RESUMO
Evidence is emerging on the roles of long noncoding RNAs (lncRNAs) as regulatory factors in a variety of viral infection processes, but the mechanisms underlying their functions in coxsackievirus group B type3 (CVB3)-induced acute viral myocarditis have not been explicitly delineated. We previously demonstrated that CVB3 infection decreases miRNA-21 expression; however, lncRNAs that regulate the miRNA-21-dependent CVB3 disease process have yet to be identified. To evaluate lncRNAs upstream of miRNA-21, differentially expressed lncRNAs in CVB3-infected mouse hearts were identified by microarray analysis and lncRNA/miRNA-21 interactions were predicted bioinformatically. MEG3 was identified as a candidate miRNA-21-interacting lncRNA upregulated in CVB3-infected mouse hearts. MEG3 expression was verified to be upregulated in HeLa cells 48 h post CVB3 infection and to act as a competitive endogenous RNA of miRNA-21. MEG3 knockdown resulted in the upregulation of miRNA-21, which inhibited CVB3 replication by attenuating P38-MAPK signaling in vitro and in vivo. Knockdown of MEG3 expression before CVB3 infection inhibited viral replication in mouse hearts and alleviated cardiac injury, which improved survival. Furthermore, the knockdown of CREB5, which was predicted bioinformatically to function upstream of MEG3, was demonstrated to decrease MEG3 expression and CVB3 viral replication. This study identifies the function of the lncRNA MEG3/miRNA-21/P38 MAPK axis in the process of CVB3 replication, for which CREB5 could serve as an upstream modulator.
Assuntos
Infecções por Coxsackievirus , Enterovirus , MicroRNAs , Miocardite , RNA Longo não Codificante , Viroses , Animais , Humanos , Camundongos , Infecções por Coxsackievirus/complicações , Infecções por Coxsackievirus/genética , Enterovirus/genética , Enterovirus Humano B/genética , Enterovirus Humano B/metabolismo , Células HeLa/virologia , MicroRNAs/genética , MicroRNAs/metabolismo , Miocardite/genética , Miocardite/metabolismo , Miocardite/virologia , RNA Longo não Codificante/genética , Replicação ViralRESUMO
Mitochondria are multifunctional organelles that produce energy and are critical for various signaling pathways. Mitochondrial antiviral signaling (MAVS) is a mitochondrial outer membrane protein essential for the anti-RNA viral immune response, which is regulated by mitochondrial dynamics and energetics; however, the molecular link between mitochondrial metabolism and immunity is unclear. Here we show in cultured mammalian cells that MAVS is activated by mitochondrial fission factor (Mff), which senses mitochondrial energy status. Mff mediates the formation of active MAVS clusters on mitochondria, independent of mitochondrial fission and dynamin-related protein 1. Under mitochondrial dysfunction, Mff is phosphorylated by the cellular energy sensor AMP-activated protein kinase (AMPK), leading to the disorganization of MAVS clusters and repression of the acute antiviral response. Mff also contributes to immune tolerance during chronic infection by disrupting the mitochondrial MAVS clusters. Taken together, Mff has a critical function in MAVS-mediated innate immunity, by sensing mitochondrial energy metabolism via AMPK signaling.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Imunidade Inata/fisiologia , Proteínas de Membrana/genética , Proteínas Mitocondriais/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Citocinas/metabolismo , Fibroblastos/imunologia , Células HeLa/virologia , Humanos , Proteínas de Membrana/metabolismo , Camundongos Knockout , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Fosforilação , Infecções por Respirovirus/imunologiaRESUMO
During internalization and trafficking, human papillomavirus (HPV) moves from the cell surface to the endosome where the transmembrane protease γ-secretase promotes insertion of the viral L2 capsid protein into the endosome membrane. Protrusion of L2 through the endosome membrane into the cytosol allows the recruitment of cytosolic host factors that target the virus to the Golgi en route for productive infection. How endosome-localized HPV is delivered to γ-secretase, a decisive infection step, is unclear. Here we demonstrate that cytosolic p120 catenin, likely via an unidentified transmembrane protein, interacts with HPV at early time-points during viral internalization and trafficking. In the endosome, p120 is not required for low pH-dependent disassembly of the HPV L1 capsid protein from the incoming virion. Rather, p120 is required for HPV to interact with γ-secretase-an interaction that ensures the virus is transported along a productive route. Our findings clarify an enigmatic HPV infection step and provide critical insights into HPV infection that may lead to new therapeutic strategies against HPV-induced diseases.
