Optical Tweezers applied to biology

La técnica de pinzas ópticas (también llamadas trampasópticas o láser) combinadas con técnicas de seguimientode partícula única permiten manipular objetos en laescala nanométrica y determinar fuerzas del orden delpico Newton. Dado que los sistemas biológicos seorganizan con esa precisión espacial mediante fuerzas deinteracción de dicho orden de magnitud, la metodologíapermite interrumpir o imitar procesos biológicos y asícomprender los mecanismos y los principios que losrigen.La trampa se genera en un punto en el espacio en el queun haz láser es enfocado, debido a la interacción de laradiación electromagnética con la partícula a atrapar.Diferentes diseños experimentales han aportado valiosainformación relacionada con el funcionamiento y laenergética de procesos realizados por motoresmoleculares, el plegamiento de diversas macromoléculasasí como la plasticidad de la membrana celular.Aquí se resumirán los fundamentos y montajes de latécnica y sus posibles aplicaciones en biología.Optical tweezers together with single particle tracking permit to manipulate objects with a nanometer precision,and determining forces in the order of the piconewton. Sincethe distances involved in the organization of biological systems are nanometric, and the organization is governed by interaction strengths in the order of the piconewton, the methodology have been widely used in the study of biological processes. The trap is generated as a consequence of the interaction of the trapped particle with a highly focused laser beam. Valuable information regarding the mechanisms and energetics related with molecular machines has been obtained using different experimental approaches. Also, this methodology has shed light on the folding of macromolecules, cell membrane rheology, among other important aspects of cellular function. Here, the fundaments and setup of the technique are briefly commented, and applications in the biological field are described.Fil: Wilke, Natalia. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Quimica Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Química Biológica de Córdoba (p); Argentin

Lipid monolayers at the air-water interface: A tool for understanding electrostatic interactions and rheology in bio-membranes

Monomolecular films of surfactants at the air-water interface are easy to prepare and to handle, and enable a broad variety of techniques to be used. As in other model-systems mimicking membranes, two-phases are observed in several experimental conditions. This Chapter compares the results found using this model membrane with other models and describes some of the techniques applicable to lipid monolayers. The factors underlying their texture when two phases coexist are summarized, with special attention to line tension, an important parameter in both nucleation and growth, as well as the final domain shape. Finally, the effects of the presence of two-phases on the observed mechanical properties of the film (elastic compressibility and shear viscosity) are detailed.Fil: Wilke, Natalia. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina.Fil: Wilke, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Química Biológica de Córdoba, Argentina.Biofísic

On the coupling between mechanical properties and electrostatics in biological membranes

Cell membrane structure is proposed as a lipid matrix with embedded proteins, and thus, their emerging mechanical and electrostatic properties are commanded by lipid behavior and their interconnection with the included and absorbed proteins, cytoskeleton, extracellular matrix and ionic media. Structures formed by lipids are soft, dynamic and viscoelastic, and their properties depend on the lipid composition and on the general conditions, such as temperature, pH, ionic strength and electrostatic potentials. The dielectric constant of the apolar region of the lipid bilayer contrasts with that of the polar region, which also differs from the aqueous milieu, and these changes happen in the nanometer scale. Besides, an important percentage of the lipids are anionic, and the rest are dipoles or higher multipoles, and the polar regions are highly hydrated, with these water molecules forming an active part of the membrane. Therefore, electric fields (both, internal and external) affects membrane thickness, density, tension and curvature, and conversely, mechanical deformations modify membrane electrostatics. As a consequence, interfacial electrostatics appears as a highly important parameter, affecting the membrane properties in general and mechanical features in particular. In this review we focus on the electromechanical behavior of lipid and cell membranes, the physicochemical origin and the biological implications, with emphasis in signal propagation in nerve cells.Fil: Galassi, Vanesa Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto Interdisciplinario de Ciencias Básicas. - Universidad Nacional de Cuyo. Instituto Interdisciplinario de Ciencias Básicas; ArgentinaFil: Wilke, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentin

Rheological properties of a two phase lipid monolayer at the air/water interface: Effect of the composition of the mixture

