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1.
Nat Commun ; 11(1): 4192, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826886

RESUMO

Bioluminescence imaging has been widely used in life sciences and biomedical applications. However, conventional bioluminescence imaging usually operates in the visible region, which hampers the high-performance in vivo optical imaging due to the strong tissue absorption and scattering. To address this challenge, here we present bioluminescence probes (BPs) with emission in the second near infrared (NIR-II) region at 1029 nm by employing bioluminescence resonance energy transfer (BRET) and two-step fluorescence resonance energy transfer (FRET) with a specially designed cyanine dye FD-1029. The biocompatible NIR-II-BPs are successfully applied to vessels and lymphatics imaging in mice, which gives ~5 times higher signal-to-noise ratios and ~1.5 times higher spatial resolution than those obtained by NIR-II fluorescence imaging and conventional bioluminescence imaging. Their capability of multiplexed imaging is also well displayed. Taking advantage of the ATP-responding character, the NIR-II-BPs are able to recognize tumor metastasis with a high tumor-to-normal tissue ratio at 83.4.


Assuntos
Trifosfato de Adenosina/metabolismo , Medições Luminescentes/métodos , Metástase Neoplásica/diagnóstico por imagem , Imagem Óptica/métodos , Animais , Técnicas Biossensoriais , Linhagem Celular Tumoral , Feminino , Transferência Ressonante de Energia de Fluorescência/instrumentação , Transferência Ressonante de Energia de Fluorescência/métodos , Xenoenxertos , Humanos , Medições Luminescentes/instrumentação , Camundongos , Imagem Óptica/instrumentação , Neoplasias Ovarianas/diagnóstico por imagem
2.
ACS Infect Dis ; 6(8): 1998-2016, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32677821

RESUMO

Since late December 2019, the coronavirus pandemic (COVID-19; previously known as 2019-nCoV) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been surging rapidly around the world. With more than 1,700,000 confirmed cases, the world faces an unprecedented economic, social, and health impact. The early, rapid, sensitive, and accurate diagnosis of viral infection provides rapid responses for public health surveillance, prevention, and control of contagious diffusion. More than 30% of the confirmed cases are asymptomatic, and the high false-negative rate (FNR) of a single assay requires the development of novel diagnostic techniques, combinative approaches, sampling from different locations, and consecutive detection. The recurrence of discharged patients indicates the need for long-term monitoring and tracking. Diagnostic and therapeutic methods are evolving with a deeper understanding of virus pathology and the potential for relapse. In this Review, a comprehensive summary and comparison of different SARS-CoV-2 diagnostic methods are provided for researchers and clinicians to develop appropriate strategies for the timely and effective detection of SARS-CoV-2. The survey of current biosensors and diagnostic devices for viral nucleic acids, proteins, and particles and chest tomography will provide insight into the development of novel perspective techniques for the diagnosis of COVID-19.


Assuntos
Betacoronavirus/química , Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Betacoronavirus/isolamento & purificação , Sistemas CRISPR-Cas , Infecções por Coronavirus/virologia , Efeito Citopatogênico Viral , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Imunoquímica/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias , Pneumonia Viral/virologia , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Análise Espectral Raman/métodos , Tomografia Computadorizada por Raios X/métodos , Sequenciamento Completo do Genoma/métodos
3.
Nat Commun ; 11(1): 3336, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620782

RESUMO

We describe theory, experiments, and analyses of three-color Förster resonance energy transfer (FRET) spectroscopy for probing sub-millisecond conformational dynamics of protein folding and binding of disordered proteins. We devise a scheme that uses single continuous-wave laser excitation of the donor instead of alternating excitation of the donor and one of the acceptors. This scheme alleviates photophysical problems of acceptors such as rapid photobleaching, which is crucial for high time resolution experiments with elevated illumination intensity. Our method exploits the molecular species with one of the acceptors absent or photobleached, from which two-color FRET data is collected in the same experiment. We show that three FRET efficiencies and kinetic parameters can be determined without alternating excitation from a global maximum likelihood analysis of two-color and three-color photon trajectories. We implement co-parallelization of CPU-GPU processing, which leads to a significant reduction of the likelihood calculation time for efficient parameter determination.


