In vitro modeling of dysfunctional glial cells in neurodegenerative diseases using human pluripotent stem cells

Most neurodegenerative diseases are characterized by a complex and mostly still unresolved pathology. This fact, together with the lack of reliable models, have precluded the development of effective therapies counteracting the disease progression. In the past few years, several studies have evidenced that lack of proper functionality of glial cells (astrocytes, microglia and oligodendrocytes) has a key role in the pathology of several neurodegenerative conditions including Alzheimer´s disease, amyotrophic lateral sclerosis and multiple sclerosis among others. However, this glial dysfunction is poorly modelled by available animal models, and we hypothesize that patientderived cells can serve as a better platform where to study this glial dysfunction. In this sense, human pluripotent stem cells (hPSCs) has revolutionized the field allowing the generation of disease-relevant neural cell types that can be used for disease modelling, drug screening and, possibly, cell transplantation purposes. In the case of the generation of oligodendrocytes (OLs) from hPSCs, we have developed a fast and robust protocol to generate surface antigen O4-positive (O4+) and myelin basic protein-positive OLs from hPSCs in only 22 days, including from patients with multiple sclerosis or amyotrophic lateral sclerosis. The generated cells resemble primary human OLs at the transcriptome level and can myelinate neurons in vivo. Using in vitro OLneuron co-cultures, effective myelination of neurons can also be demonstrated. This platform is being translated as well to the generation of the other glial cell types, allowing the derivation of patient-specific glial cells where to model disease-specific dysfunction. This methodology can be used for elucidating pathogenic pathways associated with neurodegeneration and to identify therapeutic targets susceptible of drug modulation, contributing to the development of novel and effective drugs for these devastating disorders.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Supported by PI18/01557 (to AG) and P18/1556 (to JV) grants from ISCiii of Spain co-financed by FEDER funds from European Union, and PI-0276-2018 grant (to JAGL) from Consejeria de Salud of Junta de Andalucia. JAGL held a postdoctoral contract from the I Research Plan Propio of the University of Malaga. CV and KE were supported by IWT-SBO-150031-iPSCAF and the Thierry Lathran Foundation grant – ALS-OL, and KN by FWO1166518

BNIP3 supports melanoma cell migration and vasculogenic mimicry by orchestrating the actin cytoskeleton

BNIP3 is an atypical BH3-only member of the BCL-2 family of proteins with reported pro-death as well as pro-autophagic and cytoprotective functions, depending on the type of stress and cellular context. In line with this, the role of BNIP3 in cancer is highly controversial and increased BNIP3 levels in cancer patients have been linked with both good as well as poor prognosis. In this study, using small hairpin RNA (shRNA) lentiviral transduction to stably knockdown BNIP3 (BNIP3-shRNA) expression levels in melanoma cells, we show that BNIP3 supports cancer cell survival and long-term clonogenic growth. Although BNIP3-shRNA increased mitochondrial mass and baseline levels of reactive oxygen species production, which are features associated with aggressive cancer cell behavior, it also prevented cell migration and completely abolished the ability to form a tubular-like network on matrigel, a hallmark of vasculogenic mimicry (VM). We found that this attenuated aggressive behavior of these melanoma cells was underscored by severe changes in cell morphology and remodeling of the actin cytoskeleton associated with loss of BNIP3. Indeed, BNIP3-silenced melanoma cells displayed enhanced formation of actin stress fibers and membrane ruffles, while lamellopodial protrusions and filopodia, tight junctions and adherens junctions were reduced. Moreover, loss of BNIP3 resulted in re-organization of focal adhesion sites associated with increased levels of phosphorylated focal adhesion kinase. Remarkably, BNIP3 silencing led to a drop of the protein levels of the integrin-associated protein CD47 and its downstream signaling effectors Rac1 and Cdc42. These observations underscore that BNIP3 is required to maintain steady-state levels of intracellular complexes orchestrating the plasticity of the actin cytoskeleton, which is integral to cell migration and other vital processes stimulating cancer progression. All together these results unveil an unprecedented pro-tumorigenic role of BNIP3 driving melanoma cell's aggressive features, like migration and VM

