A real-time signal processing software package for the electrophysiology laboratory.

BACKGROUND: Real-time signal processing has to date been difficult to implement in the clinical electrophysiology laboratory. To date, no open access software solutions are available in electrophysiology (EP) laboratories to facilitate real-time intraprocedural signal analysis. We aimed to develop an open access, scalable Python plug-in to allow real-time signal processing during human EP procedures. METHODS AND RESULTS: A Python-based plug in for the widely available EnsiteX mapping system was developed. This plug-in utilized the LiveSync feature of the system to allow real-time signal analysis. An open access library was developed to allow end-users to implement real-time signal analysis using this platform, implemented in the Python programming language https://github.com/anand9176/WaveWatch5000Public. CONCLUSION: We have developed and demonstrated the feasibility of a readily scalable and open-access Python-based plug in to an electroanatomic mapping system (EnSiteX) to allow real-time processing and display of electrogram (EGM) based information for the procedural electrophysiologist to view intraprocedurally in the electrophysiology laboratory. The availability, to the clinician, of traditional and novel EGM-based metrics at the time of intervention, such as atrial fibrillation ablation, allows for key mechanistic insights into critical unresolved questions regarding arrhythmia mechanism.

Infra-Red Imaging to Detect Respirator Leak in Healthcare Workers During Fit-Testing Clinic.

OBJECTIVE: This study addressed the problem of objectively detecting leaks in P2 respirators at point of use, an essential component for healthcare workers' protection. To achieve this, we explored the use of infra-red (IR) imaging combined with machine learning algorithms on the thermal gradient across the respirator during inhalation. RESULTS: The study achieved high accuracy in predicting pass or fail outcomes of quantitative fit tests for flat-fold P2 FFRs. The IR imaging methods surpassed the limitations of self fit-checking. CONCLUSIONS: The integration of machine learning and IR imaging on the respirator itself demonstrates promise as a more reliable alternative for ensuring the proper fit of P2 respirators. This innovative approach opens new avenues for technology application in occupational hygiene and emphasizes the need for further validation across diverse respirator styles. SIGNIFICANCE STATEMENT: Our novel approach leveraging infra-red imaging and machine learning to detect P2 respirator leaks represents a critical advancement in occupational safety and healthcare workers' protection.

Assessing Torque Transfer in Conduction System Pacing: Development and Evaluation of an Ex Vivo Model.

BACKGROUND: Conduction system pacing (CSP) faces challenges in achieving reliable and safe deployments. Complex interactions between tissue and lead tip can result in endocardial entanglement, a drill effect that prevents penetration. No verified ex vivo model exists to quantitatively assess this relationship. OBJECTIVES: The purpose of this study was to quantitatively characterize CSP lead tip to tissue responses for 4 commonly used leads. METHODS: CSP leads (from Medtronic, Biotronik, Boston Scientific, and Abbott) were examined for helix rotation efficiency in ex vivo ovine right ventricular septa. A custom jig was utilized for rotation measurements. Fifteen turns were executed, documenting tissue-interface changes every 90° using high-resolution photography. Response curves (input rotation vs helix rotation) were evaluated using piecewise linear regression, with a focus on output vs input response slopes and torque breakpoint events. RESULTS: We analyzed 3,840 quarter-turn CSP insertions with 4 different lead types. Helix rotations were consistently less than input: Abbott Tendril = 0.21:1, Medtronic 3830 = 0.21:1, Biotronik Solia = 0.47:1, and Boston Scientific Ingevity = 0.56:1. Torque breakpoint events were observed on average 7.22 times per insertion (95% CI: 6.08-8.35; P = NS) across all leads. In 57.8% of insertions (37 of 64), uncontrolled torque breakpoint events occurred, signaling unexpected excess helix rotations. CONCLUSIONS: Using a robust ex vivo model, we revealed a muted helix rotation response compared with input turns on the lead, and frequent torque change events during insertion. This is critical for CSP implanters, emphasizing the potential for unexpected torque breakpoint events, and suggesting the need for novel lead designs or deployment methods to enhance CSP efficiency and safety.

Uncovering Minimal Pathways in Melanoma Initiation.

