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1.
World J Microbiol Biotechnol ; 35(8): 129, 2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-31376017

RESUMO

Metal whole-cell biosensors (WCBs) have been reported as very useful tools to detect and quantify the presence of bioavailable fractions of certain metals in water and soil samples. In the current work, two bacterial WCBs able to report Cr(VI) presence and plants growing on Cr(VI)-enriched soil/medium were used to assess the potential transfer of this metal to organisms of higher trophic levels, and the risk of transfer to the food chain. To do it, the functionality of the WCBs within tissues of inoculated plants in contact with Cr(VI)-contaminated soil and water was studied in vitro and in a controlled greenhouse environment. One WCB was the previously described Ochrobactrum tritici pCHRGFP2 and the second, Nitrospirillum amazonense pCHRGFP2, is a newly engineered naturally-occurring endophytic microorganism. Three rice varieties (IAC 4440, BRS 6 CHUÍ, IRGA 425) and one maize variety (1060) were tested as hosts and subjected to Cr(VI) treatments (25 µM), with different results obtained. Inoculation of each WCB into plants exposed to Cr(VI) showed GFP expression within plant tissues. WCBs penetrated the root tissues and later colonized the shoots and leaves. In general, a higher fluorescence signal was detected in roots, together with a higher Cr content and denser WCB colonization. Best fluorescence intensities per plant biomass of shoots were obtained for plant host IRGA 425. Therefore, by analyzing colonized tissues, both WCBs allowed the detection of Cr(VI) contamination in soils and its transfer to plants commonly used in crops for human diet.


Assuntos
Técnicas Biossensoriais/métodos , Cromo/análise , Ochrobactrum/crescimento & desenvolvimento , Oryza/química , Rhodospirillaceae/crescimento & desenvolvimento , Zea mays/química , Disponibilidade Biológica , Engenharia Metabólica , Microscopia de Fluorescência , Ochrobactrum/genética , Ochrobactrum/metabolismo , Oryza/crescimento & desenvolvimento , Oryza/microbiologia , Rhodospirillaceae/genética , Rhodospirillaceae/metabolismo , Poluentes do Solo/análise , Poluentes Químicos da Água/análise , Zea mays/crescimento & desenvolvimento , Zea mays/microbiologia
2.
Soft Matter ; 15(33): 6660-6676, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31389467

RESUMO

The dynamic behavior of monoclonal antibodies (mAbs) at high concentration provides insight into protein microstructure and protein-protein interactions (PPI) that influence solution viscosity and protein stability. At high concentration, interpretation of the collective-diffusion coefficient Dc, as determined by dynamic light scattering (DLS), is highly challenging given the complex hydrodynamics and PPI at close spacings. In contrast, self-diffusion of a tracer particle by Brownian motion is simpler to understand. Herein, we develop fluorescence correlation spectroscopy (FCS) for the measurement of the long-time self-diffusion of mAb2 over a wide range of concentrations and viscosities in multiple co-solute formulations with varying PPI. The normalized self-diffusion coefficient D0/Ds (equal to the microscopic relative viscosity ηeff/η0) was found to be smaller than η/η0. Smaller ratios of the microscopic to macroscopic viscosity (ηeff/η) are attributed to a combination of weaker PPI and less self-association. The interaction parameters extracted from fits of D0/Ds with a length scale dependent viscosity model agree with previous measurements of PPI by SLS and SAXS. Trends in the degree of self-association, estimated from ηeff/η with a microviscosity model, are consistent with oligomer sizes measured by SLS. Finally, measurements of collective diffusion and osmotic compressibility were combined with FCS data to demonstrate that the changes in self-diffusion between formulations are due primarily to changes in the protein-protein friction in these systems, and not to protein-solvent friction. Thus, FCS is a robust and accessible technique for measuring mAb self-diffusion, and, by extension, microviscosity, PPI and self-association that govern mAb solution dynamics.


