Impelling TLR9: Road to perspective vaccine for visceral leishmaniasis.

Recent trends in immunotherapy have shown enthusiasm in exploring Toll-like receptors (TLRs) for designing therapeutical interventions against numerous deadly diseases. TLRs are subfamily of pathogen recognition receptor playing pivotal role in innate immunity. TLR9 is one such critical member belonging to intracellular TLRs which is associated with mounting inflammatory response in response to intruders. Explorative studies have shown CG motifs from the prokaryotic origin as activators of TLR9 culminating in the expression of NFκB. These CG rich short stranded DNA sequences have been further delineated into different classes based on their structural specificities and immunomodulatory properties. Here we discuss the progress of how activation of TLR9 can be utilized with novel parasitic CpG islands to function as potential adjuvants specifically against protozoan parasitic diseases primarily visceral leishmaniasis caused by Leishmania donovani.

Microglial TLR9: Plausible Novel Target for Therapeutic Regime Against Glioblastoma Multiforme.

An understanding of pattern recognition receptors (PRRs) and immunomodulatory approach based on activation of these receptors has provided insights critical for the management of neurological health disorders. Toll-like receptors (TLRs) are one of the most widely explored PRRs and have been exploited in the recent past for development of novel immunomodulatory therapeutic agents. Glioblastoma multiforme is characterized by significant infiltration of resident microglia and expresses all the members of the TLR family. The present report is focused on exciting findings pertaining to probable implications of TLR9 activation by unmethylated CG sequences for novel therapeutic intervention against glioblastoma multiforme, which could be a discrete step toward the effective management of neurological health issues.

Modulating neuroinflammation in neurodegeneration-related dementia: can microglial toll-like receptors pull the plug?

Neurodegeneration-associated dementia disorders (NADDs), namely Alzheimer and Parkinson diseases, are developed by a significant portion of the elderly population globally. Extensive research has provided critical insights into the molecular basis of the pathological advancements of these diseases, but an efficient curative therapy seems elusive. A common attribute of NADDs is neuroinflammation due to a chronic inflammatory response within the central nervous system (CNS), which is primarily modulated by microglia. This response within the CNS is positively regulated by cytokines, chemokines, secondary messengers or cyclic nucleotides, and free radicals. Microglia mediated immune activation is regulated by a positive feedback loop in NADDs. The present review focuses on evaluating the crosstalk between inflammatory mediators and microglia, which aggravates both the clinical progression and extent of NADDs by forming a persistent chronic inflammatory milieu within the CNS. We also discuss the role of the human gut microbiota and its effect on NADDs as well as the suitability of targeting toll-like receptors for an immunotherapeutic intervention targeting the deflation of an inflamed milieu within the CNS.

Repolarization of glioblastoma macrophage cells using non-agonistic Dectin-1 ligand encapsulating TLR-9 agonist: plausible role in regenerative medicine against brain tumor.

AIM OF THE STUDY: Glioblastoma multiforme (GBM) is the most severe forms of brain cancer, eventually becoming the leading cause of brain cancer-related death worldwide. Owing to the bleak surgical interventions and resistance to the different treatment regime, GBM is a parlous disease demanding newer therapeutical perspective for its treatment. Toll-like receptors (TLRs) are well-known members of pathogen recognition receptors (PRRs) and have been extensively explored for their therapeutic and prophylactic potential in an array of disease including cancer. Recent trends in drug delivery research has shown shift towards delivering short DNA sequences (CpG DNA) to endosomal TLR9 within immune cells (macrophages, dendritic cells, etc.) for the activation of desired inflammatory response using non-agonistic ß-glucan particles; a well-known ligand for Dectin-1 receptors. Our study is therefore focused to explore the role of nano-encapsulated CpG ODN as critical players in polarizing M2 scavenging to much desired pro-inflammatory type. MATERIALS AND METHODS: The nanoparticles entrapping CpG ODN 1826 were prepared by using a fungal polymer Schizophyllan (SPG). The constructed nanoparticles were characterized and assessed for their efficacy on rat glioblastoma cells (C6). RESULTS: The constructed Schizophyllan (SPG) nanoparticles entrapping CpG ODN 1826 (95.3%) were of 25.49 nm in diameter and thus capable of crossing blood-brain barrier. The rat glioblastoma (C6) cells evaluated for intracellular oxidative burst and cytokine levels pre- and post-incubation with nanoparticles exhibited marked elevation in the expression of intracellular ROS and IFN-γ as well as IL-1ß post treatment. CONCLUSION: The findings indicate towards potentiality of repolarizing the M2 macrophages to much desired M1 phase by inducing higgh levels of oxidative burst and inflammatory cytokines. Consequently, the apoptosis was induced in glioblastoma cells establishing the suitablity of CpG ODN carrying nanoformulations as emerging therapeutic intervention for GBM.