Cutaneous melanomas are clinically and histologically heterogeneous. Most display activating mutations in Braf or Nras and complete loss of function of one or more tumor suppressor genes. Mouse models that replicate such mutations produce fast-growing, pigmented tumors. However, mice that combine Braf activation with only heterozygous loss of Pten also produce tumors and, as we show here, in an Albino background this occurs even with Braf activation alone. Such tumors arise rarely, grow slowly, and express low levels of pigmentation genes. The timing of their appearance was consistent with a single step stochastic event, but no evidence could be found that it required de novo mutation, suggesting instead the involvement of an epigenetic transition. Single-cell transcriptomic analysis revealed such tumors to be heterogeneous, including a minor cell type we term LNM ( L ow-pigment, N eural- and extracellular M atrix-signature) that displays gene expression resembling "neural crest"-like cell subsets detected in the fast-growing tumors of more heavily-mutated mice, as well as in human biopsy and xenograft samples. We provide evidence that LNM cells pre-exist in normal skin, are expanded by Braf activation, can transition into malignant cells, and persist with malignant cells through multiple rounds of transplantation. We discuss the possibility that LNM cells not only serve as a pre-malignant state in the production of some melanomas, but also as an important intermediate in the development of drug resistance.

Percolation theory as a conceptual framework to explain spontaneous atrial fibrillation termination: a pilot study.

Atrial fibrillation (AF) strikingly possesses the ability to abruptly transition into more organized electrical activity and spontaneously terminate, even after persisting for long periods. Despite being central to the clinical behavior and treatment of AF, these phenomena remain incompletely understood. In this paper, we hypothesized that the spontaneous termination of AF may represent a type of percolation phase transition, which is more likely to occur when a domain spanning cluster of refractory sites in the atrium are connected (called a 'percolation cluster'). This was assessed in n=50 computational simulations of AF developed using the Aliev-Panfilov (APV) 2-dimensional cell model. In self-terminating simulations of AF, it was found that the average refractory cluster size, χ(p) (median: 647.7), and the largest refractory cluster size, M1 (median: 1702.3), abruptly increased just prior to AF spontaneously terminating, indicating the onset of the formation of a domain spanning percolation cluster. In contrast, simulations of sustained AF did not demonstrate an increase in χ(p) (median: 463.0) and M1 (median: 1448.2). Self-terminating AF simulations also demonstrated hallmark properties characteristic of a percolation phase transition, such as an abrupt increase in χ(p) at the critical occupation probability pc. The cluster size distribution was also shown to obey a power law for various occupation probabilities p, also indicative of a percolation phase transition. Collectively, these properties suggests that the spontaneous termination of AF could be a form of percolation phase transition, which could provide new insights for AF treatment.

Excess Bed Days and Hospitalization Costs Associated With 30-Day Complications Following Catheter Ablation of Atrial Fibrillation.

BACKGROUND: The incidence and type of complications following catheter ablation of atrial fibrillation have been extensively examined, but the impact associated with these complications on the length of stay and hospitalization costs is unknown. METHODS AND RESULTS: This cohort study included 20 117 adult patients (mean age 62.6±11.4 years, 30.3% women, median length of stay 1 day [interquartile range 1-2 days]) undergoing atrial fibrillation ablation in financial years 2011 to 2017 in Australia with available cost data from the National Hospital Cost Data Collection, which determines government reimbursement of health services provided. The primary outcome was the costs associated with complications occurring up to 30 days postdischarge adjusted for inflation to 2021 Australian dollars. We used generalized linear models to estimate the increase in length of stay and cost associated with complications, adjusting for patient characteristics. Within 30 days of hospital discharge, 1151 (5.72%) patients experienced a complication with bleeding (3.35%) and pericardial effusion (0.75%) being the most common. On average, the occurrence of a complication was associated with an adjusted 3.3 (95% CI, 3.1-3.6) excess bed days of hospital care (totaling 3851 days), and a $7812 (95% CI, $6754-$8870) increase in hospitalization cost (totaling $9.0 million). Most of the total excess cost was attributable to bleeding ($3.8 million, 41.9% of total excess cost) and pericardial effusion ($1.6 million, 18.2%). CONCLUSIONS: Complications following atrial fibrillation ablation were associated with significant increase in length of stay and hospitalization costs, most of which were attributable to bleeding and pericardial effusion. Strategies to improve procedural safety and reduce health care costs should focus on these complications.

