Cytological evaluation, culture and genomics to evaluate the microbiome in healthy rabbit external ear canals.

BACKGROUND: Lop-eared rabbits may be predisposed to otitis externa (OE) as a consequence of their ear conformation. Although otoscopy, otic cytological evaluation and culture are valuable tools in dogs and cats, published data on rabbits remain lacking. HYPOTHESIS/OBJECTIVES: This study aimed to assess the utility of otoscopy and cytological results in evaluating healthy rabbit external ear canals (EECs) and to characterise ear cytological and microbiological findings through culture techniques and metagenomic sequencing. ANIMALS: Sixty-three otitis-free client-owned rabbits. MATERIALS AND METHODS: All rabbits underwent otoscopy and ear cytological evaluation. In a subset of 12 rabbits, further bacterial and fungal culture, fungal DNA assessment and metagenomic sequencing were performed. RESULTS: Otic cytological results revealed yeast in 73%, cocci in 42.9% and rods in 28.6% of healthy rabbit EECs. Compared to upright-eared rabbits, lop-eared rabbits had more discharge and more bacteria per oil immersion field. Culture isolated eight different species yet metagenomic sequencing identified 36, belonging to the Bacillota (Firmicutes), Pseudomonadota and Actinomycetota phyla. Staphylococcus were the most commonly observed species with both methods. Ten of 12 rabbits were yeast-positive on cytological evaluation with only three yielding fungal growth identified as Yarrowia (Candida) lipolytica, Eurotium echinulatum and Cystofilobasidium infirmominiatum. CONCLUSIONS AND CLINICAL RELEVANCE: Healthy rabbit EECs lack inflammatory cells yet can host yeast and bacteria, emphasising the need to evaluate cytological results alongside the clinical signs. Lop-ear anatomy may predispose to bacterial overgrowth and OE. Notably, yeasts may be present despite a negative culture.

Seeing Keratinocyte Proteins through the Looking Glass of Intrinsic Disorder.

Epidermal keratinocyte proteins include many with an eccentric amino acid content (compositional bias), atypical ultrastructural fate (built-in protease sensitivity), or assembly visible at the light microscope level (cytoplasmic granules). However, when considered through the looking glass of intrinsic disorder (ID), these apparent oddities seem quite expected. Keratinocyte proteins with highly repetitive motifs are of low complexity but high adaptation, providing polymers (e.g., profilaggrin) for proteolysis into bioactive derivatives, or monomers (e.g., loricrin) repeatedly cross-linked to self and other proteins to shield underlying tissue. Keratohyalin granules developing from liquid-liquid phase separation (LLPS) show that unique biomolecular condensates (BMC) and proteinaceous membraneless organelles (PMLO) occur in these highly customized cells. We conducted bioinformatic and in silico assessments of representative keratinocyte differentiation-dependent proteins. This was conducted in the context of them having demonstrated potential ID with the prospect of that characteristic driving formation of distinctive keratinocyte structures. Intriguingly, while ID is characteristic of many of these proteins, it does not appear to guarantee LLPS, nor is it required for incorporation into certain keratinocyte protein condensates. Further examination of keratinocyte-specific proteins will provide variations in the theme of PMLO, possibly recognizing new BMC for advancements in understanding intrinsically disordered proteins as reflected by keratinocyte biology.

The Small Conductance Calcium-Activated Potassium Channel Inhibitors NS8593 and UCL1684 Prevent the Development of Atrial Fibrillation Through Atrial-Selective Inhibition of Sodium Channel Activity.

