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In vitro cell activation through specific IgE bound to high-affinity receptors on the basophil surface is a widely used strategy for the evaluation of IgE-mediated immediate hypersensitivity reactions to betalactams. Cellular activation requires drug conjugation to a protein to form a large enough structure displaying a certain distance between haptens to allow the cross-linking of two IgE antibodies bound to the basophil's surface, triggering their degranulation. However, no information about the size and composition of these conjugates is available. Routine in vitro diagnosis using the basophil activation test uses free amoxicillin, which is assumed to conjugate to a carrier present in blood. To standardize the methodology, we propose the use of well-controlled and defined nanomaterials functionalized with amoxicilloyl. Silica nanoparticles decorated with PAMAM-dendrimer-amoxicilloyl conjugates (NpDeAXO) of different sizes and amoxicilloyl densities (50-300 µmol amoxicilloyl/gram nanoparticle) have been prepared and chemically characterized. Two methods of synthesis were performed to ensure reproducibility and stability. Their functional effect on basophils was measured using an in-house basophil activation test (BAT) that determines CD63+ or CD203chigh activation markers. It was observed that NpDeAXO nanocomposites are not only able to specifically activate basophils but also do so in a more effective way than free amoxicillin, pointing to a translational potential diagnosis.
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A family of acylhydrazones have been prepared and characterized with the aim of investigating their potential as information storage systems. Their well-established synthetic methodologies allowed for the preparation of seven chemically stable acylhydrazones in excellent yields that have been photophysically and photochemically characterized. In addition, DFT and TD-DFT calculations have been performed to gain more insights into the structural, energetic and photophysical properties of the E/Z isomers. Our results reveal that E/Z configurational isomerization upon irradiation is highly dependent on the stabilization of the E or Z isomers due to the formation of intramolecular H bonds and the electronic/steric effects intrinsically related to their structures. In addition, Raman spectroscopy is also used to confirm the molecular structural changes after the formation of hydrogen bonds in the isomers.
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Espectrometría Raman , Modelos Moleculares , Isomerismo , Espectroscopía de Resonancia Magnética , Teoría Funcional de la DensidadRESUMEN
An "off-on" fluorescent nanoprobe for near-infrared multiphoton imaging of singlet oxygen has been developed. The nanoprobe comprises a naphthoxazole fluorescent unit and a singlet-oxygen-sensitive furan derivative attached to the surface of mesoporous silica nanoparticles. In solution, the fluorescence of the nanoprobe increases upon reaction with singlet oxygen both under one- and multiphoton excitation, with fluorescence enhancements up to 180-fold. The nanoprobe can be readily internalized by macrophage cells and is capable of imaging intracellular singlet oxygen under multiphoton excitation.
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Amyloid beta (Aß) plaques are a major pathological hallmark of Alzheimer's disease (AD) and constitute of structurally heterogenic entities (polymorphs) that have been implicated in the phenotypic heterogeneity of AD pathology and pathogenesis. Understanding amyloid aggregation has been a critical limiting factor to gain understanding of AD pathogenesis, ultimately reflected in that the underlying mechanism remains elusive. We identified a fluorescent probe in the form of a turn-off photoswitchable norbornadiene derivative (NBD1) with several microenvironment-sensitive properties that make it relevant for applications within advanced fluorescence imaging, for example, multifunctional imaging. We explored the application of NBD1 for in situ delineation of structurally heterogenic Aß plaques in transgenic AD mouse models. NBD1 plaque imaging shows characteristic broader emission bands in the periphery and more narrow emission bands in the dense cores of mature cored plaques. Further, we demonstrate in situ photoisomerization of NBD1 to quadricyclane and thermal recovery in single plaques, which is relevant for applications within both functional and super-resolution imaging. This is the first time a norbornadiene photoswitch has been used as a probe for fluorescence imaging of Aß plaque pathology in situ and that its spectroscopic and switching properties have been studied within the specific environment of senile Aß plaques. These findings open the way toward new applications of NBD-based photoswitchable fluorescent probes for super-resolution or dual-color imaging and multifunctional microscopy of amyloid plaque heterogeneity. This could allow to visualize Aß plaques with resolution beyond the diffraction limit, label different plaque types, and gain insights into their physicochemical composition.
