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1.
Chem Commun (Camb) ; 56(11): 1629-1632, 2020 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-31939471

RESUMO

This article presents a new label-free fluorescence assay based on supramolecular self-assembly of cucurbit[7]uril and specific peptide Gly-Pro-Phe-Gly for monitoring DPP4 activity in clinical samples. It also displays a good potential application in high-throughput screening of DPP4 inhibitors.


Assuntos
Dipeptidil Peptidase 4/sangue , Ensaios Enzimáticos/métodos , Laranja de Acridina/química , Animais , Hidrocarbonetos Aromáticos com Pontes/química , Dipeptidil Peptidase 4/química , Inibidores da Dipeptidil Peptidase IV/química , Feminino , Corantes Fluorescentes/química , Humanos , Imidazóis/química , Limite de Detecção , Masculino , Camundongos Endogâmicos C57BL , Oligopeptídeos/química , Espectrometria de Fluorescência/métodos
2.
BMC Res Notes ; 13(1): 29, 2020 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-31931859

RESUMO

OBJECTIVE: Bacterial meningitis is a life threatening condition that requires prompt recognition and treatment. Currently, Gram stain is widely used for the microscopic detection of bacterial pathogens in cerebrospinal fluid (CSF). In Nepal, fluorescent microscopes have been installed in laboratories as a part of the National tuberculosis control program. However, information on the utility of the acridine orange (AO) stain for the direct detection of bacteria in CSF samples in Nepal is not available. Therefore, this study aims to compare Gram stain and AO stain for the rapid detection of bacterial pathogens in CSF of clinically suspected meningitis cases in Kathmandu, Nepal. RESULTS: Bacterial pathogens were detected in 9.30% (36/387) by either of the three tests, 9.04% (35/387) by AO stain, 8.27% (32/387) by culture and 6.46% (25/387) by Gram's stain. Considering culture as a gold standard, the sensitivity of AO stain was higher than Gram stain. The specificity of AO stain was 98.87%. Detection and differentiation of the bacteria was much clear in AO staining than Gram staining. AO is a better alternative to Gram stain in the rapid detection of bacterial pathogens in CSF in the setting where fluorescent microscope is available.


Assuntos
Laranja de Acridina , Bactérias/isolamento & purificação , Líquido Cefalorraquidiano/microbiologia , Corantes Fluorescentes , Meningites Bacterianas/diagnóstico , Microscopia de Fluorescência , Laranja de Acridina/química , Bactérias/patogenicidade , Corantes , Corantes Fluorescentes/química , Violeta Genciana/química , Meningites Bacterianas/microbiologia , Microscopia , Nepal , Fenazinas/química , Sensibilidade e Especificidade , Coloração e Rotulagem
3.
Anticancer Res ; 39(11): 6365-6372, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31704869

RESUMO

BACKGROUND/AIM: Although few studies have shown the effectiveness of adjuvant therapy with acridine orange (AO) for soft tissue sarcoma (STS) patients, no study has investigated this among cases with marginal resection. The aim of the study was to evaluate the effectiveness of AO therapy directly by comparing it to marginal resection cases that did not receive AO. PATIENTS AND METHODS: This retrospective study included 19 and 33 patients with STS who received AO therapy (AO group) and marginal resection without AO therapy (non-AO group), respectively. The patients' clinical information was collected, and the clinical courses were compared. RESULTS: The local recurrence rate in the AO group was significantly lower than that in the non-AO group (p<0.05). The local recurrence-free survival curves significantly differed between the two groups (p<0.05). High grade malignancy and no treatment with AO were identified as risk factors for local recurrence (p<0.05). CONCLUSION: AO therapy strongly suppressed local recurrence after marginal resection of STS.


