Pallavi Somvanshi

Gene cloning, expression and homology modeling of hemolysin gene from Aeromonas hydrophila.

Hemolysin is a significant toxin secreted by Aeromonas hydrophila, which contributes pathogenicity of fish to humans. The complete ORF of hemolysin gene (1886 bp) was amplified using PCR. It was cloned in TA and sub-cloned in pET28a vector then transformed into Escherichia coli BL21(DE3) codon plus RP cells expressed by the induction with 1.0 mM of IPTG. The expected size of expressed protein was 68.0 kDa estimated by migration in 12% SDS-PAGE. Anti-His monoclonal antibodies were used to substantiate the recombinant protein by Western blotting. The percent similarity between hemolysin of A. hydrophila with other hemolytic toxins revealed that the hemolysin/aerolysin/cytotoxin sequence varied from 99.35 to 50.40%. Homology modeling was used to construct 3-D structure of hemolysin of A. hydrophila with the known crystal 3-D structure (PDB: 1XEZ). This protein can be used for immunoassays and it is suitable for vaccine candidate against A. hydrophila infection.

Implication of Caspase-3 as a Common Therapeutic Target for Multineurodegenerative Disorders and Its Inhibition Using Nonpeptidyl Natural Compounds

Caspase-3 has been identified as a key mediator of neuronal apoptosis. The present study identifies caspase-3 as a common player involved in the regulation of multineurodegenerative disorders, namely, Alzheimers disease (AD), Parkinsons disease (PD), Huntingtons disease (HD), and amyotrophic lateral sclerosis (ALS). The protein interaction network prepared using STRING database provides a strong evidence of caspase-3 interactions with the metabolic cascade of the said multineurodegenerative disorders, thus characterizing it as a potential therapeutic target for multiple neurodegenerative disorders. In silico molecular docking of selected nonpeptidyl natural compounds against caspase-3 exposed potent leads against this common therapeutic target. Rosmarinic acid and curcumin proved to be the most promising ligands (leads) mimicking the inhibitory action of peptidyl inhibitors with the highest Gold fitness scores 57.38 and 53.51, respectively. These results were in close agreement with the fitness score predicted using X-score, a consensus based scoring function to calculate the binding affinity. Nonpeptidyl inhibitors of caspase-3 identified in the present study expeditiously mimic the inhibitory action of the previously identified peptidyl inhibitors. Since, nonpeptidyl inhibitors are preferred drug candidates, hence, discovery of natural compounds as nonpeptidyl inhibitors is a significant transition towards feasible drug development for neurodegenerative disorders.

Molecular detection and cloning of thermostable hemolysin gene from Aeromonas hydrophila

Aeromonas hydrophila is a major bacterial pathogen associated with hemorrhagic septicemia in aquatic and terrestrial animals including humans. There is an urgent need to develop molecular and immunological assays for rapid, specific and sensitive diagnosis. A new set of primers has been designed for detection of thermostable hemolysin (TH) gene (645 bp) from A. hydrophila, and sensitivity limit for detection of TH gene was 5 pg. The TH gene was cloned, sequenced and analyzed. The G+C content was 68.06%; and phylogeny was constructed using TH protein sequences which had significant homology with those for thermostable and other hemolysins present in several bacterial pathogens. In addition, we have predicted the four and eight T-cell epitopes for MHC class I and II alleles, respectively. These results provide new insight for TH protein containing antigenic epitopes that can be used in immunoassays and also designing of thermostable vaccines.

Fragment based G-QSAR and molecular dynamics based mechanistic simulations into hydroxamic-based HDAC inhibitors against spinocerebellar ataxia

