Creation of a 3D Animation on Evanescent Wave Microscopy and its application in Virology: a tool for the study and understanding of the mechanisms of infection by microorganisms in living cells

En el estudio de los mecanismos de invasión por patógenos (virus, bacterias, parásitos), es importante abordar, a nivel avanzado y molecular, los procesos que gobiernan la fusión de membrana, como uno de los eventos biológicos clave en la infección por microorganismos y en el contexto de las Enfermedades Infecciosas. En este contexto, es importante entender cómo funcionan las proteínas de los patógenos que regulan la invasión de células y tejidos, mediante el control y promoción del proceso de fusión de membrana entre el patógeno y la célula diana. Donde el empleo de la miscroscopía de onda evanescente es importante. Las ilustraciones científicas en este tema suelen ser láminas o esquemas 2D fijos, que ilustran la función de las proteínas implicadas y la fusión de membranas entre patógeno y célula diana, con cadenas secuenciales de imágenes no dinámicas, y difícilmente se ilustra el funcionamiento de este microscopio de última generación. En este capítulo se presenta, a modo de resumen, nuestro proyecto de innovación docente ULL, cuyo objetivo ha sido la creación y generación, por vez primera, de material de Animación 3D para ilustrar de forma pedagógica, y con el mayor reigor ciéntifico, la aplicación de la tecnología de microscopía fluorescente de onda evanescente (TIRFM) en el estudio de células vivas y del mecanismo de su infección por patógenos, aplicándolo a la comprenión del proceso de fusión de membranas y, en particular, al proceso de infección temprana por el virus VIH-1.In the study of the mechanisms of invasion by pathogens (viruses, bacteria, parasites), it is important to address, at an advanced and molecular level, the processes that govern membrane fusion, as one of the key biological events in infection by microorganisms and in the context of Infectious Diseases. In this scenario, it is important to understand how the proteins of the pathogens that regulate the invasion of cells and tissues work, by controlling and promoting the process of membrane fusion between the pathogen and the target cell. In this matter, the use of evanescent wave microscopy (TIRFM) is key and very important. The scientific illustrations in this topic are usually fixed sheets or 2D-diagrams that illustrate the function of the proteins involved and the fusion of membranes between pathogen and target cell, with sequential chains of non-dynamic images, where it is difficult to illustrate how this last generation microscope works. In this chapter we briefly present our ULL teaching innovation project, whose objective has been the creation and generation, for the first time, of a 3D Animation material to illustrate in a pedagogical way, and with the greatest scientific rigor, the application of the evanescent wave fluorescence microscopy technology in the study of living cells and the mechanism of their infection by pathogens, applying it to the understanding of the membrane fusion process and, in particular, to the process of early infection by the HIV-1

Nanostructured Ti thin films by magnetron sputtering at oblique angles

The growth of Ti thin films by the magnetron sputtering technique at oblique angles and at room temperature is analysed from both experimental and theoretical points of view. Unlike other materials deposited in similar conditions, the nanostructure development of the Ti layers exhibits an anomalous behaviour when varying both the angle of incidence of the deposition flux and the deposition pressure. At low pressures, a sharp transition from compact to isolated, vertically aligned, nanocolumns is obtained when the angle of incidence surpasses a critical threshold. Remarkably, this transition also occurs when solely increasing the deposition pressure under certain conditions. By the characterization of the Ti layers, the realization of fundamental experiments and the use of a simple growth model, we demonstrate that surface mobilization processes associated to a highly directed momentum distribution and the relatively high kinetic energy of sputtered atoms are responsible for this behaviourJunta de Andalucía P12-FQM- 2265Ministerio de Ciencia e Innovación CSD2008- 00023, MAT2013-42900-P, MAT2013-40852-R, MAT2014-59772-C2-1, MAT2011- 2908

3D animations as a tool for a better teaching an understanding of viral early infection processes

