RESUMEN
The aim of this work was to isolate and characterize bacteriocins produced by 2 Lactobacillus fermentum strains isolated from artisanal Mexican Cocido cheese. Fractions (F ≤3 kDa) obtained from cell-free supernatants of Lb. fermentum strains J23 and J32 were further fractionated by reversed-phase HPLC on a C18 column. Antimicrobial activities of F ≤3 kDa and bacteriocin-containing fractions (BCF), obtained from fractionation of F ≤3 kDa against 4 indicator microorganisms, were determined by the disk diffusion method and growth inhibition in milk. Subsequently, isolated BCF were analyzed by reversed-phase HPLC tandem mass spectrometry. Results showed that BCF presented antimicrobial activity against the 4 indicator microorganisms tested. For J23, one of the fractions (F3) presented the highest activity against Escherichia coli and was also inhibitory against Staphylococcus aureus, Listeria innocua, Salmonella Typhimurium, and Salmonella Choleraesuis. Similarly, fractions F3 and F4 produced by J32 presented antimicrobial activity against all indicator microorganisms. Furthermore, generation time and growth rate showed that F3 from J23 presented significantly higher antimicrobial activity against the 4 indicator microorganisms (2 gram-positive and 2 gram-negative) when inoculated in milk compared with F3 from J32. Interestingly, this fraction presented a broader antimicrobial spectrum in milk than nisin (control). Reversed-phase HPLC tandem mass spectrometry analysis revealed the presence of several peptides in BCF; however, F3 from J23 that was the most active fraction of all presented only 1 bacteriocin. The chemical characterization of this bacteriocin suggested that it was a novel peptide with 10 hydrophobic AA residues in its sequence and a molecular weight of 2,056 Da. This bacteriocin and its producing strain, J23, may find application as a biopreservative against these indicator microorganisms in dairy products.
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Bacteriocinas , Limosilactobacillus fermentum , Animales , Antibacterianos/farmacología , Microbiología de Alimentos , Lactobacillus , Listeria , LecheAsunto(s)
COVID-19 , Traqueostomía , Humanos , Respiración Artificial , SARS-CoV-2 , Procedimientos Quirúrgicos VascularesRESUMEN
The aim of the present study was to characterize the aroma and volatile profiles of milk fermented by wild Lactococcus lactis NRRL B-50571 (FM-571) and NRRL B-50572 (FM-572) and co-fermented with both strains (co-FM). Milks fermented by these strains have been reported to have an antihypertensive effect, yet their sensory characteristics, which are of great importance for consumer acceptance of functional foods, have not been studied. In the study, volatiles were determined using solid-phase microextraction gas chromatography mass spectrometry (SPME-GC-MS) and aroma was determined by quantitative descriptive sensory analysis (QDA). Volatile compounds identified in FM-571, FM-572, and co-FM were mainly acids, alcohols, aldehydes, and ketones. FM-571 showed higher total relative volatile abundance than FM-572 or co-FM. Furthermore, the concentrations of specific amino acids (aa) were lower in FM-571 and co-FM than in FM-572. Thus, these results suggested that FM-571 or co-FM are more efficient in transforming specific aa into the corresponding volatiles than FM-572. Indeed, several alcohols and aldehydes, associated with the catabolism of these aa, were found in FM-571 and co-FM, but not in FM-572. Additionally, QDA showed that FM-571 and co-FM presented higher yeasty and cheesy aroma descriptors than FM-572. Also, total aroma intensity scores for FM-571 were higher than those for co-FM or FM-572. Thus, results suggested that the combination of these two specific wild L. lactis strains may complement amino acid catabolic routes that resulted in the enhancement or attenuation of aroma production of single strains, presenting new possibilities for the preparation of custom-made starter cultures.
RESUMEN
Toxicity is an important factor in failed drug development, and its efficient identification and prediction is a major challenge in drug discovery. We have explored the potential of microscopy images of fluorescently labeled nuclei for the prediction of toxicity based on nucleus pattern recognition. Deep learning algorithms obtain abstract representations of images through an automated process, allowing them to efficiently classify complex patterns, and have become the state-of-the art in machine learning for computer vision. Here, deep convolutional neural networks (CNN) were trained to predict toxicity from images of DAPI-stained cells pre-treated with a set of drugs with differing toxicity mechanisms. Different cropping strategies were used for training CNN models, the nuclei-cropping-based Tox_CNN model outperformed other models classifying cells according to health status. Tox_CNN allowed automated extraction of feature maps that clustered compounds according to mechanism of action. Moreover, fully automated region-based CNNs (RCNN) were implemented to detect and classify nuclei, providing per-cell toxicity prediction from raw screening images. We validated both Tox_(R)CNN models for detection of pre-lethal toxicity from nuclei images, which proved to be more sensitive and have broader specificity than established toxicity readouts. These models predicted toxicity of drugs with mechanisms of action other than those they had been trained for and were successfully transferred to other cell assays. The Tox_(R)CNN models thus provide robust, sensitive, and cost-effective tools for in vitro screening of drug-induced toxicity. These models can be adopted for compound prioritization in drug screening campaigns, and could thereby increase the efficiency of drug discovery.
