RESUMEN
The development of prophylactic agents against the SARS-CoV-2 virus is a public health priority in the search for new surrogate markers of active virus replication. Early detection markers are needed to follow disease progression and foresee patient negativization. Subgenomic RNA transcripts (with a focus on sgN) were evaluated in oro/nasopharyngeal swabs from COVID-19-affected patients with an analysis of 315 positive samples using qPCR technology. Cut-off Cq values for sgN (Cq < 33.15) and sgE (Cq < 34.06) showed correlations to high viral loads. The specific loss of sgN in home-isolated and hospitalized COVID-19-positive patients indicated negativization of patient condition, 3-7 days from the first swab, respectively. A new detection kit for sgN, gene E, gene ORF1ab, and gene RNAse P was developed recently. In addition, in vitro studies have shown that 2'-O-methyl antisense RNA (related to the sgN sequence) can impair SARS-CoV-2 N protein synthesis, viral replication, and syncytia formation in human cells (i.e., HEK-293T cells overexpressing ACE2) upon infection with VOC Alpha (B.1.1.7)-SARS-CoV-2 variant, defining the use that this procedure might have for future therapeutic actions against SARS-CoV-2.
Asunto(s)
COVID-19/virología , Proteínas de la Nucleocápside de Coronavirus/genética , SARS-CoV-2/fisiología , Replicación Viral/fisiología , Proteínas de la Nucleocápside de Coronavirus/análisis , Células Gigantes/efectos de los fármacos , Células Gigantes/virología , Células HEK293 , Humanos , Límite de Detección , Nasofaringe/virología , Fosfoproteínas/análisis , Fosfoproteínas/genética , ARN sin Sentido/farmacología , ARN Viral , Ribonucleasa P/genética , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/genética , Sensibilidad y Especificidad , Aislamiento Social , Carga Viral , Proteínas Viroporinas/genética , Replicación Viral/efectos de los fármacosRESUMEN
Genetic modifications during development of paediatric groups 3 and 4 medulloblastoma are responsible for their highly metastatic properties and poor patient survival rates. PRUNE1 is highly expressed in metastatic medulloblastoma group 3, which is characterized by TGF-ß signalling activation, c-MYC amplification, and OTX2 expression. We describe the process of activation of the PRUNE1 signalling pathway that includes its binding to NME1, TGF-ß activation, OTX2 upregulation, SNAIL (SNAI1) upregulation, and PTEN inhibition. The newly identified small molecule pyrimido-pyrimidine derivative AA7.1 enhances PRUNE1 degradation, inhibits this activation network, and augments PTEN expression. Both AA7.1 and a competitive permeable peptide that impairs PRUNE1/NME1 complex formation, impair tumour growth and metastatic dissemination in orthotopic xenograft models with a metastatic medulloblastoma group 3 cell line (D425-Med cells). Using whole exome sequencing technology in metastatic medulloblastoma primary tumour cells, we also define 23 common 'non-synonymous homozygous' deleterious gene variants as part of the protein molecular network of relevance for metastatic processes. This PRUNE1/TGF-ß/OTX2/PTEN axis, together with the medulloblastoma-driver mutations, is of relevance for future rational and targeted therapies for metastatic medulloblastoma group 3.10.1093/brain/awy039_video1awy039media15742053534001.
Asunto(s)
Proteínas Portadoras/metabolismo , Neoplasias Cerebelosas/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Meduloblastoma/metabolismo , Metástasis de la Neoplasia/fisiopatología , Fosfohidrolasa PTEN/metabolismo , Adolescente , Animales , Proteínas Portadoras/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Neoplasias Cerebelosas/patología , Niño , Preescolar , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes , Humanos , Lactante , Masculino , Meduloblastoma/patología , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Metástasis de la Neoplasia/genética , Fosfohidrolasa PTEN/genética , Monoéster Fosfórico Hidrolasas , Pirimidinonas/química , Pirimidinonas/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factores de Transcripción de la Familia Snail/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Inorganic polyphosphates (polyPs) are linear polymers composed of repeated phosphate (PO4 3-) units linked together by multiple high-energy phosphoanhydride bonds. In addition to being a source of energy, polyPs have cytoprotective and antiviral activities. Here, we investigated the antiviral activities of long-chain polyPs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In molecular docking analyses, polyPs interacted with several conserved amino acid residues in angiotensin-converting enzyme 2 (ACE2), the host receptor that facilitates virus entry, and in viral RNA-dependent RNA polymerase (RdRp). ELISA and limited proteolysis assays using nano- LC-MS/MS mapped polyP120 binding to ACE2, and site-directed mutagenesis confirmed interactions between ACE2 and SARS-CoV-2 RdRp and identified the specific amino acid residues involved. PolyP120 enhanced the proteasomal degradation of both ACE2 and RdRp, thus impairing replication of the British B.1.1.7 SARS-CoV-2 variant. We thus tested polyPs for functional interactions with the virus in SARS-CoV-2-infected Vero E6 and Caco2 cells and in primary human nasal epithelial cells. Delivery of a nebulized form of polyP120 reduced the amounts of viral positive-sense genomic and subgenomic RNAs, of RNA transcripts encoding proinflammatory cytokines, and of viral structural proteins, thereby presenting SARS-CoV-2 infection in cells in vitro.