A Protocol for FRET-Based Live-Cell Imaging in Microglia

This protocol highlights the use of FRET-based biosensors to investigate signaling events during microglia activation in real time. Understanding microglia activation has gained momentum as it can help decipher signaling mechanisms underlying the neurodegenerative process occurring in neurological disorders. Unlike more traditional methods widely employed in the microglia field, FRET allows microglia signaling events to be studied in real time with exquisite subcellular resolution. However, FRET-based live-cell imaging requires application-specific biosensors and specialized imaging systems, limiting its use in in vivo studies. For complete details on the use and execution of this protocol, please refer to Socodato et al. (2020), Portugal et al. (2017), and Socodato et al. (2018).This work was financed by FEDER (Fundo Europeu de Desenvolvimento Regional) funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT (Fundação para a Ciência e a Tecnologia)/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project POCI-01-0145-FEDER-031318 (PTDC/MED-NEU/31318/2017). The authors acknowledge the support of the following: i3S Scientific Platform: Advanced Light Microscopy (ALM), members of the national infrastructure PPBI-Portuguese Platform of BioImaging (supported by POCI-01–0145-FEDER-022122). C.C.P. and R.S. hold employment contracts financed by national funds through FCT – Fundação para a Ciência e a Tecnologia, IP, in the context of the program-contract described in paragraphs 4, 5, and 6 of art. 23 of Law no. 57/2016, of August 29th, as amended by Law no. 57/2017 of July 19th

Relationship between intact HIV-1 proviruses in circulating CD4+ T cells and rebound viruses emerging during treatment interruption.

Combination antiretroviral therapy controls but does not cure HIV-1 infection because a small fraction of cells harbor latent viruses that can produce rebound viremia when therapy is interrupted. The circulating latent virus reservoir has been documented by a variety of methods, most prominently by viral outgrowth assays (VOAs) in which CD4+ T cells are activated to produce virus in vitro, or more recently by amplifying proviral near full-length (NFL) sequences from DNA. Analysis of samples obtained in clinical studies in which individuals underwent analytical treatment interruption (ATI), showed little if any overlap between circulating latent viruses obtained from outgrowth cultures and rebound viruses from plasma. To determine whether intact proviruses amplified from DNA are more closely related to rebound viruses than those obtained from VOAs, we assayed 12 individuals who underwent ATI after infusion of a combination of two monoclonal anti-HIV-1 antibodies. A total of 435 intact proviruses obtained by NFL sequencing were compared with 650 latent viruses from VOAs and 246 plasma rebound viruses. Although, intact NFL and outgrowth culture sequences showed similar levels of stability and diversity with 39% overlap, the size of the reservoir estimated from NFL sequencing was larger than and did not correlate with VOAs. Finally, intact proviruses documented by NFL sequencing showed no sequence overlap with rebound viruses; however, they appear to contribute to recombinant viruses found in plasma during rebound

Extracellular environment contribution to astrogliosis-lessons learned from a tissue engineered 3D model of the glial scar

Glial scars are widely seen as a (bio)mechanical barrier to central nervous system regeneration. Due to the lack of a screening platform, which could allow in-vitro testing of several variables simultaneously, up to now no comprehensive study has addressed and clarified how different lesion microenvironment properties affect astrogliosis. Using astrocytes cultured in alginate gels and meningeal fibroblast conditioned medium, we have built a simple and reproducible 3D culture system of astrogliosis mimicking many features of the glial scar. Cells in this 3D culture model behave similarly to scar astrocytes, showing changes in gene expression (e.g., GFAP) and increased extra-cellular matrix production (chondroitin 4 sulfate and collagen), inhibiting neuronal outgrowth. This behavior being influenced by the hydrogel network properties. Astrocytic reactivity was found to be dependent on RhoA activity, and targeting RhoA using shRNA-mediated lentivirus reduced astrocytic reactivity. Further, we have shown that chemical inhibition of RhoA with ibuprofen or indirectly targeting RhoA by the induction of extracellular matrix composition modification with chondroitinase ABC, can diminish astrogliosis. Besides presenting the extracellular matrix as a key modulator of astrogliosis, this simple, controlled and reproducible 3D culture system constitutes a good scar-like system and offers great potential in future neurodegenerative mechanism studies, as well as in drug screenings envisaging the development of new therapeutic approaches to minimize the effects of the glial scar in the context of central nervous system disease.This work had the financial support of the Portuguese Fundação para a Ciência e Tecnologia (FCT) / Ministério da Educação e Ciência (MEC) through National Funds and, when applicable, co-financed by the FEDER via the PT2020 Partnership Agreement under the 4293 Unit I&D. DR acknowledges FCT for her PhD scholarship /SFRH/BD/64079/2009). Authors thank Dr. Michiyuki Matsuda (Kyoto University, Japan) for the RhoA FRET probe with enhanced sensitivity and Dr. Yingxiao Wang (University of California, USA) for the Src FRET probe

