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1.
Nat Commun ; 12(1): 2005, 2021 03 31.
Artículo en Inglés | MEDLINE | ID: mdl-33790271

RESUMEN

Förster resonant energy transfer (FRET) is a powerful mechanism to probe associations in situ. Simultaneously performing more than one FRET measurement can be challenging due to the spectral bandwidth required for the donor and acceptor fluorophores. We present an approach to distinguish overlapping FRET pairs based on the photochromism of the donor fluorophores, even if the involved fluorophores display essentially identical absorption and emission spectra. We develop the theory underlying this method and validate our approach using numerical simulations. To apply our system, we develop rsAKARev, a photochromic biosensor for cAMP-dependent protein kinase (PKA), and combine it with the spectrally-identical biosensor EKARev, a reporter for extracellular signal-regulated kinase (ERK) activity, to deliver simultaneous readout of both activities in the same cell. We further perform multiplexed PKA, ERK, and calcium measurements by including a third, spectrally-shifted biosensor. Our work demonstrates that exploiting donor photochromism in FRET can be a powerful approach to simultaneously read out multiple associations within living cells.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/química , Proteínas Luminiscentes/química , Algoritmos , Animales , Técnicas Biosensibles/métodos , Células COS , Chlorocebus aethiops , Células HEK293 , Células HeLa , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente/métodos , Imagen de Lapso de Tiempo/métodos
2.
Int J Mol Sci ; 22(6)2021 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-33804193

RESUMEN

We report here the synthesis and structural characterization of novel cationic (phenothiazinyl)vinyl-pyridinium (PVP) dyes, together with optical (absorption/emission) properties and their potential applicability as fluorescent labels. Convective heating, ultrasound irradiation and mechanochemical synthesis were considered as alternative synthetic methodologies proficient for overcoming drawbacks such as long reaction time, nonsatisfactory yields or solvent requirements in the synthesis of novel dye (E)-1-(3-chloropropyl)-4-(2-(10-methyl-10H-phenothiazin-3-yl)vinyl)pyridin-1-ium bromide 3d and its N-alkyl-2-methylpyridinium precursor 1c. The trans geometry of the newly synthesized (E)-4-(2-(7-bromo-10-ethyl-10H-phenothiazin-3-yl)vinyl)-1-methylpyridin-1-ium iodide 3b and (E)-1-methyl-4-(2-(10-methyl-10H-phenothiazin-3-yl)vinyl)pyridin-1-ium tetrafluoroborate 3a' was confirmed by single crystal X-ray diffraction. A negative solvatochromism of the dyes in polar solvents was highlighted by UV-Vis spectroscopy and explanatory insights were supported by molecular modeling which suggested a better stabilization of the lowest unoccupied molecular orbitals (LUMO). The photostability of the dye 3b was investigated by irradiation at 365 nm in different solvents, while the steady-state and time-resolved fluorescence properties of dye 3b and 3a' in solid state were evaluated under one-photon excitation at 485 nm. The in vitro cytotoxicity of the new PVP dyes on B16-F10 melanoma cells was evaluated by WST-1 assay, while their intracellular localization was assessed by epi-fluorescence conventional microscopy imaging as well as one- and two-photon excited confocal fluorescence lifetime imaging microscopy (FLIM). PVP dyes displayed low cytotoxicity, good internalization inside melanoma cells and intense fluorescence emission inside the B16-F10 murine melanoma cells, making them suitable staining agents for imaging applications.


Asunto(s)
Colorantes Fluorescentes/química , Compuestos de Piridinio/química , Coloración y Etiquetado/métodos , Animales , Colorantes Fluorescentes/síntesis química , Ratones , Microscopía Fluorescente , Fenotiazinas/química , Fotones , Compuestos de Piridinio/síntesis química , Solventes/química , Espectrometría de Fluorescencia/métodos
3.
J Am Chem Soc ; 143(14): 5413-5424, 2021 04 14.
Artículo en Inglés | MEDLINE | ID: mdl-33797236

