Introducción de la tecnología de transferencia génica en lubina (Dicentrarchus labrax)

RESUMEN Este trabajo abarca distintos aspectos de la metodología de la transferencia génica en lubina, tanto in vitro como in vivo. Por una parte, se ha centrado en el estudio y evaluación de la transferencia génica somática como posible sustitución a la transgénesis estable en distintas aplicaciones. Por otra parte, se ha evaluado el sistema de regulación por tetraciclina para la regulación exógena de genes en peces. Por último, se ha probado que una línea celular de lubina puede utilizarse como sistema de expresión de genes exógenos. Por primera vez se demuestra que la inyección de DNA plasmídico es eficiente para transferir genes exógenos a músculo de lubina. Los factores que favorecen la expresión son, dosis altas de plásmido, el uso de un promotor adecuado, y un estado metabolicamente activo del músculo. La expresión que se obtiene es transitoria, alcanzándose el máximo cinco días después de la inyección. Una limitación de esta técnica es la incapacidad de reproducir en los distintos individuos inyectados, la manera exacta en que la aguja de inyección entra en el paquete muscular, lo que origina una gran variabilidad en cuanto a la toma de DNA por parte de las células, y por tanto en el nivel de expresión del transgen. Esto se puede mejorar aumentando el número de animales inyectados para disminuir así el error debido a la técnica, o usando una segunda construcción para normalizar la transfección. La técnica de inyección de DNA en músculo se ha ensayado como método de vacunación en peces. Sin embargo, ésta es la primera vez, que se prueba esta técnica en peces para secretar una hormona en sangre. Como gen a inyectar se ha diseñado y construido un gen híbrido que codifica una hormona gonadotropa de cadena única de lubina. Tras su inyección en músculo de lubina se pudo detectar la proteína en plasma. Para sistemas de origen piscícola, no se dispone hasta el momento de un método que permita controlar la expresión de genes exógenos de manera fiable. Por primera vez, hemos probado la utilidad del sistema de regulación por tetraciclina para esta misión. Tras valorar diferentes versiones del sistema en células de pez in vitro y en músculo in vivo, podemos concluir que la opción más eficiente es el uso del transactivador tTA bajo el control de un promotor estable. Al igual que en células de mamíferos, hemos comprobado que las células de pez también presentan dificultades para mantener el sistema de regulación por tetraciclina de una manera funcional; como resultado, encontrar clones con un grado de regulación óptimo y un nivel bajo de expresión basal es difícil. Sin embargo, el uso de una estrategia basada en el uso del plásmido nuevo pTet-luc+-pur/GFP facilita esta tarea, y permite obtener un alto porcentaje de clones regulables. A lo largo de este trabajo se han comparado los promotores CMV, de origen viral, y el promotor de la actina ß de carpa, para dirigir la expresión de distintos genes tanto en las líneas celulares EPC y SBL, como en músculo de lubina in vivo. Los resultados nos permite concluir que el promotor de la actina ß de carpa, es más débil en cuanto al nivel de expresión génica que logra promover, y menos estable a la hora de mantenerse en una línea celular que el promotor CMV. Por último, los resultados obtenidos con la línea celular SBL permiten señalar a esta línea como un sistema de lubina apto para la expresión de genes exógenos. Estas células son capaces de secretar los productos de los genes introducidos, con lo que podrían ser utilizadas para la producción de proteínas recombinantes. __________________________________________________________________________________________________This work focuses on different aspects of the gene transfer methodology in sea bass. On one hand, the use of the somatic gene transfer has been evaluated as an alternative to the use of stable transgenesis in different applications. Somatic gene transfer has been used as method of delivery of a recombinant single-chain gonadotropin into the blood stream in sea bass. On the other hand, we have evaluated the use of the tetracycline regulated system to control exogenous gene expression in fish. The results showed that the best choice was the use of the rtTA transactivator under the control of a stable promoter. Finally, a sea bass cell line has proven to be an efficient system for the expression of exogenous gene

Empirical study on the use of public transport in the Community of Madrid as a key factor in sustainable mobility

The level of public transport use in the area of large cities is representative of the degree of sustainable mobility behavior. This article, based on a population survey of mobility in the Community of Madrid are and the approach of a set of hypotheses combining alternatives and modes of transportation in the framework of four different mobility scenarios, shows that, although the public transport system is currently perceived as sufficiently developed and accessible, is not capable of promoting an effective modal shift, mainly due to lower competitiveness and efficiency offered regarding the car. However, the existence of a favorable predisposition of Madrid society to accept measures aimed at improving sustainability in transportation can be a key factor in the modal shift in favor of more sustainable modes of transportEl grado de utilización del transporte público en el ámbito de las grandes ciudades es representativo del nivel de comportamiento sostenible en la movilidad. El presente artículo, sobre la base de una encuesta poblacional de movilidad en el ámbito de la Comunidad de Madrid y el planteamiento de un conjunto de hipótesis que relacionan alternativas disponibles y los modos de transporte utilizado en el marco de 4 escenarios de movilidad diferenciados, pone de manifiesto que si bien el sistema de transporte público se percibe actualmente como suficientemente desarrollado y accesible, no es capaz de impulsar un efectivo cambio modal, fundamentalmente debido a la menor competitividad y eficiencia que ofrece con respecto al automóvil. No obstante, la existencia de una predisposición favorable de la sociedad madrileña por la aceptación de medidas orientadas a una mejora de la sostenibilidad en el transporte puede suponer un factor clave en el cambio modal a favor de modos de transporte más sostenible