Assuntos
Laranja de Acridina/uso terapêutico , Corantes Fluorescentes/uso terapêutico , Margens de Excisão , Recidiva Local de Neoplasia , Sarcoma/tratamento farmacológico , Sarcoma/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos , Estudos de Casos e Controles , Terapia Combinada/métodos , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/prevenção & controle , Estudos Retrospectivos , Sarcoma/patologia
4.
Molecules ; 24(15)2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-31382361

RESUMO

Self-assembly of organic molecules in aqueous solutions is governed by a delicate entropy/enthalpy balance. Even small changes in their intermolecular interactions can cause critical changes in the structure of the aggregates and their spectral properties. The experimental results reported here demonstrate that protonated cations of acridine orange, acridine, and acridin-9-amine form stable J-heteroaggregates when in water. The structures of these aggregates are justified by the homonuclear 1H cross-relaxation nuclear magnetic resonance (NMR). The absorption and fluorescence of these aggregates deviate characteristically from the known H-homoaggregates of the protonated cations of acridine orange. The latter makes acridine orange a handy optical sensor for soft matter studies.


Assuntos
Laranja de Acridina/química , Água/química , Corantes Fluorescentes/química , Estrutura Molecular , Soluções , Análise Espectral
5.
Biomed Res Int ; 2019: 8275935, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31360726

RESUMO

Musculoskeletal sarcomas are rare and aggressive human malignancies affecting bones and soft tissues with severe consequences, in terms of both morbidity and mortality. An innovative technique that combines photodynamic surgery (PDS) and therapy (PDT) with acridine orange has been recently suggested, showing promising results. However, due to the low incidence of sarcoma in humans, this procedure has been attempted only in pilot studies and stronger evidence is needed. Naturally occurring tumors in cats are well-established and advantageous models for human cancers. Feline injection-site sarcoma (FISS) shares with human musculoskeletal sarcomas a mesenchymal origin and an aggressive behavior with a high relapse rate. Furthermore, wide surgical excision is not always possible due to the size and site of development. We assessed the feasibility and the effectiveness of PDS and PDT with acridine orange to prevent FISS recurrence by treating a short case series of cats. For PDS, the surgical field was irrigated with an acridine orange solution and exposed to UV light to enlighten the residual tumor tissue, and the resultant fluorescent areas were trimmed. For PDT, before wound closure, the field was again irrigated with acridine orange solution and exposed to visible light to get the antitumoral cytocidal effect. The procedure was easy to perform and well tolerated, we did not observe any major complications, and all the surgical resection margins were free of disease. Finally, at follow-up, all treated patients did not show evidence of tumor recurrence and had a significantly higher event-free survival rate in respect to a control group treated only by surgery. In conclusion, by this study we demonstrated that, in FISS, PDS and PDT with acridine orange may improve local tumor control, granting a better outcome, and we laid the foundation to validate its effectiveness for the treatment of human musculoskeletal sarcomas.


Assuntos
Laranja de Acridina/farmacologia , Neoplasias Musculares/terapia , Fotoquimioterapia , Sarcoma/terapia , Animais , Gatos , Feminino , Humanos , Masculino , Neoplasias Musculares/metabolismo , Neoplasias Musculares/patologia , Sarcoma/metabolismo , Sarcoma/patologia
6.
Int J Pharm ; 568: 118511, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31301466

RESUMO

Nucleic acid aptamers can specifically bind to target molecules on the cell membrane that mediate their entrance into the cells. Their small size, high binding affinity, specificity, good biocompatibility, stability and low immunogenicity make them ideal drug delivery systems for cancer therapy. These biopharmaceuticals have potential for the delivery of anticancer compounds to diseased tissues, increasing their effectiveness while mitigating the off-target toxicity towards healthy cells. Herein, we have studied two quadruplex-forming DNA sequences derived from the nucleolin-targeted aptamer AS1411 as supramolecular carriers for the cancer-selective delivery of acridine orange derivatives (C3, C5 and C8) in cervical cancer cells. The devised delivery strategy relied on the non-covalent association of the acridine derivatives and the G-quadruplex (G4) structures. This association is done with a high binding strength, as suggested by the obtained KD values in the 10-6-10-7 M range, leading to the thermal stabilization of the G4 structures, particularly for C8. The stability of the resulting supramolecular conjugates was evaluated in fetal bovine serum, which proved their resistance against serum nucleases up to 48 h. Previous studies showed that the tested acridine orange derivatives were cytotoxic towards cervical cancer cells (HeLa) and non-malignant cells. However, when conjugated to AS1411 derivatives, the cytotoxicity of the free ligands towards non-malignant cells was restrained. Furthermore, conjugated C3 showed an enhanced cytotoxicity against HeLa cancer cells. Confocal microscopy indicated that both G4 sequences appear to colocalize with nucleolin, suggesting their ability to recognize and bind nucleolin on the cell surface. Additionally, the results confirmed the internalization of these delivery systems into HeLa cancer cells and their sustained cell trafficking, although being able to dissociate intracellularly to deliver C8 to the nucleoli. Overall, we showed that AS1411-derived G4s can be used as a potential cancer drug delivery system for cervical cancer.


