Single-cell ionic current phenotyping elucidates non-canonical features and predictive potential of cardiomyocytes during automated drug experiments.

All new drugs must go through preclinical screening tests to determine their proarrhythmic potential. While these assays effectively filter out dangerous drugs, they are too conservative, often misclassifying safe compounds as proarrhythmic. In this study, we attempt to address this shortcoming with a novel, medium-throughput drug-screening approach: we use an automated patch-clamp system to acquire optimized voltage clamp (VC) and action potential (AP) data from human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) at several drug concentrations (baseline, 3×, 10× and 20× the effective free plasma concentrations). With our novel method, we show correlations between INa block and upstroke slowing after treatment with flecainide or quinine. Additionally, after quinine treatment, we identify significant reductions in current during voltage steps designed to isolate If and IKs. However, we do not detect any IKr block by either drug, and upon further investigation, do not see any IKr present in the iPSC-CMs when prepared for automated patch experiments (i.e. in suspension) - this is in contrast to similar experiments we have conducted with these cells using the manual patch setup. In this study, we: (1) present a proof-of-concept demonstration of a single-cell medium-throughput drug study, and (2) characterize the non-canonical electrophysiology of iPSC-CMs when prepared for experiments in a medium-throughput setting. KEY POINTS: Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) offer potential as an in vitro model to study the proarrhythmic potential of drugs, but insights from these cells are often limited by the low throughput of manual patch-clamp. In this study, we use a medium-throughput automated patch-clamp system to acquire action potential (AP) and complex voltage clamp (VC) data from single iPSC-CMs at multiple drug concentrations. A correlation between AP upstroke and INa transients was identified and drug-induced changes in ionic currents found. We also characterize the substantially altered physiology of iPSC-CMs when patched in an automated system, suggesting the need to investigate differences between manual and automated patch experiments.

Stem cell-derived cardiomyocyte heterogeneity confounds electrophysiological insights.

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) offer potential as an in vitro model for studying drug cardiotoxicity and patient-specific cardiovascular disease. The inherent electrophysiological heterogeneity of these cells limits the depth of insights that can be drawn from well-designed experiments. In this review, we provide our perspective on some sources and the consequences of iPSC-CM heterogeneity. We demonstrate the extent of heterogeneity in the literature and explain how such heterogeneity is exacerbated by patch-clamp experimental artifacts in the manual and automated set-up. Finally, we discuss how this heterogeneity, caused by both intrinsic and extrinsic factors, limits our ability to build digital twins of patient-derived cardiomyocytes.

Single-cell ionic current phenotyping explains stem cell-derived cardiomyocyte action potential morphology.

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are a promising tool to study arrhythmia-related factors, but the variability of action potential (AP) recordings from these cells limits their use as an in vitro model. In this study, we use recently published brief (10 s), dynamic voltage-clamp (VC) data to provide mechanistic insights into the ionic currents contributing to AP heterogeneity; we call this approach rapid ionic current phenotyping (RICP). Features of this VC data were correlated to AP recordings from the same cells, and we used computational models to generate mechanistic insights into cellular heterogeneity. This analysis uncovered several interesting links between AP morphology and ionic current density: both L-type calcium and sodium currents contribute to upstroke velocity, rapid delayed rectifier K+ current is the main determinant of the maximal diastolic potential, and an outward current in the activation range of slow delayed rectifier K+ is the main determinant of AP duration. Our analysis also identified an outward current in several cells at 6 mV that is not reproduced by iPSC-CM mathematical models but contributes to determining AP duration. RICP can be used to explain how cell-to-cell variability in ionic currents gives rise to AP heterogeneity. Because of its brief duration (10 s) and ease of data interpretation, we recommend the use of RICP for single-cell patch-clamp experiments that include the acquisition of APs.NEW & NOTEWORTHY We present rapid ionic current phenotyping (RICP), a current quantification approach based on an optimized voltage-clamp protocol. The method captures a rich snapshot of the ionic current dynamics, providing quantitative information about multiple currents (e.g., ICa,L, IKr) in the same cell. The protocol helped to identify key ionic determinants of cellular action potential heterogeneity in iPSC-CMs. This included unexpected results, such as the critical role of IKr in establishing the maximum diastolic potential.

