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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.
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Dano ao DNA , Replicação do DNA , Instabilidade Genômica , Neoplasias/genética , Clivagem do RNA , Precursores de RNA/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , Transporte Ativo do Núcleo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ácidos Nucleicos Heteroduplexes/genética , Ácidos Nucleicos Heteroduplexes/metabolismo , Poliadenilação , Precursores de RNA/biossíntese , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Proteínas de Ligação a RNARESUMO
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.
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Histonas , Neoplasias , Tamanho Celular , Histona Metiltransferases/metabolismo , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Lisina , Neoplasias/metabolismo , RNA Polimerase II/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
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.
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Ilhas de CpG , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Animais , Sítios de Ligação , Diferenciação Celular , Linhagem Celular , Células Cultivadas , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismoRESUMO
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.
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Exercício Físico/psicologia , Monitores de Aptidão Física/estatística & dados numéricos , Comportamentos Relacionados com a Saúde , Dispositivos Eletrônicos Vestíveis/estatística & dados numéricos , Adolescente , Adulto , Idoso , Austrália , Estudos Transversais , Dieta/psicologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sono , Mídias Sociais/estatística & dados numéricos , Inquéritos e Questionários , Adulto JovemRESUMO
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 silico prediction 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.
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Luz , Modelos Biológicos , Miócitos Cardíacos/fisiologia , Channelrhodopsins , Humanos , Optogenética , Técnicas de Patch-ClampRESUMO
Profiling compounds and genetic perturbations via high-content imaging has become increasingly popular for drug discovery, but the technique is limited to endpoint images of fixed cells. In contrast, electronic-based devices offer label-free, functional information of live cells, yet current approaches suffer from low-spatial resolution or single-well throughput. Here, we report a semiconductor 96-microplate platform designed for high-resolution real-time impedance "imaging" at scale. Each well features 4,096 electrodes at 25 µm spatial resolution while a miniaturized data interface allows 8× parallel plate operation (768 total wells) within each incubator for enhanced throughputs. New electric field-based, multi-frequency measurement techniques capture >20 parameter images including tissue barrier, cell-surface attachment, cell flatness, and motility every 15 min throughout experiments. Using these real-time readouts, we characterized 16 cell types, ranging from primary epithelial to suspension, and quantified heterogeneity in mixed epithelial and mesenchymal co-cultures. A proof-of-concept screen of 904 diverse compounds using 13 semiconductor microplates demonstrates the platform's capability for mechanism of action (MOA) profiling with 25 distinct responses identified. The scalability of the semiconductor platform combined with the translatability of the high dimensional live-cell functional parameters expands high-throughput MOA profiling and phenotypic drug discovery applications.
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High-content imaging for compound and genetic profiling is popular for drug discovery but limited to endpoint images of fixed cells. Conversely, electronic-based devices offer label-free, live cell functional information but suffer from limited spatial resolution or throughput. Here, we introduce a semiconductor 96-microplate platform for high-resolution, real-time impedance imaging. Each well features 4096 electrodes at 25 µm spatial resolution and a miniaturized data interface allows 8× parallel plate operation (768 total wells) for increased throughput. Electric field impedance measurements capture >20 parameter images including cell barrier, attachment, flatness, and motility every 15 min during experiments. We apply this technology to characterize 16 cell types, from primary epithelial to suspension cells, and quantify heterogeneity in mixed co-cultures. Screening 904 compounds across 13 semiconductor microplates reveals 25 distinct responses, demonstrating the platform's potential for mechanism of action profiling. The scalability and translatability of this semiconductor platform expands high-throughput mechanism of action profiling and phenotypic drug discovery applications.
