Role of inflammatory cytokine burst in neuro-invasion of Japanese Encephalitis virus infection: an immunotherapeutic approaches.

Japanese Encephalitis remains a significant global health concern, contributing to millions of deaths annually worldwide. Microglial cells, as key innate immune cells within the central nervous system (CNS), exhibit intricate cellular structures and possess molecular phenotypic plasticity, playing pivotal roles in immune responses during CNS viral infections. Particularly under viral inflammatory conditions, microglial cells orchestrate innate and adaptive immune responses to mitigate viral invasion and dampen inflammatory reactions. This review article comprehensively summarizes the pathophysiology of viral invasion into the CNS and the cellular interactions involved, elucidating the roles of various immune mediators, including pro-inflammatory cytokines, in neuroinflammation. Leveraging this knowledge, strategies for modulating inflammatory responses and attenuating hyperactivation of glial cells to mitigate viral replication within the brain are discussed. Furthermore, current chemotherapeutic and antiviral drugs are examined, elucidating their mechanisms of action against viral replication. This review aims to provide insights into therapeutic interventions for Japanese Encephalitis and related viral infections, ultimately contributing to improved outcomes for affected individuals.

Elucidation of marine fungi derived anthraquinones as mycobacterial mycolic acid synthesis inhibitors: an in silico approach.

Tuberculosis (TB) is a leading cause of mortality amongst infectious diseases. While the anti-TB drugs can cure TB, the non-compliance and rapidly increasing resistance is of serious concern. The study aimed to search novel potent inhibitor(s) against MabA and PKS18 targets of Mycobacterium tuberculosis (M.tb.) by virtual screening of anthraquinones from marine fungi. The target proteins MabA and PKS18 involved in M.tb. mycolic acid biosynthesis were retrieved from RCSB Protein Data Bank. Chemical structures of 100 marine fungal anthraquinones were retrieved from the PubChem database. These were filtered through Lipinski's rule of five (for druglikeness) and in silico ADME/Tox analysis (for pharmacokinetic properties) and subjected to molecular docking analysis using AutoDock 4.2. The molecular interaction revealed averufin to possess dual inhibitory potential against M.tb. MabA and PKS18 with binding energy of - 8.84 kcal/mol and - 8.23 kcal/mol, and Ki values of 1.79 and 3.12 µM respectively. Averufin exhibits improved drug-like properties, ADMET profile and binding affinity to both targets as compared to control drugs. Our study suggests that averufin a natural anthraquinone, satisfies all the in silico parameters tested and is expected to efficiently inhibit M.tb. mycolic acid pathway. It might therefore emerge as a promising dual-targeted, novel natural anti-TB lead in future.

Labda-8(17),12,14-trien-19-oic acid contained in fruits of Cupressus sempervirens suppresses benign prostatic hyperplasia in rat and in vitro human models through inhibition of androgen and STAT-3 signaling.

Fruit extract of Cupressus sempervirens (CS), which is used traditionally to treat Benign Prostatic Hyperplasia (BPH)-like urinary symptoms in patients, was scientifically validated for anti-BPH activity. The ethanolic fruit extract of CS inhibited proliferation of human BPH-stromal cells and the activity was localized to its chloroform-soluble, diterpene-rich fraction. Eight major diterpenes isolated from this fraction exhibited moderate to potent activity and the most active diterpene (labda-8(17),12,14-trien-19-oic acid) exhibited an IC50 of 37.5 µM (antiproliferative activity against human BPH-stromal cells). It significantly inhibited activation (phosphorylation) of Stat-3 in BPH-stromal cells and prevented transactivation of androgen sensitive KLK3/PSA and TMPRSS2 genes in LNCaP cells. Labda-8(17),12,14-trien-19-oic acid-rich CS fraction prevented prostatic hyperplasia in rat model and caused TUNEL labeling of stromal cells with lower expressions of IGF-I, TGF-ß and PCNA, and bcl-2/bax ratio. Human BPH tissues exhibited precise lowering of stromal component after incubation in labda-8(17),12,14-trien-19-oic acid, ex vivo. We conclude that labda-8(17),12,14-trien-19-oic acid contained in CS exhibits anti-BPH activity through inhibition of stromal proliferation and suppression of androgen action in the prostate, presenting a unique lead structure for further optimization of anti-BPH activity.