Assuntos
Laranja de Acridina/química , Aptâmeros de Nucleotídeos/química , Sistemas de Liberação de Medicamentos , Quadruplex G , Oligodesoxirribonucleotídeos/química , Laranja de Acridina/administração & dosagem , Laranja de Acridina/análogos & derivados , Aptâmeros de Nucleotídeos/administração & dosagem , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Ligantes , Oligodesoxirribonucleotídeos/administração & dosagem , Neoplasias do Colo do Útero/metabolismo
7.
Biotechniques ; 67(2): 70-73, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31238709

RESUMO

Quantitative assessment of changes in macro-autophagy is often performed through manual quantification of the number of LC3B foci in immunofluorescence microscopy images. This method is highly laborious, subject to image-field selection and foci-counting bias, and is not sensitive for analyzing changes in basal autophagy. Alternative methods such as flow cytometry and transmission electron microscopy require highly specialized, expensive instruments and time-consuming sample preparation. Immunoblots are prone to exposure-related variations and noise that prevents accurate quantification. We report a high-throughput, inexpensive, reliable and objective method for studying basal level and flux changes in late-stage autophagy using image cytometry and acridine orange staining.


Assuntos
Autofagia , Citometria por Imagem/métodos , Laranja de Acridina/análise , Linhagem Celular , Corantes Fluorescentes/análise , Humanos , Citometria por Imagem/economia
8.
Spectrochim Acta A Mol Biomol Spectrosc ; 221: 117150, 2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31176291

RESUMO

Herein, we have meticulously derived the nanosized fluorescent aggregates from pyrene Schiff base (PS) in DMSO:water (10:90) ratio. The aggregation property of PS molecule was characterized by SEM and TEM measurements, revealed the aggregated particles are in spherical shape with ~3 nm in size. Moreover, aggregates exhibit a high fluorescence quantum yield (48%) which was effectively used for the in vitro bioimaging of two different cancer cells such as A549 and MCF-7 cells in which it exhibiting excellent biocompatibility. Further, it was estimated the capability of twofold acridine orange/ethidium bromide (AO/EB) staining to identify the apoptotic associated changes in cancer cells. Additionally, the aggregates were successfully demonstrated as a luminescent probe for the perceptive biomolecule detection of bilirubin. On the other hand, the PS molecule was successfully utilized for protein binding and metal ion sensing studies. The interaction of bovine serum albumin (BSA) with PS molecule in DMSO was using fluorescence spectroscopic method and nature of interaction was also confirmed through molecular docking analysis. The PS molecule also acts as an excellent sensor for biologically important Fe3+ ion with detection limit of 336 nM. Overall, PS molecule can be a prospective material in biological field both in solution as well as aggregated forms.


Assuntos
Bilirrubina/análise , Corantes Fluorescentes/química , Ferro/análise , Pirenos/química , Soroalbumina Bovina/metabolismo , Células A549 , Laranja de Acridina , Sobrevivência Celular/efeitos dos fármacos , Etídio , Corantes Fluorescentes/toxicidade , Humanos , Limite de Detecção , Células MCF-7 , Simulação de Acoplamento Molecular , Imagem Molecular/métodos , Nanoestruturas/química , Nanoestruturas/toxicidade , Tamanho da Partícula , Bases de Schiff/química , Soroalbumina Bovina/química , Espectrometria de Fluorescência
9.
Cutan Ocul Toxicol ; 38(4): 375-383, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31223032

