Loose-patch clamp analysis applied to voltage-gated ionic currents following pharmacological ryanodine receptor modulation in murine hippocampal cornu ammonis-1 pyramidal neurons.

Introduction: The loose-patch clamp technique was first developed and used in native amphibian skeletal muscle (SkM), offering useful features complementing conventional sharp micro-electrode, gap, or conventional patch voltage clamping. It demonstrated the feedback effects of pharmacological modification of ryanodine receptor (RyR)-mediated Ca2+ release on the Na+ channel (Nav1.4) currents, initiating excitation-contraction coupling in native murine SkM. The effects of the further RyR and Ca2+-ATPase (SERCA) antagonists, dantrolene and cyclopiazonic acid (CPA), additionally implicated background tubular-sarcoplasmic Ca2+ domains in these actions. Materials and methods: We extend the loose-patch clamp approach to ion current measurements in murine hippocampal brain slice cornu ammonis-1 (CA1) pyramidal neurons. We explored the effects on Na+ currents of pharmacologically manipulating RyR and SERCA-mediated intracellular store Ca2+ release and reuptake. We adopted protocols previously applied to native skeletal muscle. These demonstrated Ca2+-mediated feedback effects on the Na+ channel function. Results: Experiments applying depolarizing 15 ms duration loose-patch clamp steps to test voltages ranging from -40 to 120 mV positive to the resting membrane potential demonstrated that 0.5 mM caffeine decreased inward current amplitudes, agreeing with the previous SkM findings. It also decreased transient but not prolonged outward current amplitudes. However, 2 mM caffeine affected neither inward nor transient outward but increased prolonged outward currents, in contrast to its increasing inward currents in SkM. Furthermore, similarly and in contrast to previous SkM findings, both dantrolene (10 µM) and CPA (1 µM) pre-administration left both inward and outward currents unchanged. Nevertheless, dantrolene pretreatment still abrogated the effects of subsequent 0.5- and 2-mM caffeine challenges on both inward and outward currents. Finally, CPA abrogated the effects of 0.5 mM caffeine on both inward and outward currents, but with 2 mM caffeine, inward and transient outward currents were unchanged, but sustained outward currents increased. Conclusion: We, thus, extend loose-patch clamping to establish pharmacological properties of murine CA1 pyramidal neurons and their similarities and contrasts with SkM. Here, evoked though not background Ca2+-store release influenced Nav and Kv excitation, consistent with smaller contributions of background store Ca2+ release to resting [Ca2+]. This potential non-canonical mechanism could modulate neuronal membrane excitability or cellular firing rates.

Loose patch clamp membrane current measurements in cornus ammonis 1 neurons in murine hippocampal slices.

Hippocampal pyramidal neuronal activity has been previously studied using conventional patch clamp in isolated cells and brain slices. We here introduce the loose patch clamping study of voltage-activated currents from in situ pyramidal neurons in murine cornus ammonis 1 hippocampal coronal slices. Depolarizing pulses of 15-ms duration elicited early transient inward, followed by transient and prolonged outward currents in the readily identifiable junctional region between the stratum pyramidalis (SP) and oriens (SO) containing pyramidal cell somas and initial segments. These resembled pyramidal cell currents previously recorded using conventional patch clamp. Shortening the depolarizing pulses to >1-2 ms continued to evoke transient currents; hyperpolarizing pulses to varying voltages evoked decays whose time constants could be shortened to <1 ms, clarifying the speed of clamping in this experimental system. The inward and outward currents had distinct pharmacological characteristics and voltage-dependent inactivation and recovery from inactivation. Comparative recordings from the SP, known to contain pyramidal cell somas, demonstrated similar current properties. Recordings from the SO and stratum radiatum demonstrated smaller inward and outward current magnitudes and reduced transient outward currents, consistent with previous conventional patch clamp results from their different interneuron types. The loose patch clamp method is thus useful for in situ studies of neurons in hippocampal brain slices.

Diagnostic and predictive ability of hyperbilirubinemia severity in cats: A multicenter retrospective study.

