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1.
Inorg Chem ; 58(18): 12422-12432, 2019 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-31483641

RESUMO

Fluorescence imaging is a powerful tool in biomedical research. It has been frequently used to uncover or better understand physiological mechanisms in disease-related processes such as cancer. The majority of chromophores used for imaging are based on a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) scaffold. However, their applications are limited due to their poor water solubility as well as poor cancer cell selectivity. To circumvent these drawbacks, we present herein the use of bis(dipyrrinato)zinc(II) complexes. As this class of compounds is associated with a quenching effect of the excited state in water, the lead compound of this study (3) was encapsulated in a polymer matrix with biotin as a targeting moiety (3-NP). This encapsulation improved the water solubility, overcame the quenching effects in water, as well as allowed selective accumulation in the lysosomes with a bright fluorescence signal in monolayer cells as well as 3D multicellular tumor spheroids (MCTS). As a benefit from the biotin targeting moiety, the nanoparticles were majorly taken up by the sodium dependent multivitamin transporter (SMVT) which is overexpressed in various cancers cells and selectively accumulated in cancerous cells over noncancerous cells.


Assuntos
Corantes Fluorescentes/química , Lisossomos/patologia , Nanopartículas/química , Neoplasias/patologia , Polímeros/química , Pirróis/química , Zinco/química , Células A549 , Linhagem Celular , Complexos de Coordenação/química , Células HeLa , Humanos , Lisossomos/ultraestrutura , Microscopia Confocal/métodos , Neoplasias/diagnóstico por imagem , Imagem Óptica/métodos
2.
Chem Commun (Camb) ; 55(73): 10916-10919, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31441466
3.
Chem Commun (Camb) ; 55(73): 10952-10955, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31441915

RESUMO

Triggering antibody-mediated innate immune mechanisms to kill cancer cells is an attractive therapeutic avenue. In this context, recruitment of endogenous antibodies to the cancer cell surface could be a viable alternative to the use of monoclonal antibodies. We report on antibody-recruiting polymers containing multiple antibody-binding hapten motifs and cyclooctynes that can covalently conjugate to azides introduced onto the glycocalyx of cancer cells by metabolic labeling with azido sugars.


Assuntos
Resinas Acrílicas/química , Anticorpos/imunologia , Azidas/metabolismo , Dinitrobenzenos/imunologia , Hexosaminas/metabolismo , Resinas Acrílicas/síntese química , Animais , Azidas/química , Linhagem Celular Tumoral , Química Click , Reação de Cicloadição , Ciclo-Octanos/síntese química , Ciclo-Octanos/química , Dinitrobenzenos/síntese química , Dinitrobenzenos/química , Fluorescência , Corantes Fluorescentes/química , Glicocálix/metabolismo , Hexosaminas/química , Humanos , Camundongos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Estudo de Prova de Conceito , Esferoides Celulares/metabolismo
4.
Anal Chim Acta ; 1078: 101-111, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31358207

RESUMO

A series of polymers and metal ions have been observed to be useful in triggering aggregation-induced emission (AIE) and AIE enhancement (AIEE) of thiolated gold nanoclusters (AuNCs). However, peptide-induced AIEE of thiolated AuNCs and their applications in biosensors have rarely been investigated. In this study, we showed that positively charged peptides induced efficient AIEE of negatively charged glutathione-capped AuNCs (GSH-AuNCs) through electrostatic attraction. In contrast to GSH-AuNCs, polyarginine (polyArg), a cationic peptide, stimulated the AIEE of the GSH-AuNCs, resulting in a 3.5-fold luminescence enhancement, 10-fold enhancement in quantum yield, 8-nm blueshift in the luminescence maximum, and a 2.1-fold increase in the mean luminescence lifetime. Four different AIEE-based biosensors with excellent selectivity and acceptable sensitivity were fabricated using cationic peptides as an AIEE-active trigger and as a biorecognition element. A heparin biosensor with a limit of detection (LOD) of 3 nM was constructed by combining AG73 peptide-mediated AIEE of the GSH-AuNCs and the specific interaction of AG73 peptides with heparin macromolecules. The concentration of human trypsin was selectively detected at a concentration as low as 1 nM using an arginine-glycine repeat peptide as an enzymatic substrate and as an AIEE-active trigger. Alkaline phosphatase (ALP)-catalyzed dephosphorylation of phosphopeptides paired with the corresponding product-mediated AIEE of the GSH-AuNCs was used for ALP sensing with an LOD of 0.3 U L-1. A peptide consisting of a cyclic RGD unit and an AIEE-active unit was designed to synthesize RGD-modified GSH-AuNC aggregates that can target αvß3 integrin receptors. These AIEE-based sensors were practically applied for the quantitative determination of heparin in human plasma, trypsin in human urine, and ALP in human plasma as well as for luminescent imaging of αvß3 integrin-overexpressing HeLa cells.


