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15 p.-7 fig.-2 tab., C-type lectin-like receptor 2 (CLEC-2) plays a crucial role in different platelet-related physiological and pathological processes. It signals through a tyrosine kinase-mediated pathway that is highly dependent on the positive feedback exerted by the platelet-derived secondary mediators, adenosine diphosphate (ADP) and thromboxane A2 (TXA2). Here, we aimed to analyze the tyrosine phosphoproteome of platelets activated with the CLEC-2 agonist rhodocytin to identify relevant phosphorylated tyrosine residues (p-Tyr) and proteins involved in platelet activation downstream of this receptor. We identified 363 differentially p-Tyr residues, corresponding to the majority of proteins previously known to participate in CLEC-2 signaling and also novel ones, including adaptors (e.g., DAPP1, Dok1/3, CASS4, Nck1/2), kinases/phosphatases (e.g., FAK1, FES, FGR, JAK2, SHIP2), and membrane proteins (e.g., G6F, JAM-A, PECAM-1, TLT-1). To elucidate the contribution of ADP and TXA2 at different points of the CLEC-2 signaling cascade, we evaluated p-Tyr levels of residues identified in the analysis and known to be essential for the catalytic activity of kinases Syk(p-Tyr525+526) and Src(p-Tyr419), and for PLCγ2 activity (p-Tyr759). We demonstrated that Syk phosphorylation at Tyr525+526 also happens in the presence of ADP and TXA2 inhibitors, which is not the case for Src-pTyr419 and PLCγ2-pTyr759. Kinetics studies for the three phosphoproteins show some differences in the phosphorylation profile. Ca2+ mobilization assays confirmed the relevance of ADP and TXA2 for full CLEC-2-mediated platelet activation. The present study provides significant insights into the intracellular events that take place following CLEC-2 activation in platelets, contributing to elucidate in detail the CLEC-2 signalosome., This study was supported by the Spanish Ministryof Economy and Competitiveness (MINECO) [grant No. SAF2016-79662-R], co-funded by the European Regional Develop-ment Fund (ERDF); and the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia [ED431C2018/21; predoctoral grant Plan I2C 2014; and CentroSingular de investigación de Galicia accreditation 2016-2019, ED431G/05], co-funded by the European Regional Development Fund (ERDF). The study also received funding from the European Union’s Horizon 2020 research andinnovation programme under the Marie Skłodowska-Curiegrant agreement No 766118. J.A.E. is supported by DeutscheForschungsgemeinschaft [DFG grant: EB177/13-1]

Lipid rafts are membrane microdomains that have been proposed to play an important role in several platelet-signalling cascades, including those mediated by the receptors Glycoprotein VI (GPVI), and C-type lectin domain family 1 member B (CLEC-2), both involved in thrombus formation. We have performed a LC-MS/MS proteomic analysis of lipid rafts isolated from platelets activated through GPVI and CLEC-2 as well as from resting platelets. Our aim was to determine the magnitude of changes in lipid rafts protein composition and to elucidate the relevance of these alterations in platelet function. A number of relevant signalling proteins were found enriched in lipid rafts following platelet activation (such as the tyrosine protein kinases Fyn, Lyn and Yes; the G proteins G(i) and G(z); and cAMP protein kinase). Interestingly, our results indicate that the relative enrichment of lipid rafts in these signalling proteins may not be a consequence of protein translocation to these domains upon platelet stimulation, but the result of a massive loss in cytoskeletal proteins after platelet activation. Thus, this study may help to better understand the effects of platelet activation in the reorganization of lipid rafts and set the basis for further proteomic studies of these membrane microdomains in platelets. Significance: We performed the first proteomic comparative analysis of lipid rafts- protein composition in platelets activated through GPVI and CLEC-2 receptors and in resting state. We identified a number of signalling proteins essential for platelet activation relatively enriched in platelets activated through both receptors, and we show that lipid rafts reorganization upon platelet activation leads to a loss in cytoskeletal proteins, highly associated to these domains in resting platelets., This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) [grant No. SAF2016-79662-R], co-funded by the European Regional Development Fund (ERDF); the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia [predoctoral grant Plan I2C 2014; and Centro Singular de investigación de Galicia accreditation 2016–2019, ED431G/05; both co-funded by the ERDF]. J.A.E. is supported by Deutsche Forschungsgemeinschat [DFG grant: EB177/13-1].

