Direct RT-PCR amplification of SARS-CoV-2 from clinical samples using a concentrated viral Lysis-Amplification Buffer prepared with IGEPAL-630. (preprint)

The pandemic of 2019 caused by the novel coronavirus (SARS-CoV-2) is still rapidly spreading worldwide. Nucleic acid amplification serves as the gold standard method for confirmation of COVID-19 infection. However, challenges faced for diagnostic laboratories from undeveloped countries includes shortage of kits and supplies to purify viral RNA. Therefore, it is urgent to validate alternative nucleic acid isolation methods for SARS-CoV-2. Our results demonstrate that a concentrated viral lysis amplification buffer (vLAB) prepared with the nonionic detergent IGEPAL enables qualitative detection of SARS-CoV-2 by direct Reverse Transcriptase-Polymerase Chain Reaction (dRT-PCR). Furthermore, vLAB was effective in inactivating SARS-CoV-2. Since this method is inexpensive and no RNA purification equipment or additional cDNA synthesis is required, this dRT-PCR with vLAB should be considered as an alternative method for COVID-19 detection.

EPAC regulates von Willebrand factor secretion from endothelial cells in a PI3K/eNOS-dependent manner during inflammation (preprint)

Coagulopathy is associated with both inflammation and infection, including infection with the novel SARS-CoV-2 (COVID-19). Endothelial cells (ECs) fine tune hemostasis via cAMP-mediated secretion of von Willebrand factor (vWF), which promote the process of clot formation. The e xchange p rotein directly a ctivated by c AMP (EPAC) is a ubiquitously expressed intracellular cAMP receptor that plays a key role in stabilizing ECs and suppressing inflammation. To assess whether EPAC could regulate vWF release during inflammation, we utilized our EPAC1 -null mouse model and revealed an increased secretion of vWF in endotoxemic mice in the absence of the EPAC1 gene. Pharmacological inhibition of EPAC1 in vitro mimicked the EPAC1 −/− phenotype. EPAC1 regulated TNFα-triggered vWF secretion from human umbilical vein endothelial cells (HUVECs) in a phosphoinositide 3-kinases (PI3K)/endothelial nitric oxide synthase (eNOS)-dependent manner. Furthermore, EPAC1 activation reduced inflammation-triggered vWF release, both in vivo and in vitro . Our data delineate a novel regulatory role of EPAC1 in vWF secretion and shed light on potential development of new strategies to controlling thrombosis during inflammation. Key Point PI3K/eNOS pathway-mediated, inflammation-triggered vWF secretion is the target of the pharmacological manipulation of the cAMP-EPAC system.