Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
1.
Anal Chem ; 94(11): 4610-4616, 2022 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-35275492

RESUMEN

The dark-field microscopy (DFM) imaging technology has the advantage of a high signal-to-noise ratio, and it is often used for real-time monitoring of plasmonic resonance scattering and biological imaging at the single-nanoparticle level. Due to the limitation of the optical diffraction limit, it is still a challenging task to accurately distinguish two or more nanoparticles whose distance is less than the diffraction limit. Here, we propose a computational strategy based on a deep learning framework (NanoNet), which will realize the effective segmentation of the scattered light spots in diffraction-limited DFM images and obtain high-resolution plasmonic light scattering imaging. A small data set of DFM and the corresponding scanning electron microscopy (SEM) image pairs are used to learn for obtaining a highly resolved semantic imaging model using NanoNet, and thus highly resolved DFM images matching the resolution of those acquired using SEM can be obtained. Our method has the ability to transform diffraction-limited DFM images to highly resolved ones without adding a complex optical system. As a proof of concept, a highly resolved DFM image of living cells through the NanoNet technique is successfully made, opening up a new avenue for high-resolution optical nanoscopic imaging.


Asunto(s)
Aprendizaje Profundo , Nanopartículas , Microscopía/métodos , Imagen Óptica
2.
Anal Chem ; 93(4): 2619-2626, 2021 02 02.
Artículo en Inglés | MEDLINE | ID: mdl-33427440

RESUMEN

Plasmonic nanoparticles, which have excellent local surface plasmon resonance (LSPR) optical and chemical properties, have been widely used in biology, chemistry, and photonics. The single-particle light scattering dark-field microscopy (DFM) imaging technique based on a color-coded analytical method is a promising approach for high-throughput plasmonic nanoparticle scatterometry. Due to the interference of high noise levels, accurately extracting real scattering light of plasmonic nanoparticles in living cells is still a challenging task, which hinders its application for intracellular analysis. Herein, we propose an automatic and high-throughput LSPR scatterometry technique using a U-Net convolutional deep learning neural network. We use the deep neural networks to recognize the scattering light of nanoparticles from background interference signals in living cells, which have a dynamic and complicated environment, and construct a DFM image semantic analytical model based on the U-Net convolutional neural network. Compared with traditional methods, this method can achieve higher accuracy, stronger generalization ability, and robustness. As a proof of concept, the change of intracellular cytochrome c in MCF-7 cells under UV light-induced apoptosis was monitored through the fast and high-throughput analysis of the plasmonic nanoparticle scattering light, providing a new strategy for scatterometry study and imaging analysis in chemistry.

3.
Circulation ; 128(17): 1897-909, 2013 Oct 22.
Artículo en Inglés | MEDLINE | ID: mdl-23995537

RESUMEN

BACKGROUND: Although methods for generating cardiomyocytes from pluripotent stem cells have been reported, current methods produce heterogeneous mixtures of cardiomyocytes and noncardiomyocyte cells. Here, we report an entirely novel system in which pluripotent stem cell-derived cardiomyocytes are purified by cardiomyocyte-specific molecular beacons (MBs). MBs are nanoscale probes that emit a fluorescence signal when hybridized to target mRNAs. METHOD AND RESULTS: Five MBs targeting mRNAs of either cardiac troponin T or myosin heavy chain 6/7 were generated. Among 5 MBs, an MB that targeted myosin heavy chain 6/7 mRNA (MHC1-MB) identified up to 99% of HL-1 cardiomyocytes, a mouse cardiomyocyte cell line, but <3% of 4 noncardiomyocyte cell types in flow cytometry analysis, which indicates that MHC1-MB is specific for identifying cardiomyocytes. We delivered MHC1-MB into cardiomyogenically differentiated pluripotent stem cells through nucleofection. The detection rate of cardiomyocytes was similar to the percentages of cardiac troponin T- or cardiac troponin I-positive cardiomyocytes, which supports the specificity of MBs. Finally, MHC1-MB-positive cells were sorted by fluorescence-activated cell sorter from mouse and human pluripotent stem cell differentiating cultures, and ≈97% cells expressed cardiac troponin T or cardiac troponin I as determined by flow cytometry. These MB-based sorted cells maintained their cardiomyocyte characteristics, which was verified by spontaneous beating, electrophysiological studies, and expression of cardiac proteins. When transplanted in a myocardial infarction model, MB-based purified cardiomyocytes improved cardiac function and demonstrated significant engraftment for 4 weeks without forming tumors. CONCLUSIONS: We developed a novel cardiomyocyte selection system that allows production of highly purified cardiomyocytes. These purified cardiomyocytes and this system can be valuable for cell therapy and drug discovery.


