RESUMEN
Here, we use single-molecule techniques to study the aggregation of α-synuclein, the protein whose misfolding and deposition is associated with Parkinson's disease. We identify a conformational change from the initially formed oligomers to stable, more compact proteinase-K-resistant oligomers as the key step that leads ultimately to fibril formation. The oligomers formed as a result of the structural conversion generate much higher levels of oxidative stress in rat primary neurons than do the oligomers formed initially, showing that they are more damaging to cells. The structural conversion is remarkably slow, indicating a high kinetic barrier for the conversion and suggesting that there is a significant period of time for the cellular protective machinery to operate and potentially for therapeutic intervention, prior to the onset of cellular damage. In the absence of added soluble protein, the assembly process is reversed and fibrils disaggregate to form stable oligomers, hence acting as a source of cytotoxic species.
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alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Animales , Células Cultivadas , Endopeptidasa K/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Humanos , Cinética , Modelos Moleculares , Neuronas/metabolismo , Estrés Oxidativo , RatasRESUMEN
Lanthanides have unique photoluminescence (PL) emission properties, including very long PL lifetimes. This makes them ideal for biological imaging applications, especially using PL lifetime imaging microscopy (PLIM). PLIM is an inherently multidimensional technique with exceptional advantages for quantitative biological imaging. Unfortunately, due to the required prolonged acquisitions times, photobleaching of lanthanide PL emission currently constitutes one of the main drawbacks of PLIM. In this study, we report a small aqueous-soluble, lanthanide antenna, 8-methoxy-2-oxo-1,2,4,5-tetrahydrocyclopenta[de]quinoline-3-phosphonic acid, PAnt, specifically designed to dynamically interact with lanthanide ions, serving as exchangeable dye aimed at mitigating photobleaching in PLIM microscopy in cellulo. Thus, self-assembled lanthanide complexes that may be photobleached during image acquisition are continuously replenished by intact lanthanide antennas from a large reservoir. Remarkably, our self-assembled lanthanide complex clearly demonstrated a significant reduction of PL photobleaching when compared to well-established lanthanide cryptates, used for bioimaging. This concept of exchangeable lanthanide antennas opens new possibilities for quantitative PLIM bioimaging.
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Elementos de la Serie de los Lantanoides , Microscopía , Luminiscencia , FotoblanqueoRESUMEN
Three achiral polycyclic aromatic fluorophoresânamely, 1-pyrenecarboxylic acid, 9-anthracenecarboxylic acid, and perylene-3,9-dicarboxylic acidâwere chosen based on their desired properties before being incorporated into the construction of a K+-carrying gamma-cyclodextrin (γ-CD)-based metal-organic framework (CD-MOF-1) and γ-CD-containing hybrid frameworks (CD-HFs). Among these fluorophores, only the pyrene-carrying one shows significant noncovalent bonding interactions with γ-CD in solution. This fluorophore is encapsulated in a CD-HF with a trigonal superstructure instead of the common cubic CD-MOF-1 found in the case of the other two fluorophores. Single-crystal X-ray diffraction analysis of the trigonal CD-HF reveals a π-stacked chiral positioning of the pyrene-carrying fluorophore inside the (γ-CD)2 tunnels and held uniformly around an enantiomorphous 32 screw axis along the c direction in the solid-state structure. This helix-like structure demonstrates an additional level of chirality over and above the point-chiral stereogenic centers of γ-CD and the axial chirality associated with the self-assembled π-stacked fluorophores. These arrangements result in specifically generated photophysical and chiroptical properties, such as the controlled emergence of circularly polarized luminescence (CPL) emission. In this manner, a complete understanding of the mechanism of chirality transfer from a chiral host (CD-HF) to an encapsulated achiral fluorophore has been achieved, an attribute which is often missing in the development of materials with CPL.