Assuntos
Glutationa/química , Ouro/química , Nanopartículas Metálicas/química , Peptídeos Cíclicos/química , Fosfopeptídeos/química , Fosfatase Alcalina/química , Técnicas Biossensoriais/métodos , Linhagem Celular Tumoral , Heparina/análise , Humanos , Hidrólise , Integrina alfaVbeta3/metabolismo , Limite de Detecção , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/metabolismo , Espectrometria de Fluorescência/métodos , Eletricidade Estática , Tripsina/análise , Tripsina/química
5.
Anal Chim Acta ; 1078: 176-181, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31358217

RESUMO

Intracellular microRNA (miRNA) analysis in single cell is highly informative and offers valuable insights to its physiological and pathological state, but it must confront the pivotal challenge of gene probe delivery and conditional release. Herein, we report an assembled DNA mini-hexahedron (DMH) that can selectively package and protect miRNA probe, target-cell-specific delivery and release it based on the target sequence recognition for intracellular miRNA detection. In brief, the DMH is self-assembled from six single-stranded oligonucleotide strands through rational design, one of which containing AS1411 sequence for specific uptake. Two fluorescent dye labeled recognition strands are inserted into two DMH edges with quencher groups through partially complementary hybridization. We find that this DMH possesses great biocompatibility, good trans-membrane ability and are able to protect the gene cargo against enzymatic degradation and protein binding. Fluorescence restoration caused by the target-mediated competitive chain replacement reaction allows to simultaneous detection of two cancer-related intracellular miRNAs with little false-positive signal, providing a powerful tool to discriminate healthy normal cell and cancerous cell. Thus, the construct opens a new avenue to circumvent the challenges in gene delivery, specific delivery and intrinsic interferences resistance.


Assuntos
DNA de Cadeia Simples/química , MicroRNAs/análise , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Linhagem Celular Tumoral , DNA de Cadeia Simples/genética , Portadores de Fármacos/química , Fluoresceínas/química , Fluorescência , Corantes Fluorescentes/química , Humanos , MicroRNAs/genética , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética
6.
Microbiol Res ; 226: 27-33, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31284941

RESUMO

Postbloom fruit drop (PFD), caused mainly by Colletotrichum abscissum, is one of the most severe citrus diseases and can causes up to 80% fruit loss in favorable climatic conditions. According to the literature, other Colletotrichum species colonize hosts using distinct strategies: intracellular hemibiotrophic or subcuticular intramural necrotrophic colonization. However, so far, for C. abscissum only the necrotrophic stage has been described and some aspects remain unclear in PFD disease cycle. To better understand the disease cycle, microscopy studies could be applied. However, even using eGFP strains (expressing green fluorescent protein), the results are unclear due to the autofluorescence of citrus leaves. To eliminate this problem and to study the interaction between C. abscissum-citrus we used a destaining and staining methodologies, and we observed that in leaves, even applying injury before inoculation, C. abscissum does not colonize adjacent tissues. Apparently, in the leaves the fungus only uses the nutrients exposed in the artificial lesions for growth, and then produces large amount of spores. However, in flowers, C. abscissum penetrated and colonized the tissues of the petals 12 h after inoculation. In the early stages of infection, we observed the development of primary biotrophic hyphae, suggesting this species as a hemibiotrophic fungus, with a short biotrophic phase during flower colonization followed by dominant necrotrophic colonization. In conclusion, the use of an eGFP strain of C. abscissum and a different methodology of destaining and staining allowed a better understanding of the morphology and mechanisms used by this citrus pathogen to colonize the host.


