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PURPOSE: Up to 30% of patients with Brugada Syndrome (BrS) carry loss-of-function (LoF) variants in the cardiac sodium channel gene SCN5A. Recent studies suggested that the SCN5A protein product Na(V)1.5 can dimerize and exert dominant negative effects. Here we sought to explore the generality of Na(V)1.5 dominant negative effects and their clinical severity. METHODS: We identified 35 LoF variants (

The COVID-19 pandemic underscored the promise of monoclonal antibody-based prophylactic and therapeutic drugs1–3, but also revealed how quickly viral escape can curtail effective options4, 5. With the emergence of the SARS-CoV-2 Omicron variant in late 2021, many clinically used antibody drug products lost potency, including EvusheldTMand its constituent, cilgavimab4, 6. Cilgavimab, like its progenitor COV2-2130, is a class 3 antibody that is compatible with other antibodies in combination4and is challenging to replace with existing approaches. Rapidly modifying such high-value antibodies with a known clinical profile to restore efficacy against emerging variants is a compelling mitigation strategy. We sought to redesign COV2-2130 to rescue in vivo efficacy against Omicron BA.1 and BA.1.1 strains while maintaining efficacy against the contemporaneously dominant Delta variant. Here we show that our computationally redesigned antibody, 2130-1-0114-112, achieves this objective, simultaneously increases neutralization potency against Delta and many variants of concern that subsequently emerged, and provides protectionin vivoagainst the strains tested, WA1/2020, BA.1.1, and BA.5. Deep mutational scanning of tens of thousands pseudovirus variants reveals 2130-1-0114-112 improves broad potency without incurring additional escape liabilities. Our results suggest that computational approaches can optimize an antibody to target multiple escape variants, while simultaneously enriching potency. Because our approach is computationally driven, not requiring experimental iterations or pre-existing binding data, it could enable rapid response strategies to address escape variants or pre-emptively mitigate escape vulnerabilities.

Background: Deprescribing is an effective means to reduce polypharmacy in elderly patients. However, geriatric day care deprescribing services are challenging to implement in rural regions. In this study, we examined whether a subacute care unit of a rural hospital could deliver a comprehensive and multidisciplinary intervention to promote deprescribing in patients and whether this intervention would succeed in achieving significant and lasting deprescribing results. Methods: We conducted a cross-sectional analysis of a deprescribing program at a rural hospital in Eastern Ontario, Canada. Participants were 11 patients, aged 65 or older, who were admitted to the hospital’s medical/surgical unit or who presented to the emergency department. Clinicians followed a structured, comprehensive and multidisciplinary approach designed to facilitate deprescribing, which concluded with an outcome evaluation at discharge and follow-up phone calls. Outcomes included the frequency and total number of medications successfully removed, reduced, substituted and restarted after discharge and emergency department visits and hospitalizations 6 months before and after the intervention. Results: Of a total 57 deprescribed medications, 38 were eliminated, 8 were switched to a safer alternative, and 11 were dose reduced. Postdischarge deprescribing reversal occurred in only 5 of 57 deprescribed medications. Among the study population, a 59.2% reduction was observed in the combined number of emergency department visits and hospitalizations 6 months after deprescribing. Conclusions: This feasibility study was successful in showing the potential added value for offering a rehabilitative, subacute care, inpatient, comprehensive and multidisciplinary approach toward patients with complex deprescribing needs. It also showed proof of concept in reducing polypharmacy-induced adverse health outcomes. Can Pharm J (Ott) 2020:153:xx-xx.

BACKGROUND: The cardiac sodium channel (SCN5A) mutation, R222Q, neutralizes a positive charge in the domain I voltage sensor. Mutation carriers display very frequent ectopy and dilated cardiomyopathy (DCM). OBJECTIVES: To describe the effect of SCN5A R222Q on murine myocyte and Purkinje fiber electrophysiology, and identify underlying mechanisms. METHODS: We generated mice carrying humanized wild-type (H) and mutant (RQ) SCN5A channels. We characterized whole heart and isolated ventricular and Purkinje myocyte properties. RESULTS: RQ/RQ mice were not viable. I(Na) from RQ/H ventricular myocytes displayed increased “window current” and hyperpolarizing shifts in both inactivation and activation compared to H/H, as previously reported in heterologous expression systems. Surprisingly, action potentials were markedly abbreviated in RQ/H myocytes (APD90: 12.6±1.3 ms versus 29.1±1.0 ms in H/H, p

