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Gene expression in human tissue has primarily been studied on the transcriptional level, largely neglecting translational regulation. Here, we analyze the translatomes of 80 human hearts to identify new translation events and quantify the effect of translational regulation. We show extensive translational control of cardiac gene expression, which is orchestrated in a process-specific manner. Translation downstream of predicted disease-causing protein-truncating variants appears to be frequent, suggesting inefficient translation termination. We identify hundreds of previously undetected microproteins, expressed from lncRNAs and circRNAs, for which we validate the protein products in vivo. The translation of microproteins is not restricted to the heart and prominent in the translatomes of human kidney and liver. We associate these microproteins with diverse cellular processes and compartments and find that many locate to the mitochondria. Importantly, dozens of microproteins are translated from lncRNAs with well-characterized noncoding functions, indicating previously unrecognized biology.
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Miocardio/metabolismo , Biosíntesis de Proteínas , Adolescente , Adulto , Anciano , Animales , Codón/genética , Femenino , Regulación de la Expresión Génica , Células HEK293 , Humanos , Lactante , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Sistemas de Lectura Abierta/genética , ARN Circular/genética , ARN Circular/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ribosomas/genética , Ribosomas/metabolismo , Adulto JovenRESUMEN
In systemic lupus erythematosus (SLE) loss of immune tolerance, autoantibody production and immune complex deposition are required but not sufficient for organ damage1. How inflammatory signals are initiated and amplified in the setting of autoimmunity remains elusive. Here, we set out to dissect layers and hierarchies of autoimmune kidney inflammation in order to identify tissue-specific cellular hubs that amplify auto-inflammatory responses. Using high-resolution single-cell profiling of kidney immune and parenchymal cells, in combination with antibody blocking and genetic deficiency, we show that tissue-resident NKp46+ innate lymphoid cells (ILC) are crucial signal amplifiers of disease-associated macrophage expansion and epithelial cell injury in lupus nephritis, downstream of autoantibody production. NKp46 signaling in a distinct subset of ILC1 instructed an unconventional immune-regulatory transcriptional program, which included the expression of the myeloid cell growth factor CSF2. CSF2 production by NKp46+ ILC promoted the population expansion of monocyte-derived macrophages. Blockade of the NKp46 receptor (using the antibody mNCR1.152) or genetic deficiency of NKp46 abrogated epithelial cell injury. The same cellular and molecular patterns were operative in human lupus nephritis. Our data support that NKp46+ ILC1 promote parenchymal cell injury by granting monocyte-derived macrophages access to epithelial cell niches. NKp46 activation in ILC1 thus constitutes a previously unrecognized, critical tissue rheostat that amplifies organ damage in autoimmune hosts, with broad implications for inflammatory pathologies and therapies.
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BACKGROUND: The elaborate patterning of coronary arteries critically supports the high metabolic activity of the beating heart. How coronary endothelial cells coordinate hierarchical vascular remodeling and achieve arteriovenous specification remains largely unknown. Understanding the molecular and cellular cues that pattern coronary arteries is crucial to develop innovative therapeutic strategies that restore functional perfusion within the ischemic heart. METHODS: Single-cell transcriptomics and histological validation were used to delineate heterogeneous transcriptional states of the developing and mature coronary endothelium with a focus on sprouting endothelium and arterial cell specification. Genetic lineage tracing and high-resolution 3-dimensional imaging were used to characterize the origin and mechanisms of coronary angiogenic sprouting, as well as to fate-map selective endothelial lineages. Integration of single-cell transcriptomic data from ischemic adult mouse hearts and human embryonic data served to assess the conservation of transcriptional states across development, disease, and species. RESULTS: We discover that coronary arteries originate from cells that have previously transitioned through a specific tip cell phenotype. We identify nonoverlapping intramyocardial and subepicardial tip cell populations with differential gene expression profiles and regulatory pathways. Esm1-lineage tracing confirmed that intramyocardial tip cells selectively contribute to coronary arteries and endocardial tunnels, but not veins. Notably, prearterial cells are detected from development stages to adulthood, increasingly in response to ischemic injury, and in human embryos, suggesting that tip cell-to-artery specification is a conserved mechanism. CONCLUSIONS: A tip cell-to-artery specification mechanism drives arterialization of the intramyocardial plexus and endocardial tunnels throughout life and is reactivated upon ischemic injury. Differential sprouting programs govern the formation and specification of the venous and arterial coronary plexus.
