Monitoring SARS-COV-2 surrogate TGEV individual virions structure survival under harsh physicochemical environments

Effective airborne transmission of coronaviruses via liquid microdroplets requires a virion structure that must withstand harsh environmental conditions. Due to the demanding biosafety requirements for the study of human respiratory viruses, it is important to develop surrogate models to facilitate their investigation. Here we explore the mechanical properties and nanostructure of transmissible gastroenteritis virus (TGEV) virions in liquid milieu and their response to different chemical agents commonly used as biocides. Our data provide two-fold results on virus stability: First, while particles with larger size and lower packing fraction kept their morphology intact after successive mechanical aggressions, smaller viruses with higher packing fraction showed conspicuous evidence of structural damage and content release. Second, monitoring the structure of single TGEV particles in the presence of detergent and alcohol in real time revealed the stages of gradual degradation of the virus structure in situ. These data suggest that detergent is three orders of magnitude more efficient than alcohol in destabilizing TGEV virus particles, paving the way for optimizing hygienic protocols for viruses with similar structure, such as SARS-CoV-

Characterization of interaction of magnetic nanoparticles with breast cancer cells

Background: Different superparamagnetic iron oxide nanoparticles have been tested for their potential use in cancer treatment, as they enter into cells with high effectiveness, do not induce cytotoxicity, and are retained for relatively long periods of time inside the cells. We have analyzed the interaction, internalization and biocompatibility of dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles with an average diameter of 15 nm and negative surface charge in MCF-7 breast cancer cells. Results: Cells were incubated with dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles for different time intervals, ranging from 0.5 to 72 h. These nanoparticles showed efficient internalization and relatively slow clearance. Time-dependent uptake studies demonstrated the maximum accumulation of dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles after 24 h of incubation, and afterwards they were slowly removed from cells. Superparamagnetic iron oxide nanoparticles were internalized by energy dependent endocytosis and localized in endosomes. Transmission electron microscopy studies showed macropinocytosis uptake and clathrin-mediated internalization depending on the nanoparticles aggregate size. MCF-7 cells accumulated these nanoparticles without any significant effect on cell morphology, cytoskeleton organization, cell cycle distribution, reactive oxygen species generation and cell viability, showing a similar behavior to untreated control cells. Conclusions: All these findings indicate that dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles have excellent properties in terms of efficiency and biocompatibility for application to target breast cancer cellsThe research leading to these results have received partial funding from the European Seventh Framework Programme (FP7/2007-2013) under the project MULTIFUN grant agreement no. 262943, and the project Nanofrontmag-CM (S2013/MIT-2850) from the Comunidad de Madrid. Additional grants were obtained from BFU 2011–29038 and CTQ2013-48767-C3-3-R from the Ministerio de Economia y Competitividad and S2009/Mat 1507 from the Comunidad de Madrid (to JLC), from EU FP7 project NAMDIATREAM (ref 246479) and from “la Caixa” / CNB International PhD Programme Fellowship

XTEND: Extending the depth of field in cryo soft X-ray tomography

We have developed a new data collection method and processing framework in full field cryo soft X-ray tomography to computationally extend the depth of field (DOF) of a Fresnel zone plate lens. Structural features of 3D-reconstructed eukaryotic cells that are affected by DOF artifacts in standard reconstruction are now recovered. This approach, based on focal series projections, is easily applicable with closed expressions to select specific data acquisition parameters.This work was partially supported by MINECO grants BFU2014-54181 to JLC and AIC-A-2011-0638, BIO2013-44647-R and BIO2016-76400-R to JMC, Madrid. Regional government grants S2013/MIT-2850 to JLC and S2010/BMD-2305 to JMC, National Science Foundation grant DMS-1114901 to GTH, the European Union through BioStruct-X Project 283570 and Horizon 2020 through grant iNEXT (INFRAIA-1-2014-2015, Proposal: 653706).S

Clathrin regulates lymphocyte migration by driving actin accumulation at the cellular leading edge