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Enfermedad de Alzheimer , Péptidos beta-Amiloides , Ratones , Animales , Péptidos beta-Amiloides/química , Placa Amiloide/patología , Modelos Animales de Enfermedad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Enfermedad de Alzheimer/diagnóstico por imagen , Enfermedad de Alzheimer/patología , Ratones Transgénicos , Colorantes FluorescentesRESUMEN
Mesenchymal condensation is a prevalent morphogenetic transition that is essential in chondrogenesis. However, the current understanding of condensation mechanisms is limited. In vivo, progenitor cells directionally migrate from the surrounding loose mesenchyme towards regions of increasing matrix adherence (the condensation centers), which is accompanied by the upregulation of fibronectin. Here, we focused on the mechanisms of cell migration during mesenchymal cell condensation and the effects of matrix adherence. Dendrimer-based nanopatterns of the cell-adhesive peptide arginine-glycine-aspartic acid (RGD), which is present in fibronectin, were used to regulate substrate adhesion. We recorded collective and single-cell migration of mesenchymal stem cells, under chondrogenic induction, using live-cell imaging. Our results show that the cell migration mode of single cells depends on substrate adhesiveness, and that cell directionality controls cell condensation and the fusion of condensates. Inhibition experiments revealed that cell-cell interactions mediated by N-cadherin (also known as CDH2) are also pivotal for directional migration of cell condensates by maintaining cell-cell cohesion, thus suggesting a fine interplay between cell-matrix and cell-cell adhesions. Our results shed light on the role of cell interactions with a fibronectin-depositing matrix during chondrogenesis in vitro, with possible applications in regenerative medicine. This article has an associated First Person interview with the first author of the paper.
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Condrogénesis , Células Madre Mesenquimatosas , Humanos , Fibronectinas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Mesodermo , Cadherinas/metabolismo , Adhesión Celular , Diferenciación CelularRESUMEN
Aim: To unveil the influence of cell-matrix adhesions in the establishment of gap junction intercellular communication (GJIC) during cell condensation in chondrogenesis. Materials & methods: Previously developed nanopatterns of the cell adhesive ligand arginine-glycine-aspartic acid were used as cell culture substrates to control cell adhesion at the nanoscale. In vitro chondrogenesis of mesenchymal stem cells was conducted on the nanopatterns. Cohesion and GJIC were evaluated in cell condensates. Results: Mechanical stability and GJIC are enhanced by a nanopattern configuration in which 90% of the surface area presents adhesion sites separated less than 70 nm, thus providing an onset for cell signaling. Conclusion: Cell-matrix adhesions regulate GJIC of mesenchymal cell condensates during in vitro chondrogenesis from a threshold configuration at the nanoscale.
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Condrogénesis , Uniones Comunicantes , Comunicación Celular/fisiología , Uniones Comunicantes/metabolismo , Ligandos , Transducción de SeñalRESUMEN
BACKGROUND: Immediate drug hypersensitivity reactions (IDHRs) to clavulanic acid (CLV) have increased in the last decades due to a higher consumption alongside amoxicillin (AX). Due to its chemical instability, diagnostic procedures to evaluate IDHRs to CLV are difficult, and current in vitro assays do not have an optimal sensitivity. The inclusion of the specific metabolites after CLV degradation, which are efficiently recognised by the immune system, could help to improve sensitivity of in vitro tests. METHODS: Recognition by dendritic cells (DCs) of CLV and the synthetic analogues of two of its hypothesised antigenic determinants (ADs) was evaluated by flow cytometry in 27 allergic patients (AP) and healthy controls (HC). Their ability to trigger the proliferation of T cells was also analysed by flow cytometry. RESULTS: The inclusion of synthetic analogues of CLV ADs, significantly increased the expression of maturation markers on DCs from AP compared to HC. A different recognition pattern could be observed with each AD, and, therefore, the inclusion of both ADs achieves an improved sensitivity. The addition of synthetic ADs analogues increased the proliferative response of CD4+ Th2 compared to the addition of native CLV. The combination of results from both ADs increased the sensitivity of proliferative assays from 19% to 65% with a specificity higher than 90%. CONCLUSIONS: Synthetic ADs from CLV are efficiently recognised by DCs with ability to activate CD4+ Th2 cells from AP. The combination of analogues from both ADs, significantly increased the sensitivity of DC maturation and T-cell proliferation compared to native CLV.