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Ciclodextrinas , Estructuras Metalorgánicas , Colorantes Fluorescentes/química , Luminiscencia , PirenosRESUMEN
The transient outward potassium current (Itof) is generated by the activation of KV4 channels assembled with KChIP2 and other accessory subunits (DPP6 and KCNE2). To test the hypothesis that these subunits modify the channel pharmacology, we analyzed the electrophysiological effects of (3-(2-(3-phenoxyphenyl)acetamido)-2-naphthoic acid) (IQM-266), a new KChIP2 ligand, on the currents generated by KV4.3/KChIP2, KV4.3/KChIP2/DPP6 and KV4.3/KChIP2/KCNE2 channels. CHO cells were transiently transfected with cDNAs codifying for different proteins (KV4.3/KChIP2, KV4.3/KChIP2/DPP6 or KV4.3/KChIP2/KCNE2), and the potassium currents were recorded using the whole-cell patch-clamp technique. IQM-266 decreased the maximum peak of KV4.3/KChIP2, KV4.3/KChIP2/DPP6 and KV4.3/KChIP2/KCNE2 currents, slowing their time course of inactivation in a concentration-, voltage-, time- and use-dependent manner. IQM-266 produced an increase in the charge in KV4.3/KChIP2 channels that was intensified when DPP6 was present and abolished in the presence of KCNE2. IQM-266 induced an activation unblocking effect during the application of trains of pulses to cells expressing KV4.3/KChIP2 and KV4.3/KChIP2/KCNE2, but not in KV4.3/KChIP2/DPP6 channels. Overall, all these results are consistent with a preferential IQM-266 binding to an active closed state of Kv4.3/KChIP2 and Kv4.3/KChIP2/KCNE2 channels, whereas in the presence of DPP6, IQM-266 binds preferentially to an inactivated state. In conclusion, DPP6 and KCNE2 modify the pharmacological response of KV4.3/KChIP2 channels to IQM-266.
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Proteínas de Interacción con los Canales Kv , Canales de Potasio Shal , Animales , Cricetinae , Cricetulus , Proteínas de Interacción con los Canales Kv/genética , Proteínas de Interacción con los Canales Kv/metabolismo , Técnicas de Placa-Clamp , Potasio/metabolismo , Canales de Potasio Shal/genética , Canales de Potasio Shal/metabolismoRESUMEN
Protein O-fucosyltransferase 1 (PoFUT1) is a GT-B fold enzyme that fucosylates proteins containing EGF-like repeats. GT-B glycosyltransferases have shown a remarkable grade of plasticity adopting closed and open conformations as a way of tuning their catalytic cycle, a feature that has not been observed for PoFUT1. Here, we analyzed Caenorhabditis elegans PoFUT1 (CePoFUT1) conformational behavior in solution by atomic force microscopy (AFM) and single-molecule fluorescence resonance energy transfer (SMF-FRET). Our results show that this enzyme is very flexible and adopts mainly compact conformations and to a lesser extend a highly dynamic population that oscillates between compact and highly extended conformations. Overall, our experiments illustrate the inherent complexity of CePoFUT1 dynamics, which might play a role during its catalytic cycle.
