RESUMO
In this paper, we explore how the concepts of autonomy and autonomous choice are understood in the context of spinal cord injury in the academic literature, both in reporting on research results and more broadly on outcomes and quality of life. We find inconsistent, framework-absent portrayals of autonomy as well as an absence of discourse that draws upon ethical constructs and theory. In response, we advance a person-centered framework for spinal cord injury research that combines both lived experience and a disability ethics approach to fill this gap.
RESUMO
Echocardiography (echo) is a skilled technical procedure that depends on the experience of the operator. The aim of this paper is to reduce user variability in data acquisition by automatically computing a score of echo quality for operator feedback. To do this, a deep convolutional neural network model, trained on a large set of samples, was developed for scoring apical four-chamber (A4C) echo. In this paper, 6,916 end-systolic echo images were manually studied by an expert cardiologist and were assigned a score between 0 (not acceptable) and 5 (excellent). The images were divided into two independent training-validation and test sets. The network architecture and its parameters were based on the stochastic approach of the particle swarm optimization on the training-validation data. The mean absolute error between the scores from the ultimately trained model and the expert's manual scores was 0.71 ± 0.58. The reported error was comparable to the measured intra-rater reliability. The learned features of the network were visually interpretable and could be mapped to the anatomy of the heart in the A4C echo, giving confidence in the training result. The computation time for the proposed network architecture, running on a graphics processing unit, was less than 10 ms per frame, sufficient for real-time deployment. The proposed approach has the potential to facilitate the widespread use of echo at the point-of-care and enable early and timely diagnosis and treatment. Finally, the approach did not use any specific assumptions about the A4C echo, so it could be generalizable to other standard echo views.
Assuntos
Ecocardiografia , Humanos , Redes Neurais de Computação , Reprodutibilidade dos TestesRESUMO
We propose a joint information approach for automatic analysis of 2D echocardiography (echo) data. The approach combines a priori images, their segmentations and patient diagnostic information within a unified framework to determine various clinical parameters, such as cardiac chamber volumes, and cardiac disease labels. The main idea behind the approach is to employ joint Independent Component Analysis of both echo image intensity information and corresponding segmentation labels to generate models that jointly describe the image and label space of echo patients on multiple apical views, instead of independently. These models are then both used for segmentation and volume estimation of cardiac chambers such as the left atrium and for detecting pathological abnormalities such as mitral regurgitation. We validate the approach on a large cohort of echoes obtained from 6,993 studies. We report performance of the proposed approach in estimation of the left-atrium volume and detection of mitral-regurgitation severity. A correlation coefficient of 0.87 was achieved for volume estimation of the left atrium when compared to the clinical report. Moreover, we classified patients that suffer from moderate or severe mitral regurgitation with an average accuracy of 82%.
Assuntos
Átrios do Coração , Cardiopatias/diagnóstico por imagem , Ecocardiografia , Humanos , Insuficiência da Valva MitralRESUMO
This chapter presents a general approach for the application of spatial intensity distribution analysis (SpIDA) to pharmacodynamic quantification of receptor tyrosine kinase homodimerization in response to direct ligand activation or transactivation by G protein-coupled receptors. A custom graphical user interface developed for MATLAB is used to extract quantal brightness and receptor density information from intensity histograms calculated from single fluorescence microscopy images. This approach allows measurement of monomer/oligomer protein mixtures within subcellular compartments using conventional confocal laser scanning microscopy. Application of quantitative pharmacological analysis to data obtained using SpIDA provides a universal method for comparing studies between cell lines and receptor systems. In addition, because of its compatibility with conventional immunostaining approaches, SpIDA is suitable not only for use in recombinant systems but also for the characterization of mechanisms involving endogenous proteins. Therefore, SpIDA enables these biological processes to be monitored directly in their native cellular environment.