Asunto(s)
Trasplante de Células/métodos , Infarto del Miocardio/terapia , Miocitos Cardíacos/citología , Células Madre Pluripotentes/citología , ARN Mensajero/aislamiento & purificación , Potenciales de Acción/fisiología , Animales , Biomarcadores , Diferenciación Celular/fisiología , Línea Celular , Células Cultivadas , Citometría de Flujo/métodos , Humanos , Ratones , Miocitos Cardíacos/fisiología , Cadenas Pesadas de Miosina/genética , Nanotecnología , Conformación de Ácido Nucleico , Células Madre Pluripotentes/fisiología , Sondas ARN/química , Sondas ARN/aislamiento & purificación , ARN Mensajero/química , Troponina I/genética , Troponina T/genética
4.
J Neural Transm (Vienna) ; 116(12): 1643-9, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19756370

RESUMEN

Rivastigmine is a potent acetyl- and butyrylcholinesterase inhibitor widely used for cognitive improvement in Alzheimer's disease (AD) therapy. However, dose-limiting adverse effects restrict its tolerability and clinical outcomes. This study explored new combined therapy, in which peripheral cholinergic adverse effects and central cognitive amelioration of rivastigmine were differentiated by a peripheral cholinoceptor antagonist anisodamine. The results demonstrated that rivastigmine (0.75 and 2.0 mg/kg) could significantly reverse the scopolamine-induced cognitive deficit in mice through passive avoidance test. Nevertheless, a high dose of rivastigmine (3.25 mg/kg) would compromise cognitive amelioration and produce obvious adverse effects, including hypersalivation, intestinal hyperperistalsis and muscle cramp. Interestingly, concomitant administration of anisodamine (10 mg/kg) effectively counteracted both the muscarinergic and nicotinergic adverse effects, while facilitating cognitive amelioration of rivastigmine (3.25 mg/kg). These findings provide an insight into the feasibility of combined therapy with cholinesterase inhibitors and peripheral cholinoceptor antagonists for the treatment of AD.


Asunto(s)
Antagonistas Colinérgicos/farmacología , Trastornos del Conocimiento/tratamiento farmacológico , Fármacos Neuroprotectores/efectos adversos , Enfermedades del Sistema Nervioso Periférico/tratamiento farmacológico , Fenilcarbamatos/efectos adversos , Alcaloides Solanáceos/farmacología , Animales , Reacción de Prevención/efectos de los fármacos , Antagonistas Colinérgicos/administración & dosificación , Cognición/efectos de los fármacos , Trastornos del Conocimiento/inducido químicamente , Quimioterapia Combinada , Intestinos/efectos de los fármacos , Intestinos/fisiología , Masculino , Ratones , Ratones Endogámicos , Calambre Muscular/inducido químicamente , Calambre Muscular/tratamiento farmacológico , Fármacos Neuroprotectores/administración & dosificación , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Fenilcarbamatos/administración & dosificación , Distribución Aleatoria , Rivastigmina , Salivación/efectos de los fármacos , Escopolamina , Alcaloides Solanáceos/administración & dosificación
5.
Neurosci Lett ; 443(3): 241-5, 2008 Oct 10.
Artículo en Inglés | MEDLINE | ID: mdl-18672024