Assuntos
Alphapapillomavirus/patogenicidade , Cateninas/metabolismo , Infecções por Papillomavirus/virologia , Internalização do Vírus , Alphapapillomavirus/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Proteínas do Capsídeo/metabolismo , Endossomos/metabolismo , Células HeLa/virologia , Humanos , Membranas Intracelulares/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/metabolismo , Transporte Proteico/fisiologia , Vírion/metabolismoRESUMO
Avian salmonellosis caused by Salmonella enterica serovar Enteritidis (S. Enteritidis) and Pullorum (S. Pullorum) remains a big threat to the poultry industry and public hygiene. AvrA is an effector involved in inhibiting inflammation. Compared to AvrA from S. Enteritidis (SE-AvrA), the AvrA from S. Pullorum (SP-AvrA) lacks ten amino acids at the C-terminal. In this study, we compared the anti-inflammatory response induced by SP-AvrA to that of SE-AvrA. Transient expression of SP-AvrA in epithelial cells resulted in significantly weaker inhibition of NF-κB pathway activation when treated with TNF-α compared to the inhibition by SE-AvrA. SP-AvrA expression in the S. Enteritidis resulted in weaker suppression of NF-κB pathway in infected HeLa cells compared to SE-AvrA expression in the cells, while SP-AvrA expressed in S. Pullorum C79-13 suppressed NF-κB activation in infected HeLa and Caco 2 BBE cells to a greater extent than did SE-AvrA because of the higher expression of SP-AvrA than SE-AvrA in S. Pullorum. Further analysis demonstrated that the inhibition of NF-κB pathway in Salmonella-infected cells corresponded to the downregulation of the p-JNK and Beclin-1 protein molecules. Our study reveals that AvrA modifies the anti-inflammatory response in a manner dependent on the Salmonella serotype through inhibition of NF-κB pathway.
Assuntos
Proteínas de Bactérias/genética , Proteína Beclina-1/metabolismo , Salmonelose Animal/metabolismo , Salmonella enterica/patogenicidade , Animais , Proteínas de Bactérias/metabolismo , Células CACO-2/virologia , Galinhas , Citocinas/metabolismo , Células HeLa/virologia , Interações Hospedeiro-Patógeno , Humanos , Interleucina-8/metabolismo , Sistema de Sinalização das MAP Quinases , NF-kappa B/metabolismo , Doenças das Aves Domésticas/microbiologia , Salmonelose Animal/microbiologia , Salmonella enterica/genética , Salmonella enteritidis/genética , Salmonella enteritidis/patogenicidade , Sorogrupo , Transfecção , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Electrochemical sensors are the tools to detect the accurate and sensitive miRs. There is the challenge to increase the power and sensitivity of the surface for the electrochemical sensor. We design a virus-like hallow structure of cuco2o4 that it holds the large amounts of p19 protein by mimicking of inherent virus (Carnation italian ringspot virus) to detect 21mir with the limit of detection (LOD = 1aM). The electrochemical measurements are performed between the potentials at -0.3 V and +0.3 V with 1 mM [Fe(CN)6] -3/-4. After dropping the cuco2o4 on the SCPE (screen carbon printed electrode), the sensor is turned on due to the high electrochemical properties. Then, p19 proteins move into the hallow structure and inhibit the exchange of electrochemical reactions between the shells and the sensor is turned off. Then, adding the duplexes of RNA/miRs cause to increase the electrochemical property of p19 due to the change of p19 conformation and the system is turned on, again. So, for the first time, a virus-like hallow structure has been used to detect the 21miR in the human serum, MCF-7, Hella cells, with high sensitivity, specificity, and reproducibility in few minutes.
