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Sample records for cell imaging screen

  1. A Versatile Cell Death Screening Assay Using Dye-Stained Cells and Multivariate Image Analysis.

    Science.gov (United States)

    Collins, Tony J; Ylanko, Jarkko; Geng, Fei; Andrews, David W

    2015-11-01

    A novel dye-based method for measuring cell death in image-based screens is presented. Unlike conventional high- and medium-throughput cell death assays that measure only one form of cell death accurately, using multivariate analysis of micrographs of cells stained with the inexpensive mix, red dye nonyl acridine orange, and a nuclear stain, it was possible to quantify cell death induced by a variety of different agonists even without a positive control. Surprisingly, using a single known cytotoxic agent as a positive control for training a multivariate classifier allowed accurate quantification of cytotoxicity for mechanistically unrelated compounds enabling generation of dose-response curves. Comparison with low throughput biochemical methods suggested that cell death was accurately distinguished from cell stress induced by low concentrations of the bioactive compounds Tunicamycin and Brefeldin A. High-throughput image-based format analyses of more than 300 kinase inhibitors correctly identified 11 as cytotoxic with only 1 false positive. The simplicity and robustness of this dye-based assay makes it particularly suited to live cell screening for toxic compounds.

  2. PopulationProfiler: A Tool for Population Analysis and Visualization of Image-Based Cell Screening Data

    OpenAIRE

    Matuszewski, Damian J.; Carolina Wählby; Jordi Carreras Puigvert; Ida-Maria Sintorn

    2016-01-01

    Image-based screening typically produces quantitative measurements of cell appearance. Large-scale screens involving tens of thousands of images, each containing hundreds of cells described by hundreds of measurements, result in overwhelming amounts of data. Reducing per-cell measurements to the averages across the image(s) for each treatment leads to loss of potentially valuable information on population variability. We present PopulationProfiler-a new software tool that reduces per-cell mea...

  3. Resonant waveguide grating imagers for single cell analysis and high throughput screening

    Science.gov (United States)

    Fang, Ye

    2015-08-01

    Resonant waveguide grating (RWG) systems illuminate an array of diffractive nanograting waveguide structures in microtiter plate to establish evanescent wave for measuring tiny changes in local refractive index arising from the dynamic mass redistribution of living cells upon stimulation. Whole-plate RWG imager enables high-throughput profiling and screening of drugs. Microfluidics RWG imager not only manifests distinct receptor signaling waves, but also differentiates long-acting agonism and antagonism. Spatially resolved RWG imager allows for single cell analysis including receptor signaling heterogeneity and the invasion of cancer cells in a spheroidal structure through 3-dimensional extracellular matrix. High frequency RWG imager permits real-time detection of drug-induced cardiotoxicity. The wide coverage in target, pathway, assay, and cell phenotype has made RWG systems powerful tool in both basic research and early drug discovery process.

  4. Expanding the Diversity of Imaging-Based RNAi Screen Applications Using Cell Spot Microarrays.

    Science.gov (United States)

    Rantala, Juha K; Kwon, Sunjong; Korkola, James; Gray, Joe W

    2013-04-11

    Over the past decade, great strides have been made in identifying gene aberrations and deregulated pathways that are associated with specific disease states. These association studies guide experimental studies aimed at identifying the aberrant genes and networks that cause the disease states. This requires functional manipulation of these genes and networks in laboratory models of normal and diseased cells. One approach is to assess molecular and biological responses to high-throughput RNA interference (RNAi)-induced gene knockdown. These responses can be revealed by immunofluorescent staining for a molecular or cellular process of interest and quantified using fluorescence image analysis. These applications are typically performed in multiwell format, but are limited by high reagent costs and long plate processing times. These limitations can be mitigated by analyzing cells grown in cell spot microarray (CSMA) format. CSMAs are produced by growing cells on small (~200 mm diameter) spots with each spot carrying an siRNA with transfection reagent. The spacing between spots is only a few hundred micrometers, thus thousands of cell spots can be arranged on a single cell culture surface. These high-density cell cultures can be immunofluorescently stained with minimal reagent consumption and analyzed quickly using automated fluorescence microscopy platforms. This review covers basic aspects of imaging-based CSMA technology, describes a wide range of immunofluorescence assays that have already been implemented successfully for CSMA screening and suggests future directions for advanced RNAi screening experiments.

  5. Functional screening with a live cell imaging-based random cell migration assay.

    Science.gov (United States)

    van Roosmalen, Wies; Le Dévédec, Sylvia E; Zovko, Sandra; de Bont, Hans; van de Water, Bob

    2011-01-01

    Cell migration, essential in cancer progression, is a complex process comprising a number of spatiotemporally regulated and well-coordinated mechanisms. In order to study (random) cell migration in the context of responses to various external cues (such as growth factors) or intrinsic cell signaling, a number of different tools and approaches have been developed. In order to unravel the key pathways and players involved in the regulation of (cancer) cell migration, a systematical mapping of the players/pathways is required. For this purpose, we developed a cell migration assay based on automatic high-throughput microscopy screen. This approach allows for screening of hundreds of genes, e.g., those encoding various kinases and phosphatases but can also be used for screening of drugs libraries. Moreover, we have developed an automatic analysis pipeline comprising of (a) automatic data acquisition (movie) and (b) automatic analysis of the acquired movies of the migrating cells. Here, we describe various facets of this approach. Since cell migration is essential in progression of cancer metastasis, we describe two examples of experiments performed on highly motile (metastatic) cancer cells.

  6. PopulationProfiler: A Tool for Population Analysis and Visualization of Image-Based Cell Screening Data.

    Directory of Open Access Journals (Sweden)

    Damian J Matuszewski

    Full Text Available Image-based screening typically produces quantitative measurements of cell appearance. Large-scale screens involving tens of thousands of images, each containing hundreds of cells described by hundreds of measurements, result in overwhelming amounts of data. Reducing per-cell measurements to the averages across the image(s for each treatment leads to loss of potentially valuable information on population variability. We present PopulationProfiler-a new software tool that reduces per-cell measurements to population statistics. The software imports measurements from a simple text file, visualizes population distributions in a compact and comprehensive way, and can create gates for subpopulation classes based on control samples. We validate the tool by showing how PopulationProfiler can be used to analyze the effect of drugs that disturb the cell cycle, and compare the results to those obtained with flow cytometry.

  7. PopulationProfiler: A Tool for Population Analysis and Visualization of Image-Based Cell Screening Data.

    Science.gov (United States)

    Matuszewski, Damian J; Wählby, Carolina; Puigvert, Jordi Carreras; Sintorn, Ida-Maria

    2016-01-01

    Image-based screening typically produces quantitative measurements of cell appearance. Large-scale screens involving tens of thousands of images, each containing hundreds of cells described by hundreds of measurements, result in overwhelming amounts of data. Reducing per-cell measurements to the averages across the image(s) for each treatment leads to loss of potentially valuable information on population variability. We present PopulationProfiler-a new software tool that reduces per-cell measurements to population statistics. The software imports measurements from a simple text file, visualizes population distributions in a compact and comprehensive way, and can create gates for subpopulation classes based on control samples. We validate the tool by showing how PopulationProfiler can be used to analyze the effect of drugs that disturb the cell cycle, and compare the results to those obtained with flow cytometry.

  8. Library synthesis, screening, and discovery of modified Zinc(II)-Bis(dipicolylamine) probe for enhanced molecular imaging of cell death.

    Science.gov (United States)

    Plaunt, Adam J; Harmatys, Kara M; Wolter, William R; Suckow, Mark A; Smith, Bradley D

    2014-04-16

    Zinc(II)-bis(dipicolylamine) (Zn-BDPA) coordination complexes selectively target the surfaces of dead and dying mammalian cells, and they have promise as molecular probes for imaging cell death. A necessary step toward eventual clinical imaging applications is the development of next-generation Zn-BDPA complexes with enhanced affinity for the cell death membrane biomarker, phosphatidylserine (PS). This study employed an iterative cycle of library synthesis and screening, using a novel rapid equilibrium dialysis assay, to discover a modified Zn-BDPA structure with high and selective affinity for vesicles containing PS. The lead structure was converted into a deep-red fluorescent probe and its targeting and imaging performance was compared with an unmodified control Zn-BDPA probe. The evaluation process included a series of FRET-based vesicle titration studies, cell microscopy experiments, and rat tumor biodistribution measurements. In all cases, the modified probe exhibited comparatively higher affinity and selectivity for the target membranes of dead and dying cells. The results show that this next-generation deep-red fluorescent Zn-BDPA probe is well suited for preclinical molecular imaging of cell death in cell cultures and animal models. Furthermore, it should be possible to substitute the deep-red fluorophore with alternative reporter groups that enable clinically useful, deep-tissue imaging modalities, such as MRI and nuclear imaging.

  9. Impact of image segmentation on high-content screening data quality for SK-BR-3 cells

    Directory of Open Access Journals (Sweden)

    Li Yizheng

    2007-09-01

    Full Text Available Abstract Background High content screening (HCS is a powerful method for the exploration of cellular signalling and morphology that is rapidly being adopted in cancer research. HCS uses automated microscopy to collect images of cultured cells. The images are subjected to segmentation algorithms to identify cellular structures and quantitate their morphology, for hundreds to millions of individual cells. However, image analysis may be imperfect, especially for "HCS-unfriendly" cell lines whose morphology is not well handled by current image segmentation algorithms. We asked if segmentation errors were common for a clinically relevant cell line, if such errors had measurable effects on the data, and if HCS data could be improved by automated identification of well-segmented cells. Results Cases of poor cell body segmentation occurred frequently for the SK-BR-3 cell line. We trained classifiers to identify SK-BR-3 cells that were well segmented. On an independent test set created by human review of cell images, our optimal support-vector machine classifier identified well-segmented cells with 81% accuracy. The dose responses of morphological features were measurably different in well- and poorly-segmented populations. Elimination of the poorly-segmented cell population increased the purity of DNA content distributions, while appropriately retaining biological heterogeneity, and simultaneously increasing our ability to resolve specific morphological changes in perturbed cells. Conclusion Image segmentation has a measurable impact on HCS data. The application of a multivariate shape-based filter to identify well-segmented cells improved HCS data quality for an HCS-unfriendly cell line, and could be a valuable post-processing step for some HCS datasets.

  10. High-content screening of drug-induced cardiotoxicity using quantitative single cell imaging cytometry on microfluidic device.

    Science.gov (United States)

    Kim, Min Jung; Lee, Su Chul; Pal, Sukdeb; Han, Eunyoung; Song, Joon Myong

    2011-01-07

    Drug-induced cardiotoxicity or cytotoxicity followed by cell death in cardiac muscle is one of the major concerns in drug development. Herein, we report a high-content quantitative multicolor single cell imaging tool for automatic screening of drug-induced cardiotoxicity in an intact cell. A tunable multicolor imaging system coupled with a miniaturized sample platform was destined to elucidate drug-induced cardiotoxicity via simultaneous quantitative monitoring of intracellular sodium ion concentration, potassium ion channel permeability and apoptosis/necrosis in H9c2(2-1) cell line. Cells were treated with cisapride (a human ether-à-go-go-related gene (hERG) channel blocker), digoxin (Na(+)/K(+)-pump blocker), camptothecin (anticancer agent) and a newly synthesized anti-cancer drug candidate (SH-03). Decrease in potassium channel permeability in cisapride-treated cells indicated that it can also inhibit the trafficking of the hERG channel. Digoxin treatment resulted in an increase of intracellular [Na(+)]. However, it did not affect potassium channel permeability. Camptothecin and SH-03 did not show any cytotoxic effect at normal use (≤300 nM and 10 μM, respectively). This result clearly indicates the potential of SH-03 as a new anticancer drug candidate. The developed method was also used to correlate the cell death pathway with alterations in intracellular [Na(+)]. The developed protocol can directly depict and quantitate targeted cellular responses, subsequently enabling an automated, easy to operate tool that is applicable to drug-induced cytotoxicity monitoring with special reference to next generation drug discovery screening. This multicolor imaging based system has great potential as a complementary system to the conventional patch clamp technique and flow cytometric measurement for the screening of drug cardiotoxicity.

  11. Electro- and photoluminescence imaging as fast screening technique of the layer uniformity and device degradation in planar perovskite solar cells

    Science.gov (United States)

    Soufiani, Arman Mahboubi; Tayebjee, Murad J. Y.; Meyer, Steffen; Ho-Baillie, Anita; Sung Yun, Jae; MacQueen, Rowan W.; Spiccia, Leone; Green, Martin A.; Hameiri, Ziv

    2016-07-01

    In this study, we provide insights into planar structure methylammonium lead triiodide (MAPbI3) perovskite solar cells (PSCs) using electroluminescence and photoluminescence imaging techniques. We demonstrate the strength of these techniques in screening relatively large area PSCs, correlating the solar cell electrical parameters to the images and visualizing the features which contribute to the variation of the parameters extracted from current density-voltage characterizations. It is further used to investigate one of the major concerns about perovskite solar cells, their long term stability and aging. Upon storage under dark in dry glovebox condition for more than two months, the major parameter found to have deteriorated in electrical performance measurements was the fill factor; this was elucidated via electroluminescence image comparisons which revealed that the contacts' quality degrades. Interestingly, by deploying electroluminescence imaging, the significance of having a pin-hole free active layer is demonstrated. Pin-holes can grow over time and can cause degradation of the active layer surrounding them.

  12. Automated analysis of siRNA screens of cells infected by hepatitis C and dengue viruses based on immunofluorescence microscopy images

    Science.gov (United States)

    Matula, Petr; Kumar, Anil; Wörz, Ilka; Harder, Nathalie; Erfle, Holger; Bartenschlager, Ralf; Eils, Roland; Rohr, Karl

    2008-03-01

    We present an image analysis approach as part of a high-throughput microscopy siRNA-based screening system using cell arrays for the identification of cellular genes involved in hepatitis C and dengue virus replication. Our approach comprises: cell nucleus segmentation, quantification of virus replication level in the neighborhood of segmented cell nuclei, localization of regions with transfected cells, cell classification by infection status, and quality assessment of an experiment and single images. In particular, we propose a novel approach for the localization of regions of transfected cells within cell array images, which combines model-based circle fitting and grid fitting. By this scheme we integrate information from single cell array images and knowledge from the complete cell arrays. The approach is fully automatic and has been successfully applied to a large number of cell array images from screening experiments. The experimental results show a good agreement with the expected behaviour of positive as well as negative controls and encourage the application to screens from further high-throughput experiments.

  13. A high-throughput three-dimensional cell migration assay for toxicity screening with mobile device-based macroscopic image analysis

    Science.gov (United States)

    Timm, David M.; Chen, Jianbo; Sing, David; Gage, Jacob A.; Haisler, William L.; Neeley, Shane K.; Raphael, Robert M.; Dehghani, Mehdi; Rosenblatt, Kevin P.; Killian, T. C.; Tseng, Hubert; Souza, Glauco R.

    2013-10-01

    There is a growing demand for in vitro assays for toxicity screening in three-dimensional (3D) environments. In this study, 3D cell culture using magnetic levitation was used to create an assay in which cells were patterned into 3D rings that close over time. The rate of closure was determined from time-lapse images taken with a mobile device and related to drug concentration. Rings of human embryonic kidney cells (HEK293) and tracheal smooth muscle cells (SMCs) were tested with ibuprofen and sodium dodecyl sulfate (SDS). Ring closure correlated with the viability and migration of cells in two dimensions (2D). Images taken using a mobile device were similar in analysis to images taken with a microscope. Ring closure may serve as a promising label-free and quantitative assay for high-throughput in vivo toxicity in 3D cultures.

  14. Live Cell Bioluminescence Imaging in Temporal Reaction of G Protein-Coupled Receptor for High-Throughput Screening and Analysis.

    Science.gov (United States)

    Hattori, Mitsuru; Ozawa, Takeaki

    2016-01-01

    G protein-coupled receptors (GPCRs) are notable targets of basic therapeutics. Many screening methods have been established to identify novel agents for GPCR signaling in a high-throughput manner. However, information related to the temporal reaction of GPCR with specific ligands remains poor. We recently developed a bioluminescence method for the quantitative detection of the interaction between GPCR and β-arrestin using split luciferase complementation. To monitor time-course variation of the interactions, a new imaging system contributes to the accurate evaluation of drugs for GPCRs in a high-throughput manner.

  15. Discovery of Small Molecules That Induce Lysosomal Cell Death in Cancer Cell Lines Using an Image-Based Screening Platform

    NARCIS (Netherlands)

    Pagliero, Romina J; D'Astolfo, Diego S; Lelieveld, Daphne; Pratiwi, Riyona D; Aits, Sonja; Jaattela, Marja; Martin, Nathaniel I; Klumperman, Judith; Egan, David A

    2016-01-01

    The lysosomal cell death (LCD) pathway is a caspase 3-independent cell death pathway that has been suggested as a possible target for cancer therapy, making the development of sensitive and specific high-throughput (HT) assays to identify LCD inducers highly desirable. In this study, we report a two

  16. Computer-aided screening system for cervical precancerous cells based on field emission scanning electron microscopy and energy dispersive x-ray images and spectra

    Science.gov (United States)

    Jusman, Yessi; Ng, Siew-Cheok; Hasikin, Khairunnisa; Kurnia, Rahmadi; Osman, Noor Azuan Bin Abu; Teoh, Kean Hooi

    2016-10-01

    The capability of field emission scanning electron microscopy and energy dispersive x-ray spectroscopy (FE-SEM/EDX) to scan material structures at the microlevel and characterize the material with its elemental properties has inspired this research, which has developed an FE-SEM/EDX-based cervical cancer screening system. The developed computer-aided screening system consisted of two parts, which were the automatic features of extraction and classification. For the automatic features extraction algorithm, the image and spectra of cervical cells features extraction algorithm for extracting the discriminant features of FE-SEM/EDX data was introduced. The system automatically extracted two types of features based on FE-SEM/EDX images and FE-SEM/EDX spectra. Textural features were extracted from the FE-SEM/EDX image using a gray level co-occurrence matrix technique, while the FE-SEM/EDX spectra features were calculated based on peak heights and corrected area under the peaks using an algorithm. A discriminant analysis technique was employed to predict the cervical precancerous stage into three classes: normal, low-grade intraepithelial squamous lesion (LSIL), and high-grade intraepithelial squamous lesion (HSIL). The capability of the developed screening system was tested using 700 FE-SEM/EDX spectra (300 normal, 200 LSIL, and 200 HSIL cases). The accuracy, sensitivity, and specificity performances were 98.2%, 99.0%, and 98.0%, respectively.

  17. Image analysis benchmarking methods for high-content screen design.

    Science.gov (United States)

    Fuller, C J; Straight, A F

    2010-05-01

    The recent development of complex chemical and small interfering RNA (siRNA) collections has enabled large-scale cell-based phenotypic screening. High-content and high-throughput imaging are widely used methods to record phenotypic data after chemical and small interfering RNA treatment, and numerous image processing and analysis methods have been used to quantify these phenotypes. Currently, there are no standardized methods for evaluating the effectiveness of new and existing image processing and analysis tools for an arbitrary screening problem. We generated a series of benchmarking images that represent commonly encountered variation in high-throughput screening data and used these image standards to evaluate the robustness of five different image analysis methods to changes in signal-to-noise ratio, focal plane, cell density and phenotype strength. The analysis methods that were most reliable, in the presence of experimental variation, required few cells to accurately distinguish phenotypic changes between control and experimental data sets. We conclude that by applying these simple benchmarking principles an a priori estimate of the image acquisition requirements for phenotypic analysis can be made before initiating an image-based screen. Application of this benchmarking methodology provides a mechanism to significantly reduce data acquisition and analysis burdens and to improve data quality and information content.

  18. An on-chip imaging droplet-sorting system: a real-time shape recognition method to screen target cells in droplets with single cell resolution

    Science.gov (United States)

    Girault, Mathias; Kim, Hyonchol; Arakawa, Hisayuki; Matsuura, Kenji; Odaka, Masao; Hattori, Akihiro; Terazono, Hideyuki; Yasuda, Kenji

    2017-01-01

    A microfluidic on-chip imaging cell sorter has several advantages over conventional cell sorting methods, especially to identify cells with complex morphologies such as clusters. One of the remaining problems is how to efficiently discriminate targets at the species level without labelling. Hence, we developed a label-free microfluidic droplet-sorting system based on image recognition of cells in droplets. To test the applicability of this method, a mixture of two plankton species with different morphologies (Dunaliella tertiolecta and Phaeodactylum tricornutum) were successfully identified and discriminated at a rate of 10 Hz. We also examined the ability to detect the number of objects encapsulated in a droplet. Single cell droplets sorted into collection channels showed 91 ± 4.5% and 90 ± 3.8% accuracy for D. tertiolecta and P. tricornutum, respectively. Because we used image recognition to confirm single cell droplets, we achieved highly accurate single cell sorting. The results indicate that the integrated method of droplet imaging cell sorting can provide a complementary sorting approach capable of isolating single target cells from a mixture of cells with high accuracy without any staining.

  19. A Novel High Content Imaging-Based Screen Identifies the Anti-Helminthic Niclosamide as an Inhibitor of Lysosome Anterograde Trafficking and Prostate Cancer Cell Invasion.

    Directory of Open Access Journals (Sweden)

    Magdalena L Circu

    Full Text Available Lysosome trafficking plays a significant role in tumor invasion, a key event for the development of metastasis. Previous studies from our laboratory have demonstrated that the anterograde (outward movement of lysosomes to the cell surface in response to certain tumor microenvironment stimulus, such as hepatocyte growth factor (HGF or acidic extracellular pH (pHe, increases cathepsin B secretion and tumor cell invasion. Anterograde lysosome trafficking depends on sodium-proton exchanger activity and can be reversed by blocking these ion pumps with Troglitazone or EIPA. Since these drugs cannot be advanced into the clinic due to toxicity, we have designed a high-content assay to discover drugs that block peripheral lysosome trafficking with the goal of identifying novel drugs that inhibit tumor cell invasion. An automated high-content imaging system (Cellomics was used to measure the position of lysosomes relative to the nucleus. Among a total of 2210 repurposed and natural product drugs screened, 18 "hits" were identified. One of the compounds identified as an anterograde lysosome trafficking inhibitor was niclosamide, a marketed human anti-helminthic drug. Further studies revealed that niclosamide blocked acidic pHe, HGF, and epidermal growth factor (EGF-induced anterograde lysosome redistribution, protease secretion, motility, and invasion of DU145 castrate resistant prostate cancer cells at clinically relevant concentrations. In an effort to identify the mechanism by which niclosamide prevented anterograde lysosome movement, we found that this drug exhibited no significant effect on the level of ATP, microtubules or actin filaments, and had minimal effect on the PI3K and MAPK pathways. Niclosamide collapsed intralysosomal pH without disruption of the lysosome membrane, while bafilomycin, an agent that impairs lysosome acidification, was also found to induce JLA in our model. Taken together, these data suggest that niclosamide promotes

  20. PROLIFERATION AS A KEY EVENT IN DEVELOPMENTAL TOXICITY: "CHEMICAL SCREENING IN HUMAN NEURAL STEM CELLS USING HIGH CONTENT IMAGING

    Science.gov (United States)

    New toxicity testing approaches will rely on in vitro assays to assess chemical effects at the cellular and molecular level. Cell proliferation is imperative to normal development, and chemical disruption of this process can be detrimental to the organism. As part of an effort to...

  1. High-throughput automated confocal microscopy imaging screen of a kinase-focused library to identify p38 mitogen-activated protein kinase inhibitors using the GE InCell 3000 analyzer.

    Science.gov (United States)

    Trask, O Joseph; Nickischer, Debra; Burton, Audrey; Williams, Rhonda Gates; Kandasamy, Ramani A; Johnston, Patricia A; Johnston, Paul A

    2009-01-01

    The integration of fluorescent microscopy imaging technologies and image analysis into high-content screening (HCS) has been applied throughout the drug discovery pipeline to identify, evaluate, and advance compounds from early lead generation through preclinical candidate selection. In this chapter we describe the development, validation, and implementation of an HCS assay to screen compounds from a kinase-focused small-molecule library to identify inhibitors of the p38 pathway using the GE InCell 3000 automated imaging platform. The assay utilized a genetically modified HeLa cell line stably expressing mitogen-activated, protein-activating protein kinase-2 fused to enhanced green fluorescent protein (MK2-EGFP) and measured the subcellular distribution of the MK2-EGFP as a direct readout of p38 activation. The MK2-EGFP translocation assay performed in 384-well glass bottom microtiter plates exhibited a robust Z-factor of 0.46 and reproducible EC50 and IC50 determinations for activators and inhibitors, respectively. A total of 32,891 compounds were screened in singlicate at 50 microM and 156 were confirmed as inhibitors of p38-mediated MK2-EGFP translocation in follow-up IC50 concentration response curves. Thirty-one compounds exhibited IC50s less than 1 microM, and at least one novel structural class of p38 inhibitor was identified using this HCA/HCS chemical biology screening approach.

  2. An integrated one-step system to extract, analyze and annotate all relevant information from image-based cell screening of chemical libraries.

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    Rabal, Obdulia; Link, Wolfgang; Serelde, Beatriz G; Bischoff, James R; Oyarzabal, Julen

    2010-04-01

    Here we report the development and validation of a complete solution to manage and analyze the data produced by image-based phenotypic screening campaigns of small-molecule libraries. In one step initial crude images are analyzed for multiple cytological features, statistical analysis is performed and molecules that produce the desired phenotypic profile are identified. A naïve Bayes classifier, integrating chemical and phenotypic spaces, is built and utilized during the process to assess those images initially classified as "fuzzy"-an automated iterative feedback tuning. Simultaneously, all this information is directly annotated in a relational database containing the chemical data. This novel fully automated method was validated by conducting a re-analysis of results from a high-content screening campaign involving 33 992 molecules used to identify inhibitors of the PI3K/Akt signaling pathway. Ninety-two percent of confirmed hits identified by the conventional multistep analysis method were identified using this integrated one-step system as well as 40 new hits, 14.9% of the total, originally false negatives. Ninety-six percent of true negatives were properly recognized too. A web-based access to the database, with customizable data retrieval and visualization tools, facilitates the posterior analysis of annotated cytological features which allows identification of additional phenotypic profiles; thus, further analysis of original crude images is not required.

  3. Automated screening versus manual screening: a comparison of the ThinPrep imaging system and manual screening in a time study.

    Science.gov (United States)

    Schledermann, Doris; Hyldebrandt, Tina; Ejersbo, Dorthe; Hoelund, Berit

    2007-06-01

    The ThinPrep Imaging System (TIS) is an automated system that assists cytotechnologists in the primary screening of ThinPrep liquid based cervical samples. Between June 1, 2004, and April 1, 2005, four experienced cytotechnologists participated in the study in which the duration of the screening procedure was timed for each of the 11,354 slides included. In every slide 22 fields of view were reviewed, and the samples that contained potentially abnormal cells were fully screened. The screening time was reduced by 42% (mean) (p < 0.001). By manual rescreening of the negative TIS samples, abnormal cells were found in 10 samples (false negative rate 0.14%). In every case the abnormal cells had been identified by the scanner, but misinterpreted by the cytotechnologist. These findings stressed the importance of carefulness in the interpretation of the marked fields and beyond that helped the cytotechnologists and pathologists to have more confidence in the automated system.

  4. Dynamic infrared imaging for skin cancer screening

    Science.gov (United States)

    Godoy, Sebastián E.; Ramirez, David A.; Myers, Stephen A.; von Winckel, Greg; Krishna, Sanchita; Berwick, Marianne; Padilla, R. Steven; Sen, Pradeep; Krishna, Sanjay

    2015-05-01

    Dynamic thermal imaging (DTI) with infrared cameras is a non-invasive technique with the ability to detect the most common types of skin cancer. We discuss and propose a standardized analysis method for DTI of actual patient data, which achieves high levels of sensitivity and specificity by judiciously selecting pixels with the same initial temperature. This process compensates the intrinsic limitations of the cooling unit and is the key enabling tool in the DTI data analysis. We have extensively tested the methodology on human subjects using thermal infrared image sequences from a pilot study conducted jointly with the University of New Mexico Dermatology Clinic in Albuquerque, New Mexico (ClinicalTrials ID number NCT02154451). All individuals were adult subjects who were scheduled for biopsy or adult volunteers with clinically diagnosed benign condition. The sample size was 102 subjects for the present study. Statistically significant results were obtained that allowed us to distinguish between benign and malignant skin conditions. The sensitivity and specificity was 95% (with a 95% confidence interval of [87.8% 100.0%]) and 83% (with a 95% confidence interval of [73.4% 92.5%]), respectively, and with an area under the curve of 95%. Our results lead us to conclude that the DTI approach in conjunction with the judicious selection of pixels has the potential to provide a fast, accurate, non-contact, and non-invasive way to screen for common types of skin cancer. As such, it has the potential to significantly reduce the number of biopsies performed on suspicious lesions.

  5. B and T cell screen

    Science.gov (United States)

    Direct immunofluorescence; E-rosetting; T and B lymphocyte assays; B and T lymphocyte assays ... identifiers are added to distinguish between T and B cells. The E-rosetting test identifies T cells ...

  6. Primary cells and stem cells in drug discovery: emerging tools for high-throughput screening.

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    Eglen, Richard; Reisine, Terry

    2011-04-01

    Many drug discovery screening programs employ immortalized cells, recombinantly engineered to express a defined molecular target. Several technologies are now emerging that render it feasible to employ more physiologically, and clinically relevant, cell phenotypes. Consequently, numerous approaches use primary cells, which retain many functions seen in vivo, as well as endogenously expressing the target of interest. Furthermore, stem cells, of either embryonic or adult origin, as well as those derived from differentiated cells, are now finding a place in drug discovery. Collectively, these cells are expanding the utility of authentic human cells, either as screening tools or as therapeutics, as well as providing cells derived directly from patients. Nonetheless, the growing use of phenotypically relevant cells (including primary cells or stem cells) is not without technical difficulties, particularly when their envisioned use lies in high-throughput screening (HTS) protocols. In particular, the limited availability of homogeneous primary or stem cell populations for HTS mandates that novel technologies be developed to accelerate their adoption. These technologies include detection of responses with very few cells as well as protocols to generate cell lines in abundant, homogeneous populations. In parallel, the growing use of changes in cell phenotype as the assay readout is driving greater use of high-throughput imaging techniques in screening. Taken together, the greater availability of novel primary and stem cell phenotypes as well as new detection technologies is heralding a new era of cellular screening. This convergence offers unique opportunities to identify drug candidates for disorders at which few therapeutics are presently available.

  7. Imaging and screening in lung cancer

    Directory of Open Access Journals (Sweden)

    Matteo Giaj Levra

    2008-12-01

    Full Text Available Lung cancer is the main cause of death for neoplasia in the world. Hence it’s growing the necessity to investigate screening tests to detect tumoral lesions at the early stages: several trials have been performed to establish the best method, target and frequence of the screening to offer. CT, X-ray, PET, sputum citology and CAD software are here analyzed, together with the associated statistics and bias.

  8. Microchip Screening Platform for Single Cell Assessment of NK Cell Cytotoxicity

    Science.gov (United States)

    Guldevall, Karolin; Brandt, Ludwig; Forslund, Elin; Olofsson, Karl; Frisk, Thomas W.; Olofsson, Per E.; Gustafsson, Karin; Manneberg, Otto; Vanherberghen, Bruno; Brismar, Hjalmar; Kärre, Klas; Uhlin, Michael; Önfelt, Björn

    2016-01-01

    Here, we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK) cells within larger populations. Human primary NK cells were distributed across a silicon–glass microchip containing 32,400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis, the numbers of NK and live or dead target cells in each well could be assessed at different time points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (≈75%) were non-cytotoxic against the leukemia cell line K562, some NK cells were able to kill several (≥3) target cells within the 12-h long experiment. In addition, the screening approach was adapted to increase the chance to find and evaluate serial killing NK cells. Even if the cytotoxic potential varied between donors, it was evident that a small fraction of highly cytotoxic NK cells were responsible for a substantial portion of the killing. We demonstrate multiple assays where our platform can be used to enumerate and characterize cytotoxic cells, such as NK or T cells. This approach could find use in clinical applications, e.g., in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy. PMID:27092139

  9. Microchip screening platform for single cell assessment of NK cell cytotoxicity

    Directory of Open Access Journals (Sweden)

    Karolin eGuldevall

    2016-04-01

    Full Text Available Here we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK cells within larger populations. Human primary NK cells were distributed across a silicon-glass microchip containing 32 400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis the numbers of NK and live or dead target cells in each well could be assessed at different time points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (≈75% were non-cytotoxic against the leukemia cell line K562, some NK cells were able to kill several (≥3 target cells within the 12 hours long experiment. In addition, the screening approach was adapted to increase the chance to find and evaluate serial killing NK cells. Even if the cytotoxic potential varied between donors it was evident that a small fraction of highly cytotoxic NK cells were responsible for a substantial portion of the killing. We demonstrate multiple assays where our platform can be used to enumerate and characterize cytotoxic cells, such as NK or T cells. This approach could find use in clinical applications, e.g. in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy.

  10. Screen printed interdigitated back contact solar cell

    Science.gov (United States)

    Baraona, C. R.; Mazaris, G. A.; Chai, A. T.

    1984-10-01

    Interdigitated back contact solar cells are made by screen printing dopant materials onto the back surface of a semiconductor substrate in a pair of interdigitated patterns. These dopant materials are then diffused into the substrate to form junctions having configurations corresponding to these patterns. Contacts having configurations which match the patterns are then applied over the junctions.

