Recombinant human scFv antibody fragments against phospholipase A2 from Naja naja and Echis carinatus snake venoms: In vivo neutralization and mechanistic insights.

Snake envenomation results in a range of clinical sequelae, and widely used animal-based conventional antivenoms exhibit several limitations including the adverse immunological effects in human snake bite victims. Therefore, human monoclonal anti-snake venom antibodies or fragments can be an alternate therapy for overcoming the existing limitations. We developed venom-neutralizing humanized scFv antibodies and analyzed biochemical mechanisms associated with the inhibition of toxicity. Tomlinson I and J human scFv antibody libraries were screened against Naja naja and Echis carinatus venoms, and seven unique scFv antibodies were obtained. Further, specific toxins of snake venom interacting with each of these scFvs were identified, and phospholipase A2 (PLA2) was found to be prominently captured by the phage-anchored scFv antibodies. Our study indicated PLA2 to be one of the abundant toxins in Naja naja and Echis carinatus venom samples. The scFvs binding to PLA2 were used to perform in vivo survival assay using the mouse model and in vitro toxin inhibition assays. scFv N194, which binds to acidic PLA2, protected 50% of mice treated with Naja naja venom. Significant prolongation of survival time and 16% survival were observed in Echis carinatus venom-challenged mice treated with scFv E113 and scFv E10, respectively. However, a combination comprised of an equal amount of two scFvs, E113 and E10, both interacting with basic PLA2, exhibited synergistically enhanced survival of 33% in Echis carinatus venom-challenged mice. No such synergistically enhanced survival was observed in the case of combinatorial treatment with anti-Naja naja scFvs, N194, and N248. These scFvs demonstrated partial inhibition of venom-induced myotoxicity, and E113 also inhibited hemolysis by 50%, which corroborates the enhanced survival during combinatorial treatment in Echis carinatus venom-challenged mice.

Effect of gamma and Ultraviolet-C sterilization on BMP-7 level of indigenously prepared demineralized freeze-dried bone allograft.

The presence of bone morphogenetic proteins in demineralized freeze-dried bone allograft (DFDBA) are responsible for developing hard tissues in intraosseous defects. The most common mode of sterilization of bone allografts, i.e., Gamma rays, have dramatic effects on the structural and biological properties of DFDBA, leading to loss of BMPs. Ultraviolet-C radiation is a newer approach to sterilize biodegradable scaffolds, which is simple to use and ensures efficient sterilization. However, UV-C radiation has not yet been effectively studied to sterilize bone allografts. This study aimed to compare and evaluate the effectiveness of Gamma and Ultraviolet-C rays in sterilizing indigenously prepared DFDBA and assess their effect on the quantity of BMP-7 present in the allograft. DFDBA samples from non-irradiated, gamma irradiated, and UV-C irradiated groups were tested for BMP-7 level and samples sterilized with gamma and UV-C rays were analysed for sterility testing. The estimated mean BMP-7 level was highest in non-irradiated DFDBA samples, followed by UV-C irradiated, and the lowest in gamma irradiated samples. Our study concluded that UV-C rays effectively sterilized DFDBA as indicated by negative sterility test and comprised lesser degradation of BMP-7 than gamma irradiation.

Dynamics of lipid displacement inside the hydrophobic cavity of a nonspecific lipid transfer protein from Solanum melongena.

Nonspecific lipid transfer proteins are multifunctional and multispecific seed proteins with a characteristic hydrophobic cavity that runs form N-terminal to the C-terminal end. They are capable of binding and transferring different lipid molecules by means of their hydrophobic cavity. Apart from the cavity, lipid molecules bind and interact at key positions on the nsLTP surface as well. The plasticity of the hydrophobic cavity is an unusual property, considered as the primary lipid binding site. Here, we report a crystal structure of nsLTP from Solanum melongena with two lauric acid molecules bound inside the cavity. It has been observed that the extent of the N-terminal entry point and plasticity of the cavity can be extended, upon binding of one or two lipid molecules inside the cavity. The MD simulation further revealed that the lipid molecule shows high mobility inside the cavity and interestingly, was able to change its orientation. An alternate lipid entry site adjacent to the N-terminal end was uncovered during simulation and Arg-84 was implicated to be a potential regulatory residue aside from Tyr-59. Collectively, this study helps to understand that changes in orientation of the lipid inside the cavity could occur intermittently besides entering the cavity via tail-in-mechanism.Communicated by Ramaswamy H. Sarma.

Structural insights into the lipid transfer mechanism of a non-specific lipid transfer protein.

The non-specific lipid transfer proteins (nsLTPs) are multifunctional seed proteins engaged in several different physiological processes. The nsLTPs are stabilized by four disulfide bonds and exhibit a characteristic hydrophobic cavity, which is the primary lipid binding site. While these proteins are known to transfer lipids between membranes, the mechanism of lipid transfer has remained elusive. Four crystal structures of nsLTP from Solanum melongena, one in the apo-state and three myristic acid bound states were determined. Among the three lipid bound states, two lipid molecules were bound on the nsLTP surface at different positions and one was inside the cavity. The lipid-dependent conformational changes leading to opening of the cavity were revealed based on structural and spectroscopic data. The surface-bound lipid represented a transient intermediate state and the lipid ultimately moved inside the cavity through the cavity gate as revealed by molecular dynamics simulations. Two critical residues in the loop regions played possible 'gating' role in the opening and closing of the cavity. Antifungal activity and membrane permeabilization effect of nsLTP against Fusarium oxysporum suggested that it could possibly involve in bleaching out the lipids. Collectively, these studies support a model of lipid transfer mechanism by nsLTP via intermediate states.