Sestrin2 Modulates AMPK Subunit Expression and Its Response to Ionizing Radiation in Breast Cancer Cells

Background: The sestrin family of stress-responsive genes (SESN1-3) are suggested to be involved in regulation of metabolism and aging through modulation of the AMPK-mTOR pathway. AMP-activated protein kinase (AMPK) is an effector of the tumour suppressor LKB1, which regulates energy homeostasis, cell polarity, and the cell cycle. SESN1/2 can interact directly with AMPK in response to stress to maintain genomic integrity and suppress tumorigenesis. Ionizing radiation (IR), a widely used cancer therapy, is known to increase sestrin expression, and acutely activate AMPK. However, the regulation of AMPK expression by sestrins in response to IR has not been studied in depth. Methods and Findings: Through immunoprecipitation we observed that SESN2 directly interacted with the AMPKa1b1c1 trimer and its upstream regulator LKB1 in MCF7 breast cancer cells. SESN2 overexpression was achieved using a Flag-tagged SESN2 expression vector or a stably-integrated tetracycline-inducible system, which also increased AMPKa1 and AMPKb1 subunit phosphorylation, and co-localized with phosphorylated AMPKa-Thr127 in the cytoplasm. Furthermore, enhanced SESN2 expression increased protein levels of LKB1 and AMPKa1b1c1, as well as mRNA levels of LKB1, AMPKa1, and AMPKb1. Treatment of MCF7 cells with IR elevated AMPK expression and activity, but this effect was attenuated in the presence of SESN2 siRNA. In addition, elevated SESN2 inhibited IR-induced mTOR signalling and sensitized MCF7 cells to IR through an AMPK-dependent mechanism

Analysis of parcel-based image classification methods for monitoring the activities of the Land Bank of Galicia (Spain)

[EN] The abandonment of agricultural plots entails a low economic productivity of the land and a higher vulnerability to wildfires and degradation of affected areas. In this sense, the local government of Galicia is promoting new methodologies based on high-resolution images in order to classify the territory in basic and generic land uses. This procedure will be used to control the sustainable management of plots belonging to the Land Bank. This paper presents an application study for maintaining and updating land use/land cover geospatial databases using parcel-oriented classification. The test is performed over two geographic areas of Galicia, in the northwest of Spain. In this region, forest and shrublands in mountain environments are very heterogeneous with many private unproductive plots, some of which are in a high state of abandonment. The dataset is made of high spatial resolution multispectral imagery, cadastral cartography employed to define the image objects (plots), and field samples used to define evaluation and training samples. A set of descriptive features is computed quantifying different properties of the objects, i.e. spectral, texture, structural, and geometrical. Additionally, the effect on the classification and updating processes of the historical land use as a descriptive feature is tested. Three different classification methodologies are analyzed: linear discriminant analysis, decision trees, and support vector machine. The overall accuracies of the classifications obtained are always above 90 % and support vector machine method is proved to provide the best performance. Forest and shrublands areas are especially undefined, so the discrimination between these two classes is low. The results enable to conclude that the use of automatic parcel-oriented classification techniques for updating tasks of land use/land cover geospatial databases, is effective in the areas tested, particularly when broad and well defined classes are required.The authors appreciate the collaboration and support provided by Xunta de Galicia, Sociedade para o Desenvolvemento Comarcal de Galícia, and Banco de Terras de Galicia. The financial support provided by the Spanish Ministerio de Ciencia e Innovación in the framework of the projects CGL2010-19591/BTE and CGL2009-14220 is also acknowledged.Hermosilla, T.; Díaz Manso, J.; Ruiz Fernández, LÁ.; Recio Recio, JA.; Fernández-Sarría, A.; Ferradáns Nogueira, P. (2012). Analysis of parcel-based image classification methods for monitoring the activities of the Land Bank of Galicia (Spain). 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