  11. Screen printed interdigitated back contact solar cell

    Science.gov (United States)

    Baraona, C. R.; Mazaris, G. A.; Chai, A. T. (Inventor)

    1984-01-01

    Interdigitated back contact solar cells are made by screen printing dopant materials onto the back surface of a semiconductor substrate in a pair of interdigitated patterns. These dopant materials are then diffused into the substrate to form junctions having configurations corresponding to these patterns. Contacts having configurations which match the patterns are then applied over the junctions.

  12. Motion as a phenotype: the use of live-cell imaging and machine visual screening to characterize transcription-dependent chromosome dynamics

    Directory of Open Access Journals (Sweden)

    Silver Pamela A

    2006-04-01

    Full Text Available Abstract Background Gene transcriptional activity is well correlated with intra-nuclear position, especially relative to the nuclear periphery, which is a region classically associated with gene silencing. Recently however, actively transcribed genes have also been found localized to the nuclear periphery in the yeast Saccharomyces cerevisiae. When genes are activated, they become associated with the nuclear pore complex (NPC at the nuclear envelope. Furthermore, chromosomes are not static structures, but exhibit constrained diffusion in real-time, live-cell studies of particular loci. The relationship of chromosome motion with transcriptional activation and active-gene recruitment to the nuclear periphery has not yet been investigated. Results We have generated a yeast strain that enables us to observe the motion of the galactose-inducible GAL gene locus relative to the nuclear periphery in real-time under transcriptionally active and repressed conditions. Using segmented geometric particle tracking, we show that the repressed GAL locus undergoes constrained diffusive movement, and that transcriptional induction with galactose is associated with an enrichment in cells with GAL loci that are both associated with the nuclear periphery and much more constrained in their movement. Furthermore, we report that the mRNA export factor Sac3 is involved in this galactose-induced enrichment of GAL loci at the nuclear periphery. In parallel, using a novel machine visual screening technique, we find that the motion of constrained GAL loci correlates with the motion of the cognate nuclei in galactose-induced cells. Conclusion Transcriptional activation of the GAL genes is associated with their tethering and motion constraint at the nuclear periphery. We describe a model of gene recruitment to the nuclear periphery involving gene diffusion and the mRNA export factor Sac3 that can be used as a framework for further experimentation. In addition, we applied to

  13. Graph cut and image intensity-based splitting improves nuclei segmentation in high-content screening

    Science.gov (United States)

    Farhan, Muhammad; Ruusuvuori, Pekka; Emmenlauer, Mario; Rämö, Pauli; Yli-Harja, Olli; Dehio, Christoph

    2013-02-01

    Quantification of phenotypes in high-content screening experiments depends on the accuracy of single cell analysis. In such analysis workflows, cell nuclei segmentation is typically the first step and is followed by cell body segmentation, feature extraction, and subsequent data analysis workflows. Therefore, it is of utmost importance that the first steps of high-content analysis are done accurately in order to guarantee correctness of the final analysis results. In this paper, we present a novel cell nuclei image segmentation framework which exploits robustness of graph cut to obtain initial segmentation for image intensity-based clump splitting method to deliver the accurate overall segmentation. By using quantitative benchmarks and qualitative comparison with real images from high-content screening experiments with complicated multinucleate cells, we show that our method outperforms other state-of-the-art nuclei segmentation methods. Moreover, we provide a modular and easy-to-use implementation of the method for a widely used platform.

  14. A Novel Technique for Performing PID Susceptibility Screening during the Solar Cell Fabrication Process

    Energy Technology Data Exchange (ETDEWEB)

    Oh, Jaewon; Dahal, Som; Dauksher, Bill; Bowden, Stuart; Tamizhmani, Govindasamy; Hacke, Peter

    2016-11-21

    Various characterization techniques have historically been developed in order to screen potential induced degradation (PID)-susceptible cells, but those techniques require final solar cells. We present a new characterization technique for screening PID-susceptible cells during the cell fabrication process. Illuminated Lock-In Thermography (ILIT) was used to image PID shunting of the cell without metallization and clearly showed PID-affected areas. PID-susceptible cells can be screened by ILIT, and the sample structure can advantageously be simplified as long as the sample has the silicon nitride antireflection coating and an aluminum back surface field.

  15. Single-cell analysis of population context advances RNAi screening at multiple levels

    NARCIS (Netherlands)

    Snijder, Berend; Sacher, Raphael; Rämö, Pauli; Liberali, Prisca; Mench, Karin; Wolfrum, Nina; Burleigh, Laura; Scott, Cameron C; Verheije, Monique H; Mercer, Jason; Moese, Stefan; Heger, Thomas; Theusner, Kristina; Jurgeit, Andreas; Lamparter, David; Balistreri, Giuseppe; Schelhaas, Mario; De Haan, Cornelis A M; Marjomäki, Varpu; Hyypiä, Timo; Rottier, Peter J M; Sodeik, Beate; Marsh, Mark; Gruenberg, Jean; Amara, Ali; Greber, Urs; Helenius, Ari; Pelkmans, Lucas

    2012-01-01

    Isogenic cells in culture show strong variability, which arises from dynamic adaptations to the microenvironment of individual cells. Here we study the influence of the cell population context, which determines a single cell's microenvironment, in image-based RNAi screens. We developed a comprehensi

  16. Fundus autofluorescence imaging in an ocular screening program.

    Science.gov (United States)

    Kolomeyer, A M; Nayak, N V; Szirth, B C; Khouri, A S

    2012-01-01

    Purpose. To describe integration of fundus autofluorescence (FAF) imaging into an ocular screening program. Methods. Fifty consecutive screening participants were included in this prospective pilot imaging study. Color and FAF (530/640 nm exciter/barrier filters) images were obtained with a 15.1MP Canon nonmydriatic hybrid camera. A clinician evaluated the images on site to determine need for referral. Visual acuity (VA), intraocular pressure (IOP), and ocular pathology detected by color fundus and FAF imaging modalities were recorded. Results. Mean ± SD age was 47.4 ± 17.3 years. Fifty-two percent were female and 58% African American. Twenty-seven percent had a comprehensive ocular examination within the past year. Mean VA was 20/39 in the right eye and 20/40 in the left eye. Mean IOP was 15 mmHg bilaterally. Positive color and/or FAF findings were identified in nine (18%) individuals with diabetic retinopathy or macular edema (n = 4), focal RPE defects (n = 2), age-related macular degeneration (n = 1), central serous retinopathy (n = 1), and ocular trauma (n = 1). Conclusions. FAF was successfully integrated in our ocular screening program and aided in the identification of ocular pathology. Larger studies examining the utility of this technology in screening programs may be warranted.

  17. Fundus Autofluorescence Imaging in an Ocular Screening Program

    Directory of Open Access Journals (Sweden)

    A. M. Kolomeyer

    2012-01-01

    Full Text Available Purpose. To describe integration of fundus autofluorescence (FAF imaging into an ocular screening program. Methods. Fifty consecutive screening participants were included in this prospective pilot imaging study. Color and FAF (530/640 nm exciter/barrier filters images were obtained with a 15.1MP Canon nonmydriatic hybrid camera. A clinician evaluated the images on site to determine need for referral. Visual acuity (VA, intraocular pressure (IOP, and ocular pathology detected by color fundus and FAF imaging modalities were recorded. Results. Mean ± SD age was 47.4 ± 17.3 years. Fifty-two percent were female and 58% African American. Twenty-seven percent had a comprehensive ocular examination within the past year. Mean VA was 20/39 in the right eye and 20/40 in the left eye. Mean IOP was 15 mmHg bilaterally. Positive color and/or FAF findings were identified in nine (18% individuals with diabetic retinopathy or macular edema (n=4, focal RPE defects (n=2, age-related macular degeneration (n=1, central serous retinopathy (n=1, and ocular trauma (n=1. Conclusions. FAF was successfully integrated in our ocular screening program and aided in the identification of ocular pathology. Larger studies examining the utility of this technology in screening programs may be warranted.

  18. A novel imaging-based high-throughput screening approach to anti-angiogenic drug discovery.

    Science.gov (United States)

    Evensen, Lasse; Micklem, David R; Link, Wolfgang; Lorens, James B

    2010-01-01

    The successful progression to the clinic of angiogenesis inhibitors for cancer treatment has spurred interest in developing new classes of anti-angiogenic compounds. The resulting surge in available candidate therapeutics highlights the need for robust, high-throughput angiogenesis screening systems that adequately capture the complexity of new vessel formation while providing quantitative evaluation of the potency of these agents. Available in vitro angiogenesis assays are either cumbersome, impeding adaptation to high-throughput screening formats, or inadequately model the complex multistep process of new vessel formation. We therefore developed an organotypic endothelial-mural cell co-culture assay system that reflects several facets of angiogenesis while remaining compatible with high-throughput/high-content image screening. Co-culture of primary human endothelial cells (EC) and vascular smooth muscle cells (vSMC) results in assembly of a network of tubular endothelial structures enveloped with vascular basement membrane proteins, thus, comprising the three main components of blood vessels. Initially, EC are dependent on vSMC-derived VEGF and sensitive to clinical anti-angiogenic therapeutics. A subsequent phenotypic VEGF-switch renders EC networks resistant to anti-VEGF therapeutics, demarcating a mature vascular phenotype. Conversely, mature EC networks remain sensitive to vascular disrupting agents. Therefore, candidate anti-angiogenic compounds can be interrogated for their relative potency on immature and mature networks and classified as either vascular normalizing or vascular disrupting agents. Here, we demonstrate that the EC-vSMC co-culture assay represents a robust high-content imaging high-throughput screening system for identification of novel anti-angiogenic agents. A pilot high-throughput screening campaign was used to define informative imaging parameters and develop a follow-up dose-response scheme for hit characterization. High

  19. High-Content Screening for Quantitative Cell Biology.

    Science.gov (United States)

    Mattiazzi Usaj, Mojca; Styles, Erin B; Verster, Adrian J; Friesen, Helena; Boone, Charles; Andrews, Brenda J

    2016-08-01

    High-content screening (HCS), which combines automated fluorescence microscopy with quantitative image analysis, allows the acquisition of unbiased multiparametric data at the single cell level. This approach has been used to address diverse biological questions and identify a plethora of quantitative phenotypes of varying complexity in numerous different model systems. Here, we describe some recent applications of HCS, ranging from the identification of genes required for specific biological processes to the characterization of genetic interactions. We review the steps involved in the design of useful biological assays and automated image analysis, and describe major challenges associated with each. Additionally, we highlight emerging technologies and future challenges, and discuss how the field of HCS might be enhanced in the future.

  20. Validation of a high-content screening assay using whole-well imaging of transformed phenotypes.

    Science.gov (United States)

    Ramirez, Christina N; Ozawa, Tatsuya; Takagi, Toshimitsu; Antczak, Christophe; Shum, David; Graves, Robert; Holland, Eric C; Djaballah, Hakim

    2011-06-01

    Automated microscopy was introduced two decades ago and has become an integral part of the discovery process as a high-content screening platform with noticeable challenges in executing cell-based assays. It would be of interest to use it to screen for reversers of a transformed cell phenotype. In this report, we present data obtained from an optimized assay that identifies compounds that reverse a transformed phenotype induced in NIH-3T3 cells by expressing a novel oncogene, KP, resulting from fusion between platelet derived growth factor receptor alpha (PDGFRα) and kinase insert domain receptor (KDR), that was identified in human glioblastoma. Initial image acquisitions using multiple tiles per well were found to be insufficient as to accurately image and quantify the clusters; whole-well imaging, performed on the IN Cell Analyzer 2000, while still two-dimensional imaging, was found to accurately image and quantify clusters, due largely to the inherent variability of their size and well location. The resulting assay exhibited a Z' value of 0.79 and a signal-to-noise ratio of 15, and it was validated against known effectors and shown to identify only PDGFRα inhibitors, and then tested in a pilot screen against a library of 58 known inhibitors identifying mostly PDGFRα inhibitors as reversers of the KP induced transformed phenotype. In conclusion, our optimized and validated assay using whole-well imaging is robust and sensitive in identifying compounds that reverse the transformed phenotype induced by KP with a broader applicability to other cell-based assays that are challenging in HTS against chemical and RNAi libraries.

  1. Optical diagnostics of osteoblast cells and osteogenic drug screening

    Science.gov (United States)

    Kolanti, Elayaraja; Veerla, Sarath C.; Khajuria, Deepak K.; Roy Mahapatra, D.

    2016-02-01

    Microfluidic device based diagnostics involving optical fibre path, in situ imaging and spectroscopy are gaining importance due to recent advances in diagnostics instrumentation and methods, besides other factors such as low amount of reagent required for analysis, short investigation times, and potential possibilities to replace animal model based study in near future. It is possible to grow and monitor tissues in vitro in microfluidic lab-on-chip. It may become a transformative way of studying how cells interact with drugs, pathogens and biomaterials in physiologically relevant microenvironments. To a large extent, progress in developing clinically viable solutions has been constrained because of (i) contradiction between in vitro and in vivo results and (ii) animal model based and clinical studies which is very expensive. Our study here aims to evaluate the usefulness of microfluidic device based 3D tissue growth and monitoring approach to better emulate physiologically and clinically relevant microenvironments in comparison to conventional in vitro 2D culture. Moreover, the microfluidic methodology permits precise high-throughput investigations through real-time imaging while using very small amounts of reagents and cells. In the present study, we report on the details of an osteoblast cell based 3D microfluidic platform which we employ for osteogenic drug screening. The drug formulation is functionalized with fluorescence and other biomarkers for imaging and spectroscopy, respectively. Optical fibre coupled paths are used to obtain insight regarding the role of stress/flow pressure fluctuation and nanoparticle-drug concentration on the osteoblast growth and osteogenic properties of bone.

  2. 1Click1View: Interactive Visualization Methodology for RNAi Cell-Based Microscopic Screening

    Directory of Open Access Journals (Sweden)

    Lukasz Zwolinski

    2013-01-01

    Full Text Available Technological advancements are constantly increasing the size and complexity of data resulting from large-scale RNA interference screens. This fact has led biologists to ask complex questions, which the existing, fully automated analyses are often not adequate to answer. We present a concept of 1Click1View (1C1V as a methodology for interactive analytic software tools. 1C1V can be applied for two-dimensional visualization of image-based screening data sets from High Content Screening (HCS. Through an easy-to-use interface, one-click, one-view concept, and workflow based architecture, visualization method facilitates the linking of image data with numeric data. Such method utilizes state-of-the-art interactive visualization tools optimized for fast visualization of large scale image data sets. We demonstrate our method on an HCS dataset consisting of multiple cell features from two screening assays.

  3. High-Throughput Screening Using Fourier-Transform Infrared Imaging

    Directory of Open Access Journals (Sweden)

    Erdem Sasmaz

    2015-06-01

    Full Text Available Efficient parallel screening of combinatorial libraries is one of the most challenging aspects of the high-throughput (HT heterogeneous catalysis workflow. Today, a number of methods have been used in HT catalyst studies, including various optical, mass-spectrometry, and gas-chromatography techniques. Of these, rapid-scanning Fourier-transform infrared (FTIR imaging is one of the fastest and most versatile screening techniques. Here, the new design of the 16-channel HT reactor is presented and test results for its accuracy and reproducibility are shown. The performance of the system was evaluated through the oxidation of CO over commercial Pd/Al2O3 and cobalt oxide nanoparticles synthesized with different reducer-reductant molar ratios, surfactant types, metal and surfactant concentrations, synthesis temperatures, and ramp rates.

  4. Image-based siRNA screen to identify kinases regulating Weibel-Palade body size control using electroporation.

    Science.gov (United States)

    Ketteler, Robin; Freeman, Jamie; Ferraro, Francesco; Bata, Nicole; Cutler, Dan F; Kriston-Vizi, Janos

    2017-03-01

    High-content screening of kinase inhibitors is important in order to identify biogenesis and function mechanisms of subcellular organelles. Here, we present a human kinome siRNA high-content screen on primary human umbilical vein endothelial cells, that were transfected by electroporation. The data descriptor contains a confocal fluorescence, microscopic image dataset. We also describe an open source, automated image analysis workflow that can be reused to perform high-content analysis of other organelles. This dataset is suitable for analysis of morphological parameters that are linked to human umbilical vein endothelial cell (HUVEC) biology.

  5. Russian Image on the Federal Republic of Germany Screen

    Directory of Open Access Journals (Sweden)

    Alexander Fedorov

    2014-06-01

    Full Text Available This article analyzed the image of Russia in the screen mirror of the Federal Republic of Germany for: determine the place and role of the image of the USSR and the Russian and Soviet / Russian characters in the movies from the beginning of the postwar German ideological confrontation to 1991 (the collapse of the Soviet Union as compared with the trends of the modern era (1992 – present; identify political, ideological, social and cultural contexts, the main stages of development, concepts, goals, objectives, authors of fiction films themes outlined above; classify and compare trends and stereotypes German films related to the Soviet / Russian theme: ideology, content model, genre modifications (including: socio-political analysis, stereotypes, ideological analysis, identification analysis, the iconographic analysis, narrative analysis, the characters etc..

  6. Screening Diabetic Retinopathy in Developing Countries using Retinal Images

    Directory of Open Access Journals (Sweden)

    D. SIVA SUNDHARA RAJA

    2015-03-01

    Full Text Available In developing countries, diabetic retinopathy (DR is the leading cause of blindness in diabetic patients due to intraocular hypertension or high glucose level. Its detection in an earlier stage is essential to prevent vision loss in type 2 diabetic patients. In this paper, the computer aided automatic screening system for diabetic retinopathy is proposed. DR can be diagnosed by detecting the abnormal lesions such as hemorrhages in retinal images and analyzing its relationship with the fovea region. The proposed method consists of the following stages, namely: retinal image enhancement and classification, hemorrhages detection and segmentation, fovea localization and Diabetic Retinopathy classification. The multi directional local histogram equalization is used to enhance the retinal image for better classification rate. The Gabor transform and Support vector machine (SVM classifier is used for retinal image classifications. The proposed method is tested on publicly available HRFand DIARETDB1datasets. The sensitivity and specificity of hemorrhages detection are 94.76% and 99.85%, respectively. Thus, the severity of Diabetic Retinopathy in Type 2 diabetic patients can be easily identified by detecting fovea region and hemorrhage lesions and analyzing the relation between them to prevent vision loss in diabetic patients.

  7. Automated cell analysis tool for a genome-wide RNAi screen with support vector machine based supervised learning

    Science.gov (United States)

    Remmele, Steffen; Ritzerfeld, Julia; Nickel, Walter; Hesser, Jürgen

    2011-03-01

    RNAi-based high-throughput microscopy screens have become an important tool in biological sciences in order to decrypt mostly unknown biological functions of human genes. However, manual analysis is impossible for such screens since the amount of image data sets can often be in the hundred thousands. Reliable automated tools are thus required to analyse the fluorescence microscopy image data sets usually containing two or more reaction channels. The herein presented image analysis tool is designed to analyse an RNAi screen investigating the intracellular trafficking and targeting of acylated Src kinases. In this specific screen, a data set consists of three reaction channels and the investigated cells can appear in different phenotypes. The main issue of the image processing task is an automatic cell segmentation which has to be robust and accurate for all different phenotypes and a successive phenotype classification. The cell segmentation is done in two steps by segmenting the cell nuclei first and then using a classifier-enhanced region growing on basis of the cell nuclei to segment the cells. The classification of the cells is realized by a support vector machine which has to be trained manually using supervised learning. Furthermore, the tool is brightness invariant allowing different staining quality and it provides a quality control that copes with typical defects during preparation and acquisition. A first version of the tool has already been successfully applied for an RNAi-screen containing three hundred thousand image data sets and the SVM extended version is designed for additional screens.

  8. Automated screening versus manual screening: a comparison of the ThinPrep imaging system and manual screening in a time study

    DEFF Research Database (Denmark)

    Schledermann, Doris; Hyldebrandt, Tina; Ejersbo, Dorthe;

    2007-01-01

    The ThinPrep Imaging System (TIS) is an automated system that assists cytotechnologists in the primary screening of ThinPrep liquid based cervical samples. Between June 1, 2004, and April 1, 2005, four experienced cytotechnologists participated in the study in which the duration of the screening ...

  9. Microfluidic cell chips for high-throughput drug screening.

    Science.gov (United States)

    Chi, Chun-Wei; Ahmed, Ah Rezwanuddin; Dereli-Korkut, Zeynep; Wang, Sihong

    2016-05-01

    The current state of screening methods for drug discovery is still riddled with several inefficiencies. Although some widely used high-throughput screening platforms may enhance the drug screening process, their cost and oversimplification of cell-drug interactions pose a translational difficulty. Microfluidic cell-chips resolve many issues found in conventional HTS technology, providing benefits such as reduced sample quantity and integration of 3D cell culture physically more representative of the physiological/pathological microenvironment. In this review, we introduce the advantages of microfluidic devices in drug screening, and outline the critical factors which influence device design, highlighting recent innovations and advances in the field including a summary of commercialization efforts on microfluidic cell chips. Future perspectives of microfluidic cell devices are also provided based on considerations of present technological limitations and translational barriers.

  10. Non-interferometric quantitative phase imaging of yeast cells

    Science.gov (United States)

    Poola, Praveen K.; Pandiyan, Vimal Prabhu; John, Renu

    2015-12-01

    Real-time imaging of live cells is quite difficult without the addition of external contrast agents. Various methods for quantitative phase imaging of living cells have been proposed like digital holographic microscopy and diffraction phase microscopy. In this paper, we report theoretical and experimental results of quantitative phase imaging of live yeast cells with nanometric precision using transport of intensity equations (TIE). We demonstrate nanometric depth sensitivity in imaging live yeast cells using this technique. This technique being noninterferometric, does not need any coherent light sources and images can be captured through a regular bright-field microscope. This real-time imaging technique would deliver the depth or 3-D volume information of cells and is highly promising in real-time digital pathology applications, screening of pathogens and staging of diseases like malaria as it does not need any preprocessing of samples.

  11. Cell-free DNA screening and sex chromosome aneuploidies.

    Science.gov (United States)

    Mennuti, Michael T; Chandrasekaran, Suchitra; Khalek, Nahla; Dugoff, Lorraine

    2015-10-01

    Cell-free DNA (cfDNA) testing is increasingly being used to screen pregnant women for fetal aneuploidies. This technology may also identify fetal sex and can be used to screen for sex chromosome aneuploidies (SCAs). Physicians offering this screening will need to be prepared to offer comprehensive prenatal counseling about these disorders to an increasing number of patients. The purpose of this article is to consider the source of information to use for counseling, factors in parental decision-making, and the performance characteristics of cfDNA testing in screening for SCAs. Discordance between ultrasound examination and cfDNA results regarding fetal sex is also discussed.

  12. An infrared image based methodology for breast lesions screening

    Science.gov (United States)

    Morais, K. C. C.; Vargas, J. V. C.; Reisemberger, G. G.; Freitas, F. N. P.; Oliari, S. H.; Brioschi, M. L.; Louveira, M. H.; Spautz, C.; Dias, F. G.; Gasperin, P.; Budel, V. M.; Cordeiro, R. A. G.; Schittini, A. P. P.; Neto, C. D.

    2016-05-01

    The objective of this paper is to evaluate the potential of utilizing a structured methodology for breast lesions screening, based on infrared imaging temperature measurements of a healthy control group to establish expected normality ranges, and of breast cancer patients, previously diagnosed through biopsies of the affected regions. An analysis of the systematic error of the infrared camera skin temperature measurements was conducted in several different regions of the body, by direct comparison to high precision thermistor temperature measurements, showing that infrared camera temperatures are consistently around 2 °C above the thermistor temperatures. Therefore, a method of conjugated gradients is proposed to eliminate the infrared camera direct temperature measurement imprecision, by calculating the temperature difference between two points to cancel out the error. The method takes into account the human body approximate bilateral symmetry, and compares measured dimensionless temperature difference values (Δ θ bar) between two symmetric regions of the patient's breast, that takes into account the breast region, the surrounding ambient and the individual core temperatures, and doing so, the results interpretation for different individuals become simple and non subjective. The range of normal whole breast average dimensionless temperature differences for 101 healthy individuals was determined, and admitting that the breasts temperatures exhibit a unimodal normal distribution, the healthy normal range for each region was considered to be the dimensionless temperature difference plus/minus twice the standard deviation of the measurements, Δ θ bar ‾ + 2σ Δ θ bar ‾ , in order to represent 95% of the population. Forty-seven patients with previously diagnosed breast cancer through biopsies were examined with the method, which was capable of detecting breast abnormalities in 45 cases (96%). Therefore, the conjugated gradients method was considered effective

  13. Analysis and recognition of touching cell images based on morphological structures.

    Science.gov (United States)

    Yu, Donggang; Pham, Tuan D; Zhou, Xiaobo

    2009-01-01

    Automated analysis and recognition of cell-nuclear phases using fluorescence microscopy images play an important role for high-content screening. A major task of automated imaging based high-content screening is to segment and reconstruct each cell from the touching cell images. In this paper we present new useful method for recognizing morphological structural models of touching cells, detecting segmentation points, determining the number of segmented cells in touching cell image, finding the related data of segmented cell arcs and reconstructing segmented cells. The conceptual frameworks are based on the morphological structures where a series of structural points and their morphological relationships are established. Experiment results have shown the efficient application of the new method for analysis and recognition of touching cell images of high-content screening.

  14. Change descriptors for determining nodule malignancy in national lung screening trial CT screening images

    Science.gov (United States)

    Geiger, Benjamin; Hawkins, Samuel; Hall, Lawrence O.; Goldgof, Dmitry B.; Balagurunathan, Yoganand; Gatenby, Robert A.; Gillies, Robert J.

    2016-03-01

    Pulmonary nodules are effectively diagnosed in CT scans, but determining their malignancy has been a challenge. The rate of change of the volume of a pulmonary nodule is known to be a prognostic factor for cancer development. In this study, we propose that other changes in imaging characteristics are similarly informative. We examined the combination of image features across multiple CT scans, taken from the National Lung Screening Trial, with individual scans of the same patient separated by approximately one year. By subtracting the values of existing features in multiple scans for the same patient, we were able to improve the ability of existing classification algorithms to determine whether a nodule will become malignant. We trained each classifier on 83 nodules determined to be malignant by biopsy and 172 nodules determined to be benign by their clinical stability through two years of no change; classifiers were tested on 77 malignant and 144 benign nodules, using a set of features that in a test-retest experiment were shown to be stable. An accuracy of 83.71% and AUC of 0.814 were achieved with the Random Forests classifier on a subset of features determined to be stable via test-retest reproducibility analysis, further reduced with the Correlation-based Feature Selection algorithm.

  15. The future of imaging screening: proceedings of the Fourth Annual ACR FORUM.

    Science.gov (United States)

    Hillman, Bruce J; Amis, E Stephen; Weinreb, Jeffrey C; Neiman, Harvey L

    2005-01-01

    The FORUM is an ACR planning activity focused on a specific topic deemed to be of long-range importance to the college. A select panel of multidisciplinary experts met in 2004 to consider the ramifications of imaging screening from diverse perspectives. Considerations included the nature of screening for disease, its technological issues, and biases associated with the perceived success of screening; potential technologies and target diseases; business and economics; insurance coverage; ethics and the law; how imaging screening could be paired with nonimaging screening; and how the lay public perceives screening. The FORUM participants considered how imaging screening might develop over the next 5 to 10 years and made recommendations to the ACR on actions it might take to benefit both the specialty and patients.

  16. High-Throughput Screening for Bioactive Molecules Using Primary Cell Culture of Transgenic Zebrafish Embryos

    Directory of Open Access Journals (Sweden)

    Haigen Huang

    2012-09-01

    Full Text Available Transgenic zebrafish embryos expressing tissue-specific green fluorescent protein (GFP can provide an unlimited supply of primary embryonic cells. Agents that promote the differentiation of these cells may be beneficial for therapeutics. We report a high-throughput approach for screening small molecules that regulate cell differentiation using lineage-specific GFP transgenic zebrafish embryonic cells. After validating several known regulators of the differentiation of endothelial and other cell types, we performed a screen for proangiogenic molecules using undifferentiated primary cells from flk1-GFP transgenic zebrafish embryos. Cells were grown in 384-well plates with 12,128 individual small molecules, and GFP expression was analyzed by means of an automated imaging system, which allowed us to screen thousands of compounds weekly. As a result, 23 molecules were confirmed to enhance angiogenesis, and 11 of them were validated to promote the proliferation of mammalian human umbilical vascular endothelial cells and induce Flk1+ cells from murine embryonic stem cells. We demonstrated the general applicability of this strategy by analyzing additional cell lineages using zebrafish expressing GFP in pancreatic, cardiac, and dopaminergic cells.

  17. Sickle cell disease: time for a targeted neonatal screening programme.

    LENUS (Irish Health Repository)

    Gibbons, C

    2015-02-01

    Ireland has seen a steady increase in paediatric sickle cell disease (SCD). In 2005, only 25% of children with SCD were referred to the haemoglobinopathy service in their first year. A non-funded screening programme was implemented. This review aimed to assess the impact screening has had. All children referred to the haemoglobinopathy service born in Ireland after 2005 were identified. Data was collected from the medical chart and laboratory system. Information was analysed using Microsoft Excel. 77 children with SCD were identified. The median age at antibiotic commencement in the screened group was 56 days compared with 447 days in the unscreened group, p = < 0.0003. 22 (28%) of infants were born in centre\\'s that do not screen and 17 (81%) were over 6 months old at referral, compared with 14 (21%) in the screened group. 6 (27%) of those in the unscreened group presented in acute crisis compared with 2 (3%) in the screened population. The point prevalence of SCD in Ireland is 0.2% in children under 15 yr of African and Asian descent. We identified delays in referral and treatment, which reflect the lack of government funded support and policy. We suggest all maternity units commence screening for newborns at risk of SCD. It is a cost effective intervention with a number needed to screen of just 4 to prevent a potentially fatal crisis.

  18. Cell based assay for hypoglycemic drugs screening

    Institute of Scientific and Technical Information of China (English)

    LiZHANG; Juan-juanHU; Guan-huaDU

    2004-01-01

    OBJECTIVE: To establish a cell based assay for hypoglyc emicdrugs. METHODS: The five cell lines, BALB/c3T3, HepG2, NIH3T3, Be17402, and L929 were incubated with insulin (0-125n mol/L) for 48 h. Their sensitivities to insulin were studied by detecting glucose consumption. The dose-response and time-response relationship between the sensitive cell line (BALB/c 3T3)

  19. On-Chip Peptide Mass Spectrometry Imaging for Protein Kinase Inhibitor Screening.

    Science.gov (United States)

    Cho, Young-Lai; Kim, Young-Pil; Son, Jin Gyeong; Son, Miyoung; Lee, Tae Geol

    2017-01-03

    Protein kinases are enzymes that are important targets for drug discovery because of their involvement in regulating the essential cellular processes. For this reason, the changes in protein kinase activity induced by each drug candidate (the inhibitor in this case) need to be accurately determined. Here, an on-chip secondary ion mass spectrometry (SIMS) imaging technique of the peptides was developed for determining protein kinase activity and inhibitor screening without a matrix. In our method, cysteine-tethered peptides adsorbed onto a gold surface produced changes in the relative peak intensities of the phosphorylated and unphosphorylated substrate peptides, which were quantitatively dependent on protein kinase activity. Using mass spectrometry imaging of multiple compartments on the gold surface in the presence of a peptide substrate, we screened 13,727 inhibitors, of which seven were initially found to have inhibitor efficiencies that surpassed 50%. Of these, we were able to identify a new breakpoint cluster region-abelson (BCR-ABL)(T315I) kinase inhibitor, henceforth referred to as KR135861. KR135861 showed no cytotoxicity and was subsequently confirmed to be superior to imatinib, a commercial drug marketed as Gleevec. Moreover, KR135861 exhibited a greater inhibitory effect on the BCR-ABL(T315I) tyrosine kinase, with an IC50 value as low as 1.3 μM. In in vitro experiments, KR135861 reduced the viability of both Ba/F3 cells expressing wild-type BCR-ABL and BCR-ABL(T315I), in contrast to imatinib's inhibitory effects only on Ba/F3 cells expressing wild-type BCR-ABL. Due to the surface sensitivity and selectivity of SIMS imaging, it is anticipated that our approach will make it easier to validate the small modifications of a substrate in relation to enzyme activity as well as for drug discovery. This mass spectrometry imaging analysis enables efficient screening for protein kinase inhibitors, thus permitting high-throughput drug screening with high accuracy

  20. Microfluidic cell culture systems with integrated sensors for drug screening

    Science.gov (United States)

    Grist, Samantha; Yu, Linfen; Chrostowski, Lukas; Cheung, Karen C.

    2012-03-01

    Cell-based testing is a key step in drug screening for cancer treatments. A microfluidic platform can permit more precise control of the cell culture microenvironment, such as gradients in soluble factors. These small-scale devices also permit tracking of low cell numbers. As a new screening paradigm, a microscale system for integrated cell culture and drug screening promises to provide a simple, scalable tool to apply standardized protocols used in cellular response assays. With the ability to dynamically control the microenvironment, we can create temporally varying drug profiles to mimic physiologically measured profiles. In addition, low levels of oxygen in cancerous tumors have been linked with drug resistance and decreased likelihood of successful treatment and patient survival. Our work also integrates a thin-film oxygen sensor with a microfluidic oxygen gradient generator which will in future allow us to create spatial oxygen gradients and study effects of hypoxia on cell response to drug treatment. In future, this technology promises to improve cell-based validation in the drug discovery process, decreasing the cost and increasing the speed in screening large numbers of compounds.

  1. Screening of nanoparticle embryotoxicity using embryonic stem cells.

    Science.gov (United States)

    Campagnolo, Luisa; Fenoglio, Ivana; Massimiani, Micol; Magrini, Andrea; Pietroiusti, Antonio

    2013-01-01

    Due to the increasing use of engineered nanoparticles in many consumer products, rapid and economic tests for evaluating possible adverse effects on human health are urgently needed. In the present chapter the use of mouse embryonic stem cells as a valuable tool to in vitro screen nanoparticle toxicity on embryonic tissues is described. This in vitro method is a modification of the embryonic stem cell test, which has been widely used to screen soluble chemical compounds for their embryotoxic potential. The test offers an alternative to animal experimentation, reducing experimental costs and ethical issues.