Assuntos
Citrus/microbiologia , Colletotrichum/citologia , Colletotrichum/crescimento & desenvolvimento , Colletotrichum/patogenicidade , Doenças das Plantas/microbiologia , Flores/microbiologia , Frutas/microbiologia , Proteínas de Fluorescência Verde , Interações Hospedeiro-Patógeno , Hifas/citologia , Hifas/crescimento & desenvolvimento , Microscopia/métodos , Microscopia Confocal/métodos , Folhas de Planta , Esporos Fúngicos/citologia
8.
Chem Commun (Camb) ; 55(65): 9629-9632, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31353368

RESUMO

Excessive accumulation of reducing agents in the ER leads to a constitutively high UPR. And the co-function of GSH, Cys and HOCl in biological processes is not well understood. To address this, a TP probe, NPCC, was developed for monitoring reductive stress in the ER. It can also distinguish cancer cells from normal cells.


Assuntos
Cumarínicos/química , Estresse do Retículo Endoplasmático/fisiologia , Retículo Endoplasmático/metabolismo , Corantes Fluorescentes/química , Pirazóis/química , Animais , Cumarínicos/síntese química , Cisteína/química , Cisteína/metabolismo , Corantes Fluorescentes/síntese química , Glutationa/química , Glutationa/metabolismo , Cabras , Células HeLa , Humanos , Ácido Hipocloroso/química , Ácido Hipocloroso/metabolismo , Camundongos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Oxirredução , Pirazóis/síntese química , Peixe-Zebra
9.
Chem Commun (Camb) ; 55(58): 8494-8497, 2019 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-31268095

RESUMO

A rational strategy was reported to construct boranil complexes (DPFB derivatives) with unique aggregation-induced emission effects by installing phenyl rings in the anil ligand as the intramolecular rotors. In view of the good biocompatibility and suitable lipophilicity, DPFB derivatives can serve as excellent fluorescent probes for specific imaging of lipid droplets in living cells and yolk lipids in zebrafish.


Assuntos
Compostos de Anilina/química , Compostos de Boro/química , Complexos de Coordenação/química , Corantes Fluorescentes/química , Gotículas Lipídicas/metabolismo , Compostos de Anilina/síntese química , Animais , Compostos de Boro/síntese química , Complexos de Coordenação/síntese química , Fluorescência , Corantes Fluorescentes/síntese química , Células HeLa , Humanos , Ligantes , Microscopia Confocal/métodos , Estrutura Molecular , Peixe-Zebra
10.
Semin Ophthalmol ; 34(4): 340-346, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31215821

RESUMO

Fuchs endothelial corneal dystrophy (FECD) is characterized by the progressive degeneration of the corneal endothelium (CE). The purpose of this article is to review the diagnostic tools available to image and assess the CE in FECD. Slit-lamp biomicroscopy with specular reflection and retroillumination are important techniques to assess the CE. Objective diagnostic tests, such as retroillumination photographic analysis, specular microscopy, in vivo confocal microscopy (IVCM), and anterior segment optical coherence tomography, are valuable tools to evaluate the CE in FECD. Specular microscopy can be performed rapidly without touching the eye but requires a clear cornea with a smooth CE. In contrast, IVCM can image all layers of the cornea, even in advanced FECD. However, IVCM is contact-based and more technically challenging. It is important to select the appropriate objective diagnostic test to image and assess the CE in managing patients with FECD.