En la enseñanza de «Mecanismos de invasión por patógenos», se desarrolla, a nivel avanzado molecular, los «Procesos de fusión de membrana» como uno de los eventos biológicos clave en la infección por microorganismos (virus como paradigma). Por tanto, es importante entender cómo ocurre la fusión de membrana a nivel molecular y dinámico, para la consecución de los objetivos de esta temática científica. Objetivos El presente proyecto de innovación docente pretende crear/generar, por vez primera, material audiovisual de animación 3D para ilustrar pedagógicamente el movimiento de las proteínas virales en el momento de la fusión virus-célula; como se ha descrito por autores del campo (Kielian y Rey, 2006; Sánchez-San Martín, Liu y Kielian, 2009; Smit et al. 2011; Wilen, Tilton y Doms, 2012; Melikyan, 2014), para garantizar la formación avanzada del alumno de Biomedicina/Enfermedades Infecciosas. Metodología Uso de tecnologías gráficas avanzadas y de softwares especializados en modelado y animación 3D, como Autodesk Maya y Zbrush. Resultados Las herramientas audiovisuales realizadas han permitido una mejor comprensión sobre cómo ocurre la fusión de membrana, a nivel molecular y de manera dinámica, en virus tipo VIH-1 (Wilen et al. 2012; Melikyan, 2014) y Dengue (Sánchez-San Martín et al. 2009; Smit et al. 2011). Discusión y conclusiones El modelado y animación con tecnologías 3D son cruciales para el desarrollo de herramientas docentes que mejoren la compresión, enseñanza y la divulgación en Ciencia de procesos biológicos complejos, necesarios para la formación de especialistas con el mayor rigor científico, como en este campo de conocimiento.The “Membrane Fusion Processes” topic has been developed at a molecular advanced level in the teaching of “Pathogen Invasion Mechanisms” as one of the key biological events in the Infection (mainly studied in viruses) and in the context of Tropical Diseases. It is important to understand how membrane fusion occurs at a molecular level and in a dynamic way, to adequately achieve the objectives of this scientific topic. 327 Índice Objetives The present teaching innovation project aims to create, for the first time, 3D animation materials to pedagogically illustrate the movement of viral proteins at the moment of virus-cell fusion, as reported (Kielian and Rey, 2006; Sánchez-San Martín, Liu and Kielian, 2009; Smit et al. 2011; Wilen, Tilton and Doms, 2012; Melikyan, 2014), and in order to guarantee advanced training of students in Biomedicine / Infectious Diseases. Methods Use of advanced graphics technologies and software specialized in 3D modeling and animation (e.g.; Autodesk Maya and Zbrush). Results The 3D animation tools developed have allowed a better understanding of how membrane fusion occurs dynamically at a molecular level, in viruses such as HIV-1 (Wilen et al. 2012; Melikyan, 2014) and Dengue (Sánchez-San Martín et al. 2009; Smit et al. 2011). Discussion and conclusions Animation and 3D technologies are crucial for the development of educational tools key for compression and teaching, as well as, for the dissemination in Science of complex biological processes that occur dynamically in time and space, which are necessary for rigorous training specialists at the highest scientific level

Antibacterial Nanostructured Ti Coatings by Magnetron Sputtering: From Laboratory Scales to Industrial Reactors

Based on an already tested laboratory procedure, a new magnetron sputtering methodology to simultaneously coat two-sides of large area implants (up to ~15 cm2) with Ti nanocolumns in industrial reactors has been developed. By analyzing the required growth conditions in a laboratory setup, a new geometry and methodology have been proposed and tested in a semi-industrial scale reactor. A bone plate (DePuy Synthes) and a pseudo-rectangular bone plate extracted from a patient were coated following the new methodology, obtaining that their osteoblast proliferation efficiency and antibacterial functionality were equivalent to the coatings grown in the laboratory reactor on small areas. In particular, two kinds of experiments were performed: Analysis of bacterial adhesion and biofilm formation, and osteoblasts–bacteria competitive in vitro growth scenarios. In all these cases, the coatings show an opposite behavior toward osteoblast and bacterial proliferation, demonstrating that the proposed methodology represents a valid approach for industrial production and practical application of nanostructured titanium coatings.EU-FEDER and the MINECO-AEI 201560E055EU-FEDER and the MINECO-AEI MAT2014-59772-C2-1-PEU-FEDER and the MINECO-AEI MAT2016-75611-REU-FEDER and the MINECO-AEI MAT2016-79866-REU-FEDER and the MINECO-AEI MAT2015-69035-REDCUniversity of Seville (Spain) V and VI PPIT-USMINECO CSIC13-4E-179