Hesperidin Induces ROS-Mediated Apoptosis along with Cell Cycle Arrest at G2/M Phase in Human Gall Bladder Carcinoma.

A natural predominant flavonoid hesperidin rich in citrus fruits exhibits multifunctional medicinal properties. The anticancerous potential of hesperidin has been widely explored; however, the gall bladder carcinoma (GBC) still remains untouched due to the unavailability of efficient experimental model. The aim of our study was to identify the apoptotic and antiproliferative potential of hesperidin in GBC. The promising efficacy of hesperidin was assessed through the generation of reactive oxygen species (ROS), cellular apoptosis, and loss of mitochondrial membrane potential (MMP) in the primary cells generated from surgically removed cancerous gall bladder tissues. Moreover, cell cycle analysis and caspases-3 activity were performed to confirm the apoptosis inducing potential of hesperidin. Results revealed that hesperidin exposure for 24 h at a dose of 200 µM reduced the cell proliferation of GBC cells significantly. In addition, hesperidin treatment further resulted in an increased ROS generation and nuclear condensation at the same dose. Caspase-3 activation and cell cycle arrest at G2/M phase were also accelerated in a dose-dependent manner. Together, these results suggest that hesperidin can be considered as a potential anticancerous compound for the treatment of GBC. Furthermore, evaluation of the pharmacological aspects of hesperidin is desirable for drug development.

In silico analysis of human Toll-like receptor 7 ligand binding domain.

Toll-like receptors recognizing pathogen-associated molecular patterns are preface actors for innate immunity. Among them TLR7 is a transmembrane protein playing very crucial role in the signaling pathways involved in innate immunity by recognizing viral ssRNA and specific small molecule agonists. The unavailability of experimental 3D structure of this receptor till date hampers the focused exploration of TLR7 interaction with its ligands. However, several proteins possessing high homology domain enabled us to construct a reliable 3D model of hTLR7 ECD, which was employed to generate the homodimer model using protein-protein docking strategy. Further molecular docking studies between developed homodimer model and ligands were performed to explore the most preferred site of hTLR7 ECD interacting with ligands. The comparative analysis of docking energies and protein-ligand interactions of all the ligands revealed resiquimod as the prominent agonist. Furthermore, molecular interactions between protein-ligand complexes suggested LRR15 and LRR16 region of hTLR7 ECD as the most preferential site for ligand binding. The Ser434 and Gly437 of LRR15 region of hTLR7 were found to be conserved with Drosophila Toll protein. The obtained complex model may lead to a better understanding of TLR7 functioning along with its inheritance from invertebrates to mammals.

HLA B27 typing in 511 children with juvenile idiopathic arthritis from India.