  2. Whole organism high-content screening by label-free, image-based Bayesian classification for parasitic diseases.

    Directory of Open Access Journals (Sweden)

    Ross A Paveley

    Full Text Available Sole reliance on one drug, Praziquantel, for treatment and control of schistosomiasis raises concerns about development of widespread resistance, prompting renewed interest in the discovery of new anthelmintics. To discover new leads we designed an automated label-free, high content-based, high throughput screen (HTS to assess drug-induced effects on in vitro cultured larvae (schistosomula using bright-field imaging. Automatic image analysis and Bayesian prediction models define morphological damage, hit/non-hit prediction and larval phenotype characterization. Motility was also assessed from time-lapse images. In screening a 10,041 compound library the HTS correctly detected 99.8% of the hits scored visually. A proportion of these larval hits were also active in an adult worm ex-vivo screen and are the subject of ongoing studies. The method allows, for the first time, screening of large compound collections against schistosomes and the methods are adaptable to other whole organism and cell-based screening by morphology and motility phenotyping.

  3. Population-based study of DNA image cytometry as screening method for esophageal cancer

    Institute of Scientific and Technical Information of China (English)

    Lin Zhao; Guo-Qing Wang; Qi Shang; You-Lin Qiao; Wen-Qiang Wei; De-Li Zhao; Chang-Qing Hao; Dong-Mei Lin; Qin-Jing Pan; Xin-Qing Li; Fu-Hua Lei; Jin-Wu Wang

    2012-01-01

    AIM: To explore the DNA image cytometry (DNA-ICM) technique as a primary screening method for esophageal squamous precancerous lesions.METHODS: This study was designed as a populationbased screening study. A total of 582 local residents aged 40 years-69 years were recruited from Linzhou in Henan and Feicheng in Shandong. However, only 452 subjects had results of liquid-based cytology, DNA-ICM and pathology. The sensitivity and specificity of DNAICM were calculated and compared with liquid-based cytology in moderate dysplasia or worse. RESULTS: Sensitivities of DNA-ICM ranging from at least 1 to 4 aneuploid cells were 90.91%, 86.36%, 79.55% and 77.27%, respectively, which were better than that of liquid-based cytology (75%). Specificities of DNA-ICM were 70.83%, 84.07%, 92.65% and 96.81%, but the specificity of liquid-based cytology was 91.91%. The sensitivity and specificity of a combination of liquid-based cytology and DNA-ICM were 84.09% and 85.78%, respectively. CONCLUSION: It is possible to use DNA-ICM technique as a primary screening method for esophageal squamous precancerous lesions.

  4. Sickle Cell Screening: Medical, Legal, Ethical, Psychological and Social Problems; A Sickle Cell Crisis.

    Science.gov (United States)

    Bowman, James E.

    In recent years, sickle cell screening programs have been initiated by community groups, health centers, hospitals, medical schools, health departments, school systems, city and State governments, various branches of the Federal Government, fraternal and social clubs, and other organizations. Problems have resulted from mass sickle cell screening,…

  5. Fluorescence imaging-based high-throughput screening of fast- and slow-cycling LOV proteins.

    Directory of Open Access Journals (Sweden)

    Fuun Kawano

    Full Text Available Light-oxygen-voltage (LOV domains function as blue light-inducible molecular switches. The photosensory LOV domains derived from plants and fungi have provided an indispensable tool for optogenetics. Here we develop a high-throughput screening system to efficiently improve switch-off kinetics of LOV domains. The present system is based on fluorescence imaging of thermal reversion of a flavin cofactor bound to LOV domains. We conducted multi site-directed random mutagenesis of seven amino acid residues surrounding the flavin cofactor of the second LOV domain derived from Avena sativa phototropin 1 (AsLOV2. The gene library was introduced into Escherichia coli cells. Then thermal reversion of AsLOV2 variants, respectively expressed in different bacterial colonies on agar plate, was imaged with a stereoscopic fluorescence microscope. Based on the mutagenesis and imaging-based screening, we isolated 12 different variants showing substantially faster thermal reversion kinetics than wild-type AsLOV2. Among them, AsLOV2-V416T exhibited thermal reversion with a time constant of 2.6 s, 21-fold faster than wild-type AsLOV2. With a slight modification of the present approach, we also have efficiently isolated 8 different decelerated variants, represented by AsLOV2-V416L that exhibited thermal reversion with a time constant of 4.3 × 10(3 s (78-fold slower than wild-type AsLOV2. The present approach based on fluorescence imaging of the thermal reversion of the flavin cofactor is generally applicable to a variety of blue light-inducible molecular switches and may provide a new opportunity for the development of molecular tools for emerging optogenetics.

  6. Screening fluorescent voltage indicators with spontaneously spiking HEK cells.

    Directory of Open Access Journals (Sweden)

    Jeehae Park

    Full Text Available Development of improved fluorescent voltage indicators is a key challenge in neuroscience, but progress has been hampered by the low throughput of patch-clamp characterization. We introduce a line of non-fluorescent HEK cells that stably express NaV 1.3 and KIR 2.1 and generate spontaneous electrical action potentials. These cells enable rapid, electrode-free screening of speed and sensitivity of voltage sensitive dyes or fluorescent proteins on a standard fluorescence microscope. We screened a small library of mutants of archaerhodopsin 3 (Arch in spiking HEK cells and identified two mutants with greater voltage-sensitivity than found in previously published Arch voltage indicators.

  7. High-content, high-throughput screening for the identification of cytotoxic compounds based on cell morphology and cell proliferation markers.

    Directory of Open Access Journals (Sweden)

    Heather L Martin

    Full Text Available Toxicity is a major cause of failure in drug discovery and development, and whilst robust toxicological testing occurs, efficiency could be improved if compounds with cytotoxic characteristics were identified during primary compound screening. The use of high-content imaging in primary screening is becoming more widespread, and by utilising phenotypic approaches it should be possible to incorporate cytotoxicity counter-screens into primary screens. Here we present a novel phenotypic assay that can be used as a counter-screen to identify compounds with adverse cellular effects. This assay has been developed using U2OS cells, the PerkinElmer Operetta high-content/high-throughput imaging system and Columbus image analysis software. In Columbus, algorithms were devised to identify changes in nuclear morphology, cell shape and proliferation using DAPI, TOTO-3 and phosphohistone H3 staining, respectively. The algorithms were developed and tested on cells treated with doxorubicin, taxol and nocodazole. The assay was then used to screen a novel, chemical library, rich in natural product-like molecules of over 300 compounds, 13.6% of which were identified as having adverse cellular effects. This assay provides a relatively cheap and rapid approach for identifying compounds with adverse cellular effects during screening assays, potentially reducing compound rejection due to toxicity in subsequent in vitro and in vivo assays.

  8. Recovery of indium from LCD screens of discarded cell phones.

    Science.gov (United States)

    Silveira, A V M; Fuchs, M S; Pinheiro, D K; Tanabe, E H; Bertuol, D A

    2015-11-01

    Advances in technological development have resulted in high consumption of electrical and electronic equipment (EEE), amongst which are cell phones, which have LCD (liquid crystal display) screens as one of their main components. These multilayer screens are composed of different materials, some with high added value, as in the case of the indium present in the form of indium tin oxide (ITO, or tin-doped indium oxide). Indium is a precious metal with relatively limited natural reserves (Dodbida et al., 2012), so it can be profitable to recover it from discarded LCD screens. The objective of this study was to develop a complete process for recovering indium from LCD screens. Firstly, the screens were manually removed from cell phones. In the next step, a pretreatment was developed for removal of the polarizing film from the glass of the LCD panels, because the adherence of this film to the glass complicated the comminution process. The choice of mill was based on tests using different equipment (knife mill, hammer mill, and ball mill) to disintegrate the LCD screens, either before or after removal of the polarizing film. In the leaching process, it was possible to extract 96.4 wt.% of the indium under the following conditions: 1.0M H2SO4, 1:50 solid/liquid ratio, 90°C, 1h, and stirring at 500 rpm. The results showed that the best experimental conditions enabled extraction of 613 mg of indium/kg of LCD powder. Finally, precipitation of the indium with NH4OH was tested at different pH values, and 99.8 wt.% precipitation was achieved at pH 7.4.

  9. Prototype of Microwave Imaging System for Breast-Cancer Screening

    DEFF Research Database (Denmark)

    Rubæk, Tonny; Zhurbenko, Vitaliy

    2009-01-01

    Microwave imaging for breast-cancer detection has received the attention of a large number of research groups in the last decade. In this paper, the imaging system currently being developed at the Technical university of Denmark is presented. This includes a description of the antenna system......, the microwave hardware, and the imaging algorithm....

  10. A low cost touch-screen interface for image collage

    Directory of Open Access Journals (Sweden)

    Idanis Díaz Bolaño

    2011-01-01

    Full Text Available En este artículo se presenta el diseño de una interfaz de bajo costo basada en interacción touch-screen para crear collage de imágenes. La interfaz fue diseñada para propósitos de rehabilitación, tales como la estimulación de desarrollo cognitivo, habilidades motoras, creatividad y expresividad. La interacción touch-screen de la interfaz se basa en la utilización de un control remoto de Wii. Para evaluar la eficiencia de la interfaz, el diseño touch-screen fue comparado con un diseño basado en interacción con el mouse. A pesar de los problemas inherentes a la baja resolución de la cámara infrarroja del Wii Remote, los resultados experimentales obtenidos muestran que la versión del sistema touch-screen permitió un mejor desempeño para realizar tareas que requieren habilidades motoras finas, tales como delinear la forma libre de una figura.

  11. Development of automatic image analysis methods for high-throughput and high-content screening

    NARCIS (Netherlands)

    Di, Zi

    2013-01-01

    This thesis focuses on the development of image analysis methods for ultra-high content analysis of high-throughput screens where cellular phenotype responses to various genetic or chemical perturbations that are under investigation. Our primary goal is to deliver efficient and robust image analysis

  12. Imaging of sickle cell disease

    Energy Technology Data Exchange (ETDEWEB)

    Crowley, J.J. [Department of Pediatric Imaging, Children`s Hospital of Michigan, Detroit (United States); Sarnaik, S. [Sickle Cell Center, Children`s Hospital of Michigan, Detroit (United States)

    1999-09-01

    Sickle cell disease is an important health care issue in the United States and in certain areas in Africa, the Middle East and India. Although a great deal of progress has been made in understanding the disease at the molecular and pathophysiologic level, specific treatment which is safe and accessible for most patients is still elusive. Going into the next millennium, the management of this disease is still largely dependent on early diagnosis and the treatment of complications with supportive care. Thus, diagnosis and evaluation of the complications of the disease are crucial in directing clinical care at the bedside. Modern imaging modalities have greatly improved, and their application in the patient with the sickling disorders has enhanced the decision - making process. The purpose of this article is to review the clinical aspects of common complications of the disease and to discuss imaging approaches which are useful in their evaluation. (orig.) With 15 figs., 102 refs.

  13. Functions of the digital image in Education: A methodological proposal for reading and writing the digital image on instructional screens

    Directory of Open Access Journals (Sweden)

    Mariella Milagros Azzato

    2011-07-01

    Full Text Available This research goes through the instructional possibilities that reading and writing the digital image have in Education. Along these lines, we are presenting this research that looks for, on one hand, to develop a methodological proposal for reading and writing the digital image, and on the other, to implement these methodologies in a course used as a study case and whose objective was to evaluate students' performance when writing screens for a learning object using the methodologies for reading and writing the digital image. The process for compiling date was based on the questionnaire technique, individual interviews and the analysis of course proposed activities. The application of the first questionnaire allowed us to determine students' knowledge level about the digital image before starting the course. The individual interview allowed us to determine the students' reading criteria gained after using the reading methodology for the digital image to analyse educational materials (Galavis, 2008; Azzato, 2009. The proposed activities for the course permitted us to value students' performance when reading and writing the digital image of a learning object. Finally, after course completion, the second questionnaire was applied in order to determine the students' acquired knowledge level about reading and writing an image on digital screens. The results obtained in each of the analysis allowed us to establish that the proposed methodologies were highly useful to write the educational image for the screens of each one of the learning objects created in the course.

  14. Secondary solid cancer screening following hematopoietic cell transplantation.

    Science.gov (United States)

    Inamoto, Y; Shah, N N; Savani, B N; Shaw, B E; Abraham, A A; Ahmed, I A; Akpek, G; Atsuta, Y; Baker, K S; Basak, G W; Bitan, M; DeFilipp, Z; Gregory, T K; Greinix, H T; Hamadani, M; Hamilton, B K; Hayashi, R J; Jacobsohn, D A; Kamble, R T; Kasow, K A; Khera, N; Lazarus, H M; Malone, A K; Lupo-Stanghellini, M T; Margossian, S P; Muffly, L S; Norkin, M; Ramanathan, M; Salooja, N; Schoemans, H; Wingard, J R; Wirk, B; Wood, W A; Yong, A; Duncan, C N; Flowers, M E D; Majhail, N S

    2015-08-01

    Hematopoietic stem cell transplant (HCT) recipients have a substantial risk of developing secondary solid cancers, particularly beyond 5 years after HCT and without reaching a plateau overtime. A working group was established through the Center for International Blood and Marrow Transplant Research and the European Group for Blood and Marrow Transplantation with the goal to facilitate implementation of cancer screening appropriate to HCT recipients. The working group reviewed guidelines and methods for cancer screening applicable to the general population and reviewed the incidence and risk factors for secondary cancers after HCT. A consensus approach was used to establish recommendations for individual secondary cancers. The most common sites include oral cavity, skin, breast and thyroid. Risks of cancers are increased after HCT compared with the general population in skin, thyroid, oral cavity, esophagus, liver, nervous system, bone and connective tissues. Myeloablative TBI, young age at HCT, chronic GVHD and prolonged immunosuppressive treatment beyond 24 months were well-documented risk factors for many types of secondary cancers. All HCT recipients should be advised of the risks of secondary cancers annually and encouraged to undergo recommended screening based on their predisposition. Here we propose guidelines to help clinicians in providing screening and preventive care for secondary cancers among HCT recipients.

  15. Secondary solid cancer screening following hematopoietic cell transplantation

    Science.gov (United States)

    Inamoto, Y; Shah, NN; Savani, BN; Shaw, BE; Abraham, AA; Ahmed, IA; Akpek, G; Atsuta, Y; Baker, KS; Basak, GW; Bitan, M; DeFilipp, Z; Gregory, TK; Greinix, HT; Hamadani, M; Hamilton, BK; Hayashi, RJ; Jacobsohn, DA; Kamble, RT; Kasow, KA; Khera, N; Lazarus, HM; Malone, AK; Lupo-Stanghellini, MT; Margossian, SP; Muffly, LS; Norkin, M; Ramanathan, M; Salooja, N; Schoemans, H; Wingard, JR; Wirk, B; Wood, WA; Yong, A; Duncan, CN; Flowers, MED; Majhail, NS

    2016-01-01

    Hematopoietic stem cell transplant (HCT) recipients have a substantial risk of developing secondary solid cancers, particularly beyond 5 years after HCT and without reaching a plateau overtime. A working group was established through the Center for International Blood and Marrow Transplant Research and the European Group for Blood and Marrow Transplantation with the goal to facilitate implementation of cancer screening appropriate to HCT recipients. The working group reviewed guidelines and methods for cancer screening applicable to the general population and reviewed the incidence and risk factors for secondary cancers after HCT. A consensus approach was used to establish recommendations for individual secondary cancers. The most common sites include oral cavity, skin, breast and thyroid. Risks of cancers are increased after HCT compared with the general population in skin, thyroid, oral cavity, esophagus, liver, nervous system, bone and connective tissues. Myeloablative TBI, young age at HCT, chronic GVHD and prolonged immunosuppressive treatment beyond 24 months were well-documented risk factors for many types of secondary cancers. All HCT recipients should be advised of the risks of secondary cancers annually and encouraged to undergo recommended screening based on their predisposition. Here we propose guidelines to help clinicians in providing screening and preventive care for secondary cancers among HCT recipients. PMID:25822223

  16. Microfluidic Approaches for Manipulating, Imaging, and Screening C. elegans

    Directory of Open Access Journals (Sweden)

    Bhagwati P. Gupta

    2016-07-01

    Full Text Available The nematode C. elegans (worm is a small invertebrate animal widely used in studies related to fundamental biological processes, disease modelling, and drug discovery. Due to their small size and transparent body, these worms are highly suitable for experimental manipulations. In recent years several microfluidic devices and platforms have been developed to accelerate worm handling, phenotypic studies and screens. Here we review major tools and briefly discuss their usage in C. elegans research.

  17. Fundus autofluorescence and colour fundus imaging compared during telemedicine screening in patients with diabetes.

    Science.gov (United States)

    Kolomeyer, Anton M; Baumrind, Benjamin R; Szirth, Bernard C; Shahid, Khadija; Khouri, Albert S

    2013-06-01

    We investigated the use of fundus autofluorescence (FAF) imaging in screening the eyes of patients with diabetes. Images were obtained from 50 patients with type 2 diabetes undergoing telemedicine screening with colour fundus imaging. The colour and FAF images were obtained with a 15.1 megapixel non-mydriatic retinal camera. Colour and FAF images were compared for pathology seen in nonproliferative and proliferative diabetic retinopathy (NPDR and PDR, respectively). A qualitative assessment was made of the ease of detecting early retinopathy changes and the extent of existing retinopathy. The mean age of the patients was 47 years, most were male (82%) and most were African American (68%). Their mean visual acuity was 20/45 and their mean intraocular pressure was 14.3 mm Hg. Thirty-eight eyes (76%) did not show any diabetic retinopathy changes on colour or FAF imaging. Seven patients (14%) met the criteria for NPDR and five (10%) for severe NPDR or PDR. The most common findings were microaneurysms, hard exudates and intra-retinal haemorrhages (IRH) (n = 6 for each). IRH, microaneurysms and chorioretinal scars were more easily visible on FAF images. Hard exudates, pre-retinal haemorrhage and fibrosis, macular oedema and Hollenhorst plaque were easier to identify on colour photographs. The value of FAF imaging as a complementary technique to colour fundus imaging in detecting diabetic retinopathy during ocular screening warrants further investigation.

  18. Including Antenna Models in Microwave Imaging for Breast-Cancer Screening

    DEFF Research Database (Denmark)

    Rubæk, Tonny; Meincke, Peter

    2006-01-01

    Microwave imaging is emerging as a tool for screening for breast cancer, but the lack of methods for including the characteristics of the antennas of the imaging systems in the imaging algorithms limits their performance. In this paper, a method for incorporating the full antenna characteristics......, in terms of the transmission-matrix representation, in a frequency-domain imaging algorithm is presented. The algorithm is tested on a simulation of the Physical-Anomaly Tomography (PAT) scanner imaging system developed at the Technical University of Denmark and is shown to have superior performance when...

  19. BioSig3D: High Content Screening of Three-Dimensional Cell Culture Models.

    Directory of Open Access Journals (Sweden)

    Cemal Cagatay Bilgin

    Full Text Available BioSig3D is a computational platform for high-content screening of three-dimensional (3D cell culture models that are imaged in full 3D volume. It provides an end-to-end solution for designing high content screening assays, based on colony organization that is derived from segmentation of nuclei in each colony. BioSig3D also enables visualization of raw and processed 3D volumetric data for quality control, and integrates advanced bioinformatics analysis. The system consists of multiple computational and annotation modules that are coupled together with a strong use of controlled vocabularies to reduce ambiguities between different users. It is a web-based system that allows users to: design an experiment by defining experimental variables, upload a large set of volumetric images into the system, analyze and visualize the dataset, and either display computed indices as a heatmap, or phenotypic subtypes for heterogeneity analysis, or download computed indices for statistical analysis or integrative biology. BioSig3D has been used to profile baseline colony formations with two experiments: (i morphogenesis of a panel of human mammary epithelial cell lines (HMEC, and (ii heterogeneity in colony formation using an immortalized non-transformed cell line. These experiments reveal intrinsic growth properties of well-characterized cell lines that are routinely used for biological studies. BioSig3D is being released with seed datasets and video-based documentation.

  20. Live cell imaging in Drosophila melanogaster.

    Science.gov (United States)

    Parton, Richard M; Vallés, Ana Maria; Dobbie, Ian M; Davis, Ilan

    2010-04-01

    Although many of the techniques of live cell imaging in Drosophila melanogaster are also used by the greater community of cell biologists working on other model systems, studying living fly tissues presents unique difficulties with regard to keeping the cells alive, introducing fluorescent probes, and imaging through thick, hazy cytoplasm. This article outlines the major tissue types amenable to study by time-lapse cinematography and different methods for keeping the cells alive. It describes various imaging and associated techniques best suited to following changes in the distribution of fluorescently labeled molecules in real time in these tissues. Imaging, in general, is a rapidly developing discipline, and recent advances in imaging technology are able to greatly extend what can be achieved with live cell imaging of Drosophila tissues. As far as possible, this article includes the latest technical developments and discusses likely future developments in imaging methods that could have an impact on research using Drosophila.

  1. Generation of human pluripotent stem cell-derived hepatocyte-like cells for drug toxicity screening.

    Science.gov (United States)

    Takayama, Kazuo; Mizuguchi, Hiroyuki

    2017-02-01

    Because drug-induced liver injury is one of the main reasons for drug development failures, it is important to perform drug toxicity screening in the early phase of pharmaceutical development. Currently, primary human hepatocytes are most widely used for the prediction of drug-induced liver injury. However, the sources of primary human hepatocytes are limited, making it difficult to supply the abundant quantities required for large-scale drug toxicity screening. Therefore, there is an urgent need for a novel unlimited, efficient, inexpensive, and predictive model which can be applied for large-scale drug toxicity screening. Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are able to replicate indefinitely and differentiate into most of the body's cell types, including hepatocytes. It is expected that hepatocyte-like cells generated from human ES/iPS cells (human ES/iPS-HLCs) will be a useful tool for drug toxicity screening. To apply human ES/iPS-HLCs to various applications including drug toxicity screening, homogenous and functional HLCs must be differentiated from human ES/iPS cells. In this review, we will introduce the current status of hepatocyte differentiation technology from human ES/iPS cells and a novel method to predict drug-induced liver injury using human ES/iPS-HLCs.

  2. A Picture You Can Handle: Infants Treat Touch-Screen Images More Like Photographs than Objects.

    Science.gov (United States)

    Ziemer, Christine J; Snyder, Makenna

    2016-01-01

    Infants actively explore their world in order to determine the different ways in which they can interact with various objects. Although research on infant perception has focused on how infants understand the differences between 2- and 3-dimensional objects, today's infants increasingly encounter 2D images with interactive qualities on smart-phone screens, tablets, and laptops. The purpose of this experiment was to examine the types of manual behaviors infants direct toward tablet images and to compare these actions to those evoked by 2D photographs or 3D when tactile feedback is controlled. Infants between the ages of 7-10 months sat on their parent's lap in front of a table with a built-in well covered by a clear, plastic sheet while the three types of displays (photographs, objects, and screen images on a tablet) were presented for 30 s each. Infants saw three examples of each type of display presented in the built-in well so that tactile feedback information from the different displays was controlled. Coders noted the proportion of trials in which infants grasped, scratched, rubbed, or patted the display. Results indicate that infants direct significantly more grasps, scratches, and rubs toward 3D objects than 2D photographs. Infants also direct more grasps to objects compared to screen images. Our data suggests that infants are treating screen images more similarly to 2D photographs than 3D objects.

  3. Automatic screening and classification of diabetic retinopathy and maculopathy using fuzzy image processing.

    Science.gov (United States)

    Rahim, Sarni Suhaila; Palade, Vasile; Shuttleworth, James; Jayne, Chrisina

    2016-12-01

    Digital retinal imaging is a challenging screening method for which effective, robust and cost-effective approaches are still to be developed. Regular screening for diabetic retinopathy and diabetic maculopathy diseases is necessary in order to identify the group at risk of visual impairment. This paper presents a novel automatic detection of diabetic retinopathy and maculopathy in eye fundus images by employing fuzzy image processing techniques. The paper first introduces the existing systems for diabetic retinopathy screening, with an emphasis on the maculopathy detection methods. The proposed medical decision support system consists of four parts, namely: image acquisition, image preprocessing including four retinal structures localisation, feature extraction and the classification of diabetic retinopathy and maculopathy. A combination of fuzzy image processing techniques, the Circular Hough Transform and several feature extraction methods are implemented in the proposed system. The paper also presents a novel technique for the macula region localisation in order to detect the maculopathy. In addition to the proposed detection system, the paper highlights a novel online dataset and it presents the dataset collection, the expert diagnosis process and the advantages of our online database compared to other public eye fundus image databases for diabetic retinopathy purposes.

  4. A screen against Leishmania intracellular amastigotes: comparison to a promastigote screen and identification of a host cell-specific hit.

    Directory of Open Access Journals (Sweden)

    Geraldine De Muylder

    2011-07-01

    Full Text Available The ability to screen compounds in a high-throughput manner is essential in the process of small molecule drug discovery. Critical to the success of screening strategies is the proper design of the assay, often implying a compromise between ease/speed and a biologically relevant setting. Leishmaniasis is a major neglected disease with limited therapeutic options. In order to streamline efforts for the design of productive drug screens against Leishmania, we compared the efficiency of two screening methods, one targeting the free living and easily cultured promastigote (insect-infective stage, the other targeting the clinically relevant but more difficult to culture intra-macrophage amastigote (mammal-infective stage. Screening of a 909-member library of bioactive compounds against Leishmania donovani revealed 59 hits in the promastigote primary screen and 27 in the intracellular amastigote screen, with 26 hits shared by both screens. This suggested that screening against the promastigote stage, although more suitable for automation, fails to identify all active compounds and leads to numerous false positive hits. Of particular interest was the identification of one compound specific to the infective amastigote stage of the parasite. This compound affects intracellular but not axenic parasites, suggesting a host cell-dependent mechanism of action, opening new avenues for anti-leishmanial chemotherapy.

  5. Stem cells and small molecule screening: haploid embryonic stem cells as a new tool

    Institute of Scientific and Technical Information of China (English)

    Bi WU; Wei LI; Liu WANG; Zhong-hua LIU; Xiao-yang ZHAO

    2013-01-01

    Stem cells can both self-renew and differentiate into various cell types under certain conditions,which makes them a good model for development and disease studies.Recently,chemical approaches have been widely applied in stem cell biology by promoting stem cell self-renewal,proliferation,differentiation and somatic cell reprogramming using specific small molecules.Conversely,stem cells and their derivatives also provide an efficient and robust platform for small molecule and drug screening.Here,we review the current research and applications of small molecules that modulate stem cell self-renewal and differentiation and improve reprogramming,as well as the applications that use stem cells as a tool for small molecule screening.Moreover,we introduce the recent advance in haploid embryonic stem cells research.Haploid embryonic stem cells maintain haploidy and stable growth over extensive passages,possess the ability to differentiate into all three germ layers in vitro and in vivo,and contribute to the germlines of chimeras when injected into blastocysts.Androgenetic haploid stem cells can also be used in place of sperm to produce fertile progeny after intracytoplasmic injection into mature oocytes.Such characteristics demonstrate that haploid stem cells are a new approach for genetic studies at both the cellular and animal levels and that they are a valuable platform for future small molecule screening.

  6. PRELIMINARY APPLICATION OF WHOLE BODY DIFFUSION WEIGHTED IMAGING IN SCREENING METASTASIS

    Institute of Scientific and Technical Information of China (English)

    Yong-jing Guan; Hua-wei Ling; Ke-min Chen

    2008-01-01

    Objective To investigate the feasibility of whole body diffusion weighted imaging (WB-DWI) in screening metastasis.Methods WB-DWI was performed in 24 patients diagnosed with various types of primary tumors. The three-dimensional maximum intensity projection reconstruction and black-and-white flip technique were used to observe metastatic lesions, and the results were compared with those of bone scintigraphy. Results By WB-DWI scanning sequence at b = 800 s/mm2, all the bone lesions found by bone scintigraphy in the cohort were well identified, and other lesions of soft tissue and organs were also well demonstrated. Its screening capability was equivalent with bone scintigraphy in screening metastases in bones (P = 0.062). Conclusion WB-DWI was practicable with the parameter settings attempted in metastases screening.

  7. Influence of metal screens on contrast in megavoltage x-ray imaging.

    Science.gov (United States)

    Droege, R T; Bjärngard, B E

    1979-01-01

    The radiographic contrast of metal screen-film detectors was investigated in order to determine the contrast capabilities of these detectors applied to megavoltage x-ray imaging. The film contrast gamma was found to be independent of the metal screen composition. Measurement of the scatter-to-primary film dose ratio in contact geometry demonstrated that a thick front screen of either 1.5 g/cm2 copper of 2.5 g/cm2 lead provides optimum contrast for the photon energies studied (60Co and 4- and 8-MV x rays). The same thickness were also found to be suitable in an air gap geometry which significantly improved the contrast compared to the contact geometry. Rear lead screens were found to provide no contrast improvement.

  8. Performance assessment of CsI(Tl) screens on various substrates for X-ray imaging

    Institute of Scientific and Technical Information of China (English)

    FENG Zhao-Dong; JIANG Peng; ZHANG Hong-Kai; ZHAO Bo-Zhen; QIN Xiu-Bo; WEI Cun-Feng; LIU Yu

    2015-01-01

    Thallium-doped cesium iodide (CsI(Tl)) screens are widely used in X-ray imaging devices because of the columnar structure of the CsI(Tl) layer,but few reports focus on the optical role of the substrate in the screen system.In this paper,four substrates including fused silica (SiO2),silver-film coated SiO2,graphite (C) and fiber optic plate (FOP) are used to fabricate CsI(Tl) screens by thermal evaporation.Their imaging performance is evaluated by relative light output (RLO),modulation transfer function (MTF),normalized noise power spectrum (NNPS) and noise equivalent quanta (NEQ).The results reveal that although CsI(Tl) film on graphite plate yields images with the lowest light output,it presents relatively higher spatial resolution and better signal-to-noise characteristics.However,films on SiO2 plate obtain low MTF but high NNPS curves,whether they are coated with silver film or not.Furthermore,scintillation screens on FOP have bright images with low NNPS and high NEQ,but have the lowest MTF.By controlling the substrate optical features,CsI(Tl) films can be tailored to suit a given application.

  9. IDIOS: An innovative index for evaluating dental imaging-based osteoporosis screening indices

    Energy Technology Data Exchange (ETDEWEB)

    Barngkgei, Imad; Al Haffar, Iyad; Khattab, Razan [Faculty of Dentistry, Damascus University, Damascus (Syrian Arab Republic); Halboub, Esam; Almashraqi, Abeer Abdulkareem [Dept. of Maxillofacial Surgery and Diagnostic Sciences, College of Dentistry, Jazan University, Jazan (Saudi Arabia)

    2016-09-15

    The goal of this study was to develop a new index as an objective reference for evaluating current and newly developed indices used for osteoporosis screening based on dental images. Its name; IDIOS, stands for Index of Dental-imaging Indices of Osteoporosis Screening. A comprehensive PubMed search was conducted to retrieve studies on dental imaging-based indices for osteoporosis screening. The results of the eligible studies, along with other relevant criteria, were used to develop IDIOS, which has scores ranging from 0 (0%) to 15 (100%). The indices presented in the studies we included were then evaluated using IDIOS. The 104 studies that were included utilized 24, 4, and 9 indices derived from panoramic, periapical, and computed tomographic/cone-beam computed tomographic techniques, respectively. The IDIOS scores for these indices ranged from 0 (0%) to 11.75 (78.32%). IDIOS is a valuable reference index that facilitates the evaluation of other dental imaging-based osteoporosis screening indices. Furthermore, IDIOS can be utilized to evaluate the accuracy of newly developed indices.

  10. Screen-imaging guidance using a modified portable video macroscope for middle cerebral artery occlusion

    Institute of Scientific and Technical Information of China (English)

    Xingbao Zhu; Xinghua Pan; Junli Luo; Yun Liu; Guolong Chen; Song Liu; Qiangjin Ruan; Xunding Deng; Dianchun Wang; Quanshui Fan

    2012-01-01

    The use of operating microscopes is limited by the focal length. Surgeons using these instruments cannot simultaneously view and access the surgical field and must choose one or the other. The longer focal length (more than 1 000 mm) of an operating telescope permits a position away from the operating field, above the surgeon and out of the field of view. This gives the telescope an advantage over an operating microscope. We developed a telescopic system using screen-imaging guidance and a modified portable video macroscope constructed from a Computar MLH-10 × macro lens, a DFK-21AU04 USB CCD Camera and a Dell laptop computer as monitor screen. This system was used to establish a middle cerebral artery occlusion model in rats. Results showed that magnification of the modified portable video macroscope was appropriate (5-20 ×) even though the Computar MLH-10 × macro lens was placed 800 mm away from the operating field rather than at the specified working distance of 152.4 mm with a zoom of 1-40 ×. The screen-imaging telescopic technique was clear, life-like, stereoscopic and matched the actual operation. Screen-imaging guidance led to an accurate, smooth, minimally invasive and comparatively easy surgical procedure. Success rate of the model establishment evaluated by neurological function using the modified neurological score system was 74.07%. There was no significant difference in model establishment time, sensorimotor deficit and infarct volume percentage. Our findings indicate that the telescopic lens is effective in the screen surgical operation mode referred to as "long distance observation and short distance operation" and that screen-imaging guidance using an modified portable video macroscope can be utilized for the establishment of a middle cerebral artery occlusion model and micro-neurosurgery.

