RESUMEN
The recognition of cytosolic nucleic acid triggers the DNA/RNA sensor-IRF3 axis-mediated production of type I interferons (IFNs), which are essential for antiviral immune responses. However, the inappropriate activation of these signaling pathways is implicated in autoimmune conditions. Here, we report that indomethacin, a widely used nonsteroidal anti-inflammatory drug, inhibits nucleic acid-triggered IFN production. We found that both DNA- and RNA-stimulated IFN expression can be effectively blocked by indomethacin. Interestingly, indomethacin also prohibits the nuclear translocation of IRF3 following cytosolic nucleic acid recognition. Importantly, in cell lines and a mouse model of Aicardi-Goutières syndrome, indomethacin administration blunts self-DNA-induced autoimmune responses. Thus, our study reveals a previously unknown function of indomethacin and provides a potential treatment for cytosolic nucleic acid-stimulated autoimmunity.
RESUMEN
BACKGROUND: Type 1 diabetes is believed to be an autoimmune condition, characterized by destruction of insulin-producing cells, due to the detrimental inflammation in pancreas. Growing evidences have indicated the important role of type I interferon in the development of type 1 diabetes. METHODS: Trex1-deficient rats were generated by using CRISPR-Cas9. The fasting blood glucose level of rat was measured by a Roche Accuchek blood glucose monitor. The levels of insulin, islet autoantibodies, and interferon-ß were measured using enzyme-linked immunosorbent assay. The inflammatory genes were detected by quantitative PCR and RNA-seq. Hematein-eosin staining was used to detect the pathological changes in pancreas, eye and kidney. The pathological features of kidney were also detected by Masson trichrome and periodic acid-Schiff staining. The distribution of islet cells, immune cells or ssDNA in pancreas was analyzed by immunofluorescent staining. RESULTS: In this study, we established a Trex1-deletion Sprague Dawley rat model, and unexpectedly, we found that the Trex1-/- rats spontaneously develop type 1 diabetes. Similar to human diabetes, the hyperglycemia in rats is accompanied by diabetic complications such as diabetic nephropathy and cataract. Mechanistical investigation revealed the accumulation of ssDNA and the excessive production of proinflammatory cytokines, including IFN-ß, in Trex1 null pancreas. These are likely contributing to the inflammation in pancreas and eventually leading to the decline of pancreatic ß cells. CONCLUSIONS: Our study links the DNA-induced chronic inflammation to the pathogenesis of type 1 diabetes, and also provides an animal model for type 1 diabetes studies.
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The suprachiasmatic nucleus (SCN) drives circadian clock coherence through intercellular coupling, which is resistant to environmental perturbations. We report that primary cilia are required for intercellular coupling among SCN neurons to maintain the robustness of the internal clock in mice. Cilia in neuromedin S-producing (NMS) neurons exhibit pronounced circadian rhythmicity in abundance and length. Genetic ablation of ciliogenesis in NMS neurons enabled a rapid phase shift of the internal clock under jet-lag conditions. The circadian rhythms of individual neurons in cilia-deficient SCN slices lost their coherence after external perturbations. Rhythmic cilia changes drive oscillations of Sonic Hedgehog (Shh) signaling and clock gene expression. Inactivation of Shh signaling in NMS neurons phenocopied the effects of cilia ablation. Thus, cilia-Shh signaling in the SCN aids intercellular coupling.
