Computationally restoring the potency of a clinical antibody against Omicron.

The COVID-19 pandemic underscored the promise of monoclonal antibody-based prophylactic and therapeutic drugs1-3 and revealed how quickly viral escape can curtail effective options4,5. When the SARS-CoV-2 Omicron variant emerged in 2021, many antibody drug products lost potency, including Evusheld and its constituent, cilgavimab4-6. Cilgavimab, like its progenitor COV2-2130, is a class 3 antibody that is compatible with other antibodies in combination4 and is challenging to replace with existing approaches. Rapidly modifying such high-value antibodies to restore efficacy against emerging variants is a compelling mitigation strategy. We sought to redesign and renew the efficacy of COV2-2130 against Omicron BA.1 and BA.1.1 strains while maintaining efficacy against the 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 subsequent variants of concern, and provides protection in vivo against the strains tested: WA1/2020, BA.1.1 and BA.5. Deep mutational scanning of tens of thousands of pseudovirus variants reveals that 2130-1-0114-112 improves broad potency without increasing escape liabilities. Our results suggest that computational approaches can optimize an antibody to target multiple escape variants, while simultaneously enriching potency. Our computational approach does not require experimental iterations or pre-existing binding data, thus enabling rapid response strategies to address escape variants or lessen escape vulnerabilities.

The electrophysiologic effects of KCNQ1 extend beyond expression of IKs: evidence from genetic and pharmacologic block.

AIMS: While variants in KCNQ1 are the commonest cause of the congenital long QT syndrome, we and others find only a small IKs in cardiomyocytes from human-induced pluripotent stem cells (iPSC-CMs) or human ventricular myocytes. METHODS AND RESULTS: We studied population control iPSC-CMs and iPSC-CMs from a patient with Jervell and Lange-Nielsen (JLN) syndrome due to compound heterozygous loss-of-function (LOF) KCNQ1 variants. We compared the effects of pharmacologic IKs block to those of genetic KCNQ1 ablation, using JLN cells, cells homozygous for the KCNQ1 LOF allele G643S, or siRNAs reducing KCNQ1 expression. We also studied the effects of two blockers of IKr, the other major cardiac repolarizing current, in the setting of pharmacologic or genetic ablation of KCNQ1: moxifloxacin, associated with a very low risk of drug-induced long QT, and dofetilide, a high-risk drug. In control cells, a small IKs was readily recorded but the pharmacologic IKs block produced no change in action potential duration at 90% repolarization (APD90). In contrast, in cells with genetic ablation of KCNQ1 (JLN), baseline APD90 was markedly prolonged compared with control cells (469 ± 20 vs. 310 ± 16 ms). JLN cells displayed increased sensitivity to acute IKr block: the concentration (µM) of moxifloxacin required to prolong APD90 100 msec was 237.4 [median, interquartile range (IQR) 100.6-391.6, n = 7] in population cells vs. 23.7 (17.3-28.7, n = 11) in JLN cells. In control cells, chronic moxifloxacin exposure (300 µM) mildly prolonged APD90 (10%) and increased IKs, while chronic exposure to dofetilide (5 nM) produced greater prolongation (67%) and no increase in IKs. However, in the siRNA-treated cells, moxifloxacin did not increase IKs and markedly prolonged APD90. CONCLUSION: Our data strongly suggest that KCNQ1 expression modulates baseline cardiac repolarization, and the response to IKr block, through mechanisms beyond simply generating IKs.

Computationally restoring the potency of a clinical antibody against SARS-CoV-2 Omicron subvariants.

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 Evusheld™ and its constituent, cilgavimab4,6. Cilgavimab, like its progenitor COV2-2130, is a class 3 antibody that is compatible with other antibodies in combination4 and 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 protection in vivo against 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.

Functional Assays Reclassify Suspected Splice-Altering Variants of Uncertain Significance in Mendelian Channelopathies.