Assuntos
Algoritmos , Transferência Ressonante de Energia de Fluorescência/métodos , Modelos Teóricos , Conformação Proteica , Dobramento de Proteína , Proteínas/química , Cor , Transferência Ressonante de Energia de Fluorescência/instrumentação , Transferência Ressonante de Energia de Fluorescência/estatística & dados numéricos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Cinética , Microscopia Confocal , Fotodegradação , Fótons , Ligação Proteica , Proteínas/metabolismo , Fatores de Tempo
4.
Nat Commun ; 11(1): 3444, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32651384

RESUMO

Imaging membrane voltage from genetically defined cells offers the unique ability to report spatial and temporal dynamics of electrical signaling at cellular and circuit levels. Here, we present a general approach to engineer electrochromic fluorescence resonance energy transfer (eFRET) genetically encoded voltage indicators (GEVIs) with positive-going fluorescence response to membrane depolarization through rational manipulation of the native proton transport pathway in microbial rhodopsins. We transform the state-of-the-art eFRET GEVI Voltron into Positron, with kinetics and sensitivity equivalent to Voltron but flipped fluorescence signal polarity. We further apply this general approach to GEVIs containing different voltage sensitive rhodopsin domains and various fluorescent dye and fluorescent protein reporters.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Potenciais de Ação/fisiologia , Animais , Proteínas Luminescentes/metabolismo , Neurônios/metabolismo , Neurociências/métodos , Rodopsina/química , Rodopsina/metabolismo
5.
Science ; 368(6496): 1253-1257, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32527832

RESUMO

Transition paths of macromolecular conformational changes such as protein folding are predicted to be heterogeneous. However, experimental characterization of the diversity of transition paths is extremely challenging because it requires measuring more than one distance during individual transitions. In this work, we used fast three-color single-molecule Förster resonance energy transfer spectroscopy to obtain the distribution of binding transition paths of a disordered protein. About half of the transitions follow a path involving strong non-native electrostatic interactions, resulting in a transition time of 300 to 800 microseconds. The remaining half follow more diverse paths characterized by weaker electrostatic interactions and more than 10 times shorter transition path times. The chain flexibility and non-native interactions make diverse binding pathways possible, allowing disordered proteins to bind faster than folded proteins.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas Intrinsicamente Desordenadas/química , Dobramento de Proteína , Imagem Individual de Molécula/métodos , Ligação Proteica , Conformação Proteica , Eletricidade Estática
6.
Nat Commun ; 11(1): 3114, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561744

RESUMO

Revealing antibody-antigen interactions at the single-molecule level will deepen our understanding of immunology. However, structural determination under crystal or cryogenic conditions does not provide temporal resolution for resolving transient, physiologically or pathologically relevant functional antibody-antigen complexes. Here, we develop a triangular DNA origami framework with site-specifically anchored and spatially organized artificial epitopes to capture transient conformations of immunoglobulin Gs (IgGs) at room temperature. The DNA origami epitopes (DOEs) allows programmed spatial distribution of epitope spikes, which enables direct imaging of functional complexes with atomic force microscopy (AFM). We establish the critical dependence of the IgG avidity on the lateral distance of epitopes within 3-20 nm at the single-molecule level. High-speed AFM imaging of transient conformations further provides structural and dynamic evidence for the IgG avidity from monovalent to bivalent in a single event, which sheds light on various applications including virus neutralization, diagnostic detection and cancer immunotherapy.


Assuntos
Afinidade de Anticorpos , Epitopos/ultraestrutura , Imunoglobulina G/ultraestrutura , Sondas Moleculares/ultraestrutura , Imagem Individual de Molécula/métodos , Complexo Antígeno-Anticorpo/ultraestrutura , DNA de Cadeia Simples/imunologia , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/ultraestrutura , Epitopos/imunologia , Epitopos/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Microscopia de Força Atômica/métodos , Simulação de Dinâmica Molecular , Sondas Moleculares/imunologia , Sondas Moleculares/metabolismo , Nanotecnologia , Relação Estrutura-Atividade
7.
Nat Commun ; 11(1): 3216, 2020 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-32587248