Asunto(s)
Cilios , Relojes Circadianos , Ritmo Circadiano , Proteínas Hedgehog , Neuronas del Núcleo Supraquiasmático , Animales , Ratones , Cilios/metabolismo , Cilios/fisiología , Relojes Circadianos/genética , Ritmo Circadiano/fisiología , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Neuronas del Núcleo Supraquiasmático/fisiología , Transducción de Señal , Regulación de la Expresión Génica , Ratones TransgénicosRESUMEN
Primary cilia transduce diverse signals in embryonic development and adult tissues. Defective ciliogenesis results in a series of human disorders collectively known as ciliopathies. The CP110-CEP97 complex removal from the mother centriole is an early critical step for ciliogenesis, but the underlying mechanism for this step remains largely obscure. Here, we reveal that the linear ubiquitin chain assembly complex (LUBAC) plays an essential role in ciliogenesis by targeting the CP110-CEP97 complex. LUBAC specifically generates linear ubiquitin chains on CP110, which is required for CP110 removal from the mother centriole in ciliogenesis. We further identify that a pre-mRNA splicing factor, PRPF8, at the distal end of the mother centriole acts as the receptor of the linear ubiquitin chains to facilitate CP110 removal at the initial stage of ciliogenesis. Thus, our study reveals a direct mechanism of regulating CP110 removal in ciliogenesis and implicates the E3 ligase LUBAC as a potential therapy target of cilia-associated diseases, including ciliopathies and cancers.
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Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Cilios/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Organogénesis , Fosfoproteínas/metabolismo , Ubiquitina/metabolismo , Animales , Línea Celular , Humanos , Ratones , Complejos Multiproteicos , Proteínas de Unión al ARN/metabolismo , Especificidad por Sustrato , Ubiquitinación , Pez CebraRESUMEN
Cyclic GMP-AMP synthase (cGAS) functions as a key sensor for microbial invasion and cellular damage by detecting emerging cytosolic DNA. Here, we report that GTPase-activating protein-(SH3 domain)-binding protein 1 (G3BP1) primes cGAS for its prompt activation by engaging cGAS in a primary liquid-phase condensation state. Using high-resolution microscopy, we show that in resting cells, cGAS exhibits particle-like morphological characteristics, which are markedly weakened when G3BP1 is deleted. Upon DNA challenge, the pre-condensed cGAS undergoes liquid-liquid phase separation (LLPS) more efficiently. Importantly, G3BP1 deficiency or its inhibition dramatically diminishes DNA-induced LLPS and the subsequent activation of cGAS. Interestingly, RNA, previously reported to form condensates with cGAS, does not activate cGAS. Accordingly, we find that DNA - but not RNA - treatment leads to the dissociation of G3BP1 from cGAS. Taken together, our study shows that the primary condensation state of cGAS is critical for its rapid response to DNA.
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ADN Helicasas , Nucleotidiltransferasas , Proteínas de Unión a Poli-ADP-Ribosa , ARN Helicasas , Proteínas con Motivos de Reconocimiento de ARN , ADN/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Nucleotidiltransferasas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Helicasas/genética , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Gránulos de EstrésRESUMEN
The detection of intracellular nucleic acids is a fundamental mechanism of host defense against infections. The dysregulated nucleic acid sensing, however, is a major cause for a number of autoimmune diseases. In this study, we report that GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) is critical for both intracellular DNA- and RNA-induced immune responses. We found that in both human and mouse cells, the deletion of G3BP1 led to the dampened cGAS activation by DNA and the insufficient binding of RNA by RIG-I. We further found that resveratrol (RSVL), a natural compound found in grape skin, suppressed both intracellular DNA- and RNA-induced type I IFN production through inhibiting G3BP1. Importantly, using experimental mouse models for Aicardi-Goutières syndrome, an autoimmune disorder found in humans, we demonstrated that RSVL effectively alleviated intracellular nucleic acid-stimulated autoimmune responses. Thus, our study demonstrated a broader role of G3BP1 in sensing different kinds of intracellular nucleic acids and presented RSVL as a potential treatment for autoimmune conditions caused by dysregulated nucleic acid sensing.