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Núcleo Celular/efectos de los fármacos , Aprendizaje Profundo , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Algoritmos , Automatización , Colorantes Fluorescentes/química , Interpretación de Imagen Asistida por Computador/métodos , Indoles/química , Redes Neurales de la ComputaciónRESUMEN
Advances in flow cytometry (FCM) increasingly demand adoption of computational analysis tools to tackle the ever-growing data dimensionality. In this study, we tested different data input modes to evaluate how cytometry acquisition configuration and data compensation procedures affect the performance of unsupervised phenotyping tools. An analysis workflow was set up and tested for the detection of changes in reference bead subsets and in a rare subpopulation of murine lymph node CD103+ dendritic cells acquired by conventional or spectral cytometry. Raw spectral data or pseudospectral data acquired with the full set of available detectors by conventional cytometry consistently outperformed datasets acquired and compensated according to FCM standards. Our results thus challenge the paradigm of one-fluorochrome/one-parameter acquisition in FCM for unsupervised cluster-based analysis. Instead, we propose to configure instrument acquisition to use all available fluorescence detectors and to avoid integration and compensation procedures, thereby using raw spectral or pseudospectral data for improved automated phenotypic analysis.
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Células Dendríticas/citología , Animales , Antígenos CD/metabolismo , Análisis por Conglomerados , Células Dendríticas/metabolismo , Citometría de Flujo/métodos , Cadenas alfa de Integrinas/metabolismo , Ganglios Linfáticos/citología , Ganglios Linfáticos/metabolismo , Ratones , Ratones Endogámicos C57BL , FenotipoRESUMEN
Caveolin-1 (CAV1) is over-expressed in prostate cancer (PCa) and is associated with adverse prognosis, but the molecular mechanisms linking CAV1 expression to disease progression are poorly understood. Extensive gene expression correlation analysis, quantitative multiplex imaging of clinical samples, and analysis of the CAV1-dependent transcriptome, supported that CAV1 re-programmes TGFß signalling from tumour suppressive to oncogenic (i.e. induction of SLUG, PAI-1 and suppression of CDH1, DSP, CDKN1A). Supporting such a role, CAV1 knockdown led to growth arrest and inhibition of cell invasion in prostate cancer cell lines. Rationalized RNAi screening and high-content microscopy in search for CAV1 upstream regulators revealed integrin beta1 (ITGB1) and integrin associated proteins as CAV1 regulators. Our work suggests TGFß signalling and beta1 integrins as potential therapeutic targets in PCa over-expressing CAV1, and contributes to better understand the paradoxical dual role of TGFß in tumour biology.
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Caveolina 1/metabolismo , Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Neoplasias de la Próstata/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Línea Celular Tumoral , Humanos , Masculino , Oncogenes , Fenotipo , Neoplasias de la Próstata/genética , Transducción de Señal , Regulación hacia ArribaRESUMEN
Embryonic stem cells (ESCs) can be established as permanent cell lines, and their potential to differentiate into adult tissues has led to widespread use for studying the mechanisms and dynamics of stem cell differentiation and exploring strategies for tissue repair. Imaging live ESCs during development is now feasible due to advances in optical imaging and engineering of genetically encoded fluorescent reporters; however, a major limitation is the low spatio-temporal resolution of long-term 3-D imaging required for generational and neighboring reconstructions. Here, we present the ESC-Track (ESC-T) workflow, which includes an automated cell and nuclear segmentation and tracking tool for 4-D (3-D + time) confocal image data sets as well as a manual editing tool for visual inspection and error correction. ESC-T automatically identifies cell divisions and membrane contacts for lineage tree and neighborhood reconstruction and computes quantitative features from individual cell entities, enabling analysis of fluorescence signal dynamics and tracking of cell morphology and motion. We use ESC-T to examine Myc intensity fluctuations in the context of mouse ESC (mESC) lineage and neighborhood relationships. ESC-T is a powerful tool for evaluation of the genealogical and microenvironmental cues that maintain ESC fitness.
