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

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

Cooperative secretions facilitate host range expansion in bacteria

The majority of emergent human pathogens are zoonotic in origin, that is, they can transmit to humans from other animals. Understanding the factors underlying the evolution of pathogen host range is therefore of critical importance in protecting human health. There are two main evolutionary routes to generalism: organisms can tolerate multiple environments or they can modify their environments to forms to which they are adapted. Here we use a combination of theory and a phylogenetic comparative analysis of 191 pathogenic bacterial species to show that bacteria use cooperative secretions that modify their environment to extend their host range and infect multiple host species. Our results suggest that cooperative secretions are key determinants of host range in bacteria, and that monitoring for the acquisition of secreted proteins by horizontal gene transfer can help predict emerging zoonoses

Combining ability of summer-squash lines with different degrees of parthenocarpy and PRSV-W resistance

The aim was to assess heterosis in a set of 16 summer-squash hybrids, and evaluate the combining capacity of the respective parental lines, which differed as to the degree of parthenocarpy and resistance to PRSV-W (Papaya Ringspot Virus-Watermelon strain). The hybrids were obtained using a partial diallel cross design (4 × 4). The lines of parental group I were 1 = ABX-037G-77-03-05-01-01-bulk, 2 = ABX-037G-77-03-05-03-10-bulk, 3 = ABX-037G-77-03-05-01-04-bulk and 4 = ABX-037G-77-03-05-05-01-bulk, and of group II, 1′ = ABX-037G-77-03-05-04-08-bulk, 2′ = ABX-037G-77-03-05-02-11-bulk, 3′ = Clarice and 4′ = Caserta. The 16 hybrids and eight parental lines were evaluated for PRSV-W resistance, parthenocarpic expression and yield in randomized complete-block designs, with three replications. Parthenocarpy and the resistance to PRSV-W were rated by means of a scale from 1 to 5, where 1 = non-parthenocarpic or high resistance to PRSV-W, and 5 = parthenocarpic or high susceptibility to PRSV-W. Both additive and non-additive gene effects were important in the expression of parthenocarpy and resistance to PRSV-W. Whereas estimates of heterosis in parthenocarpy usually tended towards a higher degree, resistance to PRSV-W was towards higher susceptibility. At least one F1 hybrid was identified with a satisfactory degree of parthenocarpy, resistance to PRSV-W and high fruit-yield

What traits are carried on mobile genetic elements, and why?

Although similar to any other organism, prokaryotes can transfer genes vertically from mother cell to daughter cell, they can also exchange certain genes horizontally. Genes can move within and between genomes at fast rates because of mobile genetic elements (MGEs). Although mobile elements are fundamentally self-interested entities, and thus replicate for their own gain, they frequently carry genes beneficial for their hosts and/or the neighbours of their hosts. Many genes that are carried by mobile elements code for traits that are expressed outside of the cell. Such traits are involved in bacterial sociality, such as the production of public goods, which benefit a cell's neighbours, or the production of bacteriocins, which harm a cell's neighbours. In this study we review the patterns that are emerging in the types of genes carried by mobile elements, and discuss the evolutionary and ecological conditions under which mobile elements evolve to carry their peculiar mix of parasitic, beneficial and cooperative genes

Spatial Evaluation and Modeling of Dengue Seroprevalence and Vector Density in Rio de Janeiro, Brazil

Dengue is a major public health problem in many tropical regions of the world, including Brazil, where Aedes aegypti is the main vector. We present a household study that combines data on dengue fever seroprevalence, recent dengue infection, and vector density, in three neighborhoods of Rio de Janeiro, Brazil, during its most devastating dengue epidemic to date. This integrated entomological–serological survey showed evidence of silent transmission even during a severe epidemic. Also, past exposure to dengue virus was highly associated with age and living in areas of high movement of individuals and social/commercial activity. No association was observed between household infestation index and risk of dengue infection in these areas. Our findings are discussed in the light of current theories regarding transmission thresholds and relative role of mosquitoes and humans as vectors of dengue viruses