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Fucosiltransferasas/química , Dominios Proteicos , Dominios y Motivos de Interacción de Proteínas , Algoritmos , Proteínas Portadoras , Fucosiltransferasas/genética , Fucosiltransferasas/metabolismo , Humanos , Microscopía de Fuerza Atómica , Modelos Moleculares , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Proteínas Recombinantes , Soluciones , Especificidad por Sustrato , Galactósido 2-alfa-L-FucosiltransferasaRESUMEN
Na+-H+ exchanger regulatory factor-1 (NHERF1) is a PDZ protein that scaffolds membrane proteins, including sodium-phosphate co-transport protein 2A (NPT2A) at the plasma membrane. NHERF1 is a phosphoprotein with 40 Ser and Thr residues. Here, using tandem MS analysis, we characterized the sites of parathyroid hormone (PTH)-induced NHERF1 phosphorylation and identified 10 high-confidence phosphorylation sites. Ala replacement at Ser46, Ser162, Ser181, Ser269, Ser280, Ser291, Thr293, Ser299, and Ser302 did not affect phosphate uptake, but S290A substitution abolished PTH-dependent phosphate transport. Unexpectedly, Ser290 was rapidly dephosphorylated and rephosphorylated after PTH stimulation, and we found that protein phosphatase 1α (PP1α), which binds NHERF1 through a conserved VxF/W PP1 motif, dephosphorylates Ser290 Mutating 257VPF259 eliminated PP1 binding and blunted dephosphorylation. Tautomycetin blocked PP1 activity and abrogated PTH-sensitive phosphate transport. Using fluorescence lifetime imaging (FLIM), we observed that PTH paradoxically and transiently elevates intracellular phosphate. Added phosphate blocked PP1α-mediated Ser290 dephosphorylation of recombinant NHERF1. Hydrogen-deuterium exchange MS revealed that ß-sheets in NHERF1's PDZ2 domain display lower deuterium uptake than those in the structurally similar PDZ1, implying that PDZ1 is more cloistered. Dephosphorylated NHERF1 exhibited faster exchange at C-terminal residues suggesting that NHERF1 dephosphorylation precedes Ser290 rephosphorylation. Our results show that PP1α and NHERF1 form a holoenzyme and that a multiprotein kinase cascade involving G protein-coupled receptor kinase 6A controls the Ser290 phosphorylation status of NHERF1 and regulates PTH-sensitive, NPT2A-mediated phosphate uptake. These findings reveal how reversible phosphorylation modifies protein conformation and function and the biochemical mechanisms underlying PTH control of phosphate transport.
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Hormona Paratiroidea/fisiología , Fosfatos/metabolismo , Fosfoproteínas/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/fisiología , Secuencia de Aminoácidos , Cristalografía por Rayos X , Furanos/farmacología , Células HEK293 , Humanos , Transporte Iónico/fisiología , Lípidos/farmacología , Fosfoproteínas/química , Fosforilación , Conformación Proteica , Receptores de Neuropéptido Y/antagonistas & inhibidores , Receptores de Neuropéptido Y/metabolismo , Serina/metabolismo , Intercambiadores de Sodio-Hidrógeno/químicaRESUMEN
The main role of mitochondria, as pivotal organelles for cellular metabolism, is the production of energy (ATP) through an oxidative phosphorylation system. During this process, the electron transport chain creates a proton gradient that drives the synthesis of ATP. One of the main features of tumoral cells is their altered metabolism, providing alternative routes to enhance proliferation and survival. Hence, it is of utmost importance to understand the relationship between mitochondrial pH, tumoral metabolism, and cancer. In this manuscript, we develop a highly specific nanosensor to accurately measure the intramitochondrial pH using fluorescence lifetime imaging microscopy (FLIM). Importantly, we have applied this nanosensor to establish differences that may be hallmarks of different metabolic pathways in breast cancer cell models, leading to the characterization of different metabophenotypes.
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Técnicas Biosensibles/métodos , Neoplasias de la Mama/metabolismo , Metabolómica/métodos , Mitocondrias/metabolismo , Nanotecnología/métodos , Femenino , Humanos , Concentración de Iones de Hidrógeno , Células MCF-7 , Metaboloma , Microscopía Fluorescente/métodos , Nanopartículas/metabolismoRESUMEN
In recent years, the prevalence of amyloid neurodegenerative diseases such as Alzheimer's disease (AD) has significantly increased in developed countries due to increased life expectancy. This amyloid disease is characterized by the presence of accumulations and deposits of ß-amyloid peptide (Aß) in neuronal tissue, leading to the formation of oligomers, fibers, and plaques. First, oligomeric intermediates that arise during the aggregation process are currently thought to be primarily responsible for cytotoxicity in cells. This work aims to provide further insights into the mechanisms of cytotoxicity by studying the interaction of Aß aggregates with Neuro-2a (N2a) neuronal cells and the effects caused by this interaction. For this purpose, we have exploited the advantages of advanced, multidimensional fluorescence microscopy techniques to determine whether different types of Aß are involved in higher rates of cellular toxicity, and we measured the cellular stress caused by such aggregates by using a fluorogenic intracellular biothiol sensor. Stress provoked by the peptide is evident by N2a cells generating high levels of biothiols as a defense mechanism. In our study, we demonstrate that Aß aggregates act as seeds for aggregate growth upon interacting with the cellular membrane, which results in cell permeability and damage and induces lysis. In parallel, these damaged cells undergo a significant increase in intracellular biothiol levels.