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Hipersensibilidad a las Drogas , Hipersensibilidad Inmediata , Amoxicilina , Proliferación Celular , Ácido Clavulánico/efectos adversos , Células Dendríticas , Epítopos/metabolismo , HumanosRESUMEN
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.
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Ciclobutanos , Colorantes Fluorescentes , Fenoles , FotonesRESUMEN
ß-lactam antibiotics (BLs) are the drugs most frequently involved in drug hypersensitivity reactions. However, current in vitro diagnostic tests have limited sensitivity, partly due to a poor understanding of in vivo drug-protein conjugates that both induce the reactions and are immunologically recognized. Dendrimeric Antigen-Silica particle composites (DeAn@SiO2), consisting on nanoparticles decorated with BL-DeAns are promising candidates for improving the in vitro clinical diagnostic practice. In this nano-inspired system biology, the synthetic dendrimer plays the role of the natural carrier protein, emulating its haptenation by drugs and amplifying the multivalence. Herein, we present the design and synthesis of new multivalent mono- and bi-epitope DeAn@SiO2, using amoxicillin and/or benzylpenicillin allergenic determinants as ligands. The homogeneous composition of nanoparticles provides high reproducibility and quality, which is critical for in vitro applications. The suitable functionalization of nanoparticles allows the anchoring of DeAn, minimizing the nonspecific interactions and facilitating the effective exposure to specific IgE; while the larger interaction area increments the likelihood of capturing specific IgE. This achievement is particularly important for improving sensitivity of current immunoassays since IgE levels in BL allergic patients are very low. Our data suggest that these new nano-based platforms provide a suitable tool for testing IgE recognition to more than one BL simultaneously. Immunochemical studies evidence that mono and bi-epitope DeAn@SiO2 composites could potentially allow the diagnosis of patients allergic to any of these drugs with a single test. These organic-inorganic hybrid materials represent the basis for the development of a single screening for BL-allergies.
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Antígenos/química , Dendrímeros/química , Inmunoglobulina E/análisis , Nanopartículas/química , Dióxido de Silicio/química , Adolescente , Adulto , Anciano , Antígenos/inmunología , Femenino , Humanos , Inmunoglobulina E/inmunología , Masculino , Persona de Mediana Edad , Dióxido de Silicio/inmunología , Adulto JovenRESUMEN
A water soluble diarylethene (DAE) derivative that displays exceptionally intense fluorescence from the colorless open form has been synthesized and characterized using UV/vis spectroscopy and fluorescence microscopy. We show that the bright emission from the open form can be rapidly switched using amplitude modulated red light, that is, by light at wavelengths longer than those absorbed by the fluorescent species. This is highly appealing in any context where undesired background fluorescence disturbs the measurement, e.g., the autofluorescence commonly observed in fluorescence microscopy. We show that this scheme is conveniently applicable using lock-in detection, and that robust amplitude modulation of the probe fluorescence is indeed possible also in cell studies using fluorescence microscopy.
