Sharpening gene editing toolbox in Arabidopsis for plants

Journal of Plant Biochemistry and Biotechnology - Arabidopsis thaliana is considered as an indispensable model system across various disciplines of modern plant biology. The short life cycle, well...     

Mutations in exons 10 and 11 of human glucokinase result in conformational variations in the active site of the structure contributing to poor substrate binding – explains hyperglycemia in type 2 diabetic patients

Mutations in the glucokinase (GK) gene play a critical role in the establishment of type 2 diabetes. In our earlier study, R308K mutation in GK in a clinically proven type 2 diabetic patient showed, structural and functional variations that contributed immensely to the hyperglycemic condition. In the extension of this work, a cohort of 30 patients with established type 2 diabetic condition were chosen and the exons 10 and 11 of GK were PCR-amplified and sequenced. The sequence alignment showed A379S, D400Y, E300A, E395A, E395G, H380N, I348N, L301M, M298I, M381G, M402R, R308K, R394P, R397S, and S398R mutations in 12 different patients. The structural analysis of these mutated GKs, showed a variable number of β-α-β units, hairpins, β-bulges, strands, helices, helix–helix interactions, β-turns, and γ-turns along with the RMSD variations when compared to wild-type GK. Molecular modeling studies revealed that the substrate showed variable binding orientations and could not fit into the active site of these mutated structures; moreover, it was expelled out of the conformations. Therefore, these structural variations in GK due to mutations could be one of the strongest reasons for the hyperglycemic levels in these type 2 diabetic patients.     

An Assessment of Growth Media Enrichment on Lipid Metabolome and the Concurrent Phenotypic Properties of Candida albicans

A critical question among the researchers working on fungal lipid biology is whether the use of an enriched growth medium can affect the lipid composition of a cell and, therefore, contribute to the observed phenotypes. One presumption is that enriched medias, such as YPD (yeast extract, peptone and dextrose), are likely to contain lipids, which may homogenize with the yeast lipids and play a role in masking the actual differences in the observed phenotypes or lead to an altered phenotype altogether. To address this issue, we compared the lipids of Candida albicans, our fungus of interest, grown in YPD or in a defined media such as YNB (yeast nitrogen base). Mass spectrometry-based lipid analyses showed differences in the levels of phospholipids, including phosphatidylinositol, phosphatidylglycerol, lyso-phospholipids; sphingolipids, such as mannosyldiinositolphosphorylceramide; and sterols, such as ergostatetraenol. Significant differences were observed in 70 lipid species between the cells grown in the two media, but the two growth conditions did not affect the morphological characteristics of C. albicans. The lipid profiles of the YNB- and YPD-grown C. albicans cells did vary, but these differences did not influence their response to the majority of the tested agents. Rather, the observed differences could be attributed to the slow growth rate of the Candida cells in YNB compared to YPD. Notably, the altered lipid changes between the two media did impact the susceptibility to some drugs. This data provided evidence that changes in media can lead to certain lipid alterations, which may affect specific pathways but, in general, do not affect the majority of the phenotypic properties of C. albicans. It was determined that either YNB or YPD may be suitable for the growth and lipid analysis of C. albicans, depending upon the experimental requirements, but additional precautions are necessary when correlating the phenotypes with the lipids.     

Gelatin grafted poly(D,L-lactide) as an inhibitor of protein aggregation: An in vitro case study

Amyloids are a group of proteins that are capable of forming aggregated amyloid fibrils, which is responsible for many neurodegenerative diseases including Alzheimer's disease (AD). In our previous study, synthesis and characterization of star-shaped poly(D,L-lactide)-b-gelatin (ss-pLG) have been reported. In the present work, we have extended our work to study ss-pLG against protein aggregation. To the best of our knowledge, this is the first report on the inhibition of amyloid fibrillation by protein grafted poly(D,L-lactide). Bovine serum albumin (BSA) was chosen as the model protein, which readily forms fibril under high temperature. We found that ss-pLG efficiently suppressed the fibril formation of BSA compared with gelatin (Gel), which was supported by Thioflavin T assay, circular dichroism (CD) spectroscopy and atomic force microscopy (AFM). In addition, ss-pLG significantly curtailed amyloid-induced hemolysis. We also found that incubation of ss-pLG with neuroblastoma cells (MC65) protected the cells from fibril-induced toxicity. The rescuing efficiency of ss-pLG was better than Gel, which could be attributed to the reduced lamella thickness in branched ss-pLG. These results suggest the significance of gelatin grafting, which probably allows gelatin to interact with the key residues of the amyloidogenic core of BSA effectively.     

Discovery of hit molecules targeting allosteric site of hepatitis C virus NS5B polymerase

Abstract

Nonstructural protein 5B (NS5B), the RNA-dependent RNA polymerase of Hepatitis C Virus (HCV), plays a key role in viral amplification and is an attractive and most explored target for discovery of new therapeutic agents for Hepatitis C. Though safe and effective, NS5B inhibitors were launched in 2013 (Sovaldi) and 2014 (Harvoni, Viekira Pak), the high price tags of these medications limit their use among poor people in developing countries. Hence, still there exists a need for cost-effective and short duration anti-HCV agents especially those targeting niche patient population who were non-respondent to earlier therapies or with comorbid conditions. The present study describes the discovery of novel non-nucleoside (NNI) inhibitors of NS5B using a series of rational drug design techniques such as virtual screening, scaffold matching and molecular docking. 2D and 3D structure based virtual screening technique identified 300 hit compounds. Top 20 hits were screened out from identified hits using molecular docking technique. Four molecules, that are representative of 20 hits were evaluated for binding affinity under in vitro conditions using surface plasmon resonance-based assay and the results emphasized that compound with CoCoCo ID: 412075 could exhibit good binding response toward NS5B and could be a potential candidate as NS5B inhibitor.