International audience; The integration of nanoparticles (NPs) into photonic devices and plasmonic sensors requires selective patterning of these NPs with fine control of their size, shape, and spatial positioning. In this article, we report on a general strategy to pattern different types of NPs. This strategy involves the functionalization of photopolymers before their patterning by two-photon laser writing to fabricate micro- and nanostructures that selectively attract colloidal NPs with suitable ligands, allowing their precise immobilization and organization even within complex 3D structures. Monolayers of NPs without aggregations are obtained and the surface density of NPs on the polymer surface can be controlled by changing either the time of immersion in the colloidal solution or the type of amine molecule chemically grafted on the polymer surface. Different types of NPs (gold, silver, polystyrene, iron oxide, colloidal quantum dots, and nanodiamonds) of different sizes are introduced showing a potential toward nanophotonic applications. To validate the great potential of our method, we successfully demonstrate the integration of quantum dots within a gold nanocube with high spatial resolution and nanometer precision. The promise of this hybrid nanosource of light (plasmonic/polymer/QDs) as optical nanoswitch is illustrated through photoluminescence measurements under polarized exciting light.

Background: Clinical management of ischemic events and prevention of vascular disease is based on antiplatelet drugs. Given the relevance of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) as a candidate target in thrombosis, the main goal of the present study was to identify novel antiplatelet agents within the existing inhibitors blocking PI3K isoforms. Methods: We performed a biological evaluation of the pharmacological activity of PI3K inhibitors in platelets. The effect of the inhibitors was evaluated in intracellular calcium release and platelet functional assays, the latter including aggregation, adhesion, and viability assays. The in vivo drug antithrombotic potential was assessed in mice undergoing chemically induced arterial occlusion, and the associated hemorrhagic risk evaluated by measuring the tail bleeding time. Results: We show that PI3K Class IA inhibitors potently block calcium mobilization in human platelets. The PI3K p110δ inhibitor Idelalisib inhibits platelet aggregation mediated by ITAM receptors GPVI and CLEC-2, preferentially by the former. Moreover, Idelalisib also inhibits platelet adhesion and aggregation under shear and adhesion to collagen. Interestingly, an antithrombotic effect was observed in mice treated with Idelalisib, with mild bleeding effects at high doses of the drug. Conclusion: Idelalisib may have antiplatelet effects with minor bleeding effects, which provides a rationale to evaluate its antithrombotic efficacy in humans.