Cypermethrin induces astrocyte damage: role of aberrant Ca(2+), ROS, JNK, P38, matrix metalloproteinase 2 and migration related reelin protein.

Cypermethrin is a synthetic type II pyrethroid, derived from a natural pyrethrin of the chrysanthemum plant. Cypermethrin-mediated neurotoxicity is well studied; however, relatively less is known of its effect on astrocyte development and migration. Astrocytes are the major components of blood brain barrier (BBB), and astrocyte damage along with BBB dysfunction impair the tight junction (TJ) proteins resulting in altered cell migration and neurodegeneration. Here, we studied the mechanism of cypermethin mediated rat astrocyte damage and BBB disruption, and determined any change in expression of proteins associated with cell migration. Through MTT assay we found that cypermethrin reduced viability of cultured rat astrocytes. Immunolabelling with astrocyte marker, glial fibrillary acidic protein, revealed alteration in astrocyte morphology. The astrocytes demonstrated an enhanced release of intracellular Ca(++) and ROS, and up-regulation in p-JNK and p-P38 levels in a time-dependent manner. Cypermethrin disrupted the BBB (in vivo) in developing rats and attenuated the expression of the extracellular matrix molecule (ECM) and claudin-5 in cultured astrocytes. We further observed an augmentation in the levels of matrix metalloproteinase 2 (MMP2), known to modulate cellular migration and disrupt the developmental ECM and BBB. We observed an increase in the levels of reelin, involved in cell migration, in cultured rat astrocytes. The reelin receptor, α3ß1integrin, and a mammalian cytosolic protein Disabled1 (Dab1) were also up-regulated. Overall, our study demonstrates that cypermethrin induces astrocyte injury via modulation in Ca(++), ROS, JNK and P38 pathways, which may alter MMP expression and reelin dependent astrocyte migration during brain development.

Down-regulated GFAPα: a major player in heavy metal induced astrocyte damage.

Exposure to a mixture of As, Pb and Cd induces apoptosis and morphological alterations in the cortical astrocytes of rat brain. The levels of the glial fibrillary acidic protein (GFAP) undergo a reduction. The GFAP exists in several isoforms, viz., α, ß, κ, δ and ϵ. However, contribution of the isoforms towards astrocyte damage is not understood. We investigated the effect of the metal mixture (MM) on the expression profiles of mRNAs encoding the GFAP isoforms in astrocytes. The MM was administered in drinking water to developing rats till postnatal day (PD) 60. We observed a fall (10.20 ± 1.04%, 18.91 ± 2.12% and 30.26 ± 3.21% at PD24, PD45 and PD60 respectively) in GFAPα. This may have been compensated by a rise in ß, κ, and ϵ. The GFAPδ remained unchanged. To determine the role of the GFAPα, we silenced its gene using SiRNA technology in the rat primary astrocytes. We observed a 23.73 ± 1.56% increase in the number of apoptotic cells. The cleaved PARP and Bax levels increased by 2.48 ± 0.14-fold and 3.73 ± 0.23-fold respectively, and the Bcl-2 and Bcl-xl decreased by 2.38 ± 0.08-fold and 1.76 ± 0.09-fold respectively. The change was comparable to the cells treated with MM. Moreover, silencing the GFAPα gene induced a reduction in the area (6.19 ± 0.18-folds), perimeter (12.65 ± 1.68-folds) and the number of processes (5.88 ± 1.5-folds) in the astrocytes, which closely matched the MM-treated ones. Taken together, these observations are the first to show that MM disturbs the composition of the GFAP isoforms, and a suppressed GFAPα promotes apoptosis in the matured rat astrocytes.

Structure-based in silico and in vitro Analysis Reveals Asiatic Acid as Novel Potential Inhibitor of Mycobacterium tuberculosis Maltosyl Transferase.

