Atul Kumar Singh

Metabolic pathway analysis and molecular docking analysis for identification of putative drug targets in Toxoplasma gondii: novel approach.

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that can infect a wide range of warm-blooded animals including humans. In humans and other intermediate hosts, toxoplasma develops into chronic infection that cannot be eliminated by host’s immune response or by currently used drugs. In most cases, chronic infections are largely asymptomatic unless the host becomes immune compromised. Thus, toxoplasma is a global health problem and the situation has become more precarious due to the advent of HIV infections and poor toleration of drugs used to treat toxoplasma infection, having severe side effects and also resistance have been developed to the current generation of drugs. The emergence of these drug resistant varieties of T. gondii has led to a search for novel drug targets. We have performed a comparative analysis of metabolic pathways of the host Homo sapiens and the pathogen T. gondii. The enzymes in the unique pathways of T. gondii, which do not show similarity to any protein from the host, represent attractive potential drug targets. We have listed out 11 such potential drug targets which are playing some important work in more than one pathway. Out of these, one important target is Glutamate dehydrogenase enzyme; it plays crucial part in oxidation reduction, metabolic process and amino acid metabolic process. As this is also present in the targets of tropical diseases of TDR (Tropical disease related Drug) target database and no PDB and MODBASE 3D structural model is available, homology models for Glutamate dehydrogenase enzyme were generated using MODELLER9v6. The model was further explored for the molecular dynamics simulation study with GROMACS, virtual screening and docking studies with suitable inhibitors against the NCI diversity subset molecules from ZINC database, by using AutoDock-Vina. The best ten docking solutions were selected (ZINC01690699, ZINC17465979, ZINC17465983, ZINC18141294_03, ZINC05462670, ZINC01572309, ZINC18055497_01, ZINC18141294, ZINC05462674 and ZINC13152284_01). Further the Complexes were analyzed through LIGPLOT. On the basis of Complex scoring and binding ability it is deciphered that these NCI diversity set II compounds, specifically ZINC01690699 (as it has minimum energy score and one of the highest number of interactions with the active site residue), could be promising inhibitors for T. gondii using Glutamate dehydrogenase as Drug target.

Bioconductive 3D nano-composite constructs with tunable elasticity to initiate stem cell growth and induce bone mineralization

Bioactive 3D composites play an important role in advanced biomaterial design to provide molecular coupling and improve integrity with the cellular environment of the native bone. In the present study, a hybrid lyophilized polymer composite blend of anionic charged sodium salt of carboxymethyl chitin and gelatin (CMChNa-GEL) reinforced with nano-rod agglomerated hydroxyapatite (nHA) has been developed with enhanced biocompatibility and tunable elasticity. The scaffolds have an open, uniform and interconnected porous structure with an average pore diameter of 157±30μm and 89.47+0.03% with four dimensional X-ray. The aspect ratio of ellipsoidal pores decrease from 4.4 to 1.2 with increase in gelatin concentration; and from 2.14 to 1.93 with decrease in gelling temperature. The samples were resilient with elastic stain at 1.2MPa of stress also decreased from 0.33 to 0.23 with increase in gelatin concentration. The crosslinker HMDI (hexamethylene diisocyanate) yielded more resilient samples at 1.2MPa in comparison to glutaraldehyde. Increased crosslinking time from 2 to 4h in continuous compression cycle show no improvement in maximum elastic stain of 1.2MPa stress. This surface elasticity of the scaffold enables the capacity of these materials for adherent self renewal and cultivation of the NTERA-2 cL.D1 (NT2/D1), pluripotent embryonal carcinoma cell with biomechanical surface, as is shown here. Proliferation with MG-63, ALP activity and Alizarin red mineralization assay on optimized scaffold demonstrated ***p<0.001 between different time points thus showing its potential for bone healing. In pre-clinical study histological bone response of the scaffold construct displayed improved activity of bone regeneration in comparison to self healing of control groups (sham) up to week 07 after implantation in rabbit tibia critical-size defect. Therefore, this nHA-CMChNa-GEL scaffold composite exhibits inherent and efficient physicochemical, mechanical and biological characteristics based on gel concentrations, gelatin mixing and gelling temperature thus points to creating bioactive 3D scaffolds with tunable elasticity for orthopedic applications.