RESUMO

Chemical tools to monitor drug-target engagement of endogenously expressed protein kinases are highly desirable for preclinical target validation in drug discovery. Here, we describe a chemical genetics strategy to selectively study target engagement of endogenous kinases. By substituting a serine residue into cysteine at the DFG-1 position in the ATP-binding pocket, we sensitize the non-receptor tyrosine kinase FES towards covalent labeling by a complementary fluorescent chemical probe. This mutation is introduced in the endogenous FES gene of HL-60 cells using CRISPR/Cas9 gene editing. Leveraging the temporal and acute control offered by our strategy, we show that FES activity is dispensable for differentiation of HL-60 cells towards macrophages. Instead, FES plays a key role in neutrophil phagocytosis via SYK kinase activation. This chemical genetics strategy holds promise as a target validation method for kinases.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes , Proteínas Proto-Oncogênicas c-fes , Transportadores de Cassetes de Ligação de ATP/química , Sistemas CRISPR-Cas , Diferenciação Celular , Linhagem Celular , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Edição de Genes , Humanos , Macrófagos/metabolismo , Mutação , Neutrófilos , Fagocitose , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-fes/química , Proteínas Proto-Oncogênicas c-fes/genética , Proteínas Proto-Oncogênicas c-fes/metabolismo , Transdução de Sinais , Quinase Syk/metabolismo
8.
Nat Commun ; 11(1): 2185, 2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32366822

RESUMO

Signal amplification in biological systems is achieved by cooperatively recruiting multiple copies of regulatory biomolecules. Nevertheless, the multiplexing capability of artificial fluorescent amplifiers is limited due to the size limit and lack of modularity. Here, we develop Cayley tree-like fractal DNA frameworks to topologically encode the fluorescence states for multiplexed detection of low-abundance targets. Taking advantage of the self-similar topology of Cayley tree, we use only 16 DNA strands to construct n-node (n = 53) structures of up to 5 megadalton. The high level of degeneracy allows encoding 36 colours with 7 nodes by site-specifically anchoring of distinct fluorophores onto a structure. The fractal topology minimises fluorescence crosstalk and allows quantitative decoding of quantized fluorescence states. We demonstrate a spectrum of rigid-yet-flexible super-multiplex structures for encoded fluorescence detection of single-molecule recognition events and multiplexed discrimination of living cells. Thus, the topological engineering approach enriches the toolbox for high-throughput cell imaging.


Assuntos
DNA/química , Fluorescência , Fractais , Oligonucleotídeos/química , Algoritmos , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Células HeLa , Humanos , Microscopia de Força Atômica/métodos , Microscopia Confocal/métodos , Nanoestruturas/química
10.
Chem Commun (Camb) ; 56(21): 3183-3186, 2020 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-32067022

RESUMO

A novel nanoarchitecture (MSN-Tb-UbR) was prepared by modifying rhodamine B-labelled Ubs (Ub-Rs) on the surface of mesoporous silica nanoparticles (MSNs) loaded with Tb3+-complexes. The MSN-Tb-UbR exhibits ratiometric sensing ability for DUB (UCH-L1) with good sensitivity and selectivity.


Assuntos
Nanopartículas/química , Dióxido de Silício/química , Ubiquitina Tiolesterase/análise , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Porosidade , Rodaminas/química , Propriedades de Superfície , Térbio/química
11.
PLoS One ; 15(2): e0219886, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32023253

RESUMO

Förster Resonance Energy Transfer (FRET) has become an immensely powerful tool to profile intra- and inter-molecular interactions. Through fusion of genetically encoded fluorescent proteins (FPs) researchers have been able to detect protein oligomerization, receptor activation, and protein translocation among other biophysical phenomena. Recently, two bright monomeric red fluorescent proteins, mRuby3 and mScarlet-I, have been developed. These proteins offer much improved physical properties compared to previous generations of monomeric red FPs that should help facilitate more general adoption of Green/Red FRET. Here we assess the ability of these two proteins, along with mCherry, to act as a FRET acceptor for the bright, monomeric, green-yellow FP mNeonGreen using intensiometric FRET and 2-photon Fluorescent Lifetime Imaging Microscopy (FLIM) FRET techniques. We first determined that mNeonGreen was a stable donor for 2-photon FLIM experiments under a variety of imaging conditions. We then tested the red FP's ability to act as FRET acceptors using mNeonGreen-Red FP tandem construct. With these constructs we found that mScarlet-I and mCherry are able to efficiently FRET with mNeonGreen in spectroscopic and FLIM FRET. In contrast, mNeonGreen and mRuby3 FRET with a much lower efficiency than predicted in these same assays. We explore possible explanations for this poor performance and determine mRuby3's protein maturation properties are a major contributor. Overall, we find that mNeonGreen is an excellent FRET donor, and both mCherry and mScarlet-I, but not mRuby3, act as practical FRET acceptors, with the brighter mScarlet-I out performing mCherry in intensiometric studies, but mCherry out performing mScarlet-I in instances where consistent efficiency in a population is critical.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/normas , Transferência Ressonante de Energia de Fluorescência/normas , Células HEK293 , Humanos , Microscopia Intravital/métodos , Microscopia de Fluorescência/métodos , Análise de Célula Única/métodos
12.
Anal Bioanal Chem ; 412(8): 1915-1923, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32030494

