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1.
Cell ; 178(3): 521-535.e23, 2019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31348885

RESUMO

Intracellular accumulation of misfolded proteins causes toxic proteinopathies, diseases without targeted therapies. Mucin 1 kidney disease (MKD) results from a frameshift mutation in the MUC1 gene (MUC1-fs). Here, we show that MKD is a toxic proteinopathy. Intracellular MUC1-fs accumulation activated the ATF6 unfolded protein response (UPR) branch. We identified BRD4780, a small molecule that clears MUC1-fs from patient cells, from kidneys of knockin mice and from patient kidney organoids. MUC1-fs is trapped in TMED9 cargo receptor-containing vesicles of the early secretory pathway. BRD4780 binds TMED9, releases MUC1-fs, and re-routes it for lysosomal degradation, an effect phenocopied by TMED9 deletion. Our findings reveal BRD4780 as a promising lead for the treatment of MKD and other toxic proteinopathies. Generally, we elucidate a novel mechanism for the entrapment of misfolded proteins by cargo receptors and a strategy for their release and anterograde trafficking to the lysosome.


Assuntos
Benzamidas/metabolismo , Compostos Bicíclicos com Pontes/farmacologia , Heptanos/farmacologia , Lisossomos/efeitos dos fármacos , Proteínas de Transporte Vesicular/metabolismo , Fator 6 Ativador da Transcrição/metabolismo , Animais , Benzamidas/química , Benzamidas/farmacologia , Compostos Bicíclicos com Pontes/uso terapêutico , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Mutação da Fase de Leitura , Heptanos/uso terapêutico , Humanos , Receptores de Imidazolinas/antagonistas & inibidores , Receptores de Imidazolinas/genética , Receptores de Imidazolinas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Rim/citologia , Rim/metabolismo , Rim/patologia , Nefropatias/metabolismo , Nefropatias/patologia , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Mucina-1/química , Mucina-1/genética , Mucina-1/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Proteínas de Transporte Vesicular/química
2.
Nature ; 608(7921): 174-180, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35732739

RESUMO

Heart failure encompasses a heterogeneous set of clinical features that converge on impaired cardiac contractile function1,2 and presents a growing public health concern. Previous work has highlighted changes in both transcription and protein expression in failing hearts3,4, but may overlook molecular changes in less prevalent cell types. Here we identify extensive molecular alterations in failing hearts at single-cell resolution by performing single-nucleus RNA sequencing of nearly 600,000 nuclei in left ventricle samples from 11 hearts with dilated cardiomyopathy and 15 hearts with hypertrophic cardiomyopathy as well as 16 non-failing hearts. The transcriptional profiles of dilated or hypertrophic cardiomyopathy hearts broadly converged at the tissue and cell-type level. Further, a subset of hearts from patients with cardiomyopathy harbour a unique population of activated fibroblasts that is almost entirely absent from non-failing samples. We performed a CRISPR-knockout screen in primary human cardiac fibroblasts to evaluate this fibrotic cell state transition; knockout of genes associated with fibroblast transition resulted in a reduction of myofibroblast cell-state transition upon TGFß1 stimulation for a subset of genes. Our results provide insights into the transcriptional diversity of the human heart in health and disease as well as new potential therapeutic targets and biomarkers for heart failure.


Assuntos
Cardiomiopatia Dilatada , Cardiomiopatia Hipertrófica , Núcleo Celular , Perfilação da Expressão Gênica , Insuficiência Cardíaca , Análise de Célula Única , Sistemas CRISPR-Cas , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/patologia , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/patologia , Estudos de Casos e Controles , Núcleo Celular/genética , Células Cultivadas , Técnicas de Inativação de Genes , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Humanos , Miocárdio/metabolismo , Miocárdio/patologia , Miofibroblastos/metabolismo , Miofibroblastos/patologia , RNA-Seq , Transcrição Gênica , Fator de Crescimento Transformador beta1
3.
Nat Methods ; 21(6): 1114-1121, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38594452

RESUMO

The identification of genetic and chemical perturbations with similar impacts on cell morphology can elucidate compounds' mechanisms of action or novel regulators of genetic pathways. Research on methods for identifying such similarities has lagged due to a lack of carefully designed and well-annotated image sets of cells treated with chemical and genetic perturbations. Here we create such a Resource dataset, CPJUMP1, in which each perturbed gene's product is a known target of at least two chemical compounds in the dataset. We systematically explore the directionality of correlations among perturbations that target the same protein encoded by a given gene, and we find that identifying matches between chemical and genetic perturbations is a challenging task. Our dataset and baseline analyses provide a benchmark for evaluating methods that measure perturbation similarities and impact, and more generally, learn effective representations of cellular state from microscopy images. Such advancements would accelerate the applications of image-based profiling of cellular states, such as uncovering drug mode of action or probing functional genomics.