Constructing bilayer and volumetric atrial models at scale.

To enable large in silico trials and personalized model predictions on clinical timescales, it is imperative that models can be constructed quickly and reproducibly. First, we aimed to overcome the challenges of constructing cardiac models at scale through developing a robust, open-source pipeline for bilayer and volumetric atrial models. Second, we aimed to investigate the effects of fibres, fibrosis and model representation on fibrillatory dynamics. To construct bilayer and volumetric models, we extended our previously developed coordinate system to incorporate transmurality, atrial regions and fibres (rule-based or data driven diffusion tensor magnetic resonance imaging (MRI)). We created a cohort of 1000 biatrial bilayer and volumetric models derived from computed tomography (CT) data, as well as models from MRI, and electroanatomical mapping. Fibrillatory dynamics diverged between bilayer and volumetric simulations across the CT cohort (correlation coefficient for phase singularity maps: left atrial (LA) 0.27 ± 0.19, right atrial (RA) 0.41 ± 0.14). Adding fibrotic remodelling stabilized re-entries and reduced the impact of model type (LA: 0.52 ± 0.20, RA: 0.36 ± 0.18). The choice of fibre field has a small effect on paced activation data (less than 12 ms), but a larger effect on fibrillatory dynamics. Overall, we developed an open-source user-friendly pipeline for generating atrial models from imaging or electroanatomical mapping data enabling in silico clinical trials at scale (https://github.com/pcmlab/atrialmtk).

Non-invasive Imaging Techniques for Monitoring Cellular Response to Treatment in Stable Vitiligo.

Punch grafting procedures, where small pieces of normal skin are transplanted into stable vitiligo patches, results in repigmentation in only half of patients treated, yet the factors that determine whether a patient responds to treatment or not are still unknown. Reflectance confocal microscopy (RCM) is adept at visualizing melanocyte migration and epidermal changes over large areas while multiphoton microscopy (MPM) can capture metabolic changes in keratinocytes. With the overall goal of identifying optical biomarkers for early treatment response, we followed 12 vitiligo lesions undergoing punch grafting. Dendritic melanocytes adjacent to the graft site were observed before clinical evidence of repigmentation in treatment responsive patients but not in treatment non-responsive patients, suggesting that the early visualization of melanocytes is indicative of a therapeutic response. Keratinocyte metabolic changes in vitiligo skin adjacent to the graft site also correlated with treatment response, indicating that a keratinocyte microenvironment that more closely resembles normal skin is more hospitable for migrating melanocytes. Taken together, these studies suggest that successful melanocyte transplantation requires both the introduction of new melanocytes and modulation of the local tissue microenvironment.

Markov modeling of phase singularity interaction effects in human atrial and ventricular fibrillation.

Atrial and ventricular fibrillation (AF/VF) are characterized by the repetitive regeneration of topological defects known as phase singularities (PSs). The effect of PS interactions has not been previously studied in human AF and VF. We hypothesized that PS population size would influence the rate of PS formation and destruction in human AF and VF, due to increased inter-defect interaction. PS population statistics were studied in computational simulations (Aliev-Panfilov), human AF and human VF. The influence of inter-PS interactions was evaluated by comparison between directly modeled discrete-time Markov chain (DTMC) transition matrices of the PS population changes, and M/M/∞ birth-death transition matrices of PS dynamics, which assumes that PS formations and destructions are effectively statistically independent events. Across all systems examined, PS population changes differed from those expected with M/M/∞. In human AF and VF, the formation rates decreased slightly with PS population when modeled with the DTMC, compared with the static formation rate expected through M/M/∞, suggesting new formations were being inhibited. In human AF and VF, the destruction rates increased with PS population for both models, with the DTMC rate increase exceeding the M/M/∞ estimates, indicating that PS were being destroyed faster as the PS population grew. In human AF and VF, the change in PS formation and destruction rates as the population increased differed between the two models. This indicates that the presence of additional PS influenced the likelihood of new PS formation and destruction, consistent with the notion of self-inhibitory inter-PS interactions.

