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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (λFRET) 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. λFRET method was validated using structurally characterized FRET standards with variable linker lengths and stoichiometries designed for this purpose. λFRET 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, λFRET analysis was unaffected by high fluorochrome spectral overlap and cellular autofluorescence. The λFRET method demonstrated outstanding performance in intra- and intermolecular FRET analysis in both fixed and live cell imaging studies. Microsc. Res. Tech., 2009. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

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. Cell. Motil. Cytoskelton 2008. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 misalignament between meiotic spindle and polar body. [Copyright &y& Elsevier]