Assuntos
Anticorpos Monoclonais/química , Fenômenos Biofísicos , Difusão , Fluorescência , Corantes Fluorescentes/química , Microscopia de Fluorescência , Modelos Químicos , Multimerização Proteica , Estabilidade Proteica , Soluções , Viscosidade
3.
Arq Gastroenterol ; 56(2): 191-196, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31460585

RESUMO

BACKGROUND: Colorectal cancer is one of the most prevalent pathologies. Its prognosis is linked to the early detection and treatment. Currently diagnosis is performed by histological analysis from polyp biopsies, followed by morphological classification. Kudo's pit pattern classification is frequently used for the differentiation of neoplastic colorectal lesions using hematoxylin-eosin stained samples. Few articles have reported this classification with image software processing, using exogenous markers over the samples. The processing of autofluorescence images is an alternative that could allow the characterization of the pits from the crypts of Lieberkühn, bypassing staining techniques. OBJECTIVE: Processing and analysis of widefield autofluorescence microscopy images obtained by fresh colon tissue samples from a murine model of colorectal cancer in order to quantify and characterize the pits morphology by measuring morphology parameters and shape descriptors. METHODS: Adult male BALB/cCmedc strain mice (n=27), ranging from 20 to 30 g, were randomly assigned to four and five groups of treated and control animals. Colon samples were collected at day zero and at fourth, eighth, sixteenth and twentieth weeks after treatmentwith azoxymethane. Two-dimensional (2D) segmentation, quantification and morphological characterization of pits by image processing applied using macro programming from FIJI. RESULTS: Type I is the pit morphology prevailing between 53 and 81% in control group weeks. III-L and III-S types were detected in reduced percentages. Between the 33 and 56% of type I was stated as the prevailing morphology for the 4th, 8th and 20th weeks of treated groups, followed by III-L type. For the 16th week, the 39% of the pits was characterized as III-L type, followed by type I. Further, pattern types as IV, III-S and II were also found mainly in that order for almost all of the treated weeks. CONCLUSION: These preliminaries outcomes could be considered an advance in two-dimensional pit characterization as the whole image processing, comparing to the conventional procedure, takes a few seconds to quantify and characterize non-pathological colon pits as well as to estimate early pathological stages of colorectal cancer.


Assuntos
Pólipos do Colo/diagnóstico por imagem , Neoplasias Colorretais/diagnóstico por imagem , Microscopia de Fluorescência , Animais , Pólipos do Colo/patologia , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos BALB C
4.
An Acad Bras Cienc ; 91(3): e20180654, 2019 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-31365653

RESUMO

Candida albicans is the most frequent fungal species that causes infections in humans. Fluconazole is the main antifungal used to treat Candida infections, and its prolonged and indiscriminate use for the last decades are the most established causes which originated resistant strains. Fungal drug resistance is associated to alterations in ERG11 gene and overexpression of multidrug resistance (MDR) transporters belonging to two families: ATP-binding cassette (ABC) and Major Facilitator Superfamily (MFS). To evaluate the role of MFS transporters in azoles resistance of C. albicans clinical strains, this study aimed to analyze four Candida albicans clinical isolates from the University Hospital in Juiz de Fora (Minas Gerais/Brazil), selected in our previous study as they were unaffected by FK506, an ABC pumps inhibitor. In a primary investigation on MFS proteins overexpression, the extrusion of fluorescent substrates (rhodamine 6G and nile red) was analyzed by fluorescence microscopy and flow cytometry. Results suggest participation of MFS transporters in azole resistance of C. albicans isolates and indicate the existence of secondary resistance mechanisms. Therefore, this study contributes to the information about Candida albicans infections in Brazil and reinforces the importance of epidemiological studies focusing on an improved understanding of the disease and further resistance reversion.


Assuntos
Antifúngicos/farmacologia , Azóis/farmacologia , Candida albicans/efeitos dos fármacos , Proteínas de Membrana Transportadoras/metabolismo , Azóis/classificação , Transporte Biológico/efeitos dos fármacos , Citometria de Fluxo , Humanos , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Microscopia de Fluorescência , Centros de Atenção Terciária
5.
Sheng Li Xue Bao ; 71(4): 555-561, 2019 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-31440752

