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BACKGROUND: Epidemiological data offer conflicting views of the natural course of binge-eating disorder (BED), with large retrospective studies suggesting a protracted course and small prospective studies suggesting a briefer duration. We thus examined changes in BED diagnostic status in a prospective, community-based study that was larger and more representative with respect to sex, age of onset, and body mass index (BMI) than prior multi-year prospective studies. METHODS: Probands and relatives with current DSM-IV BED (n = 156) from a family study of BED ('baseline') were selected for follow-up at 2.5 and 5 years. Probands were required to have BMI > 25 (women) or >27 (men). Diagnostic interviews and questionnaires were administered at all timepoints. RESULTS: Of participants with follow-up data (n = 137), 78.1% were female, and 11.7% and 88.3% reported identifying as Black and White, respectively. At baseline, their mean age was 47.2 years, and mean BMI was 36.1. At 2.5 (and 5) years, 61.3% (45.7%), 23.4% (32.6%), and 15.3% (21.7%) of assessed participants exhibited full, sub-threshold, and no BED, respectively. No participants displayed anorexia or bulimia nervosa at follow-up timepoints. Median time to remission (i.e. no BED) exceeded 60 months, and median time to relapse (i.e. sub-threshold or full BED) after remission was 30 months. Two classes of machine learning methods did not consistently outperform random guessing at predicting time to remission from baseline demographic and clinical variables. CONCLUSIONS: Among community-based adults with higher BMI, BED improves with time, but full remission often takes many years, and relapse is common.
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The in vitro oxygen microenvironment profoundly affects the capacity of cell cultures to model physiological and pathophysiological states. Cell culture is often considered to be hyperoxic, but pericellular oxygen levels, which are affected by oxygen diffusivity and consumption, are rarely reported. Here, we provide evidence that several cell types in culture actually experience local hypoxia, with important implications for cell metabolism and function. We focused initially on adipocytes, as adipose tissue hypoxia is frequently observed in obesity and precedes diminished adipocyte function. Under standard conditions, cultured adipocytes are highly glycolytic and exhibit a transcriptional profile indicative of physiological hypoxia. Increasing pericellular oxygen diverted glucose flux toward mitochondria, lowered HIF1α activity, and resulted in widespread transcriptional rewiring. Functionally, adipocytes increased adipokine secretion and sensitivity to insulin and lipolytic stimuli, recapitulating a healthier adipocyte model. The functional benefits of increasing pericellular oxygen were also observed in macrophages, hPSC-derived hepatocytes and cardiac organoids. Our findings demonstrate that oxygen is limiting in many terminally-differentiated cell types, and that considering pericellular oxygen improves the quality, reproducibility and translatability of culture models.
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Adipocitos , Diferenciación Celular , Oxígeno , Oxígeno/metabolismo , Adipocitos/metabolismo , Adipocitos/citología , Humanos , Técnicas de Cultivo de Célula/métodos , Animales , Glucólisis , Hepatocitos/metabolismo , Hipoxia de la Célula , Mitocondrias/metabolismo , Ratones , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Células Cultivadas , Glucosa/metabolismo , Macrófagos/metabolismoRESUMEN
The heart is a metabolic "omnivore" and adjusts its energy source depending on the circulating metabolites. Human cardiac organoids, a three-dimensional in vitro model of the heart wall, are a useful tool to study cardiac physiology and pathology. However, cardiac tissue naturally experiences shear stress and nutrient fluctuations via blood flow in vivo, whilst in vitro models are conventionally cultivated in a static medium. This necessitates the regular refreshing of culture media, which creates acute cellular disturbances and large metabolic fluxes. To culture human cardiac organoids in a more physiological manner, we have developed a perfused bioreactor for cultures in a 96-well plate format. The designed bioreactor is easy to fabricate using a common culture plate and a 3D printer. Its open system allows for the use of traditional molecular biology techniques, prevents flow blockage issues, and provides easy access for sampling and cell assays. We hypothesized that a perfused culture would create more stable environment improving cardiac function and maturation. We found that lactate is rapidly produced by human cardiac organoids, resulting in large fluctuations in this metabolite under static culture. Despite this, neither medium perfusion in bioreactor culture nor lactate supplementation improved cardiac function or maturation. In fact, RNA sequencing revealed little change across the transcriptome. This demonstrates that cardiac organoids are robust in response to fluctuating environmental conditions under normal physiological conditions. Together, we provide a framework for establishing an easily accessible perfusion system that can be adapted to a range of miniaturized cell culture systems.