  11. Screen-imaging guidance using a modified portable video macroscope for middle cerebral artery occlusion.

    Science.gov (United States)

    Zhu, Xingbao; Luo, Junli; Liu, Yun; Chen, Guolong; Liu, Song; Ruan, Qiangjin; Deng, Xunding; Wang, Dianchun; Fan, Quanshui; Pan, Xinghua

    2012-04-25

    The use of operating microscopes is limited by the focal length. Surgeons using these instruments cannot simultaneously view and access the surgical field and must choose one or the other. The longer focal length (more than 1 000 mm) of an operating telescope permits a position away from the operating field, above the surgeon and out of the field of view. This gives the telescope an advantage over an operating microscope. We developed a telescopic system using screen-imaging guidance and a modified portable video macroscope constructed from a Computar MLH-10 × macro lens, a DFK-21AU04 USB CCD Camera and a Dell laptop computer as monitor screen. This system was used to establish a middle cerebral artery occlusion model in rats. Results showed that magnification of the modified portable video macroscope was appropriate (5-20 ×) even though the Computar MLH-10 × macro lens was placed 800 mm away from the operating field rather than at the specified working distance of 152.4 mm with a zoom of 1-40 ×. The screen-imaging telescopic technique was clear, life-like, stereoscopic and matched the actual operation. Screen-imaging guidance led to an accurate, smooth, minimally invasive and comparatively easy surgical procedure. Success rate of the model establishment evaluated by neurological function using the modified neurological score system was 74.07%. There was no significant difference in model establishment time, sensorimotor deficit and infarct volume percentage. Our findings indicate that the telescopic lens is effective in the screen surgical operation mode referred to as "long distance observation and short distance operation" and that screen-imaging guidance using an modified portable video macroscope can be utilized for the establishment of a middle cerebral artery occlusion model and micro-neurosurgery.

  12. Probing bacterial cell biology using image cytometry.

    Science.gov (United States)

    Cass, Julie A; Stylianidou, Stella; Kuwada, Nathan J; Traxler, Beth; Wiggins, Paul A

    2017-03-01

    Advances in automated fluorescence microscopy have made snapshot and time-lapse imaging of bacterial cells commonplace, yet fundamental challenges remain in analysis. The vast quantity of data collected in high-throughput experiments requires a fast and reliable automated method to analyze fluorescence intensity and localization, cell morphology and proliferation as well as other descriptors. Inspired by effective yet tractable methods of population-level analysis using flow cytometry, we have developed a framework and tools for facilitating analogous analyses in image cytometry. These tools can both visualize and gate (generate subpopulations) more than 70 cell descriptors, including cell size, age and fluorescence. The method is well suited to multi-well imaging, analysis of bacterial cultures with high cell density (thousands of cells per frame) and complete cell cycle imaging. We give a brief description of the analysis of four distinct applications to emphasize the broad applicability of the tool.

  13. Can imaging be used for inflammatory arthritis screening?

    DEFF Research Database (Denmark)

    Østergaard, Mikkel

    2012-01-01

    This article reviews the utility of imaging in the diagnostic work-up of suspected and undifferentiated axial and peripheral inflammatory arthritis. Radiographic findings, that is, late damage but not early inflammation, are part of the classification criteria for rheumatoid arthritis (RA......), ankylosing spondylitis, spondyloarthritis (SpA), and psoriatic arthritis (PsA), and they are generally part of the early examination program in arthritis.Computed tomography visualizes calcified tissue with high resolution but is rarely used unless radiography is unclear and MRI unavailable...... of RA, PsA, and SpA.MRI and US are part of the recent American College of Rheumatology/European League against Rheumatism 2010 classification criteria for RA (can be used to count involved joints), and MRI is part of the SpondyloArthritis International Society criteria for axial and peripheral Sp...

  14. Gold silver alloy nanoparticles (GSAN): an imaging probe for breast cancer screening with dual-energy mammography or computed tomography

    Science.gov (United States)

    Naha, Pratap C.; Lau, Kristen C.; Hsu, Jessica C.; Hajfathalian, Maryam; Mian, Shaameen; Chhour, Peter; Uppuluri, Lahari; McDonald, Elizabeth S.; Maidment, Andrew D. A.; Cormode, David P.

    2016-07-01

    Earlier detection of breast cancer reduces mortality from this disease. As a result, the development of better screening techniques is a topic of intense interest. Contrast-enhanced dual-energy mammography (DEM) is a novel technique that has improved sensitivity for cancer detection. However, the development of contrast agents for this technique is in its infancy. We herein report gold-silver alloy nanoparticles (GSAN) that have potent DEM contrast properties and improved biocompatibility. GSAN formulations containing a range of gold : silver ratios and capped with m-PEG were synthesized and characterized using various analytical methods. DEM and computed tomography (CT) phantom imaging showed that GSAN produced robust contrast that was comparable to silver alone. Cell viability, reactive oxygen species generation and DNA damage results revealed that the formulations with 30% or higher gold content are cytocompatible to Hep G2 and J774A.1 cells. In vivo imaging was performed in mice with and without breast tumors. The results showed that GSAN produce strong DEM and CT contrast and accumulated in tumors. Furthermore, both in vivo imaging and ex vivo analysis indicated the excretion of GSAN via both urine and feces. In summary, GSAN produce strong DEM and CT contrast, and has potential for both blood pool imaging and for breast cancer screening.Earlier detection of breast cancer reduces mortality from this disease. As a result, the development of better screening techniques is a topic of intense interest. Contrast-enhanced dual-energy mammography (DEM) is a novel technique that has improved sensitivity for cancer detection. However, the development of contrast agents for this technique is in its infancy. We herein report gold-silver alloy nanoparticles (GSAN) that have potent DEM contrast properties and improved biocompatibility. GSAN formulations containing a range of gold : silver ratios and capped with m-PEG were synthesized and characterized using various

  15. Laser-Induced Fluorescence Detection in High-Throughput Screening of Heterogeneous Catalysts and Single Cells Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Hui Su

    2001-05-01

    Laser-induced fluorescence detection is one of the most sensitive detection techniques and it has found enormous applications in various areas. The purpose of this research was to develop detection approaches based on laser-induced fluorescence detection in two different areas, heterogeneous catalysts screening and single cell study. First, we introduced laser-induced imaging (LIFI) as a high-throughput screening technique for heterogeneous catalysts to explore the use of this high-throughput screening technique in discovery and study of various heterogeneous catalyst systems. This scheme is based on the fact that the creation or the destruction of chemical bonds alters the fluorescence properties of suitably designed molecules. By irradiating the region immediately above the catalytic surface with a laser, the fluorescence intensity of a selected product or reactant can be imaged by a charge-coupled device (CCD) camera to follow the catalytic activity as a function of time and space. By screening the catalytic activity of vanadium pentoxide catalysts in oxidation of naphthalene, we demonstrated LIFI has good detection performance and the spatial and temporal resolution needed for high-throughput screening of heterogeneous catalysts. The sample packing density can reach up to 250 x 250 subunits/cm{sub 2} for 40-{micro}m wells. This experimental set-up also can screen solid catalysts via near infrared thermography detection.

  16. Laser-Induced Fluorescence Detection in High-Throughput Screening of Heterogeneous Catalysts and Single Cells Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Su, Hui [Iowa State Univ., Ames, IA (United States)

    2001-01-01

    Laser-induced fluorescence detection is one of the most sensitive detection techniques and it has found enormous applications in various areas. The purpose of this research was to develop detection approaches based on laser-induced fluorescence detection in two different areas, heterogeneous catalysts screening and single cell study. First, we introduced laser-induced imaging (LIFI) as a high-throughput screening technique for heterogeneous catalysts to explore the use of this high-throughput screening technique in discovery and study of various heterogeneous catalyst systems. This scheme is based on the fact that the creation or the destruction of chemical bonds alters the fluorescence properties of suitably designed molecules. By irradiating the region immediately above the catalytic surface with a laser, the fluorescence intensity of a selected product or reactant can be imaged by a charge-coupled device (CCD) camera to follow the catalytic activity as a function of time and space. By screening the catalytic activity of vanadium pentoxide catalysts in oxidation of naphthalene, we demonstrated LIFI has good detection performance and the spatial and temporal resolution needed for high-throughput screening of heterogeneous catalysts. The sample packing density can reach up to 250 x 250 subunits/cm2 for 40-μm wells. This experimental set-up also can screen solid catalysts via near infrared thermography detection.

  17. Quantitative Phenotyping-Based In Vivo Chemical Screening in a Zebrafish Model of Leukemia Stem Cell Xenotransplantation

    Science.gov (United States)

    Zhang, Beibei; Shimada, Yasuhito; Kuroyanagi, Junya; Umemoto, Noriko; Nishimura, Yuhei; Tanaka, Toshio

    2014-01-01

    Zebrafish-based chemical screening has recently emerged as a rapid and efficient method to identify important compounds that modulate specific biological processes and to test the therapeutic efficacy in disease models, including cancer. In leukemia, the ablation of leukemia stem cells (LSCs) is necessary to permanently eradicate the leukemia cell population. However, because of the very small number of LSCs in leukemia cell populations, their use in xenotransplantation studies (in vivo) and the difficulties in functionally and pathophysiologically replicating clinical conditions in cell culture experiments (in vitro), the progress of drug discovery for LSC inhibitors has been painfully slow. In this study, we developed a novel phenotype-based in vivo screening method using LSCs xenotransplanted into zebrafish. Aldehyde dehydrogenase-positive (ALDH+) cells were purified from chronic myelogenous leukemia K562 cells tagged with a fluorescent protein (Kusabira-orange) and then implanted in young zebrafish at 48 hours post-fertilization. Twenty-four hours after transplantation, the animals were treated with one of eight different therapeutic agents (imatinib, dasatinib, parthenolide, TDZD-8, arsenic trioxide, niclosamide, salinomycin, and thioridazine). Cancer cell proliferation, and cell migration were determined by high-content imaging. Of the eight compounds that were tested, all except imatinib and dasatinib selectively inhibited ALDH+ cell proliferation in zebrafish. In addition, these anti-LSC agents suppressed tumor cell migration in LSC-xenotransplants. Our approach offers a simple, rapid, and reliable in vivo screening system that facilitates the phenotype-driven discovery of drugs effective in suppressing LSCs. PMID:24454867

  18. High-Throughput Screening and Optimization of Binary Quantum Dots Cosensitized Solar Cell.

    Science.gov (United States)

    Yuan, Ding; Xiao, Lina; Luo, Jianheng; Luo, Yanhong; Meng, Qingbo; Mao, Bing-Wei; Zhan, Dongping

    2016-07-20

    Quantum dots (QDs) are considered as the alternative of dye sensitizers for solar cells. However, interfacial construction and evaluation of photocatalytic nanomaterials still remains challenge through the conventional methodology involving demo devices. We propose here a high-throughput screening and optimizing method based on combinatorial chemistry and scanning electrochemical microscopy (SECM). A homogeneous TiO2 catalyst layer is coated on a FTO substrate, which is then covered by a dark mask to expose the photocatalyst array. On each photocatalyst spot, different successive ionic layer adsorption and reaction (SILAR) processes are performed by a programmed solution dispenser to load the binary PbxCd1-xS QDs sensitizers. An optical fiber is employed as the scanning tip of SECM, and the photocatalytic current is recorded during the imaging experiment, through which the optimized technical parameters are figured out. To verify the validity of the combinatorial SECM imaging results, the controlled trials are performed with the corresponding photovoltaic demo devices. The harmonious accordance proved that the methodology based on combinatorial chemistry and SECM is valuable for the interfacial construction, high-throughput screening, and optimization of QDSSCs. Furthermore, the PbxCd1-xS/CdS QDs cosensitized solar cell optimized by SECM achieves a short circuit current density of 24.47 mA/cm(2), an open circuit potential of 421 mV, a fill factor of 0.52, and a photovoltaic conversion efficiency of 5.33%.

  19. Liquid marbles for high-throughput biological screening of anchorage-dependent cells.

    Science.gov (United States)

    Oliveira, Nuno M; Correia, Clara R; Reis, Rui L; Mano, João F

    2015-01-28

    Stable liquid marbles (LM) are produced by coating liquid droplets with a hydrophobic powder. The used hydrophobic powder is produced by fluorosi-lanization of diatomaceous earth, used before to produce superhydrophobic structures. Here, the use of LM is proposed for high-throughput drug screening on anchorage-dependent cells. To provide the required cell adhesion sites inside the liquid environment of LM, surface-modified poly(l-lactic acid) microparticles are used. A simple method that takes advantage from LM appealing features is presented, such as the ability to inject liquid on LM without disrupting (self-healing ability), and to monitor color changes inside of LM. After promoting cell adhesion, a cytotoxic screening test is performed as a proof of concept. Fe(3+) is used as a model cytotoxic agent and is injected on LM. After incubation, AlamarBlue reagent is injected and used to assess the presence of viable cells, by monitoring color change from blue to red. Color intensity is measured by image processing and the analysis of pictures takes using an ordinary digital camera. The proposed method is fully validated in counterpoint to an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carbo​xymethoxyphenyl)-2-(4-sulfophenyl)-2H-te​trazolium) colorimetric assay, a well-known method used for the cytotoxicity assessment.

  20. The Image of Russia on the Western Screen: the Present Stage (1992–2013

    Directory of Open Access Journals (Sweden)

    Alexander Fedorov

    2013-01-01

    Full Text Available This article included the analysis of evolution Russian image representation on the Western (USA, UK, Germany, Canada, France, Italy screen – of the period of post-communist modern times (1992-2013: ideological and social trends, myths, stereotypes, illusions, classification of the contents’ models and modifications of genres. The main conclusion: the image of Russia in the majority of the Western fiction films of 1992–2013 is still treated as an image of something, “alien”, “different”, often hostile

  1. From Screens to Streets: The Dissemination of Guy Fawkes Image in Physical and Living Media

    Directory of Open Access Journals (Sweden)

    S. Yigit Soncul

    2014-05-01

    Full Text Available From New York and Cairo, to São Paulo and Istanbul; Guy Fawkes mask embodies the changing face of contemporary mass mobilizations around the world. The initial signification of the Guy Fawkes image that is rooted from the Gunpowder Plot of 1605, has gone under transformations through its employments in the comic book V for Vendetta (Alan Moore and David Lloyd, 1981 and the film version V for Vendetta (James McTeigue, 2006. Though, the recent utilization of the mask by the hacktivist group Anonymous has supplied a stronger link between the screen and the street. Through using Hans Belting’s image analysis approach that he had developed in the book, An Anthropology of Images (2011, this paper aims to analyze a sample of contagious embodiments of public images in twenty-first century screen culture—where one can designate a relationship of power between screens (physical media and bodies (living media. The questions that are likely to emerge from such analysis will be discussed vis-a-vis the bodily potentials for political expression on the street today.  The employment of the public images by the protestors as a rhetorical tool will be examined and positioned in this context. 

  2. In vivo cell tracking with bioluminescence imaging

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jung Eun; Kalimuthu, Senthilkumar; Ahn, Byeong Cheol [Dept. of Nuclear Medicine, Kyungpook National University School of Medicine and Hospital, Daegu (Korea, Republic of)

    2015-03-15

    Molecular imaging is a fast growing biomedical research that allows the visual representation, characterization and quantification of biological processes at the cellular and subcellular levels within intact living organisms. In vivo tracking of cells is an indispensable technology for development and optimization of cell therapy for replacement or renewal of damaged or diseased tissue using transplanted cells, often autologous cells. With outstanding advantages of bioluminescence imaging, the imaging approach is most commonly applied for in vivo monitoring of transplanted stem cells or immune cells in order to assess viability of administered cells with therapeutic efficacy in preclinical small animal models. In this review, a general overview of bioluminescence is provided and recent updates of in vivo cell tracking using the bioluminescence signal are discussed.

  3. In vivo cell tracking with bioluminescence imaging.

    Science.gov (United States)

    Kim, Jung Eun; Kalimuthu, Senthilkumar; Ahn, Byeong-Cheol

    2015-03-01

    Molecular imaging is a fast growing biomedical research that allows the visual representation, characterization and quantification of biological processes at the cellular and subcellular levels within intact living organisms. In vivo tracking of cells is an indispensable technology for development and optimization of cell therapy for replacement or renewal of damaged or diseased tissue using transplanted cells, often autologous cells. With outstanding advantages of bioluminescence imaging, the imaging approach is most commonly applied for in vivo monitoring of transplanted stem cells or immune cells in order to assess viability of administered cells with therapeutic efficacy in preclinical small animal models. In this review, a general overview of bioluminescence is provided and recent updates of in vivo cell tracking using the bioluminescence signal are discussed.

  4. Red blood cells, sickle cell (image)

    Science.gov (United States)

    Sickle cell anemia is an inherited blood disease in which the red blood cells produce abnormal pigment (hemoglobin). ... abnormal hemoglobin causes deformity of the red blood cells into crescent or sickle-shapes, as seen in this photomicrograph.

  5. Red blood cells, multiple sickle cells (image)

    Science.gov (United States)

    Sickle cell anemia is an inherited disorder in which abnormal hemoglobin (the red pigment inside red blood cells) is produced. The abnormal hemoglobin causes red blood cells to assume a sickle shape, like the ones seen in this photomicrograph.

  6. Novel method for screening of enteric film coatings properties with magnetic resonance imaging.

    Science.gov (United States)

    Dorożyński, Przemysław; Jamróz, Witold; Niwiński, Krzysztof; Kurek, Mateusz; Węglarz, Władysław P; Jachowicz, Renata; Kulinowski, Piotr

    2013-11-18

    The aim of the study is to present the concept of novel method for fast screening of enteric coating compositions properties without the need of preparation of tablets batches for fluid bed coating. Proposed method involves evaluation of enteric coated model tablets in specially designed testing cell with application of MRI technique. The results obtained in the testing cell were compared with results of dissolution studies of mini-tablets coated in fluid bed apparatus. The method could be useful in early stage of formulation development for screening of film coating properties that will shorten and simplify the development works.

  7. A review of automated image understanding within 3D baggage computed tomography security screening.

    Science.gov (United States)

    Mouton, Andre; Breckon, Toby P

    2015-01-01

    Baggage inspection is the principal safeguard against the transportation of prohibited and potentially dangerous materials at airport security checkpoints. Although traditionally performed by 2D X-ray based scanning, increasingly stringent security regulations have led to a growing demand for more advanced imaging technologies. The role of X-ray Computed Tomography is thus rapidly expanding beyond the traditional materials-based detection of explosives. The development of computer vision and image processing techniques for the automated understanding of 3D baggage-CT imagery is however, complicated by poor image resolutions, image clutter and high levels of noise and artefacts. We discuss the recent and most pertinent advancements and identify topics for future research within the challenging domain of automated image understanding for baggage security screening CT.

  8. Cost and detection rate of glaucoma screening with imaging devices in a primary care center

    Science.gov (United States)

    Anton, Alfonso; Fallon, Monica; Cots, Francesc; Sebastian, María A; Morilla-Grasa, Antonio; Mojal, Sergi; Castells, Xavier

    2017-01-01

    Purpose To analyze the cost and detection rate of a screening program for detecting glaucoma with imaging devices. Materials and methods In this cross-sectional study, a glaucoma screening program was applied in a population-based sample randomly selected from a population of 23,527. Screening targeted the population at risk of glaucoma. Examinations included optic disk tomography (Heidelberg retina tomograph [HRT]), nerve fiber analysis, and tonometry. Subjects who met at least 2 of 3 endpoints (HRT outside normal limits, nerve fiber index ≥30, or tonometry ≥21 mmHg) were referred for glaucoma consultation. The currently established (“conventional”) detection method was evaluated by recording data from primary care and ophthalmic consultations in the same population. The direct costs of screening and conventional detection were calculated by adding the unit costs generated during the diagnostic process. The detection rate of new glaucoma cases was assessed. Results The screening program evaluated 414 subjects; 32 cases were referred for glaucoma consultation, 7 had glaucoma, and 10 had probable glaucoma. The current detection method assessed 677 glaucoma suspects in the population, of whom 29 were diagnosed with glaucoma or probable glaucoma. Glaucoma screening and the conventional detection method had detection rates of 4.1% and 3.1%, respectively, and the cost per case detected was 1,410 and 1,435€, respectively. The cost of screening 1 million inhabitants would be 5.1 million euros and would allow the detection of 4,715 new cases. Conclusion The proposed screening method directed at population at risk allows a detection rate of 4.1% and a cost of 1,410 per case detected. PMID:28243057

  9. Optical cell sorting with multiple imaging modalities

    DEFF Research Database (Denmark)

    Banas, Andrew; Carrissemoux, Caro; Palima, Darwin

    2017-01-01

    techniques. Scattering forces from beams actuated via efficient phase-only efficient modulation has been adopted. This has lowered the required power for sorting cells to a tenth of our previous approach, and also makes the cell sorter safer for use in clinical settings. With the versatility of dynamically...... programmable phase spatial light modulators, a plurality of light shaping techniques, including hybrid approaches, can be utilized in cell sorting....... healthy cells. With the richness of visual information, a lot of microscopy techniques have been developed and have been crucial in biological studies. To utilize their complementary advantages we adopt both fluorescence and brightfield imaging in our optical cell sorter. Brightfield imaging has...

  10. Automatic cell counting with ImageJ.

    Science.gov (United States)

    Grishagin, Ivan V

    2015-03-15

    Cell counting is an important routine procedure. However, to date there is no comprehensive, easy to use, and inexpensive solution for routine cell counting, and this procedure usually needs to be performed manually. Here, we report a complete solution for automatic cell counting in which a conventional light microscope is equipped with a web camera to obtain images of a suspension of mammalian cells in a hemocytometer assembly. Based on the ImageJ toolbox, we devised two algorithms to automatically count these cells. This approach is approximately 10 times faster and yields more reliable and consistent results compared with manual counting.

  11. RNAi screening reveals a large signaling network controlling the Golgi apparatus in human cells.

    Science.gov (United States)

    Chia, Joanne; Goh, Germaine; Racine, Victor; Ng, Susanne; Kumar, Pankaj; Bard, Frederic

    2012-01-01

    The Golgi apparatus has many important physiological functions, including sorting of secretory cargo and biosynthesis of complex glycans. These functions depend on the intricate and compartmentalized organization of the Golgi apparatus. To investigate the mechanisms that regulate Golgi architecture, we developed a quantitative morphological assay using three different Golgi compartment markers and quantitative image analysis, and performed a kinome- and phosphatome-wide RNAi screen in HeLa cells. Depletion of 159 signaling genes, nearly 20% of genes assayed, induced strong and varied perturbations in Golgi morphology. Using bioinformatics data, a large regulatory network could be constructed. Specific subnetworks are involved in phosphoinositides regulation, acto-myosin dynamics and mitogen activated protein kinase signaling. Most gene depletion also affected Golgi functions, in particular glycan biosynthesis, suggesting that signaling cascades can control glycosylation directly at the Golgi level. Our results provide a genetic overview of the signaling pathways that control the Golgi apparatus in human cells.

  12. Quantum dot imaging for embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Gambhir Sanjiv S

    2007-10-01

    Full Text Available Abstract Background Semiconductor quantum dots (QDs hold increasing potential for cellular imaging both in vitro and in vivo. In this report, we aimed to evaluate in vivo multiplex imaging of mouse embryonic stem (ES cells labeled with Qtracker delivered quantum dots (QDs. Results Murine embryonic stem (ES cells were labeled with six different QDs using Qtracker. ES cell viability, proliferation, and differentiation were not adversely affected by QDs compared with non-labeled control cells (P = NS. Afterward, labeled ES cells were injected subcutaneously onto the backs of athymic nude mice. These labeled ES cells could be imaged with good contrast with one single excitation wavelength. With the same excitation wavelength, the signal intensity, defined as (total signal-background/exposure time in millisecond was 11 ± 2 for cells labeled with QD 525, 12 ± 9 for QD 565, 176 ± 81 for QD 605, 176 ± 136 for QD 655, 167 ± 104 for QD 705, and 1,713 ± 482 for QD 800. Finally, we have shown that QD 800 offers greater fluorescent intensity than the other QDs tested. Conclusion In summary, this is the first demonstration of in vivo multiplex imaging of mouse ES cells labeled QDs. Upon further improvements, QDs will have a greater potential for tracking stem cells within deep tissues. These results provide a promising tool for imaging stem cell therapy non-invasively in vivo.

  13. Measurements and simulations of scatter imaging as a simultaneous adjunct for screening mammography

    Science.gov (United States)

    Kern, Katie; Hassan, Laila; Peerzada, Lubna; Ur-Rehman, Mahboob; MacDonald, C. A.

    2015-03-01

    X-ray coherent scatter is dependent upon the molecular structure of the scattering material and hence allows differentiation between tissue types with potentially much higher contrast than conventional absorption-based radiography. Coherent-scatter computed tomography has been used to produce images based on the x-ray scattering properties of the tissue. However, the geometry for CT imaging requires a thin fan beam and multiple projections and is incommensurate with screening mammography. In this work we demonstrate progress in a developing a system using a wide slot beam and simple anti-scatter grid which is adequate to differentiate between scatter peaks to remove the fat background from the coherent scatter image. Adequate intensity in the coherent scatter image can be achieved at the dose commonly used for screening mammography to detect carcinoma surrogates as small as 2 mm in diameter. This technique would provide an inexpensive, low dose, simultaneous adjunct to conventional screening mammography to provide a localized map of tissue type that could be overlaid on the conventional transmission mammogram. Comparisons between phantom measurements and Monte Carlo simulations show good agreement, which allowed for detailed examination of the visibility of carcinoma under realistic conditions.

  14. Patterns of nonmasslike enhancement at screening breast MR imaging of high-risk premenopausal women.

    Science.gov (United States)

    Giess, Catherine S; Raza, Sughra; Birdwell, Robyn L

    2013-01-01

    Current U.S. recommendations for breast cancer screening of women with at least a 20%-25% lifetime risk of developing breast cancer include contrast material-enhanced magnetic resonance (MR) imaging of the breasts. The cancer detection rate in high-risk women undergoing screening MR imaging is approximately 10 times higher than that in normal-risk women undergoing screening mammography. Many of these high-risk women commence MR imaging screening while they are premenopausal, when the breasts are most influenced by cyclical hormonal changes. Healthy premenopausal breast tissue enhances in a cyclical and variable manner. This enhancement is described as background enhancement. Typically, enhancement of normal breast tissue occurs in a symmetric and diffuse pattern, and there is little diagnostic difficulty in classifying it as normal background parenchymal enhancement. However, sometimes the pattern is more focal, asymmetric, or regional. It may then be described as nonmasslike enhancement, an observation associated with both benign and malignant breast pathologic conditions. A review of the morphologic features and internal enhancement patterns in normal but nondiffuse background enhancement and abnormal nonmasslike enhancement in high-risk premenopausal women can help improve interpretive specificity and decrease false-positive interpretations. MR imaging pitfalls and interpretation strategies for localized background enhancement and pathologic nonmasslike enhancement in this high-risk population are highlighted. In evaluating nonmasslike enhancement, the use of the Breast Imaging Reporting and Data System (BI-RADS) lexicon to perform careful analysis of morphologic features, along with an understanding of the role and limitations of kinetic information, will help balance early breast cancer detection against false-positive interpretation.

  15. A chemical screen probing the relationship between mitochondrial content and cell size.

    Directory of Open Access Journals (Sweden)

    Toshimori Kitami

    Full Text Available The cellular content of mitochondria changes dynamically during development and in response to external stimuli, but the underlying mechanisms remain obscure. To systematically identify molecular probes and pathways that control mitochondrial abundance, we developed a high-throughput imaging assay that tracks both the per cell mitochondrial content and the cell size in confluent human umbilical vein endothelial cells. We screened 28,786 small molecules and observed that hundreds of small molecules are capable of increasing or decreasing the cellular content of mitochondria in a manner proportionate to cell size, revealing stereotyped control of these parameters. However, only a handful of compounds dissociate this relationship. We focus on one such compound, BRD6897, and demonstrate through secondary assays that it increases the cellular content of mitochondria as evidenced by fluorescence microscopy, mitochondrial protein content, and respiration, even after rigorous correction for cell size, cell volume, or total protein content. BRD6897 increases uncoupled respiration 1.6-fold in two different, non-dividing cell types. Based on electron microscopy, BRD6897 does not alter the percent of cytoplasmic area occupied by mitochondria, but instead, induces a striking increase in the electron density of existing mitochondria. The mechanism is independent of known transcriptional programs and is likely to be related to a blockade in the turnover of mitochondrial proteins. At present the molecular target of BRD6897 remains to be elucidated, but if identified, could reveal an important additional mechanism that governs mitochondrial biogenesis and turnover.

  16. Laser-Induced Fluorescence Detection in High-Throughput Screening of Heterogeneous Catalysts and Single Cells Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Hui Su

    2001-05-25

    Laser-induced fluorescence detection is one of the most sensitive detection techniques and it has found enormous applications in various areas. The purpose of this research was to develop detection approaches based on laser-induced fluorescence detection in two different areas, heterogeneous catalysts screening and single cell study. First, the author introduced laser-induced imaging (LIFI) as a high-throughput screening technique for heterogeneous catalysts to explore the use of this high-throughput screening technique in discovery and study of various heterogeneous catalyst systems. This scheme is based on the fact that the creation or the destruction of chemical bonds alters the fluorescence properties of suitably designed molecules. By irradiating the region immediately above the catalytic surface with a laser, the fluorescence intensity of a selected product or reactant can be imaged by a charge-coupled device (CCD) camera to follow the catalytic activity as a function of time and space. By screening the catalytic activity of vanadium pentoxide catalysts in oxidation of naphthalene, they demonstrated LIFI has good detection performance and the spatial and temporal resolution needed for high-throughput screening of heterogeneous catalysts. The sample packing density can reach up to 250 x 250 subunits/cm{sup 2} for 40-{micro}m wells. This experimental set-up also can screen solid catalysts via near infrared thermography detection. In the second part of this dissertation, the author used laser-induced native fluorescence coupled with capillary electrophoresis (LINF-CE) and microscope imaging to study the single cell degranulation. On the basis of good temporal correlation with events observed through an optical microscope, they have identified individual peaks in the fluorescence electropherograms as serotonin released from the granular core on contact with the surrounding fluid.

  17. Laser-Induced Fluorescence Detection in High-Throughput Screening of Heterogeneous Catalysts and Single Cells Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Su, Hui [Iowa State Univ., Ames, IA (United States)

    2001-01-01

    Laser-induced fluorescence detection is one of the most sensitive detection techniques and it has found enormous applications in various areas. The purpose of this research was to develop detection approaches based on laser-induced fluorescence detection in two different areas, heterogeneous catalysts screening and single cell study. First, the author introduced laser-induced imaging (LIFI) as a high-throughput screening technique for heterogeneous catalysts to explore the use of this high-throughput screening technique in discovery and study of various heterogeneous catalyst systems. This scheme is based on the fact that the creation or the destruction of chemical bonds alters the fluorescence properties of suitably designed molecules. By irradiating the region immediately above the catalytic surface with a laser, the fluorescence intensity of a selected product or reactant can be imaged by a charge-coupled device (CCD) camera to follow the catalytic activity as a function of time and space. By screening the catalytic activity of vanadium pentoxide catalysts in oxidation of naphthalene, they demonstrated LIFI has good detection performance and the spatial and temporal resolution needed for high-throughput screening of heterogeneous catalysts. The sample packing density can reach up to 250 x 250 subunits/cm2 for 40-μm wells. This experimental set-up also can screen solid catalysts via near infrared thermography detection. In the second part of this dissertation, the author used laser-induced native fluorescence coupled with capillary electrophoresis (LINF-CE) and microscope imaging to study the single cell degranulation. On the basis of good temporal correlation with events observed through an optical microscope, they have identified individual peaks in the fluorescence electropherograms as serotonin released from the granular core on contact with the surrounding fluid.

  18. Proximal surface caries detection with direct-exposure and rare earth screen/film imaging

    Energy Technology Data Exchange (ETDEWEB)

    Lundeen, R.C.; McDavid, W.D.; Barnwell, G.M.

    1988-12-01

    This laboratory study compared five imaging systems for their diagnostic accuracy in detection of proximal surface dental caries. Ten viewers provided data on radiographic detectability of carious lesions. The diagnostic accuracy of each system was determined with receiver operating characteristic (ROC) curves by comparing viewer data with the true state of the teeth as determined microscopically. D-speed film marginally outperformed the other four systems, but the three screen/film systems matched the diagnostic accuracy of E-speed film. Radiation reductions between 62% and 92% were achieved with the screen/film systems when compared to the two conventional dental films. The feasibility of designing a screen/film bite-wing cassette was shown, but the poor diagnostic accuracy of the present bite-wing system indicated a need for a new technology in caries detection.

  19. Screening of a specific peptide binding to esophageal squamous carcinoma cells from phage displayed peptide library.

    Science.gov (United States)

    Ma, Caixia; Li, Chunyan; Jiang, Dongliang; Gao, Xiaojie; Han, Juanjuan; Xu, Nan; Wu, Qiong; Nie, Guochao; Chen, Wei; Lin, Fenghuei; Hou, Yingchun

    2015-06-01

    To select a specifically binding peptide for imaging detection of human esophageal squamous cell carcinoma (ESCC), a phage-displayed 12-mer peptide library was used to screen the peptide that bind to ESCC cells specifically. After four rounds of bio-panning, the phage recovery rate gradually increased, and specific phage clones were effectively enriched. The 60 randomly selected phage clones were tested using cellular enzyme-linked immunosorbent assay (ELISA), and 41 phage clones were identified as positive clones with the over 2.10 ratio of absorbance higher than other clones, IRP and PBS controls. From the sequencing results of the positive clones, 14 peptide sequences were obtained and ESCP9 consensus sequence was identified as the peptide with best affinity to ESCC cells via competitive inhibition, fluorescence microscopy, and flow cytometry. The results indicate that the peptide ESCP9 can bind to ESCC cells specifically and sensitively, and it is a potential candidate to be developed as an useful molecule to the imaging detection and targeting therapy for ESCC.