Expansion of polyglutamine (CAG) triplets within the coding gene ataxin 2 results in transcriptional repression, forming the molecular basis of the neurodegenerative disorder named spinocerebellar ataxia type-2 (SCA2). HDAC inhibitors (HDACi) have been elements of great interest in polyglutamine disorders such as Huntington’s and Ataxia’s. In this study, we have selected hydroxamic acid derivatives as HDACi and performed fragment-based G-QSAR, molecular docking studies and molecular dynamics simulations for elucidating the dynamic mode of action of HDACi with His-Asp catalytic dyad of HDAC4. The model was statistically validated to establish its predictive robustness. The model was statistically significant with r2 value of .6297, cross-validated co-relation coefficient q2 value of .5905 and pred_r2 (predicted square co-relation coefficient) value of .85. An F-test value of 56.11 confirms absolute robustness of the model. Two combinatorial libraries comprising of 3180 compounds were created with hydroxamate moiety as the template and their pIC50 activities were predicted based on the G-QSAR model. The combinatorial library created was screened on the basis of predicted activity (pIC50), with two resultant top scoring compounds, HIC and DHC. The interaction of the compounds with His-Asp dyad in terms of H-bond interactions with His802, Asp840, Pro942, and Gly975 residues of HDAC4 was evaluated by docking and 20 ns long molecular dynamics simulations. This study provides valuable leads for structural substitutions required for hydroxamate moiety to exhibit enhanced inhibitory activity against HDAC4. The reported compounds demonstrated good binding and thus can be considered as potent therapeutic leads against ataxia.

Codon optimization of the major antigen encoding genes of diverse strains of influenza a virus

A large number of influenza A virus outbreaks and mortality occurred in the world recently, an urgent attention to develop effective and sufficient quantity of vaccines are needed. Vaccines are generally protein with immunogenic properties and are not expressed in sufficient quantity because of the codon bias, so it is necessary to optimize its codon in the expression host. Codon optimization was used to improve the protein expression in living organisms by increasing the translational efficiency of gene of interest. Two surface antigenic glycoproteins, hemagglutinin (HA) and neuraminidase (NA) are present in influenza A viruses. We have used HA and NA genes from 19 strains of influenza A viruses for codon optimization in E. coli. Both genes of the influenza virus show that the codon adaptation index (CAI) and GC content of the genes in optimized DNA were enhanced significantly (p <0.01) as compared to wild type. CAI and GC of HA in optimized DNA was enhanced by 3.2 (68.5%) and 1.2 (16.2%) fold respectively, while in NA it was increased by 3.3 (69.7%) and 1.2 (15.8%) fold respectively. Our finding demonstrates that the optimized genes could be useful for better expression in host without any truncated proteins and also helpful for protein folding and function. This work provides new insight in the synthetic biology research.

Mechanistic principles behind molecular mechanism of Rifampicin resistance in mutant RNA polymerase beta subunit of Mycobacterium tuberculosis

Evolution of drug‐resistant Mycobacterium strains threatens the TB treatment and control programs globally. Rifampicin (RIF) is an important first line antitubercular drug. Resistance to Rifampicin is caused mainly by mutations in its target RNA polymerase beta subunit protein (RpoB). RpoB contains a Rifampicin resistance determining region (RRDR) and has several potent sites for mutations. In this study, we have investigated mutations of a single site (H451) to eight different amino acids, involved in RIF resistance. Long‐term molecular dynamics simulations were performed on wild type (WT) and mutant protein structures and various structural analysis were carried out to elucidate the dynamic behavior of WT and mutant forms. Essential dynamics uncovered the difference in conformational flexibility and collective modes of motions between WT and mutants. MMPBSA calculations and interaction pattern analysis revealed the binding site relocation in some mutants. This study presents an exhaustive analysis of RIF binding to the WT and mutant RpoB and clearly highlights structural mechanism for differences in stable binding of Rifampicin with WT than the mutant targets. J. Cell. Biochem. 118: 4594–4606, 2017.

Reconstruction and visualization of carbohydrate, N-glycosylation pathways in Pichia pastoris CBS7435 using computational and system biology approaches

Pichia pastoris is an efficient expression system for production of recombinant proteins. To understand its physiology for building novel applications it is important to understand and reconstruct its metabolic network. The metabolic reconstruction approach connects genotype with phenotype. Here, we have attempted to reconstruct carbohydrate metabolism pathways responsible for high biomass density and N-glycosylation pathways involved in the post translational modification of proteins of P. pastoris CBS7435. Both these metabolic pathways play a crucial role in heterologous protein production. We report novel, missing and unannotated enzymes involved in the target metabolic pathways. A strong possibility of cellulose and xylose metabolic processes in P. pastoris CBS7435 suggests its use in the area of biofuels. The reconstructed metabolic networks can be used for increased yields and improved product quality, for designing appropriate growth medium, for production of recombinant therapeutics and for making biofuels.