Assuntos
Técnicas Eletroquímicas , Tombusvirus/metabolismo , Espectroscopia Dielétrica/métodos , Células HeLa/virologia , Humanos , Limite de Detecção , Células MCF-7/virologia , MicroRNAs/análise , Nanocompostos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Tombusvirus/genéticaRESUMO
Tetraspanins are master organizers of the cell membrane. Recent evidence suggests that tetraspanins themselves may become crowded by virus particles and that these crowds/aggregates co-internalize with the viral particles. Using microscopy, we studied human papillomavirus (HPV) type 16-dependent aggregates on the cell surface of tetraspanin overexpressing keratinocytes. We find that aggregates are (1) rich in at least two different tetraspanins, (2) three-dimensional architectures extending up to several micrometers into the cell, and (3) decorated intracellularly by filamentous actin. Moreover, in cells not overexpressing tetraspanins, we note that obscurin-like protein 1 (OBSL1), which is thought to be a cytoskeletal adaptor, associates with filamentous actin. We speculate that HPV contact with the cell membrane could trigger the formation of a large tetraspanin web. This web may couple the virus contact site to the intracellular endocytic actin machinery, possibly involving the cytoskeletal adaptor protein OBSL1. Functionally, such a tetraspanin web could serve as a virus entry platform, which is co-internalized with the virus particle.
Assuntos
Actinas/fisiologia , Proteínas do Citoesqueleto/fisiologia , Papillomavirus Humano 16/fisiologia , Tetraspanina 24/fisiologia , Tetraspanina 30/fisiologia , Endocitose , Células HaCaT/virologia , Células HeLa/ultraestrutura , Células HeLa/virologia , Células Hep G2/virologia , Humanos , Microscopia Confocal , Microscopia Eletrônica , Infecções por Papillomavirus/virologia , Plaquinas/fisiologia , Vírion/fisiologia , Vírion/ultraestrutura , Internalização do VírusRESUMO
With the aid of the single-virus tracking technique and the quantum dot-labeling strategy, the internalization of the pseudorabies virus (PrV) has been visualized in real time. The results demonstrate that macropinocytosis can be considered as a major pathway for PrV entering HeLa cells, which facilitates the development of therapeutics for virus-triggered diseases.
Assuntos
Herpesvirus Suídeo 1/fisiologia , Pinocitose , Pontos Quânticos/química , Internalização do Vírus , Animais , Células HeLa/virologia , Humanos , Microscopia Confocal/métodos , Fosfatidilinositol 3-Quinase/metabolismo , Proteína Quinase C/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Nexinas de Classificação/metabolismo , Suínos , Proteína cdc42 de Ligação ao GTP/metabolismo , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Up-Frameshift Suppressor 1 Homolog (UPF1) is a key factor for nonsense-mediated mRNA decay (NMD), a cellular process that can actively degrade mRNAs. Here, we study NMD inhibition during infection by human T-cell lymphotropic virus type I (HTLV-1) and characterise the influence of the retroviral Tax factor on UPF1 activity. Tax interacts with the central helicase core domain of UPF1 and might plug the RNA channel of UPF1, reducing its affinity for nucleic acids. Furthermore, using a single-molecule approach, we show that the sequential interaction of Tax with a RNA-bound UPF1 freezes UPF1: this latter is less sensitive to the presence of ATP and shows translocation defects, highlighting the importance of this feature for NMD. These mechanistic insights reveal how HTLV-1 hijacks the central component of NMD to ensure expression of its own genome.
Assuntos
Produtos do Gene tax/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Degradação do RNAm Mediada por Códon sem Sentido , RNA Helicases/metabolismo , Transativadores/metabolismo , Trifosfato de Adenosina/metabolismo , Produtos do Gene tax/genética , Células HeLa/virologia , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Humanos , Mutação , Domínios Proteicos , Transporte Proteico , RNA Helicases/genética , Transativadores/genéticaRESUMO