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Background: Prior research has demonstrated associations between anabolic-androgenic steroid (AAS) use and features from several childhood and adolescent psychosocial domains including body image concerns, antisocial traits, and low levels of parental care. However, prior approaches have been limited by their focus on individual features and lack of consideration of the relevant causal structure. Methods: We re-analyzed data from a previous cross-sectional cohort study of 232 male weightlifters aged 18-40, of whom 101 had used AAS. These men completed retrospective measures of features from their childhood and early adolescence, including body image concerns, eating disorder psychopathology, antisocial traits, substance use, and family relationships. Using an approach informed by principles of causal inference, we applied four machine-learning methods - lasso regression, elastic net regression, random forests, and gradient boosting - to predict AAS use. Results: The four methods yielded similar receiver operating curves, mean area under the curve (range 0.66 to 0.72), and sets of highly important features. Features related to adolescent body image concerns (especially muscle dysmorphia symptoms) were the strongest predictors. Other important features were adolescent rebellious behaviors; adolescent feelings of ineffectiveness and lack of interoceptive awareness; and low levels of paternal care. Conclusions: Applying machine learning within a causally informed approach to re-analyze data from a prior study of weightlifters, we identified six factors (most prominently those related to adolescent body image concerns) as proposed causal factors for the development of AAS use. Compared with the prior analyses, this approach achieved greater methodologic rigor and yielded stronger and broader findings.
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Immunotherapy harnesses neoantigens encoded within the human genome, but their therapeutic potential is hampered by low expression, which may be controlled by the nonsense-mediated mRNA decay (NMD) pathway. This study investigates the impact of UPF1-knockdown on the expression of non-canonical/mutant proteins, employing proteogenomic to explore UPF1 role within the NMD pathway. Additionally, we conducted a comprehensive pan-cancer analysis of UPF1 expression and evaluated UPF1 expression in Triple-Negative Breast Cancer (TNBC) tissue in-vivo. Our findings reveal that UPF1-knockdown leads to increased translation of non-canonical/mutant proteins, particularly those originating from retained-introns, pseudogenes, long non-coding RNAs, and unannotated transcript biotypes. Moreover, our analysis demonstrates elevated UPF1 expression in various cancer types, with notably heightened protein levels in patient-derived TNBC tumors compared to adjacent tissues. This study elucidates UPF1 role in mitigating transcriptional noise by degrading transcripts encoding non-canonical/mutant proteins. Targeting this mechanism may reveal a new spectrum of neoantigens accessible to the antigen presentation pathway. Our novel findings provide a strong foundation for the development of therapeutic strategies aimed at targeting UPF1 or modulating the NMD pathway.
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Evidence-based interventions vary in effectiveness for individuals with depression, which has a large public health burden. Readiness for change or treatment can be an important individual difference predictor of depression outcomes. To inform public service initiatives targeting readiness for treatment, characterizing readiness across settings and levels of care is key. However, limited data exist on the role of readiness for treatment in acute psychiatric settings and in particular, partial hospital programs which are key points in the continuity of inpatient and outpatient care. The present study assessed readiness for treatment in terms of importance, confidence, and motivation to engage in a partial hospital program and tested whether higher levels of readiness were associated with better treatment outcomes among clients with depression. Participants (N = 192) with major depressive disorder rated their readiness for treatment (Readiness Rulers), depression (Patient Health Questionnaire-9), and global improvement (Clinical Global Impression Scale-Improvement Self-Report) while enrolled in a partial hospital program. Generalized linear regression models assessed the effect of baseline readiness on outcomes at discharge, adjusted for baseline level of the outcome, age, sex, race, and ethnicity. Greater baseline readiness predicted reduced depression and better global improvement at discharge. Higher confidence and motivation to engage in treatment, but not importance, were associated with better depression outcomes. Identifying and addressing readiness for treatment by leveraging public health systems and services (e.g., help lines, family interventions) prior to or upon starting a partial hospital program may be useful to maximize gains in treatment. (PsycInfo Database Record (c) 2024 APA, all rights reserved).
