ABSTRACT
The association of mortality with early humoral response to SARS-CoV-2 infection within the first few days after onset of symptoms (DAOS) has not been thoroughly investigated partly due to a lack of sufficiently sensitive antibody testing methods. Here we report two sensitive and automated testing-on-a-probe (TOP) biosensor assays for SARS-CoV-2 viral specific total antibodies (TAb) and surrogate neutralizing antibodies (SNAb), which are suitable for clinical use. The TOP assays employ an RBD-coated quartz probe using a Cy5-Streptavidin-polysacharide conjugate to improved sensitivity and minimize interference. Disposable cartridge containing pre-dispensed reagents requires no liquid manipulation or fluidics during testing. The TOP-TAb assay exhibited higher sensitivity in the 0-7 DAOS window than a widely used FDA-EUA assay. The rapid (18 min) and automated TOP-SNAb correlated well with two well-established SARS-CoV-2 virus neutralization tests. The clinical utility of the TOP assays was demonstrated by evaluating early antibody responses in 120 SARS-CoV-2 RT-PCR positive adult hospitalized patients. Higher baseline TAb and SNAb positivity rates and more robust antibody responses were seen in patients who survived COVID-19 than those who died in the hospital. Survival analysis using the Cox Proportional Hazards Model showed that patients who were TAb and SNAb negative at initial hospital presentation were at a higher risk of in-hospital mortality. Furthermore, TAb and SNAb levels at presentation were inversely associated with SARS-CoV-2 viral load based on concurrent RT-PCR testing. Overall, the sensitive and automated TAb and SNAb assays allow detection of early SARS-CoV-2 antibodies which associate with mortality.
ABSTRACT
The Ras-related GTPase Rap1 promotes cell adhesion and migration. Although the significance of Rap1 contribution to cell migration is increasingly being recognized, little is known about the biochemical mechanisms driving this process. In the present study, we discovered a previously unidentified regulatory role of insulin-like growth factor type I (IGF-I) receptor (IGF-IR) in CRK Src homology 3 (SH3)-binding guanine-nucleotide-releasing protein (C3G)-Rap1-fascin-actin axis promoting cell movement. We demonstrate that a burst of Rap1 activity, rather than presumed hyperactivation, is imperative for the onset of cell movement. We show that while autophosphorylated IGF-IR signals to C3G to activate Rap1, subsequent IGF-IR internalization promotes gradual inactivation of Rap1 by putative Rap1 GTPase-activating protein (GAP). Additionally, IGF-IR signalling recruits active Rap1 at sites of cell motile protrusions. C3G depletion prevents IGF-I-induced fascin accumulation at actin microspikes and blocks protrusions. In the absence of IGF-IR activity, the wild-type (WT) Rap1 and the constitutively active V12Rap1 mutant remain in cell-cell contacts. Forced inactivation of Rap1 signalling by overexpressing dominant negative N17Rap1, Rap1GAP or by silencing C3G has a detrimental effect on filamentous (F)-actin and cell adhesion irrespective of IGF-IR signalling. We conclude that the basal levels of Rap1 activity holds up cell adhesion, whereas sequential regulation of C3G and GAP by IGF-IR reverses the labile Rap1 function from supporting adhesion to promoting migration.