Vibrio vulnificus causes fatal infections in humans, and antibiotics are commonly used in treatment regimens against V. vulnificus infection. However, the therapeutic effects of antibiotics are limited by multidrug resistance. In this study, we demonstrated that an antimicrobial peptide (AMP), HPA3PHis, loaded onto a gold nanoparticle-DNA aptamer (AuNP-Apt) conjugate (AuNP-Apt-HPA3PHis) is an effective therapeutic tool against V. vulnificus infection in vivo in mice. HPA3PHis induced bacterial cell death through the disruption of membrane integrity of V. vulnificus. The introduction of AuNP-Apt-HPA3PHis into V. vulnificus-infected HeLa cells dramatically reduced intracellular V. vulnificus by 90%, leading to an increase in the viability of the infected cells. Moreover, when V. vulnificus-infected mice were intravenously injected with AuNP-Apt-HPA3PHis, a complete inhibition of V. vulnificus colonization was observed in the mouse organs, leading to a 100% survival rate among the treated mice, whereas all the control mice died within 40 hours of being infected. Therefore, this study demonstrated the potential of an AMP delivered by AuNP-Apt as an effective and rapid treatment option against infection caused by a major pathogen in humans and aquatic animals.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Aptâmeros de Nucleotídeos/química , Sistemas de Liberação de Medicamentos/métodos , Vibrio vulnificus/efeitos dos fármacos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Feminino , Ouro , Células HeLa/virologia , Humanos , Nanopartículas Metálicas/administração & dosagem , Nanopartículas Metálicas/química , Camundongos Endogâmicos ICR , Testes de Sensibilidade Microbiana , Fragmentos de Peptídeos/química , Proteínas Ribossômicas/química , Vibrioses/tratamento farmacológico , Vibrioses/mortalidade , Vibrio vulnificus/patogenicidadeRESUMO
PICT-1 is a nucleolar protein with various tumor suppressor functions. Recently, PICT-1 expression was reported to be dramatically reduced in several cancers. To investigate the role of PICT-1 in uterine cervical carcinogenesis, we examined its gene mutations, protein expression, cellular localization, and effect on p53 stabilization. PCR-SSCP analysis of the entire coding region of PICT-1 showed that a polymorphism at codon 389 may increase the risk of uterine cervical cancers, and also identified a novel missense mutation. Expression of wild-type PICT-1 inhibited the degradation of p53 in the presence or absence of HPV 18 E6 viral protein in vitro, while the expression of codon 389 polymorphic PICT-1 had a diminished inhibitory effect on p53 degradation. Moreover, we observed that PICT-1 degradation was induced both independently and cooperatively by E6 and E7 proteins from high-risk HPVs, but only marginal degradation was observed with proteins from low-risk HPV. Immunohistochemical staining of tumor samples revealed that lower levels of PICT-1 were observed in samples from CIN III and cervical cancer tissues, compared to normal cervical epithelium and CIN I, II tissues (P < 0.05). The reduction of PICT-1 may therefore be an early event in uterine cervical tumorigenesis. Our results indicated that PICT-1 counteracts HPV-induced p53 degradation and that aberrant PICT-1 function may contribute towards inactivating p53. Therefore, PICT-1 may play a critical role during the pathogenesis of uterine cervical cancers.
Assuntos
Polimorfismo Genético , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Neoplasias do Colo do Útero/genética , Linhagem Celular Tumoral , Códon , Proteínas de Ligação a DNA/metabolismo , Feminino , Predisposição Genética para Doença , Células HeLa/virologia , Papillomavirus Humano 18/patogenicidade , Humanos , Pessoa de Meia-Idade , Mutação , Proteínas Oncogênicas Virais/metabolismo , Estabilidade Proteica , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Neoplasias do Colo do Útero/virologiaRESUMO
Encephalomyocarditis virus (EMCV), like hepatitis C virus (HCV), requires phosphatidylinositol 4-kinase IIIα (PI4KA) for genome replication. Here, we demonstrate that tyrphostin AG1478, a known epidermal growth factor receptor (EGFR) inhibitor, also inhibits PI4KA activity, both in vitro and in cells. AG1478 impaired replication of EMCV and HCV but not that of an EMCV mutant previously shown to escape PI4KA inhibition. This work uncovers novel cellular and antiviral properties of AG1478, a compound previously regarded only as a cancer chemotherapy agent.