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Autoinmunidad/genética , ADN Helicasas/deficiencia , ADN Helicasas/metabolismo , Espacio Intracelular/metabolismo , Ácidos Nucleicos/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/deficiencia , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Helicasas/deficiencia , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/deficiencia , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Transducción de Señal/genética , Células A549 , Animales , Autoinmunidad/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , ADN Helicasas/antagonistas & inhibidores , ADN Helicasas/genética , Fibroblastos/metabolismo , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Espacio Intracelular/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Unión a Poli-ADP-Ribosa/antagonistas & inhibidores , Proteínas de Unión a Poli-ADP-Ribosa/genética , ARN Helicasas/antagonistas & inhibidores , ARN Helicasas/genética , Proteínas con Motivos de Reconocimiento de ARN/antagonistas & inhibidores , Proteínas con Motivos de Reconocimiento de ARN/genética , Resveratrol/administración & dosificación , Transducción de Señal/inmunología , TransfecciónRESUMEN
Lack of detailed knowledge of SARS-CoV-2 infection has been hampering the development of treatments for coronavirus disease 2019 (COVID-19). Here, we report that RNA triggers the liquid-liquid phase separation (LLPS) of the SARS-CoV-2 nucleocapsid protein, N. By analyzing all 29 proteins of SARS-CoV-2, we find that only N is predicted as an LLPS protein. We further confirm the LLPS of N during SARS-CoV-2 infection. Among the 100,849 genome variants of SARS-CoV-2 in the GISAID database, we identify that ~37% (36,941) of the genomes contain a specific trio-nucleotide polymorphism (GGG-to-AAC) in the coding sequence of N, which leads to the amino acid substitutions, R203K/G204R. Interestingly, NR203K/G204R exhibits a higher propensity to undergo LLPS and a greater effect on IFN inhibition. By screening the chemicals known to interfere with N-RNA binding in other viruses, we find that (-)-gallocatechin gallate (GCG), a polyphenol from green tea, disrupts the LLPS of N and inhibits SARS-CoV-2 replication. Thus, our study reveals that targeting N-RNA condensation with GCG could be a potential treatment for COVID-19.
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Sustitución de Aminoácidos/efectos de los fármacos , COVID-19/prevención & control , Catequina/análogos & derivados , Proteínas de la Nucleocápside/genética , SARS-CoV-2/efectos de los fármacos , Replicación Viral/efectos de los fármacos , COVID-19/virología , Catequina/farmacología , Genoma Viral/genética , Humanos , Extracción Líquido-Líquido , Proteínas de la Nucleocápside/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , SARS-CoV-2/genética , Replicación Viral/genéticaRESUMEN
Dynamic assembly and disassembly of primary cilia controls embryonic development and tissue homeostasis. Dysregulation of ciliogenesis causes human developmental diseases termed ciliopathies. Cell-intrinsic regulatory mechanisms of cilia disassembly have been well-studied. The extracellular cues controlling cilia disassembly remain elusive, however. Here, we show that lysophosphatidic acid (LPA), a multifunctional bioactive phospholipid, acts as a physiological extracellular factor to initiate cilia disassembly and promote neurogenesis. Through systematic analysis of serum components, we identify a small molecular-LPA as the major driver of cilia disassembly. Genetic inactivation and pharmacological inhibition of LPA receptor 1 (LPAR1) abrogate cilia disassembly triggered by serum. The LPA-LPAR-G-protein pathway promotes the transcription and phosphorylation of cilia disassembly factors-Aurora A, through activating the transcription coactivators YAP/TAZ and calcium/CaM pathway, respectively. Deletion of Lpar1 in mice causes abnormally elongated cilia and decreased proliferation in neural progenitor cells, thereby resulting in defective neurogenesis. Collectively, our findings establish LPA as a physiological initiator of cilia disassembly and suggest targeting the metabolism of LPA and the LPA pathway as potential therapies for diseases with dysfunctional ciliogenesis.