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Cardiovascular disease is the leading cause of death worldwide. Advanced insights into disease mechanisms and therapeutic strategies require a deeper understanding of the molecular processes involved in the healthy heart. Knowledge of the full repertoire of cardiac cells and their gene expression profiles is a fundamental first step in this endeavour. Here, using state-of-the-art analyses of large-scale single-cell and single-nucleus transcriptomes, we characterize six anatomical adult heart regions. Our results highlight the cellular heterogeneity of cardiomyocytes, pericytes and fibroblasts, and reveal distinct atrial and ventricular subsets of cells with diverse developmental origins and specialized properties. We define the complexity of the cardiac vasculature and its changes along the arterio-venous axis. In the immune compartment, we identify cardiac-resident macrophages with inflammatory and protective transcriptional signatures. Furthermore, analyses of cell-to-cell interactions highlight different networks of macrophages, fibroblasts and cardiomyocytes between atria and ventricles that are distinct from those of skeletal muscle. Our human cardiac cell atlas improves our understanding of the human heart and provides a valuable reference for future studies.
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Miocardio/citología , Análisis de la Célula Individual , Transcriptoma , Adipocitos/clasificación , Adipocitos/metabolismo , Adulto , Enzima Convertidora de Angiotensina 2/análisis , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Células Epiteliales/clasificación , Células Epiteliales/metabolismo , Epitelio , Femenino , Fibroblastos/clasificación , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Atrios Cardíacos/anatomía & histología , Atrios Cardíacos/citología , Atrios Cardíacos/inervación , Ventrículos Cardíacos/anatomía & histología , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/inervación , Homeostasis/inmunología , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Miocitos Cardíacos/clasificación , Miocitos Cardíacos/metabolismo , Neuronas/clasificación , Neuronas/metabolismo , Pericitos/clasificación , Pericitos/metabolismo , Receptores de Coronavirus/análisis , Receptores de Coronavirus/genética , Receptores de Coronavirus/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad , Células del Estroma/clasificación , Células del Estroma/metabolismoRESUMEN
Myocardial fibrosis is a key pathologic feature of hypertrophic cardiomyopathy (HCM). However, the fibrotic pathways activated by HCM-causing sarcomere protein gene mutations are poorly defined. Because lysophosphatidic acid is a mediator of fibrosis in multiple organs and diseases, we tested the role of the lysophosphatidic acid pathway in HCM. Lysphosphatidic acid receptor 1 (LPAR1), a cell surface receptor, is required for lysophosphatidic acid mediation of fibrosis. We bred HCM mice carrying a pathogenic myosin heavy-chain variant (403+/-) with Lpar1-ablated mice to create mice carrying both genetic changes (403+/- LPAR1 -/-) and assessed development of cardiac hypertrophy and fibrosis. Compared with 403+/- LPAR1WT, 403+/- LPAR1 -/- mice developed significantly less hypertrophy and fibrosis. Single-nucleus RNA sequencing of left ventricular tissue demonstrated that Lpar1 was predominantly expressed by lymphatic endothelial cells (LECs) and cardiac fibroblasts. Lpar1 ablation reduced the population of LECs, confirmed by immunofluorescence staining of the LEC markers Lyve1 and Ccl21a and, by in situ hybridization, for Reln and Ccl21a. Lpar1 ablation also altered the distribution of fibroblast cell states. FB1 and FB2 fibroblasts decreased while FB0 and FB3 fibroblasts increased. Our findings indicate that Lpar1 is expressed predominantly by LECs and fibroblasts in the heart and is required for development of hypertrophy and fibrosis in an HCM mouse model. LPAR1 antagonism, including agents in clinical trials for other fibrotic diseases, may be beneficial for HCM.