Assuntos
1-Fosfatidilinositol 4-Quinase/antagonistas & inibidores , Antivirais/farmacologia , Vírus da Encefalomiocardite/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Hepacivirus/efeitos dos fármacos , Quinazolinas/farmacologia , Tirfostinas/farmacologia , 1-Fosfatidilinositol 4-Quinase/metabolismo , Relação Dose-Resposta a Droga , Vírus da Encefalomiocardite/genética , Vírus da Encefalomiocardite/fisiologia , Células HeLa/efeitos dos fármacos , Células HeLa/virologia , Hepacivirus/fisiologia , Humanos , Terapia de Alvo Molecular/métodos , Mutação , Replicação Viral/efeitos dos fármacosRESUMO
Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel and highly pathogenic human coronavirus and has quickly spread to other countries in the Middle East, Europe, North Africa and Asia since 2012. Previous studies have shown that MERS-CoV ORF4b antagonizes the early antiviral alpha/beta interferon (IFN-α/ß) response, which may significantly contribute to MERS-CoV pathogenesis; however, the underlying mechanism is poorly understood. Here, we found that ORF4b in the cytoplasm could specifically bind to TANK binding kinase 1 (TBK1) and IκB kinase epsilon (IKKε), suppress the molecular interaction between mitochondrial antiviral signaling protein (MAVS) and IKKε, and inhibit IFN regulatory factor 3 (IRF3) phosphorylation and subsequent IFN-ß production. Further analysis showed that ORF4b could also inhibit IRF3 and IRF7-induced production of IFN-ß, whereas deletion of the nuclear localization signal of ORF4b abrogated its ability to inhibit IRF3 and IRF7-induced production of IFN-ß, but not IFN-ß production induced by RIG-I, MDA5, MAVS, IKKε, and TBK-1, suggesting that ORF4b could inhibit the induction of IFN-ß in both the cytoplasm and nucleus. Collectively, these results indicate that MERS-CoV ORF4b inhibits the induction of type I IFN through a direct interaction with IKKε/TBK1 in the cytoplasm, and also in the nucleus with unknown mechanism. Viruses have evolved multiple strategies to evade or thwart a host's antiviral responses. A novel human coronavirus (HCoV), Middle East respiratory syndrome coronavirus (MERS-CoV), is distinguished from other coronaviruses by its high pathogenicity and mortality. However, virulence determinants that distinguish MERS-CoV from other HCoVs have yet to be identified. MERS-CoV ORF4b antagonizes the early antiviral response, which may contribute to MERS-CoV pathogenesis. Here, we report the identification of the interferon (IFN) antagonism mechanism of MERS-CoV ORF4b. MERS-CoV ORF4b inhibits the production of type I IFN through a direct interaction with IKKε/TBK1 in the cytoplasm, and also in the nucleus with unknown mechanism. These findings provide a rationale for the novel pathogenesis of MERS-CoV as well as a basis for developing a candidate therapeutic against this virus.
Assuntos
Núcleo Celular/virologia , Citoplasma/virologia , Interferon beta/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , Proteínas Virais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/metabolismo , Células HeLa/virologia , Interações Hospedeiro-Patógeno , Humanos , Quinase I-kappa B/metabolismo , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 7 de Interferon/metabolismo , Helicase IFIH1 Induzida por Interferon/genética , Helicase IFIH1 Induzida por Interferon/metabolismo , Interferon beta/genética , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Imunológicos , Proteínas Virais/genética , Proteínas Virais/farmacologiaRESUMO
OBJECTIVE: Human papillomavirus (HPV) infection is a prerequisite of developing cervical cancer, approximately half of which are associated with HPV type 16. There are reports that HPV can disturb the expression pattern of host miRNAs, but its mechanism is not well understood. MATERIALS AND METHODS: In this study, we scanned 11 tumorigenesis related miRNAs in Hela cells that were overexpressed with HPV type 16 E6 protein. RESULTS: We found the expression of miR-21 was upregulated by HPV type 16 E6 protein and meanwhile, the expression of miR-27a and miR-218 was downregulated. Furthermore, we identified that miR-21 overexpression could promote Hela and U2OS cells proliferation by targeting phosphatase-tensin homolog (PTEN), the result of which can be rescued by miR-21 inhibitor. In addition, E6 overexpression could also promote Hela cell migration and invasion. CONCLUSION: Our results indicate that HPV infection and subsequent transformation take place through complex regulatory patterns of gene expression in the host cells, part of which are regulated by the E6 protein.
Assuntos
Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Papillomavirus Humano 16/genética , MicroRNAs/genética , Proteínas Oncogênicas Virais/genética , Proteínas Repressoras/genética , Neoplasias do Colo do Útero/virologia , Carcinogênese/genética , Carcinogênese/patologia , Transformação Celular Neoplásica/patologia , Regulação para Baixo , Feminino , Células HeLa/patologia , Células HeLa/virologia , Humanos , Sensibilidade e Especificidade , Regulação para Cima , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologiaRESUMO
Temporal profiles of miRNA activity during productive virus infection can provide fundamental insights into host-virus interactions. Most reported miRNA targetome analyses in the context of virus infection have been performed in latently infected cells and lack reliable models for quantifying the suppression efficacy at specific miRNA target sites. Here, we identified highly competent temporal miRNA targetomes during lytic HCMV infection by using AGO-CLIP-seq together with a bioinformatic method that quantifies miRNA functionality at a specific target site, called ACE-scoring. The repression efficiency at target sites correlates with the magnitude of the ACE-score, and temporal HCMV-encoded miRNA targetomes identified by ACE-scoring were significantly enriched in functional categories involved in pathways central for HCMV biology. Furthermore, comparative analysis between human and viral miRNA targetomes supports the existence of intimate cooperation and co-targeting between them. Our holistic survey provides a valuable resource for understanding host-virus interactions during lytic HCMV infection.
