The 5′-Nor Aristeromycin Analogues of 5′-Deoxy-5′-Methylthioadenosine and 5′-Deoxy-5′-Thiophenyladenosine

To extend the potential of 5′-noraristeromycin (and its enantiomer) as potential antiviral candidates, the enantiomers of the carbocyclic 5′-nor derivatives of 5′-methylthio-5′-deoxyadenosine and 5′-phenylthio-5′-deoxyadenosine have been synthesized and evaluated. None of the compounds showed meaningful antiviral activity.     

Distinct molecular features facilitating ice-binding mechanisms in hyperactive antifreeze proteins closely related to an Antarctic sea ice bacterium

Antifreeze proteins or ice-binding proteins (IBPs) facilitate the survival of certain cellular organisms in freezing environment by inhibiting the growth of ice crystals in solution. Present study identifies orthologs of the IBP of Colwellia sp. SLW05, which were obtained from a wide range of taxa. Phylogenetic analysis on the basis of conserved regions (predicted as the ‘ice-binding domain’ [IBD]) present in all the orthologs, separates the bacterial and archaeal orthologs from that of the eukaryotes’. Correspondence analysis pointed out that the bacterial and archaeal IBDs have relatively higher average hydrophobicity than the eukaryotic members. IBDs belonging to bacterial as well as archaeal AFPs contain comparatively more strands, and therefore are revealed to be under higher evolutionary selection pressure. Molecular docking studies prove that the ice crystals form more stable complex with the bacterial as well as archaeal proteins than the eukaryotic orthologs. Analysis of the docked structures have traced out the ice-binding sites (IBSs) in all the orthologs which continue to facilitate ice-binding activity even after getting mutated with respect to the well-studied IBSs of Typhula ishikariensis and notably, all these mutations performing ice-binding using ‘anchored clathrate mechanism’ have been found to prefer polar and hydrophilic amino acids. Horizontal gene transfer studies point toward a strong selection pressure favoring independent evolution of the IBPs in some polar organisms including prokaryotes as well as eukaryotes because these proteins facilitate the polar organisms to acclimatize to the adversities in their niche, thus safeguarding their existence.     

Anti-herpes virus activities of Achyranthes aspera: An Indian ethnomedicine,and its triterpene acid

The aim of this study was to evaluate the antiviral potential of methanolic extract (ME) of Achyranthes aspera, an Indian folk medicine and one of its pure compound oleanolic acid (OA) against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). The ME possessed weak anti-herpes virus activity (EC₅₀ 64.4 μg/ml for HSV-1 and 72.8 μg/ml for HSV-2). While OA exhibited potent antiherpesvirus activity against both HSV-1 (EC₅₀ 6.8 μg/ml) and HSV-2 (EC₅₀ 7.8 μg/ml). The time response study revealed that the antiviral activity of ME and OA is highest at 2–6 h post infection. The infected and drug-treated peritoneal macrophage at specific time showed increased level of pro-inflammatory cytokines (IL6 and IL12). Further, the PCR of DNA from infected cultures treated with ME and OA, at various time intervals, failed to show amplification at 48–72 h, similar to that of HSV infected cells treated with acyclovir, indicating that the ME and OA probably inhibit the early stage of multiplication (post infection of 2–6 h). Thus, our study demonstrated that ME and OA have good anti-HSV activity, with SI values of 12, suggesting the potential use of this plant.     

Glutamate Receptor 6 Gene (<Emphasis Type="Italic">GluR6</Emphasis> or <Emphasis Type="Italic">GRIK2</Emphasis>) Polymorphisms in the Indian Population: A Genetic Association Study on Autism Spectrum Disorder

Autism is a neurodevelopmental disorder with early manifestation. It is a multifactorial disorder and several susceptible chromosomal regions for autism are identified through genome scan studies. The gene coding for glutamate receptor 6 (GluR6 or GRIK2) has been suggested as a candidate gene for autism based on its localization in the autism specific region on chromosome 6q21 and the involvement of receptor protein in cognitive functions like learning and memory. Despite its importance, so far no studies have been carried out on possible involvement of GluR6 with autism in the Indian population. Therefore in the present study, we have performed genetic analysis of three markers of GluR6 (SNP1: rs2227281, SNP2: rs2227283, SNP3: rs2235076) for possible association with autism through population, and family-based (TDT and HHRR) approaches. DSM-IV criteria and CARS/ADI-R have been utilized for diagnosis. Genotyping analysis for the SNPs has been carried out in 101 probands with autism spectrum disorder, 180 parents and 152 controls from different regions of India. Since the minor allele frequency of SNP3 was too low, the association studies have been carried out only for SNP1 and SNP2. Even though two earlier studies have shown association of these markers with autism, the present case–control and TDT, as well as HHRR analyses have not demonstrated any biased transmission of alleles or haplotypes to the affected offspring. Thus our results suggest that these markers of GluR6 are unlikely to be associated with autism in the Indian population.     

