Browsing by MeSH term "Microscopy, Confocal"
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Publication A Cre-reporter transgenic mouse expressing the far-red fluorescent protein Katushka.(Wiley, 2011-01) Diéguez-Hurtado, Rodrigo; Martín, Javier; Martínez-Corral, Inés; Martinez Garcia, Maria Dolores; Megías, Diego; Olmeda, David; Ortega Jimenez, Sagrario; Ministerio de Ciencia e Innovación (España)Cre/loxP-dependent expression of fluorescent proteins represents a powerful biological tool for cell lineage, fate-mapping, and genetic analysis. Live tissue imaging has significantly improved with the development of far-red fluorescent proteins, with optimized spectral characteristics for in vivo applications. Here, we report the generation of the first transgenic mouse line expressing the far-red fluorescent protein Katushka, driven by the hybrid CAG promoter upon Cre-mediated recombination. After germ line or tissue-specific Cre-driven reporter activation, Katushka expression is strong and ubiquitous, without toxic effects, allowing fluorescence detection in fresh and fixed samples from all tissues examined. Moreover, fluorescence can be detected by in vivo noninvasive whole-body imaging when Katuhska is expressed exclusively in a specific cell population deep within the animal body such as pancreatic beta cells. Thus, this reporter model enables early, widespread, and sensitive in vivo detection of Cre activity and should provide a versatile tool for a wide spectrum of fluorescence and live-imaging applications.Publication A new mode of DNA binding distinguishes Capicua from other HMG-box factors and explains its mutation patterns in cancer(Public Library of Science (PLOS), 2017-03-09) Forés, Marta; Simón-Carrasco, Lucía; Ajuria, Leiore; Samper, Núria; González-Crespo, Sergio; Drosten, Matthias; Jiménez, Gerardo; Barbacid, Mariano; Ministerio de Ciencia y Competitividad (España); Unión Europea. Comisión Europea. European Research Council (ERC); Comunidad de Madrid (España); Fundación AXAHMG-box proteins, including Sox/SRY (Sox) and TCF/LEF1 (TCF) family members, bind DNA via their HMG-box. This binding, however, is relatively weak and both Sox and TCF factors employ distinct mechanisms for enhancing their affinity and specificity for DNA. Here we report that Capicua (CIC), an HMG-box transcriptional repressor involved in Ras/MAPK signaling and cancer progression, employs an additional distinct mode of DNA binding that enables selective recognition of its targets. We find that, contrary to previous assumptions, the HMG-box of CIC does not bind DNA alone but instead requires a distant motif (referred to as C1) present at the C-terminus of all CIC proteins. The HMG-box and C1 domains are both necessary for binding specific TGAATGAA-like sites, do not function via dimerization, and are active in the absence of cofactors, suggesting that they form a bipartite structure for sequence-specific binding to DNA. We demonstrate that this binding mechanism operates throughout Drosophila development and in human cells, ensuring specific regulation of multiple CIC targets. It thus appears that HMG-box proteins generally depend on auxiliary DNA binding mechanisms for regulating their appropriate genomic targets, but that each sub-family has evolved unique strategies for this purpose. Finally, the key role of C1 in DNA binding also explains the fact that this domain is a hotspot for inactivating mutations in oligodendroglioma and other tumors, while being preserved in oncogenic CIC-DUX4 fusion chimeras associated to Ewing-like sarcomas.Publication A short G1 phase imposes constitutive replication stress and fork remodelling in mouse embryonic stem cells(Nature Publishing Group, 2016-02-15) Ahuja, Akshay K; Jodkowska, Karolina; Teloni, Federico; Bizard, Anna H; Zellweger, Ralph; Herrador, Raquel; Ortega Jimenez, Sagrario; Hickson, Ian D; Altmeyer, Matthias; Mendez, Juan; Lopes, Massimo; Danish National Research Foundation; Ministerio de Economía y Competitividad (España); Unión Europea. Comisión Europea. European Research Council (ERC)Embryonic stem cells (ESCs) represent a transient biological state, where pluripotency is coupled with fast proliferation. ESCs display a constitutively active DNA damage