Resumen: El aprendizaje organizacional (AO) ha sido estudiado como una capacidad o proceso que permite mejorar la performance de las organizaciones. Sin embargo, el campo de estudio del AO se caracteriza por contar con posturas y definiciones heterogéneas o no explicitadas, a partir de las cuales se construyen diversos modelos de medición. El objetivo de este trabajo es proponer una conceptualización del fenómeno del AO desde una perspectiva multidimensional que articule aportes y elementos de las distintas definiciones, y señalar sus implicancias metodológicas para la medición del fenómeno. Para cumplir con ese objetivo, se realiza una revisión sistemática de la literatura en las bases de datos Scopus y Google Scholar. Los 36 artículos seleccionados se analizan en profundidad en un proceso iterativo de lectura, codificación y análisis comparativo. De lo anterior se extraen ocho elementos centrales, descritos y articulados en una propuesta de definición de AO. Finalmente, se discute la postura ontológica y epistemológica que sustenta la conceptualización del fenómeno propuesta y se señalan las implicancias en términos metodológicos para la medición del AO. La principal contribución de este artículo es el desarrollo de una definición multidimensional de AO con una mirada ontológica y epistemológica del fenómeno, con vistas a su medición. De esta manera se busca superar definiciones reduccionistas, y reconocer su complejidad. Abstract: Organizational learning (OL) has been studied as a capability or process for improving organizations' performance. However, this field of study is characterized by heterogeneous or unspecified positions and definitions from which various measurement models are built. This paper aims to propose an OL conceptualization from a multidimensional perspective that brings together insights and elements from the different definitions and point out its methodological implications for measuring the phenomenon. For this, literature is reviewed systematically in the Scopus and Google Scholar databases. The 36 selected articles are examined in depth in an iterative reading, coding, and comparative analysis process. Eight central elements are extracted, described, and assembled in a proposed definition of OL. Finally, the ontological and epistemological position that supports the proposed conceptualization is discussed, pointing out its methodological implications for OL measurement. The main contribution is the development of a multidimensional definition of OL with an ontological and epistemological view for its measurement. Thus, it seeks to overcome reductionist definitions and recognize its complexity. Resumo: A aprendizagem organizacional (AO) vem sendo estudada como uma capacidade ou processo que permite melhorar a performance das organizações. Contudo, o campo de estudo da AO é caracterizado por contar com posicionamentos e definições heterogêneos ou não explícitos, a partir dos quais são construídos diversos modelos de medição. O objetivo deste trabalho é propor uma conceituação do fenômeno da AO sob uma perspectiva multidimensional que articule contribuições e elementos das diferentes definições, e indicar suas implicâncias metodológicas para avaliar o fenômeno. Para atingir esse objetivo, é realizada uma revisão sistemática da literatura nas bases de dados da Scopus e do Google Scholar. Os 36 artigos selecionados são analisados em profundidade em um processo iterativo de leitura, codificação e análise comparativa. A partir disso, são extraídos oito elementos centrais, descritos e articulados em uma proposta de definição de AO. Por último, é discutido o posicionamento ontológico e epistemológico que subsidia a conceituação do fenômeno proposta e são apresentadas as implicâncias em termos metodológicos para avaliar a AO. A principal contribuição deste artigo é o desenvolvimento de uma definição multidimensional da AO com um olhar ontológico e epistemológico do fenômeno, com vistas à sua medição. Dessa maneira, pretende-se superar definições reducionistas e reconhecer sua complexidade.

Obesity is one of the main health problems in industrialized countries. The contribution of multiple factors developed in obesity can hardly be modeled in vitro. In this context, the development of animal models mimicking human obesity could be essential. The aim of the present study was to compare platelets from a diet-induced obesity (DIO) rat model with their lean control group in order to elucidate platelet dysfunction mechanisms in obesity and correlate the results with previous data from morbid obese patients. In parallel, we also established a blood collection and platelet isolation methodology to study the DIO rat model at biochemical and functional level. Optimal blood collection was obtained from vena cava and platelet isolation was based on a serial of centrifugations avoiding platelet activation. Our results show that the DIO rat model simulate obesity pathologically since weight gain, fasting glucose and platelet counts are increased in obese rats. Interestingly, platelet levels of the active form of Src (pTyr419) showed a tendency to increase in DIO rats pointing towards a potential dysfunction in Src family kinases-related signalling pathways in obesity. Moreover, platelets from DIO rats adhere more to collagen compared with the control group, pointing towards Glycoprotein VI (GPVI) as one of the dysregulated receptors in obesity, in agreement with our recent studies in humans. These results confirm that obesity, in line with human studies, present a platelet dysregulation, and highlight the relevance of considering novel antithrombotic drug targets in these patients, such as GPVI.

Resumen El presente estudio muestra un caso clinico de reversion de una artrodesis con un implante total a una paciente de 63 anos que habia sido intervenida en dos ocasiones de artrodesis fallidas. La paciente presentaba dolor persistente a nivel de la articulacion de Chopart, asi como a nivel del maleolo externo, en especial durante la marcha, dolor a la palpacion directa a nivel de los ligamentos laterales del tobillo y dolor a nivel de articulacion astragalo-escafoidea y calcaneo-cuboidea cuando realizaba inversion forzada. En el presente articulo se introduce un metodo de tratamiento para revertir una artrodesis de la primera articulacion metatarso-falangica mediante la colocacion de un implante total tipo Toefit-Plus™ para devolver la movilidad de la articulacion. Se consiguio alivio del dolor con una movilidad de la primera articulacion metatarso-falangica dentro de limites normales, con un primer radio ligeramente insuficiente, siendo necesario un tratamiento posquirurgico con soportes plantares para mejorar la funcionalidad. En el examen clinico de rango de movimiento activo se lograron 40 de dorsiflexion, y en cuanto a la funcionalidad del extensor largo del primer dedo, volvio a su estado natural despues de la intervencion. El mayor conocimiento de las ventajas y desventajas de esta tecnica nos permitiria que mas pacientes pudieran verse favorecidos ante situaciones desfavorables con artrodesis metatarso-falangicas que no han dado el resultado esperado, por lo que es necesario profundizar en el estudio de mas casos.