RESUMO

Due to its important role in tumor development and treatment, hyaluronidase (HAase) has been widely investigated in vitro and in vivo. However, such investigation was limited by the absence of sensitive and in situ detection methods. Herein, a hyaluronic acid (HA) hydrogel based on the fluorescence resonance energy transfer (FRET) effect was constructed for the detection of HAase. FITC and AuNPs were covalently coupled with two HA derivatives respectively to form a fluorescent donor-acceptor pair. In the presence of HAase, the hydrogel established by cross-linking of HA derivatives was hydrolyzed specifically. The FRET effect in the hydrogel disappeared and the fluorescence intensity increased proportionally with the changes in the concentration of the HAase. Experiments proved that the HAase sensing system had a wide response range (0.5-100 U/mL), good anti-interference, and excellent biocompatibility. When the hydrogel was used for 3D culture of lung cancer cells, in situ fluorescent response could be achieved. Graphical abstract.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Ácido Hialurônico/química , Hialuronoglucosaminidase/análise , Hidrogéis/química , Células A549 , Fluorescência , Humanos , Limite de Detecção
13.
PLoS One ; 15(2): e0228543, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32045426

RESUMO

Two molecules, 7-(diethylamino)coumarin-3-carbohydrazide (DCCH) and fluorescein-5-thiosemicarbazide (FTSC) were investigated in different solvents, under varying pH conditions regarding their spectroscopic properties for the usage as a Förster Resonance Energy Transfer (FRET) pair to study the molecular interaction between cellulosic surfaces. All the relevant spectroscopic properties to determine the Förster distance were measured and the performance as a FRET system was checked. From the results, it is clear that the environmental conditions need to be accurately controlled as both, but especially the FTSC dyes are sensitive to changes. For high enough concentrations positive FRET systems were observed in DMF, DMSO, H2O, THF and alkaline DMF. However due to the low quantum yield of the unmodified DCCH throughout the investigated parameter range and the strong environmental dependency of FTSC, both dyes are not preferable for being used in a FRET system for studying interaction between cellulosic surfaces.


Assuntos
Cumarínicos/química , Fluoresceínas/química , Transferência Ressonante de Energia de Fluorescência/métodos , Hidrazinas/química , Solventes/química , Análise Espectral/métodos , Transferência de Energia/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Solventes/farmacologia , Espectrometria de Fluorescência/métodos , Espectrofotometria Ultravioleta/métodos
14.
Biochim Biophys Acta Biomembr ; 1862(6): 183216, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32067963

RESUMO

Surfactant protein SP-B is absolutely required for the generation of functional pulmonary surfactant, a unique network of multilayered membranes, which stabilizes the respiratory air-liquid interface. It has been proposed that SP-B assembles into hydrophobic rings and tubes that facilitate the rapid transfer of phospholipids from membrane stores into the interface and the formation of multilayered films, ensuring the stability of the alveoli against physical forces leading to their collapse. To elucidate the molecular organization of SP-B-promoted multilamellar membrane structures, time-resolved Förster Resonance Energy Transfer (FRET) experiments between BODIPY-PC or BODIPY-derivatized SP-B (BODIPY/SP-B), as donor probes, and octadecylrhodamine B, as acceptor probe, were performed in liposomes containing SP-B or BODIPY/SP-B. Our results show that both SP-B and fluorescently labeled SP-B oligomers mediate the connection of adjacent bilayers. Furthermore, by applying rational models to the FRET data, we have been able to provide quantitative details of the structure of SP-B-induced multilayered membrane arrays at the nanometer scale, defining interactions between SP-B rings as key elements for connecting surfactant membranes. The data sustain the structural model and the mechanism of action of SP-B assemblies to sustain the crucial surfactant function.


