RESUMEN
Senecavirus A (SVA), a picornavirus, causes vesicular diseases and epidemic transient neonatal losses in swine, resulting in a multifaceted economic impact on the swine industry. SVA counteracts host antiviral response through multiple strategies facilitatng viral infection and transmission. However, the mechanism of how SVA modulates interferon (IFN) response remains elusive. Here, we demonstrate that SVA 3C protease (3Cpro) blocks the transduction of Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway to antagonize type I IFN response. Mechanistically, 3Cpro selectively cleaves and degrades STAT1 and STAT2 while does not target JAK1, JAK2, and IRF9, through its protease activity. Notably, SVA 3Cpro cleaves human and porcine STAT1 on a Leucine (L)-Aspartic acid (D) motif, specifically L693/D694. In the case of STAT2, two cleavage sites were identified: glutamine (Q) 707 was identified in both human and porcine, while the second cleavage pattern differed, with residues 754-757 (Valine-Leucine-Glutamine-Serine motifs) in human STAT2 and Q758 in porcine STAT2. These cleavage patterns by SVA 3Cpro partially differ from previously reported classical motifs recognized by other picornaviral 3Cpro, highlighting the distinct characteristics of SVA 3Cpro. Together, these results reveal a mechanism by which SVA 3Cpro antagonizes IFN-induced antiviral response but also expands our knowledge about the substrate recognition patterns for picornaviral 3Cpro.IMPORTANCESenecavirus A (SVA), the only member in the Senecavirus genus within the Picornaviridae family, causes vesicular diseases in pigs that are clinically indistinguishable from foot-and-mouth disease (FMD), a highly contagious viral disease listed by the World Organization for Animal Health (WOAH). Interferon (IFN)-mediated antiviral response plays a pivotal role in restricting and controlling viral infection. Picornaviruses evolved numerous strategies to antagonize host antiviral response. However, how SVA modulates the JAK-STAT signaling pathway, influencing the type I IFN response, remains elusive. Here, we identify that 3Cpro, a protease of SVA, functions as an antagonist for the IFN response. 3Cpro utilizes its protease activity to cleave STAT1 and STAT2, thereby diminishing the host IFN response to promote SVA infection. Our findings underscore the significance of 3Cpro as a key virulence factor in the antagonism of the type I signaling pathway during SVA infection.
Asunto(s)
Cisteína Endopeptidasas , Infecciones por Picornaviridae , Picornaviridae , Factor de Transcripción STAT1 , Factor de Transcripción STAT2 , Transducción de Señal , Proteínas Virales , Animales , Porcinos , Factor de Transcripción STAT2/metabolismo , Humanos , Infecciones por Picornaviridae/virología , Infecciones por Picornaviridae/inmunología , Infecciones por Picornaviridae/metabolismo , Factor de Transcripción STAT1/metabolismo , Cisteína Endopeptidasas/metabolismo , Proteínas Virales/metabolismo , Proteasas Virales 3C , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/inmunología , Células HEK293 , Interferón Tipo I/antagonistas & inhibidores , Interferón Tipo I/metabolismo , Línea Celular , Quinasas Janus/metabolismo , Quinasas Janus/antagonistas & inhibidoresRESUMEN
African swine fever, one of the major viral diseases of swine, poses an imminent threat to the global pig industry. The high-efficient replication of the causative agent African swine fever virus (ASFV) in various organs in pigs greatly contributes to the disease. However, how ASFV manipulates the cell population to drive high-efficient replication of the virus in vivo remains unclear. Here, we found that the spleen reveals the most severe pathological manifestation with the highest viral loads among various organs in pigs during ASFV infection. By using single-cell-RNA-sequencing technology and multiple methods, we determined that macrophages and monocytes are the major cell types infected by ASFV in the spleen, showing high viral-load heterogeneity. A rare subpopulation of immature monocytes represents the major population infected at late infection stage. ASFV causes massive death of macrophages, but shifts its infection into these monocytes which significantly arise after the infection. The apoptosis, interferon response, and antigen-presentation capacity are inhibited in these monocytes which benefits prolonged infection of ASFV in vivo. Until now, the role of immature monocytes as an important target by ASFV has been overlooked due to that they do not express classical monocyte marker CD14. The present study indicates that the shift of viral infection from macrophages to the immature monocytes is critical for maintaining prolonged ASFV infection in vivo. This study sheds light on ASFV tropism, replication, and infection dynamics, and elicited immune response, which may instruct future research on antiviral strategies.