Assuntos
Processamento de Imagem Assistida por Computador , Humanos , Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodos
4.
ACS Cent Sci ; 10(5): 1105-1114, 2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38799654

RESUMO

Cyclooxygenase-2 (COX-2) is an enzyme that plays a pivotal role in peripheral inflammation and pain via the prostaglandin pathway. In the central nervous system (CNS), COX-2 is implicated in neurodegenerative and psychiatric disorders as a potential therapeutic target and biomarker. However, clinical studies with COX-2 have yielded inconsistent results, partly due to limited mechanistic understanding of how COX-2 activity relates to CNS pathology. Therefore, developing COX-2 positron emission tomography (PET) radiotracers for human neuroimaging is of interest. This study introduces [11C]BRD1158, which is a potent and uniquely fast-binding, selective COX-2 PET radiotracer. [11C]BRD1158 was developed by prioritizing potency at COX-2, isoform selectivity over COX-1, fast binding kinetics, and free fraction in the brain. Evaluated through in vivo PET neuroimaging in rodent models with human COX-2 overexpression, [11C]BRD1158 demonstrated high brain uptake, fast target-engagement, functional reversibility, and excellent specific binding, which is advantageous for human imaging applications. Lastly, post-mortem samples from Huntington's disease (HD) patients and preclinical HD mouse models showed that COX-2 levels were elevated specifically in disease-affected brain regions, primarily from increased expression in microglia. These findings indicate that COX-2 holds promise as a novel clinical marker of HD onset and progression, one of many potential applications of [11C]BRD1158 human PET.

5.
Biochemistry ; 52(7): 1137-48, 2013 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-23346953

RESUMO

The design and application of an effective, new class of multifunctional small molecule inhibitors of amyloid self-assembly are described. Several compounds based on the diaryl hydrazone scaffold were designed. Forty-four substituted derivatives of this core structure were synthesized using a variety of benzaldehydes and phenylhydrazines and characterized. The inhibitor candidates were evaluated in multiple assays, including the inhibition of amyloid ß (Aß) fibrillogenesis and oligomer formation and the reverse processes, the disassembly of preformed fibrils and oligomers. Because the structure of the hydrazone-based inhibitors mimics the redox features of the antioxidant resveratrol, the radical scavenging effect of the compounds was evaluated by colorimetric assays against 2,2-diphenyl-1-picrylhydrazyl and superoxide radicals. The hydrazone scaffold was active in all of the different assays. The structure-activity relationship revealed that the substituents on the aromatic rings had a considerable effect on the overall activity of the compounds. The inhibitors showed strong activity in fibrillogenesis inhibition and disassembly, and even greater potency in the inhibition of oligomer formation and oligomer disassembly. Supporting the quantitative fluorometric and colorimetric assays, size exclusion chromatographic studies indicated that the best compounds practically eliminated or substantially inhibited the formation of soluble, aggregated Aß species, as well. Atomic force microscopy was also applied to monitor the morphology of Aß deposits. The compounds also possessed the predicted antioxidant properties; approximately 30% of the synthesized compounds showed a radical scavenging effect equal to or better than that of resveratrol or ascorbic acid.


Assuntos
Amiloide/antagonistas & inibidores , Antioxidantes/farmacologia , Hidrazonas/química , Hidrazonas/farmacologia , Relação Estrutura-Atividade , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Antioxidantes/química , Compostos de Bifenilo/química , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Hidrazonas/síntese química , Microscopia de Força Atômica , Estrutura Molecular , Picratos/química , Superóxidos/química
6.
Nat Protoc ; 18(7): 1981-2013, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37344608