Signalling by senescent melanocytes hyperactivates hair growth.

Niche signals maintain stem cells in a prolonged quiescence or transiently activate them for proper regeneration1. Altering balanced niche signalling can lead to regenerative disorders. Melanocytic skin nevi in human often display excessive hair growth, suggesting hair stem cell hyperactivity. Here, using genetic mouse models of nevi2,3, we show that dermal clusters of senescent melanocytes drive epithelial hair stem cells to exit quiescence and change their transcriptome and composition, potently enhancing hair renewal. Nevus melanocytes activate a distinct secretome, enriched for signalling factors. Osteopontin, the leading nevus signalling factor, is both necessary and sufficient to induce hair growth. Injection of osteopontin or its genetic overexpression is sufficient to induce robust hair growth in mice, whereas germline and conditional deletions of either osteopontin or CD44, its cognate receptor on epithelial hair cells, rescue enhanced hair growth induced by dermal nevus melanocytes. Osteopontin is overexpressed in human hairy nevi, and it stimulates new growth of human hair follicles. Although broad accumulation of senescent cells, such as upon ageing or genotoxic stress, is detrimental for the regenerative capacity of tissue4, we show that signalling by senescent cell clusters can potently enhance the activity of adjacent intact stem cells and stimulate tissue renewal. This finding identifies senescent cells and their secretome as an attractive therapeutic target in regenerative disorders.

Design, Synthesis, In Vitro and In Vivo Characterization of CDC42 GTPase Interaction Inhibitors for the Treatment of Cancer.

CDC42 GTPases (RHOJ, CDC42, and RHOQ) are overexpressed in multiple tumor types and activate pathways critical for tumor growth, angiogenesis, and metastasis. Recently, we reported the discovery of a novel lead compound, ARN22089, which blocks the interaction of CDC42 GTPases with specific downstream effectors. ARN22089 blocks tumor growth in BRAF mutant mouse melanoma models and patient-derived xenografts (PDXs) in vivo. ARN22089 also inhibits tumor angiogenesis in three-dimensional vascularized microtumor models in vitro. Notably, ARN22089 belongs to a novel class of trisubstituted pyrimidines. Based on these results, we describe an extensive structure-activity relationship of ∼30 compounds centered on ARN22089. We discovered and optimized two novel inhibitors (27, ARN25062, and 28, ARN24928), which are optimal back-up/follow-up leads with favorable drug-like properties and in vivo efficacy in PDX tumors. These findings further demonstrate the potential of this class of CDC42/RHOJ inhibitors for cancer treatment, with lead candidates ready for advanced preclinical studies.

Personalized 3D-printed frames to reduce leak from N95 filtering facepiece respirators: A prospective crossover trial in health care workers.

Correctly fitting N95 filtering facepiece respirators (FFRs) have become increasingly important in health care throughout the COVID-19 pandemic. We evaluated the hypothesis that personalized 3D-printed frames could improve N95 FFRs quantitative fit test pass rates and test scores in health care workers (HCWs). HCWs were recruited at a tertiary hospital in Adelaide, Australia (ACTRN 12622000388718). A mobile iPhone camera + app was used to produce 3D scans of volunteers' faces, which were then imported into a software program to produce personalized virtual scaffolds suited to each user's face and their unique anatomical features. These virtual scaffolds were printed on a commercially available 3D printer, producing plastic (and then silicone-coated, biocompatible) frames that can be fitted inside existing hospital supply N95 FFR. The primary endpoint was improved pass rates on quantitative fit testing, comparing participants wearing an N95 FFR alone (control 1) with participants wearing the frame + N95 FFR (intervention 1). The secondary endpoint was the fit factor (FF) in these groups, and R-COMFI respirator comfort and tolerability survey scores. N = 66 HCWs were recruited. The use of intervention 1 increased overall fit test pass rates to 62/66 (93.8%), compared to 27/66 (40.9%) for controls. (OR for pFF pass 20.89 (95%CI: 6.77, 64.48, p < 0.001.) Average FF increased, with the use of intervention 1-179.0 (95%CI: 164.3,193.7), compared to 85.2 (95%CI: 70.4,100.0) with control 1. Pass rates and FF were improved with intervention 1 compared to control 1 for all stages of the fit-test: bending, talking, side-to-side, and up-down motion. (p < 0.001 all stages). Tolerability and comfort of the frame were evaluated with the validated R-COMFI respirator comfort score, showing improvement with the frame compared to N95 FFR alone (p = 0.006). Personalized 3D-printed face frames decrease leakage, improve fit testing pass rates and FF, and provide improved comfort compared to the N95 FFR alone. Personalized 3D-printed face frames represent a rapidly scalable new technology to decrease FFR leakage in HCW and potentially the wider population.