  20. Label-free cardiac contractility monitoring for drug screening applications based on compact high-speed lens-free imaging

    Science.gov (United States)

    Pauwelyn, Thomas; Reumers, Veerle; Vanmeerbeeck, Geert; Stahl, Richard; Janssens, Stefan; Lagae, Liesbet; Braeken, Dries; Lambrechts, Andy

    2015-03-01

    Cardiotoxicity is the major cause of drug withdrawal from the market, despite rigorous toxicity testing during the drug development process. Existing safety screening techniques, some of which are based on simplified cellular assays, others on electrical (impedance) or optical (fluorescent microscopy) measurements, are either too limited in throughput or offer too poor predictability of toxicity to be applied on large numbers of compounds in the early stage of drug development. We present a compact optical system for direct (label-free) monitoring of fast cellular movements that enable low cost and high throughput drug screening. Our system is based on a high-speed lens-free in-line holographic microscope. When compared to a conventional microscope, the system can combine adequate imaging resolution (5.5 μm pixel pitch) with a large field-of-view (63.4 mm2) and high speed (170 fps) to capture physical cell motion in real-time. This combination enables registration of cardiac contractility parameters such as cell contraction frequency, total duration, and rate and duration of both contraction and relaxation. The system also quantifies conduction velocity, which is challenging in existing techniques. Additionally, to complement the imaging hardware we have developed image processing software that extracts all the contractility parameters directly from the raw interference images. The system was tested with varying concentration of the drug verapamil and at 100 nM, showed a decrease in: contraction frequency (-23.3% +/- 13%), total duration (-21% +/- 5%), contraction duration (-19% +/- 6%) and relaxation duration (-21% +/- 8%). Moreover, contraction displacement ceased at higher concentrations.

  1. Demonstration of a visual cell-based assay for screening glucose transporter 4 translocation modulators in real time

    Indian Academy of Sciences (India)

    Maleppillil Vavachan Vijayakumar; Amrendra Kumar Ajay; Manoj Kumar Bhat

    2010-12-01

    Insulin-stimulated translocation of glucose transporter 4 (GLUT4) to cell membrane leading to glucose uptake is the rate-limiting step in diabetes. It is also a defined target of antidiabetic drug research. Existing GLUT4 translocation assays are based on time-consuming immunoassays and are hampered by assay variability and low sensitivity. We describe a real-time, visual, cell-based qualitative GLUT4 translocation assay using CHO-HIRc-myc-GLUT4eGFP cells that stably express myc- and eGFP-tagged GLUT4 in addition to human insulin receptor (HIRc). GLUT4 translocation is visualized by live cell imaging based on GFP fluorescence by employing a cooled charge-coupled device camera attached to a fluorescent microscope. This video imaging method and further quantitative analysis of GLUT4 on the cell membrane provide rapid and foolproof visual evidence that this method is suitable for screening GLUT4 translocation modulators.

  2. Cost and detection rate of glaucoma screening with imaging devices in a primary care center

    Directory of Open Access Journals (Sweden)

    Anton A

    2017-02-01

    Full Text Available Alfonso Anton,1–4 Monica Fallon,3,5 Francesc Cots,2 María A Sebastian,6 Antonio Morilla-Grasa,4 Sergi Mojal,3 Xavier Castells2 1Medicine School, Universidad Internacional de Cataluña, 2Servei d’Estudies, Parc de Salut Mar, 3Instituto Hospital del Mar de Investigaciones Médicas (IMIM, 4Glaucoma Department, Instituto Catalán de Retina (ICR, 5Universidad Autónoma de Barcelona, 6Centro de Atención Primaria Larrard, Barcelona, Spain Purpose: To analyze the cost and detection rate of a screening program for detecting glaucoma with imaging devices. Materials and methods: In this cross-sectional study, a glaucoma screening program was applied in a population-based sample randomly selected from a population of 23,527. Screening targeted the population at risk of glaucoma. Examinations included optic disk tomography (Heidelberg retina tomograph [HRT], nerve fiber analysis, and tonometry. Subjects who met at least 2 of 3 endpoints (HRT outside normal limits, nerve fiber index ≥30, or tonometry ≥21 mmHg were referred for glaucoma consultation. The currently established (“conventional” detection method was evaluated by recording data from primary care and ophthalmic consultations in the same population. The direct costs of screening and conventional detection were calculated by adding the unit costs generated during the diagnostic process. The detection rate of new glaucoma cases was assessed. Results: The screening program evaluated 414 subjects; 32 cases were referred for glaucoma consultation, 7 had glaucoma, and 10 had probable glaucoma. The current detection method assessed 677 glaucoma suspects in the population, of whom 29 were diagnosed with glaucoma or probable glaucoma. Glaucoma screening and the conventional detection method had detection rates of 4.1% and 3.1%, respectively, and the cost per case detected was 1,410 and 1,435€, respectively. The cost of screening 1 million inhabitants would be 5.1 million euros and would allow

  3. Immunomagnetic Nano-Screening Chip for Circulating Tumor Cells Detection in Blood

    Science.gov (United States)

    Horton, A. P.; Lane, N.; Tam, J.; Sokolov, K.; Garner, H. R.; Uhr, J. W.; Zhang, X. J.

    2010-03-01

    We present a novel method towards diagnose cancer at an early stage via a blood test. Early diagnosis is high on the future agenda of oncologists because of significant evidence that it will result in a higher cure rate. Capture of circulating tumor cells (CTCs) which are known to escape from carcinomas at an early stage offers such an opportunity. We design, fabricate and optimize the nanomagnetic-screening chip that captures the CTCs in microfluid, and further integrate the nano-chip with the new multispectral imaging system so that it can quantify different tumor markers and automate the entire instrument. Specifically, hybrid plasmonic (Fe2O3-core Au shell) nanoparticles, conjugated a collection of antibodies especially chosen to target breast cancer CTCs, with high magnetic susceptibility will be used for effective immunomagnetic CTC isolation. Greatly increased sensitivity over previous attempts is demonstrated by decreasing the length scale for interactions between the magnetic-nanoparticle-tagged CTCs and the isolative magnetic field, while increasing the effective cross-sectional area over which this interaction takes place. The screening chip is integrated with a novel hyperspectral microscopic imaging (HMI) platform capable of recording the entire emission spectra in a single pass evaluation. The combined system will precisely quantify up to 10 tumor markers on CTCs.

  4. Purified human pancreatic duct cell culture conditions defined by serum-free high-content growth factor screening.

    Directory of Open Access Journals (Sweden)

    Corinne A Hoesli

    Full Text Available The proliferation of pancreatic duct-like CK19+ cells has implications for multiple disease states including pancreatic cancer and diabetes mellitus. The in vitro study of this important cell type has been hampered by their limited expansion compared to fibroblast-like vimentin+ cells that overgrow primary cultures. We aimed to develop a screening platform for duct cell mitogens after depletion of the vimentin+ population. The CD90 cell surface marker was used to remove the vimentin+ cells from islet-depleted human pancreas cell cultures by magnetic-activated cell sorting. Cell sorting decreased CD90+ cell contamination of the cultures from 34±20% to 1.3±0.6%, yielding purified CK19+ cultures with epithelial morphology. A full-factorial experimental design was then applied to test the mitogenic effects of bFGF, EGF, HGF, KGF and VEGF. After 6 days in test conditions, the cells were labelled with BrdU, stained and analyzed by high-throughput imaging. This screening assay confirmed the expected mitogenic effects of bFGF, EGF, HGF and KGF on CK19+ cells and additionally revealed interactions between these factors and VEGF. A serum-free medium containing bFGF, EGF, HGF and KGF led to CK19+ cell expansion comparable to the addition of 10% serum. The methods developed in this work should advance pancreatic cancer and diabetes research by providing effective cell culture and high-throughput screening platforms to study purified primary pancreatic CK19+ cells.

  5. Development of x-ray imaging technique for liquid screening at airport

    Energy Technology Data Exchange (ETDEWEB)

    Sulaiman, Nurhani binti, E-mail: nhani.sulaiman@gmail.com; Srisatit, Somyot, E-mail: somyot.s@chula.ac.th [Department of Nuclear Engineering, Faculty of Engineering, Chulalongkorn University, Phayathai Rd., Patumwan, Bangkok 10330 (Thailand)

    2016-01-22

    X-ray imaging technology is a viable option to recognize flammable liquids for the purposes of aviation security. In this study, an X-ray imaging technology was developed whereby, the image viewing system was built with the use of a digital camera coupled with a gadolinium oxysulfide (GOS) fluorescent screen. The camera was equipped with a software for remote control setting of the camera via a USB cable which allows the images to be captured. The image was analysed to determine the average grey level using a software designed by Microsoft Visual Basic 6.0. The data was obtained for various densities of liquid thickness of 4.5 cm, 6.0 cm and 7.5 cm respectively for X-ray energies ranging from 70 to 200 kVp. In order to verify the reliability of the constructed calibration data, the system was tested with a few types of unknown liquids. The developed system could be conveniently employed for security screening in order to discriminate between a threat and an innocuous liquid.

  6. A versatile knowledge-based clinical imaging annotation system for breast cancer screening

    Science.gov (United States)

    Gemo, Monica; Gouze, Annabelle; Debande, Benoît; Grivegnée, André; Mazy, Gilbert; Macq, Benoît

    2007-03-01

    Medical information is evolving towards more complex multimedia data representation, as new imaging modalities are made available by sophisticated devices. Features such as segmented lesions can now be extracted through analysis techniques and need to be integrated into clinical patient data. The management of structured information extracted from multimedia has been addressed in knowledge based annotation systems providing methods to attach interpretative semantics to multimedia content. Building on these methods, we develop a new clinical imaging annotation system for computer aided breast cancer screening. The proposed system aims at more consistent, efficient and standardised data mark-up of digital and digitalised radiology images. The objective is to provide detailed characterisation of abnormalities as an aid in the diagnostic task through integrated annotation management. The system combines imaging analysis results and radiologist diagnostic information about suspicious findings by mapping well-established visual and low-level descriptors into pathology specific profiles. The versatile characterisation allows differentiating annotation descriptors for different types of findings. Our approach of semi-automatic integrated annotations supports increased quality assurance in screening practice. This is achieved through detailed and objective patient imaging information while providing user-friendly means for their manipulation that is oriented to relieving the radiologist's workload.

  7. A flow cytometer-based whole cell screening toolbox for directed hydrolase evolution through fluorescent hydrogels.

    Science.gov (United States)

    Lülsdorf, Nina; Pitzler, Christian; Biggel, Michael; Martinez, Ronny; Vojcic, Ljubica; Schwaneberg, Ulrich

    2015-05-21

    A high throughput whole cell flow cytometer screening toolbox was developed and validated by identifying improved variants (1.3-7-fold) for three hydrolases (esterase, lipase, cellulase). The screening principle is based on coupled enzymatic reaction using glucose derivatives which yield upon hydrolysis a fluorescent-hydrogel-layer on the surface of E. coli cells.

  8. Pluripotent stem cell derived hepatocyte like cells and their potential in toxicity screening.

    Science.gov (United States)

    Greenhough, Sebastian; Medine, Claire N; Hay, David C

    2010-12-30

    Despite considerable progress in modelling human liver toxicity, the requirement still exists for efficient, predictive and cost effective in vitro models to reduce attrition during drug development. Thousands of compounds fail in this process, with hepatotoxicity being one of the significant causes of failure. The cost of clinical studies is substantial, therefore it is essential that toxicological screening is performed early on in the drug development process. Human hepatocytes represent the gold standard model for evaluating drug toxicity, but are a limited resource. Current alternative models are based on immortalised cell lines and animal tissue, but these are limited by poor function, exhibit species variability and show instability in culture. Pluripotent stem cells are an attractive alternative as they are capable of self-renewal and differentiation to all three germ layers, and thereby represent a potentially inexhaustible source of somatic cells. The differentiation of human embryonic stem cells and induced pluripotent stem cells to functional hepatocyte like cells has recently been reported. Further development of this technology could lead to the scalable production of hepatocyte like cells for liver toxicity screening and clinical therapies. Additionally, induced pluripotent stem cell derived hepatocyte like cells may permit in vitro modelling of gene polymorphisms and genetic diseases.

  9. Microfluidics for cell-based high throughput screening platforms - A review.

    Science.gov (United States)

    Du, Guansheng; Fang, Qun; den Toonder, Jaap M J

    2016-01-15

    In the last decades, the basic techniques of microfluidics for the study of cells such as cell culture, cell separation, and cell lysis, have been well developed. Based on cell handling techniques, microfluidics has been widely applied in the field of PCR (Polymerase Chain Reaction), immunoassays, organ-on-chip, stem cell research, and analysis and identification of circulating tumor cells. As a major step in drug discovery, high-throughput screening allows rapid analysis of thousands of chemical, biochemical, genetic or pharmacological tests in parallel. In this review, we summarize the application of microfluidics in cell-based high throughput screening. The screening methods mentioned in this paper include approaches using the perfusion flow mode, the droplet mode, and the microarray mode. We also discuss the future development of microfluidic based high throughput screening platform for drug discovery.

  10. Thermographic image analysis as a pre-screening tool for the detection of canine bone cancer

    Science.gov (United States)

    Subedi, Samrat; Umbaugh, Scott E.; Fu, Jiyuan; Marino, Dominic J.; Loughin, Catherine A.; Sackman, Joseph

    2014-09-01

    Canine bone cancer is a common type of cancer that grows fast and may be fatal. It usually appears in the limbs which is called "appendicular bone cancer." Diagnostic imaging methods such as X-rays, computed tomography (CT scan), and magnetic resonance imaging (MRI) are more common methods in bone cancer detection than invasive physical examination such as biopsy. These imaging methods have some disadvantages; including high expense, high dose of radiation, and keeping the patient (canine) motionless during the imaging procedures. This project study identifies the possibility of using thermographic images as a pre-screening tool for diagnosis of bone cancer in dogs. Experiments were performed with thermographic images from 40 dogs exhibiting the disease bone cancer. Experiments were performed with color normalization using temperature data provided by the Long Island Veterinary Specialists. The images were first divided into four groups according to body parts (Elbow/Knee, Full Limb, Shoulder/Hip and Wrist). Each of the groups was then further divided into three sub-groups according to views (Anterior, Lateral and Posterior). Thermographic pattern of normal and abnormal dogs were analyzed using feature extraction and pattern classification tools. Texture features, spectral feature and histogram features were extracted from the thermograms and were used for pattern classification. The best classification success rate in canine bone cancer detection is 90% with sensitivity of 100% and specificity of 80% produced by anterior view of full-limb region with nearest neighbor classification method and normRGB-lum color normalization method. Our results show that it is possible to use thermographic imaging as a pre-screening tool for detection of canine bone cancer.

  11. Langerhans cell histiocytosis of bone: MR imaging

    Energy Technology Data Exchange (ETDEWEB)

    George, J.C. [Indiana Univ., Indianapolis, IN (United States). Dept. of Radiology; Buckwalter, K.A. [Indiana Univ., Indianapolis, IN (United States). Dept. of Radiology; Cohen, M.D. [Indiana Univ., Indianapolis, IN (United States). Dept. of Radiology; Edwards, M.K. [Indiana Univ., Indianapolis, IN (United States). Dept. of Radiology; Smith, R.R. [Indiana Univ., Indianapolis, IN (United States). Dept. of Radiology

    1994-03-01

    Magnetic resonance (MR) images of 12 pathologically proven lesions of Langerhans cell histiocytosis (LCH) of bone were reviewed retrospectively. MR identified all lesions, three of which were not identified on plain radiographs. In all cases, MR showed greater abnormality than did plain radiographs. With one exception, all lesions were hypointense on T1-weighted images and hyperintense on T2-weighted images. The lesions and associated soft tissue abnormalities were very conspicuous on short TI inversion sequences and T1-weighted post-contrast images. Follow-up MR studies in two patients after chemotherapy showed decreased size and enhancement of lesions compared with baseline studies. (orig.)

  12. Langerhans cell histiocytosis of bone: MR imaging.

    Science.gov (United States)

    George, J C; Buckwalter, K A; Cohen, M D; Edwards, M K; Smith, R R

    1994-01-01

    Magnetic resonance (MR) images of 12 pathologically proven lesions of Langerhans cell histiocytosis (LCH) of bone were reviewed retrospectively. MR identified all lesions, three of which were not identified on plain radiographs. In all cases, MR showed greater abnormality than did plain radiographs. With one exception, all lesions were hypointense on T1-weighted images and hyperintense on T2-weighted images. The lesions and associated soft tissue abnormalities were very conspicuous on short TI inversion sequences and T1-weighted post-contrast images. Follow-up MR studies in two patients after chemotherapy showed decreased size and enhancement of lesions compared with baseline studies.

  13. Bioluminescence imaging in live cells and animals.

    Science.gov (United States)

    Tung, Jack K; Berglund, Ken; Gutekunst, Claire-Anne; Hochgeschwender, Ute; Gross, Robert E

    2016-04-01

    The use of bioluminescent reporters in neuroscience research continues to grow at a rapid pace as their applications and unique advantages over conventional fluorescent reporters become more appreciated. Here, we describe practical methods and principles for detecting and imaging bioluminescence from live cells and animals. We systematically tested various components of our conventional fluorescence microscope to optimize it for long-term bioluminescence imaging. High-resolution bioluminescence images from live neurons were obtained with our microscope setup, which could be continuously captured for several hours with no signs of phototoxicity. Bioluminescence from the mouse brain was also imaged noninvasively through the intact skull with a conventional luminescence imager. These methods demonstrate how bioluminescence can be routinely detected and measured from live cells and animals in a cost-effective way with common reagents and equipment.

  14. Active screen plasma nitriding enhances cell attachment to polymer surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Kaklamani, Georgia, E-mail: g.kaklamani@bham.ac.uk [University of Birmingham, College of Engineering and Physical Sciences, School of Metallurgy and Materials, Edgbaston, Birmingham B15 2TT (United Kingdom); Bowen, James; Mehrban, Nazia [University of Birmingham, College of Engineering and Physical Sciences, School of Chemical Engineering, Edgbaston, Birmingham B15 2TT (United Kingdom); Dong, Hanshan [University of Birmingham, College of Engineering and Physical Sciences, School of Metallurgy and Materials, Edgbaston, Birmingham B15 2TT (United Kingdom); Grover, Liam M. [University of Birmingham, College of Engineering and Physical Sciences, School of Chemical Engineering, Edgbaston, Birmingham B15 2TT (United Kingdom); Stamboulis, Artemis [University of Birmingham, College of Engineering and Physical Sciences, School of Metallurgy and Materials, Edgbaston, Birmingham B15 2TT (United Kingdom)

    2013-05-15

    Active screen plasma nitriding (ASPN) is a well-established technique used for the surface modification of materials, the result of which is often a product with enhanced functional performance. Here we report the modification of the chemical and mechanical properties of ultra-high molecular weight poly(ethylene) (UHMWPE) using 80:20 (v/v) N{sub 2}/H{sub 2} ASPN, followed by growth of 3T3 fibroblasts on the treated and untreated polymer surfaces. ASPN-treated UHMWPE showed extensive fibroblast attachment within 3 h of seeding, whereas fibroblasts did not successfully attach to untreated UHMWPE. Fibroblast-coated surfaces were maintained for up to 28 days, monitoring their metabolic activity and morphology throughout. The chemical properties of the ASPN-treated UHMWPE surface were studied using X-ray photoelectron spectroscopy, revealing the presence of C-N, C=N, and C≡N chemical bonds. The elastic modulus, surface topography, and adhesion properties of the ASPN-treated UHMWPE surface were studied over 28 days during sample storage under ambient conditions and during immersion in two commonly used cell culture media.

  15. Pain and stress assessment after retinopathy of prematurity screening examination: Indirect ophthalmoscopy versus digital retinal imaging

    Directory of Open Access Journals (Sweden)

    Moral-Pumarega M

    2012-08-01

    Full Text Available Abstract Background Increasingly, neonatal clinics seek to minimize painful experiences and stress for premature infants. Fundoscopy performed with a binocular indirect ophthalmoscope is the reference examination technique for screening of retinopathy of prematurity (ROP, and it is associated with pain and stress. Wide-field digital retinal imaging is a recent technique that should be evaluated for minimizing infant pain and stress. Methods The purpose of the study was to assess and compare the impact of using a binocular indirect ophthalmoscope (BIO, or wide-field digital retinal imaging (WFDRI on pain and stress in infants undergoing ROP screening examination. This was a comparative evaluation study of two screening procedures. Ophthalmologic examinations (N = 70 were performed on 24 infants with both BIO and WFDRI. Pain assessments were performed with two specific neonatal scales (Crying, requires oxygen, increased vital signs, expression and sleeplessness, CRIES and, Premature infant pain profile, PIPP just prior to the examination, and 30 seconds, 1 hour, and 24 hours later after ending the examination. Results Changes over time were significantly different between BIO and WFDRI with both scales (PIPP score, p = .007, and CRIES score, p = .001. Median PIPP score (interquartile interval at baseline was 4 (3–5. At 30 seconds the score was 8 (6–9 for BIO and 6 (5–7 for WFDRI, respectively. The increase in PIPP score between baseline and 30 seconds was significantly lower with WFDRI (p = .006. The median increase in CRIES score from baseline to 30 seconds was 1 point lower for WFDRI than for BIO (p  Conclusions A transient short-term pain and stress response occurs with both BIO and WFDRI. Infants examined for screening of ROP with digital retinal imaging present less pain and stress at 30 seconds following completion of the exam when compared with binocular indirect ophthalmoscopy.

  16. Use of cryopreserved cell aliquots in the high-throughput screening of small interfering RNA libraries.

    Science.gov (United States)

    Swearingen, Elissa A; Fajardo, Flordeliza; Wang, Xiangyun; Watson, J E Vivienne; Quon, Kim C; Kassner, Paul D

    2010-06-01

    Screening small interfering RNA (siRNA) libraries holds the potential to elucidate gene function as well as discover new targets for the therapeutic treatment of disease. Since the inception of siRNA as a discovery tool, there have been progressive improvements in siRNA design algorithms, the transfection reagents used to deliver them, and the assay formats used to monitor phenotypic changes. These changes have helped to improve the quality of the data emerging from siRNA screens. One variable that introduces inconsistency into high-throughput screening (HTS) of siRNA libraries is the state of the cells used in the assays. Multiple factors can contribute to differences in transfection efficiency as well as the basic cell biology, which can lead to differences in the genes identified in siRNA screens. The authors have developed a system using frozen cell aliquots to use in siRNA HTS, so that a major source of variability introduced into cell-based screens can be standardized. In addition, by transiently transfecting plasmids into cell lines and then freezing these cells down to use in siRNA transfection experiments, they have used this same technology to create new cell lines. This process of using aliquots of frozen cells is logistically advantageous in an HTS setting, as it reduces the time spent maintaining cell lines, as well as reducing possible downtime in screening due to lack of cells or poor cell health.

  17. PET imaging of adoptive progenitor cell therapies.

    Energy Technology Data Exchange (ETDEWEB)

    Gelovani, Juri G.

    2008-05-13

    Objectives. The overall objective of this application is to develop novel technologies for non-invasive imaging of adoptive stem cell-based therapies with positron emission tomography (PET) that would be applicable to human patients. To achieve this objective, stem cells will be genetically labeled with a PET-reporter gene and repetitively imaged to assess their distribution, migration, differentiation, and persistence using a radiolabeled reporter probe. This new imaging technology will be tested in adoptive progenitor cell-based therapy models in animals, including: delivery pro-apoptotic genes to tumors, and T-cell reconstitution for immunostimulatory therapy during allogeneic bone marrow progenitor cell transplantation. Technical and Scientific Merits. Non-invasive whole body imaging would significantly aid in the development and clinical implementation of various adoptive progenitor cell-based therapies by providing the means for non-invasive monitoring of the fate of injected progenitor cells over a long period of observation. The proposed imaging approaches could help to address several questions related to stem cell migration and homing, their long-term viability, and their subsequent differentiation. The ability to image these processes non-invasively in 3D and repetitively over a long period of time is very important and will help the development and clinical application of various strategies to control and direct stem cell migration and differentiation. Approach to accomplish the work. Stem cells will be genetically with a reporter gene which will allow for repetitive non-invasive “tracking” of the migration and localization of genetically labeled stem cells and their progeny. This is a radically new approach that is being developed for future human applications and should allow for a long term (many years) repetitive imaging of the fate of tissues that develop from the transplanted stem cells. Why the approach is appropriate. The novel approach to

  18. Geant4 simulation of the response of phosphor screens for X-ray imaging

    Energy Technology Data Exchange (ETDEWEB)

    Pistrui-Maximean, S.A. [Laboratory of Nondestructive Testing using Ionizing Radiation, INSA-Lyon Scientific and Technical University, Bat. Antoine de Saint Exupery, 69621 Villeurbanne Cedex (France)]. E-mail: simona.pistrui@insa-lyon.fr; Freud, N. [Laboratory of Nondestructive Testing using Ionizing Radiation, INSA-Lyon Scientific and Technical University, Bat. Antoine de Saint Exupery, 69621 Villeurbanne Cedex (France); Letang, J.M. [Laboratory of Nondestructive Testing using Ionizing Radiation, INSA-Lyon Scientific and Technical University, Bat. Antoine de Saint Exupery, 69621 Villeurbanne Cedex (France); Koch, A. [Thales Electron Devices, 38430 Moirans (France); Munier, B. [Thales Electron Devices, 38430 Moirans (France); Walenta, A.H. [Department of Detectors and Electronics, FB Physik, University of Siegen, 57068 Siegen (Germany); Montarou, G. [Corpuscular Physics Laboratory, Blaise Pascal University, 63177 Aubiere Cedex (France); Babot, D. [Laboratory of Nondestructive Testing using Ionizing Radiation, INSA-Lyon Scientific and Technical University, Bat. Antoine de Saint Exupery, 69621 Villeurbanne Cedex (France)

    2006-07-01

    In order to predict and optimize the response of phosphor screens, it is important to understand the role played by the different physical processes inside the scintillator layer. A simulation model based on the Monte Carlo code Geant4 was developed to determine the Modulation Transfer Function (MTF) of phosphor screens for energies used in X-ray medical imaging and nondestructive testing applications. The visualization of the dose distribution inside the phosphor layer gives an insight into how the MTF is progressively degraded by X-ray and electron transport. The simulation model allows to study the influence of physical and technological parameters on the detector performances, as well as to design and optimize new detector configurations. Preliminary MTF measurements have been carried out and agreement with experimental data has been found in the case of a commercial screen (Kodak Lanex Fine) at an X-ray tube potential of 100 kV. Further validation with other screens (transparent or granular) at different energies is under way.

  19. High-throughput screening of high Monascus pigment-producing strain based on digital image processing.

    Science.gov (United States)

    Xia, Meng-lei; Wang, Lan; Yang, Zhi-xia; Chen, Hong-zhang

    2016-04-01

    This work proposed a new method which applied image processing and support vector machine (SVM) for screening of mold strains. Taking Monascus as example, morphological characteristics of Monascus colony were quantified by image processing. And the association between the characteristics and pigment production capability was determined by SVM. On this basis, a highly automated screening strategy was achieved. The accuracy of the proposed strategy is 80.6 %, which is compatible with the existing methods (81.1 % for microplate and 85.4 % for flask). Meanwhile, the screening of 500 colonies only takes 20-30 min, which is the highest rate among all published results. By applying this automated method, 13 strains with high-predicted production were obtained and the best one produced as 2.8-fold (226 U/mL) of pigment and 1.9-fold (51 mg/L) of lovastatin compared with the parent strain. The current study provides us with an effective and promising method for strain improvement.

  20. Size measurement of radioactive aerosol particles in intense radiation fields using wire screens and imaging plates

    Energy Technology Data Exchange (ETDEWEB)

    Oki, Yuichi; Tanaka, Toru; Takamiya, Koichi; Ishi, Yoshihiro; UesugI, Tomonori; Kuriyama, Yasutoshi; Sakamoto, Masaaki; Ohtsuki, Tsutomu [Kyoto University Research Reactor Institute, Osaka (Japan); Nitta, Shinnosuke [Graduate School of Engineering, Kyoto University, Kyoto (Japan); Osada, Naoyuki [Advanced Science Research Center, Okayama University, Okayama (Japan)

    2016-09-15

    Very fine radiation-induced aerosol particles are produced in intense radiation fields, such as high-intensity accelerator rooms and containment vessels such as those in the Fukushima Daiichi nuclear power plant (FDNPP). Size measurement of the aerosol particles is very important for understanding the behavior of radioactive aerosols released in the FDNPP accident and radiation safety in high-energy accelerators. A combined technique using wire screens and imaging plates was developed for size measurement of fine radioactive aerosol particles smaller than 100 nm in diameter. This technique was applied to the radiation field of a proton accelerator room, in which radioactive atoms produced in air during machine operation are incorporated into radiation-induced aerosol particles. The size of 11C-bearing aerosol particles was analyzed using the wire screen technique in distinction from other positron emitters in combination with a radioactive decay analysis. The size distribution for 11C-bearing aerosol particles was found to be ca. 70 μm in geometric mean diameter. The size was similar to that for 7Be-bearing particles obtained by a Ge detector measurement, and was slightly larger than the number-based size distribution measured with a scanning mobility particle sizer. The particle size measuring method using wire screens and imaging plates was successfully applied to the fine aerosol particles produced in an intense radiation field of a proton accelerator. This technique is applicable to size measurement of radioactive aerosol particles produced in the intense radiation fields of radiation facilities.

  1. Computer-aided diagnostics of screening mammography using content-based image retrieval

    Science.gov (United States)

    Deserno, Thomas M.; Soiron, Michael; de Oliveira, Júlia E. E.; de A. Araújo, Arnaldo

    2012-03-01

    Breast cancer is one of the main causes of death among women in occidental countries. In the last years, screening mammography has been established worldwide for early detection of breast cancer, and computer-aided diagnostics (CAD) is being developed to assist physicians reading mammograms. A promising method for CAD is content-based image retrieval (CBIR). Recently, we have developed a classification scheme of suspicious tissue pattern based on the support vector machine (SVM). In this paper, we continue moving towards automatic CAD of screening mammography. The experiments are based on in total 10,509 radiographs that have been collected from different sources. From this, 3,375 images are provided with one and 430 radiographs with more than one chain code annotation of cancerous regions. In different experiments, this data is divided into 12 and 20 classes, distinguishing between four categories of tissue density, three categories of pathology and in the 20 class problem two categories of different types of lesions. Balancing the number of images in each class yields 233 and 45 images remaining in each of the 12 and 20 classes, respectively. Using a two-dimensional principal component analysis, features are extracted from small patches of 128 x 128 pixels and classified by means of a SVM. Overall, the accuracy of the raw classification was 61.6 % and 52.1 % for the 12 and the 20 class problem, respectively. The confusion matrices are assessed for detailed analysis. Furthermore, an implementation of a SVM-based CBIR system for CADx in screening mammography is presented. In conclusion, with a smarter patch extraction, the CBIR approach might reach precision rates that are helpful for the physicians. This, however, needs more comprehensive evaluation on clinical data.

  2. Red blood cells, spherocytosis (image)

    Science.gov (United States)

    Spherocytosis is a hereditary disorder of the red blood cells (RBCs), which may be associated with a mild anemia. Typically, the affected RBCs are small, spherically shaped, and lack the light centers seen ...

  3. Can laptops be left inside passenger bags if motion imaging is used in X-ray security screening?

    Directory of Open Access Journals (Sweden)

    Marcia eMendes

    2013-10-01

    Full Text Available This paper describes a study where a new X-ray machine for security screening featuring motion imaging (i.e. 5 views of a bag are shown as an image sequence was evaluated and compared to single view imaging available on conventional X-ray screening systems. More specifically, it was investigated whether with this new technology X-ray screening of passenger bags could be enhanced to such an extent that laptops could be left inside passenger bags, without causing a significant impairment in threat detection performance. An X-ray image interpretation test was created in four different versions, manipulating the factors packing condition (laptop and bag separate vs. laptop in bag and display condition (single vs. motion imaging. There was a highly significant and large main effect of packing condition. When laptops and bags were screened separately, threat item detection was substantially higher. For display condition, a medium effect was observed. Detection could be slightly enhanced through the application of motion imaging. There was no interaction between display and packing condition, implying that the high negative effect of leaving laptops in passenger bags could not be fully compensated by motion imaging. Additional analyses were carried out to examine effects depending on different threat categories (guns, improvised explosive devices, knives, others, the placement of the threat items (in bag vs. in laptop and viewpoint (easy vs. difficult view. In summary, although motion imaging provides an enhancement, it is not strong enough to allow leaving laptops in bags for security screening.

  4. High-Throughput 3D Tumor Spheroid Screening Method for Cancer Drug Discovery Using Celigo Image Cytometry.

    Science.gov (United States)

    Kessel, Sarah; Cribbes, Scott; Déry, Olivier; Kuksin, Dmitry; Sincoff, Eric; Qiu, Jean; Chan, Leo Li-Ying

    2016-06-01

    Oncologists have investigated the effect of protein or chemical-based compounds on cancer cells to identify potential drug candidates. Traditionally, the growth inhibitory and cytotoxic effects of the drugs are first measured in 2D in vitro models, and then further tested in 3D xenograft in vivo models. Although the drug candidates can demonstrate promising inhibitory or cytotoxicity results in a 2D environment, similar effects may not be observed under a 3D environment. In this work, we developed an image-based high-throughput screening method for 3D tumor spheroids using the Celigo image cytometer. First, optimal seeding density for tumor spheroid formation was determined by investigating the cell seeding density of U87MG, a human glioblastoma cell line. Next, the dose-response effects of 17-AAG with respect to spheroid size and viability were measured to determine the IC50 value. Finally, the developed high-throughput method was used to measure the dose response of four drugs (17-AAG, paclitaxel, TMZ, and doxorubicin) with respect to the spheroid size and viability. Each experiment was performed simultaneously in the 2D model for comparison. This detection method allowed for a more efficient process to identify highly qualified drug candidates, which may reduce the overall time required to bring a drug to clinical trial.