Assuntos
Técnicas de Diagnóstico Oftalmológico , Epitélio Posterior/diagnóstico por imagem , Distrofia Endotelial de Fuchs/diagnóstico por imagem , Humanos , Microscopia Confocal/métodos , Microscopia com Lâmpada de Fenda/métodos , Tomografia de Coerência Óptica/métodos
11.
Anal Chim Acta ; 1074: 123-130, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159932

RESUMO

Abnormal levels of Cys, Hcy and GSH are associated with various diseases, thus monitoring biothiols is of great significance. In this work, a dual-emission responsive near-infrared fluorescent probe NIR-NBD for detecting Hcy and Cys/GSH was developed based on the conjugation of a dicyanoisophorone based fluorophore (NIR-OH) and 7-nitrobenzofurazan (NBD). To our surprise, the addition of Hcy induced significant fluorescence enhancement at both 549 and 697 nm; while Cys/GSH resulted in major fluorescence emission at 697 nm. The detection limit was determined to be 33.2 nM for Cys, 33.5 nM for Hcy, and 34.4 nM for GSH. Therefore, the probe can be used for discriminative detection of Hcy and Cys/GSH. Moreover, fluorescence imaging of HeLa cells indicated that the probe was cell membrane permeable and could be used for visualizing Hcy and Cys/GSH in living cells.


Assuntos
4-Cloro-7-nitrobenzofurazano/análogos & derivados , Aminofenóis/química , Cisteína/análise , Corantes Fluorescentes/química , Glutationa/análise , Homocisteína/análise , 4-Cloro-7-nitrobenzofurazano/síntese química , 4-Cloro-7-nitrobenzofurazano/toxicidade , Aminofenóis/síntese química , Aminofenóis/toxicidade , Fluorescência , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/toxicidade , Células HeLa , Humanos , Limite de Detecção , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos
12.
Anal Chim Acta ; 1074: 43-53, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159938

RESUMO

This work evaluates the possibility of placement of high-resolution imaging and single-cell analysis via laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) within precision medicine by assessing the suitability of LA-ICP-MS as a micro-analytical technique for the localization and quantification of membranous receptors in heterogeneous cell samples that express both the membrane-bound receptors C-X-C chemokine receptor type 4 (CXCR4) and epidermal growth factor receptor (EGFR). Staining of the breast cancer cell lines MDA-MB-231 X4 and MDA-MB-468 was achieved using receptor-specific hybrid tracers, containing both a fluorophore and a DTPA single-lanthanide chelate. Prior to LA-ICP-MS imaging, fluorescence confocal microscopy (FCM) imaging was performed to localize the receptors, hereby enabling direct comparison. Based on the different expression levels of CXCR4 and EGFR, a distinction could be made between the cell lines using both imaging modalities. Furthermore, FCM and LA-ICP-MS demonstrated complementary characteristics, as a more distinct discrimination could be made between both cell lines based on the EGFR-targeting hybrid tracer via LA-ICP-MS, due to the intrinsic CXCR4-related green fluorescent protein (GFP) signal present in the MDA-MB-231 X4 cells. Employing state-of-the-art LA-ICP-MS instrumentation in bidirectional area scanning mode for sub-cellular imaging of MDA-MB-231 X4 cells enabled the specific binding of the CXCR4-targeting hybrid tracer to the cell membrane to be clearly demonstrated. The stretching of cells over the glass substrate led to a considerably higher signal response for pixels at the cell edges, relative to the more central pixels. The determination of the expression levels of CXCR4 and EGFR for the MDA-MB-468 cell line was performed using LA-ICP-MS single-cell analysis (sc-LA-ICP-MS) and external calibration, based on the quantitative ablation of Ho-spiked dried gelatin droplet standards. Additionally, a second calibration approach was applied based on spot ablation of highly homogeneous dried gelatin gels in combination with the determination of the ablated volume using atomic force microscopy (AFM) and yielded results which were in good agreement with the expression levels determined via flow cytometry (FC) and mass cytometry (MC). Hybrid tracers enable a direct comparison between (i) FCM and LA-ICP-MS imaging for the evaluation of the microscopic binding pattern and between (ii) FC, MC and sc-LA-ICP-MS for the quantification of receptor expression levels in single cells.


