RESUMEN
Cell morphology is a fundamental feature used to evaluate patient specimens in pathologic analysis. However, traditional cytopathology analysis of patient effusion samples is limited by low tumor cell abundance coupled with the high background of nonmalignant cells, restricting the ability of downstream molecular and functional analyses to identify actionable therapeutic targets. We applied the Deepcell platform that combines microfluidic sorting, brightfield imaging, and real-time deep learning interpretations based on multidimensional morphology to enrich carcinoma cells from malignant effusions without cell staining or labels. Carcinoma cell enrichment was validated with whole genome sequencing and targeted mutation analysis, which showed a higher sensitivity for detection of tumor fractions and critical somatic variant mutations that were initially at low levels or undetectable in presort patient samples. Our study demonstrates the feasibility and added value of supplementing traditional morphology-based cytology with deep learning, multidimensional morphology analysis, and microfluidic sorting.
Asunto(s)
Líquidos Corporales , Carcinoma , Derrame Pleural Maligno , Humanos , Inteligencia Artificial , Derrame Pleural Maligno/diagnóstico , Derrame Pleural Maligno/patologíaRESUMEN
BACKGROUND: Acne vulgaris is one of the most common dermatological diseases. Some topical treatments for acne used in combination, such as blue light and topical antibiotics (such as metronidazole) by needle-free jet injection (NFJI), are becoming prevalent in clinical practice, but the efficacy remains uncertain. METHODS: In order to investigate the effect of blue light combined with metronidazole by NFJI in the treatment of acne, the 251 enrolled patients were randomly assigned into the blue light group, metronidazole (MNZ) group, and MNZ + blue light group, and then received 6-weeks' treatment. A variety of objective and subjective methods such as clinical pictures, skin barrier physiological parameters (including trans-epidermal water loss (TEWL), stratum corneum hydration, facail surface sebum, erythema and pigmentation), the Investigator Global Assessment score, acne lesion count assessment, Patients' Self-Assessment, and VAS score were used to evaluate the efficacy and side effects of the treatments. RESULTS: Compared to the baseline, the MNZ + blue light group showed significant improvement in acne lesion count reduction, TEWL, straum corneum hydration, facial surface sebum and erythema (p < 0.05). The MNZ + blue light group showed significant differences compared with the MNZ group and blue light group in terms of acne lesion count reduction and erythema (p < 0.05) Compared to the MNZ group, the MNZ + blue light group demonstrated significant improvement in TEWL and sebum (p < 0.05). While compared to the blue light group, the MNZ + blue light group showed significant improvement in hydration (p < 0.05). There was no statistically significant difference among the three groups in pigmentation (p > 0.05). CONCLUSION: The combination of MNZ by NFJI and blue light has a synergistic effect and can relieve acne skin lesion within 6 weeks in the treatment of moderate and moderate-to-severe facial acne vulgaris, meanwhile, this method has a good safety.
Asunto(s)
Acné Vulgar , Metronidazol , Humanos , Metronidazol/efectos adversos , Resultado del Tratamiento , Fototerapia , Acné Vulgar/terapia , Acné Vulgar/tratamiento farmacológico , Inyecciones a ChorroRESUMEN
Because China produces the most crayfish in the world, safe solutions must be improved to mitigate the risks of ongoing heavy metal stressors accumulation. This study aimed to use Saccharomyces cerevisiae as a bioremediation agent to counteract the harmful effect of cadmium (Cd) on crayfish (Procambarus clarkia). Our study used three concentrations of S. cerevisiae on crayfish feed to assess their Cd toxicity remediation effect by measuring total antioxidant capacity (TAC) and the biomarkers related to oxidative stress like malondialdehyde (MDA), protein carbonyl derivates (PCO), and DNA-protein crosslink (DPC). A graphite furnace atomic absorption spectroscopy device was used to determine Cd contents in crayfish. Furthermore, the mRNA expression levels of lysozyme (LSZ), metallothionein (MT), and prophenoloxidase (proPO) were evaluated before and following the addition of S. cerevisiae. The results indicated that S. cerevisae at 5% supplemented in fundamental feed exhibited the best removal effect, and Cd removal rates at days 4th, 8th, 12th, and 21st were 12, 19, 29.7, and 66.45%, respectively, which were significantly higher than the basal diet of crayfish. The addition of S. cerevisiae increased TAC levels. On the other hand, it decreased MDA, PCO, and DPC, which had risen due to Cd exposure. Furthermore, it increased the expression of proPO, which was reduced by Cd exposure, and decreased the expression of LSZ and MT, acting in the opposite direction of Cd exposure alone. These findings demonstrated that feeding S. cerevisiae effectively reduces the Cd from crayfish and could be used to develop Cd-free crayfish-based foods.