The mechanisms underlying atrial-selective prolongation of effective refractory period (ERP) and suppression of atrial fibrillation (AF) by NS8593 and UCL1684, small conductance calcium-activated potassium (SK) channel blockers, are poorly defined. The purpose of the study was to confirm the effectiveness of these agents to suppress AF and to probe the underlying mechanisms. Transmembrane action potentials and pseudoelectrocardiograms were recorded from canine isolated coronary-perfused canine atrial and ventricular wedge preparations. Patch clamp techniques were used to record sodium channel current (INa) in atrial and ventricular myocytes and human embryonic kidney cells. In both atria and ventricles, NS8593 (3-10 µM) and UCL1684 (0.5 µM) did not significantly alter action potential duration, suggesting little to no SK channel inhibition. Both agents caused atrial-selective: (1) prolongation of ERP secondary to development of postrepolarization refractoriness, (2) reduction of Vmax, and (3) increase of diastolic threshold of excitation (all are sodium-mediated parameters). NS8593 and UCL1684 significantly reduced INa density in human embryonic kidney cells as well as in atrial but not in ventricular myocytes at physiologically relevant holding potentials. NS8593 caused a shift of steady-state inactivation to negative potentials in atrial but not ventricular cells. NS8593 and UCL1684 prevented induction of acetylcholine-mediated AF in 6/6 and 8/8 preparations, respectively. This anti-AF effect was associated with strong rate-dependent depression of excitability. The SK channel blockers, NS8593 and UCL1684, are effective in preventing the development of AF due to potent atrial-selective inhibition of INa, causing atrial-selective prolongation of ERP secondary to induction of postrepolarization refractoriness.

Coagulopathy Characterized by Rotational Thromboelastometry in a Porcine Pediatric ECMO Model.

Venoarterial extracorporeal membrane oxygenation (VA-ECMO) is used to support patients with reversible cardiopulmonary insufficiency. Although it is a lifesaving technology, bleeding, inflammation, and thrombosis are well-described complications of ECMO. Adult porcine models of ECMO have been used to recapitulate the physiology and hemostatic consequences of ECMO cannulation in adults. However, these models lack the unique physiology and persistence of fetal forms of coagulation factors and fibrinogen as in human infants. We aimed to describe physiologic and coagulation parameters of piglets cannulated and supported with VA-ECMO. Four healthy piglets (5.7-6.4 kg) were cannulated via jugular vein and carotid artery by cutdown and supported for a maximum of 20 hours. Heparin was used with a goal activated clotting time of 180-220 seconds. Arterial blood gas (ABG) was performed hourly, and blood was transfused from an adult donor to maintain hematocrit (Hct) > 24%. Rotational thromboelastometry (ROTEM) was performed at seven time points. All animals achieved adequate flow with a patent circuit throughout the run (pre- and post-oxygenator pressure gradient <10 mmHg). There was slow but significant hemorrhage at cannulation, arterial line, and bladder catheter sites. All animals required the maximum blood transfusion volume available. All animals became anemic after exhaustion of blood for transfusion. ABG showed progressively declining Hct and adequate oxygenation. ROTEM demonstrated decreasing fibrin-only ROTEM (FIBTEM) clot firmness. Histology was overall unremarkable. Pediatric swine are an important model for the study of pediatric ECMO. We have demonstrated the feasibility of such a model while providing descriptions of physiologic, hematologic, and coagulation parameters throughout. Weak whole-blood clot firmness by ROTEM suggested defects in fibrinogen, and there was a clinical bleeding tendency in all animals studied. This model serves as an important means to study the complex derangements in hemostasis during ECMO.

Structural Basis of Protein Kinase R Autophosphorylation.

The RNA-activated protein kinase, PKR, is a key mediator of the innate immunity response to viral infection. Viral double-stranded RNAs induce PKR dimerization and autophosphorylation. The PKR kinase domain forms a back-to-back dimer. However, intermolecular ( trans) autophosphorylation is not feasible in this arrangement. We have obtained PKR kinase structures that resolves this dilemma. The kinase protomers interact via the known back-to-back interface as well as a front-to-front interface that is formed by exchange of activation segments. Mutational analysis of the front-to-front interface support a functional role in PKR activation. Molecular dynamics simulations reveal that the activation segment is highly dynamic in the front-to-front dimer and can adopt conformations conducive to phosphoryl transfer. We propose a mechanism where back-to-back dimerization induces a conformational change that activates PKR to phosphorylate a "substrate" kinase docked in a front-to-front geometry. This mechanism may be relevant to related kinases that phosphorylate the eukaryotic initiation factor eIF2α.

Structural and Functional Studies of Bacterial Enolase, a Potential Target against Gram-Negative Pathogens.