Assuntos
Corantes Fluorescentes/química , Receptores CXCR4/análise , Calibragem , Linhagem Celular Tumoral , Cetuximab/química , Quelantes/química , Receptores ErbB/análise , Citometria de Fluxo , Fluoresceínas/química , Fluorescência , Humanos , Elementos da Série dos Lantanídeos/química , Terapia a Laser , Limite de Detecção , Espectrometria de Massas/métodos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Ácido Pentético/análogos & derivados , Peptídeos Cíclicos/química , Análise de Célula Única/métodos
13.
Chemistry ; 25(45): 10566-10570, 2019 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-31197892

RESUMO

A family of three neutral iridium(III) tetrazolato complexes are investigated as bacterial imaging agents. The complexes offer a facile tuning of the emission colour from green (520 nm) to red (600 nm) in aqueous media, while keeping the excitation wavelength unchanged. The three complexes do not inhibit the bacterial growth of Bacillus Cereus, used as a model in this study, and exhibit extremely fast cellular uptake. After a minute incubation time, the nontoxic complexes show subcellular localisation in spherical structures identified as lipid vacuoles. Confocal Raman imaging has been exploited for the first time on live bacteria, to provide direct and label-free mapping of the lipid-enriched organelles within B. cereus, complementing the use of luminescent probes. Examination of the Raman spectra not only confirmed the presence of lipophilic inclusions in B. cereus but offered additional information about their chemical composition, suggesting that the lipid vacuoles may contain polyhydroxybutyrate (PHB).


Assuntos
Bacillus cereus/metabolismo , Complexos de Coordenação/química , Irídio/química , Lipídeos/química , Microscopia Confocal/métodos , Complexos de Coordenação/metabolismo , Substâncias Luminescentes/química , Análise Espectral Raman
14.
Chem Commun (Camb) ; 55(54): 7852-7855, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31215553
15.
Nat Commun ; 10(1): 2620, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197165

RESUMO

Conventional drug screens and treatments often ignore the underlying complexity of brain network dysfunctions, resulting in suboptimal outcomes. Here we ask whether we can correct abnormal functional connectivity of the entire brain by identifying and combining multiple neuromodulators that perturb connectivity in complementary ways. Our approach avoids the combinatorial complexity of screening all drug combinations. We develop a high-speed platform capable of imaging more than 15000 neurons in 50ms to map the entire brain functional connectivity in large numbers of vertebrates under many conditions. Screening a panel of drugs in a zebrafish model of human Dravet syndrome, we show that even drugs with related mechanisms of action can modulate functional connectivity in significantly different ways. By clustering connectivity fingerprints, we algorithmically select small subsets of complementary drugs and rapidly identify combinations that are significantly more effective at correcting abnormal networks and reducing spontaneous seizures than monotherapies, while minimizing behavioral side effects. Even at low concentrations, our polytherapy performs superior to individual drugs even at highest tolerated concentrations.


Assuntos
Epilepsias Mioclônicas/tratamento farmacológico , Modelos Biológicos , Rede Nervosa/efeitos dos fármacos , Fenômenos Fisiológicos do Sistema Nervoso/efeitos dos fármacos , Neurotransmissores/farmacologia , Algoritmos , Animais , Animais Geneticamente Modificados , Comportamento Animal/efeitos dos fármacos , Encéfalo/citologia , Encéfalo/diagnóstico por imagem , Encéfalo/efeitos dos fármacos , Encéfalo/fisiologia , Mapeamento Encefálico/métodos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Sinergismo Farmacológico , Quimioterapia Combinada/métodos , Epilepsias Mioclônicas/genética , Epilepsias Mioclônicas/patologia , Ensaios de Triagem em Larga Escala/métodos , Humanos , Microscopia Confocal/métodos , Rede Nervosa/diagnóstico por imagem , Rede Nervosa/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Neurotransmissores/uso terapêutico , Peixe-Zebra
16.
Nat Commun ; 10(1): 2704, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221964