Introduction: Cardiovascular sequelae of targeted cancer therapies may provide novel insights into cardiovascular biology and disease pathogenesis, including atrial fibrillation (AF). The Bruton tyrosine kinase (BTK) inhibitor ibrutinib has revolutionized treatment for B-cell malignancies but increases the incidence of AF compared with conventional chemotherapy. Recent data in mouse models have shown that ibrutinib mediated-AF likely results not from inhibition of BTK (“on target” effect), but from off-target inhibition of a different kinase, C-terminal Src kinase (CSK). The signaling pathway by which CSK inhibition results in AF remains unknown and thus may represent a novel mechanism for AF molecular pathogenesis. Methods: To study how CSK inhibition results in AF in a human derived model, we created atrial specific cardiomyocytes from human induced pluripotent stem cells (hiPSC-aCMs) from a healthy control. We studied cellular monolayers of hiPSC-aCMs using extracellular field potential (EFP) recordings obtained by the Nanion CardioExcyte 96 system after 24 hour exposure to vehicle, ibrutinib, or the second generation BTK inhibitor acalabrutinib. Results: Chamber specificity of hiPSC-aCMs was confirmed by Western blot analysis of subtype-specific markers. Treatment with ibrutinib for 24 hours resulted in striking increase in beat-to-beat variability in spontaneous contraction, an in vitro correlate of arrhythmogenic behavior (mean coefficient-of-variance/second following treatment 0.835 for ibrutinib vs. 0.198 for acalabrutinib vs. 0.165 for DMSO vehicle control, p Conclusion: These experiments provide a novel method of screening for arrhythmogenic potential of targeted cancer therapies and validate atrial-specific hiPSC-CMs as a model system for the study of CSK mediated AF.

Background: While the cardiac sodium channel ( SCN5A ) variants S1103Y and R1193Q have been associated with LQT3 and drug-induced arrhythmia, they are too common to be highly arrhythmogenic: S1103Y allele frequency is 8.1% in Africans and R1193Q 6.1% in East Asians. We have previously reported that S1103Y had no effect on QT in a large biobank or on action potential duration (APD) in iPSC-CMs. Here, we identify a mechanism underlying control of normal baseline repolarization with these variants and study whether the mechanism is generalizable. Methods: Late sodium current (I Na-L ), I Kr , and sensitivity to the blockers dofetilide (I Kr ) and GS967 (I Na-L ) were measured in natural or genome-edited iPSC-CMs carrying S1103Y, R1193Q, or R518X, a common KCNQ1 nonsense variant in the Jervell Lange-Nielsen (JLN) syndrome and unaffected family members. Results: Although APD was unaffected by S1103Y, I Na-L was increased 2~16-fold in SY/YY compared to SS cells (pKr was increased in SY/YY ([SY] 1.2 pA/pF, n=17, pKr and I Na-L and increased dofetilide sensitivity. In R518X JLN iPSC-CMs, I Ks was absent as expected but I Kr was increased (1.9 pA/pF, n=5), and editing R518X to wild-type increased I Ks (1.3 pA/pF, n=10) and dramatically decreased I Kr (0.2 pA/pF, n=4). Conclusions: S1103Y and R1193Q increase I Na-L but unexpectedly do not affect baseline repolarization. We hypothesize that increased I Kr through as yet undetermined mechanisms maintains normal repolarization but at the cost of increased sensitivity to I Kr block. The findings with R518X suggest this may be a general mechanism. Our data show that preposition to genetically- or drug-mediated arrhythmias is determined by a complex interaction among multiple repolarizing currents that is not fully captured by heterologous expression systems.

IntroductionUp to 30% of patients with Brugada Syndrome (BrS) carry loss-of-function (LoF) variants in the cardiac sodium channel geneSCN5A. Recent studies have suggested that theSCN5Aprotein product NaV1.5 can form dimers and exert dominant negative effects.MethodsWe identified 35 LoF variants (SCN5Avariants were studied in HEK293T cells alone or in heterozygous co-expression with WTSCN5Ausing automated patch clamp. To assess clinical risk, we compared the prevalence of dominant negative vs. putative haploinsufficient (frameshift/splice site) variants in a BrS case consortium and the gnomAD population database.ResultsIn heterozygous expression with WT, 32/35 LoF variants and 6/15 partial LoF showed reduction to Na, demonstrating a dominant negative effect. Carriers of dominant negative LoF missense variants had an enriched disease burden compared to putative haploinsufficient variant carriers (2.7-fold enrichment in BrS cases, p=0.019).ConclusionsMostSCN5Amissense LoF variants exert a dominant negative effect. Cohort analyses reveal that this class of variant confers an especially high burden of BrS.