BACKGROUND: Total serum bilirubin concentration (TBIL) can provide useful information on several pathophysiological conditions in cats. Nevertheless, whether the variable severity classification of hyperbilirubinemia can reliably indicate certain disease processes or predict a biliary obstruction (BO) has not been investigated. HYPOTHESIS/OBJECTIVE: Determine if hyperbilirubinemia of variable severity can assist clinicians to identify BO, which often is considered a surgical emergency. ANIMALS: Two-hundred sixteen client-owned cats. METHODS: Data were retrospectively collected from all cats (January 2015-August 2022) with an increased TBIL (>0.58 mg/dL [>10 µmol/L]) presented to 3 referral centers in the United Kingdom (UK). Presenting clinical features and diagnostic outcomes were collected. The predictive ability of TBIL to indicate BO was evaluated by multivariable binary logistic regression modeling and receiver operating characteristic (ROC) curves. RESULTS: Median TBIL was 1.73 mg/dL (range, 0.59-26.15; 29.5 µmol/L; range, 10.1-447.1) with severity classification of hyperbilirubinemia categorized as mild (>0.58-2.92 mg/dL; >10-50 µmol/L; 68.1%), moderate (>2.92-5.85 mg/dL; >50-100 µmol/L; 17.6%), severe (>5.85-11.70 mg/dL; >100-200 µmol/L; 9.7%) and very severe (>11.70 mg/dL; >200 µmol/L; 4.6%). Biliary obstruction was present in 17 (7.9%) cats, all of which received recommendation for emergency surgery. Median TBIL in cats with BO (9.69 mg/dL; 165.7 µmol/L) differed significantly from those without obstruction (1.51 mg/dL; 25.8 µmol/L; P < .01). The optimal TBIL cut-off to discriminate between cats with and without BO was ≥3.86 mg/dL (≥66 µmol/L) with a sensitivity of 94.1% and specificity of 82.4%. Using multivariable logistic regression, as age increased, the odds of BO increased significantly (odds ratio, 1.20; 95% confidence interval, 1.01-1.42; P = .04). CONCLUSIONS AND CLINICAL IMPORTANCE: As part of a thorough clinical assessment, the severity classification of hyperbilirubinemia has the potential to predict the likelihood of a BO and to discriminate between cats that may or may not require surgery for BO at a suggested cut-off of ≥3.86 mg/dL (≥66 µmol/L). Alongside TBIL, age is also useful when assessing for the likelihood of BO in a cat presented with hyperbilirubinemia.

On the low-frequency dispersion observed in dielectrophoresis spectra.

The foundation of dielectrophoresis (DEP) as a tool for biological investigation is the use of the Clausius-Mossotti (C-M) factor to model the observed behaviour of cells experiencing DEP across a frequency range. Nevertheless, it is also the case that at lower frequencies, the DEP spectrum deviates from predictions; there exists a rise in DEP polarisability, which varies in frequency and magnitude with different cell types and medium conductivities. In order to evaluate the origin of this effect, we have studied DEP spectra from five cell types (erythrocytes, platelets, neurons, HeLa cancer cells and monocytes) in several conditions including medium conductivity and cell treatment. Our results suggest the effect manifests as a low-pass dispersion whose cut-off frequency varies with membrane conductance and capacitance as determined using the DEP spectrum; the effect also varies as a logarithm of medium conductivity and Debye length. These together suggest that the values of membrane capacitance and conductance depend not only on the impedance of the membrane itself, but also of the surrounding double layer. The amplitude of the effect in different cell types compared to the C-M factor was found to correlate with the depolarisation factors for the cells' shapes, suggesting that this ratio may be useful as an indicator of cell shape for DEP modelling.

Clinical supervisors' and students' perspectives on preparedness for veterinary workplace clinical training: An international study.

BACKGROUND: The alignment of student and workplace supervisors' perspectives on student preparedness for veterinary workplace clinical training (WCT) is unknown, yet misalignment could negatively impact workplace learning. The aim of this study was to quantify the relative importance of WCT preparedness characteristics according to students and supervisors and to identify differences. METHODS: A survey was completed by 657 veterinary students and 244 clinical supervisors from 25 veterinary schools, from which rankings of the preparedness characteristics were derived. Significant rank differences were assessed using confidence intervals and permutation tests. RESULTS: 'Honesty, integrity and dependability' was the most important characteristic according to both groups. The three characteristics with the largest rank differences were: students' awareness of their own and others' mental wellbeing and the importance of self-care; being willing to try new practical skills with support (students ranked both of these higher); and having a clinical reasoning framework for common problems (supervisors ranked higher). LIMITATIONS: Using pooled data from many schools means that the results are not necessarily representative of the perspectives at any one institution. CONCLUSION: There are both similarities and differences in the perspectives of students and supervisors regarding which characteristics are more important for WCT. This provides insights that can be used by educators, curriculum developers and admissions tutors to improve student preparedness for workplace learning.