In blood banks, platelets are stored until 7 days after a pathogen reduction technology (PRT) treatment, Mirasol® (vitamin B2 plus UVB light) in the present case. The storage time under these conditions may have an impact on platelets and their releasate leading to potential adverse reactions following transfusion to patients. The aim of this study was to analyze the proteome of extracellular vesicles generated by platelets at different storage days (2 and 7) to gain deeper information on the platelet concentrates state at those moments. EVs were isolated by a centrifugation-based approach and characterized by dynamic light scattering and transmission electron microscopy. Proteomic analysis was by LC-MS/MS and quantification by SWATH. In this way, 151 proteins were found up-regulated at day 7 of storage. This group includes CCL5 and Platelet Factor 4, chemokines with power to attract neutrophils and monocytes, which could generate transfusion adverse reactions. In addition, other glycoproteins and platelet activation markers were also found elevated at day 7. Proteins related to glycolysis and lactate production were found altered with high fold changes, showing a deregulation of platelet metabolism at day 7. The obtained results provide novel information about possible effects of platelet-derived EVs on transfusion adverse reactions. Significance We performed the first proteomic analysis of extracellular vesicles derived from platelets upon storage at different time points on blood bank conditions after Mirasol® treatment. We identified a high number of proteins related to platelet activation and platelet storage lesion that could have a role in possible transfusion adverse reactions.

Unwanted platelet activation is associated with numerous diseases, mainly thrombosis-related. In this context, proteomics has emerged as a novel tool with potential for drug target discovery and to scrutinize the effects of antiplatelet drugs. Areas covered: The present review presents the main findings of platelet proteomic studies to date in the context of drug target discovery and perspectives for the future ahead. It includes data and evidences obtained from literature searches on PubMed as well as commentaries derived from the authors' experience and opinions. Expert commentary: Platelet proteomics applied to drug target discovery is a young field. Recent studies have shown promising data, especially in the context of coronary artery disease. However, challenges remain such as establishing definitive guidelines for blood collection and platelet isolation, essential to guarantee data reproducibility. Recent advances in quantitative platelet proteomics should lead to novel studies with higher clinical impact in the near future.

Dilated cardiomyopathy (DCM) is a severe heart disease characterized by progressive ventricular dilation and impaired systolic function of the left ventricle. We recently identified a novel pathogenic mutation in the LMNA gene in a family affected by DCM showing sudden death background. We now aimed to identify potential biomarkers of disease status, as well as sudden death predictors, in members of this family. We analysed plasma samples from 14 family members carrying the mutation, four of which (with relevant clinical symptoms) were chosen for the proteomic analysis. Plasma samples from these four patients and from four sex- and age-matched healthy controls were processed for their enrichment in low- and medium-abundance proteins (ProteoMiner™) prior to proteomic analysis by 2D-DIGE and MS. 111 spots were found to be differentially regulated between mutation carriers and control groups, 83 of which were successfully identified by MS, corresponding to 41 different ORFs. Some proteins of interest were validated either by turbidimetry or western blot in family members and healthy controls. Actin, alpha-1-antytripsin, clusterin, vitamin-D binding protein and antithrombin-III showed increased levels in plasma from the diseased group. We suggest following these proteins as putative biomarkers for the evaluation of DCM status in LMNA mutation carriers. Biological significance We developed a proteomic analysis of plasma samples from a family showing history of dilated cardiomyopathy caused by a LMNA mutation, which may lead to premature death or cardiac transplant. We identified a number of proteins augmented in mutation carriers that could be followed as potential biomarkers for dilated cardiomyopathy on these patients.