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Genomic regulation of cardiomyocyte differentiation is central to heart development and function. This study uses genetic loss-of-function human-induced pluripotent stem cell-derived cardiomyocytes to evaluate the genomic regulatory basis of the non-DNA-binding homeodomain protein HOPX. We show that HOPX interacts with and controls cardiac genes and enhancer networks associated with diverse aspects of heart development. Using perturbation studies in vitro, we define how upstream cell growth and proliferation control HOPX transcription to regulate cardiac gene programs. We then use cell, organoid, and zebrafish regeneration models to demonstrate that HOPX-regulated gene programs control cardiomyocyte function in development and disease. Collectively, this study mechanistically links cell signaling pathways as upstream regulators of HOPX transcription to control gene programs underpinning cardiomyocyte identity and function.
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Células Madre Pluripotentes Inducidas , Miocitos Cardíacos , Animales , Humanos , Miocitos Cardíacos/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Pez Cebra/metabolismo , Diferenciación Celular/genética , Proliferación CelularRESUMEN
Cardiomyocytes (CMs) lost during ischemic cardiac injury cannot be replaced due to their limited proliferative capacity, which leads to progressive heart failure. Calcium (Ca2+) is an important signal transducer that regulates key cellular processes, but its role in regulating CM proliferation is incompletely understood. A drug screen targeting proteins involved in CM calcium cycling in human embryonic stem cell-derived cardiac organoids (hCOs) revealed that only the inhibition of L-Type Calcium Channel (LTCC), but not other Ca2+ regulatory proteins (SERCA or RYR), induced the CM cell cycle. Furthermore, overexpression of Ras-related associated with Diabetes (RRAD), an endogenous inhibitor of LTCC, induced CM cell cycle activity in vitro, in human cardiac slices, and in vivo. Mechanistically, LTCC inhibition by RRAD induces the cell cycle in CMs by modulating calcineurin activity and translocating Hoxb13 to the CM nucleus. Together, this represents a robust pathway for regenerative strategies.
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Terpenes are the most extensive and varied group of naturally occurring compounds mostly found in plants, including cannabis, and have an array of potential therapeutic benefits for pathological conditions. The endocannabinoid system can potently modulate anxiety in humans, rodents, and zebrafish. The 'entourage effect' suggests terpenes may target cannabinoid CB1 and CB2 receptors, among others, but this requires further investigation. In this study we first tested for anxiety-altering effects of the predominant 'Super-Class' terpenes, bisabolol (0.001%, 0.0015%, and 0.002%) and terpinolene (TPL; 0.01%, 0.05%, and 0.1%), in zebrafish with the open field test. Bisabolol did not have an effect on zebrafish behaviour or locomotion. However, TPL caused a significant increase in time spent in the inner zone and decrease in time spent in the outer zone of the arena indicating an anxiolytic (anxiety decreasing) effect. Next, we assessed whether CB1 and CB2 receptor antagonists, rimonabant and AM630 (6-Iodopravadoline) respectively, could eliminate or reduce the anxiolytic effects of TPL (0.1%) and ß-caryophyllene (BCP; 4%), another super-class terpene previously shown to be anxiolytic in zebrafish. Rimonabant and AM630 were administered prior to terpene exposure and compared to controls and fish exposed to only the terpenes. AM630, but not rimonabant, eliminated the anxiolytic effects of both BCP and TPL. AM630 modulated locomotion on its own, which was potentiated by terpenes. These findings suggest the behavioural effects of TPL and BCP on zebrafish anxiety-like behaviour are mediated by a selective preference for CB2 receptor sites. Furthermore, the CB2 pathways mediating the anxiolytic response are likely different from those altering locomotion.