Asunto(s)
Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Porcinos , Animales , Virus de la Fiebre Porcina Africana/fisiología , Bazo/patología , Replicación Viral , Macrófagos/patologíaRESUMEN
Cuproptosis is a recently identified controlled process of cell death that functions in tumor development and treatment. Long non-coding RNAs (lncRNAs) are RNA molecules longer than 200 nucleotides that bind to transcription factors and regulate tumor invasion, penetration, metastasis, and prognosis. However, there are limited data on the function of cuproptosis-associated lncRNAs in pancreatic adenocarcinoma. Utilizing data retrieved from the cancer genome atlas database, we devised a risk prediction model of cuproptosis-associated lncRNAs in pancreatic adenocarcinoma, determined their prognostic significance and relationship with tumor immunity, and screened potential therapeutic drugs. Overall, 178 patients were randomized to a training or test group. We then obtained 6 characteristic cuproptosis-associated lncRNAs from the training group, based on which we constructed the risk prediction model, calculated the risk score, and verified the test group results. Subsequently, we performed differential gene analysis, tumor immunoassays, functional enrichment analysis, and potential drug screening. Finally, we found that the prediction model was highly reliable for the prognostic assessment of pancreatic adenocarcinoma patients. Generally, low risk patients had better outcomes than high risk patients. A tumor immunoassay showed that immunotherapy may benefit high risk patients more as there is a greater likelihood that the tumors could escape the immune system in low-risk patients. Through drug screening, we identified ten drugs that may have therapeutic effects on patients with pancreatic adenocarcinoma. In conclusion, this study constructed a risk prediction model of cuproptosis-associated lncRNAs, which can reliably predict the prognosis of pancreatic adenocarcinoma patients, provided a clinical reference for determining treatment approach, and provided some insights into the associations between lncRNAs and cuproptosis. This provides useful insight to aid in the development of therapeutic drugs for pancreatic adenocarcinoma.
Asunto(s)
Adenocarcinoma , Apoptosis , Neoplasias Pancreáticas , ARN Largo no Codificante , Muerte Celular Regulada , Humanos , Adenocarcinoma/metabolismo , Cobre/metabolismo , Inmunoterapia , Neoplasias Pancreáticas/metabolismo , Pronóstico , Muerte Celular Regulada/fisiología , ARN Largo no Codificante/metabolismo , Neoplasias PancreáticasRESUMEN
Cyclic GMP-AMP synthase (cGAS) plays a key role in the innate immune responses to both DNA and RNA virus infection. Here, we found that enterovirus 71 (EV-A71), Seneca Valley virus (SVV), and foot-and-mouth disease virus (FMDV) infection triggered mitochondria damage and mitochondrial DNA (mtDNA) release in vitro and vivo. These responses were mediated by picornavirus 2B proteins which induced mtDNA release during viral replication. SVV infection caused the opening of mitochondrial permeability transition pore (mPTP) and led to voltage-dependent anion channel 1 (VDAC1)- and BCL2 antagonist/killer 1 (Bak) and Bak/BCL2-associated X (Bax)-dependent mtDNA leakage into the cytoplasm, while EV-A71 and FMDV infection induced mPTP opening and resulted in VDAC1-dependent mtDNA release. The released mtDNA bound to cGAS and activated cGAS-mediated antiviral immune response. cGAS was essential for inhibiting EV-A71, SVV, and FMDV replication by regulation of IFN-ß production. cGAS deficiency contributed to higher mortality of EV-A71- or FMDV-infected mice. In addition, we found that SVV 2C protein was responsible for decreasing cGAS expression through the autophagy pathway. The 9th and 153rd amino acid sites in 2C were critical for induction of cGAS degradation. Furthermore, we also show that EV-A71, CA16, and EMCV 2C antagonize the cGAS-stimulator of interferon genes (STING) pathway through interaction with STING, and highly conserved amino acids Y155 and S156 were critical for this inhibitory effect. In conclusion, these data reveal novel mechanisms of picornaviruses to block the antiviral effect mediated by the cGAS-STING signaling pathway, which will provide insights for developing antiviral strategies against picornaviruses.
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Virus de la Fiebre Aftosa , Infecciones por Picornaviridae , Animales , Ratones , Antivirales/metabolismo , ADN Mitocondrial/genética , Virus de la Fiebre Aftosa/genética , Inmunidad Innata , Interferón beta/metabolismo , Mitocondrias/metabolismo , Nucleotidiltransferasas/metabolismo , Infecciones por Picornaviridae/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
The antioxidant enzymes play the crucial role in inhibiting mutton spoilage. In this study, visible near-infrared (Vis-NIR) hyperspectral imaging (HSI) combined with entropy weight method (EWM) was developed for the first time to evaluate the antioxidant properties of Tan mutton. The comprehensive index of antioxidant enzymes (AECI) consisting of peroxidase (49.34%), catalase (37.97%) and superoxidase (12.69%) was constructed by the EWM. Partial least squares regression, least squares support vector machine and artificial neural networks (ANN) were developed based on characteristic wavelengths extracted by successful projections algorithm, uninformative variable selection, iteratively retains informative variables (IRIV), regression coefficient and competitive adaptive reweighted sampling (CARS). The textural features (TF) were extracted by the gray level co-occurrence matrix and fused with the spectral data to establish models. Visualization of the changes in antioxidant enzyme activity was constructed from the optimal model. In addition, two-dimensional correlation spectra (2D-COS) with AECI as a perturbation variable was used to identify spectral features, revealing chemical bond changes order under the characteristic peaks at 612-799-473-708-559 nm. The results showed that the IRIV-CARS-TF-ANN model performed the best, with prediction set coefficient of determination (RP2) of 0.8813, which improved 2.12%, 1.11% and 2.77% over the RP2 of full band, IRIV and IRIV-CARS, respectively. It was suggested that fusion data of HSI may effectively predict the activity of antioxidant enzymes in Tan mutton.