GJ Express: an improvement initiative to decrease sedation and anesthesia for gastrojejunostomy tube exchanges.

BACKGROUND: Overuse of sedation and anesthesia causes delays in gastrojejunostomy tube (GJ) exchanges, increased risk of complications, unnecessary use of resources, preventable hospital admissions, and an adverse impact on patient and family experience. Our hospital was over-utilizing sedation and anesthesia, and we aimed to decrease this use from 78% to 20% within two years. METHODS: An interdisciplinary quality improvement team comprehensively evaluated current processes for GJ tube exchanges through a retrospective chart review for baseline data with prospective time series analysis after improvement implementation. The primary outcome measure was the percentage of pediatric patients that utilized sedation or anesthesia for routine GJ tube exchanges. RESULTS: A statistical process control p-chart was used to calculate and show changes over time for patients (n = 45 patients average). The median percent of pediatric GJ tube exchanges performed with sedation or anesthesia decreased from 77.8% to 11.3%. Most patients (76%) were covered by Medicaid programs; with low reimbursement rates, decreased anesthesiologist billing revenue does not have a negative financial impact. CONCLUSIONS: An interprofessional improvement initiative that engaged patients and families, incorporated pediatric-specific staff services, and developed systematic weaning was associated with a significant decrease in the overuse of sedation and anesthesia for GJ tube exchanges. IMPACT: We believe that this work is highly relevant and impactful for medical centers caring for children who require gastrojejunostomy tubes, an increasingly common approach to management of children with feeding issues. There is very little literature available on the use of sedation or anesthesia for changing these tubes. While large children's medical centers in the USA usually do not utilize sedation or anesthesia, there are likely many serious outliers, especially when children receive care outside of a pediatric specific institution. This paper brings awareness to this serious issue and provides information about how we changed care to achieve higher patient safety and lower medical costs.

Communities of practice to facilitate change in health professions education: A realist synthesis.

BACKGROUND: Communities of practice could contribute to transformations in health professions education to meet complex and emerging challenges. However, little is known about the underlying mechanisms of communities of practice in this setting, and how context influences outcomes. OBJECTIVE: To understand when, why and how communities of practice with health professions education faculty work to facilitate higher education change. DESIGN: A realist synthesis according to the RAMESES standards and steps described by Pawson and colleagues. REVIEW METHODS: Early scoping of the literature informed the development of an initial program theory to describe underlying assumptions about how communities of practice in higher education, implemented with health professions education faculty, were likely to work. The theory was tested and further refined through a realist synthesis. A systematic search for evidence using search terms 'faculty', 'communities of practice' and 'higher education' and related terms was supplemented with citation tracking and hand searching of significant authors and journals. Following study appraisal, data were extracted and synthesised from 21 manuscripts describing 16 communities of practice. The realist synthesis focused on identifying patterns in context-mechanism-outcome interactions, and the alignment with substantive theory. RESULTS: From the included manuscripts, ten context-mechanism-outcome configurations were identified that describe a range of individual, interpersonal and institutional outcomes of communities of practice with health professions education faculty and context-mechanism interactions that contribute to achieving these outcomes. CONCLUSIONS: This study expands theoretical understandings of how and why communities of practice work. There is value in communities of practice in the higher education sector, primarily in the field of health professions education. Communities of practice implemented in the context of complex change with participants who have a desire to participate can facilitate change in health professions education, including institutional level changes, through reflection, experiential learning and creating a shared agenda for change. Findings from this study can be used by policy and decision-makers within health education to best apply communities of practice to achieve meaningful outcomes.

Logic-based modeling of biological networks with Netflux.

Molecular signaling networks drive a diverse range of cellular decisions, including whether to proliferate, how and when to die, and many processes in between. Such networks often connect hundreds of proteins, genes, and processes. Understanding these complex networks is greatly aided by computational modeling, but these tools require extensive programming knowledge. In this article, we describe a user-friendly, programming-free network simulation tool called Netflux (https://github.com/saucermanlab/Netflux). Over the last decade, Netflux has been used to construct numerous predictive network models that have deepened our understanding of how complex biological networks make cell decisions. Here, we provide a Netflux tutorial that covers how to construct a network model and then simulate network responses to perturbations. Upon completion of this tutorial, you will be able to construct your own model in Netflux and simulate how perturbations to proteins and genes propagate through signaling and gene-regulatory networks.

