Jeyachandran Sivakamavalli

Improvement on dissolution rate of inclusion complex of Rifabutin drug with β-cyclodextrin

The effect of β-cyclodextrin (β-CD) on the improvement of solubility and antimicrobial activity of poorly water soluble drug Rifabutin (RFB) was studied. The solid inclusion complex is prepared under different methods and it is characterized by FT-IR, XRD, DSC and SEM methods. Solubility type, stability constant, stoichiometric ratio were investigated from phase solubility diagram of inclusion complex (RFB with β-CD). The dissolution profiles of the inclusion complexes were carried out and obvious increase in dissolution rate was observed when compared with pure RFB drug. Inclusion complexation process was further confirmed by molecular docking studies using PatchDock server. The in vitro antimicrobial and antibiofilm activity of RFB sensible microorganisms was significantly increased by on inclusion complexation process. This trend of inclusion complexation of poorly water soluble drugs is highly recognized as a successful and useful approach for the application in pharmaceutical field.

Purification, characterization and functional analysis of a novel β-1, 3-glucan binding protein from green tiger shrimp Penaeus semisulcatus

A β-1, 3-Glucan binding protein (β-GBP) was isolated from green tiger shrimp Penaeus semisulcatus and purified using laminarin precipitation and affinity chromatography on laminarin-Sepharose 6B column respectively. P. semisulcatus β-GBP exhibits a single band with a molecular weight of 112 kDa on SDS-PAGE and pI of 5.9 in isoelectric focusing (IEF). Negative staining of P. semisulcatus β-GBP showed large aggregates with crystalline surface when viewed by Electron Microscopy. Circular dichroism spectra of P. semisulcatus β-GBP showed broad negative minimum wavelength extending from 200 to 250 nm can be attributed to the presence of β-sheets in its secondary structure. P. semisulcatus β-GBP comprises the specific binding affinity with the polysaccharide β-1, 3-glucans (laminarin), this recognition and binding leads to the activation of prophenoloxidase cascade. Interestingly, P. semisulcatus β-GBP also involved in the agglutination of bakers yeast, bacteria, erythrocytes (RBCs) and enhances the PO activity. Herein, we have investigated the importance of β-GBP in innate immune response of P. semisulcatus and they implicate the evolutionary link with similar proteins found in other invertebrates.

Fluorometric sensing of Pb2+ and CrO42− ions through host–guest inclusion for human lung cancer live cell imaging

The formation of an inclusion complex between 1,5-dihydroxyanthraquinone (1,5-DHAQ; 1) and β-cyclodextrin (β-CD) in aqueous media has been studied by UV-visible and fluorescence spectroscopy. A solid inclusion complex (β-CD:1,5-DHAQ; 2) has been prepared and characterized by FT-IR, XRD, DSC and SEM analyses. The chemosensor probes 1 and 2 showed selective recognition and sensing ability towards the Pb2+ and CrO42− ions. The association constants (Ka) of 2·Pb2+ and Stern–Volmer quenching constant (Ksv) of 2·CrO42− were obtained to be 1.6 × 103 M−1 and 1.9 × 106 M−1 in water, and the corresponding detection limits were calculated to be 9.0 × 10−8 and 3.9 × 10−8 M according to fluorescence titration analysis. Theoretical studies on molecular docking and density functional theory (DFT) calculations have been performed to prove the binding of Pb2+ and CrO42− ions with chemosensor probes 1 and 2. Furthermore, bio-imaging indicated that these probes 1 and 2 have good cell permeability and are suitable for monitoring intracellular uptake of Pb2+ and CrO42− ions in living cells (human lung cancer A549) by confocal microscopy.

Bifunctional role of a pattern recognition molecule β-1,3 glucan binding protein purified from mangrove crab Episesarma tetragonum

β-1,3-Glucan binding protein (β-GBP) was purified from the haemolymph of Episesarma tetragonum by affinity chromatography with epoxy-activated laminarin-sepharose CL-6B column. E. tetragonum β-GBP exhibits a single band with a molecular weight of 100 kDa on SDS-PAGE and a pI of 5.9 in isoelectric focusing (IEF). The circular dichroism (CD) spectra result of E. tetragonum β-GBP indicates that the negative ellipticity bands near 220 nm and 208 nm correspond to the β-sheets in the secondary structure. Functional analysis results demonstrate that the purified E. tetragonum β-GBP agglutinates fungal cells (Candida albicans) containing β-glucan. This recognition and binding specificity leads to the activation of the prophenoloxidase (ProPO) cascade and enhance the phenoloxidase (PO) activity in a dose-dependent manner. Our finding discloses new insights in the ProPO activation and fungal agglutination of purified E. tetragonum β-GBP. It seems to play a significant role in microbial uncovering mechanism in invertebrates.