RESUMO

Purpose: A comet assay is one of the genotoxicity methods for evaluating the potential of chemicals to induce DNA strand breaks. To investigate the usefulness of comet assays for evaluating the genotoxic potential of ophthalmic solutions, a three-dimensional (3D) reconstructed human corneal epithelial model (3D corneal model) was exposed to conditions mimicking topical ocular instillation administration. Methods: The 3D corneal model was exposed to acridine orange, ethidium bromide, hydrogen peroxide, 1,1'-dimethyl-4,4'-bipyridinium dichloride (paraquat), 4-nitroquinoline 1-oxide (4-NQO), acrylamide and methyl methanesulfonate (MMS). To mimic the ocular surface condition to which ophthalmic solutions are administered, the exposure time was set to 1 minute. Likewise, human corneal epithelial (HCE-T) cells, as monolayer cultured cells, were exposed to the same chemicals, for comparison. Results: In the 3D corneal model, the amount of DNA fragments was statistically significantly increased in cells treated with each of the test chemicals except acrylamide. In HCE-T cells, the amount of DNA fragments was statistically significantly increased in acridine orange-, ethidium bromide-, hydrogen peroxide-, 4-NQO- and MMS-treated cells but not in paraquat- or acrylamide-treated cells. In the 3D corneal model, the lowest concentrations at which we observed DNA damage were about 100 times higher than the concentrations in HCE-T cells. Since the 3D corneal model is morphologically similar to human corneal tissue, form a multilayer and having tight junctions, it may be that the test chemicals only permeated about 1% into the 3D corneal model. Conclusion: These results suggest that the comet assay using 3D cell culture models may reflect in vivo conditions better than do monolayer cultured cells, and that the comet assay may be useful for the evaluation of genotoxic potential of topical ophthalmic solution.


Assuntos
Ensaio Cometa/métodos , Epitélio Anterior/efeitos dos fármacos , Soluções Oftálmicas/toxicidade , 4-Nitroquinolina-1-Óxido/toxicidade , Laranja de Acridina/toxicidade , Acrilamida/toxicidade , Administração Oftálmica , Linhagem Celular , Córnea , Dano ao DNA , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Epitélio Anterior/citologia , Epitélio Anterior/metabolismo , Etídio/toxicidade , Humanos , Peróxido de Hidrogênio/toxicidade , Técnicas In Vitro , Metanossulfonato de Metila/toxicidade , Paraquat/toxicidade , Quinolonas/toxicidade
10.
Oncogene ; 38(20): 3886-3902, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30692638

RESUMO

Palbociclib is a selective inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6) approved for the treatment of some cancers. The main mechanism of action of palbociclib is to induce cell cycle arrest and senescence on responsive cells. Here, we report that palbociclib concentrates in intracellular acidic vesicles, where it can be readily observed due to its intrinsic fluorescence, and it is released from these vesicles upon dilution or washing out of the extracellular medium. This reversible storage of drugs into acidic vesicles is generally known as lysosomal trapping and, based on this, we uncover novel properties of palbociclib. In particular, a short exposure of cells to palbociclib is sufficient to produce a stable cell-cycle arrest and long-term senescence. Moreover, after washing out the drug, palbociclib-treated cells release the drug to the medium and this conditioned medium is active on susceptible cells. Interestingly, cancer cells resistant to palbociclib also accumulate and release the drug producing paracrine senescence on susceptible cells. Finally, other lysosomotropic drugs, such as chloroquine, interfere with the accumulation of palbociclib into lysosomes, thereby reducing the minimal dose of palbociclib required for cell-cycle arrest and senescence. In summary, lysosomal trapping explains the prolonged temporal activity of palbociclib, the paracrine activity of exposed cells, and the cooperation with lysosomotropic drugs. These are important features that may help to improve the therapeutic dosing and efficacy of palbociclib. Finally, two other clinically approved CDK4/6 inhibitors, ribociclib and abemaciclib, present a similar behavior as palbociclib, suggesting that lysosomal trapping is a property common to all three clinically-approved CDK4/6 inhibitors.