Assuntos
Infecções por Citomegalovirus/virologia , Citomegalovirus/genética , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno , MicroRNAs , Citomegalovirus/patogenicidade , Perfilação da Expressão Gênica/métodos , Células HeLa/virologia , Humanos , Interferons/metabolismo , Janus Quinases/metabolismo , MicroRNAs/genética , Reprodutibilidade dos Testes , Fatores de Transcrição STAT/metabolismo , Análise de Sequência de RNA/métodos , Transdução de Sinais/genéticaRESUMO
Objetivo. Conocer las necesidades percibidas de salud mental de migrantes centroamericanos indocumentados en tránsito por la ciudad de Tapachula, Chiapas. Material y métodos. Estudio cualitativo realizado en Casa de Migrantes de Tapachula, Chiapas. Se realizaron 20 entrevistas semiestructuradas a diez mujeres y diez hombres migrantes. Se exploró el estado de salud mental y las expectativas de atención. Se retomaron nociones teórico-metodológicas de la fenomenología sociológica. Resultados. Los migrantes presentaban signos y síntomas de daños en su salud mental relacionados con experiencias vividas en el lugar de origen y en el tránsito por México. La percepción sobre su salud mental es influida por el modelo biomédico hegemónico. Las expectativas de servicios se relacionaron con la satisfacción de necesidades básicas. Conclusiones. Es necesario fortalecer la respuesta del sistema de atención en salud mental a partir de estrategias de cooperación y emprender acciones que promuevan la superación de una construcción biomédica de salud mental que estigmatiza, medicaliza, segrega y dificulta el acceso a servicios.
Objective. To identify the perception and needs in mental health of Central American migrants in transit through Tapachula, Chiapas. Materials and methods. Qualitative study in a migrant shelter in Tapachula, Chiapas. In 20 semi-structured interviews with migrant men and women, we explored their perceptions on mental health and expectations on care. We used basic notions of phenomenology to guide the analysis. Results. Migrants had several mental health problems related to the conditions at their country of origin and due to their initial transit through Mexico.Their perception on mental health problems was heavily influenced by the biomedical health paradigm. The expectations they had on the provision of services were related to the satisfaction of basic needs. Conclusions. It is necessary to strengthen the governmental response to mental health needs through collaborative strategies. Also, actions are needed to further the understanding of mental health in order to transcend the biomedical notions that stigmatize, segregate and create a barrier to accessing services.
Assuntos
Humanos , Genética Reversa/métodos , Rhinovirus/genética , Rhinovirus/patogenicidade , Clonagem Molecular , DNA Complementar/síntese química , Células HeLa/virologia , Reação em Cadeia da Polimerase/métodos , RNA Viral/isolamento & purificação , Rhinovirus/crescimento & desenvolvimento , TransfecçãoRESUMO
Contact between the human immunodeficiency virus (HIV-1) and its target cell is initiated by the interaction of viral gp120 with cellular CD4. An assembled peptide (CD4bs-M) that presents the CD4 binding site of gp120 was previously shown to inhibit the gp120-CD4 interaction. Here, we demonstrate that CD4bs-M selectively enhances infection of cells with HIV-1, whereas infection with herpes simplex virus remains largely unaffected. The effects of CD4bs-M variants containing D-amino acids, or prolines at selected positions, point to the importance of side chain orientation and spatial orientation of this fragment. Furthermore, CD4bs-M was shown to assemble into amyloid-like fibrils that capture HIV-1 particles, which likely contributes to the infection-enhancing effect. Beyond infection enhancement, CD4bs-M enabled HIV-1 infection of CD4-negative cells, suggesting that binding of the peptide to gp120 facilitates interaction of gp120 with coreceptors, which might in turn enhance HIV-1 entry.
