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[Background]: Fibroblast growth factors (Fgfs) are required for survival and organ formation during embryogenesis. Fgfs often execute their functions redundantly. Previous analysis of Fgf3 mutants revealed effects on inner ear formation and embryonic survival with incomplete penetrance. [Results]: Here, we show that presence of a neomycin resistance gene (neo) replacing the Fgf3 coding region leads to reduced survival during embryogenesis and an increased penetrance of inner ear defects. Fgf3neo/neo mutants showed reduced expression of Fgf4, which is positioned in close proximity to the Fgf3 locus in the mouse genome. Conditional inactivation of Fgf4 during inner ear development on a Fgf3 null background using Fgf3/4 cis mice revealed a redundant requirement between these Fgfs during otic placode induction. In contrast, inactivation of Fgf3 and Fgf4 in the pharyngeal region where both Fgfs are also co-expressed using a Foxg1-Cre driver did not affect development of the pharyngeal arches. However, these mutants showed reduced perinatal survival. [Conclusions]: These results highlight the importance of Fgf signaling during development. In particular, different members of the Fgf family act redundantly to guarantee inner ear formation and embryonic survival., Consejería de Educación, Junta de Castilla y León, Grant/Award Number: CSI143P20; Programa Estratégico Instituto de Biología y Genética Molecular (IBGM), Escalera de Excelencia, Junta de Castilla y León, Grant/Award Numbers: CCVC8485, CLU-2019-02; MEC, Grant/Award Number: BFU2004-00860/BFI

The glass cutting problem proposed by Saint Gobain for the 2018 ROADEF challenge includes some specific constraints that prevent the direct application of procedures developed for the standard cutting problem. On the one hand, the sheets to be cut have defects that make them unique and they must be used in a given order. On the other hand, pieces are grouped in stacks and the pieces in each stack must be cut in order. There are also some additional characteristics due to the technology being used, especially the requirement for a three-stage guillotine cutting process. Taking into account the sequencing constraints on sheets and pieces, we have developed a beam search algorithm, using a tree structure in which at each level the partial solution is increased by adding some new elements until a complete solution is built. We have developed a randomized constructive algorithm for building these new elements and explored several alternatives for the local and the global evaluation. An improvement procedure, specifically designed for the problem, has also been added. The computational study, using the datasets provided by the company, shows the efficiency of the proposed algorithm for short and long running times.

Container loading problems (CLP), in which a set of boxes have to be loaded into containers or onto trucks, are at the core of many transportation problems. Good solutions to these packing problems are crucial for the efficient use of logistic resources. However, to produce solutions that are useful in practice, besides the basic conditions ensuring that boxes do not exceed the container dimensions and do not overlap each other, other physical and logistic constraints have to be included when solving the CLP. In this study, we focus on a special type of logistic constraint: complete shipment constraints, which ensure that either all or none of the boxes in each customer order are loaded. Although these constraints have seldom been considered in the literature, they arise very often in practice. Customers do not want to receive incomplete parts of their orders in successive deliveries and retailers do not want to have to store incomplete orders and wait until the remaining boxes arrive to deliver the orders to the customers. We include these constraints in an integer linear formulation of the CLP and develop four heuristic strategies to deal with them efficiently. An extensive computational study compares the relative performance of these strategies and shows that when a good strategy is used the impact of these constraints on the solution of the CLP is quite small.

Excitability in astroglia is controlled by Ca2+ fluxes from intracellular organelles, mostly from the endoplasmic reticulum (ER). Astrocytic ER possesses inositol 1,4,5-trisphosphate receptors (InsP3R) that can be activated upon stimulation through a vast number of metabotropic G-protein-coupled receptors. By contrast, the role of Ca2+-gated Ca2+ release channels is less explored in astroglia. Here we address this process by monitoring Ca2+ dynamics directly in the cytosol and the ER of astroglial cells. Cultured astrocytes exhibited spontaneous and high-K-evoked cytosolic Ca2+ transients, both of them reversibly abolished by external Ca2+ removal, addition of plasma membrane channel blockers or ER Ca2+ depletion with SERCA inhibitors. Resting astrocyte [Ca2+]ER averaged 400 μM and maximal stimulation with ATP provoked a complete and reversible ER discharge. Direct monitoring of Ca2+ in the lumen of ER showed that high-K induced a Ca2+ release from the ER, and its amplitude was proportional to the [K]. Furthermore, by combining the low affinity GAP3 indicator targeted to the ER with the high affinity cytosolic Rhod-2, we simultaneously imaged ER- and cytosolic-Ca2+ signals, in astrocytes in culture and in situ. Plasma membrane Ca2+ entry triggered a fast ER Ca2+ release coordinated with an increase in cytosolic Ca2+. Thus, we identify a Ca2+-induced Ca2+-release (CICR) mechanism that is likely to participate in spontaneous astroglial oscillations, providing a graded amplification of the cytosolic Ca2+ signal., This work was supported by grants from The Spanish MINECO (BFU2017-83066-P) and the Junta de Castilla y León (GR175). Macarena Rodriguez-Prados and Jonathan Rojo-Ruiz were supported by fellowships from the Spanish MINECO.

