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1.
PLoS Comput Biol ; 19(12): e1011733, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-38113280

RESUMEN

High throughput experimental approaches are increasingly allowing for the quantitative description of cellular and organismal phenotypes. Distilling these large volumes of complex data into meaningful measures that can drive biological insight remains a central challenge. In the quantitative study of development, for instance, one can resolve phenotypic measures for single cells onto their lineage history, enabling joint consideration of heritable signals and cell fate decisions. Most attempts to analyze this type of data, however, discard much of the information content contained within lineage trees. In this work we introduce a generalized metric, which we term the branch edit distance, that allows us to compare any two embryos based on phenotypic measurements in individual cells. This approach aligns those phenotypic measurements to the underlying lineage tree, providing a flexible and intuitive framework for quantitative comparisons between, for instance, Wild-Type (WT) and mutant developmental programs. We apply this novel metric to data on cell-cycle timing from over 1300 WT and RNAi-treated Caenorhabditis elegans embryos. Our new metric revealed surprising heterogeneity within this data set, including subtle batch effects in WT embryos and dramatic variability in RNAi-induced developmental phenotypes, all of which had been missed in previous analyses. Further investigation of these results suggests a novel, quantitative link between pathways that govern cell fate decisions and pathways that pattern cell cycle timing in the early embryo. Our work demonstrates that the branch edit distance we propose, and similar metrics like it, have the potential to revolutionize our quantitative understanding of organismal phenotype.


Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Diferenciación Celular/genética , Proteínas de Caenorhabditis elegans/metabolismo , Interferencia de ARN , Ciclo Celular/genética , Linaje de la Célula/genética
2.
BMC Bioinformatics ; 16: 189, 2015 Jun 09.
Artículo en Inglés | MEDLINE | ID: mdl-26051157

RESUMEN

BACKGROUND: Imaging and image analysis advances are yielding increasingly complete and complicated records of cellular events in tissues and whole embryos. The ability to follow hundreds to thousands of cells at the individual level demands a spatio-temporal data infrastructure: tools to assemble and collate knowledge about development spatially in a manner analogous to geographic information systems (GIS). Just as GIS indexes items or events based on their spatio-temporal or 4D location on the Earth these tools would organize knowledge based on location within the tissues or embryos. Developmental processes are highly context-specific, but the complexity of the 4D environment in which they unfold is a barrier to assembling an understanding of any particular process from diverse sources of information. In the same way that GIS aids the understanding and use of geo-located large data sets, software can, with a proper frame of reference, allow large biological data sets to be understood spatially. Intuitive tools are needed to navigate the spatial structure of complex tissue, collate large data sets and existing knowledge with this spatial structure and help users derive hypotheses about developmental mechanisms. RESULTS: Toward this goal we have developed WormGUIDES, a mobile application that presents a 4D developmental atlas for Caenorhabditis elegans. The WormGUIDES mobile app enables users to navigate a 3D model depicting the nuclear positions of all cells in the developing embryo. The identity of each cell can be queried with a tap, and community databases searched for available information about that cell. Information about ancestry, fate and gene expression can be used to label cells and craft customized visualizations that highlight cells as potential players in an event of interest. Scenes are easily saved, shared and published to other WormGUIDES users. The mobile app is available for Android and iOS platforms. CONCLUSION: WormGUIDES provides an important tool for examining developmental processes and developing mechanistic hypotheses about their control. Critically, it provides the typical end user with an intuitive interface for developing and sharing custom visualizations of developmental processes. Equally important, because users can select cells based on their position and search for information about them, the app also serves as a spatially organized index into the large body of knowledge available to the C. elegans community online. Moreover, the app can be used to create and publish the result of exploration: interactive content that brings other researchers and students directly to the spatio-temporal point of insight. Ultimately the app will incorporate a detailed time lapse record of cell shape, beginning with neurons. This will add the key ability to navigate and understand the developmental events that result in the coordinated and precise emergence of anatomy, particularly the wiring of the nervous system.