Lymphocyte migration, which is essential for effective immune responses, belongs to the so-called amoeboid migration. The lymphocyte migration is up to 100 times faster than between mesenchymal and epithelial cell types. Migrating lymphocytes are highly polarized in three well-defined structural and functional zones: uropod, medial zone, and leading edge (LE). The actiomyosin-dependent driving force moves forward the uropod, whereas massive actin rearrangements protruding the cell membrane are observed at the LE. These actin rearrangements resemble those observed at the immunological synapse driven by clathrin, a protein normally involved in endocytic processes. Here, we used cell lines as well as primary lymphocytes to demonstrate that clathrin and clathrin adaptors colocalize with actin at the LE of migrating lymphocytes, but not in other cellular zones that accumulate both clathrin and actin. Moreover, clathrin and clathrin adaptors, including Hrs, the clathrin adaptor for multivesicular bodies, drive local actin accumulation at the LE. Clathrin recruitment at the LE resulted necessary for a complete cell polarization and further lymphocyte migration in both 2D and 3D migration models. Therefore, clathrin, including the clathrin population associated to internal vesicles, controls lymphocyte migration by regulating actin rearrangements occurring at the LE.This work was supported by the grants from the Spanish Ministry of Science and Technology (MICINN; BFU2011-29450 to E.V.) and Ministry of Economy and Competitiveness (MINECO; SAF2014-56716-REDT and BFU2014-59585-R to E.V., SAF2011-25834 to F.S.M., SAF2014-58895-JIN to A.C.A and BFU2014-54181-P to J.L.C.), the Madrid regional government (INDISNET-S2011/BMD-2332 to F.S.M.) and the European Research Council (ERC-2011-AdG 294340-GENTRIS to F.S.M.). We are grateful to the “Centro de Transfusión” of the “Comunidad Autónoma de Madrid” for providing the Buffy Coats

Desarrollo de herramientas para la caracterización de la chaperonina de Haloferax mediterranei

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 27-04-2004In this work we tned to perform the methodology for the further characterization of the Huloferm nte~literrur~eic'hs aperonin. We found three chaperonin's genes in H i~re(liliterrrirlei:c pr~ric prlh and cpnc, with a high homology with other haloarchaeas. The clonation and expression of these genes in E. coli allowed us to study the'special folding conditions of the chaperonin subunit. The reconstitution of the archaeal chaperonin increasing the salt concentration showed ir1 vitro differences between the subunit CPNA and CPNB. CPNA was able to form nngs and filaments wile the CPNB subunit could not. We analyzed the sequence's differences between the subunits in order to locate aminoacidical changes at the interactions points in the chaperonin ring, described using the crystallization of the thermosome. We found two significant changes that could decreased the strength in the theoretical CPNB ring. We developed a native electrophoretic system in halophilic conditions, capable to separate the macromolecular panicles like ribosomes or chaperonins from monomeric proteins. This system was used to study the ring formation ~indedr ifferent conditions during the reconstitution. The structural analysis of the hornoligomeric CPNA ring by TEM iniage processing showed two groups of particles. CPNA fomied irr vitro tetradecameric or hexadecamenc chaperonins, while the iiative H. n~erliterrrinei seems to be hexadecamenc. CPNB, witch is included in the native ring, could be iniplicated in the eight symmetry because of it stnictural differences with CPNA. Both, the native and the reconstituted CPNA chaperonins, could form filaments due the apical domain interaction between the particles. Hydrophobic residues seems to be implied in the apical lid contacts

Primary Education schools and pedagogical renewal: Reviewing experiences

In this article we review the support of several representative Primary Education schools in Spain (2015-16) for a pedagogical renewal that move away from the traditional model. Method: The methodology focuses on the study of outstanding bibliography, interviews with significant members, observation of centres and analysis of the content of educational projects. Results: The centers selected for analysis show multiple common pedagogical aspects, such as the participation of the educational community, the promotion of creativity among the students, as well as curricular, methodological and organizational flexibility. Discussion and Conclusion: Despite the uniqueness of each project, all show multiple common pedagogical aspects. This is due to the direct influence of the Spanish pedagogical renewal developed throughout the 20th century which has shaped the current renewing map of which this article analyses specific example