Introduction: External loop recorders (ELRs) are recommended for the investigation of syncope and palpitations. This study aimed to compare rates of arrhythmia detection between primary care (PC) and hospital-based cardiac unit (HBCU) fitted ELRs. Methods: Data were captured from January to December 2015. Twenty-eight general practitioner practices and 1 hospital took part. Patients were divided into those with ELR fitted in PC or HBCU. All ELR data were analyzed by a cardiac physiologist. Results: A total of 560 ELR recordings were analyzed; 219 (PC) versus 341 (HBCU). There was no difference between the baseline characteristics (all Ps > .05). The predominant indication for ELR in each group were palpitations; between-group variation was observed for syncope ( P = .0004). There were no significant between-group differences in the number of recordings per patient; however, PC group wore the ELR for less time (median 7 days vs median 14 days; P < .0001). There were no differences in arrhythmia detection between PC- and HBCU-fitted ELRs (16.2% [n = 39] vs 21.7% [n = 74], respectively; P = .28). PC placement of ELRs was highest in very remote rural communities ( P = .005) and correlated with distance from HBCU ( r = 0.39; P = .04). Conclusions: This study showed no difference in detection of arrhythmias between PC and HBCU fitted ELRs. This suggests adequate ELR recording can be completed by suitably trained staff in PC. Furthermore, ELRs were fitted for less time in PC without an adverse effect on diagnostic yield. ELR usage increased with increasing distance from the specialist center and rurality suggesting improved local access to arrhythmia detection services.

RationalePartial or complete loss of function variants inSCN5Aare the most common genetic cause of the arrhythmia disorder Brugada Syndrome (BrS1). However, the pathogenicity ofSCN5Avariants is often unknown or disputed; 80% of the 1,390SCN5Amissense variants observed in at least one individual to date are variants of uncertain significance (VUS). The designation of VUS is a barrier to the use of sequence data in clinical care.ObjectiveWe selected 83 variants for study: 10 previously studied control variants, 10 suspected benign variants, and 63 suspected Brugada Syndrome-associated variants, selected on the basis of their frequency in the general population and in patients with Brugada Syndrome. We used high-throughput automated patch clamping to study the function of the 83 variants, with the goal of reclassifying variants with functional data.Methods and ResultsTen previously studied variants had functional properties concordant with published manual patch clamp data. All 10 suspected benign variants had wildtype-like function. 22 suspected BrS variants had loss of channel function (ConclusionsHigh-throughput automated patch clamping enabled the reclassification of the majority of tested VUS’s inSCN5A.

Hereditary hemochromatosis (HH) is an autosomal recessive disorder of excess iron absorption. The most common form, HH1, is caused by loss of function variants in HFE. HFE encodes a cell surface protein that binds to the Transferrin Receptor (TfR1), reducing TfR1’s affinity for the transferrin/iron complex and thereby limiting cellular iron uptake. Two common missense alleles for HH1 have been identified, HFE C282Y and HFE H63D; H63D is considered to be a less penetrant allele. When we deployed Phenotype Risk Scores (PheRS), a method that aggregates multiple symptoms together in Electronic Health Records (EHRs), we identified HFE E168Q as a novel variant associated with HH. E168Q is on the same haplotype as H63D, and in a crystal structure HFE E168 lies at the interface of the HFE-TfR1 interaction and makes multiple salt bridge connections with TfR1. In in vitro cell surface abundance experiments, the HFE E168Q+H63D double mutation surprisingly increased cell surface abundance of HFE by 10-fold compared to wildtype. In coimmunoprecipitation experiments, however, HFE C282Y, E168Q, and E168Q+H63D completely abolished the interaction between HFE and TfR1, while H63D alone only partially reduced binding. These findings provide mechanistic insight to validate the PheRS result that HFE E168Q is an HH1-associated allele and lead to the reclassification of E168Q from a variant of uncertain significance to a pathogenic variant, according to ACMG guidelines. HFE E168Q results in loss of HFE function by disrupting the HFE-TfR1 interaction. In addition, some disease manifestations attributed to H63D may reflect the functional effects of E168Q.

