Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

[EN] Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action

Fast Decision Algorithms in Low-Power Embedded Processors for Quality-of-Service Based Connectivity of Mobile Sensors in Heterogeneous Wireless Sensor Networks

When a mobile wireless sensor is moving along heterogeneous wireless sensor networks, it can be under the coverage of more than one network many times. In these situations, the Vertical Handoff process can happen, where the mobile sensor decides to change its connection from a network to the best network among the available ones according to their quality of service characteristics. A fitness function is used for the handoff decision, being desirable to minimize it. This is an optimization problem which consists of the adjustment of a set of weights for the quality of service. Solving this problem efficiently is relevant to heterogeneous wireless sensor networks in many advanced applications. Numerous works can be found in the literature dealing with the vertical handoff decision, although they all suffer from the same shortfall: a non-comparable efficiency. Therefore, the aim of this work is twofold: first, to develop a fast decision algorithm that explores the entire space of possible combinations of weights, searching that one that minimizes the fitness function; and second, to design and implement a system on chip architecture based on reconfigurable hardware and embedded processors to achieve several goals necessary for competitive mobile terminals: good performance, low power consumption, low economic cost, and small area integration

Estimation of social cohesion in the Spanish municipalities after the implantation of the high speed railway

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Estimación de la cohesión social en los municipios españoles tras la implantación de la Alta Velocidad ferroviaria

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A Short Region of Connexin43 Reduces Human Glioma Stem Cell Migration, Invasion, and Survival through Src, PTEN, and FAK

[EN] Connexin43 (CX43), a protein that forms gap junction channels and hemichannels in astrocytes, is downregulated in high-grade gliomas. Its relevance for glioma therapy has been thoroughly explored; however, its positive effects on proliferation are counterbalanced by its effects onmigration and invasion. Here,weshowthat a cell-penetrating peptide based onCX43(TAT-Cx43266-283) inhibited c-Src and focal adhesion kinase (FAK) and upregulated phosphatase and tensinhomolog inglioma stem cells (GSCs) derived from patients. Consequently, TAT-Cx43266-283 reduced GSC motility, as analyzed by time-lapse microscopy, and strongly reduced their invasive ability. Interestingly, we investigated the effects of TAT-Cx43266-283 on freshly removed surgical specimens as undissociated glioblastoma blocks, which revealed a dramatic reduction in the growth, migration, and survival of these cells. In conclusion, a region of CX43 (amino acids 266–283) exerts an important anti-tumor effect in patient-derived glioblastoma models that includes impairment of GSC migration and invasion

Estimation of social cohesion in the Spanish municipalities after the implantation of the high speed railway

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The invasion of glioblastoma stem cells is reduced by the sequence of the connexin43 that interacts with c-Src

Resumen del póster presentado al XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Salamanca del 5 al 8 de septiembre de 2016.Glioblastoma stem cells (GSC) constitute a niche of cells with self-renewal ability and resistance to conventional therapies. Thus, they are responsible for relapse in these aggressive brain tumors when their resection is not complete. The expression of connexin43 (Cx43), the main gap junction channel-forming protein, and c-Src, a non receptor tyrosine kinase, is inversely related in glioblastoma cells. Interestingly, restoring Cx43 in GSC reverses GSC phenotype. The sequence responsible for this effect is the region that interacts with c-Src (amino acids 266-283). The Focal Adhesion Kinase (FAK) requires the c-Src-mediated phosphorylation in tyrosines 576 and 577 in order to be fully active and promote cell migration. In this study, fresh human tumour biopsies were disaggregated to obtain human primary GSCs. These cells were treated with a cell-penetrating peptide containing the Cx43-Src interacting sequence (Tat-Cx43266-283). The migration was analyzed by tracking individual cell trajectories in cultures recorded by Time-Lapse Live-cell Imaging. Matrigel-treated transwell inserts were used to analyze the invasion and the levels of phosphorylation of FAK tyrosines 576 and 577 were evaluated by western Blot. Our results showed that Tat-Cx43266-283 reduced the migration and invasion of human primary GSC. The analysis of c-Src and FAK proteins suggests that the mechanism by which this reduction takes place includes the inhibition of c-Src/FAK axis. In conclusion, Tat-Cx43266-283 inhibits c-Src with the subsequent reduction in FAK phosphorylation required to establish appropriate focal adhesions for migration. All together, these results indicate an important antivasive role of the sequence of Cx43 that interacts with c-Src in glioma stem cells.Peer reviewe

Recomendaciones prácticas en relación a la gestión de la geodiversidad y el patrimonio geológico en la red de reservas de la biosfera

