Development of an optimized colorimetric RT-LAMP for SARS-CoV-2 assay with enhanced procedure controls for remote diagnostics.

The coronavirus pandemic accentuated the need for molecular diagnostic tests. A technique highly used to this end is the Polymerase Chain Reaction (PCR)-a sensitive and specific technique commonly used as the gold standard for molecular diagnostics. However, it demands highly trained personnel and high-maintenance equipment and is relatively time-consuming. An alternative is the Loop-Mediated Isothermal Amplification (LAMP) technique, which doesn't need sample purification or expensive equipment, and is similar to PCR when compared in sensitivity and specificity. In this paper, we developed an optimized colorimetric Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) Point-of-Care test using a portable device to diagnose COVID-19. Variables such as concentration of primers, magnesium sulfate, betaine, hydrochloride guanidine, Bst, and temperature of the reactions were tested. We also created a pipetting quality control system-using a combination of dyes-to avoid false negatives due to a lack of samples added to the reaction test tube. Mineral oil was incorporated in the composition of the RT-LAMP reactions to avoid evaporation when a heating lid isn't available. The final RT-LAMP test is tenfold more sensitive when compared to the WarmStart Colorimetric Master mix from New England Biolabs with a sensitivity of 5 copies per µL.

Polybrominated diphenyl ethers BDE-47 and BDE-99 modulate murine melanoma cell phenotype in vitro.

Cancer is one of the leading causes of mortality worldwide. Even with the advances of pharmaceutical industry and treatments, the mortality rate for various types of cancer remains high. In particular, phenotypic alterations of tumor cells concerning drug efflux, migratory and invasive capabilities may represent a hurdle for cancer treatment and contribute to poor prognosis. In the present study, we investigated the effects of polybrominated diphenyl ethers (PBDEs) used as flame retardants on phenotypic features of melanoma cells that are important for cancer. Murine melanoma B16-F1 (less metastatic) and B16-F10 (more metastatic) cells were exposed to 0.01-1.0 nM of BDE-47 (2,2',4,4'-tetrabromodiphenyl ether), BDE-99 (2,2',4,4',5-pentabromodiphenyl ether), and the mixture of both (at 0.01 nM) for 24 h (acute exposure) and 15 days (chronic exposure). The polybrominated diphenyl ethers (PBDEs) did not affect cell viability but led to increased drug efflux transporter activity, cell migration, and colony formation, as well as overexpression of Abcc2 (ATP-binding cassette subfamily C member 2), Mmp-2 (matrix metalloproteinase-2), Mmp-9 (matrix metalloproteinase-9), and Tp53 (tumor protein p53) genes and downregulation of Timp-3 (tissue inhibitor of metalloproteinase 3) gene in B16-F10 cells. These effects are consistent with increased aggressiveness and malignancy of tumors due to exposure to the flame retardants and raise some concerns on the effects such chemicals may have on melanoma treatment and cancer prognosis.