Prevalence of adverse events in pronated intubated adult COVID-19 patients: A systematic review with meta-analysis.

AIM: To present the pooled estimated prevalence of adverse events in pronated intubated adult COVID-19 patients. DESIGN: A systematic review and meta-analysis. DATA SOURCES: This study used the Cochrane Library, CINAHL, Embase, LILACS, Livivo, PubMed, Scopus, and Web of Science databases as data sources. METHODS: The studies were meta-analysed using JAMOVI 1.6.15 software. A random-effects model was used to identify the global prevalence of adverse events, confidence intervals and the heterogeneity data. Risk of bias was assessed using the Joanna Briggs Institute tool, and the certainty of evidence was assessed using the Grading of Recommendations Assessment, Development, and Evaluation approach. RESULTS: Of the 7904 studies identified, 169 were included for full reading, and 10 were included in the review. The most prevalent adverse events were pressure injuries (59%), haemodynamic instability (23%), death (17%) and device loss or traction (9%). CONCLUSION: The most prevalent adverse events in mechanically ventilated pronated patients with COVID-19 are pressure injuries, presence of haemodynamic instability, death and device loss or traction. IMPLICATIONS FOR THE PATIENT CARE: The evidence identified in this review can help improve the quality and safety of patient care by helping to design care protocols to avoid the development of adverse events that can cause permanent sequelae in these patients. IMPACT: This systematic review addressed the adverse events related to prone position in intubated adult COVID-19 patients. We identified that the most prevalent adverse events in these patients were pressure injuries, haemodynamic instability, device loss or traction and death. The results of this review may influence the clinical practice of nurses who work in intensive care units and, consequently, the nursing care provided not only to COVID-19 patients but for all intubated patients due to other reasons in intensive care units. REPORTING METHOD: This systematic review adhered to the PRISMA reporting guideline. PATIENT OR PUBLIC CONTRIBUTION: As this is a systematic review, we analysed data from primary studies conducted by many researchers. Thus, there was no patient or public contribution in this review.

Optimization of a cardiomyocyte model illuminates role of increased INa,L in repolarization reserve.

Cardiac ion currents may compensate for each other when one is compromised by a congenital or drug-induced defect. Such redundancy contributes to a robust repolarization reserve that can prevent the development of lethal arrhythmias. Most efforts made to describe this phenomenon have quantified contributions by individual ion currents. However, it is important to understand the interplay between all major ion-channel conductances, as repolarization reserve is dependent on the balance between all ion currents in a cardiomyocyte. Here, a genetic algorithm was designed to derive profiles of nine ion-channel conductances that optimize repolarization reserve in a mathematical cardiomyocyte model. Repolarization reserve was quantified using a previously defined metric, repolarization reserve current, i.e., the minimum constant current to prevent normal action potential repolarization in a cell. The optimization improved repolarization reserve current up to 84% compared to baseline in a human adult ventricular myocyte model and increased resistance to arrhythmogenic insult. The optimized conductance profiles were not only characterized by increased repolarizing current conductances but also uncovered a previously unreported behavior by the late sodium current. Simulations demonstrated that upregulated late sodium increased action potential duration, without compromising repolarization reserve current. The finding was generalized to multiple models. Ultimately, this computational approach, in which multiple currents were studied simultaneously, illuminated mechanistic insights into how the metric's magnitude could be increased and allowed for the unexpected role of late sodium to be elucidated.NEW & NOTEWORTHY Genetic algorithms are typically used to fit models or extract desired parameters from data. Here, we use the tool to produce a ventricular cardiomyocyte model with increased repolarization reserve. Since arrhythmia mitigation is dependent on multiple cardiac ion-channel conductances, study using a comprehensive, unbiased, and systems-level approach is important. The use of this optimization strategy allowed us to find robust profiles that illuminated unexpected mechanistic determinants of key ion-channel conductances in repolarization reserve.

Main Mechanisms of Remote Monitoring Programs for Cardiac Rehabilitation and Secondary Prevention: A SYSTEMATIC REVIEW.