AIMS: The present study aimed to search for novel potent inhibitor(s) against the recently discovered maltosyltransferase (GlgE) target of M.tb. BACKGROUND: GlgE belongs to an α-amylase family and catalyzes the elongation of cytosolic branched α-glucan. Inactivation of M.tb. GlgE results in DNA damage and rapid death of M.tb. due to the accumulation of a toxic altosyl donor, maltose-1-phosphate (M1P), suggesting that GlgE is an intriguing target for inhibitor design. METHODS: 1000 natural compounds were compiled from public databases and literature through virtual screening, of which 25 compounds were found to satisfy all drug-likeness properties and ADME/ toxicity criteria, followed by molecular docking with GlgE. Compound(s) showing the lowest binding energy was further subjected to molecular dynamics simulation (MDS) and in vitro analysis. RESULTS: Molecular docking analysis allowed the selection of 5 compounds withsignificant binding affinity to GlgE targets. Amongst these compounds, asiatic acid exhibited the lowest binding energy (-12.61 kcal/mol). The results of 20-ns MDS showed that asiatic acid formed a stable complex with GlgE. Additionally, asiatic acid exhibited in vitro anti-mycobacterial activity against M.tb. H37Ra, M. bovis BCG, and M. smegmatis strains. CONCLUSION: The study reveals asiatic acid as a promising anti-mycobacterial agent that might emerge as a novel natural anti-TB lead molecule in the future.

Identification of Novel Inhibitor of Enoyl-Acyl Carrier Protein Reductase (InhA) Enzyme in Mycobacterium tuberculosis from Plant-Derived Metabolites: An In Silico Study.

Mycobacterium tuberculosis (M.tb.) enoyl-acyl carrier protein (ACP) reductase (InhA) is validated as a useful target for tuberculosis therapy and is considered an attractive enzyme to drug discovery. This study aimed to identify the novel inhibitor of the InhA enzyme, a potential target of M.tb. involved in the type II fatty acid biosynthesis pathway that controls mycobacterial cell envelope synthesis. We compiled 80 active compounds from Ruta graveolens and citrus plants belonging to the Rutaceae family for pharmacokinetics and molecular docking analyses. The chemical structures of the 80 phytochemicals and the 3D structure of the target protein were retrieved from the PubChem database and RCSB Protein Data Bank, respectively. The evaluation of druglikeness was performed based on Lipinski's Rule of Five, while the computed phytochemical properties and molecular descriptors were used to predict the ADMET of the compounds. Amongst these, 11 pharmacokinetically-screened compounds were further examined by performing molecular docking analysis with an InhA target using AutoDock 4.2. The docking results showed that gravacridonediol, a major glycosylated natural alkaloid from Ruta graveolens, might possess a promising inhibitory potential against InhA, with a binding energy (B.E.) of -10.80 kcal/mole and inhibition constant (Ki) of 600.24 nM. These contrast those of the known inhibitor triclosan, which has a B.E. of -6.69 kcal/mole and Ki of 12.43 µM. The binding efficiency of gravacridonediol was higher than that of the well-known inhibitor triclosan against the InhA target. The present study shows that the identified natural compound gravacridonediol possesses drug-like properties and also holds promise in inhibiting InhA, a key target enzyme of M.tb.

Particulate ß-glucan activates early and delayed phagosomal maturation and autophagy within macrophage in a NOX-2 dependent manner.

AIMS: Macrophage is known to readily engulf any particulate material they encounter, including invading microbes and nano- or micro-particles. While recent studies show that some microparticles (MP) are immunogenic even without drug-cargo, the mechanism underlying this phenomenon is yet unclear. Phagocytosis induces NADPH oxidase-2 (NOX-2) mediated ROS generation that is reported to regulate antibacterial autophagy. We therefore, investigated the role of NOX-2 derived ROS in phagosomal maturation and autophagy induction in response to phagocytic uptake of two kinds of polymeric biodegradable and biocompatible microparticles: yeast-derived ß-glucan particles (YDGP) and poly-(D, L-Lactic Acid) microparticles (PMP). MAIN METHODS: J774A.1 macrophage wereas exposed to polymeric particles and the immune responses: ROS, phagosomal maturation and autophagy induction, were examined by assays including NBT, DCFH-DA, NADPH-Oxidase activity, Lysotracker and Acridine Orange. Further, the LC3 and NOX-2 expression were validated by RT-PCR, immunofluorescence assay and Western blotting. Antimicrobial activity of both MP was examined by CFU counting after administration to Mycobacterium tuberculosis and Salmonella typhimurium infected macrophage. KEY FINDINGS: YDGP induces phagosomal maturation and acidic vesicle accumulation at 30 min and 24 h post-exposure, much more proficiently than that by PMP. YDGP exposure also induced NOX-2 dependent expression of light chain 3 (LC3-II), further confirmed as autophagy activation via autophagic flux assay with autophagolysosome inhibitor bafilomycin A1. Additionally, YDGP displayed superior anti-microbial activity than that by PMP. SIGNIFICANCE: The induction of NOX-2-dependent autophagy and antimicrobial activity exhibited by particulate glucans has significant implications in harnessing these drug delivery vehicles as potential 'value-added' autophagy-mediated therapeutics in future.