The enthesitis-related arthritis (ERA) category of juvenile idiopathic arthritis (JIA) is the most common category in India. HLA B27 has a high prevalence in ERA, and ILAR classification includes it in exclusion criteria for other categories, but due to its cost, it is not routinely done. We undertook this study to assess the prevalence of HLA B27 in ERA and other groups of juvenile arthritis in India. Consecutive patients of JIA ERA and select patients from other categories were recruited from a single tertiary care hospital over a span of 3 years. HLA B27 was tested using PCR. Five hundred and eleven children were studied: 312 had ERA, and 199 had other categories (29 oligoarthritis, 107 polyarthritis, 44 systemic onset JIA, 9 psoriatic arthritis and 10 undifferentiated). The prevalence of HLA B27 was highest in the ERA group (87 %) and correlated with the presence of sacroiliitis. Prevalence was 10.3 % in oligoarthritis, 16 % in polyarticular rheumatoid factor (RF)-positive arthritis, 26 % in RF-negative polyarticular arthritis, 66 % in psoriatic arthritis and 40 % in the unclassified and 0 % in systemic onset category. Twenty-seven children had a change in category of JIA as per ILAR owing to HLA B27 testing positive, most commonly in the RF-negative polyarthritis group. Only six of these had clinical features suggestive of Spondyloarthropathy. There is high prevalence of HLA B27 in ERA. Though HLA B27 testing helps in correct classification, a minority of these patients have features suggestive of spondyloarthropathy like back pain, enthesitis or sacroiliitis.

Cross talk between Leishmania donovani CpG DNA and Toll-like receptor 9: an immunoinformatics approach.

The precise and potential contribution of Toll-like receptors (TLRs) signaling pathways in fighting parasitic infections of Leishmania spp., an intracellular protozoan parasite, has gained significant attention during the last decades. Although it is well established that TLR9 recognizes CpG motifs in microbial genomes, the specificity of the CpG DNA pattern of Leishmania parasite interacting with endosomal TLR9 is still unknown. Hence in our study to identify the CpG DNA pattern of Leishmania donovani acting as ligand for TLR9, consecutive homology searches were performed using known CpG ODN 2216 as initial template until a consistent CpG pattern in L. donovani was found. A reliable model of TLR9 ectodomains (ECDs) as well as CpG DNA patterns was predicted to develop the 3D structural complexes of TLR9 ECD-CpG DNA utilizing molecular modeling and docking approaches. The results revealed the preferential specificity of L. donovani CpG DNA to TLR9 compared to control ODN and other CpG patterns. The interface between TLR9 and L. donovani CpG DNA was also found to be geometrically complementary with the LRR11 region of TLR9, acting as the critical region for ligand recognition. The L. donovani CpG pattern identified can be employed to derive a platform for development of an innate immunomodulatory agent for deadly disease.

Antimicrobial and immunomodulatory efficacy of extracellularly synthesized silver and gold nanoparticles by a novel phosphate solubilizing fungus Bipolaris tetramera.

BACKGROUND: Particulates of nanometers size have occupied a significant area in the field of medicinal and agricultural purposes due to their large surface-to-volume ratio and exceptional physicochemical, electronic and mechanical properties. Myconanotechnology, an interface between mycology and nanotechnology is budding nowadays for nanoparticle-fabrication using fungus or its metabolites. In the present study, we have isolated and characterized a novel phosphate solubilizing fungus B. tetramera KF934408 from rhizospheric soil. This phosphatase releasing fungus was subjected to extracellular synthesis of metal nanoparticles by redox reaction. RESULTS: Silver (AgNPs) and gold nanoparticles (AuNPs) were characterized by dynamic light scattering and transmission electron microscopic analysis. The formulated AgNPs were irregular shaped with a size ranging between 54.78 nm to 73.49 nm whereas AuNPs were spherical or hexagonal, with a size of 58.4 and 261.73 nm, respectively. The nanoparticles were assessed for their antibacterial and antifungal efficacy. The results showed effective antimicrobial activity of AgNPs against Bacillus cereus, Staphylococcus aureus, Enterobacter aeroginosa and Trichoderma sp. at higher concentrations, however, AuNPs possessed only moderate antibacterial efficacy while they found no antifungal activity. Cytotoxicity analysis of nanoparticles on J774 and THP1 α cell lines revealed the dose dependence in case of AgNPs, while AuNPs were non-toxic at both low and high doses. Furthermore, significant elevation of intracellular ROS was observed after 4 h of incubation with both the nanoparticles. The capping of fungal proteins on the particulates might be involved in the activities demonstrated by these inert metal nanoparticles. CONCLUSION: In conclusion, the findings showed that the metal nanoparticles synthesized by fungus B. tetramera could be used as an antimicrobial agents as well as cost effective and nontoxic immunomodulatory delivery vehicle.