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Cadmio , Saccharomyces cerevisiae , Animales , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Cadmio/metabolismo , Astacoidea/metabolismo , Hemocitos/metabolismo , Estrés Oxidativo , Antioxidantes/metabolismoRESUMEN
Cells are the singular building blocks of life, and a comprehensive understanding of morphology, among other properties, is crucial to the assessment of underlying heterogeneity. We developed Computational Sorting and Mapping of Single Cells (COSMOS), a platform based on Artificial Intelligence (AI) and microfluidics to characterize and sort single cells based on real-time deep learning interpretation of high-resolution brightfield images. Supervised deep learning models were applied to characterize and sort cell lines and dissociated primary tissue based on high-dimensional embedding vectors of morphology without the need for biomarker labels and stains/dyes. We demonstrate COSMOS capabilities with multiple human cell lines and tissue samples. These early results suggest that our neural networks embedding space can capture and recapitulate deep visual characteristics and can be used to efficiently purify unlabeled viable cells with desired morphological traits. Our approach resolves a technical gap in the ability to perform real-time deep learning assessment and sorting of cells based on high-resolution brightfield images.
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Inteligencia Artificial , Aprendizaje Profundo , Humanos , Movimiento Celular , Línea Celular , Separación Celular , ColorantesRESUMEN
Salmonella enteritidis can cause significant morbidity and mortality in humans and economic loss in the animal industry. Improving the innate immunity is an effective method to prevent S. enteritidis infection. Pediococcus pentosaceus is a Gram-positive coccus which had probiotics properties. Numerous previously published studies reported that probiotics were beneficial to gut microbiota by changing the intestinal flora structure and inhibiting the harmful microbial growth to enhance the innate immunity. We investigated the immunological effects of P. pentosaceus on Salmonella-infected chickens by the following experiment. A total of 120 broilers from AA line were fed and divided into 2 groups (treated and control groups) for the experiment from day 1. The control group was fed with the basic diet, while the treated group was fed with the basic diet adding P. pentosaceus microcapsule with the bacterial concentration of 1 g/kg in the feed and bacterial counts 2.5 × 109 CFU/g. All the birds were given with 0.5 ml of S. enteritidis bacterial suspension (109 CFU/ml) through oral cavity at day 9. The number of dead birds was recorded and used in the analysis. The bacterial culture method and quantitative real-time PCR analysis were used to evaluate the effects of P. pentosaceus on chickens infected with S. enteritidis and to ascertain the mechanism of the effect. The results showed that the P. pentosaceus could restrain the pathogenicity of S. enteritidis and reduce the death rate from 44.4% to 23.3%. The flora in the caecum exhibited "rising-declining" trends, and the gene (TLR4, MyD88, TRAF6 NF-κB, IFN-ß, TNF-a, IL6, and IL8) expression pattern was different between the experimental and control group. P. pentosaceus as a probiotic may competitively inhibit the growth of S. enteritidis and control the inflammatory response through regulating the gene expression which involved in the toll-like receptor pathway and inflammation pathway.