Inhalable particles containing isoniazid and rifabutin as adjunct therapy for safe, efficacious and relapse-free cure of experimental animal tuberculosis in one month.

We investigated the preclinical efficacy and safety/tolerability of biodegradable polymeric particles containing isoniazid (INH) and rifabutin (RFB) dry powder for inhalation (DPI) as an adjunct to oral first-line therapy. Mice and guinea pigs infected with Mycobacterium tuberculosis H37Rv (Mtb) were treated with ∼80 and ∼300 µg of the DPI, respectively, for 3-4 weeks starting 3, 10, and 30 days post-infection. Adjunct combination therapy eliminated culturable Mtb from the lungs and spleens of all but one of 52 animals that received the DPI. Relapse-free cure was not achieved in one mouse that received DPI + oral, human-equivalent doses (HED) of four drugs used in the Directly Observed Treatment, Short Course (DOTS), starting 30 days post-infection. Oral doses (20 mg/Kg/day, each) of INH + RFB reduced Mtb burden from ∼106 to ∼103 colony-forming units. Combining half the oral dose with DPI prevented relapse of infection four weeks after stopping the treatment. The DPI was safe in rodents, guinea pigs, and monkeys at 1, 10, and 100 µg/day doses over 90 days. In conclusion, we show the efficacy and safety/tolerability of the DPI as an adjunct to oral chemotherapy in three different animal models of TB.

Association of DNA Repair Genes XRCC1 and APE-1 with the Risk of Cervical Cancer in North Indian population.

BACKGROUNDS: Cervical cancer (CC) is one of the leading cause of death in women worldwide, HPV infection is the major risk factor in the disease development, 0and however other risk factor such as chemical carcinogens, genetic susceptibility and altered immune system are also a cause of the disease progression. In the light of the above statement we studied the base excision repair pathway (BER). METHODS: We identified and studied the association of Single Nucleotide polymorphisms in the DNA repair genes of XRCC1 (Arg194Trp, Arg399G,) and APE-1Asp/148Glu to the susceptibility of cervical cancer (CC) in North Indian population. In our study of cases (n=102). Controls (n=109) were recruited from among women without cervical abnormalities. Genotypes were determined by PCR-CTPP method, Taking DNA from peripheral blood in a case control study. RESULTS: A positive association was observed between the polymorphisms of XRCC1 genes, that is, in codons 194 (P=0.03, odds ratio (OR) =2.39, 95% confidence interval (CI)=5.2-1.1), 280 (P=0.01, OR=4.1, 95% CI=11.5-1.3) and 399 (P=0.01, OR=3.4, 95% CI=8.6-1.3) while APE-1 genotype GG (p=0.03,odds ratio(OR)=0.2,95% confidence interval (CI)=0.97-0.004) we observed a statistically significant protective role in developing cervical cancer. CONCLUSION: Our results suggested that, XRCC1 gene is an important candidate gene for susceptibility to cervical cancer. Although the sample size was small, the present study indicate a statistical association between cervical cancer and XRCC1 SNPs. Future studies are needed that may provide a better understanding of the association between gene polymorphism and cervical carcinoma risk.
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Inhalable microparticles containing isoniazid and rifabutin target macrophages and 'stimulate the phagocyte' to achieve high efficacy.

Macrophage responses to infection with Mycobacterium tuberculosis (MTB) and treatment with soluble isoniazid (INH) plus rifabutin (RFB) versus microparticles containing equivalent amounts of drugs were compared. It was investigated whether macrophages driven to alternative activation upon infection with MTB could be rescued to display the classical activation phenotype. It was established that microparticles sustain high levels of drugs in cytosol of macrophages for longer period as compared to soluble drugs. Microparticles co-localized with intracellular bacteria, and induced a variety of innate bactericidal responses, including induction of free radicals, alteration of mitochondrial membrane potential and apoptosis. The data strongly suggest that additional benefit may be derived from the nature of the drug delivery system, which fulfils Koch's dictum 'stimulate the phagocyte' for curing tuberculosis.