Retrospective study and outcome of 307 cats with feline infectious peritonitis treated with legally sourced veterinary compounded preparations of remdesivir and GS-441524 (2020-2022).

OBJECTIVES: Feline infectious peritonitis (FIP) is a serious disease that arises due to feline coronavirus infection. The nucleoside analogues remdesivir and GS-441524 can be effective in its treatment, but most studies have used unregulated products of unknown composition. The aim of the present study was to describe the treatment of FIP using legally sourced veterinary-prescribed regulated veterinary compounded products containing known amounts of remdesivir (injectable) or GS-441524 (oral tablets). METHODS: Cats were recruited via email advice services, product sales contacts and study publicity. Cats were excluded if they were deemed unlikely to have FIP, were not treated exclusively with the veterinary compounded products, or if there was a lack of cat and/or treatment (including response) data. Extensive cat and treatment data were collected. RESULTS: Among the 307 cats recruited, the predominant type of FIP was most commonly abdominal effusive (49.5%) and then neurological (14.3%). Three treatment protocols were used; remdesivir alone (33.9%), remdesivir followed by GS-441524 (55.7%) and GS-441524 alone (10.4%). The median (range) initial treatment period duration and longest follow-up time point after starting treatment were 84 (1-330) days and 248 (1-814) days, respectively. The most common side effect was injection pain (in 47.8% of those given subcutaneous remdesivir). Of the 307 cats, 33 (10.8%) relapsed, 15 (45.5%) during and 18 (54.5%) after the initial treatment period. At the longest follow-up time point after completion of the initial treatment period, 84.4% of cats were alive. The cats achieving a complete response within 30 days of starting treatment were significantly more likely to be alive at the end of the initial treatment period than those cats that did not. CONCLUSIONS AND RELEVANCE: Legally sourced remdesivir and GS-441524 products, either alone or used sequentially, were very effective in the treatment of FIP in this group of cats. Variable protocols precluded statistical comparison of treatment regimens.

Clinical and Non-Clinical Cardiovascular Disease Associated Pathologies in Parkinson's Disease.

Despite considerable breakthroughs in Parkinson's disease (PD) research, understanding of non-motor symptoms (NMS) in PD remains limited. The lack of basic level models that can properly recapitulate PD NMS either in vivo or in vitro complicates matters. Even so, recent research advances have identified cardiovascular NMS as being underestimated in PD. Considering that a cardiovascular phenotype reflects sympathetic autonomic dysregulation, cardiovascular symptoms of PD can play a pivotal role in understanding the pathogenesis of PD. In this study, we have reviewed clinical and non-clinical published papers with four key parameters: cardiovascular disease risks, electrocardiograms (ECG), neurocardiac lesions in PD, and fundamental electrophysiological studies that can be linked to the heart. We have highlighted the points and limitations that the reviewed articles have in common. ECG and pathological reports suggested that PD patients may undergo alterations in neurocardiac regulation. The pathological evidence also suggested that the hearts of PD patients were involved in alpha-synucleinopathy. Finally, there is to date little research available that addresses the electrophysiology of in vitro Parkinson's disease models. For future reference, research that can integrate cardiac electrophysiology and pathological alterations is required.

Virtual medical research mentoring.