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Cilios/efectos de los fármacos , Lisofosfolípidos/farmacología , Neurogénesis/efectos de los fármacos , Epitelio Pigmentado de la Retina/efectos de los fármacos , Transducción de Señal , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Cilios/genética , Cilios/metabolismo , Células HEK293 , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Humanos , Lisofosfolípidos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Células-Madre Neurales/citología , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Neurogénesis/genética , Unión Proteica , Interferencia de ARN , Receptores del Ácido Lisofosfatídico/genética , Receptores del Ácido Lisofosfatídico/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismoRESUMEN
Primary cilia protrude from the cell surface and have diverse roles during development and disease, which depends on the precise timing and control of cilia assembly and disassembly. Inactivation of assembly often causes cilia defects and underlies ciliopathy, while diseases caused by dysfunction in disassembly remain largely unknown. Here, we demonstrate that CEP55 functions as a cilia disassembly regulator to participate in ciliopathy. Cep55-/- mice display clinical manifestations of Meckel-Gruber syndrome, including perinatal death, polycystic kidneys, and abnormalities in the CNS. Interestingly, Cep55-/- mice exhibit an abnormal elongation of cilia on these tissues. Mechanistically, CEP55 promotes cilia disassembly by interacting with and stabilizing Aurora A kinase, which is achieved through facilitating the chaperonin CCT complex to Aurora A. In addition, CEP55 mutation in Meckel-Gruber syndrome causes the failure of cilia disassembly. Thus, our study establishes a cilia disassembly role for CEP55 in vivo, coupling defects in cilia disassembly to ciliopathy and further suggesting that proper cilia dynamics are critical for mammalian development.
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Aurora Quinasa A/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cilios/metabolismo , Animales , Puntos de Control del Ciclo Celular , Proteínas de Ciclo Celular/deficiencia , Células Cultivadas , Centrosoma/metabolismo , Centrosoma/ultraestructura , Chaperonina con TCP-1/metabolismo , Cilios/ultraestructura , Trastornos de la Motilidad Ciliar/patología , Encefalocele/patología , Estabilidad de Enzimas , Marcación de Gen , Células HEK293 , Humanos , Ratones , Mitosis , Fenotipo , Enfermedades Renales Poliquísticas/patología , Unión Proteica , Retinitis Pigmentosa/patología , Receptor Smoothened/metabolismoRESUMEN
The presence of DNA in the cytoplasm is normally a sign of microbial infections and is quickly detected by cyclic GMP-AMP synthase (cGAS) to elicit anti-infection immune responses. However, chronic activation of cGAS by self-DNA leads to severe autoimmune diseases for which no effective treatment is available yet. Here we report that acetylation inhibits cGAS activation and that the enforced acetylation of cGAS by aspirin robustly suppresses self-DNA-induced autoimmunity. We find that cGAS acetylation on either Lys384, Lys394, or Lys414 contributes to keeping cGAS inactive. cGAS is deacetylated in response to DNA challenges. Importantly, we show that aspirin can directly acetylate cGAS and efficiently inhibit cGAS-mediated immune responses. Finally, we demonstrate that aspirin can effectively suppress self-DNA-induced autoimmunity in Aicardi-Goutières syndrome (AGS) patient cells and in an AGS mouse model. Thus, our study reveals that acetylation contributes to cGAS activity regulation and provides a potential therapy for treating DNA-mediated autoimmune diseases.