RESUMO

Attachment of lipid tails to oligonucleotides has emerged as a powerful technology in constructing cell membrane-anchorable nucleic acid-based probes. In practice, however, conventional lipid-conjugated oligonucleotides fail to distinguish among different cell membranes. Herein, a phosphorylated lipid-conjugated oligonucleotide (DNA-lipid-P) is reported for alkaline phosphatase (ALP)-dependent cell membrane adhesion. In the absence of ALP, DNA-lipid-P with its poor hydrophobicity shows only weak interaction with cell membrane. However, in the presence of the highly expressed plasma membrane-associated ALP, DNA-lipid-P is converted to lipid-conjugated oligonucleotide (DNA-lipid) by enzymatic dephosphorylation. As a result of such conversion, the generated DNA-lipid has greater hydrophobicity than DNA-lipid-P and is thus able to insert into cell membranes in situ. Accordingly, DNA-lipid-P enables selective anchoring on cell membranes with elevated ALP level. Since elevated ALP level is a critical index of some diseases and even cancers, DNA-lipid-P holds promise for cell membrane engineering and disease diagnostics at the molecular level.


Assuntos
Fosfatase Alcalina/metabolismo , Membrana Celular/metabolismo , Sondas Moleculares/metabolismo , Oligonucleotídeos/metabolismo , Linhagem Celular Tumoral , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lipídeos/química , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Sondas Moleculares/química , Oligonucleotídeos/química , Compostos Organofosforados/química , Fosforilação
17.
Methods Mol Biol ; 1966: 27-38, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31041737

RESUMO

Immunohistochemistry using formalin-fixed, paraffin-embedded tissue, chromogen label, and light microscopy has traditionally been used to semiquantify estrogen receptor (ER) to guide diagnosis and management of breast cancer. Quantitation of ER for this purpose currently only assesses levels of the ER-alpha subtype. Considerable variability in results reported has been due to protocol and fixation variability, intraobserver and interobserver variability, and different scoring systems and thresholds for scoring ER positivity. Results can also vary with low expression levels of ER. ER-beta expression is reduced in breast and ovarian cancers and requires quantitation.Herein we describe a novel approach to quantifying ERß using older mouse ovarian surface epithelium, where ERß is expressed at lower levels than ERα and is therefore harder to detect. We use an antibody highly specific to the ERß1 isoform, together with immunofluorescence, confocal microscopy, and imaging and statistical software to achieve clear, reproducible, and unbiased quantitation of ERß.


Assuntos
Receptor beta de Estrogênio/análise , Regulação da Expressão Gênica , Imuno-Histoquímica/métodos , Microscopia Confocal/métodos , Ovário/metabolismo , Epitélio/metabolismo , Receptor beta de Estrogênio/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Microscopia Intravital/métodos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo
18.
Methods Mol Biol ; 1966: 137-149, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31041744

RESUMO

Increases in levels of protoporphyrin IX (PPIX; a heme precursor) may be driven by xenobiotic induction of aminolevulinic acid synthase 1 (ALAS1) expression. ALAS1 is the rate-limiting enzyme of heme biosynthesis and may be upregulated to satisfy the increased need for heme in CYP450 enzymes. Therefore, a high-throughput fluorescence spectroscopy method that detects PPIX would enable the screening of drugs that increase ALAS1 through nuclear hormone receptor-mediated induction of transcription that may cause toxicity or even provide utility in the diagnosis or treatment of cancers that have elevated cellular PPIX levels. This chapter describes a high-throughput plate-based imaging technique for determining cellular protoporphyrin levels by using the GE Healthcare InCell 6000 confocal imaging system to detect the presence and location of PPIX in each cell and may be adapted for use with other imaging systems. Laser excitation and a scientific-grade complementary metal oxide semiconductor (CMOS) camera generate short exposure times, decreasing photobleaching in the target cells that may result in inaccurate measurements of PPIX and increasing screening throughput. Nuclear staining was detected by using a laser with 405-nm excitation and 455-nm emission wavelengths, and the presence of PPIX was measured using 405-nm excitation and 706-nm emission wavelengths. Image analysis involving top-hat segmentation on both nuclear and PPIX staining was performed by using the InCell Analyzer Workstation software. This assay may be adapted to screen for PPIX formation, degradation, and transportation effectors. Indeed, the inclusion of PPIX transport inhibition would be expected to further widen the linear range of fluorescence and improve the method.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Microscopia Confocal/métodos , Protoporfirinas/análise , Espectrometria de Fluorescência/métodos , Células Hep G2 , Humanos , Interpretação de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos
19.
Analyst ; 144(11): 3643-3648, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31073567