RESUMO

The aim of the present study was to establish a cell model of volume-regulated anion channel subunit LRRC8A and investigate the physiological characteristics of LRRC8A. The eukaryotic expression vectors of LRRC8A and YFP-H148Q/I152L were constructed and transfected into Fischer rat thyroid (FRT) cells by Lipofectamine 2000. The FRT cell lines co-expressing LRRC8A and YFP-H148Q/I152L were obtained by antibiotic screening. The expression of LRRC8A and YFP-H148Q/I152L in FRT cells was detected by the inverted fluorescence microscope. The fluorescence quenching kinetic experiment was done to verify the function and effectiveness of the cell model. Then the cell model was utilized to study the physiological characteristics of LRRC8A, such as the characteristics of anion transport, the opening of LRRC8A by osmotic pressure, the effect of anion transport velocity, and the effect of chloride channel inhibitors on LRRC8A anion channel. The results of the inverted fluorescence microscope showed that LRRC8A was expressed on the cell membrane and YFP-H148Q/I152L was expressed in the cytoplasm. The results of fluorescence quenching kinetic test showed that under the condition of low osmotic state, LRRC8A could transport some kinds of anions, such as iodine and chloride ions. Osmotic pressure played a key role in the regulation of LRRC8A volume-regulated anion channel opening. Chloride channel inhibitors inhibited ion transport of LRRC8A channel in a dose-dependent manner. It is suggested that LRRC8A has the characteristics of classic volume-regulated anion channels by using the cell model of FRT cells co-expressing LRRC8A and YFP-H148Q/I152L.


Assuntos
Canais de Cloreto/antagonistas & inibidores , Transporte de Íons , Proteínas de Membrana/fisiologia , Animais , Ânions , Células Cultivadas , Microscopia de Fluorescência , Ratos , Ratos Endogâmicos F344 , Glândula Tireoide/citologia , Transfecção
6.
J Agric Food Chem ; 67(35): 9934-9941, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31402655

RESUMO

A fluorescence microscopic method for characterizing size, quantity, and oxidation of lipid droplets (LDs) in HepG2 cells was developed. LDs were induced by palmitic (PA), oleic (OA), or linoleic acids (LA) and stained with two fluorescent probes for neutral lipids and lipid peroxides. Each fatty acid increased the number of LDs and oxidized LDs (oxLDs) and the degree of LD oxidation time dependently, as well as increased intracellular triglyceride hydroperoxides. LDs induced by LA without 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) showed the most significant oxidation degree over PA and OA, especially in large LDs (area ≥ 3 µm2, oxLD/LD = 52.3 ± 21.7%). Under this condition, two food-derived antioxidants were evaluated, and both of them significantly improved the LD characteristics. Moreover, chlorogenic acid reduced the quantity of large LDs by 74.0-87.6% in a dose-dependent manner. The proposed method provides a new approach to evaluate the effect of dietary antioxidants on LD characteristics.


Assuntos
Antioxidantes/metabolismo , Hepatócitos/química , Hepatócitos/metabolismo , Gotículas Lipídicas/metabolismo , Microscopia de Fluorescência/métodos , Antioxidantes/química , Ácido Clorogênico/química , Ácido Clorogênico/metabolismo , Fluorescência , Células Hep G2 , Humanos , Gotículas Lipídicas/química , Oxirredução
7.
Chem Commun (Camb) ; 55(65): 9681-9684, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31347618

RESUMO

Here, we report a convenient, fast labeling strategy for the imaging of cell surface sialic acids (SAs, nine-carbon monosaccharides located at the terminals of cell surface sugar chains). This strategy is based on the synthesis of sticky, furry and fluorescent "wool-balls", which are wound into nanoclusters from p-benzoquinone/ethylenediamine polymer "wires". With abundant amino groups at the surface, the wool-balls can easily stick to the C-7 aldehyde group generated at the ends of periodate treated SAs in less than 30 min.


Assuntos
Benzoquinonas/química , Etilenodiaminas/química , Corantes Fluorescentes/química , Nanopartículas/química , Polímeros/química , Ácidos Siálicos/análise , Animais , Benzoquinonas/síntese química , Linhagem Celular Tumoral , Etilenodiaminas/síntese química , Fluorescência , Humanos , Camundongos , Microscopia de Fluorescência/métodos , Neuraminidase/química , Tamanho da Partícula , Polímeros/síntese química , Células RAW 264.7 , Bases de Schiff/síntese química , Bases de Schiff/química , Ácidos Siálicos/química
9.
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
10.
J Laryngol Otol ; 133(8): 696-699, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31290382