Enolase is a glycolytic metalloenzyme involved in carbon metabolism. The advantage of targeting enolase lies in its essentiality in many biological processes such as cell wall formation and RNA turnover and as a plasminogen receptor. We initially used a DARTS assay to identify enolase as a target in Escherichia coli. The antibacterial activities of α-, ß-, and γ-substituted seven-member ring tropolones were first evaluated against four strains representing a range of Gram-negative bacteria. We observed that the chemical properties and position of the substituents on the tropolone ring play an important role in the biological activity of the investigated compounds. Both α- and ß-substituted phenyl derivatives of tropolone were the most active with minimum inhibitory concentrations in the range of 11-14 µg/mL. The potential inhibitory activity of the synthetic tropolones was further evaluated using an enolase inhibition assay, X-ray crystallography, and molecular docking simulations. The catalytic activity of enolase was effectively inhibited by both the naturally occurring ß-thujaplicin and the α- and ß-substituted phenyl derivatives of tropolones with IC50 values in range of 8-11 µM. Ligand binding parameters were assessed by isothermal titration calorimetry and differential scanning calorimetry techniques and agreed with the in vitro data. Our studies validate the antibacterial potential of tropolones with careful consideration of the position and character of chelating moieties for stronger interaction with metal ions and residues in the enolase active site.

A pilot study of the in vitro antimicrobial activity and in vivo residual activity of chlorhexidine and acetic acid/boric acid impregnated cleansing wipes.

BACKGROUND: Topical antimicrobials are recommended for first line treatment of surface and superficial infections in dogs. This is especially important given the increasing prevalence of antimicrobial resistant infections. Antimicrobial wipes have become popular, but there are a lack of controlled studies assessing their in vitro antimicrobial and in vivo residual activity. We aimed to assess the antimicrobial efficacy of two commercial antimicrobial wipes against frequently isolated pathogens. Ten clinical and one reference isolate each of meticillin-susceptible Staphylococcus pseudintermedius (MSSP), meticillin-resistant S. pseudintermedius (MRSP), Escherichia coli (EC), extended spectrum beta-lactamase (ESBL) producing E. coli (ESBL-EC), Pseudomonas aeruginosa (PA) and Malassezia pachydermatis (MP) were tested using a modified Kirby-Bauer technique. Each isolate was tested against 6 mm discs of chlorhexidine (CHX) and acetic acid/boric acid (AABA) wipes, and positive and negative controls either overnight (bacteria) or for 3 days (Malassezia). Healthy dogs were treated with the wipes and distilled water on a randomised flank (n = 5 each). Hair samples (1 cm; 0.1 g) taken at days 0, 1 and 3 were inoculated with an isolate of each organism. Zones of inhibition (ZI) were measured. RESULTS: All isolates produced confluent growth with AABA and control wipes, except for the cleansing wipes and MP (median ZI 12 mm; 95% CI 8.2-15.8). The median (95% CI) CHX wipe ZIs (mm) were: MP 48.0 (47.0-49.0), MSSP 15.6 (14.2-17.0), MRSP 14.0 (13.6-14.4), EC 13.6 (12.0-15.2) and ESBL-EC 10.0 (9.4-10.6). PA showed confluent growth. The differences between the bacterial isolates was significant (Kruskal-Wallis p < 0.0001; post-tests MSSP = MRSP = EC > EBSL-EC > PA). Confluent growth was visible with all the hair samples. CONCLUSION: CHX but not AABA showed in vitro efficacy against MSSP, MRSP, EC and MP. ESBL-EC were less susceptible and there was no activity against PA. There was no residual activity on hair. Additional studies are required to determine efficacy of these products in clinically affected patients.

Biofilm production of Pseudomonas spp. isolates from canine otitis in three different enrichment broths.

BACKGROUND: Pseudomonas spp. are commonly isolated from dogs with clinical otitis and have been shown to produce biofilm. There is a paucity of studies demonstrating biofilm growth in veterinary medicine. HYPOTHESIS/OBJECTIVES: To compare biofilm production of Pseudomonas spp. isolated from dogs with otitis using three different enrichment broths at two different time points. Speciation was performed. ANIMALS: One hundred isolates from 98 dogs with clinical otitis were assessed for biofilm production. METHODS AND MATERIALS: One hundred isolates were assessed for biofilm production using a microtitre plate assay. Biofilm production in Luria-Bertani Broth (LBB), Mueller-Hinton Broth (MHB) and Tryptic Soy Broth (TSB) were assessed after 18 and 24 h of incubation. RESULTS: At 18 h, biofilm production was demonstrated in 87% of LBB, 91% of TSB and 93% of MHB grown isolates. By 24 h, this was 92% of LBB, 96% of TSB and 99% of MHB isolates. Biofilm production was significantly increased after 24 h incubation compared to 18 h. A significant difference was noted in biofilm production between LBB and MHB (P = 0.0349), but not between LBB and TSB (P = 0.3727) or MHB and TSB (P = 0.3687) at 24 h incubation. Two isolates were speciated as P. fluorescens and 98 as P. aeruginosa. CONCLUSION AND CLINICAL IMPORTANCE: Not all enrichment broths were equivalent to one another and 24 h incubation was superior to 18 h. Biofilm production was high in this population of Pseudomonas spp. isolates.

