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In this work, Ag nanoparticles decorated with NiFe2O4/CuWO4 heterostructure were synthesized using the step-wise precipitation method. The influence of varying Ag loading on the NiFe2O4/CuWO4 heterostructure and its electrochemical OER performance was extensively studied in 1 M KOH electrolyte. The obtained LSV profile was analyzed to determine the overpotential, Tafel slope, and onset potential. The heterostructure with an optimal Ag loading of 5 wt% required the least overpotential (1.60 V vs. RHE) for generating a current density of 10 mA cm-2 with a lower Tafel slope of 44.5 mV dec-1, indicating its faster OER kinetics. Furthermore, the composite remained stable over a period of 24 hours with a minimum rise in the overpotential after the stability test. The enhanced OER performance of the as-prepared catalyst can be attributed to the presence of multiple metallic elements in the Ag-loaded NiFe2O4/CuWO4 composite, which created a diverse array of oxygen-vacant sites with varying reactivity, enhancing the charge-transfer kinetics; and thus contributing to the overall efficiency of OER. Therefore, optimizing the Ag concentration and engineering a microstructure represents an encouraging strategy for developing cost-effective catalysts for next-generation energy-conversion applications.
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Natural products possess unique and broader intricacies in the chemical space and have been essential for drug discovery. The crucial factor for drug discovery success is not the size of the library but rather its structural diversity. Although reports on the number of new structurally diverse natural products (NPs) have declined recently, researchers follow the next logical step: synthesizing natural product hybrids and their analogues using the most potent tool, diversity-oriented synthesis (DOS). Here, we use weed Parthenium hysterophorus as a source of parthenin for synthesis of novel dispiro-pyrrolizidino/thiopyrrolizidino-oxindolo/indanedione natural product hybrids of parthenin via chemo-, regio-, and stereoselective azomethine ylide cycloaddition. All synthesized compounds were characterized through a detailed analysis of one-dimensional (1D) and two-dimensional (2D) NMR and HRMS data, and the stereochemistries of the compounds were confirmed by X-ray diffraction analysis. All compounds were evaluated for their cytotoxicity against four cell lines (HCT-116, A549, Mia-Paca-2, and MCF-7), and compound 6 inhibited the HCT-116 cells with an IC50 of 5.0 ± 0.08 µM.
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We report a high rate of seropositivity against SARS-CoV-2 in wild felines in India. Seropositivity was determined by microneutralization and plaque reduction neutralization assays in captive Asiatic lions, leopards, and Bengal tigers. The rate of seropositivity was positively correlated with that of the incidence in humans, suggesting the occurrence of large spillover events.
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COVID-19 , Leões , Panthera , Tigres , Animais , Gatos , Humanos , SARS-CoV-2 , Estudos Retrospectivos , COVID-19/epidemiologia , Índia/epidemiologiaRESUMO
BACKGROUND/OBJECTIVES: Chronic low-grade inflammation/meta-inflammation in adipose tissue leads to obesity-associated metabolic complications. Despite growing understanding, the roles of immune cell subsets, their interrelationship, and chronological events leading to progression of obesity-associated insulin resistance (IR) remains unclear. METHODS: We carried out temporal immunometabolic profiling of adipose tissue from C57BL/6 mice fed a high-fat diet (HFD) for 4, 8, 12, 16, and 20 weeks. We used clodronate sodium liposomes (CLODs) to deplete macrophages and disodium cromoglycate sodium liposomes (DSCGs) to stabilize mast cells. RESULTS: In the temporal HFD settings, mice showed progressive glucose intolerance, insulin resistance, and adipose tissue senescence. Histochemistry analysis of epididymal white adipose tissue (eWAT) using picro-sirius red and Masson's trichrome staining showed extensive collagen deposition in the 16th and 20th weeks. Flow cytometry analysis of the stromal vascular fraction (SVF) from eWAT revealed T-cell subsets as early-phase components and pro-inflammatory macrophages, as well as mast cells as the later phase components during obesity progression. In our therapeutic strategies, macrophage depletion by CLOD and mast stabilization by DSCG attenuated obesity, adipose tissue fibrosis, and improved whole-body glucose homeostasis. In addition, mast cell stabilization also attenuated senescence (p53 and X-gal staining) in eWAT, signifying the role of mast cells over macrophages during obesity. CONCLUSION: New-generation mast cell stabilizers can be exploited for the treatment of obesity-associated metabolic complications.