Cytogenomics of hexavalent chromium (Cr 6+) exposed cells: a comprehensive review.

The altered cellular gene expression profile is being hypothesized as the possible molecular basis navigating the onset or progress of various morbidities. This hypothesis has been evaluated here in respect of Cr 6+ induced toxicity. Several studies using gene microarray show selective and strategic dysregulations of cellular genes and pathways induced by Cr 6+. Relevant literature has been reviewed to unravel these changes in different test systems after exposure to Cr 6+ and also to elucidate association if any, of the altered cytogenomics with Cr 6+ induced toxicity or carcinogenicity. The aim was to verify the hypothesis for critical role of altered cytogenomics in onset of Cr 6+ induced biological/clinical effects by identifying genes modulated commonly by the toxicant irrespective of test system or test concentrations/doses, and by scrutinizing their importance in regulation of the flow of mechanistically linked events crucial for resultant morbidities. Their probability as biomarkers to monitor the toxicant induced biological changes is speculative. The modulated genes have been found to cluster under the pathways that manage onset of oxidative stress, DNA damage, apoptosis, cell-cycle regulation, cytoskeleton, morphological changes, energy metabolism, biosynthesis, oncogenes, bioenergetics, and immune system critical for toxicity. In these studies, the identity of genes has been found to differ remarkably; albeit the trend of pathways' dysregulation has been found to remain similar. We conclude that the intensity of dysregulation of genes or pathways involved in mechanistic events forms a sub-threshold or threshold level depending upon the dose and type (including speciation) of the toxicant, duration of exposure, type of target cells, and niche microenvironment of cells, and the intensity of sub-threshold or threshold level of the altered cytogenomics paves way in toxicant exposed cells eventually either to opt for reversal to differentiation and growth, or to result in toxicity like dedifferentiation and apoptosis, respectively.

High therapeutic efficacy of 5-Fluorouracil-loaded exosomes against colon cancer cells.

The successful chemotherapeutic regime required for the clinical management of different cancers largely depends on the efficient drug delivery within the cancer cells. Exosomes have emerged as an enticing candidate for exploring their role as delivery vehicles. Exosomes are reported to be intrinsically nanosized vesicles competent for efficient delivery across the cellular membrane. In the present study, we assessed the feasibility of an autologous exosome-based drug delivery platform for delivering 5-Fluorouracil (5-FU) against human colon cancer HCT116 cells. Autologous exosomes have shown probable tropism toward the tumor microenvironment, which makes them the most competitive vehicle for drug delivery. It was observed that the autologous exosomes loaded with 5-FU showed an enhanced rate of drug release under acidic conditions. The result of the cell viability assay showed that treatment of 5-FU-loaded exosomes (equivalent to 5 µg 5-FU) resulted in enhanced cytotoxic effect in HCT116 cells as compared to an equivalent amount of free 5-FU (5 µg), which elucidated the efficient delivery of the 5-FU by exosomes inside the cancer cells. Subsequently, 5-FU-loaded exosomes led to increased nuclear condensation and fragmentation along with increased ROS production. In addition, 5-FU-loaded exosomes caused enhanced dissipation of mitochondrial membrane potential and caspase-3 activation, resulting in increased apoptosis induction. Our study also revealed that 5-FU-loaded exosomes upsurged the arrest in the cell cycle at the G0/G1 stage in HCT-116 cells and it was found to be associated with decreased CDK4 and Cyclin D1 expression concomitantly with the upregulation of CDK inhibitor, p21Cip1 expression. Thus, the findings from the present study highlight the advantages of autologous exosomes as a natural drug carrier which could efficiently deliver chemotherapeutic drugs to cancer cells.

Identification of novel S-adenosyl-L-homocysteine hydrolase inhibitors through homology-model-based virtual screening, synthesis, and biological evaluation.