Results of a curtailed randomized controlled trial, evaluating the efficacy and safety of azimilide in patients with implantable cardioverter-defibrillators: The SHIELD-2 trial.

BACKGROUND: Frequent hospital attendances in patients with implantable cardioverter-defibrillators (ICDs) result in significant morbidity and health care costs. Current drugs to reduce ICD shocks and hospital visits have limited efficacy and considerable toxicity. We evaluated the efficacy and safety of azimilide, a novel oral class III antiarrhythmic, for use in ICD patients. METHODS: A total of 240 patients were enrolled in a prospective, randomized, double-blind, placebo-controlled trial to evaluate the effect of oral azimilide 75 mg daily in ICD patients with previously documented ventricular tachycardia or ventricular fibrillation, and a left ventricular ejection fraction ≤40%. The primary outcome metric was the adjudicated time-to-first unplanned cardiovascular (CV) hospitalization, or CV emergency department (ED) visit, or CV death. The trial was prematurely discontinued due to withdrawal of study sponsorship. RESULTS: Azimilide demonstrated numerical but statistically nonsignificant reductions in the primary composite outcome (odds ratio [OR] 0.79, 95% CI 0.44-1.44), unplanned CV hospitalizations (OR 0.75, 95% CI 0.41-1.38), ED visits (OR 0.68, 95% CI 0.35-1.31), and all-cause shocks (OR 0.58, 95% CI 0.32-1.05). The incidence of adverse events was lower in the azimilide group. Neutropenia was not observed (absolute neutrophil count <1000 µ/L), and there was one possible torsade de pointes case that led to a successful ICD discharge. CONCLUSION: The SHIELD-2 trial was statistically underpowered due to early trial termination and did not meet its primary objective. Despite this limitation, azimilide showed promise as a safe and effective drug in reducing all-cause shocks, unplanned hospitalizations, and ED visits in ICD patients.

Climate Change and European Water Bodies, a Review of Existing Gaps and Future Research Needs: Findings of the ClimateWater Project.

There is general agreement among scientists that global temperatures are rising and will continue to increase in the future. It is also agreed that human activities are the most important causes of these climatic variations, and that water resources are already suffering and will continue to be greatly impaired as a consequence of these changes. In particular, it is probable that areas with limited water resources will expand and that an increase of global water demand will occur, estimated to be around 35-60% by 2025 as a consequence of population growth and the competing needs of water uses. This will cause a growing imbalance between water demand (including the needs of nature) and supply. This urgency demands that climate change impacts on water be evaluated in different sectors using a cross-cutting approach (Contestabile in Nat Clim Chang 3:11-12, 2013). These issues were examined by the EU FP7-funded Co-ordination and support action "ClimateWater" (bridging the gap between adaptation strategies of climate change impacts and European water policies). The project studied adaptation strategies to minimize the water-related consequences of climate change and assessed how these strategies should be taken into consideration by European policies. This article emphasizes that knowledge gaps still exist about the direct effects of climate change on water bodies and their indirect impacts on production areas that employ large amounts of water (e.g., agriculture). Some sectors, such as ecohydrology and alternative sewage treatment technologies, could represent a powerful tool to mitigate climate change impacts. Research needs in these still novel fields are summarized.

Implementation and faculty perception of outpatient medical student workplace-based assessments.