Asunto(s)
Antioxidantes , Carne Roja , Algoritmos , Entropía , Imágenes Hiperespectrales , Análisis de los Mínimos Cuadrados , Espectroscopía Infrarroja Corta/métodos , Ovinos , AnimalesRESUMEN
Foot-and-mouth disease virus (FMDV) is a highly contagious virus that causes severe vesicular disease of cloven-hoofed animals. Various endocytosis mechanisms are involved in the entry of FMDV after binding to the integrin and heparan sulfate (HS) receptors. However, the mechanism of FMDV using other unknown receptors to enter the cells remains unclear. Here, we reported that the endocytosis and endosomal pathways are employed by FMDV to invade the Chinese hamster ovary cell line (CHO-677) without the integrin and HS receptors. We demonstrated that the internalization of FMDV into CHO-677 cells was abrogated by chlorpromazine, an inhibitor of clathrin-mediated endocytosis. Knockdown of the clathrin heavy chain decreased the viral protein abundance. Incubation of the CHO-677 cells with the inhibitors of caveolae-mediated endocytosis or transfection by caveolin-1 siRNA also limited FMDV replication. In addition, we determined that the acidic environment and the existence of dynamin were essential for FMDV infection in CHO-677 cells. The endosomal proteins Rab5 (early endosome) and Rab7 (late endosome), but not Rab11 (recycling endosome), were utilized by FMDV during infection. These data provide a new entry model of FMDV by unknown receptors which will help to better understand the pathogenesis mediated by FMDV.
Asunto(s)
Virus de la Fiebre Aftosa , Enfermedades de la Boca , Enfermedades de los Roedores , Cricetinae , Animales , Clatrina/metabolismo , Virus de la Fiebre Aftosa/genética , Virus de la Fiebre Aftosa/metabolismo , Células CHO , Caveolina 1/metabolismo , Cricetulus , ARN Interferente Pequeño , Cadenas Pesadas de Clatrina/metabolismo , Clorpromazina , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión al GTP rab5/metabolismo , Internalización del Virus , Endocitosis , Dinaminas/metabolismo , Integrinas/metabolismo , Heparitina Sulfato , Proteínas Virales/metabolismo , Enfermedades de la Boca/veterinariaRESUMEN
African swine fever (ASF)-an aggressive infectious disease caused by the African swine fever virus (ASFV)-is significantly unfavorable for swine production. ASFV has a complex structure and encodes 150-167 proteins; however, the function of most of these proteins is unknown. This study identified ASFV MGF360-9L as a negative regulator of the interferon (IFN)-ß signal. Further evidence showed that MGF360-9L interacts with signal transducer and activator of transcription (STAT) 1 and STAT2 and degrades STAT1 and STAT2 through apoptosis and ubiquitin-proteasome pathways, respectively. Subsequently, the activation of IFN-ß signaling was inhibited. Naturally isolated or genetically manipulated live attenuated viruses are known to protect against the virulent parental ASFV strains. Therefore, through homologous recombination, we deleted MGF360-9L from the virulent ASFV CN/GS/2018 strain to construct a recombinant strain, ASFV-Δ360-9L. Compared with the parent ASFV CN/GS/2018 strain, the replication level of ASFV-Δ360-9L decreased in primary porcine alveolar macrophage cultures at 24 h postinfection, but the difference is unlikely to be biologically relevant. Notably, ASFV-Δ360-9L was partially attenuated in pigs. To our knowledge, this study is the first to uncover the function of MGF360-9L during ASFV infection. MGF360-9L inhibits IFN-ß signaling through the targeted degradation of STAT1 and STAT2. Furthermore, MGF360-9L is a key virulence gene of ASFV. Our findings reveal a new mechanism by which ASFV inhibits host antiviral response; this might facilitate the development of live attenuated ASFV vaccines. IMPORTANCE African swine fever-an acute, febrile, hemorrhagic, highly contacting, and highly lethal disease caused by African swine fever virus (ASFV)-jeopardizes the global pig industry. Understanding the mechanism ASFV employs to evade host defense during infection is essential for developing targeted drugs and vaccines against ASFV. To our knowledge, this study identifies the mechanism of innate immunity against by MGF360-9L and the effect of MGF360-9L on ASFV pathogenicity. The results showed that MGF360-9L may help ASFV escape the host immunity by degrading STAT1 and STAT2 and thus inhibiting IFN-ß signaling. MGF360-9L is also an important virulence factor of ASFV. The deletion of MGF360-9L reduces ASFV virulence in pigs. This study explored a new mechanism of ASFV against innate immunity and identified a new ASFV virulence factor; these findings may guide the development of live attenuated ASFV vaccines.