Nano-Rifabutin entrapment within glucan microparticles enhances protection against intracellular Mycobacterium tuberculosis.

Recently, yeast-derived glucan particles (GP) have emerged as novel drug delivery agents that provide for receptor-mediated uptake by phagocytic cells expressing ß-glucan receptors. In our previous study, we prepared GP loaded with high payload (40.5 + 1.9%) of rifabutin (RB) nano-particles [(RB-NPs)-GP]. We investigated the anti-mycobacterial efficacy and cellular activation responses within Mycobacterium tuberculosis (M. tuberculosis) infected J774 macrophage cells following exposure to the (RB-NPs)-GP formulation. The exposure was seen to augment a robust innate immune response including the induction of reactive oxygen and nitrogen species, autophagy and apoptosis within M. tuberculosis infected macrophage. Further, the efficacy testing of these particles in murine macrophage exhibited that the (RB-NPs)-GP formulation enhanced the efficacy of RB drug by ∼2.5 fold. The study suggests that the set of innate responses conducive to killing intracellular bacteria evoked by (RB-NPs)-GP play a pivotal role in impeding the intracellular M. tuberculosis survival, resulting in enhanced efficacy of the formulation. Our results establish that the (RB-NPs)-GP formulation not only activate M. tuberculosis infected, immune-suppressed macrophage, but also adds significantly to the efficacy of loaded drug, and thus forms a promising approach that should be explored further as an alternative or adjunct form of TB therapy. Highlights Nano-Rifabutin loaded Glucan microparticles [(RB-NPs)-GP] administered to M. tuberculosis infected macrophage. (RB-NPs)-GP induces appropriate innate immune responses in host macrophage. Mycobactericidal Effect of Rifabutin was markedly enhanced by its nano-entrapment in GP. Intracellular drug delivery supplements the innate response in M. tuberculosis infected macrophage.

A hand-held apparatus for "nose-only" exposure of mice to inhalable microparticles as a dry powder inhalation targeting lung and airway macrophages.

Microparticles containing isoniazid and rifabutin were aerosolised using a simple apparatus fabricated from a 15-ml centrifuge tube. The dose available for inhalation by rodents was determined by collecting microparticles emitted at the delivery port. The dose available for inhalation was proportional to durations of exposure ranging from 10 to 90 s (10.5-13.5 CV%) and the weight of powder taken for fluidization (10-50 mg, r2=0.982). The apparatus was then used to administer inhalations of microparticles to mice. Other groups of mice received free rifabutin orally, or by i.v. injection. Rifabutin was estimated in serum and tissues of dosed mice by HPLC. When approximately 20 mg of microparticles were loaded in the apparatus, approximately 2.5 mg were collected at the delivery port in 30 s of operation. Mice inhaled approximately 300 microg of the 2.5 mg emitted at the delivery port. Airway and lung macrophages of mice receiving inhalations for 30 s accumulated 0.38 microg of rifabutin, while the amount in blood serum of these mice was 0.62 microg. In mice receiving 83 microg rifabutin i.v. or orally, the intracellular amounts were 0.06 and 0.07 microg respectively, while the amounts in serum were 1.02 and 0.80 microg. These observations confirmed that inhalation of microparticles targeted airway and lung macrophages.

Circulating MicroRNA-21 Expression as a Novel Serum Biomarker for Oral Sub-Mucous Fibrosis and Oral Squamous Cell Carcinoma