Summary Appropriate cristae remodeling is a determinant of mitochondrial function and bioenergetics and thus represents a crucial process for cellular metabolic adaptations. Here, we show that mitochondrial cristae architecture and expression of the master cristae-remodeling protein OPA1 in proopiomelanocortin (POMC) neurons, which are key metabolic sensors implicated in energy balance control, is affected by fluctuations in nutrient availability. Genetic inactivation of OPA1 in POMC neurons causes dramatic alterations in cristae topology, mitochondrial Ca2+ handling, reduction in alpha-melanocyte stimulating hormone (α-MSH) in target areas, hyperphagia, and attenuated white adipose tissue (WAT) lipolysis resulting in obesity. Pharmacological blockade of mitochondrial Ca2+ influx restores α-MSH and the lipolytic program, while improving the metabolic defects of mutant mice. Chemogenetic manipulation of POMC neurons confirms a role in lipolysis control. Our results unveil a novel axis that connects OPA1 in POMC neurons with mitochondrial cristae, Ca2+ homeostasis, and WAT lipolysis in the regulation of energy balance., Graphical abstract, Highlights • Nutritional state shapes mitochondrial cristae and OPA1 expression in POMC neurons • OPA1 deletion in POMC neurons alters mitochondrial Ca2+ handling and α-MSH release • POMC OPA1-deficient mice show impaired fat lipolysis and metabolic health • Pharmacological restoration of Ca2+ recovered molecular and metabolic alterations, Gómez-Valadés et al. report that mitochondrial cristae dynamically reshape in POMC neurons in response to nutritional state. With genetic targeting, they reveal that the cristae-remodeling protein OPA1 is essential to sustain cristae structure and mitochondrial Ca2+ homeostasis in POMC neurons, ensuring appropriate modulation of α-MSH release, the lipolytic program in adipose tissue and metabolic health.

In Spain, water use in agriculture is expected to become limited by resources in the future. It is pertinent to study the effect of decreased irrigation on the presence of pests, plant damage, and arthropod communities in a super-intensive olive orchard examined from 2017 to 2019. Arthropods were studied with visual and vacuum sampling methods in two irrigation treatments (T1—control and T2—Regulated Deficit Irrigation (RDI)). Univariate analyses showed that the total arthropod abundance was significantly greater in T1 than in T2 in 2018 and 2019, mostly due to Diptera Nematocera. Visual sampling revealed that the feeding damage produced by Eriophyidae (Trombidiformes) was significantly lower in T2 in 2018 and 2019: 10–40% of shoots were affected in the late season compared with 50–60% affected for T1. The feeding symptoms caused by Palpita unionalis Hübner (Lepidoptera: Crambidae) and Zelleria oleastrella (Milliere) (Lepidoptera: Yponomeutidae) were significantly less for T2 than for T1. Multivariate principal response curves showed significant differences between irrigation strategies in the 2018 and 2019 data for both sampling methods. In conclusion, irrigation schemes with restricted water use (T2—RDI) help to reduce the abundance of several types of pests in olive crops, especially of those that feed on the plants’ new sprouts.

We consider the multicontainer loading problem of a company that has to serve its customers by first putting the products on pallets and then loading pallets onto trucks. When a large number of units of a product have to be shipped, the company requires that homogeneous pallets, with only one product, are built first, then weakly heterogeneous pallets, in which each layer corresponds to a single product, and finally strongly heterogeneous pallets with the remaining units of the products. To be useful in practice, the solutions have to satisfy five types of constraints: geometric constraints, so that pallets are completely inside the trucks and do not overlap; weight constraints, limiting the total weight a truck can bear and the maximum weight supported by each axle; constraints limiting the position of the centre of gravity of the cargo; dynamic stability constraints, to avoid cargo displacement when the truck is moving; and constraints ensuring that the delivery dates of products are respected. We have developed a Greedy Randomized Adaptive Search Procedure, including some improvement methods tailored to the problem, among them an adaptation of ejection chains. The approach has been tested on a benchmark of real problems and it has been shown to be capable of finding high-quality, realistic solutions in short computing times. We also provide a comparison with an integer programming formulation that justifies the use of a metaheuristic algorithm.

The adenohypophysis contains five secretory cell types (somatotrophs, lactotrophs, thyrotrophs, corticotrophs, and gonadotrophs), each secreting a different hormone, and controlled by different hypothalamic releasing hormones (HRHs). Exocytic secretion is regulated by cytosolic Ca2+ signals ([Ca2+]C), which can be generated either by Ca2+ entry through the plasma membrane and/or by Ca2+ release from the endoplasmic reticulum (ER). In addition, Ca2+ entry signals can eventually be amplified by ER release via calcium-induced calcium release (CICR). We have investigated the contribution of ER Ca2+ release to the action of physiological agonists in pituitary gland. Changes of [Ca2+] in the ER ([Ca2+]ER) were measured with the genetically encoded low-affinity Ca2+ sensor GAP3 targeted to the ER. We used a transgenic mouse strain that expressed erGAP3 driven by a ubiquitous promoter. Virtually all the pituitary cells were positive for the sensor. In order to mimick the physiological environment, intact pituitary glands or acute slices from the transgenic mouse were used to image [Ca2+]ER. [Ca2+]C was measured simultaneously with Rhod-2. Luteinizing hormone-releasing hormone (LHRH) or thyrotropin releasing hormone (TRH), two agonists known to elicit intracellular Ca2+ mobilization, provoked robust decreases of [Ca2+]ER and concomitant rises of [Ca2+]C. A smaller fraction of cells responded to thyrotropin releasing hormone (TRH). By contrast, depolarization with high K+ triggered a rise of [Ca2+]C without a decrease of [Ca2+]ER, indicating that the calcium-induced calcium-release (CICR) via ryanodine receptor amplification mechanism is not present in these cells. Our results show the potential of transgenic ER Ca2+ indicators as novel tools to explore intraorganellar Ca2+ dynamics in pituitary gland in situ.