RESUMEN

Two naturally occurring tropane alkaloids, anisodamine and scopolamine, structurally dissimilar in one OH group, are well established as muscarinic acetylcholine receptor (mAChR) antagonists in clinic and basic research. However, experimental evidence for central effects of anisodamine is limited and conflicting compared with that of scopolamine. In the present study, Morris water maze test, long-term potentiation (LTP) recording and receptor radioligand binding assays were used to explore the disparity in neuropsychopharmacological influences of anisodamine versus scopolamine and possible mechanisms. Anisodamine, at 10-40-fold higher doses than those of scopolamine, did not produce any spatial cognitive deficits as scopolamine, but tended to improve cognition at the repeated high doses. LTP in vivo was then adopted to predict BBB permeability of the muscarinic antagonists following systemic drug administration. Contrary to scopolamine, anisodamine did not influence the formation of LTP in the CA(1) region of rat hippocampus at 40-fold higher dose than that of scopolamine. Additionally, receptor radioligand binding assays (RRLBA) revealed that the binding affinity of anisodamine to mice brain mAChR was much lower than that of scopolamine. The findings suggested that anisodamine did not impair cognition nor depress LTP primarily due to its poor BBB permeability. This work enlarged knowledge of structure-activity relationship among tropane alkaloids, meanwhile providing evidence for more reasonable drug prescription in clinic.


Asunto(s)
Depuradores de Radicales Libres/farmacología , Neurofarmacología , Psicofarmacología , Escopolamina/farmacología , Alcaloides Solanáceos/farmacología , Animales , Conducta Animal/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Depuradores de Radicales Libres/química , Hipocampo/efectos de los fármacos , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Ratones Endogámicos , Distribución Aleatoria , Tiempo de Reacción/efectos de los fármacos , Escopolamina/química , Alcaloides Solanáceos/química
6.
Eur J Pharmacol ; 531(1-3): 47-53, 2006 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-16442097

RESUMEN

14-Benzoyltalatisamine is a potent and selective blocker of the delayed rectifier K+ channel found in a computational virtual screening study. The compound was found to block the K+ channel from the extracellular side. However, it is unclear whether 14-benzoyltalatisamine shares the same block mechanism with tetraethylammonium (TEA). In order to elucidate how the hit compound found by the virtual screening interacts with the outer vestibule of the K+ channel, the effects of 14-benzoyltalatisamine and TEA on the delayed rectifier K+ current of rat dissociated hippocampal neurons were compared using whole-cell voltage-clamp recording. External application of 14-benzoyltalatisamine and TEA reversibly inhibited the current with IC50 values of 10.1+/-2.2 microM and 1.05+/-0.21 mM, respectively. 14-Benzoyltalatisamine exerted voltage-dependent inhibition, markedly accelerated the decay of the current, and caused a significant hyperpolarizing shift of the steady-state activation curve, whereas TEA caused voltage-independent inhibition, without affecting the kinetic parameters of the current. The blockade by 14-benzoyltalatisamine, but not by TEA, was significantly diminished in a high K+ (60 mM) external solution. The potency of 14-benzoyltalatisamine was markedly reduced in the presence of 15 mM TEA. The results suggest that 14-benzoyltalatisamine bind to the external pore entry of the delayed rectifier K+ channel with partial insertion into the selectivity filter, which is in conformity with that predicted by the molecular docking model in the virtual screening.


