RESUMEN
African swine fever (ASF) is one of the most severe infectious diseases of pigs. In this study, a loop-mediated isothermal amplification (LAMP) assay coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system was established in one tube for the detection of the African swine fever virus (ASFV) p72 gene. The single-stranded DNA-fluorophore quencher reporter and CRISPR-derived RNA were screened and selected for the CRISPR detection system. In combination with LAMP amplification assay, the detection limit for the LAMP-CRISPR assay can reach 7 copies/µl of p72 gene per reaction. Furthermore, this method displays no cross-reactivity with other porcine DNA or RNA viruses. The performance of the LAMP-CRISPR assay was compared with real-time qPCR tests for clinical samples; a good consistency between the LAMP-CRISPR assay and real-time qPCR was observed. The method shed a light on the convenient, portable, low cost, highly sensitive and specific detection of ASFV, demonstrating a great application potential for monitoring on-site ASFV in the field.
Asunto(s)
Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Enfermedades de los Porcinos , Fiebre Porcina Africana/diagnóstico , Virus de la Fiebre Porcina Africana/genética , Animales , Sistemas CRISPR-Cas , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico/métodos , Técnicas de Amplificación de Ácido Nucleico/veterinaria , Sensibilidad y Especificidad , Porcinos , Enfermedades de los Porcinos/genéticaRESUMEN
Hepatitis B virus (HBV) infection is a leading cause of liver cirrhosis, liver cancer, and liver failure, affecting 350 million people worldwide. Currently available anti-HBV drugs include (PEGylated-) interferon-α and nucleos(t)ide analogs, which can cause significant side effects and drug-resistance in many cases of long-term treatment. The lack of a reliable and robust in vitro infection system is a major barrier for understanding the HBV life cycle and discovering novel therapeutic targets. In the present study, we demonstrate that overexpression of the hepatitis B surface antigen binding protein (SBP) in HepG2 cells (HepG2-SBP) resulted in their susceptibility to HBV infection. HepG2-SBP cells supported the uptake of the viral surface protein (HBsAg-preS), HBV-pseudotyped virus, and live HBV in patient sera. Moreover, SBP-mediated HBsAg-preS uptake, and HBV pseudotyped virus infections were efficiently blocked by preS1- and SBP-specific antibodies. These observations suggest that SBP is involved in HBV entry and that HepG2-SBP cells can serve as a cellular model to study the post-binding steps of HBV infection.
Asunto(s)
Proteínas Portadoras/metabolismo , Antígenos de Superficie de la Hepatitis B/metabolismo , Virus de la Hepatitis B/fisiología , Receptores Virales/metabolismo , Internalización del Virus , Células Hep G2 , HumanosRESUMEN
HER2 is an orphan receptor tyrosine kinase of the EGFR families and is considered to be a key tumor driver gene [1]. Breast cancer and gastric cancer with HER2 amplification can be effectively treated by its neutralizing antibody, Herceptin. In clinic, Immunohistochemistry (IHC) was used as the primary screening method to diagnose HER2 amplification [2]. However, recent evidence suggested that the frequently used rabbit HER2 antibody 4B5 cross reacted with another family member HER4 [3]. IHC staining with 4B5 also indicated that there was strong non-specific cytoplasmic and nuclear signals in normal gastric mucosal cells and some gastric cancer samples. Using a protein lysate array which covers 85% of the human proteome, we have confirmed that the 4B5 bound to HER4 and a nuclear protein ZSCAN18 besides HER2. The non-specific binding accounts for the unexpected cytoplasmic and nuclear staining of 4B5 of normal gastric epithelium. Finally, we have developed a novel mouse HER2 monoclonal antibody UMAB36 with similar sensitivity to 4B5 but only reacted to HER2 across the 17,000 proteins on the protein chip. In 129 breast cancer and 158 gastric cancer samples, UMAB36 showed 100% sensitivity and specificity comparing to the HER2 FISH reference results with no unspecific staining in the gastric mucosa layer. Therefore, UMAB36 could provide as an alternative highly specific IHC reagent for testing HER2 amplification in gastric cancer populations.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Receptor ErbB-2/inmunología , Especificidad de Anticuerpos , Línea Celular Tumoral , Reacciones Cruzadas , Humanos , Inmunohistoquímica , Análisis por Matrices de Proteínas , Neoplasias Gástricas/inmunologíaRESUMEN
The reprogramming factors that induce pluripotency have been identified primarily from embryonic stem cell (ESC)-enriched, pluripotency-associated factors. Here, we report that, during mouse somatic cell reprogramming, pluripotency can be induced with lineage specifiers that are pluripotency rivals to suppress ESC identity, most of which are not enriched in ESCs. We found that OCT4 and SOX2, the core regulators of pluripotency, can be replaced by lineage specifiers that are involved in mesendodermal (ME) specification and in ectodermal (ECT) specification, respectively. OCT4 and its substitutes attenuated the elevated expression of a group of ECT genes, whereas SOX2 and its substitutes curtailed a group of ME genes during reprogramming. Surprisingly, the two counteracting lineage specifiers can synergistically induce pluripotency in the absence of both OCT4 and SOX2. Our study suggests a "seesaw model" in which a balance that is established using pluripotency factors and/or counteracting lineage specifiers can facilitate reprogramming.
Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Factores de Transcripción/metabolismo , Animales , Células Madre Embrionarias/metabolismo , Fibroblastos/metabolismo , Factor de Transcripción GATA3/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Ratones , Modelos Biológicos , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Estómago/citologíaRESUMEN
BACKGROUND: An antibody with cross-reactivity can create unexpected side effects or false diagnostic reports if used for clinical purposes. ERCC1 is being explored as a predictive diagnostic biomarker for cisplatin-based chemotherapy. High ERCC1 expression is linked to drug resistance on cisplatin-based chemotherapy. 8F1 is one of the most commonly used monoclonal antibodies for evaluating ERCC1 expression levels in lung cancer patient tissues, but it has been noted that this antibody cross-reacts with an unknown protein. RESULTS: By using a high density protein microarray chip technology, we discovered that 8F1 not only reacts with its authentic target, ERCC1, but also cross-reacts with an unrelated nuclear membrane protein, PCYT1A. The cross-reactivity is due to a common epitope presented on these two unrelated proteins. Similar to the subcellular localization of ERCC1, IHC tests demonstrated that PCYT1A is localized mainly on nuclear membrane. In this study, we also discovered that the PCYT1A gene expression level is significantly higher than the ERCC1 gene expression level in a certain population of lung cancer patient tissue samples. To develop the best monoclonal antibody for ERCC1 IHC analysis, 18 monoclonal antibodies were generated and 6 of them were screened against our protein microarray chip. Two clones showed high mono-specificity on the protein microarray chip test and both worked for the IHC application. CONCLUSION: In summary, the 8F1 clone is not suitable for ERCC1 IHC assay due to its cross-reactivity with PCYT1A protein. Two newly generated monoclonal antibodies, 4F9 and 2E12, demonstrated ultra-specificity against ERCC1 protein and superior performance for IHC analyses.
Asunto(s)
Anticuerpos Monoclonales/química , Biomarcadores de Tumor/inmunología , Proteínas de Unión al ADN/inmunología , Endonucleasas/inmunología , Análisis por Matrices de Proteínas/métodos , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Biomarcadores de Tumor/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/química , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Citidililtransferasa de Colina-Fosfato/inmunología , Citidililtransferasa de Colina-Fosfato/metabolismo , Reacciones Cruzadas , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Células HEK293 , Humanos , Inmunohistoquímica/métodos , Neoplasias Pulmonares/química , Neoplasias Pulmonares/metabolismoRESUMEN
As a critical apoptosis executioner, caspase-3 becomes activated and then enters into the nucleus to exert its function. However, the molecular mechanism of this nuclear entry of active caspase-3 is still unknown. In this study, we revealed that caspase-3 harbors a crm-1-independent nuclear export signal (NES) in its small subunit. Using reverse-caspase-3 as the study model, we found that the function of the NES in caspase-3 was not disturbed by the conformational changes during induced caspase-3 activation. Mutations disrupting the cleavage activity or p3-recognition site resulted in a defect in the nuclear entry of active caspase-3. We provide evidence that the p3-mediated specific cleavage activity of active caspase-3 abrogated the function of the NES. In conclusion, our results demonstrate that during caspase-3 activation, NES is constitutively present. p3-mediated specific cleavage activity abrogates the NES function in caspase-3, thus facilitating the nuclear entry of active caspase-3.