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Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Ensaios de Triagem em Larga Escala/métodos , Diagnóstico por Imagem , Impedância Elétrica , EletrodosRESUMO
Increased dispersion of repolarization has been suggested to underlie increased arrhythmogenesis in human heart failure (HF). However, no detailed repolarization mapping data were available to support the presence of increased dispersion of repolarization in failing human heart. In the present study, we aimed to determine the existence of enhanced repolarization dispersion in the right ventricular (RV) endocardium from failing human heart and examine its association with arrhythmia inducibility. RV free wall preparations were dissected from five failing and five nonfailing human hearts, cannulated and coronary perfused. RV endocardium was optically mapped from an â¼6.3 × 6.3 cm(2) field of view. Action potential duration (APD), dispersion of APD, and conduction velocity (CV) were quantified for basic cycle lengths (BCL) ranging from 2,000 ms to the functional refractory period. We found that RV APD was significantly prolonged within the failing group compared with the nonfailing group (560 ± 44 vs. 448 ± 39 ms, at BCL = 2,000 ms, P < 0.05). Dispersion of APD was increased in three failing hearts (161 ± 5 vs. 86 ± 19 ms, at BCL = 2,000 ms). APD alternans were induced by rapid pacing in these same three failing hearts. CV was significantly reduced in the failing group compared with the nonfailing group (81 ± 11 vs. 98 ± 8 cm/s, at BCL = 2,000 ms). Arrhythmias could be induced in two failing hearts exhibiting an abnormally steep CV restitution and increased dispersion of repolarization due to APD alternans. Dispersion of repolarization is enhanced across the RV endocardium in the failing human heart. This dispersion, together with APD alternans and abnormal CV restitution, could be responsible for the arrhythmia susceptibility in human HF.
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Displasia Arritmogênica Ventricular Direita/fisiopatologia , Sistema de Condução Cardíaco/fisiopatologia , Insuficiência Cardíaca/fisiopatologia , Disfunção Ventricular Direita/fisiopatologia , Remodelação Ventricular/fisiologia , Potenciais de Ação/fisiologia , Adulto , Idoso , Suscetibilidade a Doenças/fisiopatologia , Técnicas Eletrofisiológicas Cardíacas , Feminino , Insuficiência Cardíaca/cirurgia , Transplante de Coração , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Tempo , Imagens com Corantes Sensíveis à VoltagemRESUMO
The effect of malnutrition beyond morbidity and mortality has become a critical area of investigation in older people with an increased focus on quality-of-life (QoL), but as yet the relationship between malnutrition and QoL remains to be reviewed in older people from aged care settings. The current study conducted a systematic review and meta-analyses of studies published between the years 1995 and 2020 examining the relationship between nutritional status and QoL or the effects of a nutrition-based intervention on QoL in older people in residential aged care. Based on searches of the databases MEDLINE, PsycINFO, Emcare, and Embase, 21 studies were identified. Meta-analyses of the cross-sectional and quasi-experimental studies revealed a significant positive relationship between nutritional status and QoL and that nutritional intervention significantly improved QoL. By contrast, meta-analysis of randomised controlled trials revealed a non-significant but improved trend post-intervention in QoL. Although the effect sizes were small, the present findings indicate that nutrition-based interventions improve QoL in older people in residential aged care and align with previous reviews based on findings from other aged settings. Future research is needed to determine causality and to better identify and control for confounding factors which may influence both nutritional status and QoL.
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Desnutrição , Qualidade de Vida , Idoso , Estudos Transversais , Humanos , Estado NutricionalRESUMO
Induced pluripotent stem cell (iPSC) and gene editing technologies have revolutionized the field of in vitro disease modeling, granting us access to disease-pertinent human cells of the central nervous system. These technologies are particularly well suited for the study of diseases with strong monogenic etiologies. Epilepsy is one of the most common neurological disorders in children, with approximately half of all genetic cases caused by mutations in ion channel genes. These channelopathy-associated epilepsies are clinically diverse, mechanistically complex, and hard to treat. Here, we review the genetic links to epilepsy, the opportunities and challenges of iPSC-based approaches for developing in vitro models of channelopathy-associated disorders, the available tools for effective phenotyping of iPSC-derived neurons, and discuss the potential therapeutic approaches for these devastating diseases.
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Canalopatias , Epilepsia , Células-Tronco Pluripotentes Induzidas , Criança , Epilepsia/genética , Epilepsia/terapia , Humanos , Mutação , NeurôniosRESUMO
The ability to precisely edit the genome of human induced pluripotent stem cell (iPSC) lines using CRISPR/Cas9 has enabled the development of cellular models that can address genotype to phenotype relationships. While genome editing is becoming an essential tool in iPSC-based disease modeling studies, there is no established quality control workflow for edited cells. Moreover, large on-target deletions and insertions that occur through DNA repair mechanisms have recently been uncovered in CRISPR/Cas9-edited loci. Yet the frequency of these events in human iPSCs remains unclear, as they can be difficult to detect. We examined 27 iPSC clones generated after targeting 9 loci and found that 33% had acquired large, on-target genomic defects, including insertions and loss of heterozygosity. Critically, all defects had escaped standard PCR and Sanger sequencing analysis. We describe a cost-efficient quality control strategy that successfully identified all edited clones with detrimental on-target events and could facilitate the integrity of iPSC-based studies.