Rapid plasma membrane repair in response to pore‐forming toxins is crucial for cell survival, but the molecular mechanisms employed by eukaryotic nucleated cells to maintain membrane integrity and the specificities of such pathways remain poorly understood. Here, we have explored the permeabilization elicited by the Bordetella pertussis adenylate cyclase toxin, a 200‐kDa protein toxin with α‐helical pore‐forming domain that forms pores of tunable size, and evaluated the response of target macrophages to such toxin poration. We show here that the response and the fate of target macrophages depend on toxin pore width. We find that the toxin′s hemolysin moiety induces a transient membrane permeabilization by forming wide enough pores allowing Ca2+ influx into the target cell cytosol. This activates a Ca2+‐dependent cellular response involving exocytosis and endocytosis steps eliminating toxin pores and restoring membrane integrity. In contrast, the full‐length native toxin, at low concentrations, forms very small pores that cause insidious perturbation of cell ion homeostasis that escapes control by the macrophage membrane repair response, eventually leading to cell death. Our data reveal that permeability to Ca2+ and ATP are key elements in the membrane repair pathway for eliminating α‐helical pores of bacterial origin., This study was supported by grants from the Basque Government (Grupos Consolidados IT849‐13 and IT1264‐19) and grants from the Spanish Ministerio de Economía y Competitividad (BFU2014‐53469P to MTA A, and BFU‐2017‐82758‐P (AEI/FEDER, UE) to HO, AE was recipient of a fellowship from the University of the Basque Country (UPV/EH). DGB was recipient of a fellowship from the Bizkaia Biophysics Foundation.

Producción Científica, Sarcopenia, the loss of muscle mass and strength associated with age, has been linked to impairment of the cytosolic Ca2+ peak that triggers muscle contraction, but mechanistic details remain unknown. Here we explore the hypothesis that a reduction in sarcoplasmic reticulum (SR) Ca2+ concentration ([Ca2+]SR) is at the origin of this loss of Ca2+ homeostasis. We engineered Drosophila melanogaster to express the Ca2+ indicator GAP3 targeted to muscle SR, and we developed a new method to calibrate the signal into [Ca2+]SR in vivo. [Ca2+]SR fell with age from ∼600 µM to 50 µM in close correlation with muscle function, which declined monotonically when [Ca2+]SR was, Ministerio de Economía, Industria y Competitividad (grant BFU2017-83066-P), Junta de Castilla y León (project GR175)

Resumen: Objetivo: Buscar elementos comparativos para el control de calidad en unidades de cirugía mayor ambulatoria (CMA). Método: Estudio descriptivo comparativo del índice de ambulatorización (IA) y el índice de sustitución (IS) en el Servicio de Cirugía del Hospital Santa Cristina, de Madrid (España), respecto a indicadores clave (IC) del Sistema Nacional de Salud (SNS). Resultados: Se analizaron 7817 procedimientos de CMA (entre 2006 y 2014) y se obtuvo un IA anual medio del 54%, superior (p

We present a randomized clinical trial to compare postoperative pain, complications, feeling of a foreign body, and recurrence between heavyweight and lightweight meshes in patients with bilateral groin hernia. Sixty-seven patients with bilateral hernia were included in our study. In each patient, the side of the lightweight mesh was decided by random numbers table. Pain score was measured by visual analogue scale, on 1st, 3rd, 5th, and 7th postoperative day, and one year after the surgery. There were no statistically significative differences between both meshes in postoperative complications. About differences of pain average, there were statistically significant differences only on the 1st postoperative day (P

In a setting that prioritises the achievement of Sustainable Development Goals (SDG), sustainable development cannot be achieved without the contribution of female talent. This paper is focused on studying the reasons for the gender gap and the offered strategies to reduce it. The debate on the reasons for the low enrolment rates of girls in technological degrees remains open in the literature. This study investigates the causes of why girls do not choose a computer engineering degree and makes a comparison between ex-ante (secondary school) and ex-post (higher education). Based on a survey of students from both secondary school (229) and higher education (171), a quantitative study on gender differences was performed. The chi-squared test was applied to compute the corresponding p-value. Gender significant differences concerning goals were found (such as, at secondary school, girls preferred to help people, whereas boys preferred to be rich or to have a lot of holidays), and degree preferences (secondary school girls showed great interest in degrees, such as psychology, criminology and medicine, while boys showed great interest in sports sciences and engineering). However, these differences cannot be attributed to the existence of gender stereotypes, to the poor social image of workers in that field, or to the goals to be achieved within this profession. Some proposals are offered to reduce the gap.

Renault, a large car manufacturer with factories all over the world, has a production system in which not every factory produces all the parts required to assemble a vehicle. Every day, large quantities of car parts are sent from one factory to another, defining very large truck/container transportation problems. The main challenge faced by the Renault logistics platforms is to load the items into trucks and containers as efficiently as possible so as to minimize the number of vehicles sent. Therefore, the problem to be solved is a multicontainer loading problem in which, besides the usual geometric constraints preventing items from overlapping and exceeding the dimensions of the container, there are many other constraints, concerning the way in which items are put into layers, layers into stacks and stacks into containers, limiting the total weight and the weight supported by the items. In this paper we propose a GRASP algorithm, including constructive procedures to build solutions satisfying all the constraints, randomization strategies to produce diversity of solutions, and improvement moves to obtain high-quality solutions in short computing times. The algorithm has been tested on a set of real instances provided by the company and the results are competitive with the best results known, including some new improved solutions.