RESUMO

OBJECTIVE: To explore the use of fluorescence lifetime imaging microscopy in thyroid tissues, and to investigate how different thyroid lesions affect fluorescence lifetime. METHOD: Fluorescence lifetime measurements were taken of fresh frozen thyroid surgical specimens stained with fluorescein isothiocyanate tagged anti-thyroglobulin monoclonal antibodies. RESULTS: The mean fluorescence lifetime measurements in 12 patients - 3 with multinodular goitre, 4 with follicular adenoma, 4 with papillary thyroid carcinoma and 1 with follicular carcinoma - were 3.16 ns (range, 2.66-3.52 ns), 3.75 ns (range, 2.99-4.57 ns), 2.97 ns (range, 2.57-3.21 ns) and 3.61 ns, respectively. The fluorescence lifetime of follicular adenoma patients was higher than that of papillary thyroid carcinoma patients by 26 per cent (p = 0.058). The fluorescence lifetime in the follicular carcinoma patient was similar to the follicular adenoma group, but higher than in the papillary thyroid carcinoma group by 22 per cent (p = 0.01). CONCLUSION: Fluorescence lifetime measurements varied in different thyroid pathologies, possibly because of tissue-scale structural influences.


Assuntos
Adenoma/diagnóstico por imagem , Bócio Nodular/diagnóstico por imagem , Glândula Tireoide/diagnóstico por imagem , Neoplasias da Glândula Tireoide/diagnóstico por imagem , Adenoma/metabolismo , Feminino , Fluoresceína-5-Isotiocianato/farmacologia , Técnica Direta de Fluorescência para Anticorpo , Bócio Nodular/metabolismo , Humanos , Masculino , Microscopia de Fluorescência , Tireoglobulina/antagonistas & inibidores , Glândula Tireoide/patologia
11.
Photochem Photobiol Sci ; 18(8): 2003-2011, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31268087

RESUMO

Here we report the activatable photosensitizer BromoAcroB, a brominated BODIPY dye incorporating a reactive acrolein warhead. The acrolein moiety serves as an intramolecular switch, deactivating the BODIPY dye in its singlet and triplet excited states via internal conversion. Thiolate addition to this moiety disables the intramolecular quenching mechanism restoring the photosensitizing properties of the parent dye, characterized by a quantum yield of singlet oxygen photosensitization of 0.69 ± 0.02. In cell cultures, and upon thiol adduct formation, BromoAcroB induced light-dependent cell death in MRC-5 and HeLa cell lines. Using fluorescence microscopy and upon measuring the low yet non-negligible emission of the activated compound, we show that the phototoxicity of the dormant photosensitizer correlated with the quantity of BromoAcroB adducts generated. BromoAcroB thus serves as a dormant photosensitizer sensitive to intracellular electrophile response. Our results highlight the effective control of a triplet state process by modulation of an unsaturated moiety on the BODIPY scaffold and underscore the mechanistic opportunities arising for controlled singlet oxygen production in cells specifically sensitive to electrophile stress.


Assuntos
Acroleína/farmacologia , Compostos de Boro/farmacologia , Corantes/farmacologia , Cisteína/farmacologia , Fármacos Fotossensibilizantes/farmacologia , Oxigênio Singlete/farmacologia , Acroleína/química , Compostos de Boro/química , Morte Celular/efeitos dos fármacos , Corantes/síntese química , Corantes/química , Cisteína/química , Células HeLa , Humanos , Luz , Microscopia de Fluorescência , Estrutura Molecular , Fármacos Fotossensibilizantes/síntese química , Fármacos Fotossensibilizantes/química , Teoria Quântica , Oxigênio Singlete/química
12.
Chem Commun (Camb) ; 55(59): 8583-8586, 2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31274135

RESUMO

We present a near-infrared (NIR) fluorescent probe, NR-HNO, which was successfully applied to visualizing H2S/NO "crosstalk" by the fluorescence detection of nitroxyl with a fast response time (5 min) and a large Stokes shift (131 nm) in living cells and tissue; it was also used to image nitroxyl in live mice.


