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2.
Front Microbiol ; 14: 996287, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36846749

RESUMO

Bacillus cereus sensu lato (Bcsl) strains are widely explored due to their capacity to antagonize a broad range of plant pathogens. These include B. cereus sp. UW85, whose antagonistic capacity is attributed to the secondary metabolite Zwittermicin A (ZwA). We recently isolated four soil and root-associated Bcsl strains (MO2, S-10, S-25, LSTW-24) that displayed different growth profiles and in-vitro antagonistic effects against three soilborne plant pathogens models: Pythium aphanidermatum (oomycete) Rhizoctonia solani (basidiomycete), and Fusarium oxysporum (ascomycete). To identify genetic mechanisms potentially responsible for the differences in growth and antagonistic phenotypes of these Bcsl strains, we sequenced and compared their genomes, and that of strain UW85 using a hybrid sequencing pipeline. Despite similarities, specific Bcsl strains had unique secondary metabolite and chitinase-encoding genes that could potentially explain observed differences in in-vitro chitinolytic potential and anti-fungal activity. Strains UW85, S-10 and S-25 contained a (~500 Kbp) mega-plasmid that harbored the ZwA biosynthetic gene cluster. The UW85 mega-plasmid contained more ABC transporters than the other two strains, whereas the S-25 mega-plasmid carried a unique cluster containing cellulose and chitin degrading genes. Collectively, comparative genomics revealed several mechanisms that can potentially explain differences in in-vitro antagonism of Bcsl strains toward fungal plant pathogens.

3.
Drug Dev Res ; 83(2): 222-224, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-32216115

RESUMO

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.


Assuntos
Leishmania donovani , Leishmaniose Visceral , Vacinas , Ilhas de CpG , Humanos , Leishmania donovani/genética , Leishmania donovani/metabolismo , Leishmaniose Visceral/prevenção & controle , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismo
4.
Comput Struct Biotechnol J ; 19: 6201-6211, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34900133

RESUMO

Farmers apply broiler chicken litter to soils to enrich organic matter and provide crops with nutrients, following varying periods of stockpiling. However, litter frequently harbors fecal-derived microbial pathogens and associated antibiotic resistance genes (ARGs), and may be a source of microbial contamination of produce. We coupled a cutting-edge Loop Genomics long-read 16S rRNA amplicon-sequencing platform with high-throughput qPCR that targeted a suite of ARGs, to assess temporal (five time points over a 60-day period) and spatial (top, middle and bottom layers) microbiome and resistome dynamics in a broiler litter stockpile. We focused on potentially pathogenic species from the Enterobacteriaceae, Enterococcaceae and Staphylococcaceae families associated with food-borne disease. Bacterial diversity was significantly lower in the middle of the stockpile, where targeted pathogens were lowest and Bacillaceae were abundant. E. coli was the most abundant Enterobacteriaceae species, and high levels of the opportunistic pathogen Enterococcus faecium were detected. Correlation analyses revealed that the latter was significantly associated with aminoglycoside (aac(6')-Ib(aka aacA4), aadA5), tetracycline (tetG), vancomycin (vanC), phenicol (floR) and MLSB (mphB) resistance genes. Staphylococcaceae were primarily non-pathogenic, but extremely low levels of the opportunistic pathogen S. aureus were detected, as was the opportunistic pathogen S. saprophyticus, which was linked to vancomycin (vanSA, vanC1), MLSB (vatE, ermB) and tetracycline (tetK) resistance genes. Collectively, we found that stockpile microbiomes and resistomes are strongly dictated by temporal fluctuations and spatial heterogeneity. Insights from this study can be exploited to improve stockpile management practice to support sustainable antimicrobial resistance mitigation policies in the future.