Cryo X-ray nano-tomography of vaccinia virus infected cells

We have performed full-field cryo X-ray microscopy in the water window photon energy range on vaccinia virus (VACV) infected cells to produce tomographic reconstructions. PtK2 cells were infected with a GFP-expressing VACV strain and frozen by plunge fast freezing. The infected cells were selected by light fluorescence microscopy of the GFP marker and subsequently imaged in the X-ray microscope under cryogenic conditions. Tomographic tilt series of X-ray images were used to yield three-dimensional reconstructions showing different cell organelles (nuclei, mitochondria, filaments), together with other structures derived from the virus infection. Among them, it was possible to detect viral factories and two types of viral particles related to different maturation steps of VACV (immature and mature particles), which were compared to images obtained by standard electron microscopy of the same type of cells. In addition, the effect of radiation damage during X-ray tomographic acquisition was analyzed. Thin sections studied by electron microscopy revealed that the morphological features of the cells do not present noticeable changes after irradiation. Our findings show that cryo X-ray nano-tomography is a powerful tool for collecting three-dimensional structural information from frozen, unfixed, unstained whole cells with sufficient resolution to detect different virus particles exhibiting distinct maturation levels. © 2011 Elsevier Inc.This work was supported by Grants BFU2008-2328, BFU2011-29038 and Consolider CSD2007-00010 from the MICINN, and S009/MAT-1507 from the CM (to J.L.C) and SAF.2008-02036 (to M.E.). The X-ray microscopy work was funded partly by the Human Frontier Science Program (HFSP) Research Grant Ref. RGP0053/2005-C, and by the European Community’s Seventh Framework Programme (FP712007-2013) under grant agreement n° 226716.Peer Reviewe

Monitoring SARS-CoV-2 Surrogate TGEV Individual Virions Structure Survival under Harsh Physicochemical Environments

Effective airborne transmission of coronaviruses via liquid microdroplets requires a virion structure that must withstand harsh environmental conditions. Due to the demanding biosafety requirements for the study of human respiratory viruses, it is important to develop surrogate models to facilitate their investigation. Here we explore the mechanical properties and nanostructure of transmissible gastroenteritis virus (TGEV) virions in liquid milieu and their response to different chemical agents commonly used as biocides. Our data provide two-fold results on virus stability: First, while particles with larger size and lower packing fraction kept their morphology intact after successive mechanical aggressions, smaller viruses with higher packing fraction showed conspicuous evidence of structural damage and content release. Second, monitoring the structure of single TGEV particles in the presence of detergent and alcohol in real time revealed the stages of gradual degradation of the virus structure in situ. These data suggest that detergent is three orders of magnitude more efficient than alcohol in destabilizing TGEV virus particles, paving the way for optimizing hygienic protocols for viruses with similar structure, such as SARS-CoV-2.P. J. De Pablo acknowledges support by grants from the Spanish Ministry of Economy, Industry and Competitiveness projects (FIS2017- 89549-R and FIS2017–90701- REDT), the Human Frontiers Science Program (HFSPO RGP0012/2018) and Fondo SUPERA COVID19 Santander CRUE project TEXGRAF. J. Martín-Benito acknowledges support by grants from the Spanish Ministry of Science and Innovation projects (PID2020–117752RB-I00 and BFU2017–90018-R), CSIC project (PIE202020E079–6) and Fondo SUPERA COVID19 Santander CRUE project TEXGRAF. We acknowledge access to the cryoEM CNB-CSIC facility in the context of the CRIOMECORR project (ESFRI2019–01-CSIC16). The professional editing service NB Revisions was used for technical preparation of the text prior to submission