HIV-1 Nef Targets HDAC6 to Assure Viral Production and Virus Infection

HIV Nef is a central auxiliary protein in HIV infection and pathogenesis. Our results indicate that HDAC6 promotes the aggresome/autophagic degradation of the viral polyprotein Pr55Gag to inhibit HIV-1 production. Nef counteracts this antiviral activity of HDAC6 by inducing its degradation and subsequently stabilizing Pr55Gag and Vif viral proteins. Nef appears to neutralize HDAC6 by an acidic/endosomal-lysosomal processing and does not need the downregulation function, since data obtained with the non-associated cell-surface Nef-G2A mutant – the cytoplasmic location of HDAC6 – together with studies with chemical inhibitors and other Nef mutants, point to this direction. Hence, the polyproline rich region P72xxP75 (69–77 aa) and the di-Leucin motif in the Nef-ExxxLL160-165 sequence of Nef, appear to be responsible for HDAC6 clearance and, therefore, required for this novel Nef proviral function. Nef and Nef-G2A co-immunoprecipitate with HDAC6, whereas the Nef-PPAA mutant showed a reduced interaction with the anti-HIV-1 enzyme. Thus, the P72xxP75 motif appears to be responsible, directly or indirectly, for the interaction of Nef with HDAC6. Remarkably, by neutralizing HDAC6, Nef assures Pr55Gag location and aggregation at plasma membrane, as observed by TIRFM, promotes viral egress, and enhances the infectivity of viral particles. Consequently, our results suggest that HDAC6 acts as an anti-HIV-1 restriction factor, limiting viral production and infection by targeting Pr55Gag and Vif. This function is counteracted by functional HIV-1 Nef, in order to assure viral production and infection capacities. The interplay between HIV-1 Nef and cellular HDAC6 may determine viral infection and pathogenesis, representing both molecules as key targets to battling HIV

Moléculas motoras y tráfico de membrana: elaboración de una animación 3D y su incorporación a la metodología docente

En el área de conocimiento Biomedicina, respecto de la dinámica de tráfico o transporte vesicular mediado por moléculas motoras, el alumno necesita comprender los objetivos mediante la adquisición de conocimiento por visualización espacio-temporal en 3D, de dicho proceso biológico complejo. En este sentido, la creación de material gráfico de animación 3D para ilustrar de forma pedagógica, y con el mayor rigor científico, las funciones y procesos que ocurren en el interior de las células del sistema inmune, relacionados con molécu- las motoras en el transporte y movimiento de vesículas intracelulares, es de importancia para la compresión de estos procesos y funciones biológicas. Este movimiento motor, por ejemplo, localizado en la membrana plasmática celular, representa la mayor ruta de entrada e infección de muchos patógenos asociados con las Enfermedades Infecciosas (de virus y bacterias) consideradas un problema mayor de salud pública (VIH-1, Zika, Dengue, malaria, etc.). En este ca- pítulo se presenta el proyecto desarrollado a tal efecto, mediante la incorporación de tecnologías gráficas avanzadas al servicio de la animación 3D. Este nuevo material didáctico ha sido realizado por el equipo de científicos, modeladores y expertos en tecnologías gráficas 3D del Laboratorio de Diseño y Fabricación Digital de la ULL (SciArt3D-FabLabULL), que cuenta entre sus miembros con científicos y con expertos en tecnologías gráficas 3D del Laboratorio de Diseño y Fabricación Digital de la ULL.In Biomedicine, regarding the traffic dynamics or vesicular transport mediated by molecular motors, the student needs to understand this complex biological processes through the acquisition of knowledge by spatiotemporal visualization in 3D. In this sense, the creation of graphic material of 3D animation to illustrate in a pedagogi- cal way, and with the highest scientific rigor, the functions and processes that occur inside the cells of the immune system, related to molecular motors in transport and movement of intracellular vesicles, is of importance for the understanding of these biological processes and functions. This motor movement, for example, occurred at cell-plasma membrane, represents a major route of entry and infection of many pathogens associated with Infectious Diseases (i.e., viruses and bacteria), which are considered a major public health problem (HIV-1, Zika, Dengue, malaria, etc.). In this chapter, the project developed for this purpose is presented, through the incorporation of advanced graphic technologies in a new 3D animation. This new didactic material has been made by the team of scientists, modelers and experts in 3D graphic technologies of the ULL Digital Design and Manufacturing Laboratory (SciArt3D-FabLabULL), a team of scientists and experts in 3D graphic technologies from the ULL Digital Design and Manufacturing Laboratory

Contribution of the HIV-1 Envelope Glycoprotein to AIDS Pathogenesis and Clinical Progression