Asunto(s)
Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Animales , Virus de la Fiebre Porcina Africana/genética , Macrófagos , Transducción de Señal , Porcinos , Proteínas Virales/genética , Factores de Virulencia/genética , Quinasas Janus/metabolismo , Factores de Transcripción STAT/metabolismoRESUMEN
Osteosarcoma is an aggressive malignancy with rapid development and poor prognosis. microRNA-19 (miR-19) plays an important role in several biological processes. Sprouty-related EVH1 domain protein 2 (SPRED2) is a suppressor of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signaling to inhibit tumor development and progression by promoting autophagy. In this study, we investigated the roles of miR-19, SPRED2, and autophagy in osteosarcoma. We detected the expression of miR-19, SPRED2, epithelial-mesenchymal transition (EMT) markers, and autophagy-related proteins via quantitative real-time polymerase chain reaction or western blot. To evaluate the function of miR-19 and SPRED2, we used MTT and colony formation assays to detect cell proliferation, Transwell, and wound-healing assays to detect cell invasion and migration. Targetscan and luciferase reporter assays confirmed the relationship between SPRED2 and miR-19. The expression of miR-19 was significantly upregulated in osteosarcoma, while SPRED2 was downregulated. miR-19 inhibitor reduced cell proliferation, invasion, migration, and EMT, while its cell biological effects were partially reversed by addition of autophagy inhibitor 3-methyladenine (3-MA) or SPRED2 siRNA in osteosarcoma. SPRED2, a suppressor of ERK/MAPK pathway that is known to trigger autophagy, was identified as a direct target of miR-19. SPRED2 overexpression increased cell proliferation, invasion, migration, and EMT by promoting autophagy, and the effects could be inhibited by 3-MA. Collectively, these findings reveal an underlying mechanism for development of osteosarcoma. miR-19 was upregulated in osteosarcoma cells, and negatively regulated SPRED2, thus promoting the malignant transformation of osteosarcoma cells via inhibiting SPRED2-induced autophagy. Therefore, miR-19/SPRED2 may be a potential target for the treatment of osteosarcoma.
Asunto(s)
Autofagia/genética , Movimiento Celular/genética , Transición Epitelial-Mesenquimal/genética , MicroARNs/metabolismo , Osteosarcoma/genética , Proteínas Represoras/metabolismo , Secuencia de Bases , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/genética , Invasividad Neoplásica , Regulación hacia Arriba/genéticaRESUMEN
OBJECTIVE: To compare the effectiveness of traditional and hybrid teaching strategies in pathophysiology and to conduct a survey of students' opinions about the hybrid teaching strategy. METHODS: A hybrid pathophysiology course was developed by combining traditional lectures, case- or problem-based learning, group discussion and several quizzes. A total of 167 students were assigned to the hybrid teaching group and 118 students assigned to the traditional lecture group. RESULTS: Compared with students who received traditional lectures, no students in the hybrid teaching class failed the final examination. The percentage of students with high scores was significantly higher in the hybrid teaching class. In addition, 73.7% of students in the hybrid teaching class expressed substantial interest in pathophysiology during the course, and 83% of these students felt they had received essential training and acquired the ability to solve clinical case problems. CONCLUSION: The hybrid teaching strategy is an advanced approach that encourages students to actively learn teaching materials and solve practical clinical problems, and that promotes student interest in pathophysiology.
Asunto(s)
Evaluación Educacional , Aprendizaje Basado en Problemas , Humanos , Aprendizaje , Estudiantes , Encuestas y CuestionariosRESUMEN
The feasibility of combining spectral and textural information from hyperspectral imaging to improve the prediction of the C16:0 and C18:1 n9 contents for lamb was explored. 29 and 22 optimal wavelengths were selected for the C16:0 and C18:1 n9 contents, respectively, by conducting the variable combination population analysis-iteratively retaining informative variables (VCPA-IRIV) algorithm. To extract the textural features of images, a gray-level co-occurrence matrix (GLCM) analysis was implemented in the first principal component image. The least squares support vector machine (LSSVM) model and the partial least squares regression (PLSR) model were developed to predict the C16:0 and C18:1 n9 contents from the spectra and the fusion data. The distribution map was visualized using the best model with the imaging process. The results showed that the combination of the spectral and textural information of hyperspectral imaging coupled with the VCPA-IRIV algorithm had strong potential for the prediction and visualization of the C16:0 and C18:1 n9 contents of lamb.
