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Platelet-Rich Plasma (PRP) is enriched in molecular messengers with restorative effects on altered tissue environments. Upon activation, platelets release a plethora of growth factors and cytokines, either in free form or encapsulated in exosomes, which have been proven to promote tissue repair and regeneration. Translational research on the potential of exosomes as a safe nanosystem for therapeutic cargo delivery requires standardizing exosome isolation methods along with their molecular and morphological characterization. With this aim, we isolated and characterized the exosomes released by human PRP platelets. Western blot analysis revealed that CaCl2-activated platelets (PLT-Exos-Ca2+) released more exosomes than non-activated ones (PLT-Exos). Moreover, PLT-Exos-Ca2+ exhibited a molecular signature that meets the most up-to-date biochemical criteria for platelet-derived exosomes and possessed morphological features typical of exosomes as assessed by transmission electron microscopy. Array analysis of 105 analytes including growth factors and cytokines showed that PLT-Exos-Ca2+ exhibited lower levels of most analytes compared to PLT-Exos, but relatively higher levels of those consistently validated as components of the protein cargo of platelet exosomes. In summary, the present study provides new insights into the molecular composition of human platelet-derived exosomes and validates a method for isolating highly pure platelet exosomes as a basis for future preclinical studies in regenerative medicine and drug delivery.

Background: Replacement of radioligand binding assays with antibody-antigen interaction-based approaches for quantitative analysis of G protein-coupled receptor (GPCR) levels requires the use of purified protein standards containing the antigen. GPCRs in general and cannabinoid CB1 receptor in particular show a progressive tendency to aggregate and precipitate in aqueous solution outside of their biological context due to the low solubility that the hydrophobic nature imprinted by their seven transmembrane domains. This renders full-length recombinant GPCRs useless for analytical purposes, a problem that can be overcome by engineering soluble recombinant fragments of the receptor containing the antigen. Results: Here we generated highly soluble and stable recombinant protein constructs GST-CB1(41)(4-)(472 )and GST-CB1(414-442) containing much of the human CB1 receptor C-terminal tail for use as standard and negative control, respectively, in quantitative Western blot analysis of CB1 receptor expression on crude synaptosomes of the adult rat brain cortex. To this end we used three different antibodies, all raised against a peptide comprising the C-terminal residues 443-473 of the mouse CB1 receptor that corresponds to residues 442-472 in the human homolog. Estimated values of CB1 receptor density obtained by quantitative Western blot were of the same order of magnitude but slightly higher than values obtained by the radioligand saturation binding assay. Conclusions: Collectively, here we provide a suitable Western blot-based design as a simple, cost-effective and radioactivity-free alternative for the quantitative analysis of CB1 receptor expression, and potentially of any GPCR, in a variety of biological samples. The discrepancies between the results obtained by quantitative Western blot and radioligand saturation binding techniques are discussed in the context of their particular theoretical bases and methodological constraints.

[EN]The production of artificial anti-CB1 antibodies in nanoparticle format is described using the solid-phase imprinting approach. Instead of whole protein imprinting, a linear C-terminus sequence of the receptor comprising 15 amino acids (458-KVTMSVSTDTSAEAL-472) has been used as template, in accordance with the epitope imprinting approach. This sequence is located intracellularly, and it is involved in coupling to G(i/o) proteins, being responsible for CB1 receptor desensitisation and internalisation. Developed molecularly imprinted materials were found to be in the nanometre scale, with a particle size of 126.4 +/- 10.5 nm at pH 3 (25 oC) and spherical shape. It was also observed that the size was sensible to temperature changes being reduced to 106.3 +/- 15.2 nm at 35 degrees C. Lower critical solution temperature of this polymer was found to be approximate to 33.4 degrees C. The affinity and selectivity of the artificial antibody were assessed through dot blot and Western blot experiments. For the latter, recombinant fusion proteins GST-CB1(414-472) and GST-CB1(414-442) were produced to work respectively as target and negative control proteins. The control protein did not carry the target epitope for being devoid of last 30 amino acids at the C-terminus. The results demonstrated that the anti-CB1 material recognised selectively the target protein, thanks to the presence of the 15-amino acid sequence selected as epitope, which revealed that binding occurred at the C-terminus of the receptor itself. The methodology presented may pave the way for the development of novel imprinted nanomaterials for other proteins included in the superfamily of the G-protein-coupled receptors (GPCR).