PURPOSE: The objective of this report was to identify the main mechanisms of home-based remote monitoring programs for cardiac rehabilitation (RM CR) and examine how these mechanisms vary by context. METHODS: This was a systematic review using realist synthesis. To be included, articles had to be published in English between 2010 and November 2020 and contain specific data related to mechanisms of effect of programs. MEDLINE All (1946-) via Ovid, Embase (1974-) via Ovid, APA PsycINFO (1806-), CINAHL via EBSCO, Scopus databases, and gray literature were searched. RESULTS: From 13 747 citations, 91 focused on cardiac conditions, with 23 reports including patients in CR. Effective RM CR programs more successfully adapted to different patient home settings and broader lives, incorporated individualized patient health data, and had content designed specifically for patients in cardiac rehabilitation. Relatively minor but common technical issues could significantly reduce perceived benefits. Patients and families were highly receptive to the programs and viewed themselves as fortunate to receive such services. The RM CR programs could be improved via incorporating more connectivity to other patients. No clear negative effects on perceived utility or outcomes occurred by patient age, ethnicity, or sex. Overall, the programs were seen to best suit highly motivated patients and consolidated rather than harmed existing relationships with health care professionals and teams. CONCLUSIONS: Remote monitoring CR programs are perceived by patients to be beneficial and attractive. Future RM CR programs should consider adaptability to different home settings, incorporate individualized health data, and contain content specific to patient needs.

Relaxational dynamics of the T-number conversion of virus capsids.

We extend a recently proposed kinetic theory of virus capsid assembly based on Model A kinetics and study the dynamics of the interconversion of virus capsids of different sizes triggered by a quench, that is, by sudden changes in the solution conditions. The work is inspired by in vitro experiments on functionalized coat proteins of the plant virus cowpea chlorotic mottle virus, which undergo a reversible transition between two different shell sizes (T = 1 and T = 3) upon changing the acidity and salinity of the solution. We find that the relaxation dynamics are governed by two time scales that, in almost all cases, can be identified as two distinct processes. Initially, the monomers and one of the two types of capsids respond to the quench. Subsequently, the monomer concentration remains essentially constant, and the conversion between the two capsid species completes. In the intermediate stages, a long-lived metastable steady state may present itself, where the thermodynamically less stable species predominate. We conclude that a Model A based relaxational model can reasonably describe the early and intermediate stages of the conversion experiments. However, it fails to provide a good representation of the time evolution of the state of assembly of the coat proteins in the very late stages of equilibration when one of the two species disappears from the solution. It appears that explicitly incorporating the nucleation barriers to assembly and disassembly is crucial for an accurate description of the experimental findings, at least under conditions where these barriers are sufficiently large.

Rapid ionic current phenotyping (RICP) identifies mechanistic underpinnings of iPSC-CM AP heterogeneity.

As a renewable, easily accessible, human-derived in vitro model, human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) are a promising tool for studying arrhythmia-related factors, including cardiotoxicity and congenital proarrhythmia risks. An oft-mentioned limitation of iPSC-CMs is the abundant cell-to-cell variability in recordings of their electrical activity. Here, we develop a new method, rapid ionic current phenotyping (RICP), that utilizes a short (10 s) voltage clamp protocol to quantify cell-to-cell heterogeneity in key ionic currents. We correlate these ionic current dynamics to action potential recordings from the same cells and produce mechanistic insights into cellular heterogeneity. We present evidence that the L-type calcium current is the main determinant of upstroke velocity, rapid delayed rectifier K+ current is the main determinant of the maximal diastolic potential, and an outward current in the excitable range of slow delayed rectifier K+ is the main determinant of action potential duration. We measure an unidentified outward current in several cells at 6 mV that is not recapitulated by iPSC-CM mathematical models but contributes to determining action potential duration. In this way, our study both quantifies cell-to-cell variability in membrane potential and ionic currents, and demonstrates how the ionic current variability gives rise to action potential heterogeneity. Based on these results, we argue that iPSC-CM heterogeneity should not be viewed simply as a problem to be solved but as a model system to understand the mechanistic underpinnings of cellular variability.

Leak current, even with gigaohm seals, can cause misinterpretation of stem cell-derived cardiomyocyte action potential recordings.

