RESUMO
The core protein (Cp) of hepatitis B virus (HBV) assembles pregenomic RNA (pgRNA) and viral DNA polymerase to form nucleocapsids where the reverse transcriptional viral DNA replication takes place. Core protein allosteric modulators (CpAMs) inhibit HBV replication by binding to a hydrophobic "HAP" pocket at Cp dimer-dimer interfaces to misdirect the assembly of Cp dimers into aberrant or morphologically "normal" capsids devoid of pgRNA. We report herein that a panel of CpAM-resistant Cp with single amino acid substitution of residues at the dimer-dimer interface not only disrupted pgRNA packaging, but also compromised nucleocapsid envelopment, virion infectivity and covalently closed circular (ccc) DNA biosynthesis. Interestingly, these mutations also significantly reduced the secretion of HBeAg. Biochemical analysis revealed that the CpAM-resistant mutations in the context of precore protein (p25) did not affect the levels of p22 produced by signal peptidase removal of N-terminal 19 amino acid residues, but significantly reduced p17, which is produced by furin cleavage of C-terminal arginine-rich domain of p22 and secreted as HBeAg. Interestingly, p22 existed as both unphosphorylated and phosphorylated forms. While the unphosphorylated p22 is in the membranous secretary organelles and the precursor of HBeAg, p22 in the cytosol and nuclei is hyperphosphorylated at the C-terminal arginine-rich domain and interacts with Cp to disrupt capsid assembly and viral DNA replication. The results thus indicate that in addition to nucleocapsid assembly, interaction of Cp at dimer-dimer interface also plays important roles in the production and infectivity of progeny virions through modulation of nucleocapsid envelopment and uncoating. Similar interaction at reduced p17 dimer-dimer interface appears to be important for its metabolic stability and sensitivity to CpAM suppression of HBeAg secretion.
Assuntos
Antígenos E da Hepatite B/metabolismo , Vírus da Hepatite B/fisiologia , Hepatite B/virologia , Multimerização Proteica , Proteínas do Core Viral/química , Montagem de Vírus , Replicação Viral , Replicação do DNA , DNA Viral , Células Hep G2 , Humanos , Nucleocapsídeo , Proteínas do Core Viral/metabolismoRESUMO
OBJECTIVES: To develop and evaluate an artificial intelligence (AI) system that can automatically calculate the glomerular filtration rate (GFR) from dynamic renal imaging without manually delineating the regions of interest (ROIs) of kidneys and the corresponding background. METHODS: This study was a single-center retrospective analysis of the data of 14,634 patients who underwent 99mTc-DTPA dynamic renal imaging. Two systems based on convolutional neural networks (CNN) were developed and evaluated: sGFRa predicts the radioactive counts of ROIs and calculates GFR using the Gates equation and sGFRb directly predicts GFR from dynamic renal imaging without using other information. The root-mean-square error (RMSE), mean absolute error (MAE), mean absolute percentage error (MAPE), and R2 were used to evaluate the performance of our approach. RESULTS: sGFRa achieved an RMSE of 5.05, MAE of 4.03, MAPE of 6.07%, and R2 of 0.93 for total GFR while sGFRb achieved an RMSE of 7.61, MAE of 5.92, MAPE of 8.92%, and R2 of 0.85 for total GFR. The accuracy of sGFRa and sGFRb in determining the stage of chronic kidney disease was 87.41% and 82.44%, respectively. CONCLUSIONS: The findings of sGFRa show that automatic GFR calculation based on CNN and using dynamic renal imaging is feasible and efficient and, additionally, can aid clinical diagnosis. Furthermore, the promising results of sGFRb demonstrate that CNN can predict GFR from dynamic renal imaging without additional information. KEY POINTS: ⢠Our CNN-based AI systems can automatically calculate GFR from dynamic renal imaging without manually delineating the ROIs of kidneys and the corresponding background. ⢠sGFRa accurately predicted the radioactive counts of ROIs and calculated GFR using the Gates method. ⢠sGFRb-predicted GFR directly without any parameters related to the Gates equation.
Assuntos
Renografia por Radioisótopo , Pentetato de Tecnécio Tc 99m , Humanos , Taxa de Filtração Glomerular , Renografia por Radioisótopo/métodos , Inteligência Artificial , Estudos Retrospectivos , Compostos Radiofarmacêuticos , Rim/diagnóstico por imagemRESUMO
Covalently closed circular DNA (cccDNA) of hepadnaviruses exists as an episomal minichromosome in the nucleus of an infected hepatocyte and serves as the template for the transcription of viral mRNAs. It had been demonstrated by others and us that interferon alpha (IFN-α) treatment of hepatocytes induced a prolonged suppression of human and duck hepatitis B virus cccDNA transcription, which is associated with the reduction of cccDNA-associated histone modifications specifying active transcription (H3K9ac or H3K27ac), but not the histone modifications marking constitutive (H3K9me3) or facultative (H3K27me3) heterochromatin formation. In our efforts to identify IFN-induced cellular proteins that mediate the suppression of cccDNA transcription by the cytokine, we found that downregulating the expression of signal transducer and activator of transcription 1 (STAT1), structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1), or promyelocytic leukemia (PML) protein increased basal level of cccDNA transcription activity and partially attenuated IFN-α suppression of cccDNA transcription. In contrast, ectopic expression of STAT1, SMCHD1, or PML significantly reduced cccDNA transcription activity. SMCHD1 is a noncanonical SMC family protein and implicated in epigenetic silencing of gene expression. PML is a component of nuclear domain 10 (ND10) and is involved in suppressing the replication of many DNA viruses. Mechanistic analyses demonstrated that STAT1, SMCHD1, and PML were recruited to cccDNA minichromosomes and phenocopied the IFN-α-induced posttranslational modifications of cccDNA-associated histones. We thus conclude that STAT1, SMCHD1, and PML may partly mediate the suppressive effect of IFN-α on hepadnaviral cccDNA transcription.IMPORTANCE Pegylated IFN-α is the only therapeutic regimen that can induce a functional cure of chronic hepatitis B in a small, but significant, fraction of treated patients. Understanding the mechanisms underlying the antiviral functions of IFN-α in hepadnaviral infection may reveal molecular targets for development of novel antiviral agents to improve the therapeutic efficacy of IFN-α. By a loss-of-function genetic screening of individual IFN-stimulated genes (ISGs) on hepadnaviral mRNAs transcribed from cccDNA, we found that downregulating the expression of STAT1, SMCHD1, or PML significantly increased the level of viral RNAs without altering the level of cccDNA. Mechanistic analyses indicated that those cellular proteins are recruited to cccDNA minichromosomes and induce the posttranslational modifications of cccDNA-associated histones similar to those induced by IFN-α treatment. We have thus identified three IFN-α-induced cellular proteins that suppress cccDNA transcription and may partly mediate IFN-α silencing of hepadnaviral cccDNA transcription.
Assuntos
DNA Circular/metabolismo , Hepadnaviridae/efeitos dos fármacos , Hepadnaviridae/genética , Interferon-alfa/metabolismo , Interferon-alfa/farmacologia , Animais , Antivirais/metabolismo , Antivirais/farmacologia , Linhagem Celular , Galinhas , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , DNA Viral/genética , Epigênese Genética , Infecções por Hepadnaviridae/virologia , Vírus da Hepatite B do Pato/efeitos dos fármacos , Vírus da Hepatite B , Hepatite B Crônica/virologia , Hepatócitos/virologia , Código das Histonas , Histonas/metabolismo , Humanos , Interferon-alfa/genética , Proteína da Leucemia Promielocítica/metabolismo , Processamento de Proteína Pós-Traducional , RNA Viral , Fator de Transcrição STAT1/metabolismo , Transcrição Gênica , Replicação ViralRESUMO
BACKGROUND: We aimed to construct an artificial intelligence (AI) guided identification of suspicious bone metastatic lesions from the whole-body bone scintigraphy (WBS) images by convolutional neural networks (CNNs). METHODS: We retrospectively collected the 99mTc-MDP WBS images with confirmed bone lesions from 3352 patients with malignancy. 14,972 bone lesions were delineated manually by physicians and annotated as benign and malignant. The lesion-based differentiating performance of the proposed network was evaluated by fivefold cross validation, and compared with the other three popular CNN architectures for medical imaging. The average sensitivity, specificity, accuracy and the area under receiver operating characteristic curve (AUC) were calculated. To delve the outcomes of this study, we conducted subgroup analyses, including lesion burden number and tumor type for the classifying ability of the CNN. RESULTS: In the fivefold cross validation, our proposed network reached the best average accuracy (81.23%) in identifying suspicious bone lesions compared with InceptionV3 (80.61%), VGG16 (81.13%) and DenseNet169 (76.71%). Additionally, the CNN model's lesion-based average sensitivity and specificity were 81.30% and 81.14%, respectively. Based on the lesion burden numbers of each image, the area under the receiver operating characteristic curve (AUC) was 0.847 in the few group (lesion number n ≤ 3), 0.838 in the medium group (n = 4-6), and 0.862 in the extensive group (n > 6). For the three major primary tumor types, the CNN-based lesion identifying AUC value was 0.870 for lung cancer, 0.900 for prostate cancer, and 0.899 for breast cancer. CONCLUSION: The CNN model suggests potential in identifying suspicious benign and malignant bone lesions from whole-body bone scintigraphic images.
Assuntos
Neoplasias Ósseas/secundário , Osso e Ossos/diagnóstico por imagem , Diagnóstico por Computador , Redes Neurais de Computação , Cintilografia , Neoplasias Ósseas/diagnóstico por imagem , Osso e Ossos/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
In order to identify host cellular DNA metabolic enzymes that are involved in the biosynthesis of hepatitis B virus (HBV) covalently closed circular (ccc) DNA, we developed a cell-based assay supporting synchronized and rapid cccDNA synthesis from intracellular progeny nucleocapsid DNA. This was achieved by arresting HBV DNA replication in HepAD38 cells with phosphonoformic acid (PFA), a reversible HBV DNA polymerase inhibitor, at the stage of single-stranded DNA and was followed by removal of PFA to allow the synchronized synthesis of relaxed circular DNA (rcDNA) and subsequent conversion into cccDNA within 12 to 24 h. This cccDNA formation assay allows systematic screening of the effects of small molecular inhibitors of DNA metabolic enzymes on cccDNA synthesis but avoids cytotoxic effects upon long-term treatment. Using this assay, we found that all the tested topoisomerase I and II (TOP1 and TOP2, respectively) poisons as well as topoisomerase II DNA binding and ATPase inhibitors significantly reduced the levels of cccDNA. It was further demonstrated that these inhibitors also disrupted cccDNA synthesis during de novo HBV infection of HepG2 cells expressing sodium taurocholate cotransporting polypeptide (NTCP). Mechanistic analyses indicate that whereas TOP1 inhibitor treatment prevented the production of covalently closed negative-strand rcDNA, TOP2 inhibitors reduced the production of this cccDNA synthesis intermediate to a lesser extent. Moreover, small interfering RNA (siRNA) knockdown of topoisomerase II significantly reduced cccDNA amplification. Taking these observations together, our study demonstrates that topoisomerase I and II may catalyze distinct steps of HBV cccDNA synthesis and that pharmacologic targeting of these cellular enzymes may facilitate the cure of chronic hepatitis B.IMPORTANCE Persistent HBV infection relies on stable maintenance and proper functioning of a nuclear episomal form of the viral genome called cccDNA, the most stable HBV replication intermediate. One of the major reasons for the failure of currently available antiviral therapeutics to cure chronic HBV infection is their inability to eradicate or inactivate cccDNA. We report here a chemical genetics approach to identify host cellular factors essential for the biosynthesis and maintenance of cccDNA and reveal that cellular DNA topoisomerases are required for both de novo synthesis and intracellular amplification of cccDNA. This approach is suitable for systematic screening of compounds targeting cellular DNA metabolic enzymes and chromatin remodelers for their ability to disrupt cccDNA biosynthesis and function. Identification of key host factors required for cccDNA metabolism and function will reveal molecular targets for developing curative therapeutics of chronic HBV infection.
Assuntos
DNA Topoisomerases/metabolismo , DNA Circular/metabolismo , Vírus da Hepatite B/genética , Antivirais/farmacologia , DNA Topoisomerases/genética , DNA Viral/genética , Foscarnet/farmacologia , Genoma Viral/genética , Células Hep G2 , Hepatite B/genética , Vírus da Hepatite B/metabolismo , Hepatócitos/virologia , Humanos , RNA Interferente Pequeno/metabolismo , Vírion/metabolismo , Replicação Viral/genéticaRESUMO
Hepatitis B virus (HBV) core protein consists of an N-terminal assembly domain and a C-terminal domain (CTD) with seven conserved serines or threonines that are dynamically phosphorylated/dephosphorylated during the viral replication cycle. Sulfamoylbenzamide derivatives are small molecular core protein allosteric modulators (CpAMs) that bind to the heteroaryldihydropyrimidine (HAP) pocket between the core protein dimer-dimer interfaces. CpAM binding alters the kinetics and pathway of capsid assembly and can result in the formation of morphologically "normal" capsids devoid of viral pregenomic RNA (pgRNA) and DNA polymerase. In order to investigate the mechanism underlying CpAM inhibition of pgRNA encapsidation, we developed an immunoblotting assay that can resolve core protein based on its phosphorylation status and demonstrated, for the first time, that core protein is hyperphosphorylated in free dimers and empty capsids from both mock-treated and CpAM-treated cells but is hypophosphorylated in pgRNA- and DNA-containing nucleocapsids. Interestingly, inhibition of pgRNA encapsidation by a heat shock protein 90 (HSP90) inhibitor prevented core protein dephosphorylation. Moreover, core proteins with point mutations at the wall of the HAP pocket, V124A and V124W, assembled empty capsids and nucleocapsids with altered phosphorylation status. The results thus suggest that core protein dephosphorylation occurs in the assembly of pgRNA and that interference with the interaction between core protein subunits at dimer-dimer interfaces during nucleocapsid assembly alters not only capsid structure, but also core protein dephosphorylation. Hence, inhibition of pgRNA encapsidation by CpAMs might be due to disruption of core protein dephosphorylation during nucleocapsid assembly.IMPORTANCE Dynamic phosphorylation of HBV core protein regulates multiple steps of viral replication. However, the regulatory function was mainly investigated by phosphomimetic mutagenesis, which disrupts the natural dynamics of core protein phosphorylation/dephosphorylation. Development of an immunoblotting assay capable of resolving hyper- and hypophosphorylated core proteins allowed us to track the phosphorylation status of core proteins existing as free dimers and the variety of intracellular capsids and to investigate the role of core protein phosphorylation/dephosphorylation in viral replication. Here, we found that disruption of core protein interaction at dimer-dimer interfaces during nucleocapsid assembly (by CpAMs or mutagenesis) inhibited core protein dephosphorylation and pgRNA packaging. Our work has thus revealed a novel function of core protein dephosphorylation in HBV replication and the mechanism by which CpAMs, a class of compounds that are currently in clinical trials for treatment of chronic hepatitis B, induce the assembly of empty capsids.
Assuntos
Genoma Viral , Vírus da Hepatite B/fisiologia , Hepatite B/virologia , Precursores de RNA/metabolismo , RNA Viral/metabolismo , Proteínas do Core Viral/metabolismo , Montagem de Vírus , Regulação Alostérica , Capsídeo/metabolismo , Células Cultivadas , Hepatite B/genética , Hepatite B/metabolismo , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Fosforilação , Precursores de RNA/genética , RNA Viral/genética , Proteínas do Core Viral/genética , Replicação ViralRESUMO
Interferon-induced transmembrane proteins (IFITMs) are restriction factors that inhibit the infectious entry of many enveloped RNA viruses. However, we demonstrated previously that human IFITM2 and IFITM3 are essential host factors facilitating the entry of human coronavirus (HCoV) OC43. In a continuing effort to decipher the molecular mechanism underlying IFITM differential modulation of HCoV entry, we investigated the roles of structural motifs important for IFITM protein posttranslational modifications, intracellular trafficking, and oligomerization in modulating the entry of five HCoVs. We found that three distinct mutations in IFITM1 or IFITM3 converted the host restriction factors to enhance entry driven by the spike proteins of severe acute respiratory syndrome coronavirus (SARS-CoV) and/or Middle East respiratory syndrome coronavirus (MERS-CoV). First, replacement of IFITM3 tyrosine 20 with either alanine or aspartic acid to mimic unphosphorylated or phosphorylated IFITM3 reduced its activity to inhibit the entry of HCoV-NL63 and -229E but enhanced the entry of SARS-CoV and MERS-CoV. Second, replacement of IFITM3 tyrosine 99 with either alanine or aspartic acid reduced its activity to inhibit the entry of HCoV-NL63 and SARS-CoV but promoted the entry of MERS-CoV. Third, deletion of the carboxyl-terminal 12 amino acid residues from IFITM1 enhanced the entry of MERS-CoV and HCoV-OC43. These findings suggest that these residues and structural motifs of IFITM proteins are key determinants for modulating the entry of HCoVs, most likely through interaction with viral and/or host cellular components at the site of viral entry to modulate the fusion of viral envelope and cellular membranes.IMPORTANCE The differential effects of IFITM proteins on the entry of HCoVs that utilize divergent entry pathways and membrane fusion mechanisms even when using the same receptor make the HCoVs a valuable system for comparative investigation of the molecular mechanisms underlying IFITM restriction or promotion of virus entry into host cells. Identification of three distinct mutations that converted IFITM1 or IFITM3 from inhibitors to enhancers of MERS-CoV or SARS-CoV spike protein-mediated entry revealed key structural motifs or residues determining the biological activities of IFITM proteins. These findings have thus paved the way for further identification of viral and host factors that interact with those structural motifs of IFITM proteins to differentially modulate the infectious entry of HCoVs.
Assuntos
Antígenos de Diferenciação/metabolismo , Coronavirus/metabolismo , Proteínas de Membrana/metabolismo , Mutação de Sentido Incorreto , Multimerização Proteica , Proteínas de Ligação a RNA/metabolismo , Internalização do Vírus , Motivos de Aminoácidos , Substituição de Aminoácidos , Antígenos de Diferenciação/genética , Linhagem Celular Tumoral , Coronavirus/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Ligação a RNA/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Hepatitis B virus (HBV) core protein assembles viral pre-genomic (pg) RNA and DNA polymerase into nucleocapsids for reverse transcriptional DNA replication to take place. Several chemotypes of small molecules, including heteroaryldihydropyrimidines (HAPs) and sulfamoylbenzamides (SBAs), have been discovered to allosterically modulate core protein structure and consequentially alter the kinetics and pathway of core protein assembly, resulting in formation of irregularly-shaped core protein aggregates or "empty" capsids devoid of pre-genomic RNA and viral DNA polymerase. Interestingly, in addition to inhibiting nucleocapsid assembly and subsequent viral genome replication, we have now demonstrated that HAPs and SBAs differentially modulate the biosynthesis of covalently closed circular (ccc) DNA from de novo infection and intracellular amplification pathways by inducing disassembly of nucleocapsids derived from virions as well as double-stranded DNA-containing progeny nucleocapsids in the cytoplasm. Specifically, the mistimed cuing of nucleocapsid uncoating prevents cccDNA formation during de novo infection of hepatocytes, while transiently accelerating cccDNA synthesis from cytoplasmic progeny nucleocapsids. Our studies indicate that elongation of positive-stranded DNA induces structural changes of nucleocapsids, which confers ability of mature nucleocapsids to bind CpAMs and triggers its disassembly. Understanding the molecular mechanism underlying the dual effects of the core protein allosteric modulators on nucleocapsid assembly and disassembly will facilitate the discovery of novel core protein-targeting antiviral agents that can more efficiently suppress cccDNA synthesis and cure chronic hepatitis B.
Assuntos
DNA Circular/biossíntese , Vírus da Hepatite B/fisiologia , Hepatite B Crônica/virologia , Replicação Viral/efeitos dos fármacos , Antivirais/farmacologia , Linhagem Celular , DNA Circular/genética , DNA Viral , DNA Polimerase Dirigida por DNA/metabolismo , Hepatócitos/virologia , Humanos , Nucleocapsídeo/efeitos dos fármacos , Nucleocapsídeo/genética , Reação em Cadeia da Polimerase em Tempo Real , Replicação Viral/fisiologiaRESUMO
Induction of interferon and proinflammatory cytokines is a hallmark of the infection of many different viruses. However, hepatitis B virus (HBV) does not elicit a detectable cytokine response in infected hepatocytes. In order to investigate the molecular mechanism underlying the innate immune evasion, a functional cyclic GMP-AMP (cGAMP) synthase (cGAS)-stimulator of interferon genes (STING) pathway was reconstituted in a human hepatoma cell line supporting tetracycline-inducible HBV replication. It was demonstrated that induction of HBV replication neither activated nor inhibited this cytosolic DNA sensing pathway. However, human hepatoma cells, as well as immortalized mouse hepatocytes, express low levels of STING, which upon activation by cGAMP, the natural ligand of STING, led to induction of a proinflammatory cytokine response. Treatment of immortalized mouse hepatocytes supporting HBV replication with either cGAMP or a small molecule pharmacologic STING agonist significantly reduced viral DNA in a STING- and Janus kinase 1-dependent manner. Moreover, cGAMP treatment was able to induce inflammatory cytokine gene expression and inhibit the transcription of covalently closed circular DNA in HBV-infected human hepatoma cells expressing sodium taurocholate cotransporting polypeptide, an essential receptor for HBV infection of hepatocytes. The studies reported here and previously (F. Guo et al., Antimicrob Agents Chemother 59:1273-1281, 2015, https://doi.org/10.1128/AAC.04321-14) thus support the notion that pharmacological activation of STING in macrophages and hepatocytes induces host innate responses that can efficiently control HBV replication. Hence, despite not playing a significant role in host innate immune response to HBV infection of hepatocytes, STING is potentially a valuable target for immunotherapy of chronic hepatitis B.
Assuntos
Vírus da Hepatite B/crescimento & desenvolvimento , Hepatócitos/imunologia , Interferons/biossíntese , Macrófagos/imunologia , Proteínas de Membrana/metabolismo , Replicação Viral/genética , Animais , Antivirais/farmacologia , Linhagem Celular Tumoral , Células Hep G2 , Vírus da Hepatite B/imunologia , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/virologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Interferons/imunologia , Macrófagos/metabolismo , Macrófagos/virologia , Proteínas de Membrana/agonistas , Camundongos , Nucleotidiltransferases/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/metabolismoRESUMO
Apoptotic and necrotic macrophages have long been known for their existence in atherosclerotic lesions. However, the mechanisms underlying the choice of their death pattern have not been fully elucidated. Here, we report the effects of PS-341, a potent and specific proteasome inhibitor, on the cell death of primary bone marrow-derived macrophages (BMDMs) in vitro. The results showed that PS-341 could not induce macrophage apoptosis or promote TNF-induced macrophage apoptosis, on the other hand, PS-341 could significantly inhibit macrophage necroptosis induced by TNF and pan-caspase inhibitor z-VAD treatment. Remarkably, high-dose of PS-341 showed similar inhibitory effects on macrophage necroptosis comparable to that of kinase inhibition of RIP1 through specific inhibitor Nec-1 or inhibition of RIP3 via specific genetical ablation. Furthermore, the degradation of cellular inhibitor of apoptosis proteins (cIAPs) was suppressed by PS-341, which could antagonize the activation of RIP1 kinase via post-translational mechanism. Further evidences demonstrated reduced levels of both RIP1 and RIP 3 upon PS-341 treatment, concomitantly, a more strong association of RIP1 with cIAPs and less with RIP3 was found following PS-341 treatment, these findings suggested that PS-341 may disrupt the formation of RIP1-RIP3 complex (necrosome) through stabilizing cIAPs. Collectively, our results indicated that the proteasome-mediated degradation of cIAPs could be inhibited by PS-341 and followed by limited RIP1 and RIP3 kinase activities, which were indispensable for necroptosis, thus eliciting a significant necroptosis rescue in BMDMs in vitro. Overall, our study has identified a new role of PS-341 in the cell death of BMDMs and provided a novel insight into the atherosclerotic inflammation caused by proteasome-mediated macrophage necroptosis.
Assuntos
Apoptose/efeitos dos fármacos , Bortezomib/administração & dosagem , Macrófagos/metabolismo , Macrófagos/patologia , Inibidores de Proteassoma/administração & dosagem , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Apoptose/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Necrose , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
The level of intracellular keratin 8(KRT-8) is associated with liver diseases, whose expression is increased in hepatitis C virus(HCV)-infected patients with hepatocarcinoma and in cultural cells infected with HCV. However, it is not clear whether KRT-8 will impact HCV replication. In this paper, the HCV replication was analyzed in response to high expression and silence of KRT-8. The inhibitory activities against wild-type and mutant HCV were also analyzed by silence of KRT-8 or combined with known anti-HCV drug telaprevir. Results showed that the protein level of KRT-8 was increased in proportion with the HCV replication. The high expression was found to facilitate HCV replication, while the silence of KRT-8 was able to inhibit HCV replication and enhanced the anti-HCV activity of telaprevir. It also inhibited A156 T and D168 V mutant HCV, which are resistant to protease inhibitors. These results suggest that KRT-8 is a co-factor for HCV replication. Down-regulation of KRT-8 can inhibit wild type and mutant HCV replication to enhance the anti-HCV activity of known anti-HCV drugs. Therefore, KRT-8 may be a new target in the development of anti-HCV agents.
Assuntos
Hepacivirus/fisiologia , Queratina-8/metabolismo , Replicação Viral , Antivirais/farmacologia , Carcinoma Hepatocelular/virologia , Células Cultivadas , Replicação do DNA , Humanos , Oligopeptídeos/farmacologia , Inibidores de Proteases/farmacologiaRESUMO
BACKGROUND: Over the past a few years, evidences indicate that adenosine triphosphate (ATP) is an energy source for the binding, maturation, assembly, and budding process of many enveloped viruses. Our previous studies suggest that the F1-ATP synthase beta subunit (ATPsyn ß, BP53) of the shrimp Litopenaeus vannamei (L. vannamei) might serve as a potential receptor for white spot syndrome virus (WSSV)'s infection. METHODS: BP53 was localized on the surface of shrimp hemocytes and gill epithelial cells by immunofluorescence assay and immunogold labeling technique. Cell surface ATP synthesis was demonstrated by an in vitro bioluminescent luciferase assay. Furthermore, the expression of bp53 after WSSV infection was investigated by RT-PCR test. In addition, RNAi was developed to knock down endogenous bp53. RESULTS: BP53 is present on shrimp cell surface of hemocytes and gill epithelia. The synthesized ATP was detectable in the extracellular supernatant by using a bioluminescence assay, and the production declined post WSSV binding and infection. Knocking down endogenous bp53 resulted in a 50% mortality of L. vannamei. CONCLUSION: These results suggested that BP53, presenting on cell surface, likely served as one of the receptors for WSSV infection in shrimp. Correspondingly, WSSV appears to disturb the host energy metabolism through interacting with host ATPsyn ß during infection. This work firstly showed that host ATP production is required and consumed by the WSSV for binding and proceeds with infection process.
Assuntos
Trifosfato de Adenosina/biossíntese , Penaeidae/metabolismo , Penaeidae/virologia , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Interações Hospedeiro-Patógeno , Penaeidae/enzimologia , Penaeidae/genética , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismoRESUMO
Cytokines play a critical role in innate and adaptive immunity. Astakines represent a group of invertebrate cytokines that are related to vertebrate prokineticin and function in promoting hematopoiesis in crustaceans. We have identified an astakine from the white shrimp Litopeneaus vannamei and named it LvAST in a previous research. In the present research, we investigated the interactions among LvAST, the envelope protein VP37 of white spot syndrome virus (i.e., WSSV), and the ß subunit of F1-ATP synthase (ATPsyn-ß) of the white shrimp (i.e., BP53) using binding assays and co-precipitations. We also examined the effects of LvAST on shrimp susceptibility to WSSV. We found that LvAST and VP37 competitively bound to BP53, but did not bind to each other. Shrimps that had been injected with recombinant LvAST exhibited significantly lower mortality and longer survival time in experimental infections by WSSV. In contrast, shrimps whose LvAST gene expression had been inhibited by RNA interference showed significantly higher WSSV infection intensity and shorter survival time following viral challenges. These results suggested that LvAST and WSSV both likely use ATPsyn-ß as a receptor and LvAST plays a role in shrimp defense against WSSV infection. This represented the first research showing the involvement of astakines in host antiviral immunity.
Assuntos
Imunidade Adaptativa , Proteínas de Artrópodes/genética , Penaeidae/genética , Penaeidae/imunologia , Fator de Crescimento do Endotélio Vascular Derivado de Glândula Endócrina/genética , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Proteínas de Artrópodes/metabolismo , Clonagem Molecular , Penaeidae/virologia , Interferência de RNA , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fator de Crescimento do Endotélio Vascular Derivado de Glândula Endócrina/metabolismoRESUMO
ABSTRACT: A 68 Ga-DOTATATE PET/CT scan was conducted to locate the causative tumor responsible for suspected tumor-induced osteomalacia in a 56-year-old woman. The PET/CT images showed a focus in the right occipital region. Subsequent MRI showed an extra-axial nodule in the right occipital region, mimicking a meningioma. Although rare, an intracranial phosphaturic mesenchymal tumor was still suspected because of the typical clinical settings. Finally, phosphaturic mesenchymal tumor was confirmed by the postoperative pathology.
Assuntos
Neoplasias Meníngeas , Mesenquimoma , Neoplasias de Tecido Conjuntivo , Compostos Organometálicos , Cintilografia , Neoplasias de Tecidos Moles , Feminino , Humanos , Pessoa de Meia-Idade , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Neoplasias de Tecido Conjuntivo/etiologia , Tomografia por Emissão de Pósitrons , Neoplasias Meníngeas/complicaçõesRESUMO
RF-amide related peptides (RFRP) have been proposed as critical regulators of gonadotropin secretion in mammals. This study was designed to construct a DNA vaccine and investigate the effect of vaccine encoding RFRP-3 on reproduction physiology in ewe. A recombinant vaccine was constructed using two copies of the RFRP-3 gene and HBsAg-S that generate a fusion protein to induce an immunology response. Results showed this recombinant vaccine could produce a significant antibody titer in the treated animals (P < 0.05). The specific RFRP-3 antibody response induced by the vaccine was detected at week 2 with a peak at week 6 after the initial immunization. Furthermore, we found that ewes inoculated with pVAX-tPA-HBsAg-S-2RFRP-asd vaccine significantly raised the concentration of GnRH, LH and E2 in serum compared to the control group. LH and E2 concentration in the treated ewes (Group T) was significantly higher than that in control ewes (Group C) at weeks 10, 12 and 14 after the initial immunization, respectively (P < 0.05). Therefore, RFRP-3 can be used as a target for DNA immunization to promote reproductive hormone secretion in ewes and RFRP-3 gene immunization might be a candidate tool to regulate mammal reproduction.
Assuntos
Neuropeptídeos , Vacinas de DNA , Animais , Feminino , Hormônio Liberador de Gonadotropina/farmacologia , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/farmacologia , Mamíferos , Neuropeptídeos/genética , Reprodução/fisiologia , Ovinos , Vacinas de DNA/farmacologiaRESUMO
A key step of hepatitis B virus (HBV) replication is the selective packaging of pregenomic RNA (pgRNA) by core protein (Cp) dimers, forming a nucleocapsid where the reverse transcriptional viral DNA replication takes place. One approach in the development of new anti-HBV drugs is to disrupt the assembly of HBV nucleocapsids by misdirecting Cp dimers to assemble morphologically normal capsids devoid of pgRNA. In this study, we built upon our previous discovery of benzamide-derived HBV capsid assembly modulators by exploring fused bicyclic scaffolds with an exocyclic amide that is ß, γ to the fused ring, and identified 1,2,3,4-tetrahydroquinoxaline derived phenyl ureas as a novel scaffold. Structure-activity relationship studies showed that a favorable hydrophobic substitution can be tolerated at the 2-position of the 1,2,3,4-tetrahydroquinoxaline core, and the resulting compound 88 demonstrated comparable or improved antiviral potencies in mouse and human hepatocyte-derived HBV-replicating cell lines compared to our previously reported benzamide compound, 38017 (8). In addition, a novel bis-urea series based on 1,2,3,4-tetrahydroquinoxaline was also found to inhibit HBV DNA replication with sub-micromolar EC50 values. The mode of action of these compounds is consistent with specific inhibition of pgRNA encapsidation into nucleocapsids in hepatocytes.
Assuntos
Vírus da Hepatite B , Hepatite B , Humanos , Animais , Camundongos , Vírus da Hepatite B/metabolismo , Replicação Viral , Montagem de Vírus , Replicação do DNA , RNA Viral/genética , DNA Viral , Nucleocapsídeo/metabolismo , Antivirais/química , Benzamidas/farmacologia , Hepatite B/tratamento farmacológicoRESUMO
A previous study reported that ginsenoside-Rd reduced the production of tumor necrosis factor-α by inhibiting nuclear factor-κB in lipopolysaccharide-activated N9 microglia in vitro. The aim of the present study was to confirm the anti-inflammatory effects and mechanisms of ginsenoside-Rd in animal experiments involving acute inflammation. The results indicated that ginsenoside-Rd at doses ranging from 12.5 to 50 mg/kg i.m. significantly inhibited the swelling of hind paws in rats for 1-6 h after the carrageenan injection. The levels of proinflammatory cytokines and proinflammatory mediators were markedly reduced by ginsenoside-Rd. Ginsenoside-Rd, when administered intramuscularly at 12.5, 25, and 50 mg/kg doses, showed signicant inhibition of carrageenan-induced production of interleukin-1ß (6.91%, 45.75%, and 55.18%, respectively), tumor necrosis factor-α (37.99%, 56.39%, and 47.38%, respectively), prostaglandin E(2) (22.92%, 30.12%, and 36.36%, respectively), and nitric oxide (28.27%, 44.53%, and 53.42%, respectively). In addition, ginsenoside-Rd (12.5, 25, and 50 mg/kg i.m.) effectively decreased the levels of nuclear factor-κB (6.77%, 20.28%, and 41.03%, respectively) and phosphorylation of IκBα (13.23%, 26.92%, and 41.80%, respectively) in the carrageenan-inflamed paw tissues. These results suggest that ginsenoside-Rd has significant anti-inflammatory effects in vivo, which might be due to its blocking of the nuclear factor-κB signaling pathway. Thus, it may be possible to develop ginsenoside-Rd as a useful agent for inflammatory diseases.
Assuntos
Anti-Inflamatórios/farmacologia , Carragenina , Ginsenosídeos/farmacologia , Inflamação/prevenção & controle , Animais , Anti-Inflamatórios/administração & dosagem , Citocinas/metabolismo , Dinoprostona/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Ginsenosídeos/administração & dosagem , Proteínas I-kappa B/metabolismo , Inflamação/induzido quimicamente , Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Injeções Intramusculares , Masculino , Inibidor de NF-kappaB alfa , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Fosforilação , Ratos , Ratos Wistar , Fatores de TempoRESUMO
ABSTRACT: Soft tissue aneurysmal bone cyst is very rare. Herein, we report FDG PET/CT findings of aneurysmal bone cyst in a 19-year-old man. On conventional image, it presented as a paravertebral soft tissue mass with heterogeneous enhancement and rim eggshell-like calcification. On PET/CT, this solitary lesion had intense FDG uptake with an SUV max of 10.33. The final pathology supported a diagnosis of aneurysmal bone cyst. Our case suggests that soft tissue aneurysmal bone cyst should be regarded as a differential diagnosis of solitary paravertebral mass with intense FDG uptake.
Assuntos
Cistos Ósseos Aneurismáticos , Fluordesoxiglucose F18 , Adulto , Cistos Ósseos Aneurismáticos/diagnóstico por imagem , Cistos Ósseos Aneurismáticos/patologia , Humanos , Masculino , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Tomografia por Emissão de Pósitrons , Tomografia Computadorizada por Raios X/métodos , Adulto JovemRESUMO
The most common sites of metastasis for pheochromocytoma are the lymph nodes, bones, lungs, and liver. Peritoneal metastasis or implantation is very rare. We present a case of recurrent malignant pheochromocytoma with unusual multiple peritoneal carcinomatosis on I-MIBG SPECT/CT after laparoscopic adrenalectomy.