BACKGROUND: Medical research is important for professional advancement, and mentoring is a key means by which students and early-career doctors can engage in research. Contrasting international research collaborations, research mentoring programmes are often geographically limited. As the COVID-19 pandemic has led to increased use of online technology for classes and conferences, a virtual, international approach to medical research mentoring may be valuable. APPROACH: We hereby describe our experience at the Cardiovascular Analytics Group, a virtual international medical research mentoring group established in 2015. We make use of virtual platforms in multi-level mentoring with peer mentoring and emphasise active participation, early leadership, an open culture, accessible research support and a distributed research workflow. EVALUATION: With 63 active members from 14 different countries, the Group has been successful in training medical students and early-career medical graduates in academic medicine. Our members have led over 100 peer-reviewed publications of original research and reviews since 2015, winning 13 research prizes during this time. IMPLICATIONS: Our accessible-distributed model of virtual international medical research collaboration and multi-level mentoring is viable and efficient and caters to the needs of contemporary healthcare. Others should consider building similar models to improve medical research mentoring globally.

Rating and ranking preparedness characteristics important for veterinary workplace clinical training: a novel application of pairwise comparisons and the Elo algorithm.

Quantitatively eliciting perspectives about a large number of similar entities (such as a list of competences) is a challenge for researchers in health professions education (HPE). Traditional survey methods may include using Likert items. However, a Likert item approach that generates absolute ratings of the entities may suffer from the "ceiling effect," as ratings cluster at one end of the scale. This impacts on researchers' ability to detect differences in ratings between the entities themselves and between respondent groups. This paper describes the use of pairwise comparison (this or that?) questions and a novel application of the Elo algorithm to generate relative ratings and rankings of a large number of entities, on a unidimensional scale. A study assessing the relative importance of 91 student "preparedness characteristics" for veterinary workplace clinical training (WCT) is presented as an example of this method in action. The Elo algorithm uses pairwise comparison responses to generate an importance rating for each preparedness characteristic on a scale from zero to one. This is continuous data with measurement variability which, by definition, spans an entire spectrum and is not susceptible to the ceiling effect. The output should allow for the detection of differences in perspectives between groups of survey respondents (such as students and workplace supervisors) which Likert ratings may be insensitive to. Additional advantages of the pairwise comparisons are their low susceptibility to systematic bias and measurement error, they can be quicker and arguably more engaging to complete than Likert items, and they should carry a low cognitive load for respondents. Methods for evaluating the validity and reliability of this survey design are also described. This paper presents a method that holds great potential for a diverse range of applications in HPE research. In the pursuit quantifying perspectives on survey items which are measured on a relative basis and a unidimensional scale (e.g., importance, priority, probability), this method is likely to be a valuable option.

Feedback contributions to excitation-contraction coupling in native functioning striated muscle.

Skeletal and cardiac muscle excitation-contraction coupling commences with Nav1.4/Nav1.5-mediated, surface and transverse (T-) tubular, action potential generation. This initiates feedforward, allosteric or Ca2+-mediated, T-sarcoplasmic reticular (SR) junctional, voltage sensor-Cav1.1/Cav1.2 and ryanodine receptor-RyR1/RyR2 interaction. We review recent structural, physiological and translational studies on possible feedback actions of the resulting SR Ca2+ release on Nav1.4/Nav1.5 function in native muscle. Finite-element modelling predicted potentially regulatory T-SR junctional [Ca2+]TSR domains. Nav1.4/Nav1.5, III-IV linker and C-terminal domain structures included Ca2+ and/or calmodulin-binding sites whose mutations corresponded to specific clinical conditions. Loose-patch-clamped native murine skeletal muscle fibres and cardiomyocytes showed reduced Na+ currents (INa) following SR Ca2+ release induced by the Epac and direct RyR1/RyR2 activators, 8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphate and caffeine, abrogated by the RyR inhibitor dantrolene. Conversely, dantrolene and the Ca2+-ATPase inhibitor cyclopiazonic acid increased INa. Experimental, catecholaminergic polymorphic ventricular tachycardic RyR2-P2328S and metabolically deficient Pgc1ß-/- cardiomyocytes also showed reduced INa accompanying [Ca2+]i abnormalities rescued by dantrolene- and flecainide-mediated RyR block. Finally, hydroxychloroquine challenge implicated action potential (AP) prolongation in slowing AP conduction through modifying Ca2+ transients. The corresponding tissue/organ preparations each showed pro-arrhythmic, slowed AP upstrokes and conduction velocities. We finally extend discussion of possible Ca2+-mediated effects to further, Ca2+, K+ and Cl-, channel types. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Ageing and the Autonomic Nervous System.