We address the multi container loading problem of a company that serves its customers’ orders by building pallets with the required products and loading them into trucks. The problem is solved by using integer linear models. To be useful in practice, our models consider three types of constraints: geometric constraints, so that pallets lie completely inside the trucks and do not overlap; weight constraints, defining the maximum weights supported by a truck and by each axle, as well as the position of the centre of gravity of the cargo; and dynamic stability constraints. These last constraints forbid empty spaces between pallets to avoid cargo displacement when the truck is moving, and limit differences between the heights of adjacent pallets to prevent tall pallets tipping over short ones. We also consider extensions of the models to the case of heavy loads, requiring a special configuration of the pallets in the truck, and to the case in which the orders must be served over a set of time periods to meet delivery dates. The computational study that we performed on a large number of real instances with up to 44 trucks shows that the proposed models are able to obtain optimal solutions in most cases and very small gaps when optimality could not be proven.

We present a randomized clinical trial to compare postoperative pain, complications, feeling of a foreign body, and recurrence between heavyweight and lightweight meshes in patients with bilateral groin hernia. Sixty-seven patients with bilateral hernia were included in our study. In each patient, the side of the lightweight mesh was decided by random numbers table. Pain score was measured by visual analogue scale, on 1st, 3rd, 5th, and 7th postoperative day, and one year after the surgery. There were no statistically significative differences between both meshes in postoperative complications. About differences of pain average, there were statistically significant differences only on the 1st postoperative day (P0.01) and the 7th postoperative day (P0.05). In the review after a year, there were no statistically significative differences in any parameter. In our study, we did not find statistically significative differences between lightweight and heavyweight meshes in postoperative pain, complications, feeling of a foreign body, and recurrence, except pain on 1st and 7th postoperative day.

Cytosolic Ca2+ signals are often amplified by massive calcium release from the endoplasmic reticulum (ER). This calcium-induced calcium release (CICR) occurs by activation of an ER Ca2+ channel, the ryanodine receptor (RyR), which is facilitated by both cytosolic- and ER Ca2+ levels. Caffeine sensitizes RyR to Ca2+ and promotes ER Ca2+ release at basal cytosolic Ca2+ levels. This outcome is frequently used as a readout for the presence of CICR. By monitoring ER luminal Ca2+ with the low-affinity genetic Ca2+ probe erGAP3, we find here that application of 50 mM caffeine rapidly reduces the Ca2+ content of the ER in HeLa cells by ∼50%. Interestingly, this apparent ER Ca2+ release does not go along with the expected cytosolic Ca2+ increase. These results can be explained by Ca2+ chelation by caffeine inside the ER. Ca2+-overloaded mitochondria also display a drop of the matrix Ca2+ concentration upon caffeine addition. In contrast, in the cytosol, with a low free Ca2+ concentration (10−7 M), no chelation is observed. Expression of RyR3 sensitizes the responses to caffeine with effects both in the ER (increase in Ca2+ release) and in the cytosol (increase in Ca2+ peak) at low caffeine concentrations (0.3–1 mM) that have no effects in control cells. Our results illustrate the fact that simultaneous monitoring of both cytosolic- and ER Ca2+ are necessary to understand the action of caffeine and raise concerns against the use of high concentrations of caffeine as a readout of the presence of CICR., This work was supported by grants from The Spanish MINECO [BFU2017-83066-P] and the Junta de Castilla y León [GR175]. J.R.-R., M.R.-P., and A.D.-L. were supported by fellowships from the Spanish MINECO.

Producción Científica, Aequorins are excellent tools for measuring intra-organellar Ca2+ and assessing its role in physiological and pathological functions. Here we review targeting strategies to express aequorins in various organelles. We address critical topics such as probe affinity tuning as well as normalization and calibration of the signal. We also focus on bioluminescent Ca2+ imaging in nucleus or mitochondria of living cells. Finally, recent advances with a new chimeric GFP-aequorin protein (GAP), which can be used either as luminescent or fluorescent Ca2+ probe, are presented. GAP is robustly expressed in transgenic flies and mice, where it has proven to be a suitable Ca2+ indicator for monitoring physiological Ca2+ signaling ex vivo and in vivo., Ministerio de Economía, Industria y Competitividad (Project BFU2014-53469P), Instituto de Salud Carlos III (TerCel, RD16/0011/0003)

Producción Científica, GFP-Aequorin Protein (GAP) can be used to measure [Ca2 +] inside intracellular organelles, both by luminescence and by fluorescence. The low-affinity variant GAP3 is adequate for ratiometric imaging in the endoplasmic reticulum and Golgi apparatus, and it can be combined with conventional synthetic indicators for simultaneous measurements of cytosolic Ca2 +. GAP is bioorthogonal as it does not have mammalian homologues, and it is robust and functionally expressed in transgenic flies and mice, where it can be used for Ca2 + measurements ex vivo and in vivo to explore animal models of health and disease. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech., Ministerio de Economía, Industria y Competitividad (Project BFU2014-53469P), Instituto de Salud Carlos III (TerCel; RD16/0011/0003)

International audience; Mitochondrial Ca2+ homeostasis is crucial for regulating vital functions such as respiration or apoptosis. Targeted aequorins are excellent probes to measure subcellular Ca2+. Ca2+ concentration in mitochondria ([Ca2+]M) is low at rest (about 10-7 M) and can increase to the micromolar or even approach the millimolar range, upon cell activation. Here we describe a new quantitative luminescent protocol to directly measure mitochondrial Ca2+ uptake, optimized for high throughput. The sensitivity of the method allows detection of changes in either the capacity or the affinity of mitochondrial Ca2+ transport.

This paper deals with the problem of a distribution company that has to serve its customers by putting first the products on pallets and then loading the pallets onto trucks. We approach the problem by developing and solving integer linear models. We start with basic models, that include the essential features of the problem, such as respecting the dimensions of the truck, and not exceeding the total weight capacity and the maximum weigh capacity on each axle. Then, we add progressively new conditions to consider the weight and volume of pallet bases and to include other desirable features for the solutions to be useful in practice, such as the position of the center of gravity and the minimization of the number of pallets. The models have been tested on a large set of real instances involving up to 46 trucks and kindly provided to us by a distribution company. The results show that in most cases the optimal solution can be obtained in small running times. Moreover, when optimality cannot be proven, the gap is very small, so we obtain high quality solutions for all the instances that we tested.

Producción Científica, Signal transduction in sensory neurons of the mammalian vomeronasal organ (VNO) involves the opening of the canonical transient receptor potential channel Trpc2, a Ca2+-permeable cation channel that is activated by diacylglycerol and inhibited by Ca2+-calmodulin. There has been a long-standing debate about the extent to which the second messenger inositol 1,4,5-trisphosphate (InsP3) and type 3 InsP3 receptor (InsP3R3) are involved in the opening of Trpc2 channels and in sensory activation of the VNO. To address this question, we investigated VNO function of mice carrying a knockout mutation in the Itpr3 locus causing a loss of InsP3R3. We established a new method to monitor Ca2+ in the endoplasmic reticulum of vomeronasal sensory neurons (VSNs) by employing the GFP-aequorin protein sensor erGAP2. We also performed simultaneous InsP3 photorelease and Ca2+ monitoring experiments, and analysed Ca2+ dynamics, sensory currents, and action potential or field potential responses in InsP3R3-deficient VSNs. Disruption of Itpr3 abolished or minimized the Ca2+ transients evoked by photoactivated InsP3, but there was virtually no effect on sensory activation of VSNs. Therefore, InsP3R3 is dispensable for primary chemoelectrical transduction in mouse VNO. We conclude that InsP3R3 is not required for gating of Trpc2 in VSNs., Deutsche Forschungsgemeinschaf (grant CH920/2-1), Sonderforschungsbereich 894 (project A17), Transregio - SFB (projects P09 and P10), Ministerio de Economía, Industria y Competitividad (grant BFU2014-53469P)

A distribution company has to send products, packed into shippers, from the warehouse to retail shops. The number of different shipper types is regarded as a parameter given by the user, who is looking for a balance between transportation costs and stock and procurement costs. The problem is to decide the sizes of the shipper types to keep at the warehouse so as to minimize the cost of meeting the forecasted demand over the planning horizon. In this paper, we describe an integer linear programming formulation for the problem and obtaining feasible solutions. Other models, based on multiknapsack and p-median and facility location models, are for obtaining lower bounds. We study several ways of reducing the set of possible shipper types in order to obtain models of a manageable size. The feasible solutions obtained are improved by using a reduced model and metaheuristic algorithm. A computational study conducted on real instances provided by the company is presented and discussed.

Producción Científica, One of the more intriguing subjects in neuroscience is how a precursor or stem cell is induced to differentiate into a neuron. Neurogenesis begins early in brain development and suddenly becomes a very intense process, which is related with the influence of Retinoic Acid. Here, using a biological test (F9-1.8 cells) in chick embryos, we show that ―in vivo‖ embryonic cerebrospinal fluid regulates mesencephalic-rombencephalic Isthmic Retinoic Acid synthesis and this effect has a direct influence on mesencephalic neuroepithelial precursors, inducing a significant increase in neurogenesis. This effect is mediated by the Retinol Binding Protein present in the embryonic cerebrospinal fluid. The knowledge of embryonic neurogenetic stimulus could be useful in the control of adult brain neurogenesis., Ministerio de Educación y Ciencia (BFU207/6516), Junta de Castilla y León (Consejería de Educación, GR195)

When the offeree come up with changes in the offer terms, these can be legally treated as a new offer and this one must be accepted by the first offeror to valide the contract (BGB Proposed modification of the french Civil Code). However, it can be said that an acceptance introducing no substantial changes in the initial offer terms, is effective (proposed modification of the Spanish Civil Code, art. 2 : 208 PECL, art. II. 4 : 208 : DCFR). Legal texts contemplating effectiveness of the modified acceptance want to protect the expectations of the contract that the offer could arise in the offeree stimulating the validation of the contract when the differences are unimportant or accessory. However, the reluctance to take all the consequences from this position are the cause of the introduction of some exceptions that defeat this target., Quand le destinataire d’une offre propose des modifications au contenu de la dite offre, on peut réglementer que c’est une contre-offre et qu’elle doit être acceptée par l’originaire offrant pour conclure le contrat (BGB, proposition de modification du Code civil français, -rapport Catala-). Néanmoins, on peut affirmer que l’acceptation introduisant des modifications non substantielles au contenu de l’offre émise est efficace (Proposition espagnole de modification du Code civil, art. 2 : 208 PECL, art. II. 4 : 208 : DCFR etc.). Les textes qui envisagent la possibilité de l’efficacité de l’acceptation modifiée veulent protéger les attentes du contrat que l’offre a pu susciter chez le bénéficiaire de cette dernière, en favorisant la conclusion du contrat lorsque les divergences sont insignifiantes ou accessoires. Toutefois, les réticences à porter aux dernières conséquences de cette position sont dans l’origine de l’ajout de quelques exceptions qui frustrent l’objectif., Alonso-Pérez Maria-Teresa. Contre-offre versus acceptation modifiée. In: Revue internationale de droit comparé. Vol. 66 N°1,2014. pp. 53-66.

BACKGROUND: Organ dysfunction scores, based on physiological parameters, have been created to describe organ failure. In a general pediatric intensive care unit (PICU) population, the PEdiatric Logistic Organ Dysfunction-2 score (PELOD-2) score had both a good discrimination and calibration, allowing to describe the clinical outcome of critically ill children throughout their stay. This score is increasingly used in clinical trials in specific subpopulation. Our objective was to assess the performance of the PELOD-2 score in a subpopulation of critically ill children requiring plasma transfusions.METHODS: This was an ancillary study of a prospective observational study on plasma transfusions over a 6-week period, in 101 PICUs in 21 countries. All critically ill children who received at least one plasma transfusion during the observation period were included. PELOD-2 scores were measured on days 1, 2, 5, 8, and 12 after plasma transfusion. Performance of the score was assessed by the determination of the discrimination (area under the ROC curve: AUC) and the calibration (Hosmer-Lemeshow test).RESULTS: Four hundred and forty-three patients were enrolled in the study (median age and weight: 1 year and 9.1 kg, respectively). Observed mortality rate was 26.9 % (119/443). For PELOD-2 on day 1, the AUC was 0.76 (95 % CI 0.71-0.81) and the Hosmer-Lemeshow test was p = 0.76. The serial evaluation of the changes in the daily PELOD-2 scores from day 1 demonstrated a significant association with death, adjusted for the PELOD-2 score on day 1.CONCLUSIONS: In a subpopulation of critically ill children requiring plasma transfusion, the PELOD-2 score has a lower but acceptable discrimination than in an entire population. This score should therefore be used cautiously in this specific subpopulation.

This paper deals with the problem of a logistics company that has to serve its customers by first putting the products on pallets and then loading the pallets into trucks. Besides the standard geometric constraints of products not overlapping each other and not exceeding the dimensions of pallets and trucks, in this real problem, there are many other constraints, related to the total weight of the load, the maximum weight supported by each axle, and the distribution of the load inside the truck. Although the problem can be decomposed into two phases, pallet loading and truck loading, we have taken a combined approach, building and placing pallets at the same time. For each position in the truck, a pallet is built and tailored for that position according to the constraints of height and weight. We have developed a GRASP algorithm, in which the constructive algorithm is randomized and an improvement phase is added to obtain high-quality solutions. The algorithm has been tested on two sets of real instances with different characteristics, involving up to 44 trucks. The results show that solutions with an optimal or near optimal number of trucks are obtained in very short computing times.

In mice, social behaviors such as mating and aggression are mediated by pheromones and related chemosignals. The vomeronasal organ (VNO) detects olfactory information from other individuals by sensory neurons tuned to respond to specific chemical cues. Receptors expressed by vomeronasal neurons are implicated in selective detection of these cues. Nearly 400 receptor genes have been identified in the mouse VNO, but the tuning properties of individual receptors remain poorly understood, in part due to the lack of a robust heterologous expression system. Here we develop a herpes virus-based amplicon delivery system to overexpress three types of vomeronasal receptor genes and to characterize cell responses to their proposed ligands. Through Ca imaging in native VNO cells we show that virus-induced overexpression of V1rj2, V2r1b or Fpr3 caused a pronounced increase of responsivity to sulfated steroids, MHC-binding peptide or the synthetic hexapeptide W-peptide, respectively. Other related ligands were not recognized by infected individual neurons, indicating a high degree of selectivity by the overexpressed receptor. Removal of G-protein signaling eliminates Ca responses, indicating that the endogenous second messenger system is essential for observing receptor activation. Our results provide a novel expression system for vomeronasal receptors that should be useful for understanding the molecular logic of VNO ligand detection. Functional expression of vomeronasal receptors and their deorphanization provides an essential requirement for deciphering the neural mechanisms controlling behavior., This work was supported by Deutsche Forschungsgemeinschaft grants CH 920/2-1 (PC) and Sonderforschungsbereich 894 projects A16 (TL-Z) and A17 (FZ), a University of Saarland HOMFORexzellent grant (PC), the Volkswagen Foundation (TL-Z).

In this paper, we present precise time and energy models for an intra-only HEVC video encoder. These models are a step forward to understand and estimate the computational complexity and energy demands of an HEVC encoder, which in turn opens the path to finely tuning the computational resources that are dedicated to this purpose. Our models estimate the complexity and energy consumed by the HEVC encoder, in a frame by frame basis, considering two factors: the quantification parameter used to encode each frame and the spatial information of that frame. Our experimental validation demonstrates the accuracy of these models, which report errors that are, on average, below 10% for full HD videos, and 5% for 832 × 480 videos.

7 páginas, 5 figuras.-- El pdf es la versión post-print del artículo., STIM1 and Orai1 are the main players in capacitative calcium entry (CCE). STIM1 senses [Ca2+] inside the endoplasmic reticulum (ER) and, when it decreases, opens Orai1, a store-operated calcium channel (SOC) in the plasma membrane that promotes Ca2+ entry and increases cytosolic Ca2+. The final destination of the entering Ca2+ is the ER, which refills very efficiently (capacitatively) with it. We propose here that SERCA is the third element of CCE, to which is tightly coupled to favour rapid Ca2+ pumping from the high Ca2+ microdomains, generated at the SOC's mouth, to the ER. We find that, on depletion of the intracellular Ca2+ stores, SERCA co-localizes with STIM1 at puncta. Adequate coupling of CCE and ER Ca2+ pumping requires correct proportions of STIM1, Orai1 and SERCA. Overexpression of Orai1 decreased modestly Ca2+ entry, but produced a dramatic fall of Ca2+ uptake into ER, which was rescued by STIM1 co-expression or by increasing external Ca2+. In permeabilized cells, Ca2+ uptake into the ER was indistinguishable in the Orai1-expressing and in the control cells. We propose that excess Orai1 uncouples SERCA from Ca2+ entry in the intact cell by disturbing the fine topology of Ca2+ pumping complexes within the ER-plasma membrane junctions., This work was supported by grants from the Spanish Ministerio de Ciencia e Innovación (MICINN, BFI2007-60157 and SAF2009- 03175-E) and the Instituto de Salud Carlos III. IMM was supported by a fellowship from the Spanish MICINN.

et al., Simultaneous control of different functions by calcium signals is possible because of subcellular compartmentalization. Targeted chemiluminescent Ca 2+ probes, such as aequorins (AEQs) are optimal for detecting signals originating in different subcellular domains, but imaging is difficult because of low photon yield causing poor spatiotemporal resolution. To overcome this problem, we have co-expressed two spectrally distinct AEQs in different subcellular locations within the same cells. Seven chimeric proteins containing either green- or red-emitting AEQs, with different targeting sequences and Ca2+ affinities, have been designed and tested. We show here evidence for physical and functional independence of the different probes. Cytosolic Ca2+ signals were mirrored in the nucleus, but amplified inside mitochondria and endoplasmic reticulum, and had different time courses in the various locations. Our results demonstrate that these novel tools permit simultaneous and independent monitoring of [Ca2+] in different subcellular domains of the same cell. © 2007 Springer-Verlag., We thank the Spanish Ministerio de Educación y Ciencia (MEC; BFU2004-02765/BFI, and BFU2005-02078) and Junta de Castilla y León (VA-088/A06) for financial support. IMMand BD held predoctoral fellowships from MEC and Junta de Comunidades de Castilla-La Mancha, respectively.

Changes in the cytosolic Ca2+ concentration ([Ca2+]c) are essential for triggering neurotransmitter release from presynaptic nerve terminals. Calcium-induced Ca2+ release (CICR) from the endoplasmic reticulum (ER) may amplify the [Ca2+]c signals and facilitate neurotransmitter release in sympathetic neurons. In adrenal chromaffin cells, functional triads are formed by voltage-operated Ca2+ channels (VOCCs), CICR sites and mitochondria. In fact, mitochondria take up most of the Ca2+ load entering the cells and are essential for shaping [Ca2+]c signals and exocytosis. Here we have investigated the existence of such functional triads in sympathetic neurons. The mitochondrial Ca2+ concentration ([Ca2+]m) in soma and neurites of individual mouse superior cervical ganglion (SCG) neurons was monitored by bioluminescence imaging of targeted aequorins. In soma, Ca2+ entry through VOCCs evoked rapid, near millimolar [Ca2+]m increases in a subpopulation of mitochondria containing about 40% of the aequorin. Caffeine evoked a similar [Ca2+]m increase in a mitochondrial pool containing about 30% of the aequorin and overlapping with the VOCC-sensitive pool. These observations suggest the existence of functional triads similar to the ones described in chromaffin cells. In neurites, mitochondria were able to buffer [Ca2+]c increases resulting from activation of VOCCs but not those mediated by caffeine-induced Ca2+ release from the ER. The weaker Ca2+ buffering by mitochondria in neurites could contribute to facilitate Ca2+-induced exocytosis at the presynaptic sites.

Producción Científica, We have recently described a new class of genetically encoded Ca2+ indicators composed of two jellyfish proteins, a variant of green fluorescent protein (GFP) and the calcium binding protein apoaequorin, named GAP (Rodriguez-García et al., 2014). GAP is a unique dual-mode Ca2+ indicator, able to function either as a fluorescent or a luminescent probe, depending on whether the photoprotein aequorin is in its apo-state or reconstituted with its cofactor coelenterazine. We describe here a novel application of GAP as a low affinity bioluminescent indicator, suitable for measurements of [Ca2+] in ER or in Golgi apparatus. We used the low affinity variant, GAP1, which carries mutations in two EF-hands of aequorin, reconstituted with coelenterazine n. In comparison to previous bioluminescent aequorin fusions, the decay rate of GAP1 was decreased 8 fold and the affinity for Ca2+ was lowered one order of magnitude. This improvement allows long-term measurements in high Ca2+ environments avoiding fast aequorin consumption. GAP1 was targeted to the ER of various cell types, where it monitored resting Ca2+ concentrations in the range from 400 to 600 μM. ER could be emptied of calcium by stimulation with ATP, carbachol or histamine in intact cells, and by challenge with inositol tris-phosphate in permeabilized cells. GAP1 was also targeted to the Golgi apparatus where it was able to precisely monitor long-term calcium dynamics. GAP1 provides a novel and robust indicator applicable to bioluminescent high-throughput quantitative assays., Ministerio de Economía, Industria y Competitividad (Project BFU2014-534698P), Instituto de Salud Carlos III (RD06/0010/0000 and RD12/0019/0036)

High Ca2+ content in the Golgi apparatus (Go) is essential for protein processing and sorting. In addition, the Go can shape the cytosolic Ca2+ signals by releasing or sequestering Ca2+. We generated two new aequorin-based Ca2+ probes to specifically measure Ca2+ in the cis/cis-to-medial-Go (cGo) or the trans-Go (tGo). Ca2+ homoeostasis in these compartments and in the endoplasmic reticulum (ER) has been studied and compared. Moreover, the relative size of each subcompartment was estimated from aequorin consumption. We found that the cGo accumulates Ca2+ to high concentrations (150-300 μM) through the sarco plasmic/endoplasmic reticulum Ca2+-ATPase (SERCA). The tGo, in turn, is divided into two subcompartments: tGo1 and tGo2. The subcompartment tGo1 contains 20% of the aequorin and has a high internal [Ca2+]; Ca2+ is accumulated in this subcompartment via the secretory pathway Ca2+-ATPase 1 (SPCA-1) at a very high affinity (K50=30 nM). The subcompartment tGo2 contains 80% of aequorin, has a lower [Ca2+] and no SPCA-1 activity; Ca2+ uptake happens through SERCA and is slower than in tGo1. The two tGo subcompartments, tGo1 and tGo2, are diffusionally isolated. Inositol trisphosphate mobilizes Ca2+ from the cGo and tGo2, but not from tGo1, whereas caffeine releases Ca2+ from all the Golgi regions, and nicotinic acid dinucleotide phosphate and cADP ribose from none., This work was supported by a grant from the Spanish Ministerio de Economía y Competitividad (MEC) [grant numbers BFU2010–17379] and the Instituto de Salud Carlos III [grant numbers RD06/0010/0000 and RD12/0019/0036]. F.J.A. was supported by an FPI fellowship from MEC.

Endomembranes modify the progression of the cytosolic Ca2+ wave and contribute to generate Ca2+ microdomains, both in the cytosol and inside the own organella. The concentration of Ca2+ in the cytosol ([Ca2+]C), the mitochondria ([Ca2+]M) and the nucleus ([Ca2+]N) are similar at rest, but may become very different during cell activation. Mitochondria avidly take up Ca2+ from the high [Ca2+]C microdomains generated during cell activation near Ca2+ channels of the plasma membrane and/or the endomembranes and prevent propagation of the high Ca2+ signal to the bulk cytosol. This shaping of [Ca2+]C signaling is essential for independent regulation of compartmentalized cell functions. On the other hand, a high [Ca2+]M signal is generated selectively in the mitochondria close to the active areas, which tunes up respiration to the increased local needs. The progression of the [Ca2+]C signal to the nucleus may be dampened by mitochondria, the nuclear envelope or higher buffering power inside the nucleoplasm. On the other hand, selective [Ca2+]N signals could be generated by direct release of stored Ca2+ into the nucleoplasm. Ca2+ release could even be restricted to subnuclear domains. Putative Ca2+ stores include the nuclear envelope, their invaginations inside the nucleoplasm (nucleoplasmic reticulum) and nuclear microvesicles. Inositol trisphosphate, cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate have all been reported to produce release of Ca2+ into the nucleoplasm, but contribution of these mechanisms under physiological conditions is still uncertain. © 2006 Elsevier Ltd. All rights reserved., This work was funded by grants from the Ministerio de Educación y Ciencia (BFU2004-02765/BFI and BFU2005-05464) and from Junta de Castilla y León (VA016A05). Pablo Chamero holded a predoctoral fellowship from the Basque government.

2-DE followed by MALDI-TOF was used to purify and identify a Drosophila protein (catalogued as SSP 6002) that showed marked differences in the level of expression in the different imaginal discs of third instar larvae. Fingerprinting showed that the spot of interest was the heat shock 23 polypeptide (hsp23). We characterized the kinetics of its induction by heat shock in wing imaginal discs and raised an antiserum against the denatured protein, which recognizes a single unphosphorylated spot on 2-D gels. The difference in its expression in discs was corroborated by analyzing its level in the imaginal discs of postbithorax mutants. We also investigated the developmental expression of hsp23 in imaginal discs with antiserum raised against the native protein. Its spatial and temporal pattern of expression is related to the proneural territories and maintained even under heat shock conditions. In addition, its pattern of expression is regulated by transcription factors and signaling pathways (notch and epidermal growth factor receptor) involved in proneural specification. © 2005 Wiley-VCH Verlag GmbH & Co. KGaA., Comision Asesora de Investigación Científica y Técnica and by an institutional grant from Fundación Ramón Areces