BACKGROUND: There is growing interest in use of entrustable professional activity (EPA)-grounded workplace-based assessments (WBAs) to assess medical students through direct observation in the clinical setting. However, there has been very little reflection on how these tools are received by the faculty using them to deliver feedback. Faculty acceptance of WBAs is fundamentally important to sustained utilisation in the clinical setting, and understanding faculty perceptions of the WBA as an adjunct for giving targeted feedback is necessary to guide future faculty development in this area. APPROACH: Use of a formative EPA-grounded WBA was implemented in the ambulatory setting during the paediatrics clerkship following performance-driven training and frame-of-reference training with faculty. Surveys and semi-structured interviews with faculty members explored how faculty perceived the tool and its impact on feedback delivery. EVALUATION: Faculty reported providing more specific, task-oriented feedback following implementation of the WBA, as well as greater timeliness of feedback and greater satisfaction with opportunities to provide feedback, although these later two findings did not reach significance. Themes from the interviews reflected the benefits of WBAs, persistent barriers to the provision of feedback and suggestions for improvement of the WBA. IMPLICATIONS: EPA-grounded WBAs are feasible to implement in the outpatient primary care setting and improve feedback delivery around core EPAs. The WBAs positively impacted the way faculty conceptualise feedback and provide learners with more actionable, behaviour-based feedback. Findings will inform modifications to the WBA and future faculty development and training to allow for sustainable WBA utilisation in the core clerkship.

Perspective on Antiarrhythmic Drug Combinations.

Physicians use multiple drugs in combination to treat hypertension, heart failure, diabetes mellitus, angina, hyperlipidemia, and many other cardiovascular conditions and risk factors. However, administering antiarrhythmic drugs (AAD) in combination is rarely discussed. Yet, the possibility of increasing efficacy and/or tolerance and/or safety of AADs (by adding mechanisms, offsetting adverse mechanisms, and/or using lower doses) exists. Unfortunately, this topic has not been reviewed in any contemporary cardiac literature of which we are aware, although information regarding AAD combinations has been published. In conclusion, and accordingly, this review discusses the possibility of using AAD combinations for both ventricular arrhythmias and atrial fibrillation, in which the rationale for such combinations, considerations for such combinations, and supporting literature are covered.

Potential costs and consequences associated with medication error at hospital discharge: an expert judgement study.

OBJECTIVES: Assessing the cost-effectiveness of complex pharmaceutical care interventions and medication error outcomes is hindered by lack of available data on actual outcomes consequent to errors that were intercepted for patient safety reasons. Expert judgement is an approach to acquire data regarding unknown parameters in an economic model which are otherwise insufficient or not possible to obtain. The aim of this paper is to describe a method to approach this problem using findings from a single intervention study and to calculate the potential costs and consequences associated with discharge medication error. METHODS: Using data from a previous intervention study, the hypothetical consequences of medication error(s) at hospital discharge, in terms of diagnosis, healthcare resource utilisation and impact on health-related quality of life, were identified by expert judgement of anonymised cases. Primary healthcare utilisation costs were derived from published tariffs, inpatient costs were derived by simulation in the hospital discharge activity database test environment and the difference between adjudicated baseline and posterror health state was expressed as quality-adjusted life year (QALY) decrement. RESULTS: Four experts provided judgement on 81 cases. Of these, 75 were judged to have potential clinical consequences. Between 56 and 69 of the 81 cases were variably judged to require remedial healthcare utilisation. The mean calculated cost per case (representing an individual patient), based on all 81 cases, was €1009.58, 95% CI 726.64 to 1585.67. The mean QALY loss was 0.03 (95% CI 0.01 to 0.05). CONCLUSION: An expert judgement process proved feasible and useful to estimate financial cost and QALY loss associated with hospital discharge medication error. These estimates will be employed in model-based economic evaluation. This method could be transferred to other prospective observational patient safety research which seeks to assess value for money of complex interventions.

Mechanisms underlying the antiarrhythmic effect of ARumenamide-787 in experimental models of the J wave syndromes and hypothermia.