5.
Microbiome ; 9(1): 178, 2021 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-34454634

RESUMO

BACKGROUND: Therapeutic and growth-promoting antibiotics are frequently used in broiler production. Indirect evidence indicates that these practices are linked to the proliferation of antimicrobial resistance (AMR), the spread of antibiotic-resistant bacteria from food animals to humans, and the environment, but there is a lack of comprehensive experimental data supporting this. We investigated the effects of growth promotor (bacitracin) and therapeutic (enrofloxacin) antibiotic administration on AMR in broilers for the duration of a production cycle, using a holistic approach that integrated both culture-dependent and culture-independent methods. We specifically focused on pathogen-harboring families (Enterobacteriaceae, Enterococcaceae, and Staphylococcaceae). RESULTS: Antibiotic-resistant bacteria and antibiotic resistance genes were ubiquitous in chicken cloaca and litter regardless of antibiotic administration. Environment (cloaca vs. litter) and growth stage were the primary drivers of variation in the microbiomes and resistomes, with increased bacterial diversity and a general decrease in abundance of the pathogen-harboring families with age. Bacitracin-fed groups had higher levels of bacitracin resistance genes and of vancomycin-resistant Enterococcaceae (total Enterococcaceae counts were not higher). Although metagenomic analyses classified 28-76% of the Enterococcaceae as the commensal human pathogens E. faecalis and E. faecium, culture-based analysis suggested that approximately 98% of the vancomycin-resistant Enterococcaceae were avian and not human-associated, suggesting differences in the taxonomic profiles of the resistant and non-resistant strains. Enrofloxacin treatments had varying effects, but generally facilitated increased relative abundance of multidrug-resistant Enterobacteriaceae strains, which were primarily E. coli. Metagenomic approaches revealed a diverse array of Staphylococcus spp., but the opportunistic pathogen S. aureus and methicillin resistance genes were not detected in culture-based or metagenomic analyses. Camphylobacteriaceae were significantly more abundant in the cloacal samples, especially in enrofloxacin-treated chickens, where a metagenome-assembled C. jejuni genome harboring fluoroquinolone and ß-lactam resistance genes was identified. CONCLUSIONS: Within a "farm-to-fork, one health" perspective, considering the evidence that bacitracin and enrofloxacin used in poultry production can select for resistance, we recommend their use be regulated. Furthermore, we suggest routine surveillance of ESBL E. coli, vancomycin-resistant E. faecalis and E. faecium, and fluoroquinolone-resistant C. jejuni strains considering their pathogenic nature and capacity to disseminate AMR to the environment. Video Abstract.


Assuntos
Antibacterianos/uso terapêutico , Galinhas , Farmacorresistência Bacteriana , Microbiota , Animais , Cloaca/microbiologia , Farmacorresistência Bacteriana/genética , Escherichia coli , Estudos Longitudinais , Staphylococcus aureus
6.
Environ Sci Technol ; 55(10): 6814-6827, 2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-33904706

RESUMO

Treated-wastewater (TW) irrigation transfers antibiotic-resistant bacteria (ARB) to soil, but persistence of these bacteria is generally low due to resilience of the soil microbiome. Nonetheless, wastewater-derived bacteria and associated antibiotic resistance genes (ARGs) may persist below detection levels and potentially proliferate under copiotrophic conditions. To test this hypothesis, we exposed soils from microcosm, lysimeter, and field experiments to short-term enrichment in copiotroph-stimulating media. In microcosms, enrichment stimulated growth of multidrug-resistant Escherichia coli up to 2 weeks after falling below detection limits. Lysimeter and orchard soils irrigated in-tandem with either freshwater or TW were subjected to culture-based, qPCR and shotgun metagenomic analyses prior, and subsequent, to enrichment. Although native TW- and freshwater-irrigated soil microbiomes and resistomes were similar to each other, enrichment resulted in higher abundances of cephalosporin- and carbapenem-resistant Enterobacteriaceae and in substantial differences in the composition of microbial communities and ARGs. Enrichment stimulated ARG-harboring Bacillaceae in the freshwater-irrigated soils, whereas in TWW-irrigated soils, ARG-harboring γ-proteobacterial families Enterobacteriaceae and Moraxellaceae were more profuse. We demonstrate that TW-derived ARB and associated ARGs can persist at below detection levels in irrigated soils and believe that similar short-term enrichment strategies can be applied for environmental antimicrobial risk assessment in the future.


Assuntos
Solo , Águas Residuárias , Irrigação Agrícola , Antagonistas de Receptores de Angiotensina , Inibidores da Enzima Conversora de Angiotensina , Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos/genética , Genes Bacterianos , Humanos , Microbiologia do Solo , Águas Residuárias/análise
7.
Int J Neurosci ; 131(6): 591-598, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32250189

RESUMO

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.


