Cambios temporales en los transportes y la distribución de las masas de agua a lo largo de la sección 20º W de CAIBOX (Atlántico NE)

24 páginas, 3 tablas, 7 figuras[EN] The CAIBOX cruise was conducted from 25 July to 14 August 2009. Three consecutive transects (zonal, meridional, and transverse) formed a closed box to the west of the Strait of Gibraltar. This study aimed to analyze the thermohaline properties, volume transports, and water mass distributions (percentages) along the meridional section (30–41.5º N, 20º W). We identified the main geostrophic current (Azores Current) and its associated volume transport and interannual changes. Data from previous cruises (AZORES I, A16N, CLIVAR, OACES, and CHAOS) with similar tracks were employed to compare with the CAIBOX meridional section. All but one (CHAOS) were summer cruises. We estimated a mean transport for the Azores Current at 20º W of 9.3 ± 2.6 Sv. There appears to be an inverse relation between the position of this current and its associated transport, with relatively high (low) transports when the current is located roughly south (north) of 35º N. Regarding water masses, an increase of 14.4% was found for Mediterranean Water compared with the 1993, 1998, and 2003 cruises; however, Labrador Sea Water decreased its contribution and southward spreading between 1998 and 2009[ES] Entre el 25 de julio y el 14 de agosto de 2009 se llevó a cabo la campaña CAIBOX, que constó de tres transectos consecutivos (zonal, occidental y transversal) conformando una caja cerrada al oeste del estrecho de Gibraltar. El objetivo principal del presente trabajo fue el estudio de las propiedades termohalinas, los transportes (en términos de volumen) y la distribución de las masas de agua (en porcentajes) a lo largo de la sección occidental (30–41.5º N, 20º W). Se identificó la corriente geostrófica principal (Corriente de las Azores), el transporte asociado a ella y sus cambios interanuales. Adicionalmente se consideraron los datos de cinco campañas anteriores (AZORES I, A16N, CLIVAR, OACES y CHAOS) con secciones similares a las de la campaña CAIBOX. Todas excepto una (CHAOS) fueron campañas estivales. El transporte medio para la Corriente de las Azores a 20º W se estimó en 9.3 ± 2.6 Sv. Parece existir cierta relación inversa entre la posición de dicha corriente y su transporte asociado, con transportes relativamente altos (bajos) cuando la corriente se sitúa al sur (norte) de 35º N. En cuanto a las masas de agua, se observa un incremento de 14.4% del Agua Mediterránea en comparación con las campañas de 1993, 1998 y 2003; sin embargo, el Agua de Labrador ha disminuido su contribución y su propagación hacia el sur entre 1998 y 2009.An important part of the data presented in this paper was obtained through the CAIBEX project: Shelf-ocean Exchanges in the Canaries-Iberia Large Marine Ecosystem (CTM2007-66408-C02/MAR), supported by the Spanish Ministry of Education and Science. The first author, LIC, was funded by a FPU predoctoral fellowship from the National Human Resources Formation Program, within the framework of the 2008–2011 National Scientific Research, Development and Technological Innovation Plan of the Spanish Ministry of Education. The fourth author, NVR, was supported by the Isabel Barreto Program (IN840E) from the Xunta de Galicia.Peer reviewe

Site-Specific Integration of Foreign DNA into Minimal Bacterial and Human Target Sequences Mediated by a Conjugative Relaxase