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ADN/inmunología , Nucleotidiltransferasas/metabolismo , Autotolerancia/inmunología , Acetilación , Secuencia de Aminoácidos , Animales , Aspirina/farmacología , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/metabolismo , Enfermedades Autoinmunes del Sistema Nervioso/genética , Enfermedades Autoinmunes del Sistema Nervioso/inmunología , Enfermedades Autoinmunes del Sistema Nervioso/metabolismo , Autoinmunidad , Línea Celular , ADN/genética , ADN/metabolismo , Modelos Animales de Enfermedad , Exodesoxirribonucleasas/metabolismo , Células HEK293 , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Mutación , Malformaciones del Sistema Nervioso/genética , Malformaciones del Sistema Nervioso/inmunología , Malformaciones del Sistema Nervioso/metabolismo , Nucleotidiltransferasas/antagonistas & inhibidores , Nucleotidiltransferasas/química , Nucleotidiltransferasas/genética , Células THP-1RESUMEN
Defective ciliogenesis causes human developmental diseases termed ciliopathies. Microtubule (MT) asters originating from centrosomes in mitosis ensure the fidelity of cell division by positioning the spindle apparatus. However, the function of microtubule asters in interphase remains largely unknown. Here, we reveal an essential role of MT asters in transition zone (TZ) assembly during ciliogenesis. We demonstrate that the centrosome protein FSD1, whose biological function is largely unknown, anchors MT asters to interphase centrosomes by binding to microtubules. FSD1 knockdown causes defective ciliogenesis and affects embryonic development in vertebrates. We further show that disruption of MT aster anchorage by depleting FSD1 or other known anchoring proteins delocalizes the TZ assembly factor Cep290 from centriolar satellites, and causes TZ assembly defects. Thus, our study establishes FSD1 as a MT aster anchorage protein and reveals an important function of MT asters anchored by FSD1 in TZ assembly during ciliogenesis.
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Axonema/metabolismo , Cilios/metabolismo , Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Axonema/genética , Centrosoma/metabolismo , Cilios/genética , Humanos , Mitosis , Proteínas del Tejido Nervioso/genética , Huso Acromático/genética , Huso Acromático/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Many infections and stress signals can rapidly activate the NLRP3 inflammasome to elicit robust inflammatory responses. This activation requires a priming step, which is thought to be mainly for upregulating NLRP3 transcription. However, recent studies report that the NLRP3 inflammasome can be activated independently of transcription, suggesting that the priming process has unknown essential regulatory steps. Here, we report that JNK1-mediated NLRP3 phosphorylation at S194 is a critical priming event and is essential for NLRP3 inflammasome activation. We show that NLRP3 inflammasome activation is disrupted in NLRP3-S194A knockin mice. JNK1-mediated NLRP3 S194 phosphorylation is critical for NLRP3 deubiquitination and facilitates its self-association and the subsequent inflammasome assembly. Importantly, we demonstrate that blocking S194 phosphorylation prevents NLRP3 inflammasome activation in cryopyrin-associated periodic syndromes (CAPS). Thus, our study reveals a key priming molecular event that is a prerequisite for NLRP3 inflammasome activation. Inhibiting NLRP3 phosphorylation could be an effective treatment for NLRP3-related diseases.
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Inflamasomas/genética , Macrófagos/inmunología , Proteína Quinasa 8 Activada por Mitógenos/genética , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Choque Séptico/genética , Secuencia de Aminoácidos , Animales , Enzimas Desubicuitinizantes/genética , Enzimas Desubicuitinizantes/inmunología , Escherichia coli/química , Femenino , Regulación de la Expresión Génica , Células HEK293 , Humanos , Inflamasomas/inmunología , Lipopolisacáridos/farmacología , Macrófagos/patología , Masculino , Ratones , Ratones Transgénicos , Proteína Quinasa 8 Activada por Mitógenos/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/deficiencia , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Fosforilación , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Choque Séptico/inducido químicamente , Choque Séptico/mortalidad , Choque Séptico/patología , Transducción de Señal , Análisis de SupervivenciaRESUMEN
Embryonic stem (ES) cells are pluripotent cells that are capable of giving rise to any type of cells in the body and possess unlimited self-renewal potential. However, the exact regulatory mechanisms that govern the self-renewal ability of ES cells remain elusive. To understand the immediate early events during ES cell differentiation, we performed a proteomics study and analyzed the proteomic difference in murine ES cells before and after a 6-h spontaneous differentiation. We found that the expression level of glutathione peroxidase-1 (GPx-1), an antioxidant enzyme, is dramatically decreased upon the differentiation. Both knockdown of GPx-1 expression with shRNA and inhibiting GPx-1 activity by inhibitor led to the differentiation of ES cells. Furthermore, we showed that during early differentiation, the quick degradation of GPx-1 was mediated by proteasome. Thus, our data indicated that GPx-1 is a key regulator of self-renewal of murine embryonic stem cells.
