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We recently established a long-term SARS-CoV-2 infection model using lung-cancer xenograft mice and identified mutations that arose in the SARS-CoV-2 genome during long-term propagation. Here, we applied our model to the SARS-CoV-2 Delta variant, which has increased transmissibility and immune escape compared with ancestral SARS-CoV-2. We observed limited mutations in SARS-CoV-2 Delta during long-term propagation, including two predominant mutations: R682W in the spike protein and L330W in the nucleocapsid protein. We analyzed two representative isolates, Delta-10 and Delta-12, with both predominant mutations and some additional mutations. Delta-10 and Delta-12 showed lower replication capacity compared with SARS-CoV-2 Delta in cultured cells; however, Delta-12 was more lethal in K18-hACE2 mice compared with SARS-CoV-2 Delta and Delta-10. Mice infected with Delta-12 had higher viral titers, more severe histopathology in the lungs, higher chemokine expression, increased astrocyte and microglia activation, and extensive neutrophil infiltration in the brain. Brain tissue hemorrhage and mild vacuolation were also observed, suggesting that the high lethality of Delta-12 was associated with lung and brain pathology. Our long-term infection model can provide mutant viruses derived from SARS-CoV-2 Delta and knowledge about the possible contributions of emergent mutations to the properties of new variants.
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COVID-19 , SARS-CoV-2 , Humanos , Animales , Ratones , Xenoinjertos , SARS-CoV-2/genética , EncéfaloRESUMEN
SARS-CoV-2 Omicron has the largest number of mutations among all the known SARS-CoV-2 variants. The presence of these mutations might explain why Omicron is more infectious and vaccines have lower efficacy to Omicron than other variants, despite lower virulence of Omicron. We recently established a long-term in vivo replication model by infecting Calu-3 xenograft tumors in immunodeficient mice with parental SARS-CoV-2 and found that various mutations occurred majorly in the spike protein during extended replication. To investigate whether there are differences in the spectrum and frequency of mutations between parental SARS-CoV-2 and Omicron, we here applied this model to Omicron. At 30 days after infection, we found that the virus was present at high titers in the tumor tissues and had developed several rare sporadic mutations, mainly in ORF1ab with additional minor spike protein mutations. Many of the mutant isolates had higher replicative activity in Calu-3 cells compared with the original SARS-CoV-2 Omicron virus, suggesting that the novel mutations contributed to increased viral replication. Serial propagation of SARS-CoV-2 Omicron in cultured Calu-3 cells resulted in several rare sporadic mutations in various viral proteins with no mutations in the spike protein. Therefore, the genome of SARS-CoV-2 Omicron seems largely stable compared with that of the parental SARS-CoV-2 during extended replication in Calu-3 cells and xenograft model. The sporadic mutations and modified growth properties observed in Omicron might explain the emergence of Omicron sublineages. However, we cannot exclude the possibility of some differences in natural infection.
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COVID-19 , Neoplasias Pulmonares , Mutación , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Replicación Viral , Animales , Replicación Viral/genética , SARS-CoV-2/genética , SARS-CoV-2/patogenicidad , Ratones , Humanos , COVID-19/virología , Neoplasias Pulmonares/virología , Neoplasias Pulmonares/genética , Glicoproteína de la Espiga del Coronavirus/genética , Modelos Animales de Enfermedad , Línea Celular TumoralRESUMEN
Peptides are promising therapeutic agents for COVID-19 because of their specificity, easy synthesis, and ability to be fine-tuned. We previously demonstrated that a cell-permeable peptide corresponding to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike C-terminal domain (CD) inhibits the interaction between viral spike and nucleocapsid proteins that results in SARS-CoV-2 replication in vitro. Here, we used docking studies to design R-t-Spike CD(D), a more potent short cell-penetrating peptide composed of all D-form amino acids and evaluated its inhibitory effect against the replication of SARS-CoV-2 S clade and other variants. R-t-Spike CD(D) was internalized into Vero cells and Calu-3 cells and suppressed the replication of SARS-CoV-2 S clade, delta variant, and omicron variant with higher potency than the original peptide. In hemizygous K18-hACE2 mice, intratracheal administration of R-t-Spike CD(D) effectively delivered the peptide to the trachea and lungs, whereas intranasal administration delivered the peptide mostly to the upper respiratory system and stomach, and a small amount to the lungs. Administration by either route reduced viral loads in mouse lungs and turbinates. Furthermore, intranasally administered R-t-Spike CD(D) mitigated pathological change in the lungs and increased the survival of mice after infection with the SARS-CoV-2 S clade or delta variant. Our data suggest that R-t-Spike CD(D) has potential as a therapeutic agent against SARS-CoV-2 infection.
