Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 12 de 12
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Elife ; 122023 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-37083703

RESUMO

Resolving fundamental molecular and functional processes underlying human synaptic development is crucial for understanding normal brain function as well as dysfunction in disease. Based upon increasing evidence of species-divergent features of brain cell types, coupled with emerging studies of complex human disease genetics, we developed the first automated and quantitative high-content synaptic phenotyping platform using human neurons and astrocytes. To establish the robustness of our platform, we screened the effects of 376 small molecules on presynaptic density, neurite outgrowth, and cell viability, validating six small molecules that specifically enhanced human presynaptic density in vitro. Astrocytes were essential for mediating the effects of all six small molecules, underscoring the relevance of non-cell-autonomous factors in synapse assembly and their importance in synaptic screening applications. Bromodomain and extraterminal (BET) inhibitors emerged as the most prominent hit class and global transcriptional analyses using multiple BET inhibitors confirmed upregulation of synaptic gene expression. Through these analyses, we demonstrate the robustness of our automated screening platform for identifying potent synaptic modulators, which can be further leveraged for scaled analyses of human synaptic mechanisms and drug discovery efforts.


Assuntos
Neurogênese , Neurônios , Humanos , Neurogênese/fisiologia , Neurônios/fisiologia , Sinapses/fisiologia , Crescimento Neuronal , Astrócitos
2.
Mol Biol Cell ; 33(6): ar54, 2022 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-34910584

RESUMO

Patient stem cell-derived models enable imaging of complex disease phenotypes and the development of scalable drug discovery platforms. Current preclinical methods for assessing cellular activity do not, however, capture the full intricacies of disease-induced disturbances and instead typically focus on a single parameter, which impairs both the understanding of disease and the discovery of effective therapeutics. Here, we describe a cloud-based image processing and analysis platform that captures the intricate activity profile revealed by GCaMP fluorescence recordings of intracellular calcium changes and enables the discovery of molecules that correct 153 parameters that define the amyotrophic lateral sclerosis motor neuron disease phenotype. In a high-throughput screen, we identified compounds that revert the multiparametric disease profile to that found in healthy cells, a novel and robust measure of therapeutic potential quite distinct from unidimensional screening. This platform can guide the development of therapeutics that counteract the multifaceted pathological features of diseased cellular activity.


Assuntos
Esclerose Lateral Amiotrófica , Descoberta de Drogas , Esclerose Lateral Amiotrófica/genética , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos , Humanos , Neurônios , Fenótipo
4.
Cell Rep ; 32(5): 107995, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32755587

RESUMO

Cellular responses to stimuli can evolve over time, resulting in distinct early and late phases in response to a single signal. DNA damage induces a complex response that is largely orchestrated by the transcription factor p53, whose dynamics influence whether a damaged cell will arrest and repair the damage or will initiate cell death. How p53 responses and cellular outcomes evolve in the presence of continuous DNA damage remains unknown. Here, we have found that a subset of cells switches from oscillating to sustained p53 dynamics several days after undergoing damage. The switch results from cell cycle progression in the presence of damaged DNA, which activates the caspase-2-PIDDosome, a complex that stabilizes p53 by inactivating its negative regulator MDM2. This work defines a molecular pathway that is activated if the canonical checkpoints fail to halt mitosis in the presence of damaged DNA.


Assuntos
Pontos de Checagem do Ciclo Celular , Quebras de DNA de Cadeia Dupla , Proteína Supressora de Tumor p53/metabolismo , Células A549 , Caspase 2/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Humanos , Células MCF-7 , Mitose , Modelos Biológicos , Proteólise , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Raios Ultravioleta
5.
J Thromb Haemost ; 18(10): 2701-2711, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32662223

RESUMO

BACKGROUND: The mechanisms that regulate platelet biogenesis remain unclear; factors that trigger megakaryocytes (MKs) to initiate platelet production are poorly understood. Platelet formation begins with proplatelets, which are cellular extensions originating from the MK cell body. OBJECTIVES: Proplatelet formation is an asynchronous and dynamic process that poses unique challenges for researchers to accurately capture and analyze. We have designed an open-source, high-content, high-throughput, label-free analysis platform. METHODS: Phase-contrast images of live, primary MKs are captured over a 24-hour period. Pixel-based machine-learning classification done by ilastik generates probability maps of key cellular features (circular MKs and branching proplatelets), which are processed by a customized CellProfiler pipeline to identify and filter structures of interest based on morphology. A subsequent reinforcement classification, by CellProfiler Analyst, improves the detection of cellular structures. RESULTS: This workflow yields the percent of proplatelet production, area, count of proplatelets and MKs, and other statistics including skeletonization information for measuring proplatelet branching and length. We propose using a combination of these analyzed metrics, in particular the area measurements of MKs and proplatelets, when assessing in vitro proplatelet production. Accuracy was validated against manually counted images and an existing algorithm. We then used the new platform to test compounds known to cause thrombocytopenia, including bromodomain inhibitors, and uncovered previously unrecognized effects of drugs on proplatelet formation, thus demonstrating the utility of our analysis platform. CONCLUSION: This advance in creating unbiased data analysis will increase the scale and scope of proplatelet production studies and potentially serve as a valuable resource for investigating molecular mechanisms of thrombocytopenia.