[EN] The transient receptor potential vanilloid 1 (TRPV1) participates in synaptic functions in the brain. In the dentate gyrus, post-synaptic TRPV1 in the granule cell (GC) dendritic spines mediates a type of long-term depression (LTD) of the excitatory medial perforant path (MPP) synapses independent of pre-synaptic cannabinoid CB1 receptors. As CB1 receptors also mediate LTD at these synapses, both CB1 and TRPV1 might be influencing the activity of each other acting from opposite synaptic sites. We tested this hypothesis in the MPP–GC synapses of mice lacking TRPV1 (TRPV1-/-). Unlike wild-type (WT) mice, low-frequency stimulation (10min at 10Hz) of TRPV1-/- MPP fibers elicited a form of long-term potentiation (LTP) that was dependent on (1) CB1 receptors, (2) the endocannabinoid 2-arachidonoylglycerol (2-AG), (3) rearrangement of actin filaments, and (4) nitric oxide signaling. These functional changes were associated with an increase in the maximum binding efficacy of guanosine-5′-O-(3-[35S]thiotriphosphate) ([35S]GTPgS) stimulated by the CB1 receptor agonist CP 55,940, and a significant decrease in receptor basal activation in the TRPV1-/- hippocampus. Finally, TRPV1-/- hippocampal synaptosomes showed an augmented level of the guanine nucleotide-binding (G) Gai1, Gai2, and Gai3 protein alpha subunits. Altogether, the lack of TRPV1 modifies CB1 receptor signaling in the dentate gyrus and causes the shift from CB1 receptor-mediated LTD to LTP at the MPP–GC synapses.

176 p., Egun, ezaguna da terminal sinaptiko glutamatergiko eta GABAergikoetan espresatzen den CB1 hartzailearen aktibazioak aktibitate neuronal eszitatzaile-inhibitzailearen balantzea erregulatzen duela. Horregatik, bi terminal mota hauetan aurkitzen den CB1 hartzailearen seinaleztapen kanonikoaren (G¿i/o proteinen akoplamentua) azterketa zehatza beharrezkoa da agonista kannabinoideen efektuak garunean zehar karakterizatzeko. Lan honetan, CB1 hartzailea neurona glutamatergiko edo GABAergikoetan espresatzen duen sagu transgenikoak erabiliz, kortex frontaleko eta hipokanpoko bi terminal sinaptiko hauetan CB1 hartzailearen seinaleztapen kanonikoa hartzailearen espresio mailarekin positiboki erlazionatzen dela frogatu dugu. Gainera, kolesterola CB1 hartzailearen funtzio kanonikoa negatiboki modulatzen duen konposatu alosteriko gisa identifikatu dugu.

The transient receptor potential vanilloid 1 (TRPV1) is a non-selective ligand-gated cation channel involved in synaptic transmission, plasticity, and brain pathology. In the hippocampal dentate gyrus, TRPV1 localizes to dendritic spines and dendrites postsynaptic to excitatory synapses in the molecular layer (ML). At these same synapses, the cannabinoid CB1 receptor (CB1R) activated by exogenous and endogenous cannabinoids localizes to the presynaptic terminals. Hence, as both receptors are activated by endogenous anandamide, co-localize, and mediate long-term depression of the excitatory synaptic transmission at the medial perforant path (MPP) excitatory synapses though by different mechanisms, it is plausible that they might be exerting a reciprocal influence from their opposite synaptic sites. In this anatomical scenario, we tested whether the absence of TRPV1 affects the endocannabinoid system. The results obtained using biochemical techniques and immunoelectron microscopy in a mouse with the genetic deletion of TRPV1 show that the expression and localization of components of the endocannabinoid system, included CB1R, change upon the constitutive absence of TRPV1. Thus, the expression of fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) drastically increased in TRPV1(-/-) whole homogenates. Furthermore, CB1R and MAGL decreased and the cannabinoid receptor interacting protein 1a (CRIP1a) increased in TRPV1(-/-) synaptosomes. Also, CB1R positive excitatory terminals increased, the number of excitatory terminals decreased, and CB1R particles dropped significantly in inhibitory terminals in the dentate ML of TRPV1(-/-) mice. In the outer 2/3 ML of the TRPV1(-/-) mutants, the proportion of CB1R particles decreased in dendrites, and increased in excitatory terminals and astrocytes. In the inner 1/3 ML, the proportion of labeling increased in excitatory terminals, neuronal mitochondria, and dendrites. Altogether, these observations indicate the exis