The vertebrate nervous system is divided into central (CNS) and peripheral (PNS) components. In turn, the PNS is divided into the autonomic (ANS) and enteric (ENS) nervous systems. Ageing implicates time-related changes to anatomy and physiology in reducing organismal fitness. In the case of the CNS, there exists substantial experimental evidence of the effects of age on individual neuronal and glial function. Although many such changes have yet to be experimentally observed in the PNS, there is considerable evidence of the role of ageing in the decline of ANS function over time. As such, this chapter will argue that the ANS constitutes a paradigm for the physiological consequences of ageing, as well as for their clinical implications.

Framing Heartaches: The Cardiac ECM and the Effects of Age.

The cardiac extracellular matrix (ECM) is involved in several pathological conditions, and age itself is also associated with certain changes in the heart: it gets larger and stiffer, and it develops an increased risk of abnormal intrinsic rhythm. This, therefore, makes conditions such as atrial arrythmia more common. Many of these changes are directly related to the ECM, yet the proteomic composition of the ECM and how it changes with age is not fully resolved. The limited research progress in this field is mainly due to the intrinsic challenges in unravelling tightly bound cardiac proteomic components and also the time-consuming and costly dependency on animal models. This review aims to give an overview of the composition of the cardiac ECM, how different components aid the function of the healthy heart, how the ECM is remodelled and how it is affected by ageing.

Attendance-related Healthcare Resource Utilisation and Costs in Patients With Brugada Syndrome in Hong Kong: A Retrospective Cohort Study.

Understanding health care resource utilisation and its associated costs are important for identifying areas of improvement regarding resource allocations. However, there is limited research exploring this issue in the setting of Brugada syndrome (BrS).This was a retrospective territory-wide study of BrS patients from Hong Kong. Healthcare resource utilisation for accident and emergency (A&E), inpatient and specialist outpatient attendances were analyzed over a 19-year period, with their associated costs presented in US dollars. A total of 507 BrS patients with a mean presentation age of 49.9 ± 16.3 years old were included. Of these, 384 patients displayed spontaneous type 1 electrocardiographic (ECG) Brugada pattern and 77 patients had presented with ventricular tachycardia/ventricular fibrillation (VT/VF). At the individual patient level, the median annualized costs were $110 (52-224) at the (A&E) setting, $6812 (1982-32414) at the inpatient setting and $557 (326-1001) for specialist outpatient attendances. Patients with initial VT/VF presentation had overall greater costs in inpatient ($20161 [9147-189215] vs $5290 [1613-24937],P < 0.0001) and specialist outpatient setting ($776 [438-1076] vs $542 [293-972],P = 0.015) compared to those who did not present VT. In addition, patients without Type 1 ECG pattern had greater median costs in the specialist outpatient setting ($7036 [3136-14378] vs $4895 [2409-10554],p=0.019). There is a greater health care demand in the inpatient and specialist outpatient settings for BrS patients. The most expensive attendance type was inpatient setting stay at $6812 per year. The total median annualized cost of BrS patients without VT/VF presentation was 78% lower compared to patients with VT/VF presentation.

Predicting Stroke and Mortality in Mitral Regurgitation: A Machine Learning Approach.

We hypothesized that an interpretable gradient boosting machine (GBM) model considering comorbidities, P-wave and echocardiographic measurements, can better predict mortality and cerebrovascular events in mitral regurgitation (MR). Patients from a tertiary center were analyzed. The GBM model was used as an interpretable statistical approach to identify the leading indicators of high-risk patients with either outcome of CVAs and all-cause mortality. A total of 706 patients were included. GBM analysis showed that age, systolic blood pressure, diastolic blood pressure, plasma albumin levels, mean P-wave duration (PWD), MR regurgitant volume, left ventricular ejection fraction (LVEF), left atrial dimension at end-systole (LADs), velocity-time integral (VTI) and effective regurgitant orifice were significant predictors of TIA/stroke. Age, sodium, urea and albumin levels, platelet count, mean PWD, LVEF, LADs, left ventricular dimension at end systole (LVDs) and VTI were significant predictors of all-cause mortality. The GBM demonstrates the best predictive performance in terms of precision, sensitivity c-statistic and F1-score compared to logistic regression, decision tree, random forest, support vector machine, and artificial neural networks. Gradient boosting model incorporating clinical data from different investigative modalities significantly improves risk prediction performance and identify key indicators for outcome prediction in MR.