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Ansiolíticos , Cannabinoides , Humanos , Animales , Terpenos/farmacología , Ansiolíticos/farmacología , Rimonabant , Pez Cebra , Receptores de Cannabinoides , Ansiedad/inducido químicamente , Ansiedad/tratamiento farmacológico , Cannabinoides/farmacologíaRESUMEN
Recombinant adeno-associated viruses (rAAVs) have emerged as one of the most promising gene therapy vectors that have been successfully used in pre-clinical models of heart disease. However, this has not translated well to humans due to species differences in rAAV transduction efficiency. As a result, the search for human cardiotropic capsids is a major contemporary challenge. We used a capsid-shuffled rAAV library to perform directed evolution in human iPSC-derived cardiomyocytes (hiPSC-CMs). Five candidates emerged, with four presenting high sequence identity to AAV6, while a fifth divergent variant was related to AAV3b. Functional analysis of the variants was performed in vitro using hiPSC-CMs, cardiac organoids, human cardiac slices, non-human primate and porcine cardiac slices, as well as mouse heart and liver in vivo. We showed that cell entry was not the best predictor of transgene expression efficiency. The novel variant rAAV.KK04 was the best-performing vector in human-based screening platforms, exceeding the benchmark rAAV6. None of the novel capsids demonstrate a significant transduction of liver in vivo. The range of experimental models used revealed the value of testing for tropism differences under the conditions of human specificity, bona fide, myocardium and cell type of interest.
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The cardiac endothelium influences ventricular chamber development by coordinating trabeculation and compaction. However, the endothelial-specific molecular mechanisms mediating this coordination are not fully understood. Here, we identify the Sox7 transcription factor as a critical cue instructing cardiac endothelium identity during ventricular chamber development. Endothelial-specific loss of Sox7 function in mice results in cardiac ventricular defects similar to non-compaction cardiomyopathy, with a change in the proportions of trabecular and compact cardiomyocytes in the mutant hearts. This phenotype is paralleled by abnormal coronary artery formation. Loss of Sox7 function disrupts the transcriptional regulation of the Notch pathway and connexins 37 and 40, which govern coronary arterial specification. Upon Sox7 endothelial-specific deletion, single-nuclei transcriptomics analysis identifies the depletion of a subset of Sox9/Gpc3-positive endocardial progenitor cells and an increase in erythro-myeloid cell lineages. Fate mapping analysis reveals that a subset of Sox7-null endothelial cells transdifferentiate into hematopoietic but not cardiomyocyte lineages. Our findings determine that Sox7 maintains cardiac endothelial cell identity, which is crucial to the cellular cross-talk that drives ventricular compaction and coronary artery development.
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Vasos Coronarios , Células Endoteliales , Animales , Ratones , Vasos Coronarios/metabolismo , Células Endoteliales/metabolismo , Miocitos Cardíacos/metabolismo , Regulación de la Expresión Génica , Endotelio/metabolismo , Factores de Transcripción SOXF/genética , Factores de Transcripción SOXF/metabolismoRESUMEN
Recent studies in non-human model systems have shown therapeutic potential of nucleoside-modified messenger RNA (modRNA) treatments for lysosomal storage diseases. Here, we assessed the efficacy of a modRNA treatment to restore the expression of the galactosidase alpha (GLA), which codes for α-Galactosidase A (α-GAL) enzyme, in a human cardiac model generated from induced pluripotent stem cells (iPSCs) derived from two individuals with Fabry disease. Consistent with the clinical phenotype, cardiomyocytes from iPSCs derived from Fabry-affected individuals showed accumulation of the glycosphingolipid Globotriaosylceramide (GB3), which is an α-galactosidase substrate. Furthermore, the Fabry cardiomyocytes displayed significant upregulation of lysosomal-associated proteins. Upon GLA modRNA treatment, a subset of lysosomal proteins were partially restored to wild-type levels, implying the rescue of the molecular phenotype associated with the Fabry genotype. Importantly, a significant reduction of GB3 levels was observed in GLA modRNA-treated cardiomyocytes, demonstrating that α-GAL enzymatic activity was restored. Together, our results validate the utility of iPSC-derived cardiomyocytes from affected individuals as a model to study disease processes in Fabry disease and the therapeutic potential of GLA modRNA treatment to reduce GB3 accumulation in the heart.