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COVID-19 , Péptidos de Penetración Celular , Chlorocebus aethiops , Animales , Ratones , Péptidos de Penetración Celular/farmacología , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Células VeroRESUMEN
BACKGROUND: CD83 is known to regulate lymphocyte maturation, activation, homeostasis, and antibody response to immunization and infection. While CD83 has a major part in B cell function, its role in influenza A virus infection has not yet been investigated. METHODS: We investigated the role of CD83 using C57BL/6J wild type mice and CD83 knockout (KO) mice after intraperitoneal administration of the influenza A/WSN/1933 virus. We analyzed cells of the peritoneal cavity, splenocytes, and cells of the bone marrow with FACS to investigate CD83 expression and cell population change in response to the virus infection. ELISA was performed with sera and peritoneal cavity fluids to detect A/WSN/1933 virus-specific IgG and the subclasses of IgG. RESULTS: FACS analysis data showed a transient but distinct induction of CD83 expression in the peritoneal B cells of wild type mice. CD83 KO mice exhibited a delayed recovery of B cells in the bone marrow after influenza virus infection and overall, a smaller T cell population compared to wild type mice. The peritoneal cavity and serum of the wild type mice contained a high titer of IgG within 14 days after infection, whereas the CD83 KO mice had a very low titer of IgG. CONCLUSIONS: These results show the importance of CD83 in lymphocytes homeostasis and antibody production during influenza A virus infection.
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Antígenos CD/genética , Antígenos CD/inmunología , Regulación de la Expresión Génica/inmunología , Inmunoglobulinas/genética , Inmunoglobulinas/inmunología , Virus de la Influenza A/inmunología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Infecciones por Orthomyxoviridae/inmunología , Animales , Anticuerpos Antivirales/sangre , Formación de Anticuerpos , Linfocitos B/inmunología , Células de la Médula Ósea/inmunología , Femenino , Inmunoglobulina G/sangre , Inmunoglobulina G/clasificación , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Cavidad Peritoneal/citología , Bazo/citología , Antígeno CD83RESUMEN
Mucin1 (MUC1) is a highly glycosylated transmembrane protein that plays a crucial role in the lubrication and protection of normal epithelial cells. However, MUC1 has emerged as a potential target for cancer therapy because it is overexpressed and functions in several types of cancers. Recently, we produced a monoclonal antibody (the anti-hMUC1 antibody) specific to the extracellular region of the MUC1 subunit MUC1-C to evaluate the utility of using anti-MUC1 antibodies in pancreatic cancer models. The anti-hMUC1 antibody recognized the MUC1-C protein in pancreatic cancer cells. Based on immunostaining and confocal image analyses, the anti-hMUC1 antibody initially bound to the cell membrane then was internalized in cancer cells that express MUC1. The anti-hMUC1 antibody suppressed epidermal growth factor (EGF)-mediated extracellular signalâ»regulated kinase (ERK) phosphorylation and cyclin D1 expression. When the anti-hMUC1 antibody was injected into a xenograft mouse model and traced using an in vivo imaging system, we observed that the anti-hMUC1 antibody was localized to MUC1-expressing pancreatic tumors. Importantly, the anti-hMUC1 monoclonal antibody suppressed pancreatic tumor growth in mice. According to immunohistochemistry analysis using a pancreatic cancer tissue array and the anti-hMUC1 antibody, MUC1 was highly expressed in human pancreatic cancer tissues compared to normal tissues. Therefore, we conclude that the anti-hMUC1 antibody specifically targets MUC1 and suppresses its function in pancreatic cancer in vitro and in vivo and can be further developed as a promising targeted therapy to treat pancreatic cancer.