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Linaje de la Célula/fisiología , Rastreo Celular/métodos , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/fisiología , Imagenología Tridimensional/métodos , Microscopía Fluorescente/métodos , Reconocimiento de Normas Patrones Automatizadas/métodos , Algoritmos , Diferenciación Celular/fisiología , Células Cultivadas , Humanos , Aprendizaje Automático , Microscopía Confocal/métodos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Programas Informáticos , Flujo de TrabajoRESUMEN
Although the recent Zika virus (ZIKV) epidemic in the Americas and its link to birth defects have attracted a great deal of attention, much remains unknown about ZIKV disease epidemiology and ZIKV evolution, in part owing to a lack of genomic data. Here we address this gap in knowledge by using multiple sequencing approaches to generate 110 ZIKV genomes from clinical and mosquito samples from 10 countries and territories, greatly expanding the observed viral genetic diversity from this outbreak. We analysed the timing and patterns of introductions into distinct geographic regions; our phylogenetic evidence suggests rapid expansion of the outbreak in Brazil and multiple introductions of outbreak strains into Puerto Rico, Honduras, Colombia, other Caribbean islands, and the continental United States. We find that ZIKV circulated undetected in multiple regions for many months before the first locally transmitted cases were confirmed, highlighting the importance of surveillance of viral infections. We identify mutations with possible functional implications for ZIKV biology and pathogenesis, as well as those that might be relevant to the effectiveness of diagnostic tests.
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Filogenia , Infección por el Virus Zika/transmisión , Infección por el Virus Zika/virología , Virus Zika/genética , Virus Zika/aislamiento & purificación , Animales , Brasil/epidemiología , Colombia/epidemiología , Culicidae/virología , Brotes de Enfermedades/estadística & datos numéricos , Genoma Viral/genética , Mapeo Geográfico , Honduras/epidemiología , Humanos , Metagenoma/genética , Epidemiología Molecular , Mosquitos Vectores/virología , Mutación , Vigilancia en Salud Pública , Puerto Rico/epidemiología , Estados Unidos/epidemiología , Virus Zika/clasificación , Virus Zika/patogenicidad , Infección por el Virus Zika/diagnóstico , Infección por el Virus Zika/epidemiologíaRESUMEN
The common and persistent failures to translate promising preclinical drug candidates into clinical success highlight the limited effectiveness of disease models currently used in drug discovery. An apparent reluctance to explore and adopt alternative cell- and tissue-based model systems, coupled with a detachment from clinical practice during assay validation, contributes to ineffective translational research. To help address these issues and stimulate debate, here we propose a set of principles to facilitate the definition and development of disease-relevant assays, and we discuss new opportunities for exploiting the latest advances in cell-based assay technologies in drug discovery, including induced pluripotent stem cells, three-dimensional (3D) co-culture and organ-on-a-chip systems, complemented by advances in single-cell imaging and gene editing technologies. Funding to support precompetitive, multidisciplinary collaborations to develop novel preclinical models and cell-based screening technologies could have a key role in improving their clinical relevance, and ultimately increase clinical success rates.
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Técnicas de Cultivo de Célula/métodos , Descubrimiento de Drogas/métodos , Modelos Biológicos , Animales , Línea Celular Transformada , Evaluación Preclínica de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/fisiología , Preparaciones Farmacéuticas/administración & dosificaciónRESUMEN
Rab8 is a small Ras-related GTPase that regulates polarized membrane transport to the plasma membrane. Here, we developed a high-content analysis (HCA) tool to dissect Rab8-mediated actin and focal adhesion reorganization that revealed that Rab8 activation significantly induced Rac1 and Tiam1 to mediate cortical actin polymerization and RhoA-dependent stress fibre disassembly. Rab8 activation increased Rac1 activity, whereas its depletion activated RhoA, which led to reorganization of the actin cytoskeleton. Rab8 was also associated with focal adhesions, promoting their disassembly in a microtubule-dependent manner. This Rab8 effect involved calpain, MT1-MMP (also known as MMP14) and Rho GTPases. Moreover, we demonstrate the role of Rab8 in the cell migration process. Indeed, Rab8 is required for EGF-induced cell polarization and chemotaxis, as well as for the directional persistency of intrinsic cell motility. These data reveal that Rab8 drives cell motility by mechanisms both dependent and independent of Rho GTPases, thereby regulating the establishment of cell polarity, turnover of focal adhesions and actin cytoskeleton rearrangements, thus determining the directionality of cell migration.