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Tecido Adiposo/imunologia , Tecido Adiposo/metabolismo , Envelhecimento/patologia , Dieta Hiperlipídica , Fibrose/patologia , Mastócitos/patologia , Obesidade/imunologia , Obesidade/metabolismo , Tecido Adiposo/patologia , Animais , Modelos Animais de Doenças , Inflamação/metabolismo , Resistência à Insulina , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/patologiaRESUMO
The ubiquitin proteasome system (UPS) is the major pathway for intracellular protein degradation in most organs, including the heart. UPS controls many fundamental biological processes such as cell cycle, cell division, immune responses, antigen presentation, apoptosis, and cell signaling. The UPS not only degrades substrates but also regulates activity of gene transcription at the post-transcription level. Emerging evidence suggests that impairment of UPS function is sufficient to cause a number of cardiac diseases, including heart failure, cardiomyopathies, hypertrophy, atrophy, ischemia-reperfusion, and atherosclerosis. Alterations in the expression of UPS components, changes in proteasomal peptidase activities and increased ubiquitinated and oxidized proteins have also been detected in diabetic cardiomyopathy (DCM). However, the pathophysiological role of the UPS in DCM has not been examined. Recently, in vitro and in vivo studies have proven highly valuable in assessing effects of various stressors on the UPS and, in some cases, suggesting a causal link between defective protein clearance and disease phenotypes in different cardiac diseases, including DCM. Translation of these findings to human disease can be greatly strengthened by corroboration of discoveries from experimental model systems using human heart tissue from well-defined patient populations. This review will summarize the general role of the UPS in different cardiac diseases, with major focus on DCM, and on recent advances in therapeutic development.
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Cardiopatias/enzimologia , Cardiopatias/terapia , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Complicações do Diabetes/enzimologia , Complicações do Diabetes/terapia , Humanos , Ubiquitina/metabolismoRESUMO
Expression levels of A disintegrin and metalloproteases (ADAMs) (10 and 17) and Th17-related cytokines [interleukin (IL) 17A, IL-17F, IL-33, IL-23, IL-23R] were investigated by quantitative real time polymerase chain reaction in gastric biopsies of patients with different gastroduodenal pathologies in the presence and absence of Helicobacter pylori infection. Patients with gastric cancer (GC) (n = 70, intestinal-type 38 and diffuse type 32), peptic ulcer disease [n = 50, duodenal ulcer (DU) 16 and gastric ulcer (GU) 34] and functional dyspepsia (n = 120) were included in the study. Further, the expression levels of ADAMs and Th17 cytokines were correlated with H. pylori cytotoxin-associated genes pathogenicity island (cagPAI) status. Expression levels of ADAMs (10 and 17) and Th17-related cytokines (IL-17A, IL-23, IL-23R) were significantly higher in H. pylori-positive than in H. pylori-negative gastric biopsies. Significant increase in ADAM17 and Th17 cytokines (IL-17A and IL-23) expressions was observed in patients with GU and intestinal-type GC in the presence of H. pylori infection and in strains harbouring intact cagPAI. Expression levels of IL-17A, IL-23 and ADAM17 were strongly correlated with GU and intestinal-type GC and weakly with DU and diffuse-type GC in the presence of H. pylori infection. Higher expression levels of ADAM17 and Th17 cytokines (IL-17A and IL-23), and their strong correlation with GU and intestinal-type GC patients in the presence of H. pylori and its intact cagPAI status, suggest a possible role of strain specificity in the pathogenesis of these diseases.