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Anticuerpos Monoclonales/administración & dosificación , Antineoplásicos Inmunológicos/administración & dosificación , Mucina-1/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Regulación hacia Arriba/efectos de los fármacos , Animales , Anticuerpos Monoclonales/farmacología , Antineoplásicos Inmunológicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Neoplasias Pancreáticas/inmunología , Fosforilación/efectos de los fármacos , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Blood-brain barrier (BBB) breakdown and inflammation are critical events in ischemic stroke, contributing to aggravated brain damage. The BBB mainly consists of microvascular endothelial cells sealed by tight junctions to protect the brain from blood-borne substances. Thus, the maintenance of BBB integrity may be a potential target for neuroprotection. Sac-1004, a pseudo-sugar derivative of cholesterol, enhances the endothelial barrier by the stabilization of the cortical actin ring. RESULTS: Here, we report on the protective effects of Sac-1004 on cerebral ischemia-reperfusion (I/R) injury. Treatment with Sac-1004 significantly blocked the interleukin-1ß-induced monolayer hyperpermeability of human brain microvascular endothelial cells (HBMECs), loss of tight junctions, and formation of actin stress fiber. Sac-1004 suppressed the expression of adhesion molecules, adhesion of U937 cells, and activation of nuclear factor-κB in HBMECs. Using a rat model of transient focal cerebral ischemia, it was shown that Sac-1004 effectively ameliorated neurological deficits and ischemic damage. In addition, Sac-1004 decreased BBB leakage and rescued tight junction-related proteins. Moreover, the staining of CD11b and glial fibrillary acidic protein showed that Sac-1004 inhibited glial activation. CONCLUSIONS: Taken together, these results demonstrate that Sac-1004 has neuroprotective activities through maintaining BBB integrity, suggesting that it is a great therapeutic candidate for stroke.
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Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/diagnóstico por imagen , Isquemia Encefálica/diagnóstico por imagen , Isquemia Encefálica/tratamiento farmacológico , Daño por Reperfusión/tratamiento farmacológico , Saponinas/uso terapéutico , Animales , Barrera Hematoencefálica/metabolismo , Isquemia Encefálica/metabolismo , Permeabilidad Capilar/efectos de los fármacos , Permeabilidad Capilar/fisiología , Endotelio Vascular/diagnóstico por imagen , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Humanos , Inflamación/diagnóstico por imagen , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Saponinas/farmacologíaRESUMEN
The maintenance of endothelial barrier is critical for the vascular homeostasis and is maintained by the interaction of adherens junction (AJ) and tight junction (TJ) proteins between adjacent cells. This interaction is stabilized by actin cytoskeleton forming cortical actin ring. Here, we developed a novel vascular leakage blocker, Sac-1004 and investigated its mechanism of action in endothelial cells (ECs). Sac-1004 inhibited endothelial hyperpermeability induced by vascular endothelial growth factor, histamine and thrombin via stabilization of cortical actin ring and AJ proteins at the cell-cell junction. Treatment of Sac-1004 in ECs increased cAMP levels and activated Rac, both of which are known to strengthen endothelial barrier. Furthermore, Sac-1004 induced phosphorylation of cortactin and its localization at cell membrane that is essential for the stabilization of cortical actin ring. These effects of Sac-1004 on ECs were significantly abrogated by dideoxyadenosine (adenylyl cyclase inhibitor) and NSC23766 (Rac inhibitor). Taken together, our findings indicate that Sac-1004 blocks vascular leakage by enhancing endothelial integrity via the cAMP/Rac/cortactin pathway and imply the potential usefulness of Sac-1004 in the development of therapeutic means for vascular leakage-related diseases.
