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Abstract Or122: Noncoding regulation of epicardial gene expression and epithelial-to-mesenchymal transition during heart development
During organogenesis, epicardial cells undergo epithelial-to-mesenchymal transition (EMT), contributing essential cell types and paracrine signalling to the growing heart. The epicardium is integral to heart regeneration in lower vertebrates and neonatal mammalian injured hearts. That said, prospects to harness the epicardium for therapeutic applications to effect adult heart repair, heavily depend on improved insight into its intrinsic properties in development. Cell fate decisions underpinning EMT are directed by transcription factors such as Wilms’ tumour 1 (WT1). Whilst a requirement for Wt1 in heart development is established, the mechanisms underpinning its activation remain elusive. We identified two e volutionary c onserved r egions (ECRs) shared in mouse and human, located within intron 1 of Wt1 locus. We hypothesised these regulatory sequences direct locus activation and EMT to support normal heart development. Here, we used CRISPR/Cas9 gene-editing technology to generate mice carrying a sequence deletion containing one ECR or a deletion comprising both ECRs. Extensive survival analysis, high-resolution episcopic microscopy (HREM), qPCR, immunostaining, confocal microscopy and epicardial explants were used to characterise heart formation. Mendelian ratios indicated an overall underrepresentation of Wt1 ΔECR/ΔECR mutants in adulthood. HREM revealed smaller hearts, incidence of myocardial non-compaction and spongy interventricular septum with muscular and membranous defects, tricuspid hypoplasia and enlarged aortic valves, as well as abnormal patterning of coronary artery stems in mutant hearts. Expression of Wt1 was markedly reduced in Wt1 ΔECR/ΔECR hearts but not kidneys, suggesting intronic enhancers are cardiac-specific. Whole-mount and tissue immunostaining revealed abnormal coronary vessel and innervation patterning in the subepicardium, as well as reduced EMT and myocardial non-compaction/hyper-trabeculation in Wt1 ΔECR/ΔECR . Collectively, we demonstrated a requirement for novel Wt1 intronic enhancers regulating locus activity and essential epicardial EMT-associated biological processes in normal heart development. Importantly, observation of septum and semilunar valve defects in Δ ECR hearts suggests an hitherto unrecognised role for WT1-driven EMT, opening new avenues of research to improve our understanding of congenital heart disease affecting at least 1:150 live births, with remarkably two thirds of cases having unknown aetiology.
Evaluating the transcriptional regulators of arterial gene expression via a catalogue of characterized arterial enhancers.
The establishment and growth of the arterial endothelium requires the coordinated expression of numerous genes. However, regulation of this process is not yet fully understood. Here, we combined in silico analysis with transgenic mice and zebrafish models to characterize arterial-specific enhancers associated with eight key arterial identity genes (Acvrl1/Alk1, Cxcr4, Cxcl12, Efnb2, Gja4/Cx37, Gja5/Cx40, Nrp1 and Unc5b). Next, to elucidate the regulatory pathways upstream of arterial gene transcription, we investigated the transcription factors binding each arterial enhancer compared to a similar assessment of non-arterial endothelial enhancers. These results found that binding of SOXF and ETS factors was a common occurrence at both arterial and pan-endothelial enhancers, suggesting neither are sufficient to direct arterial specificity. Conversely, FOX motifs independent of ETS motifs were over-represented at arterial enhancers. Further, MEF2 and RBPJ binding was enriched but not ubiquitous at arterial enhancers, potentially linked to specific patterns of behaviour within the arterial endothelium. Lastly, there was no shared or arterial-specific signature for WNT-associated TCF/LEF, TGFβ/BMP-associated SMAD1/5 and SMAD2/3, shear stress-associated KLF4 or venous-enriched NR2F2. This cohort of well characterized and in vivo-verified enhancers can now provide a platform for future studies into the interaction of different transcriptional and signalling pathways with arterial gene expression.
From Cadavers to Neural Networks: A Narrative Review on Artificial Intelligence Tools in Anatomy Teaching
The application of artificial intelligence (AI) in anatomy teaching is gaining attention due to its potential to support personalized learning and its ability to provide customized, real-time feedback. While the potential impact of complete AI integration in medical education remains unclear, there is a suspicion that it could revolutionize pedagogical and assessment practices. Traditional anatomy teaching strategies that use donated human resources hinder continuous learning due to accessibility and ethical challenges. Existing resources, such as anatomy atlases, may not provide knowledge of spatial relationships. AI-powered applications enable students to access more flexible and accessible learning material beyond physical classrooms. This review critically evaluates current advances and the possible impacts of AI in learning anatomy based on the reported empirical original studies. Additionally, it recognizes the challenges and provides possible solutions for them. Most of the initiatives to integrate AI in anatomy teaching are directed towards the development of customized anatomy chatbots and their integration with virtual reality (VR). Although the crucial role of medical imaging in the anatomy curriculum is recognized, currently, no AI application has been developed to target this field. This review discusses the currently available AI tools for anatomy teaching. Additionally, the knowledge gaps and future directions of AI in medical education, especially anatomy education, are also discussed. With the present advances in AI technologies, their application in anatomical education is still deficient. This review paper provides an overview of recent tools used in anatomy teaching and learning.