Translation of biological and sedimentological point data towards habitat suitability maps of biological communities and EUNIS level 5 maps. Part 2: From habitat suitability maps of biological communities towards EUNIS level 5 maps

The full coverage habitat suitability maps of the macrobenthic communities serve as an input to apply the EUNIS classification on the Belgian Continental Shelf and to translate the maps into EUNIS habitat types (EUNIS level 5 maps). The whole analysis was performed within a GIS (Geographic Information System). The habitat suitability maps were classified by means of the natural breaks classification scheme. Two derivative maps were generated, respectively exceeding probabilities of 60% and 70%. Subsequently, the derived habitat suitability maps were translated into EUNIS habitat types. A large proportion of the Belgian shelf is covered and assigned to EUNIS classes. Each defined EUNIS habitat type has a habitat suitability percentage exceeding 60%. So far, only the Macoma balthica community matches within the current EUNIS classification. The other communities do not exactly match classes within the EUNIS classification. As such, only temporary codes are created and those need an expert review. The Habitat model (Degraer et al., in prep.) does not foresee transitional communities; as such they cannot be mapped. Once these are defined, a complete full coverage EUNIS map can be attained

Are you approaching me? Motor execution influences perceived action orientation

Human observers are especially sensitive to the actions of conspecifics that match their own actions. This has been proposed to be critical for social interaction, providing the basis for empathy and joint action. However, the precise relation between observed and executed actions is still poorly understood. Do ongoing actions change the way observers perceive others' actions? To pursue this question, we exploited the bistability of depth-ambiguous point-light walkers, which can be perceived as facing towards the viewer or as facing away from the viewer. We demonstrate that point-light walkers are perceived more often as facing the viewer when the observer is walking on a treadmill compared to when the observer is performing an action that does not match the observed behavior (e.g., cycling). These findings suggest that motor processes influence the perceived orientation of observed actions: Acting observers tend to perceive similar actions by conspecifics as oriented towards themselves. We discuss these results in light of the possible mechanisms subtending action-induced modulation of perception

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells (hPSCs) is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming

Recognizing the seafloor’s characteristics using habitat signatures

Seafloor images become increasingly available, both derived from video or photographs and from acoustic remote sensing. Very-high resolution acoustic imagery has indeed the potential of depicting a recognisable sign on an image that relates to a physical and biological nature, i.e. its habitat signature. Still, most of this information is stored at institutes or universities and no up-to-date comprehensive compilation is yet available. Moreover, the acoustic imagery often remains hard to interpret; this is mainly because of the multitude of factors influencing the image and the lack of reference material. When an interpreter studies a remote sensing image, he indeed needs to refer to particular textures and patterns that are recognisable on the image and relate that to reality. Ground truthing remains crucial; still comparison with a large number of similar cases is a necessity. In the framework of marine environmental issues this becomes increasingly important and the need for sound interpretations is real. To anticipate on this need, a web-based catalogue of seabed habitat signatures is being built in the framework of the MESH project (Mapping European Seabed Habitats), for both scientists and non-scientists. The catalogue contains a collection of images produced by different remote sensing techniques (acoustic and optically derived images, photographs and video). As such, the results of the different techniques can be compared and can strengthen interpretations in view of seabed assessments. The catalogue has a comprehensive list of metadata per habitat signature, both in terms of its physical and biological environment and the conditions under which the signatures were generated. The web catalogue is easy manageable. Habitats can be searched using their own name or by typing a key word or choosing a EUNIS code or making a query on physical factors. For every habitat one or more significant locations in the Mesh area are chosen and every location displays all the signatures available. Every location is identified by its coordinates (lat., long.) to be easily positioned on the MESH webGIS (http://www.searchmesh.net/webGIS). The signatures are presented as little thumbnails to let the web user have an easy overview. These link to a page where a description of the image, an enlarged image and all technical data referred to it can be found. The catalogue will largely increase the visibility of how the seafloor looks like, but above all it is hoped that it will assist in the interpretation of newly acquired data in view of - 48 - environmental assessments. Any potential contributor to this catalogue is invited to share their images to a wider European community. The web-catalogue is developed at Ifremer (http://www.ifremer.fr/meshmalo/ essai_signatures). RCMG is responsible for the input of imagery related to the Belgian part of the North Sea