Genotyping of Aeromonas hydrophila by Box elements

PCR-based DNA fingerprinting techniques were evaluated to genotype eight diseased, particularly normal and environmental isolates of Aeromonas hydrophila. PCR-based fingerprinting method has an advantage of having repetitive sequence also called Box elements that are interspersed throughout the genome in diverse bacterial species. The BOX-PCR fingerprinting technique was evaluated for the discrimination of different isolates of A. hydrophila. All the studied isolates have shown major banding patterns ranged from 500–3000 bp. These finding could be advantageous to investigate the strain level specific fingerprints of A. hydrophila as potential genotypic markers.

Mechanistic Insights into the Binding of Class IIa HDAC Inhibitors toward Spinocerebellar Ataxia Type-2: A 3D-QSAR and Pharmacophore Modeling Approach

Spinocerebellar ataxia (SCA-2) type-2 is a rare neurological disorder among the nine polyglutamine disorders, mainly caused by polyQ (CAG) trinucleotide repeats expansion within gene coding ataxin-2 protein. The expanded trinucleotide repeats within the ataxin-2 protein sequesters transcriptional cofactors i.e., CREB-binding protein (CBP), Ataxin-2 binding protein 1 (A2BP1) leading to a state of hypo-acetylation and transcriptional repression. Histone de-acetylases inhibitors (HDACi) have been reported to restore transcriptional balance through inhibition of class IIa HDACs, that leads to an increased acetylation and transcription as demonstrated through in-vivo studies on mouse models of Huntingtons. In this study, 61 di-aryl cyclo-propanehydroxamic acid derivatives were used for developing three dimensional (3D) QSAR and pharmacophore models. These models were then employed for screening and selection of anti-ataxia compounds. The chosen QSAR model was observed to be statistically robust with correlation coefficient (r2) value of 0.6774, cross-validated correlation coefficient (q2) of 0.6157 and co-relation coefficient for external test set (pred_r2) of 0.7570. A high F-test value of 77.7093 signified the robustness of the model. Two potential drug leads ZINC 00608101 (SEI) and ZINC 00329110 (ACI) were selected after a coalesce procedure of pharmacophore based screening using the pharmacophore model ADDRR.20 and structural analysis using molecular docking and dynamics simulations. The pharmacophore and the 3D-QSAR model generated were further validated for their screening and prediction ability using the enrichment factor (EF), goodness of hit (GH), and receiver operating characteristics (ROC) curve analysis. The compounds SEI and ACI exhibited a docking score of −10.097 and −9.182 kcal/mol, respectively. An evaluation of binding conformation of ligand-bound protein complexes was performed with MD simulations for a time period of 30 ns along with free energy binding calculations using the g_mmpbsa technique. Prediction of inhibitory activities of the two lead compounds SEI (7.53) and ACI (6.84) using the 3D-QSAR model reaffirmed their inhibitory characteristics as potential anti-ataxia compounds.

Homology Modeling and Prediction of Catalytic Amino Acid in the Neurotoxin from Indian Cobra (Naja naja)

The neurotoxin secreted by Indian cobra (Naja naja) binds to acetylcholine receptor of nerve cells, which leads to a lump in the nerve impulse, ceases breathing, thereby causing the death of a person due to suffocation. These neuro- toxins are small peptide with approximately 7 kDa. Homology modeling was performed to generate the 3D structure of neurotoxins (A, B, C and D) of N. naja, using the known protein template crystal structure (PDB: 2CTX). The validation of 3D structure was done using PROCHECK. Furthermore, the prediction of catalytic amino acid residues in the active site domain of the 3-D structure of neurotoxin was identified. The 3-D structures of neurotoxin and catalytic amino acid residue may be used to target and design the antivenom drugs against the Indian cobra.