A Low-Cost Modular Imaging System for Rapid, Multiplexed Immunofluorescence Detection in Clinical Tissues.

To image 4-plex immunofluorescence-stained tissue samples at a low cost with cellular level resolution and sensitivity and dynamic range required to detect lowly and highly abundant targets, here we describe a robust, inexpensive (<$9000), 3D printable portable imaging device (Tissue Imager). The Tissue Imager can immediately be deployed on benchtops for in situ protein detection in tissue samples. Applications for this device are broad, ranging from answering basic biological questions to clinical pathology, where immunofluorescence can detect a larger number of markers than the standard H&E or chromogenic immunohistochemistry (CIH) staining, while the low cost also allows usage in classrooms. After characterizing our platform's specificity and sensitivity, we demonstrate imaging of a 4-plex immunology panel in human cutaneous T-cell lymphoma (CTCL) formalin-fixed paraffin-embedded (FFPE) tissue samples. From those images, positive cells were detected using CellProfiler, a popular open-source software package, for tumor marker profiling. We achieved a performance on par with commercial epifluorescence microscopes that are >10 times more expensive than our Tissue Imager. This device enables rapid immunofluorescence detection in tissue sections at a low cost for scientists and clinicians and can provide students with a hands-on experience to understand engineering and instrumentation. We note that for using the Tissue Imager as a medical device in clinical settings, a comprehensive review and approval processes would be required.

Efficacy and safety of oral ritlecitinib for the treatment of active nonsegmental vitiligo: A randomized phase 2b clinical trial.

BACKGROUND: Vitiligo is a chronic autoimmune disorder characterized by depigmented patches of the skin. OBJECTIVE: To evaluate the efficacy and safety of ritlecitinib, an oral JAK3 (Janus kinase)/TEC (tyrosine kinase expressed in hepatocelluar carcinoma) inhibitor, in patients with active nonsegmental vitiligo in a phase 2b trial (NCT03715829). METHODS: Patients were randomized to once-daily oral ritlecitinib ± 4-week loading dose (200/50 mg, 100/50 mg, 30 mg, or 10 mg) or placebo for 24 weeks (dose-ranging period). Patients subsequently received ritlecitinib 200/50 mg daily in a 24-week extension period. The primary efficacy endpoint was percent change from baseline in Facial-Vitiligo Area Scoring Index at week 24. RESULTS: A total of 364 patients were treated in the dose-ranging period. Significant differences from placebo in percent change from baseline in Facial-Vitiligo Area Scoring Index were observed for the ritlecitinib 50 mg groups with (-21.2 vs 2.1; P < .001) or without (-18.5 vs 2.1; P < .001) a loading dose and ritlecitinib 30 mg group (-14.6 vs 2.1; P = .01). Accelerated improvement was observed after treatment with ritlecitinib 200/50 mg in the extension period (n = 187). No dose-dependent trends in treatment-emergent or serious adverse events were observed across the 48-week treatment. LIMITATIONS: Patients with stable vitiligo only were excluded. CONCLUSIONS: Oral ritlecitinib was effective and well tolerated over 48 weeks in patients with active nonsegmental vitiligo.

Role of interatrial conduction in atrial fibrillation: Mechanistic insights from renewal theory-based fibrillatory dynamic analysis.