  5. Investigation of optimum energies for chest imaging using film-screen and computed radiography.

    Science.gov (United States)

    Honey, I D; Mackenzie, A; Evans, D S

    2005-05-01

    The purpose of the study was to compare the image quality of film-screen (FS) and computed radiography (CR) for adult chest examinations across a range of beam energies. A series of images of the CDRAD threshold contrast detail detection phantom were acquired for a range of tube potential and exposure levels with both CR and FS. The phantom was placed within 9 cm of Perspex to provide attenuation and realistic levels of scatter in the image. Hardcopy images of the phantom were scored from a masked light-box by two scorers. Threshold contrast indices were used to calculate a visibility index (VI). The relationships between dose and image quality for CR and for FS are fundamentally different. The improvements in VIs obtained using CR at 75 kVp and 90 kVp were found to be statistically significant compared with 125 kVp at matched effective dose levels. The relative performance of FS and CR varies as a function of energy owing to the different k-edges of each system. When changing from FS to CR, the use of lower tube potentials may allow image quality to be maintained whilst reducing effective dose. A tube voltage of 90 kVp is indicated by this work, but may require clinical verification.

  6. Image-based phenotyping for non-destructive screening of different salinity tolerance traits in rice

    KAUST Repository

    Hairmansis, Aris

    2014-08-14

    Background Soil salinity is an abiotic stress wide spread in rice producing areas, limiting both plant growth and yield. The development of salt-tolerant rice requires efficient and high-throughput screening techniques to identify promising lines for salt affected areas. Advances made in image-based phenotyping techniques provide an opportunity to use non-destructive imaging to screen for salinity tolerance traits in a wide range of germplasm in a reliable, quantitative and efficient way. However, the application of image-based phenotyping in the development of salt-tolerant rice remains limited. Results A non-destructive image-based phenotyping protocol to assess salinity tolerance traits of two rice cultivars (IR64 and Fatmawati) has been established in this study. The response of rice to different levels of salt stress was quantified over time based on total shoot area and senescent shoot area, calculated from visible red-green-blue (RGB) and fluorescence images. The response of rice to salt stress (50, 75 and 100 mM NaCl) could be clearly distinguished from the control as indicated by the reduced increase of shoot area. The salt concentrations used had only a small effect on the growth of rice during the initial phase of stress, the shoot Na+ accumulation independent phase termed the ‘osmotic stress’ phase. However, after 20 d of treatment, the shoot area of salt stressed plants was reduced compared with non-stressed plants. This was accompanied by a significant increase in the concentration of Na+ in the shoot. Variation in the senescent area of the cultivars IR64 and Fatmawati in response to a high concentration of Na+ in the shoot indicates variation in tissue tolerance mechanisms between the cultivars. Conclusions Image analysis has the potential to be used for high-throughput screening procedures in the development of salt-tolerant rice. The ability of image analysis to discriminate between the different aspects of salt stress (shoot ion

  7. A Multi-Modality CMOS Sensor Array for Cell-Based Assay and Drug Screening.

    Science.gov (United States)

    Chi, Taiyun; Park, Jong Seok; Butts, Jessica C; Hookway, Tracy A; Su, Amy; Zhu, Chengjie; Styczynski, Mark P; McDevitt, Todd C; Wang, Hua

    2015-12-01

    In this paper, we present a fully integrated multi-modality CMOS cellular sensor array with four sensing modalities to characterize different cell physiological responses, including extracellular voltage recording, cellular impedance mapping, optical detection with shadow imaging and bioluminescence sensing, and thermal monitoring. The sensor array consists of nine parallel pixel groups and nine corresponding signal conditioning blocks. Each pixel group comprises one temperature sensor and 16 tri-modality sensor pixels, while each tri-modality sensor pixel can be independently configured for extracellular voltage recording, cellular impedance measurement (voltage excitation/current sensing), and optical detection. This sensor array supports multi-modality cellular sensing at the pixel level, which enables holistic cell characterization and joint-modality physiological monitoring on the same cellular sample with a pixel resolution of 80 μm × 100 μm. Comprehensive biological experiments with different living cell samples demonstrate the functionality and benefit of the proposed multi-modality sensing in cell-based assay and drug screening.

  8. Use and imaging performance of CMOS flat panel imager with LiF/ZnS(Ag) and Gadox scintillation screens for neutron radiography

    Science.gov (United States)

    Cha, B. K.; kim, J. Y.; Kim, T. J.; Sim, C.; Cho, G.; Lee, D. H.; Seo, C.-W.; Jeon, S.; Huh, Y.

    2011-01-01

    In digital neutron radiography system, a thermal neutron imaging detector based on neutron-sensitive scintillating screens with CMOS(complementary metal oxide semiconductor) flat panel imager is introduced for non-destructive testing (NDT) application. Recently, large area CMOS APS (active-pixel sensor) in conjunction with scintillation films has been widely used in many digital X-ray imaging applications. Instead of typical imaging detectors such as image plates, cooled-CCD cameras and amorphous silicon flat panel detectors in combination with scintillation screens, we tried to apply a scintillator-based CMOS APS to neutron imaging detection systems for high resolution neutron radiography. In this work, two major Gd2O2S:Tb and 6LiF/ZnS:Ag scintillation screens with various thickness were fabricated by a screen printing method. These neutron converter screens consist of a dispersion of Gd2O2S:Tb and 6LiF/ZnS:Ag scintillating particles in acrylic binder. These scintillating screens coupled-CMOS flat panel imager with 25x50mm2 active area and 48μm pixel pitch was used for neutron radiography. Thermal neutron flux with 6x106n/cm2/s was utilized at the NRF facility of HANARO in KAERI. The neutron imaging characterization of the used detector was investigated in terms of relative light output, linearity and spatial resolution in detail. The experimental results of scintillating screen-based CMOS flat panel detectors demonstrate possibility of high sensitive and high spatial resolution imaging in neutron radiography system.

  9. Effects of image compression and degradation on an automatic diabetic retinopathy screening algorithm

    Science.gov (United States)

    Agurto, C.; Barriga, S.; Murray, V.; Pattichis, M.; Soliz, P.

    2010-03-01

    Diabetic retinopathy (DR) is one of the leading causes of blindness among adult Americans. Automatic methods for detection of the disease have been developed in recent years, most of them addressing the segmentation of bright and red lesions. In this paper we present an automatic DR screening system that does approach the problem through the segmentation of features. The algorithm determines non-diseased retinal images from those with pathology based on textural features obtained using multiscale Amplitude Modulation-Frequency Modulation (AM-FM) decompositions. The decomposition is represented as features that are the inputs to a classifier. The algorithm achieves 0.88 area under the ROC curve (AROC) for a set of 280 images from the MESSIDOR database. The algorithm is then used to analyze the effects of image compression and degradation, which will be present in most actual clinical or screening environments. Results show that the algorithm is insensitive to illumination variations, but high rates of compression and large blurring effects degrade its performance.

  10. A recessive genetic screen for components of the RNA interference pathway in mouse embryonic stem cells.

    Science.gov (United States)

    Trombly, Melanie I; Wang, Xiaozhong

    2010-01-01

    Several key components of the RNA interference (RNAi) pathway were identified in genetic screens performed in nonmammalian model organisms. To identify components of the mammalian RNAi pathway, we developed a recessive genetic screen in mouse embryonic stem (ES) cells. Recessive genetic screens are feasible in ES cells that are Bloom-syndrome protein (Blm-) deficient. Therefore, we constructed a reporter cell line in Blm-deficient ES cells to isolate RNAi mutants through a simple drug-selection scheme. This chapter describes how we used retroviral gene traps to mutagenize the reporter cell line and select for RNAi mutants. Putative RNAi mutants were confirmed using a separate functional assay. The location of the gene trap was then identified using molecular techniques such as Splinkerette PCR. Our screening strategy successfully isolated several mutant clones of Argonaute2, a vital component of the RNAi pathway.

  11. DHM (Digital Holography Microscope) for imaging cells

    Energy Technology Data Exchange (ETDEWEB)

    Emery, Yves [Lyncee Tec SA, PSE-A, 1015 Lausanne (Switzerland); Cuche, Etienne [Lyncee Tec SA, PSE-A, 1015 Lausanne (Switzerland); Colomb, Tristan [STI-IOA-EPFL, 1015 Lausanne (Switzerland); Depeursinge, Christian [STI-IOA-EPFL, 1015 Lausanne (Switzerland); Rappaz, Benjamin [SV-BM-EPFL, 1015 Lausanne (Switzerland); Marquet, Pierre [CNP-CHUV, Site de Cery, 1008 Prilly (Switzerland); Magistretti, Pierre [SV-BM-EPFL, 1015 Lausanne (Switzerland)

    2007-04-15

    Light interaction with a sample modifies both intensity and phase of the illuminating wave. Any available supports for image recording are only sensitive to intensity, but Denis Gabor [P. Marquet, B. Rappaz, P. Magistretti, et. al. Digital Holography for quantitative phase-contrast imaging, Optics Letters, 30, 5, pp 291-93 (2005)] invented in 1948 a way to encode the phase as an intensity variation: the {sup h}ologram{sup .} Digital Holographic Microscopy (DHM) [D. Gabor, A new microscopic principle, Nature, 1948] implements digitally this powerful hologram. Characterization of various pollen grains and of morphology changes of neurones associated with hypotonic shock demonstrates the potential of DHM for imaging cells.

  12. Multiparameter fluorescence spectroscopic imaging of cell function

    Science.gov (United States)

    Bright, Gary R.

    1994-08-01

    The ability to quantitate physiological parameters in single living cells using fluorescence spectroscopic imaging has expanded our understanding of many cell regulatory processes. Previous studies have focussed on the measurement of single parameters, such as the concentration of calcium, and more recently two parameters, such as calcium and pH using fluorescence ratio imaging. The complexity of the interrelationships among cell biochemical reactions suggests a need to extend the measurement scheme to several parameters. Expansion of the number of parameters involves several complexities associated with fluorescent probe selection and instrumentation design as well as the processing and management of the data. A system has been assembled which provides maximum flexibility in multiparameter fluorescence imaging measurements. The system provides multiple combinations of excitation, dichroic mirror, and emission wavelengths. It has automatic acquisition of any number of parameters. The number of parameters is primarily limited by the selection of fluorescent probes with nonoverlapping spectra. We demonstrate the utility of the system by the coordinated monitoring of stimulated changes in the concentrations of calcium, magnesium, and pH using fluorescence ratio imaging coupled with a conventional transmitted light image of single smooth muscle cells. The results demonstrate coordinated changes in some instances but uncoordinated changes in others.

  13. Advances in Tumor Screening, Imaging, and Avatar Technologies for High-Grade Serous Ovarian Cancer

    Directory of Open Access Journals (Sweden)

    Anders eOhman

    2014-11-01

    Full Text Available The majority of high-grade serous ovarian carcinoma cases are detected in advanced stages when treatment options are limited. Surgery is less effective at eradicating the disease when it is widespread, resulting in high rates of disease relapse and chemoresistance. Current screening techniques are ineffective for early tumor detection and consequently, BRCA mutations carriers, with an increased risk for developing high-grade serous ovarian cancer, elect to undergo risk-reducing surgery. While prophylactic surgery is associated with a significant reduction in the risk of cancer development, it also results in surgical menopause and significant adverse side effects. The development of efficient early-stage screening protocols and imaging technologies is critical to improving the outcome and quality of life for current patients and women at increased risk. In addition, more accurate animal models are necessary in order to provide relevant in vivo testing systems and advance our understanding of the disease origin and progression. Moreover, both genetically engineered and tumor xenograft animal models enable the preclinical testing of novel imaging techniques and molecularly targeted therapies as they become available. Recent advances in xenograft technologies have made possible the creation of avatar mice, personalized tumorgrafts, which can be used as therapy testing surrogates for individual patients prior to or during treatment. High-grade serous ovarian cancer may be an ideal candidate for use with avatar models based on key characteristics of the tumorgraft platform. This review explores multiple strategies, including novel imaging and screening technologies in both patients and animal models, aimed at detecting cancer in the early stages and improving the disease prognosis.

  14. Evaluation of an Automated Information Extraction Tool for Imaging Data Elements to Populate a Breast Cancer Screening Registry.

    Science.gov (United States)

    Lacson, Ronilda; Harris, Kimberly; Brawarsky, Phyllis; Tosteson, Tor D; Onega, Tracy; Tosteson, Anna N A; Kaye, Abby; Gonzalez, Irina; Birdwell, Robyn; Haas, Jennifer S

    2015-10-01

    Breast cancer screening is central to early breast cancer detection. Identifying and monitoring process measures for screening is a focus of the National Cancer Institute's Population-based Research Optimizing Screening through Personalized Regimens (PROSPR) initiative, which requires participating centers to report structured data across the cancer screening continuum. We evaluate the accuracy of automated information extraction of imaging findings from radiology reports, which are available as unstructured text. We present prevalence estimates of imaging findings for breast imaging received by women who obtained care in a primary care network participating in PROSPR (n = 139,953 radiology reports) and compared automatically extracted data elements to a "gold standard" based on manual review for a validation sample of 941 randomly selected radiology reports, including mammograms, digital breast tomosynthesis, ultrasound, and magnetic resonance imaging (MRI). The prevalence of imaging findings vary by data element and modality (e.g., suspicious calcification noted in 2.6% of screening mammograms, 12.1% of diagnostic mammograms, and 9.4% of tomosynthesis exams). In the validation sample, the accuracy of identifying imaging findings, including suspicious calcifications, masses, and architectural distortion (on mammogram and tomosynthesis); masses, cysts, non-mass enhancement, and enhancing foci (on MRI); and masses and cysts (on ultrasound), range from 0.8 to1.0 for recall, precision, and F-measure. Information extraction tools can be used for accurate documentation of imaging findings as structured data elements from text reports for a variety of breast imaging modalities. These data can be used to populate screening registries to help elucidate more effective breast cancer screening processes.

  15. Quantitative phenotyping-based in vivo chemical screening in a zebrafish model of leukemia stem cell xenotransplantation.

    Directory of Open Access Journals (Sweden)

    Beibei Zhang

    Full Text Available Zebrafish-based chemical screening has recently emerged as a rapid and efficient method to identify important compounds that modulate specific biological processes and to test the therapeutic efficacy in disease models, including cancer. In leukemia, the ablation of leukemia stem cells (LSCs is necessary to permanently eradicate the leukemia cell population. However, because of the very small number of LSCs in leukemia cell populations, their use in xenotransplantation studies (in vivo and the difficulties in functionally and pathophysiologically replicating clinical conditions in cell culture experiments (in vitro, the progress of drug discovery for LSC inhibitors has been painfully slow. In this study, we developed a novel phenotype-based in vivo screening method using LSCs xenotransplanted into zebrafish. Aldehyde dehydrogenase-positive (ALDH+ cells were purified from chronic myelogenous leukemia K562 cells tagged with a fluorescent protein (Kusabira-orange and then implanted in young zebrafish at 48 hours post-fertilization. Twenty-four hours after transplantation, the animals were treated with one of eight different therapeutic agents (imatinib, dasatinib, parthenolide, TDZD-8, arsenic trioxide, niclosamide, salinomycin, and thioridazine. Cancer cell proliferation, and cell migration were determined by high-content imaging. Of the eight compounds that were tested, all except imatinib and dasatinib selectively inhibited ALDH+ cell proliferation in zebrafish. In addition, these anti-LSC agents suppressed tumor cell migration in LSC-xenotransplants. Our approach offers a simple, rapid, and reliable in vivo screening system that facilitates the phenotype-driven discovery of drugs effective in suppressing LSCs.

  16. Terahertz imaging for early screening of diabetic foot syndrome: A proof of concept

    Science.gov (United States)

    Hernandez-Cardoso, G. G.; Rojas-Landeros, S. C.; Alfaro-Gomez, M.; Hernandez-Serrano, A. I.; Salas-Gutierrez, I.; Lemus-Bedolla, E.; Castillo-Guzman, A. R.; Lopez-Lemus, H. L.; Castro-Camus, E.

    2017-01-01

    Most people with diabetes suffer some deterioration of the feet. Diabetic foot syndrome causes ulceration in about 15% of cases and such deterioration leads to amputation in about 2.5% of diabetic patients, diminishing their quality of life and generating extraordinary costs for patients and public health systems. Currently, there is no objective method for the detection of diabetic foot syndrome in its early stages. We propose terahertz imaging as a method for the evaluation of such deterioration. This screening method could aid the prevention and medical treatment of this condition in the future. PMID:28165050

  17. Terahertz imaging for early screening of diabetic foot syndrome: A proof of concept

    Science.gov (United States)

    Hernandez-Cardoso, G. G.; Rojas-Landeros, S. C.; Alfaro-Gomez, M.; Hernandez-Serrano, A. I.; Salas-Gutierrez, I.; Lemus-Bedolla, E.; Castillo-Guzman, A. R.; Lopez-Lemus, H. L.; Castro-Camus, E.

    2017-02-01

    Most people with diabetes suffer some deterioration of the feet. Diabetic foot syndrome causes ulceration in about 15% of cases and such deterioration leads to amputation in about 2.5% of diabetic patients, diminishing their quality of life and generating extraordinary costs for patients and public health systems. Currently, there is no objective method for the detection of diabetic foot syndrome in its early stages. We propose terahertz imaging as a method for the evaluation of such deterioration. This screening method could aid the prevention and medical treatment of this condition in the future.

  18. Surface impedance based microwave imaging method for breast cancer screening: contrast-enhanced scenario.

    Science.gov (United States)

    Güren, Onan; Çayören, Mehmet; Ergene, Lale Tükenmez; Akduman, Ibrahim

    2014-10-01

    A new microwave imaging method that uses microwave contrast agents is presented for the detection and localization of breast tumours. The method is based on the reconstruction of breast surface impedance through a measured scattered field. The surface impedance modelling allows for representing the electrical properties of the breasts in terms of impedance boundary conditions, which enable us to map the inner structure of the breasts into surface impedance functions. Later a simple quantitative method is proposed to screen breasts against malignant tumours where the detection procedure is based on weighted cross correlations among impedance functions. Numerical results demonstrate that the method is capable of detecting small malignancies and provides reasonable localization.

  19. Time-stretch microscopy on a DVD for high-throughput imaging cell-based assay.

    Science.gov (United States)

    Tang, Anson H L; Yeung, P; Chan, Godfrey C F; Chan, Barbara P; Wong, Kenneth K Y; Tsia, Kevin K

    2017-02-01

    Cell-based assay based on time-stretch imaging is recognized to be well-suited for high-throughput phenotypic screening. However, this ultrafast imaging technique has primarily been limited to suspension-cell assay, leaving a wide range of solid-substrate assay formats uncharted. Moreover, time-stretch imaging is generally restricted to intrinsic biophysical phenotyping, but lacks the biomolecular signatures of the cells. To address these challenges, we develop a spinning time-stretch imaging assay platform based on the functionalized digital versatile disc (DVD). We demonstrate that adherent cell culture and biochemically-specific cell-capture can now be assayed with time-stretch microscopy, thanks to the high-speed DVD spinning motion that naturally enables on-the-fly cellular imaging at an ultrafast line-scan rate of >10MHz. As scanning the whole DVD at such a high speed enables ultra-large field-of-view imaging, it could be favorable for scaling both the assay throughput and content as demanded in many applications, e.g. drug discovery, and rare cancer cell screening.

  20. Imaging Tumor Cell Movement In Vivo

    OpenAIRE

    Entenberg, David; Kedrin, Dmitriy; Wyckoff, Jeffrey; Sahai, Erik; Condeelis, John; Segall, Jeffrey E

    2013-01-01

    This unit describes the methods that we have been developing for analyzing tumor cell motility in mouse and rat models of breast cancer metastasis. Rodents are commonly used both to provide a mammalian system for studying human tumor cells (as xenografts in immunocompromised mice) as well as for following the development of tumors from a specific tissue type in transgenic lines. The Basic Protocol in this unit describes the standard methods used for generation of mammary tumors and imaging th...

  1. Nonlinear 3-D Microwave Imaging for Breast-Cancer Screening: Log, Phase, and Log-Phase Formulation

    DEFF Research Database (Denmark)

    Jensen, Peter Damsgaard; Rubæk, Tonny; Mohr, Johan Jacob;

    2011-01-01

    The imaging algorithm used in the 3-D microwave imaging system for breast cancer screening, currently being developed at the Technical University of Denmark, is based on an iterative Newton-type algorithm. In this algorithm, the distribution of the electromagnetic constitutive parameters is updat...

  2. Calcium Imaging of Sonoporation of Mammalian Cells

    Science.gov (United States)

    Sabens, David; Aehle, Matthew; Steyer, Grant; Kourennyi, Dmitri; Deng, Cheri X.

    2006-05-01

    Ultrasound mediated delivery of compounds is a relatively recent development in drug delivery and gene transfection techniques. Due to the lack of methods for real-time monitoring of sonoporation at the cellular level, the efficiency of drug/gene delivery and sonoporation associated side effects, such as the loss of cell viability and enhanced apoptosis, have been studied only through post US exposure analyses, requiring days for cell incubation. Furthermore, because microporation appears to be transient in nature, it was not possible to correlate transfection with microporation on an individual cellular basis. By studying the role of calcium in the cell and using fluorescent calcium imaging to study sonoporation it is possible to quantify both cell porosity and sonoporation side effects. Since both post sonoporation cell survival and delivery efficiency are related to the dynamic process of the cell membrane poration, calcium imaging of sonoporation will provide important knowledge to obtain improved understanding of sonoporation mechanism. Our experimental results demonstrated the feasibility of calcium imaging of sonoporation in Chinese Hamster Ovary (CHO) cells. We have measured the changes in the intracellular calcium concentration using Fura-2, a fluorescent probe, which indicate influx or flow of Calcium across the cell membrane. Analysis of data identified key aspects in the dynamic sonoporation process including the formation of pores in the cell membrane, and the relative temporal duration of the pores and their resealing. These observations are obtained through the analysis of the rate the calcium concentration changes within the cells, making it possible to visualize membrane opening and repair in real-time through such changes in the intracellular calcium concentration.

  3. Live imaging RNAi screen reveals genes essential for meiosis in mammalian oocytes.

    Science.gov (United States)

    Pfender, Sybille; Kuznetsov, Vitaliy; Pasternak, Michał; Tischer, Thomas; Santhanam, Balaji; Schuh, Melina

    2015-08-13

    During fertilization, an egg and a sperm fuse to form a new embryo. Eggs develop from oocytes in a process called meiosis. Meiosis in human oocytes is highly error-prone, and defective eggs are the leading cause of pregnancy loss and several genetic disorders such as Down's syndrome. Which genes safeguard accurate progression through meiosis is largely unclear. Here we develop high-content phenotypic screening methods for the systematic identification of mammalian meiotic genes. We targeted 774 genes by RNA interference within follicle-enclosed mouse oocytes to block protein expression from an early stage of oocyte development onwards. We then analysed the function of several genes simultaneously by high-resolution imaging of chromosomes and microtubules in live oocytes and scored each oocyte quantitatively for 50 phenotypes, generating a comprehensive resource of meiotic gene function. The screen generated an unprecedented annotated data set of meiotic progression in 2,241 mammalian oocytes, which allowed us to analyse systematically which defects are linked to abnormal chromosome segregation during meiosis, identifying progression into anaphase with misaligned chromosomes as well as defects in spindle organization as risk factors. This study demonstrates how high-content screens can be performed in oocytes, and allows systematic studies of meiosis in mammals.

  4. Recent Advances in Morphological Cell Image Analysis

    Directory of Open Access Journals (Sweden)

    Shengyong Chen

    2012-01-01

    Full Text Available This paper summarizes the recent advances in image processing methods for morphological cell analysis. The topic of morphological analysis has received much attention with the increasing demands in both bioinformatics and biomedical applications. Among many factors that affect the diagnosis of a disease, morphological cell analysis and statistics have made great contributions to results and effects for a doctor. Morphological cell analysis finds the cellar shape, cellar regularity, classification, statistics, diagnosis, and so forth. In the last 20 years, about 1000 publications have reported the use of morphological cell analysis in biomedical research. Relevant solutions encompass a rather wide application area, such as cell clumps segmentation, morphological characteristics extraction, 3D reconstruction, abnormal cells identification, and statistical analysis. These reports are summarized in this paper to enable easy referral to suitable methods for practical solutions. Representative contributions and future research trends are also addressed.

  5. Genome-wide genetic screening with chemically mutagenized haploid embryonic stem cells

    OpenAIRE

    Forment, Josep V.; Herzog, Mareike; Coates, Julia; Konopka, Tomasz; Gapp, Bianca V.; Nijman, Sebastian M.; Adams, David J; Keane, Thomas M.; Jackson, Stephen P.

    2016-01-01

    This is the author accepted manuscript. In model organisms, classical genetic screening via random mutagenesis provides key insights into the molecular bases of genetic interactions, helping to define synthetic lethality, synthetic viability and drug-resistance mechanisms. The limited genetic tractability of diploid mammalian cells, however, precludes this approach. Here, we demonstrate the feasibility of classical genetic screening in mammalian systems by using haploid cells, chemical mut...

  6. A cell-based screen reveals that the albendazole metabolite, albendazole sulfone, targets Wolbachia.

    OpenAIRE

    Serbus, Laura R.; Frederic Landmann; Bray, Walter M.; Pamela M White; Jordan Ruybal; R Scott Lokey; Alain Debec; William Sullivan

    2012-01-01

    Wolbachia endosymbionts carried by filarial nematodes give rise to the neglected diseases African river blindness and lymphatic filariasis afflicting millions worldwide. Here we identify new Wolbachia-disrupting compounds by conducting high-throughput cell-based chemical screens using a Wolbachia-infected, fluorescently labeled Drosophila cell line. This screen yielded several Wolbachia-disrupting compounds including three that resembled Albendazole, a widely used anthelmintic drug that targe...

  7. Genetic synthetic lethality screen at the single gene level in cultured human cells

    OpenAIRE

    Simons, Arnold H.; Dafni, Naomi; Dotan, Iris; Oron, Yoram; Canaani, Dan

    2001-01-01

    Recently, we demonstrated the feasibility of a chemical synthetic lethality screen in cultured human cells. We now demonstrate the principles for a genetic synthetic lethality screen. The technology employs both an immortalized human cell line deficient in the gene of interest, which is complemented by an episomal survival plasmid expressing the wild-type cDNA for the gene of interest, and the use of a novel GFP-based double-label fluorescence system. Dominant negative genetic suppressor elem...

  8. An integrated microfluidic biosensor for the rapid screening of foodborne pathogens by surface plasmon resonance imaging

    Science.gov (United States)

    Zordan, Michael D.; Grafton, Meggie M. G.; Leary, James F.

    2011-03-01

    The rapid detection of foodborne pathogens is of vital importance to keep the food supply rid of contamination. Previously we have demonstrated the design of a hybrid optical device that performs real-time surface plasmon resonance (SPR) and epi-fluorescence imaging. Additionally we have developed a biosensor array chip that is able to specifically detect the presence of two known pathogens. This biosensor detects the presence of the pathogen strains by the selective capture of whole pathogens by peptide ligands functionalized to the spots of the array. We have incorporated this biosensor array into a self contained PDMS microfluidic chip. The enclosure of the biosensor array by a PDMS microfluidic chip allows for a sample to be screened for many strains of pathogens simultaneously in a safe one time use biochip. This disposable optical biochip is inserted into with the hybrid SPR/epi-fluorescence imaging device to form an integrated system for the detection of foodborne pathogens. Using this integrated system, we can selectively detect the presence of E. coli 0157:H7 or S. enterica in a simultaneously in real-time. Additionally, we have modeled the mechanical properties of the microfluidic biochip in order to manipulate the flow conditions to achieve optimal pathogen capture by the biosensor array. We have developed an integrated system that is able to screen a sample for multiple foodborne pathogens simultaneously in a safe, rapid and label-free manner.

  9. Droplet microfluidic technology for single-cell high-throughput screening.

    Science.gov (United States)

    Brouzes, Eric; Medkova, Martina; Savenelli, Neal; Marran, Dave; Twardowski, Mariusz; Hutchison, J Brian; Rothberg, Jonathan M; Link, Darren R; Perrimon, Norbert; Samuels, Michael L

    2009-08-25

    We present a droplet-based microfluidic technology that enables high-throughput screening of single mammalian cells. This integrated platform allows for the encapsulation of single cells and reagents in independent aqueous microdroplets (1 pL to 10 nL volumes) dispersed in an immiscible carrier oil and enables the digital manipulation of these reactors at a very high-throughput. Here, we validate a full droplet screening workflow by conducting a droplet-based cytotoxicity screen. To perform this screen, we first developed a droplet viability assay that permits the quantitative scoring of cell viability and growth within intact droplets. Next, we demonstrated the high viability of encapsulated human monocytic U937 cells over a period of 4 days. Finally, we developed an optically-coded droplet library enabling the identification of the droplets composition during the assay read-out. Using the integrated droplet technology, we screened a drug library for its cytotoxic effect against U937 cells. Taken together our droplet microfluidic platform is modular, robust, uses no moving parts, and has a wide range of potential applications including high-throughput single-cell analyses, combinatorial screening, and facilitating small sample analyses.

  10. Women's attitude towards prenatal screening for red blood cell antibodies, other than RhD

    Directory of Open Access Journals (Sweden)

    van der Schoot CE

    2008-11-01

    Full Text Available Abstract Background Since July 1998 all Dutch women (± 200,000/y are screened for red cell antibodies, other than anti-RhesusD (RhD in the first trimester of pregnancy, to facilitate timely treatment of pregnancies at risk for hemolytic disease of the fetus and newborn (HDFN. Evidence for benefits, consequences and costs of screening for non-RhD antibodies is still under discussion. The screening program was evaluated in a nation-wide study. As a part of this evaluation study we investigated, according to the sixth criterium of Wilson and Jüngner, the acceptance by pregnant women of the screening program for non-RhD antibodies. Methods Controlled longitudinal survey, including a prenatal and a postnatal measurement by structured questionnaires. Main outcome measures: information satisfaction, anxiety during the screening process (a.o. STAI state inventory and specific questionnaire modules, overall attitude on the screening program. Univariate analysis was followed by standard multivariate analysis to identify significant predictors of the outcome measures. Participants: 233 pregnant women, distributed over five groups, according to the screening result. Results Satisfaction about the provided information was moderate in all groups. All screen- positive groups desired more supportive information. Anxiety increased in screen- positives during the screening process, but decreased to basic levels postnatally. All groups showed a strongly positive balance between perceived utility and burden of the screening program, independent on test results or background characteristics. Conclusion Women highly accept the non-RhD antibody screening program. However, satisfaction about provided information is moderate. Oral and written information should be provided by obstetric care workers themselves, especially to screen-positive women.

  11. Museum as an integrated imaging device: visualization of ancient Kyoto cityscape from folding screen artifact

    Science.gov (United States)

    Miyata, Kimiyoshi; Oyabu, Umi; Kojima, Michihiro

    2012-03-01

    Museums hold cultural resources such as artworks, historical artifacts, and folklore materials. The National Museum of Japanese History holds over 200,000 of the cultural resources. A role of museum is to exhibit the cultural resources, therefore a museum could be referred to as a visualization device for the information society. In this research, visualization of a history image from cultural resources with interactive user interface will be mentioned. The material focused on is the oldest extant version of a genre of folding screen paintings that depict the thriving city of Kyoto in the four seasons, named Rekihaku's "Scenes In and Around Kyoto" designated as a nationally important cultural property in Japan. Over 1,400 people and a lot of residences, temples, and houses are drawn, and those are also information resource telling us about city scenes and people's life in Kyoto at that time. Historical researches were done by using a high resolution digital image obtained by a large scaled scanner, and scanned images are used for computer programs to visualize a history image of ancient Kyoto. Combinations between real materials and information provided by using the computer programs are also described in this research.

  12. Cell classification using big data analytics plus time stretch imaging (Conference Presentation)

    Science.gov (United States)

    Jalali, Bahram; Chen, Claire L.; Mahjoubfar, Ata

    2016-09-01

    We show that blood cells can be classified with high accuracy and high throughput by combining machine learning with time stretch quantitative phase imaging. Our diagnostic system captures quantitative phase images in a flow microscope at millions of frames per second and extracts multiple biophysical features from individual cells including morphological characteristics, light absorption and scattering parameters, and protein concentration. These parameters form a hyperdimensional feature space in which supervised learning and cell classification is performed. We show binary classification of T-cells against colon cancer cells, as well classification of algae cell strains with high and low lipid content. The label-free screening averts the negative impact of staining reagents on cellular viability or cell signaling. The combination of time stretch machine vision and learning offers unprecedented cell analysis capabilities for cancer diagnostics, drug development and liquid biopsy for personalized genomics.

  13. Noninvasive Imaging of Administered Progenitor Cells

    Energy Technology Data Exchange (ETDEWEB)

    Steven R Bergmann, M.D., Ph.D.

