Sequence specificity, energetics and mechanism of mismatch recognition by DNA damage sensing protein Rad4/XPC.

The ultraviolet (UV) radiation-induced DNA lesions play a causal role in many prevalent genetic skin-related diseases and cancers. The damage sensing protein Rad4/XPC specifically recognizes and repairs these lesions with high fidelity and safeguards genome integrity. Despite considerable progress, the mechanistic details of the mode of action of Rad4/XPC in damage recognition remain obscure. The present study investigates the mechanism, energetics, dynamics, and the molecular basis for the sequence specificity of mismatch recognition by Rad4/XPC. We dissect the following three key molecular events that occur as Rad4/XPC tries to recognize and bind to DNA lesions/mismatches: (a) the association of Rad4/XPC with the damaged/mismatched DNA, (b) the insertion of a lesion-sensing ß-hairpin of Rad4/XPC into the damage/mismatch site and (c) the flipping of a pair of nucleotide bases at the damage/mismatch site. Using suitable reaction coordinates, the free energy surfaces for these events are determined using molecular dynamics (MD) and umbrella sampling simulations on three mismatched (CCC/CCC, TTT/TTT and TAT/TAT mismatches) Rad4-DNA complexes. The study identifies the key determinants of the sequence-dependent specificity of Rad4 for the mismatches and explores the ramifications of specificity in the aforementioned events. The results unravel the molecular basis for the high specificity of Rad4 towards CCC/CCC mismatch and lower specificity for the TAT/TAT mismatch. A strong correlation between the depth of ß-hairpin insertion into the DNA duplex and the degree of coupling between the hairpin insertion and the flipping of bases is also observed. The interplay of the conformational flexibility of mismatched bases, the depth of ß-hairpin insertion, Rad4-DNA association energetics and the Rad4 specificity explored here complement recent experimental FRET studies on Rad4-DNA complexes.

Niosome-based hydrogel of resveratrol for topical applications: An effective therapy for pain related disorder(s).

The present work endeavors for development and evaluation of resveratrol loaded niosomal hydrogel system for its anti-inflammatory action. Niosomes were prepared by thin film hydration and ether injection methods employing Span 80 as a surfactant at three different levels. Best optimized formulation was selected on the basis of entrapment efficiency (% EE), mean particle size, sedimentation volume, and microscopy. The vesicular and spherical nature of the niosomes was confirmed by optical microscopy and transmission electron microscope (TEM). Further, resveratrol entrapped niosomal gel was prepared by gelling in Carbopol 934, and evaluated for pH, viscosity, and in vitro release, employing dialysis membrane method. The in vitro release data after fitting to various models revealed it to follow Korsmeyer-Pappas model. Ex vivo permeation studies witnessed high permeation and deposition of resveratrol in skin when compared to plain resveratrol. Dermatokinetic studies elaborated that niosomal gel enhanced the biological half-life and reduced Tmax of the drug, in both the skin layers. Finally, in vivo anti-inflammatory activity of niosomal gel was evaluated by carrageenan induced paw edema model and compared with standard sodium diclofenac gel. The resveratrol entrapped niosomal gel sufficiently reduced the edema and revealed prolonged therapeutic action vis-a-vis the marketed anti-inflammatory gel formulation. Our findings suggest that a topical drug delivery system using niosomal hydrogel could lead to expansion in the anti-inflammatory use of resveratrol.