Astrocytic striatal GABA transporter activity governs dopamine release and shows maladaptive downregulation in early parkinsonism
SummaryStriatal dopamine (DA) is critical for action and learning. Recent data show DA release is under tonic inhibition by striatal GABA. Ambient striatal GABA tone on striatal projection neurons can be governed by plasma membrane GABA uptake transporters (GATs) on astrocytes. However, whether striatal GATs and astrocytes determine DA output are unknown. We reveal that DA release in mouse dorsolateral striatum, but not nucleus accumbens core, is governed by GAT-1 and GAT-3. These GATs are partly localized to astrocytes, and are enriched in dorsolateral striatum compared to accumbens core. In a mouse model of early parkinsonism, GATs were downregulated and tonic GABAergic inhibition of DA release augmented, with corresponding attenuation of GABA co-release from dopaminergic axons. These data define previously unappreciated and important roles for GATs and astrocytes in determining DA release in striatum, and reveal a maladaptive plasticity in early parkinsonism that impairs DA output in vulnerable striatal regions.HighlightsGABA transporters set the level of GABA inhibition of DA output in dorsal striatumAstrocytes facilitate DA release levels by limiting tonic GABA inhibitionTonic inhibition of DA release is augmented in a mouse model of early parkinsonismDA and GABA co-release are reduced in a mouse model of early parkinsonism
Rapid modulation of striatal cholinergic interneurons and dopamine release by satellite astrocytes.
Astrocytes are increasingly appreciated to possess underestimated and important roles in modulating neuronal circuits. Astrocytes in striatum can regulate dopamine transmission by governing the extracellular tone of axonal neuromodulators, including GABA and adenosine. However, here we reveal that striatal astrocytes occupy a cell type-specific anatomical and functional relationship with cholinergic interneurons (ChIs), through which they rapidly excite ChIs and govern dopamine release via nicotinic acetylcholine receptors on subsecond timescales. We identify that ChI somata are in unexpectedly close proximity to astrocyte somata, in mouse and human, forming a "soma-to-soma" satellite-like configuration not typically observed for other striatal neurons. We find that transient depolarization of astrocytes in mouse striatum reversibly regulates ChI excitability by decreasing extracellular calcium. These findings reveal a privileged satellite astrocyte-interneuron interaction for striatal ChIs operating on subsecond timescales via regulation of extracellular calcium dynamics to shape downstream striatal circuit activity and dopamine signaling.
Circulating neuropeptide Y dynamics and performance during exercise in heart failure patients with contemporary medical and device therapy.
High cardiac sympathetic drive and release of the sympathetic cotransmitter neuropeptide Y (NPY) are significant features of congestive heart failure (CHF), in which resting venous NPY levels are known to be associated with mortality. However, whether circulating NPY levels increase during exercise in CHF when they are already elevated is controversial. We sought to establish the dynamics of circulating NPY levels in CHF patients treated with contemporary medical therapy and devices in relationship to indices of performance linked to long-term prognosis. CHF patients (n = 15) underwent cardiopulmonary exercise testing with venous blood sampling at rest, peak exercise and recovery. These patients had significantly higher resting venous NPY levels compared with an age- and sex-matched control group of patients (n = 16) with normal left ventricular function (40 ± 6.9 vs. 9.0 ± 4.6 pg/mL, respectively; P
Nanoscale Biodegradable Printing for Designed Tuneability of Vaccine Delivery Kinetics.
Two-photon polymerization (2PP) 3D printing enables top-down biomaterial synthesis with nanoscale spatial resolution for de novo design of monodisperse injectable drug delivery systems. Spatiotemporal Controlled Release Inks of Biocompatible polyEsters (SCRIBE) is a novel poly(lactic-co-glycolic acid)-triacrylate resin family with sub-micron resolution and tuneable hydrolysis that addresses the limitations of current 2PP resins. SCRIBE enables the direct printing of hollow microparticles with tuneable chemistry and complex geometries inaccessible to molding techniques, which are used to engineer controlled protein release in vitro and in vivo. SCRIBE microparticles are used to modulate antibody titers and class switching as a function of antigen release rate and extend these findings to enable a single-injection vaccine formulation with extended antibody induction kinetics. Demonstrating how the chemistry and computer-aided design of 2PP-printed microparticles can be used to tune responses to biomacromolecule release in vivo opens significant opportunities for a new generation of drug delivery vehicles.