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Cardiomiopatía Hipertrófica , Receptores del Ácido Lisofosfatídico/genética , Animales , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/patología , Proteínas Portadoras , Modelos Animales de Enfermedad , Células Endoteliales/patología , Fibrosis , Hipertrofia/patología , RatonesRESUMEN
Skeletal muscle cellular development requires the integrated assembly of mitochondria and other organelles adjacent to the sarcomere in support of muscle contractile performance. However, it remains unclear how interactions among organelles and with the sarcomere relates to the development of muscle cell function. Here, we combine 3D volume electron microscopy, proteomic analyses, and live cell functional imaging to investigate the postnatal reorganization of mitochondria-organelle interactions in skeletal muscle. We show that while mitochondrial networks are disorganized and loosely associated with the contractile apparatus at birth, contact sites among mitochondria, lipid droplets and the sarcoplasmic reticulum are highly abundant in neonatal muscles. The maturation process is characterized by a transition to highly organized mitochondrial networks wrapped tightly around the muscle sarcomere but also to less frequent interactions with both lipid droplets and the sarcoplasmic reticulum. Concomitantly, expression of proteins involved in mitochondria-organelle membrane contact sites decreases during postnatal development in tandem with a decrease in abundance of proteins associated with sarcomere assembly despite an overall increase in contractile protein abundance. Functionally, parallel measures of mitochondrial membrane potential, NADH redox status, and NADH flux within intact cells revealed that mitochondria in adult skeletal muscle fibres maintain a more activated electron transport chain compared with neonatal muscle mitochondria. These data demonstrate a developmental redesign reflecting a shift from muscle cell assembly and frequent inter-organelle communication toward a muscle fibre with mitochondrial structure, interactions, composition and function specialized to support contractile function. KEY POINTS: Mitochondrial network organization is remodelled during skeletal muscle postnatal development. The mitochondrial outer membrane is in frequent contact with other organelles at birth and transitions to more close associations with the contractile apparatus in mature muscles. Mitochondrial energy metabolism becomes more activated during postnatal development. Understanding the developmental redesign process within skeletal muscle cells may help pinpoint specific areas of deficit in muscles with developmental disorders.
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NAD , Proteómica , Humanos , Adulto , Recién Nacido , NAD/metabolismo , Mitocondrias/metabolismo , Músculo Esquelético/metabolismo , Mitocondrias Musculares/metabolismo , Gotas Lipídicas/metabolismoRESUMEN
The pathophysiology of sickle cell disease (SCD) is driven by chronic inflammation fueled by damage associated molecular patterns (DAMPs). We show that elevated cell-free DNA (cfDNA) in patients with SCD is not just a prognostic biomarker, it also contributes to the pathological inflammation. Within the elevated cfDNA, patients with SCD had a significantly higher ratio of cell-free mitochondrial DNA (cf-mtDNA)/cell-free nuclear DNA compared with healthy controls. Additionally, mitochondrial DNA in patient samples showed significantly disproportionately increased hypomethylation compared with healthy controls, and it was increased further in crises compared with steady-state. Using flow cytometry, structured illumination microscopy, and electron microscopy, we showed that circulating SCD red blood cells abnormally retained their mitochondria and, thus, are likely to be the source of the elevated cf-mtDNA in patients with SCD. Patient plasma containing high levels of cf-mtDNA triggered the formation of neutrophil extracellular traps (NETs) that was substantially reduced by inhibition of TANK-binding kinase 1, implicating activation of the cGAS-STING pathway. cf-mtDNA is an erythrocytic DAMP, highlighting an underappreciated role for mitochondria in sickle pathology. These trials were registered at www.clinicaltrials.gov as #NCT00081523, #NCT03049475, and #NCT00047996.