Asunto(s)
Caspasa 3/metabolismo , Núcleo Celular/metabolismo , Señales de Exportación Nuclear/fisiología , Transporte Activo de Núcleo Celular/fisiología , Secuencia de Aminoácidos , Caspasa 3/química , Caspasa 3/genética , Dominio Catalítico/genética , Análisis Mutacional de ADN , Activación Enzimática/genética , Células HeLa , Humanos , Carioferinas/metabolismo , Modelos Biológicos , Señales de Exportación Nuclear/genética , Unión Proteica , Conformación Proteica , Procesamiento Proteico-Postraduccional/fisiología , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteína Exportina 1Asunto(s)
Diferenciación Celular/genética , Reprogramación Celular/genética , Fibroblastos/metabolismo , Redes Reguladoras de Genes/genética , Células Madre Pluripotentes/fisiología , Adulto , Técnicas de Cultivo de Célula , Ensayo de Unidades Formadoras de Colonias , Fibroblastos/citología , Prepucio/citología , Humanos , Masculino , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Células Madre Pluripotentes/citología , ARN Interferente Pequeño , Transactivadores/genética , Transactivadores/metabolismo , Transfección , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Mesenchymal stem cells (MSCs) have already been proved to be multipotent. Our goal was to evaluate the differentiating ability of rat MSCs into insulin-secreting cells in vitro to cure diabetes resulting from abnormal function of pancreatic islets. MSCs were identified by Fluorescence-activated cell sorting (FACS). Pdx1 is a transcription factor involved in the early endocrine development. Betacellulin (BTC) is a growth factor involved in beta-cell maturation. MSCs were transfected with plasmids carrying rat Pdx1 and BTC genes. Coexpression of Pdx1 and BTC significantly increased the number of nestin-positive epithelium-like progenitors and islet-like spheroids which differentiated from MSCs. In Pdx1- and BTC-expressed (Pdx1+ + BTC+) MSCs, insulin and Glut-2 mRNA levels significantly rose. The number of islet-like cells was also evidently augmented. In response to glucose, Pdx1+ + BTC+ MSCs released insulin and C-peptide. It is concluded that genetic manipulation of transcription factor Pdx1 and growth factor BTC in combination with appropriate differentiating culture could induce MSCs into the pancreatic lineage in vitro and produce islet-like spheroids that could secrete increased levels of insulin in response to glucose.
Asunto(s)
Diferenciación Celular/fisiología , Proteínas de Homeodominio/biosíntesis , Células Secretoras de Insulina/metabolismo , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Proteínas de Filamentos Intermediarios/biosíntesis , Células Madre Mesenquimatosas/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Esferoides Celulares/metabolismo , Transactivadores/biosíntesis , Animales , Betacelulina , Células Cultivadas , Epitelio/metabolismo , Expresión Génica , Proteínas de Homeodominio/genética , Células Secretoras de Insulina/citología , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas de Filamentos Intermediarios/genética , Células Madre Mesenquimatosas/citología , Proteínas del Tejido Nervioso/genética , Nestina , Ratas , Ratas Sprague-Dawley , Esferoides Celulares/citología , Transactivadores/genéticaRESUMEN
Severe acute respiratory syndrome coronavirus (SARS-CoV) is the pathogen of SARS, which caused a global panic in 2003. We describe here the screening of Chinese herbal medicine-based, novel small molecules that bind avidly with the surface spike protein of SARS-CoV and thus can interfere with the entry of the virus to its host cells. We achieved this by using a two-step screening method consisting of frontal affinity chromatography-mass spectrometry coupled with a viral infection assay based on a human immunodeficiency virus (HIV)-luc/SARS pseudotyped virus. Two small molecules, tetra-O-galloyl-beta-D-glucose (TGG) and luteolin, were identified, whose anti-SARS-CoV activities were confirmed by using a wild-type SARS-CoV infection system. TGG exhibits prominent anti-SARS-CoV activity with a 50% effective concentration of 4.5 microM and a selective index of 240.0. The two-step screening method described here yielded several small molecules that can be used for developing new classes of anti-SARS-CoV drugs and is potentially useful for the high-throughput screening of drugs inhibiting the entry of HIV, hepatitis C virus, and other insidious viruses into their host cells.
Asunto(s)
Antivirales/farmacología , Flavonoides/farmacología , Taninos Hidrolizables/farmacología , Plantas Medicinales/química , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/efectos de los fármacos , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/patogenicidad , Animales , Antivirales/química , Antivirales/metabolismo , Línea Celular , China , Chlorocebus aethiops , Cromatografía de Afinidad , VIH-1/genética , VIH-1/metabolismo , Humanos , Taninos Hidrolizables/química , Luciferasas/genética , Luciferasas/metabolismo , Luteolina , Espectrometría de Masas , Glicoproteínas de Membrana/metabolismo , Pruebas de Sensibilidad Microbiana , Plantas Medicinales/metabolismo , Glicoproteína de la Espiga del Coronavirus , Células Vero , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Heptad repeat regions (HR1 and HR2) are highly conserved sequences located in the glycoproteins of enveloped viruses. They form a six-helix bundle structure and are important in the process of virus fusion. Peptides derived from the HR regions of some viruses have been shown to inhibit the entry of these viruses. SARS-CoV was also predicted to have HR1 and HR2 regions in the S2 protein. Based on this prediction, we designed 25 peptides and screened them using a HIV-luc/SARS pseudotyped virus assay. Two peptides, HR1-1 and HR2-18, were identified as potential inhibitors, with EC(50) values of 0.14 and 1.19microM, respectively. The inhibitory effects of these peptides were validated by the wild-type SARS-CoV assay. HR1-1 and HR2-18 can serve as functional probes for dissecting the fusion mechanism of SARS-CoV and also provide the potential of further identifying potent inhibitors for SARS-CoV entry.