Visual Dyslexia
Purpose: To revive the idea which was previously generally accepted, that learning to sequence written text visually is the crucial first step in learning to read. But in the last few years the phonological theory of dyslexia (PT) has become dominant. It asserts that DD is due to a psycho-linguistic deficit causing difficulty with grasping the concept of phonemes. This ignores a century of research showing that the development of a network of visual magnocellular neurons (M- cells) that signal the timing of visual events precedes and is essential for the development of phonological awareness. Recent Findings: We review the cerebral structures with which the visual M- system is associated in reading alphabetic and other scripts and show in each case how their development is impaired in DD. The simplest way to improve M- cell function involves viewing text through yellow or blue filters; we discuss how these may work. Even simpler is improving children’s nutrition; and we discuss some aspects of this. The genes which are associated with the M- system also play an important role in regulating the immune system; hence we discuss how DD is associated with autoimmune conditions such as asthma and eczema and disordered control of inflammation. Summary: There is now a very great weight of evidence demonstrating that visual sequencing of text mediated by the visual magnocellular timing system is the first essential for learning to read, and that impaired development of this system is a major cause of Developmental Dyslexia.
Metabolic flexibility and reverse remodelling of the failing human heart.
BACKGROUND AND AIMS: Cardiac resynchronization therapy (CRT) produces long-term reverse remodelling which requires greater adenosine triphosphate delivery to the contractile machinery. Whilst the heart retains some metabolic flexibility in non-ischaemic cardiomyopathy, whether this correlates with reverse remodelling is unknown. This study investigated whether CRT acutely changes cardiac substrate uptake, and whether this translates to favourable reverse remodelling. METHODS: The effect of CRT on cardiac substrate uptake was assessed via direct coronary flow and arteriovenous measurements, with metabolomic/lipidomic analysis on infusions of insulin/glucose and intralipid. Cardiac function was assessed with left ventricular pressure-volume loops during implantation, and cardiac magnetic resonance before and 6 months following CRT, with and without biventricular pacing. RESULTS: Regardless of substrate infusion, CRT acutely improved stroke work without increasing O2 uptake on both insulin/glucose (by 34%, P = .05) and intralipid (by 36%, P = .03). This was followed by increased fatty acid (FA) uptake on insulin/glucose (R = 0.89, P = .03) and increased β-hydroxybutyrate uptake (R = 0.81, P = .05) during intralipid infusion. After 6 months, there was a 48% (P < .001) reduction in left ventricular end diastolic volume, beyond that achievable by acutely shortening or lengthening QRS duration. Reverse remodelling significantly correlated with increased FA uptake with CRT on insulin/glucose (R = 0.71, P = .05) driven by long and medium chain uptake, and increased ketone uptake with CRT on intralipid (R = 0.79, P = .05). CONCLUSIONS: CRT acutely alters the metabolic phenotype of non-ischaemic cardiomyopathy towards a more physiological picture of FA uptake which correlates with reverse remodelling. Retained metabolic flexibility may therefore be critical for subsequent reverse remodelling.
BMP signaling promotes zebrafish heart regeneration via alleviation of replication stress.
In contrast to mammals, adult zebrafish achieve complete heart regeneration via proliferation of cardiomyocytes. Surprisingly, we found that regenerating cardiomyocytes experience DNA replication stress, which represents one reason for declining tissue regeneration during aging in mammals. Pharmacological inhibition of ATM and ATR kinases revealed that DNA damage response signaling is essential for zebrafish heart regeneration. Manipulation of Bone Morphogenetic Protein (BMP)-Smad signaling using transgenics and mutants showed that BMP signaling alleviates cardiomyocyte replication stress. BMP signaling also rescues neonatal mouse cardiomyocytes, human fibroblasts and human hematopoietic stem and progenitor cells (HSPCs) from replication stress. DNA fiber spreading assays indicate that BMP signaling facilitates re-start of replication forks after replication stress-induced stalling. Our results identify the ability to overcome replication stress as key factor for the elevated zebrafish heart regeneration capacity and reveal a conserved role for BMP signaling in promotion of stress-free DNA replication.