AIMS: Human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have become an essential tool to study arrhythmia mechanisms. Much of the foundational work on these cells, as well as the computational models built from the resultant data, has overlooked the contribution of seal-leak current on the immature and heterogeneous phenotype that has come to define these cells. The aim of this study is to understand the effect of seal-leak current on recordings of action potential (AP) morphology. METHODS AND RESULTS: Action potentials were recorded in human iPSC-CMs using patch clamp and simulated using previously published mathematical models. Our in silico and in vitro studies demonstrate how seal-leak current depolarizes APs, substantially affecting their morphology, even with seal resistances (Rseal) above 1 GΩ. We show that compensation of this leak current is difficult due to challenges with obtaining accurate measures of Rseal during an experiment. Using simulation, we show that Rseal measures (i) change during an experiment, invalidating the use of pre-rupture values, and (ii) are polluted by the presence of transmembrane currents at every voltage. Finally, we posit that the background sodium current in baseline iPSC-CM models imitates the effects of seal-leak current and is increased to a level that masks the effects of seal-leak current on iPSC-CMs. CONCLUSION: Based on these findings, we make recommendations to improve iPSC-CM AP data acquisition, interpretation, and model-building. Taking these recommendations into account will improve our understanding of iPSC-CM physiology and the descriptive ability of models built from such data.

Effectiveness, feasibility, and acceptability of behaviour change tools used by family doctors: a global systematic review.

BACKGROUND: Priority patients in primary care include people from low-income, rural, or culturally and linguistically diverse communities, and First Nations people. AIM: To describe the effectiveness, feasibility, and acceptability of behaviour change tools that have been tested by family doctors working with priority patients. DESIGN AND SETTING: A global systematic review. METHOD: Five databases were searched for studies published from 2000 to 2021, of any design, that tested the effectiveness or feasibility of tangible, publicly available behaviour change tools used by family doctors working with priority patients. The methodological quality of each study was appraised using the Mixed Methods Appraisal Tool. RESULTS: Thirteen of 4931 studies screened met the eligibility criteria, and described 12 tools. The health-related behaviours targeted included smoking, diet and/or physical activity, alcohol and/or drug use, and suicidal ideation. Six tools had an online/web/app-based focus; the remaining six utilised only printed materials and/or in-person training. The effectiveness of the tools was assessed in 11 studies, which used diverse methods, with promising results for enabling behaviour change. The nine studies that assessed feasibility found that the tools were easy to use and enhanced the perceived quality of care. CONCLUSION: Many of the identified behaviour change tools were demonstrated to be effective at facilitating change in a target behaviour and/or feasible for use in practice. The tools varied across factors, such as the mode of delivery and the way the tool was intended to influence behaviour. There is clear opportunity to build on existing tools to enable family doctors to assist priority patients towards achieving healthier lifestyles.

Reconstructing the dietary habits and trophic positions of the Longipterygidae (Aves: Enantiornithes) using neontological and comparative morphological methods.

The Longipterygidae are a unique clade among the enantiornithines in that they exhibit elongate rostra (≥60% total skull length) with dentition restricted to the distal tip of the rostrum, and pedal morphologies suited for an arboreal lifestyle (as in other enantiornithines). This suite of features has made interpretations of this group's diet and ecology difficult to determine due to the lack of analogous taxa that exhibit similar morphologies together. Many extant bird groups exhibit rostral elongation, which is associated with several disparate ecologies and diets (e.g., aerial insectivory, piscivory, terrestrial carnivory). Thus, the presence of rostral elongation in the Longipterygidae only somewhat refines trophic predictions of this clade. Anatomical morphologies do not function singularly but as part of a whole and thus, any dietary or ecological hypothesis regarding this clade must also consider other features such as their unique dentition. The only extant group of dentulous volant tetrapods are the chiropterans, in which tooth morphology and enamel thickness vary depending upon food preference. Drawing inferences from both avian bill proportions and variations in the dental morphology of extinct and extant taxa, we provide quantitative data to support the hypothesis that the Longipterygidae were animalivorous, with greater support for insectivory.

Need for Tympanostomy Tubes in Children With Recurrent Acute Otitis Media Without Middle Ear Effusion.