Background: Interatrial conduction has been postulated to play an important role in atrial fibrillation (AF). The pathways involved in interatrial conduction during AF remain incompletely defined. Objective: We recently showed physiological assessment of fibrillatory dynamics could be performed using renewal theory, which determines rates of phase singularity formation (λf) and destruction (λd). Using the renewal approach, we aimed to understand the role of the interatrial septum and other electrically coupled regions during AF. Method: RENEWAL-AF is a prospective multicenter observational study recruiting AF ablation patients (ACTRN 12619001172190). We studied unipolar electrograms obtained from 16 biatrial locations prior to ablation using a 16-electrode Advisor HD Grid catheter. Renewal rate constants λf and λd were calculated, and the relationships between these rate constants in regions of interatrial connectivity were examined. Results: Forty-one AF patients (28.5% female) were recruited. A positive linear correlation was observed between λf and λd (1) across the interatrial septum (λf r2 = 0.5, P < .001, λd r2 = 0.45, P < .001), (2) in regions connected by the Bachmann bundle (right atrial appendage-left atrial appendage λf r2 = 0.29, P = .001; λd r2 = 0.2, P = .008), and (3) across the inferior interatrial routes (cavotricuspid isthmus-left atrial septum λf r2 = 0.67, P < .001; λd r2 = 0.55, P < .001). Persistent AF status and left atrial volume were found to be important effect modifiers of the degree of interatrial renewal rate statistical correlation. Conclusion: Our findings support the role of interseptal statistically determined electrical disrelation in sustaining AF. Additionally, renewal theory identified preferential conduction through specific interatrial pathways during fibrillation. These findings may be of importance in identifying clinically significant targets for ablation in AF patients.

The inspection paradox: An important consideration in the evaluation of rotor lifetimes in cardiac fibrillation.

Background and Objective: Renewal theory is a statistical approach to model the formation and destruction of phase singularities (PS), which occur at the pivots of spiral waves. A common issue arising during observation of renewal processes is an inspection paradox, due to oversampling of longer events. The objective of this study was to characterise the effect of a potential inspection paradox on the perception of PS lifetimes in cardiac fibrillation. Methods: A multisystem, multi-modality study was performed, examining computational simulations (Aliev-Panfilov (APV) model, Courtmanche-Nattel model), experimentally acquired optical mapping Atrial and Ventricular Fibrillation (AF/VF) data, and clinically acquired human AF and VF. Distributions of all PS lifetimes across full epochs of AF, VF, or computational simulations, were compared with distributions formed from lifetimes of PS existing at 10,000 simulated commencement timepoints. Results: In all systems, an inspection paradox led towards oversampling of PS with longer lifetimes. In APV computational simulations there was a mean PS lifetime shift of +84.9% (95% CI, ± 0.3%) (p < 0.001 for observed vs overall), in Courtmanche-Nattel simulations of AF +692.9% (95% CI, ±57.7%) (p < 0.001), in optically mapped rat AF +374.6% (95% CI, ± 88.5%) (p = 0.052), in human AF mapped with basket catheters +129.2% (95% CI, ±4.1%) (p < 0.05), human AF-HD grid catheters 150.8% (95% CI, ± 9.0%) (p < 0.001), in optically mapped rat VF +171.3% (95% CI, ±15.6%) (p < 0.001), in human epicardial VF 153.5% (95% CI, ±15.7%) (p < 0.001). Conclusion: Visual inspection of phase movies has the potential to systematically oversample longer lasting PS, due to an inspection paradox. An inspection paradox is minimised by consideration of the overall distribution of PS lifetimes.

Alchemical Free Energy Calculations to Investigate Protein-Protein Interactions: the Case of the CDC42/PAK1 Complex.

Here, we show that alchemical free energy calculations can quantitatively compute the effect of mutations at the protein-protein interface. As a test case, we have used the protein complex formed by the small Rho-GTPase CDC42 and its downstream effector PAK1, a serine/threonine kinase. Notably, the CDC42/PAK1 complex offers a wealth of structural, mutagenesis, and binding affinity data because of its central role in cellular signaling and cancer progression. In this context, we have considered 16 mutations in the CDC42/PAK1 complex and obtained excellent agreement between computed and experimental data on binding affinity. Importantly, we also show that a careful analysis of the side-chain conformations in the mutated amino acids can considerably improve the computed estimates, solving issues related to sampling limitations. Overall, this study demonstrates that alchemical free energy calculations can conveniently be integrated into the design of experimental mutagenesis studies.