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Anemia de Células Falciformes/sangre , Ácidos Nucleicos Libres de Células/sangre , Metilación de ADN , ADN Mitocondrial/sangre , Adulto , Anciano , Biomarcadores/sangre , Trampas Extracelulares/metabolismo , Femenino , Humanos , Inflamación/sangre , Masculino , Proteínas de la Membrana/metabolismo , Persona de Mediana Edad , Nucleotidiltransferasas/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: Remodeling of the extracellular matrix (ECM) is a hallmark of heart failure (HF). Our previous analysis of the secretome of murine cardiac fibroblasts returned ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5) as one of the most abundant proteases. ADAMTS5 cleaves chondroitin sulfate proteoglycans such as versican. The contribution of ADAMTS5 and its substrate versican to HF is unknown. METHODS: Versican remodeling was assessed in mice lacking the catalytic domain of ADAMTS5 (Adamts5ΔCat). Proteomics was applied to study ECM remodeling in left ventricular samples from patients with HF, with a particular focus on the effects of common medications used for the treatment of HF. RESULTS: Versican and versikine, an ADAMTS-specific versican cleavage product, accumulated in patients with ischemic HF. Versikine was also elevated in a porcine model of cardiac ischemia/reperfusion injury and in murine hearts after angiotensin II infusion. In Adamts5ΔCat mice, angiotensin II infusion resulted in an aggravated versican build-up and hyaluronic acid disarrangement, accompanied by reduced levels of integrin ß1, filamin A, and connexin 43. Echocardiographic assessment of Adamts5ΔCat mice revealed a reduced ejection fraction and an impaired global longitudinal strain on angiotensin II infusion. Cardiac hypertrophy and collagen deposition were similar to littermate controls. In a proteomics analysis of a larger cohort of cardiac explants from patients with ischemic HF (n=65), the use of ß-blockers was associated with a reduction in ECM deposition, with versican being among the most pronounced changes. Subsequent experiments in cardiac fibroblasts confirmed that ß1-adrenergic receptor stimulation increased versican expression. Despite similar clinical characteristics, patients with HF treated with ß-blockers had a distinct cardiac ECM profile. CONCLUSIONS: Our results in animal models and patients suggest that ADAMTS proteases are critical for versican degradation in the heart and that versican accumulation is associated with impaired cardiac function. A comprehensive characterization of the cardiac ECM in patients with ischemic HF revealed that ß-blockers may have a previously unrecognized beneficial effect on cardiac chondroitin sulfate proteoglycan content.
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Proteína ADAMTS5/metabolismo , Matriz Extracelular/metabolismo , Insuficiencia Cardíaca/metabolismo , Proteoglicanos/metabolismo , Animales , Insuficiencia Cardíaca/patología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , ProteómicaRESUMEN
Lysyl oxidase (LOX) is a copper-binding enzyme that cross-links elastin and collagen. The dominant LOX variation contributes to familial thoracic aortic aneurysm. Previously reported murine Lox mutants had a mild phenotype and did not dilate without drug-induced provocation. Here, we present a new, more severe mutant, Loxb2b370.2Clo (c.G854T; p.Cys285Phe), whose mutation falls just N-terminal to the copper-binding domain. Unlike the other mutants, the C285F Lox protein was stably produced/secreted, and male C57Bl/6J Lox+/C285F mice exhibit increased systolic blood pressure (BP; p < 0.05) and reduced caliber aortas (p < 0.01 at 100mmHg) at 3 months that independently dilate by 6 months (p < 0.0001). Multimodal imaging reveals markedly irregular elastic sheets in the mutant (p = 2.8 × 10−8 for breaks by histology) that become increasingly disrupted with age (p < 0.05) and breeding into a high BP background (p = 6.8 × 10−4). Aortic dilation was amplified in males vs. females (p < 0.0001 at 100mmHg) and ameliorated by castration. The transcriptome of young Lox mutants showed alteration in dexamethasone (p = 9.83 × 10−30) and TGFß-responsive genes (p = 7.42 × 10−29), and aortas from older C57Bl/6J Lox+/C285F mice showed both enhanced susceptibility to elastase (p < 0.01 by ANOVA) and increased deposition of aggrecan (p < 0.05). These findings suggest that the secreted Lox+/C285F mutants produce dysfunctional elastic fibers that show increased susceptibility to proteolytic damage. Over time, the progressive weakening of the connective tissue, modified by sex and blood pressure, leads to worsening aortic disease.