RESUMO

Using fluorescent probes to detect endogenous hydrogen peroxide, which is associated with many diseases in the human body, remains an essential technique. Cyanine fluorochromes are a class of dyes that have attracted much attention and are widely used in the synthesis of fluorescent probes. In this article, a novel near-infrared (NIR) fluorescence probe for the detection of hydrogen peroxide was constructed and successfully applied to imaging endogenous hydrogen peroxide in vivo. Notably, probe 1 was designed by connecting 4-(bromomethyl)benzeneboronic acid pinacol ester as the sensing unit to the IR-780 hemicyanine skeleton, which exhibits excellent properties like NIR fluorescence emission over 700 nm. Probe 1 has satisfactory sensitivity to hydrogen peroxide with a low detection limit of 0.14 µM (S/N = 3), attributed to a responding mechanism that leads to the oxidation of phenylboronic acid pinacol ester and thereby releases fluorophore 2. Moreover, probe 1 displays excellent selectivity towards hydrogen peroxide over other substances. Taking advantage of these properties, the probe proved to be cell-permeable. Based on the results of N-acetylcysteine and rotenone together, probe 1 is capable of clearly visualizing endogenously produced hydrogen peroxide in living HepG2 cells and mice. The superior performance of the probe, as a reliable chemical tool, makes it of great potential application for exploring the role played by hydrogen peroxide in biological systems.


Assuntos
Ácidos Borônicos/química , Corantes Fluorescentes/química , Peróxido de Hidrogênio/análise , Indóis/química , Acetilcisteína/farmacologia , Animais , Antioxidantes/farmacologia , Ácidos Borônicos/síntese química , Ácidos Borônicos/toxicidade , Fluorescência , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/toxicidade , Células Hep G2 , Humanos , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Indóis/síntese química , Indóis/toxicidade , Limite de Detecção , Camundongos Endogâmicos BALB C , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Oxirredução , Rotenona/farmacologia , Temperatura Ambiente
20.
Int J Mol Sci ; 20(10)2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31091708

RESUMO

We performed a three-dimensional (3D) analysis of the microvascular network of the cerebral cortex of twitcher mice (an authentic model of Krabbe disease) using a restricted set of indexes that are able to describe the arrangement of the microvascular tree in CD31-stained sections. We obtained a near-linear graphical "fingerprint" of the microangioarchitecture of wild-type and twitcher animals that describes the amounts, spatial dispersion, and spatial relationships of adjacent classes of caliber-filtered microvessels. We observed significant alterations of the microangioarchitecture of the cerebral cortex of twitcher mice, whereas no alterations occur in renal microvessels, which is keeping with the observation that kidney is an organ that is not affected by the disease. This approach may represent an important starting point for the study of the microvascular changes that occur in the central nervous system (CNS) under different physiopathological conditions.


Assuntos
Córtex Cerebral/diagnóstico por imagem , Processamento de Imagem Assistida por Computador/métodos , Imagem Tridimensional/métodos , Leucodistrofia de Células Globoides/diagnóstico por imagem , Microvasos/diagnóstico por imagem , Animais , Córtex Cerebral/irrigação sanguínea , Camundongos , Microscopia Confocal/métodos
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