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Permeabilidad Capilar/efectos de los fármacos , Permeabilidad Capilar/fisiología , AMP Cíclico/fisiología , Endotelio Vascular/efectos de los fármacos , Pregnenolona/análogos & derivados , Saponinas/farmacología , Transducción de Señal/fisiología , Proteínas de Unión al GTP rac/fisiología , Células Cultivadas , Cortactina/fisiología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Pregnenolona/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
Introduction: The spectrum of SARS-CoV-2 mutations have increased over time, resulting in the emergence of several variants of concern. Persistent infection is assumed to be involved in the evolution of the variants. Calu-3 human lung cancer cells persistently grow without apoptosis and release low virus titers after infection. Methods: We established a novel in vivo long-term replication model using xenografts of Calu-3 human lung cancer cells in immunodeficient mice. Virus replication in the tumor was monitored for 30 days and occurrence of mutations in the viral genome was determined by whole-genome deep sequencing. Viral isolates with mutations were selected after plaque forming assays and their properties were determined in cells and in K18-hACE2 mice. Results: After infection with parental SARS-CoV-2, viruses were found in the tumor tissues for up to 30 days and acquired various mutations, predominantly in the spike (S) protein, some of which increased while others fluctuated for 30 days. Three viral isolates with different combination of mutations produced higher virus titers than the parental virus in Calu-3 cells without cytopathic effects. In K18-hACE2 mice, the variants were less lethal than the parental virus. Infection with each variant induced production of cross-reactive antibodies to the receptor binding domain of parental SARS-CoV-2 S protein and provided protective immunity against subsequent challenge with parental virus. Discussion: These results suggest that most of the SARS-CoV-2 variants acquired mutations promoting host adaptation in the Calu-3 xenograft mice. This model can be used in the future to further study SARS-CoV-2 variants upon long-term replication in vivo.
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SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Animales , Humanos , Ratones , COVID-19/virología , Neoplasias Pulmonares , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Línea Celular TumoralRESUMEN
The coronavirus disease 2019 pandemic, elicited by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is ongoing. Currently accessible antigen-detecting rapid diagnostic tests are limited by their low sensitivity and detection efficacy due to evolution of SARS-CoV-2 variants. Here, we produced and characterized an anti-SARS-CoV-2 nucleocapsid (N) protein-specific monoclonal antibody (mAb), 2A7H9. Monoclonal antibody 2A7H9 and a previously developed mAb, 1G10C4, have different specificities. The 2A7H9 mAb detected the N protein of S clade, delta, iota, and mu but not omicron, whereas the 1G10C4 antibody recognized the N protein of all variants under study. In a sandwich enzyme-linked immunosorbent assay, recombinant N protein bound to the 1G10C4 mAb could be detected by both 1G10C4 and 2A7H9 mAbs. Similarly, N protein bound to the 2A7H9 mAb was detected by both mAbs, confirming the existence of dimeric N protein. While the 1G10C4 mAb detected omicron and mu with higher efficiency than S clade, delta, and iota, the 2A7H9 mAb efficiently detected all the strains except omicron, with higher affinity to S clade and mu than others. Combined use of 1G10C4 and 2A7H9 mAb resulted in the detection of all the strains with considerable sensitivity, suggesting that antibody combinations can improve the simultaneous detection of virus variants. Therefore, our findings provide insights into the development and improvement of diagnostic tools with broader specificity and higher sensitivity to detect rapidly evolving SARS-CoV-2 variants.
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COVID-19 , Proteínas de la Nucleocápside , Humanos , Anticuerpos Monoclonales , SARS-CoV-2/genética , COVID-19/diagnóstico , Anticuerpos Antivirales , Ensayo de Inmunoadsorción Enzimática/métodos , Proteínas Recombinantes , Glicoproteína de la Espiga del CoronavirusRESUMEN
Influenza is a major cause of highly contagious respiratory illness resulting in high mortality and morbidity worldwide. Annual vaccination is an effective way to prevent infection and complication from constantly mutating influenza strains. Vaccination utilizes preemptive inoculation with live virus, live attenuated virus, inactivated virus, or virus segments for optimal immune activation. The route of administration also affects the efficacy of the vaccination. Here, we evaluated the effects of inoculation with ultraviolet (UV)-inactivated or live influenza A virus strains and compared their effectiveness and cross protection when intraperitoneal and intramuscular routes of administration were used in mice. Intramuscular or intraperitoneal inoculation with UV-inactivated Influenza A/WSN/1933 provided some protection against intranasal challenge with a lethal dose of live Influenza A/WSN/1933 but only when a high dose of the virus was used in the inoculation. By contrast, inoculation with a low dose of live virus via either route provided complete protection against the same intranasal challenge. Intraperitoneal inoculation with live or UV-inactivated Influenza A/Philippines/2/1982 and intramuscular inoculation with UV-inactivated Influenza A/Philippines/2/1982 failed to produce cross-reactive antibodies against Influenza A/WSN/1933. Intramuscular inoculation with live Influenza A/Philippines/2/1982 induced small amounts of cross-reactive antibodies but could not suppress the cytokine storm produced upon intranasal challenge with Influenza A/WSN/1993. None of the tested inoculation conditions provided observable cross protection against intranasal challenge with a different influenza strain. Taken together, vaccination efficacy was affected by the state and dose of the vaccine virus and the route of administration. These results provide practical data for the development of effective vaccines against influenza virus.