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Enfermedad de Fabry , Células Madre Pluripotentes Inducidas , Humanos , Miocitos Cardíacos , ARN , Enfermedad de Fabry/genética , Enfermedad de Fabry/terapia , ARN MensajeroRESUMEN
Here, we provide a protocol for next-generation human cardiac organoid modeling containing markers of vascularized tissues. We describe steps for cardiac differentiation, harvesting cardiac cells, and generating vascularized human cardiac organoids. We then detail downstream analysis of functional parameters and fluorescence labeling of human cardiac organoids. This protocol is useful for high throughput disease modeling, drug discovery, and providing mechanistic insight into cell-cell and cell-matrix interactions. For complete details on the use and execution of this protocol, please refer to Voges et al.1 and Mills et al.2.
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Comunicación Celular , Organoides , Humanos , Diferenciación Celular , Descubrimiento de Drogas , CorazónRESUMEN
Climate change is leading to species redistributions. In the tundra biome, shrubs are generally expanding, but not all tundra shrub species will benefit from warming. Winner and loser species, and the characteristics that may determine success or failure, have not yet been fully identified. Here, we investigate whether past abundance changes, current range sizes and projected range shifts derived from species distribution models are related to plant trait values and intraspecific trait variation. We combined 17,921 trait records with observed past and modelled future distributions from 62 tundra shrub species across three continents. We found that species with greater variation in seed mass and specific leaf area had larger projected range shifts, and projected winner species had greater seed mass values. However, trait values and variation were not consistently related to current and projected ranges, nor to past abundance change. Overall, our findings indicate that abundance change and range shifts will not lead to directional modifications in shrub trait composition, since winner and loser species share relatively similar trait spaces.
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Ecosistema , Tundra , Semillas , Cambio Climático , FenotipoRESUMEN
Crosstalk between cardiac cells is critical for heart performance. Here we show that vascular cells within human cardiac organoids (hCOs) enhance their maturation, force of contraction, and utility in disease modeling. Herein we optimize our protocol to generate vascular populations in addition to epicardial, fibroblast, and cardiomyocyte cells that self-organize into in-vivo-like structures in hCOs. We identify mechanisms of communication between endothelial cells, pericytes, fibroblasts, and cardiomyocytes that ultimately contribute to cardiac organoid maturation. In particular, (1) endothelial-derived LAMA5 regulates expression of mature sarcomeric proteins and contractility, and (2) paracrine platelet-derived growth factor receptor ß (PDGFRß) signaling from vascular cells upregulates matrix deposition to augment hCO contractile force. Finally, we demonstrate that vascular cells determine the magnitude of diastolic dysfunction caused by inflammatory factors and identify a paracrine role of endothelin driving dysfunction. Together this study highlights the importance and role of vascular cells in organoid models.
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Células Endoteliales , Miocitos Cardíacos , Humanos , Miocitos Cardíacos/metabolismo , Pericitos/metabolismo , Transducción de Señal , Organoides/metabolismoRESUMEN
Extracellular matrix (ECM) provides fundamental support for epithelial tissues and controls cell function. The chemistry and mechanical properties of ECM components, including stiffness, elasticity, and fibrillar organization, influence epithelial tissue responses. Here we present a protocol describing the culture and transfer of epithelial acini from Matrigel to collagen gel and an approach to axially align the collagen fibrils by the external gel stretching. This protocol uses the acini of MCF10A cells and needs to be modified for different cell lines. For complete details on the use and execution of this protocol, please refer to Katsuno-Kambe et al. (2021).1.
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Colágeno , Matriz Extracelular , Matriz Extracelular/metabolismo , Colágeno/química , ElasticidadRESUMEN
Muscle contraction is driven by the molecular machinery of the sarcomere. As phosphorylation is a critical regulator of muscle function, the identification of regulatory kinases is important for understanding sarcomere biology. Pathogenic variants in alpha kinase 3 (ALPK3) cause cardiomyopathy and musculoskeletal disease, but little is known about this atypical kinase. Here we show that ALPK3 is an essential component of the M-band of the sarcomere and define the ALPK3-dependent phosphoproteome. ALPK3 deficiency impaired contractility both in human cardiac organoids and in the hearts of mice harboring a pathogenic truncating Alpk3 variant. ALPK3-dependent phosphopeptides were enriched for sarcomeric components of the M-band and the ubiquitin-binding protein sequestosome-1 (SQSTM1) (also known as p62). Analysis of the ALPK3 interactome confirmed binding to M-band proteins including SQSTM1. In human pluripotent stem cell-derived cardiomyocytes modeling cardiomyopathic ALPK3 mutations, sarcomeric organization and M-band localization of SQSTM1 were abnormal suggesting that this mechanism may underly disease pathogenesis.