Many biologically relevant monolayers show coexistence of discrete domains of a long-range ordered condensed phase dispersed in a continuous, disordered, liquid-expanded phase. In this work, we determined the viscous and elastic components of the compressibility modulus and the shear viscosity of monolayers exhibiting phase coexistence with the aim at elucidating the contribution of each phase to the observed monolayer mechanical properties. To this purpose, mixed monolayers with different proportions of distearoylphosphatidylcholine (DSPC) and dimyristoylphosphatidylcholine (DMPC) were prepared and their rheological properties were analyzed. The relationship between the phase diagram of the mixture at 10 mN m-1 and the rheological properties was studied. We found that the monolayer shear viscosity is highly dependent on the presence of domains and on the domain density. In turn, the monolayer compressibility is only influenced by the presence of domains for high domain densities. For monolayers that look homogeneous on the micrometer scale (DSPC amount lower that 23 mol %), all the analyzed rheological properties remain similar to those observed for pure DMPC monolayers, indicating that in this proportion range the DSPC molecules contribute as DMPC to the surface rheology in spite of having hydrocarbon chains four carbons longer.Fil: Wilke, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Vega Mercado, Franco. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Maggio, Bruno. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentin

Evidence of tryglyceride-phase incorporation into artificial bilayers for studying lipid droplets biogenesis

Lipid Droplets (LD) are intracelular structures consisting on an apolar lipid core - composed mainly of triglycerides (TG) and steryl esters- which is surrounded by a phospholipid and protein monolayer. LDs originate in the ER bilayer, where TG synthesis concludes. The mechanisms underlying TG nucleation, size maduration and budding-off from the ER membrane are a matter of current investigations and the role of dewetting from cytosolic-bilayer interface appears to play a critical role. In order to contrast the nano-sized "blisters" of TG that some authors predict1, here we formed free-standing bilayers by transferring films of a monolayer of mixed phosphatidylcholine(EPC)/TG in coexistence with TG microlenses (i.e. an excluded TG phase floating in the surface). These membranes were characterized by adding them the solvatochromic fluorescent probe Nile Red (NR) and observing them under spectral confocal microscope. Such bilayers exhibit fluorescence emission spectra comparable of bilayers of vesicles with similar composition (POPC and TO). By comparison with literature data and fluorescence spectra of EPC and TG monolayers, the peaks could be assigned to different phases, namely 1) PC membranes (λemmax=630 nm ) bilayer and bilayer) and 2) TG isotropic phase (λemmax=570 nm ). No microscopic structures could be observed at λ emmax=570 nm. Diffusion of NR under this TG phase was characterized using FRAP analysis yielding values (D=2 μm2s ) typical of model bilayer membranes, suggesting that the probe is diffusing in a 2D structure. This system appears appropiate for describing which is the distribution of the TG phase, that is, homogeneously among the intrabilayer space or in nanoscopic "blisters", by evaluating diffusion times obtained by FCS and FRAP.Fil: Caruso, Benjamin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; ArgentinaFil: Mangiarotti, Agustín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Wilke, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Perillo, Maria Angelica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; ArgentinaXLVII Reunión Anual de la Sociedad Argentina de BiofísicaLa PlataArgentinaSociedad Argentina de BiofísicaUniversidad Nacional de La plata. Facultad de Ciencias Médica

Zn2+-dependent surface behavior of diacylglycerol pyrophosphate and its mixtures with phosphatidic acid at different pHs

Diacylglycerol pyrophosphate (DGPP) is a minor lipid that attenuates the phosphatidic acid (PA) signal, and also DGPP itself would be a signaling lipid. DGPP is an anionic phospholipid with a pyrophosphate group attached to diacylglycerol that was shown to respond to changes of pH, thus affecting the surface organization of DGPP and their interaction with PA. In this work, we have investigated how the presence of Zn2+, modulates the surface organization of DGPP and its interaction with PA at acid and basic pHs. Both lipids formed expanded monolayers at pH 5 and 8. At pH 5, monolayers formed by DGPP became stiffer when Zn2+was added to the subphase,  while the surface potential decreased. At this pH, Zn2+ induced a phase transition from an expanded to a condensed phase state in monolayers formed by PA. Conversely, at pH 8 the effects induced by the presence of Zn2+ on the surface behaviors of the pure lipids were smaller. Thus, the interaction of the bivalent cation with both lipids was modulated by pH and by the ionization state of the polar head-groups. Mixed monolayers of PA and DGPP showed a non-ideal behavior and were not affected by the presence of Zn2+ at pH 8. This could be explained considering that when mixed, the lipids formed a close packed monolayer that could not be further modified by the cation. Our results indicate that DGPP and PA exhibit expanded and condensed phase states depending on pH, on the proportion of each lipid in the film and on the presence of Zn2+. This may have implications for a possible role of DGPP as a signaling lipid molecule.Fil: Villasuso, Ana Laura. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Wilke, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Maggio, Bruno. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Machado, Estela Edelmira. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentin

Implementación de actividades de laboratorios en el nivel educativo primario

En este trabajo comentamos la experiencia adquirida durante el proyecto de articulación financiado por la Facultad de Ciencias Químicas de la Universidad Nacional de Córdoba durante el 2017 titulado "Implementación de actividades de laboratorios en Ciencias Naturales en el Nivel Educativo Primario". El proyecto se focalizó en la implementación de un laboratorio ambulante (que pueda ser transportado a las aulas) que abarque las distintas disciplinas de las Ciencias Naturales y en el diseño de Actividades didácticas en los niveles de 4to, 5to y 6to grado, en conjunto con docentes de diferentes escuelas, aprovechando el material de Laboratorio y equipamiento suministrado por la Presidencia de la Nación en el "Plan Nacional de enseñanza de las Ciencias Naturales para Escuela Primaria (año 2014), Modulo B". Se abordó la problemática a través de talleres con docentes de cuatro Escuelas Provinciales de la Provincia de Córdoba de nivel primario. Aquí describimos el desarrollo del proyecto, el porcentaje de su cumplimiento y los obstáculos atravesados. El resultado de la experiencia del trabajo conjunto se plasmó en el libro "Guía de actividades de laboratorios en ciencias naturales para escuelas primarias", escrito por las Dras. María Laura Fanani y Natalia Wilke, el cual tiene como objetivo servir de consulta para el desarrollo de las actividades de laboratorio en futuros ciclos lectivos.Fil: Fanani, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Wilke, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentin

Surface charge density and fatty acids enhance the membrane permeation rate of CPP–cargo complexes

The CPP-effect makes reference to the process by which the membrane translocation rate of a cargo is enhanced by chemical functionalization with cell-penetrating peptides (CPPs). In this work we combine a simple kinetic model with free-energy calculations to explore the energetic basis of the CPP-effect. Two polyglicines are selected as model hydrophilic cargoes, and nona-arginine as a prototypical CPP. We assess the cargo carrying efficiency of nona-arginine by comparing the adsorption and insertion energies of the cargoes, the cargo-free CPPs, and the CPP-cargo complexes, into lipid membranes of varying composition. We also analyze the effect of modifying the type and concentration of anionic lipids, and the implication of these factors on the translocation rate of the CPP-cargo complex. Of particular interest is the evaluation of the catalytic role of palmitic acid (palmitate) as a promoter of the CPP-effect. We also analyse the influence of the size of the cargo on the membrane adsorption and insertion energies. Our results show that the efficiency of nona-arginine as a transmembrane carrier of simple hydrophilic molecules is modulated by the size of the cargo, and is strongly enhanced by increasing the concentration of anionic lipids and of ionized fatty acids in the membrane.Fil: Via, Matías Alejandro. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza; ArgentinaFil: Wilke, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Mayorga, Luis Segundo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: del Popolo, Mario Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto Interdisciplinario de Ciencias Básicas. - Universidad Nacional de Cuyo. Instituto Interdisciplinario de Ciencias Básicas; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; Argentin

Complex Spatial Dynamics of Oncolytic Viruses In Vitro: Mathematical and Experimental Approaches

Oncolytic viruses replicate selectively in tumor cells and can serve as targeted treatment agents. While promising results have been observed in clinical trials, consistent success of therapy remains elusive. The dynamics of virus spread through tumor cell populations has been studied both experimentally and computationally. However, a basic understanding of the principles underlying virus spread in spatially structured target cell populations has yet to be obtained. This paper studies such dynamics, using a newly constructed recombinant adenovirus type-5 (Ad5) that expresses enhanced jellyfish green fluorescent protein (EGFP), AdEGFPuci, and grows on human 293 embryonic kidney epithelial cells, allowing us to track cell numbers and spatial patterns over time. The cells are arranged in a two-dimensional setting and allow virus spread to occur only to target cells within the local neighborhood. Despite the simplicity of the setup, complex dynamics are observed. Experiments gave rise to three spatial patterns that we call “hollow ring structure”, “filled ring structure”, and “disperse pattern”. An agent-based, stochastic computational model is used to simulate and interpret the experiments. The model can reproduce the experimentally observed patterns, and identifies key parameters that determine which pattern of virus growth arises. The model is further used to study the long-term outcome of the dynamics for the different growth patterns, and to investigate conditions under which the virus population eliminates the target cells. We find that both the filled ring structure and disperse pattern of initial expansion are indicative of treatment failure, where target cells persist in the long run. The hollow ring structure is associated with either target cell extinction or low-level persistence, both of which can be viewed as treatment success. Interestingly, it is found that equilibrium properties of ordinary differential equations describing the dynamics in local neighborhoods in the agent-based model can predict the outcome of the spatial virus-cell dynamics, which has important practical implications. This analysis provides a first step towards understanding spatial oncolytic virus dynamics, upon which more detailed investigations and further complexity can be built