response (DDR), but its molecular determinants have remained elusive. Here we show in cultured ESCs and mouse embryos that H2AX phosphorylation is dependent on Ataxia telangiectasia and Rad3 related (ATR) and is associated with chromatin loading of the ssDNA-binding proteins RPA and RAD51. Single-molecule analysis of replication intermediates reveals massive ssDNA gap accumulation, reduced fork speed and frequent fork reversal. All these marks of replication stress do not impair the mitotic process and are rapidly lost at differentiation onset. Delaying the G1/S transition in ESCs allows formation of 53BP1 nuclear bodies and suppresses ssDNA accumulation, fork slowing and reversal in the following S-phase. Genetic inactivation of fork slowing and reversal leads to chromosomal breakage in unperturbed ESCs. We propose that rapid cell cycle progression makes ESCs dependent on effective replication-coupled mechanisms to protect genome integrity.Publication Biofilm formation avoids complement immunity and phagocytosis of Streptococcus pneumoniae(American Society for Microbiology (ASM), 2013-07) Domenech, Mirian; Ramos-Sevillano, Elisa; García, Ernesto; Moscoso, Miriam; Yuste, Jose Enrique; Ministerio de Economía y Competitividad (España); Centro de Investigación Biomedica en Red - CIBERStreptococcus pneumoniae is a frequent member of the microbiota of the human nasopharynx. Colonization of the nasopharyngeal tract is a first and necessary step in the infectious process and often involves the formation of sessile microbial communities by this human pathogen. The ability to grow and persist as biofilms is an advantage for many microorganisms, because biofilm-grown bacteria show reduced susceptibility to antimicrobial agents and hinder recognition by the immune system. The extent of host protection against biofilm-related pneumococcal disease has not been determined yet. Using pneumococcal strains growing as planktonic cultures or as biofilms, we have investigated the recognition of S. pneumoniae by the complement system and its interactions with human neutrophils. Deposition of C3b, the key complement component, was impaired on S. pneumoniae biofilms. In addition, binding of C-reactive protein and the complement component C1q to the pneumococcal surface was reduced in biofilm bacteria, demonstrating that pneumococcal biofilms avoid the activation of the classical complement pathway. In addition, recruitment of factor H, the downregulator of the alternative pathway, was enhanced by S. pneumoniae growing as biofilms. Our results also show that biofilm formation diverts the alternative complement pathway activation by a PspC-mediated mechanism. Furthermore, phagocytosis of pneumococcal biofilms was also impaired. The present study confirms that biofilm formation in S. pneumoniae is an efficient means of evading both the classical and the PspC-dependent alternative complement pathways the host immune system.Publication Clonal dynamics in osteosarcoma defined by RGB marking(Nature Publishing Group, 2018) Gambera, Stefano; Abarrategi, Ander; Garcia-Castro, Javier; Morales-Molina, Alvaro; Roma, Josep; Alfranca, Arantzazu; Gonzalez-Camacho, Fernando; Instituto de Salud Carlos III; Comunidad de MadridOsteosarcoma is a type of bone tumour characterized by considerable levels of phenotypic heterogeneity, aneuploidy, and a high mutational rate. The life expectancy of osteosarcoma patients has not changed during the last three decades and thus much remains to be learned about the disease biology. Here, we employ a RGB-based single-cell tracking system to study the clonal dynamics occurring in a de novo-induced murine osteosarcoma model. We show that osteosarcoma cells present initial polyclonal dynamics, followed by clonal dominance associated with adaptation to the microenvironment. Interestingly, the dominant clones are composed of subclones with a similar tumour generation potential when they are re-implanted in mice. Moreover, individual spontaneous metastases are clonal or oligoclonal, but they have a different cellular origin than the dominant clones present in primary tumours. In summary, we present evidence that osteosarcomagenesis can follow a neutral evolution model, in which different cancer clones coexist and propagate simultaneously.Publication Combined deficiency of Notch1 and Notch3 causes pericyte dysfunction, models CADASIL, and results in arteriovenous malformations.