The present study describes a successful application of computational approaches to identify novel Leishmania donovani (Ld) AdoHcyase inhibitors utilizing the differences for Ld AdoHcyase NAD(+) binding between human and Ld parasite. The development and validation of the three-dimensional (3D) structures of Ld AdoHcyase using the L. major AdoHcyase as template has been carried out. At the same time, cloning of the Ld AdoHcyase gene from clinical strains, its overexpression and purification have been performed. Further, the model was used in combined docking and molecular dynamics studies to validate the binding site of NAD in Ld. The hierarchical structure based virtual screening followed by the synthesis of five active hits and enzyme inhibition assay has resulted in the identification of novel Ld AdoHcyase inhibitors. The most potent inhibitor, compound 5, may serve as a "lead" for developing more potent Ld AdoHcy hydrolase inhibitors as potential antileishmanial agents.

Ethanolic extract of Coleus aromaticus leaves impedes the proliferation and instigates apoptotic cell death in liver cancer HepG2 cells through repressing JAK/STAT cascade.

Liver cancer or hepatocellular carcinoma (HCC) has become a leading cause for cancer burden across the globe, and incidences have tripled since the last two decades. Poor diagnosis of primary liver cancer and limited treatment strategies aggravate the challenges. Researchers globally have shown a steep inclination toward the exploration of plant-based compounds for their nutraceutical and anticancer potential to fit into the role of novel chemotherapeutics. Coleus aromaticus is a well-known culinary herb that earlier has been reported for several medicinal attributes. The current investigation deals with exploring the anticancer potential of ethanolic leaf extract of C. aromaticus (CoL-EtOH) against hepatocellular carcinoma HepG2 cell line. The observations made it evident that CoL-EtOH extract impeded the viability of HepG2 at 400 µg/ml (p < .01). Additionally, the extract also succeeded in escalating ROS production (p < .01) which aided dissipation of mitochondrial membrane potential and disruption of nuclear morphology. CoL-EtOH further activated caspase-8, -9, and -3 which was reaffirmed by increase in apoptosis at 400 µg/ml (p < .01). Moreover, post treatment with CaLEt-OH extract significantly reduced the expression of JAK-1 & STAT-3 genes (p < .01) along with regulated expression of Mcl1, Bcl-2, cyclinD1, p21, and p27 within HepG2 cells. This evidence portrays the promising anticancer potential of CoL-EtOH projecting it as a novel chemotherapeutic agent against HCC. PRACTICAL APPLICATIONS: The herb Coleus aromaticus belonging to Lamiaceae family and Coleus genus is known by various names in different regions of the world and several language-specific vernacular names. The herb has been used in therapeutic and medicinal applications as well as in culinary preparations. Various attributes of the nutritional strength and functional characteristics of the leaves in terms of carotenoids, minerals, phenols, dietary fiber, and antioxidant activity have been reported by several researchers. Carvacrol and thymol are majorly found in the plant, while chlorogenic acid and rosmarinic acid etc. as the phenolic components. The herb has been used in therapeutic and medicinal implications as well as in culinary preparations.

Enhanced field emission and improved supercapacitor obtained from plasma-modified bucky paper.

The surface morphology of bucky papers (BPs) made from single-walled carbon nanotubes (CNTs) is modified by plasma treatment resulting in the formation of vertical microstructures on the surface. The shapes of these structures are either pillarlike or conelike depending on whether the gas used during plasma treatment is Ar or CH(4) . A complex interplay between different factors, such as the electric field within the plasma sheath, polarization of the CNT, intertubular cohesive forces, and ion bombardment, result in the formation of these structures. The roles played by these factors are quantitatively and qualitatively analyzed. The final material is flexible, substrate-free, composite-free, made only of CNTs, and has discrete vertically aligned structures on its surface. It shows enhanced field emission and electrochemical charge-storage capabilities. The field enhancement factor is increased by 6.8 times, and the turn-on field drops by 3.5 times from an initial value of 0.35 to 0.1 V µm(-1) as a result of the treatment. The increase in Brunauer-Emmett-Teller surface area results in about a fourfold improvement in the specific capacitance of the BP electrodes. Capacitance values before and after the treatments are 75 and 290 F g(-1) , respectively. It is predicted that this controlled surface modification technique could be put to good use in several applications based on macroscopic CNT films.