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Vacunas contra la Influenza , Gripe Humana , Infecciones por Orthomyxoviridae , Orthomyxoviridae , Administración Intranasal , Animales , Anticuerpos Antivirales , Modelos Animales de Enfermedad , Humanos , Ratones , Vacunación/métodos , Vacunas de Productos InactivadosRESUMEN
Stress granule formation is induced by numerous environmental stressors, including sodium arsenite treatment and viral infection. Accordingly, stress granules can inhibit viral propagation and function as part of the antiviral host response to numerous viral infections. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antagonizes stress granule formation, in part, via interaction between SARS-CoV-2 nucleocapsid (N) protein and Ras-GTPase-activating SH3-domain-binding protein 1 (G3BP1). However, it is unclear whether there are differential effects in different cell types. In this study, we assessed interaction between the N protein of SARS-CoV-2 S clade and G3BP1/2 in Vero and Calu-3 cells and investigated the effect of various SARS-CoV-2 strains on sodium arsenite-induced stress granule formation. Our data show that SARS-CoV-2 S clade N protein interacts with both G3BP1 and G3BP2 more strongly in Calu-3 vs. Vero cells. Consistent with this observation, infection with SARS-CoV-2 S clade induces stress granule formation in Vero but not in Calu-3 cells. However, infection with SARS-CoV-2 S clade, as well as other SARS-CoV-2 variants, inhibits sodium arsenite-induced stress granule formation in both cell lines. Taken together, our results show differential effects of SARS-CoV-2 infection on stress granule formation that is dependent on host cell type, rather than virus strain type.
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Human coronavirus OC43 (HCoV-OC43) is the coronavirus most associated with "common colds", infections of the upper respiratory tract. Previously, we reported that direct interactions of nucleocapsid (N) protein and C-terminal domain of Spike protein (Spike CD) are essential for replication of SARS-CoV-2 and MERS-CoV. Thus, we developed a novel ELISA-based strategy targeting these specific interactions to detect SARS-CoV-2 and MERS-CoV. Here, we investigated whether the same principles apply to HCoV-OC43. We discovered that the S protein of HCoV-OC43 interacts with N protein and that cell penetrating Spike CD peptide inhibits virus protein expression and replication of HCoV-OC43. The interaction between HCoV-OC43 S and N proteins were recapitulated with a recombinant HCoV-OC43 Spike CD fusion protein and a recombinant HCoV-OC43 N fusion protein in vitro. By producing an anti-HCoV-OC43 N protein-specific monoclonal antibody, we established a virus detection system based on the interaction between recombinant Spike CD and N protein of HCoV-OC43. We suggest that the interaction between Spike CD and N protein is conserved in coronaviruses and therefore could be a target for therapeutics against both novel coronavirus and its variants.
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COVID-19 , Coronavirus Humano OC43 , Coronavirus del Síndrome Respiratorio de Oriente Medio , Proteínas de la Nucleocápside de Coronavirus , Humanos , Proteínas de la Nucleocápside , SARS-CoV-2 , Glicoproteína de la Espiga del CoronavirusRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces coronavirus disease 2019 (COVID-19) and may increase the risk of adverse outcomes in lung cancer patients. In this study, we investigated the expression and function of mucin 1 (MUC1) after SARS-CoV-2 infection in the lung epithelial cancer cell line Calu-3. MUC1 is a major constituent of the mucus layer in the respiratory tract and contributes to pathogen defense. SARS-CoV-2 infection induced MUC1 C-terminal subunit (MUC1-C) expression in a STAT3 activation-dependent manner. Inhibition of MUC1-C signaling increased apoptosis-related protein levels and reduced proliferation-related protein levels; however, SARS-CoV-2 replication was not affected. Together, these results suggest that increased MUC1-C expression in response to SARS-CoV-2 infection may trigger the growth of lung cancer cells, and COVID-19 may be a risk factor for lung cancer patients. [BMB Reports 2021; 54(8): 425-430].