Assuntos
Megacariócitos , Trombocitopenia , Plaquetas , Células Cultivadas , Humanos , Trombopoese
6.
Nat Methods ; 17(2): 241, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31969730

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

7.
Nat Methods ; 16(12): 1247-1253, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31636459

RESUMO

Segmenting the nuclei of cells in microscopy images is often the first step in the quantitative analysis of imaging data for biological and biomedical applications. Many bioimage analysis tools can segment nuclei in images but need to be selected and configured for every experiment. The 2018 Data Science Bowl attracted 3,891 teams worldwide to make the first attempt to build a segmentation method that could be applied to any two-dimensional light microscopy image of stained nuclei across experiments, with no human interaction. Top participants in the challenge succeeded in this task, developing deep-learning-based models that identified cell nuclei across many image types and experimental conditions without the need to manually adjust segmentation parameters. This represents an important step toward configuration-free bioimage analysis software tools.


Assuntos
Núcleo Celular/ultraestrutura , Processamento de Imagem Assistida por Computador/métodos , Ciência de Dados , Humanos , Microscopia de Fluorescência/métodos
8.
Cytometry A ; 95(9): 952-965, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31313519

RESUMO

Identifying nuclei is often a critical first step in analyzing microscopy images of cells and classical image processing algorithms are most commonly used for this task. Recent developments in deep learning can yield superior accuracy, but typical evaluation metrics for nucleus segmentation do not satisfactorily capture error modes that are relevant in cellular images. We present an evaluation framework to measure accuracy, types of errors, and computational efficiency; and use it to compare deep learning strategies and classical approaches. We publicly release a set of 23,165 manually annotated nuclei and source code to reproduce experiments and run the proposed evaluation methodology. Our evaluation framework shows that deep learning improves accuracy and can reduce the number of biologically relevant errors by half. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.


Assuntos
Núcleo Celular , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Linhagem Celular , Confiabilidade dos Dados , Aprendizado Profundo , Fluorescência , Humanos , Citometria por Imagem/métodos
10.
Mol Cell ; 71(4): 581-591.e5, 2018 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-30057196

RESUMO

Biological signals need to be robust and filter small fluctuations yet maintain sensitivity to signals across a wide range of magnitudes. Here, we studied how fluctuations in DNA damage signaling relate to maintenance of long-term cell-cycle arrest. Using live-cell imaging, we quantified division profiles of individual human cells in the course of 1 week after irradiation. We found a subset of cells that initially establish cell-cycle arrest and then sporadically escape and divide. Using fluorescent reporters and mathematical modeling, we determined that fluctuations in the oscillatory pattern of the tumor suppressor p53 trigger a sharp switch between p21 and CDK2, leading to escape from arrest. Transient perturbation of p53 stability mimicked the noise in individual cells and was sufficient to trigger escape from arrest. Our results show that the self-reinforcing circuitry that mediates cell-cycle transitions can translate small fluctuations in p53 signaling into large phenotypic changes.


Assuntos
Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células Epiteliais/metabolismo , Modelos Estatísticos , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Divisão Celular/efeitos da radiação , Linhagem Celular Transformada , Proliferação de Células/efeitos da radiação , Quinase 2 Dependente de Ciclina/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Dano ao DNA , Células Epiteliais/citologia , Células Epiteliais/efeitos da radiação , Raios gama , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Estabilidade Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/efeitos da radiação , Imagem com Lapso de Tempo , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Proteína Vermelha Fluorescente
11.
PLoS Biol ; 16(7): e2005970, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29969450

RESUMO

CellProfiler has enabled the scientific research community to create flexible, modular image analysis pipelines since its release in 2005. Here, we describe CellProfiler 3.0, a new version of the software supporting both whole-volume and plane-wise analysis of three-dimensional (3D) image stacks, increasingly common in biomedical research. CellProfiler's infrastructure is greatly improved, and we provide a protocol for cloud-based, large-scale image processing. New plugins enable running pretrained deep learning models on images. Designed by and for biologists, CellProfiler equips researchers with powerful computational tools via a well-documented user interface, empowering biologists in all fields to create quantitative, reproducible image analysis workflows.


Assuntos
Processamento de Imagem Assistida por Computador , Software , Animais , Núcleo Celular/metabolismo , DNA/metabolismo , Aprendizado Profundo , Humanos , Imageamento Tridimensional , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
12.
Science ; 336(6087): 1440-4, 2012 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-22700930

RESUMO

Cells transmit information through molecular signals that often show complex dynamical patterns. The dynamic behavior of the tumor suppressor p53 varies depending on the stimulus; in response to double-strand DNA breaks, it shows a series of repeated pulses. Using a computational model, we identified a sequence of precisely timed drug additions that alter p53 pulses to instead produce a sustained p53 response. This leads to the expression of a different set of downstream genes and also alters cell fate: Cells that experience p53 pulses recover from DNA damage, whereas cells exposed to sustained p53 signaling frequently undergo senescence. Our results show that protein dynamics can be an important part of a signal, directly influencing cellular fate decisions.


Assuntos
Senescência Celular/genética , Quebras de DNA de Cadeia Dupla , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Apoptose/genética , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , Reparo do DNA , Raios gama , Humanos , Imidazóis/metabolismo , Imidazóis/farmacologia , Modelos Biológicos , Proteínas Nucleares/genética , Piperazinas/metabolismo , Piperazinas/farmacologia , Proteína da Leucemia Promielocítica , Análise de Célula Única , Fatores de Transcrição/genética , Ativação Transcricional , Proteínas Supressoras de Tumor/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...