Identification, Isolation and Amplification of BRCA1 Gene Involved on Breast Cancer.

Cancer is a disease that begins in the cells of the body which is characterized by uncontrolled, uncoordinated and undesirable cell division. If a cell accumulates critical mutations in five or six of the protooncogenes, tumour suppressor genes and DNA repair genes are likely to result in a fully malignant cell, capable of forming a tumour. In this work we described the isolation and amplification of the BRCA1 gene. Primers were designed and synthesised later used to amplify the BRCA1 gene. The total new workflow includes all steps from purified DNA to data analysis, and includes PCR for all amplicons covering the gene, PCR cleanup, cycle sequencing, electrophoresis, and data analysis. To simplify workflows and decrease the time-to-result, we focused on the method “one sample, one assay” approach. The success of this workflow was the 24-well plate design, which contained prespotted PCR primers covering the gene and also included multiplex nontemplate controls. The workflow was developed using a Genetic Analyzer and bands were observed.

Phytochemical screening and insilico approach for the identification of anti stress compounds from medicinal plants.

In India, many forms of alternative medicines are available for those who cannot be helped by conventional medicine. Ayurvedha and Herbal medicine are two important forms of alternative medicine that is widely available in India. This work was mainly concerned with the identification of the therapeutic properties of Indian medicinal plant extracts. Everyone knows that medicinal plants have disease curing properties and this is due to the compounds presents in the extracts used for the treatment. So we identified the compounds of different medicinal plants like Celastrus paniculatus, Withania somnifera, Convolvulus pluricaulis, Rauvoifia Serpentina etc which are used as medicine for Stress in Ayurvedha from previous literature. After identification using chemsketch software these compounds were designed and screened for antistress property. The proteins responsible for stress in Homo sapiens were collected using Protein databank (PDB). Active sites were identified and used for docking with the compounds. Then the compounds were docked to the Calcium channel in order find better inhibitor. Among 33 compounds 10 compounds showed best docking results for each protein. ADMET studies were performed using Molinspiration and OSIRIS server.

Docking studies of emblica officinalis compounds with DNA polymerase.

For understanding the molecular mechanism one can also adopt in silico approach where the interactions such as Protein-protein interactions, Protein-compound interactions, DNA-compound interactions, DNA-protein interactions are commonly used. Here we focused on the fate of these interactions for some of the compounds of Emblica officinalis with normal DNA polymerase β. Efficacy of these compounds involves design/taking from the databases of these molecules that are probable to act on the bimolecular target. DNA polymerase beta protein PDB (1DK3) was used to dock with compounds like Quercetin, Myricetin and 3,7,3,4-Tetra Hydroxy Flavone. All these compounds showed best binding studies with DNA polymerase beta.

Computational modeling and drug designing of Lipoprotein Lipase (LPL) involved in ischemic stroke.

Homology modeling and flexible docking of Lipoprotein Lipase has been studied in silico approach. Blast result was found to have similarity with Lipoprotein Lipase of 83% identity with 1LPA. Active site of LPL protein was identified by CASTP. Large potential drugs were designed for identifying molecules that can likely bind to protein target of interest. The different drug derivatives designed were used for docking with the generated structure, among the 10 derivatives designed, 3rd derivative showed highest docking result. The drug derivatives were docked to the protein by hydrogen bonding interactions and these interactions play an important role in the binding studies. Our investigations may be helpful for further studies.

A study on nitrogen fixing and phosphate solubilizing microorganisms on growth and Physiology of Plumbago Zeylanica.L.

Organisms that are commonly used as biofertilizers component are nitrogen fixers (N-fixer), potassium solubilizer (K-solubilizer) and phosphorus solubilizer (P- solubilizer), or with the combination of molds or fungi. Most of the bacteria included in biofertilizer have close relationship with plant roots. In this work we have selected plumbago zeylanica.L plant to study the effect of Azotobacter on the growth of roots, stem, and leaves. Also biochemical characterization was done to identify the effect of Azotobacter in Plumbago. The maximum shoot length was recorded in T4 plants (43.51) on 90th days of plant growth after transplanting the plants. There was a significant increase at 5 % level in the root length from 30th days to 90th days in all the treatments. The maximum number of leaves were found in T4 treatment followed by T3 and T2. Minimum numbers of leaves were found in T1 (1083). On 60th day and 90th day also the total chlorophyll content was maximum in T4 treated plants followed by T3, T2 plants. The amount of reducing sugars (μg/g) in shoots of T4, T3 and T2 plants on 30th, 60th and 90th days were significantly high when compared to T1 plants. The content of protein in roots of T2, T3 and T4 plants on 30th, 60th and 90th days were significantly high when compared to protein content of T1 plants.

Molecular identification of bacteria isolated from meat sample.

Contamination of food with bacteria moulds, yeasts, viruses, protozoa or parasites could occur within food premises and result in unfit or unsafe food. Microbial contamination is frequently the result of ignorance, carelessness or negligence by the food operator, or due to lack of space or inappropriate design of food areas. In this work we have identified meat contaminating bacteria through molecular techniques. The bacterium identified through morphological and biochemical tests was sporosarcina halofila. This was confirmed after amplification of genomic DNA with suitable primers. Plasmid was isolated and it was identified to be 11kb. PCR amplified product was 100bp and primers were designed accordingly to the suitable organism.

Modelling and drug designing of glutamate receptor involved in alzheimer’s disease.

