Protocol to identify flavonoid antagonists of the SARS-CoV-2 main protease.

Flavonoids are naturally occurring metabolites of plants that can inhibit the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro), which is required for viral replication. Here, we present a protocol to identify flavonoid antagonists of the SARS-CoV-2 Mpro. We describe steps for the expression and purification of Mpro and a kinetic enzymatic assay for Mpro activity using a dequenching fluorescence resonance energy transfer peptide substrate. We then detail procedures for using this enzymatic assay to test flavonoid antagonism and reversible inhibition. For complete details on the use and execution of this protocol, please refer to Lin et al.1.

Targeting Cysteine Oxidation in Thrombotic Disorders.

Oxidative stress increases the risk for clinically significant thrombotic events, yet the mechanisms by which oxidants become prothrombotic are unclear. In this review, we provide an overview of cysteine reactivity and oxidation. We then highlight recent findings on cysteine oxidation events in oxidative stress-related thrombosis. Special emphasis is on the signaling pathway induced by a platelet membrane protein, CD36, in dyslipidemia, and by protein disulfide isomerase (PDI), a member of the thiol oxidoreductase family of proteins. Antioxidative and chemical biology approaches to target cysteine are discussed. Lastly, the knowledge gaps in the field are highlighted as they relate to understanding how oxidative cysteine modification might be targeted to limit thrombosis.

Lower glycemia levels in subjects with excessive erythrocytosis during the oral glucose tolerance test living in conditions of severe hypoxia.

Background: Previous studies showed that residents of higher elevations have lower glucose levels. Our objective in this study is to determine the basal and postprandial glucose levels in apparently healthy permanent residents of the miner population center of La Rinconada located 5100 meters (m) above sea level. Method: Forty male permanent residents of the Rinconada miner population center were studied. The oral glucose tolerance test was used to evaluate basal and postprandial glycemia levels at 1, 2, and 3 h. Results: The individuals had a mean age of 43.95 ± 8.54 years. Basal glycemia in subjects without excessive erythrocytosis (EE) was 73.3 ± 7.9 mg/dL, while levels in patients with EE were 57.98 ± 7.38 mg/dL. In the postprandial period, at 1 h after oral glucose overload, a mean value of 76.35 ± 13.53 mg/dL was observed in subjects with EE compared to 94.68 ± 9.98 mg/dL in subjects without EE. After 2 h, subjects with EE had a glycemia level of 72.91 ± 9.17 mg/dL EE compared to 90.73 ± 13.86 mg/dL without EE. At 3 h, the average glycemia level in subjects with EE was 70.77 ± 8.73 mg/dL compared to 87.79 ± 14.16 mg/dL in those without EE. Conclusion: These findings suggest that under hypoxic conditions, glycemia levels are lower in both subjects with and without EE, having obtained lower levels in subjects with EE in relation to those with normal values of Hb and Hct. The results of this study indicate that in the conditions of severe hypoxia, blood glucose levels are below the values considered normal for sea level.

Oxidized LDL regulates efferocytosis through the CD36-PKM2-mtROS pathway.

Macrophage efferocytosis, the process by which phagocytes engulf and remove apoptotic cells (ACs), plays a critical role in maintaining tissue homeostasis. Efficient efferocytosis prevents secondary necrosis, mitigates chronic inflammation, and impedes atherosclerosis progression. However, the regulatory mechanisms of efferocytosis under atherogenic conditions remain poorly understood. We previously demonstrated that oxidized LDL (oxLDL), an atherogenic lipoprotein, induces mitochondrial reactive oxygen species (mtROS) in macrophages via CD36. In this study, we demonstrate that macrophage mtROS facilitate continual efferocytosis through a positive feedback mechanism. However, oxLDL disrupts continual efferocytosis by dysregulating the internalization of ACs. This disruption is mediated by an overproduction of mtROS. Mechanistically, oxLDL/CD36 signaling promotes the translocation of cytosolic PKM2 to mitochondria, facilitated by the chaperone GRP75. Mitochondrial PKM2 then binds to Complex III of the electron transport chain, inducing mtROS production. This study elucidates a novel regulatory mechanism of efferocytosis in atherosclerosis, providing potential therapeutic targets for intervention. SUMMARY: Macrophages clear apoptotic cells through a process called efferocytosis, which involves mitochondrial ROS. However, the atherogenic oxidized LDL overstimulates mitochondrial ROS via the CD36-PKM2 pathway, disrupting continual efferocytosis. This finding elucidates a novel molecular mechanism that explains defects in efferocytosis, driving atherosclerosis progression.