Asunto(s)
Aconitina/análogos & derivados , Canales de Potasio de Tipo Rectificador Tardío/fisiología , Bloqueadores de los Canales de Potasio/farmacología , Aconitina/química , Aconitina/farmacología , Animales , Relación Dosis-Respuesta a Droga , Potenciales de la Membrana/efectos de los fármacos , Estructura Molecular , Potasio/farmacología , Bloqueadores de los Canales de Potasio/química , Células Piramidales/efectos de los fármacos , Células Piramidales/fisiología , Ratas , Ratas Sprague-Dawley , Tetraetilamonio/farmacología , Factores de Tiempo
7.
Stem Cell Reports ; 5(6): 1239-1249, 2015 Dec 08.
Artículo en Inglés | MEDLINE | ID: mdl-26651608

RESUMEN

Isolation of ventricular cardiomyocytes (vCMs) has been challenging due to the lack of specific surface markers. Here we show that vCMs can be purified from differentiating mouse embryonic stem cells (mESCs) using molecular beacons (MBs) targeting specific intracellular mRNAs. We designed MBs (IRX4 MBs) to target mRNA encoding Iroquois homeobox protein 4 (Irx4), a transcription factor specific for vCMs. To purify mESC vCMs, IRX4 MBs were delivered into cardiomyogenically differentiating mESCs, and IRX4 MBs-positive cells were FACS-sorted. We found that, of the cells isolated, ~98% displayed vCM-like action potentials by electrophysiological analyses. These MB-purified vCMs continuously maintained their CM characteristics as verified by spontaneous beating, Ca(2+) transient, and expression of vCM-specific proteins. Our study shows the feasibility of isolating pure vCMs via cell sorting without modifying host genes. The homogeneous and functional ventricular CMs generated via the MB-based method can be useful for disease investigation, drug discovery, and cell-based therapies.


Asunto(s)
Separación Celular/métodos , Células Madre Embrionarias/citología , Ventrículos Cardíacos/citología , Proteínas de Homeodominio/genética , Miocitos Cardíacos/citología , Potenciales de Acción , Animales , Secuencia de Bases , Diferenciación Celular , Células Cultivadas , Citometría de Flujo , Ratones , Sondas de Oligonucleótidos/genética , ARN Mensajero/genética
8.
Sheng Li Xue Bao ; 56(5): 651-5, 2004 Oct 25.
Artículo en Zh | MEDLINE | ID: mdl-15497049

RESUMEN

To explore the electrophysiological characteristics of embryonic cardiomyocytes, single embryonic cardiomyocytes were obtained from mice at different periods by a collegenase B digestion approach, whole cell patch clamp recording technique was used to record I(f) and I(Ca-L), and spontaneous action potential was also recorded. The morphological and spontaneous contractile properties of the isolated cells appeared to be typical embryonic cardiomyocytes when the cells were assessed by phase-contrast microscope. Whole cell recording of isolated cells is easily performed by the whole cell patch clamp technique. Elelctrophysiological properties of I(f) and I(Ca-L) from embryonic cardiomyocytes have been proved to be similar to those from adult pacemaker cells or cardiomyocytes. The established method of isolation is simple, stable, effective and reliable. It allows to obtain as early as 8.5-day embryonic myocytes. The electrophysiological recording of embryonic cardiomyocytes will provide a useful model for exploring the electrophysiological characteristics of embryonic cardiomyocytes and the possible mechanism underlying some heart diseases.


Asunto(s)
Separación Celular/métodos , Miocitos Cardíacos/citología , Miocitos Cardíacos/fisiología , Potenciales de Acción/fisiología , Animales , Células Cultivadas , Electrofisiología , Embrión de Mamíferos , Ratones , Técnicas de Placa-Clamp
9.
Int J Cardiol ; 168(1): 41-52, 2013 Sep 20.
Artículo en Inglés | MEDLINE | ID: mdl-23044428