Asunto(s)
Caenorhabditis elegans/crecimiento & desarrollo , Sistema Nervioso/citología , Análisis de la Célula Individual/métodos , Programas Informáticos , Interfaz Usuario-Computador , Animales , Bases de Datos Factuales
3.
Cytometry A ; 85(7): 642-9, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24939722

RESUMEN

Primary patient samples are the gold standard for molecular investigations of tumor biology yet are difficult to acquire, heterogeneous in nature and variable in size. Patient-derived xenografts (PDXs) comprised of primary tumor tissue cultured in host organisms such as nude mice permit the propagation of human tumor samples in an in vivo environment and closely mimic the phenotype and gene expression profile of the primary tumor. Although PDX models reduce the cost and complexity of acquiring sample tissue and permit repeated sampling of the primary tumor, these samples are typically contaminated by immune, blood, and vascular tissues from the host organism while also being limited in size. For very small tissue samples (on the order of 10(3) cells) purification by fluorescence-activated cell sorting (FACS) is not feasible while magnetic activated cell sorting (MACS) of small samples results in very low purity, low yield, and poor viability. We developed a platform for imaging cytometry integrated with micropallet array technology to perform automated cell sorting on very small samples obtained from PDX models of pancreatic and colorectal cancer using antibody staining of EpCAM (CD326) as a selection criteria. These data demonstrate the ability to automate and efficiently separate samples with very low number of cells.


Asunto(s)
Antígenos de Neoplasias/análisis , Moléculas de Adhesión Celular/análisis , Neoplasias Colorrectales/patología , Citometría de Flujo/métodos , Xenoinjertos/citología , Neoplasias Pancreáticas/patología , Animales , Procesamiento Automatizado de Datos , Molécula de Adhesión Celular Epitelial , Humanos , Procesamiento de Imagen Asistido por Computador , Ratones , Ratones Desnudos , Coloración y Etiquetado , Células Tumorales Cultivadas
4.
bioRxiv ; 2024 Jan 22.
Artículo en Inglés | MEDLINE | ID: mdl-38328064

RESUMEN

Here we describe embGAN, a deep learning pipeline that addresses the challenge of automated cell detection and tracking in label-free 3D time lapse imaging. embGAN requires no manual data annotation for training, learns robust detections that exhibits a high degree of scale invariance and generalizes well to images acquired in multiple labs on multiple instruments.

5.
Anal Chem ; 85(10): 4991-7, 2013 May 21.
Artículo en Inglés | MEDLINE | ID: mdl-23590517

RESUMEN

Microfluidic systems show great promise for single-cell analysis; however, as these technologies mature, their utility must be validated by studies of biologically relevant processes. An important biomedical application of these systems is characterization of tumor cell heterogeneity. In this work, we used a robust microfluidic platform to explore the heterogeneity of enzyme activity in single cells treated with a chemotherapeutic drug. Using chemical cytometry, we measured peptide degradation in the U937 acute myeloid leukemia (AML) cell line in the presence and absence of the aminopeptidase inhibitor Tosedostat (CHR-2797). The analysis of 99 untreated cells revealed rapid and consistent degradation of the peptide reporter within 20 min of loading. Results from drug-treated cells showed inhibited, but ongoing degradation of the reporter. Because the device operates at an average sustained throughput of 37 ± 7 cells/h, we were able to sample cells over the course of this time-dependent degradation. In data from 498 individual drug-treated cells, we found a linear dependence of degradation rate on amount of substrate loaded superimposed upon substantial heterogeneity in peptide processing in response to inhibitor treatment. Importantly, these data demonstrated the potential of microfluidic systems to sample biologically relevant analytes and time-dependent processes in large numbers of single cells.


Asunto(s)
Antineoplásicos/farmacología , Leucemia Mieloide Aguda/patología , Técnicas Analíticas Microfluídicas/métodos , Péptidos/metabolismo , Proteolisis/efectos de los fármacos , Secuencia de Aminoácidos , Línea Celular Tumoral , Humanos , Cinética , Péptidos/química
6.
bioRxiv ; 2023 May 24.
Artículo en Inglés | MEDLINE | ID: mdl-37292606

RESUMEN

High throughput experimental approaches are increasingly allowing for the quantitative description of cellular and organismal phenotypes. Distilling these large volumes of complex data into meaningful measures that can drive biological insight remains a central challenge. In the quantitative study of development, for instance, one can resolve phenotypic measures for single cells onto their lineage history, enabling joint consideration of heritable signals and cell fate decisions. Most attempts to analyze this type of data, however, discard much of the information content contained within lineage trees. In this work we introduce a generalized metric, which we term the branch distance, that allows us to compare any two embryos based on phenotypic measurements in individual cells. This approach aligns those phenotypic measurements to the underlying lineage tree, providing a flexible and intuitive framework for quantitative comparisons between, for instance, Wild-Type (WT) and mutant developmental programs. We apply this novel metric to data on cell-cycle timing from over 1300 WT and RNAi-treated Caenorhabditis elegans embryos. Our new metric revealed surprising heterogeneity within this data set, including subtle batch effects in WT embryos and dramatic variability in RNAi-induced developmental phenotypes, all of which had been missed in previous analyses. Further investigation of these results suggests a novel, quantitative link between pathways that govern cell fate decisions and pathways that pattern cell cycle timing in the early embryo. Our work demonstrates that the branch distance we propose, and similar metrics like it, have the potential to revolutionize our quantitative understanding of organismal phenotype.