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Tejido Elástico , Proteína-Lisina 6-Oxidasa , Animales , Aorta/metabolismo , Presión Sanguínea , Cobre , Dilatación Patológica/patología , Tejido Elástico/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína-Lisina 6-Oxidasa/genética , Proteína-Lisina 6-Oxidasa/metabolismoRESUMEN
BACKGROUND: Acute cellular rejection (ACR) is associated with complications after kidney transplantation, such as graft dysfunction and graft loss. Early risk assessment is therefore critical for the improvement of transplantation outcomes. In this work, we retrospectively analyzed a pre-transplant HLA antigen bead assay data set that was acquired by the e:KID consortium as part of a systems medicine approach. RESULTS: The data set included single antigen bead (SAB) reactivity profiles of 52 low-risk graft recipients (negative complement dependent cytotoxicity crossmatch, PRA < 30%) who showed detectable pre-transplant anti-HLA 1 antibodies. To assess whether the reactivity profiles provide a means for ACR risk assessment, we established a novel approach which differs from standard approaches in two aspects: the use of quantitative continuous data and the use of a multiparameter classification method. Remarkably, it achieved significant prediction of the 38 graft recipients who experienced ACR with a balanced accuracy of 82.7% (sensitivity = 76.5%, specificity = 88.9%). CONCLUSIONS: The resultant classifier achieved one of the highest prediction accuracies in the literature for pre-transplant risk assessment of ACR. Importantly, it can facilitate risk assessment in non-sensitized patients who lack donor-specific antibodies. As the classifier is based on continuous data and includes weak signals, our results emphasize that not only strong but also weak binding interactions of antibodies and HLA 1 antigens contain predictive information. TRIAL REGISTRATION: ClinicalTrials.gov NCT00724022 . Retrospectively registered July 2008.
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Rechazo de Injerto/diagnóstico , Prueba de Histocompatibilidad/métodos , Trasplante de Riñón , Enfermedad Aguda , Adulto , Anciano , Femenino , Antígenos HLA/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Isoanticuerpos/sangre , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Estudios Retrospectivos , Medición de Riesgo , Sensibilidad y EspecificidadRESUMEN
Bioluminescence resonance energy transfer (BRET) has been widely used for studying dynamic processes in biological systems such as protein-protein interactions and other signaling events. Aside from acting as a reporter, BRET can also turn on functions in living systems. Herein, we report the application of BRET to performing a biorthogonal reaction in living cells; namely, releasing functional molecules through energy transfer to a coumarin molecule, a process termed bioluminolysis. An efficient BRET from Nanoluc-Halotag chimera protein (H-Luc) to a coumarin substrate yields the excited state of coumarin, which in turn triggers hydrolysis to uncage a target molecule. Compared to the conventional methods, this novel uncaging system requires no external light source and shows fast kinetics (t1/2 <2â min). We applied this BRET uncaging system to release a potent kinase inhibitor, ibrutinib, in living cells, highlighting its broad utility in controlling the supply of bioactive small molecules inâ vivo.
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Cumarinas/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Luciferasas/metabolismo , Mediciones Luminiscentes/métodos , Proteínas Luminiscentes/metabolismo , Pirazoles/metabolismo , Pirimidinas/metabolismo , Adenina/análogos & derivados , Células HeLa , Humanos , Piperidinas , Unión ProteicaRESUMEN
Nucleic acid templated reactions have attracted attention as an important technology to sense oligonucleotides and to translate nucleic acid-based instructions into diverse outputs. Great progress has been made in accelerating the reaction in order to improve signal amplification, reaching rates where substrate turnover rather than chemical reaction is rate limiting. Herein we explore the utility of architectures inspired by three-way junction that yield a cleavage of a strand thus accelerating substrate turnover. We demonstrate that such design can overcome product inhibition in templated reactions and operate close to the rate of hybridization.