IntroductionA urinary catheter constitutes a one-point patient restraint, can induce deconditioning and may lead to patient mortality. An audit performed at Winchester District Memorial Hospital revealed that 20% of patients had a urinary catheter, of whom 31% did not meet the criteria for catheterisation. The main objective of this study was to use the Influencer Change Model and the Choosing Wisely Canada toolkit to create a bundle of interventions that would reduce the unnecessary use of urinary catheters in hospitalised patients.MethodsIn a rural teaching hospital, a time-series quasi-experiment was employed to decrease inappropriate use of urinary catheters. Both the Choosing Wisely Canada toolkit for appropriate use of urinary catheters and the Influencer change management approach were used to create effective interventions.ResultsThis study revealed that there was no improvement in appropriate urinary catheter use during Plan-Do-Study-Act (PDSA) cycle 1. There was gradual improvement during PDSA cycle 2, with the percentage of inappropriate urinary catheter use dropping from an initial 31% before any interventions to less than 5% by the end of this study.Discussion/conclusionThis study aimed to reduce the inappropriate use of urinary catheters in a rural hospital with limited resources. The findings indicate that by using a change model, such as the Influencer Change Model, it is possible to promote better patient care through empowering healthcare staff to implement accepted protocols more stringently and thereby to decrease the inappropriate use of urinary catheters to 0%.

Twenty years beyond its signing, the Good Friday Agreement remains the cornerstone of ‘peace’ in Northern Ireland, even as it has faced political, social, and cultural challenges. Despite the lack of renewed violence in the region since 1998, the Good Friday Agreement left many issues unaddressed, hampered by the region’s reality as a ‘deeply divided society’ and ultimately a ‘negative peace’. This article seeks to address the reasons peace has failed to flourish in the region, claiming that a ‘peace process’ ultimately concerned with governmental structures and paramilitary ceasefire was inadequate to truly resolve the conflict, resulting in the endurance of tensions into the present. As a result, the legacy of the Good Friday Agreement is a social stalemate from which the region cannot progress. Northern Ireland remains polarised by many of the same differences visible at the start of the Troubles half a century ago, as old divisions play out in new ways. This has resulted in a ‘culture war’, further dividing the populace. Current political instability in both Northern Ireland and the United Kingdom continue to challenge the tentative peace in the region, raising doubts that such divisions can be reasonably overcome. Publisher PDF

Background: The calcium-activated chloride channel ANO1 is highly expressed in cancer. Results: Inhibition of ANO1 activity alone is not sufficient to inhibit cancer cell proliferation, suggesting a novel function of ANO1 protein in cancer. Conclusion: The ANO1 inhibitor CaCCinh-A01 inhibits cancer cell proliferation by facilitating degradation of ANO1. Significance: Our results may provide a new targeting approach for antitumor therapy in ANO1-amplified cancers., ANO1, a calcium-activated chloride channel, is highly expressed and amplified in human cancers and is a critical survival factor in these cancers. The ANO1 inhibitor CaCCinh-A01 decreases proliferation of ANO1-amplified cell lines; however, the mechanism of action remains elusive. We explored the mechanism behind the inhibitory effect of CaCCinh-A01 on cell proliferation using a combined experimental and in silico approach. We show that inhibition of ANO1 function is not sufficient to diminish proliferation of ANO1-dependent cancer cells. We report that CaCCinh-A01 reduces ANO1 protein levels by facilitating endoplasmic reticulum-associated, proteasomal turnover of ANO1. Washout of CaCCinh-A01 rescued ANO1 protein levels and resumed cell proliferation. Proliferation of newly derived CaCCinh-A01-resistant cell pools was not affected by CaCCinh-A01 as compared with the parental cells. Consistently, CaCCinh-A01 failed to reduce ANO1 protein levels in these cells, whereas ANO1 currents were still inhibited by CaCCinh-A01, indicating that CaCCinh-A01 inhibits cell proliferation by reducing ANO1 protein levels. Furthermore, we employed in silico methods to elucidate novel biological functions of ANO1 inhibitors. Specifically, we derived a pharmacophore model to describe inhibitors capable of promoting ANO1 degradation and report new inhibitors of ANO1-dependent cell proliferation. In summary, our data demonstrate that inhibition of the channel activity of ANO1 is not sufficient to inhibit ANO1-dependent cell proliferation, indicating that the role of ANO1 in cancer only partially depends on its function as a channel. Our results provide an impetus for gaining a deeper understanding of ANO1 modulation in cells and introduce a new targeting approach for antitumor therapy in ANO1-amplified cancers.

