methodology: Modelled structure of WSP of Uzifly was retrieved from our laboratory archive. The proteins involved in the Ethanol-induced toxicity and the proteins involved in its cytoprotective process in HepG2 cell line were chosen based on the literature study. The six proteins like CYP2E1, Superoxide dismutase, Catalase, Death-associated protein kinase1, Alcohol dehydrogenases (Alpha/beta/gamma) and Cytochrome-C which are involved in the Ethanol-induced toxicity and the proteins involved in its cytoprotective process in HepG2 cell line were retrieved from PDB database with ID: PDB (3T3Z), PDB (2C9V), PDB (1DGG), PDB (2YAK), PDB (1U3W) and PDB (3NWV) respectively. Docking study was processed using ZDOCK and the best poses of protein were sorted using rDock. Finally, the atomic level interaction was studied for the best-scored protein-protein complex. The best complex was further subjected to molecular dynamics simulation to study its stability using standard dynamics cascade tool. Results: From the results, it was observed that three proteins such as Cytochrome-C, CYP2E1 and Superoxide dismutase have more favourable shape complementarity for WSP binding to exhibit the cytoprotective process. However, the interaction analysis was done only for the top complex, Cytochrome-C-WSP. Time dependent parameter analysis of best complex Cytochrome-C-WSP showed that root-mean-square deviation (RMSD) values initially deviated but it was stabilized at the end of 1ns dynamics. The radius of gyration (Rg) during dynamics was within the limit. Conclusion: This insilico study revealed that WSP has cytoprotective potential and therapeutical application.

Improvised microinjection technique for mosquito vectors.

Background & objectives: Bio-manipulation technique is of primary importance during the development of transgenic mosquitoes. The study describes the variable factors that influence the viability of medically important mosquito vectors during microinjection. Methods: Three mosquito vectors belonging to the genus Aedes, Anopheles and Culex were microinjected at different developmental stages of their life cycle viz., egg, larvae, pupae and adult. Results: The improvisations revealed an increased survivability of biomanipulated mosquitoes during the embryonic and adult microinjection. The study of injecting larvae and pupae resulted in poor survivability. Interpretation & conclusions: The microinjection protocol was successfully tested on three important mosquito vectors. The critical period after biomanipulation which contributes heavily for the survivability factor was evaluated. The results provide a common protocol for biomanipulation of three mosquito vectors with enhanced survivability.

Wolbachia endosymbiont infection in two Indian butterflies and female-biased sex ratio in the Red Pierrot, Talicada nyseus.

The maternally inherited obligate bacteria Wolbachia is known to infect various lepidopteran insects. However, so far only a few butterfly species harbouring this bacterium have been thoroughly studied. The current study aims to identify the infection status of these bacteria in some of the commonly found butterfly species in India. A total of nine butterfly species belonging to four different families were screened using PCR with Wolbachia-specific wsp and ftsZ primers. The presence of the Wolbachia super group ‘B’ in the butterflies Red Pierrot, Talicada nyseus (Guerin) (Lepidoptera: Lycaenidae) and Blue Mormon, Papilio polymnestor Cramer (Papilionidae), is documented for the first time in India. The study also gives an account on the lifetime fecundity and female-biased sex ratio in T. nyseus, suggesting a putative role for Wolbachia in the observed female-biased sex ratio distortion.

Screening and characterization of protease from bacillus sp.

Protease production by alkalophilic Bacillus sp were isolated from different regions of Bangalore and used to screen for the protease production by using casein and skim milk agar plate assay. The agro wastes are also used to screen and produce protease. The agro wastes are rice bran, paddy straw and pigeon pea waste, among these substrate rice bran showed maximum synthesis of protease. At 48 hr it showed maximum 0.13 IU protease production in rice bran medium was used for partial purification studies. Fermented medium were used for salt precipitation, dialysis. The dayalyste were used for studies on effect of Ph, temperature, inhibitors and metal ions. The optimum pH 10, temperature 650C were optimum and the inhibitors and metal ion studies, metalions like Mn2+ and Ca2+ were found to be potent enhancers. The complete loss of enzyme activity was found in presence of PMSF.

Frequency of infection with A and B supergroup Wolbachia in insects and pests associated with mulberry and silkworm.

Wolbachia is a ubiquitous, Gram-negative,vertically transmitted, alpha-proteobacterium that causes an array of reproductive abnormalities including cytoplasmic incompatibility, feminization of genetic males, parthenogenesis in a number of insect species, among others. Wolbachia is now being exploited as an agent for pest and vector control. Previous surveys indicated that it is commonly seen in 16-76% of arthropods. In this paper, using polymerase chain reaction assay based on specific amplification of the ftsZ -A and -B supergroup Wolbachia gene fragments, we found that 30% of insects and pests screened were positive for Wolbachia. Among them 66.7% harbour double Wolbachia infection, while 33.3 % harbour single Wolbachia infection. These results indicate widespread infection with both double and single Wolbachia, and provide a wealth of information to exploit this endobacterium for the management of pests and vectors.