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Péptidos beta-Amiloides/metabolismo , Membrana Celular/metabolismo , Neuronas/metabolismo , Agregación Patológica de Proteínas/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Línea Celular , Membrana Celular/patología , Permeabilidad de la Membrana Celular , Ratones , Neuronas/patología , Agregado de Proteínas , Agregación Patológica de Proteínas/patologíaRESUMEN
Ferritin, a soluble and highly robust protein with subunits packed into well-defined helices, is a key component of the iron regulatory system in the brain and thus is widely recognized as a crucial protein for iron metabolism, but may also bear possible implications in some neurodegenerative disorders. Here, we present evidence of how human recombinant apoferritin can convert into an unusual structure from its folded native state; that is, amyloid fibrils analogue to those found in pathological disorders such as Alzheimer's and Parkinson's diseases. An extensive combination of advanced microscopy, spectroscopy and scattering techniques concur to reveal that apoferritin fibrils possess a common double stranded twisted ribbon structure which can result in a mesoscopic right-handed chirality. We highlight a direct connection between the chirality and morphology of the resulting amyloid fibrils, and the initial protein subunits composition, advancing our understanding on the possible role of misfolding in some ferritin-related pathologies and posing new bases for the design of chiral 1D functional nanostructures.
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Amiloide/química , Apoferritinas/química , Agregado de Proteínas , Animales , Humanos , Modelos Moleculares , Conformación Proteica , EstereoisomerismoRESUMEN
In the last few years, quantum dot (QD) nanoparticles have been employed for bioimaging and sensing due to their excellent optical features. Most studies have used photoluminescence (PL) intensity-based techniques, which have some drawbacks, especially when working with nanoparticles in intracellular media, such as fluctuations in the excitation power, fluorophore concentration dependence, or interference from cell autofluorescence. Some of those limitations can be overcome with the use of time-resolved spectroscopy and fluorescence lifetime imaging microscopy (FLIM) techniques. In this work, CdSe/ZnS QDs with long decay times were modified with aminophenylboronic acid (APBA) to achieve QD-APBA conjugates, which can act as glucose nanosensors. The attachment of the boronic acid moiety on the surface of the nanoparticle quenched the PL average lifetime of the QDs. When glucose bonded to the boronic acid, the PL was recovered and its lifetime was enhanced. The nanosensors were satisfactorily applied to the detection of glucose into MDA-MB-231 cells with FLIM. The long PL lifetimes of the QD nanoparticles made them easily discernible from cell autofluorescence, thereby improving selectivity in their sensing applications. Since the intracellular levels of glucose are related to the metabolic status of cancer cells, the proposed nanosensors could potentially be used in cancer diagnosis.
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Técnicas Biosensibles , Glucosa/aislamiento & purificación , Neoplasias/diagnóstico , Puntos Cuánticos/química , Ácidos Borónicos/química , Compuestos de Cadmio/química , Humanos , Microscopía Fluorescente , Nanopartículas/química , Neoplasias/química , Compuestos de Selenio/química , Sulfuros/química , Compuestos de Zinc/químicaRESUMEN
In this manuscript, we report the first synthesis of an organic monomolecular emitter, which behaves as a circularly polarized luminescence (CPL)-based ratiometric probe. The enantiopure helical ortho-oligo(phenylene)ethynylene ( o-OPE) core has been prepared by a new and efficient macrocyclization reaction. The combination of such o-OPE helical skeleton and a pyrene couple leads to two different CPL emission features in a single structure whose ratio linearly responds to silver(I) concentration.