Assuntos
Adjuvantes Imunológicos , Neoplasias Encefálicas/tratamento farmacológico , Citocinas/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Lectinas Tipo C , Macrófagos/efeitos dos fármacos , Nanopartículas , Oligodesoxirribonucleotídeos , Sizofirano , Receptor Toll-Like 9/agonistas , Adjuvantes Imunológicos/administração & dosagem , Animais , Linhagem Celular Tumoral , Citocinas/metabolismo , Interferon gama/efeitos dos fármacos , Interferon gama/metabolismo , Interleucina-1beta/efeitos dos fármacos , Interleucina-1beta/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Sizofirano/administração & dosagem
8.
Int Immunopharmacol ; 90: 107181, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33249044

RESUMO

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.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Antígenos de Protozoários/administração & dosagem , Leishmania donovani/imunologia , Leishmaniose Visceral/prevenção & controle , Nanopartículas , Oligodesoxirribonucleotídeos/administração & dosagem , Vacinas Protozoárias/administração & dosagem , Sizofirano/administração & dosagem , Adjuvantes Imunológicos/química , Animais , Antígenos de Protozoários/química , Modelos Animais de Doenças , Composição de Medicamentos , Interações Hospedeiro-Patógeno , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Imunogenicidade da Vacina , Leishmania donovani/patogenicidade , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Masculino , Mesocricetus , Oligodesoxirribonucleotídeos/química , Vacinas Protozoárias/química , Sizofirano/química , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/parasitologia , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/metabolismo , Vacinação
9.
Cell Mol Neurobiol ; 41(7): 1391-1393, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32691190

RESUMO

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.


Assuntos
Glioblastoma/tratamento farmacológico , Microglia/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor Toll-Like 9/metabolismo , Animais , Glioblastoma/metabolismo , Humanos , Microglia/efeitos dos fármacos , Receptor Toll-Like 9/genética
10.
Front Microbiol ; 11: 1814, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32849421

RESUMO

Several factors influenced the sugarcane production, and among them, higher use of nitrogen and depletion of soil nutrient constitutes a significant concern in China. Sugarcane-legume intercropping may help to regulate the microbial structure and functions. In the present study, sugarcane rhizosphere soils of three cropping systems: Sugarcane only (S-only), sugarcane with peanut (S + P), and sugarcane + soybean (S + S) were sampled in tillering, elongation, and maturation stages from two different experimental fields. High-throughput sequencing technologies applied to assess the effects of different cropping systems on the structure of nitrogenase (nifH) gene communities. A total of 3818 OTUs (operational taxonomic units) were acquired from all soil samples. Intercropping systems noticeably increased the relative abundance of Proteobacteria in the tillering stage. The increased microbial diversity in the rhizosphere was mainly due to soil organic carbon and total soil N. In contrast, intercropping has no significant negative impact on microbial abundance, but sugarcane growth stages influence it significantly, and two bacteria (Bradyrhizobium and Pseudacidovorax) showed significant shift during plant growth. The results provide insight into the microbial structure of Proteobacteria in the sugarcane legume-intercropping field, and how microbial community behaves in different growth stages. It can boost the microbial activity of the soil, and that could be a new strategy to stimulate soil fertility without causing any negative impact on crop production.

11.
Eur J Med Chem ; 185: 111833, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31734024

RESUMO

Herein we present and describe the design and synthesis of novel phenantrene derivatives substituted with either amino or amido side chains and their biological activity. Antiproliferative activities were assessed in vitro on a panel of human cancer cell lines. Tested compounds showed moderate activity against cancer cells in comparison with 5-fluorouracile. Among all tested compounds, some compounds substituted with cyano groups showed a pronounced and selective activity in the nanomolar range of inhibitory concentrations against HeLa and HepG2. The strongest selective activity against HeLa cells was observed for acrylonitriles 8 and 11 and their cyclic analogues 15 and 17 substituted with two cyano groups with a corresponding IC50 = 0.33, 0.21, 0.65 and 0.45 µM, respectively. Compounds 11 showed the most pronounced selectivity being almost non cytotoxic to normal fibroblasts. Additionally, mode of biological action analysis was performed in silico and in vitro by Western blot analysis of HIF-1-α relative expression for compounds 8 and 11.