Exenatide once a week versus placebo as a potential disease-modifying treatment for people with Parkinson's disease in the UK: a phase 3, multicentre, double-blind, parallel-group, randomised, placebo-controlled trial.
BACKGROUND: GLP-1 receptor agonists have neurotrophic properties in in-vitro and in-vivo models of Parkinson's disease and results of epidemiological studies and small randomised trials have suggested possible benefits for risk and progression of Parkinson's disease. We aimed to establish whether the GLP-1 receptor agonist, exenatide, could slow the rate of progression of Parkinson's disease. METHODS: We did a phase 3, multicentre, double-blind, parallel-group, randomised, placebo-controlled trial at six research hospitals in the UK. Participants were aged 25-80 years with a diagnosis of Parkinson's disease, were at Hoehn and Yahr stage 2·5 or less when on dopaminergic treatment, and were on dopaminergic treatment for at least 4 weeks before enrolment. Participants were randomly assigned (1:1) using a web-based system with minimisation according to Hoehn and Yahr stage and study site to receive extended-release exenatide 2 mg by subcutaneous pen injection once per week over 96 weeks, or visually identical placebo. All participants and all research team members at study sites were masked to randomisation allocation. The primary outcome was the Movement Disorder Society-sponsored revision of the Unified Parkinson's Disease Rating Scale (MDS-UPDRS) part III score, off dopaminergic medication at 96 weeks, analysed in the intention-to-treat population using a linear mixed modelling approach. This study is registered with ISRCTN (14552789), EudraCT (2018-003028-35), and ClinicalTrials.gov (NCT04232969). FINDINGS: Between Jan 23, 2020, and April 23, 2022, 215 participants were screened for eligibility, of whom 194 were randomly assigned to exenatide (n=97) or placebo (n=97). 56 (29%) participants were female and 138 (71%) were male. 92 participants in the exenatide group and 96 in the placebo group had at least one follow-up visit and were included in analyses. At 96 weeks, MDS-UPDRS III OFF-medication scores had increased (worsened) by a mean of 5·7 points (SD 11·2) in the exenatide group, and by 4·5 points (SD 11·4) points in the placebo group (adjusted coefficient for the effect of exenatide 0·92 [95% CI -1·56 to 3·39]; p=0·47). Nine (9%) participants in the exenatide group had at least one serious adverse event compared with 11 (11%) in the placebo group. INTERPRETATION: Our findings suggest that exenatide is safe and well tolerated. We found no evidence to support exenatide as a disease-modifying treatment for people with Parkinson's disease. Studies with agents that show better target engagement or in specific subgroups of patients are needed to establish whether there is any support for the use of GLP-1 receptor agonists for Parkinson's disease. FUNDING: National Institute for Health and Care Research and Cure Parkinson's.
Ac/Ds transposition for CRISPR/dCas9-SID4x epigenome modulation in zebrafish.
Due to its genetic amenability coupled with advances in genome editing, zebrafish is an excellent model to examine the function of (epi)genomic elements. Here, we repurposed the Ac/Ds maize transposition system to efficiently characterise zebrafish cis-regulated elements, also known as enhancers, in F0-microinjected embryos. We further used the system to stably express guide RNAs enabling CRISPR/dCas9-interference (CRISPRi) perturbation of enhancers without disrupting the underlying genetic sequence. In addition, we probed the phenomenon of antisense transcription at two neural crest gene loci. Our study highlights the utility of Ac/Ds transposition as a new tool for transient epigenome modulation in zebrafish.
Macrophages directly contribute collagen to scar formation during zebrafish heart regeneration and mouse heart repair.
Canonical roles for macrophages in mediating the fibrotic response after a heart attack include extracellular matrix turnover and activation of cardiac fibroblasts to initiate collagen deposition. Here we reveal that macrophages directly contribute collagen to the forming post-injury scar. Unbiased transcriptomics shows an upregulation of collagens in both zebrafish and mouse macrophages following heart injury. Adoptive transfer of macrophages, from either collagen-tagged zebrafish or adult mouse GFPtpz-collagen donors, enhances scar formation via cell autonomous production of collagen. In zebrafish, the majority of tagged collagen localises proximal to the injury, within the overlying epicardial region, suggesting a possible distinction between macrophage-deposited collagen and that predominantly laid-down by myofibroblasts. Macrophage-specific targeting of col4a3bpa and cognate col4a1 in zebrafish significantly reduces scarring in cryoinjured hosts. Our findings contrast with the current model of scarring, whereby collagen deposition is exclusively attributed to myofibroblasts, and implicate macrophages as direct contributors to fibrosis during heart repair.