Thermodynamic Efficiency of Somatic Exocytosis of Serotonin

Through somatic exocytosis neurons liberate immense amounts of transmitter molecules that modulate the functioning of the nervous system. A stream of action potentials triggers an ATP-dependent transport of transmitter-containing vesicles to the plasma membrane, that ends with a large-scale exocytosis. It is commonly assumed that biological processes use metabolic energy with a high thermodynamic efficiency, meaning that most energy generates work with minor dissipation. However, the intricate ultrastructure underlying the pathway for the vesicle flow necessary for somatic exocytosis challenges this possibility. To study this problem here we first applied thermodynamic theory to quantify the efficiency of somatic exocytosis of the vital transmitter serotonin. Then we correlated the efficiency to the ultrastructure of the transport pathway of the vesicles. Exocytosis was evoked in cultured Retzius neurons of the leech by trains of 10 impulses delivered at 20 Hz. The kinetics of exocytosis was quantified from the gradual fluorescence increase of FM1-43 dye as it became incorporated into vesicles that underwent their exo-endocytosis cycle. By fitting a model of the vesicle transport carried by motor forces to the kinetics of exocytosis, we calculated the thermodynamic efficiency of the ATP expenses per vesicle, as the power of the transport divided by total energy ideally produced by the hydrolysis of ATP during the process. The efficiency was remarkably low (0.1-6.4%) and the values formed a W-shape distribution with the transport distances of the vesicles. Electron micrographs and fluorescent staining of the actin cortex indicated that the slopes of the W chart could be explained by the interaction of vesicles with the actin cortex and the calcium-releasing endoplasmic reticulum. We showed that the application of thermodynamic theory permitted to predict aspects of the intracellular structure. Our results suggest that the distribution of subcellular structures that are essential for somatic exocytosis abates the thermodynamic efficiency of the transport by hampering vesicle mobilization. It is remarkable that the modulation of the nervous system occurs at the expenses of an efficient use of metabolic energy

Cycling of Dense Core Vesicles Involved in Somatic Exocytosis of Serotonin by Leech Neurons

We studied the cycling of dense core vesicles producing somatic exocytosis of serotonin. Our experiments were made using electron microscopy and vesicle staining with fluorescent dye FM1-43 in Retzius neurons of the leech, which secrete serotonin from clusters of dense core vesicles in a frequency-dependent manner. Electron micrographs of neurons at rest or after 1 Hz stimulation showed two pools of dense core vesicles. A perinuclear pool near Golgi apparatuses, from which vesicles apparently form, and a peripheral pool with vesicle clusters at a distance from the plasma membrane. By contrast, after 20 Hz electrical stimulation 47% of the vesicle clusters were apposed to the plasma membrane, with some omega exocytosis structures. Dense core and small clear vesicles apparently originating from endocytosis were incorporated in multivesicular bodies. In another series of experiments, neurons were stimulated at 20 Hz while bathed in a solution containing peroxidase. Electron micrographs of these neurons contained gold particles coupled to anti-peroxidase antibodies in dense core vesicles and multivesicular bodies located near the plasma membrane. Cultured neurons depolarized with high potassium in the presence of FM1-43 displayed superficial fluorescent spots, each reflecting a vesicle cluster. A partial bleaching of the spots followed by another depolarization in the presence of FM1-43 produced restaining of some spots, other spots disappeared, some remained without restaining and new spots were formed. Several hours after electrical stimulation the FM1-43 spots accumulated at the center of the somata. This correlated with electron micrographs of multivesicular bodies releasing their contents near Golgi apparatuses. Our results suggest that dense core vesicle cycling related to somatic serotonin release involves two steps: the production of clear vesicles and multivesicular bodies after exocytosis, and the formation of new dense core vesicles in the perinuclear region

