Screening of Atherosclerotic Druggable Targets from the Proteome Network of Differentially Expressed Genes.

Differently expressed genes of atherosclerotic sample analysis are helpful to sort the prominent genes that influence the plaque formation and progression. Scientific evidence-based protein-protein interaction network (PPIN) studies were used to find hub proteins in complex disease conditions. Druggable capacity is one of the important parameters to confirm as a successful drug target. Construction of protein interaction network and principal node analysis (PNA) on atherosclerotic data sets lead to screen the hub proteins. Furthermore, druggable property of protein pocket confirms the targetability of susceptible target candidates and for target selection. Differentially expressed genes are identified through GEO2R analyzer on data sets of various atherosclerotic samples. STRING database and Cytoscape are employed to construct PPIN. Targets were identified by PNA such as centrality measures and clustering algorithm. Gene Ontology enrichment also used as one of the screening parameters to filter the candidates related to atherosclerotic terms. Topological evaluation of target protein was successfully done by ITASSER and GROMACS, respectively. Grid-based principle of DoGSiteScorer is utilized for druggability analysis. Six proteins such as integrin alpha L (ITGAL), metallothionein 1F (MT1F), metallothionein 1X (MT1X), P-selectin glycoprotein ligand-1 (SELPLG), solute carrier family 30 A, zinc transporter protein (SLC30A1), and TNFSF13B are screened as potential biomarkers through network-based analysis. Among the six, ITGAL, SELPLG, SLC30A1, and TNSF13B are identified as better prioritized atherosclerotic targets through druggability efficiency.

Nitric Oxide Inhibition Assay and the Respective Target Identification of an Aptamer Designed to Control Atherosclerosis.

INTRODUCTION: Aptamers are emerging newer therapeutics and diagnostics whichcan be designed to bind any kind of target proteins. Vascular endothelial damage by the excess amount of nitric oxide production in systemic circulation leads to the secretion of inflammatory chemoattractants and cell adhesions are the prime pro-atherogenic events in the formation of plagues at atrial intimal layers due to oxidation - sensitive mechanisms. Nitric oxide inhibition assay is one of the valuable qualitative anti-atherosclerosis matrices. METHODS: In this research, Nitric oxide inhibition efficiency of an ssDNA aptamer on cell lines was studied, and the respective targets of that aptamer were identified by network analysis. The aptamer used here was originally designed for Selectin P Ligand Protein to control the atherogenic process. 20 nM of aptamer solution in LipofectamineTM 2000 sshowshighest level 70.5% inhibition of nitric oxide liberation on 24 hours cultured medium of lipopolysaccharide stimulated murine macrophage RAW 264.7 cell lines. RESULTS: Protein interaction network analysis of the nitric oxide synthesis pathway interactors and the molecular docking analysis with network resulted in proteins such as AKT serine/threonine kinase 1, calmodulin, estrogen receptor 1, and nitric oxide synthase-3, which confirmed that the G - quadruplex Model of 18-mer sequence effectively bound to the active sites of estrogen receptor 1, and nitric oxide synthase-3. CONCLUSION: The aptamer designed for atherosclerotic target has also exertedsignificant nitric oxide inhibition to control the atherogenic events through the proteins such as, AKT1, NOS3 and ESR1.

In-silico strategies for identification of potent inhibitor for MMP-1 to prevent metastasis of breast cancer.

Matrix Metalloproteinase-1 (MMP-1) has been often upregulated in advanced breast cancers, known to participate in ECM degradation, migration, invasion, thus leading to metastasis. Due to these effects, the condition is often reported to inversely correlate with survival in advanced breast cancers. In the present study, in-silico method was adopted based on selective non zinc binding inhibitors of MMP-1. ADME properties were predicted for PASS filtered compounds and docking calculations were performed using Glide XP and IFD protocols of Schrodinger program. We identified six ligands as potent inhibitors and validated by observing structures and the interactions of MMP-1. The identified hits were validated using molecular dynamics simulation studies. Electronic structure analysis was performed for two top hit compounds myricetin and quercetin using density function theory (DFT) at B3LYP/6-31**G level to understand their molecular reactivity. Finally, one compound myricetin has emerged as the structurally stable compound with -7.801 kcal/mol and reasonable pose inside the binding site. Molecular dynamics results indicated that myricetin forms a stable interaction with the key amino acid residues such as Glu209, Glu219, Tyr240 and Pro238. In addition, it did not form any binding with the catalytic zinc at its active site. The interaction pattern of myricetin at its substrate binding site exhibited to be potent MMP-1 inhibitor. DFT study also showed that it has more potent inhibitory effect and solubility. These factors altogether show that myricetin could be considered as the best among the compounds evaluated in inhibiting MMP-1 thereby preventing metastasis of breast cancer. Communicated by Ramaswamy H. Sarma.