7.
bioRxiv ; 2023 Apr 18.
Artículo en Inglés | MEDLINE | ID: mdl-37131635

RESUMEN

Immune cells live intensely physical lifestyles characterized by structural plasticity, mechanosensitivity, and force exertion. Whether specific immune functions require stereotyped patterns of mechanical output, however, is largely unknown. To address this question, we used super-resolution traction force microscopy to compare cytotoxic T cell immune synapses with contacts formed by other T cell subsets and macrophages. T cell synapses were globally and locally protrusive, which was fundamentally different from the coupled pinching and pulling of macrophage phagocytosis. By spectrally decomposing the force exertion patterns of each cell type, we associated cytotoxicity with compressive strength, local protrusiveness, and the induction of complex, asymmetric interfacial topographies. These features were further validated as cytotoxic drivers by genetic disruption of cytoskeletal regulators, direct imaging of synaptic secretory events, and in silico analysis of interfacial distortion. We conclude that T cell-mediated killing and, by implication, other effector responses are supported by specialized patterns of efferent force.

8.
Anal Bioanal Chem ; 402(3): 1065-72, 2012 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-22086401

RESUMEN

There is a need for a technology that can be incorporated into routine laboratory procedures to obtain a continuous, quantitative, fluorescence-based measurement of the dynamic behaviors of numerous individual living cells in parallel, while allowing other manipulations, such as staining, rinsing, and even retrieval of targeted cells. Here, we report a simple, low-cost microarray platform that can trap cells for dynamic single-cell analysis of mammalian cells. The elasticity of polydimethylsiloxane (PDMS) was utilized to trap tens of thousands of cells on an array. The PDMS microwell array was stretched by a tube through which cells were loaded on the array. Cells were trapped on the array by removal of the tube and relaxation of the PDMS. Once that was accomplished, the cells remained trapped on the array without continuous application of an external force and permitted subsequent manipulations, such as staining, rinsing, imaging, and even isolation of targeted cells. We demonstrate the utility of this platform by multicolor analysis of trapped cells and monitoring in individual cells real-time calcium flux after exposure to the calcium ionophore ionomycin. Additionally, a proof of concept for target cell isolation was demonstrated by using a microneedle to locally deform the PDMS membrane in order to retrieve a particular cell from the array.


Asunto(s)
Análisis de la Célula Individual/instrumentación , Análisis de Matrices Tisulares/instrumentación , Animales , Calcio/metabolismo , Línea Celular , Dimetilpolisiloxanos/química , Diseño de Equipo , Ionomicina/metabolismo , Análisis de la Célula Individual/economía , Análisis de Matrices Tisulares/economía
9.
Genetics ; 221(4)2022 07 30.
Artículo en Inglés | MEDLINE | ID: mdl-35766819

RESUMEN

Light microscopes are the cell and developmental biologists' "best friend," providing a means to see structures and follow dynamics from the protein to the organism level. A huge advantage of Caenorhabditis elegans as a model organism is its transparency, which coupled with its small size means that nearly every biological process can be observed and measured with the appropriate probe and light microscope. Continuous improvement in microscope technologies along with novel genome editing techniques to create transgenic probes have facilitated the development and implementation of a dizzying array of methods for imaging worm embryos, larvae, and adults. In this review, we provide an overview of the molecular and cellular processes that can be visualized in living worms using light microscopy. A partial inventory of fluorescent probes and techniques successfully used in worms to image the dynamics of cells, organelles, DNA, and protein localization and activity is followed by a practical guide to choosing between various imaging modalities, including widefield, confocal, lightsheet, and structured illumination microscopy. Finally, we discuss the available tools and approaches, including machine learning, for quantitative image analysis tasks, such as colocalization, segmentation, object tracking, and lineage tracing. Hopefully, this review will inspire worm researchers who have not yet imaged their worms to begin, and push those who are imaging to go faster, finer, and longer.