Thermodynamic Efficiency of Somatic Exocytosis of Serotonin

Through somatic exocytosis neurons liberate immense amounts of transmitter molecules that modulate the functioning of the nervous system. A stream of action potentials triggers an ATP-dependent transport of transmitter-containing vesicles to the plasma membrane, that ends with a large-scale exocytosis. It is commonly assumed that biological processes use metabolic energy with a high thermodynamic efficiency, meaning that most energy generates work with minor dissipation. However, the intricate ultrastructure underlying the pathway for the vesicle flow necessary for somatic exocytosis challenges this possibility. To study this problem here we first applied thermodynamic theory to quantify the efficiency of somatic exocytosis of the vital transmitter serotonin. Then we correlated the efficiency to the ultrastructure of the transport pathway of the vesicles. Exocytosis was evoked in cultured Retzius neurons of the leech by trains of 10 impulses delivered at 20 Hz. The kinetics of exocytosis was quantified from the gradual fluorescence increase of FM1-43 dye as it became incorporated into vesicles that underwent their exo-endocytosis cycle. By fitting a model of the vesicle transport carried by motor forces to the kinetics of exocytosis, we calculated the thermodynamic efficiency of the ATP expenses per vesicle, as the power of the transport divided by total energy ideally produced by the hydrolysis of ATP during the process. The efficiency was remarkably low (0.1–6.4%) and the values formed a W-shape distribution with the transport distances of the vesicles. Electron micrographs and fluorescent staining of the actin cortex indicated that the slopes of the W chart could be explained by the interaction of vesicles with the actin cortex and the calcium-releasing endoplasmic reticulum. We showed that the application of thermodynamic theory permitted to predict aspects of the intracellular structure. Our results suggest that the distribution of subcellular structures that are essential for somatic exocytosis abates the thermodynamic efficiency of the transport by hampering vesicle mobilization. It is remarkable that the modulation of the nervous system occurs at the expenses of an efficient use of metabolic energy

Análisis por elementos finitos de losas de edificación sometidas a punzonamiento

Comunicación presentada en el VII Congreso Trienal de la Asociación Científico-Técnica del Hormigón Estructural (ACHE), A Coruña, 20-22 junio 2017.Uno de los principales problemas en las estructuras de edificación es el debido al punzonamiento en el encuentro losa-pilar. En el presente trabajo se expone un modelo numérico no lineal basado en método de los elementos finitos, y desarrollado en ABAQUS, para el análisis de la rotura por punzonamiento en losas de hormigón armado. El objeto es disponer de una herramienta de simulación que permita el estudio de forjados existentes, para la evaluación de su capacidad a punzonamiento y de un posible refuerzo. También el estudio de forjados de nueva construcción, donde se pueda optimizar la geometría y las cuantías.One of the main building structure problems is the phenomenon of punching in the slab-column connection. In this paper a nonlinear numerical model based on finite element method is exposed, and developed in ABAQUS, for the analysis of punching failure of reinforced concrete slabs. The goal is to achieve simulation tool that allows the study of existing slabs, to assess their ability to punching and a possible reinforcement. The study also concerns new constructions, where geometry and ratios can be optimized

Vacuum tribological behaviour of self lubricant quasicrystalline composite coatings

High temperature resistant self-lubricant coatings are needed in space vehicles for components that operate at high temperatures and/or under vacuum. Thick composite lubricant coatings containing quasicrystalline alloys (QC) as the hard phase for wear resistance, have been deposited by thermal spray. The coatings also comprise lubricating materials (silver and BaF2-CaF2 eutectic) and NiCr as the tough component. This paper describes the vacuum tribological properties of TH103, a coating belonging to this family, with excellent microstructural quality. The coating was deposited by HVOF and tested under vacuum on a pin-on-disc tribometer. Different loads, linear speeds and pin materials were studied. The pin scars and disc wear tracks were characterized by EDS-SEM. A minimum mean steady friction coefficient of 0.32 was obtained employing a X-750 Ni superalloy pin in vacuum conditions under 10 N load and 15 cm/s linear speed, showing moderate wear of the disc and low wear of the pin

Biophysics of active vesicle transport, an intermediate step that couples excitation and exocytosis of serotonin in the neuronal soma

Transmitter exocytosis from the neuronal soma is evoked by brief trains of high frequency electrical activity and continues for several minutes. Here we studied how active vesicle transport towards the plasma membrane contributes to this slow phenomenon in serotonergic leech Retzius neurons, by combining electron microscopy, the kinetics of exocytosis obtained from FM1-43 dye fluorescence as vesicles fuse with the plasma membrane, and a diffusion equation incorporating the forces of local confinement and molecular motors. Electron micrographs of neurons at rest or after stimulation with 1 Hz trains showed cytoplasmic clusters of dense core vesicles at 1.5±0.2 and 3.7±0.3 µm distances from the plasma membrane, to which they were bound through microtubule bundles. By contrast, after 20 Hz stimulation vesicle clusters were apposed to the plasma membrane, suggesting that transport was induced by electrical stimulation. Consistently, 20 Hz stimulation of cultured neurons induced spotted FM1-43 fluorescence increases with one or two slow sigmoidal kinetics, suggesting exocytosis from an equal number of vesicle clusters. These fluorescence increases were prevented by colchicine, which suggested microtubule-dependent vesicle transport. Model fitting to the fluorescence kinetics predicted that 52-951 vesicles/cluster were transported along 0.60-6.18 µm distances at average 11-95 nms−1 velocities. The ATP cost per vesicle fused (0.4-72.0), calculated from the ratio of the ΔGprocess/ΔGATP, depended on the ratio of the traveling velocity and the number of vesicles in the cluster. Interestingly, the distance-dependence of the ATP cost per vesicle was bistable, with low energy values at 1.4 and 3.3 µm, similar to the average resting distances of the vesicle clusters, and a high energy barrier at 1.6-2.0 µm. Our study confirms that active vesicle transport is an intermediate step for somatic serotonin exocytosis by Retzius neurons and provides a quantitative method for analyzing similar phenomena in other cell types