BACKGROUND: Brugada (BrS) and early repolarization syndromes (ERS), the so-called J wave syndromes (JWS), are associated with life-threatening ventricular arrhythmias. Pharmacologic approaches to therapy are currently limited. In this study, we examine the effects of ARumenamide-787 (AR-787) to suppress the electrocardiographic and arrhythmic manifestations of JWS and hypothermia. METHODS: We studied the effects of AR-787 on INa and IKr in HEK-293 cells stably expressing the α- and ß1-subunits of the cardiac (NaV1.5) sodium channel and hERG channel, respectively. In addition, we studied its effect on Ito, INa and ICa in dissociated canine ventricular myocytes along with action potentials and ECG from coronary-perfused right (RV) and left (LV) ventricular wedge preparations. The Ito agonist, NS5806 (5-10 µM), ICa blocker, verapamil (2.5 µM), and INa blocker, ajmaline (2.5 µM), were used to mimic the genetic defects associated with JWS and to induce the electrocardiographic and arrhythmic manifestations of JWS (prominent J waves/ST segment elevation, phase 2 reentry and polymorphic VT/VF) in canine ventricular wedge preparations. RESULTS: AR-787 (1, 10 and 50 µM) exerted pleiotropic effects on cardiac ion channels. The predominant effect was inhibition of the transient outward current (Ito) and enhancement of the sodium channel current (INa), with lesser effects to inhibit IKr and augment calcium channel current (ICa). AR-787 diminished the electrocardiographic J wave and prevented and/or suppressed all arrhythmic activity in canine RV and LV experimental models of BrS, ERS and hypothermia. CONCLUSIONS: Our findings point to AR-787 as promising candidate for the pharmacologic treatment of JWS and hypothermia.

UK-based multicentre cross-sectional study assessing advice requests veterinary dermatologists received from general practitioners.

BACKGROUND: There is little currently little information available regarding the nature of the advice requests veterinary dermatologists receive from general practitioners. Collation of such data could direct continuing veterinary development in the future. METHODS: Dermatologists completed hand-written recording sheets during or after enquiries. Information recorded included the route of enquiry, nature of advice, material provided, practice type and location, animal signalment, presenting signs, diagnosis/differential diagnosis, treatment and referral recommendations, time taken and if charges were made. RESULTS: Twelve dermatology services recorded 768 advice requests over a 6-month period. Most requests were submitted via email and related to canine dermatology (81%). An average of 9.5 minutes was spent replying to requests. Charges were made in 2% of cases. Advice regarding otitis was most commonly sought, followed by pruritus, alopecia and crusting. The most frequently discussed diagnoses included allergy, otitis, pyoderma, demodicosis, dermatophytosis and neoplasia. Antibiotics, anti-pruritics and topical otic medications were the most commonly discussed therapeutics. LIMITATIONS: This is an initial study and therefore there are limitations involving the depth of the data. Additional studies should be completed which identify why advice is sought, decision-making regarding referral, and if advice should be charged similarly to other disciplines. CONCLUSIONS: These findings highlight that veterinarians mostly frequently seek advice on management of common dermatological problems, including allergy, otitis and pyoderma.

Nuclear magnetic resonance structure and dynamics of the response regulator Sma0114 from Sinorhizobium meliloti.

Receiver domains control intracellular responses triggered by signal transduction in bacterial two-component systems. Here, we report the solution nuclear magnetic resonance structure and dynamics of Sma0114 from the bacterium Sinorhizobium meliloti, the first such characterization of a receiver domain from the HWE-kinase family of two-component systems. The structure of Sma0114 adopts a prototypical α(5)/ß(5) Rossman fold but has features that set it apart from other receiver domains. The fourth ß-strand of Sma0114 houses a PFxFATGY sequence motif, common to many HWE-kinase-associated receiver domains. This sequence motif in Sma0114 may substitute for the conserved Y-T coupling mechanism, which propagates conformational transitions in the 455 (α4-ß5-α5) faces of receiver domains, to prime them for binding downstream effectors once they become activated by phosphorylation. In addition, the fourth α-helix of the consensus 455 face in Sma0114 is replaced with a segment that shows high flexibility on the pico- to nanosecond time scale by (15)N relaxation data. Secondary structure prediction analysis suggests that the absence of helix α4 may be a conserved property of the HWE-kinase-associated family of receiver domains to which Sma0114 belongs. In spite of these differences, Sma0114 has a conserved active site, binds divalent metal ions such as Mg(2+) and Ca(2+) that are required for phosphorylation, and exhibits micro- to millisecond active-site dynamics similar to those of other receiver domains. Taken together, our results suggest that Sma0114 has a conserved active site but differs from typical receiver domains in the structure of the 455 face that is used to effect signal transduction following activation.

A carvedilol analogue, VK-II-86, prevents hypokalaemia-induced ventricular arrhythmia through novel multi-channel effects.

