RESUMO
The emergence of a polybasic cleavage motif for the protease furin in the SARS-CoV-2 spike protein has been established as a major factor for enhanced viral transmission in humans. The peptide region N-terminal to that motif is extensively mutated in major variants of concern including Alpha, Delta and Omicron. Besides furin, spike proteins from these variants appear to rely on other proteases for maturation, including TMPRSS2 that may share the same cleavage motif. Glycans found near the cleavage site have raised questions about proteolytic processing and the consequences of variant-borne mutations. Here, with a suite of chemical tools, we establish O-linked glycosylation as a major determinant of SARS-CoV-2 spike cleavage by the host proteases furin and TMPRSS2, and as a likely driving force for the emergence of common mutations in variants of concern. We provide direct evidence that the glycosyltransferase GalNAc-T1 primes glycosylation at Thr678 in the living cell, and this glycosylation event is suppressed by many, but not all variant mutations. A novel strategy for rapid bioorthogonal modification of Thr678-containing glycopeptides revealed that introduction of a negative charge completely abrogates furin activity. In a panel of synthetic glycopeptides containing elaborated O-glycans, we found that the sole incorporation of N-acetylgalactosamine did not substantially impact furin activity, but the presence of sialic acid in elaborated O-glycans reduced furin rate by up to 65%. Similarly, O-glycosylation with a sialylated trisaccharide had a negative impact on spike cleavage by TMPRSS2. With a chemistry-centered approach, we firmly establish O-glycosylation as a major determinant of spike maturation and propose that a disruption of O-GalNAc glycosylation is a substantial driving force for the evolution of variants of concern. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/508093v3_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@71b54eorg.highwire.dtl.DTLVardef@1361b4aorg.highwire.dtl.DTLVardef@139bdd9org.highwire.dtl.DTLVardef@1df192b_HPS_FORMAT_FIGEXP M_FIG C_FIG
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Background@#Pedicled perforator flaps can present postoperative complications similar to those encountered in free flap surgery. Beyond a clinical evaluation, there is still no reliable technical aid for the early prediction of vascular issues. The aim of this study was to assess the support of near-infrared spectroscopy technology as an intraoperative tool to anticipate postsurgical flap ischemia. @*Methods@#We prospectively enrolled 13 consecutive patients who were referred to our hospital from March 2017 to July 2018 and required a reconstructive procedure with a pedicled fasciocutaneous perforator flap. We measured flap peripheral capillary oxygen saturation (SpO2) in each patient with a Somanetics INVOS 5100C Cerebral/Somatic Oximeter (Medtronic), both before and after transposition. Patient demographics, operative data, and complications were then recorded during the following 6 months. We analyzed the data using the Wilcoxon signed-rank test and linear regression. @*Results@#The mean flap SpO2 before and after transposition was 92%±3% and 78%±19%, respectively. The mean change in SpO2 was 14%±17%, with a range of 0% to 55%. The change in saturation and mean saturation ratio were significantly different between patients with and without postoperative flap necrosis. @*Conclusions@#An immediate quantitative analysis of flap peripheral capillary SpO2 after transposition has never before been described. In our experience, an intraoperative drop in SpO2 equal to or greater than 15%–20% predicted vascular complications in pedicled perforator flaps. Conversely, flap size and rotation angle were not correlated with the risk of flap necrosis.
RESUMO
Background@#Pedicled perforator flaps can present postoperative complications similar to those encountered in free flap surgery. Beyond a clinical evaluation, there is still no reliable technical aid for the early prediction of vascular issues. The aim of this study was to assess the support of near-infrared spectroscopy technology as an intraoperative tool to anticipate postsurgical flap ischemia. @*Methods@#We prospectively enrolled 13 consecutive patients who were referred to our hospital from March 2017 to July 2018 and required a reconstructive procedure with a pedicled fasciocutaneous perforator flap. We measured flap peripheral capillary oxygen saturation (SpO2) in each patient with a Somanetics INVOS 5100C Cerebral/Somatic Oximeter (Medtronic), both before and after transposition. Patient demographics, operative data, and complications were then recorded during the following 6 months. We analyzed the data using the Wilcoxon signed-rank test and linear regression. @*Results@#The mean flap SpO2 before and after transposition was 92%±3% and 78%±19%, respectively. The mean change in SpO2 was 14%±17%, with a range of 0% to 55%. The change in saturation and mean saturation ratio were significantly different between patients with and without postoperative flap necrosis. @*Conclusions@#An immediate quantitative analysis of flap peripheral capillary SpO2 after transposition has never before been described. In our experience, an intraoperative drop in SpO2 equal to or greater than 15%–20% predicted vascular complications in pedicled perforator flaps. Conversely, flap size and rotation angle were not correlated with the risk of flap necrosis.