Apoptosis induction and inhibition of hyperplasia formation by 2-[piperidinoethoxyphenyl]-3-[4-hydroxyphenyl]-2H-benzo(b)pyran in rat uterus.

OBJECTIVE: The study was undertaken to explore the antiproliferative mechanism of action of 2-[piperidinoethoxyphenyl]-3-[4-hydroxyphenyl]-2H-benzo(b)pyran (K-1) in estradiol-induced rat uterine hyperplasia. STUDY DESIGN: Adult ovariectomized rats received vehicle or estradiol alone (20 µg/kg) or estradiol along with K-1 (100 or 200 µg/kg) for 14 days. Uterine histomorphometric analysis and immunoblotting were performed. Caspase-3 activity and terminal deoxynucleotidyl transferase-mediated nick end-labeling staining were performed to analyze the apoptotic potential of compound. RESULTS: Compound inhibited estradiol-induced uterine weight and histomorphometric changes pertaining to endometrial growth and down-regulated the expression of estrogen response element and activator protein-1 regulated genes and transcription factors. The compound significantly induced apoptosis, interfered with Akt activation, decreased X-linked inhibitor of apoptosis protein expression leading to an increased cleavage of caspase-9, caspase-3, poly(adenosine diphosphate-ribose) polymerase, increased Bax/Bcl2 ratio, and caspase-3 activity. CONCLUSION: K-1 inhibits endometrial proliferation via nonclassical estrogen receptor signaling mechanisms. It interfered with Akt activation and induced apoptosis via the intrinsic pathway and inhibited estradiol-induced hyperplasia formation in rat uterus.

Leishmanial CpG DNA nanovesicles: A propitious prophylactic approach against visceral leishmaniasis.

Unmethylated CpG motifs with phosphothioate backbone trigger TLR9 to elicit innate immune response characterized by the production of Th1 cytokines. The use of CpG DNA as an adjuvant has established its role in potentiating the humoral and cell mediated vaccine specific immune response. However, none of the synthetic oligodeoxynucleotides (ODNs) know and used till date are associated with the parasite itself. Our group identified a novel CG rich sequence of 14 base pairs from Leishmania donovani genome (Ld CpG ODN) and established it as a TLR9 agonist. The present study was designed to ascertain the adjuvanticity of Ld CpG ODN with soluble leishmanial antigen in experimental model of L. donovani. During the study Schizophyllan (SPG), a fungal polymer was used for encapsulating Ld CpG ODN for efficient endosomal delivery. The synthesized nanovehicles were of nearly 100 nm and localized within endosomes as confirmed by confocal microscopy. Immunization studies displayed the superior ability of synthesized nanovehicles co-administered with parasite antigen in augmenting innate immune response in comparison to ODN, nanoparticles or soluble antigen alone. The response included generation of ROS, NO and iNOS expression followed by proinflammatory cytokine milieu with reduced parasitic load within liver, spleen and bone marrow. These immune-tailored particles in combination with parasitic antigens elicited significant generation of cell mediated response owing to the presence of high levels of CD8+ T-cells and lymphocyte proliferation. Moreover, vaccination regime with synthesized adjuvant also activated humoral immunity by escalating the levels of IgG2 followed by reduced levels of anti-leishmanial IgG and IgG1 antibodies. The findings support the efficacy of Ld CpG ODN as a potential adjuvant against visceral leishmaniasis.

Elucidation of the Chemopreventive Role of Stigmasterol Against Jab1 in Gall Bladder Carcinoma.