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Improving muscle function has great potential to improve the quality of life. To identify novel regulators of skeletal muscle metabolism and function, we performed a proteomic analysis of gastrocnemius muscle from 73 genetically distinct inbred mouse strains, and integrated the data with previously acquired genomics and >300 molecular/phenotypic traits via quantitative trait loci mapping and correlation network analysis. These data identified thousands of associations between protein abundance and phenotypes and can be accessed online (https://muscle.coffeeprot.com/) to identify regulators of muscle function. We used this resource to prioritize targets for a functional genomic screen in human bioengineered skeletal muscle. This identified several negative regulators of muscle function including UFC1, an E2 ligase for protein UFMylation. We show UFMylation is up-regulated in a mouse model of amyotrophic lateral sclerosis, a disease that involves muscle atrophy. Furthermore, in vivo knockdown of UFMylation increased contraction force, implicating its role as a negative regulator of skeletal muscle function.
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Proteoma , Proteómica , Ratones , Animales , Humanos , Proteoma/metabolismo , Calidad de Vida , Músculo Esquelético/metabolismo , FenotipoRESUMEN
Aim: Adult mammalian cardiomyocytes are incapable of significant proliferation, limiting regeneration after myocardial injury. Overexpression of the transcription factor Myc has been shown to drive proliferation in the adult mouse heart, but only when combined with Cyclin T1. As constitutive HRas activity has been shown to stabilise Cyclin T1 in vivo, we aimed to establish whether Myc and HRas could also act cooperatively to induce proliferation in adult mammalian cardiomyocytes in vivo. Methods and results: Using a genetically modified mouse model, we confirmed that constitutive HRas activity (HRas G 12 V ) increased Cyclin T1 expression. HRas G 12 V and constitutive Myc expression together co-operate to drive cell-cycle progression of adult mammalian cardiomyocytes. However, stimulation of endogenous cardiac proliferation by the ectopic expression of HRas G 12 V and Myc also induced cardiomyocyte death, while Myc and Cyclin T1 expression did not. Conclusion: Co-expression of Cyclin T1 and Myc may be a therapeutically tractable approach for cardiomyocyte neo-genesis post injury, while cell death induced by HRas G 12 V and Myc expression likely limits this option as a regenerative therapeutic target.
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Initial controlled trials of the serotonergic antidepressant fluvoxamine showed promise for treatment of mild to moderate COVID-19 in outpatients, although more recent outpatient data have been less encouraging. Turning to studies of hospitalized patients, a retrospective cohort study by Hoertel and associates in 2021 found a markedly reduced risk of intubation or death among patients hospitalized with COVID-19 who were receiving serotonergic antidepressants at the time of admission vs. those not receiving antidepressants. In an attempt to replicate these latter findings, we performed a similarly designed study of 500 individuals hospitalized with COVID-19 in a large academic hospital system who were taking a serotonergic antidepressant at the time of admission compared with two groups (N = 573 and N = 593) not receiving an antidepressant. In analyses controlling for demographic and clinical variables, we found no significant difference in effect between the antidepressant group and either of the two comparison groups [hazard ratios (95% CI) for intubation or death 1.1 (0.83-1.5) and 1.1 (0.86-1.5); and for death alone 1.3 (0.93-1.8) and 1.1 (0.85-1.7)]. Examining the results of our study, along with those of Hoertel et al. and three additional retrospective cohort studies in inpatients published in the interim, the data permit only very limited conclusions, with the findings on the effect of serotonergic antidepressants ranging from a strongly protective effect to no effect. Although there are numerous threats to validity that might account for this wide range of findings, we could not identify any principal factor or set of factors that could clearly explain the differences.