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Células-Tronco Pluripotentes Induzidas , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Homozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Controle de QualidadeRESUMO
Mutations in the TECPR2 gene are the cause of an ultra-rare neurological disorder characterized by intellectual disability, impaired speech, motor delay, and hypotonia evolving to spasticity, central sleep apnea, and premature death (SPG49 or HSAN9; OMIM: 615031). Little is known about the biological function of TECPR2, and there are currently no available disease-modifying therapies for this disease. Here we describe implementation of an antisense oligonucleotide (ASO) exon-skipping strategy targeting TECPR2 c.1319delT (p.Leu440Argfs∗19), a pathogenic variant that results in a premature stop codon within TECPR2 exon 8. We used patient-derived fibroblasts and induced pluripotent stem cell (iPSC)-derived neurons homozygous for the p.Leu440Argfs∗19 mutation to model the disease in vitro. Both patient-derived fibroblasts and neurons showed lack of TECPR2 protein expression. We designed and screened ASOs targeting sequences across the TECPR2 exon 8 region to identify molecules that induce exon 8 skipping and thereby remove the premature stop signal. TECPR2 exon 8 skipping restored in-frame expression of a TECPR2 protein variant (TECPR2ΔEx8) containing 1,300 of 1,411 amino acids. Optimization of ASO sequences generated a lead candidate (ASO-005-02) with â¼27 nM potency in patient-derived fibroblasts. To examine potential functional rescue induced by ASO-005-02, we used iPSC-derived neurons to analyze the neuronal localization of TECPR2ΔEx8 and showed that this form of TECPR2 retains the distinct, punctate neuronal expression pattern of full-length TECPR2. Finally, ASO-005-02 had an acceptable tolerability profile in vivo following a single 20-mg intrathecal dose in cynomolgus monkeys, showing some transient non-adverse behavioral effects with no correlating histopathology. Broad distribution of ASO-005-02 and induction of TECPR2 exon 8 skipping was detected in multiple central nervous system (CNS) tissues, supporting the potential utility of this therapeutic strategy for a subset of patients suffering from this rare disease.
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This study compared the effects of ATP-regulated potassium channel (K(ATP)) openers, diazoxide and pinacidil, on diseased and normal human atria and ventricles. We optically mapped the endocardium of coronary-perfused right (n=11) or left (n=2) posterior atrial-ventricular free wall preparations from human hearts with congestive heart failure (CHF, n=8) and non-failing human hearts without (NF, n=3) or with (INF, n=2) infarction. We also analyzed the mRNA expression of the K(ATP) targets K(ir)6.1, K(ir)6.2, SUR1, and SUR2 in the left atria and ventricles of NF (n=8) and CHF (n=4) hearts. In both CHF and INF hearts, diazoxide significantly decreased action potential durations (APDs) in atria (by -21±3% and -27±13%, p<0.01) and ventricles (by -28±7% and -28±4%, p<0.01). Diazoxide did not change APD (0±5%) in NF atria. Pinacidil significantly decreased APDs in both atria (-46 to -80%, p<0.01) and ventricles (-65 to -93%, p<0.01) in all hearts studied. The effect of pinacidil on APD was significantly higher than that of diazoxide in both atria and ventricles of all groups (p<0.05). During pinacidil perfusion, burst pacing induced flutter/fibrillation in all atrial and ventricular preparations with dominant frequencies of 14.4±6.1 Hz and 17.5±5.1 Hz, respectively. Glibenclamide (10 µM) terminated these arrhythmias and restored APDs to control values. Relative mRNA expression levels of K(ATP) targets were correlated to functional observations. Remodeling in response to CHF and/or previous infarct potentiated diazoxide-induced APD shortening. The activation of atrial and ventricular K(ATP) channels enhances arrhythmogenicity, suggesting that such activation may contribute to reentrant arrhythmias in ischemic hearts.