RESUMEN

BACKGROUND: Human pluripotent stem cells (hPSCs) hold great promise for treating ischemic heart disease. However, current protocols for differentiating hPSCs either result in low yields or require expensive cytokines. METHODS: Here we developed a novel two dimensional (2D) stepwise differentiation system that generates a high yield of cardiomyocytes (CMs) from hPSCs without using special cytokines. Initially, undifferentiated hPSCs were transferred onto Matrigel-coated plates without forming embryoid bodies (EBs) for a few days and were cultured in bFGF-depleted human embryonic stem cells (hESCs) medium. When linear cell aggregation appeared in the margins of the hPSC colonies, the medium was changed to DMEM supplemented with 10% fetal bovine serum (FBS). Thereafter when cell clusters became visible, the medium was changed to DMEM with 20% FBS. RESULTS AND CONCLUSIONS: At about two weeks of culture, contracting clusters began to appear and the number of contracting clusters continuously increased, reaching approximately 70% of all clusters. These clusters were dissociated by two-step enzyme treatment to monolayered CMs, of which ~90% showed CM phenotypes confirmed by an α-myosin heavy chain reporter system. Electrophysiologic studies demonstrated that the hPSC-derived CMs showed three major CM action potential types with 61 to 78% having a ventricular-CM phenotype. This differentiation system showed a clear spatiotemporal role of the surrounding endodermal cells for differentiation of mesodermal cell clusters into CMs. In conclusion, this system provides a novel platform to generate CMs from hPSCs at high yield without using cytokines and to study the development of hPSCs into CMs.


Asunto(s)
Diferenciación Celular/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Células Madre Pluripotentes Inducidas/trasplante , Miocitos Cardíacos/fisiología , Miocitos Cardíacos/trasplante , Cultivo Primario de Células/métodos , Animales , Humanos , Masculino , Infarto del Miocardio/patología , Infarto del Miocardio/cirugía , Células Madre Pluripotentes/fisiología , Células Madre Pluripotentes/trasplante , Cultivo Primario de Células/tendencias , Ratas Desnudas
10.
Neurosci Lett ; 461(1): 25-9, 2009 Sep 11.
Artículo en Inglés | MEDLINE | ID: mdl-19463897

RESUMEN

A large amount of in vitro studies demonstrate suppression of M-current in hippocampal neurons by Kv7/M channel blocker results in depolarization of membrane potential and release of neurotransmitters, such as acetylcholine and glutamate, suggesting that Kv7/M channel may play important roles in regulating synaptic plasticity. In the present study, we examined the in vivo effect of Kv7/M channel inhibition on the long-term potentiation (LTP) induction at basal dendrites in hippocampal CA1 area of urethane-anaesthetized rats. The Kv7/M channel was inhibited by intraperitoneal injection of XE991 (10mg/kg) and the LTP of field excitatory postsynaptic potential (fEPSP) was induced by supra-threshold high frequency stimulation (S1 HFS). A weak protocol which was just below the threshold for evoking LTP was used as sub-threshold high frequency stimulation (S2 HFS). XE991 did not significantly alter the slope of fEPSP and the magnitude of LTP induced by S1 HFS, suggesting that Kv7/M channel inhibition had little or no effect on glutamatergic transmission under basal conditions. However, XE991 could make S2 HFS evoke LTP even after the application of the muscarinic cholinergic (mACh) receptor antagonist scopolamine, suggesting that Kv7/M channel inhibition lowered the threshold for LTP induction and the effect was independent of muscarinic activation. Based on the above findings, we concluded that the facilitating effect of XE991 on LTP induction is not mediated by its ability to enhance the release of acetylcholine; therefore, Kv7/M channel blockers may provide a therapeutic benefit to cholinergic deficiency-related cognitive impairment, e.g., Alzheimer's disease.


Asunto(s)
Antracenos/farmacología , Hipocampo/efectos de los fármacos , Canal de Potasio KCNQ2/antagonistas & inhibidores , Canal de Potasio KCNQ3/antagonistas & inhibidores , Potenciación a Largo Plazo , Bloqueadores de los Canales de Potasio/farmacología , Receptores Muscarínicos/fisiología , Animales , Dendritas/fisiología , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores , Hipocampo/fisiología , Masculino , Antagonistas Muscarínicos/farmacología , Ratas , Ratas Wistar , Escopolamina/farmacología , Sinapsis/fisiología
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA