RESUMO
Nuclear entrance and stability of porcine circovirus type 2 (PCV2), the smallest virus in mammals, are crucial for its infection and replication. However, the mechanisms are not fully understood. Here, we found that the PCV2 virion maintains self-stability via the host importin 5 (IPO5) during infection. Coimmunoprecipitation combined with mass spectrometry and glutathione S-transferase pulldown assays showed that the capsid protein (Cap) of PCV2 binds directly to IPO5. Fine identification demonstrated that the N-terminal residue arginine24 of Cap is the most critical to efficient binding to the proline709 residue of IPO5. Detection of replication ability further showed that IPO5 supports PCV2 replication by promoting the nuclear import of incoming PCV2 virions. Knockdown of IPO5 delayed the nuclear transport of incoming PCV2 virions and significantly decreased the intracellular levels of overexpressed PCV2 Cap, which was reversed by treatment with a proteasome inhibitor or by rescuing IPO5 expression. Cycloheximide treatment showed that IPO5 increases the stability of the PCV2 Cap protein. Taken together, our findings demonstrated that during infection, IPO5 facilitates PCV2 replication by directly binding to the nuclear localization signal of Cap to block proteasome degradation. IMPORTANCE Circovirus is the smallest virus to cause immune suppression in pigs. The capsid protein (Cap) is the only viral structural protein that is closely related to viral infection. The nuclear entry and stability of Cap are necessary for PCV2 replication. However, the molecular mechanism maintaining the stability of Cap during nuclear trafficking of PCV2 is unknown. Here, we report that IPO5 aggregates within the nuclear periphery and combines with incoming PCV2 capsids to promote their nuclear entry. Concurrently, IPO5 inhibits the degradation of newly synthesized Cap protein, which facilitates the synthesis of virus proteins and virus replication. These findings highlight a mechanism whereby IPO5 plays a dual role in PCV2 infection, which not only enriches our understanding of the virus replication cycle but also lays the foundation for the subsequent development of antiviral drugs.
Assuntos
Proteínas do Capsídeo , Infecções por Circoviridae , Circovirus , Carioferinas , Doenças dos Suínos , Animais , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Infecções por Circoviridae/veterinária , Circovirus/metabolismo , Suínos , Vírion/metabolismo , Carioferinas/metabolismo , Doenças dos Suínos/virologiaRESUMO
BACKGROUND: The recurrence of hepatocellular carcinoma (HCC) after radiofrequency ablation (RFA) remains a major clinical problem. Cells that survive the sublethal heat stress that is induced by incomplete RFA are the main source of HCC relapse. Heat stress has long been reported to increase intracellular reactive oxygen species (ROS) generation. Although ROS can induce apoptosis, a pro-survival effect of ROS has also been demonstrated. However, the role of ROS in HCC cells exposed to sublethal heat stress remains unclear. METHODS: HepG2 and HuH7 cells were used for this experiment. Insufficient RFA was performed in cells and in a xenograft model. ROS and antioxidant levels were measured. Apoptosis was analyed by Annexin-V/PI staining and flow cytometry. Protein expression was measured using western blotting. Colocalization of lysosomes and mitochondria was analyzed to assess mitophagy. Corresponding activators or inhibitors were applied to verify the function of specific objectives. RESULTS: Here,we showed that sublethal heat stress induced a ROS burst, which caused acute oxidative stress. This ROS burst was generated by mitochondria, and it was initiated by upregulated NOX4 expression in the mitochondria. N-acetylcysteine (NAC) decreased HCC cell survival under sublethal heat stress conditions in vivo and in vitro. NOX4 triggers the production of mitochondrial ROS (mtROS), and NOX4 inhibitors or siNOX4 also decreased HCC cell survival under sublethal heat stress conditions in vitro. Increased mtROS trigger PINK1-dependent mitophagy to eliminate the mitochondria that are damaged by sublethal heat stress and to protect cells from apoptosis. Nrf2 expression was elevated in response to this ROS burst and mediated the ROS burst-induced increase in PINK1 expression after sublethal heat stress. CONCLUSION: These data confirmed that the ROS burst that occurs after iRFA exerted a pro-survival effect. NOX4 increased the generation of ROS by mitochondria. This short-term ROS burst induced PINK1-dependent mitophagy to eliminate damaged mitochondria by increasing Nrf2 expression.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Ablação por Radiofrequência , Humanos , Mitofagia , Carcinoma Hepatocelular/patologia , Espécies Reativas de Oxigênio/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Regulação para Cima , Sobrevivência Celular , Proteínas Quinases/metabolismo , Neoplasias Hepáticas/patologia , Recidiva Local de Neoplasia/patologia , Mitocôndrias/metabolismo , NADPH Oxidase 4/metabolismoRESUMO
In this study, keratins were extracted from pig nail waste through the reduction method using L-cysteine as a reductant. Curcumin was successively incorporated in a mixed solution including keratin, gelatin, and glycerin to prepare different kinds of keratin/gelatin/glycerin/curcumin composite films. The morphology of the keratin/ gelatin/glycerin/curcumin composite films were examined using scanning electron microscopy. The structures and the molecular interactions between curcumin, keratin, and pectin were examined using Fourier transform infrared spectroscopy and X-ray diffraction, and the thermal properties were determined through thermogravimetric analysis. The tensile strengths of keratin/gelatin/glycerin/curcumin and keratin/gelatin/curcumin composite films are 13.73 and 12.45 MPa, respectively, and their respective elongations at break are 56.7% and 4.6%. In addition, compared with the control group (no film wrapped on the surface of tomato), the ratio of weight loss of the keratin (7.0%)/gelatin (10%)/glycerin (2.0%)/curcumin (1.0%) experimental groups is 8.76 ± 0.2%, and the hardness value of the tomatoes wrapped with composite films is 11.2 ± 0.39 kg/cm3. Finally, the composite films have a superior antibacterial effect against Staphylococcus aureus and Escherichia coli because of the addition of curcumin. As the concentration of curcumin reaches 1.0%, the antibacterial activity effect of the film is significantly improved. The diameter of the inhibition zone of E. coli is (12.16 ± 0.53) mm, and that of S. aureus is (14.532 ± 0.97) mm. The multifunctional keratin/gelatin/glycerin/curcumin bioactive films have great potential application in the food packaging industry.
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Curcumina , Solanum lycopersicum , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Curcumina/química , Curcumina/farmacologia , Cisteína/farmacologia , Escherichia coli , Embalagem de Alimentos , Gelatina/química , Gelatina/farmacologia , Glicerol/farmacologia , Queratinas/química , Pectinas/farmacologia , Substâncias Redutoras/farmacologia , Espectroscopia de Infravermelho com Transformada de Fourier , Staphylococcus aureus , SuínosRESUMO
The 5' cap methylation of viral RNA plays important roles in RNA stability, efficient translation, and immune evasion. Thus, RNA cap methylation is an attractive target for antiviral discovery and development of new live attenuated vaccines. For coronaviruses, RNA cap structure is first methylated at the guanine-N-7 (G-N-7) position by nonstructural protein 14 (nsp14), which facilitates and precedes the subsequent ribose 2'-O methylation by the nsp16-nsp10 complex. Using porcine epidemic diarrhea virus (PEDV), an Alphacoronavirus, as a model, we showed that G-N-7 methyltransferase (G-N-7 MTase) of PEDV nsp14 methylated RNA substrates in a sequence-unspecific manner. PEDV nsp14 can efficiently methylate RNA substrates with various lengths in both neutral and alkaline pH environments and can methylate cap analogs (GpppA and GpppG) and single-nucleotide GTP but not ATP, CTP, or UTP. Mutations to the S-adenosyl-l-methionine (SAM) binding motif in the nsp14 abolished the G-N-7 MTase activity and were lethal to PEDV. However, recombinant rPEDV-D350A with a single mutation (D350A) in nsp14, which retained 29.0% of G-N-7 MTase activity, was viable. Recombinant rPEDV-D350A formed a significantly smaller plaque and had significant defects in viral protein synthesis and viral replication in Vero CCL-81 cells and intestinal porcine epithelial cells (IPEC-DQ). Notably, rPEDV-D350A induced significantly higher expression of both type I and III interferons in IPEC-DQ cells than the parental rPEDV. Collectively, our results demonstrate that G-N-7 MTase activity of PEDV modulates viral replication, gene expression, and innate immune responses.IMPORTANCE Coronaviruses (CoVs) include a wide range of important human and animal pathogens. Examples of human CoVs include severe acute respiratory syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), and the most recently emerged SARS-CoV-2. Examples of pig CoVs include porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine enteric alphacoronavirus (SeACoV). There are no vaccines or antiviral drugs for most of these viruses. All known CoVs encode a bifunctional nsp14 protein which possesses ExoN and guanine-N-7 methyltransferase (G-N-7 MTase) activities, responsible for replication fidelity and RNA cap G-N-7 methylation, respectively. Here, we biochemically characterized G-N-7 MTase of PEDV nsp14 and found that G-N-7 MTase-deficient PEDV was defective in replication and induced greater responses of type I and III interferons. These findings highlight that CoV G-N-7 MTase may be a novel target for rational design of live attenuated vaccines and antiviral drugs.
Assuntos
Exorribonucleases/metabolismo , Interferon Tipo I/biossíntese , Interferons/biossíntese , Vírus da Diarreia Epidêmica Suína/fisiologia , Capuzes de RNA/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Chlorocebus aethiops , Exorribonucleases/genética , Expressão Gênica , Guanina/metabolismo , Imunidade Inata , Metilação , Mutação , Vírus da Diarreia Epidêmica Suína/enzimologia , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/patogenicidade , RNA Viral/metabolismo , S-Adenosilmetionina/metabolismo , Suínos , Células Vero , Proteínas não Estruturais Virais/genética , Replicação Viral , Interferon lambdaRESUMO
The transport of circovirus capsid protein into nucleus is essential for viral replication in infected cell. However, the role of nucleolar shuttle proteins during porcine circovirus 3 capsid protein (PCV3 Cap) import is still not understood. Here, we report a previously unidentified nucleolar localization signal (NoLS) of PCV3 Cap, which hijacks the nucleolar phosphoprotein nucleophosmin-1 (NPM1) to facilitate nucleolar localization of PCV3 Cap. The NoLS of PCV3 Cap and serine-48 residue of N-terminal oligomerization domain of NPM1 are essential for PCV3 Cap/NPM1 interaction. In addition, charge property of serine-48 residue of NPM1 is critical for nucleolar localization and interaction with PCV3 Cap. Taken together, our findings demonstrate for the first time that NPM1 interacts with PCV3 Cap and is responsible for its nucleolar localization.
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Proteínas do Capsídeo/metabolismo , Circovirus/metabolismo , Proteínas Nucleares/metabolismo , Animais , Sítios de Ligação , Proteínas do Capsídeo/genética , Linhagem Celular , Circovirus/genética , Eletroforese em Gel de Poliacrilamida , Técnicas de Silenciamento de Genes , Immunoblotting , Microscopia Confocal , Nucleofosmina , Serina , SuínosRESUMO
A relatively stable and flexible capsid is critical to the viral life cycle. However, the capsid dynamics and cytosol trafficking of porcine circovirus type 2 (PCV2) during its infectious cycle are poorly understood. Here, we report the structural stability and conformation flexibility of PCV2 virions by genome labeling and the use of three monoclonal antibodies (MAbs) against the native capsid of PCV2. Genome labeling showed that the infectivity of the PCV2 virion was not affected by conjugation with deoxy-5-ethynylcytidine (EdC). Heat stability experiments indicated that PCV2 capsids started to disassemble at 65°C, causing binding incompetence for all antibodies, and the viral genome was released without capsid disassembly upon heating at 60°C. Antibody binding experiments with PCV2 showed that residues 186 to 192 were concealed in the early endosomes of epithelial PK-15 and monocytic 3D4/31 cells with or without chloroquine treatment and then exposed in PK-15 cytosol and the 3D4/31 nucleus. Viral propagation and localization experiments showed that PCV2 replication and cytosol trafficking were not significantly affected by microtubule depolymerization in monocytic 3D4/31 cells treated with nocodazole. These findings demonstrated that nuclear targeting of viral capsids involved conformational changes, the PCV2 genome was released from the assembled capsid, and the transit of PCV2 particles was independent of microtubules in 3D4/31 cells.IMPORTANCE Circovirus is the smallest virus known to replicate autonomously. Knowledge of viral genome release may provide understanding of viral replication and a method to artificially inactivate viral particles. Currently, little is known about the release model of porcine circovirus type 2 (PCV2). Here, we report the release of the PCV2 genome from assembled capsid and the intracellular trafficking of infectious PCV2 by alterations in the capsid conformation. Knowledge of PCV2 capsid stability and dynamics is essential to understanding its infectious cycle and lays the foundation for discovering powerful targets for therapeutic and prophylactic intervention.
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Infecções por Circoviridae/virologia , Circovirus/fisiologia , Montagem de Vírus , Internalização do Vírus , Capsídeo/química , Capsídeo/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Células Cultivadas , Endossomos , Genoma Viral , Interações Hospedeiro-Patógeno , Microtúbulos/metabolismo , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Mammalian orthoreoviruses (MRVs) infect almost all mammals, and there are some reports on MRVs in China. In this study, a novel strain was identified, which was designated as HLJYC2017. The results of genetic analysis showed that MRV HLJYC2017 is a reassortant strain. According to biological information analysis, different serotypes of MRV contain specific amino acid insertions and deletions in the σ1 protein. Neutralizing antibody epitope analysis revealed partial cross-protection among MRV1, MRV2, and MRV3 isolates from China. L3 gene recombination in MRV was identified for the first time in this study. The results of this study provide valuable information on MRV reassortment and evolution.
Assuntos
Antígenos Virais/genética , Proteínas do Capsídeo/genética , Orthoreovirus de Mamíferos/genética , Vírus Reordenados/genética , Infecções por Reoviridae/epidemiologia , Infecções por Reoviridae/veterinária , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Proteínas do Capsídeo/imunologia , China/epidemiologia , Quirópteros , Cervos , Fezes/virologia , Expressão Gênica , Mutação INDEL , Camundongos , Epidemiologia Molecular , Orthoreovirus de Mamíferos/classificação , Orthoreovirus de Mamíferos/imunologia , Orthoreovirus de Mamíferos/isolamento & purificação , Filogenia , RNA Viral/genética , Vírus Reordenados/classificação , Vírus Reordenados/imunologia , Vírus Reordenados/isolamento & purificação , Infecções por Reoviridae/imunologia , Infecções por Reoviridae/virologia , Sorogrupo , SuínosRESUMO
Apoptosis is an essential strategy of host defense responses and is used by viruses to maintain their life cycles. However, the apoptotic signals involved in virus replication are poorly known. In the present study, we report the molecular mechanism of apoptotic induction by the viral protein ORF4, a newly identified viral protein of porcine circovirus type 2 (PCV2). Apoptosis detection revealed not only that the activity of caspase-3 and -9 is increased in PCV2-infected and ORF4-transfected cells but also that cytochrome c release from the mitochondria to the cytosol is upregulated. Subsequently, ORF4 protein colocalization with adenine nucleotide translocase 3 (ANT3) was observed using structured illumination microscopy. Moreover, coimmunoprecipitation and pulldown analyses confirmed that the ORF4 protein interacts directly with mitochondrial ANT3 (mtANT3). Binding domain analysis further confirmed that N-terminal residues 1 to 30 of the ORF4 protein, comprising a mitochondrial targeting signal, are essential for the interaction with ANT3. Knockdown of ANT3 markedly inhibited the apoptotic induction of both ORF4 protein and PCV2, indicating that ANT3 plays an important role in ORF4 protein-induced apoptosis during PCV2 infection. Taken together, these data indicate that the ORF4 protein is a mitochondrial targeting protein that induces apoptosis by interacting with ANT3 through the mitochondrial pathway.IMPORTANCE The porcine circovirus type 2 (PCV2) protein ORF4 is a newly identified viral protein; however, little is known about its functions. Apoptosis is an essential strategy of the host defense response and is used by viruses to maintain their life cycles. In the present study, we report the molecular mechanism of the apoptosis induced by the ORF4 protein. The ORF4 protein contains a mitochondrial targeting signal and is an unstable protein that is degraded by the proteasome-dependent pathway. Viral protein ORF4 triggers caspase-3- and -9-dependent cellular apoptosis in mitochondria by directly binding to ANT3. We conclude that the ORF4 protein is a mitochondrial targeting protein and reveal a mechanism whereby circovirus recruits ANT3 to induce apoptosis.
Assuntos
Translocador 3 do Nucleotídeo Adenina/metabolismo , Apoptose/genética , Circovirus/genética , Circovirus/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Translocador 3 do Nucleotídeo Adenina/genética , Animais , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular , Infecções por Circoviridae/imunologia , Infecções por Circoviridae/virologia , Citocromos c/metabolismo , Células HEK293 , Humanos , Mitocôndrias/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , SuínosRESUMO
Viral coinfection or superinfection in host has caused public health concern and huge economic losses of farming industry. The influence of viral coinfection on cellular protein abundance is essential for viral pathogenesis. Based on a coinfection model for porcine circovirus type 2 (PCV2) and classical swine fever virus (CSFV) developed previously by our laboratory, isobaric tags for relative and absolute quantitation (iTRAQ)-coupled LC-MS/MS proteomic profiling was performed to explore the host cell responses to PCV2-CSFV coinfection. Totally, 3932 proteins were identified in three independent mass spectrometry analyses. Compared with uninfected cells, 304 proteins increased (fold change >1.2) and 198 decreased (fold change <0.833) their abundance in PCV2-infected cells (p < 0.05), 60 and 61 were more and less abundant in CSFV-infected cells, and 196 and 158 were more and less abundant, respectively in cells coinfected with PCV2 and CSFV. Representative differentially abundant proteins were validated by quantitative real-time PCR, Western blotting and confocal laser scanning microscopy. Bioinformatic analyses confirmed the dominant role of PCV2, and indicated that mitochondrial dysfunction, nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated oxidative stress response and apoptosis signaling pathways might be the specifical targets during PCV2-CSFV coinfection.
Assuntos
Cromatografia Líquida/métodos , Infecções por Circoviridae/metabolismo , Circovirus/química , Vírus da Febre Suína Clássica/química , Peste Suína Clássica/metabolismo , Coinfecção/metabolismo , Proteínas Virais/análise , Animais , Linhagem Celular , Circovirus/patogenicidade , Vírus da Febre Suína Clássica/patogenicidade , Análise por Conglomerados , Marcação por Isótopo , Modelos Biológicos , Suínos , Espectrometria de Massas em Tandem/métodos , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
Stimulator of interferon gene (STING) plays an important role in the cyclic GMP-AMP synthase (cGAS)-mediated activation of type I IFN responses. In this study, we identified and cloned canine STING gene. Full-length STING encodes a 375 amino acid product that shares the highest similarity with feline STING. Highest levels of mRNA of canine STING were detected in the spleen and lungs while the lowest levels in the heart and muscle. Analysis of its cellular localization showed that STING is localizes to the endoplasmic reticulum. STING overexpression induced the IFN response via the IRF3 and NF-κB pathways and up-regulated the expression of ISG15 and viperin. However, knockdown of STING did not inhibit the IFN-ß response triggered by poly(dA:dT), poly(I:C), or SeV. Finally, overexpression of STING significantly inhibited the replication of canine influenza virus H3N2. Collectively, our findings indicate that STING is involved in the regulation of the IFN-ß pathway in canine.
Assuntos
Interferons , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/fisiologia , Sequência de Aminoácidos , Animais , Citocinas/metabolismo , Cães , Retículo Endoplasmático/metabolismo , Expressão Gênica , Técnicas de Silenciamento de Genes , Vetores Genéticos , Coração , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Fator Regulador 3 de Interferon/metabolismo , Interferon beta , Pulmão/metabolismo , Proteínas de Membrana/farmacologia , Músculos/metabolismo , NF-kappa B/metabolismo , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de Proteína , Baço/metabolismo , Ubiquitinas/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Qinghai Lake is a major migratory-bird breeding site that has experienced several highly pathogenic avian influenza virus (AIV) epizootics. Plateau pikas (Ochotona curzoniae) have previously been implicated in the ecology of avian influenza virus in this region. We first isolated an H9N2 AIV (A/Pika/Menyuan/01/2008) from plateau pikas between November 2008 and October 2009. Sequence analysis showed that the A/Pika/Menyuan/01/2008 AIV was closely related to the H9N2 AIV strain (A/Turkey/Wisconsin/ 1/1966). Our findings suggested that plateau pikas may contribute to AIV epidemiology in the Qinghai Lake region.
Assuntos
Doenças das Aves/transmissão , Reservatórios de Doenças/veterinária , Vírus da Influenza A Subtipo H9N2/genética , Vírus da Influenza A Subtipo H9N2/isolamento & purificação , Lagomorpha/virologia , Animais , Animais Selvagens/virologia , Doenças das Aves/virologia , Embrião de Galinha , China , Reservatórios de Doenças/virologia , Vetores de Doenças , Vírus da Influenza A Subtipo H9N2/classificação , Lagos , Filogenia , Proteínas Virais/genéticaRESUMO
We present a facile strategy to prepare grape-like silica-based hierarchical porous interlocked microcapsules (HPIMs) by polystyrene colloidal crystals templates, whose structure is the subtle integration of open mouthed structure, hierarchical porous nanostructure and interlocked architecture. HPIMs are fabricated by replicating colloidal crystals templates that have a hexagonal close-packed structure; thus, theoretically, each microcapsule has 12 open mouths, and these open mouths with mesoporous microcapsule wall construct the hierarchical porous structure. Furthermore, the interlocked architecture of the microcapsules can endow HPIMs with excellent mechanical stability and recyclability. By adjusting sulfonation time, the morphology, shell thickness, and even mesporous size of the HPIMs can be precisely controlled. In addition, HPIMs with various compositions are obtained via this method, such as silica and aminopropyl polysilsesquioxane (APSQ). All these unique features derived from a readily available method will give products with a broader range of applications.
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During investigations into the outbreak of duck Tembusu virus (DTMUV) infection in 2011 in China, a DTMUV strain (DTMUV-AH2011) was isolated from the affected ducks. The length of the genome of the DTMUV-AH2011 strain was found to be 11,064 nucleotides and to possess 10,278 nucleotides of one open reading frame (ORF), flanked by 94 nucleotides of the 5' non-translated region (NTR) and 692 nucleotides of the 3' NTR. In comparison with five fully sequenced TMUV genomes, the genome of DTMUV-AH2011 had a 74 nucleotide insertion in the 3' NTR. Comparison of the DTMUV-AH2011 fully deduced amino acid sequences with those of other Tembusu virus strains reported recently in China showed they had a highly conserved polyprotein precursor, sharing 98.9% amino acid identities, at least. The overall divergences of amino acid substitutions were randomly distributed among viral proteins except for the protein NS4B, the protein NS4B was unchanged. Knowledge of the biological characters of DTMUV and the potential role of the insertion in the 3' NTR in RNA replication will be useful for further studies of the mechanisms of virus replication and pathogenesis.
Assuntos
Surtos de Doenças/veterinária , Patos/virologia , Infecções por Flavivirus/epidemiologia , Flavivirus/genética , Genoma Viral/genética , Mutagênese Insercional/genética , Doenças das Aves Domésticas/virologia , Regiões 3' não Traduzidas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , China/epidemiologia , Infecções por Flavivirus/genética , Dados de Sequência Molecular , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
Due to the difficulty in obtaining highly branched rhamnogalacturonan-I (RG-I) type pectin, the relationship between the extent of RG-I branching (EB) of pectin and prebiotic/immunomodulatory activity has not been systematically investigated. Moreover, it is only possible to establish a structure-activity relationship using pectin that is highly purified and accurately characterized. In this study, a homogeneous highly branched RG-I type pectin (LBP-P4, a final product) with dual proliferative effects on Bifidobacterium and macrophage was effectively purified for the first time using enzyme hydrolysis combined with ultrafiltration. The RG-I content and EB of LBP-P4 reached 97.32 and 77.12, respectively. Its two branches were composed of arabinan and arabinogalactan-II, containing â 5)-Araf-(1â, â3)-Araf-(1â, â3,6)-Galp-(1â and â6)-Galp-(1â residues). The structure-activity relationship analysis indicated that strong prebiotic/immunomodulatory activity of LBP-P4 was depended on its high EB, which was further confirmed by the molecular docking simulation between low/high branched pectin with ß-1,6-galactosidase, α-l-arabinanase, and Toll-like receptor 4 (TLR4).
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Arabinogalactan exhibits many biological activities, which is the candidate for functional food ingredients. However, there is limited research on the arabinogalactan from Moringa Oleifera leaf, and its structure needs to be more accurately characterized. This study investigated structural characteristics and immunomodulatory activity of a high-purity polysaccharide from Moringa oleifera leaf (i.e. MOLP-PE) to further explore arabinogalactan from Moringa Oleifera leaf and its potential application area. The results showed that MOLP-PE was a unique type II arabinogalactan: the main chain consisted of â 3, 4)-α-D-Galp-(1â, â3)-ß-D-Galp-(1â and â2, 4)-ß-D-Rhap-(1â, with branches at the C-4 position of â3, 4)-α-D-Galp-(1â and â2, 4)-ß-D-Rhap-(1â, consisting of â5)-α-L-Araf-(1â, â3)-α-L-Araf-(1â, â6)-ß-D-Galp-(1â and â4)-ß-D-GalpA-(1â. Compared with arabinogalactan from larch, galactan and arabinan, MOLP-PE exhibited stronger ability in stimulating proliferation, phagocytosis and cytokines release of macrophages and bound with Toll-like receptor 4 closer via more binding sites, which might be due to its higher contents of 1,3-linked-Galp and 1,5-linked-Araf. These findings elucidated that MOLP-PE, as type II arabinogalactan with a unique structure, could be exploited as an immunomodulatory food ingredient.
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The establishment of high sensitive detection method for various pathogenic microorganisms remains constantly concerned. In the present study, multi-probe strategy was first systematically investigated followed by establishing a highly sensitive TaqMan real-time fluorescent quantitative PCR (qPCR) method for detecting African swine fever virus (ASFV). Briefly, four probes based on the B646L gene of ASFV were designed and the effects of different combinations of the probes in a single TaqMan qPCR assay on the detection sensitivity were investigated. As less as 0.5-5 copies/µl of the ASFV gene was detected by the established TaqMan qPCR assay. Furthermore, plasmid harboring the B646L in water samples could be concentrated 1000 times by ultrafiltration to enable a highly sensitive detection of trace viral nucleic acids. Moreover, no cross-reactivity was observed with other common clinical swine viruses such as PCV2, PCV3, PCV4, PEDV, PDCoV, CSFV, PRRSV, and PRV. When detecting 173 clinical porcine serum samples, the coincidence rate between the developed method and WOAH (World Organization of Animal Health) recommended method was 100%. This study might provide an integrated strategy to achieve higher detection sensitivity of trace pathogenic microorganisms and applicably sensitive TaqMan-based qPCR assays.
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Introduction: Variant pseudorabies virus (PRV) is a newly emerged zoonotic pathogen that can cause human blindness. PRV can take advantage of its large genome and multiple non-essential genes to construct recombinant attenuated vaccines carrying foreign genes. However, a major problem is that the foreign genes in recombinant PRV are only integrated into the genome for independent expression, rather than assembled on the surface of virion. Methods: We reported a recombinant PRV with deleted gE/TK genes and an inserted porcine circovirus virus 2 (PCV2) Cap gene into the extracellular domain of the PRV gE gene using the Cre-loxP recombinant system combined with the CRISPR-Cas9 gene editing system. This recombinant PRV (PRV-Cap), with the envelope-embedded Cap protein, exhibits a similar replication ability to its parental virus. Results: An immunogenicity assay revealed that PRV-Cap immunized mice have 100% resistance to lethal PRV and PCV2 attacks. Neutralization antibody and ELISPOT detections indicated that PRV-Cap can enhance neutralizing antibodies to PRV and produce IFN-γ secreting T cells specific for both PRV and PCV2. Immunological mechanistic investigation revealed that initial immunization with PRV-Cap stimulates significantly early activation and expansion of CD69+ T cells, promoting the activation of CD4 Tfh cell dependent germinal B cells and producing effectively specific effector memory T and B cells. Booster immunization with PRV-Cap recalled the activation of PRV-specific IFN-γ+IL-2+CD4+ T cells and IFN-γ+TNF-α+CD8+ T cells, as well as PCV2-specific IFN-γ+TNF-α+CD8+ T cells. Conclusion: Collectively, our data suggested an immunological mechanism in that the recombinant PRV with envelope-assembled PCV2 Cap protein can serve as an excellent vaccine candidate for combined immunity against PRV and PCV2, and provided a cost-effective method for the production of PRV- PCV2 vaccine.