RESUMO

In image-based profiling, software extracts thousands of morphological features of cells from multi-channel fluorescence microscopy images, yielding single-cell profiles that can be used for basic research and drug discovery. Powerful applications have been proven, including clustering chemical and genetic perturbations on the basis of their similar morphological impact, identifying disease phenotypes by observing differences in profiles between healthy and diseased cells and predicting assay outcomes by using machine learning, among many others. Here, we provide an updated protocol for the most popular assay for image-based profiling, Cell Painting. Introduced in 2013, it uses six stains imaged in five channels and labels eight diverse components of the cell: DNA, cytoplasmic RNA, nucleoli, actin, Golgi apparatus, plasma membrane, endoplasmic reticulum and mitochondria. The original protocol was updated in 2016 on the basis of several years' experience running it at two sites, after optimizing it by visual stain quality. Here, we describe the work of the Joint Undertaking for Morphological Profiling Cell Painting Consortium, to improve upon the assay via quantitative optimization by measuring the assay's ability to detect morphological phenotypes and group similar perturbations together. The assay gives very robust outputs despite various changes to the protocol, and two vendors' dyes work equivalently well. We present Cell Painting version 3, in which some steps are simplified and several stain concentrations can be reduced, saving costs. Cell culture and image acquisition take 1-2 weeks for typically sized batches of ≤20 plates; feature extraction and data analysis take an additional 1-2 weeks.This protocol is an update to Nat. Protoc. 11, 1757-1774 (2016): https://doi.org/10.1038/nprot.2016.105.


Assuntos
Técnicas de Cultura de Células , Processamento de Imagem Assistida por Computador , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência , Mitocôndrias , Software
7.
Midwifery ; 115: 103483, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36115272

RESUMO

IMPORTANCE: Perineal injury is the most common complication of vaginal delivery, and labor and delivery (L&D) nurses are crucial in managing and educating women following perineal trauma. OBJECTIVE: The aims of this study were to assess L&D nurse experience, knowledge, and self-perception of preparedness in caring for women with obstetric anal sphincter injuries (OASIS) and to compare pre- and post-test scores using a computer-based learning module (CBL) for OASIS nurse education. STUDY DESIGN: All L&D nurses were invited to complete a voluntary, self-assessment questionnaire inquiring about prior experience, training, and education and current clinical practice in caring for patients with OASIS. They were also asked to answer ten knowledge-based questions about OASIS. The primary outcome was change in pretest and posttest knowledge-based scores after completion of CBL. RESULTS: Forty-one L&D nurses voluntarily responded to the self-assessment survey. Of respondents, 20% answered they were "very comfortable", 48% "comfortable", 23% "neutral," and 8% "uncomfortable" for caring for women with OASIS post-delivery. Fifty-three percent of reported having no formal education in nursing school about OASIS and 35% reported no formal training while at work. The average pretest knowledge test score was 66.3% and 93.5% (p < 0.001) after completion of the CBL. CONCLUSIONS: Most L&D nurses in this study reported having very limited formal nursing education in OASIS. Regardless of this lack of formal training, the majority of L&D nurses in this sample described themselves as comfortable caring for patients with OASIS post-delivery. Completion of a CBL was associated with higher OASIS knowledge scores.


Assuntos
Educação em Enfermagem , Lacerações , Complicações do Trabalho de Parto , Humanos , Gravidez , Feminino , Estados Unidos , Canal Anal/lesões , Autoavaliação (Psicologia) , Períneo/lesões , Parto Obstétrico/efeitos adversos , Computadores , Complicações do Trabalho de Parto/etiologia , Estudos Retrospectivos , Fatores de Risco
8.
bioRxiv ; 2020 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-32637960

RESUMO

Drug repurposing is the only method capable of delivering treatments on the shortened time-scale required for patients afflicted with lung disease arising from SARS-CoV-2 infection. Mucin-1 (MUC1), a membrane-bound molecule expressed on the apical surfaces of most mucosal epithelial cells, is a biochemical marker whose elevated levels predict the development of acute lung injury (ALI) and respiratory distress syndrome (ARDS), and correlate with poor clinical outcomes. In response to the pandemic spread of SARS-CoV-2, we took advantage of a high content screen of 3,713 compounds at different stages of clinical development to identify FDA-approved compounds that reduce MUC1 protein abundance. Our screen identified Fostamatinib (R788), an inhibitor of spleen tyrosine kinase (SYK) approved for the treatment of chronic immune thrombocytopenia, as a repurposing candidate for the treatment of ALI. In vivo , Fostamatinib reduced MUC1 abundance in lung epithelial cells in a mouse model of ALI. In vitro , SYK inhibition by Fostamatinib promoted MUC1 removal from the cell surface. Our work reveals Fostamatinib as a repurposing drug candidate for ALI and provides the rationale for rapidly standing up clinical trials to test Fostamatinib efficacy in patients with COVID-19 lung injury.