Bioactive Copper-Doped Glass Scaffolds Can Stimulate Endothelial Cells in Co-Culture in Combination with Mesenchymal Stem Cells

Bioactive glass (BG) scaffolds are being investigated for bone tissue engineering applications because of their osteoconductive and angiogenic nature. However, to increase the in vivo performance of the scaffold, including enhancing the angiogenetic growth into the scaffolds, some researchers use different modifications of the scaffold including addition of inorganic ionic components to the basic BG composition. In this study, we investigated the in vitro biocompatibility and bioactivity of Cu²⁺-doped BG derived scaffolds in either BMSC (bone-marrow derived mesenchymal stem cells)-only culture or co-culture of BMSC and human dermal microvascular endothelial cells (HDMEC). In BMSC-only culture, cells were seeded either directly on the scaffolds (3D or direct culture) or were exposed to ionic dissolution products of the BG scaffolds, kept in permeable cell culture inserts (2D or indirect culture). Though we did not observe any direct osteoinduction of BMSCs by alkaline phosphatase (ALP) assay or by PCR, there was increased vascular endothelial growth factor (VEGF) expression, observed by PCR and ELISA assays. Additionally, the scaffolds showed no toxicity to BMSCs and there were healthy live cells found throughout the scaffold. To analyze further the reasons behind the increased VEGF expression and to exploit the benefits of the finding, we used the indirect method with HDMECs in culture plastic and Cu²⁺-doped BG scaffolds with or without BMSCs in cell culture inserts. There was clear observation of increased endothelial markers by both FACS analysis and acetylated LDL (acLDL) uptake assay. Only in presence of Cu²⁺-doped BG scaffolds with BMSCs, a high VEGF secretion was demonstrated by ELISA; and typical tubular structures were observed in culture plastics. We conclude that Cu²⁺-doped BG scaffolds release Cu²⁺, which in turn act on BMSCs to secrete VEGF. This result is of significance for the application of BG scaffolds in bone tissue engineering approaches.     

Molecular docking studies shows tivozanib and lapatinib as potential inhibitors of EML4-ALK translocation mediated fusion protein in non small cell lung cancer

Identification of activating mutations in non-small cell lung cancers (NSCLC) has been a focus in recent years. This led to successful evidence of using tyrosine kinase inhibitors (TKIs) over the standard platinum doublet based chemotherapy as the first line treatment in the metastatic setting.The rearrangements of fusion protein EML4-ALK in NSCLC lead to the use of crizotinib for this class of tumors. Preclinical and Phase 1 clinical studies show that ceritinib is more effective against both crizotinib sensitive and resistant tumors. Although robust responses to crizotinib are observed in NSCLC harboring ALK mutations, majority of tumors eventually become resistant, posing a major challenge in treatment course. Thus, there is a need for the identification and development of second-generation of ALK inhibitors. Computer aided molecular docking data show Tivozanib and Lapatinib bind EML4-ALK with high score. Tivozanib is in clinical trials for renal cell cancer and Lapatinib is a known dual tyrosine kinase inhibitor effective in breast cancer patients with HER2 over-expression. Additional data on these compounds for use in EML4-ALK positive NSCLC will provide evidence for use in patients treated with crizotinib. Data shows the importance of computer aided molecular docking in developing candidates with improved activity for further consideration in vitro and in vivo validation.     

Identification of Crucial Amino Acids of Bacterial Peptide Deformylases Affecting Enzymatic Activity in Response to Oxidative Stress

Peptide deformylase (PDF) is an essential bacterial metalloprotease involved in deformylation of N-formyl group from nascent polypeptide chains during protein synthesis. Iron-containing variants of this enzyme from Salmonella enterica serovar Typhimurium (sPDF) and Mycobacterium tuberculosis (mPDF), although inhibited by oxidizing agents like H₂O₂, exhibited strikingly different 50% inhibitory concentrations (IC₅₀s) that ranged from nanomolar (sPDF) to millimolar (mPDF) levels. Furthermore, the metal dissociation rate was higher in sPDF than mPDF. We hypothesized that a restriction in entry of environmental oxygen or oxidizing agents into the active site of mPDF might be the cause for such discrepancies between two enzymes. Since the active-site residues of the two proteins are similar, we evaluated the role of the oxidation-prone noncatalytic residue(s) in the process. Amino acid sequence analysis revealed that Cys-130 of sPDF corresponds to Met-145 of mPDF and that they are away from the active sites. Swapping methionine with cysteine in mPDF, the M145C protein displayed a drastic decrease in the IC₅₀ for H₂O₂ and an increased metal dissociation rate compared to the wild type. Matrix-assisted laser desorption ionization (MALDI) analysis of a trypsin-digested fragment containing Cys-145 of the M145C protein also indicated its increased susceptibility to oxidation. To incorporate residues identical to those of mPDF, we created a double mutant of sPDF (C130M-V63C) that showed increased IC₅₀ for H₂O₂ compared to the wild type. Interestingly, the oxidation state of cysteines in C130M-V63C was unaffected during H₂O₂ treatment. Taken together, our results unambiguously established the critical role of noncatalytic cysteine/methionine for enzymatic sensitivity to H₂O₂ and, thus, for conferring behavioral distinction of bacterial PDFs under oxidative stress conditions.     

Immunotoxicity of phosphamidon following subchronic exposure in albino rats

Effect of subchronic doses of phosphamidon exposure on humoral and cell mediated immune (CMI) responses were studied in male albino rats using SRBC, ovalbumin and KLH as antigens. Humoral immune responses were assessed by estimating antibody titre against antigen and splenic plaque forming cells (PFC) assay. CMI responses were studied by using leucocyte migration inhibition (LMI), macrophage migration inhibition (MMI) and delayed type hypersensitivity (DTH) response. Results obtained in the present study revealed marked suppression of humoral and CMI responses in a dose dependent pattern. Hence, suppression of immune responses by phosphamidon even at subchronic doses is clearly an important aspect for its safety evaluation.