A significant challenge and potential high-value application of computer-aided drug design is the accurate prediction of protein–ligand binding affinities. Free energy perturbation (FEP) using molecular dynamics (MD) sampling is among the most suitable approaches to achieve accurate binding free energy predictions, due to the rigorous statistical framework of the methodology, correct representation of the energetics, and thorough treatment of the important degrees of freedom in the system (including explicit waters). Recent advances in sampling methods and force fields coupled with vast increases in computational resources have made FEP a viable technology to drive hit-to-lead and lead optimization, allowing for more efficient cycles of medicinal chemistry and the possibility to explore much larger chemical spaces. However, previous FEP applications have focused on systems with high-resolution crystal structures of the target as starting points—something that is not always available in drug discovery projects. As such, the ability to apply FEP on homology models would greatly expand the domain of applicability of FEP in drug discovery. In this work we apply a particular implementation of FEP, called FEP+, on congeneric ligand series binding to four diverse targets: a kinase (Tyk2), an epigenetic bromodomain (BRD4), a transmembrane GPCR (A2A), and a protein–protein interaction interface (BCL-2 family protein MCL-1). We apply FEP+ using both crystal structures and homology models as starting points and find that the performance using homology models is generally on a par with the results when using crystal structures. The robustness of the calculations to structural variations in the input models can likely be attributed to the conformational sampling in the molecular dynamics simulations, which allows the modeled receptor to adapt to the “real” conformation for each ligand in the series. This work exemplifies the advantages of using all-atom simulation methods with full system flexibility and offers promise for the general application of FEP to homology models, although additional validation studies should be performed to further understand the limitations of the method and the scenarios where FEP will work best.

With the continued rise of phenotypic- and genotypic-based screening projects, computational methods to analyze, process, and ultimately make predictions in this field take on growing importance. Here we show how automated machine learning workflows can produce models that are predictive of differential gene expression as a function of a compound structure using data from A673 cells as a proof of principle. In particular, we present predictive models with an average accuracy of greater than 70% across a highly diverse ∼1000 gene expression profile. In contrast to the usual in silico design paradigm, where one interrogates a particular target-based response, this work opens the opportunity for virtual screening and lead optimization for desired multitarget gene expression profiles.

Background Genome‐wide association studies have implicated variants in SCN 10A , which encodes Nav1.8, as modulators of cardiac conduction. Follow‐up work has indicated the SCN 10A sequence includes an intronic enhancer for SCN 5A . Yet the role of the Nav1.8 protein in the myocardium itself is still unclear. To investigate this , we use homozygous knockout mice ( Scn10a −/− ) generated by disruption of exons 4 and 5, leaving the Scn5a enhancer intact. Methods and Results We previously reported that pharmacologic blockade of Nav1.8 in wild‐type animals blunts action potential prolongation by ATX ‐ II at slow drive rates (≤1 Hz). Here we present evidence of the same blunting in Scn10a −/− compared to wild‐type ventricular myocytes, supporting the conclusion that Nav1.8 contributes to late sodium current at slow rates. In contrast to earlier studies, we found no differences in electrocardiographic parameters between genotypes. Low‐dose ATX ‐ II exposure in lightly anesthetized animals and Langendorff‐perfused hearts prolonged QT c and generated arrhythmias to the same extent in wild‐type and Scn10a −/− . RNA sequencing failed to identify full‐length Scn10a transcripts in wild‐type or knockout isolated ventricular myocytes. However, loss of late current in Scn10a −/− myocytes was replicated independently in a blinded set of experiments. Conclusions While Scn10a transcripts are not detectible in ventricular cardiomyocytes, gene deletion results in reproducible loss of late sodium current under extreme experimental conditions. However, there are no identifiable consequences of this Scn10a deletion in the intact mouse heart at usual rates. These findings argue that common variants in SCN 10A that affect ventricular conduction do so by modulating SCN 5A .

The fossil pollen contents found in the charred dottle residue from 20 clay pipe bowls recovered from a cesspit at a house in Amsterdam, the Netherlands (which was owned and used by the artist Rembrandt Harmenszoon van Rijn), were collected and studied. The clay pipes date from approximately the same period when Rembrandt lived in the house and used the cesspit. We believe that some of the pollen in the dottle residue may have come from the tobacco, some possibly from airborne sources at Rembrandt's house and/or from debris in the cesspit in which the pipes were discarded. The results are compared with a similar study of pipe dottle from Native American clay pipes conducted more than 25 years ago. We also briefly review the origin and spread of domestic tobacco in an effort to determine potential sources of the tobacco imported and used in the Netherlands during the lifetime of Rembrandt and the period when he occupied the house. The pollen content of modern brands of commercial pipe tobacco is studied and the potential for using pollen found in tobacco products as evidence in forensic circumstances is reviewed.