Assuntos
Antígenos CD4/metabolismo , Proteína gp120 do Envelope de HIV/química , HIV-1/patogenicidade , Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Chlorocebus aethiops , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Células HeLa/virologia , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Células Vero/virologiaRESUMO
Human cytomegalovirus (HCMV) is a common and usually asymptomatic virus agent in healthy individuals. Initiation of HCMV productive infection depends on expression of the major immediate early (MIE) genes. The transcription of HCMV MIE genes is regulated by a diverse set of transcription factors. It was previously reported that productive HCMV infection is triggered probably by elevation of the plasma hydroxycorticoid level. However, it is poorly understood whether the transcription of MIE genes is directly regulated by glucocorticoid. Here, we found that the dexamethasone (DEX), a synthetic glucocorticoid, facilitates the transcription of HCMV MIE genes through the MIE promoter and enhancer in a glucocorticoid receptor (GR)-dependent manner. By competitive EMSA and reporter assays, we revealed that an NF-I like protein is involved in DEX-mediated transcriptional activation of the MIE promoter. Thus, this study supports a notion that the increased level of hydroxycorticoid in the third trimester of pregnancy reactivates HCMV virus production from the latent state.
Assuntos
Citomegalovirus/efeitos dos fármacos , Citomegalovirus/genética , Dexametasona/farmacologia , Genes Precoces , Fatores de Transcrição NFI/genética , Receptores de Glucocorticoides/genética , Sequência de Bases , Citomegalovirus/patogenicidade , Elementos Facilitadores Genéticos , Regulação Viral da Expressão Gênica , Glucocorticoides/farmacologia , Células HeLa/efeitos dos fármacos , Células HeLa/virologia , Humanos , Dados de Sequência Molecular , Fatores de Transcrição NFI/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Sequências Repetitivas de Ácido Nucleico , Elementos de Resposta/genéticaRESUMO
HeLa cell culture is the most widely used system for in vitro studies of the basic biology of human rhinovirus (HRV). It is also useful for making sufficient quantities of virus for experiments that require highly concentrated and purified virus. This chapter describes the protocols for producing a large amount of HeLa cells in suspension culture, using these cells to grow a large quantity of virus of HeLa-adapted HRV-A and -B serotypes, and making highly concentrated virus stock and highly purified virions. These purified HRV virions are free of cellular components and suitable for experiments that are sensitive to cellular contaminations.
Assuntos
Células HeLa/virologia , Rhinovirus/crescimento & desenvolvimento , Vírion/isolamento & purificação , Virologia/métodos , Técnicas de Cultura de Células/métodos , Criopreservação , HumanosRESUMO
Infectivity is a fundamental property of viral pathogens such as human rhinoviruses (HRVs). This chapter describes two methods for measuring the infectivity of HRV-A and -B serotypes: end point dilution (TCID50) assay and plaque assay. End point dilution assay is a quantal, not quantitative, assay that determines the dilution of the sample at which 50 % of the aliquots have infectious virus. It can be used for all the HRV-A and -B serotypes and related clinical isolates that grow in cell culture and induce cytopathic effect (CPE), degenerative changes in cells that are visible under a microscope. Plaque assay is a quantitative assay that determines the number of infectious units of a virus in a sample. After an infectious unit of virus infects one cell, the infected cell produces progeny viruses that then infect and kill a circle of adjacent cells. This circle of dead cells detaches from the dish and thus leaves a clear hole in a cell monolayer. Plaque assay works only for HeLa-adapted HRV-A and -B serotypes that can make visible plaques on the cell monolayer. Currently the end point dilution assay and plaque assay have not been developed for the newly discovered HRV-C.
Assuntos
Rhinovirus/patogenicidade , Ensaio de Placa Viral/métodos , Linhagem Celular , Criopreservação/métodos , Células HeLa/virologia , Humanos , Rhinovirus/crescimento & desenvolvimento , SorogrupoRESUMO
We describe standard methods for propagation, purification, quality control, and physicochemical characterization of human rhinoviruses, using HRV-A2 as an example. Virus is propagated in HeLa-OHIO cells grown in suspension culture and purified via sucrose density gradient centrifugation. Purity and homogeneity of the preparations are assessed with SDS-polyacrylamide gel electrophoresis (SDS-PAGE), capillary electrophoresis (CE), gas-phase electrophoretic mobility molecular analysis (GEMMA), and electron microscopy (EM). We also briefly describe usage of these methods for the characterization of subviral particles as well as for the analysis of their complexes with antibodies and soluble recombinant receptor mimics.