Improved hemodialysis with hemocompatible polyethersulfone hollow fiber membranes: In vitro performance

We show that addition of nanozeolite (NZ) and vitamin E D-α-Tocopherol polyethylene glycol succinate (TPGS or T) considerably improves the performance of polyethersulfone (PES or P) hollow fiber membrane (HFM) for hemodialysis. Nanocomposite HFMs were manufactured using PES as a polymer, TPGS as an additive and NZ as a filler to give a composite membrane called PT-NZ. HFMs were spun by dry-wet spinning principle based on liquid-liquid phase separation. TPGS and NZ were successfully incorporated in HFMs, as confirmed by EDX elemental mapping. The resultant PT-NZ HFMs had improved hemocompatibility: lower percent hemolysis (0.28% in batch mode and 0.32% in continuous mode), lower platelet adhesion, higher coagulation time and lower protein adsorption (16.34 µg/cm2 ), compared with P, PT, and commercial (F60S) HFMs. The ultrafiltration coefficient of PT-NZ HFM-based module (274.59 mL/m2 /h/mmHg) was ∼1.5-times higher than that of F60S membranes (151.67 mL/m2 /h/mmHg), and the solute rejection of both the membranes was comparable. The toxin clearance performance of lab-scale PT-NZ HFM-based hemodialyzer with uremic toxin spiked goat blood was remarkably higher (five times) than that of F60S. Hence, the synthesized PT-NZ HFMs are a potentially attractive membrane material for hemodialysis application, particularly due to decreased treatment time and minimal side reactions.

Odanacatib restores trabecular bone of skeletally mature female rabbits with osteopenia but induces brittleness of cortical bone: a comparative study of the investigational drug with PTH, Estrogen and Alendronate †

Cathepsin K (CK), a lysosomal cysteine protease, is highly expressed in mature osteoclasts and degrades type 1 collagen. Odanacatib (ODN) is a selective and reversible CK inhibitor that inhibits bone loss in preclinical and clinical studies. Although an antiresorptive, ODN does not suppress bone formation, which led us to hypothesize that ODN may display restorative effect on the osteopenic bones. In a curative study, skeletally mature New Zealand rabbits were ovarectomized (OVX) and after induction of bone loss were given a steady-state exposure of ODN (9 mM/d) for 14 weeks. Sham-operated and OVX rabbits treated with alendronate (ALD), 17b-estradiol (E2), or parathyroid hormone (PTH) served as various controls. Efficacy was evaluated by assessing bone mineral density (BMD), bone microarchitecture (using micro-computed tomography), fluorescent labeling of bone, and biomechanical strength. Skeletal Ca/P ratio was measured by scanning electron microscopy (SEM) with X-ray microanalysis, crystallinity by X-ray diffraction, and bone mineral density distribution (tissue mineralization) by backscattered SEM. Between the sham and ODN-treated osteopenic groups, lumbar and femur metaphyseal BMD, Ca/P ratio, trabecular microstructure and geometric indices, vertebral compressive strength, trabecular lining cells, cortical parameters (femoral area and thickness and periosteal deposition), and serum P1NP were largely comparable. Skeletal improvements in ALD-treated or E2-treated groups fell significantly short of the sham/ODN/PTH group. However, the ODN group displayed reduced ductility and enhanced brittleness of central femur, which might have been contributed by higher crytallinity and tissue mineralization. Rabbit bone marrow stromal cells expressed CK and when treated with ODN displayed increased formation of mineralized nodules and decreased apoptosis in serum-deficient medium compared with control. In vivo, ODN did not suppress remodeling but inhibited osteoclast activity more than ALD. Taken together, we show that ODN reverses BMD, skeletal architecture, and compressive strength in osteopenic rabbits; however, it increases crystallinity and tissue mineralization, thus leading to increased cortical bone brittleness.

Prediction and analysis of paralogous proteins in Trichomonas vaginalis genome.