Assuntos
Nanoestruturas/química , Alvéolos Pulmonares/química , Proteína B Associada a Surfactante Pulmonar/química , Surfactantes Pulmonares/química , Animais , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Bicamadas Lipídicas/química , Lipossomos/química , Alvéolos Pulmonares/ultraestrutura , Proteína B Associada a Surfactante Pulmonar/metabolismo
15.
Chemistry ; 26(26): 5780-5783, 2020 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-32092185

RESUMO

Activity of acid sphingomyelinase has been implicated in a number of diseases like acute lung injury, sepsis or metastasis of melanoma cells. Here, we present a sphingomyelinase FRET probe based on FAM/BODIPY dyes for real-time monitoring of acid sphingomyelinase. The probe gives rise to a tremendous increase in fluorescence of the fluorescein FRET donor upon cleavage and we show that this is, to a significant part, due to cleavage-associated phase transition, suggesting a more systematic consideration of such effects for future probe development. The probe allows for the first time to monitor relative sphingomyelinase activities of intact living cells by flow cytometry.


Assuntos
Compostos de Boro/química , Transferência Ressonante de Energia de Fluorescência/métodos , Esfingomielina Fosfodiesterase/química , Citometria de Fluxo , Fluorescência , Humanos , Esfingomielina Fosfodiesterase/metabolismo
16.
Biochem Soc Trans ; 48(1): 61-70, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32104883

RESUMO

Multiple intra-cellular signalling pathways rely on calcium and 3'-5' cyclic adenosine monophosphate (cAMP) to act as secondary messengers. This is especially true in cardiomyocytes which act as the force-producing units of the cardiac muscle and are required to react rapidly to environmental stimuli. The specificity of functional responses within cardiomyocytes and other cell types is produced by the organellar compartmentation of both calcium and cAMP. In this review, we assess the role of molecular localisation and relative contribution of active and passive processes in producing compartmentation. Active processes comprise the creation and destruction of signals, whereas passive processes comprise the release or sequestration of signals. Cardiomyocytes display a highly articulated membrane structure which displays significant cell-to-cell variability. Special attention is paid to the way in which cell membrane caveolae and the transverse-axial tubule system allow molecular localisation. We explore the effects of cell maturation, pathology and regional differences in the organisation of these processes. The subject of signal compartmentation has had a significant amount of attention within the cardiovascular field and has undergone a revolution over the past two decades. Advances in the area have been driven by molecular imaging using fluorescent dyes and genetically encoded constructs based upon fluorescent proteins. We also explore the use of scanning probe microscopy in the area. These techniques allow the analysis of molecular compartmentation within specific organellar compartments which gives researchers an entirely new perspective.


Assuntos
Compartimento Celular/fisiologia , Miócitos Cardíacos/metabolismo , Transdução de Sinais/fisiologia , Animais , Sinalização do Cálcio , Cavéolas/metabolismo , AMP Cíclico/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Insuficiência Cardíaca/metabolismo
17.
J Vis Exp ; (155)2020 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-32065139

RESUMO

Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as atomic force microscopy have limited temporal resolution, while Förster resonance energy transfer (FRET) only allow conformations to be inferred. Fluorescence microscopy, on the other hand, allows real-time in situ visualization of single molecules in various flow conditions. Our protocol describes the steps to capture conformational changes of single biomolecules under different shear flow environments using fluorescence microscopy. The shear flow is created inside microfluidic channels and controlled by a syringe pump. As demonstrations of the method, von Willebrand factor (VWF) and lambda DNA are labeled with biotin and fluorophore and then immobilized on the channel surface. Their conformations are continuously monitored under variable shear flow using total internal reflection (TIRF) and confocal fluorescence microscopy. The reversible unraveling dynamics of VWF are useful for understanding how its function is regulated in human blood, while the conformation of lambda DNA offers insights into the biophysics of macromolecules. The protocol can also be widely applied to study the behavior of polymers, especially biopolymers, in varying flow conditions and to investigate the rheology of complex fluids.