(Nature Publishing Group, 2015-11-13) Kofler, Natalie M; Cuervo, Henar; Uh, Minji K; Murtomäki, Aino; Kitajewski, JanPericytes regulate vessel stability and pericyte dysfunction contributes to retinopathies, stroke, and cancer. Here we define Notch as a key regulator of pericyte function during angiogenesis. In Notch1(+/-); Notch3(-/-) mice, combined deficiency of Notch1 and Notch3 altered pericyte interaction with the endothelium and reduced pericyte coverage of the retinal vasculature. Notch1 and Notch3 were shown to cooperate to promote proper vascular basement membrane formation and contribute to endothelial cell quiescence. Accordingly, loss of pericyte function due to Notch deficiency exacerbates endothelial cell activation caused by Notch1 haploinsufficiency. Mice mutant for Notch1 and Notch3 develop arteriovenous malformations and display hallmarks of the ischemic stroke disease CADASIL. Thus, Notch deficiency compromises pericyte function and contributes to vascular pathologies.Publication Distance-dependent cellular palmitoylation of de-novo-designed sequences and their translocation to plasma membrane subdomains.(The Company of Biologists, 2002-08-01) Navarro-Lerida, Inmaculada; Alvarez-Barrientos, Alberto; Gavilanes, Francisco; Rodriguez-Crespo, Ignacio; Comunidad de Madrid (España); Spanish DGIUsing recursive PCR, we created an artificial protein sequence that consists of a consensus myristoylation motif (MGCTLS) followed by the triplet AGS repeated nine times and fused to the GFP reporter. This linker-GFP sequence was utilized as a base to produce multiple mutants that were used to transfect COS-7 cells. Constructs where a 'palmitoylable' cysteine residue was progressively moved apart from the myristoylation site to positions 3, 9, 15 and 21 of the protein sequence were made, and these mutants were used to investigate the effect of protein myristoylation on subsequent palmitoylation, subcellular localization, membrane association and caveolin-1 colocalization. In all cases, dual acylation of the GFP chimeras correlated with translocation to Triton X-100-insoluble cholesterol/sphingomyelin-enriched subdomains. Whereas a strong Golgi labeling was observed in all the myristoylated chimeras, association with the plasma membrane was only observed in the dually acylated constructs. Taking into account the conflicting data regarding the existence and specificity of cellular palmitoyl-transferases, our results provide evidence that de-novo-designed sequences can be efficiently S-acylated with palmitic acid in vivo, strongly supporting the hypothesis that non-enzymatic protein palmitoylation can occur within mammalian cells. Additionally, this palmitoylation results in the translocation of the recombinant construct to low-fluidity domains in a myristate-palmitate distance-dependent manner.Publication Endothelial adhesion receptors are recruited to adherent leukocytes by inclusion in preformed tetraspanin nanoplatforms.(Rockefeller University Press, 2008-11-03) Barreiro, Olga; Zamai, Moreno; Yanez-Mo, Maria; Tejera, Emilio; Lopez-Romero, Pedro; Monk, Peter N; Gratton, Enrico; Caiolfa, Valeria R; Sanchez-Madrid, Francisco; Ministerio de Ciencia e Innovación (España); Ministerio de Educación y Ciencia (España); Fundación Lilly; National Institutes of Health (Estados Unidos); Unión Europea; Centro de Investigación Biomédica en Red - CIBERCV (Enfermedades Cardiovasculares); Ministerio de Sanidad y Consumo (España); Fundación ProCNICVCAM-1 and ICAM-1, receptors for leukocyte integrins, are recruited to cell-cell contact sites on the apical membrane of activated endothelial cells. In this study, we show that this recruitment is independent of ligand engagement, actin cytoskeleton anchorage, and heterodimer formation. Instead, VCAM-1 and ICAM-1 are recruited by inclusion within specialized preformed tetraspanin-enriched microdomains, which act as endothelial adhesive platforms (EAPs). Using advanced analytical fluorescence techniques, we have characterized the diffusion properties at the single-molecule level, nanoscale