RESUMEN

Methods for tracking RNA inside living cells without perturbing their natural interactions and functions are critical within biology and, in particular, to facilitate studies of therapeutic RNA delivery. We present a stealth labeling approach that can efficiently, and with high fidelity, generate RNA transcripts, through enzymatic incorporation of the triphosphate of tCO, a fluorescent tricyclic cytosine analogue. We demonstrate this by incorporation of tCO in up to 100% of the natural cytosine positions of a 1.2 kb mRNA encoding for the histone H2B fused to GFP (H2B:GFP). Spectroscopic characterization of this mRNA shows that the incorporation rate of tCO is similar to cytosine, which allows for efficient labeling and controlled tuning of labeling ratios for different applications. Using live cell confocal microscopy and flow cytometry, we show that the tCO-labeled mRNA is efficiently translated into H2B:GFP inside human cells. Hence, we not only develop the use of fluorescent base analogue labeling of nucleic acids in live-cell microscopy but also, importantly, show that the resulting transcript is translated into the correct protein. Moreover, the spectral properties of our transcripts and their translation product allow for their straightforward, simultaneous visualization in live cells. Finally, we find that chemically transfected tCO-labeled RNA, unlike a state-of-the-art fluorescently labeled RNA, gives rise to expression of a similar amount of protein as its natural counterpart, hence representing a methodology for studying natural, unperturbed processing of mRNA used in RNA therapeutics and in vaccines, like the ones developed against SARS-CoV-2.


Asunto(s)
Fluorescencia , Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/química , Imagen Molecular , ARN Mensajero/análisis , ARN Mensajero/metabolismo , /tratamiento farmacológico , Línea Celular Tumoral , Citosina/análogos & derivados , Citosina/análisis , Citosina/síntesis química , Citosina/química , Colorantes Fluorescentes/síntesis química , Proteínas Fluorescentes Verdes/metabolismo , Histonas/metabolismo , Humanos , Estructura Molecular , ARN Mensajero/química , ARN Mensajero/uso terapéutico , Espectrometría de Fluorescencia
4.
Int J Mol Sci ; 22(5)2021 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-33806656

RESUMEN

Ligand-protein binding is responsible for the vast majority of bio-molecular functions. Most experimental techniques examine the most populated ligand-bound state. The determination of less populated, intermediate, and transient bound states is experimentally challenging. However, hidden bound states are also important because these can strongly influence ligand binding and unbinding processes. Here, we explored the use of a classical optical spectroscopic technique, red-edge excitation shift spectroscopy (REES) to determine the number, population, and energetics associated with ligand-bound states in protein-ligand complexes. We describe a statistical mechanical model of a two-level fluorescent ligand located amongst a finite number of discrete protein microstates. We relate the progressive emission red shift with red-edge excitation to thermodynamic parameters underlying the protein-ligand free energy landscape and to photo-physical parameters relating to the fluorescent ligand. We applied the theoretical model to published red-edge excitation shift data from small molecule inhibitor-kinase complexes. The derived thermodynamic parameters allowed dissection of the energetic contribution of intermediate bound states to inhibitor-kinase interactions.


Asunto(s)
Proteínas/química , Espectrometría de Fluorescencia/métodos , Polarización de Fluorescencia/métodos , Colorantes Fluorescentes/química , Ligandos , Bibliotecas de Moléculas Pequeñas/química , Termodinámica
5.
Nat Commun ; 12(1): 1707, 2021 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-33731708

RESUMEN

Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease of immunocompromised humans, caused by the opportunistic fungal pathogen Aspergillus fumigatus. Inadequacies in current diagnostic procedures mean that early diagnosis of the disease, critical to patient survival, remains a major clinical challenge, and is leading to the empiric use of antifungal drugs and emergence of azole resistance. A non-invasive procedure that allows both unambiguous detection of IPA and its response to azole treatment is therefore needed. Here, we show that a humanised Aspergillus-specific monoclonal antibody, dual labelled with a radionuclide and fluorophore, can be used in immunoPET/MRI in vivo in a neutropenic mouse model and 3D light sheet fluorescence microscopy ex vivo in the infected mouse lungs to quantify early A. fumigatus lung infections and to monitor the efficacy of azole therapy. Our antibody-guided approach reveals that early drug intervention is critical to prevent complete invasion of the lungs by the fungus, and demonstrates the power of molecular imaging as a non-invasive procedure for tracking IPA in vivo.