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Células Madre Embrionarias/citología , Células Madre Embrionarias/enzimología , Glutatión Peroxidasa/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Línea Celular , Proliferación Celular/efectos de los fármacos , Células Madre Embrionarias/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Técnicas de Silenciamiento del Gen , Glutatión Peroxidasa/antagonistas & inhibidores , Glutatión Peroxidasa/genética , Leupeptinas/farmacología , Ratones , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/enzimología , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , ARN Interferente Pequeño/genética , Tiomalatos/farmacología , Glutatión Peroxidasa GPX1RESUMEN
Distribution of macroalgal community was investigated monthly in the intertidal zone of Yangma Island, Yantai, Shandong Province, China during April 2010 to March 2011. Macroalgae sampling was conducted at two sites (A and B) along Yangma Island coastline. The relationships between macroalgae species composition and biomass and environment variables were studied. In total, 35 macroalgae species were identified, including 24 Rhodophyta (68.6% of the total number), 6 Chlorophyta (17.1% of the total number) and 5 Phaeophyta (14.3% of the total number). Brown algae and green algae dominated in summer, and red algae and brown algae dominated in other seasons. Sargassum thunbergii was the dominant species all the year. The biomass of macroalgae was higher in summer and lower in winter. The highest biomass of macroalgae was observed in June. The lowest biomass of macroalgae was observed in January at A sampling site and in November at B sampling site with the characters of macroalgae in the temperate seas. Seawater temperature, nutrients and pH could have significant effects on the variations of macroalgae biomass in the Yangma Island intertidal zone.
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Ambiente , Algas Marinas/crecimiento & desarrollo , Biomasa , China , Chlorophyta/crecimiento & desarrollo , Islas , Rhodophyta/crecimiento & desarrollo , Estaciones del Año , Agua de MarRESUMEN
An optical monitoring method is proposed for the rapid, non destructive measurements of chlorophyll concentration (Chl-a) in the surface sediments of emerged tidal flat, and it can be further applied in remote sensing work. Hyperspectral reflectance of intertidal sediments were measured in day time at the tidal flats of the Sishili Bay, the Northern Yellow Sea, and surface sediments (3 mm) were sampled for the in-door measurements of Chl-a. On the basis of the reflectance at 650, 675 and 700 nm, the indices of normalized difference index of microbenthos (NDI-MPB) and trough depth (T-depth) were proposed for the measurements of microphytobenthos biomass. T-depth can be used to remove the linear background spectral noises and indicate the existence of microphytobenthos; Good linear relationship was observed between NDI-MPB and Chl-a content in sediments (2.22-49.36 mg x m(-2), r > 0.99), which may be used to monitor the biomass of microphy to benthos.
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Clorofila/análisis , Sedimentos Geológicos/microbiología , Fitoplancton/metabolismo , Agua de Mar/microbiología , Análisis Espectral , Océanos y Mares , Hojas de la Planta/química , Hojas de la Planta/crecimiento & desarrollo , Tecnología de Sensores Remotos/métodos , Dispersión de RadiaciónRESUMEN
OBJECTIVE: To establish Gankyrin knocking down 4T1-luc cell model and detect the effects of Gankyrin expression on breast cancer metastasis. METHODS: 4T1-luc cells carrying shGankyrin construct were established by lentivirus infection and antibiotic screening. Western blotting and real-time PCR were used to check the expression levels of Gankyrin. In vivo imaging system was used to monitor the effects of Gankyrin knocked down on cell growth and tumor metastasis after the in situ implantation of Gankyrin knocking down 4T1-luc cells in BALB/c mice. RESULTS: The cell expression decreased at the protein and mRNA levels. Gankyrin mRNA expression in different shGankyrin 4T1-luc cells was respectively 4.9%, 25.1% and 69.8% versus the control cells. ShGankyrin#2 4T1-luc cells were chosen for in situ implantation into BAL/c mice because luminescent intensity was consistent with cell numbers. The photon flux of lung metastatic tumor induced by Gankyrin knocking down 4T1-luc cell was 3.02 × 10(6), while that of lung metastasis induced by control cells was 10.9 × 10(6). The differences between two groups were significant. In pathology, Gankyrin was detected positive in lung metastasis tumors induced by control group. However, Gankyrin was negative in the Gankyrin knockdown group. CONCLUSIONS: Lentivirus infection may be effectively used to establish Gankyrin knocking down 4T1-luc cell model. Because of its involvement in the in vivo pulmonary metastasis of breast cancers, Gankyrin should be a novel target for tumor therapy.
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Neoplasias Mamarias Experimentales/genética , Complejo de la Endopetidasa Proteasomal/genética , Proteínas Proto-Oncogénicas/genética , Animales , Línea Celular , Línea Celular Tumoral , Femenino , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Ratones Endogámicos BALB C , Metástasis de la NeoplasiaRESUMEN
In this study, genomic sequences of pestiviruses available in GenBank were aligned to design three primer pairs and TaqMan probes: two targeting the NS5A region of the viral genome of classical swine fever virus (CSFV) for the differentiation of wild-type CSFV and hog cholera lapinized vaccine (HCLV) vaccine, and one targeting the 5'-untranslated region of bovine viral diarrhea virus 1 (BVDV-1). With these primers and probes, a triplex TaqMan real-time RT-PCR assay was developed for differentiating wild-type CSFV, the HCLV strain, and BVDV-1. The detection limit of the assay was 4.5 TCID(50) for wild-type CSFV, 10 TCID(50) for HCLV-strain CSFV, and 3.2 TCID(50) for BVDV-1. The triplex real-time RT-PCR had at least 98% (248 samples) agreement with other RT-PCR methods. The assay provides a sensitive tool for simultaneous detection and differentiation of wild-type CSFV and HCLV from BVDV-1.
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Virus de la Fiebre Porcina Clásica/clasificación , Virus de la Diarrea Viral Bovina Tipo 1/clasificación , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Animales , Secuencia de Bases , Línea Celular , Virus de la Fiebre Porcina Clásica/genética , Virus de la Fiebre Porcina Clásica/aislamiento & purificación , ADN Complementario/genética , ADN Viral/genética , Virus de la Diarrea Viral Bovina Tipo 1/genética , Virus de la Diarrea Viral Bovina Tipo 1/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Sensibilidad y Especificidad , Vacunas ViralesRESUMEN
Low efficacy of gene-based vaccines due to inefficient gene delivery and expression has been major bottleneck of their applications. Efforts have been made to improve the efficacy, such as gene gun and electroporation, but the strategies are difficult to put into practical use. In this study, we developed and evaluated an adenovirus-delivered, alphavirus replicon-vectored vaccine (chimeric vector-based vaccine) expressing the E2 gene of classical swine fever virus (CSFV) (rAdV-SFV-E2). Rabbits immunized with rAdV-SFV-E2 developed CSFV-specific antibodies as early as 9 days and as long as 189 days and completely protected from challenge with C-strain. Pigs immunized with rAdV-SFV-E2 (n=5) developed robust humoral and cell-mediated responses to CSFV and were completely protected from subsequent lethal CSFV infection clinically and virologically. The level of immunity and protection induced by rAdV-SFV-E2 was comparable to that provided by the currently used live attenuated vaccine, C-strain. In contrast, both the conventional alphavirus replicon-vectored vaccine pSFV1CS-E2 and conventional adenovirus-vectored vaccine rAdV-E2 provided incomplete protection. The chimeric vector-based vaccine represents the first gene-based vaccine that is able to confer sterile immunity and complete protection against CSFV. The new-concept vaccination strategy may also be valuable in vaccine development against other pathogens.
Asunto(s)
Adenoviridae/genética , Alphavirus/genética , Virus de la Fiebre Porcina Clásica/inmunología , Peste Porcina Clásica/prevención & control , Vectores Genéticos , Vacunas de ADN/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Antivirales/sangre , Peste Porcina Clásica/inmunología , Peste Porcina Clásica/mortalidad , Peste Porcina Clásica/patología , Virus de la Fiebre Porcina Clásica/genética , Inmunidad Celular , Conejos , Análisis de Supervivencia , Porcinos , Vacunación/métodos , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genética , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/genéticaRESUMEN
BACKGROUND: Currently, larger domestic pigs are only animals widely used in vaccine evaluation and pathogenicity study of classical swine fever virus (CSFV). This study was aimed to create an alternative animal experimental infection model of CSFV. RESULTS: Twenty specific-pathogen-free Bama miniature pigs were randomly divided into two groups and rooms, infected and non-infected, and the pigs in the infected group were inoculated intramuscularly with 104, 105 or 106 TCID50 (median tissue culture infective dose) CSFV Shimen strain (n = 5 × 3) or left uninoculated to serve as in-contact pigs (n = 3). The uninfected control pigs (n = 2) were housed in a separate room. Clinical signs, body temperature, viraemia, tissue antigen distribution, pathological changes and seroconversion were monitored. Clinical signs were observed as early as 2 days post-inoculation (dpi) in all infected pigs (though mild in contact pigs), but not non-infected control pigs. All inoculated pigs showed viraemia by 6 dpi. The in-contact pigs showed lower levels of viraemia. At 10 dpi, seroconversion was noted in five of the 15 inoculated pigs. All inoculated or one in-contact pigs died by 15 dpi. CONCLUSIONS: These results show that Bama miniature pigs support productive CSFV infection and display clinical signs and pathological changes consistent with CSFV infections observed in larger domestic pigs.
Asunto(s)
Anticuerpos Antivirales/análisis , Virus de la Fiebre Porcina Clásica/patogenicidad , Peste Porcina Clásica/virología , Viremia/virología , Animales , Anticuerpos Antivirales/inmunología , Línea Celular , Peste Porcina Clásica/inmunología , Peste Porcina Clásica/mortalidad , Peste Porcina Clásica/patología , Peste Porcina Clásica/fisiopatología , Virus de la Fiebre Porcina Clásica/fisiología , Ensayo de Inmunoadsorción Enzimática , Inyecciones Intramusculares , Modelos Animales , Reacción en Cadena en Tiempo Real de la Polimerasa , Tasa de Supervivencia , Porcinos , Porcinos Enanos , Vacunas Virales/inmunología , Viremia/inmunología , Viremia/mortalidad , Viremia/patología , Viremia/fisiopatologíaRESUMEN
A loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for the rapid and specific detection of HCLV vaccine strain against classical swine fever. Four primers were designed for amplification of NS5B gene region with Bst DNA polymerase at a constant temperature of 65°C. The products showed ladder-like pattern on 2% agarose gel, and can be visualised after addition of SYBR Green I dye. The detection limit of the assay was 5 copies of the HCLV genome per reaction. No cross-reaction with other porcine viruses including different wild-type CSFV strains and the bovine viral diarrhoea virus was observed. The agreement between the LAMP and TaqMan real-time RT-PCR assays was 94.4% for the detection of 72 batches of HCLV vaccine. The assay provides a rapid tool for the control of vaccine quality and can be an accompanying assay of the LAMP for wild-type CSFV described previously for differential diagnosis.