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The high prevalence of allergy to ß-lactam antibiotics is a worldwide issue. Accuracy of diagnostic methods is important to prove tolerance or allergy, with skin test considered the best validated in vivo method for diagnosing immediate reactions to ß-lactams. Although drug provocation test is the reference standard, it cannot be performed in highly risk reactions or in those with positive skin tests. For skin tests, the inclusion of major and minor determinants of benzylpenicillin (BP) is recommended. Commercial skin test reagents have changed along time, including as minor determinants benzylpenicillin, benzylpenicilloate (BPO), and benzylpenilloate (PO). Major determinants consists of multivalent conjugates of benzylpenicilloyl coupled through amide bond to a carrier polymer, such as penicilloyl-polylysine (PPL) or benzylpenicilloyl-octalysine (BP-OL). The chemical stability of such reagents has influenced the evolution of the composition of the commercial kits, as this requirement is necessary for improving the quality and standardization of the product. In this work, we provide a detailed study of the chemical stability of BP determinants. We observed that those structures suffer from an epimerization process in C-5 at different rates. Butylamine-Benzylpenicilloyl conjugates (5R,6R)-Bu-BPO and (5S,6R)-Bu-BPO were selected as a simple model for mayor determinant to evaluate the role of the different epimers in the immunoreactivity with sera from penicillin-allergic patients. In vitro immunoassays indicate that any change in the chemical structure of the antigenic determinant of BP significantly affects IgE recognition. The inclusion of stereochemically pure compounds or mixtures may have important implications for both the reproducibility and sensitivity of in vivo and in vitro diagnostic tests.
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Nanotechnology is science, engineering and technology conducted at the nanoscale, which is about 1-100 nm. It has led to the development of nanomaterials, which behave very differently from materials with larger scales and can have a wide range of applications in biomedicine. The physical and chemical properties of materials of such small compounds depend mainly on the size, shape, composition and functionalization of the system. Nanoparticles, carbon nanotubes, liposomes, polymers, dendrimers and nanogels, among others, can be nanoengineeried for controlling all parameters, including their functionalization with ligands, which provide the desired interaction with the immunological system, that is dendritic cell receptors to activate and/or modulate the response, as well as specific IgE, or effector cell receptors. However, undesired issues related to toxicity and hypersensitivity responses can also happen and would need evaluation. There are wide panels of accessible structures, and controlling their physico-chemical properties would permit obtaining safer and more efficient compounds for clinical applications goals, either in diagnosis or treatment. The application of dendrimeric antigens, nanoallergens and nanoparticles in allergy diagnosis is very promising since it can improve sensitivity by increasing specific IgE binding, mimicking carrier proteins or enhancing signal detection. Additionally, in the case of immunotherapy, glycodendrimers, liposomes, polymers and nanoparticles have shown interest, behaving as platforms of allergenic structures, adjuvants or protectors of allergen from degradation or having a depot capacity. Taken together, the application of nanotechnology to allergy shows promising facts facing important goals related to the improvement of diagnosis as well as specific immunotherapy.
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Hipersensibilidad , Nanoestructuras , Nanotubos de Carbono , Alérgenos , Humanos , Hipersensibilidad/diagnóstico , Hipersensibilidad/terapia , NanotecnologíaRESUMEN
The use of aminals in dynamic covalent chemistry is slightly underexplored, probably due to their inherent instability. Here we report the spontaneous [2+2] macrocyclization of tetrakis(aminals). Their unexpected stability and structural modularity, the dynamic nature of the connections and their water tolerance make them appealing systems for future applications as stimulus-responsive materials.