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Calpaína/metabolismo , Adhesiones Focales/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Metaloproteinasa 14 de la Matriz/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Citoesqueleto de Actina/metabolismo , Movimiento Celular , Polaridad Celular , Células HeLa , Humanos , ARN Interferente Pequeño/genética , Fibras de Estrés/metabolismo , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T , Proteínas de Unión al GTP rab/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
High-content screening (HCS) allows the exploration of complex cellular phenotypes by automated microscopy and is increasingly being adopted for small interfering RNA genomic screening and phenotypic drug discovery. We introduce a series of cell-based evaluation metrics that have been implemented and validated in a mono-parametric HCS for regulators of the membrane trafficking protein caveolin 1 (CAV1) and have also proved useful for the development of a multiparametric phenotypic HCS for regulators of cytoskeletal reorganization. Imaging metrics evaluate imaging quality such as staining and focus, whereas cell biology metrics are fuzzy logic-based evaluators describing complex biological parameters such as sparseness, confluency, and spreading. The evaluation metrics were implemented in a data-mining pipeline, which first filters out cells that do not pass a quality criterion based on imaging metrics and then uses cell biology metrics to stratify cell samples to allow further analysis of homogeneous cell populations. Use of these metrics significantly improved the robustness of the monoparametric assay tested, as revealed by an increase in Z' factor, Kolmogorov-Smirnov distance, and strict standard mean difference. Cell biology evaluation metrics were also implemented in a novel supervised learning classification method that combines them with phenotypic features in a statistical model that exceeded conventional classification methods, thus improving multiparametric phenotypic assay sensitivity.
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Evaluación Preclínica de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Línea Celular Tumoral , Lógica Difusa , Humanos , Microscopía Confocal , Microscopía Fluorescente , Curva ROC , Reproducibilidad de los ResultadosRESUMEN
The biology of caveolin-1 (Cav1)/caveolae is intimately linked to actin dynamics and adhesion receptors. Caveolar domains are organized in hierarchical levels of complexity from curved or flattened caveolae to large, higher-order caveolar rosettes. We report that stress fibers controlled by Abl kinases and mDia1 determine the level of caveolar domain organization, which conditions the subsequent inward trafficking of caveolar domains induced upon loss of cell adhesion from the extracellular matrix. Abl-deficient cells have fewer stress fibers, a smaller pool of stress-fiber co-aligned Cav1 and increased clustering of Cav1/caveolae at the cell surface. Defective caveolar linkage to stress fibers prevents the formation of big caveolar rosettes upon loss of cell adhesion, correlating with a lack of inward trafficking. Live imaging of stress fibers and Cav1 showed that the actin-linked Cav1 pool loses its spatial organization in the absence of actin polymerization and is dragged and clustered by depolymerizing filaments. We identified mDia1 as the actin polymerization regulator downstream of Abl kinases that controls the stress-fiber-linked Cav1 pool. mDia1 knockdown results in Cav1/caveolae clustering and defective inward trafficking upon loss of cell adhesion. By contrast, cell elongation imposed by the excess of stress fibers induced by active mDia1 flattens caveolae. Furthermore, active mDia1 rescues the actin co-aligned Cav1 pool and Cav1 inward trafficking upon loss of adhesion in Abl-deficient cells. Thus, caveolar domain organization and trafficking are tightly coupled to adhesive and stress fiber regulatory pathways.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Caveolas/metabolismo , Caveolina 1/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Caveolas/fisiología , Caveolas/ultraestructura , Caveolina 1/genética , Adhesión Celular , Clonación Molecular , Forminas , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Células HeLa , Humanos , Ratones , Microscopía Electrónica , Plásmidos/genética , Plásmidos/metabolismo , Polimerizacion , Estructura Terciaria de Proteína , Transporte de Proteínas , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Fibras de Estrés/metabolismo , Fibras de Estrés/fisiologíaRESUMEN
To assess possible effects on subcellular organization after cryopreservation, we compared vitrified and slowly frozen oocytes in terms of their post-warm/thaw morphology, meiotic spindle configuration, and DNA integrity. DNA integrity of cryopreserved oocytes was not altered after the procedures, but vitrification was more effective than slow cooling, as shown by higher survival rate and spindle assessment despite a higher misalignment between meiotic spindle and polar body.