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Pollos/microbiología , Pediococcus pentosaceus/inmunología , Enfermedades de las Aves de Corral/microbiología , Enfermedades de las Aves de Corral/terapia , Probióticos/uso terapéutico , Salmonelosis Animal/microbiología , Salmonelosis Animal/terapia , Salmonella enteritidis/patogenicidad , Animales , Proteínas Aviares/genética , Proteínas Aviares/inmunología , Ciego/inmunología , Ciego/microbiología , Microbioma Gastrointestinal/genética , Microbioma Gastrointestinal/inmunología , Expresión Génica , Inmunidad Innata , Enfermedades de las Aves de Corral/inmunología , Salmonelosis Animal/inmunología , Transducción de Señal/genética , Transducción de Señal/inmunología , Receptores Toll-Like/genética , Receptores Toll-Like/inmunologíaRESUMEN
Soft lithography of polydimethylsiloxane (PDMS), an elastomeric polymer, has enabled rapid and inexpensive fabrication of microfluidic devices for various biotechnology applications. However, concerns remain about adsorption of compounds on PDMS surfaces because of its porosity and hydrophobicity. Here, the adsorption of 2 small fluorescent dyes of different hydrophobicity (calcein and 5- (and 6-)carboxytetramethylrhodamine (TMR)) on PDMS surface has been systematically characterized, and PDMS adsorption has been compared with 2 traditional substrates: glass and polystyrene. To characterize adsorption in a regimen that is more relevant to microfluidic applications, the adsorption and desorption of the 2 compounds in PDMS microfluidic channels under flow conditions were also studied. Results showed that there was minimal adsorption of the hydrophilic compound calcein on PDMS, whereas the more hydrophobic TMR adsorbed on PDMS up to 4 times of that on glass or polystyrene. Under flow conditions, the desorption profiles and times needed to drop desorbed compound concentrations to negligible levels (desorption time constant, 10-42 s) were characterized. In the worst case scenario, after a 4-min exposure to TMR, 4 min of continuous wash resulted in compound concentrations in the microchannels to drop to values below 2 x 10(- 5) of the initial concentration.
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Dimetilpolisiloxanos/farmacocinética , Evaluación Preclínica de Medicamentos/métodos , Adsorción , Fluoresceínas/farmacocinética , Colorantes Fluorescentes/farmacocinética , Vidrio/química , Interacciones Hidrofóbicas e Hidrofílicas , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Modelos Biológicos , Poliestirenos/farmacocinética , Bibliotecas de Moléculas Pequeñas/análisis , Bibliotecas de Moléculas Pequeñas/química , Propiedades de SuperficieRESUMEN
Agrin is a proteoglycan secreted by the motor neuron's growing axon terminal upon contact with the muscle during embryonic development. It was long thought that agrin's role was to trigger the clustering of acetylcholine receptors (AChRs) to nascent synapse sites. However, agrin-predating, protosynaptic AChR clusters are present well before innervation in the embryo and in myotube cultures, yet no role has been conclusively ascribed to agrin. We used a microfluidic device to focally deliver agrin to protosynaptic AChR clusters in micropatterned myotube cultures. The distribution of AChRs labeled with fluorescent bungarotoxin was imaged at various time points over >24 h. We find that a 4-h focal application of agrin (100 nM) preferentially reduces AChR loss at agrin-exposed clusters by 17% relative to the agrin-deprived clusters on the same myotube. In addition, the focal application increases the addition of AChRs preferentially at the clusters by 10% relative to the agrin-exposed, noncluster areas. Taken together, these findings suggest that a focal agrin stimulus can play a key stabilizing role in the aggregation of AChRs at the early stages of synapse formation. This methodology is generally applicable to various developmental processes and cell types, including neurons and stem cells.
Asunto(s)
Agrina/farmacología , Técnicas Analíticas Microfluídicas , Receptores Colinérgicos/metabolismo , Animales , Bovinos , Células Cultivadas , Fluorescencia , Fibras Musculares Esqueléticas/metabolismoRESUMEN
The objective of the study is to compare the effects of free-range (FR) and cage-range (CR) breeding on gut microbiota and flavor compounds of Caoke (C) and Partridge Shank chickens (Q). A total of 120 experimental chickens were assigned to FR group and CR group; each group contain both 30 Caoke chickens and 30 Partridge Shank chickens. At 154 d old, 12 chickens of each group were selected and their cecal contents were extracted and examined for the composition of gut microbiota by illumina sequencing of the V3 region of the 16S rDNA genes, and flavor compounds were analyzed through headspace-solid-phase microextraction (HS-SPME) method. The results showed that, except for acids, the amount of flavor substances in the FR group was higher than those in the CR group, especially the content of Hexanal and D-limonene. Meanwhile, the higher concentrations of carbonyls including (E,E)-2,4-decadienal, (E)-2-decenal, (E)-2-octenal, and pentanal were in the FR chicken meat, but the differences in concentrations compared with CR were not significant. High levels of ethyl hexanoate and ß-ocimene were only detected in FR groups. The Firmicutes had the highest proportion of chicken cecal microbiota, whereas the Fusobacteria was only detected in the cecal samples of Q chicken in FR group. Actinobacteria was more prevalent in FR groups than in CR groups. Meanwhile, in Q chickens, the proportions of Bacteroidetes and Proteobacteria in FR group were higher than those in CR group. Using MG-RAST Subsystem Technology, we found that some genes were associated with the formation of precursors of flavor compounds or with the metabolism and degradation of aromatic compounds. Overall, CR and FR breeding influenced the gut microbiota and flavor compounds, potentially because of the changes in diet and living conditions.