Assuntos
Antineoplásicos/farmacocinética , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Piperazinas/farmacocinética , Piridinas/farmacocinética , Laranja de Acridina/química , Aminopiridinas/farmacocinética , Benzimidazóis/farmacocinética , Linhagem Celular Tumoral , Senescência Celular/efeitos dos fármacos , Cloroquina/farmacologia , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Citocinas/metabolismo , Corantes Fluorescentes/química , Humanos , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Inibidores de Proteínas Quinases/farmacocinética , Purinas/farmacocinética
11.
Reprod Domest Anim ; 54(2): 160-166, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30168871

RESUMO

The sperm chromatin structure assay is a method for assessment of sperm DNA fragmentation, a parameter reported to be negatively related to field fertility in several mammal species. This method calculates a DNA fragmentation index (DFI) whose high values indicate abnormal chromatin structure. In this study, running from March 2010 until June 2017, the aim was to assess sperm DFI in stored liquid extended semen from two different pig breeds, Norwegian Landrace (NL; n = 693) and Norwegian Duroc (ND; n = 655), and to evaluate the influence on total number of piglets born (TNB). There was a significantly higher median DFI (p < 0.0001) in ejaculates from the 478 ND boars compared to the 452 NL boars. Data from 19,496 NL litters and 3,877 ND litters of the same boars were retrieved. For either breed, sow herd (p < 0.0001), parity (p < 0.05) and DFI (p < 0.05) showed significant effects on TNB. The DFI was negatively correlated to TNB in both breeds. The boars with the 5% lowest TNB had a least square means DFI of 3.05% and 2.24% in NL and ND, respectively, compared to 1.67% and 1.23% for the boars with the 5% highest TNB (p < 0.01). The DFI and the motility of the same semen samples were negatively correlated (p < 0.0001), and the high and low TNB groups showed significant differences in motility. However, this difference could not be used for practical prediction of TNB group (92.1% vs. 89.7%; p = 0.0038 and 92.3% vs. 89.5%; p = 0.018; NL and ND, respectively). In conclusion, our results indicate that sperm DNA integrity in semen with good motility and morphology may be an additional prediction parameter for fertility in pigs.


Assuntos
Cromatina/química , Fragmentação do DNA/efeitos dos fármacos , Fertilidade , Análise do Sêmen/veterinária , Espermatozoides/fisiologia , Laranja de Acridina , Animais , Cruzamento , Cromatina/efeitos dos fármacos , Feminino , Citometria de Fluxo , Tamanho da Ninhada de Vivíparos , Masculino , Paridade , Gravidez , Preservação do Sêmen/veterinária , Espermatozoides/efeitos dos fármacos , Suínos
12.
Chem Biol Interact ; 297: 141-154, 2019 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-30419219

RESUMO

The present study reports the regulation of cytotoxicity of Cu doped ZnO nanoparticles in macrophages (RAW 264.7) due to altered physiochemical properties changes like electrical properties by controlled doping of Cu in ZnO. Cu-doped ZnO nanoparticles were prepared by High Energy Ball Milling technique (HEBM) and formed single phase Zn1-xCuxO (x = 0.0, 0.01, 0.02, 0.03) were called as pure ZnO, Cu1%, 2%, 3% respectively. Hexagonal wurtzite structure with size range of 22-26 nm was verified. FE-SEM with EDX analysis indicated the Cu doping effect on the surface morphology of ZnO. Zeta potential of Zn1-xCuxO was found to be elevated with increase in doping percentage of Cu (-36.6 mV to +18.2 mV). Dielectric constant was found to be decreased with increasing doping percentage. Increase in doping percentage enhanced cytotoxicity of Zn1-xCuxO in macrophages with LC50 of 62 µg/ml, 51 µg/ml, 40 µg/ml, 32 µg/ml. Granularity change of macrophages suggested doping influenced cellular uptake as consequence of zeta potential and dielectric properties changes. 3% Cu doped ZnO shown a higher ROS signal and apoptosis than 2% and 1% Cu doping with exhibition of ROS scavenging nature leading to apoptosis of prepared Cu doped ZnO nanoparticles. Our findings revealed mechanism of cytotoxicity of Zn1-xCuxO as a consequence of alteration in electric properties eliciting ROS scavenging leading to higher apoptosis with increasing doping percentage of Cu in ZnO.