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Adenocarcinoma/patología , COVID-19/complicaciones , Neoplasias Pulmonares/patología , Mucina-1/fisiología , Adenocarcinoma/complicaciones , COVID-19/virología , Línea Celular Tumoral , Humanos , Neoplasias Pulmonares/complicaciones , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/fisiología , Replicación Viral/fisiologíaRESUMEN
Human coronavirus OC43 (HCoV-OC43) is one of the coronaviruses causing a mild common cold, but few studies have been made on this strain. Here, we identified the molecular mechanisms involved in HCoV-OC43-induced apoptosis and its implications for viral reproduction in Vero cells and MRC-5 cells. HCoV-OC43 infection induced apoptosis that was accompanied by cleavage of caspase-3 and PARP, degradation of cyclin D1, and cell cycle arrest at S and G2M phases. Dephosphorylation of STAT1 and STAT3, induced by HCoV-OC43 infection, was also associated with HCoV-OC43-mediated apoptosis. The pan-caspase inhibitor effectively prevented HCoV-OC43-induced apoptosis and reduced viral replication, suggesting that apoptosis contributes to viral replication. Collectively our results indicate that HCoV-OC43 induces caspase-dependent apoptosis to promote viral replication in Vero cells and MRC-5 cells.
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Apoptosis , Coronavirus Humano OC43/fisiología , Replicación Viral , Animales , Inhibidores de Caspasas/farmacología , Caspasas/metabolismo , Línea Celular , Proliferación Celular , Chlorocebus aethiops , Coronavirus Humano OC43/efectos de los fármacos , Humanos , Interferón alfa-2/farmacología , Fosforilación , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/metabolismo , Células Vero , Carga ViralRESUMEN
Severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is responsible for the current coronavirus disease 2019 (COVID-19) pandemic. Signaling pathways that are essential for virus production have potential as therapeutic targets against COVID-19. In this study, we investigated cellular responses in two cell lines, Vero and Calu-3, upon SARS-CoV-2 infection and evaluated the effects of pathway-specific inhibitors on virus production. SARS-CoV-2 infection induced dephosphorylation of STAT1 and STAT3, high virus production, and apoptosis in Vero cells. However, in Calu-3 cells, SARS-CoV-2 infection induced long-lasting phosphorylation of STAT1 and STAT3, low virus production, and no prominent apoptosis. Inhibitors that target STAT3 phosphorylation and dimerization reduced SARS-CoV-2 production in Calu-3 cells, but not in Vero cells. These results suggest a necessity to evaluate cellular consequences upon SARS-CoV-2 infection using various model cell lines to find out more appropriate cells recapitulating relevant responses to SARS-CoV-2 infection in vitro.
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SARS-CoV-2 infections continue to spread quickly by human-to-human transmission around the world. Therefore, developing methods to rapidly detect SARS-CoV-2 with high sensitivity are still urgently needed. We produced a monoclonal antibody that specifically detects the N protein of SARS-CoV-2 and recognizes N protein in cell lysates of SARS-CoV-2-infected Vero cells but not in cell lysates of MERS-CoV- or HCoV-OC43-infected Vero cells. This antibody recognized N protein in SARS-CoV-2 clades S, GR, and GH and recognized N protein in all the SARS-CoV-2 clades to â¼300 pfu. Previously, we reported that the coronavirus N protein interacts with the C-terminal domain of the spike protein (Spike CD). In this study, we developed an ELISA-based "bait and prey" system to confirm the interaction between SARS-CoV-2 Spike CD and N protein using recombinant fusion proteins. Furthermore, this system can be modified to quantitatively detect SARS-CoV-2 in culture media of infected cells by monitoring the interaction between the recombinant Spike CD fusion protein and the viral N protein, which is captured by the N protein-specific antibody. Therefore, we conclude that our N protein-specific monoclonal antibody and our ELISA-based bait and prey system could be used to diagnose SARS-CoV-2 infections.
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Design and synthesis of cholesterol-derived anti-apoptotic agents were described. The synthesized cholesterol analogs designed on the structural basis of ginsenoside Rk1 inhibited the undesirable apoptosis of human endothelial cells, which are induced by a vascular injury. In particular, analogue 1 possessing 4,6-di-O-acetyl-2,3-dideoxyhex-2-enopyran linked to hydroxyl group of cholesterol exhibited the most effective anti-apoptotic activities at both 5 and 10 µg/ml.