Assuntos
Diazóxido/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Átrios do Coração/efeitos dos fármacos , Insuficiência Cardíaca/fisiopatologia , Ventrículos do Coração/efeitos dos fármacos , Canais KATP/metabolismo , Pinacidil/farmacologia , Potenciais de Ação/efeitos dos fármacos , Adolescente , Adulto , Arritmias Cardíacas/fisiopatologia , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/metabolismo , Feminino , Átrios do Coração/metabolismo , Átrios do Coração/fisiopatologia , Ventrículos do Coração/metabolismo , Ventrículos do Coração/fisiopatologia , Humanos , Canais KATP/genética , Masculino , Pessoa de Meia-Idade , Isquemia Miocárdica/fisiopatologia , RNA Mensageiro/genética , Vasodilatadores/farmacologia , Adulto JovemRESUMO
Surface electrode recordings cannot delineate the activation within the human or canine sinoatrial node (SAN) because they are intramural structures. Thus, the site of origin of excitation and conduction pathway(s) within the SAN of these mammals remains unknown. Canine right atrial preparations (n=7) were optically mapped. The SAN 3D structure and protein expression were mapped using immunohistochemistry. SAN optical action potentials had diastolic depolarization and multiple upstroke components that corresponded to the separate excitations of the node and surface atrial layers. Pacing-induced SAN exit block eliminated atrial optical action potential components but retained SAN optical action potential components. Excitation originated in the SAN (cycle length, 557+/-72 ms) and slowly spread (1.2 to 14 cm/sec) within the SAN, failing to directly excite the crista terminalis and intraatrial septum. After a 49+/-22 ms conduction delay within the SAN, excitation reached the atrial myocardium via superior and/or inferior sinoatrial exit pathways 8.8+/-3.2 mm from the leading pacemaker site. The ellipsoidal 13.7+/-2.8/4.9+/-0.6 mm SAN structure was functionally insulated from the atrium. This insulation coincided with connexin43-negative regions at the borders of the node, connective tissue, and coronary arteries. During normal sinus rhythm, the canine SAN is functionally insulated from the surrounding atrial myocardium except for 2 (or more) narrow superior and inferior sinoatrial exit pathways separated by 12.8+/-4.1 mm. Conduction failure in these sinoatrial exit pathways leads to SAN exit block and is a modulator of heart rate.
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Função Atrial , Frequência Cardíaca , Miócitos Cardíacos/fisiologia , Nó Sinoatrial/fisiologia , Potenciais de Ação , Animais , Estimulação Cardíaca Artificial , Conexina 43/análise , Cães , Técnicas Eletrofisiológicas Cardíacas , Imunofluorescência , Átrios do Coração/citologia , Técnicas In Vitro , Miócitos Cardíacos/química , Dispositivos Ópticos , Processamento de Sinais Assistido por Computador , Nó Sinoatrial/química , Nó Sinoatrial/citologia , Fatores de TempoRESUMO
The atrioventricular node (AVN) has mystified generations of investigators over the last century and continues today to be at the epicenter of debates among anatomists, experimentalists, and electrophysiologists. Over the years, discrepancies have remained in regard to correlating components of AVN structure to function, as evidenced by studies from microelectrodes, optical mapping, and the electrophysiology laboratory. Historically, the AVN has been defined by classical histological methods; however, with recent advances in molecular biology techniques, a more precise characterization of structure is becoming attainable. Distinct molecular compartments are becoming apparent based on connexin staining and genotyping, providing new insight into previously characterized functional aspects of the AVN and its surrounding structures. Advances in optical mapping have provided a unique opportunity for correlating structure and function--unmasking properties of the native AVN pacemaker and providing further insight into basic mechanisms involved in AV conduction. Additionally, procurement of explanted human hearts have provided a unique opportunity to further characterize the human AVN structurally and functionally with both molecular biology techniques and optical mapping. With the elucidation of basic elements of both structure and function via molecular investigation and optical mapping, new opportunities are becoming apparent in utilizing the unique properties of the AVN for pursuing novel clinical applications relevant to clinical electrophysiology.
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Nó Atrioventricular/anatomia & histologia , Nó Atrioventricular/fisiologia , Animais , Humanos , Imagens com Corantes Sensíveis à VoltagemRESUMO
Chromatin modifications regulate genome function by recruiting proteins to the genome. However, the protein composition at distinct chromatin modifications has yet to be fully characterized. In this study, we used natural protein domains as modular building blocks to develop engineered chromatin readers (eCRs) selective for DNA methylation and histone tri-methylation at H3K4, H3K9 and H3K27 residues. We first demonstrated their utility as selective chromatin binders in living cells by stably expressing eCRs in mouse embryonic stem cells and measuring their subnuclear localization, genomic distribution and histone-modification-binding preference. By fusing eCRs to the biotin ligase BASU, we established ChromID, a method for identifying the chromatin-dependent protein interactome on the basis of proximity biotinylation, and applied it to distinct chromatin modifications in mouse stem cells. Using a synthetic dual-modification reader, we also uncovered the protein composition at bivalently modified promoters marked by H3K4me3 and H3K27me3. These results highlight the ability of ChromID to obtain a detailed view of protein interaction networks on chromatin.