Asunto(s)
Ácido Oléico/análisis , Ácido Palmítico/análisis , Carne Roja/análisis , Algoritmos , Animales , Imágenes Hiperespectrales/métodos , Imágenes Hiperespectrales/veterinaria , Análisis de los Mínimos Cuadrados , Análisis de Componente Principal , Oveja Doméstica , Máquina de Vectores de SoporteRESUMEN
Colorectal cancer (CRC) is one of deadly malignancies that affects humans globally. Herein, the effects of MALAT1 on CRC cellular functions were investigated. RT-qPCR measured expression of MALAT1 in human cell lines for colorectal Cancer. Radiation-resistance CRC cells (CRC-IR) were generated by increasing treatments of irradiation. Cell transfection upregulated or silenced genes in CRC-IR cells so as to study the correlation between MALAT1/miR-101-3p expression and cellular resistance to irradiation through evaluation of CCK-8, FCM apoptosis, Transwell migration and invasion and western blot assays for cell viability,apoptosis, migration and invasion and EMT. MALAT1 was upregulated in radio-resistance cell lines compared to normal CRC cells and upregulation promoted cell viability. In addition, decreased MALAT1 inhibited cell proliferation and metastasis and promoted apoptosis of CRC-IR cells. The luciferase assays confirmed that MALAT1 targeted and regulated miR-101-3p expression in radio-resistance cells. MiR-101-3p counteracted the effect exerted by MALAT1 in CRC-IR cells, indicating that MALAT1 added to the radio-resistance in vitro while miR-101-3p mimics could decrease the resistance to irradiation in CRC. In this study we have demonstrated that MALAT1 could regulate the radio-resistance in colorectal cancer via sponging miR-101-3p. Eventually, these outcomes unearthed a novel axis lncRNA MALAT1/miR-101-3p,which might be a prospective treatment to regulate radio-therapy in the near future.
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Neoplasias Colorrectales/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , ARN Largo no Codificante/genética , Tolerancia a Radiación/genética , Apoptosis/genética , Secuencia de Bases , Línea Celular Tumoral , Proliferación Celular/genética , Supervivencia Celular/genética , Neoplasias Colorrectales/patología , Regulación hacia Abajo/genética , Humanos , MicroARNs/metabolismo , ARN Largo no Codificante/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
Osteosarcoma (OS) is a primary malignant tumor with a complex etiology. Therefore, research into the pathogenesis of osteosarcoma is considered a priority. Circular RNAs play important roles in cell metabolism and in the immune response and are closely associated with cancer treatment. However, research into the association of circular RNAs with osteosarcoma is limited. In the present study, CircSAMD4A was validated by RTqPCR and agarose gel electrophoresis. CircSAMD4A and miR3423p expression was detected by RTqPCR. The relative protein expression levels were measured by western blot analysis. MTT assay and flow cytometry were used to detect cell cytotoxicity and apoptosis, respectively. Transwell assay was applied to assess cell migration and invasion. Dualluciferase reporter assay was used to determine the association among CircSAMD4A, Frizzled7 (FZD7) and miR3423p. In vivo, subcutaneous tumor formation assay was performed in an experiment with nude mice. The results revealed that the expression levels of CircSAMD4A and FZD7 were upregulated, while those of miR3423p were downregulated in OS tissues and cells. The inhibition of CircSAMD4A suppressed cell progression and epithelialmesenchymal transition (EMT), and promoted cell apoptosis in OS. The reduction of miR3423p reversed the effects of CircSAMD4A downregulation on cell cytotoxicity, migration, invasion, apoptosis and EMT in OS, while FZD7 overexpression blocked the effect of miR3423p upregulation on OS progression. The suppressive effect of shCircSAMD4A on tumor growth was thus verified in OS. Overall, the present study demonstrated that CircSAMD4A affected cell cytotoxicity, invasion, apoptosis, migration and EMT by regulating the miR3423p/FDZ7 axis in OS, thereby providing a novel regulatory mechanism and a potential therapeutic target for OS.