Trichomonas vaginalis causes trichomoniasis, second most sexually transmitted disease. The genome sequence draft of T. vaginalis was published by The Institute of Genomic Research reveals an abnormally large genome size of 160 Mb. It was speculated that a significant portion of the proteome contains paralogous proteins. The present study was aimed at identification and analysis of the paralogous proteins. The all against all search approach is used to identify the paralogous proteins. The dataset of proteins was retrieved from TIGR and TrichDB FTP server. The BLAST-P program performed all against all database searches against the protein database of Trichomonas vaginalis available at NCBI genome database. In the present study about 50,000 proteins were searched where 2,700 proteins were found to be paralogous under the rigid selection criteria. The Pfam database search has identified significant number of paralogous proteins which were further categorized among different 1496 paralogous protein in pfam families, 1027 paralogous protein contains domain, 60 proteins were having different repeats and 1092 paralogous protein sequences of clans. Such identification and functional annotation of paralogous proteins will also help in removing paralogous proteins from possible drug targets in future. Presence of huge number of paralogous proteins across wide range of gene families and domains may be one of the possible mechanisms involved in the T. vaginalis genome expansion and evolution.

Porosity and compatibility of novel polysulfone-/vitamin E-TPGS-grafted composite membrane

Polysulfone (Psf) hollow fiber membranes are widely used for hemodialysis. Despite its popularity as a biomaterial, the hydrophobicity of the polymer is a major concern for blood contact applications. Various blends of the polymer with hydrophilic ligands have been reported in the literature, to achieve desired surface property. To increase hydrophilicity, we report here a hydrophilic polysulfone polymer by covalently coupling vitamin E tocopheryl polyethylene glycol 1000 succinate (Vit E-TPGS or TPGS) and Psf. 1H NMR confirmed grafting of TPGS to Psf. With a high degree of TPGS substitution, the Psf-g-TPGS was completely hydrophilic, which resulted in no fiber formation alone. Hence, composite membranes were prepared by mixing plain Psf and Psf-g-TPGS in 1.8:1 ratio, which also resulted in a hydrophilic character. This can be tuned toward slightly hydrophilic with a higher ratio. The porosity and biocompatibility of the Psf-g-TPGS (M-III, PTC) were compared against those of unmodified Psf membrane (M-I, Psf) and unmodified Psf-TPGS blend membrane (M-II, PTB). The in vitro cellular compatibility was tested in hepatocarcinoma cell line (HepG2); the cells grown on membrane surface were examined by SEM. Results confirmed that the modified membrane is hydrophilic, is non-toxic, and may have improved efficiency in hemodialysis. Hemocompatibility of M-III, PTC had a slightly better performance over M-I; M-I showed better performance in the cellular attachment, which shows the promising role of the grafted hydrophilic polymer for related biocompatibility applications.

Functionally coated polyethersulfone hollow fiber membranes: A substrate for enhanced HepG2/C3A functions

Hollow fiber membrane (HFM) based liver assist systems are a life-saving bridge for patients until a donor organ is available for transplantation or until liver regeneration. However, liver cell attachment and functional maintenance on HFM surface is a major challenge in bio-artificial liver (BAL) support systems. In the present study, novel glutaraldehyde (GTA)-crosslinked gelatin (gel)-coated polyethersulfone (X-gel-PT) HFMs were manufactured using triple orifice spinneret by the dry-wet spinning method. HFMs were characterized for morphology, outer surface roughness, hydrophilicity, tensile strength, thermal stability, BET surface area and pore volume measurements, permeability and rejection. Fourier transform infrared spectroscopy, and transmission electron microscopy confirmed the GTA-crosslinked gel-coating in the X-gel-PT HFMs, which provided the desirable extracellular matrix-like environment to the HepG2/C3A cells. The results of in-vitro hemocompatibility tests showed the better suitability of the developed HFMs for the blood-contact application. X-gel-PT HFMs showed significantly better cellular attachment and proliferation of HepG2/C3A cells on day 3 and 6, as shown by scanning electron and confocal microscopy. Significantly high urea synthesis and albumin secretion seen indicated the improved functional and metabolic activity of HepG2/C3A cells. Thus, the developed X-gel-PT HFMs is a suitable substrate for the hepatocyte culture, mass culture, and development of BAL support system.