Protein Network Studies on PCOS Biomarkers With S100A8, Druggability Assessment, and RNA Aptamer Designing to Control Its Cyst Migration Effect.

The prevalence of polycystic ovary syndrome (PCOS) has been gradually increasing among adult females worldwide. Laparoscopy drilling on ovary is the only available temporary solution with a high incidence of reoccurrence. S100A8 with S100A9 complex is believed to facilitate the cyst migration in PCOS condition. The high evident protein interaction network studies between PCOS biomarkers, cancer invasion markers, and the interactors of S100A8 confirm that this protein has strong interaction with other selective PCOS biomarkers, which may be associative in the immature cyst invasion process. Through the network studies, intensive structural and pathway analysis, S100A8 is identified as a targetable protein. In this research, the non-SELEX in silico method is adapted to construct RNA Library based on the consensus DNA sequence of Glucocorticoid Response Element (GRE) and screened the best nucleotide fragments which are bound within the active sites of the target protein. Selected sequences are joined as a single strand and screened the one which competitively binds with minimal energy. In vitro follow-up of this computational research, the designed RNA aptamer was used to infect the MCF7 cell line through Lipofectamine 2000 mediated delivery to study the anti-cell migration effect. Wound Scratch assay confirms that the synthesized 18-mer oligo has significant inhibition activity toward tumor cell migration at the cellular level.

Process development for the degradation of textile azo dyes (mono-, di-, poly-) by advanced oxidation process - Ozonation: Experimental & partial derivative modelling approach.

The present study focuses on modelling the removal of reactive azo dyes (Reactive Orange 16, Reactive Red 120 and Direct Red 80) by ozonolytic degradation. The process was optimised using One Variable at a Time (OVAT) approach followed by Response Surface Methodology (RSM). The operational parameters influencing the process of degradation, i.e. initial dye concentration (mg/L), pH and ozone exposure time were modelled using Central Composite Design (CCD). Under the optimal condition (Initial dye concentration = 2000 mg/L, pH = 11.0, Ozone exposure time = 10 min), the highest desirable response (i.e. Concentration of the degraded dye) for the degradation of RO 16, RR 120 and DR 80 are 1289.35 mg/L, 1224.98 mg/L and 1039.87 mg/L, respectively. The high correlation coefficients, 0.9814 (RO 16), 0.9815 (RR 120) and 0.9685 (DR 80) indicates the closeness of the results predicted by RSM with the experimental results. The rate of degradation for all the three dyes at the optimal condition followed pseudo-first order kinetics with the rate of reaction as 141 mg/L.min, 197.2 mg/L.min and 216.6 mg/Lmin. The predicted model was also evaluated by partial derivative-based equation modelling and experimental approach. The reliability and applicability of the developed process were confirmed by degrading the synthetic mixed dye effluent.

Immunomodulating Properties of Carrageenan from Tichocarpus crinitus.

Several in vivo immunotropic effects of κ/ß-carrageenan isolated from the red algae Tichocarpus crinitus were studied, by orally administering it at 100 mg/kg/day to mice for 7 days. Serum levels of IFN-γ, IL-12, IL-1ß, and IL-4 were measured. Carrageenan's ability to influence development of LPS-induced inflammation was also assessed. Oral administration of κ/ß-carrageenan increased serum levels of all the studied cytokines at least twice in comparison to the intact mice, while intraperitoneal LPS injection at 1 mg/kg increased concentration of only the pro-inflammatory cytokines: IFN-γ, IL-12, and IL-1ß. Furthermore, κ/ß-carrageenan demonstrated a higher efficacy at inducing IFN-γ production than LPS. Previous 7-day-long oral carrageenan administration impaired development of LPS-induced inflammation: level of IL-1ß dropped below that found in intact mice, while IFN-γ and IL-12 concentrations were at least 40% lower than in mice with LPS-induced inflammation. Murine peritoneal macrophages were also affected by the oral administration of the κ/ß-carrageenan: their motility was increased, and morphology altered. In sum, we have demonstrated that κ/ß-carrageenan, when administered orally, is not only not immunologically inert, but at the dose of 100 mg/kg possesses pharmacologically exploitable effects.

Report on biopharmaceutical profile of recent biotherapeutics and insilco docking studies on target bindings of known aptamer biotherapeutics.