Assessment by self-organizing maps of element release from sediments in contact with acidified seawater in laboratory leaching test conditions

Carbon capture and storage (CCS) is gaining interest as a significant global option to reduce emissions of CO2. CCS development requires an assessment of the potential risks associated with CO2 leakages from storage sites. Laboratory leaching tests have proved to be a useful tool to study the potential mobilization of metals from contaminated sediment in a decreased-pH environment that mimics such a leakage event. This work employs a self-organizing map (SOM) tool to interpret and analyze the release of dissolved organic carbon (DOC), As, Cd, Cr, Cu, Ni, Pb, and Zn from equilibrium, column, and pH-dependent leaching tests. In these tests, acidified seawater is used for simulating different CO2 leakage scenarios. Classification was carried out detailing the mobilization of contaminants for environments of varying pH, liquid-to-solid ratio, and type of contact of the laboratory leaching tests. Component planes in the SOMs allow visualization of the results and the determination of the worst case of element release. The pH-dependent leaching test with initial addition of either base or acid was found to mobilize the highest concentrations of metals.This work was supported by the Spanish Ministry of Economy and Competitiveness under Project CTM 2011-28437-C02-01, ERDF included. MCMT was funded by the Spanish Ministry of Economy and Competitiveness by F.P.I. fellowship N° BES-2012-053816

Experience with the use of Rituximab for the treatment of rheumatoid arthritis in a tertiary Hospital in Spain: RITAR study

There is evidence supporting that there are no relevant clinical differences between dosing rituximab 1000 mg or 2000 mg per cycle in rheumatoid arthritis (RA) patients in clinical trials, and low-dose cycles seem to have a better safety profile. Our objective was to describe the pattern of use of rituximab in real-life practice conditions. Methods: Rituximab for RA in clinical practice (RITAR) study is a retrospective cohort study from 2005 to 2015. Eligibility criteria were RA adults treated with rituximab for active articular disease. Response duration was the main outcome defined as months elapsed from the date of rituximab first infusion to the date of flare. A multivariable analysis was performed to determine the variables associated with response duration. Results: A total of 114 patients and 409 cycles were described, 93.0% seropositive and 80.7% women. Rituximab was mainly used as second-line biological therapy. On demand retreatment was used in 94.6% of cases versus fixed 6 months retreatment in 5.4%. Median response duration to on demand rituximab cycles was 10 months (interquartile range, 7–13). Multivariable analysis showed that age older than 65 years, number of rituximab cycles, seropositivity, and first- or second-line therapy were associated with longer response duration. The dose administered at each cycle was not significantly associated with response duration. Conclusions: Our experience suggests that 1000 mg rituximab single infusion on demand is a reasonable schedule for long-term treatment of those patients with good response after the first cycles, especially in seropositive patients and when it is applied as a first- or second-line biological therap

Analysis of environmental benefits associated with the incorporation of Waelz slag into fired bricks using LCA

A comparative cradle-to-grave LCA shows that incorporating Waelz slag into ceramic bricks generates lower impact on climate change and reduces the impact on freshwater ecotoxicity and fossil depletion. These benefits are attributable to impact savings due to avoiding the landfilling of the slag and reduced fuel demand during the manufacturing stage. However, due to the higher SO2 and HF emissions generated in the firing of slag containing bricks, these benefits are offset by higher impacts on human toxicity and terrestrial acidification categories. The aggregated results suggest very limited environmental benefits in this practice even taking into account different end-of-life scenarios

Gonadotropin actions in European sea bass: endocrine roles and biotechnological applications

