Drug-induced cardiotoxicity arises primarily when a compound alters the electrophysiological properties of cardiomyocytes. Features of intracellular action potentials (iAPs) are powerful biomarkers that predict proarrhythmic risks. In the last decade, a number of vertical nanoelectrodes have been demonstrated to achieve parallel and minimally-invasive iAP recordings. However, the large variability in success rate and signal strength have hindered nanoelectrodes from being broadly adopted for proarrhythmia drug assessment. In this work, we develop vertically-aligned nanocrown electrodes that are mechanically robust and achieve > 99% success rates in obtaining intracellular access through electroporation. We validate the accuracy of nanocrown electrode recordings by simultaneous patch clamp recording from the same cell. Finally, we demonstrate that nanocrown electrodes enable prolonged iAP recording for continual monitoring of the same cells upon the sequential addition of four incremental drug doses. Our technology development provides an advancement towards establishing an iAP screening assay for preclinical evaluation of drug-induced arrhythmogenicity.
Ischemia-reperfusion injury (IRI) is an important cause of adverse prognosis after recanalization in patients with acute occlusion of major intracranial artery (AOMIA). Here, we provided data indicating that gradual flow restoration (GFR) would be superior to rapid flow restoration (RFR) in alleviating cerebral IRIs in middle cerebral artery occlusion (MCAO) rats. A total of 94 MCAO rats with 15, 30 and 60-minute occlusion were randomly assigned to receive either GFR or RFR intervention. There were significant differences between GFR and RFR group in mean neurological severity score (1.02 versus 1.28; p < 0.05), median infarct ratio (0.016 versus 0.12; p < 0.001), median neuronal apoptosis ratio (1.81 versus 14.46; p < 0.001), and mean histopathological abnormality score (0.92 versus 1.66; p < 0.001). In addition, these differences were mainly distributed in 30-minute and 60-minute occlusion rats, not in 15-minute occlusion rats. These results indicated that GFR rather than RFR could effectively alleviate cerebral IRIs in MCAO rats, especially in rats with longer occlusion duration, suggesting that GFR may be particularly applicable to AOMIA patients who are presented to neurointerventionalists in the later-time of recanalization therapy window.
Nanoscale membrane curvature is now understood to play an active role in essential cellular processes such as endocytosis, exocytosis and actin dynamics. Previous studies have shown that membrane curvatures directly affect protein functions and intracellular signaling. However, few methods are able to precisely manipulate membrane curvature in live cells. Here, we report the development of a new method of generating nanoscale membrane curvature in live cells that is controllable, reversible, and capable of precise spatial and temporal manipulation. For this purpose, we make use of BAR domain proteins, a family of well-studied membrane-remodeling and membrane-sculpting proteins. Specifically, we engineered two optogenetic systems, opto-FBAR and opto-IBAR, that allow light-inducible formation of positive and negative membrane curvature respectively. Using opto-FBAR, blue light activation results in the formation of tubular membrane invaginations (positive curvature), controllable down to the subcellular level. Using opto-IBAR, blue light illumination results in the formation of membrane protrusions or filopodia (negative curvature). These systems present a novel approach for light-inducible manipulation of nanoscale membrane curvature in live cells. Highlights• Opto-FBAR enables light-inducible positive membrane curvature formation.• Opto-IBAR enables light-inducible negative membrane curvature formation.• Light-inducible activation enables precise spatial and temporal control.• Opto-BAR systems present a new approach for studying membranes in live cells.
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