Communicated by Ramaswamy H. Sarma     

Synthesis and solution conformation studies of the modified nucleoside N(4),2'-O-dimethylcytidine (m(4)Cm) and its analogues

The dimethylated ribosomal nucleoside m(4)Cm and its monomethylated analogues Cm and m(4)C were synthesized. The conformations (syn vs anti) of the three modified nucleosides and cytidine were determined by CD and 1D NOE difference spectroscopy. The ribose sugar puckers were determined by the use of proton coupling constants. The position of modification (e.g., O vs N methylation) was found to have an effect on the sugar pucker of cytidine.     

Molecular modeling,docking and ADMET studies towards development of novel Disopyramide analogs for potential inhibition of human voltage gated sodium channel proteins

The sodium “channelopathies” are the first among the ion channel diseases identified and have attracted widespread clinical and scientific interests. Human voltage gated sodium channels are sites of action of several antiarrhythmic drugs, local anesthetics and related antiepileptic drugs. The present study aims to optimize the activity of Disopyramide, by modification in its structures which may improve the drug action by reducing its side effects. Herein, we have selected Human voltage-gated sodium channel protein type 5 as a potent molecular target. Nearly eighty analogs of Disopyramide are designed and optimized. Thirty are selected for energy minimization using Discovery studio and the LigPrep 2.5. Prior to docking, the active sites of all the proteins are identified. The processing, optimization and minimization of all the proteins is done in Protein preparation wizard. The docking study is performed using the GLIDE. Finally top five ranked lead molecules with better dock scores are identified as having strong binding affinity to 2KAV protein than Disopyramide based on XP G scores. These five leads are further docked with other similar voltage gated sodium channel proteins (PDB IDs: 2KBI, 4DCK, 2L53 and 4DJC) and the best scoring analog with each protein is identified. Drug likeliness and comparative bioactivity analysis for all the analogs is done using QikProp 3.4. Results have shown that the top five lead molecules would have the potential to act as better drugs as compared to Disopyramide and would be of interest as promising starting point for designing compounds against various Sodium channelopathies.     

Genetic variation at the growth hormone (GH1) and growth hormone receptor (GHR) loci as a risk factor for hypertension and stroke

An increased prevalence of both hypertension and cerebrovascular stroke is apparent in growth hormone (GH) deficiency whilst hypertension is a frequent complication in acromegaly. This has suggested a possible link between GH, stature and arterial function. Since the risk of both hypertension and stroke also appears to be inversely correlated with adult height, we have instigated an exploratory study to assess whether inter-individual variation in the genes encoding human growth hormone (GH1) and the GH receptor (GHR) might be associated with an increased risk of hypertension and stroke. GH1 promoter haplotypes were found to differ significantly not only between hypertensive patients (n=111) and controls (n=121) but also between stroke patients (n=155) and controls (n=158). Intriguingly, the association between GH1 promoter haplotype and risk of hypertension was much greater in females than in males. An inverse correlation between height and central systolic blood pressure was apparent in both hypertensive patients and normal controls but was much stronger in individuals carrying at least one GH1 promoter risk haplotype. The GH1 genotype therefore constitutes a risk factor for hypertension that interacts with stature. A strong association was found between the presence of at least one GH1 risk haplotype and a family history of stroke at an early age (odds ratio: 9.07, 95% confidence interval: 1.14–72.22). Three novel GH variants (Arg16His, Phe176Cys, Cys189Arg) were identified during the course of this study. Although two exhibited markedly reduced biological activity in vitro, their clinical significance remains unclear. No association was found between GHR genotype and either hypertension or stroke, nor was any interaction noted between GHR and GH1 genotypes in terms of a disease association. However, an association between GHRd3 genotype and hypertension was observed among stroke patients, particularly females. Elevated HDL was found to be a risk factor for hypertension in individuals lacking a copy of the GHRd3 allele. Weak associations with GHR genotype were also noted for peripheral systolic and diastolic blood pressure in hypertensive patients. Although the underlying mechanisms are still unclear, our findings are consistent with a complex relationship between height, hypertension, GH1 promoter haplotype, GHR polymorphism and the risk of stroke.     

Molecular designing and in silico evaluation of darunavir derivatives as anticancer agents

Darunavir is a synthetic nonpeptidic protease inhibitor which has been tested for anticancer properties. To deduce and enhance the anticancer activity of the Darunavir, we have modified its reactive moiety in an effective way. We designed 9 analogues in ChemBioOffice 2010 and minimized using the LigPrep tool of Schrödinger 2011. These analogues can obstruct the activity of other signalling pathways which are implicated in many tumors. Results of the QikProp showed that all the analogues lied in the specified range of all the pharmacokinetic (ADMET) properties required to become the successful drug. Docking study was performed to test its anticancer activity against the biomarkers of the five main types of cancers i.e. bone, brain, breast, colon and skin cancer. Grid was generated for each oncoproteins by specifying the active site amino acids. The binding model of best scoring analogue with each protein was assessed from their G-scores and disclosed by docking analysis using the XP visualizer tool. An analysis of the receptor-ligand interaction studies revealed that these nine Darunavir analogues are active against all cancer biomarkers and have the features to prove themselves as anticancer drugs, further to be synthesized and tested against the cell lines.