Accumulated Toxicity, disease recurrence and drug resistivity problems have been observed due to the synthetic and semisynthetic therapeutic practices, which alternatively led to focus on Bio-therapeutics production than xenobiotics. Quick plasma clearance and high potency are the reasons for trending research with huge pharma market of numerous Bio-therapeutics than ever before. Researchers proved that most of the nano and micro Bio-therapeutics have multiple beneficial therapeutic effects. We have analyzed the past, and present scenario of some notable clinically approved Bio-therapeutics to identify the future formulation needs with advanced techniques. Protein-related drugs are the foremost Bio-therapeutics such as antibodies, enzymes, and short, fragmented polypeptides show aggregation properties during storage, naked peptide moieties are resisted by the polar cell membrane, and also the antidrug antibodies were reported. Even though Nucleic acid nano-bodies are excellent target binders than proteins, they had only a few minutes of half-life. Maintaining homogeneousness upon storage of Bio-therapeutics is still a significant challenge in industrial-scale formulation. Notably, plant systems are identified as most useful cost-effective hosts to produce human enzymes than animal systems without any possible viral loads. Irrespective of numerous advancements in routes of administration and additives, subcutaneous is still a golden one to achieve better dynamics. Additionally, the interactions and effective bonds made by each class of well-known aptamer biotherapeutics which are considered as future drugs were studied.

Fractional factorial design modelling on degradation of Direct Red 81 dye by advanced oxidation process - ozonation: reaction kinetics.

In the present work, the degradation of Direct Red 81 by ozonation was studied. The interactive effects of the influencing factors (dye concentration = 500-2,000 mg/L; time = 10-30 min; pH = 7.0-11.0) on degradation efficiency was critically examined through experimental design optimization by central composite design under the response surface methodology. The high correlation coefficients (R2 = 0.976 & adjusted R2 = 0.958) obtained by analysis of variance (ANOVA) demonstrated close fit between the experimental and the predicted values. Optimized conditions under specified cost-driven restraints were obtained for the highest desirability (i.e. degradation of 1,210.59 mg/L dye) at pH = 11.0, initial dye concentration = 2,000 mg/L and ozone exposure time of 27.16 min. The degradation of Direct Red 81 was confirmed through Fourier transform infrared spectroscopy (FTIR) analysis and UV-Vis spectrometry.

Human cytosolic glutathione-S-transferases: quantitative analysis of expression, comparative analysis of structures and inhibition strategies of isozymes involved in drug resistance.

Glutathione-S-transferase (GST) inhibition is a strategy to overcome drug resistance. Several isoforms of human GSTs are present and they are expressed in almost all the organs. Specific expression levels of GSTs in various organs are collected from the human transcriptome data and analysis of the organ-specific expression of GST isoforms is carried out. The variations in the level of expressions of GST isoforms are statistically significant. The GST expression differs in diseased conditions as reported by many investigators and some of the isoforms of GSTs are disease markers or drug targets. Structure analysis of various isoforms is carried out and literature mining has been performed to identify the differences in the active sites of the GSTs. The xenobiotic binding H site is classified into H1, H2, and H3 and the differences in the amino acid composition, the hydrophobicity and other structural features of H site of GSTs are discussed. The existing inhibition strategies are compared. The advent of rational drug design, mechanism-based inhibition strategies, availability of high-throughput screening, target specific, and selective inhibition of GST isoforms involved in drug resistance could be achieved for the reversal of drug resistance and aid in the treatment of diseases.

Utilization of Agro-Industry Residue for Rhamnolipid Production by P. aeruginosa AMB AS7 and Its Application in Chromium Removal.

Coconut oil sludge and oil cake was utilized as carbon source for biosurfactant production by Pseudomonas aeruginosa AMB AS7. The results of optimization study revealed that 1.5% (w/v) of coconut oil cake, 2% (w/v) of coconut oil sludge, pH 7.2, 37 °C, and 120 rpm were the optimum conditions for biosurfactant production. The yield coefficient of biosurfactant on biomass (Y P/X ) was 1.29 g/g. Besides, the results indicated that aeration of 0.5 vvm and agitation of 450 rpm in bioreactor resulted in high volumetric productivity of biosurfactant (r p ) and specific product formation rate (q p ) of 0.115 g/(L h) and 0.0131 g/(g h), respectively in medium containing 2% (w/v) coconut oil sludge. The maximum biosurfactant concentration of 5.53 g/L was obtained during 60 h of cultivation. The emulsification index (EI24) against coconut oil was found to be 88.42 ± 0.5%, and cell surface hydrophobicity of P. aeruginosa AMB AS7 was obtained 32.4 ± 0.9%. FTIR and GC-MS analysis revealed that the biosurfactant is rhamnolipid with anionic charge. The critical micelle concentration (CMC) of rhamnolipid was found to be 50 mg/L. It was found that 66.95% of chromium from aqueous solution can be removed using rhamnolipid at its CMC.

Synthesis of Feruloyl Ester Using Bacillus subtilis AKL 13 Lipase Immobilized on Celite® 545.