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Allergic reactions to antibiotics are a major concern in the clinic. ß-lactam antibiotics are the class most frequently reported to cause hypersensitivity reactions. One of the mechanisms involved in this outcome is the modification of proteins by covalent binding of the drug (haptenation). Hence, interest in identifying the corresponding serum and cellular protein targets arises. Importantly, haptenation susceptibility and extent can be modulated by the context, including factors affecting protein conformation or the occurrence of other posttranslational modifications. We previously identified the glycolytic enzyme α-enolase as a target for haptenation by amoxicillin, both in cells and in the extracellular milieu. Here, we performed an in vitro study to analyze amoxicillin haptenation of α-enolase using gel-based and activity assays. Moreover, the possible interplay or interference between amoxicillin haptenation and acetylation of α-enolase was studied in 1D- and 2D-gels that showed decreased haptenation and displacement of the haptenation signal to lower pI spots after chemical acetylation of the protein, respectively. In addition, the peptide containing lysine 239 was identified by mass spectrometry as the amoxicillin target sequence on α-enolase, thus suggesting a selective haptenation under our conditions. The putative amoxicillin binding site and the surrounding interactions were investigated using the α-enolase crystal structure and molecular docking. Altogether, the results obtained provide the basis for the design of novel diagnostic tools or approaches in the study of amoxicillin-induced allergic reactions.
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Over the past two decades, integrated photonic sensors have been of major interest to the optical biosensor community due to their capability to detect low concentrations of molecules with label-free operation. Among these, interferometric sensors can be read-out with simple, fixed-wavelength laser sources and offer excellent detection limits but can suffer from sensitivity fading when not tuned to their quadrature point. Recently, coherently detected sensors were demonstrated as an attractive alternative to overcome this limitation. Here we show, for the first time, to the best of our knowledge, that this coherent scheme provides sub-nanogram per milliliter limits of detection in C-reactive protein immunoassays and that quasi-balanced optical arm lengths enable operation with inexpensive Fabry-Perot-type lasers sources at telecom wavelengths.
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Técnicas Biosensibles/instrumentación , Proteína C-Reactiva/análisis , Inmunoensayo/instrumentación , Interferometría/instrumentación , Silicio/química , Óptica y Fotónica , Procesos FotoquímicosRESUMEN
The combination of two two-photon-induced processes in a Förster resonance energy transfer (FRET)-operated photochromic fluorene-dithienylethene dyad lays the foundation for the observation of a quartic dependence of the fluorescence signal on the excitation light intensity. While this photophysical behavior is predicted for a four-photon absorbing dye, the herein proposed approach opens the way to use two-photon absorbing dyes, reaching the same performance. Hence, the spatial resolution limit, being a critical parameter for applications in fluorescence imaging or data storage with common two-photon absorbing dyes, is dramatically improved.
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Tackling the first stages of the chondrogenic commitment is essential to drive chondrogenic differentiation to healthy hyaline cartilage and minimize hypertrophy. During chondrogenesis, the extracellular matrix continuously evolves, adapting to the tissue adhesive requirements at each stage. Here, we take advantage of previously developed nanopatterns, in which local surface adhesiveness can be precisely tuned, to investigate its effects on prechondrogenic condensation. Fluorescence live cell imaging, immunostaining, confocal microscopy and PCR analysis are used to follow the condensation process on the nanopatterns. Cell tracking parameters, condensate morphology, cell-cell interactions, mechanotransduction and chondrogenic commitment are evaluated in response to local surface adhesiveness. Results show that only condensates on the nanopatterns of high local surface adhesiveness are stable in culture and able to enter the chondrogenic pathway, thus highlighting the importance of controlling cell-substrate adhesion in the tissue engineering strategies for cartilage repair.
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Comunicación Celular , Condrogénesis , Cartílago Hialino/metabolismo , Mecanotransducción Celular , Células Madre Mesenquimatosas/metabolismo , Adulto , Línea Celular , Femenino , Humanos , Cartílago Hialino/citología , Células Madre Mesenquimatosas/citología , Ingeniería de TejidosRESUMEN
Boronic acid-derived salicylidenehydrazone complex (BASHY) dyes with a polymethine backbone were designed to yield efficient red-emitting and two-photon absorbing fluorophores that can be used as markers for astrocytes. The dyes are chemically stable in aqueous solution and do not undergo photodecomposition. Their photophysical properties can be electronically fine-tuned and thereby adapted to potentially different imaging situations and requirements.