Assuntos
Receptores ErbB/metabolismo , Imagem Molecular/métodos , Neurônios/metabolismo , Receptor trkB/metabolismo , Receptores Dopaminérgicos/metabolismo , Software , Apomorfina/farmacologia , Linhagem Celular , Receptores ErbB/genética , Receptores ErbB/ultraestrutura , Imunofluorescência , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Ligantes , Microscopia Confocal , Microscopia de Fluorescência , Imagem Molecular/estatística & dados numéricos , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Multimerização Proteica , Quinazolinas/farmacologia , Receptor trkB/genética , Receptor trkB/ultraestrutura , Receptores Dopaminérgicos/genética , Receptores Dopaminérgicos/ultraestrutura , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/ultraestrutura , Ativação Transcricional , Tirfostinas/farmacologiaRESUMO
This chapter presents a general approach for the application of spatial intensity distribution analysis (SpIDA) to the pharmacodynamic quantification of receptor tyrosine kinase homodimerization in response to direct ligand activation or transactivation by G-protein-coupled receptors. Intensity histograms are generated from single fluorescence microscopy images. These histograms are then fit with Poissonian distributions to obtain density maps and quantal brightness values of the labeled proteins underlying the images. This approach allows resolving monomer/oligomer protein mixtures within subcellular compartments using conventional confocal laser scanning microscopy. The application of quantitative pharmacological analysis to data obtained using SpIDA provides a universal method for comparing studies between cell lines and receptor systems. In contrast to methods based on resonance energy transfer, SpIDA is suitable not only for use in recombinant systems but also for the characterization of mechanisms involving endogenous proteins. Therefore, SpIDA enables these biological processes to be monitored directly in their native cellular environment.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Ativação Transcricional , Animais , Linhagem Celular , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Processamento de Imagem Assistida por Computador/estatística & dados numéricos , Distribuição de Poisson , Multimerização Proteica , Receptores Proteína Tirosina Quinases/genética , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de SinaisRESUMO
Fluorescence microscopy is widely used in the life sciences, but largely for qualitative imaging. Here we apply a bioanalytical technique, fluorescence image moment analysis, to demonstrate how the distribution of the fluorescent molecules can be measured directly from confocal microscopy images. We measured the oligomerization state of EGF-eGPF receptors expressed in CHO-K1 cells in situ.
Assuntos
Receptores ErbB/química , Ligantes , Animais , Células CHO , Análise por Conglomerados , Cricetinae , Cricetulus , Receptores ErbB/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Microscopia de FluorescênciaRESUMO
Measuring protein interactions is key to understanding cell signaling mechanisms, but quantitative analysis of these interactions in situ has remained a major challenge. Here, we present spatial intensity distribution analysis (SpIDA), an analysis technique for image data obtained using standard fluorescence microscopy. SpIDA directly measures fluorescent macromolecule densities and oligomerization states sampled within single images. The method is based on fitting intensity histograms calculated from images to obtain density maps of fluorescent molecules and their quantal brightness. Because spatial distributions are acquired by imaging, SpIDA can be applied to the analysis of images of chemically fixed tissue as well as live cells. However, the technique does not rely on spatial correlations, freeing it from biases caused by subcellular compartmentalization and heterogeneity within tissue samples. Analysis of computer-based simulations and immunocytochemically stained GABA(B) receptors in spinal cord samples shows that the approach yields accurate measurements over a broader range of densities than established procedures. SpIDA is applicable to sampling within small areas (6 µm(2)) and reveals the presence of monomers and dimers with single-dye labeling. Finally, using GFP-tagged receptor subunits, we show that SpIDA can resolve dynamic changes in receptor oligomerization in live cells. The advantages and greater versatility of SpIDA over current techniques open the door to quantificative studies of protein interactions in native tissue using standard fluorescence microscopy.
Assuntos
Simulação por Computador , Multimerização Proteica/fisiologia , Receptores de GABA-B/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de GABA-B/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Medula Espinal/citologia , Medula Espinal/metabolismoRESUMO
Cell signaling involves dynamic changes in protein oligomerization leading to the formation of different signaling complexes and modulation of activity. Spatial intensity distribution analysis (SpIDA) is an image analysis method that can directly measure oligomerization and trafficking of endogenous proteins in single cells. Here, we show the use of SpIDA to quantify dimerization/activation and surface transport of receptor protein kinases--EGF receptor and TrkB--at early stages of their transactivation by several G protein-coupled receptors (GPCRs). Transactivation occurred on the same timescale and was directly limited by GPCR activation but independent of G-protein coupling types. Early receptor protein kinase transactivation and internalization were not interdependent for all receptor pairs tested, revealing heterogeneity between groups of GPCRs. SpIDA also detected transactivation of TrkB by dopamine receptors in intact neurons. By allowing for time and space resolved quantification of protein populations with heterogeneous oligomeric states, SpIDA provides a unique approach to undertake single cell multivariate quantification of signaling processes involving changes in protein interactions, trafficking, and activity.