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Cromatina , Histonas , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas/genética , Proteômica/métodos , Animais , Células Cultivadas , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Metilação de DNA/genética , Células-Tronco Embrionárias , Histonas/química , Histonas/genética , Histonas/metabolismo , CamundongosRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Cardiac stimulation via sympathetic neurons can potentially trigger arrhythmias. We present approaches to study neuron-cardiomyocyte interactions involving optogenetic selective probing and all-optical electrophysiology to measure activity in an automated fashion. Here we demonstrate the utility of optical interrogation of sympathetic neurons and their effects on macroscopic cardiomyocyte network dynamics to address research targets such as the effects of adrenergic stimulation via the release of neurotransmitters, the effect of neuronal numbers on cardiac behavior, and the applicability of optogenetics in mechanistic in vitro studies. As arrhythmias are emergent behaviors that involve the coordinated activity of millions of cells, we image at macroscopic scales to capture complex dynamics. We show that neurons can both decrease and increase wave stability and re-entrant activity in culture depending on their induced activity-a finding that may help us understand the often conflicting results seen in experimental and clinical studies.
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Adeno-associated viruses (AAVs) provide advantages in long-term, cardiac-specific gene expression. However, AAV serotype specificity data is lacking in experimental models relevant to cardiac electrophysiology and cardiac optogenetics. We aimed to identify the optimal AAV serotype (1, 6, or 9) in pursuit of scalable rodent and human models using genetic modifications in cardiac electrophysiology and optogenetics, in particular, as well as to elucidate the mechanism of virus uptake. In vitro syncytia of primary neonatal rat ventricular cardiomyocytes (NRVMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were infected with AAVs 1, 6, and 9 containing the transgene for eGFP or channelrhodopsin-2 (ChR2) fused to mCherry. In vivo adult rats were intravenously injected with AAV1 and 9 containing ChR2-mCherry. Transgene expression profiles of rat and human cells in vitro revealed that AAV1 and 6 significantly outperformed AAV9. In contrast, systemic delivery of AAV9 in adult rat hearts yielded significantly higher levels of ChR2-mCherry expression and optogenetic responsiveness. We tracked the mechanism of virus uptake to purported receptor-mediators for AAV1/6 (cell surface sialic acid) and AAV9 (37/67 kDa laminin receptor, LamR). In vitro desialylation of NRVMs and hiPSC-CMs with neuraminidase (NM) significantly decreased AAV1,6-mediated gene expression, but interestingly, desialylation of hiPSC-CMs increased AAV9-mediated expression. In fact, only very high viral doses of AAV9-ChR2-mCherry, combined with NM treatment, yielded consistent optogenetic responsiveness in hiPSC-CMs. Differences between the in vitro and in vivo performance of AAV9 could be correlated to robust LamR expression in the intact heart (neonatal rat hearts as well as adult human and rat hearts), but no expression in vitro in cultured cells (primary rat cells and hiPS-CMs). The dynamic nature of LamR expression and its dependence on environmental factors was further corroborated in intact adult human ventricular tissue. The combined transgene expression and cell surface receptor data may explain the preferential efficiency of AAV1/6 in vitro and AAV9 in vivo for cardiac delivery and mechanistic knowledge of their action can help guide cardiac optogenetic efforts. More broadly, these findings are relevant to future efforts in gene therapy for cardiac electrophysiology abnormalities in vivo as well as for genetic modifications of cardiomyocytes by viral means in vitro applications such as disease modeling or high-throughput drug testing.
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Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) is currently the method of choice to determine binding sites of chromatin-associated factors in a genome-wide manner. Here, we describe a method to investigate the binding preferences of mammalian DNA methyltransferases (DNMT) based on ChIP-seq using biotin-tagging. Stringent ChIP of DNMT proteins based on the strong interaction between biotin and avidin circumvents limitations arising from low antibody specificity and ensures reproducible enrichment. DNMT-bound DNA fragments are ligated to sequencing adaptors, amplified and sequenced on a high-throughput sequencing instrument. Bioinformatic analysis gives valuable information about the binding preferences of DNMTs genome-wide and around promoter regions. This method is unconventional due to the use of genetically engineered cells; however, it allows specific and reliable determination of DNMT binding.