Asunto(s)
Anticuerpos Monoclonales Humanizados/inmunología , Antifúngicos/uso terapéutico , Aspergillus fumigatus/inmunología , Pulmón/efectos de los fármacos , Pulmón/diagnóstico por imagen , Radiofármacos/inmunología , Animales , Anticuerpos Antifúngicos/química , Anticuerpos Antifúngicos/inmunología , Anticuerpos Monoclonales Humanizados/química , Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/patogenicidad , Azoles/uso terapéutico , Radioisótopos de Cobre/química , Monitoreo de Drogas , Colorantes Fluorescentes/química , Humanos , Aspergilosis Pulmonar Invasiva/diagnóstico por imagen , Aspergilosis Pulmonar Invasiva/tratamiento farmacológico , Aspergilosis Pulmonar Invasiva/microbiología , Pulmón/microbiología , Imagen por Resonancia Magnética , Ratones , Microscopía Fluorescente , Tomografía de Emisión de Positrones , Radioinmunodetección , Radiofármacos/química
6.
Methods Mol Biol ; 2259: 227-246, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687719

RESUMEN

Carbonylation is a nonenzymatic irreversible posttranslational protein modification and the main hallmark of protein oxidative damage. Elevated levels of protein carbonyl groups have been detected in age-related and metabolic diseases such as obesity, diabetes, Alzheimer, Parkinson, and several other oxidative stress-related maladies. Interestingly, many studies have shown that only a subset of proteins is carbonylated under the conditions of oxidative stress, demonstrating that carbonylation is a highly selective process. As a consequence, identifying and quantifying the disease-induced changes on a certain carbonylome are crucial to understanding the etiology and progression of numerous diseases and then designing adequate prevention/palliation strategies. However, the low abundance of carbonylated proteins in vivo, the enormous diversity of reactive species, and their relative lability make the analysis of carbonylated proteins a challenging task for redox proteomic technology. Therefore, we present a proteomic approach based on the labeling of carbonyls formed in vivo on proteins using the fluorescein 5-thiosemicarbazide (FTSC) tag to detect the subset of carbonylated proteins among a complex mixture of proteins regardless of the nature of carbonyl adduct, isolation and relative quantification of carbonylated proteins in 2D gel electrophoresis, and protein identification by LC-MS/MS analysis. This method has been successfully used for the evaluation of in vivo protein carbonylation in very diverse animal tissues (plasma, liver, kidney, skeletal muscle, and adipose tissue) and species (from fish to mammalian) and has also been applied in different research fields (from food technology to nutrition), demonstrating its robustness and reliability.


Asunto(s)
Electroforesis en Gel Bidimensional/métodos , Carbonilación Proteica , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Animales , Cromatografía Liquida/métodos , Fluoresceínas/química , Colorantes Fluorescentes/química , Humanos , Focalización Isoeléctrica/métodos , Oxidación-Reducción , Proteoma/análisis
7.
Molecules ; 26(5)2021 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-33669118

RESUMEN

The goal of the work reported here was to amplify the fluorescent properties of 4-aryliden-5(4H)-oxazolones by suppression of the hula-twist non-radiative deactivation pathway. This aim was achieved by simultaneous bonding of a Pd center to the N atom of the heterocycle and the ortho carbon of the arylidene ring. Two different 4-((Z)-arylidene)-2-((E)-styryl)-5(4H)-oxazolones, the structures of which are closely related to the chromophore of the Kaede protein and substituted at the 2- and 4-positions of the arylidene ring (1a OMe; 1b F), were used as starting materials. Oxazolones 1a and 1b were reacted with Pd(OAc)2 to give the corresponding dinuclear orthometalated palladium derivates 2a and 2b by regioselective C-H activation of the ortho-position of the arylidene ring. Reaction of 2a (2b) with LiCl promoted the metathesis of the bridging carboxylate by chloride ligands to afford dinuclear 3a (3b). Mononuclear complexes containing the orthopalladated oxazolone and a variety of ancillary ligands (acetylacetonate (4a, 4b), hydroxyquinolinate (5a), aminoquinoline (6a), bipyridine (7a), phenanthroline (8a)) were prepared from 3a or 3b through metathesis of anionic ligands or substitution of neutral weakly bonded ligands. All species were fully characterized and the X-ray determination of the molecular structure of 7a was carried out. This structure has strongly distorted ligands due to intramolecular interactions. Fluorescence measurements showed an increase in the quantum yield (QY) by up to one order of magnitude on comparing the free oxazolone (QY < 1%) with the palladated oxazolone (QY = 12% for 6a). This fact shows that the coordination of the oxazolone to the palladium efficiently suppresses the hula-twist deactivation pathway.