BACKGROUND: Rare protein-altering variants in SCN5A, KCNQ1, and KCNH2 are major causes of Brugada syndrome and the congenital long QT syndrome. While splice-altering variants lying outside 2-bp canonical splice sites can cause these diseases, their role remains poorly described. We implemented 2 functional assays to assess 12 recently reported putative splice-altering variants of uncertain significance and 1 likely pathogenic variant without functional data observed in Brugada syndrome and long QT syndrome probands. METHODS: We deployed minigene assays to assess the splicing consequences of 10 variants. Three variants incompatible with the minigene approach were introduced into control induced pluripotent stem cells by CRISPR genome editing. We differentiated cells into induced pluripotent stem cell-derived cardiomyocytes and studied splicing outcomes by reverse transcription-polymerase chain reaction. We used the American College of Medical Genetics and Genomics functional assay criteria (PS3/BS3) to reclassify variants. RESULTS: We identified aberrant splicing, with presumed disruption of protein sequence, in 8/10 variants studied using the minigene assay and 1/3 studied in induced pluripotent stem cell-derived cardiomyocytes. We reclassified 8 variants of uncertain significance to likely pathogenic, 1 variant of uncertain significance to likely benign, and 1 likely pathogenic variant to pathogenic. CONCLUSIONS: Functional assays reclassified splice-altering variants outside canonical splice sites in Brugada Syndrome- and long QT syndrome-associated genes.

Dominant negative effects of SCN5A missense variants.

PURPOSE: Up to 30% of patients with Brugada syndrome (BrS) carry loss-of-function (LoF) variants in the cardiac sodium channel gene SCN5A encoding for the protein NaV1.5. Recent studies suggested that NaV1.5 can dimerize, and some variants exert dominant negative effects. In this study, we sought to explore the generality of missense variant NaV1.5 dominant negative effects and their clinical severity. METHODS: We identified 35 LoF variants (<10% of wild type [WT] peak current) and 15 partial LoF variants (10%-50% of WT peak current) that we assessed for dominant negative effects. SCN5A variants were studied in HEK293T cells, alone or in heterozygous coexpression with WT SCN5A using automated patch clamp. To assess the clinical risk, we compared the prevalence of dominant negative vs putative haploinsufficient (frameshift, splice, or nonsense) variants in a BrS consortium and the Genome Aggregation Database population database. RESULTS: In heterozygous expression with WT, 32 of 35 LoF and 6 of 15 partial LoF variants showed reduction to <75% of WT-alone peak current, showing a dominant negative effect. Individuals with dominant negative LoF variants had an elevated disease burden compared with the individuals with putative haploinsufficient variants (2.7-fold enrichment in BrS cases, P = .019). CONCLUSION: Most SCN5A missense LoF variants exert a dominant negative effect. This class of variant confers an especially high burden of BrS.

Evaluating an inpatient deprescribing initiative at a rural community hospital in Ontario.

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.

External Loop Recorders: Primary Care Placement Is Noninferior to Hospital-Based Cardiac Unit.

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.

High-Throughput Reclassification of SCN5A Variants.

Partial or complete loss-of-function variants in SCN5A are the most common genetic cause of the arrhythmia disorder Brugada syndrome (BrS1). However, the pathogenicity of SCN5A variants is often unknown or disputed; 80% of the 1,390 SCN5A missense variants observed in at least one individual to date are variants of uncertain significance (VUSs). The designation of VUS is a barrier to the use of sequence data in clinical care. We selected 83 variants: 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 individuals 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. The ten previously studied controls had functional properties concordant with published manual patch clamp data. All 10 suspected benign variants had wild-type-like function. 22 suspected BrS variants had loss of channel function (<10% normalized peak current) and 22 variants had partial loss of function (10%-50% normalized peak current). The previously unstudied variants were initially classified as likely benign (n = 2), likely pathogenic (n = 10), or VUSs (n = 61). After the patch clamp studies, 16 variants were benign/likely benign, 45 were pathogenic/likely pathogenic, and only 12 were still VUSs. Structural modeling identified likely mechanisms for loss of function including altered thermostability and disruptions to alpha helices, disulfide bonds, or the permeation pore. High-throughput patch clamping enabled reclassification of the majority of tested VUSs in SCN5A.

Improving urinary catheterisation practices in a rural hospital in Ontario.

INTRODUCTION: A 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. METHODS: In 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. RESULTS: This 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/CONCLUSION: This 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%.

SCN5A variant R222Q generated abnormal changes in cardiac sodium current and action potentials in murine myocytes and Purkinje cells.