9.
Cell Rep Med ; 1(8): 100137, 2020 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-33294858

RESUMO

Drug repurposing has the advantage of identifying potential treatments on a shortened timescale. In response to the pandemic spread of SARS-CoV-2, we took advantage of a high-content screen of 3,713 compounds at different stages of clinical development to identify FDA-approved compounds that reduce mucin-1 (MUC1) protein abundance. Elevated MUC1 levels predict the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) and correlate with poor clinical outcomes. Our screen identifies fostamatinib (R788), an inhibitor of spleen tyrosine kinase (SYK) approved for the treatment of chronic immune thrombocytopenia, as a repurposing candidate for the treatment of ALI. In vivo, fostamatinib reduces MUC1 abundance in lung epithelial cells in a mouse model of ALI. In vitro, SYK inhibition by the active metabolite R406 promotes MUC1 removal from the cell surface. Our work suggests fostamatinib as a repurposing drug candidate for ALI.

10.
Circ Arrhythm Electrophysiol ; 8(5): 1255-64, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26226998

RESUMO

BACKGROUND: Heterozygous human NKX2-5 homeodomain (DNA-binding domain) missense mutations are highly penetrant for varied congenital heart defects, including progressive atrioventricular (AV) block requiring pacemaker implantation. We recently replicated this genetic defect in a murine knockin model, in which we demonstrated highly penetrant, pleiotropic cardiac anomalies. In this study, we examined postnatal AV conduction in the knockin mice. METHODS AND RESULTS: A murine knockin model (Arg52Gly, Nkx2-5(+/R52G)) in a 129/Sv background was analyzed by histopathology, surface, and telemetry ECG, and in vivo electrophysiology studies, comparing with control Nkx2-5(+/+) mice at diverse postnatal stages, ranging from postnatal day 1 (P1) to 17 months. PR prolongation (first degree AV block) was present at 4 weeks, 7 months, and 17 months of age, but not at P1 in the mutant mice. Advanced AV block was also occasionally demonstrated in the mutant mice. Electrophysiology studies showed that AV nodal function and right ventricular effective refractory period were impaired in the mutant mice, whereas sinus nodal function was not affected. AV nodal size was significantly smaller in the mutant mice than their controls at 4 weeks of age, corresponding to the presence of PR prolongation, but not P1, suggesting, at least in part, that the conduction abnormalities are the result of a morphologically atrophic AV node. CONCLUSIONS: The highly penetrant and progressive AV block phenotype seen in human heterozygous missense mutations in NKX2-5 homeodomain was replicated in mice by knocking in a comparable missense mutation.


Assuntos
Bloqueio Atrioventricular/genética , Modelos Animais de Doenças , Cardiopatias Congênitas/fisiopatologia , Mutação de Sentido Incorreto/genética , Animais , Bloqueio Atrioventricular/fisiopatologia , Eletrocardiografia , Técnicas Eletrofisiológicas Cardíacas , Heterozigoto , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Fenótipo , Fatores de Transcrição
11.
Biochem Mol Biol Educ ; 41(3): 156-62, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23281241

RESUMO

An undergraduate laboratory exercise is described to demonstrate the biochemical applications of electron paramagnetic resonance (EPR) spectroscopy. The ß93 cysteine residue of hemoglobin is labeled by the covalent binding of 3-maleimido-proxyl (5-MSL) and 2,2,5,5-tetramethyl-1-oxyl-3-methyl methanethiosulfonate (MTSL), respectively. The excess spin label is removed by gel-exclusion chromatography. Changes in the mobility of the reporter groups attached to the protein are monitored by EPR spectroscopy. While the spectral parameters of the rigidly attached 5-MSL provide information on the rotation of the whole spin labeled protein, MTSL bound by a more flexible linkage describes the local environment of the cysteine residue in the interior of the protein structure. Students can study the known crystal structure of hemoglobin in comparison to the results they obtain by analyzing the EPR spectra. Overall, the exercise introduces them to laboratory techniques such as protein labeling, gel filtration, EPR spectroscopy, as well as familiarizes them with the online Protein Data Bank as a research resource and PyMOL software as a structure visualization tool.


Assuntos
Currículo , Óxidos N-Cíclicos/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Hemoglobinas/química , Marcadores de Spin , Estudantes , Sítios de Ligação , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica
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