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Hibridación de Ácido Nucleico/métodos , Ácidos Nucleicos/química , Ácidos Nucleicos de Péptidos/química , Cumarinas/química , Fluorescencia , Colorantes Fluorescentes/química , Oligonucleótidos/química , Vancomicina/químicaRESUMEN
BACKGROUND: Out-of-hospital cardiac arrest survival rates in the UK are poor, and non-medically trained individuals have been identified to perform substandard cardiopulmonary resuscitation (CPR). Millions watch televised medical dramas and, for many, these comprise their only education on CPR. This study aims to investigate the quality of CPR portrayed on these programmes and whether this has an effect on public knowledge. METHODS: Prospective observational study of 30 consecutive episodes of three popular medical dramas. Public knowledge of CPR and viewing habits were assessed with a survey of non-medically trained personnel. RESULTS: 90 episodes were reviewed with 39 resuscitation attempts shown. Chest compression rates varied from 60 to 204 compressions per minute with a median of 122 (95% CI 113 to 132). Depth varied from 1.5 to 7.5 cm with a median of 3 (3.15-4.31). Rate and depth were significantly different from the UK Resuscitation Council Guidelines (2010) (p<0.05, t-test). Survey participants (n=160, 80% response rate) documented what they thought was the correct rate and depth of chest compressions and were scored accordingly. Those who documented watching medical dramas regularly scored significantly worse than those who watched occasionally (p<0.05, Mann-Whitney test). CONCLUSION: Televised medical dramas depict CPR inaccurately and laypersons may be less well informed about the correct technique the more they tune into these programmes. While there may be other confounding variables, given the popularity of television medical dramas, the poor depiction may be significantly contributing to poor public CPR knowledge and represent a potential new avenue of public education.
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Reanimación Cardiopulmonar/métodos , Difusión de la Información/métodos , Paro Cardíaco Extrahospitalario , Televisión , Reanimación Cardiopulmonar/estadística & datos numéricos , Conocimientos, Actitudes y Práctica en Salud , Encuestas Epidemiológicas , Humanos , Películas Cinematográficas , Paro Cardíaco Extrahospitalario/mortalidad , Estudios Prospectivos , Opinión Pública , Tasa de Supervivencia , Reino UnidoRESUMEN
Templated reactions proceed by bringing reagents in close proximity through their interaction with a template thus raising their effective concentrations. Templated reactions empower chemists to perform reactions at low concentrations in complex environments. Herein, we discuss our work on templated reactions leveraged on ruthenium photocatalysis. Over the past five years, we have used this reaction to uncage reporter molecules and sense or image nucleic acids or proteins of interest. The ruthenium photocatalysis chemistry has proven to be extremely robust and compatible with complex biological environments.
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Rutenio/química , Moldes Genéticos , CatálisisRESUMEN
Nucleic acid-templated reactions have attracted significant attention for nucleic acid sensing and imaging. The level of signal amplification obtained from templated reactions is a function of the template turnover, wherein the template acts as the catalyst. Herein, we report the application of a pyridinium linker that immolates upon photocatalytic reduction with a ruthenium complex to yield the fastest nucleic acid templated reaction reported to date. We show that the templated reaction turnover is limited by the duplex dissociation kinetics beyond probes longer than a 6-mer and proceeded fastest for a 5-mer PNA probe. Using a beacon architecture that masks the catalytic template, we show that this methodology can be used for nucleic acid sensing extending the analyte recognition beyond a 5-mer. The system proceeds with a catalytic efficiency of 105 M-1 s-1 and achieves turnover frequency of >100 h-1.
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Ácidos Nucleicos/análisis , Compuestos Organometálicos/química , Compuestos de Piridinio/química , Rutenio/química , Estructura MolecularRESUMEN
Herein, we describe selective imaging of hydrogen peroxide using a precipitating dye conjugated to a boronic acid-based immolative linker. We achieved visualization of endogenous hydrogen peroxide in phagosomes by solid-state two-photon fluorescence imaging in living cells with exceptionally high spatial resolution.
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Fluorescencia , Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/química , Peróxido de Hidrógeno/análisis , Animales , Ácidos Borónicos/química , Línea Celular , Colorantes Fluorescentes/síntesis química , Células HeLa , Humanos , Ratones , Microscopía de Fluorescencia por Excitación Multifotónica , Estructura MolecularRESUMEN
Two bioluminogenic caged coelenterazine derivatives (bGalCoel and bGalNoCoel) were designed and synthesized to detect ß-galactosidase activity and expression by means of bioluminescence imaging. Our approach addresses the instability of coelenterazine by introducing ß-galactose caging groups to block the auto-oxidation of coelenterazine. Both probes contain ß-galactosidase cleavable caging groups at the carbonyl group of the imidazo-pyrazinone moiety. One of the probes in particular, bGalNoCoel, displayed a fast cleavage profile, high stability, and high specificity for ß-galactosidase over other glycoside hydrolases. bGalN-oCoel could detect ß-galactosidase activity in living HEK-293T cell cultures that expressed a mutant Gaussia luciferase. It was determined that coelenterazine readily diffuses in and out of cells after uncaging by ß-galactosidase. We showed that this new caged coelenterazine derivative, bGalNoCoel, could function as a dual-enzyme substrate and detect enzyme activity across two separate cell populations.