    2012-12-03

    The objective of this research grant was to develop an approach for labeling progenitor cells, specifically those that we had identified as being able to replace ischemic heart cells, so that the distribution could be followed non-invasively. In addition, the research was aimed at determining whether administration of progenitor cells resulted in improved myocardial perfusion and function. The efficiency and toxicity of radiolabeling of progenitor cells was to be evaluated. For the proposed clinical protocol, subjects with end-stage ischemic coronary artery disease were to undergo a screening cardiac positron emission tomography (PET) scan using N-13 ammonia to delineate myocardial perfusion and function. If they qualified based on their PET scan, they would undergo an in-hospital protocol whereby CD34+ cells were stimulated by the administration of granulocytes-colony stimulating factor (G-CSF). CD34+ cells would then be isolated by apharesis, and labeled with indium-111 oxine. Cells were to be re-infused and subjects were to undergo single photon emission computed tomography (SPECT) scanning to evaluate uptake and distribution of labeled progenitor cells. Three months after administration of progenitor cells, a cardiac PET scan was to be repeated to evaluate changes in myocardial perfusion and/or function. Indium oxine is a radiopharmaceutical for labeling of autologous lymphocytes. Indium-111 (In-111) decays by electron capture with a t{sub ½} of 67.2 hours (2.8 days). Indium forms a saturated complex that is neutral, lipid soluble, and permeates the cell membrane. Within the cell, the indium-oxyquinolone complex labels via indium intracellular chelation. Following leukocyte labeling, ~77% of the In-111 is incorporated in the cell pellet. The presence of red cells and /or plasma reduces the labeling efficacy. Therefore, the product needed to be washed to eliminate plasma proteins. This repeated washing can damage cells. The CD34 selected product was a 90

  14. Newborn screening for sickle cell disease in Jamaica: logistics and experience with umbilical cord samples.

    Science.gov (United States)

    Serjeant, G R; Serjeant, B E; Mason, K P; Gardner, R; Warren, L; Gibson, F; Coombs, M

    2017-01-01

    The study aims to describe the logistics and results of a programme for newborn screening for sickle cell disease based on samples from the umbilical cord. Samples were dried on Guthrie cards and analysed by high pressure liquid chromatography. All suspected clinically significant abnormal genotypes were confirmed by age 4-6 weeks with family studies and then recruited to local sickle cell clinics. The programme has screened 66,833 samples with the sickle cell trait in 9.8 % and the HbC trait in 3.8 %. Sickle cell syndromes occurred in 407 babies (204 SS, 148 SC, 35 Sbeta(+) thalassaemia, 6 Sbeta(o) thalassaemia, 6 sickle cell-variants, 8 sickle cell-hereditary persistence of fetal haemoglobin) and HbC syndromes in 42 (22 CC, 14 Cbeta(+) thalassaemia, 1 Cbeta(o) thalassaemia, 5 HbC- hereditary persistence of fetal haemoglobin). Focusing on the year 2015, screening was performed in 15,408, compliance with sample collection was 98.1 %, and maternal contamination occurred in 335 (2.6 %) but in only 0.05 % did diagnostic confusion require patient recall and further tests. This model of newborn screening for sickle cell disease is accurate, robust and economic. It is hoped that it may be helpful for other societies with high prevalence of abnormal haemoglobins and limited resources, who are planning to embark on newborn screening for sickle cell disease.

  15. Split-screen display system and standardized methods for ultrasound image acquisition and multi-frame data processing

    Science.gov (United States)

    Selzer, Robert H. (Inventor); Hodis, Howard N. (Inventor)

    2011-01-01

    A standardized acquisition methodology assists operators to accurately replicate high resolution B-mode ultrasound images obtained over several spaced-apart examinations utilizing a split-screen display in which the arterial ultrasound image from an earlier examination is displayed on one side of the screen while a real-time "live" ultrasound image from a current examination is displayed next to the earlier image on the opposite side of the screen. By viewing both images, whether simultaneously or alternately, while manually adjusting the ultrasound transducer, an operator is able to bring into view the real-time image that best matches a selected image from the earlier ultrasound examination. Utilizing this methodology, dynamic material properties of arterial structures, such as IMT and diameter, are measured in a standard region over successive image frames. Each frame of the sequence has its echo edge boundaries automatically determined by using the immediately prior frame's true echo edge coordinates as initial boundary conditions. Computerized echo edge recognition and tracking over multiple successive image frames enhances measurement of arterial diameter and IMT and allows for improved vascular dimension measurements, including vascular stiffness and IMT determinations.

  16. Monitoring stem cells in phase contrast imaging

    Science.gov (United States)

    Lam, K. P.; Dempsey, K. P.; Collins, D. J.; Richardson, J. B.

    2016-04-01

    Understanding the mechanisms behind the proliferation of Mesenchymal Stem cells (MSCs) can offer a greater insight into the behaviour of these cells throughout their life cycles. Traditional methods of determining the rate of MSC differentiation rely on population based studies over an extended time period. However, such methods can be inadequate as they are unable to track cells as they interact; for example, in autologous cell therapies for osteoarthritis, the development of biological assays that could predict in vivo functional activity and biological action are particularly challenging. Here further research is required to determine non-histochemical biomarkers which provide correlations between cell survival and predictive functional outcome. This paper proposes using a (previously developed) advanced texture-based analysis algorithm to facilitate in vitro cells tracking using time-lapsed microscopy. The technique was adopted to monitor stem cells in the context of unlabelled, phase contrast imaging, with the goal of examining the cell to cell interactions in both monoculture and co-culture systems. The results obtained are analysed using established exploratory procedures developed for time series data and compared with the typical fluorescent-based approach of cell labelling. A review of the progress and the lessons learned are also presented.

  17. DIAGNOSTIC VALUE OF WHOLE BODY DIFFUSION WEIGHTED IMAGING FOR SCREENING PRIMARY TUMORS OF PATIENTS WITH METASTASES

    Institute of Scientific and Technical Information of China (English)

    Tai-fu Gu; Xin-lan Xiao; Fei Sun; Jian-hua Yin; Hai Zhao

    2008-01-01

    Objective To evaluate the values of whole body diffusion weighted imaging (DWI) in screening primary unknown tumor in patients with metastases.Methods Totally, 34 patients with metastases of primary unknown tumors were scanned with whole body DWI, and conventional magnetic resonance (MR) imaging was performed if suspected lesions were detected. All the metastases including 27 cases of osseous metastases, 2 brain metastases, 2 liver metastases, 1 pulmonary multiple metastasis, 1 neck metastasis and 1 malignant ascites, were diagnosed by computed tomography, single photon emission computed tomography, or MR imaging. For the proven primary tumors diagnosed by biopsy or pathology of surgical specimens, apparent diffusion coefficient (ADC) values of the primary and metastatic lesions were measured respectively. The sensitivity and specificity of this technique for screening primary tumors were evaluated. Results We found 24 cases with suspected primary lesions, in which 23 lesions were proved to be primary tumors, and 1 was proved to be benign lesion. And no definite primary lesion was found in 10 cases on whole body DWI, but in which 1 case was diagnosed with primary tumor by biopsy later, and the other 9 cases remained unknown within follow-up of over half a year. The difference was not significant in ADC values between primary and metastatic lesions (P>0.05). The sensitivity and specificity of whole body DWI for searching primary tumors was 95.8% and 90.0%, respectively. Conclusion Combined with conventional MR scanning, whole body DWI can help to search primary lesions of patients with metastases.

  18. Super-resolution imaging strategies for cell biologists using a spinning disk microscope.

    Science.gov (United States)

    Hosny, Neveen A; Song, Mingying; Connelly, John T; Ameer-Beg, Simon; Knight, Martin M; Wheeler, Ann P

    2013-01-01

    In this study we use a spinning disk confocal microscope (SD) to generate super-resolution images of multiple cellular features from any plane in the cell. We obtain super-resolution images by using stochastic intensity fluctuations of biological probes, combining Photoactivation Light-Microscopy (PALM)/Stochastic Optical Reconstruction Microscopy (STORM) methodologies. We compared different image analysis algorithms for processing super-resolution data to identify the most suitable for analysis of particular cell structures. SOFI was chosen for X and Y and was able to achieve a resolution of ca. 80 nm; however higher resolution was possible >30 nm, dependant on the super-resolution image analysis algorithm used. Our method uses low laser power and fluorescent probes which are available either commercially or through the scientific community, and therefore it is gentle enough for biological imaging. Through comparative studies with structured illumination microscopy (SIM) and widefield epifluorescence imaging we identified that our methodology was advantageous for imaging cellular structures which are not immediately at the cell-substrate interface, which include the nuclear architecture and mitochondria. We have shown that it was possible to obtain two coloured images, which highlights the potential this technique has for high-content screening, imaging of multiple epitopes and live cell imaging.

  19. Super-resolution imaging strategies for cell biologists using a spinning disk microscope.

    Directory of Open Access Journals (Sweden)

    Neveen A Hosny

    Full Text Available In this study we use a spinning disk confocal microscope (SD to generate super-resolution images of multiple cellular features from any plane in the cell. We obtain super-resolution images by using stochastic intensity fluctuations of biological probes, combining Photoactivation Light-Microscopy (PALM/Stochastic Optical Reconstruction Microscopy (STORM methodologies. We compared different image analysis algorithms for processing super-resolution data to identify the most suitable for analysis of particular cell structures. SOFI was chosen for X and Y and was able to achieve a resolution of ca. 80 nm; however higher resolution was possible >30 nm, dependant on the super-resolution image analysis algorithm used. Our method uses low laser power and fluorescent probes which are available either commercially or through the scientific community, and therefore it is gentle enough for biological imaging. Through comparative studies with structured illumination microscopy (SIM and widefield epifluorescence imaging we identified that our methodology was advantageous for imaging cellular structures which are not immediately at the cell-substrate interface, which include the nuclear architecture and mitochondria. We have shown that it was possible to obtain two coloured images, which highlights the potential this technique has for high-content screening, imaging of multiple epitopes and live cell imaging.

  20. Screening ToxCast™ Phase I Chemicals in a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) Assay

    Science.gov (United States)

    An Adherent Cell Differentiation and Cytotoxicity (ACDC) in vitro assay with mouse embryonic stem cells was used to screen the ToxCast Phase I chemical library for effects on cellular differentiation and cell number. The U.S. Environmental Protection Agency (EPA) established the ...

  1. Imaging in haematopoietic stem cell transplantation

    Energy Technology Data Exchange (ETDEWEB)

    Evans, A.; Steward, C.G.; Lyburn, I.D.; Grier, D.J

    2003-03-01

    Haematopoietic stem cell transplantation (SCT) is used to treat a wide range of malignant and non-malignant haematological conditions, solid malignancies, and metabolic and autoimmune diseases. Although imaging has a limited role before SCT, it is important after transplantation when it may support the clinical diagnosis of a variety of complications. It may also be used to monitor the effect of therapy and to detect recurrence of the underlying disease if the transplant is unsuccessful. We present a pictorial review of the imaging of patients who have undergone SCT, based upon 15 years experience in a large unit performing both adult and paediatric transplants.

  2. Fluorescence imaging technology (FI) for high-throughput screening of selenide-modified nano-TiO2 catalysts.

    Science.gov (United States)

    Wang, Liping; Lee, Jianchao; Zhang, Meijuan; Duan, Qiannan; Zhang, Jiarui; Qi, Hailang

    2016-02-18

    A high-throughput screening (HTS) method based on fluorescence imaging (FI) was implemented to evaluate the catalytic performance of selenide-modified nano-TiO2. Chemical ink-jet printing (IJP) technology was reformed to fabricate a catalyst library comprising 1405 (Ni(a)Cu(b)Cd(c)Ce(d)In(e)Y(f))Se(x)/TiO2 (M6Se/Ti) composite photocatalysts. Nineteen M6Se/Tis were screened out from the 1405 candidates efficiently.

  3. A small molecule screen for enhanced homing of systemically infused cells

    Science.gov (United States)

    Tong, Zhixiang; Perrault, Christelle; Benhamou, Brigitte; Zhang, Jidong; Stratton, Tara; Han, Edward; Safaee, Helia; Musabeyezu, Juliet; Yang, Zijiang; Multon, Marie-Christine; Rothblatt, Jonathan; Deleuze, Jean-Francois; Lin, Charles P.; Karp, Jeffrey M.

    2015-01-01

    SUMMARY Poor homing of systemically infused cells to disease sites may limit the success of exogenous cell-based therapy. In this study, we screened 9,000 signal transduction modulators to identify hits that increase mesenchymal stromal cell (MSC) surface expression of homing ligands that bind to ICAM-1, such as CD11a. Pretreatment of MSCs with Ro-31-8425, an identified hit from this screen, increased MSC firm adhesion to an ICAM-1-coated substrate in-vitro, and enabled targeted delivery of systemically administered MSCs to inflamed sites in-vivo in a CD11a (and other ICAM-1-binding domains)-dependent manner. This resulted in a heightened anti-inflammatory response. This represents a new strategy for engineering cell homing to enhance therapeutic efficacy and validates CD11a/ICAM-1 as potential targets. Altogether, this multi-step screening process may significantly improve clinical outcomes of cell-based therapies. PMID:25732817

  4. Identification of novel compounds inhibiting chikungunya virus-induced cell death by high throughput screening of a kinase inhibitor library.

    Directory of Open Access Journals (Sweden)

    Deu John M Cruz

    Full Text Available Chikungunya virus (CHIKV is a mosquito-borne arthrogenic alphavirus that causes acute febrile illness in humans accompanied by joint pains and in many cases, persistent arthralgia lasting weeks to years. The re-emergence of CHIKV has resulted in numerous outbreaks in the eastern hemisphere, and threatens to expand in the foreseeable future. Unfortunately, no effective treatment is currently available. The present study reports the use of resazurin in a cell-based high-throughput assay, and an image-based high-content assay to identify and characterize inhibitors of CHIKV-infection in vitro. CHIKV is a highly cytopathic virus that rapidly kills infected cells. Thus, cell viability of HuH-7 cells infected with CHIKV in the presence of compounds was determined by measuring metabolic reduction of resazurin to identify inhibitors of CHIKV-associated cell death. A kinase inhibitor library of 4,000 compounds was screened against CHIKV infection of HuH-7 cells using the resazurin reduction assay, and the cell toxicity was also measured in non-infected cells. Seventy-two compounds showing ≥50% inhibition property against CHIKV at 10 µM were selected as primary hits. Four compounds having a benzofuran core scaffold (CND0335, CND0364, CND0366 and CND0415, one pyrrolopyridine (CND0545 and one thiazol-carboxamide (CND3514 inhibited CHIKV-associated cell death in a dose-dependent manner, with EC50 values between 2.2 µM and 7.1 µM. Based on image analysis, these 6 hit compounds did not inhibit CHIKV replication in the host cell. However, CHIKV-infected cells manifested less prominent apoptotic blebs typical of CHIKV cytopathic effect compared with the control infection. Moreover, treatment with these compounds reduced viral titers in the medium of CHIKV-infected cells by up to 100-fold. In conclusion, this cell-based high-throughput screening assay using resazurin, combined with the image-based high content assay approach identified compounds against

  5. Molecular imaging of stem cell transplantation for neurodegenerative diseases.

    Science.gov (United States)

    Wang, Ping; Moore, Anna

    2012-01-01

    Cell replacement therapy with stem cells holds tremendous therapeutic potential for treating neurodegenerative diseases. Over the last decade, molecular imaging techniques have proven to be of great value in tracking transplanted cells and assessing the therapeutic efficacy. This current review summarizes the role and capabilities of different molecular imaging modalities including optical imaging, nuclear imaging and magnetic resonance imaging in the field of stem cell therapy for neurodegenerative disorders. We discuss current challenges and perspectives of these techniques and encompass updated information such as theranostic imaging and optogenetics in stem cell-based treatment of neurodegenerative diseases.

  6. A sandwiched microarray platform for benchtop cell-based high throughput screening

    Science.gov (United States)

    Wu, Jinhui; Wheeldon, Ian; Guo, Yuqi; Lu, Tingli; Du, Yanan; Wang, Ben; He, Jiankang; Hu, Yiqiao; Khademhosseini, Ali

    2010-01-01

    The emergence of combinatorial chemistries and the increased discovery of natural compounds have led to the production of expansive libraries of drug candidates and vast numbers of compounds with potentially interesting biological activities. Despite broad interest in high throughput screening (HTS) across varied fields of biological research, there has not been an increase in accessible HTS technologies. Here, we present a simple microarray sandwich system suitable for screening chemical libraries in cell-based assays at the benchtop. The microarray platform delivers chemical compounds to isolated cell cultures by ‘sandwiching’ chemical-laden arrayed posts with cell-seeded microwells. In this way, an array of sealed cell-based assays was generated without cross-contamination between neighboring assays. After chemical exposure, cell viability was analyzed by fluorescence detection of cell viability indicator assays on a per microwell basis in a standard microarray scanner. We demonstrate the efficacy of the system by generating four hits from toxicology screens towards MCF-7 human breast cancer cells. Three of the hits were identified in a combinatorial screen of a library of natural compounds in combination with verapamil, a P-glycoprotein inhibitor. A fourth hit, 9-methoxy-camptothecin, was identified by screening the natural compound library in the absence of verapamil. The method developed here miniaturizes existing HTS systems and enables the screening of a wide array of individual or combinatorial libraries in a reproducible and scalable manner. We anticipate broad application of such a system as it is amenable to combinatorial drug screening in a simple, robust and portable platform. PMID:20965560

  7. A Novel Multiparametric Drug-Scoring Method for High-Throughput Screening of 3D Multicellular Tumor Spheroids Using the Celigo Image Cytometer.

    Science.gov (United States)

    Cribbes, Scott; Kessel, Sarah; McMenemy, Scott; Qiu, Jean; Chan, Leo Li-Ying

    2017-01-01

    Three-dimensional (3D) tumor models have been increasingly used to investigate and characterize cancer drug compounds. The ability to perform high-throughput screening of 3D multicellular tumor spheroids (MCTS) can highly improve the efficiency and cost-effectiveness of discovering potential cancer drug candidates. Previously, the Celigo Image Cytometer has demonstrated a novel method for high-throughput screening of 3D multicellular tumor spheroids. In this work, we employed the Celigo Image Cytometer to examine the effects of 14 cancer drug compounds on 3D MCTS of the glioblastoma cell line U87MG in 384-well plates. Using parameters such as MCTS diameter and invasion area, growth and invasion were monitored for 9 and 3 d, respectively. Furthermore, fluorescent staining with calcein AM, propidium iodide, Hoechst 33342, and caspase 3/7 was performed at day 9 posttreatment to measure viability and apoptosis. Using the kinetic and endpoint data generated, we created a novel multiparametric drug-scoring system for 3D MCTS that can be used to identify and classify potential drug candidates earlier in the drug discovery process. Furthermore, the combination of quantitative and qualitative image data can be used to delineate differences between drugs that induce cytotoxic and cytostatic effects. The 3D MCTS-based multiparametric scoring method described here can provide an alternative screening method to better qualify tested drug compounds.

  8. Cell extraction combined with off-line HPLC for screening active compounds from Coptis chinensis.

    Science.gov (United States)

    Tang, Cheng; Wu, Xiao-Dan; Yu, Ya-Ming; Duan, Hongquan; Zhou, Jing; Xu, Liang

    2016-04-01

    Cell membrane chromatography is a useful tool for screening active compounds from natural products. As the reason of separation mechanism, traditional cell membrane chromatography could not be used for screening the active compounds absorbed through the cell membrane and influencing the cell signal transduction pathway. In this work, we establish a new method named cell extraction combined with off-line HPLC for screening the compounds penetrating the cell membrane. This is the first time 3 T3-L1 adipocyte culture has been combined with HPLC technology. Compared with other cell membrane chromatography methods, there is good resolution and no further analysis by other chromatographic steps is required. On co-incubating crude extracts of Coptis chinensis with cells and analyzing the compounds extracted by the cells, active compounds such as berberine were detected. Glucose consumption tests showed that berberine could increase glucose consumption by insulin-resistant 3 T3-L1 adipocytes. The levels of intracellular berberine correlated with its activity. The results indicate that the developed method could be an alternative method for screening active compounds from natural products.

  9. Fabrication of Cu{sub 2}ZnSnS{sub 4} screen printed layers for solar cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Zhihua; Wang, Yanyan; Xu, Dong; Zhang, Yafei [National Key laboratory of Micro/Nano Fabrication Technology, Key Laboratory for Thin film and Microfabrication of Ministry of Education, Research Institute of Micro/Nano Science and Technology, Shanghai Jiaotong University, Shanghai (China)

    2010-12-15

    Cu{sub 2}ZnSnS{sub 4} layers have been successfully fabricated on flexible polyimide substrates by screen printing. The microparticles of Cu{sub 2}ZnSnS{sub 4}, which is the main raw material for preparing screen printing paste, were prepared by wet ball milling and sintering methods. X-ray diffraction characterization results show the screen printed Cu{sub 2}ZnSnS{sub 4} layers were well indexed to single phase Cu{sub 2}ZnSnS{sub 4}. Scanning electronic microscopy images show the diameters and lengths of the as-prepared Cu{sub 2}ZnSnS{sub 4} microparticles are about 30-50 and 150-200 nm, respectively. The bandgap, sheet resistance, carrier concentration, and Hall mobility of the screen printed Cu{sub 2}ZnSnS{sub 4} layers were about 1.49 eV, 2.42 x 10{sup 3} {omega}, 3.81 x 10{sup 18} cm{sup -3}, and 12.61 cm{sup 2} V{sup -1} s{sup -s} at 25 C, respectively. To evaluate the photoresponse of the screen printed Cu{sub 2}ZnSnS{sub 4} layers, complete photovoltaic cells with the structure of polyimide/Mo/Cu{sub 2}ZnSnS{sub 4}/CdS/ZnO:Al/Al-grid were further fabricated. The short-circuit current density, open-circuit voltage, fill factor, and efficiency of a typical photovoltaic cell with an active area of 0.15 cm{sup 2} were 4.76 mA/cm{sup 2}, 386 mV, 0.27, and 0.49%, respectively. (author)

  10. High throughput screening to investigate the interaction of stem cells with their extracellular microenvironment

    OpenAIRE

    Ankam, Soneela; Teo, Benjamin KK; Kukumberg, Marek; Yim, Evelyn KF

    2013-01-01

    Stem cells in vivo are housed within a functional microenvironment termed the “stem cell niche.” As the niche components can modulate stem cell behaviors like proliferation, migration and differentiation, evaluating these components would be important to determine the most optimal platform for their maintenance or differentiation. In this review, we have discussed methods and technologies that have aided in the development of high throughput screening assays for stem cell research, including ...

  11. Engineering cell-compatible paper chips for cell culturing, drug screening, and mass spectrometric sensing.

    Science.gov (United States)

    Chen, Qiushui; He, Ziyi; Liu, Wu; Lin, Xuexia; Wu, Jing; Li, Haifang; Lin, Jin-Ming

    2015-10-28

    Paper-supported cell culture is an unprecedented development for advanced bioassays. This study reports a strategy for in vitro engineering of cell-compatible paper chips that allow for adherent cell culture, quantitative assessment of drug efficiency, and label-free sensing of intracellular molecules via paper spray mass spectrometry. The polycarbonate paper is employed as an excellent alternative bioscaffold for cell distribution, adhesion, and growth, as well as allowing for fluorescence imaging without light scattering. The cell-cultured paper chips are thus amenable to fabricate 3D tissue construction and cocultures by flexible deformation, stacks and assembly by layers of cells. As a result, the successful development of cell-compatible paper chips subsequently offers a uniquely flexible approach for in situ sensing of live cell components by paper spray mass spectrometry, allowing profiling the cellular lipids and quantitative measurement of drug metabolism with minimum sample pretreatment. Consequently, the developed paper chips for adherent cell culture are inexpensive for one-time use, compatible with high throughputs, and amenable to label-free and rapid analysis.

  12. Bioorthogonal probes for imaging sterols in cells.

    Science.gov (United States)

    Jao, Cindy Y; Nedelcu, Daniel; Lopez, Lyle V; Samarakoon, Thilani N; Welti, Ruth; Salic, Adrian

    2015-03-01

    Cholesterol is a fundamental lipid component of eukaryotic membranes and a precursor of potent signaling molecules, such as oxysterols and steroid hormones. Cholesterol and oxysterols are also essential for Hedgehog signaling, a pathway critical in embryogenesis and cancer. Despite their importance, the use of imaging sterols in cells is currently very limited. We introduce a robust and versatile method for sterol microscopy based on C19 alkyne cholesterol and oxysterol analogues. These sterol analogues are fully functional; they rescue growth of cholesterol auxotrophic cells and faithfully recapitulate the multiple roles that sterols play in Hedgehog signal transduction. Alkyne sterol analogues incorporate efficiently into cellular membranes and can be imaged with high resolution after copper(I)-catalyzed azide-alkyne cycloaddition reaction with fluorescent azides. We demonstrate the use of alkyne sterol probes for visualizing the subcellular distribution of cholesterol and for two-color imaging of sterols and choline phospholipids. Our imaging strategy should be broadly applicable to studying the role of sterols in normal physiology and disease.

  13. Combinatorial electrochemical cell array for high throughput screening of micro-fuel-cells and metal/air batteries

    Science.gov (United States)

    Jiang, Rongzhong

    2007-07-01

    An electrochemical cell array was designed that contains a common air electrode and 16 microanodes for high throughput screening of both fuel cells (based on polymer electrolyte membrane) and metal/air batteries (based on liquid electrolyte). Electrode materials can easily be coated on the anodes of the electrochemical cell array and screened by switching a graphite probe from one cell to the others. The electrochemical cell array was used to study direct methanol fuel cells (DMFCs), including high throughput screening of electrode catalysts and determination of optimum operating conditions. For screening of DMFCs, there is about 6% relative standard deviation (percentage of standard deviation versus mean value) for discharge current from 10to20mA/cm2. The electrochemical cell array was also used to study tin/air batteries. The effect of Cu content in the anode electrode on the discharge performance of the tin/air battery was investigated. The relative standard deviations for screening of metal/air battery (based on zinc/air) are 2.4%, 3.6%, and 5.1% for discharge current at 50, 100, and 150mA/cm2, respectively.

  14. Combinatorial electrochemical cell array for high throughput screening of micro-fuel-cells and metal/air batteries.

    Science.gov (United States)

    Jiang, Rongzhong

    2007-07-01

    An electrochemical cell array was designed that contains a common air electrode and 16 microanodes for high throughput screening of both fuel cells (based on polymer electrolyte membrane) and metal/air batteries (based on liquid electrolyte). Electrode materials can easily be coated on the anodes of the electrochemical cell array and screened by switching a graphite probe from one cell to the others. The electrochemical cell array was used to study direct methanol fuel cells (DMFCs), including high throughput screening of electrode catalysts and determination of optimum operating conditions. For screening of DMFCs, there is about 6% relative standard deviation (percentage of standard deviation versus mean value) for discharge current from 10 to 20 mAcm(2). The electrochemical cell array was also used to study tin/air batteries. The effect of Cu content in the anode electrode on the discharge performance of the tin/air battery was investigated. The relative standard deviations for screening of metal/air battery (based on zinc/air) are 2.4%, 3.6%, and 5.1% for discharge current at 50, 100, and 150 mAcm(2), respectively.

  15. A new level set model for cell image segmentation

    Science.gov (United States)

    Ma, Jing-Feng; Hou, Kai; Bao, Shang-Lian; Chen, Chun

    2011-02-01

    In this paper we first determine three phases of cell images: background, cytoplasm and nucleolus according to the general physical characteristics of cell images, and then develop a variational model, based on these characteristics, to segment nucleolus and cytoplasm from their relatively complicated backgrounds. In the meantime, the preprocessing obtained information of cell images using the OTSU algorithm is used to initialize the level set function in the model, which can speed up the segmentation and present satisfactory results in cell image processing.

  16. A new level set model for cell image segmentation

    Institute of Scientific and Technical Information of China (English)

    Ma Jing-Feng; Hou Kai; Bao Shang-Lian; Chen Chun

    2011-01-01

    In this paper we first determine three phases of cell images: background, cytoplasm and nucleolus according to the general physical characteristics of cell images, and then develop a variational model, based on these characteristics, to segment nucleolus and cytoplasm from their relatively complicated backgrounds. In the meantime, the preprocessing obtained information of cell images using the OTSU algorithm is used to initialize the level set function in the model, which can speed up the segmentation and present satisfactory results in cell image processing.

  17. Quantitative digital image analysis of chromogenic assays for high throughput screening of alpha-amylase mutant libraries.

    Science.gov (United States)

    Shankar, Manoharan; Priyadharshini, Ramachandran; Gunasekaran, Paramasamy

    2009-08-01

    An image analysis-based method for high throughput screening of an alpha-amylase mutant library using chromogenic assays was developed. Assays were performed in microplates and high resolution images of the assay plates were read using the Virtual Microplate Reader (VMR) script to quantify the concentration of the chromogen. This method is fast and sensitive in quantifying 0.025-0.3 mg starch/ml as well as 0.05-0.75 mg glucose/ml. It was also an effective screening method for improved alpha-amylase activity with a coefficient of variance of 18%.

  18. Li-Ion Cell Lot Testing and Flight Screening Results

    Science.gov (United States)

    2011-02-01

    than \\c7<. Results arc shown in Table 4. Table 4 Moli-Energ) Li-Ion Cell Weights Alter Vaeuun Leak Tesi Initial Final Final Weight Weight...All tested cells were within 95 *5! o\\ their initial capacity. Note: Cells C009 and C023 were misplaced following being weighed after the vac- uum

  19. Improvement of an X-ray imaging detector based on a scintillating guides screen

    CERN Document Server

    Badel, X; Linnros, J; Kleimann, P; Froejdh, C; Petersson, C S

    2002-01-01

    An X-ray imaging detector has been developed for dental applications. The principle of this detector is based on application of a silicon charge coupled device covered by a scintillating wave-guide screen. Previous studies of such a detector showed promising results concerning the spatial resolution but low performance in terms of signal to noise ratio (SNR) and sensitivity. Recent results confirm the wave-guiding properties of the matrix and show improvement of the detector in terms of response uniformity, sensitivity and SNR. The present study is focussed on the fabrication of the scintillating screen where the principal idea is to fill a matrix of Si pores with a CsI scintillator. The photoluminescence technique was used to prove the wave-guiding property of the matrix and to inspect the filling uniformity of the pores. The final detector was characterized by X-ray evaluation in terms of spatial resolution, light output and SNR. A sensor with a spatial resolution of 9 LP/mm and a SNR over 50 has been achie...

  20. Assessment of stereoscopic optic disc images using an autostereoscopic screen – experimental study

    Directory of Open Access Journals (Sweden)

    Vaideanu Daniella

    2008-07-01

    Full Text Available Abstract Background Stereoscopic assessment of the optic disc morphology is an important part of the care of patients with glaucoma. The aim of this study was to assess stereoviewing of stereoscopic optic disc images using an example of the new technology of autostereoscopic screens compared to the liquid shutter goggles. Methods Independent assessment of glaucomatous disc characteristics and measurement of optic disc and cup parameters whilst using either an autostereoscopic screen or liquid crystal shutter goggles synchronized with a view switching display. The main outcome measures were inter-modality agreements between the two used modalities as evaluated by the weighted kappa test and Bland Altman plots. Results Inter-modality agreement for measuring optic disc parameters was good [Average kappa coefficient for vertical Cup/Disc ratio was 0.78 (95% CI 0.62–0.91 and 0.81 (95% CI 0.6–0.92 for observer 1 and 2 respectively]. Agreement between modalities for assessing optic disc characteristics for glaucoma on a five-point scale was very good with a kappa value of 0.97. Conclusion This study compared two different methods of stereo viewing. The results of assessment of the different optic disc and cup parameters were comparable using an example of the newly developing autostereoscopic display technologies as compared to the shutter goggles system used. The Inter-modality agreement was high. This new technology carries potential clinical usability benefits in different areas of ophthalmic practice.

  1. Image-Based Single Cell Profiling: High-Throughput Processing of Mother Machine Experiments

    Science.gov (United States)

    Sachs, Christian Carsten; Grünberger, Alexander; Helfrich, Stefan; Probst, Christopher; Wiechert, Wolfgang; Kohlheyer, Dietrich; Nöh, Katharina

    2016-01-01

    Background Microfluidic lab-on-chip technology combined with live-cell imaging has enabled the observation of single cells in their spatio-temporal context. The mother machine (MM) cultivation system is particularly attractive for the long-term investigation of rod-shaped bacteria since it facilitates continuous cultivation and observation of individual cells over many generations in a highly parallelized manner. To date, the lack of fully automated image analysis software limits the practical applicability of the MM as a phenotypic screening tool. Results We present an image analysis pipeline for the automated processing of MM time lapse image stacks. The pipeline supports all analysis steps, i.e., image registration, orientation correction, channel/cell detection, cell tracking, and result visualization. Tailored algorithms account for the specialized MM layout to enable a robust automated analysis. Image data generated in a two-day growth study (≈ 90 GB) is analyzed in ≈ 30 min with negligible differences in growth rate between automated and manual evaluation quality. The proposed methods are implemented in the software molyso (MOther machine AnaLYsis SOftware) that provides a new profiling tool to analyze unbiasedly hitherto inaccessible large-scale MM image stacks. Conclusion Presented is the software molyso, a ready-to-use open source software (BSD-licensed) for the unsupervised analysis of MM time-lapse image stacks. molyso source code and user manual are available at https://github.com/modsim/molyso. PMID:27661996

  2. Current MR imaging of renal cell carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Oh, Sae Lin; Sung, Seuk Jae [Dept. of Radiology, Anam Hospital, Korea University College of Medicine, Seoul (Korea, Republic of)

    2016-08-15

    Renal cell carcinoma (RCC) consists of approximately 85-90% of renal masses, and its incidence is increasing due to widespread use of modern imaging modalities such as ultrasonography or computed tomography. Computed tomography has served an important role in the diagnosis and staging of RCC; however, recent advances in magnetic resonance imaging (MRI) techniques have considerably improved our ability to predict tumor biology beyond the morphologic assessment. Multiparametric MRI protocols include standard sequences tailored for the morphologic evaluation and acquisitions that provide information about the tumor microenvironment such as diffusion-weighted imaging and dynamic contrast-enhanced MRI. The role of multiparametric MRI in the evaluation of RCC now extends to preoperative characterization of RCC subtypes, histologic grade, and quantitative assessment of tumor response to targeted therapies in patients with metastatic disease. Herein, the clinical applications and recent advances in MRI applied to RCC are reviewed along with its merits and demerits. We aimed to review MRI techniques and image analysis that can improve the management of patients with RCC. Familiarity with the advanced MRI techniques and various imaging findings of RCC would also facilitate optimal clinical recommendations for patients.