Asunto(s)
Fenómenos Biológicos , Caenorhabditis elegans , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/genética , Colorantes Fluorescentes/química , Microscopía/métodos
10.
Nat Commun ; 11(1): 20, 2020 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-31911639

RESUMEN

Force exertion is an integral part of cellular behavior. Traction force microscopy (TFM) has been instrumental for studying such forces, providing spatial force measurements at subcellular resolution. However, the applications of classical TFM are restricted by the typical planar geometry. Here, we develop a particle-based force sensing strategy for studying cellular interactions. We establish a straightforward batch approach for synthesizing uniform, deformable and tuneable hydrogel particles, which can also be easily derivatized. The 3D shape of such particles can be resolved with superresolution (<50 nm) accuracy using conventional confocal microscopy. We introduce a reference-free computational method allowing inference of traction forces with high sensitivity directly from the particle shape. We illustrate the potential of this approach by revealing subcellular force patterns throughout phagocytic engulfment and force dynamics in the cytotoxic T-cell immunological synapse. This strategy can readily be adapted for studying cellular forces in a wide range of applications.


Asunto(s)
Comunicación Celular , Linfocitos T Citotóxicos/química , Linfocitos T Citotóxicos/inmunología , Animales , Línea Celular , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Microscopía de Fuerza Atómica , Fagocitosis , Linfocitos T Citotóxicos/citología , Tracción
12.
Nat Cell Biol ; 20(4): 393-399, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29556089

RESUMEN

Phagocytosis of dying cells is critical in development and immunity1-3. Although proteins for recognition and engulfment of cellular debris following cell death are known4,5, proteins that directly mediate phagosome sealing are uncharacterized. Furthermore, whether all phagocytic targets are cleared using the same machinery is unclear. Degeneration of morphologically complex cells, such as neurons, glia and melanocytes, produces phagocytic targets of various shapes and sizes located in different microenvironments6,7. Thus, such cells offer unique settings to explore engulfment programme mechanisms and specificity. Here, we report that dismantling and clearance of a morphologically complex Caenorhabditis elegans epithelial cell requires separate cell soma, proximal and distal process programmes. Similar compartment-specific events govern the elimination of a C. elegans neuron. Although canonical engulfment proteins drive cell soma clearance, these are not required for process removal. We find that EFF-1, a protein previously implicated in cell-cell fusion 8 , specifically promotes distal process phagocytosis. EFF-1 localizes to phagocyte pseudopod tips and acts exoplasmically to drive phagosome sealing. eff-1 mutations result in phagocytosis arrest with unsealed phagosomes. Our studies suggest universal mechanisms for dismantling morphologically complex cells and uncover a phagosome-sealing component that promotes cell process clearance.


Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Glicoproteínas de Membrana/metabolismo , Neuronas/metabolismo , Fagocitos/metabolismo , Fagocitosis , Fagosomas/metabolismo , Seudópodos/metabolismo , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/genética , Caenorhabditis elegans/ultraestructura , Proteínas de Caenorhabditis elegans/genética , Muerte Celular , Glicoproteínas de Membrana/genética , Mutación , Neuronas/patología , Fagocitos/ultraestructura , Fagosomas/genética , Fagosomas/ultraestructura , Seudópodos/genética , Seudópodos/ultraestructura , Transducción de Señal
13.
Dev Cell ; 43(4): 530-540.e4, 2017 11 20.
Artículo en Inglés | MEDLINE | ID: mdl-29161596

RESUMEN

Single-cell measurements have broadened our understanding of heterogeneity in biology, yet have been limited to mostly observational studies of normal or globally perturbed systems. Typically, perturbations are utilized in an open-ended approach wherein an endpoint is assayed during or after the biological event of interest. Here we describe ShootingStar, a platform for perturbation analysis in vivo, which combines live imaging, real-time image analysis, and automated optical perturbations. ShootingStar builds a quantitative record of the state of the sample being analyzed, which is used to automate the identification of target cells for perturbation, as well as to validate the impacts of the perturbation. We used ShootingStar to dissect the cellular basis of development, morphogenesis, and polarity in the lateral line of Danio rerio and the embryo of Caenorhabditis elegans. ShootingStar can be extended to diverse optical manipulations and enables more robust and informative single-cell perturbations in complex tissues.


