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.

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.

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.

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.

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.

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.

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.

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.

Thrombosis in infants in the neonatal intensive care unit: Analysis of a large national database.

BACKGROUND: Thrombosis in the neonatal population is rare, but increasing. Its incidence and management are not well understood. OBJECTIVES: To investigate the incidence, associated factors, and management of thrombosis in the neonatal intensive care unit (NICU) population. PATIENTS/METHODS: We performed a retrospective cohort study of infants admitted to a Pediatrix Medical Group-affiliated NICU from 1997 through 2015. We determined the prevalence of venous and arterial thrombosis, and assessed demographic characteristics and known risk factors. Categorical variables were compared with the Pearson χ2 test and continuous variables with Wilcoxon rank-sum tests. Stepwise logistic regression was used to identify associated factors. The primary outcome was incidence of thrombosis. Secondary analyses investigated correlations between clinical and demographic characteristics and thrombosis. RESULTS: Among 1 158 755 infants, we identified 2367 (0.20%) diagnosed with thrombosis. In a multivariable regression analysis, prematurity, male sex, congenital heart disease, sepsis, ventilator support, vasopressor receipt, central venous catheter, invasive procedures, and receipt of erythropoietin were associated with increased risk of thrombosis, while Black race and Hispanic ethnicity were associated with reduced risk. The majority of infants diagnosed with thrombosis (73%) received no anticoagulation, but anticoagulant use in infants with thrombosis was higher than those without (27% versus 0.2%, P < .001). Thrombosis in infants was associated with higher mortality (11% versus 2%, P < .001) and longer hospital stays (57 days, [interquartile range (IQR) 28--100] versus 10 days, [IQR 6--22], P < .001). CONCLUSIONS: In the largest national study to date, we found that thrombosis in NICU patients is associated with prematurity, low birth weight, sepsis, and invasive procedures.

Acacetin suppresses the electrocardiographic and arrhythmic manifestations of the J wave syndromes.

BACKGROUND: J wave syndromes (JWS), including Brugada (BrS) and early repolarization syndromes (ERS), are associated with increased risk for life-threatening ventricular arrhythmias. Pharmacologic approaches to therapy are currently very limited. Here, we evaluate the effects of the natural flavone acacetin. METHODS: The effects of acacetin on action potential (AP) morphology and transient outward current (Ito) were first studied in isolated canine RV epicardial myocytes using whole-cell patch clamp techniques. Acacetin's effects on transmembrane APs, unipolar electrograms and transmural ECGs were then studied in isolated coronary-perfused canine RV and LV wedge preparations as well as in whole-heart, Langendorff-perfused preparations from which we recorded a 12 lead ECG and unipolar electrograms. Using floating glass microelectrodes we also recorded transmembrane APs from the RVOT of the whole-heart model. The Ito agonist NS5806, sodium channel blocker ajmaline, calcium channel blocker verapamil or hypothermia (32°C) were used to pharmacologically mimic the genetic defects and conditions associated with JWS, thus eliciting prominent J waves and provoking VT/VF. RESULTS: Acacetin (5-10 µM) reduced Ito density, AP notch and J wave area and totally suppressed the electrocardiographic and arrhythmic manifestation of both BrS and ERS, regardless of the experimental model used. In wedge and whole-heart models of JWS, increasing Ito with NS5806, decreasing INa or ICa (with ajmaline or verapamil) or hypothermia all resulted in accentuation of epicardial AP notch and ECG J waves, resulting in characteristic BrS and ERS phenotypes. Phase 2-reentrant extrasystoles originating from the RVOT triggered VT/VF. The J waves in leads V1 and V2 were never associated with a delay of RVOT activation and always coincided with the appearance of the AP notch recorded from RVOT epicardium. All repolarization defects giving rise to VT/VF in the BrS and ERS models were reversed by acacetin, resulting in total suppression of VT/VF. CONCLUSIONS: We present experimental models of BrS and ERS capable of recapitulating all of the ECG and arrhythmic manifestations of the JWS. Our findings provide definitive support for the repolarization but not the depolarization hypothesis proposed to underlie BrS and point to acacetin as a promising new pharmacologic treatment for JWS.

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.

The Developing Balance of Thrombosis and Hemorrhage in Pediatric Surgery: Clinical Implications of Age-Related Changes in Hemostasis.

Bleeding and thrombosis in critically ill infants and children is a vexing clinical problem. Despite the relatively low incidence of bleeding and thrombosis in the overall pediatric population relative to adults, these critically ill children face unique challenges to hemostasis due to extreme physiologic derangements, exposure of blood to foreign surfaces and membranes, and major vascular endothelial injury or disruption. Caring for pediatric patients on extracorporeal support, recovering from solid organ transplant or invasive surgery, and after major trauma is often complicated by major bleeding or clotting events. As our ability to care for the youngest and sickest of these children increases, the gaps in our understanding of the clinical implications of developmental hemostasis have become increasingly important. We review the current understanding of the development and function of the hemostatic system, including the complex and overlapping interactions of coagulation proteins, platelets, fibrinolysis, and immune mediators from the neonatal period through early childhood and to young adulthood. We then examine scenarios in which our ability to effectively measure and treat coagulation derangements in pediatric patients is limited. In these clinical situations, adult therapies are often extrapolated for use in children without taking age-related differences in pediatric hemostasis into account, leaving clinicians confused and impacting patient outcomes. We discuss the limitations of current coagulation testing in pediatric patients before turning to emerging ideas in the measurement and management of pediatric bleeding and thrombosis. Finally, we highlight opportunities for future research which take into account this developing balance of bleeding and thrombosis in our youngest patients.

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.

Functional and structural basis of E. coli enolase inhibition by SF2312: a mimic of the carbanion intermediate.

Many years ago, the natural secondary metabolite SF2312, produced by the actinomycete Micromonospora, was reported to display broad spectrum antibacterial properties against both Gram-positive and Gram-negative bacteria. Recent studies have revealed that SF2312, a natural phosphonic acid, functions as a potent inhibitor of human enolase. The mechanism of SF2312 inhibition of bacterial enolase and its role in bacterial growth and reproduction, however, have remained elusive. In this work, we detail a structural analysis of E. coli enolase bound to both SF2312 and its oxidized imide-form. Our studies support a model in which SF2312 acts as an analog of a high energy intermediate formed during the catalytic process. Biochemical, biophysical, computational and kinetic characterization of these compounds confirm that altering features characteristic of a putative carbanion (enolate) intermediate significantly reduces the potency of enzyme inhibition. When SF2312 is combined with fosfomycin in the presence of glucose-6 phosphate, significant synergy is observed. This suggests the two agents could be used as a potent combination, targeting distinct cellular mechanism for the treatment of bacterial infections. Together, our studies rationalize the structure-activity relationships for these phosphonates and validate enolase as a promising target for antibiotic discovery.