Plasma-derived extracellular vesicles (EVs) have been extensively described as putative biomarkers in different diseases. Interestingly, increased levels of EVs subpopulations are well known to associate with obesity. The goal of this study is to identify EVs-derived biomarkers in plasma from obese patients in order to predict the development of pathological events associated with obesity. Samples are obtained from 22 obese patients and their lean-matched controls are divided into two cohorts: one for a 2D fluorescence difference gel electrophoresis (2D-DIGE)-based study, and the other one for a label free LC–MS/MS-based approach. EVs are isolated following a serial ultracentrifugation protocol. Twenty-two and 23 differentially regulated features are detected from 2D-DIGE and label free LC–MS/MS, respectively; most of them involve in the coagulation and complement cascades. Remarkably, there is an upregulation of complement C4, complement C3, and fibrinogen in obese patients following both approaches, the latter two also validated by 2D-western-blotting in an independent cohort. These results correlate with a proinflammatory and prothrombotic state of those individuals. On the other hand, a downregulation of adiponectin leading to an increased risk of suffering cardiovascular diseases has been shown. The results suggest the relevance of plasma-derived-EVs proteins as a source of potential biomarkers for the development of atherothrombotic events in obesity., This work was supported by the Spanish Ministry of Economy and Competitiveness; grant No. SAF2016-79662-R), co-funded by the European Regional Development Fund. Financial support from the Conseller ́ıa de Cultura, Educaci ́on e Ordenaci ́on Universitaria, Xunta de Galicia (Centro Singular de investigaci ́on de Galicia accreditation 2016–2019,ED431G/05), and the European Regional Development Fund is also grate-fully acknowledged. The Proteomics Laboratory CSIC/UAB is a member ofProteored, PRB2-ISCIII and is supported by Grant PT13/0001, of the PEI+D+i 2013–2016, funded by ISCIII and FEDER.

© 2014 American Chemical Society. Surface-enhanced Raman scattering (SERS) spectroscopy was employed in the present work to elucidate the adsorption of two diisocyanide molecules: 1,4-phenylenediisocyanide (PDI) and 1,4′-terphenylenediisocyanide (TPDI) on nanogaps of Ag and Au nanoparticles (NPs). The main goal of this work was the study of the adsorption of these molecules and more specifically the coordination mechanism and the orientation of these molecules on the metal surface as well as the study of the effect of the above adsorbates on the aggregation and formation of interparticle junctions. To get more insight into the above phenomena, we have investigated different experimental variables such as the numbers of phenylene rings (monophenylene vs terphenylene), the nature of the metal (Au and Ag), the excitation wavelength (532 and 785 nm), and the surface coverage provided by different adsorbate concentrations. The experiments carried out at these conditions revealed the existence concentration of critical values at 0.1 and 3 μM corresponding to changes in the coordination and a complete surface coverage of interparticle junctions. Finally, we have also deduced the great importance of charge-transfer processes occurring between the metal surface and diisocyanide molecules. This charge transfer exhibits a strong dependence on the surface coverage., This work has been supported by the Spanish Ministerio de Economia y Competitividad ́ (grant FIS2010-15405). S. SanchezCortes acknowledges Overseas Research Fellowship organized by Catalysis Research Center, Hokkaido University.

The adsorption mechanism of linear aliphatic α,ω-dithiols with chain lengths of 6, 8 and 10 carbon atoms on silver and gold nanoparticles has been studied by surface-enhanced Raman scattering (SERS) spectroscopy. SERS spectra provided the structural marker bands of these compounds and they were employed to obtain information about the adsorption and coordination mechanism, the orientation, conformational order, and packing of the aliphatic chains of dithiols on the metal nanoparticle surface. The effect of the type of metal (silver or gold) and the extent of surface coverage on all the above mentioned properties is discussed. It was found that the adsorption of dithiols on Au nanoparticles leads to a more disordered structure of the aliphatic chains of dithiols in comparison with the adsorption on Ag nanoparticles. The interaction through both thiol groups makes the adsorption of dithiols on metal surfaces substantially different from that of monothiols; in particular, the orientation of dithiols is perpendicular, while monothiols adopt a tilted orientation. Dithiols may act as linkers between metal nanoparticles and induce the formation of nanogaps with a controllable interparticle distance. The nanogaps thus formed are able to produce hot spots exhibiting a large intensification of electromagnetic field in these points which has been proved by the observation of intense SERS spectra of dithiols until a concentration of 10-8 M, corresponding to a large Raman enhancement factor of 5 × 106. © 2014 the Partner Organisations., This work has been supported by the Spanish Ministerio de Economía y Competitividad (MINECO, grant FIS2010-15405) and Comunidad de Madrid through the MICROSERES II network (grant S2009/TIC-1476), by the Agency of the Ministry of Education of Slovak Republic for the Structural Funds of the European Union, Operational program Education (Doctorand, ITMS code: 26110230013 and KVARK, ITMS code: 26110230084) and Operational program Research and Development (NanoBioSens (ITMS code: 26220220107) and CEVA II (ITMS code: 26220120040)), by the Slovak Research and Development Agency under the contract APVV-0242-11, and by the project CELIM (316310) funded by 7FP EU.