BACKGROUND: Plant sterols have proven a potent anti-proliferative and apoptosis inducing agent against several carcinomas including breast and prostate cancers. Jab1 has been reported to be involved in the progression of numerous carcinomas. However, antiproliferative effects of sterols against Jab1 in gall bladder cancer have not been explored yet. OBJECTIVE: In the current study, we elucidated the mechanism of action of stigmasterol regarding apoptosis induction mediated via downregulation of Jab1 protein in human gall bladder cancer cells. METHODS: In our study, we performed MTT and Trypan blue assay to assess the effect of stigmasterol on cell proliferation. In addition, RT-PCR and western blotting were performed to identify the effect of stigmasterol on Jab1 and p27 expression in human gall bladder cancer cells. We further performed cell cycle, Caspase-3, Hoechst and FITC-Annexin V analysis, to confirm the apoptosis induction in stigmasterol treated human gall bladder cancer cells. RESULTS: Our results clearly indicated that stigmasterol has up-regulated the p27 expression and down-regulated Jab1 gene. These modulations of genes might occur via mitochondrial apoptosis signaling pathway. Caspase-3 gets activated with the apoptotic induction. Increase in apoptotic cells and DNA were confirmed through annexin V staining, Hoechst staining, and cell cycle analysis. CONCLUSION: Thus, these results strongly suggest that stigmasterol has the potential to be considered as an anticancerous therapeutic agent against Jab1 in gall bladder cancer.

Pharmacophore-based virtual screening approach for identification of potent natural modulatory compounds of human Toll-like receptor 7.

Toll-like receptor 7 (TLR7) is a transmembrane glycoprotein playing very crucial role in the signaling pathways involved in innate immunity and has been demonstrated to be useful in fighting against infectious disease by recognizing viral ssRNA & specific small molecule agonists. In order to find novel human TLR7 (hTLR7) modulators, computational ligand-based pharmacophore modeling approach was used to identify the molecular chemical features required for the modulation of hTLR7 protein. A training set of 20 TLR7 agonists with their known experimental activity was used to create pharmacophore model using 3D-QSAR pharmacophore generation (HypoGen algorithm) module in Discovery Studio. The best developed hypothesis consists of four pharmacophoric features namely, one hydrogen bond donor (HBD), one ring aromatic (RA), and two hydrophobic (HY) character. The developed hypothesis was then validated by different methods such as cost analysis, test set method, and Fischer's test method for consistency. Hence, this validated model was further employed for screening of natural hit compounds from InterBioScreen Natural product database, consisting of more than 60,000 natural compounds and derivatives. The screened hit compounds were subsequently filtered by Lipinski's rule of 5, ADME and toxicity parameters and molecular docking studies to remove the false positive rates. Finally, molecular docking analysis led to identification of the (3a'S,6a'R)-3'-(3,4-dihydroxybenzyl)-5'-(3,4-dimethoxyphenethyl)-5-ethyl-3',3a'-dihydro-2'H-spiro[indoline-3,1'-pyrrolo[3,4-c]pyrrole]-2,4',6'(5'H,6a'H)-trione (Compound ID: STOCK1N-65837) as potent hTLR7 modulator due to its better docking score and molecular interactions compared to other compounds. The result of virtual screening was further validated using molecular dynamics (MD) simulation analysis. Thus, a 30 ns MD simulation analysis revealed high stability and effective binding of STOCK1N-65837 within the binding site of hTLR7. Therefore, the present study provides confidence for the utility of the selected chemical feature based pharmacophore model to design novel TLR7 modulators with desired biological activity.

Jab1-siRNA Induces Cell Growth Inhibition and Cell Cycle Arrest in Gall Bladder Cancer Cells via Targeting Jab1 Signalosome.

BACKGROUND: The aberrant alteration in Jab1 signalosome (COP9 Signalosome Complex Subunit 5) has been proven to be associated with the progression of several carcinomas. However the specific role and mechanism of action of Jab1 signalosome in carcinogenesis of gall bladder cancer (GBC) are poorly understood. OBJECTIVE: The main objective of our study was to elucidate the role and mechanism of Jab1 signalosome in gall bladder cancer by employing siRNA. METHODS: Jab1 overexpression was identified in gall bladder cancer tissue sample. The role of Jab1-siRNA approach in cell growth inhibition and apoptotic induction was then examined by RT-PCR, Western Blotting, MTT, ROS, Hoechst and FITC/Annexin-V staining. RESULTS: In the current study, we have shown that overexpression of Jab1 stimulated the proliferation of GBC cells; whereas downregulation of Jab1 by using Jab1-siRNA approach resulted incell growth inhibition and apoptotic induction. Furthermore, we found that downregulation of Jab1 induces cell cycle arrest at G1 phase and upregulated the expression of p27, p53 and Bax gene. Moreover, Jab1-siRNA induces apoptosis by enhancing ROS generation and caspase-3 activation. In addition, combined treatment with Jab1-siRNA and gemicitabine demonstrated an enhanced decline in cell proliferation which further suggested increased efficacy of gemcitabine at a very lower dose (5µM) in combination with Jab1-siRNA. CONCLUSION: In conclusion, our study strongly suggests that targeting Jab1 signalosome could be a promising therapeutic target for the treatment of gall bladder cancer.