Assuntos
Apoptose/efeitos dos fármacos , Cobre/química , Macrófagos/efeitos dos fármacos , Nanopartículas/química , Espécies Reativas de Oxigênio/metabolismo , Óxido de Zinco/toxicidade , Laranja de Acridina/química , Animais , Brometos/química , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Condutividade Elétrica , Fluorescência , Camundongos , Nanotecnologia , Tamanho da Partícula , Células RAW 264.7 , Relação Estrutura-Atividade , Propriedades de Superfície , Óxido de Zinco/síntese química , Óxido de Zinco/química
14.
J Mater Sci Mater Med ; 30(1): 4, 2018 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-30569403

RESUMO

The purpose of our study is to prepare a biomimetic porous silk fibroin (SF)/biphasic calcium phosphate (BCP) scaffold, and evaluate its performance in bone tissue regeneration. The differences in pore size, porosity, mechanical strength and biocompatibility of four different fibroin-containing scaffolds (0, 20, 40, and 60% SF) were studied in vitro. After inoculation with MC3T3-E1 cells, the ectopic bone formation ability of the SF/BCP bionic scaffold was evaluated in a rat model. The SEM and CT demonstrated that compared with pure BCP group (0% SF), the pore size and porosity of SF/BCP scaffolds were proportional to SF content, of which 40% of SF and 60% of SF groups were more suitable for cell growth. The compressive strength of SF/BCP scaffold was greater than that of the pure BCP scaffold, and showed a trend of first increasing and then decreasing with the increase of SF content, among which 40% of SF group had the maximum compressive strength (40.80 + 0.68) MPa. The SF/BCP scaffold had good biocompatibility, under the electron microscope, the cells can be smoothly attached to and propagated on the scaffold. After loading the osteoblasts, it showed excellent osteogenic capacity in the rat model. The SF/BCP scaffold can highly simulate the micro-environment of natural bone formation and can meet the requirements of tissue engineering. The SF/BCP biomimetic porous scaffold has excellent physical properties and biocompatibility. It can highly simulate the natural bone matrix composition and microenvironment, and can promote the adhesion and proliferation of osteoblasts. The SF/BCP scaffold has good ectopic osteogenesis after loading with osteoblasts, which can meet the requirements of scaffold materials in tissue engineering, and has broad application prospects in clinical application.


Assuntos
Biomimética , Regeneração Óssea/fisiologia , Fibroínas/química , Hidroxiapatitas/química , Tecidos Suporte , Células 3T3 , Laranja de Acridina , Fosfatase Alcalina/metabolismo , Animais , Materiais Biocompatíveis , Proliferação de Células , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Teste de Materiais , Camundongos , Microscopia Eletrônica de Varredura , Imagem Óptica , Osteogênese , Ratos , Coloração e Rotulagem
15.
Sci Rep ; 8(1): 17357, 2018 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-30478455

RESUMO

To understand the burgeoning challenges of metastasis, a microchannel of 35 µm diameter, constricted to 7 µm for a distance of 200 µm in a total length of 3 mm, was designed and fabricated using a mask aligner made of polydimethylsiloxane (PDMS) to mimic in vivo capillaries. A thin glass cover-slide was mounted on top to monitor the motion of single or aggregated malignant HeLa cells (size 17-30 µm) microscopically through the constricted microchannel at a constant flow rate of 30 µl/h. Quantitative deconvolution of high-speed videographs of a single cell of 30 µm revealed cellular deformation while passing through constriction, having elongation index, average transit velocity and entry time of 2.67, 18 mm/s and 5.1 ms, respectively. Morphological analysis of live and apoptotic cells by dual staining with Acridine Orange/Ethidium Bromide demonstrated retention of a significant viable cell population after exit through the constriction and a viability index of 50% was quantified by dye exclusion assay. The cumulative data for microfluidic parameters, morphology and relevant metastatic MMP2 gene expression efficiency measured by real-time polymerase chain reaction revealed retention of virulence potency that could possibly cause metastasis, would be beneficial in developing futuristic MEMS device for cancer theranostics.