This is an open-access article distributed under the terms of the Creative Commons Attribution License.[Background]: Bacterial conjugation is a mechanism for horizontal DNA transfer between bacteria which requires cell to cell contact, usually mediated by self-transmissible plasmids. A protein known as relaxase is responsible for the processing of DNA during bacterial conjugation. TrwC, the relaxase of conjugative plasmid R388, is also able to catalyze site-specific integration of the transferred DNA into a copy of its target, the origin of transfer (oriT), present in a recipient plasmid. This reaction confers TrwC a high biotechnological potential as a tool for genomic engineering. [Methodology/Principal Findings]: We have characterized this reaction by conjugal mobilization of a suicide plasmid to a recipient cell with an oriT-containing plasmid, selecting for the cointegrates. Proteins TrwA and IHF enhanced integration frequency. TrwC could also catalyze integration when it is expressed from the recipient cell. Both Y18 and Y26 catalytic tyrosil residues were essential to perform the reaction, while TrwC DNA helicase activity was dispensable. The target DNA could be reduced to 17 bp encompassing TrwC nicking and binding sites. Two human genomic sequences resembling the 17 bp segment were accepted as targets for TrwC-mediated site-specific integration. TrwC could also integrate the incoming DNA molecule into an oriT copy present in the recipient chromosome. [Conclusions/Significance]: The results support a model for TrwC-mediated site-specific integration. This reaction may allow R388 to integrate into the genome of non-permissive hosts upon conjugative transfer. Also, the ability to act on target sequences present in the human genome underscores the biotechnological potential of conjugative relaxase TrwC as a site-specific integrase for genomic modification of human cells.This work was supported by grant BIO2008-00133 from the Spanish Ministry of Science and Innovation to ML. CGP was a recipient of a predoctoral fellowship from the University of Cantabria, Spain.Peer reviewe

Primary skin fibroblasts as a model of Parkinson's disease

Parkinson's disease is the second most frequent neurodegenerative disorder. While most cases occur sporadic mutations in a growing number of genes including Parkin (PARK2) and PINK1 (PARK6) have been associated with the disease. Different animal models and cell models like patient skin fibroblasts and recombinant cell lines can be used as model systems for Parkinson's disease. Skin fibroblasts present a system with defined mutations and the cumulative cellular damage of the patients. PINK1 and Parkin genes show relevant expression levels in human fibroblasts and since both genes participate in stress response pathways, we believe fibroblasts advantageous in order to assess, e.g. the effect of stressors. Furthermore, since a bioenergetic deficit underlies early stage Parkinson's disease, while atrophy underlies later stages, the use of primary cells seems preferable over the use of tumor cell lines. The new option to use fibroblast-derived induced pluripotent stem cells redifferentiated into dopaminergic neurons is an additional benefit. However, the use of fibroblast has also some drawbacks. We have investigated PARK6 fibroblasts and they mirror closely the respiratory alterations, the expression profiles, the mitochondrial dynamics pathology and the vulnerability to proteasomal stress that has been documented in other model systems. Fibroblasts from patients with PARK2, PARK6, idiopathic Parkinson's disease, Alzheimer's disease, and spinocerebellar ataxia type 2 demonstrated a distinct and unique mRNA expression pattern of key genes in neurodegeneration. Thus, primary skin fibroblasts are a useful Parkinson's disease model, able to serve as a complement to animal mutants, transformed cell lines and patient tissues

Design of Novel Relaxase Substrates Based on Rolling Circle Replicases for Bioconjugation to DNA Nanostructures

During bacterial conjugation and rolling circle replication, HUH endonucleases, respectively known as relaxases and replicases, form a covalent bond with ssDNA when they cleave their target sequence (nic site). Both protein families show structural similarity but limited amino acid identity. Moreover, the organization of the inverted repeat (IR) and the loop that shape the nic site differs in both proteins. Arguably, replicases cleave their target site more efficiently, while relaxases exert more biochemical control over the process. Here we show that engineering a relaxase target by mimicking the replicase target, results in enhanced formation of protein-DNA covalent complexes. Three widely different relaxases, which belong to MOBF, MOBQ and MOBP families, can properly cleave DNA sequences with permuted target sequences. Collaterally, the secondary structure that the permuted targets acquired within a supercoiled plasmid DNA resulted in poor conjugation frequencies underlying the importance of relaxase accessory proteins in conjugative DNA processing. Our results reveal that relaxase and replicase targets can be interchangeable in vitro. The new Rep substrates provide new bioconjugation tools for the design of sophisticated DNA-protein nanostructures.This work was financed by grants BFU2014-55534-C2-1-P from the Spanish Ministry of Economy and Competitiveness and 612146/FP7-ICT- 2013 and 282004/FP7-HEALTH.2011.2.3.1-2 from the European Union Seventh Framework Programme to FC and grant BFU2014-55534-C2-2-P from the Spanish Ministry of Economy and Competitiveness to GM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Exchange of functional domains between a bacterial conjugative relaxase and the integrase of the human adeno-associated virus