Sociedad Geológica de España. Comisión de Patrimonio Geológico. Reunión Nacional (12º. 2017. Mahón, España)La Red Mundial de las Reservas de la Biosfera, está reconocida por el Programa Hombre y Biosfera (MaB) de la UNESCO. Esta red tiene como objetivo conciliar la conservación de la naturaleza con el desarrollo socio-económico sostenible, tomando como base la investigación, la capacitación, la interpretación y la educación para la sostenibilidad de la población residente. Los 48 espacios que constituyen la Red de Reservas de la Biosfera Estatal (RRRBE) abarcan un número importante de Lugares de Interés Geológico (LIG) inventariados a diferentes escalas, siendo 50 de ellos de importancia internacional (Geosites). Pese a que en la RRRBE se han implementado diversas acciones de gestión sobre este patrimonio geológico, lamentablemente, todavía no han sido realizadas con una metodología estandarizada englobada en una estrategia de gestión planificada. En este trabajo se propone una serie de recomendaciones para conseguir este objetivo.Área de Patrimonio Geológico y Minero, Instituto Geológico y Minero de España, EspañaÁrea de Geología Ambiental y Geomatemáticas, Instituto Geológico y Minero de España, EspañaBiosfera XXI, Estudios Ambientales, EspañaServicio de la Reserva de la Biosfera de Urdaibai, Departamento de Medio Ambiente, Planificación Territorial y Vivienda del Gobierno Vasco, Españ

A short region of connexin43 reduces human glioma stem cell migration, invasion, and survival through Src, PTEN, and FAK

Connexin43 (CX43), a protein that forms gap junction channels and hemichannels in astrocytes, is downregulated in high-grade gliomas. Its relevance for glioma therapy has been thoroughly explored; however, its positive effects on proliferation are counterbalanced by its effects on migration and invasion. Here, we show that a cell-penetrating peptide based on CX43 (TAT-Cx43) inhibited c-Src and focal adhesion kinase (FAK) and upregulated phosphatase and tensin homolog in glioma stem cells (GSCs) derived from patients. Consequently, TAT-Cx43 reduced GSC motility, as analyzed by time-lapse microscopy, and strongly reduced their invasive ability. Interestingly, we investigated the effects of TAT-Cx43 on freshly removed surgical specimens as undissociated glioblastoma blocks, which revealed a dramatic reduction in the growth, migration, and survival of these cells. In conclusion, a region of CX43 (amino acids 266–283) exerts an important anti-tumor effect in patient-derived glioblastoma models that includes impairment of GSC migration and invasion.This work was supported by the Ministerio de Economía y Competitividad, Spain (FEDER BFU2015-70040-R), Junta de Castilla y León, Spain (FEDER SA026U16), and Fundación Ramón Areces. M.J.-R. was a fellowship recipient from the Junta de Castilla y León and the European Social Fund.Peer Reviewe

A c-Src Inhibitor Peptide Based on Connexin43 Exerts Neuroprotective Effects through the Inhibition of Glial Hemichannel Activity

The non-receptor tyrosine kinase c-Src is an important mediator in several signaling pathways related to neuroinflammation. Our previous study showed that cortical injection of kainic acid (KA) promoted a transient increase in c-Src activity in reactive astrocytes surrounding the neuronal lesion. As a cell-penetrating peptide based on connexin43 (Cx43), specifically TAT-Cx43266–283, inhibits Src activity, we investigated the effect of TAT-Cx43266–283 on neuronal death promoted by cortical KA injections in adult mice. As expected, KA promoted neuronal death, estimated by the reduction in NeuN-positive cells and reactive gliosis, characterized by the increase in glial fibrillary acidic protein (GFAP) expression. Interestingly, TAT-Cx43266–283 injected with KA diminished neuronal death and reactive gliosis compared to KA or KA+TAT injections. In order to gain insight into the neuroprotective mechanism, we used in vitro models. In primary cultured neurons, TAT-Cx43266–283 did not prevent neuronal death promoted by KA, but when neurons were grown on top of astrocytes, TAT-Cx43266–283 prevented neuronal death promoted by KA. These observations demonstrate the participation of astrocytes in the neuroprotective effect of TAT-Cx43266–283. Furthermore, the neuroprotective effect was also present in non-contact co-cultures, suggesting the contribution of soluble factors released by astrocytes. As glial hemichannel activity is associated with the release of several factors, such as ATP and glutamate, that cause neuronal death, we explored the participation of these channels on the neuroprotective effect of TAT-Cx43266–283. Our results confirmed that inhibitors of ATP and NMDA receptors prevented neuronal death in co-cultures treated with KA, suggesting the participation of astrocyte hemichannels in neurotoxicity. Furthermore, TAT-Cx43266–283 reduced hemichannel activity promoted by KA in neuron-astrocyte co-cultures as assessed by ethidium bromide (EtBr) uptake assay. In fact, TAT-Cx43266–283 and dasatinib, a potent c-Src inhibitor, strongly reduced the activation of astrocyte hemichannels. In conclusion, our results suggest that TAT-Cx43266–283 exerts a neuroprotective effect through the reduction of hemichannel activity likely mediated by c-Src in astrocytes. These data unveil a new role of c-Src in the regulation of Cx43-hemichannel activity that could be part of the mechanism by which astroglial c-Src participates in neuroinflammation