Assuntos
Sobrevivência Celular/fisiologia , Metástase Neoplásica/patologia , Neoplasias do Colo do Útero/patologia , Laranja de Acridina/administração & dosagem , Apoptose/fisiologia , Linhagem Celular Tumoral , Etídio/administração & dosagem , Feminino , Células HeLa , Humanos , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/métodos
16.
Sci Rep ; 8(1): 16245, 2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30390005

RESUMO

Automated blood cell counters can distinguish cells based on their size and the presence or absence of a nucleus. However, most vertebrates have nucleated blood cells that cannot be counted automatically. We established an alternative automatic method for counting peripheral blood cells by staining cells with the fluorescent dye acridine orange (AO) and analysing cell populations using flow cytometry (FCM). As promising new animal models, we chose Xenopus laevis and three inbred strains of X. tropicalis. We compared the haematological phenotypes, including blood cell types, cell sizes, cellular structure, and erythrocyte lifespans/turnover rate among X. laevis and the three inbred strains of X. tropicalis. Each cell type from X. laevis was sorted according to six parameters: forward- and side-scattered light emission, AO red and green fluorescence intensity, and cellular red and green fluorescence. Remarkably, the erythrocyte count was the highest in the Golden line, suggesting that genetic factors were associated with the blood cells. Furthermore, immature erythrocytes in anaemic X. laevis could be separated from normal blood cells based on red fluorescence intensity. These results show that FCM with AO staining allows for an accurate analysis of peripheral blood cells from various species.


Assuntos
Células Sanguíneas , Separação Celular/métodos , Citometria de Fluxo/métodos , Coloração e Rotulagem/métodos , Xenopus laevis/sangue , Laranja de Acridina/química , Animais , Animais Endogâmicos/sangue , Animais Selvagens/sangue , Contagem de Células Sanguíneas/métodos , Corantes Fluorescentes/química , Modelos Animais , Especificidade da Espécie
17.
Analyst ; 144(1): 274-283, 2018 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-30398257

RESUMO

Sensing of pyrophosphate (PPi) is helpful to better understand many life processes and diagnose various early-stage diseases. However, many traditional reported methods based on artificial receptors for sensing of PPi exhibit some disadvantages including difficulties in designing appropriate binding sites and complicated multi-step assembly/functionalization. Thus, it is significantly important and a big challenge to know how to use a simple molecular self-assembly or an interaction system to solve the above-mentioned limits to achieve the quantitative analysis of specific substances in the system. Based on the natural connection and similarity (such as stimulus responsiveness) between sensing and logic computing, in this study, the Boolean logic tree of molecular self-assembly system based on the cobalt oxyhydroxide (CoOOH) nanoplatform is constructed and applied to organize and connect "plug and play" molecular events (fluorescent dye, acridine orange and anion, PPi). By using molecules as inputs and the corresponding fluorescence signal as the output, the CoOOH-based molecular self-assembly system can be programmed for three-input fluorescent Boolean logic computation, fluorescent three-state logic computation, detection of PPi (linear range from 50 to 6400 nM with a detection limit of 20 nM) and even for imaging in living cancer cells and in vivo (in systems such as Zebrafish and Carassius auratus). Our approach adds a new dimension for expanding molecular logic computing and sensing systems, which will not only provide more opportunities for developing novel logic computing paradigms, but also be helpful in promoting the development and applications of intelligent molecular computing and sensing systems.


Assuntos
Cobalto/química , Difosfatos/sangue , Lógica , Nanoestruturas/química , Óxidos/química , Laranja de Acridina/química , Animais , Corantes Fluorescentes/química , Carpa Dourada , Humanos , Limite de Detecção , Microscopia de Fluorescência/métodos , Peixe-Zebra
18.
Biochim Biophys Acta Gen Subj ; 1862(12): 2824-2834, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30251671

RESUMO

BACKGROUND: The fluorescent dye 10-N-nonyl acridine orange (NAO) is widely used as a mitochondrial marker. NAO was reported to have cytotoxic effects in cultured eukaryotic cells when incubated at high concentrations. Although the biochemical response of NAO-induced toxicity has been well identified, the underlying molecular mechanism has not yet been explored in detail. METHODS: We use optical techniques, including fluorescence confocal microscopy and lifetime imaging microscopy (FLIM) both in model membranes built up as giant unilamellar vesicles (GUVs) and cultured cells. These experiments are complemented with computational studies to unravel the molecular mechanism that makes NAO cytotoxic. RESULTS: We have obtained direct evidence that NAO promotes strong membrane adhesion of negatively charged vesicles. The attractive forces are derived from van der Waals interactions between anti-parallel H-dimers of NAO molecules from opposing bilayers. Semi-empirical calculations have confirmed the supramolecular scenario by which anti-parallel NAO molecules form a zipper of bonds at the contact region. The membrane remodeling effect of NAO, as well as the formation of H-dimers, was also confirmed in cultured fibroblasts, as shown by the ultrastructure alteration of the mitochondrial cristae. CONCLUSIONS: We conclude that membrane adhesion induced by NAO stacking accounts for the supramolecular basis of its cytotoxicity. GENERAL SIGNIFICANCE: Mitochondria are a potential target for cancer and gene therapies. The alteration of the mitochondrial structure by membrane remodeling agents able to form supramolecular assemblies via adhesion properties could be envisaged as a new therapeutic strategy.