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Pollos , Microbioma Gastrointestinal/genética , Vivienda para Animales , Carne , Odorantes/análisis , Crianza de Animales Domésticos , Animales , Bacterias/genética , Ciego/microbiología , Pollos/microbiología , Pollos/fisiología , Carne/análisis , Carne/clasificación , Aves de Corral , ARN Ribosómico 16S/genéticaRESUMEN
To date, no report has demonstrated the use of beneficial microbes for contributing to the flavour characteristics and gut microbiota diversity of chicken. Here, we selected six probiotics obtained from our laboratory and supplemented them in six different combinations to 420 newborn male Qingjiaoma chickens under the same controlled living environment (60 birds, no probiotic supplements). The results showed that chicken supplemented with Bacillus species showed beneficial effects in body weight. Acetate is the major fermentation production in the chicken caecum, and chicken supplemented with Pediococcus pentosaceus had the average higher short chain fatty acids (SCFAs) contents. In chicken caecal microflora, the abundance of Bacteroidetes bacteria was positively correlated with the content of propionate, butyrate, and isobutyrate, whereas an increase in acetate content was positively correlated to the abundance of Firmicutes. Compared to chickens without probiotic supplement, chickens supplemented with P. pentosaceus had more characteristic flavour compounds in the sampled breast meat, especially higher concentrations of (E)-2-heptenal, (E,E)-2,4-nonadienal, and certain C6-C9 unsaturated fatty acids. This resulted in a stronger chicken-fatty or fatty odour which directly improved the flavour. These findings suggest that probiotics can improve chicken meat flavour and increase gut microbiota diversity.
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Ácidos Grasos/análisis , Microbioma Gastrointestinal/efectos de los fármacos , Carne/análisis , Probióticos/farmacología , Alimentación Animal/análisis , Animales , Animales Recién Nacidos , Bacillus/fisiología , Peso Corporal/efectos de los fármacos , Pollos , Suplementos Dietéticos , Masculino , Pediococcus pentosaceusRESUMEN
The isolation and purification of axon guidance molecules has enabled in vitro studies of the effects of axon guidance molecule gradients on numerous neuronal cell types. In a typical experiment, cultured neurons are exposed to a chemotactic gradient and their growth is recorded by manual identification of the axon tip position from two or more micrographs. Detailed and statistically valid quantification of axon growth requires evaluation of a large number of neurons at closely spaced time points (e.g. using a time-lapse microscopy setup). However, manual tracing becomes increasingly impractical for recording axon growth as the number of time points and/or neurons increases. We present a software tool that automatically identifies and records the axon tip position in each phase-contrast image of a time-lapse series with minimal user involvement. The software outputs several quantitative measures of axon growth, and allows users to develop custom measurements. For, example analysis of growth velocity for a dissociated E13 mouse cortical neuron revealed frequent extension and retraction events with an average growth velocity of 0.05 +/- 0.14 microm/min. Comparison of software-identified axon tip positions with manually identified axon tip positions shows that the software's performance is indistinguishable from that of skilled human users.
Asunto(s)
Inteligencia Artificial , Conos de Crecimiento/fisiología , Conos de Crecimiento/ultraestructura , Interpretación de Imagen Asistida por Computador/métodos , Microscopía de Contraste de Fase/métodos , Microscopía por Video/métodos , Reconocimiento de Normas Patrones Automatizadas/métodos , Animales , Axones/fisiología , Axones/ultraestructura , Aumento de la Célula , Células Cultivadas , Ratones , Técnica de SustracciónRESUMEN
The roles of nitric oxide are primarily undefined in astrocytes, cells that are active partners in synaptic transmission. Because nitric oxide synthases are present in astrocytes, we imaged the formation of nitric oxide in cultured murine cortical astrocytes using DAF-FM (4-amino-5-methylamino-2',7'-difluorofluorescein diacetate). We demonstrated that physiological concentrations of ATP induced a Ca2+-dependent production of nitric oxide. We then investigated the roles of nitric oxide in astrocytic Ca2+ signaling by exogenous application of a nitric oxide donor and found that nitric oxide induced an influx of external Ca2+. Because these observations raise the possibility that nitric oxide-dependent Ca2+ influx could lead to the refilling of internal stores with Ca2+, we directly monitored the Ca2+ levels of the cytosol and of internal stores while manipulating nitric oxide. Cultures were coloaded with mag-fluo-4 and X-rhod-1 to differentially load the internal stores and cytosol, respectively. ATP induced a cytosolic increase in Ca2+ that results from the IP3-dependent release of Ca2+ from internal stores, detected as a simultaneous reduction in mag-fluo-4 and an increase in X-rhod-1 fluorescence. To monitor store refilling, we measured the recovery of mag-fluo-4 fluorescence after removal of ATP. When nitric oxide signaling was blocked by the nitric oxide scavenger 2-phenyl-4,4,5,5-ketramethyl-imidazoline-1-oxyl-3-oxide or by the nitric oxide synthase inhibitor NG-monomethyl-l-arginine, fluorescence recovery was significantly reduced. These data suggest that transmitters that induce Ca2+ signaling in astrocytes lead to the Ca2+-dependent synthesis of nitric oxide. This in turn stimulates a Ca2+ influx pathway that is, in part, responsible for the refilling of internal Ca2+ stores.
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Astrocitos/metabolismo , Señalización del Calcio/fisiología , Calcio/metabolismo , GMP Cíclico/análogos & derivados , Óxido Nítrico/metabolismo , Adenosina Trifosfato/farmacología , Animales , Astrocitos/citología , Bloqueadores de los Canales de Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , GMP Cíclico/farmacología , Colorantes Fluorescentes , Ratones , Neurotransmisores/farmacología , Donantes de Óxido Nítrico/farmacología , S-Nitroso-N-Acetilpenicilamina/farmacologíaRESUMEN
The ability to culture cells in vitro has revolutionized hypothesis testing in basic cell and molecular biology research and has become a standard methodology in drug screening and toxicology assays. However, the traditional cell culture methodology--consisting essentially of the immersion of a large population of cells in a homogeneous fluid medium--has become increasingly limiting, both from a fundamental point of view (cells in vivo are surrounded by complex spatiotemporal microenvironments) and from a practical perspective (scaling up the number of fluid handling steps and cell manipulations for high-throughput studies in vitro is prohibitively expensive). Microfabrication technologies have enabled researchers to design, with micrometer control, the biochemical composition and topology of the substrate, the medium composition, as well as the type of neighboring cells surrounding the microenvironment of the cell. In addition, microtechnology is conceptually well suited for the development of fast, low-cost in vitro systems that allow for high-throughput culturing and analysis of cells under large numbers of conditions. Here we review a variety of applications of microfabrication in cell culture studies, with an emphasis on the biology of various cell types.
Asunto(s)
Técnicas Biosensibles/instrumentación , Técnicas de Cultivo de Célula/instrumentación , Fenómenos Fisiológicos Celulares , Microfluídica/métodos , Micromanipulación/instrumentación , Estimulación Física/métodos , Animales , Técnicas Biosensibles/métodos , Adhesión Celular/fisiología , Técnicas de Cultivo de Célula/métodos , Diseño de Equipo , Humanos , Microelectrodos , Microfluídica/instrumentación , Micromanipulación/métodos , Miniaturización , Estimulación Física/instrumentaciónRESUMEN
Large-scale surveys of single-cell gene expression have the potential to reveal rare cell populations and lineage relationships but require efficient methods for cell capture and mRNA sequencing. Although cellular barcoding strategies allow parallel sequencing of single cells at ultra-low depths, the limitations of shallow sequencing have not been investigated directly. By capturing 301 single cells from 11 populations using microfluidics and analyzing single-cell transcriptomes across downsampled sequencing depths, we demonstrate that shallow single-cell mRNA sequencing (~50,000 reads per cell) is sufficient for unbiased cell-type classification and biomarker identification. In the developing cortex, we identify diverse cell types, including multiple progenitor and neuronal subtypes, and we identify EGR1 and FOS as previously unreported candidate targets of Notch signaling in human but not mouse radial glia. Our strategy establishes an efficient method for unbiased analysis and comparison of cell populations from heterogeneous tissue by microfluidic single-cell capture and low-coverage sequencing of many cells.