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Citocinas/biossíntese , Desintegrinas/biossíntese , Infecções por Helicobacter/patologia , Helicobacter pylori/crescimento & desenvolvimento , Metaloproteases/biossíntese , Úlcera Péptica/patologia , Neoplasias Gástricas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Citocinas/genética , Desintegrinas/genética , Feminino , Mucosa Gástrica/patologia , Humanos , Mucosa Intestinal/patologia , Masculino , Metaloproteases/genética , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Cell cycle regulation is one of the hallmarks of virus-mediated oncogenesis. Epstein-Barr virus (EBV)-induced lymphomas express a repertoire of essential viral latent proteins that regulate expression of cell cycle-related proteins to dysregulate this process, thereby facilitating the proliferation of infected cells. We now demonstrate that the essential EBV latent protein 3C (EBNA3C) stabilizes cyclin D2 to regulate cell cycle progression. More specifically, EBNA3C directly binds to cyclin D2 and they colocalize together in nuclear compartments. We show that EBNA3C regulates the promoter of cyclin D2 through cooperation with master transcription factor Bcl6 and enhances its stability by inhibiting its ubiquitin-dependent degradation. EBNA3C also promoted cell proliferation in the presence of cyclin D2, suggesting that cyclin D2 contributes to EBNA3C-mediated cell cycle progression. These results provide new clues as to the role of this essential viral latent protein and its ability to regulate expression of cellular factors, which drives the oncogenic process.IMPORTANCE Epstein-Barr virus (EBV) is the first identified human tumor virus and is associated with a range of human cancers. During EBV-induced lymphomas, the essential viral latent proteins modify the expression of cell cycle-related proteins to disturb the cell cycle process, thereby facilitating the proliferative process. The essential EBV nuclear antigen 3C (EBNA3C) plays an important role in EBV-mediated B-cell transformation. Here we show that EBNA3C stabilizes cyclin D2 to regulate cell cycle progression. More specifically, EBNA3C directly binds to cyclin D2, and they colocalize together in nuclear compartments. EBNA3C enhances cyclin D2 stability by inhibiting its ubiquitin-dependent degradation and significantly promotes cell proliferation in the presence of cyclin D2. Our results provide novel insights into the function of EBNA3C on cell progression by regulating the cyclin D2 protein and raise the possibility of the development of new anticancer therapies against EBV-associated cancers.
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Proliferação de Células/genética , Ciclina D2/genética , Antígenos Nucleares do Vírus Epstein-Barr/genética , Regulação da Expressão Gênica , Linfócitos B/virologia , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Transformação Celular Viral , Herpesvirus Humano 4/fisiologia , HumanosRESUMO
BACKGROUND/AIMS: Systemic hyperlipidemia and intracellular lipid accumulation induced by chronic high fat diet (HFD) leads to enhanced fatty acid oxidation (FAO) and ketogenesis. The present study was aimed to determine whether activation of peroxisome proliferator-activated receptor-γ (PPAR-γ) by surplus free fatty acids (FA) in hyperlipidemic condition, has a positive feedback regulation over FAO and ketogenic enzymes controlling lipotoxicity and cardiac apoptosis. METHODS: 8 weeks old C57BL/6 wild type (WT) or PPAR-γ-/- mice were challenged with 16 weeks 60% HFD to induce obesity mediated type 2 diabetes mellitus (T2DM) and diabetic cardiomyopathy. Treatment course was followed by echocardiographic measurements, glycemic and lipid profiling, immunoblot, qPCR and immunohistochemistry (IHC) analysis of PPAR-γ and following mitochondrial metabolic enzymes 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS2), mitochondrial ß- hydroxy butyrate dehydrogenase (BDH1) and pyruvate dehydrogenase kinase isoform 4 (PDK4). In vivo model was translated in vitro, with neonatal rat cardiomyocytes (NRCM) treated with PPAR-γ agonist/antagonist and PPAR-γ overexpression adenovirus in presence of palmitic acid (PA). Apoptosis was determined in vivo from left ventricular heart by TUNEL assay and immunoblot analysis. RESULTS: We found exaggerated circulating ketone bodies production and expressions of the related mitochondrial enzymes HMGCS2, BDH1 and PDK4 in HFD-induced diabetic hearts and in PA-treated NRCM. As a mechanistic approach we found HFD mediated activation of PPAR-γ is associated with the above-mentioned mitochondrial enzymes. HFD-fed PPAR-γ-/-mice display decreased hyperglycemia, hyperlipidemia associated with increased insulin responsiveness as compared to HFD-fed WT mice PPAR-γ-/-HFD mice demonstrated a more robust functional recovery after diabetes induction, as well as significantly reduced myocyte apoptosis and improved cardiac function. CONCLUSIONS: PPAR-γ has been described previously to regulate lipid metabolism and adipogenesis. The present study suggests for the first time that increased PPAR-γ expression by HFD is responsible for cardiac dysfunction via upregulation of mitochondrial enzymes HMGCS2, BDH1 and PDK4. Targeting PPAR-γ and its downstream mitochondrial enzymes will provide novel strategies in preventing metabolic and myocardial dysfunction in diabetes mellitus.