The modeling of the conformational properties of conjugated polymers entails a unique challenge for classical force fields. Conjugation imposes strong constraints upon bond rotation. Planar configurations are favored, but the concomitantly shortened bond lengths result in moieties being brought into closer proximity than usual. The ensuing steric repulsions are particularly severe in the presence of side chains, straining angles, and stretching bonds to a degree infrequently found in nonconjugated systems. We herein demonstrate the resulting inaccuracies by comparing the LMP2-calculated inter-ring torsion potentials for a series of substituted stilbenes and bithiophenes to those calculated using standard classical force fields. We then implement adjustments to the OPLS-2005 force field in order to improve its ability to model such systems. Finally, we show the impact of these changes on the dihedral angle distributions, persistence lengths, and conjugation length distributions observed during molecular dynamics simulations of poly[2-methoxy-5-(2'-ethylhexyloxy)-p-phenylene vinylene] (MEH-PPV) and poly 3-hexylthiophene (P3HT), two of the most widely used conjugated polymers.

Bromodomains (BRDs) are epigenetic interaction domains currently recognized as emerging drug targets for development of anticancer or anti-inflammatory agents. In this study, development of a selective ligand of the fifth BRD of polybromo protein-1 (PB1(5)) related to switch/sucrose nonfermenting (SWI/SNF) chromatin remodeling complexes is presented. A compound collection was evaluated by consensus virtual screening and a hit was identified. The biophysical study of protein–ligand interactions was performed using X-ray crystallography and isothermal titration calorimetry. Collective data supported the hypothesis that affinity improvement could be achieved by enhancing interactions of the complex with the solvent. The derived SAR along with free energy calculations and a consensus hydration analysis using WaterMap and SZmap algorithms guided rational design of a set of novel analogues. The most potent analogue demonstrated high affinity of 3.3 μM and an excellent selectivity profile, thus comprising a promising lead for the development of chemical probes targeting PB1(5).

The hydroxamic acid moiety is an effective metal-binding warhead for a variety of metalloenzyme targets of interest in drug-discovery. For the zinc-containing histone deacetylase enzymes in particular, this chemical group has been widely incorporated and studied in the clinic. An alternative chemical functionality for binding zinc is the α-aminocarbonyl motif, which has been shown to bind to histone deacetylase enzymes. The current article explores the minimal binding site theoretical approach combined with structural knowledge to explore the ideal chemical substitution pattern of the α-aminocarbonyl motif within HDAC8. The metal-binding strength of the group is predicted to be highly tunable to chemical substitution at the carbonyl and the α-amino carbon. A fixed receptor model approach with a dispersion-corrected density functional, clearly discerned the effect of different substituents at both these positions using either a flexible or partially fixed ligand optimized in the presence of a fixed receptor model of the HDAC8 binding site. An electron donating substituent such as methyl at the Cα in combination with NMe2 substitution at the carbonyl position, similar to observed crystal structures, result in the optimal energetic profile for binding the zinc atom in the HDAC8 enzyme.

Background— The D1275N SCN5A mutation has been associated with a range of unusual phenotypes, including conduction disease and dilated cardiomyopathy, as well as atrial and ventricular tachyarrhythmias. However, when D1275N is studied in heterologous expression systems, most studies show near-normal sodium channel function. Thus, the relationship of the variant to the clinical phenotypes remains uncertain. Methods and Results— We identified D1275N in a patient with atrial flutter, atrial standstill, conduction disease, and sinus node dysfunction. There was no major difference in biophysical properties between wild-type and D1275N channels expressed in Chinese hamster ovary cells or tsA201 cells in the absence or presence of β1 subunits. To determine D1275N function in vivo, the Scn5a locus was modified to knock out the mouse gene, and the full-length wild-type (H) or D1275N (DN) human SCN5A cDNAs were then inserted at the modified locus by recombinase mediated cassette exchange. Mice carrying the DN allele displayed slow conduction, heart block, atrial fibrillation, ventricular tachycardia, and a dilated cardiomyopathy phenotype, with no significant fibrosis or myocyte disarray on histological examination. The DN allele conferred gene-dose–dependent increases in SCN5A mRNA abundance but reduced sodium channel protein abundance and peak sodium current amplitudes (H/H, 41.0±2.9 pA/pF at −30 mV; DN/H, 19.2±3.1 pA/pF, P P Conclusions— Although D1275N produces near-normal currents in multiple heterologous expression experiments, our data establish this variant as a pathological mutation that generates conduction slowing, arrhythmias, and a dilated cardiomyopathy phenotype by reducing cardiac sodium current.