Asunto(s)
Apoptosis/fisiología , Movimiento Celular/fisiología , Transición Epitelial-Mesenquimal/fisiología , MicroARNs/metabolismo , Proteínas Represoras/metabolismo , Animales , Apoptosis/genética , Western Blotting , Línea Celular Tumoral , Movimiento Celular/genética , Transición Epitelial-Mesenquimal/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Inmunoprecipitación , Ratones , Ratones Desnudos , MicroARNs/genética , Unión Proteica , Proteínas Represoras/genéticaRESUMEN
Under different circumstances, the alteration of several viral genes may give an evolutionary advantage to the virus to maintain its prevalence in nature. In this study, a 70-nucleotide deletion in the small fragment (S fragment) of the viral 5'-untranslated region (5'-UTR) together with one amino acid insertion in the leader protein (Lpro) that naturally occurred in several serotype O foot-and-mouth disease virus (FMDV) strains in China was identified. The properties of two field serotype O FMDV strains, with or without the 70-nucleotide deletion in the S fragment and the amino acid insertion in Lpro, were compared in vitro and in vivo Clinical manifestations of FMD were clearly observed in cattle and pigs infected by the virus without the mutations. However, the virus with the mentioned mutations caused FMD outcomes only in pigs, not in cattle. To determine the role of the 70-nucleotide deletion in the S fragment and the single amino acid insertion in Lpro in the pathogenicity and host range of FMDV, four recombinant viruses, with complete genomes and a 70-nucleotide deletion in the S fragment, a single amino acid insertion in Lpro, or both mutations, were constructed and rescued. It showed that deletion of 70 nucleotides in the S fragment or insertion of one amino acid (leucine) at position 10 of Lpro partly decreased the viral pathogenicity of Mya-98 lineage virus in cattle and pigs. However, the virus with dual mutations caused clinical disease only in pigs, not in cattle. This suggested that the S fragment and Lpro are significantly associated with the virulence and host specificity of FMDV. The naturally occurring dual mutation in the S fragment and Lpro is a novel determinant of viral pathogenicity and host range for serotype O FMDV.IMPORTANCE FMD is probably the most important livestock disease in the world due to the severe economic consequences caused. The alteration of several viral genes may give the virus selective advantage to maintain its prevalence in nature. Here, we identified that a 70-nucleotide deletion in the S fragment combined with a single leucine insertion in the leader protein (Lpro) is a novel determinant of restricted growth on bovine cells, which significantly contributes to the altered virulence of serotype O FMDV in cattle. A synergistic and additive effect of the 70-nucleotide deletion in the S fragment and the single leucine insertion in Lpro on the virulence and host specificity of the virus was determined. These results will benefit efforts to understand the vial pathogenicity mechanism and molecular characteristics of FMDV.
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Endopeptidasas/genética , Virus de la Fiebre Aftosa/genética , Fiebre Aftosa/virología , Virulencia/genética , Regiones no Traducidas 5' , Animales , Bovinos , Cricetinae , Virus de la Fiebre Aftosa/patogenicidad , Virus de la Fiebre Aftosa/fisiología , Eliminación de Gen , Especificidad del Huésped , Leucina/genética , Mutación , Porcinos , Proteínas no Estructurales Virales/genética , Replicación ViralRESUMEN
Foot-and-mouth disease virus (FMDV) is the causative agent of foot-and-mouth disease. It is characterized by genetic instability and different antigenic properties. The nonstructural protein 3A is a primary determinant of the tropism and virulence of Cathay topotype FMDVs. However, several other determinants are also speculated to be involved in viral tropism and virulence. Deletion of 43 nucleotides (nt) in the pseudoknot (PK) region of the 5' untranslated region (UTR) has been found to coexist with the identified 3A deletion in Cathay topotype FMDV genomes. In this study, we isolated an O/ME-SA/PanAsia lineage FMDV strain, O/GD/CHA/2015, that includes an 86-nt deletion in the PK region and shows a porcinophilic phenotype. To investigate the potential role of the PK region in viral pathogenicity, we generated a recombinant FMDV strain with an incomplete PK region and compared its virulence and pathogenesis to the intact FMDV strain in swine and bovines. Deletion of the 86 nt in the PKs had no major effects on the pathogenicity of the virus in swine but significantly attenuated its ability to infect bovine cells and cattle, indicating that the PK region is a newly discovered determinant of viral tropism and virulence. The role of the 43-nt deletion existing in the Cathay topotype FMDV was also investigated by evaluating the infection properties of genetically engineered viruses. Consistently, the 43-nt deletion in the PK region significantly decreased the pathogenicity of the virus in bovines. Overall, our findings suggest that the PK region deletion occurred naturally in the FMDV genome and that the PK region is highly associated with viral host range and functions as a novel determinant for FMDV pathogenesis.IMPORTANCE This study demonstrates that the deletion in the PK region occurred naturally in the FMDV genome. The isolated O/ME-SA/PanAsia lineage FMDV with an 86-nt deletion in the PK region showed a pig-adapted characteristic that could cause clinical signs in swine but not bovines. Compared to the wild-type FMDV strain, which possesses full infection capacity in both swine and bovines, the recombinant virus with the 86-nt deletion in the PK region is deficient in causing disease in bovines. Deletion of the previously reported 43 nt in the PK region also led to significantly decreased pathogenicity of FMDV in bovines. This study indicates that the PK region is a novel determinant of the tropism and virulence of FMDV.