Endonucleases of the HUH family are specialized in processing single-stranded DNA in a variety of evolutionarily highly conserved biological processes related to mobile genetic elements. They share a structurally defined catalytic domain for site-specific nicking and strand-transfer reactions, which is often linked to the activities of additional functional domains, contributing to their overall versatility. To assess if these HUH domains could be interchanged, we created a chimeric protein from two distantly related HUH endonucleases, containing the N-terminal HUH domain of the bacterial conjugative relaxase TrwC and the C-terminal DNA helicase domain of the human adeno-associated virus (AAV) replicase and site-specific integrase. The purified chimeric protein retained oligomerization properties and DNA helicase activities similar to Rep68, while its DNA binding specificity and cleaving-joining activity at oriT was similar to TrwC. Interestingly, the chimeric protein could catalyse site-specific integration in bacteria with an efficiency comparable to that of TrwC, while the HUH domain of TrwC alone was unable to catalyze this reaction, implying that the Rep68 C-terminal helicase domain is complementing the TrwC HUH domain to achieve site-specific integration into TrwC targets in bacteria. Our results illustrate how HUH domains could have acquired through evolution other domains in order to attain new roles, contributing to the functional flexibility observed in this protein superfamily.This work was supported by the Medical Research Council (MRC) grant MR/N022890/1 to EH and grant 1001764 to RML; National Institutes of Health (NIH) grant RO1-GM09285 to CRE; Spanish Ministry of Economy and competitiveness (MINECO) grant BIO2013-46414-P to ML and AFM is supported by a Doc.Mobility fellowship from the Swiss National Science Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Inhibitory transmission in locus coeruleus neurons expressing GABAA receptor epsilon subunit has a number of unique properties.

International audienceFast inhibitory synaptic transmission in the brain relies on ionotropic GABA(A) receptors (GABA(A)R). Eighteen genes code for GABA(A)R subunits, but little is known about the epsilon subunit. Our aim was to identify the synaptic transmission properties displayed by native receptors incorporating epsilon. Immunogold localization detected epsilon at synaptic sites on locus coeruleus (LC) neurons. In situ hybridization revealed prominent signals from epsilon, and mRNAs, some low beta1 and beta3 signals, and no gamma signal. Using in vivo extracellular and in vitro patch-clamp recordings in LC, we established that neuron firing rates, GABA-activated currents, and mIPSC charge were insensitive to the benzodiazepine flunitrazepam (FLU), in agreement with the characteristics of recombinant receptors including an epsilon subunit. Surprisingly, LC provided binding sites for benzodiazepines, and GABA-induced currents were potentiated by diazepam (DZP) in the micromolar range. A number of GABA(A)R ligands significantly potentiated GABA-induced currents, and zinc ions were only active at concentrations above 1 muM, further indicating that receptors were not composed of only alpha and beta subunits, but included an epsilon subunit. In contrast to recombinant receptors including an epsilon subunit, GABA(A)R in LC showed no agonist-independent opening. Finally, we determined that mIPSCs, as well as ensemble currents induced by ultra-fast GABA application, exhibited surprisingly slow rise times. Our work thus defines the signature of native GABA(A)R with a subunit composition including epsilon: differential sensitivity to FLU and DZP and slow rise time of currents. We further propose that alpha(3,) beta(1/3,) and epsilon subunits compose GABA(A)R in LC

Distribución de masas de agua y transportes en la sección Caibox - 20ºW

Póster.-- XV Seminario Ibérico de Química Marina, Vigo, 22-24 de febrero de 2010N

Informe de la campaña CAIBOX, 2ª Fase de CAIBEX: Shelf-ocean exchanges in the Canaries-Iberia Large Marine Ecosystem

25 páginas, 2 tabla, 10 figura

Calibrating stable carbon isotopes in planktonic foraminifera off the NW Iberian margin: nutrient approach

42th CIESM Congress, Cascais, 7-11 October 2019The interpretation of the carbon isotopic (δ C) signal of planktonic foraminifera in sediment records to reconstruct past nutrient availability is complex and poorly understood, especially in coastal upwelling areas, because the isotopic fractions of carbon incorporated into the shells may be derived from different sources and affected by different processes. To minimize the existing uncertainties, we measured δ C from various species of planktonic foraminifera from core-top sediments and plankton net samples collected off the NW Iberian Margin and compared the results directly with water column nutrient content for the purpose of calibrationPeer reviewe