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Criopreservación/métodos , Fragmentación del ADN , Meiosis , Oocitos/citología , Vitrificación , Supervivencia Celular , Femenino , Humanos , Oocitos/fisiología , Huso AcromáticoRESUMEN
Brick1 (Brk1) is the less-studied component of the Wave/Scar pathway involved in the branched nucleation of actin fibers. The clinical relevance of Brk1 is emphasized by correlative data showing that Von Hippel-Lindau (VHL) patients that also lose the BRK1 gene are protected against the development of tumors. This contrasts with recent evidence suggesting that the Wave complex may function as an invasion suppressor in epithelial cancers. Here, we show that the downregulation of Brk1 results in abnormal actin stress fiber formation and vinculin distribution and loss of Arp2/3 and Wave proteins at the cellular protrusions. Brk1 is required for cell proliferation and cell transformation by oncogenes. In addition, Brk1 downregulation results in defective directional migration and invasive growth in renal cell carcinoma cells as well as in other tumor cell types. Finally, genetic ablation of Brk1 results in dramatic defects in embryo compaction and development, suggesting an essential role for this protein in actin dynamics. Thus, genetic loss or inhibition of BRK1 is likely to be protective against tumor development due to proliferation and motility defects in affected cells.
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Actinas/metabolismo , Transformación Celular Neoplásica , Proteínas del Citoesqueleto/fisiología , Citoesqueleto/metabolismo , Desarrollo Embrionario/fisiología , Animales , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Movimiento Celular/genética , Movimiento Celular/fisiología , Proliferación Celular , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Regulación hacia Abajo , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Ratones Noqueados , Ratones SCID , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We report a highly specific, sensitive, and robust method for analyzing fluorescence resonance energy transfer (FRET) based on spectral laser scanning confocal microscopy imaging. The lambda FRET (lambdaFRET) algorithm comprises imaging of a FRET sample at multiple emission wavelengths rendering a FRET spectrum, which is separated into its donor and acceptor components to obtain a pixel-based calculation of FRET efficiency. The method uses a novel off-line precalibration procedure for spectral bleed-through correction based on the acquisition of reference reflection images, which simplifies the method and reduces variability. LambdaFRET method was validated using structurally characterized FRET standards with variable linker lengths and stoichiometries designed for this purpose. LambdaFRET performed better than other well-established methods, such as acceptor photobleaching and sensitized emission-based methods, in terms of specificity, reproducibility, and sensitivity to distance variations. Moreover, lambdaFRET analysis was unaffected by high fluorochrome spectral overlap and cellular autofluorescence. The lambdaFRET method demonstrated outstanding performance in intra- and intermolecular FRET analysis in both fixed and live cell imaging studies.
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Transferencia Resonante de Energía de Fluorescencia/métodos , Microscopía Confocal/métodos , Proteínas Bacterianas/análisis , Línea Celular Tumoral , Rayos gamma , Proteínas Fluorescentes Verdes/análisis , Humanos , Receptores de Hialuranos/análisis , Receptores de Hialuranos/metabolismo , Proteínas Luminiscentes/análisis , Proteínas de Microfilamentos/análisis , Proteínas de Microfilamentos/metabolismo , Unión Proteica , Sensibilidad y EspecificidadRESUMEN
The adhesion molecule CD44 and the membrane-type matrix metalloproteinase MT1-MMP act coordinately in tumor cells to promote cell invasion through a yet unclear mechanism. We are interested in studying the interplay between CD44 and MT1-MMP in carcinoma cells embedded in HA containing three-dimensional collagen I matrices (3D HA-Col I) by time-lapse confocal microscopy imaging. Here we report the in vivo interaction between CD44 and MT1-MMP, revealed by fluorescence resonance energy transfer (FRET) microscopy. MT1-MMP interacts with CD44 preferentially at the trailing edge of the invading tumor cells during rear retraction and on membrane fragments released during the invasion process. A fluorescent biosensor designed to monitor the proteolytic processing of CD44 by live cell imaging demonstrates that cleavage of the CD44 extracellular domain is enriched in the retracting rear ends of invasive tumor cells. Invasion assays showed that MT1-MMP mediates CD44-dependent tumor-cell invasion, whereas CD44 is not essential for MT1-MMP-mediated invasion of 3D HA-Col I matrices. Together, our results support a role for MT1-MMP in cell retraction during CD44-mediated cell invasion.