Purification, characterization and functional role of lectin from green tiger shrimp Penaeus semisulcatus

The present study reports the purification and characterization of immune molecule lectin from the green tiger shrimp Penaeus semiculcatus, which involves the non-self-recognition mechanism to destroy the infectious pathogens that continuously threaten their survival. P. semisulcatus lectin (Ps-Lec) was purified by affinity chromatography with mannose coupled sepharose CL-4B column and showed the 37 and 118kDa subunits in SDS-PAGE. The surface morphology of purified Ps-Lec exhibits the crystalline and aggregated nature in Transmission electron microscopy (TEM) analysis. Functional analysis of purified Ps-Lec showed that the broad spectrum of bacterial agglutination activity against Gram-positive and Gram-negative bacteria. In addition, Ps-Lec had the ability to involve in the haemoagglutination activity, which agglutinates the several vertebrate erythrocytes tested, and the haemagglutination titres were observed under light microscopy. Interestingly, Ps-Lec also exhibits the fungal aggregation activity against Candida albicans, this is the first report regarding the β-glucan recognition ability of fungal aggregation of Ps-Lec. Moreover, phenoloxidase activity was triggered by the Ps-Lec when compared to control. The present study reveals that Ps-Lec is an important multifunctional humoral defence protein plays a significant role against the bacterial and fungal cells.

Enzymatic elucidation of haemocyanin from Kuruma shrimp Marsupenaeus japonicus and its molecular recognition mechanism towards pathogens.

Haemocyanin is an important non-specific immune protein present in the hemolymph of invertebrates, which have the ability to recognize the microbial pathogens and trigger the innate immune system. In this study, we isolated and purified the haemocyanin using gel filtration chromatography and investigated its microbial recognition mechanism against the invading pathogens. Kuruma shrimp Marsupenaeus japonicus haemocyanin showed the single band with a molecular weight of 76 kDa on SDS-PAGE and its molecular mass was analysed through the MALDI. Pathogen recognition mechanism of M. japonicus haemocyanin was detected through bacterial agglutination, agglutination inhibition and prophenoloxidase activity. M. japonicus haemocyanin agglutinate all human blood RBC types and showed the bacterial agglutination against all tested Gram positive Staphylococcus aureus, Enterococcus faecalis and Bacillus subtilis and Gram negative Pseudomonas aeruginosa, Proteus vulgaris and Vibrio parahaemolyticus at the concentrations ranging from 30 to 50 μg/ml. Agglutination was inhibited by 50–200 mM of N-acetylneuraminic acid, a-D-glucose, D-galactose and D-xylose. Our results suggest that, 76 kDa subunit of M. japonicus haemocyanin recognize the pathogenic surface proteins which are present on the outer membrane of the bacteria and mediates the bacterial agglutination through haemocytes. This bacterial agglutination was visualized through Confocal Laser Scanning Microscopy (CLSM). This present study would be helpful to explore the importance of haemocyanin in innate immune response of M. japonicus and its eliciting pathogen recognition mechanism leads to the development of innate immunity in crustaceans.

Homology modeling, molecular dynamics, and docking studies of pattern-recognition transmembrane protein-lipopolysaccharide and β-1,3 glucan-binding protein from Fenneropenaeus indicus

Lipopolysaccharide and β-1,3 glucan-binding protein (LGBP) is a family of pattern-recognition transmembrane proteins (PRPs) which plays a vital role in the immune mechanism of crustaceans in adverse conditions. Fenneropenaeus indicus LGBP-deduced amino acid has conserved potential recognition motif for β-1,3 linkages of polysaccharides and putative RGD (Arg-Gly-Asp) cell adhesion sites for the activation of innate defense mechanism. In order to understand the stimulating activity of β-1,3 glucan (β-glucan) and its interaction with LGBP, a 3D model of LGBP is generated. Molecular docking is performed with this model, and the results indicate Arg71 with strong hydrogen bond from RGD domain of LGBP. Moreover, from the docking studies, we also suggest that Arg34, Lys68, Val135, and Ala146 in LGBP are important amino acid residues in binding as they have strong bonding interaction in the active site of LGBP. In our in vitro studies, yeast agglutination results suggest that shrimp F. indicus LGBP possesses sugar binding and recognition sites in its structure, which is responsible for agglutination reaction. Our results were synchronized with the already reported evidence both in vivo and in vitro experiments. This investigation may be valuable for further experimental investigation in the synthesis of novel immunomodulator.

