RESUMEN
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes COVID-19 and is responsible for the ongoing pandemic. Screening of potential antiviral drugs against SARS-CoV-2 depend on in vitro experiments, which are based on the quantification of the virus titer. Here, we used virus-induced cytopathic effects (CPE) in brightfield microscopy of SARS-CoV-2-infected monolayers to quantify the virus titer. Images were classified using deep transfer learning (DTL) that fine-tune the last layers of a pre-trained Resnet18 (ImageNet). To exclude toxic concentrations of potential drugs, the network was expanded to include a toxic score (TOX) that detected cell death (CPETOXnet). With this analytic tool, the inhibitory effects of chloroquine, hydroxychloroquine, remdesivir, and emetine were validated. Taken together we developed a simple method and provided open access implementation to quantify SARS-CoV-2 titers and drug toxicity in experimental settings, which may be adaptable to assays with other viruses. The quantification of virus titers from brightfield images could accelerate the experimental approach for antiviral testing.
Asunto(s)
Antivirales/farmacología , Aprendizaje Profundo , Evaluación Preclínica de Medicamentos/métodos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Aprendizaje Automático , SARS-CoV-2/efectos de los fármacos , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Alanina/análogos & derivados , Animales , COVID-19 , Chlorocebus aethiops , Proteínas de la Nucleocápside de Coronavirus , Fosfoproteínas , Células Vero , Carga Viral/efectos de los fármacosRESUMEN
USP18 (Ubiquitin-like specific protease 18) is an enzyme cleaving ubiquitin from target proteins. USP18 plays a pivotal role in antiviral and antibacterial immune responses. On the other hand, ubiquitination participates in the regulation of several ion channels and transporters. USP18 sensitivity of transporters has, however, never been reported. The present study thus explored, whether USP18 modifies the activity of the peptide transporters PEPT1 and PEPT2, and whether the peptide transporters are sensitive to the ubiquitin ligase Nedd4-2. To this end, cRNA encoding PEPT1 or PEPT2 was injected into Xenopus laevis oocytes without or with additional injection of cRNA encoding USP18. Electrogenic peptide (glycine-glycine) transport was determined by dual electrode voltage clamp. As a result, in Xenopus laevis oocytes injected with cRNA encoding PEPT1 or PEPT2, but not in oocytes injected with water or with USP18 alone, application of the dipeptide gly-gly (2 mM) was followed by the appearance of an inward current (Igly-gly). Coexpression of USP18 significantly increased Igly-gly in both PEPT1 and PEPT2 expressing oocytes. Kinetic analysis revealed that coexpression of USP18 increased maximal Igly-gly. Conversely, overexpression of the ubiquitin ligase Nedd4-2 decreased Igly-gly. Coexpression of USP30 similarly increased Igly-gly in PEPT1 expressing oocytes. In conclusion, USP18 sensitive cellular functions include activity of the peptide transporters PEPT1 and PEPT2.
Asunto(s)
Dipéptidos/metabolismo , Endopeptidasas/metabolismo , Simportadores/metabolismo , Animales , Transporte Biológico , Dipéptidos/farmacología , Endopeptidasas/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Femenino , Humanos , Inyecciones , Canal de Potasio KCNQ1/genética , Canal de Potasio KCNQ1/metabolismo , Mediciones Luminiscentes/métodos , Potenciales de la Membrana/efectos de los fármacos , Ubiquitina-Proteína Ligasas Nedd4 , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Oocitos/fisiología , Técnicas de Placa-Clamp , Transportador de Péptidos 1 , Canales de Potasio con Entrada de Voltaje/genética , Canales de Potasio con Entrada de Voltaje/metabolismo , ARN Complementario/administración & dosificación , ARN Complementario/genética , Conejos , Simportadores/genética , Ubiquitina Tiolesterasa , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Xenopus , Xenopus laevisRESUMEN
The chloride channel ClC-Kb is expressed in the basolateral cell membrane of the distal nephron and participates in renal NaCl reabsorption. Loss-of-function mutations of ClC-Kb lead to classic Bartter syndrome, a rare salt-wasting disorder. Recently, we identified the ClC-Kb(T481S) polymorphism, which confers a strong gain-of-function effect on the ClC-Kb chloride channel. The present study has been performed to explore the prevalence of the mutation and its functional significance in renal salt handling and blood pressure regulation. As evident from electrophysiological analysis with the 2-electrode voltage-clamp technique, heterologous expression of ClC-Kb(T481S) in Xenopus oocytes gave rise to a current that was 7-fold larger than the current produced by wild-type ClC-Kb. The prevalence of the mutant allele was significantly higher in an African population from Ghana (22%) than in whites (12%). As tested in 1 white population, carriers of ClC-Kb(T481S) were associated with significantly higher systolic (by approximately 6.0 mm Hg) and diastolic (by approximately 4.2 mm Hg) blood pressures and significantly higher prevalence (45% versus 25%) of hypertensive (> or =140/90 mm Hg) blood pressure levels. Individuals carrying ClC-Kb(T481S) had significantly higher plasma Na+ concentrations and significantly decreased glomerular filtration rate. In conclusion, the mutation ClC-Kb(T481S) of the renal epithelial Cl- channel ClC-Kb strongly activates ClC-Kb chloride channel function in vitro and may predispose to the development of essential hypertension in vivo.