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Movimiento Celular , Polaridad Celular , Receptores de Hialuranos/metabolismo , Metaloproteinasa 14 de la Matriz/metabolismo , Invasividad Neoplásica/patología , Línea Celular Tumoral , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Genes Reporteros , Humanos , Receptores de Hialuranos/química , Proteínas de Microfilamentos/metabolismo , Unión Proteica , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Transporte de Proteínas , Seudópodos/enzimología , Fracciones Subcelulares/metabolismoRESUMEN
MT1-MMP plays a key role in endothelial function, as underscored by the angiogenic defects found in MT1-MMP deficient mice. We have studied the molecular interactions that underlie the functional regulation of MT1-MMP. At lateral endothelial cell junctions, MT1-MMP colocalizes with tetraspanin CD151 (Tspan 24) and its associated partner alpha3beta1 integrin. Biochemical and FRET analyses show that MT1-MMP, through its hemopexin domain, associates tightly with CD151, thus forming alpha3beta1 integrin/CD151/MT1-MMP ternary complexes. siRNA knockdown of HUVEC CD151 expression enhanced MT1-MMP-mediated activation of MMP2, and the same activation was seen in ex vivo lung endothelial cells isolated from CD151-deficient mice. However, analysis of collagen degradation in these experimental models revealed a diminished MT1-MMP enzymatic activity in confined areas around the cell periphery. CD151 knockdown affected both MT1-MMP subcellular localization and its inclusion into detergent-resistant membrane domains, and prevented biochemical association of the metalloproteinase with the integrin alpha3beta1. These data provide evidence for a novel regulatory role of tetraspanin microdomains on the collagenolytic activity of MT1-MMP and indicate that CD151 is a key regulator of MT1-MMP in endothelial homeostasis.
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Antígenos CD/química , Células Endoteliales/citología , Regulación de la Expresión Génica , Metaloproteinasa 14 de la Matriz/metabolismo , Animales , Células Cultivadas , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Activación Enzimática , Transferencia Resonante de Energía de Fluorescencia , Homeostasis , Humanos , Integrina alfa3beta1/metabolismo , Pulmón/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones , Tetraspanina 24RESUMEN
Neurological signs and symptoms are very important to establish a correct neurological diagnosis. We present here a Colombian female patient, 60 years-old, who had ischaemic stroke in the left cerebral media artery. It produced right hemiplegia, motor aphasia, "central" facial palsy and atrophy of right platysma muscle. This latter finding, described originally by Joseph Babinski as "The Babinski Sign" was observed only two years and seven months after the ictus even when she had, previously, been evaluated by several neurologists. The underdiagnosis of clinical signs like the one described here may lead to erroneous diagnosis that will, ultimately, affect neurorehabilitation measures.
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Hemiplejía/diagnóstico , Reflejo de Babinski/diagnóstico , Errores Diagnósticos , Parálisis Facial/diagnóstico , Femenino , Hemiplejía/etiología , Humanos , Persona de Mediana Edad , Accidente Cerebrovascular/complicacionesRESUMEN
La evaluación semiológica apropiada es uno de los aspectos más importantes para realizar un adecuado diagnóstico neurológico. Presentamos una paciente colombiana de 60 años de edad, quien padeció un infarto de la arteria cerebral media izquierda, originándole hemiplejía derecha, afasia motora, parálisis facial "central" derecha y atrofia del músculo platisma derecho. Este último hallazgo, originalmente descrito por Joseph Babinski, el cual es el verdadero signo de Babinski, no fue identificado sino hasta dos años y siete meses después de haberse presentado el ictus, aunque había sido evaluada, previamente, por diferentes especialistas en ciencias neurológicas. La no identificación de signos como el mencionado aquí lleva, en ocasiones, a realizar diagnósticos erróneos o incompletos afectando no sólo la localización apropiada de las lesiones sino, también, las eventuales medidas que se deben tomar en la neurorrehabilitación de estos pacientes.
Neurological signs and symptoms are very important to establish a correct neurological diagnosis. We present here a Colombian female patient, 60 yearsold, who had ischaemic stroke in the left cerebral media artery. It produced right hemiplegia, motor aphasia, "central" facial palsy and atrophy of right platysma muscle. This latter finding, described originally by Joseph Babinski as "The Babinski Sign" was observed only two years and seven months after the ictus even when she had, previously, been evaluated by several neurologists. The underdiagnosis of clinical signs like the one described here may lead to erroneous diagnosis that will, ultimately, affect neurorehabilitation measures.