The lipophilic antioxidants, glyceryl ferulate and feruloyl glyceryl linoleate, were synthesized using lipase from Bacillus subtilis AKL 13. The extracellular lipase was produced by cultivation of the strain in modified minimal medium and the enzyme was recovered by fractionation at 80% ammonium salt saturation. The concentrated enzyme with the specific activity of (4647±66) U/mg was immobilized on Celite® 545 and crosslinked using glutaraldehyde. The prepared enzyme catalyst was used for esterification of ferulic and linoleic acids with glycerol separately in hexane butane solvent system at 50 °C and 3.144×g agitation. The maximum ester conversion of 94% of feruloyl glyceryl linoleate was achieved at 48 h, whereas only 35% of glyceryl ferulate was synthesized. The reaction products were characterized using RP-HPLC, FTIR, 1H NMR, 13C NMR and fluorescence spectrophotometry. The kinetic parameters of esterification reaction were determined according to ping-pong bi-bi model. The Km and υmax were found to be 69.37 and 3.46 mmol, and 0.387 and 1.02 mmol/(min·g) for glyceryl ferulate and feruloyl glyceryl linoleate, respectively. The kinetic parameters were simulated in MATLAB and the experimental data were in good agreement. Furthermore, 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity of the blend of feruloyl ester and palm oil was higher than of the plain palm oil and was closer to α-tocopherol.

Optimisation of radiolysis of Reactive Red 120 dye in aqueous solution using ionising (60)Co gamma radiation by response surface methodology.

In recent years, employing radiation technology is gaining great interest in degradation of industrial effluents. In this work the possibility of using gamma irradiation to degrade Reactive Red 120 (C.I.292775) was explored. The effects of pH, dose of gamma irradiation and concentration of dye were examined and their interaction were also established based on their response. For the analysis and optimisation of variables, three factor three level Box-Wilson face centred central composite design (CCF) was used. Analysis of variance with R(2) = 0.9988, adjusted R(2) = 0.9981 and the adequate precision value of 122.303 indicates that the CCF model can be used. The coefficient of variation (0.54%) indicates the reliability of the model. The dose of gamma irradiation (kGy) and the concentration of dye (mg/L) showed significant effects on the degradation of RR 120, while a difference of 6 to 10% degradation was observed in extending the pH towards the acid or alkali range from pH 7.00. The maximum concentration of dye degraded was observed as 347.509 mg/L at initial pH: 7.0, dose of gamma irradiation: 5.94 kGy and initial concentration of dye: 500 mg/L. This predicted value was found to be in agreement with the experimental value on the optimised conditions.

Kinetic studies on degradation of Reactive Red 120 dye in immobilized packed bed reactor by Bacillus cohnii RAPT1.

The degradation of Reactive Red 120 using Bacillus cohnii RAPT1 immobilized on polyurethane was studied. Initial experiments indicated that the percentage removal of dye in immobilized batch was significantly higher than batch (without immobilization). The optimum process parameters such as effect of dye concentration, time of immobilization on Poly Urethane Foam, initial inoculum size, pH and temperature for removal of dye were investigated and was found as 200ppm, 36h, 300*10(6) colony forming units/ml, 8.0 and 35°C respectively. Under optimum conditions, 100% removal of dye was obtained within 4h. The kinetics of biodegradation for the batch with free cells and immobilised packed batch was found to be IInd order with kinetic constant and initial rate of reaction as 0.0408, 0.084L/(mgday) and 1632, 3360 (mg/Lday) respectively.

Exploration of Sitagliptin as a potential inhibitor for the M1 Alanine aminopeptidase enzyme in Plasmodium falciparum using computational docking.

Plasmodium falciparum has limited capacity for de novo amino acid synthesis and rely on degradation of host hemoglobin to maintain protein metabolism and synthesis of proteins. M1 alanine aminopeptidase enzyme of the parasite involved in the terminal degradation of host hemoglobin was subjected to in silico screening with low molecular weight protease inhibitors. The km (avg) of the enzyme M1 alanine aminopeptidase for the substrate DL - Alanine ß Napthylamide Hydrochloride was estimated as 322.05µM. The molecular interactions between the enzyme and the substrate and the mechanism of enzyme action were analyzed which paved way for inhibition strategies. Among all the inhibitors screened, Sitagliptin was found to be most potent inhibitor with ki of 0.152 µM in its best orientation whereas the ki(avg) was 2.0055 µM. The ki of Sitagliptin is lower than the km of M1 alanine aminopeptidase for the substrate DL - Alanine ß Napthylamide Hydrochloride (322.05 µM) and Ki of the known inhibitor Bestatin. Therefore Sitagliptin may serve as a potent competitive inhibitor of the enzyme M1 alanine aminopeptidase of Plasmodium falciparum.