In the absence of antiviral therapy, HIV-1 infection progresses to a wide spectrum of clinical manifestations that are the result of an entangled contribution of host, immune and viral factors. The contribution of these factors is not completely established. Several investigations have described the involvement of the immune system in the viral control. In addition, distinct HLA-B alleles, HLA-B27, -B57-58, were associated with infection control. The combination of these elements and antiviral host restriction factors results in different clinical outcomes. The role of the viral proteins in HIV-1 infection has been, however, less investigated. We will review contributions dedicated to the pathogenesis of HIV-1 infection focusing on studies identifying the function of the viral envelope glycoprotein (Env) in the clinical progression because of its essential role in the initial events of the virus life-cycle. Some analysis showed that inefficient viral Envs were dominant in non-progressor individuals. These poorly-functional viral proteins resulted in lower cellular activation, viral replication and minor viral loads. This limited viral antigenic production allows a better immune response and a lower immune exhaustion. Thus, the properties of HIV-1 Env are significant in the clinical outcome of the HIV-1 infection and AIDS pathogenesis.This work was funded by the Spanish AIDS network “Red Temática Cooperativa de Investigación en SIDA” RD12/0017/0002, RD12/0017/0028, RD12/0017/0034, RD16/0025/0011, RDCIII16/0002/0005 and RD16/0025/0041 as part of the Plan Nacional R + D+I and co-funded by the Spanish “Instituto de Salud Carlos III (ISCIII)-Subdirección General de Evaluación y el Fondo Europeo de Desarrollo Regional (FEDER)”. J.B. is a researcher from “Fundació Institut de Recerca en Ciències de la Salut Germans Trias i Pujol” supported by the Health Department of the Catalonian Government/Generalitat de Catalunya and ISCIII grant Nos. PI17/01318 and PI20/00093 (to J.B.). Work in C.C. Lab was supported by grants SAF (2010-17226) and (2016-77894-R) from MINECO (Spain), FIS (PI13/02269, ISCIII) and PI20/00093. A.-V.F.’s Lab is supported by the European Regional Development Fund (ERDF), PID2021-123031OB-I00 (“Ministerio de Ciencia e Innovación”, Spain), RTI2018-093747-B-100 (“Ministerio de Ciencia, Innovación y Universidades”, Spain), ProID2020010093 (“Agencia Canaria de Investigación, Innovación y Sociedad de la Información” and the European Social Fund), UNLL10-3E-783 (ERDF and “Fundación CajaCanarias”) and “SEGAI-ULL”. S.-P.Y. is funded by “Fundación Doctor Manuel Morales” (La Palma, Spain) and “Contrato Pre-doctoral Ministerio-ULL Formación de Doctores” (2019 Program) (“Ministerio de Ciencia, Innovación y Universidades”, Spain). R.-C.R. is funded by RD16/0025/0011 and ProID2020010093 (“Agencia Canaria de Investigación, Innovación y Sociedad de la Información” and European Social Fund). J.-G.L. is funded by the “Juan de la Cierva de Incorporación” Spanish Program (IJC2019-038902-I) (“Ayudas Juan de la Cierva de incorporación; Agencia Estatal de Investigación. Ministerio de Ciencia e Innovación”).S

The Characteristics of the HIV-1 Env Glycoprotein Are Linked With Viral Pathogenesis