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Diabetes Mellitus Tipo 2/metabolismo , Cardiomiopatias Diabéticas/metabolismo , Dieta Hiperlipídica/efeitos adversos , Ácidos Graxos/metabolismo , Corpos Cetônicos/metabolismo , PPAR gama/metabolismo , Disfunção Ventricular Esquerda/metabolismo , Animais , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/patologia , Cardiomiopatias Diabéticas/sangue , Cardiomiopatias Diabéticas/etiologia , Cardiomiopatias Diabéticas/patologia , Ácidos Graxos/sangue , Hipertrofia Ventricular Esquerda/sangue , Hipertrofia Ventricular Esquerda/etiologia , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/patologia , Corpos Cetônicos/sangue , Masculino , Camundongos Endogâmicos C57BL , Oxirredução , Disfunção Ventricular Esquerda/sangue , Disfunção Ventricular Esquerda/etiologia , Disfunção Ventricular Esquerda/patologiaRESUMO
The molecular pathways activated in response to acute cathepsin G (CG) exposure, as well as the mechanisms involved in activation of signaling pathways that culminate in myocyte detachment and apoptosis remain unclear. This study aimed to determine the changes in gene expression patterns associated with time dependent CG exposure to neonatal rat cardiomyocytes (NRCMs). Microarray analysis revealed a total of 451, 572 and 1127 differentially expressed genes after CG exposure at 1, 4 and 8â¯h respectively. A total of 54 overlapped genes at each time point were mapped by Ingenuity Pathway Analysis (IPA). The top up-regulated genes included Hamp, SMAD6, NR4A1, FOSL2, ID3 and SLAMF7, and down-regulated genes included CYR61, GDF6, Olr640, Vom2r36, DUSP6 and MMP20. Our data suggest that there are multiple deregulated pathways associated with cardiomyocyte death after CG exposure, including JAK/Stat signaling, IL-9 signaling and Nur77 signaling. In addition, we also generated the molecular network of expressed gene and found most of the molecules were connected to ERK1/2, caspase, BCR (complex) and Cyclins. Our study reveals the ability to assess time-dependent changes in gene expression patterns in NRCMs associated with CG exposure. The global gene expression profiles may provide insight into the cellular mechanism that regulates CG dependent myocyte apoptosis. In future, the pathways important in CG response, as well as the genes found to be differentially expressed might represent the therapeutic targets for myocyte survival in heart failure.
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Catepsina G/farmacologia , Redes Reguladoras de Genes/efeitos dos fármacos , Miócitos Cardíacos/citologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , Animais Recém-Nascidos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Ratos , Software , Fatores de TempoRESUMO
Helicobacter pylori and Epstein-Barr virus (EBV) are two well-known contributors to cancer and can establish lifelong persistent infection in the host. This leads to chronic inflammation, which also contributes to development of cancer. Association with H. pylori increases the risk of gastric carcinoma, and coexistence with EBV enhances proliferation of infected cells. Further, H. pylori-EBV coinfection causes chronic inflammation in pediatric patients. We have established an H. pylori-EBV coinfection model system using human gastric epithelial cells. We showed that H. pylori infection can increase the oncogenic phenotype of EBV-infected cells and that the cytotoxin-associated gene (CagA) protein encoded by H. pylori stimulated EBV-mediated cell proliferation in this coinfection model system. This led to increased expression of DNA methyl transferases (DNMTs), which reprogrammed cellular transcriptional profiles, including those of tumor suppressor genes (TSGs), through hypermethylation. These findings provide new insights into a molecular mechanism whereby cooperativity between two oncogenic agents leads to enhanced oncogenic activity of gastric cancer cells.IMPORTANCE We have studied the cooperativity between H. pylori and EBV, two known oncogenic agents. This led to an enhanced oncogenic phenotype in gastric epithelial cells. We now demonstrate that EBV-driven epigenetic modifications are enhanced in the presence of H. pylori, more specifically, in the presence of its CagA secretory antigen. This results in increased proliferation of the infected gastric cells. Our findings now elucidate a molecular mechanism whereby expression of cellular DNA methyl transferases is induced influencing infection by EBV. Hypermethylation of the regulatory genomic regions of tumor suppressor genes results in their silencing. This drastically affects the expression of cell cycle, apoptosis, and DNA repair genes, which dysregulates their associated processes, and promotion of the oncogenic phenotype.