organization, and specific intradomain molecular interactions of EAPs in living primary endothelial cells. This study provides compelling evidence for the existence of EAPs as physical entities at the plasma membrane, distinct from lipid rafts. Scanning electron microscopy of immunogold-labeled samples treated with a specific tetraspanin-blocking peptide identify nanoclustering of VCAM-1 and ICAM-1 within EAPs as a novel mechanism for supramolecular organization that regulates the leukocyte integrin-binding capacity of both endothelial receptors during extravasation.Publication ESC-Track: A computer workflow for 4-D segmentation, tracking, lineage tracing and dynamic context analysis of ESCs(Future Medicine, 2017-05) Fernandez-de-Manuel, Laura; Diaz-Diaz, Covadonga; Jimenez-Carretero, Daniel; Torres, Miguel; Montoya, Maria; Ministerio de Economía y Competitividad (España); Unión Europea. Comisión Europea; Comunidad de Madrid (España); Fundación ProCNIC; Instituto de Salud Carlos IIIEmbryonic stem cells (ESCs) can be established as permanent cell lines, and their potential to differentiate into adult tissues has led to widespread use for studying the mechanisms and dynamics of stem cell differentiation and exploring strategies for tissue repair. Imaging live ESCs during development is now feasible due to advances in optical imaging and engineering of genetically encoded fluorescent reporters; however, a major limitation is the low spatio-temporal resolution of long-term 3-D imaging required for generational and neighboring reconstructions. Here, we present the ESC-Track (ESC-T) workflow, which includes an automated cell and nuclear segmentation and tracking tool for 4-D (3-D + time) confocal image data sets as well as a manual editing tool for visual inspection and error correction. ESC-T automatically identifies cell divisions and membrane contacts for lineage tree and neighborhood reconstruction and computes quantitative features from individual cell entities, enabling analysis of fluorescence signal dynamics and tracking of cell morphology and motion. We use ESC-T to examine Myc intensity fluctuations in the context of mouse ESC (mESC) lineage and neighborhood relationships. ESC-T is a powerful tool for evaluation of the genealogical and microenvironmental cues that maintain ESC fitness.Publication In vivo inhibition of c-MYC in myeloid cells impairs tumor-associated macrophage maturation and pro-tumoral activities(Public Library of Science (PLOS), 2012) Pello, Oscar M; Chevre, Raphael; Laoui, Damya; De Juan, Alba; Lolo, Fidel Nicolas; Andres-Manzano, Maria J.; Serrano, Manuel; Van Ginderachter, Jo A; Andres, Vicente; Ministerio de Ciencia e Innovación (España); Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF); Instituto de Salud Carlos III; Ministerio de Economía y Competitividad (España); Unión Europea. Comisión Europea; Fundación ProCNICAlthough tumor-associated macrophages (TAMs) are involved in tumor growth and metastasis, the mechanisms controlling their pro-tumoral activities remain largely unknown. The transcription factor c-MYC has been recently shown to regulate in vitro human macrophage polarization and be expressed in macrophages infiltrating human tumors. In this study, we exploited the predominant expression of LysM in myeloid cells to generate c-Myc(fl/fl) LysM(cre/+) mice, which lack c-Myc in macrophages, to investigate the role of macrophage c-MYC expression in cancer. Under steady-state conditions, immune system parameters in c-Myc(fl/fl) LysM(cre/+) mice appeared normal, including the abundance of different subsets of bone marrow hematopoietic stem cells, precursors and circulating cells, macrophage density, and immune organ structure. In a model of melanoma, however, TAMs lacking c-Myc displayed a delay in maturation and showed an attenuation of pro-tumoral functions (e.g., reduced expression of VEGF, MMP9, and HIF1α) that was associated with impaired tissue remodeling and angiogenesis and limited tumor growth in c-Myc(fl/fl) LysM(cre/+) mice. Macrophage c-Myc deletion also diminished fibrosarcoma growth. These data identify c-Myc as a positive regulator of the pro-tumoral program of TAMs and suggest c-Myc inactivation as an attractive target for anti-cancer