Assuntos
Antineoplásicos/farmacologia , Fenantrenos/farmacologia , Tiofenos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Células Hep G2 , Humanos , Estrutura Molecular , Fenantrenos/síntese química , Fenantrenos/química , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/química , Células Tumorais Cultivadas
12.
Protein Expr Purif ; 159: 1-9, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30831225

RESUMO

Arginase catalyzes the first committed step in the biosynthesis of polyamines that enable cell growth and hence potential drug target for the treatment of leishmaniasis. The arginase from Leishmania donovani (LdARG) was cloned, overexpressed and characterized. Analysis of the deduced amino acid sequence of LdARG with homologous enzyme from other trypanosomatids arginases identified a non-conserved 12 residues long segment VWGLIERTFLSA from position 161-172. This counter segment in L. mexicana arginase exhibits a different conformation compared with human arginase I. The pH and temperature optima of LdARG were 9.0 and 37 °C, respectively. Biochemical studies revealed that the KM for the substrate L-arginine was 24.76 ±â€¯0.06 mM. Molecular modeling of LdARG studies revealed that the glutamic acid residue at position 288 plays a role in substrate binding. The importance of this glutamic acid residue was validated by constructing a mutant variant of LdARG (E288Q-LdARG) by replacing glutamic acid with glutamine through site-directed mutagenesis. The KM value of mutant variant for L-arginine was found to be 107 ±â€¯0.18 mM. The increase in KM value of E288Q-LdARG as compared to LdARG suggested that substrate binding was significantly affected which could be exploited further. Studies on biochemical and structural characterization of recombinant LdARG will help in evaluating this enzyme as a potential drug target for visceral leishmaniasis.


Assuntos
Arginase/metabolismo , Leishmania donovani/metabolismo , Poliaminas/química , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Arginase/genética , Sítios de Ligação , Biocatálise , Inibidores Enzimáticos/metabolismo , Ácido Glutâmico/química , Glutamina/química , Cinética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Conformação Proteica , Proteínas de Protozoários/genética , Proteínas Recombinantes/genética , Termodinâmica
13.
J Biomol Struct Dyn ; 37(18): 4721-4736, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-30661449

RESUMO

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.


Assuntos
Fatores Imunológicos/química , Receptor 7 Toll-Like/agonistas , Receptor 7 Toll-Like/química , Algoritmos , Desenho de Fármacos , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Relação Quantitativa Estrutura-Atividade , Receptor 7 Toll-Like/imunologia
14.
Antiviral Res ; 158: 143-146, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30125616

RESUMO

We tested 65 highly pathogenic avian influenza (HPAI) A(H5N1) viruses, isolated from avian species in India between 2006 and 2015, for susceptibility to the FDA approved neuraminidase (NA) inhibitors (NAIs), oseltamivir and zanamivir using a phenotypic fluorescence-based assay. The overall incidence of resistant variants among HPAI A(H5N1) viruses was 7.69% (5/65). The NA inhibition assay identified 3 viruses resistant to oseltamivir (N294S substitution, N2 numbering) and 2 cross-resistant to oseltamivir and zanamivir (E119A or I117V+E119A substitutions), all of which belonged to hemagglutinin (HA) clade 2.2 (5/17) and predominantly circulated in Indian poultry during 2006-2010. In comparison to E119A substitution alone, viruses with I117V+E119A double substitutions showed greater reduction in susceptibility to both oseltamivir and zanamivir. The NAI resistance-associated NA markers, identified in this study, were as a result of naturally occurring mutations. Of note, 48 viruses of HA clade 2.3.2.1 that circulated in Indian poultry during 2011-2015 were susceptible to both oseltamivir and zanamivir. It is essential to monitor NAI susceptibility among human and avian HPAI A(H5N1) viruses that would provide baseline data to develop strategies for pandemic preparedness and therapeutic interventions.