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Proliferação de Células , Coinfecção/microbiologia , Coinfecção/virologia , Epigênese Genética , Células Epiteliais/fisiologia , Helicobacter pylori/crescimento & desenvolvimento , Herpesvirus Humano 4/crescimento & desenvolvimento , Linhagem Celular , Transformação Celular Neoplásica , Células Epiteliais/microbiologia , Células Epiteliais/virologia , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/patologia , Perfilação da Expressão Gênica , Infecções por Helicobacter/complicações , Infecções por Helicobacter/patologia , Humanos , Modelos BiológicosRESUMO
Diabetes increases the risk of Cardio-vascular disease (CVD). CVD is more prevalent in type 2 diabetes (T2D) than type 1 diabetes (T1D), but the mortality risk is higher in T1D than in T2D. The pathophysiology of CVD in T1D is poorly defined. To learn more about biological pathways that are potentially involved in T1D with cardiac dysfunction, we sought to identify differentially expressed genes in the T1D heart. Our study used T1D mice with severe hyperglycemia along with significant deficits in echocardiographic measurements. Microarray analysis of heart tissue RNA revealed that the T1D mice differentially expressed 10 genes compared to control. Using Ingenuity Pathway Analysis (IPA), we showed that these genes were significantly involved in ketogenesis, cardiovascular disease, apoptosis and other toxicology functions. Of these 10 genes, the 3-Hydroxy-3-Methylglutaryl-CoA Synthase 2 (HMGCS2) was the highest upregulated gene in T1D heart. IPA analysis showed that HMGCS2 was center to many biological networks and pathways. Our data also suggested that apart from heart, the expression of HMGCS2 was also different in kidney and spleen between control and STZ treated mice. In conclusion, The HMGCS2 molecule may potentially be involved in T1D induced cardiac dysfunction.
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Doenças Cardiovasculares/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Hidroximetilglutaril-CoA Sintase/metabolismo , Corpos Cetônicos/biossíntese , Animais , Doenças Cardiovasculares/diagnóstico , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/fisiopatologia , Biologia Computacional/métodos , Diabetes Mellitus Tipo 1/genética , Ecocardiografia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Hemodinâmica , Humanos , Hidroximetilglutaril-CoA Sintase/genética , Masculino , Camundongos , Anotação de Sequência Molecular , TranscriptomaRESUMO
Epstein-Barr virus (EBV), a gamma herpes virus is associated with B-cell malignancies. EBNA-3C is critical for in vitro primary B-cell transformation. Interestingly, the N terminal domain of EBNA3C which contains residues 130-159, interacts with various cellular proteins, such as p53, Mdm2, CyclinD1/Cdk6 complex, and E2F1. In the current reverse genetics study, we deleted the residues 130-159 aa within EBNA3C open reading frame (ORF) by BACmid recombinant engineering methodology. Our experiments demonstrated that deletion of the 130-159 aa showed a reduction in cell proliferation. Also, this recombinant virus showed with higher infectivity of human peripheral blood mononuclear cells (PBMCs) compared to wild type EBV. PBMCs- infected with recombinant EBV deleted for 130-159 residues have differential expression patterns for the p53/Mdm2, CyclinD1/Cdk6 and pRb/E2F1 pathways compared to wild type EBV-infected PBMCs. PBMCs infected with recombinant virus showed increased apoptotic cell death which further resulted in activation of polymerase 1 (PARP1), an important contributor to apoptotic signaling. Interestingly, cells infected with this recombinant virus showed a dramatic decrease in chromosomal instability, indicated by the presence of increased multinucleation and micronucleation. In addition infection with recombinant virus have increased cells in G0/G1 phase and decreased cells in S-G2M phase when compared to wild type infected cells. Thus, these differences in signaling activities due to 29 amino acid residues of EBNA3C is of particular significance in deregulation of cell proliferation in EBV-infected cells.
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Apoptose/genética , Proliferação de Células/genética , Ciclina D1/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Herpesvirus Humano 4/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Linfócitos B/metabolismo , Linhagem Celular , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Transformação Celular Viral , Fator de Transcrição E2F1/metabolismo , Ativação Enzimática , Infecções por Vírus Epstein-Barr , Antígenos Nucleares do Vírus Epstein-Barr/genética , Células HEK293 , Humanos , Leucócitos Mononucleares/virologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Deleção de Sequência/genéticaRESUMO
BACKGROUND: Transforming growth factor-beta 1 (TGF-ß1), a multifunctional cytokine, acts as a key factor for Epstein-Barr virus (EBV) reactivation. We investigated the role of TGF-ß1 in latent and lytic stages of EBV in relation to Helicobacter pylori infection among patients with gastric cancer (GC) and peptic ulcer disease (PUD). METHOD: Gastric mucosal TGF-ß1 expression was determined in 95 EBV positive patients with gastroduodenal pathology [GC 40, PUD 19 and non-ulcer dyspepsia (NUD) 36] by quantitative real time PCR. Presence of H. pylori infection was diagnosed when either culture or any two of three tests (RUT, histopathology and specific ureA PCR) were positive. Serum level of TGF-ß1 was detected among 60 patients using ELISA. RESULTS: Mucosal TGF-ß1 mRNA expression was detected in 85 of 95 EBV positive patients and it was significantly higher in patients with GC (p=0.042). TGF-ß1 expression tended to be higher among H. pylori non-infected than infected patients (3.80±6.24 vs. 2.07±2.50, p=0.085). Both mRNA and serum level had significant association with lytic stage of EBV in absence of H. pylori infection when compared with its presence (5.21±4.00 vs. 2.29±2.89, p=0.040 and 842.00 [669.55] vs. 662.63 [628.76], p=0.049; respectively). CONCLUSION: TGF-ß1 expression was significantly associated with GC. TGF-ß1 was higher both at expression and translational levels in lytic EBV infection without H. pylori suggests that H. pylori infection might play important role in preventing EBV reactivation through attenuated TGF-ß1 expression. This might be a "wise host defense against EBV reactivation".