therapy.Publication Intensity distribution segmentation in ultrafast Doppler combined with scanning laser confocal microscopy for assessing vascular changes associated with ageing in murine hippocampi(Nature Publishing Group, 2022) Anzibar Fialho, Maximiliano; Vázquez Alberdi, Lucia; Martínez, Mariana; Calero, Miguel; Baranger, Jerome; Tanter, Mickael; Damián, Juan Pablo; Negreira, Carlos; Rubido, Nicolás; Kun, Alejandra; Brum, Javier; Agencia Nacional de Investigación e Innovación (Uruguay)The hippocampus plays an important role in learning and memory, requiring high-neuronal oxygenation. Understanding the relationship between blood flow and vascular structure-and how it changes with ageing-is physiologically and anatomically relevant. Ultrafast Doppler ([Formula: see text]Doppler) and scanning laser confocal microscopy (SLCM) are powerful imaging modalities that can measure in vivo cerebral blood volume (CBV) and post mortem vascular structure, respectively. Here, we apply both imaging modalities to a cross-sectional and longitudinal study of hippocampi vasculature in wild-type mice brains. We introduce a segmentation of CBV distribution obtained from [Formula: see text]Doppler and show that this mice-independent and mesoscopic measurement is correlated with vessel volume fraction (VVF) distribution obtained from SLCM-e.g., high CBV relates to specific vessel locations with large VVF. Moreover, we find significant changes in CBV distribution and vasculature due to ageing (5 vs. 21 month-old mice), highlighting the sensitivity of our approach. Overall, we are able to associate CBV with vascular structure-and track its longitudinal changes-at the artery-vein, venules, arteriole, and capillary levels. We believe that this combined approach can be a powerful tool for studying other acute (e.g., brain injuries), progressive (e.g., neurodegeneration) or induced pathological changes.Publication Localization of the cannabinoid CB1 receptor and the 2-AG synthesizing (DAGLα) and degrading (MAGL, FAAH) enzymes in cells expressing the Ca(2+)-binding proteins calbindin, calretinin, and parvalbumin in the adult rat hippocampus.(Frontiers Media, 2014-06-27) Rivera, Patricia; Arrabal, Sergio; Cifuentes, Manuel; Grondona, Jesús M; Pérez-Martín, Margarita; Rubio, Leticia; Vargas, Antonio; Serrano, Antonia; Pavón, Francisco-Javier; Suárez, Juan; Rodríguez de Fonseca, Fernando; [Rivera,P; Arrabal,S; Vargas,A; Serrano,A; Pavón,FJ; Suárez,J; Rodriguez de Fonseca,F] Laboratorio de Investigación, Instituto de Investigación Biomédica (IBIMA), Universidad de Málaga-Hospital Regional Universitario de Málaga (UGC Salud Mental), Málaga, Spain. [Rivera,P; Arrabal,S; Serrano,A; Pavón,FJ; Suárez,J; Rodriguez de Fonseca,F] CIBER OBN, Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación, Madrid, Spain. [Cifuentes,M; Grondona,JM; Pérez-Martín,M] Departamento de Biología Celular, Genética y Fisiología, Facultad de Ciencias, Instituto de Investigación Biomédica (IBIMA), Universidad de Málaga, Málaga, Spain. [Cifuentes,M] CIBER BBN, Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación, Madrid, Spain. [Rubio,L] Departamento de Anatomía y Medicina Legal, Facultad de Medicina, Universidad de Málaga, Málaga, Spain.The retrograde suppression of the synaptic transmission by the endocannabinoid sn-2-arachidonoylglycerol (2-AG) is mediated by the cannabinoid CB1 receptors and requires the elevation of intracellular Ca(2+) and the activation of specific 2-AG synthesizing (i.e., DAGLα) enzymes. However, the anatomical organization of the neuronal substrates that express 2-AG/CB1 signaling system-related molecules associated with selective Ca(2+)-binding proteins (CaBPs) is still unknown. For this purpose, we used double-label immunofluorescence and confocal laser scanning microscopy for the characterization of the expression of the 2-AG/CB1 signaling system (CB1 receptor, DAGLα, MAGL, and FAAH) and the CaBPs calbindin D28k, calretinin, and parvalbumin in the rat hippocampus. CB1, DAGLα, and MAGL labeling was mainly localized in fibers and neuropil, which were differentially organized depending on the hippocampal CaBPs-expressing cells. CB(+) 1 fiber terminals localized in all hippocampal principal cell layers were tightly attached