Structural dynamics and interactions of Xeroderma pigmentosum complementation group A (XPA98–210) with damaged DNA

Nucleotide excision repair (NER) in higher organisms repair massive DNA abrasions caused by ultraviolet rays, and various mutagens, where Xeroderma pigmentosum group A (XPA) protein is known to be involved in damage recognition step. Any mutations in XPA cause classical Xeroderma pigmentosum disease. The extent to which XPA is required in the NER is still unclear. Here, we present the comparative study on the structural and conformational changes in globular DNA binding domain of XPA_98–210 in DNA bound and DNA free state. Atomistic molecular dynamics simulation was carried out for both XPA_98–210 systems using AMBER force fields. We observed that XPA_98–210 in presence of damaged DNA exhibited more structural changes compared to XPA_98–210 in its free form. When XPA is in contact with DNA, we found marked stability of the complex due to the formation of characteristic longer antiparallel β-sheets consisting mainly lysine residues.     

A unique family of Mrr-like modification-dependent restriction endonucleases

Mrr superfamily of homologous genes in microbial genomes restricts modified DNA in vivo. However, their biochemical properties in vitro have remained obscure. Here, we report the experimental characterization of MspJI, a remote homolog of Escherichia coli’s Mrr and show it is a DNA modification-dependent restriction endonuclease. Our results suggest MspJI recognizes ^mCNNR (R = G/A) sites and cleaves DNA at fixed distances (N₁₂/N₁₆) away from the modified cytosine at the 3′ side (or N₉/N₁₃ from R). Besides 5-methylcytosine, MspJI also recognizes 5-hydroxymethylcytosine but is blocked by 5-glucosylhydroxymethylcytosine. Several other close homologs of MspJI show similar modification-dependent endonuclease activity and display substrate preferences different from MspJI. A unique feature of these modification-dependent enzymes is that they are able to extract small DNA fragments containing modified sites on genomic DNA, for example ∼32 bp around symmetrically methylated CG sites and ∼31 bp around methylated CNG sites. The digested fragments can be directly selected for high-throughput sequencing to map the location of the modification on the genomic DNA. The MspJI enzyme family, with their different recognition specificities and cleavage properties, provides a basis on which many future methods can build to decode the epigenomes of different organisms.     

Antimicrobial and Antioxidant Property of a True Mangrove Rhizophora apiculata Bl.

Mangroves are abundant in bioactive natural substances that fight off pathogenic diseases. Different parts of R. apiculata, an abundant mangrove found in Bhitarkanika National Park, India were extracted with methanol and a mixture of solvents methanol/ethanol/chloroform (60 : 20 : 20) to evaluate their antimicrobial properties. The combination solvent extract of bark had the highest zone of inhibition (ZOI) of 18.62 mm against Pseudomonas aeruginosa and a ZOI of 17.41 mm against Streptococcus mitis. Bark extracts had the highest DPPH (43 %) and FRAP (96 %) activities. The combination solvent bark extract of R. apiculata had the highest ZOI of 20.42 mm (lowest MIC of 2.12 μg/ml) against Candida albicans and ZOI of 15.33 mm (MIC of 3.02 μg/mL) against Penicillium chrysogenum. Combination bark extracts of R. apiculata contained flavanols than methanolic extracts. The crude extract of R. apiculata bark made with a mixture of solvents containing more active ingredients could be used in novel drug formulation.     

Fc gamma receptor polymorphisms in systemic lupus erythematosus and their correlation with the clinical severity of the disease

Receptors for the Fc domains of IgG (Fc γ R) play a critical role in linking humoral and cellular immune responses. The various Fc γ R genes may contribute to differences in infectious and immune related diseases in various ethnic populations. Polymorphisms of Fc γ R mainly Fc γ R IIA, IIB, IIIA, IIIB have been identified as genetic factors influencing susceptibility to disease or disease course of a prototype autoimmune disease like Systemic Lupus Erythematosus (SLE). Activated and inhibitory Fc γ Rs seem to play an important role in the pathogenesis of SLE, in initiation of autoimmunity, the subsequent development of inflammatory lesions and finally immune clearance mechanisms. This review focuses on the role of Fc γ R polymorphism and their association with clinical manifestations and initiation of autoantibody production, inflammatory handling of immune complexes and disease development in SLE patients.     

Levels of oxidative damage and proinflammatory cytokines are enhanced in patients with active vitiligo

Vitiligo is an autoimmune depigmenting skin disease characterised by loss of melanocytes wherein oxidative stress is proposed to be the initial triggering factor with subsequent immune dysregulation. This study aimed to evaluate the relationship, if any, between the generation of reactive oxygen species (ROS), markers of oxidative damage and circulating cytokines in patients with active vitiligo. The generation of ROS in erythrocytes and neutrophils was significantly higher in patients with active vitiligo than healthy controls. Alongside, markers of oxidative stress-mediated damage namely lipid peroxidation, DNA damage and protein carbonylation were evaluated. Patients with active vitiligo demonstrated increased lipid and DNA damage but minimal protein damage. There was a significant decline in the free radical scavenging capacity of active vitiligo cases. A positive correlation existed between baseline levels of ROS and lipid peroxidation as also DNA damage. Patients with active vitiligo demonstrated an increase in several proinflammatory (IL-6, TNF-α, IL-1β, IFN-γ and IL-8) and some anti-inflammatory/immunoregulatory (IL-5 and IL-10) cytokines. Importantly, the levels of IFN-γ and IL-10 consistently correlated with the generation of ROS, markers of damage and their free radical scavenging capacity. Taken together, patients with active vitiligo demonstrated an enhanced generation of ROS in erythrocytes and neutrophils which mediated lipid peroxidation, DNA damage and coupled with a decline in their antioxidant capacity created a pro-oxidant milieu that favoured tissue damage and potential generation of neoantigens, accounting for disease progression.     

Kinetic modeling as a tool to understand the influence of cell culture process parameters on the glycation of monoclonal antibody biotherapeutics

Glycation, the nonenzymatic reaction between the reducing sugar glucose and the primary amine residues on amino acid side chains, commonly occurs in the cell culture supernatant during production of therapeutic monoclonal antibodies (mAbs). While glycation has the potential to impact efficacy and pharmacokinetic properties for mAbs, the most common undesirable impact of glycation is on the distribution of charged species, often a release specification for commercial processes. Existing empirical approaches are usually insufficient to rationalize the effects of cell line and process changes on glycation. To address this gap, we developed a kinetic model for estimating mAb glycation levels during the cell culture process. The rate constant for glycation, including temperature and pH dependence, was estimated by fitting the kinetic model to time-course glycation data from bioreactors operated at different process settings that yielded a wide range of glycation values. The parameter values were further validated by independently estimating glycation rate constants using cell-free incubation studies at various temperatures. The model was applied to another mAb, by re-estimating the activation energy to account for effect of a glycation “hotspot”. The model was further utilized to study the role of temperature shift as an approach to reduce glycation levels in the manufacturing process for mAb2. While a downshift in temperature resulted in lowering of glycation levels for mAb2, the model helped elucidate that this effect was caused due to contribution from changes in glucose consumption, mAb secretion and temperature, instead of a direct impact of temperature alone on the kinetic rate of glycation.     

Impact of lifestyle on circadian orientation and sleep behaviour

Sleep and Biological Rhythms - Individual variability in preferred sleep timing is popularly described as chronotype. This study was conducted on 2105 subjects living in remote (500; without...