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Infecções por Vírus Epstein-Barr/genética , Neoplasias Gástricas/genética , Fator de Crescimento Transformador beta1/genética , Adulto , Idoso , Ensaio de Imunoadsorção Enzimática , Infecções por Vírus Epstein-Barr/virologia , Feminino , Regulação Neoplásica da Expressão Gênica , Infecções por Helicobacter/genética , Infecções por Helicobacter/microbiologia , Herpesvirus Humano 4/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Masculino , Pessoa de Meia-Idade , Úlcera Péptica/genética , Úlcera Péptica/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/virologia , Fator de Crescimento Transformador beta1/sangue , Ativação Viral/fisiologiaRESUMO
Epstein-Barr virus (EBV) is an oncogenic gammaherpes virus which is linked to pathogenesis of several human lymphatic malignancies. The EBV essential latent antigen EBNA3C is critical for efficient conversion of primary human B-lymphocytes to lymphoblastic cell lines and for continued LCL growth. EBNA3C, an EBV latent antigen with oncogenic potential can bind and regulate the functions of a wide range of cellular transcription factors. In our current reverse genetics study, we deleted the full length EBNA3C, and independently the RBP-Jκ and Nm23-H1 binding sites within EBNA3C using BACmid recombinant engineering methodology. Our experiments demonstrated that deletion of the EBV EBNA3C open reading frame (ORF) and more specifically the residues 621-675 which binds Nm23H1 and SUMO-1 showed a significant reduction in the ability of the cells to proliferate. Furthermore, they exhibited lower infectivity of human peripheral blood mononuclear cells (PBMCs). We also showed that recombinant EBV with deletions of the EBNA3C ORF, as well as a recombinant with residues 621-675 within EBNA3C ORF deleted had diminished abilities to activate CD40. Our study also revealed that the full length (1-992) and 621-675 aa deletions of EBNA3C when compared to wild type EBV infected PBMCs had differential expression patterns for the phosphorylation of MAP kinases specifically p38, JNK and ERK. Regulation of ß-catenin also differed among wild type and EBNA3C deleted mutants. These temporal differences in signaling activities of these recombinant viruses in PBMCs is likely important in defining their functional importance in EBV-mediated B-cell transformation.
Assuntos
Proliferação de Células , Transformação Celular Viral , Infecções por Vírus Epstein-Barr/virologia , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Ativação Linfocitária , Linfócitos/virologia , Sítios de Ligação , Antígenos CD40/metabolismo , Infecções por Vírus Epstein-Barr/imunologia , Antígenos Nucleares do Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/imunologia , Células HEK293 , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/imunologia , Herpesvirus Humano 4/patogenicidade , Interações Hospedeiro-Patógeno , Humanos , Linfócitos/imunologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Fases de Leitura Aberta , Cultura Primária de Células , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteína SUMO-1/metabolismo , Transdução de Sinais , Fatores de Tempo , TransfecçãoRESUMO
AIM: This study was carried out to determine the presence of blaTEM , blaSHV and blaCTX-M genes in extended-spectrum ß-lactamase (ESBL) producing Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) at a tertiary care referral hospital in Northeast India. MATERIALS AND METHODS: A total of 270 E. coli and 219 K. pneumoniae isolates were recovered during the period between August 2009 and July 2010. Kirby-Bauer disk diffusion method was performed to determine the antibiotic resistance pattern. Screening and phenotypic confirmatory test for ESBL production were performed using standard disc diffusion methods. Each of the initial ESBL screening test isolate was investigated for the presence of blaTEM , blaSHV and blaCTX-M genes via polymerase chain reaction (PCR) using gene-specific primers. RESULTS: Phenotypic confirmatory test able to detect ESBL production in 73.58% of E. coli and 67.24% of K. pneumoniae. However, PCR amplification showed the presence of one or more ESBL genes in each of the initial ESBL screening positive isolate. Among three ESBL genotypes, the most prevalent genotype was found to be blaCTX-M in E. coli (88.67%) and blaTEM in K. pneumoniae (77.58%) ESBL producing isolates. Majority of ESBL producing isolates possess more than one ESBL genes. CONCLUSION: This study constituted a primer report on high prevalence of blaTEM and blaCTX-M genes in ESBL producing isolates of E. coli and K. pneumoniae and denotes the need of more extensive studies on these antibiotic genes to determine the magnitude of the problem of antibiotic resistance exiting in this locality.