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Línea Celular Tumoral/química , Galactosa/síntesis química , Imidazoles/química , Imidazoles/síntesis química , Luciferasas/química , Mediciones Luminiscentes/métodos , Pirazinas/química , Pirazinas/síntesis química , beta-Galactosidasa/química , Animales , Técnicas Biosensibles/métodos , Galactosa/química , Expresión Génica , Humanos , Luminiscencia , Especificidad por SustratoRESUMEN
With the current surge of spatial transcriptomics (ST) studies, researchers are exploring the deep interactive cell-play directly in tissues, in situ. However, with the current technologies, measurements consist of mRNA transcript profiles of mixed origin. Recently, applications have been proposed to tackle the deconvolution process, to gain knowledge about which cell types (SC) are found within. This is usually done by incorporating metrics from single-cell (SC) RNA, from similar tissues. Yet, most existing tools are cumbersome, and we found them hard to integrate and properly utilize. Therefore, we present AntiSplodge, a simple feed-forward neural-network-based pipeline designed to effective deconvolute ST profiles by utilizing synthetic ST profiles derived from real-life SC datasets. AntiSplodge is designed to be easy, fast and intuitive while still being lightweight. To demonstrate AntiSplodge, we deconvolute the human heart and verify correctness across time points. We further deconvolute the mouse brain, where spot patterns correctly follow that of the underlying tissue. In particular, for the hippocampus from where the cells originate. Furthermore, AntiSplodge demonstrates top of the line performance when compared to current state-of-the-art tools. Software availability: https://github.com/HealthML/AntiSplodge/.
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Pathogenic variants in genes that cause dilated cardiomyopathy (DCM) and arrhythmogenic cardiomyopathy (ACM) convey high risks for the development of heart failure through unknown mechanisms. Using single-nucleus RNA sequencing, we characterized the transcriptome of 880,000 nuclei from 18 control and 61 failing, nonischemic human hearts with pathogenic variants in DCM and ACM genes or idiopathic disease. We performed genotype-stratified analyses of the ventricular cell lineages and transcriptional states. The resultant DCM and ACM ventricular cell atlas demonstrated distinct right and left ventricular responses, highlighting genotype-associated pathways, intercellular interactions, and differential gene expression at single-cell resolution. Together, these data illuminate both shared and distinct cellular and molecular architectures of human heart failure and suggest candidate therapeutic targets.
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Displasia Ventricular Derecha Arritmogénica , Cardiomiopatía Dilatada , Insuficiencia Cardíaca , Análisis de la Célula Individual , Transcriptoma , Displasia Ventricular Derecha Arritmogénica/genética , Atlas como Asunto , Cardiomiopatía Dilatada/genética , Núcleo Celular/genética , Insuficiencia Cardíaca/genética , Ventrículos Cardíacos , Humanos , RNA-SeqRESUMEN
In systems neuroscience, neural activity that represents movements or sensory stimuli is often characterized by spatial tuning curves that may change in response to training, attention, altered mechanics, or the passage of time. A vital step in determining whether tuning curves change is accounting for estimation uncertainty due to measurement noise. In this study, we address the issue of tuning curve stability using methods that take uncertainty directly into account. We analyze data recorded from neurons in primary motor cortex using chronically implanted, multielectrode arrays in four monkeys performing center-out reaching. With the use of simulations, we demonstrate that under typical experimental conditions, the effect of neuronal noise on estimated preferred direction can be quite large and is affected by both the amount of data and the modulation depth of the neurons. In experimental data, we find that after taking uncertainty into account using bootstrapping techniques, the majority of neurons appears to be very stable on a timescale of minutes to hours. Lastly, we introduce adaptive filtering methods to explicitly model dynamic tuning curves. In contrast to several previous findings suggesting that tuning curves may be in constant flux, we conclude that the neural representation of limb movement is, on average, quite stable and that impressions to the contrary may be largely the result of measurement noise.