Metabolic channeling of phe for lignin biosynthesis in maritime pine

Phenylalanine (Phe) is the main precursor of phenylpropanoids biosynthesis in plants. This vast family of Phe-derived compounds can represent more than 30% of captured photosynthetic carbon, playing essential roles in plants such as cell wall components, defense molecules, pigments and flavors. In addition to its physiological importance, phenylpropanoids and particularly lignin, a component of wood, are targets in plant biotechnology. The arogenate pathway has been proposed as the main pathway for Phe biosynthesis in plants (Maeda et al., 2010). The final step in Phe biosynthesis, catalyzed by the enzyme arogenate dehydratase (ADT), has been considered as a key regulatory point in Phe biosynthesis, due to its key branch position in the pathway, the multiple isoenzymes identified in plants and the existence of a feedback inhibition mechanism by Phe. So far, the regulatory mechanisms underlying ADT genes expression have been poorly characterized, although a strong regulation of the Phe metabolic flux should be expected depending on its alternative use for protein biosynthesis versus phenylpropanoid biosynthesis. This second fate involves a massive carbon flux compared to the first one. Here we report our current research activities in the transcriptional regulation of ADT genes by MYB transcription factors in Pinus pinaster. The conifers channels massive amounts of photosynthetic carbon for phenylpropanoid biosynthesis during wood formation. We have identified the complete ADT gene family in maritime pine (El-Azaz et al., 2016) and a set of ADT isoforms specifically related with the lignification process. The potential control of transcription factors previously reported as key regulators in pine wood formation (Craven-Bartle et al., 2013) will be presented.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Nitrogen metabolism in forest trees

NITROGEN METABOLISM IN FOREST TREES Francisco Cánovas, Concepción Ávila, Fernando N de la Torre, Rafael Cañas, Belén Pascual. Universidad de Málaga Email:[email protected] Forests are essential components of the ecosystems covering approximately one-third of the Earth’s land area and playing a fundamental role in the regulation of terrestrial carbon sinks. Forest trees are also of significant economic importance, as they are used for timber and paper production worldwide. A sustainable management of forest resources is needed to preserve natural forest and to meet the increasing international demands in the production of wood and the other forest-derived products. New advances and developments in biotechnology will contribute to accelerate the domestication of important traits for forest productivity. It is critical to identify the fundamental constraints on forest productivity to addressing these constraints with modern genomic tools. Nitrogen availability extremely low in forest ecosystems, and consequently, forest trees have evolved adaptive mechanism and biotic interactions to guarantee the strict economy of this essential nutrient. Nitrogen assimilation and recycling play a key role in the tree growth and biomass production and we firmly believe that knowledge on nitrogen metabolism will lead to approaches aimed at increasing forest productivity. In our laboratory, we are interested in studying nitrogen metabolism and its regulation the conifer maritime pine (Pinus pinaster Aiton), a forest tree species of great economic and ecological importance in the Mediterranean area and relevant model for conifer genomic research in Europe. Current research efforts are focused on improving the understanding of the response of conifer trees to ammonium availability and the transcriptional control of ammonium assimilation into amino acids. An overview and update of our research programme will be presented and discussed. Research supported by Spanish Ministry of Economy and Competitiveness and Junta de Andalucía (Grants BIO2012-33797, PLE2009-016 and research group BIO-114).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Vacuum Tribological Behaviour of Self-Lubricating Quasicrystalline Composite Coatings

High-temperature-resistant self-lubricating coatings are needed in space vehicles for components that operate at high temperatures and/or under vacuum. Thick composite lubricant coatings containing quasicrystalline alloys as the hard phase for wear resistance can be deposited by a thermal spray technique. The coatings also contain lubricating materials (silver and BaF2CaF2 eutectic) and NiCr as the tough component. This paper describes the vacuum tribological properties of TH103, a coating of this type, with a very good microstructural quality. The coating was deposited by high-velocity oxygen fuel spraying and tested under vacuum using a pin-on-disc tribometer. Different loads, linear speeds, and pin materials were studied. The pin scars and disc wear tracks were characterised using a combination of scanning electron microscopy and energy dispersive spectrometry. A minimum mean steady friction coefficient of 0.32 was obtained when employing an X750 Ni superalloy pin in vacuum conditions under 10 N load and 15 cm/s linear speed, showing moderate wear of the disc and low wear of the pi