Assuntos
Infecções por Escherichia coli/microbiologia , Escherichia coli/enzimologia , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/enzimologia , beta-Lactamases/genética , Primers do DNA/genética , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/epidemiologia , Humanos , Índia/epidemiologia , Infecções por Klebsiella/epidemiologia , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase , Prevalência , Centros de Atenção TerciáriaRESUMO
The growing prevalence of carbapenem resistance in Enterobacteriaceae worldwide is a major concern. New Delhi metallo-ß-lactamase (NDM)-mediated carbapenem resistance has been identified in Enterobacteriaceae from numerous countries including those of the Indian subcontinent. Currently, seven NDM ß-lactamase variants (NDM-1 to -7) have been identified. This study evaluated the detection and molecular characterisation of NDM variants in Enterobacteriaceae at a tertiary care hospital in India. A total of 464 isolates were tested; 57 (12.3%) were resistant or showed reduced susceptibility to imipenem and meropenem. All carbapenem-resistant isolates were blaNDM-positive by PCR, but 13 isolates bore variants that differed in sequence from blaNDM-1. NDM-5, NDM-6 and NDM-7 were identified in two, eight and three isolates, respectively. blaNDM variants were located on plasmids of >100kb with IncF, IncA/C and untypeable replicon types. Genes encoding the 16S rRNA methyltransferases RmtB, RmtC and ArmA as well as those for AmpC ß-lactamases were also located on the same plasmids as blaNDM in different combinations. The prevalence of NDM-5 to -7 variants was significantly higher in Escherichia coli (P=0.015) and they were more frequently isolated from the urology ward (P=0.037) than NDM-1. The mortality rate was comparable between patients infected with isolates positive for blaNDM-1 and blaNDM variants [25% (11/44) vs. 23% (3/13)]. Expression of blaNDM variants in E. coli using the same promoter showed that NDM-7 conferred higher resistance to imipenem. The diverse genotypic features of blaNDM indicate rapid evolution of NDM resulting from their wide spread in the Indian subcontinent.
Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Infecções por Enterobacteriaceae/tratamento farmacológico , Enterobacteriaceae/genética , Regulação Bacteriana da Expressão Gênica , Plasmídeos/metabolismo , beta-Lactamases/genética , Adulto , Idoso , Antibacterianos/uso terapêutico , Pré-Escolar , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/enzimologia , Enterobacteriaceae/crescimento & desenvolvimento , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/mortalidade , Feminino , Humanos , Índia , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Metiltransferases/genética , Metiltransferases/metabolismo , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Tipagem de Sequências Multilocus , Análise de Sobrevida , Centros de Atenção Terciária , beta-Lactamases/metabolismo , beta-Lactamas/uso terapêuticoRESUMO
CagL is a pilus protein of Helicobacter pylori that interacts with host cellular α5ß1 integrins through its arginine-glycine-aspartate (RGD) motif, guiding proper positioning of the T4SS and translocation of CagA. Deletion or sequence variations of cagL significantly diminished the ability of H. pylori to induce secretion of IL-8 by the host cell. Therefore, this study was undertaken to investigate the association of cagL and its amino acid sequence polymorphisms with gastric cancer (GC), peptic ulcer disease (PUD), and non-ulcer dyspepsia (NUD) as there are no such studies from India. In total, 200 adult patients (NUD 120, PUD 30, GC 50) who underwent an upper gastrointestinal endoscopy were enrolled. H. pylori infection was diagnosed by rapid urease test, culture, histopathology, and PCR. The collected isolates were screened for cagL genotype by PCR and assessed for amino acid sequence polymorphisms using sequence translation. The prevalence of H. pylori infection in study population was 52.5%. Most of the isolates were cagL genopositive (86.6%), and all had RGD motif in their amino acid sequences. D58 and K59 polymorphisms in cagL-genopositive strains were significantly higher in GC patients (P < 0.05). Combined D58K59 polymorphism was associated with higher risk of GC (3.8-fold) when compared to NUD. In conclusion, H. pylori cagL amino acid polymorphisms such as D58K59 are correlated with a higher risk of GC in the Indian population. Further studies are required to know the exact role of particular cagL amino acid polymorphisms in the pathogenicity of H. pylori infection.