Background: Circulating miRNAs (miRs) in the biofluids such as serum and plasma act as potential biomarkers for early diagnosis, treatment and prognosis. In the present study, an attempt made to see the expression of miR-21 in serum of 20 cases of Oral sub-mucous fibrosis (OSMF), 20 cases of Oral squamous cell carcinoma and 40 healthy volunteers. The expression of miR-21 was evaluated in relation to different demographical and clinicopathological features such as sex, tobacco, pan-masala, alcohol, smoking and clinical staging respectively with an aim to identify correlation with oral pre-cancer and cancer stages. Materials and Methods: The relative expression level of miR-21 was determined by quantitative real-time RT-PCR (qRT-PCR) in the sera of 20 OSCC, 20 OSMF patients and 40 healthy subjects as a control. Association between expression of miR-21 and OSCC clinical stages and demographical parameters such as sex, pan-masala, tobacco, smoking, alcohol have also been analyzed in detail. Results: The results obtained by t-test revealed significant increase in the expression level of miR-21 in OSCC as compared to OSMF. The study also revealed the positive correlation between higher miR-21 expression and pan-masala chewers as shown by t-test. The statistical test, ANOVA has also indicated a positive correlation between up-regulation of miR-21 in the clinical stages of the OSCC. Conclusion: The results of present study indicated up-regulation of circulating miR-21 in serum of OSCC as compared to OSMF (p=0.001), this study also elucidated the positive correlation between miR-21 expression in OSCC/OSMF patients, only one demographical parameter (Pan-masala) and negative correlation for other parameters such as sex, tobacco, smoking, alcohol etc. Other findings suggested a significant increase (p=0.000) in the expression of miR-21 in clinical staging (I-IV) of oral cancer. More studies are needed to validate it as potential diagnostic and prognostic biomarker for OSMF and OSCC for better management.

Inhalable microparticles containing large payload of anti-tuberculosis drugs.

Microparticles containing large payloads of two anti-tuberculosis (TB) drugs were prepared and evaluated for suitability as a dry powder inhalation targeting alveolar macrophages. A solution containing one part each of isoniazid and rifabutin, plus two parts poly(lactic acid) (L-PLA) was spray-dried. Drug content and in vitro release were assayed by HPLC, and DSC was used to elucidate release behaviour. Particle size was measured by laser scattering and aerosol characteristics by cascade impaction using a Lovelace impactor. Microparticles were administered to mice using an in-house inhalation apparatus or by intra-tracheal instillation. Drugs in solution were administered orally and by intra-cardiac injection. Flow cytometry and HPLC were used to investigate the specificity and magnitude of targeting macrophages. Microparticles having drug content approximately 50% (w/w), particle size approximately 5 microm and satisfactory aerosol characteristics (median mass aerodynamic diameter, MMAD=3.57 microm; geometric standard deviation, GSD=1.41 microm; fine particle fraction, FPF(<4.6 microm)=78.91+/-8.4%) were obtained in yields of >60%. About 70% of the payload was released in vitro in 10 days. Microparticles targeted macrophages and not epithelial cells on inhalation. Drug concentrations in macrophages were approximately 20 times higher when microparticles were inhaled rather than drug solutions administered. Microparticles were thus deemed suitable for enhanced targeted drug delivery to lung macrophages.

Particulate beta-glucan induces early and late phagosomal maturation in murine macrophages.

Beta-glucans are carbohydrates (glucose polymers) found in the cell walls of fungi, yeast, algae, lichens, and plants such as oats and barley. Beta-glucans bind to glucan receptor on phagocytic cells and modify these cells to become "immunologically active" by generating a variety of innate immune responses. Particulate beta-glucan has been specifically shown to engage dectin-1 receptor, which leads to the recruitment and activation of nicotinamide adenine dinucleotide phosphate oxidase-2 (NOX-2) and release of antimicrobial reactive oxygen species (ROS). Here, we report that yeast-derived beta-glucan particles (YDGP) of a specific size are easily phagocytosed by macrophages and induce the production of ROS. Furthermore, the present work also demonstrates that phagosomal maturation, appearance of acidic vesicular compartments (AVO), and light chain protein-3 (LC3) accumulation within murine macrophages, occur at early and delayed time points after the phagocytic uptake of YDGP. These data suggest that particulate glucans can be exploited to trigger autophagy within phagocytes and, thereby, have implications in antimicrobial therapy.

Preparation and characterization of beta-glucan particles containing a payload of nanoembedded rifabutin for enhanced targeted delivery to macrophages.