Asunto(s)
Complejos de Coordinación/química , Colorantes Fluorescentes/química , Proteínas Luminiscentes/química , Oxazolona/química , Paladio/química , Complejos de Coordinación/síntesis química , Cristalografía por Rayos X , Colorantes Fluorescentes/síntesis química , Modelos Moleculares , Estructura Molecular
8.
Molecules ; 26(5)2021 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-33669142

RESUMEN

Water-soluble fluorescent carbon dots (CDs) were synthesized by a hydrothermal method using citric acid as the carbon source and ethylenediamine as the nitrogen source. The repeated and scale-up synthetic experiments were carried out to explore the feasibility of macroscopic preparation of CDs. The CDs/Fe3+ composite was prepared by the interaction of the CDs solution and Fe3+ solution. The optical properties, pH dependence and stability behavior of CDs or the CDs/Fe3+ composite were studied by ultraviolet spectroscopy and fluorescence spectroscopy. Following the principles of fluorescence quenching after the addition of Fe3+ and then the fluorescence recovery after the addition of asorbic acid, the fluorescence intensity of the carbon dots was measured at λex = 360 nm, λem = 460 nm. The content of ascorbic acid was calculated by quantitative analysis of the changing fluorescence intensity. The CDs/Fe3+ composite was applied to the determination of different active molecules, and it was found that the composite had specific recognition of ascorbic acid and showed an excellent linear relationship in 5.0-350.0 µmol·L-1. Moreover, the detection limit was 3.11 µmol·L-1. Satisfactory results were achieved when the method was applied to the ascorbic acid determination in jujube fruit. The fluorescent carbon dots composites prepared in this study may have broad application prospects in a rapid, sensitive and trace determination of ascorbic acid content during food processing.


Asunto(s)
Ácido Ascórbico/análisis , Carbono/química , Colorantes Fluorescentes/química , Puntos Cuánticos/química , Colorantes Fluorescentes/síntesis química , Frutas/química , Ziziphus/química
9.
Molecules ; 26(5)2021 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-33669147

RESUMEN

An aniline-functionalized naphthalene dialdehyde Schiff base fluorescent probe L with aggregation-induced enhanced emission (AIEE) characteristics was synthesized via a simple one-step condensation reaction and exhibited excellent sensitivity and selectivity towards copper(II) ions in aqueous media with a fluorescence " turn-off " phenomenon. The detection limit of the probe is 1.64 × 10-8 mol·L-1. Furthermore, according to the results of the UV-vis/fluorescence titrations, Job's plot method and 1H-NMR titrations, a 1:2 stoichiometry was identified. The binding constant between L and Cu2+ was calculated to be Ka = 1.222 × 103. In addition, the AIEE fluorescent probe L could be applied to detection in real water samples with satisfactory recoveries in the range 99.10-102.90% in lake water and 98.49-102.37% in tap water.


Asunto(s)
Cobre/análisis , Colorantes Fluorescentes/química , Naftalenos/química , Contaminantes Químicos del Agua/análisis , Cristalografía por Rayos X , Colorantes Fluorescentes/síntesis química , Iones/análisis , Modelos Moleculares , Estructura Molecular , Naftalenos/síntesis química , Bases de Schiff/síntesis química , Bases de Schiff/química
10.
Molecules ; 26(4)2021 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-33669590

RESUMEN

Nitroreductases belong to a member of flavin-containing enzymes that can reduce nitroaromatic compounds to amino derivatives with NADH as an electron donor. NTR activity is known to be elevated in the cancerous environment and is considered an advantageous target in therapeutic prodrugs for the treatment of cancer. Here, we developed a ratiometric fluorescent molecule for observing NTR activity in living cells. This can provide a selective and sensitive response to NTR with a distinct increase in fluorescence ratio (FI530/FI630) as well as color changes. We also found a significant increase in NTR activity in cervical cancer HeLa and lung cancer A549 cells compared to non-cancerous NIH3T3. We proposed that this new ratiometric fluorescent molecule could potentially be used as a NTR-sensitive molecular probe in the field of cancer diagnosis and treatment development related to NTR activity.