Asunto(s)
Comunicación Celular/fisiología , Ciclo Celular/fisiología , Embrión no Mamífero/metabolismo , Desarrollo Embrionario/fisiología , Animales , Caenorhabditis elegans/embriología , Pez Cebra/embriología
14.
Dev Cell ; 41(2): 195-203.e3, 2017 04 24.
Artículo en Inglés | MEDLINE | ID: mdl-28441532

RESUMEN

Formation and resolution of multicellular rosettes can drive convergent extension (CE) type cell rearrangements during tissue morphogenesis. Rosette dynamics are regulated by both planar cell polarity (PCP)-dependent and -independent pathways. Here we show that CE is involved in ventral nerve cord (VNC) assembly in Caenorhabditis elegans. We show that a VANG-1/Van Gogh and PRKL-1/Prickle containing PCP pathway and a Slit-independent SAX-3/Robo pathway cooperate to regulate, via rosette intermediaries, the intercalation of post-mitotic neuronal cell bodies during VNC formation. We show that VANG-1 and SAX-3 are localized to contracting edges and rosette foci and act to specify edge contraction during rosette formation and to mediate timely rosette resolution. Simultaneous loss of both pathways severely curtails CE resulting in a shortened, anteriorly displaced distribution of VNC neurons at hatching. Our results establish rosette-based CE as an evolutionarily conserved mechanism of nerve cord morphogenesis and reveal a role for SAX-3/Robo in this process.


Asunto(s)
Polaridad Celular/fisiología , Morfogénesis/fisiología , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Receptores Inmunológicos/metabolismo , Transducción de Señal , Animales , Animales Modificados Genéticamente , Axones/metabolismo , Caenorhabditis elegans , Movimiento Celular/fisiología , Proteínas Roundabout
15.
Dev Cell ; 34(5): 592-607, 2015 Sep 14.
Artículo en Inglés | MEDLINE | ID: mdl-26321128

RESUMEN

Elucidating the mechanism of cell lineage differentiation is critical for our understanding of development and fate manipulation. Here we combined systematic perturbation and direct lineaging to map the regulatory landscape of lineage differentiation in early C. elegans embryogenesis. High-dimensional phenotypic analysis of 204 essential genes in 1,368 embryos revealed that cell lineage differentiation follows a canalized landscape with barriers shaped by lineage distance and genetic robustness. We assigned function to 201 genes in regulating lineage differentiation, including 175 switches of binary fate choices. We generated a multiscale model that connects gene networks and cells to the experimentally mapped landscape. Simulations showed that the landscape topology determines the propensity of differentiation and regulatory complexity. Furthermore, the model allowed us to identify the chromatin assembly complex CAF-1 as a context-specific repressor of Notch signaling. Our study presents a systematic survey of the regulatory landscape of lineage differentiation of a metazoan embryo.


Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriología , Diferenciación Celular/genética , Linaje de la Célula/genética , Regulación del Desarrollo de la Expresión Génica/genética , Redes Reguladoras de Genes/genética , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Desarrollo Embrionario/genética , Modelos Biológicos , Transducción de Señal/genética
16.
Integr Biol (Camb) ; 6(7): 673-84, 2014 Jul 24.
Artículo en Inglés | MEDLINE | ID: mdl-24871928

RESUMEN

Wnt/ß-catenin signaling is of significant interest due to the roles it plays in regulating development, tissue regeneration and disease. Transcriptional reporters have been widely employed to study Wnt/ß-catenin signal transduction in live cells and whole organisms and have been applied to understanding embryonic development, exploring oncogenesis and developing therapeutics. Polyclonal heterogeneity in reporter cell lines has historically been seen as a challenge to be overcome in the development of novel cell lines and reporter-based assays, and monoclonal reporter cell lines are commonly employed to reduce this variability. A375 cell lines infected with a reporter for Wnt/ß-catenin signaling were screened over short (<6) and long (>25) generational timescales. To characterize phenotypic divergence over these time-scales, a microfabricated cell array-based screen was developed enabling characterization of 1119 clonal colonies in parallel. This screen revealed phenotypic divergence after <6 generations at a similar scale to that observed in monoclonal cell lines cultured for >25 generations. Not only were reporter dynamics observed to diverge widely, but monoclonal cell lines were observed with seemingly opposite signaling phenotypes. Additionally, these observations revealed a generational-dependent trend in Wnt signaling in A375 cells that provides insight into the pathway's mechanisms of positive feedback and self-inhibition.