Assuntos
Receptores ErbB/metabolismo , Neurônios/metabolismo , Multimerização Proteica/fisiologia , Receptor trkB/metabolismo , Receptores Dopaminérgicos/metabolismo , Ativação Transcricional/fisiologia , Animais , Células CHO , Cricetinae , Cricetulus , Receptores ErbB/genética , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Neurônios/citologia , Receptor trkB/genética , Receptores Dopaminérgicos/genéticaRESUMO
Fluorescence microscopy methods including total internal reflection fluorescence and confocal laser scanning microscopy have played a major role in modern cell biology research by permitting imaging of fluorescently tagged macromolecules in living cells. These methods are often used to examine the initial events in signal transduction, which involve interactions occurring between membrane receptors and ligands such as antibodies and growth factors. Most quantitative biophysical applications using these fluorescence imaging methods, including ligand binding assays, are based on the assumption that the fluorophore label of interest has equal access to all areas of the membrane on the cell. Our findings suggest that there is limited accessibility of fluorophores (25±2%)(-) under the basal membrane of adherent CHO-K1 cells expressing epidermal growth factor receptor plated on a bare glass in standard two-dimensional tissue cultures. The authors present a detailed study of the extent to which a small fluorescent dye molecule (Alexa 647) is able to propagate under the basal membrane of cells plated on a variety of biologically compatible substrates: fibronectin, bovine serum albumin, poly-d-lysine, collagen I, collagen IV, Geltrex™, and fibronectin such as binding polymer. For nonspecific dye propagation the best overall accessibility was achieved using a thin layer preparation of a commercially available basement membrane matrix, Geltrex™ (67±8%). Coupling of a specific high affinity ligand (epidermal growth factor) to the dye did result in a moderate increase in propagation for most substrates examined. Despite the overall increase in propagation for most substrates (60%-80%), large areas under the central regions of the adherent cells still remained inaccessible to the fluorescently labeled ligand. More importantly, the presence of the specific ligand did not result in consistent increase in ligand propagation. Taken together these results suggest that the reduced accessibility is not exclusively due to steric effects, and the chemistry of both the ligand and the substrate may be important when working under conditions of reduced dimensionality.
Assuntos
Adesão Celular/fisiologia , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Animais , Células CHO , Cricetinae , Cricetulus , AMP Cíclico/análogos & derivados , AMP Cíclico/química , Receptores ErbB/química , Receptores ErbB/metabolismo , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Currently, the molecular mechanism for membrane fusion remains unconfirmed. The most compelling suggested mechanism is the stalk hypothesis, which states that membrane fusion proceeds via stalk formation/hemifusion, among other steps. Because the stalk would have a very high radius of curvature, small lipophilic molecules could enhance fusion by lowering the energy barrier to stalk formation. We previously showed that the general anesthetic halothane is capable of inducing membrane fusion in 1,2-dileoyl-sn-3-glycero-3-phospocholine (DOPC) vesicles. In the present study, we examined other small molecules, general anesthetics (chloroform, isoflurane, enflurane, and sevoflurane), to determine whether they exhibit fusion properties with model lipid membranes similar to those of halothane. We employed both two-photon excitation fluorescence cross-correlation spectroscopy (TPE-FCCS) and steady-state fluorescence dequenching (FD) assays. Using volatile general anesthetics as novel fusion agents, we also aimed to gain a better understanding of the membrane fusion mechanism at a molecular level. We found that lipid mixing or lipid rearrangement, which is required for the formation of the fusion-state intermediates and the fusion pore, rather than the association of lipid vesicles, is rate-limiting. In addition, halothane and chloroform were found to induce lipid mixing (rearrangement) to a greater extent than isoflurane, enflurane, and sevoflurane. Finally, it is proposed that the efficiency of these general anesthetics as fusion agents is related to their partition coefficients, water solubilities, polarities, and molecular volumes, all of which affect the ability of each anesthetic to perturb the contacting bilayer membranes of fusing vesicles.
Assuntos
Anestésicos Gerais/química , Anestésicos Gerais/farmacologia , Fusão de Membrana/efeitos dos fármacos , Fótons , Cinética , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Conformação Molecular , Espectrometria de FluorescênciaRESUMO
A robust method to directly measure ligand-receptor binding interactions using fluorescence cross-correlation spectroscopy (FCCS) is described. The example receptor systems demonstrated here are the human micro-opioid receptor, a representative G protein-coupled receptor (GPCR), and Streptavidin, but these general protocols can be extended for the analysis of many membrane receptors. We present methods for the preparation of GPCR-containing membrane nanopatches that appear to have the shapes of nanovesicles, labeling of proteins in membrane vesicles, in addition to the coupling of quantum dots (QDs) to peptide ligands. Further, we demonstrate that reliable binding information can be obtained from these partially purified receptors.