Assuntos
Compostos de Benzilideno/química , Corantes Fluorescentes/química , Sulfeto de Hidrogênio/metabolismo , Óxido Nítrico/metabolismo , Óxidos de Nitrogênio/análise , Animais , Compostos de Benzilideno/efeitos da radiação , Linhagem Celular Tumoral , Corantes Fluorescentes/efeitos da radiação , Humanos , Rim/metabolismo , Luz , Limite de Detecção , Fígado/metabolismo , Camundongos , Microscopia de Fluorescência/métodos , Nitritos/química , Óxidos de Nitrogênio/metabolismo , Espectrometria de Fluorescência/métodos
13.
Chem Commun (Camb) ; 55(63): 9379-9382, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31317975

RESUMO

Small-molecule natural products have been an essential source of pharmaceuticals to treat human diseases, but very little is known about their behavior inside dynamic, live human cells. Here, we demonstrate the first structure-activity-distribution relationship (SADR) study of complex natural products, the anti-cancer antimycin-type depsipeptides, using the emerging bioorthogonal Stimulated Raman Scattering (SRS) Microscopy. Our results show that the intracellular enrichment and distribution of these compounds are driven by their potency and specific protein targets, as well as the lipophilic nature of compounds.


Assuntos
Antimicina A/análogos & derivados , Antineoplásicos/química , Depsipeptídeos/química , Antimicina A/química , Antimicina A/metabolismo , Antimicina A/farmacologia , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Depsipeptídeos/metabolismo , Depsipeptídeos/farmacologia , Células HeLa , Humanos , Células MCF-7 , Microscopia de Fluorescência , Análise Espectral Raman , Relação Estrutura-Atividade
14.
J Photochem Photobiol B ; 197: 111545, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31326847

RESUMO

Proper waste utilization in order to promote value added product is a promising scientific practice in recent era. Inspiring from the recurring trend, we propose a single step oxidative pyrolysis derived fluorescent carbon dots (C-dots) from Allium sativum peel, which is a natural, nontoxic, and waste raw material. Because of its excellent optical properties, and photostability this C-dots have been used in versatile area of applications. Due to its immediate water dispersing character, C-dots reinforced Poly(acrylic acid) (PAA) films revealed improvement in uniaxial stretching behavior and can be used as transparent sunlight conversion film. The nanocomposite film has been tested against rigorous simulated sunlight which proved almost identical sunlight conversion behavior with no photo-bleachable character which is definitely added an extra quality of transparent polymer films. Moreover, the C-dots dispersion has been used as in vitro biomarker for living cells owing to its ease in solubility, biocompatibility, non-cytotoxicity and bright fluorescence even in subcutaneous environment. For this case, adipose derived mesenchymal stem cells (ADMSCs) have been chosen and injected to rabbit ear skin to perform two-photon imaging experiment. The present work opens a new avenue towards the large-scale synthesis of bio-waste based fluorescent C-dots, paving the way for their versatile applications.


Assuntos
Allium/química , Nitrogênio/química , Fotodegradação/efeitos da radiação , Pontos Quânticos/química , Enxofre/química , Luz Solar , Resinas Acrílicas/química , Tecido Adiposo/citologia , Allium/metabolismo , Animais , Materiais Biocompatíveis/química , Carbono/química , Sobrevivência Celular/efeitos dos fármacos , Frutas/química , Frutas/metabolismo , Química Verde , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Microscopia de Fluorescência , Pontos Quânticos/toxicidade , Coelhos , Pele/efeitos dos fármacos , Pele/patologia , Solubilidade
15.
Biol Res ; 52(1): 36, 2019 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-31300048

RESUMO

BACKGROUND: Recent evidences indicated that some local anaesthetic agents played a role in inhibiting the proliferation of cancer cells; Whether ropivacaine is able to promote apoptosis of hepatocellular carcinoma (HCC) cells is still unclear. The aim of this study was to investigate the effect of ropivacaine on the apoptosis of HCC cells. METHODS: In the present study, we treated the HCC cell lines, Bel7402 and HLE with ropivacaine. MTT, DAPI stain, trypan blue exclusion dye assay, flow cytometry, electron microscopy, computational simulation, laser confocal microscope, Western blotting, and enzyme activity analysis of caspase-3 were applied to detect the growth and apoptosis of HCC cells and to explore the role mechanism of ropivacaine. RESULTS: Ropivacaine was able to inhibit proliferation and promote apoptosis of HCC cells in a dose- and time-dependent manner. Ropivacaine also has a trait to inhibit the migration of HCC cells; ropivacaine damaged the mitochondria of HCC cells. The results also indicated that ropivacaine was able to interact with caspase-3, promote cytoplasmic caspase-3 migration into the nucleus, stimulate cleavage of caspase-3 and PARP-1, caspase-9 proteins, inhibit the expression of Bcl-2, promote expression of Apaf-1 and mitochondria release cytochrome C, and activate the activity of caspase-3. CONCLUSIONS: Ropivacaine has a novel role in promoting apoptosis of HCC cells; The role mechanism of ropivacaine maybe involve in damaging the function of mitochondria and activating the caspase-3 signalling pathway in HCC cells. Our findings provide novel insights into the local anaesthetic agents in the therapy of HCC patients.