Assuntos
Morte Celular , Bicamadas Lipídicas , Laranja de Acridina/análogos & derivados , Laranja de Acridina/química , Animais , Membrana Celular/metabolismo , Células Cultivadas , Dimerização , Fibroblastos/citologia , Corantes Fluorescentes/química , Camundongos , Microscopia Confocal , Microscopia de Fluorescência
19.
Sci Rep ; 8(1): 14078, 2018 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-30232360

RESUMO

Ethidium bromide (EB) and acridine orange (AO) bind to nucleic acids and are thus considered as potential mutagens. In this study, the effects of EB and AO on the germination behaviours of white, yellow, red, and purple maize seeds were investigated. The results indicate that low concentrations of EB (50 µg mL-1) and AO (500 µg mL-1) promote germination, particularly for the white and yellow seeds. However, high concentrations of EB (0.5 mg mL-1) and AO (5 mg mL-1) significantly inhibit germination, with the level of inhibition decreasing in the following order: white > yellow > red > purple. In addition, EB and AO induce H2O2 production in a concentration-dependent manner. The effects of these mutagens on seed germination were partly reversed by dimethyl thiourea, a scavenger of reactive oxygen species (ROS), and diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, while the effects were enhanced by treatment with H2O2 and 3-amino-1,2,4-triazole, a specific inhibitor of catalase. In addition, AO and EB profoundly increased NADPH oxidase activities in germinating seeds. The treatment of seeds with EB and AO did not affect the growth or drought tolerance of the resultant seedlings. The findings suggest that the mechanism of mutagen toxicity is related to the induction of ROS production.


Assuntos
Germinação/efeitos dos fármacos , Mutagênicos/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Zea mays/fisiologia , Laranja de Acridina/toxicidade , Relação Dose-Resposta a Droga , Secas , Etídio/toxicidade , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , NADPH Oxidases/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Zea mays/efeitos dos fármacos
20.
J Biochem Mol Toxicol ; 32(10): e22206, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30091159

RESUMO

Ginger (Zingiber officinale Roscoe), a monocotyledonous herb, is widely used as an herbal medicine owing to the phytoconstituents it possesses. In the current study, the quantity of [6]-gingerol, the major phenolic ketone, in the fresh ginger and dried ginger rhizome was found to be 6.11 µg/mg and 0.407 µg/mg. Furthermore, [6]-gingerol was assessed for its antiapoptotic effects in human gastric adenocarcinoma (AGS) cells evidenced by acridine orange/ethidium bromide staining technique and Annexin-V assay. An increase in reactive oxygen species (ROS) generation led to a decrease in mitochondrial membrane potential (MMP) and subsequent induction of apoptosis. Results disclose that perturbations in MMP are associated with deregulation of Bax/Bcl-2 ratio at protein level, which leads to upregulation of cytochrome-c triggering the caspase cascade. These enduringly suggest that [6]-gingerol can be effectively used for targeting the mitochondrial energy metabolism to manage gastric cancer cells.


Assuntos
Adenocarcinoma/patologia , Apoptose/efeitos dos fármacos , Catecóis/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Álcoois Graxos/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Gástricas/patologia , Laranja de Acridina/química , Adenocarcinoma/enzimologia , Adenocarcinoma/metabolismo , Anexina A5/metabolismo , Caspases/metabolismo , Catecóis/análise , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Citocromos c/metabolismo , Etídio/química , Álcoois Graxos/análise , Gengibre/química , Humanos , Extratos Vegetais/química , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Neoplasias Gástricas/enzimologia , Neoplasias Gástricas/metabolismo , Regulação para Cima , Proteína X Associada a bcl-2/metabolismo
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