Cardiac sodium channel complexes and arrhythmia: structural and functional roles of the ß1 and ß3 subunits.

In cardiac myocytes, the voltage-gated sodium channel NaV 1.5 opens in response to membrane depolarisation and initiates the action potential. The NaV 1.5 channel is typically associated with regulatory ß-subunits that modify gating and trafficking behaviour. These ß-subunits contain a single extracellular immunoglobulin (Ig) domain, a single transmembrane α-helix and an intracellular region. Here we focus on the role of the ß1 and ß3 subunits in regulating NaV 1.5. We catalogue ß1 and ß3 domain specific mutations that have been associated with inherited cardiac arrhythmia, including Brugada syndrome, long QT syndrome, atrial fibrillation and sudden death. We discuss how new structural insights into these proteins raises new questions about physiological function.

P-wave durations from automated electrocardiogram analysis to predict atrial fibrillation and mortality in heart failure.

BACKGROUND: P-wave indices have been used to predict incident atrial fibrillation (AF), stroke, and mortality. However, such indices derived from automated ECG measurements have not been explored for their predictive values in heart failure (HF). We investigated whether automated P-wave indices can predict adverse outcomes in HF. METHODS: This study included consecutive Chinese patients admitted to a single tertiary centre, presenting with HF but without prior AF, and with at least one baseline ECG, between 1 January 2010 and 31 December 2016, with last follow-up of 31 December 2019. RESULTS: A total of 2718 patients were included [median age: 77.4, interquartile range (IQR): (66.9-84.3) years; 47.9 males]. After a median follow-up of 4.8 years (IQR: 1.9-9.0 years), 1150 patients developed AF (8.8/year), 339 developed stroke (2.6/year), 563 developed cardiovascular mortality (4.3/year), and 1972 had all-cause mortality (15.1/year). Compared with 101-120 ms as a reference, maximum P-wave durations predicted new-onset AF at ≤90 ms [HR: 1.17(1.11, 1.50), P < 0.01], 131-140 ms [HR: 1.29(1.09, 1.54), P < 0.001], and ≥141 ms [HR: 1.52(1.32, 1.75), P < 0.001]. Similarly, they predicted cardiovascular mortality at ≤90 ms [HR: 1.50(1.08, 2.06), P < 0.001] or ≥141 ms [HR: 1.18(1.15, 1.45), P < 0.001], and all-cause mortality at ≤90 ms [HR: 1.26(1.04, 1.51), P < 0.001], 131-140 ms [HR: 1.15(1.01, 1.32), P < 0.01], and ≥141 ms [HR: 1.31(1.18, 1.46), P < 0.001]. These remained significant after adjusting for significant demographics, past co-morbidities, P-wave dispersion, and maximum P-wave amplitude. CONCLUSIONS: Extreme values of maximum P-wave durations (≤90 ms and ≥141 ms) were significant predictors of new-onset AF, cardiovascular mortality, and all-cause mortality.

Exploring UK veterinary students' use of online resources as a tool for studying small animal internal medicine.

BACKGROUND: Despite an increase in the use of online resources, their use by veterinary students, especially in the study of small animal internal medicine (SAIM), remains poorly understood. METHODS: A questionnaire-based study consisting of 26 survey items and 263 responses from seven UK universities investigated the use of online resources among clinical veterinary students studying SAIM and whether this was affected by age, gender, year of study or entry status. RESULTS: Random internet searches were the preferred method of clarifying queries, except for graduate-entry students and age categories 18-21 years and 28 years and over who preferred traditional textbooks. Online searches were preferred over emailing instructors or contacting instructors in person. Despite 73.3% of participants not automatically trusting information from online sources, only 47.1% fact-checked, although this was higher among the graduate-entry group (62.5%). Frequent social media use was reported; however, only 44.8% of students reported using social media to discuss SAIM. Video clip usage was high, and 82.9% of students reported video clips were useful for understanding SAIM concepts. CONCLUSION: Online resources are a useful tool to complement traditional resources; however, a level of academic oversight may be required to ensure appropriate and effective use of these resources.