RESUMEN
Mechanisms of protein recognition have been extensively studied for single-domain proteins, but are less well characterized for dynamic multidomain systems. Ubiquitin chains represent a biologically important multidomain system that requires recognition by structurally diverse ubiquitin-interacting proteins. Ubiquitin chain conformations in isolation are often different from conformations observed in ubiquitin-interacting protein complexes, indicating either great dynamic flexibility or extensive chain remodelling upon binding. Using single-molecule fluorescence resonance energy transfer, we show that Lys 63-, Lys 48- and Met 1-linked diubiquitin exist in several distinct conformational states in solution. Lys 63- and Met 1-linked diubiquitin adopt extended 'open' and more compact 'closed' conformations, and ubiquitin-binding domains and deubiquitinases (DUBs) select pre-existing conformations. By contrast, Lys 48-linked diubiquitin adopts predominantly compact conformations. DUBs directly recognize existing conformations, but may also remodel ubiquitin chains to hydrolyse the isopeptide bond. Disruption of the Lys 48-diubiquitin interface changes conformational dynamics and affects DUB activity. Hence, conformational equilibria in ubiquitin chains provide an additional layer of regulation in the ubiquitin system, and distinct conformations observed in differently linked polyubiquitin may contribute to the specificity of ubiquitin-interacting proteins.
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Modelos Moleculares , Ubiquitina/química , Ubiquitina/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
The development of new fluorescent probes for cellular imaging is currently a very active field because of the large potential in understanding cell physiology, especially targeting anomalous behaviours due to disease. In particular, red-emitting dyes are keenly sought, as the light in this spectral region presents lower interferences and a deeper depth of penetration in tissues. In this work, we have synthesized a red-emitting, dual probe for the multiplexed intracellular detection of biothiols and phosphate ions. We have prepared a fluorogenic construct involving a silicon-substituted fluorescein for red emission. The fluorogenic reaction is selectively started by the presence of biothiols. In addition, the released fluorescent moiety undergoes an excited-state proton transfer reaction promoted by the presence of phosphate ions, which modulates its fluorescence lifetime, τ, with the total phosphate concentration. Therefore, in a multidimensional approach, the intracellular levels of biothiols and phosphate can be detected simultaneously using a single fluorophore and with spectral clearing of cell autofluorescence interferences. We have applied this concept to different cell lines, including photoreceptor cells, whose levels of biothiols are importantly altered by light irradiation and other oxidants.
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Fosfatos/análisis , Fluoresceína , Colorantes Fluorescentes , Protones , Espectrometría de FluorescenciaRESUMEN
The simultaneous detection of relevant metabolites in living organisms by using one molecule introduces an approach to understanding the relationships between these metabolites in healthy and deregulated cells. Fluorescent probes of low toxicity are remarkable tools for this type of analysis of biological systems in vivo. As a proof of concept, different naturally occurring compounds, such as biothiols and phosphate anions, were the focus for this work. The 2,4-dinitrobenzenesulfinate (DNBS) derivative of 9-[1-(4-tert-butyl-2-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (Granada Green; GG) were designed and synthesized. This new sulfinyl xanthene derivative can act as a dual sensor for the aforementioned analytes simultaneously. The mechanism of action of this derivative implies thiolysis of the sulfinyl group of the weakly fluorescent DNBS-GG by biological thiols at near-neutral pHâ values, thus releasing the fluorescent GG moiety, which simultaneously responds to phosphate anions through its fluorescence-decay time. The new dual probe was tested in solution by using steady-state and time-resolved fluorescence and intracellularly by using fluorescence-lifetime imaging microscopy (FLIM) in human epithelioid cervix carcinoma (HeLa) cells.
