The human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel (I Kr ). Drug-mediated or medical conditionmediated disruption of hERG function is the primary cause of acquired long-QT syndrome, which predisposes affected individuals to ventricular arrhythmias and sudden death. Fentanyl abuse poses a serious health concern, with abuse and death rates rising over recent years. As fentanyl has a propensity to cause sudden death, we investigated its effects on the hERG channel. The effects of norfentanyl, the main metabolite, and naloxone, an antidote used in fentanyl overdose, were also examined. Currents of hERG channels stably expressed in HEK293 cells were recorded using the whole-cell voltage-clamp method. When hERG tail currents were analyzed upon -50 mV repolarization after a 50 mV depolarization, fentanyl and naloxone blocked hERG current (I hERG ) with IC 50 values of 0.9 and 74.3 mM, respectively, whereas norfentanyl did not block. However, fentanyl-mediated block of I hERG was voltage dependent. When a voltage protocol that mimics a human ventricular action potential (AP) was used, fentanyl blocked I hERG with an IC 50 of 0.3 mM. Furthermore, fentanyl (0.5 mM) prolonged AP duration and blocked I Kr in ventricular myocytes isolated from neonatal rats. The concentrations of fentanyl used in this study were higher than seen with clinical use but overlap with postmortem overdose concentrations. Although mechanisms of fentanyl-related sudden death need further investigation, blockade of hERG channels may contribute to the death of individuals with high-concentration overdose or compromised cardiac repolarization.

Lung ultrasound (LUS) is an accurate thoracic imaging technique distinguished by its handheld size, low-cost, and lack of radiation. User dependence and poor access to training have limited the impact and dissemination of LUS outside of acute care hospital environments. Automated interpretation of LUS using deep learning can overcome these barriers by increasing accuracy while allowing point-of-care use by non-experts. In this multicenter study, we seek to automate the clinically vital distinction between A line (normal parenchyma) and B line (abnormal parenchyma) on LUS by training a customized neural network using 272,891 labelled LUS images. After external validation on 23,393 frames, pragmatic clinical application at the clip level was performed on 1162 videos. The trained classifier demonstrated an area under the receiver operating curve (AUC) of 0.96 (±0.02) through 10-fold cross-validation on local frames and an AUC of 0.93 on the external validation dataset. Clip-level inference yielded sensitivities and specificities of 90% and 92% (local) and 83% and 82% (external), respectively, for detecting the B line pattern. This study demonstrates accurate deep-learning-enabled LUS interpretation between normal and abnormal lung parenchyma on ultrasound frames while rendering diagnostically important sensitivity and specificity at the video clip level.

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The voltage-gated potassium channel Kv1.5 belongs to the Shaker superfamily. Kv1.5 is composed of four subunits, each comprising 613 amino acids, which make up the N terminus, six transmembrane segments (S1-S6), and the C terminus. We recently demonstrated that, in HEK cells, extracellularly applied proteinase K (PK) cleaves Kv1.5 channels at a single site in the S1-S2 linker. This cleavage separates Kv1.5 into an N-fragment (N terminus to S1) and a C-fragment (S2 to C terminus). Interestingly, the cleavage does not impair channel function. Here, we investigated the role of the N terminus and S1 in Kv1.5 expression and function by creating plasmids encoding various fragments, including those that mimic PK-cleaved products. Our results disclosed that although expression of the pore-containing fragment (Frag(304-613)) alone could not produce current, coexpression with Frag(1-303) generated a functional channel. Immunofluorescence and biotinylation analyses uncovered that Frag(1-303) was required for Frag(304-613) to traffic to the plasma membrane. Biochemical analysis revealed that the two fragments interacted throughout channel trafficking and maturation. In Frag(1-303)+(304-613)-coassembled channels, which lack a covalent linkage between S1 and S2, amino acid residues 1-209 were important for association with Frag(304-613), and residues 210-303 were necessary for mediating trafficking of coassembled channels to the plasma membrane. We conclude that the N terminus and S1 of Kv1.5 can attract and coassemble with the rest of the channel ( Frag(304-613)) to form a functional channel independently of the S1-S2 linkage.

Introduction: 690 Discussion: 1481 Abbreviations: ANOVA, analysis of variance; APD, action potential duration; FBS, fetal bovine serum; HEK, human embryonic kidney; hERG, human ether a-go-go related gene; IC 50 , half maximal inhibitory concentration; I hERG , hERG current; I Kr , rapidly activating delayed rectifier potassium current; [K + ] o , extracellular potassium concentration; LQTS, long QT syndrome; MEM, minimum essential medium; S.D., standard deviation; τ f-deact , fast time constant of deactivation; τ s-deact , slow time constant of deactivation; WT, wild-type.