to calbindin(+) cells (granular and pyramidal neurons), and calretinin(+) and parvalbumin(+) interneurons. DAGLα neuropil labeling was selectively found surrounding calbindin(+) principal cells in the dentate gyrus and CA1, and in the calretinin(+) and parvalbumin(+) interneurons in the pyramidal cell layers of the CA1/3 fields. MAGL(+) terminals were only observed around CA1 calbindin(+) pyramidal cells, CA1/3 calretinin(+) interneurons and CA3 parvalbumin(+) interneurons localized in the pyramidal cell layers. Interestingly, calbindin(+) pyramidal cells expressed FAAH specifically in the CA1 field. The identification of anatomically related-neuronal substrates that expressed 2-AG/CB1 signaling system and selective CaBPs should be considered when analyzing the cannabinoid signaling associated with hippocampal functions.Publication Multifunctional Magnetic and Upconverting Nanobeads as Dual Modal Imaging Tools(2017-11-02) Materia, Maria Elena; Pernia Leal, Manuel; Scotto, Marco; Balakrishnan, Preethi Bala; Kumar Avugadda, Sahitya; Garc�a-Mart�n, Mar�a L; Cohen, Bruce E; Chan, Emory M; Pellegrino, TeresaWe report the fabrication of aqueous multimodal imaging nanocomposites based on superparamagnetic nanoparticles (MNPs) and two different sizes of photoluminescent upconverting nanoparticles (UCNPs). The controlled and simultaneous incorporation of both types of nanoparticles (NPs) was obtained by controlling the solvent composition and the addition rate of the destabilizing solvent. The magnetic properties of the MNPs remained unaltered after their encapsulation into the polymeric beads as shown by the T2 relaxivity measurements. The UCNPs maintain photoluminescent properties even when embedded with the MNPs into the polymer bead. Moreover, the light emitted by the magnetic and upconverting nanobeads (MUCNBs) under NIR excitation (?exc = 980 nm) was clearly observed through different thicknesses of agarose gel or through a mouse skin layer. The comparison with magnetic and luminescent nanobeads based on red-emitting quantum dots (QDs) demonstrated that while the QD-based beads show significant autofluorescence background from the skin, the signal obtained by the MUCNBs allows a decrease in this background. In summary, these results indicate that MUCNBs are good magnetic and optical probes for in vivo multimodal imaging sensors.Publication Photophysical properties and bioimaging application of an aminonaphthalimide-squaraine non-conjugated system(Elsevier, 2021-10-28) Stamentovic, Vladimir; Collado, Daniel; Perez-Inestrosa, Ezequiel; [Stamentovic,V; Collado,D; Perez-Inestrosa,E] Universidad de Málaga-IBIMA, Departamento de Química Orgánica, Málaga, Spain. [Collado,D; Perez-Inestrosa,E] Centro Andaluz de Nanomedicina y Biotecnología-BIONAND, Campanillas, Málaga, Spain.An aminonaphthalimide-squaraine non-conjugated system was designed and synthetized with the purpose of preparing fluorescent molecule in the 650–700 nm region that could operate via energy transfer (ET) between covalently linked naphthalimide and squaraine chromophores. The photophysical properties of the new fluorescent system were explored with the aim of understanding the ET in one- and two-photon excitation modes. The spectroscopic techniques employed in the characterization includes; absorption, fluorescence, quantum yields and fluorescence lifetime measurements in different solvents. The effect of polarity of solvents on efficiencies of ET were evaluated using one- and two-photon excited fluorescence. The optical behavior of the non-conjugated system was compared with its individual squaraine and naphthalimide moieties. The two-photon absorption (TPA) spectrum of the molecule was obtained between 750 and 1040 nm, with the largest two-photon cross section (δTPA)above 4200 GM. Finally, the applicability of the molecule for fluorescence imaging in the one- and two-photon excitation mode was demonstrated in N13 Microglial cells. The in vitro and in vivo confocal microscopy studies indicated that the non-conjugated system efficiently accumulated in the cytoplasm suggesting it could be utilized as a subcellular probe.Publication SHIP-1 Couples to the Dectin-1 hemITAM and Selectively Modulates Reactive Oxygen Species Production in