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Colorantes Fluorescentes/química , Nitrocompuestos/química , Fosfatos/química , Compuestos de Sulfhidrilo/química , Compuestos de Sulfonio/química , Neoplasias del Cuello Uterino/química , Xantenos/química , Xantinas/química , Femenino , Fluorescencia , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Microscopía Fluorescente , Estructura Molecular , Fosfatos/análisis , Compuestos de Sulfhidrilo/análisisRESUMEN
Dyes with near-red emission are of great interest because of their undoubted advantages for use as probes in living cells. In-depth knowledge of their photophysics is essential for employment of such dyes. In this article, the photophysical behavior of a new silicon-substituted xanthene, 7-hydroxy-5,5-dimethyl-10-(o-tolyl)dibenzo[b,e]silin-3(5H)-one (2-Me TM), was explored by means absorption, steady-state, and time-resolved fluorescence. First, the near-neutral pH, ground-state acidity constant of the dye, pKN-A, was determined by absorbance and steady-state fluorescence at very low buffer concentrations. Next, we determined whether the addition of phosphate buffer promoted the excited-state proton-transfer (ESPT) reaction among the neutral and anion form of 2-Me TM in aqueous solutions at near-neutral pH. For this analysis, both the steady-state fluorescence method and time-resolved emission spectroscopy (TRES) were employed. The TRES experiments demonstrated a remarkably favored conversion of the neutral form to the anion form. Then, the values of the excited-state rate constants were determined by global analysis of the fluorescence decay traces recorded as a function of pH, and buffer concentration. The revealed kinetic parameters were consistent with the TRES results, exhibiting a higher rate constant for deprotonation than for protonation, which implies an unusual low value of the excited-state acidity constant pK*N-A and therefore an enhanced photoacid behavior of the neutral form. Finally, we determined whether 2-Me TM could be used as a sensor inside live cells by measuring the intensity profile of the probe in different cellular compartments of HeLa 229 cells.
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Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Luz , Compuestos de Organosilicio/química , Protones , Silicio/química , Permeabilidad de la Membrana Celular , Células HeLa/citología , Células HeLa/metabolismo , Humanos , Estructura Molecular , Compuestos de Organosilicio/metabolismo , Fenómenos Físicos , Xantenos/química , Xantenos/metabolismoRESUMEN
The UV-vis electronic absorption and fluorescence emission properties of 8-halogenated (Cl, Br, I) difluoroboron dipyrrin (or 8-haloBODIPY) dyes and their 8-(C, N, O, S) substituted analogues are reported. The nature of the meso-substituent has a significant influence on the spectral band positions, the fluorescence quantum yields, and lifetimes. As a function of the solvent, the spectral maxima of all the investigated dyes are located within a limited wavelength range. The spectra of 8-haloBODIPYs display the narrow absorption and fluorescence emission bands and the generally quite small Stokes shifts characteristic of classic difluoroboron dipyrrins. Conversely, fluorophores with 8-phenylamino (7), 8-benzylamino (8), 8-methoxy (9), and 8-phenoxy (10) groups emit in the blue range of the visible spectrum and generally have larger Stokes shifts than common BODIPYs, whereas 8-(2-phenylethynyl)BODIPY (6) has red-shifted spectra compared to ordinary BODIPY dyes. Fluorescence lifetimes for 6, 8, and 10 have been measured for a large set of solvents and the solvent effect on their absorption and emission maxima has been analyzed using the generalized Catalán solvent scales. Restricted rotation about the C8-N bond in 7 and 8 has been observed via temperature dependent (1)H NMR spectroscopy, whereas for 10 the rotation about the C8-O bond is not hindered. The crystal structure of 8 demonstrates that the short C8-N bond has a significant double character and that this N atom exhibits a trigonal planar geometry. The crystal structure of 10 shows a short C8-O bond and an intramolecular C-H···π interaction. Quantum-chemical calculations have been performed to assess the effect of the meso-substituent on the spectroscopic properties.