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Regiones no Traducidas 5' , Secuencia de Bases , Virus de la Fiebre Aftosa/genética , Genoma Viral , Eliminación de Secuencia , Proteínas no Estructurales Virales/genética , Tropismo Viral/genética , Animales , Bovinos , Línea Celular , Cricetinae , Fiebre Aftosa/genética , Fiebre Aftosa/metabolismo , Virus de la Fiebre Aftosa/patogenicidad , Porcinos , Proteínas no Estructurales Virales/metabolismoRESUMEN
BACKGROUND: LncRNA taurine upregulated gene 1 (TUG1) was reported to act as a possible oncogene in osteosarcoma (OS) development. However, the underlying molecular basis of TUG1 involved in the progression of OS remains to be thoroughly investigated. METHODS: The expressions of TUG1 and miR-212-3p in OS tissues and cells were examined by RT-qPCR. Cell proliferation, apoptosis, caspase-3 activity, protein levels of BCL2, Bax, and forkhead box A1 (FOXA1) were detected by colony formation assay, MTT assay, flow cytometry analysis, caspase-3 activity assay, and western blot. Luciferase reporter assay, RNA immunoprecipitation (RIP) assay, and RT-qPCR were used to explore the interaction between TUG1, FOXA1 and miR-212-3p. Tumor xenograft mouse model was used to confirm the biological role of TUG in OS in vivo. RESULTS: Elevated TUG1 and FOXA1 expression and reduced miR-212-3p expression were observed in OS tissues and cells. TUG1 knockdown suppressed OS cell proliferation and promoted apoptosis. TUG1 functioned as a ceRNA of miR-212-3p and suppressed miR-212-3p expression. miR-212-3p inhibition reversed the effect of TUG1 knockdown on OS cell proliferation and apoptosis. In addition, FOXA1 was identified as a target of miR-212-3p and TUG1 functioned as a ceRNA to upregulate FOXA1 by sponging miR-212-3p in OS cells. FOXA1 up-regulation abolished the effects of miR-212-3p on OS cell proliferation and apoptosis. CONCLUSION: TUG1 promoted OS cell proliferation and suppressed apoptosis by regulating the miR-212-3p/FOXA1 axis. Therefore, TUG1/miR-212-3p/FOXA1 axis may be a promising therapeutic target in OS treatment.
Asunto(s)
Apoptosis/genética , Regulación Neoplásica de la Expresión Génica , Factor Nuclear 3-alfa del Hepatocito/genética , MicroARNs/genética , Osteosarcoma/genética , Osteosarcoma/patología , ARN Largo no Codificante/metabolismo , Transducción de Señal , Animales , Secuencia de Bases , Línea Celular Tumoral , Proliferación Celular/genética , Regulación hacia Abajo/genética , Femenino , Técnicas de Silenciamiento del Gen , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Humanos , Ratones Endogámicos BALB C , Ratones Desnudos , MicroARNs/metabolismo , ARN Largo no Codificante/genética , Regulación hacia Arriba/genéticaRESUMEN
Recent studies have shown that long non-coding RNAs (lncRNAs) have critical roles in tumorigenesis, including osteosarcoma. The lncRNA taurine-upregulated gene 1 (TUG1) was reported to be involved in the progression of osteosarcoma. Here, we investigated the role of TUG1 in osteosarcoma cells and the underlying mechanism. TUG1 expression was measured in osteosarcoma cell lines and human normal osteoblast cells by quantitative real-time PCR (qRT-PCR). The effects of TUG1 on osteosarcoma cells were studied by RNA interference in vitro and in vivo. The mechanism of competing endogenous RNA (ceRNA) was determined using bioinformatic analysis and luciferase assays. Our data showed that TUG1 knockdown inhibited cell proliferation and colony formation, and induced G0/G1 cell cycle arrest and apoptosis in vitro, and suppressed tumor growth in vivo. Besides, we found that TUG1 acted as an endogenous sponge to directly bind to miR-9-5p and downregulated miR-9-5p expression. Moreover, TUG1 overturned the effect of miR-9-5p on the proliferation, colony formation, cell cycle arrest, and apoptosis in osteosarcoma cells, which involved the derepression of POU class 2 homeobox 1 (POU2F1) expression. In conclusion, our study elucidated a novel TUG1/miR-9-5p/POU2F1 pathway, in which TUG1 acted as a ceRNA by sponging miR-9-5p, leading to downregulation of POU2F1 and facilitating the tumorigenesis of osteosarcoma. These findings may contribute to the lncRNA-targeted therapy for human osteosarcoma.