Assuntos
Bioensaio/métodos , Membrana Celular/metabolismo , Nanotecnologia , Pontos Quânticos , Receptores Opioides/metabolismo , Estreptavidina/química , Biotinilação , Células Cultivadas , Corantes Fluorescentes , Humanos , Rim/citologia , Rim/metabolismo , Nanoestruturas , Receptores Opioides/análise , Estreptavidina/metabolismoRESUMO
Imaging of living cells and tissue is now common in many fields of the life and physical sciences, and is instrumental in revealing a great deal about cellular dynamics and function. It is crucial when performing such experiments that cell viability is at the forefront of any measurement to ensure that the physiological and biological processes that are under investigation are not altered in any way. Many cells and tissues are not normally exposed to light during their life cycle, so it is important for microscopy applications to minimize light exposure, which can cause phototoxicity. To ensure minimal light exposure, it is crucial that microscope systems are optimized to collect as much light as possible. This can be achieved using superior-quality optical components and state-of-the-art detectors. This Commentary discusses how to set up a suitable environment on the microscope stage to maintain living cells. There is also a focus on general and imaging-platform-specific ways to optimize the efficiency of light throughput and detection. With an efficient optical microscope and a good detector, the light exposure can be minimized during live-cell imaging, thus minimizing phototoxicity and maintaining cell viability. Brief suggestions for useful microscope accessories as well as available fluorescence tools are also presented. Finally, a flow chart is provided to assist readers in choosing the appropriate imaging platform for their experimental systems.
Assuntos
Microscopia/métodos , Animais , Proliferação de Células , Sobrevivência Celular , Humanos , Processamento de Imagem Assistida por Computador , Microscopia Confocal , Microscopia de Fluorescência , Microscopia de Contraste de FaseRESUMO
Fluorescent labels are often used in bioassays as a means to detect and characterize ligand-receptor binding. This is due in part to the inherently high sensitivity of fluorescence-based technology and the relative accessibility of the technique. There is often little concern raised as to whether or not the fluorescent label itself affects the ligand-receptor binding dynamics and equilibrium. This may be particularly important when considering nanoparticle labels. In this study, we examine the affects of nanoparticle (quantum dots and polymer nanospheres) fluorescent labels on the streptavidin-biotin binding system. Since the nanoparticle labels are larger than the species they tag, one could anticipate significant perturbation of the binding equilibrium. We demonstrate, using fluorescence cross-correlation spectroscopy, that although the binding equilibria do change, the relative changes are largely predictable. We suggest that the nanoparticles' mesoscopic size and surface tension effects can be used to explain changes in streptavidin-biotin binding.
Assuntos
Corantes Fluorescentes/química , Microscopia de Fluorescência/métodos , Nanopartículas/química , Nanopartículas/ultraestrutura , Coloração e Rotulagem/métodos , Tamanho da Partícula , Sensibilidade e EspecificidadeRESUMO
Current ligand-receptor binding assays for G-protein coupled receptors cannot directly measure the system's dissociation constant, Kd, without purification of the receptor protein. Accurately measured Kd's are essential in the development of a molecular level understanding of ligand-receptor interactions critical in rational drug design. Here we report the introduction of two-photon excitation fluorescence cross-correlation spectroscopy (TPE-FCCS) to the direct analysis of ligand-receptor interactions of the human micro opioid receptor (hMOR) for both agonists and antagonists. We have developed the use of fluorescently distinct, dye-labeled hMOR-containing cell membrane nanopatches ( approximately 100-nm radius) and ligands, respectively, for this assay. We show that the output from TPE-FCCS data sets can be converted to the conventional Hill format, which provides Kd and the number of active receptors per nanopatch. When ligands are labeled with quantum dots, this assay can detect binding with ligand concentrations in the subnanomolar regime. Interestingly, conjugation to a bulky quantum dot did not adversely affect the binding propensity of the hMOR pentapeptide ligand, Leu-enkephalin.
Assuntos
Membrana Celular/metabolismo , Reagentes de Ligações Cruzadas/química , Fluoresceína/química , Nanoestruturas/química , Fótons , Receptores Opioides/análise , Receptores Opioides/metabolismo , Animais , Linhagem Celular , Encefalina Leucina/química , Encefalina Leucina/metabolismo , Humanos , Ligantes , Estrutura Molecular , Naloxona/química , Naltrexona/química , Antagonistas de Entorpecentes , Pontos Quânticos , Receptores Opioides/agonistas , Soluções , Spodoptera , TitulometriaRESUMO
Semiconductor nanocrystals (quantum dots) have been increasingly employed in measuring the dynamic behavior of biomacromolecules using fluorescence correlation spectroscopy. This poses a challenge, because quantum dots display their own dynamic behavior in the form of intermittent photoluminescence, also known as blinking. In this review, the manifestation of blinking in correlation spectroscopy will be explored, preceded by an examination of quantum dot blinking in general.