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Lanthanide photoluminescence (PL) emission has attracted much attention for technological and bioimaging applications because of its particularly interesting features, such as narrow emission bands and very long PL lifetimes. However, this emission process necessitates a preceding step of energy transfer from suitable antennas. While biocompatible applications require luminophores that are stable in aqueous media, most lanthanide-based emitters are quenched by water molecules. Previously, we described a small luminophore, 8-methoxy-2-oxo-1,2,4,5-tetrahydrocyclopenta[de]quinoline-3-phosphonic acid (PAnt), which is capable of dynamically coordinating with Tb(III) and Eu(III), and its exchangeable behavior improved their performance in PL lifetime imaging microscopy (PLIM) compared with conventional lanthanide cryptate imaging agents. Herein, we report an in-depth photophysical and time-dependent density functional theory (TD-DFT) computational study that reveals different sensitization mechanisms for Eu(III) and Tb(III) in stable complexes formed in water. Understanding this unique behavior in aqueous media enables the exploration of different applications in bioimaging or novel emitting materials.
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Controlling the nano- and micropatterning of metal structures is an important requirement for various technological applications in photonics and biosensing. This work presents a method for controllably creating silver micropatterns by laser-induced photosculpting. Photosculpting is driven by plasmonic interactions between pulsed laser radiation and silver nanorods (AgNRs) in aqueous suspension; this process leads to optical binding forces transporting the AgNRs in the surroundings, while electronic thermalization results in photooxidation, melting, and ripening of the AgNRs into well-defined 3D structures. This work call these structures Airy castles due to their structural similarity with a diffraction-limited Airy disk. The photosculpted Airy castles contain emissive Ag nanoclusters, allowing for the visualization and examination of the aggregation process using luminescence microscopy. This work comprehensively examines the factors that define the photosculpting process, namely, the concentration and shape of the AgNRs, as well as the energy, power, and repetition rate of the laser. Finally, this work investigates the potential applications by measuring the metal-enhanced luminescence of a europium-based luminophore using Airy castles.
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Foxp3 acetylation is essential to regulatory T (Treg) cell stability and function, but pharmacologically increasing it remains an unmet challenge. Here, we report that small-molecule compounds that inhibit TIP60, an acetyltransferase known to acetylate Foxp3, unexpectedly increase Foxp3 acetylation and Treg induction. Utilizing a dual experimental/computational approach combined with a newly developed FRET-based methodology compatible with flow cytometry to measure Foxp3 acetylation, we unraveled the mechanism of action of these small-molecule compounds in murine and human Treg induction cell cultures. We demonstrate that at low-mid concentrations they activate TIP60 to acetylate P300, a different acetyltransferase, which in turn increases Foxp3 acetylation, thereby enhancing Treg cell induction. These results reveal a potential therapeutic target relevant to autoimmunity and transplant.
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Few-layer black phosphorus (FLBP), a technologically important 2D material, faces a major hurdle to consumer applications: spontaneous degradation under ambient conditions. Blocking the direct exposure of FLBP to the environment has remained the key strategy to enhance its stability, but this can also limit its utility. In this paper, a more ambitious approach to handling FLBP is reported where not only is FLBP oxidation blocked, but it is also repaired postoxidation. Our approach, inspired by nature, employs the antioxidant molecule ß-carotene that protects plants against photooxidative damages to act as a protecting and repairing agent for FLBP. The mechanistic role of ß-carotene is established by a suite of spectro-microscopy techniques, in combination with computational studies and biochemical assays. Transconductance studies on FLBP-based field effect transistor (FET) devices further affirm the protective and reparative effects of ß-carotene. The outcomes indicate the potential for deploying a plethora of natural antioxidant molecules to enhance the stability of other environmentally sensitive inorganic nanomaterials and expedite their translation for technological and consumer applications.