Assuntos
Proteínas de Bactérias/genética , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Neoplasias Gástricas/microbiologia , Adulto , Sequência de Aminoácidos , Dispepsia/microbiologia , Endoscopia Gastrointestinal , Genótipo , Infecções por Helicobacter/epidemiologia , Helicobacter pylori/isolamento & purificação , Helicobacter pylori/patogenicidade , Humanos , Índia/epidemiologia , Masculino , Pessoa de Meia-Idade , Úlcera Péptica/microbiologia , Reação em Cadeia da Polimerase , Polimorfismo Genético , Prevalência , Risco , Análise de Sequência de DNARESUMO
BACKGROUND & OBJECTIVES: Mutations in the oncogene and tumour suppressor genes play an important role in carcinogenesis. We investigated the association of p53 and K-ras gene mutation and Helicobacter pylori infection in patients with gastric cancer (GC) and peptic ulcer disease (PUD) attending a tertiary care hospital in north India. METHODS: In total, 348 adult patients [62 GC, 45 PUD and 241 non-ulcer dyspepsia (NUD)] who underwent an upper gastrointestinal endoscopy were enrolled. H. pylori infection was diagnosed by rapid urease test, culture, histopathology and PCR. Mutation in the exon 5-8 of p53 gene was analyzed by PCR-single stranded conformational polymorphism (SSCP) and confirmed by sequence analysis. K-ras gene codon 12 mutation was analyzed by PCR-based restriction fragment length polymorphism. RESULTS: Overall p53 gene mutation was found in 4.6 per cent of the study population, and its distribution in GC, PUD and NUD was 21, 4.4 and 0.4 per cent, respectively. p53 gene mutation was significantly higher in patients with GC than PUD (P<0.05) and NUD (P<0.001). No difference in p53 gene mutation was observed between H. pylori infected and non-infected individuals. K-ras gene mutation was absent in all the patients. INTERPRETATION & CONCLUSIONS: Our results show that p53 gene mutation may be associated with gastric carcinogenesis independent to H. pylori infection and absence of K-ras gene mutation questions its role in the pathogenesis of GC and PUD in Indian patients.
Assuntos
Genes ras/genética , Infecções por Helicobacter/genética , Helicobacter pylori , Úlcera Péptica/genética , Úlcera Péptica/microbiologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/microbiologia , Proteína Supressora de Tumor p53/genética , Primers do DNA/genética , Humanos , Mutação/genética , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Polimorfismo Conformacional de Fita Simples , Análise de Sequência de DNARESUMO
Helicobacter pylori and Epstein-Barr virus (EBV) infections are common worldwide. Although H. pylori infection is a major factor in gastroduodenal diseases, its role in association with EBV infection is unknown. Objective: To study the association of H. pylori infection and EBV DNA load in patients with gastroduodenal diseases. Methods: Biopsy samples were collected from 200 adult patients [non-ulcer dyspepsia (NUD) 100, peptic ulcer disease (PUD) 50, gastric carcinoma (GC) 50] undergoing upper gastrointestinal endoscopy. H. pylori infection was diagnosed by rapid urease test, culture, histopathology, PCR and Q-PCR. EBV DNA was detected by non-polymorphic Epstein-Barr nuclear antigen-1 (EBNA-1) gene based Q-PCR. Results: In patients with GC and PUD, EBV DNA was detected more often than NUD (GC versus NUD = 90 percent versus 37 percent, p < 0.001; PUD versus NUD = 70 percent versus 37 percent, p < 0.001). The dual prevalence of H. pylori infection and EBV DNA was significantly higher in patients with GC and PUD than in those with NUD. Median copy number of EBV DNA was considerably higher in GC and PUD than NUD (p < 0.01). The copy number of EBV DNA was significantly higher in H. pylori infected patients (p = 0.015). The number of ureA gene copies was also found to be significantly higher in PUD and NUD with presence of EBV DNA. However, in GC no significant difference was seen between EBV positive and negative status. Conclusion: There was a trend for higher EBV DNA load in H. pylori positive individuals suggesting a probable role of H. pylori in modulating the conversion of EBV to its lytic phase.