Asunto(s)
Proteínas Wnt/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismo , Línea Celular Tumoral , Células Clonales , Humanos , Procesamiento de Imagen Asistido por Computador , Cinética , Proteínas Luminiscentes/química , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente/métodos , Proteína Fluorescente Roja
17.
Lab Chip ; 13(23): 4625-34, 2013 Dec 07.
Artículo en Inglés | MEDLINE | ID: mdl-24113577

RESUMEN

Crypts are the basic structural and functional units of colonic epithelium and can be isolated from the colon and cultured in vitro into multi-cell spheroids termed "colonoids". Both crypts and colonoids are ideal building blocks for construction of an in vitro tissue model of the colon. Here we proposed and tested a microengineered platform for capture and in vitro 3D culture of colonic crypts and colonoids. An integrated platform was fabricated from polydimethylsiloxane which contained two fluidic layers separated by an array of cylindrical microwells (150 µm diameter, 150 µm depth) with perforated bottoms (30 µm opening, 10 µm depth) termed "microstrainers". As fluid moved through the array, crypts or colonoids were retained in the microstrainers with a >90% array-filling efficiency. Matrigel as an extracellular matrix was then applied to the microstrainers to generate isolated Matrigel pockets encapsulating the crypts or colonoids. After supplying the essential growth factors, epidermal growth factor, Wnt-3A, R-spondin 2 and noggin, 63 ± 13% of the crypts and 77 ± 8% of the colonoids cultured in the microstrainers over a 48-72 h period formed viable 3D colonoids. Thus colonoid growth on the array was similar to that under standard culture conditions (78 ± 5%). Additionally the colonoids displayed the same morphology and similar numbers of stem and progenitor cells as those under standard culture conditions. Immunofluorescence staining confirmed that the differentiated cell-types of the colon, goblet cells, enteroendocrine cells and absorptive enterocytes, formed on the array. To demonstrating the utility of the array in tracking the colonoid fate, quantitative fluorescence analysis was performed on the arrayed colonoids exposed to reagents such as Wnt-3A and the γ-secretase inhibitor LY-411575. The successful formation of viable, multi-cell type colonic tissue on the microengineered platform represents a first step in the building of a "colon-on-a-chip" with the goal of producing the physiologic structure and organ-level function of the colon for controlled experiments.


Asunto(s)
Técnicas de Cultivo de Célula/instrumentación , Colágeno/química , Colon/citología , Mucosa Intestinal/citología , Laminina/química , Proteoglicanos/química , Análisis de Matrices Tisulares/instrumentación , Alanina/análogos & derivados , Alanina/química , Alanina/farmacología , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Azepinas/química , Azepinas/farmacología , Células Cultivadas , Combinación de Medicamentos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Ratones , Ratones Transgénicos , Imagen de Lapso de Tiempo , Proteína Wnt3A/antagonistas & inhibidores , Proteína Wnt3A/metabolismo
18.
Langmuir ; 23(11): 6053-62, 2007 May 22.
Artículo en Inglés | MEDLINE | ID: mdl-17465581

RESUMEN

The adsorption characteristics of three proteins [bovine serum albumin (BSA), myoglobin (Mb), and cytochrome c (CytC)] onto self-assembled monolayers of mercaptoundecanoic acid (MUA) on both gold nanoparticles (AuNP) and gold surfaces (Au) are described. The combination of quartz crystal microbalance measurements with dissipation (QCM-D) and pH titrations of the zeta-potential provide information on layer structure, surface coverage, and potential. All three proteins formed adsorption layers consisting of an irreversibly adsorbed fraction and a reversibly adsorbed fraction. BSA showed the highest affinity for the MUA/Au, forming an irreversibly adsorbed rigid monolayer with a side-down orientation and packing close to that expected in the jamming limit. In addition, BSA showed a large change in the adsorbed mass due to reversibly bound protein. The data indicate that the irreversibly adsorbed fraction of CytC is a monolayer structure, whereas the irreversibly adsorbed Mb is present in form of a bilayer. The observation of stable BSA complexes on MUA/AuNPs at the isoelectric point by zeta-potential measurements demonstrates that BSA can sterically stabilize MUA/AuNP. On the other hand, MUA/AuNP coated with either Mb or CytC formed a reversible flocculated state at the isoelectric point. The colloidal stability differences may be correlated with weaker binding in the reversibly bound overlayer in the case of Mb and CytC as compared to BSA.


Asunto(s)
Ácidos Grasos/química , Oro/química , Nanopartículas del Metal/química , Proteínas/química , Adsorción , Animales , Técnicas Biosensibles , Bovinos , Materiales Biocompatibles Revestidos/química , Citocromos c/química , Electroquímica , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Mioglobina/química , Cuarzo , Albúmina Sérica Bovina/química , Propiedades de Superficie
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