Examine the characterization of biofilm formation and inhibition by targeting SrtA mechanism in Bacillus subtilis: a combined experimental and theoretical study.

AbstractBacillus subtilis is one of the well-known biofilm-forming organisms associated with plants, animals, and also used as a model organism for all Bacillus sp. In B. subtilis, SrtA enzyme plays the imperative roles in mechanism of signaling pathway and microbial adherence toward the host. SrtA is highly considered as a universal drug target for all Gram positive pathogens. Because of unresolved 3D structure of SrtA in Gram positive bacteria including B. subtilis, we developed a homology model protein using structural alignments of similar SrtA from B. anthracis. While the structural model of SrtA is analyzed because of its significance in biofilm formation by screening the suitable active site based compounds and analyzing the ability of bacterial biofilm inhibition. Druggability site based screening able to retrieve the active compounds against SrtA and checked the activity of the screened compounds through experimental biochemical assays and in situ microscopic analysis. Here in this study we concluded the computationally screened SrtA inhibitors showed high level of biofilm inhibition despite difficulties in bacterial membrane rigidification. Hence this study leads a way to the new compounds that may be useful to treat the bacterial infectionsFigureBacillus subtilis is one of the well-known biofilm-forming organisms referred to as model organism for all gram positive pathogens. From this organism, the universal drug target SrtA is evaluated for its biofilm forming ability and inhibitors are screened against the same, through in silico and in vitro methods. Druggability region based SrtA inhibitors are successful by showing anti microbial and anti biofilm activity. This study shows prominent common SrtA inhibitors against the gram positive pathogens

Synthesis and characterization of chitosan-ZnO composite and its antibiofilm activity against aquatic bacteria

In the present study, chitosan-ZnO composite was synthesized by stirring of chitosan purified from mud crab Scylla serrata shells with zinc chloride and sodium hydroxide. The physicochemical characteristics of chitosan-ZnO composite were studied using UV-Vis spec, Fourier transform infrared, X-ray diffraction and scanning electron microscopy. Chitosan-ZnO composite formation was confirmed by the functional groups stretching and bending vibrations in Fourier transform infrared. Scanning electron microscopy results showed the synthesized chitosan-ZnO composite was rod-like structure. X-ray diffraction results represent the hexagonal shape and crystalline size (30–60 nm) of zinc oxide in the chitosan-ZnO composite. Antibacterial activity of chitosan-ZnO composite demonstrated the effective growth control of a Gram-negative Vibrio parahaemolyticus and Gram-positive Bacillus lechiniformis bacteria isolated from aquatic environments. Light microscopy and confocal laser scanning microscopy also evidenced the antibiofilm activity of chitosan-ZnO composite against the V. parahaemolyticus and B. lechiniformis at the concentration of 40 and 60 µg/ml, respectively.

Virtual screening of LPXTG competitive SrtA inhibitors targeting signal transduction mechanism in Bacillus anthracis: a combined experimental and theoretical study.

Abstract Members of the sortase enzyme super family decorate the surfaces of Bacillus anthracis cell wall with proteins that play key roles in microbial pathogenesis and its biofilm formation. Bacillus anthracis Sortase-A (Ba-SrtA) is a potential target for new therapeutics as it is required for B. anthracis survival and replication within macrophages. An understanding of the binding site pocket and substrate recognition mechanism by SrtA enzymes may serve to be beneficial in the rational development of sortase inhibitors. Here, the LPXTG signal peptide-based competitive inhibitors are screened against the Ba-SrtA and compounds with reasonable inhibition, specificity, and mechanisms of inactivation of SrtA have been covered. The screened compounds are experimentally validated against the phylogenetically similar Gram-positive pathogen B. cereus. In situ microscopic visualizations suggest that these screened compounds showed the microbial and biofilm inhibitory activity against B. cereus. It facilitates the further development of these molecules into useful anti-infective agents to treat infections caused by B. anthracis and other Gram-positive pathogens. These results provide insight into basic design principles for generating new clinically relevant lead molecules. It also provides an alternative strategy where a screened ligand molecule can be used in combination to battle increasingly against the Gram-positive pathogens.