Asunto(s)
Pruebas de Enzimas/métodos , Colorantes Fluorescentes/química , Sondas Moleculares/química , Nitrorreductasas/metabolismo , Células A549 , Animales , Muerte Celular , Cromatografía Líquida de Alta Presión , Endocitosis , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Ratones , Sondas Moleculares/síntesis química , Células 3T3 NIH , Espectrometría de Fluorescencia
11.
Molecules ; 26(4)2021 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-33673272

RESUMEN

Selective recognition of nucleotides with synthetic receptors is an emerging direction to solve a series of nucleic acid-related challenges in biochemistry. Towards this goal, a new aza-cyclophane with two different dyes, naphthalimide and pyrene, connected through a triamine linker has been synthesized and studied for the ability to bind and detect nucleoside triphosphates in an aqueous solution. The receptor shows Foerster resonance energy transfer (FRET) in fluorescence spectra upon excitation in DMSO, which is diminished dramatically in the presence of water. According to binding studies, the receptor has a preference to bind ATP (adenosine triphosphate) and CTP (cytidine triphosphate) with a "turn-on" fluorescence response. Two separate emission bands of dyes allow one to detect nucleotides in a ratiometric manner in a broad concentration range of 10-5-10-3 M. Spectroscopic measurements and quantum chemical calculations suggest the formation of receptor-nucleotide complexes, which are stabilized by dispersion interactions between a nucleobase and dyes, while hydrogen bonding interactions of nucleobases with the amine linkers are responsible for selectivity.


Asunto(s)
Éteres Cíclicos/química , Naftalimidas/química , Nucleótidos/química , Piperidinas/química , Pirenos/química , Adenosina Trifosfato , Colorantes Fluorescentes/química , Enlace de Hidrógeno , Estructura Molecular , Soluciones/química , Espectrometría de Fluorescencia , Agua/química
12.
Molecules ; 26(4)2021 Feb 22.
Artículo en Inglés | MEDLINE | ID: mdl-33671600

RESUMEN

The synthesis of fluorine-containing small molecules has had numerous benefits of improving the quality and efficiency of many applications of these compounds. For example, fluorine adds promising functionalities in various areas of imaging (MRI, PET, and NIR); gives cell-targeting properties; and has demonstrated improvements in cell permeability, solubility, and other pharmacologic properties. For these and other numerous reasons, fluorination of molecules has grown in popularity in various fields of chemistry. Many reports show the effects observed from increasing the number of fluorine atoms on a fluorophore scaffold. This report will cover the most significant applications and improvements that fluorine has contributed to in various dye scaffolds such as BODIPY, rhodamine, phthalocyanine, and cyanine in the recent decade.


Asunto(s)
Colorantes Fluorescentes/síntesis química , Flúor/química , Colorantes Fluorescentes/química , Estructura Molecular
13.
Molecules ; 26(4)2021 Feb 20.
Artículo en Inglés | MEDLINE | ID: mdl-33672662

RESUMEN

A new series of tetrahedral heteroleptic copper(I) complexes exhibiting efficient thermally-activated delayed fluorescence (TADF) in green to orange electromagnetic spectral regions has been developed by using D-A type N^N ligand and P^P ligands. Their structures, electrochemical, photophysical, and electroluminescence properties have been characterized. The complexes exhibit high photoluminescence quantum yields (PLQYs) of up to 0.71 at room temperature in doped film and the lifetimes are in a wide range of 4.3-24.1 µs. Density functional theory (DFT) calculations on the complexes reveal the lowest-lying intraligand charge-transfer excited states that are localized on the N^N ligands. Solution-processed organic light emitting diodes (OLEDs) based on one of the new emitters show a maximum external quantum efficiency (EQE) of 7.96%.


Asunto(s)
Complejos de Coordinación/química , Cobre/química , Colorantes Fluorescentes/química , Teoría Cuántica , Temperatura , Ligandos , Estructura Molecular , Soluciones
14.
J Vis Exp ; (168)2021 02 14.
Artículo en Inglés | MEDLINE | ID: mdl-33645588

RESUMEN

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an "eat-me" signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.


