Atrial Arrhythmogenic Substrates: The Role of Structure and Molecular Remodeling

Atrial tachyarrhythmias, specifically atrial flutter: AFl) and fibrillation: AF), affect over 2.2 million Americans, leading to more hospitalizations than any other cardiac arrhythmia. These arrhythmias are defined by the presence of reentrant circuits of excitation leading to high atrial rates and uncoordinated activation of the ventricles. The underlying mechanisms of AFl/AF have proven complex and, despite a century of research, no one effective treatment has been developed. Surgical ablation and pharmacological therapies are both fraught with risks and potential pro-arrhythmic side effects. Electrical cardioversion, on the other hand, is extremely effective in terminating these arrhythmias, but the high-energy shocks required for termination cause substantial pain to the patient. In this dissertation, we first investigate the underlying molecular and structural mechanisms of AF in two clinically-relevant models - the human and canine hearts. We identify structural and molecular substrates responsible for the generation and maintenance of AFl/AF. We then explore the application of a novel low-voltage defibrillation therapy to a rabbit model of atrial tachyarrhythmias and show significant reductions in the defibrillation threshold for both AF and AFl. We utilize a variety of experimental techniques, such as high throughput quantitative PCR, optical coherence tomography, and optical mapping. Only truly integrative approaches to arrhythmia research, combining a variety of experimental models and techniques, can continue to unravel the complexities of underlying molecular, structural, and electrophysiological mechanisms and develop effective, safe therapies, as we demonstrate in this dissertation with regards to atrial tachyarrhythmias

Users\u27 experiences of wearable activity trackers: A cross-sectional study

Background Wearable activity trackers offer considerable promise for helping users to adopt healthier lifestyles. This study aimed to explore users’ experience of activity trackers, including usage patterns, sharing of data to social media, perceived behaviour change (physical activity, diet and sleep), and technical issues/barriers to use. Methods A cross-sectional online survey was developed and administered to Australian adults who were current or former activity tracker users. Results were analysed descriptively, with differences between current and former users and wearable brands explored using independent samples t-tests, Mann-Whitney, and chi square tests. Results Participants included 200 current and 37 former activity tracker users (total N = 237) with a mean age of 33.1 years (SD 12.4, range 18–74 years). Fitbit (67.5%) and Garmin devices (16.5%) were most commonly reported. Participants typically used their trackers for sustained periods (5–7 months) and most intended to continue usage. Participants reported they had improved their physical activity (51–81%) more commonly than they had their diet (14–40%) or sleep (11–24%), and slightly more participants reported to value the real time feedback (89%) compared to the long-term monitoring (78%). Most users (70%) reported they had experienced functionality issues with their devices, most commonly related to battery life and technical difficulties. Conclusions Results suggest users find activity trackers appealing and useful tools for increasing perceived physical activity levels over a sustained period

Parents on the Same Page:A Mixed-Methods Investigation of the Acceptability and Appropriateness of Tuning in to Kids Together

In this study, we examined the acceptability and appropriateness of Tuning in to Kids Together (TIK-Together) from the perspective of program facilitators. TIK-Together is newly a modified version of Tuning in to Kids that specifically focuses on the coparenting relationship (i.e., how parents work together to raise their children) and requires parents to both attend sessions. Through a mixed-method design, Australian facilitators who participated in the TIK-Together pilot study provided their perspectives on the acceptability (i.e., affective attitudes, burden, program benefits) and appropriateness of the program in their communities. It is essential to involve stakeholders, such as program facilitators, in pilot studies as they can provide valuable feedback from their first-hand experience with the program. Facilitators reported that TIK-Together was enjoyable and rewarding, and although delivery was straightforward, additional preparation time was required to manage program materials. Facilitators observed several program benefits, including coparents becoming more aligned and collaborative in their approach to parenting. Certain program components were particularly helpful for parents, including activities that were experiential, interactive, and reflective. Overall, facilitators deemed TIK-Together appropriate for their communities; however, it is not suitable for parents who are uncomfortable or unwilling to work alongside each other in sessions. Given the increased focus on a coparent approach to parenting interventions, the findings provide important insights for researchers and practitioners.</p

Computational Optogenetics: Empirically-Derived Voltage- and Light-Sensitive Channelrhodopsin-2 Model

Channelrhodospin-2 (ChR2), a light-sensitive ion channel, and its variants have emerged as new excitatory optogenetic tools not only in neuroscience, but also in other areas, including cardiac electrophysiology. An accurate quantitative model of ChR2 is necessary for in silicoprediction of the response to optical stimulation in realistic tissue/organ settings. Such a model can guide the rational design of new ion channel functionality tailored to different cell types/tissues. Focusing on one of the most widely used ChR2 mutants (H134R) with enhanced current, we collected a comprehensive experimental data set of the response of this ion channel to different irradiances and voltages, and used these data to develop a model of ChR2 with empirically-derived voltage- and irradiance- dependence, where parameters were fine-tuned via simulated annealing optimization. This ChR2 model offers: 1) accurate inward rectification in the current-voltage response across irradiances; 2) empirically-derived voltage- and light-dependent kinetics (activation, deactivation and recovery from inactivation); and 3) accurate amplitude and morphology of the response across voltage and irradiance settings. Temperature-scaling factors (Q10) were derived and model kinetics was adjusted to physiological temperatures. Using optical action potential clamp, we experimentally validated model-predicted ChR2 behavior in guinea pig ventricular myocytes. The model was then incorporated in a variety of cardiac myocytes, including human ventricular, atrial and Purkinje cell models. We demonstrate the ability of ChR2 to trigger action potentials in human cardiomyocytes at relatively low light levels, as well as the differential response of these cells to light, with the Purkinje cells being most easily excitable and ventricular cells requiring the highest irradiance at all pulse durations. This new experimentally-validated ChR2 model will facilitate virtual experimentation in neural and cardiac optogenetics at the cell and organ level and provide guidance for the development of in vivo tools