Glutamate receptors dysfunction plays an important role in the pathogenesis and disturbance which is probably a secondary phenomenon to other neurochemical, genetic or metabolic changes, and essential to the development of Alzheimer Disease. Glutamate receptors are synaptic receptors, which are located on the membranes of neuronal cells. Glutamate is used to assemble proteins and also it is abundant in many areas of the body, but it also functions as a neurotransmitter and is particularly abundant in the nervous system. In this work we have modeled a three dimensional structure for Glutamate [NMDA] receptor subunit using MODELLER7V7 software with 2RC7 (Crystal Structure of the NR3A Ligand Binding Core Complex with Glycine) as template. With the aid of Molecular dynamics and Molecular simulations studies it was identified that the generated structure was reliable. This structure was used to identify better inhibitor using docking studies. The drug derivatives were docked to the Glutamate receptor structure into the active site containing residues such as ASP21, LEU30, TYR31, HIS59, and MET60. Among the 21 derivatives 14 were docked and 3rd drug derivative showed better docking energy than the others. Our experimental studies can be further used to develop a better drug for Alzheimer disease.

Homology modeling and drug designing of 18KDA antigen in mycobacterium leprae.

Most of the antigens of Mycobacterium leprae that have been identified are members of stress protein families. 18kDa antigen of M. leprae is an important antigen in the immune response to leprosy. This protein antigen of M. leprae is related to the family of small heat shock protein. To predict the structure of 18kDa antigen and to understand the mechanisms of inhibitors interaction, a threedimensional model was generated based on the Crystal Structure and assembly of eukaryotic small heat shock protein (PDB: 1GME) by using MODELLER7v7. The structure having a least modeller objective function was used as a starting point for picoseconds-duration molecular dynamics simulations. With the aid of the molecular dynamics and minimization methods, the final refined model was obtained and was further assessed by ERRAT, WHATCHECK and PROCHECK, which suggested that the refined model was reliable. Docking studies were performed by using the models with 2-mercaptoethanol and 3-amino- 5-methylhexanoic acid inhibitors. The results indicate that the 3-amino-5,5-diphenylpentanoic acid has more affinity than the other drug derivatives. The docking studies also suggest that MET-03, ARG-04, ASP-31, ALA-32, TRP-33, ARG-34, GLU-35 ARG-89, GLN-90 LEU-91 and VAL-92 are important determinant residues in binding with ligands. From the docking studies, we also suggest that GLU-35, in 18kDa protein domain is an important residue in binding.

Computational approach in determination of curcumin as an anti bacterial drug.

Turmeric has anti microbial activity due to Curcuminoid compounds. Staphylococcus aureus inhibition was done by using Curcumin, a principle compound present in Turmeric. Curcumin acts on Sortase, a membrane protein and inhibits its growth. The purpose of this work is to find the mode of action of Curcumin on Sortase using Bioinformatic tools. In the present study, we have selected Curcumin and docked to the Sortase of Staphylococcus aureus. Homology modeling of Sortase enzyme has been performed based on the crystal structure of the 1IJA A by using Modeller software. With the aid of the molecular mechanics and molecular dynamics methods, the final model is obtained and further assessed by procheck and verify 3D graph programs, which showed that the final refined model is reliable. With this model, a flexible docking study of Sortase enzyme with Curcumin was performed which showed best docking result. It was found that VAL 205, LYS206 of Sortase are the amino acids which play important role in strong hydrogen bonding interaction with Curcumin. To the best of our knowledge VAL 205, LYS 206 are conserved in the domain and may be important for structural integrity or maintaining the hydrophobicity of the inhibitor-binding pocket. Our results may be helpful for further experimental investigations.

Docking studies on xanthones of mangosteen as cox-2 inhibitors.

The prostaglandins found in most of the tissues and organs are synthesized by sequential oxidation of cyclooxygenases (COX-1 and COX-2). Prostaglandins synthesized by COX-1 are responsible for the protection of gastrointestinal tract and by COX-2 are responsible for inflammation and pain. The objective of this investigation was to characterize and determine the effect of α-mangostin, β-mangostin and γ-mangostin on COX-1 and COX-2. We have carried out the docking of α, β and γ-mangostin inhibitors into the three dimensional structure of COX-1 and COX-2 enzymes using GOLD software. The inhibitor binding positions and affinity were evaluated using GOLD scoring fitness functions. We identified that amino acid residues Leu52, Arg49, Val33 in COX-1 and Ala18, Ser23, Asp38, Cys22 in COX-2 are important for inhibitor recognition via hydrogen bonding interactions. These hydrogen bonding interactions play an important role for stability of the complex. This information can be exploited to design Mangostin based inhibitors. Our results may be helpful for further experimental investigations.

Virtual screening of flavonoids as inhibitory agents of P-glycoprotein.

Flavonoids are constituents of fruits, vegetables, and plant derived beverages, as well as components in herbal dietary supplements. The objective of this investigation was to characterize and determine the effect of the Flavonoids on P-glycoprotein (P-gp) which is an important protein involved in multidrug resistance (MDR). Homology modeling of Pglycoprotein (Human) has been performed based on the crystal structure of the 2HYD (Chain A; Structure of a bacterial multidrug ABC transporter) by using Modeller software. With the aid of the molecular mechanics and molecular dynamics methods, the final model is obtained and is further assessed by procheck and verify 3D graph programs, which showed that the final refined model is reliable. With this model, a flexible docking study of P-glycoprotein with a group of Flavonoids which were selected from the previous publications was performed. The results indicated that GLN- 47, TYR -53, SER -83, ILE- 87, GLY -100, ARG -154 in P- glycoprotein are important determinant residues in binding as they have strong hydrogen bonding with Flavonoids. These hydrogen binding interactions play an important role for stability of the complex. Among the 13 Flavonoids docked, Acetylgenistin showed best docking result with Pglycoprotein. Our results may be helpful for further experimental investigations.