Asunto(s)
Biomarcadores de Tumor/análisis , Neoplasias Óseas/patología , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Factor 1 de Transcripción de Unión a Octámeros/metabolismo , Osteosarcoma/patología , ARN Largo no Codificante/genética , Apoptosis , Western Blotting , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Ciclo Celular , Proliferación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Citometría de Flujo , Humanos , Factor 1 de Transcripción de Unión a Octámeros/genética , Osteosarcoma/genética , Osteosarcoma/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Histone deacetylase inhibitors have been reported to induce tumor cell growth arrest, differentiation, and apoptosis. This study aimed to investigate the effects of one histone deacetylase inhibitor - sodium butyrate (SB) - on osteosarcoma (OS) cell proliferation and apoptosis and also the molecular mechanisms by which SB exerts regulatory effects on OS cells. U2OS and MG63 cells were treated with SB at various concentrations. Then, cell proliferation and apoptosis were determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide and flow cytometry assays, respectively; the expression of Ki67, Bax, Bcl-2, MDM2, and p53 proteins was determined by using Western blot assay. The results showed that SB suppressed proliferation in a concentration-dependent manner and promoted apoptosis of OS cells. In addition, SB enhanced p53 expression and decreased MDM2 expression, indicating that SB can regulate MDM2-p53 feedback loop. p53 inhibited proliferation and promoted apoptosis, whereas MDM2 promoted proliferation and suppressed apoptosis, which indicated that functional effect of SB on OS cell lines at least in part depended on the MDM2-p53 signaling. We also explored the effect of SB on OS cells in vivo and found that SB suppressed the growth of OS cells with no noticeable effect on activity and body weight of mice in vivo. These findings will offer new clues for OS development and progression and offer SB as a potent targeted agent for OS treatment.
RESUMEN
Foot-and-mouth disease virus (FMDV) is the causative agent of a highly contagious disease in livestock. The viral proteinase L(pro) of FMDV is involved in pathogenicity, and mutation of the L(pro) SAP domain reduces FMDV pathogenicity in pigs. To determine the gene expression profiles associated with decreased pathogenicity in porcine cells, we performed transcriptome analysis using next-generation sequencing technology and compared differentially expressed genes in SK6 cells infected with FMDV containing L(pro) with either a wild-type or mutated version of the SAP domain. This analysis yielded 1,853 genes that exhibited a ≥ 2-fold change in expression and was validated by real-time quantitative PCR detection of several differentially expressed genes. Many of the differentially expressed genes correlated with antiviral responses corresponded to genes associated with transcription factors, immune regulation, cytokine production, inflammatory response, and apoptosis. Alterations in gene expression profiles may be responsible for the variations in pathogenicity observed between the two FMDV variants. Our results provided genes of interest for the further study of antiviral pathways and pathogenic mechanisms related to FMDV L(pro).
Asunto(s)
Virus de la Fiebre Aftosa/fisiología , Fiebre Aftosa/genética , Serina Endopeptidasas/biosíntesis , Proteínas Virales/biosíntesis , Animales , Línea Celular , Fiebre Aftosa/virología , Virus de la Fiebre Aftosa/genética , Virus de la Fiebre Aftosa/patogenicidad , Perfilación de la Expresión Génica , Regulación Viral de la Expresión Génica , Genotipo , Interacciones Huésped-Patógeno , Mutación , Dominios y Motivos de Interacción de Proteínas , Reacción en Cadena en Tiempo Real de la Polimerasa , Serina Endopeptidasas/genética , Porcinos , Factores de Transcripción , Proteínas Virales/genética , Replicación Viral/genéticaRESUMEN
The efficacy of an inactivated foot-and-mouth disease (FMD) vaccine is mainly dependent on the integrity of the foot-and-mouth disease virus (FMDV) particles. At present, the standard method to quantify the active component, the 146S antigen, of FMD vaccines is sucrose density gradient (SDG) analysis. However, this method is highly operator dependent and difficult to automate. In contrast, the enzyme-linked immunosorbent assay (ELISA) is a time-saving technique that provides greater simplicity and sensitivity. To establish a valid method to detect and quantify the 146S antigen of a serotype O FMD vaccine, a double-antibody sandwich (DAS) ELISA was compared with an SDG analysis. The DAS ELISA was highly correlated with the SDG method (R2 = 0.9215, P<0.01). In contrast to the SDG method, the DAS ELISA was rapid, robust, repeatable and highly sensitive, with a minimum quantification limit of 0.06 µg/mL. This method can be used to determine the effective antigen yields in inactivated vaccines and thus represents an alternative for assessing the potency of FMD vaccines in vitro. But it still needs to be prospectively validated by analyzing a new vaccine preparation and determining the proper protective dose followed by an in vivo vaccination-challenge study to confirm the ELISA findings.