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. 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. INa 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 (action potential durations at 90% repolarization: 12.6 ± 1.3 ms vs 29.1 ± 1.0 ms in H/H, P < .01, n = 10 each). We identified a large [K+]o-dependent outward gating pore current in RQ/H but not H/H myocytes, and decreasing [K+]o elicited early afterdepolarizations (EADs) and triggered activity in isolated myocytes and ectopic beats in whole hearts. Further, RQ/H Purkinje cells displayed striking, consistent low-voltage EADs. In vivo, however, RQ/H mice displayed little ectopy and contractile function was normal. CONCLUSION: While SCN5A R222Q increases plateau inward sodium current, action potentials were unexpectedly shortened, likely reflecting an outward gating-pore current. Low extracellular potassium increased this pore current, and was arrhythmogenic in vitro and ex vivo.

Azithromycin Causes a Novel Proarrhythmic Syndrome.

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 (IC50=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 INa in cardiomyocytes. Mild block occurred for K+ currents representing IKr (CHO cells expressing hERG; IC50=219±21 µmol/L) and IKs (CHO cells expressing KCNQ1+KCNE1; IC50=184±12 µmol/L), whereas azithromycin suppressed L-type Ca++ currents (rabbit ventricular myocytes, IC50=66.5±4 µmol/L) and IK1 (HEK cells expressing Kir2.1, IC50=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.

Contrasting Nav1.8 Activity in Scn10a-/- Ventricular Myocytes and the Intact Heart.

BACKGROUND: Genome-wide association studies have implicated variants in SCN10A, which encodes Nav1.8, as modulators of cardiac conduction. Follow-up work has indicated the SCN10A sequence includes an intronic enhancer for SCN5A. 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 QTc 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 SCN10A that affect ventricular conduction do so by modulating SCN5A.

Grief intensity, psychological well-being, and the intimate partner relationship in the subsequent pregnancy after a perinatal loss.

OBJECTIVE: To examine the construct validity of the Perinatal Grief Intensity Scale (PGIS) and the associations of grief intensity with psychological well-being and the quality of intimate partner relationships of women in the subsequent pregnancy after perinatal loss. The consequences of intense grief due to perinatal loss may include significant couple relationship issues, depression, anxiety, and post-traumatic stress that may extend into the subsequent healthy pregnancy. DESIGN AND SETTING: A correlational, descriptive research design was used to collect survey data in this cross-sectional, web-based study. PARTICIPANTS: Participants were 227 currently pregnant women who experienced perinatal loss in their immediate past pregnancies. METHODS: Instruments included the Pregnancy Outcome Questionnaire (pregnancy-specific anxiety), Impact of Event Scale (post-traumatic stress), Center for Epidemiologic Studies-Depression Scale (depression symptoms), the Autonomy and Relatedness Inventory (quality of the intimate partner relationship), and the Perinatal Grief Intensity Scale (perinatal grief intensity). RESULTS: As hypothesized, greater grief intensity was associated with higher pregnancy-specific anxiety, depression symptoms, and post-traumatic stress as well as poorer quality of the intimate partner relationship. CONCLUSIONS: Support for the construct validity of the PGIS was demonstrated by its significant associations in the expected directions with pregnancy-specific anxiety, depression symptoms, post-traumatic stress, and the quality of the intimate partner relationship. The scale may be useful to health care providers in identifying mothers in need of follow-up for intense grief and other clinically relevant symptoms after perinatal loss.

Blocking Scn10a channels in heart reduces late sodium current and is antiarrhythmic.

RATIONALE: Although the sodium channel locus SCN10A has been implicated by genome-wide association studies as a modulator of cardiac electrophysiology, the role of its gene product Nav1.8 as a modulator of cardiac ion currents is unknown. OBJECTIVE: We determined the electrophysiological and pharmacological properties of Nav1.8 in heterologous cell systems and assessed the antiarrhythmic effect of Nav1.8 block on isolated mouse and rabbit ventricular cardiomyocytes. METHODS AND RESULTS: We first demonstrated that Scn10a transcripts are identified in mouse heart and that the blocker A-803467 is highly specific for Nav1.8 current over that of Nav1.5, the canonical cardiac sodium channel encoded by SCN5A. We then showed that low concentrations of A-803467 selectively block "late" sodium current and shorten action potentials in mouse and rabbit cardiomyocytes. Exaggerated late sodium current is known to mediate arrhythmogenic early afterdepolarizations in heart, and these were similarly suppressed by low concentrations of A-803467. CONCLUSIONS: Scn10a expression contributes to late sodium current in heart and represents a new target for antiarrhythmic intervention.

Increased late sodium current contributes to long QT-related arrhythmia susceptibility in female mice.