Dendritic Cells in Response to Candida albicans(American Association of Immunologists (AAI), 2015-11-01) Blanco-Menendez, Noelia; del Fresno, Carlos; Fernandes, Sandra; Calvo, Enrique; Conde-Garrosa, Ruth; Kerr, William G; Sancho, David; Ministerio de Economía y Competitividad (España); Unión Europea. Comisión Europea. European Research Council (ERC); Fundación ProCNIC; National Institutes of Health (Estados Unidos)Dectin-1 (Clec7a) is a paradigmatic C-type lectin receptor that binds Syk through a hemITAM motif and couples sensing of pathogens such as fungi to induction of innate responses. Dectin-1 engagement triggers a plethora of activating events, but little is known about the modulation of such pathways. Trying to define a more precise picture of early Dectin-1 signaling, we explored the interactome of the intracellular tail of the receptor in mouse dendritic cells. We found unexpected binding of SHIP-1 phosphatase to the phosphorylated hemITAM. SHIP-1 colocalized with Dectin-1 during phagocytosis of zymosan in a hemITAM-dependent fashion. Moreover, endogenous SHIP-1 relocated to live or heat-killed Candida albicans-containing phagosomes in a Dectin-1-dependent manner in GM-CSF-derived bone marrow cells (GM-BM). However, SHIP-1 absence in GM-BM did not affect activation of MAPK or production of cytokines and readouts dependent on NF-κB and NFAT. Notably, ROS production was enhanced in SHIP-1-deficient GM-BM treated with heat-killed C. albicans, live C. albicans, or the specific Dectin-1 agonists curdlan or whole glucan particles. This increased oxidative burst was dependent on Dectin-1, Syk, PI3K, phosphoinositide-dependent protein kinase 1, and NADPH oxidase. GM-BM from CD11c∆SHIP-1 mice also showed increased killing activity against live C. albicans that was dependent on Dectin-1, Syk, and NADPH oxidase. These results illustrate the complexity of myeloid C-type lectin receptor signaling, and how an activating hemITAM can also couple to intracellular inositol phosphatases to modulate selected functional responses and tightly regulate processes such as ROS production that could be deleterious to the host.Publication Study of protein haptenation by amoxicillin through the use of a biotinylated antibiotic.(Public Library of Science (PLOS), 2014-03-03) Ariza, Adriana; Collado, Daniel; Vida, Yolanda; Montañez, María I; Pérez-Inestrosa, Ezequiel; Blanca, Miguel; Torres, María José; Cañada, F Javier; Pérez-Sala, Dolores; [Ariza,A; Cañada,FJ; Pérez-Sala,D] Department of Chemical and Physical Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain. [Ariza,A; Torres,MJ] Research Laboratory Carlos Haya Hospital-IBIMA, Málaga, Spain. [Collado,D; VidaY; Pérez-Inestrosa,E] Department of Organic Chemistry, University of Málaga, Malaga, Spain. [Collado,D; Vida,Y; Montañez,MI; Pérez-Inestrosa,E] BIONAND-Andalusian Centre for Nanomedicine and Biotechnology, Parque Tecnológico de Andalucía, Málaga, Spain. [Blanca,M] Allergy Service, Hospital Carlos Haya, Málaga, Spain.Allergic reactions towards β-lactam antibiotics pose an important clinical problem. The ability of small molecules, such as a β-lactams, to bind covalently to proteins, in a process known as haptenation, is considered necessary for induction of a specific immunological response. Identification of the proteins modified by β-lactams and elucidation of the relevance of this process in allergic reactions requires sensitive tools. Here we describe the preparation and characterization of a biotinylated amoxicillin analog (AX-B) as a tool for the study of protein haptenation by amoxicillin (AX). AX-B, obtained by the inclusion of a biotin moiety at the lateral chain of AX, showed a chemical reactivity identical to AX. Covalent modification of proteins by AX-B was reduced by excess AX and vice versa, suggesting competition for binding to the same targets. From an immunological point of view, AX and AX-B behaved similarly in RAST inhibition studies with sera of patients with non-selective allergy towards β-lactams, whereas, as expected, competition by AX-B was poorer with sera of AX-selective patients, which recognize AX lateral chain. Use of AX-B followed by biotin detection allowed the observation of human serum albumin (HSA) modification by