Assuntos
Antivirais/farmacologia , Farmacorresistência Viral/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Neuraminidase/antagonistas & inibidores , Substituição de Aminoácidos , Animais , Aves/virologia , Farmacorresistência Viral/genética , Índia , Virus da Influenza A Subtipo H5N1/genética , Mutagênese , Oseltamivir/farmacologia , Proteínas Virais/antagonistas & inibidores , Zanamivir/farmacologia
15.
Oncotarget ; 8(24): 38650-38667, 2017 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-28418856

RESUMO

Protease-activated receptor-2 (PAR2) plays a central role in cancer; however, the molecular machinery of PAR2-instigated tumors remains to be elucidated. We show that PAR2 is a potent inducer of ß-catenin stabilization, a core process in cancer biology, leading to its transcriptional activity. Novel association of low-density lipoprotein-related protein 6 (LRP6), a known coreceptor of Frizzleds (Fz), with PAR2 takes place following PAR2 activation. The association between PAR2 and LRP6 was demonstrated employing co-immunoprecipitation, bioluminescence resonance energy transfer (BRET), and confocal microscopy analysis. The association was further supported by ZDOCK protein-protein server. PAR2-LRP6 interaction promotes rapid phosphorylation of LRP6, which results in the recruitment of Axin. Confocal microscopy of PAR2-driven mammary gland tumors in vivo, as well as in vitro confirms the association between PAR2 and LRP6. Indeed, shRNA silencing of LRP6 potently inhibits PAR2-induced ß-catenin stabilization, demonstrating its critical role in the induced path. We have previously shown a novel link between protease-activated receptor-1 (PAR1) and ß-catenin stabilization, both in a transgenic (tg) mouse model with overexpression of human PAR1 (hPar1) in the mammary glands, and in cancer epithelial cell lines. Unlike in PAR1-Gα13 axis, both Gα12 and Gα13 are equally involved in PAR2-induced ß-catenin stabilization. Disheveled (DVL) is translocated to the cell nucleus through the DVL-PDZ domain. Collectively, our data demonstrate a novel PAR2-LRP6-Axin interaction as a key axis of PAR2-induced ß-catenin stabilization in cancer. This newly described axis enhances our understanding of cancer biology, and opens new avenues for future development of anti-cancer therapies.


Assuntos
Biomarcadores Tumorais/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Receptores Acoplados a Proteínas G/metabolismo , beta Catenina/química , Sequência de Aminoácidos , Apoptose , Proteína Axina/genética , Proteína Axina/metabolismo , Biomarcadores Tumorais/genética , Proliferação de Células , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/antagonistas & inibidores , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Neoplasias/genética , Fosforilação , Conformação Proteica , RNA Interferente Pequeno/genética , Receptor PAR-2 , Receptores Acoplados a Proteínas G/genética , Homologia de Sequência , Transdução de Sinais , Células Tumorais Cultivadas , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
16.
Future Microbiol ; 11(2): 195-214, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26855259

RESUMO

AIM: Wolbachia is a promising antifilarial chemotherapeutic target. Translation initiation factor-1 (Tl IF-1) is an essential factor in prokaryotes. Functional characterization of Wolbachia's novel proteins/enzymes is necessary for the development of adulticidal drugs. MATERIALS & METHODS: Mutant, Wol Tl IF-1 R45D was constructed by site directed mutagenesis. Fluorimetry and size exclusion chromatography were used to determine the biophysical characteristics. Mobility shift assay and fluorescence resonance energy transfer were used to investigate the functional aspect of Wol Tl IF-1 with its mutant. RESULTS: Both wild and mutant were in monomeric native conformations. Wild exhibits nonspecific binding with ssRNA/ssDNA fragments under electrostatic conditions and showed annealing and displacement of RNA strands in comparison to mutant. CONCLUSION: Point mutation impaired RNA chaperone activity of the mutant and its interaction with nucleotides.