Rationale: Although multiple lines of evidence suggest that variable expression of the cardiac sodium channel gene SCN5A plays a role in susceptibility to arrhythmia, little is known about its transcriptional regulation. Objective: We used in silico and in vitro experiments to identify possible noncoding sequences important for transcriptional regulation of SCN5A . The results were extended to mice in which a putative regulatory region was deleted. Methods and Results: We identified 92 noncoding regions highly conserved (>70%) between human and mouse SCN5A orthologs. Three conserved noncoding sequences (CNS) showed significant (>5-fold) activity in luciferase assays. Further in vitro studies indicated one, CNS28 in intron 1, as a potential regulatory region. Using recombinase-mediated cassette exchange (RMCE), we generated mice in which a 435–base pair region encompassing CNS28 was removed. Animals homozygous for the deletion showed significant increases in SCN5A transcripts, Na V 1.5 protein abundance, and sodium current measured in isolated ventricular myocytes. ECGs revealed a significantly shorter QRS (10.7±0.2 ms in controls versus 9.7±0.2 ms in knockouts), indicating more rapid ventricular conduction. In vitro analysis of CNS28 identified a short 3′ segment within this region required for regulatory activity and including an E-box motif. Deletion of this segment reduced reporter activity to 3.6%±0.3% of baseline in CHO cells and 16%±3% in myocytes (both P P Conclusions: These findings establish that regulation of cardiac sodium channel expression modulates channel function in vivo, and identify a noncoding region underlying this regulation.

Our previous works have demonstrated the ability of our localized orbital correction (LOC) methodology to greatly improve the accuracy of various thermochemical properties at the stationary points of the density functional theory (DFT) reaction coordinate (RC). Herein, we extend this methodology from stationary points to the entire RC connecting any stationary points by developing continuous localized orbital corrections (CLOCs). We show that the resultant method, DFT-CLOC, is capable of producing RCs with far greater accuracy than uncorrected DFT and yet requires negligible computational cost beyond the uncorrected DFT calculations. Various post-Hartree−Fock (post-HF) reaction coordinate profiles were used, including a sigmatropic shift, Diels−Alder reaction, electrocyclization, carbon radical, and three hydrogen radical reactions to show that this method is robust across multiple reaction types of general interest.

The urea cycle is the primary means of nitrogen metabolism in humans and other ureotelic organisms. There are five key enzymes in the urea cycle: carbamoyl-phosphate synthetase 1 (CPS1), ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS1), argininosuccinate lyase (ASL), and arginase 1 (ARG1). Additionally, a sixth enzyme, N-acetylglutamate synthase (NAGS), is critical for urea cycle function, providing CPS1 with its necessary cofactor. Deficiencies in any of these enzymes result in elevated blood ammonia concentrations, which can have detrimental effects, including central nervous system dysfunction, brain damage, coma, and death. Functional variants, which confer susceptibility for disease or dysfunction, have been described for enzymes within the cycle; however, a comprehensive screen of all the urea cycle enzymes has not been performed. We examined the exons and intron/exon boundaries of the five key urea cycle enzymes, NAGS, and two solute carrier transporter genes (SLC25A13 and SLC25A15) for sequence alterations using single-stranded conformational polymorphism (SSCP) analysis and high-resolution melt profiling. SSCP was performed on a set of DNA from 47 unrelated North American individuals with a mixture of ethnic backgrounds. High-resolution melt profiling was performed on a nonoverlapping DNA set of either 47 or 100 unrelated individuals with a mixture of backgrounds. We identified 33 unarchived polymorphisms in this screen that potentially play a role in the variation observed in urea cycle function. Screening all the genes in the pathway provides a catalog of variants that can be used in investigating candidate diseases.