Assuntos
Infecções por Circoviridae , Circovirus , Herpesvirus Suídeo 1 , Animais , Circovirus/imunologia , Circovirus/genética , Camundongos , Herpesvirus Suídeo 1/imunologia , Herpesvirus Suídeo 1/genética , Infecções por Circoviridae/imunologia , Infecções por Circoviridae/prevenção & controle , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Vacinas Virais/imunologia , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/genética , Suínos , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/sangue , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/genética , Vacinas Sintéticas/imunologia , Pseudorraiva/imunologia , Pseudorraiva/prevenção & controle , Feminino , Camundongos Endogâmicos BALB CRESUMO
The bursa of Fabricius, the acknowledged central humoral immune organ, plays a vital role in B lymphocyte differentiation. However, there are few reports of the molecular basis of the mechanism on immune induction and potential antitumor activity of bursal-derived peptides. In this paper, a novel bursal-derived pentapeptide-II (BPP-II, MTLTG) was isolated and exerted immunomodulatory functions on antibody responses in vitro. Gene microarray analyses demonstrated that BPP-II regulated expression of 2478 genes in a mouse-derived hybridoma cell line. Immune-related gene ontology functional procedures were employed for further functional analysis. Furthermore, the majority of BPP-II-regulated pathways were associated with immune responses and tumor processes. Moreover, BPP-II exhibited immunomodulatory effects on antigen-specific immune responses in vivo, including enhancement of avian influenza virus (H9N2 subtype)-specific antibody and cytokine production and modification of T cell immunophenotypes and lymphocyte proliferation. Finally, BPP-II triggered p53 expression and stabilization and selectively inhibited tumor cell proliferation. These data identified the multifunctional factor, BPP-II, as a novel biomaterial representing an important linking between the humoral central immune system and immune induction, including antitumor. Information generated in this study elucidates further the mechanisms involved in humoral immune system and represents the potential basis of effective immunotherapeutic strategies for treating human tumors and immune improvement.
Assuntos
Fatores Imunológicos/farmacologia , Vírus da Influenza A Subtipo H9N2/metabolismo , Neoplasias/imunologia , Oligopeptídeos/farmacologia , Linfócitos T/imunologia , Animais , Anticorpos Antivirais/imunologia , Bolsa de Fabricius/química , Bolsa de Fabricius/imunologia , Linhagem Celular Tumoral , Galinhas/imunologia , Citocinas/imunologia , Feminino , Humanos , Fatores Imunológicos/química , Fatores Imunológicos/imunologia , Fatores Imunológicos/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Oligopeptídeos/química , Oligopeptídeos/imunologia , Oligopeptídeos/isolamento & purificação , Proteína Supressora de Tumor p53/imunologiaRESUMO
Stroke is an acute cerebrovascular disease that can lead to disability and death. This study aimed to investigate the relationship between socioeconomic status (SES) and stroke. SES was evaluated by two variables: poverty to income ratio (PIR) and education level. In this multi-subject study, we collected data from the National Health and Nutrition Examination Survey (NHANES) database between 2009 and 2018, and finally 22,792 adults (≥20 years old) were included in the study. We proceeded with weighted multivariate logistic regression analysis as well as subgroup analysis. When analyzing the effect of PIR on stroke alone, the results showed that an increase in PIR levels was associated with a decrease in stroke incidence (OR = 0.764 95% CI: (0.711, 0.820), p < 0.001). The multivariate analysis presented a decline in stroke incidence in the highest quartile PIR group compared to the lowest quartile PIR group (OR = 0.296 95% CI: (0.214, 0.409), Pï¼0.001). Our results indicated that PIR is a protective factor for stroke, but there are exceptions in this relationship among different people. Hence, it is imperative that policymakers, healthcare providers, and clinicians take into account the inequality distribution of SES among adults while developing and executing stroke prevention and treatment strategies.
RESUMO
This study investigates the potential correlation between urinary caffeine levels and the occurrence of stroke, a serious cerebrovascular disease that can lead to disability or death. The data used in this study was obtained from the National Health and Nutrition Examination Survey conducted between 2009 and 2014. The study analyzed a total of 5,339 individuals, divided into a control group (n = 5,135) and a stroke group (n = 162). The researchers utilized multiple logistic regression and smoothed curve fitting to examine the relationship between urinary caffeine and caffeine metabolites and the incidence of stroke. The study found that higher urinary caffeine levels were associated with a lower risk of stroke in Mexican American participants (odds ratio [OR] = 0.886, 95% confidence interval [CI]: (0.791, 0.993), P = 0.037). After adjusting for certain participant characteristics, it was also found that higher urinary paraxanthine levels were associated with a lower risk of stroke incidence (OR = 0.991, 95% CI (0.984, 0.999), P = 0.027). Meanwhile, the highest urinary paraxanthine levels group had 43.7% fewer strokes than the lowest level group (OR = 0.563, 95% CI (0.341, 0.929), P = 0.025). In this study, we showed a negative link between urine paraxanthine levels and the risk of stroke. Meanwhile, urinary caffeine levels were negatively associated with the incidence of stroke in Mexican Americans, but no correlation in other populations. Our findings may have predictive and diagnostic implications in clinical practice. Further extensive prospective investigations are still needed to validate our conclusions.