Phytochemical Study of Aegle marmelos: Chromatographic Elucidation of Polyphenolics and Assessment of Antioxidant and Cytotoxic Potential.

BACKGROUND: The antioxidant potential of medicinal plants has been illustrated through many reports clearly depicting that plants are a rich source of antioxidants, making them a great resource of novel drugs and health-care products. OBJECTIVES: The current study is, therefore, focused toward the assessment of antioxidant properties along with the presence of phytochemicals in leaves of 18 varieties/accessions of Aegle marmelos. MATERIALS AND METHODS: The antioxidant activities were initially measured using superoxide radical scavenging method, 2, 2-diphenyl-1-picrylhydrazyl (DPPH), and ferric-reducing ability of plasma assays. Further, thin-layer chromatography (TLC), high-performance TLC, and column chromatography were performed to isolate the potentially active fraction and anti-inflammatory activity of crude, and the isolated fraction was tested on J774 macrophage cell line. RESULTS: The maximum inhibition of superoxide anions was shown by Pant Aparna. Additionally, Pant Aparna extract was most efficient, exhibiting 92.0% inhibition in scavenging the DPPH radicals. The content of total carotenoids was found to be higher in Pant Aparna among all the varieties/accessions. Furthermore, the crude extract and the fraction A. marmelos methanolic fraction 21 (AMMF21) were found to be nontoxic and significant reactive oxygen species, and NO inhibition was observed in a concentration-dependent manner. Moreover, the methanolic extract of variety Pant Aparna showed promising in vitro antioxidant activity, indicating its potency for therapeutic applications. CONCLUSION: In brief, this is the first ever report on Pant Aparna as the best variety in terms of phytocompounds and identification of potential antioxidant activity. In addition, the AMMF21 fraction of methanolic extract possessing best antioxidant activity on macrophage cells indicates its use as a novel phytotherapeutic agent. SUMMARY: Our study identifies the best variety/accession of Aegle marmelos possessing the potential antioxidant and reactive oxygen species scavenging activity possessed by the methanolic crude extract of variety Pant Aparna along with the fraction A. marmelos methanolic fraction 21 isolated through column chromatography on J774 murine macrophage cell lineThe high-performance thin-layer chromatography fingerprinting profile obtained acts as a diagnostic tool to identify and determine the quality and purity of this extract and fraction in future studiesOn the basis of the results obtained, the above variety should be taken further to exploit its immense potential for other biological activities of medicinal importancePant Aparna is an outstanding variety of A. marmelos and should be extensively studied for isolation of a novel and potential therapeutic agent. Abbreviations used: AMMF21: Aegle marmelos methanolic fraction 21, DPPH: (2, 2-diphenyl-1-picrylhydrazyl), FRAP: Ferric-reducing ability of plasma, HP-TLC: High-performance-thin-layer chromatography, TLC: Thin-layer chromatography, TCA: Trichloroacetic acid, TPTZ: 2,4,6-Tripyridyl-s-triazine, DNPH: 2,4-dinitrophenyl hydrazine, NBT: Nitroblue tetrazolium, NADH: Nicotinamide adenine dinucleotide, PMS: Phenazine metho-sulfate, DMEM: Dulbecco's modified Eagle medium; MTT: (3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide, DCFDA: 2',7'-dichlorofluorescein diacetate, LPS: Lipopolysaccharide, NED: N-(1-Naphthyl) ethylenediamine.