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Apoptosis/efectos de los fármacos , Colesterol/análogos & derivados , Colesterol/farmacología , Ginsenósidos/química , Vasos Sanguíneos/lesiones , Vasos Sanguíneos/patología , Colesterol/química , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Humanos , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Colorectal cancer is one of the leading causes of cancer related death due to a poor prognosis. In this study, we investigated the effect of Gomisin G on colon cancer growth and examined the underlying mechanism of action. We found that Gomisin G significantly suppressed the viability and colony formation of LoVo cells. Gomisin G reduced the phosphorylation level of AKT implying that Gomisin G suppressed the PI3K-AKT signaling pathway. Gomisin G also induced apoptosis shown by Annexin V staining and an increased level of cleaved poly-ADP ribose polymerase (PARP) and Caspase-3 proteins. Furthermore, Gomisin G remarkably triggered the accumulation of cells at the sub-G1 phase which represents apoptotic cells. In addition, the level of cyclin D1 and phosphorylated retinoblastoma tumor suppressor protein (Rb) was also reduced by the treatment with Gomisin G thus curtailing cell cycle progression. These findings show the suppressive effect of Gomisin G by inhibiting proliferation and inducing apoptosis in LoVo cells. Taken together, these results suggest Gomisin G could be developed as a potential therapeutic compound against colon cancer.
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Middle East respiratory syndrome coronavirus (MERS-CoV) uses the spike (S) glycoprotein to recognize and enter target cells. In this study, we selected two epitope peptide sequences within the receptor binding domain (RBD) of the MERS-CoV S protein. We used a complex consisting of the epitope peptide of the MERS-CoV S protein and CpG-DNA encapsulated in liposome complex to immunize mice, and produced the monoclonal antibodies 506-2G10G5 and 492-1G10E4E2. The western blotting data showed that both monoclonal antibodies detected the S protein and immunoprecipitated the native form of the S protein. Indirect immunofluorescence and confocal analysis suggested strong reactivity of the antibodies towards the S protein of MERS-CoV virus infected Vero cells. Furthermore, the 506-2G10G5 monoclonal antibody significantly reduced plaque formation in MERS-CoV infected Vero cells compared to normal mouse IgG and 492-1G10E4E2. Thus, we successfully produced a monoclonal antibody directed against the RBD domain of the S protein which could be used in the development of diagnostics and therapeutic applications in the future. [BMB Reports 2019; 52(6): 397-402].
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Coronavirus del Síndrome Respiratorio de Oriente Medio/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Chlorocebus aethiops , Islas de CpG/inmunología , ADN , Epítopos/inmunología , Humanos , Liposomas , Ratones , Coronavirus del Síndrome Respiratorio de Oriente Medio/patogenicidad , Péptidos/inmunología , Unión Proteica , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células VeroRESUMEN
Intraperitoneal inoculation with live influenza A virus confers protection against intranasal infections in mice and ferrets. However, the responses of peritoneal cells to influenza A virus have not been investigated. Here we show that intraperitoneal inoculation with A/WSN/1933 (H1N1) virus induced virus-reactive IgG production in the peritoneal cavity in mice. The infection resulted in substantial but transient B cell and macrophage depletion along with massive neutrophil infiltration, but virus growth was not detected. Influenza A viruses bound to α-2,6-linked sialic acids of B cells and macrophages and induced apoptotic death of peritoneal cavity cells. However, re-infection with A/WSN/1933 virus did not have adverse effects on immune cells most likely because of the neutralizing antibodies produced in response to the first exposure. Infection of BALB/c mice with A/WSN/1933 induced cross-protection against an otherwise lethal intraperitoneal dose of A/Hongkong/4801/2014 (H3N2) virus. This information suggests that immunological responses in the peritoneal cavity can induce effective defense against future virus infection. Considering the unexpected potent immunoregulatory activity of the peritoneal cells against influenza viruses, we suggest that comparative studies on various immune reactions after infection through different routes may contribute to better selection of vaccination routes in development of efficacious influenza vaccines.