BACKGROUND AND PURPOSE: QT prolongation and intracellular Ca2+ loading with diastolic Ca2+ release via ryanodine receptors (RyR2) are the predominant mechanisms underlying hypokalaemia-induced ventricular arrhythmia. We investigated the antiarrhythmic actions of two RyR2 inhibitors: dantrolene and VK-II-86, a carvedilol analogue lacking antagonist activity at ß-adrenoceptors, in hypokalaemia. EXPERIMENTAL APPROACH: Surface ECG and ventricular action potentials (APs) were recorded from whole-heart murine Langendorff preparations. Ventricular arrhythmia incidence was compared in hearts perfused with low [K+ ], and those pretreated with dantrolene or VK-II-86. Whole-cell patch clamping was used in murine and canine ventricular cardiomyocytes to study effects of dantrolene and VK-II-86 on AP parameters in low [K+ ] and effects of VK-II-86 on the inward rectifier current (IK1 ), late sodium current (INa_L ) and the L-type Ca2+ current (ICa ). Effects of VK-II-86 on IKr were investigated in transfected HEK-293 cells. A fluorogenic probe quantified the effects of VK-II-86 on oxidative stress in hypokalaemia. KEY RESULTS: Dantrolene reduced the incidence of ventricular arrhythmias induced by low [K+ ] in explanted murine hearts by 94%, whereas VK-II-86 prevented all arrhythmias. VK-II-86 prevented hypokalaemia-induced AP prolongation and depolarization but did not alter AP parameters in normokalaemia. Hypokalaemia was associated with decreased IK1 and IKr , and increased INa-L , and ICa . VK-II-86 prevented all hypokalaemia-induced changes in ion channel activity and oxidative stress. CONCLUSIONS AND IMPLICATIONS: VK-II-86 prevents hypokalaemia-induced arrhythmogenesis by normalizing calcium homeostasis and repolarization reserve. VK-II-86 may provide an effective treatment in hypokalaemia and other arrhythmias caused by delayed repolarization or Ca2+ overload.

Applying the Digital Health Social Justice Guide.

Introduction: Digital health, the use of apps, text-messaging, and online interventions, can revolutionize healthcare and make care more equitable. Currently, digital health interventions are often not designed for those who could benefit most and may have unintended consequences. In this paper, we explain how privacy vulnerabilities and power imbalances, including racism and sexism, continue to influence health app design and research. We provide guidelines for researchers to design, report and evaluate digital health studies to maximize social justice in health. Methods: From September 2020 to April 2021, we held five discussion and brainstorming sessions with researchers, students, and community partners to develop the guide and the key questions. We additionally conducted an informal literature review, invited experts to review our guide, and identified examples from our own digital health study and other studies. Results: We identified five overarching topics with key questions and subquestions to guide researchers in designing or evaluating a digital health research study. The overarching topics are: 1. Equitable distribution; 2. Equitable design; 3. Privacy and data return; 4. Stereotype and bias; 5. Structural racism. Conclusion: We provide a guide with five key topics and questions for social justice digital health research. Encouraging researchers and practitioners to ask these questions will help to spark a transformation in digital health toward more equitable and ethical research. Future work needs to determine if the quality of studies can improve when researchers use this guide.

Clinical genomic profiling in the management of patients with soft tissue and bone sarcoma.

There are more than 70 distinct sarcomas, and this diversity complicates the development of precision-based therapeutics for these cancers. Prospective comprehensive genomic profiling could overcome this challenge by providing insight into sarcomas' molecular drivers. Through targeted panel sequencing of 7494 sarcomas representing 44 histologies, we identify highly recurrent and type-specific alterations that aid in diagnosis and treatment decisions. Sequencing could lead to refinement or reassignment of 10.5% of diagnoses. Nearly one-third of patients (31.7%) harbor potentially actionable alterations, including a significant proportion (2.6%) with kinase gene rearrangements; 3.9% have a tumor mutational burden ≥10 mut/Mb. We describe low frequencies of microsatellite instability (<0.3%) and a high degree of genome-wide loss of heterozygosity (15%) across sarcomas, which are not readily explained by homologous recombination deficiency (observed in 2.5% of cases). In a clinically annotated subset of 118 patients, we validate actionable genetic events as therapeutic targets. Collectively, our findings reveal the genetic landscape of human sarcomas, which may inform future development of therapeutics and improve clinical outcomes for patients with these rare cancers.