Probenecid (PB), a drug employed to reduce the excretion of other drugs in urine and also employed as masking agent in sport doping, is characterized for the first time in this work by vibrational spectroscopy. Previously, FTIR and FT-Raman spectra were assigned on the basis of density functional theory calculations. Afterwards, a detection protocol of PB was developed on the basis of surface-enhanced Raman scattering (SERS) spectroscopy. To accomplish this, the experimental conditions of SERS measurement were previously optimized by varying parameters such as the metal, its reduction protocol to fabricate the nanoparticles and the pH. The adsorption isotherms of PB on the metal nanoparticles were obtained, and the adsorption mechanism of this drug on Ag nanoparticles was deduced from the analysis of SERS spectra. Finally, a quantitative SERS study was carried out on the basis of the calibration curve deduced from the adsorption isotherms. The results shown in this study demonstrate that PB can be detected by SERS at concentrations as low as 1.2 µg/ml. Copyright © 2013 John Wiley & Sons, Ltd.

International audience; A new methodology is developed to determine the shape distribution of gold nanoparticles (NPs) from optical spectroscopic measurements. Indeed, the morphology of Au colloids is deduced by fitting their absorption spectra with an effective medium theory which takes into account the nanoparticle shape distribution. The same procedure is applied to ellipsometric measurements recorded on photoresist films which contain Au NPs. Three spaces (L2, r2, P2) are introduced to interpret the NPs shape distribution. In the P2 space, the sphericity, the prolacity and the oblacity estimators are proposed to quantify the shape of NPs. The r2 space enables the determination of the NP aspect ratio distribution. The distributions determined from optical spectroscopy were found to be in very good agreement with the shape distributions obtained by transmission electron microscopy. We found that fitting absorption or ellipsometric spectra with an adequate effective medium theory, provides a robust tool for measuring the shape and concentration of metallic NPs.

International audience; This paper reports the complete ellipsometric characterization of gold nanoparticles (NPs) embedded in a photoresist films. The effective dielectric function of nanocomposite films as well as the shape distribution and the volume fraction of NPs are extracted from ellipsometric measurements by introducing an effective medium theory which takes into account the NP shape distribution and the intrinsic confinement effect. This theory remains valid as long as the nanoparticle interaction is negligible. We show that the magnitude of the confinement depends on the nanoparticle shape and the environment through chemical damping. This suggests that the NP shape distribution can be directly estimated by ellipsometry, while the determination of absolute radius distribution requires transmission electron microscopy measurements. The imaginary part of the effective dielectric function exhibits a strong asymmetric surface plasmon band, while a large variation of the real part occurs close to the resonance. The redshift and the broadening of the plasmon band as the gold volume fraction increases are correlated to the evolution of NP shape distribution. This evolution is attributed to a competition between the nucleation and the coalescence of NPs. This unambiguously demonstrates that ellipsometry combined with a shape-distributed effective medium theory is a powerful alternative tool to transmission electron microscopy for the NP shape analysis.

International audience; The preparation method of nanoporous poly(methyl methacrylate)( PMMA) containing silver nanoparticles self-organized in rings is highly adaptable, yielding a variety of materials of controllable pore size (ranging from 200 nm to over 5 mu m), nanoparticle size (20 to 200 nm), surface coverage and silver precursor concentration. In this article, it is optimized aiming at a best performance as enhancers for Surface Enhanced Raman Spectroscopy. The versatility of these sensors is shown by multi-wavelength excitation experiment at 457, 488, 514 and 633 nm. Finally, the efficiency of 2,2'-bipyridine, 4,4'-bipyridine and trans-bis-(4,4'-bipyridyl) ethylene as SERS probe molecules is compared using PMMA/Ag-rings. Ag+ ions are shown to play an important role in the adsorption of the target molecules on these surfaces leading to a SERS enhancement factor of 3.783 108 at 488 nm for trans-bis-(4,4'-bipyridyl) ethylene.