Asunto(s)
Bioensayo/métodos , Células Madre Pluripotentes Inducidas/metabolismo , Macrófagos/metabolismo , Neuroblastoma/patología , Fagocitosis , Animales , Muerte Celular , Línea Celular Tumoral , Análisis de Datos , Colorantes Fluorescentes/química , Células Madre Embrionarias Humanas/citología , Humanos , Concentración de Iones de Hidrógeno , Células Madre Pluripotentes Inducidas/citología , Control de Calidad , Reproducibilidad de los Resultados , Imagen de Lapso de Tiempo
15.
Nat Protoc ; 16(4): 2023-2050, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33674788

RESUMEN

Advanced in vitro kidney models are of great importance to the study of renal physiology and disease. Kidney tubuloids can be established from primary cells derived from adult kidney tissue or urine. Tubuloids are three-dimensional multicellular structures that recapitulate tubular function and have been used to study infectious, malignant, metabolic, and genetic diseases. For tubuloids to more closely represent the in vivo kidney, they can be integrated into an organ-on-a-chip system that has a more physiological tubular architecture and allows flow and interaction with vasculature or epithelial and mesenchymal cells from other organs. Here, we describe a detailed protocol for establishing tubuloid cultures from tissue and urine (1-3 weeks), as well as for generating and characterizing tubuloid cell-derived three-dimensional tubular structures in a perfused microfluidic multi-chip platform (7 d). The combination of the two systems yields a powerful in vitro tool that better recapitulates the complexity of the kidney tubule with donor-specific properties.


Asunto(s)
Túbulos Renales/crecimiento & desarrollo , Dispositivos Laboratorio en un Chip , Organoides/crecimiento & desarrollo , Perfusión , Técnicas de Cultivo de Tejidos/métodos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Fraccionamiento Celular , Niño , Preescolar , Impedancia Eléctrica , Femenino , Colorantes Fluorescentes/química , Humanos , Lactante , Masculino , Proteínas de Transporte de Membrana/metabolismo , Microfluídica , Persona de Mediana Edad , Ratas , Adulto Joven
16.
Methods Mol Biol ; 2265: 73-80, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704706

RESUMEN

Melanoma cells have high glycolytic capacity. Glucose uptake is a key rate-limiting step in glucose utilization. Here we describe a simple protocol for measuring direct glucose uptake in living melanoma cells by flow cytometry.


Asunto(s)
Citometría de Flujo/métodos , Glucosa/metabolismo , Melanoma/metabolismo , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/química , Transporte Biológico , Técnicas de Cultivo de Célula/métodos , Línea Celular Tumoral , Desoxiglucosa/análogos & derivados , Desoxiglucosa/química , Fluorescencia , Colorantes Fluorescentes/química , Humanos
17.
Int J Mol Sci ; 22(4)2021 Feb 14.
Artículo en Inglés | MEDLINE | ID: mdl-33672992

RESUMEN

The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200-250 nm laterally, ~500-700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4',6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.


Asunto(s)
Cromosomas de las Plantas/genética , Hordeum/genética , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Imagen Individual de Molécula/métodos , Cromosomas de las Plantas/química , Cromosomas de las Plantas/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Colorantes Fluorescentes/química , Hordeum/citología , Indoles/química , Metafase/genética , Reproducibilidad de los Resultados
18.
Nat Commun ; 12(1): 1773, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33741995

RESUMEN

The exploration of artificial luminogens with bright emission has been fully developed with the advancement of synthetic chemistry. However, many of them face problems like weakened emission in the aggregated state as well as poor renewability and sustainability. Therefore, the development of renewable and sustainable luminogens with anti-quenching function in the solid state, as well as to unveil the key factors that influence their luminescence behavior become highly significant. Herein, a new class of natural rosin-derived luminogens with aggregation-induced emission property (AIEgens) have been facilely obtained with good biocompatibility and targeted organelle imaging capability as well as photochromic behavior in the solid state. Mechanistic study indicates that the introduction of the alicyclic moiety helps suppress the excited-state molecular motion to enhance the solid-state emission. The current work fundamentally elucidates the role of alicyclic moiety in luminogen design and practically demonstrates a new source to large-scalely obtain biocompatible AIEgens.