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Corteza Cerebral/crecimiento & desarrollo , Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , ARN Mensajero/análisis , Análisis de Secuencia de ARN/métodos , Transducción de Señal/genética , Animales , Corteza Cerebral/metabolismo , Diseño de Equipo , Humanos , Ratones , Técnicas Analíticas Microfluídicas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Automated patch clamping addresses the need for high-throughput screening of chemical entities that alter ion channel function. As a result, there is considerable utility in the pharmaceutical screening arena for novel platforms that can produce relevant data both rapidly and consistently. Here we present results that were obtained with an innovative microfluidic automated patch clamp system utilizing a well-plate that eliminates the necessity of internal robotic liquid handling. Continuous recording from cell ensembles, rapid solution switching, and a bench-top footprint enable a number of assay formats previously inaccessible to automated systems. An electro-pneumatic interface was employed to drive the laminar flow of solutions in a microfluidic network that delivered cells in suspension to ensemble recording sites. Whole-cell voltage clamp was applied to linear arrays of 20 cells in parallel utilizing a 64-channel voltage clamp amplifier. A number of unique assays requiring sequential compound applications separated by a second or less, such as rapid determination of the agonist EC(50) for a ligand-gated ion channel or the kinetics of desensitization recovery, are enabled by the system. In addition, the system was validated via electrophysiological characterizations of both voltage-gated and ligand-gated ion channel targets: hK(V)2.1 and human Ether-à-go-go-related gene potassium channels, hNa(V)1.7 and 1.8 sodium channels, and (α1) hGABA(A) and (α1) human nicotinic acetylcholine receptor receptors. Our results show that the voltage dependence, kinetics, and interactions of these channels with pharmacological agents were matched to reference data. The results from these IonFlux™ experiments demonstrate that the system provides high-throughput automated electrophysiology with enhanced reliability and consistency, in a user-friendly format.
Asunto(s)
Automatización/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Canales Iónicos/efectos de los fármacos , Técnicas Analíticas Microfluídicas , Animales , Células CHO , Canales de Cloruro/efectos de los fármacos , Cricetinae , Cricetulus , Canales de Potasio Éter-A-Go-Go/efectos de los fármacos , Células HEK293 , Humanos , Canal de Potasio Kv.1.2/efectos de los fármacos , Canal de Sodio Activado por Voltaje NAV1.7/efectos de los fármacos , Canal de Sodio Activado por Voltaje NAV1.8/efectos de los fármacos , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/farmacología , Receptores Colinérgicos/efectos de los fármacos , Receptores de GABA-A/efectos de los fármacos , Reproducibilidad de los Resultados , Bloqueadores de los Canales de Sodio/farmacología , SolucionesRESUMEN
Ion channel assays are essential in drug discovery, not only for identifying promising new clinical compounds, but also for minimizing the likelihood of potential side effects. Both applications demand optimized throughput, cost, and predictive accuracy of measured membrane current changes evoked or modulated by drug candidates. Several competing electrophysiological technologies are available to address this demand, but important gaps remain. We describe the industrial application of a novel microfluidic-based technology that combines compounds, cells, and buffers on a single, standard well plate. Cell trapping, whole cell, and compound perfusion are accomplished in interconnecting microfluidic channels that are coupled to pneumatic valves, which emancipate the system from robotics, fluidic tubing, and associated maintenance. IonFlux™ is a state-of-the-art, compact system with temperature control and continuous voltage clamp for potential application in screening for voltage- and ligand-gated ion channel modulators. Here, ensemble recordings of the IonFlux system were validated with the human Ether-à-go-go related gene (hERG) channel (stably expressed in a Chinese hamster ovary cell line), which has established biophysical and pharmacological characteristics in other automated planar patch systems. We characterized the temperature dependence of channel activation and its reversal potential. Concentration response characteristics of known hERG blockers and control compounds obtained with the IonFlux system correlated with literature and internal data obtained on this cell line with the QPatch HT system. Based on the biophysical and pharmacological data, we conclude that the IonFlux system offers a novel, versatile, automated profiling, and screening system for ion channel targets with the benefit of temperature control.