A comparative study of different plasmonic nanoparticles with different morphologies (nanospheres and triangular nanoprisms) and metals (Ag and Au) was done in this work and applied to the ultrasensitive detection of aminoglutethimide (AGI) drug by surface enhanced Raman spectroscopy (SERS) and plasmon resonance. AGI is an aromatase inhibitor used as an antitumoral drug with remarkable pharmacological interest and also in illegal sport doping. The application of very sensitive spectroscopic techniques based on the localization of an electromagnetic field on plasmonic nanoparticles confirms the previous study of the adsorption of drugs onto a metal surface due to the near field character of these techniques. The adsorption of AGI on the above substrates was investigated at different pH values and surface coverages, and the results were analyzed on the basis of AGI/metal affinity, considering the interaction mechanism, the existence of two binding sites in AGI, and the influence of the interface on the adsorption in terms of surface charge due to the presence of other ions linked to the surface. Finally, a comparative quantitative detection of AGI was performed on both spherical and triangular nanoprism nanoparticles, and a limit of detection lower than those reported so far was deduced on the latter nanoparticles.

International audience

Upon stimulation, platelets release a high number of proteins (the releasate). There are clear indications that these proteins are involved in the pathogenesis of several diseases, such as atherosclerosis. In the present study we compared the platelet releasate following platelet activation with two major endogenous agonists: thrombin and collagen. Proteome analysis was based on 2D-DIGE and LC-MS/MS. Firstly, we showed the primary role of thrombin and collagen receptors in platelet secretion by these agonists; moreover, we demonstrated that GPVI is the primary responsible for collagen-induced platelet activation/aggregation. Proteomic analysis allowed the detection of 122 protein spots differentially regulated between both conditions. After excluding fibrinogen spots, down-regulated in the releasate of thrombin-activated platelets, 84 differences remained. From those, we successfully identified 42, corresponding to 37 open-reading frames. Many of the differences identified correspond to post-translational modifications, primarily, proteolysis induced by thrombin. Among others, we show vitamin K-dependent protein S, an anticoagulant plasma protein, is up-regulated in thrombin samples. Our results could have pathological implications given that platelets might be playing a differential role in various diseases and biological processes through the secretion of different subsets of granule proteins and microvesicles following a predominant activation of certain receptors.

International audience; Fabrication of freestanding, three-dimensional microstructures having gold nanoparticles as building blockshas been achieved using two-photon lithography. The reported constructions hold structural coherencewithout the need of a polymeric network to embed the nanoparticles. The Au nanoparticles areproduced by radical-mediated photoreduction and immediately self-arrange in micrometre-sizedcylinders, which are used as the building units of the 3D structure writing. The manufactured structurespresent plasmonic activity, as well as excellent properties as substrates for Surface-Enhanced RamanSpectroscopy, in key issues such as reproducibility in intensity or enhancement factor. Such is calculatedto be above 8 106 for the non-resonant probe molecule trans-1,2-bis-4-pyridylethylene. The optionof repeated use of the substrates is confirmed, and they are successfully applied for detection of thefungicide thiabendazole.

Linear α,ω-dithiols have been used as linkers to control the aggregation of silver nanoparticles. The characterization of the resulting nanoparticle clusters thus formed was carried out using plasmon resonance spectroscopy and transmission electron microscopy in both independent and correlated measurements. The obtained nanoparticle assemblies present morphologies that vary according to the dithiol length, displaying controllable-size interparticle gaps (plasmonic hot spots) with potential application in the detection of a large list of hydrophobic analytes. © 2013 American Chemical Society., ACKNOWLEDGMENTS This work has been supported by the Spanish Ministerio de Economía y Competitividad (grant FIS2010-15405), by the Comunidad de Madrid, through MICROSERES II network (grant S2009/TIC-1476). I.I.-L. also acknowledges CSIC and FSE 2007- 2013 for a JAE-CSIC predoctoral grant.