Asunto(s)
Materiales Biocompatibles/química , Colorantes Fluorescentes/química , Luminiscencia , Resinas de Plantas/química , Animales , Materiales Biocompatibles/farmacología , Células COS , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Escherichia coli/efectos de los fármacos , Colorantes Fluorescentes/farmacología , Microscopía Confocal , Estructura Molecular , Movimiento (Física) , Imagen Óptica/métodos , Orgánulos/química , Orgánulos/metabolismo , Resinas de Plantas/farmacología , Relación Estructura-Actividad
19.
Front Immunol ; 12: 635558, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33679789

RESUMEN

The long-term pandemic of coronavirus disease 2019 (COVID-19) requires sensitive and accurate diagnostic assays to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and SARS-CoV-2 antibodies in infected individuals. Currently, RNA of SARS-CoV-2 virus is mainly detected by reverse transcription-polymerase chain reaction (RT-PCR)-based nucleic acid assays, while SARS-CoV-2 antigen and antibody are identified by immunological assays. Both nucleic acid assays and immunological assays rely on the luminescence signals of specific luminescence probes for qualitative and quantitative detection. The exploration of novel luminescence probes will play a crucial role in improving the detection sensitivity of the assays. As innate probes, aggregation-induced emission (AIE) luminogens (AIEgens) exhibit negligible luminescence in the free state but enhanced luminescence in the aggregated or restricted states. Moreover, AIEgen-based nanoparticles (AIE dots) offer efficient luminescence, good biocompatibility and water solubility, and superior photostability. Both AIEgens and AIE dots have been widely used for high-performance detection of biomolecules and small molecules, chemical/biological imaging, and medical therapeutics. In this review, the availability of AIEgens and AIE dots in nucleic acid assays and immunological assays are enumerated and discussed. By building a bridge between AIE materials and COVID-19, we hope to inspire researchers to use AIE materials as a powerful weapon against COVID-19.


Asunto(s)
/diagnóstico , Colorantes Fluorescentes/química , Nanopartículas/química , Humanos
20.
Int J Nanomedicine ; 16: 1473-1485, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33654397

RESUMEN

Purpose: The near-infrared fluorescent dye indocyanine green (ICG) has shown great potential in the photodynamic therapy (PDT) and photothermal therapy (PTT) of cancer. However, its disadvantages of instability in aqueous solution, short half-life, and non-targeting accumulation limit the effectiveness of ICG PDT/PTT. To overcome the disadvantages of ICG in tumor treatment, we designed PEGylated-human serum albumin (PHSA)-ICG-TAT. In this nanoparticle, PEG4000, the HSA package, and nuclear targeting peptide TAT (human immunodeficiency virus 1 [HIV-1]-transactivator protein) were used to improve the water solubility of ICG, prolong the life span of ICG in vivo, and target the nuclei of tumor cells, respectively. Methods: The PHSA-ICG-TAT was characterized in terms of morphology and size, ultraviolet spectrum, dispersion stability, singlet oxygen and cellular uptake, and colocalization using transmission electron microscopy and dynamic light scattering, and fluorescence assay, respectively. Subsequently, the anti-tumor effect of PHSA-ICG-TAT was investigated via in vitro and in vivo experiments, including cell viability, apoptosis, comet assays, histopathology, and inhibition curves. Results: The designed ICG-loaded nanoparticle had a higher cell uptake rate and stronger PDT/PTT effect than free ICG. The metabolism of PHSA-ICG-TAT in normal mice revealed that there was no perceptible toxicity. In vivo imaging of mice showed that PHSA-ICG-TAT had a good targeting effect on tumors. PHSA-ICG-TAT was used for the phototherapy of tumors, and significantly suppressed the tumor growth. The tumor tissue sections showed that the cell gap and morphology of the tumor tissue had been obviously altered after treatment with PHSA-ICG-TAT. Conclusion: These results indicate that the PHSA-ICG-TAT had a significant therapeutic effect against tumors.


Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Núcleo Celular/metabolismo , Nanopartículas/química , Fotoquimioterapia , Fármacos Fotosensibilizantes/uso terapéutico , Animales , Neoplasias de la Mama/patología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Dispersión Dinámica de Luz , Femenino , Fluorescencia , Colorantes Fluorescentes/química , Humanos , Verde de Indocianina/química , Ratones Endogámicos BALB C , Nanopartículas/ultraestructura , Fármacos Fotosensibilizantes/farmacología , Polietilenglicoles/química , Albúmina Sérica Humana/química
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