Comunicación presentada en el 10th International Symposium on Reproductive Physiology of Fish, celebrado en Olhao, Portugal, del 25 al 30 de mayo de 2014The follicle stimulating hormone (Fsh) and the luteinizing hormone (Lh) are central endocrine regulators of gonad function in vertebrates. These gonadotropins act by binding and activating their specific receptors that are located in certain cell types of the gonads. In fish, the differential roles of these hormones are being progressively elucidated with the development of different tools for their study. In the case of European sea bass Dicentrarchus labrax, the isolation of the genes coding for the gonadotropin subunits and receptors allowed in first instance for expression studies. Later, to overcome the limitation of native hormones, recombinant dimeric gonadotropins have been generated, which show different functional characteristics depending on the cell system and DNA construct. In addition, single gonadotropin beta-subunits have been produced and used as antigens for antibody production. This last approach has allowed the development of detection methods for native gonadotropins being the European sea bass one of the few species where both gonadotropins can be detected in their native form. The generation of antibodies has also allowed for their cellular localization and co-localization with other factors, helping to a better understanding of their function. By administration of the recombinant gonadotropins to gonad tissues cultured in vitro we have studied their effects on steroidogenesis, the activated intracellular pathways and their target genes. Their administration in vivo has also been tested both for basic studies and as a biotechnological approach. In this frame, it is known from different studies in mammals that exogenous administration of native hormones in cases of deficiency, including assisted reproduction, can be unsatisfactory due to their rapid clearance from circulation or limited availability. Other than the production of recombinant hormones gene-based therapies by using somatic gene transfer are offered as an alternative. Such an approach has been tested in sea bass for gonadotropin delivery in vivo. The hormones produced by the injected genes were functional and have allowed for different studies on gonadotropin action in spermatogenesis. All together, the use of gene therapy for hormone replacement in fish is a real alternative to the production of recombinant gonadotropins for in vivo use, due to the low cost of production and the high persistence of the injected DNA, and has a broad range of potential applications such as its use in out-of-season breeding programs or reproductive dysfunctions in fish species.Supported by MINECO (AGL2011-28890) and GV (ACOMP/2013/085).Peer Reviewe

Molecular evolution of aphids and their primary (buchnera sp.) and secondary endosymbionts: implications for the role of symbiosis in insect evolution

Aphids maintain an obligate, endosymbiotic association with Buchnera sp., a bacterium closely related to Escherichia coli. Bacteria are housed in specialized cells of organ-like structures called bacteriomes in the hemocoel of the aphid and are maternally transmitted. Phylogenetic studies have shown that the association had a single origin, dated about 200-250 million years ago, and that host and endosymbiont lineages have evolved in parallel since then. However, the pattern of deepest branching within the aphid family remains unsolved, which thereby hampers an appraisal of, for example, the role played by horizontal gene transfer in the early evolution of Buchnera. The main role of Buchnera in this association is the biosynthesis and provisioning of essential amino acids to its aphid host. Physiological and metabolic studies have recently substantiated such nutritional role. In addition, genetic studies of Buchnera from several aphids have shown additional modifications, such as strong genome reduction, high A+T content compared to free-living bacteria, differential evolutionary rates, a relative increase in the number of non-synonymous substitutions, and gene amplification mediated by plasmids. Symbiosis is an active process in insect evolution as revealed by the intermediate values of the previous characteristics showed by secondary symbionts compared to free-living bacteria and Buchnera.The authors thank the Servicio Central de Soporte a la Investigación Científica (Universitat de València) for bioinformatics and sequencing facilities. The work has been supported by grants GV-3216/95 from Generalitat Valenciana (Spain), BFM2000-1383 from MEC (Spain) and FEDER 2FD1997-1006 (Spain).Peer reviewe

The PHQ-PD as a screening tool for panicdisorder in theprimarcarsetting in Spain.

INTRODUCTION Panic disorder is a common anxiety disorder and is highly prevalent in Spanish primary care centres. The use of validated tools can improve the detection of panic disorder in primary care populations, thus enabling referral for specialized treatment. The aim of this study is to determine the accuracy of the Patient Health Questionnaire-Panic Disorder (PHQ-PD) as a screening and diagnostic tool for panic disorder in Spanish primary care centres. METHOD We compared the psychometric properties of the PHQ-PD to the reference standard, the Structured Clinical Interview for DSM-IV Axis I Disorders (SCID-I) interview. General practitioners referred 178 patients who completed the entire PHQ test, including the PHQ-PD, to undergo the SCID-I. The sensitivity, specificity, positive and negative predictive values and positive and negative likelihood ratios of the PHQ-PD were assessed. RESULTS The operating characteristics of the PHQ-PD are moderate. The best cut-off score was 5 (sensitivity .77, specificity .72). Modifications to the questionnaire's algorithms improved test characteristics (sensitivity .77, specificity .72) compared to the original algorithm. The screening question alone yielded the highest sensitivity score (.83). CONCLUSION Although the modified algorithm of the PHQ-PD only yielded moderate results as a diagnostic test for panic disorder, it was better than the original. Using only the first question of the PHQ-PD showed the best psychometric properties (sensitivity). Based on these findings, we suggest the use of the screening questions for screening purposes and the modified algorithm for diagnostic purposes

Transdiagnostic cognitive behavioral therapy versus treatment as usual in adult patients with emotional disorders in the primary care setting (PsicAP study) : protocol for a randomized controlled trial

Demand for primary care (PC) services in Spain exceeds available resources. Part of this strong demand is due to the high prevalence of emotional disorders (EDs)-anxiety, depression, and somatic symptom disorders-and related comorbidities such as pain or chronic illnesses. EDs are often under- or misdiagnosed by general practitioners (GPs) and, consequently, treatment is frequently inadequate. We aim to compare the short- and long-term effectiveness of group-delivered transdiagnostic cognitive behavioral therapy (TD-CBT) versus treatment as usual (TAU) in the treatment of EDs in the PC setting in Spain. We also aim to compare the effect of these treatments on disability, quality of life, cognitive-emotional factors, and treatment satisfaction. Here we present the study design of a two-arm, single-blind, randomized controlled trial (N=1126) to compare TAU to TD-CBT for EDs. TAU will consist primarily of pharmacological treatment and practical advice from the GP while TD-CBT will be administered in seven 90-minute group sessions held over a period ranging from 12 to 14 weeks. Psychological assessments are carried out at baseline (ie, pretreatment); posttreatment; and at 3-, 6-, and 12-month follow-up. The study is conducted in approximately 26 PC centers from the National Health System in Spain. This study was initiated in December 2013 and will remain open to new participants until recruitment and follow-up has been completed. We expect all posttreatment evaluations to be completed by December 2017, and follow-up will end in December 2018. We expect the TD-CBT group to have better results compared to TAU on all posttreatment measures and that this improvement will be maintained during follow-up. This project could serve as a model for use in other areas or services of the National Health System in Spain and even in other countries. International Standard Randomized Controlled Trial Number (ISRCTN): 58437086; http://www.isrctn.com/ISRCTN58437086 (Archived by WebCite at http://www.webcitation.org/6mbYjQSn3

Identification of CRF66_BF, a New HIV-1 Circulating Recombinant Form of South American Origin

Circulating recombinant forms (CRFs) are important components of the HIV-1 pandemic. Among 110 reported in the literature, 17 are BF1 intersubtype recombinant, most of which are of South American origin. Among these, all 5 identified in the Southern Cone and neighboring countries, except Brazil, derive from a common recombinant ancestor related to CRF12_BF, which circulates widely in Argentina, as deduced from coincident breakpoints and clustering in phylogenetic trees. In a HIV-1 molecular epidemiological study in Spain, we identified a phylogenetic cluster of 20 samples from 3 separate regions which were of F1 subsubtype, related to the Brazilian strain, in protease-reverse transcriptase (Pr-RT) and of subtype B in integrase. Remarkably, 14 individuals from this cluster (designated BF9) were Paraguayans and only 4 were native Spaniards. HIV-1 transmission was predominantly heterosexual, except for a subcluster of 6 individuals, 5 of which were men who have sex with men. Ten additional database sequences, from Argentina (n = 4), Spain (n = 3), Paraguay (n = 1), Brazil (n = 1), and Italy (n = 1), branched within the BF9 cluster. To determine whether it represents a new CRF, near full-length genome (NFLG) sequences were obtained for 6 viruses from 3 Spanish regions. Bootscan analyses showed a coincident BF1 recombinant structure, with 5 breakpoints, located in p17 gag , integrase, gp120, gp41-rev overlap, and nef, which was identical to that of two BF1 recombinant viruses from Paraguay previously sequenced in NFLGs. Interestingly, none of the breakpoints coincided with those of CRF12_BF. In a maximum likelihood phylogenetic tree, all 8 NFLG sequences grouped in a strongly supported clade segregating from previously identified CRFs and from the CRF12_BF "family" clade. These results allow us to identify a new HIV-1 CRF, designated CRF66_BF. Through a Bayesian coalescent analysis, the most recent common ancestor of CRF66_BF was estimated around 1984 in South America, either in Paraguay or Argentina. Among Pr-RT sequences obtained by us from HIV-1-infected Paraguayans living in Spain, 14 (20.9%) of 67 were of CRF66_BF, suggesting that CRF66_BF may be one of the major HIV-1 genetic forms circulating in Paraguay. CRF66_BF is the first reported non-Brazilian South American HIV-1 CRF_BF unrelated to CRF12_BF.This work was funded through Acción Estratégica en Salud Intramural (AESI), Instituto de Salud Carlos III, projects PI16CIII/00033 and PI19CIII/00042; Red de Investigación en SIDA (RIS), Instituto de Salud Carlos III, Subdirección General de Evaluación y Fondo Europeo de Desarrollo Regional (FEDER), Plan Nacional I+D+I, project RD16ISCIII/0002/0004; and scientific agreements with Consellería de Sanidade, Government of Galicia (MVI 1004/16) and Osakidetza-Servicio Vasco de Salud, Government of Basque Country (MVI 1001/16).S