In this work we have investigated the detection of aminoglutethimide (AGI) drug based on its adsorption on gold nanoparticles employing Surface-Enhanced Raman Spectroscopy (SERS) and plasmon resonance. The adsorption of AGI on Au was studied at different experimental conditions, such as the excitation wavelength, the pH and the concentration, aimed to optimize the trace detection of this drug with possible application in the anti-doping analysis or in bioanalysis. © 2011 The Royal Society of Chemistry., This work has been supported by the Spanish Ministerio de Ciencia e Innovacion (grants FIS2007-63065 and FIS2010-15405) and Comunidad de Madrid through the MICROSERES II network (grant S2009/TIC-1476).

8 pags.; 8 figs.; 3 tabs., The adsorption of beta2-adrenergic agonist (βAA) drugs clenbuterol, salbutamol, and terbutaline on metal surfaces has been investigated in this work by means of surface-enhanced Raman scattering (SERS). To assist in this investigation, a previous vibrational (IR and normal Raman) characterization of these drugs was performed, supported by ab initio density functional theory calculations. The application of SERS was aimed to apply this highly sensitive technique, based on localized surface plasmon resonance, in the detection of βAA at trace concentrations and as a possible alternative method which can be postulated in routine antidoping analysis. The adsorption of these drugs was studied in depth at different experimental conditions: on Au and Ag, at different pHs, and with varying adsorbate concentration. Moreover, plasmon resonance spectroscopy was employed to investigate the adsorption of these drugs on the metal nanoparticles as well as their aggregation. It was found that the adsorption of these molecules is more effective on gold nanoparticles and at acidic pH, based on the direct interaction of the aromatic or aliphatic moieties through ionic or coordination bonds with the metal. These drugs followed a Langmuir adsorption model from which the adsorption constant and the limit of detection can be determined. © 2010 American Chemical Society., This work has been supported by the Spanish Ministerio de Ciencia e Innovacion (Grant FIS2007-63065) and Comunidad de Madrid through the MICROSERES II network (Grant S2009/TIC-1476). I.I.-L. also acknowledges a Ph.D. scholarship from CSIC.

The controlled tuning of interparticle distance at the nanoscale level is a major challenge for nanofabrication of surface-enhanced Raman scattering (SERS) active clusters and their application to molecular sensing. In fact, the geometrical properties of the narrow gaps between nanoparticles play a key role in determining the local field enhancement (and therefore, the SERS enhancement factor) and the spatial enhancement distribution in the gap region. Besides, very short interparticle distances may block the access of the analyte to the hot zone. In this paper, we report the synthesis of silver colloid NP clusters with interparticle distances fine tuned in the ≤2 nm range, by exploiting the chemical properties of linear α,ω-aliphatic diamines as molecular linkers with varying chain length. The bifunctional diamines also form intermolecular cavities within their self-assembled monolayers, suitable to host molecular analytes for nanosensing applications, as evidenced by SERS detection of organochlorine insecticides at the trace level. In this regard, the extension of the aliphatic chain played a crucial role in determining the SAM conformation and thus the final sensitivity of the functionalized SERS substrate. © 2010 Springer Science+Business Media, LLC., Supported by the Spanish Ministerio de Ciencia e Innovación (grant no. FIS2007-63065, FIS2009-11264, and Consolider-Ingenio project EMET CSD2008-00066, Comunidad de Madrid through the MICROSERES network (grant S-0505/TIC-0191), and CSIC

The adsorption and self-assembly of alpha,omega-aliphatic diamines on silver nanoparticles is studied in this work by surface-enhanced Raman scattering (SERS) spectroscopy and plasmon resonance. These bifunctional diamines can act as linkers of metal nanoparticles (NPs) inducing the formation of hot spots (HS), i.e. interparticle junctions or gaps between metal NPs, which are points where a huge intensification of the electromagnetic field occurs. In addition, the dicationic nature of these diamines leads to the formation of cavities just at the induced hot spots which can be applied to molecular recognition of analytes. The influence of the surface coverage and the aliphatic chain length in diamines on their self-assembly was tested by the vibrational spectra and correlated to the different plasmon resonances of the dimers detected in the extinction spectra. These factors can be used for tuning the plasmon resonance of dimers formed by two metal nanoparticles where interparticle hot spots are formed. Finally, the analytical potential of these functionalized Ag nanoparticles is demonstrated for the trace detection of the pesticide aldrin.