concentrations 100-fold lower that when using AX followed by immunological detection. Incubation of human serum with AX-B led to the haptenation of all of the previously identified major AX targets. In addition, some new targets could be detected. Interestingly, AX-B allowed the detection of intracellular protein adducts, which showed a cell type-specific pattern. This opens the possibility of following the formation and fate of AX-B adducts in cells. Thus, AX-B may constitute a valuable tool for the identification of AX targets with high sensitivity as well as for the elucidation of the mechanisms involved in allergy towards β-lactams.Publication Validation of the 1,4-butanediol thermoplastic polyurethane as a novel material for 3D bioprinting applications(Wiley, 2020-10-20) Chocarro-Wrona, Carlos; de Vicente, Juan; Antich, Cristina; Jiménez, Gema; Martínez-Moreno, Daniel; Carrillo, Esmeralda; Montañez, Elvira; Gálvez-Martín, Patricia; Perán, Macarena; López-Ruiz, Elena; Marchal, Juan Antonio; [Chocarro-Wrona,C; Antich,C; Jiménez,G; Martínez-Moreno,D; Carrillo,E; Perán,M; López-Ruiz,E; Marchal,JA] Biosanitary Research Institute of Granada (ibs.GRANADA), University Hospitals of Granada-University of Granada, Granada, Spain. [Chocarro-Wrona,C; Antich,C; Jiménez,G; Martínez-Moreno,D; Carrillo,E; Perán,M; López-Ruiz,E; Marchal,JA] Biopathology and Regenerative Medicine Institute (IBIMER), Centre for Biomedical Research (CIBM), University of Granada, Granada, Spain. [Chocarro-Wrona,C; Antich,C; Martínez-Moreno,D; Carrillo,E; Marchal,JA] Department of Human Anatomy and Embryology, Faculty of Medicine, University of Granada, Granada, Spain. [Chocarro-Wrona,C; de Vicente,J; Antich,C; Jiménez,G; Martínez-Moreno,D; Carrillo,E; Perán,M; López-Ruiz,E; Marchal,JA] Excellence Research Unit “Modeling Nature” (MNat), University of Granada, Granada, Spain. [de Vicente,J] Department of Applied Physics, Faculty of Sciences, University of Granada, Granada, Spain. [Montañez,E] Biomedical Research Institute of Málaga (IBIMA), Málaga. [Montañez,E] Department of Orthopedic Surgery and Traumatology, Virgen de la Victoria University Hospital, Málaga, Spain. [Gálvez-Martín,P] Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, University of Granada, Granada, Spain. [Gálvez-Martín,P] Advanced Therapies Area, Bioibérica S.A.U, Barcelona, Spain. [Perán,M; López-Ruiz,E] Department of Health Sciences, University of Jaén, Jaén, SpainTissue engineering (TE) seeks to fabricate implants that mimic the mechanical strength, structure, and composition of native tissues. Cartilage TE requires the development of functional personalized implants with cartilage-like mechanical properties capable of sustaining high load-bearing environments to integrate into the surrounding tissue of the cartilage defect. In this study, we evaluated the novel 1,4-butanediol thermoplastic polyurethane elastomer (b-TPUe) derivative filament as a 3D bioprinting material with application in cartilage TE. The mechanical behavior of b-TPUe in terms of friction and elasticity were examined and compared with human articular cartilage, PCL, and PLA. Moreover, infrapatellar fat pad-derived human mesenchymal stem cells (MSCs) were bioprinted together with scaffolds. in vitro cytotoxicity, proliferative potential, cell viability, and chondrogenic differentiation were analyzed by Alamar blue assay, SEM, confocal microscopy, and RT-qPCR. Moreover, in vivo biocompatibility and host integration were analyzed. b-TPUe demonstrated a much closer compression and shear behavior to native cartilage than PCL and PLA, as well as closer tribological properties to cartilage. Moreover, b-TPUe bioprinted scaffolds were able to maintain proper proliferative potential, cell viability, and supported MSCs chondrogenesis. Finally, in vivo studies revealed no toxic effects 21 days after scaffolds implantation, extracellular matrix deposition and integration within the surrounding tissue. This is the first study that validates the biocompatibility of b-TPUe for 3D bioprinting. Our findings indicate that this biomaterial can be exploited for the automated biofabrication of artificial tissues with tailorable mechanical properties including the great potential for cartilage TE applications.