Islet encapsulated implantable composite hollow fiber membrane based device: A bioartificial pancreas

Islets from xeno-sources and islet like clusters derived from autologus stem cells have emerged as alternatives to cadaveric pancreas used for treatment of type 1 diabetes. However, the immuno-isolation of these islets from the host immune system suffers from the issue of biocompatibility and hypoxia. To overcome the issues of immunobarrier biocompatibility, we developed a Polysulfone (Psf)/TPGS composite hollow fiber membrane (HFM) using a hollow fiber spinning pilot plant specially developed for this purpose. Important structural variables such as fiber material, dope composition, dimensions, surface characteristics etc., were precisely engineered and tuned for bioartificial pancreas application. The HFMs were characterized for their morphology, molecular diffusion, selectivity and protein absorption. The optimized Polysulfone(Psf)/TPGS composite HFMs, which contained TPGS, exhibited uniformed structure with low insulin adsorption and high permeability of insulin. The HFM was further studied for the encapsulation and in-vitro growth with porcine and differentiated islets isolated from human umbilical cord Whartons jelly. To prove their efficacy under in-vivo conditions, the Polysulfone(Psf)/TPGS composite HFMs were encapsulated with either of these isolated cells (porcine islets or islet like cell clusters derived from mesenchymal stem cells isolated from human umbilical cord Whartons jelly) and they were transplanted in experimental STZ induced diabetic mice. The results showed restoration of normoglycemia for 30days, indicating their ability to respond efficiently to high glucose without immune-rejection. Thus, these results indicate that Polysulfone (Psf)/TPGS composite HFMs can be used as an implantable, immune-competent bioartificial pancreas as a therapy for type 1 diabetes.

Cross-correlative 3D micro-structural investigation of human bone processed into bone allografts.

Bone allografts (BA) are a cost-effective and sustainable alternative in orthopedic practice as they provide a permanent solution for preserving skeletal architecture and function. Such BA however, must be processed to be disease free and immunologically safe as well as biologically and clinically useful. Here, we have demonstrated a processing protocol for bone allografts and investigated the micro-structural properties of bone collected from osteoporotic and normal human donor samples. In order to characterize BA at different microscopic levels, a combination of techniques such as Solid State Nuclear Magnetic Resonance (ssNMR), Scanning Electron Microscope (SEM), micro-computed tomography (μCT) and Thermal Gravimetric Analysis (TGA) were used for delineating the ultra-structural property of bone. ssNMR revealed the extent of water, collagen fine structure and crystalline order in the bone. These were greatly perturbed in the bone taken from osteoporotic bone donor. Among the processing methods analyzed, pasteurization at 60 °C and radiation treatment appeared to substantially alter the bone integrity. SEM study showed a reduction in Ca/P ratio and non-uniform distribution of elements in osteoporotic bones. μ-CT and MIMICS (Materialize Interactive Medical Image Control System) demonstrated that pasteurization and radiation treatment affects the BA morphology and cause a shift in the HU unit. However, the combination of all these processes restored all-important parameters that are critical for BA integrity and sustainability. Cross-correlation between the various probes we used quantitatively demonstrated differences in morphological and micro-structural properties between BA taken from normal and osteoporotic human donor. Such details could also be instrumental in designing an appropriate bone scaffold. For the best restoration of bone microstructure and to be used as a biomaterial allograft, a step-wise processing method is recommended that preserves all critical parameters of bone, showing a significant advancements over currently existing methods.

Molecular Dynamic Simulation and Inhibitor Prediction of Cysteine Synthase Structured Model as a Potential Drug Target for Trichomoniasis

In our presented research, we made an attempt to predict the 3D model for cysteine synthase (A2GMG5_TRIVA) using homology-modeling approaches. To investigate deeper into the predicted structure, we further performed a molecular dynamics simulation for 10 ns and calculated several supporting analysis for structural properties such as RMSF, radius of gyration, and the total energy calculation to support the predicted structured model of cysteine synthase. The present findings led us to conclude that the proposed model is stereochemically stable. The overall PROCHECK G factor for the homology-modeled structure was −0.04. On the basis of the virtual screening for cysteine synthase against the NCI subset II molecule, we present the molecule 1-N, 4-N-bis [3-(1H-benzimidazol-2-yl) phenyl] benzene-1,4-dicarboxamide (ZINC01690699) having the minimum energy score (−13.0 Kcal/Mol) and a log P value of 6 as a potential inhibitory molecule used to inhibit the growth of T. vaginalis infection.