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Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Canales de Potasio Éter-A-Go-Go/fisiología , Microfluídica/métodos , Técnicas de Placa-Clamp/instrumentación , Bloqueadores de los Canales de Potasio/farmacología , Animales , Células CHO , Cricetinae , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/instrumentación , Evaluación Preclínica de Medicamentos/métodos , Humanos , Microfluídica/instrumentación , Técnicas de Placa-Clamp/métodosRESUMEN
Investigation of biochemical cues in isolation or in combinations in cell culture systems is crucial for unraveling the mechanisms that govern neural development and repair. The most widely used experimental paradigms that elicit axon guidance in vitro utilize as the source of the gradient a pulsatile pipette, transfected cells, or a loaded gel, producing time-varying gradients of poor reproducibility which are not well suited for studying slow-growing mammalian cells. Although microfluidic device design have allowed for generating stable, complex gradients of diffusible molecules, the flow-induced shear forces in a microchannel has made it impossible to maintain viable mammalian neuronal cultures for sufficiently long times. In this paper, we describe axonal responses of mouse cortical neurons in a "neuron-benign" gradient-generator device based on an open chamber that can establish highly stable gradients of diffusible molecules for at least 6 h with negligible shear stress, and also allows the neurons to thrive for at least 2 weeks. Except for the period when the gradient is on, the cells in the gradient are under the same conditions as the cells on the control surfaces, which ensure a consistent set of micro-environmental variables. The gradient stability and uniformity over the cell culture surface achieved by the device, together with our software platform for acquiring, post-processing and quantitatively analyzing the large number of images allowed us to extract valuable information even from small datasets. We report a directed response of primary mammalian neurons (from E14 embryonic mice cortex) to a diffusible gradient of netrin in vitro. We infer from our studies that a large majority (â¼73%) of the neurons that extend axons during the gradient application grow towards the netrin source, and our data analysis also indicates that netrin acts as a growth factor for this same population of neurons.
Asunto(s)
Técnicas Analíticas Microfluídicas/instrumentación , Neuronas/fisiología , Animales , Axones/efectos de los fármacos , Axones/fisiología , Técnicas de Cultivo de Célula , Rastreo Celular , Diseño de Equipo , Femenino , Ratones , Factores de Crecimiento Nervioso/farmacología , Netrina-1 , Neurogénesis , Neuronas/efectos de los fármacos , Embarazo , Transducción de Señal , Proteínas Supresoras de Tumor/farmacologíaRESUMEN
Single molecule-based protocols have been gaining popularity as a way to visualize DNA replication at the global genomic- and locus-specific levels. These protocols take advantage of the ability of many organisms to incorporate nucleoside analogs during DNA replication, together with a method to display stretched DNA on glass for immunostaining and microscopy. We describe here a microfluidic platform that can be used to stretch and to capture labeled DNA molecules for replication analyses. This platform consists of parallel arrays of three-sided, 3- or 4-microm high, variable-width capillary channels fabricated from polydimethylsiloxane by conventional soft lithography, and of silane-modified glass coverslips to reversibly seal the open side of the channels. Capillary tension in these microchannels facilitates DNA loading, stretching and glass coverslip deposition from microliter-scale DNA samples. The simplicity and extensibility of this platform should facilitate DNA replication analyses using small samples from a variety of biological and clinical sources.
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Replicación del ADN , ADN/química , ADN/genética , Técnicas Analíticas Microfluídicas/métodos , Mapeo Cromosómico/métodos , ADN/análisis , Genes , Genómica/métodos , Vidrio , Indicadores y Reactivos , Miniaturización/métodos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , SilicioRESUMEN
Werner syndrome is an autosomal recessive genetic instability and cancer predisposition syndrome with features of premature aging. Several lines of evidence have suggested that the Werner syndrome protein WRN plays a role in DNA replication and S-phase progression. In order to define the exact role of WRN in genomic replication we examined cell cycle kinetics during normal cell division and after methyl-methane-sulfonate (MMS) DNA damage or hydroxyurea (HU)-mediated replication arrest following acute depletion of WRN from human fibroblasts. Loss of WRN markedly extended the time cells needed to complete the cell cycle after either of these genotoxic treatments. Moreover, replication track analysis of individual, stretched DNA fibers showed that WRN depletion significantly reduced the speed at which replication forks elongated in vivo after MMS or HU treatment. These results establish the importance of WRN during genomic replication and indicate that WRN acts to facilitate fork progression after DNA damage or replication arrest. The data provide a mechanistic basis for a better understanding of WRN-mediated maintenance of genomic stability and for predicting the outcomes of DNA-targeting chemotherapy in several adult cancers that silence WRN expression.