Specific and selective anti-CB1 antibodies are among the most powerful research tools to unravel the complex biological processes mediated by the CB1 receptor in both physiological and pathological conditions. However, low performance of antibodies remains a major source of inconsistency between results from different laboratories. Using a variety of techniques, including some of the most commonly accepted ones for antibody specificity testing, we identified three of five commercial antibodies against different regions of CB1 receptor as the best choice for specific end-use purposes. Specifically, an antibody against a long fragment of the extracellular amino tail of CB1 receptor (but not one against a short sequence of the extreme amino-terminus) detected strong surface staining when applied to live cells, whereas two different antibodies against an identical fragment of the extreme carboxy-terminus of CB1 receptor (but not one against an upstream peptide) showed acceptable performance on all platforms, although they behaved differently in immunohistochemical assays depending on the tissue fixation procedure used and showed different specificity in Western blot assays, which made each of them particularly suitable for one of those techniques. Our results provide a framework to interpret past and future results derived from the use of different anti-CB1 antibodies in the context of current knowledge about the CB1 receptor at the molecular level, and highlight the need for an adequate validation for specific purposes, not only before antibodies are placed on the market, but also before the decision to discontinue them is made.

Specific and selective anti-CB1 antibodies are among the most powerful research tools to unravel the complex biological processes mediated by the CB1 receptor in both physiological and pathological conditions. However, low performance of antibodies remains a major source of inconsistency between results from different laboratories. Using a variety of techniques, including some of the most commonly accepted ones for antibody specificity testing, we identified three of five commercial antibodies against different regions of CB1 receptor as the best choice for specific end-use purposes. Specifically, an antibody against a long fragment of the extracellular amino tail of CB1 receptor (but not one against a short sequence of the extreme amino-terminus) detected strong surface staining when applied to live cells, whereas two different antibodies against an identical fragment of the extreme carboxy-terminus of CB1 receptor (but not one against an upstream peptide) showed acceptable performance on all platforms, although they behaved differently in immunohistochemical assays depending on the tissue fixation procedure used and showed different specificity in Western blot assays, which made each of them particularly suitable for one of those techniques. Our results provide a framework to interpret past and future results derived from the use of different anti-CB1 antibodies in the context of current knowledge about the CB1 receptor at the molecular level, and highlight the need for an adequate validation for specific purposes, not only before antibodies are placed on the market, but also before the decision to discontinue them is made.

Currently, Cerebrovascular accident (CVA) also known as stroke, is the third leading cause of disability and death worldwide. CVA is a sudden alteration of cerebral circulation, which results in neural cell death and consequently a temporary or permanent loss in certain neuronal functions. Therefore, in addition to working on prevention, in clinical practice is essential to have effective treatments aimed at avoiding neuronal dead. Unfortunately, the efficacy of the currently used therapeutic strategies is limited and, in most cases, patients suffering from stroke do not recover an adequate quality of life. This article presents cell therapy as a future alternative of interest in the treatment of chronic patients of CVA. This new therapy aims to replace the lost neuronal cells that are the main cause of disability in patients. Specifically, the review article presents recent progress on the use of teratocarcinoma-derived Ntera2/D1 neuronlike cells (NT2N cells), as graft source for cell transplantation in stroke, discussing the safety and efficacy demonstrated by preclinical and clinical trials made to date.; Gaur egun, istripu zerebrobaskularra (IZB) mundu osoan bigarren heriotza-kausa eta ezgaitasunen hirugarren eragilea da. IZB, garuneko zirkulazioaren bat-bateko asaldura da, non, neuronen galera baten ondorioz, aldi baterako edo betirako garunaren eskualde jakin baten funtzioa eraldatzen den. Hortaz, praktika klinikoan, prebentzioan lan egiteaz gain, ezinbestekoa da funtzio neurologikoen galeraren oinarrian dagoen neuronen heriotzaren kontra zuzendutako tratamendu eraginkorrak izatea. Tamalez, gaur egun erabiltzen diren estrategia terapeutikoen eraginkortasuna mugatua da, eta, kasu gehienetan, IZB izandako pazienteek ez dute bizi-kalitate egokia berreskuratzen. Artikulu honetan, gaixoen ezgaitasunaren arrazoia den neuronen galerari aurre egiteko zelula hauen ordezkapena itu duen terapia zelularra aurkezten da. Zehazki, Ntera2/D1 edo NT2 deituriko zelula-lerrotik