AIMS: Female gender is a risk factor for long QT-related arrhythmias, but the underlying mechanisms remain uncertain. Here, we tested the hypothesis that gender-dependent function of the post-depolarization 'late' sodium current (I(Na-L)) contributes. METHODS AND RESULTS: Studies were conducted in mice in which the canonical cardiac sodium channel Scn5a locus was disrupted, and expression of human wild-type SCN5A cDNA substituted. Baseline QT intervals were similar in male and female mice, but exposure to the sodium channel opener anemone toxin ATX-II elicited polymorphic ventricular tachycardia in 0/9 males vs. 6/9 females. Ventricular I(Na-L) and action potential durations were increased in myocytes isolated from female mice compared with those from males before and especially after treatment with ATX-II. Further, ATX-II elicited potentially arrhythmogenic early afterdepolarizations in myocytes from 0/5 male mice and 3/5 female mice. CONCLUSION: These data identify variable late I(Na) as a modulator of gender-dependent arrhythmia susceptibility.

Striking In vivo phenotype of a disease-associated human SCN5A mutation producing minimal changes in vitro.

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 ß1 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<0.001 vs. H/H; DN/DN, 9.3±1.1 pA/pF, P<0.001 versus H/H). 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.

Lessons from a patient experience survey in a randomized surgical trial of treatment of stress urinary incontinence in women.

INTRODUCTION AND HYPOTHESIS: To understand the patient burden of study procedures/measures at completion of a randomized controlled trial (RCT) requiring extensive testing and follow-up visits. METHODS: A survey sent after completing the 2-year visit of an RCT comparing Burch colposuspension and fascial retropubic sling to treat stress urinary incontinence assessed degree of bother for seven study procedures, eight study-related factors, and possible motivations to participate in the study. RESULTS: A total of 450 study participants (88%) returned the survey. Urodynamic testing was the most bothersome procedure, followed by 24-h pad test and Q-tip test. Self-administered questionnaires were the least bothersome. Main reasons to participate in the study were to help others, obtain better knowledge about the condition, and be guided by a committed team of investigators/study coordinators. CONCLUSIONS: At the end of a large multicenter RCT, we learned from a confidential patient survey that the most burdensome activities involved invasive procedures, frequent visits, and multiple forms to fill out.

Informatic and functional approaches to identifying a regulatory region for the cardiac sodium channel.

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<0.05), and mutation of individual sites in the E-box restored activity to 62%±4% and 57%±2% of baseline in CHO cells and myocytes, respectively (both P<0.05). CONCLUSIONS: These findings establish that regulation of cardiac sodium channel expression modulates channel function in vivo, and identify a noncoding region underlying this regulation.

The importance of understanding military culture.

Social workers can make a significant contribution to military service members and their families, but first it is essential that the worldview, the mindset, and the historical perspective of life in the military are understood. Unless we understand how the unique characteristics of the military impact the service members and their families, we cannot work effectively with them. In addition, unless we understand their language, their structure, why they join, their commitment to the mission, and the role of honor and sacrifice in military service, we will not be able to adequately intervene and offer care to these families.

Molecular defects in human carbamoy phosphate synthetase I: mutational spectrum, diagnostic and protein structure considerations.

Deficiency of carbamoyl phosphate synthetase I (CPSI) results in hyperammonemia ranging from neonatally lethal to environmentally induced adult-onset disease. Over 24 years, analysis of tissue and DNA samples from 205 unrelated individuals diagnosed with CPSI deficiency (CPSID) detected 192 unique CPS1 gene changes, of which 130 are reported here for the first time. Pooled with the already reported mutations, they constitute a total of 222 changes, including 136 missense, 15 nonsense, 50 changes of other types resulting in enzyme truncation, and 21 other changes causing in-frame alterations. Only ∼10% of the mutations recur in unrelated families, predominantly affecting CpG dinucleotides, further complicating the diagnosis because of the "private" nature of such mutations. Missense changes are unevenly distributed along the gene, highlighting the existence of CPSI regions having greater functional importance than other regions. We exploit the crystal structure of the CPSI allosteric domain to rationalize the effects of mutations affecting it. Comparative modeling is used to create a structural model for the remainder of the enzyme. Missense changes are found to directly correlate, respectively, with the one-residue evolutionary importance and inversely correlate with solvent accessibility of the mutated residue. This is the first large-scale report of CPS1 mutations spanning a wide variety of molecular defects highlighting important regions in this protein.