ß-glucan particles (GP) are polymeric carbohydrates, mainly found as components of cell wall fungi, yeast, bacteria and also in cereals such as barley and oat, and have been recently shown to have application in macrophage-targeted drug delivery. The aim of this study was to prepare and characterize GP containing a large payload of Rifabutin (RB), an anti-tuberculosis drug effective against MDR-TB at lower MIC than Rifampicin. GP were prepared from yeast cells by acidic and alkaline extraction were either spray dried or lyophilized, prior to RB loading and alginate sealing. The FTIR and 13C-NMR spectra of the GP confirmed a ß-(1→3) linked glucan structure, with a triple-helical conformation. The spray dried GP exhibited better characteristics in terms of uniformity, size range (2.9 to 6.1 µm) and more than 75 % particles were below 3.5 µm. The RP-HPLC analysis of spray dried GP revealed drug entrapment and drug loading up to 81.46 ± 4.9 % and ~40.5 ± 1.9 %, respectively, as compared to those dried by lyophilization. Electron microscopy showed nearly spherical and porous nature of GP, and the presence of drug 'nanoprecipitates' filling the pore spaces. The formulation showed adequate thermal stability for pharmaceutical application. The particles were readily phagocytosed by macrophage(s) within 5 min of exposure. Drug release occurred in a sustained manner via diffusion, as the release kinetics best fit for drug release was obtained using Higuchi's equation. Thus, the spray dried GP-based-formulation technology holds promise for enhanced targeted delivery of anti-TB drug(s) to macrophage within a therapeutic window for the clearance of intracellular bacteria.

Identification of Ellagic acid analogues as potent inhibitor of protein Kinase CK2:A chemopreventive role in oral Cancer.

Over expression of Protein kinase (CK2) suppresses apoptosis induced by a variety of agents, whereas down-regulation of CK2 sensitizes cells to induction of apoptosis. In this study, we have built quantitative structure activity relationship (QSAR) models, which were trained and tested on experimentally verified 38 enzyme׳s inhibitors having inhibitory value IC50 in µM. These inhibitors were docked at the active site of CK2 (PDB id: 2ZJW) using AutoDock software, which resulted in energy-based descriptors such as binding energy, intermol energy, torsional energy, internal energy and docking energy. For QSAR modeling, Multiple Linear Regression (MLR) model was engendered using energy-based descriptors yielding correlation coefficient r(2) of 0.4645. To assess the predictive performance of QSAR models, different cross-validation procedures were adopted. Our results suggests that ligand-receptor binding interactions for CK2 employing QSAR modeling seems to be a promising approach for prediction of IC50 value of a new ligand molecule against CK2.Further, twenty analogues of ellagic acid were docked with CK2 structure. After docking, two compounds CID 46229200 and CID 10003463 had lower docking energy even lower than standard control Ellagic acid with CK2 was selected as potent candidate drugs for Oral cancer. The biological activity of two compounds in terms of IC50 was predicted based on QSAR model, which could be used as a guideline for anticancerous activity of compounds before their synthesis.

Disorganized muscle protein-1 (DIM-1) of filarial parasite Brugia malayi: cDNA cloning, expression, purification, structural modeling and its potential as vaccine candidate for human filarial infection.

We have recently identified disorganized muscle protein-1 (DIM-1) in one of the proinflammatory fractions of the human filaria Brugia malayi adult worm. The present study was undertaken to characterize B. malayi DIM-1 (DIM-1bm) and explore its vaccine potential. In this study we cloned and expressed the DIM-1bm gene, investigated its sequence homology with other nematodes, constructed in silico structural model, purified the recombinant DIM-1bm (rDIM-1bm) protein, and studied the effect of immunization with rDIM-1bm on the establishment of B. malayi infection in Mastomys coucha. DIM-1bm showed similarity with DIM-1 of Caenorhabditis elegans, Ascaris suum and Loa loa. Structural modeling revealed three immunoglobulin domains in DIM-1bm indicating that it is a member of immunoglobulin superfamily (IgSF) and 'blastn' results showed that DIM-1bm coding sequence (CDS) have almost no homology with human and mouse nucleotide sequences. Immunization with rDIM-1bm partially protected M. coucha against establishment of infection as inferred by a low recovery of microfilariae (37-64%) and parasite burden (∼50%). The enhanced activity of macrophages, and IFN-γ and NO responses, and elevated levels of specific IgG, IgG1, IgG2a and IgG2b correlated with parasitological findings. This is the first report on cloning, expression, structural modeling and purification of rDIM-1bm and its ability to partially prevent establishment of B. malayi infection. DIM-1bm's almost complete lack of homology with the human counterpart makes it an attractive protein for exploring its vaccine potential.