The understanding of HIV-1 pathogenesis and clinical progression is incomplete due to the variable contribution of host, immune, and viral factors. The involvement of viral factors has been investigated in extreme clinical phenotypes from rapid progressors to long-term non-progressors (LTNPs). Among HIV-1 proteins, the envelope glycoprotein complex (Env) has been concentrated on in many studies for its important role in the immune response and in the first steps of viral replication. In this study, we analyzed the contribution of 41 Envs from 24 patients with different clinical progression rates and viral loads (VLs), LTNP-Elite Controllers (LTNP-ECs); Viremic LTNPs (vLTNPs), and non-controller individuals contemporary to LTNPs or recent, named Old and Modern progressors. We studied the Env expression, the fusion and cell-to-cell transfer capacities, as well as viral infectivity. The sequence and phylogenetic analysis of Envs were also performed. In every functional characteristic, the Envs from subjects with viral control (LTNP-ECs and vLTNPs) showed significant lower performance compared to those from the progressor individuals (Old and Modern). Regarding sequence analysis, the variable loops of the gp120 subunit of the Env (i.e., V2, V4, and mainly V5) of the progressor individuals showed longer and more glycosylated sequences than controller subjects. Therefore, HIV-1 Envs from virus of patients presenting viremic control and the non-progressor clinical phenotype showed poor viral functions and shorter sequences, whereas functional Envs were associated with virus of patients lacking virological control and with progressor clinical phenotypes. These correlations support the role of Env genotypic and phenotypic characteristics in the in vivo HIV-1 infection and pathogenesis.This work is supported by Spanish AIDS network “Red Temática Cooperativa de Investigación en SIDA” RD12/0017/0002, RD12/0017/0028, RD12/0017/0034, RD16/0025/0011, RDCIII16/0002/0005, and RD16/0025/0041 as part of the Plan Nacional R + D + I and cofunded by Spanish “Instituto de Salud Carlos III (ISCIII)-Subdirección General de Evaluación y el Fondo Europeo de Desarrollo Regional (FEDER).” JB is a researcher from “Fundació Institut de Recerca en Ciències de la Salut Germans Trias i Pujol” supported by the Health Department of the Catalan Government/Generalitat de Catalunya and ISCIII grant numbers PI17/01318 and PI20/00093 (to JB). Work in CL-G and CC lab was supported by grants SAF (2010-17226) and (2016-77894-R) from MINECO (Spain) and FIS (PI 13/02269 and PI20/00093, ISCIII). AV-F’s Lab is supported by the European Regional Development Fund (ERDF), RTI2018-093747-B-100 (“Ministerio de Ciencia e Innovación,” Spain), “Ministerio de Ciencia, Innovación y Universidades” (Spain), ProID2020010093 (“Agencia Canaria de Investigación, Innovación y Sociedad de la Información” and European Social Fund), UNLL10-3E-783 (ERDF and “Fundación CajaCanarias”) and “SEGAI-ULL.” SP-Y is funded by “Fundación Doctor Manuel Morales” (La Palma, Spain) and “Contrato Predoctoral Ministerio-ULL Formación de Doctores” (2019 Program; “Ministerio de Ciencia, Innovación y Universidades,” Spain). RC-R is funded by RD16/0025/0011 and ProID2020010093 (“Agencia Canaria de Investigación, Innovación y Sociedad de la Información” and European Social Fund). JE-H is funded by the Cabildo Tenerife “Agustin de Betancourt” 2017 Program.S

Gelsolin activity controls efficient early HIV-1 infection

HIV-1 entry into target lymphocytes requires the activity of actin adaptors that stabilize and reorganize cortical F-actin, like moesin and filamin-A. These alterations are necessary for the redistribution of CD4-CXCR4/CCR5 to one pole of the cell, a process that increases the probability of HIV-1 Envelope (Env)-CD4/co-receptor interactions and that generates the tension at the plasma membrane necessary to potentiate fusion pore formation, thereby favouring early HIV-1 infection. However, it remains unclear whether the dynamic processing of F-actin and the amount of cortical actin available during the initial virus-cell contact are required to such events

Nanocolumnar coatings on implants exhibiting antibacterial properties

Trabajo presentado en la 2nd International Conference on Nanomaterials Applied to Life Sciences 2020 (NALS 2020), celebrada en Madrid (España), del 29 al 31 de enero de 2020Addressing the problem of infection from the very first stage, i.e. inhibiting the formation of the bacterial biofilm, is a crucial step to prevent implant rejection. Nanocolumnar coatings exhibiting antibacterial properties have been fabricated by oblique deposition with magnetron sputtering [1]. The formation of nanocolumns (Fig.1) is the result of the effects of atomic shadowing when the atoms reach the surface along an inclined direction [2]. This technique is environmentally friendly: it is carried out at RT and does not involve chemical products (no recycling problems). Such methodology have been tested in a semiindustrial scale reactor, successfully coating in a single step the two sides of fixation plates for bone fractures [3]. Several in vitro experiments have been performed: analysis of bacterial adhesion and biofilm formation, analysis of osteoblast proliferation and mitochondrial activity, and osteoblasts–bacteria competitive growth scenarios, the latter also named “Race for the Surface” competition. In all these cases, the coatings show an opposite behavior toward osteoblast and bacterial proliferation [1,3]. Moreover, they are effective against Gram positive (S. aureus) and Gram negative (E. coli) bacteria [4]. Finally, when a synergic route is followed and the coatings are functionalized with Te nanorods, the antibacterial properties are enhanced, since Te adds contact-killing (Fig. 2), i.e. bactericidal effect, whilst the biocompatibility is preserved [4].MINECO and Fundación Domingo Martínez for funding. J.M.G.-M. thanks the Fulbright Commissio