OBJECTIVE: Children with recurrent acute otitis media (RAOM) presenting without middle ear effusion (MEE) do not meet indications for surgical intervention as outlined by Clinical Practice Guidelines (CPGs). The objective of this study was to determine which patients presenting with RAOM without MEE ultimately received tympanostomy tubes. STUDY DESIGN: Case series. SETTING: Single academic pediatric otolaryngology clinic. METHODS: Children (0-12 years) presenting with RAOM and no MEE were identified from October 2017 to December 2019. As per CPGs, no surgery was offered initially. Patients were given a semiurgent return appointment should they experience another suspected otitis media episode. If MEE was observed, tympanostomy tube insertion was offered. Patients were followed for 1-year following enrollment. RESULTS: One-hundred and twenty-four patients were included. The median age was 3.15 years old (interquartile range: 4.10). Seventy-five (60%) patients did not require additional follow-up and thus did not require tympanostomy tubes. Forty-nine (40%) patients were seen again; of these, 11 patients received tympanostomy tubes. Therefore, of patients presenting with no MEE, 91% did not require tympanostomy tubes. Patients who had surgery were younger on initial assessment than those who did not (mean difference 2.68 years, 95% confidence interval: 2.14-3.23). CONCLUSION: This study demonstrates the practical effect of adhering to CPGs for RAOM and suggests that many children may not require tympanostomy tube placement within the 1st year after the consultation if they did not initially present with MEE.

A potential requirement for Smad3 phosphorylation in Notch-mediated EMT in colon cancer.

Colorectal cancer (CRC) remains a challenging disease to treat due to several factors including stemness and epithelial to mesenchymal transition (EMT). Dysfunctional signaling pathways such as Notch and TGF-ß contribute to these phenomena. We previously found that cells expressing constitutively active Notch1 also had increased expression of Smad3, an important member of the TGF-ß signaling pathway. We hypothesized that Smad3, mediates the Notch-induced stemness and EMT observed in CRC cells. The human colorectal carcinoma cell line HCT-116, stably transduced with constitutively active Notch-1 (ICN) or a GFP-vector control was treated with different combinations of TGF-ß1, DAPT (a Notch inhibitor), or SIS3 (a Smad3 inhibitor). Western blot analysis was performed to determine the effects of Smad3 stimulation and inhibition on Notch and potential downstream EMT-related targets, CD44, Slug and Snail. Smad3 inhibition induced a decrease in Notch1 and Notch3 receptor expression and effectively inhibited CD44, Slug, and Snail expression. Colosphere forming ability was also reduced in cells with inhibited Smad3. These results indicate a key role of TGF-ß signaling in Notch1-induced tumorigenesis, and suggest a potential use for Smad3 inhibitors in combination with Notch1 inhibitors that are already in use for CRC treatments.

A Retrospective Review of Hospitalized Patients Receiving a Higher than Maximum Dose of Acetaminophen.

BACKGROUND: Although the 3 to 4 gram per 24 hours dose recommended for daily use are generally safe, case reports and some series raise concerns about nonacute excessive doses in some individuals. OBJECTIVE: To assess the safety of dosing more than 4 grams of acetaminophen in a 24-hour period in hospitalized patients and develop a method to evaluate the ongoing practice of acetaminophen dosing. Methods: We performed a retrospective chart review of supratherapeutic doses of acetaminophen over a 2-year period. Outcomes included death and the need for liver transplant. A "best practices alert" (BPA) was then developed in our EMR when more than 4 grams of acetaminophen was either prescribed or administered in a 24- hour period. Twelve months of alerts were then retrospectively reviewed and evaluated. RESULTS: 152 cases of dosing more than 4 grams were initially identified. No cases of death related to liver failure or liver transplant were found in any of these patients. 482 cases were identified after a BPA was put in place where the alert was overridden. There were no deaths and no cases that required liver transplantation due to liver failure. The majority of overrides were due to the allowed window of timing for nursing administration of acetaminophen for scheduled doses and overlap with as needed dosing. CONCLUSION: Supratherapeutic dosing of acetaminophen in our patients did not lead to death or liver transplant. A BPA in our EMR has allowed better evaluation of patterns of acetaminophen use at our university health system.