ChromID identifies the protein interactome at chromatin marks

ISSN:1546-1696ISSN:1087-015

Efficient pre-mRNA cleavage prevents replication-stress-associated genome instability

Cellular mechanisms that safeguard genome integrity are often subverted in cancer. To identify cancer-related genome caretakers, we employed a convergent multi-screening strategy coupled to quantitative image-based cytometry and ranked candidate genes according to multivariate readouts reflecting viability, proliferative capacity, replisome integrity, and DNA damage signaling. This unveiled regulators of replication stress resilience, including components of the pre-mRNA cleavage and polyadenylation complex. We show that deregulation of pre-mRNA cleavage impairs replication fork speed and leads to excessive origin activity, rendering cells highly dependent on ATR function. While excessive formation of RNA:DNA hybrids under these conditions was tightly associated with replication-stress-induced DNA damage, inhibition of transcription rescued fork speed, origin activation, and alleviated replication catastrophe. Uncoupling of pre-mRNA cleavage from co-transcriptional processing and export also protected cells from replication-stress-associated DNA damage, suggesting that pre-mRNA cleavage provides a mechanism to efficiently release nascent transcripts and thereby prevent gene gating-associated genomic instability

H1N1 vaccination in Sjögren’s syndrome triggers polyclonal B cell activation and promotes autoantibody production

Objectives: Vaccination of patients with rheumatic disease has been reported to result in lower antibody titres than in healthy individuals. However, studies primarily include patients on immunosuppressive therapy. Here, we investigated the immune response of treatment-naïve patients diagnosed with primary Sjögren’s syndrome (pSS) to an H1N1 influenza vaccine. Methods: Patients with Sjögren’s syndrome without immunomodulatory treatment and age-matched and gender-matched healthy controls were immunised with an H1N1 influenza vaccine and monitored for serological and cellular immune responses. Clinical symptoms were monitored with a standardised form. IgG class switch and plasma cell differentiation were induced in vitro in purified naïve B cells of untreated and hydroxychloroquine-treated patients and healthy controls. Gene expression was assessed by NanoString technology. Results: Surprisingly, treatment-naïve patients with Sjögren’s syndrome developed higher H1N1 IgG titres of greater avidity than healthy controls on vaccination. Notably, off-target B cells were also triggered resulting in increased anti-EBV and autoantibody titres. Endosomal toll-like receptor activation of naïve B cells in vitro revealed a greater propensity of patient-derived cells to differentiate into plasmablasts and higher production of class switched IgG. The amplified plasma cell differentiation and class switch could be induced in cells from healthy donors by preincubation with type 1 interferon, but was abolished in hydroxychloroquine-treated patients and after in vitro exposure of naïve B cells to chloroquine. Conclusions: This comprehensive analysis of the immune response in autoimmune patients to exogenous stimulation identifies a mechanistic basis for the B cell hyperactivity in Sjögren’s syndrome, and suggests that caution is warranted when considering vaccination in non-treated autoimmune patients

The histone methyltransferase SETD2 negatively regulates cell size

Cell size varies between cell types but is tightly regulated by cell intrinsic and extrinsic mechanisms. Cell size control is important for cell function, and changes in cell size are frequently observed in cancer. Here, we uncover a role for SETD2 in regulating cell size. SETD2 is a lysine methyltransferase and a tumor suppressor protein involved in transcription, RNA processing and DNA repair. At the molecular level, SETD2 is best known for associating with RNA polymerase II through its Set2-Rbp1 interacting (SRI) domain and methylating histone H3 on lysine 36 (H3K36) during transcription. Using multiple independent perturbation strategies, we identify SETD2 as a negative regulator of global protein synthesis rates and cell size. We provide evidence that overexpression of the H3K36 demethylase KDM4A or the oncohistone H3.3K36M also increase cell size. In addition, ectopic overexpression of a decoy SRI domain increased cell size, suggesting that the relevant substrate is engaged by SETD2 via its SRI domain. These data add a central role of SETD2 in regulating cellular physiology and warrant further studies on separating the different functions of SETD2 in cancer development

Isoform‐specific localization of DNMT3A regulates DNA methylation fidelity at bivalent CpG islands

DNA methylation is a prevalent epigenetic modification involved in transcriptional regulation and essential for mammalian development. While the genome-wide distribution of this mark has been studied to great detail, the mechanisms responsible for its correct deposition, as well as the cause for its aberrant localization in cancers, have not been fully elucidated. Here, we have compared the activity of individual DNMT3A isoforms in mouse embryonic stem and neuronal progenitor cells and report that these isoforms differ in their genomic binding and DNA methylation activity at regulatory sites. We identify that the longer isoform DNMT3A1 preferentially localizes to the methylated shores of bivalent CpG island promoters in a tissue-specific manner. The isoform-specific targeting of DNMT3A1 coincides with elevated hydroxymethylcytosine (5-hmC) deposition, suggesting an involvement of this isoform in mediating turnover of DNA methylation at these sites. Through genetic deletion and rescue experiments, we demonstrate that this isoform-specific recruitment plays a role in de novo DNA methylation at CpG island shores, with potential implications on H3K27me3-mediated regulation of developmental genes