Assuntos
Microscopia de Fluorescência/métodos , Resistência ao Cisalhamento/fisiologia , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos
18.
Curr Protoc Neurosci ; 91(1): e91, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32068967

RESUMO

Astrocytes are morphologically complex cells with numerous close contacts with neurons at the level of their somata, branches, and branchlets. The smallest astrocyte processes make discrete contacts with synapses at scales that cannot be observed by standard light microscopy. At such contact points, astrocytes are thought to perform both homeostatic and neuromodulatory roles-functions that are proposed to be determined by their close spatial apposition. To study such spatial interactions, we previously developed a Förster resonance energy transfer (FRET)-based approach, which enables observation and tracking of the static and dynamic proximity of astrocyte processes with synapses. The approach is compatible with standard imaging techniques such as confocal microscopy and permits assessment of the most proximate contacts between astrocytes and neurons in live tissues. In this protocol article we describe the approach to analyze the contacts between striatal astrocyte processes and corticostriatal neuronal projection terminals onto medium spiny neurons. We report the required protocols in detail, including adeno-associated virus microinjections, acute brain slice preparation, imaging, and post hoc FRET quantification. The article provides a detailed description that can be used to characterize and study astrocyte process proximity to synapses in living tissue. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Förster resonance energy transfer imaging in cultured cells Basic Protocol 2: Förster resonance energy transfer imaging with the neuron-astrocyte proximity assay in acute brain slices.


Assuntos
Astrócitos/citologia , Transferência Ressonante de Energia de Fluorescência/métodos , Microscopia Confocal/métodos , Neurônios/citologia , Animais , Encéfalo/citologia , Comunicação Celular , Células Cultivadas , Dependovirus/genética , Feminino , Vetores Genéticos , Células HEK293 , Humanos , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microinjeções
19.
Methods Appl Fluoresc ; 8(2): 024006, 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-32032967

RESUMO

Fluorescence Lifetime Imaging Microscopy (FLIM) is a robust tool to measure Förster Resonance Energy Transfer (FRET) between two fluorescent proteins, mainly when using genetically-encoded FRET biosensors. It is then possible to monitor biological processes such as kinase activity with a good spatiotemporal resolution and accuracy. Therefore, it is of interest to improve this methodology for future high content screening purposes. We here implement a time-gated FLIM microscope that can image and quantify fluorescence lifetime with a higher speed than conventional techniques such as Time-Correlated Single Photon Counting (TCSPC). We then improve our system to perform automatic screen analysis in a 96-well plate format. Moreover, we use a FRET biosensor of AURKA activity, a mitotic kinase involved in several epithelial cancers. Our results show that our system is suitable to measure FRET within our biosensor paving the way to the screening of novel compounds, potentially allowing to find new inhibitors of AURKA activity.


Assuntos
Aurora Quinase A/análise , Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Microscopia de Fluorescência/métodos , Aurora Quinase A/metabolismo , Linhagem Celular Tumoral , Humanos
20.
J Agric Food Chem ; 68(7): 2249-2255, 2020 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-31986034

RESUMO

Ochratoxin A (OTA) is an intrinsically fluorescent phenolic mycotoxin that contaminates a wide range of food products and is a serious health threat to animals and humans. An OTA binding aptamer (OTABA) that folds into an antiparallel G-quadruplex (GQ) in the absence and presence of target OTA has been incorporated into a vast variety of aptasensor platforms for OTA detection. The development of a simple, aptamer-based approach would allow for detection of the toxin without the use of complex analytical instrumentation, which has been the gold standard for OTA detection thus far. However, to date, none of the aptasensor platforms have utilized the natural fluorescence of the phenolic toxin for detection. Herein, we report that OTA binding to OTABA involves π-stacking interactions that lead to GQ-to-toxin energy transfer (ET), which affords a "turn-on" fluorescence self-signaling platform in which the emission of the aptamer-target complex is enhanced in comparison to the free toxin alone. Selective excitation of the GQ-OTA complex at 256 nm leads to a 4-fold enhancement in OTA fluorescence. The GQ-OTA ET detection platform boasts a limit of detection ∼2 ng/mL, which is comparable to a previously demonstrated fluorescence resonance energy transfer immunoassay platform for OTA detection, and displays excellent OTA selectivity and recovery from red wine samples.


Assuntos
Aptâmeros de Nucleotídeos/química , Transferência Ressonante de Energia de Fluorescência/métodos , Ocratoxinas/análise , Vinho/análise , Transferência Ressonante de Energia de Fluorescência/instrumentação , Contaminação de Alimentos/análise , Limite de Detecção
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