Assuntos
Anestésicos Locais/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Caspase 3/metabolismo , Neoplasias Hepáticas/patologia , Ropivacaina/farmacologia , Apoptose/fisiologia , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citometria de Fluxo , Humanos , Neoplasias Hepáticas/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Mitocôndrias/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
16.
Nat Commun ; 10(1): 2781, 2019 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-31273194

RESUMO

Recent advances in optical clearing and light-sheet microscopy have provided unprecedented access to structural and molecular information from intact tissues. However, current light-sheet microscopes have imposed constraints on the size, shape, number of specimens, and compatibility with various clearing protocols. Here we present a multi-immersion open-top light-sheet microscope that enables simple mounting of multiple specimens processed with a variety of clearing protocols, which will facilitate wide adoption by preclinical researchers and clinical laboratories. In particular, the open-top geometry provides unsurpassed versatility to interface with a wide range of accessory technologies in the future.


Assuntos
Microscopia de Fluorescência/métodos , Animais , Encéfalo/diagnóstico por imagem , Desenho de Equipamento , Humanos , Imagem Tridimensional/instrumentação , Imagem Tridimensional/métodos , Pulmão/diagnóstico por imagem , Linfonodos/diagnóstico por imagem , Masculino , Camundongos , Microscopia de Fluorescência/instrumentação , Próstata/diagnóstico por imagem
17.
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
18.
Anal Chim Acta ; 1078: 112-118, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31358208

RESUMO

Herein, an array-based in situ fluorescence assay is proposed for high-throughput analysis and localization of multiplex matrix metalloproteinases (MMPs) activities in cell monolayers and tissue sections. Five specific MMPs (MMP-2, -3, -7, -9, and -14) peptide substrates containing FAM/Dabcyl fluorescent resonance energy transfer (FRET) pair are directly spotted on the surface of cell monolayers or tissue sections, and hydrolyzed by localized MMPs, resulting in fluorescence recovery of FAM. MMPs activities are determined by the fluorescence intensity of stained cells/tissues due to the cellular internalization of peptide fragments with FAM moiety. We demonstrate that the array-based in situ fluorescence assay is suitable for identifying the MMPs expression patterns of cells, as well as determining the secreted MMPs activities in cell monolayer with high sensitivity (as low as hundreds of cells per square centimeter). The feasibility of the assay is further confirmed by evaluating inhibition potencies of six compounds toward five MMPs. Profiling of five MMPs activities in the localized parts of 32 thyroid tissues is performed without separation or extraction procedures, demonstrating the good practicality of the method.


Assuntos
Ensaios Enzimáticos/métodos , Metaloproteinases da Matriz/análise , Microscopia de Fluorescência/métodos , Peptídeos/metabolismo , Linhagem Celular Tumoral , Fluoresceínas/química , Fluorescência , Corantes Fluorescentes/química , Humanos , Hidrólise , Inibidores de Metaloproteinases de Matriz/farmacologia , Metaloproteinases da Matriz/metabolismo , Peptídeos/química , Estudo de Prova de Conceito , Glândula Tireoide/metabolismo
19.
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
20.
Chem Commun (Camb) ; 55(57): 8329-8332, 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31257378

RESUMO

Cell imaging heavily depends on fluorescent labels typically incompatible with Raman microscopy. The europium(iii) complex based on dipicolinic acid (DPA) presented here is an exception from this rule. Although its luminescence bands are very narrow, their intensity is comparable to the background Raman bands. This makes it complementary to less luminous compounds referred to as Raman tags. Through several examples we show that the complex provides a morphological context in otherwise unstained cells, thus acting as a spectral-counterstaining agent.


Assuntos
Complexos de Coordenação/química , Análise Espectral Raman , Candida albicans/metabolismo , Parede Celular/química , Células Epiteliais/química , Células Epiteliais/citologia , Európio/química , Células HeLa , Humanos , Microscopia de Fluorescência , Ácidos Picolínicos/química
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