Assuntos
Arginina , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fator de Iniciação 1 em Procariotos/genética , Fator de Iniciação 1 em Procariotos/metabolismo , Wolbachia/genética , Wolbachia/metabolismo , Animais , Proteínas de Bactérias/química , Evolução Biológica , Brugia Malayi/microbiologia , DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Mutagênese Sítio-Dirigida , Filogenia , Mutação Puntual , Fator de Iniciação 1 em Procariotos/química , Ligação Proteica , RNA/metabolismo , Alinhamento de Sequência
17.
Interdiscip Sci ; 8(1): 41-52, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26264054

RESUMO

Parasitic MAPKs exhibiting significant divergence with humans and playing an imperative role in parasitic metabolic activities have been exploited from several years as important targets for development of novel therapeutics. In addition, the emergence of the drug-resistant variants of parasitic diseases in the recent years has aroused a great need for the development of potent inhibitors against them. In the present study, we selected the metabolically active MAPKs LmxMPK4, PfMAP2 and TbMAPK5 of the three parasitic protozoans Leishmania mexicana, Plasmodium falciparum and Trypanosoma brucei, respectively. The homology modeling technique was used to develop the 3D structures of these proteins, and the same was validated by PROCHECK, ERRAT, ProQ and ProSA web servers to check the reliability. Ten phytoligands were employed for molecular docking studies with these proteins to search for potent phytoligand as a broad spectrum inhibitor. In this regard, two phytoligands (aspidocarpine for LmxMPK4 and TbMAPK5 and cubebin for PfMAP2) were found to be more effective inhibitors, in terms of robust binding energy, strong inhibition constant and better interactions between protein-ligand complexes. Furthermore, predicted ADME and toxicity properties suggested that these identified phytoligands exhibited comparable results to control drugs potentiating them as persuasive therapeutic agents for Leishmania, Trypanosoma and Plasmodium sp.


Assuntos
Simulação por Computador , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Parasitos/enzimologia , Compostos Fitoquímicos/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Sequência de Aminoácidos , Animais , Antiparasitários/química , Antiparasitários/farmacologia , Humanos , Ligantes , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Parasitos/efeitos dos fármacos , Filogenia , Compostos Fitoquímicos/química , Reprodutibilidade dos Testes , Alinhamento de Sequência , Homologia Estrutural de Proteína , Termodinâmica
18.
Biotechnol Appl Biochem ; 63(3): 441-50, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-25817271

RESUMO

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.


Assuntos
Simulação por Computador , Receptor 7 Toll-Like/química , Receptor 7 Toll-Like/metabolismo , Animais , Drosophila melanogaster , Humanos , Ligantes , Camundongos , Simulação de Acoplamento Molecular , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Estrutura Quaternária de Proteína , Alinhamento de Sequência
19.
Biochem Biophys Res Commun ; 459(3): 424-9, 2015 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-25735984

RESUMO

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.


Assuntos
DNA de Protozoário/genética , Leishmania donovani/genética , Leishmania donovani/imunologia , Oligodesoxirribonucleotídeos/genética , Receptor Toll-Like 9/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Biologia Computacional , Ilhas de CpG , DNA de Protozoário/química , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Leishmania donovani/patogenicidade , Ligantes , Camundongos , Modelos Moleculares , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Conformação Proteica , Receptor Cross-Talk , Transdução de Sinais , Receptor Toll-Like 9/química
20.
Interdiscip Sci ; 2014 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-25519156

RESUMO

Parasitic MAPKs exhibiting significant divergence with humans and playing an imperative role in parasitic metabolic activities have been exploited from several years as important targets for development of novel therapeutics. In addition, the emergence of the drug resistant variants of parasitic diseases in the recent years has aroused a great need for the development of potent inhibitors against them. In the present study we selected the metabolically active MAPKs LmxMPK4, PfMAP2 and TbMAPK5 of the three parasitic protozoans Leishmania mexicana, Plasmodium falciparum and Trypanosoma brucei respectively. The homology modeling technique was used to develop the 3D structures of these proteins and the same was validated by PROCHECK, ERRAT, ProQ and ProSA web servers to check the reliability. Ten phytoligands were employed for molecular docking studies with these proteins to search for potent phytoligand as a broad spectrum inhibitor. In this regard two phytoligands (Aspidocarpine for LmxMPK4 & TbMAPK5 and Cubebin for PfMAP2) were found to be more effective inhibitors, in term of robust binding energy, strong inhibition constant and better interactions between protein-ligand complexes. Furthermore predicted ADME & Toxicity properties suggested that these identified phytoligands exhibited comparable results to control drugs potentiating them as persuasive therapeutic agents for Leishmania, Trypanosoma and Plasmodium sp.

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