Currently, Cerebrovascular accident (CVA) also known as stroke, is the third leading cause of disability and death worldwide. CVA is a sudden alteration of cerebral circulation, which results in neural cell death and consequently a temporary or permanent loss in certain neuronal functions. Therefore, in addition to working on prevention, in clinical practice is essential to have effective treatments aimed at avoiding neuronal dead. Unfortunately, the efficacy of the currently used therapeutic strategies is limited and, in most cases, patients suffering from stroke do not recover an adequate quality of life. This article presents cell therapy as a future alternative of interest in the treatment of chronic patients of CVA. This new therapy aims to replace the lost neuronal cells that are the main cause of disability in patients. Specifically, the review article presents recent progress on the use of teratocarcinoma-derived Ntera2/D1 neuronlike cells (NT2N cells), as graft source for cell transplantation in stroke, discussing the safety and efficacy demonstrated by preclinical and clinical trials made to date.; Gaur egun, istripu zerebrobaskularra (IZB) mundu osoan bigarren heriotza-kausa eta ezgaitasunen hirugarren eragilea da. IZB, garuneko zirkulazioaren bat-bateko asaldura da, non, neuronen galera baten ondorioz, aldi baterako edo betirako garunaren eskualde jakin baten funtzioa eraldatzen den. Hortaz, praktika klinikoan, prebentzioan lan egiteaz gain, ezinbestekoa da funtzio neurologikoen galeraren oinarrian dagoen neuronen heriotzaren kontra zuzendutako tratamendu eraginkorrak izatea. Tamalez, gaur egun erabiltzen diren estrategia terapeutikoen eraginkortasuna mugatua da, eta, kasu gehienetan, IZB izandako pazienteek ez dute bizi-kalitate egokia berreskuratzen. Artikulu honetan, gaixoen ezgaitasunaren arrazoia den neuronen galerari aurre egiteko zelula hauen ordezkapena itu duen terapia zelularra aurkezten da. Zehazki, Ntera2/D1 edo NT2 deituriko zelula-lerrotik

Beste Medikuntza-arloetan ez bezala, Psikiatrian gaixotasun psikiatrikoei buruzko hipotesi etiologikoak ebi-dentzia farmakologikoetan oinarritzen dira. Horrela, 1950eko hamarkadan depresioaren kontrako farmakoak garatzen hasi zi-ren, behin ikusita zer-nolako eraginak zituzten tuberkulosiaren kontrako farmako batzuek. Lehenengo antidepresiboek neuro-transmisio monoaminergikoa bultzatzen zutenez, hipotesi monoaminergikoa sortu zen, eta depresioari buruzko ikerkuntzak serotoninaren, noradrenalinaren eta dopaminaren (monoaminen) gutxiagotzea hartu zuen oinarri. Orduz geroztik hipotesi mo-noaminergikoak, eta bere bariazioek, farmako antidepresiboen garapena baldintzatu dute. Hala ere, azkeneko urteetan egin-dako ikerketek erakutsi dute estresak eta depresioak neuronen atrofia eta galera eragiten dutela, eta horrekin batera kortex eta sistema linbikoaren bolumena murrizten dela. Ebidentzia berri hauen arabera, depresioa ez litzateke izango garuneko gune zehatz bateko asaldura hutsa, baizik eta gogo aldartea, kognizioa eta oroimenak kontrolatzen dituzten zirkuitu neuronalen asaldura. Depresioari buruzko azkeneko ikerketa preklinikoek eta klinikoek agerian jarri dute ketaminak (anestesiko disoziati-boa) eragin antidepresibo harrigarriak dituela, azkarrak eta iraunkorrak. Oinarrizko ikerketek ketaminak sinapsi konexioak oso azkar handitzen dituela frogatu dute, depresioan antzemandako neurona-atrofia leheneratzen dutela. Ebidentzia hauek farmako antidepresibo berrien bilaketa berpiztu dute, eta hainbat mekanismo berri proposatu dira helburu horretarako.; Unlike in other areas of Medicine, in Psychiatry, etiological hypotheses are based on pharmacological evidences. In this way, the development of antidepressant drugs began in the 1950s, once the effects of several drugs against tu-berculosis were observed. Since the first antidepressants potentiated monoaminergic neurotransmission, the monoaminergic hypothesis was generated, and the research on depression was based on the de