The stoichiometric reduction of N-carbophenoxypyridinium tetraphenylborate (6) by CpRu(P-P)H (Cp = eta(5)-cyclopentadienyl; P-P = dppe, 1,2-bis(diphenylphosphino)ethane, or dppf, 1,1'-bis(diphenylphosphino)ferrocene), and Cp*Ru(P-P)H (Cp* = eta(5)-pentamethylcyclopentadienyl; P-P = dppe) gives mixtures of 1,2- and 1,4-dihydropyridines. The stoichiometric reduction of 6 by Cp*Ru(dppf)H (5) gives only the 1,4-dihydropyridine, and 5 catalyzes the exclusive formation of the 1,4-dihydropyridine from 6, H(2), and 2,2,6,6-tetramethylpiperidine. In the stoichiometric reductions, the ratio of 1,4 to 1,2 product increases as the Ru hydrides become better one-electron reductants, suggesting that the 1,4 product arises from a two-step (e(-)/H(*)) hydride transfer. Calculations at the UB3LYP/6-311++G(3df,3pd)//UB3LYP/6-31G* level support this hypothesis, indicating that the spin density in the N-carbophenoxypyridinium radical (13) resides primarily at C4, while the positive charge in 6 resides primarily at C2 and C6. The isomeric dihydropyridines thus result from the operation of different mechanisms: the 1,2 product from a single-step H(-) transfer and the 1,4 product from a two-step (e(-)/H(*)) transfer.

Background— The widely used macrolide antibiotic azithromycin increases risk of cardiovascular and sudden cardiac death, although the underlying mechanisms are unclear. Case reports, including the one we document here, demonstrate that azithromycin can cause rapid, polymorphic ventricular tachycardia in the absence of QT prolongation, indicating a novel proarrhythmic syndrome. We investigated the electrophysiological effects of azithromycin in vivo and in vitro using mice, cardiomyocytes, and human ion channels heterologously expressed in human embryonic kidney (HEK 293) and Chinese hamster ovary (CHO) cells. Methods and Results— In conscious telemetered mice, acute intraperitoneal and oral administration of azithromycin caused effects consistent with multi-ion channel block, with significant sinus slowing and increased PR, QRS, QT, and QTc intervals, as seen with azithromycin overdose. Similarly, in HL-1 cardiomyocytes, the drug slowed sinus automaticity, reduced phase 0 upstroke slope, and prolonged action potential duration. Acute exposure to azithromycin reduced peak SCN5A currents in HEK cells (IC 50 =110±3 μmol/L) and Na + current in mouse ventricular myocytes. However, with chronic (24 hour) exposure, azithromycin caused a ≈2-fold increase in both peak and late SCN5A currents, with findings confirmed for I Na in cardiomyocytes. Mild block occurred for K + currents representing I Kr (CHO cells expressing hERG; IC 50 =219±21 μmol/L) and I Ks (CHO cells expressing KCNQ1+KCNE1; IC 50 =184±12 μmol/L), whereas azithromycin suppressed L-type Ca ++ currents (rabbit ventricular myocytes, IC 50 =66.5±4 μmol/L) and I K1 (HEK cells expressing Kir2.1, IC 50 =44±3 μmol/L). Conclusions— Chronic exposure to azithromycin increases cardiac Na + current to promote intracellular Na + loading, providing a potential mechanistic basis for the novel form of proarrhythmia seen with this macrolide antibiotic.

CPSI deficiency is an inborn error of metabolism caused by mutations in the first, rate-determining enzyme of the urea cycle. Our mutation detection data from this disorder suggest that a significant number of mutant alleles cause RNA instability, most likely through the nonsense-mediated decay pathway. We identified 26 non-consanguinous CPSID patients with an available RNA source (liver tissue or cell line) and screened both genomic DNA and RNA for the identification and classification of mutations. Out of 52 total alleles screened from these patients, 21 (40%) have strong evidence for RNA processing mutations demonstrated by absent/minimal heterozygosity in patient cDNA sequences despite heterozygous genomic changes. These 21 alleles are a heterogenous group primarily composed of splicing defects and frameshifts that form premature termination codons which should subsequently elicit the nonsense-mediated decay pathway. This study provides evidence for the high prevalence of RNA instability mutations in genetic disease and underscores the importance of accounting for them in mutation-screening strategies.

“When did you last see a love story in which the honeymoon ended with a passionate political debate?” —Playboy (“Movies” 26)

This study evaluated current hospital-based palliative care programs using recommendations from the Center to Advance Palliative Care (CAPC) as a framework. Seven hospitals located in Buffalo, New York were included based on the existence of a hospital-based palliative care program. Data was collected from August through October of 2013 by means of key informant interviews with nine staff members from these hospitals using a guide comprised of questions based on CAPC’s recommendations. A gap analysis was conducted to analyze the current state of each hospital’s program based upon CAPC’s definition of a quality palliative care program. The findings identify challenges facing both existing/evolving palliative care programs, and establish a foundation for strategies to attain best practices not yet implemented. This study affirms the growing availability of palliative care services among these selected hospitals along with opportunities to improve the scope of services in line with national recommendations.