RESUMO
Real-time reverse-transcription polymerase chain reaction (RT-PCR) is currently the most popular method for early COVID-19 diagnostics. However, loop-mediated isothermal amplification (LAMP) is superior to real-time RT-PCR in rapidity and simplicity, since it does not require expensive laboratory equipment and trained personnel. LAMP-based diagnostic kits for COVID-19 testing already exist, but corresponding tests are not yet widely available. The method has great potential for mass application. Here, we discuss the technical and methodological aspects of its widespread adoption.
RESUMO
An organism naturally responds to hypoxia via stabilization of hypoxia-inducible factor (HIF). There are three isoforms of HIFα subunits whose stability is regulated by three isozymes of HIF prolyl hydroxylase (PHD1-3). Despite intense studies on recombinant enzyme isoforms using homogeneous activity assay, there is no consensus on the PHD isoform preference for the HIF isoform as a substrate. This work provides a new approach to the problem of substrate specificity using cell-based reporters expressing the enzyme and luciferase-labeled substrate pair encoded in the same expression vector. The cell is used as a microbioreactor for running the reaction between the overexpressed enzyme and substrate. Using this novel approach, no PHD3 activity toward HIF3 was demonstrated, indirectly pointing to the hydroxylation of the second proline in 564PYIP567 (HIF1) catalyzed by this isozyme. The use of "paired" enzyme-substrate reporters to evaluate the potency of "branched tail" oxyquinoline inhibitors of HIF PHD allows higher precision in revealing the optimal structural motif for each enzyme isoform.
Assuntos
Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Linhagem Celular Tumoral , Genes Reporter , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , RNA Mensageiro/metabolismo , RNA Ribossômico 18S/metabolismo , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
We studied the relationship between microcirculation parameters and functional status of HepaRG cells in spheroids and chose an optimal regimen within the physiologically permissible limits of mechanical impact for the cells that maintains the expression of functional genes of the liver.