  3. Cell Wall Biology: Perspectives from Cell Wall Imaging

    Institute of Scientific and Technical Information of China (English)

    Kieran J.D.Lee; Susan E.Marcus; J.Paul Knox

    2011-01-01

    Polysaccharide-rich plant cell walls are important biomaterials that underpin plant growth,are major repositories for photosynthetically accumulated carbon,and,in addition,impact greatly on the human use of plants. Land plant cell walls contain in the region of a dozen major polysaccharide structures that are mostly encompassed by cellulose,hemicelluloses,and pectic polysaccharides. During the evolution of land plants,polysaccharide diversification appears to have largely involved structural elaboration and diversification within these polysaccharide groups. Cell wall chemistry is well advanced and a current phase of cell wall science is aimed at placing the complex polysaccharide chemistry in cellular contexts and developing a detailed understanding of cell wall biology. Imaging cell wall glycomes is a challenging area but recent developments in the establishment of cell wall molecular probe panels and their use in high throughput procedures are leading to rapid advances in the molecular understanding of the spatial heterogeneity of individual cell walls and also cell wall differences at taxonomic levels. The challenge now is to integrate this knowledge of cell wall heterogeneity with an understanding of the molecular and physiological mechanisms that underpin cell wall properties and functions.

  4. Screen-film versus full-field digital mammography: Radiation dose and image quality in a large teaching hospital

    Directory of Open Access Journals (Sweden)

    Stantić Tomislav J.

    2013-01-01

    Full Text Available The objective of this paper is to measure the radiation dose and image quality in conventional screen-film mammography and full-field digital mammography in women referred to mammography examination. Participants underwent bilateral, two-view screen-film mammography or full-field digital mammography. The visibility of anatomical regions and overall clinical image quality was rated by experienced radiologists. Total of 387 women and 1548 mammograms were enrolled in the study. Image quality was assessed in terms of image quality score, whereas patient dose assessment was performed in terms of mean glandular dose. Average mean glandular dose for cranio-caudal projection was 1.5 mGy and 2.1 mGy in full-field digital mammography and screen-film mammography, respectively. For medio-lateral oblique projection, corresponding values were 2.3 and 2.1 mGy. Overall image quality criteria scoring was 0.82 and 0.99 for screen-film and digital systems, respectively. The scores were in the range from 0.11 to 1.0 for different anatomical structures. Overall, full-field digital mammography was superior both in terms of image quality and dose over the screen-film mammography. The results have indicated that phantom dose values can assist in setting the optimisation activities in mammography and for comparison between mammography units. To obtain accurate diagnostic information with an acceptable radiation dose to breast, it is necessary to periodically perform patient dose and image quality surveys in all mammography units.

  5. An industrial multi-crystalline EWT solar cell with screen printed metallization

    Energy Technology Data Exchange (ETDEWEB)

    Schoenecker, A.; De Moor, H.H.C.; Burgers, A.R.; Weeber, A.W.; Hoornstra, J.; Sinke, W.C. [ECN Renewable Energy, Solar and Wind, Petten (Netherlands); Michiels, P.P.; Steeman, R.A. [Shell Solar Energy, Helmond (Netherlands)

    1997-07-01

    The first results from the processing of an emitter wrap-through solar cell (EWT cell) based on 10x10 cm{sup 2} multicrystalline silicon solar cell technology with screen printing technique are shown. The processing bottlenecks such as laser cutting without material deterioration and the geometrical limitations due to screen printing techniques and emitter series resistance are addressed. The characterisation of the EWT solar cells clearly shows the expected higher photocurrent but fill factor and open circuit voltage need further improvements. Nevertheless it was possible to develop a stable processing scheme and cell design that allowed the processing of a number of EWT cells with reasonable efficiencies to make the first modules. Thus the advantages of a complete backside contacted module could be demonstrated by the first standard 36 cell EWT demonstration module. 8 figs., 6 refs.

  6. Adapting Cell-Based Assays to the High Throughput Screening Platform: Problems Encountered and Lessons Learned.

    Science.gov (United States)

    Maddox, Clinton B; Rasmussen, Lynn; White, E Lucile

    2008-06-01

    In recent years, cell-based phenotypic assays have emerged as an effective and robust addition to the array of assay technologies available for drug discovery in the high throughput screening arena. Previously, biochemical target-based assays have been the technology of choice. With the emergence of stem cells as a basis for a new screening technology, it is important to keep in mind the lessons that have been learned from the adaptation of existing stable cell lines onto the high throughput screening drug discovery platform, with special consideration being given to assay miniaturization, liquid handling complications and instrument-introduced artifacts. We present an overview of the problems encountered with the implementation of multiple cell-based assays at the High Throughput Screening Center at Southern Research Institute as well as empirically defined effective solutions to these problems. These include examples of artifacts induced by temperature differences throughout the screening campaign, cell plating conditions including the effect of room temperature incubation on assay consistency, DMSO carry-over, and incubator induced artifacts.

  7. A large scale screen for neural stem cell markers in Xenopus retina.

    Science.gov (United States)

    Parain, Karine; Mazurier, Nicolas; Bronchain, Odile; Borday, Caroline; Cabochette, Pauline; Chesneau, Albert; Colozza, Gabriele; El Yakoubi, Warif; Hamdache, Johanna; Locker, Morgane; Gilchrist, Michael J; Pollet, Nicolas; Perron, Muriel

    2012-04-01

    Neural stem cell research suffers from a lack of molecular markers to specifically assess stem or progenitor cell properties. The organization of the Xenopus ciliary marginal zone (CMZ) in the retina allows the spatial distinction of these two cell types: stem cells are confined to the most peripheral region, while progenitors are more central. Despite this clear advantage, very few genes specifically expressed in retinal stem cells have been discovered so far in this model. To gain insight into the molecular signature of these cells, we performed a large-scale expression screen in the Xenopus CMZ, establishing it as a model system for stem cell gene profiling. Eighteen genes expressed specifically in the CMZ stem cell compartment were retrieved and are discussed here. These encode various types of proteins, including factors associated with proliferation, mitotic spindle organization, DNA/RNA processing, and cell adhesion. In addition, the publication of this work in a special issue on Xenopus prompted us to give a more general illustration of the value of large-scale screens in this model species. Thus, beyond neural stem cell specific genes, we give a broader highlight of our screen outcome, describing in particular other retinal cell markers that we found. Finally, we present how these can all be easily retrieved through a novel module we developed in the web-based annotation tool XenMARK, and illustrate the potential of this powerful searchable database in the context of the retina.

  8. A High-Throughput Small Molecule Screen for C. elegans Linker Cell Death Inhibitors

    Science.gov (United States)

    Schwendeman, Andrew R.; Shaham, Shai

    2016-01-01

    Programmed cell death is a ubiquitous process in metazoan development. Apoptosis, one cell death form, has been studied extensively. However, mutations inactivating key mammalian apoptosis regulators do not block most developmental cell culling, suggesting that other cell death pathways are likely important. Recent work in the nematode Caenorhabditis elegans identified a non-apoptotic cell death form mediating the demise of the male-specific linker cell. This cell death process (LCD, linker cell-type death) is morphologically conserved, and its molecular effectors also mediate axon degeneration in mammals and Drosophila. To develop reagents to manipulate LCD, we established a simple high-throughput screening protocol for interrogating the effects of small molecules on C. elegans linker cell death in vivo. From 23,797 compounds assayed, 11 reproducibly block linker cell death onset. Of these, five induce animal lethality, and six promote a reversible developmental delay. These results provide proof-of principle validation of our screening protocol, demonstrate that developmental progression is required for linker cell death, and suggest that larger scale screens may identify LCD-specific small-molecule regulators that target the LCD execution machinery. PMID:27716809

  9. Human Skin-Derived Stem Cells as a Novel Cell Source for In Vitro Hepatotoxicity Screening of Pharmaceuticals

    OpenAIRE

    Rodrigues, Robim M.; De Kock, Joery; Branson, Steven; Vinken, Mathieu; Meganathan, Kesavan; Chaudhari, Umesh; Sachinidis, Agapios; Govaere, Olivier; Roskams, Tania; De Boe, Veerle; Vanhaecke, Tamara; Rogiers, Vera

    2014-01-01

    Human skin-derived precursors (hSKP) are postnatal stem cells with neural crest properties that reside in the dermis of human skin. These cells can be easily isolated from small (fore) skin segments and have the capacity to differentiate into multiple cell types. In this study, we show that upon exposure to hepatogenic growth factors and cytokines, hSKP acquire sufficient hepatic features that could make these cells suitable in vitro tools for hepatotoxicity screening of new chemical entities...

  10. Embryonic stem cell biology: insights from molecular imaging.

    Science.gov (United States)

    Sallam, Karim; Wu, Joseph C

    2010-01-01

    Embryonic stem (ES) cells have therapeutic potential in disorders of cellular loss such as myocardial infarction, type I diabetes and neurodegenerative disorders. ES cell biology in living subjects was largely poorly understood until incorporation of molecular imaging into the field. Reporter gene imaging works by integrating a reporter gene into ES cells and using a reporter probe to induce a signal detectable by normal imaging modalities. Reporter gene imaging allows for longitudinal tracking of ES cells within the same host for a prolonged period of time. This has advantages over postmortem immunohistochemistry and traditional imaging modalities. The advantages include expression of reporter gene is limited to viable cells, expression is conserved between generations of dividing cells, and expression can be linked to a specific population of cells. These advantages were especially useful in studying a dynamic cell population such as ES cells and proved useful in elucidating the biology of ES cells. Reporter gene imaging identified poor integration of differentiated ES cells transplanted into host tissue as well as delayed donor cell death as reasons for poor long-term survival in vivo. This imaging technology also confirmed that ES cells indeed have immunogenic properties that factor into cell survival and differentiation. Finally, reporter gene imaging improved our understanding of the neoplastic risk of undifferentiated ES cells in forming teratomas. Despite such advances, much remains to be understood about ES cell biology to translate this technology to the bedside, and reporter gene imaging will certainly play a key role in formulating this understanding.

  11. Microfluidic Cell Volume Biosensor for High Throughput Drug Screening

    Science.gov (United States)

    2005-01-01

    method is non- invasive, and it provides real-time measurement of changes in cell volume for both adherent and suspended cells. The microfluidic nature of...channels as well as the release of organic osmolytes, such as taurine [11]. Upon returning to isotonic solution at the end of RVD we observed cell...Haussinger, Biochem. J, 313,697 (1996). [2] Sachs, F. Nature Structural Biology 9, 636-637 (2002). [3] F. Schliess, and D. Haussinger, Bio. Chem. 383

  12. Coded illumination for motion-blur free imaging of cells on cell-phone based imaging flow cytometer

    Science.gov (United States)

    Saxena, Manish; Gorthi, Sai Siva

    2014-10-01

    Cell-phone based imaging flow cytometry can be realized by flowing cells through the microfluidic devices, and capturing their images with an optically enhanced camera of the cell-phone. Throughput in flow cytometers is usually enhanced by increasing the flow rate of cells. However, maximum frame rate of camera system limits the achievable flow rate. Beyond this, the images become highly blurred due to motion-smear. We propose to address this issue with coded illumination, which enables recovery of high-fidelity images of cells far beyond their motion-blur limit. This paper presents simulation results of deblurring the synthetically generated cell/bead images under such coded illumination.

  13. An open source based high content screening method for cell biology laboratories investigating cell spreading and adhesion.

    Directory of Open Access Journals (Sweden)

    Andre Schmandke

    Full Text Available BACKGROUND: Adhesion dependent mechanisms are increasingly recognized to be important for a wide range of biological processes, diseases and therapeutics. This has led to a rising demand of pharmaceutical modulators. However, most currently available adhesion assays are time consuming and/or lack sensitivity and reproducibility or depend on specialized and expensive equipment often only available at screening facilities. Thus, rapid and economical high-content screening approaches are urgently needed. RESULTS: We established a fully open source high-content screening method for identifying modulators of adhesion. We successfully used this method to detect small molecules that are able to influence cell adhesion and cell spreading of Swiss-3T3 fibroblasts in general and/or specifically counteract Nogo-A-Δ20-induced inhibition of adhesion and cell spreading. The tricyclic anti-depressant clomipramine hydrochloride was shown to not only inhibit Nogo-A-Δ20-induced cell spreading inhibition in 3T3 fibroblasts but also to promote growth and counteract neurite outgrowth inhibition in highly purified primary neurons isolated from rat cerebellum. CONCLUSIONS: We have developed and validated a high content screening approach that can be used in any ordinarily equipped cell biology laboratory employing exclusively freely available open-source software in order to find novel modulators of adhesion and cell spreading. The versatility and adjustability of the whole screening method will enable not only centers specialized in high-throughput screens but most importantly also labs not routinely employing screens in their daily work routine to investigate the effects of a wide range of different compounds or siRNAs on adhesion and adhesion-modulating molecules.

  14. Utility of Early Screening Magnetic Resonance Imaging for Extensive Hip Osteonecrosis in Pediatric Patients Treated With Glucocorticoids

    Science.gov (United States)

    Kaste, Sue C.; Pei, Deqing; Cheng, Cheng; Neel, Michael D.; Bowman, W. Paul; Ribeiro, Raul C.; Metzger, Monika L.; Bhojwani, Deepa; Inaba, Hiroto; Campbell, Patrick; Rubnitz, Jeffrey E.; Jeha, Sima; Sandlund, John T.; Downing, James R.; Relling, Mary V.; Pui, Ching-Hon; Howard, Scott C.

    2015-01-01

    Purpose Hip osteonecrosis frequently complicates treatment with glucocorticoids. When extensive (affecting ≥ 30% of the epiphyseal surface), 80% of joints collapse within 2 years, so interventions are needed to prevent this outcome. Patients and Methods This prospective cohort magnetic resonance imaging (MRI) screening study included all consecutive children treated for acute lymphoblastic leukemia on a single protocol. Hip MRI was performed at 6.5 and 9 months from diagnosis (early screening) and at completion of chemotherapy (final evaluation) to determine whether screening could identify extensive hip osteonecrosis before symptom development. Results Of 498 patients, 462 underwent screening MRI. Extensive asymptomatic osteonecrosis was identified by early screening in 26 patients (41 hips); another four patients (seven hips) were detected after the screening period, such that screening sensitivity was 84.1% and specificity was 99.4%. The number of joints screened to detect one lesion was 20.1 joints for all patients, 4.4 joints for patients older than 10 years, and 198 joints for patients ≤ 10 years old (P < .001). Of the 40 extensive lesions in patients older than 10 years, 19 required total hip arthroplasty and none improved. Of eight extensive lesions in younger patients, none required arthroplasty and four improved. Conclusion In patients age 10 years old or younger who require prolonged glucocorticoid therapy, screening for extensive hip osteonecrosis is unnecessary because their risk is low and lesions tend to heal. In children older than 10 years, early screening successfully identifies extensive asymptomatic lesions in patients who would be eligible for studies of interventions to prevent or delay joint collapse. PMID:25605853

  15. Systems analysis of quantitative shRNA-library screens identifies regulators of cell adhesion

    Directory of Open Access Journals (Sweden)

    Huang XiaoDong

    2008-06-01

    Full Text Available Abstract Background High throughput screens with RNA interference technology enable loss-of-function analyses of gene activities in mammalian cells. While the construction of genome-scale shRNA libraries has been successful, results of large-scale screening of those libraries can be difficult to analyze because of the relatively high noise levels and the fact that not all shRNAs in a library are equally effective in silencing gene expression. Results We have screened a library consisting of 43,828 shRNAs directed against 8,500 human genes for functions that are necessary in cell detachment induced by a constitutively activated c-Abl tyrosine kinase. To deal with the issues of noise and uncertainty of knockdown efficiencies, we employed an analytical strategy that combines quantitative data analysis with biological knowledge, i.e. Gene Ontology and pathway information, to increase the power of the RNAi screening technique. Using this strategy we found 16 candidate genes to be involved in Abl-induced disruption of cell adhesion, and verified that the knockdown of IL6ST is associated with enhanced cell attachment. Conclusion Our results suggest that the power of genome-wide quantitative shRNA screens can be significantly increased when analyzed using a systems biology-based approach to identify functional gene networks.

  16. A Seoul-Fluor-based bioprobe for lipid droplets and its application in image-based high throughput screening.

    Science.gov (United States)

    Kim, Eunha; Lee, Sanghee; Park, Seung Bum

    2012-02-25

    We developed a novel fluorescent bioprobe (SF44) that can specifically visualize the cellular lipid droplets in in vitro and in vivo systems and illustrated the mechanistic rationale of its fluorogenic property. Its application to image-based high throughput screening led us to the identification of a new small-molecule modulator of lipid droplet formation.

  17. Normalizing for individual cell population context in the analysis of high-content cellular screens

    Directory of Open Access Journals (Sweden)

    Knapp Bettina

    2011-12-01

    Full Text Available Abstract Background High-content, high-throughput RNA interference (RNAi offers unprecedented possibilities to elucidate gene function and involvement in biological processes. Microscopy based screening allows phenotypic observations at the level of individual cells. It was recently shown that a cell's population context significantly influences results. However, standard analysis methods for cellular screens do not currently take individual cell data into account unless this is important for the phenotype of interest, i.e. when studying cell morphology. Results We present a method that normalizes and statistically scores microscopy based RNAi screens, exploiting individual cell information of hundreds of cells per knockdown. Each cell's individual population context is employed in normalization. We present results on two infection screens for hepatitis C and dengue virus, both showing considerable effects on observed phenotypes due to population context. In addition, we show on a non-virus screen that these effects can be found also in RNAi data in the absence of any virus. Using our approach to normalize against these effects we achieve improved performance in comparison to an analysis without this normalization and hit scoring strategy. Furthermore, our approach results in the identification of considerably more significantly enriched pathways in hepatitis C virus replication than using a standard analysis approach. Conclusions Using a cell-based analysis and normalization for population context, we achieve improved sensitivity and specificity not only on a individual protein level, but especially also on a pathway level. This leads to the identification of new host dependency factors of the hepatitis C and dengue viruses and higher reproducibility of results.

  18. First siRNA library screening in hard-to-transfect HUVEC cells.

    Science.gov (United States)

    Zumbansen, Markus; Altrogge, Ludger M; Spottke, Nicole Ue; Spicker, Sonja; Offizier, Sheila M; Domzalski, Sandra Bs; St Amand, Allison L; Toell, Andrea; Leake, Devin; Mueller-Hartmann, Herbert A

    2009-10-29

    Meaningful RNAi-based data for target gene identification are strongly dependent on the use of a biologically relevant cell type and efficient delivery of highly functional siRNA reagents into the selected cell type. Here we report the use of the Amaxa(R) Nucleofector(R) 96-well Shuttle(R) System for siRNA screening in primary cells. Lonza's Clonetics(R) HUVEC-Human Umbilical Vein Endothelial Cells were transfected with Thermo Scientific Dharmacon siGENOME(R) siRNA Libraries targeting protein kinases and cell cycle related genes and screened for genes important for cell viability. Of the 37 primary hits, down-regulation of 33 led to reduced proliferation or increased cell death, while down-regulation of two allowed for better cell viability. The validated four genes out of the 16 strongest primary hits (COPB2, PYCS, CDK4 and MYC) influenced cell proliferation to varying degrees, reflecting differing importance for survival of HUVEC cells. Our results demonstrate that the Nucleofector(R) 96-well Shuttle(R) System allows the delivery of siRNA libraries in cell types previously considered to be difficult to transfect. Thus, identification and validation of gene targets can now be conducted in primary cells, as the selection of cell types is not limited to those accessible by lipid-mediated transfection.

  19. Convenient cell fusion assay for rapid screening for HIV entry inhibitors

    Science.gov (United States)

    Jiang, Shibo; Radigan, Lin; Zhang, Li

    2000-03-01

    Human immunodeficiency viruses (HIV)-induced cell fusion is a critical pathway of HIV spread from infected cells to uninfected cells. A rapid and simple assay was established to measure HIV-induce cell fusion. This study is particularly useful to rapid screen for HIV inhibitors that block HIV cell-to-cell transmission. Present study demonstrated that coculture of HIV-infected cells with uninfected cells at 37 degree(s)C for 2 hours resulted in the highest cell fusion rate. Using this cell fusion assay, we have identified several potent HIV inhibitors targeted to the HIV gp41 core. These antiviral agents can be potentially developed as antiviral drugs for chemotherapy and prophylaxis of HIV infection and AIDS.

  20. A novel multiplex cell viability assay for high-throughput RNAi screening.

    Directory of Open Access Journals (Sweden)

    Daniel F Gilbert

    Full Text Available Cell-based high-throughput RNAi screening has become a powerful research tool in addressing a variety of biological questions. In RNAi screening, one of the most commonly applied assay system is measuring the fitness of cells that is usually quantified using fluorescence, luminescence and absorption-based readouts. These methods, typically implemented and scaled to large-scale screening format, however often only yield limited information on the cell fitness phenotype due to evaluation of a single and indirect physiological indicator. To address this problem, we have established a cell fitness multiplexing assay which combines a biochemical approach and two fluorescence-based assaying methods. We applied this assay in a large-scale RNAi screening experiment with siRNA pools targeting the human kinome in different modified HEK293 cell lines. Subsequent analysis of ranked fitness phenotypes assessed by the different assaying methods revealed average phenotype intersections of 50.7±2.3%-58.7±14.4% when two indicators were combined and 40-48% when a third indicator was taken into account. From these observations we conclude that combination of multiple fitness measures may decrease false-positive rates and increases confidence for hit selection. Our robust experimental and analytical method improves the classical approach in terms of time, data comprehensiveness and cost.

  1. A novel multiplex cell viability assay for high-throughput RNAi screening.

    Science.gov (United States)

    Gilbert, Daniel F; Erdmann, Gerrit; Zhang, Xian; Fritzsche, Anja; Demir, Kubilay; Jaedicke, Andreas; Muehlenberg, Katja; Wanker, Erich E; Boutros, Michael

    2011-01-01

    Cell-based high-throughput RNAi screening has become a powerful research tool in addressing a variety of biological questions. In RNAi screening, one of the most commonly applied assay system is measuring the fitness of cells that is usually quantified using fluorescence, luminescence and absorption-based readouts. These methods, typically implemented and scaled to large-scale screening format, however often only yield limited information on the cell fitness phenotype due to evaluation of a single and indirect physiological indicator. To address this problem, we have established a cell fitness multiplexing assay which combines a biochemical approach and two fluorescence-based assaying methods. We applied this assay in a large-scale RNAi screening experiment with siRNA pools targeting the human kinome in different modified HEK293 cell lines. Subsequent analysis of ranked fitness phenotypes assessed by the different assaying methods revealed average phenotype intersections of 50.7±2.3%-58.7±14.4% when two indicators were combined and 40-48% when a third indicator was taken into account. From these observations we conclude that combination of multiple fitness measures may decrease false-positive rates and increases confidence for hit selection. Our robust experimental and analytical method improves the classical approach in terms of time, data comprehensiveness and cost.

  2. Transcranial Doppler screening in sickle cell disease: The implications of using peak systolic criteria

    Institute of Scientific and Technical Information of China (English)

    Lena; N; Naffaa; Yasmeen; K; Tandon; Neville; Irani

    2015-01-01

    AIM: To compare time average maximum mean velocity(TAMV) and peak systolic velocity(PSV) criteria of Trans Cranial Doppler(TCD) in their ability to predict abnormalities on magnetic resonance imaging(MRI)/magnetic resonance angiogram(MRA) in patients with sickle cell disease. METHODS: A retrospective evaluation was performed of the outcomes in all patients with a Transcranial Doppler examination at our institution since the implementation of the hospital picture archiving and communication system(PACS) system in January 2003 through December 2012. All ultrasound imaging exams were performed by the same technologist with a 3 MHz transducer. Inclusion criteria was based upon the Transcranial Doppler procedure code in our PACS which had an indication of sickle cell disease in the history. The patient’s age and gender along with the vessel with the highest time averaged mean velocity as well as the highest peak systolic velocity was recorded for analysis. A subset of the study cohort also had subsequent MR imaging and Angiograms performed within 6 mo of the TCD examination. MRI results were categorized as having a disease related abnormality(vessel narrowing, collateral formation/moya-moya, or abnormal fluid attenuation inversion recovery signal in parenchyma indicative of prior stroke) or normal. The MRI results formed the comparison standards for TCD exams in evaluating intracranial injury. Sensitivity and specificity for the two TCD criteria(TAMV and PSV) were calculated to determine which could be a better predictor for intracranial vasculopathy /clinically occult strokes.RESULTS: The study cohort for our institution was 110patients with a total of 291 TCD examinations. These patients had a mean age of 7.6 years with a range from 2-18 years of age. Sixty-two of the 110 patients(56%) had two or more TCD exams. Thirty-seven patients(34%) had at least one MRI following a TCD examination. Of the 291 TCD examinations, 46(16%) were conditional or abnormal by TAMV

  3. High content screening for G protein-coupled receptors using cell-based protein translocation assays

    DEFF Research Database (Denmark)

    Grånäs, Charlotta; Lundholt, Betina Kerstin; Heydorn, Arne

    2005-01-01

    G protein-coupled receptors (GPCRs) have been one of the most productive classes of drug targets for several decades, and new technologies for GPCR-based discovery promise to keep this field active for years to come. While molecular screens for GPCR receptor agonist- and antagonist-based drugs...... as valuable discovery tools for several years. The application of high content cell-based screening to GPCR discovery has opened up additional possibilities, such as direct tracking of GPCRs, G proteins and other signaling pathway components using intracellular translocation assays. These assays provide...... the capability to probe GPCR function at the cellular level with better resolution than has previously been possible, and offer practical strategies for more definitive selectivity evaluation and counter-screening in the early stages of drug discovery. The potential of cell-based translocation assays for GPCR...

  4. Screening and Establishment of Human Lung Cancer Cell Lines 
with Organ-specific Metastasis Potential

    Directory of Open Access Journals (Sweden)

    Qinghua ZHOU

    2014-03-01

    Full Text Available Background and objective Cancer metastasis is not only the malignant marker and characteristics, but also the main cause of failure to cure and lose their life in the patients with lung cancer. Lung cancer metastasis has organ-specific characteristics. The most common sites of lung cancer metastasis are mediastinal lymph node, brain, bone, liver and adrenal gland. The aim of this study is to screen and establish lung cancer cell model with organ-specific metastasis potential with human high-metastatic large cell lung cancer cell line L9981 established by our laboratory previously, and to provide cell models for studying the mechanisms and signal regulation of organ-specific metastasis of lung cancer. Materials and methods The parent lung cancer cell line, L9981-Luc, was inoculated in the armpit of nude mice. The live animal imaging system, IVIS-200, was used to detect the lung cancer organ-specific metastasis every week. When the organ-specific metastasis were established, the nude mices bearing the lung cancer were sacrificed when they became moribund. Under sterile conditions, the organs (mediastinal lymph nodes, lung, spinal column and brain with lung cancer organ-specific metastasis were removed and the metastasized nodules were dissected free of connective tissue and blood clots, and rinsed twice with medium. The metastasized nodules were finely minced using sterile scalpel blades in medium, and the cells were seeded in tissue culture dishes. Then, the cells with organ-specific metastasis potential were reinoculated into the armpit of nude mice, respectively. This processes were repeated to establish the organ-specific metastatic sublines of L9981-Luc cell line more than 10 times. Finally, the organ-specific metastasis sublines of L9981-Luc were screened and established, which the four cell lines have the characteristics only metastasized to brian, lung, bone and mediastinal lymph node. Results A group of organ-specific metastasis cell

  5. Adaptation of a Cell-Based High Content Screening System for the In-Depth Analysis of Celiac Biopsy Tissue.

    Science.gov (United States)

    Cooper, Sarah E J; Mohamed, Bashir M; Elliott, Louise; Davies, Anthony Mitchell; Feighery, Conleth F; Kelly, Jacinta; Dunne, Jean

    2015-01-01

    The IN Cell Analyzer 1000 possesses several distinguishing features that make it a valuable tool in research today. This fully automated high content screening (HCS) system introduced quantitative fluorescent microscopy with computerized image analysis for use in cell-based analysis. Previous studies have focused on live cell assays, where it has proven to be a powerful and robust method capable of providing reproducible, quantitative data. Using HCS as a tool to investigate antigen expression in duodenal biopsies, we developed a novel approach to tissue positioning and mapping. We adapted IN Cell Analyzer 1000's image acquisition and analysis software for the investigation of tissue transglutaminase (tTG) and smooth muscle alpha-actin (SM α-actin) staining in paraffin-embedded duodenal tissue sections from celiac patients and healthy controls. These innovations allowed a quantitative analysis of cellular structure and protein expression. The results from routine biopsy material indicated the intensity of protein expression was altered in celiac disease compared to normal biopsy material.

  6. Potential of near-infrared hyperspectral reflectance imaging for screening of farm feed contamination

    Science.gov (United States)

    Wang, Wenbo; Paliwal, Jitendra

    2005-09-01

    With the outbreak of Bovine Spongiform Encephalopathy (BSE) (commonly known as mad cow disease) in 1987 in the United Kingdom and a recent case discovered in Alberta, more and more emphasis is placed on food and farm feed quality and safety issues internationally. The disease is believed to be spread through farm feed contamination by animal byproducts in the form of meat-and-bone-meal (MBM). The paper reviewed the available techniques necessary to the enforcement of legislation concerning the feed safety issues. The standard microscopy method, although highly sensitive, is laborious and costly. A method to routinely screen farm feed contamination certainly helps to reduce the complexity of safety inspection. A hyperspectral imaging system working in the near-infrared wavelength region of 1100-1600 nm was used to study the possibility of detection of ground broiler feed contamination by ground pork. Hyperspectral images of raw broiler feed, ground broiler feed, ground pork, and contaminated feed samples were acquired. Raw broiler feed samples were found to possess comparatively large spectral variations due to light scattering effect. Ground feed adulterated with 1%, 3%, 5%, and 10% of ground pork was tested to identify feed contamination. Discriminant analysis using Mahalanobis distance showed that the model trained using pure ground feed samples and pure ground pork samples resulted in 100% false negative errors for all test replicates of contaminated samples. A discriminant model trained with pure ground feed samples and 10% contamination level samples resulted in 12.5% false positive error and 0% false negative error.

  7. High-Throughput Method for Automated Colony and Cell Counting by Digital Image Analysis Based on Edge Detection.

    Directory of Open Access Journals (Sweden)

    Priya Choudhry

    Full Text Available Counting cells and colonies is an integral part of high-throughput screens and quantitative cellular assays. Due to its subjective and time-intensive nature, manual counting has hindered the adoption of cellular assays such as tumor spheroid formation in high-throughput screens. The objective of this study was to develop an automated method for quick and reliable counting of cells and colonies from digital images. For this purpose, I developed an ImageJ macro Cell Colony Edge and a CellProfiler Pipeline Cell Colony Counting, and compared them to other open-source digital methods and manual counts. The ImageJ macro Cell Colony Edge is valuable in counting cells and colonies, and measuring their area, volume, morphology, and intensity. In this study, I demonstrate that Cell Colony Edge is superior to other open-source methods, in speed, accuracy and applicability to diverse cellular assays. It can fulfill the need to automate colony/cell counting in high-throughput screens, colony forming assays, and cellular assays.

  8. Beyond White Blood Cell Monitoring : Screening in the Initial Phase of Clozapine Therapy

    NARCIS (Netherlands)

    Cohen, Dan; Bogers, Jan P. A. M.; van Dijk, Daniel; Bakker, Bert; Schulte, Peter F. J.

    2012-01-01

    Objective: Clozapine is the preferred option for treatment-resistant schizophrenia. However, since 1975, clozapine has been known to cause agranulocytosis. In the clozapine screening guidelines, white blood cell count is mandatory. In the past 20 years, after its reintroduction, 3 other serious side

  9. Screening and analysis of differentially expressed genes of dermal papillae cells with aggregative behavior

    Institute of Scientific and Technical Information of China (English)

    宋志强; 郝飞; 杨卫兵; 王继文; 邹锋

    2004-01-01

    Objective: To screen and clone differentially expressed genes of dermal papillae cells (DPC) with aggregative behavior, and to explore the molecular mechanism of their aggregation. Methods: Total RNAs were extracted from DPC with and without aggregative behavior and double strand cDNAs were synthesized by using SMART cDNA synthesis, respectively. The cDNA fragments of differentially expressed genes in DPCs with aggregative behavior were isolated by suppression subtractive hybridization. Positive clones were screened by PCR method and verified by cDNA dot blot, Northern blot and then analyzed through homologous retrieving. Results: A subtractive cDNA library of DPC with aggregative behavior has been successfully constructed. The result of screening and cloning of the library showed that, DPC with aggregative behavior could expresse genes related to homologous aggregation, proliferation and cycle control, including known genes (capping protein, paladin, vascular endothelial growth factor), hematopoietic stem/progenitor cells (HSPC) related clone (HSPC011 and HSPC016) and a new gene. Conclusion: The construction of subtracted library of DPC lays solid foundation for screening and cloning new and specific genes related to aggregative behavior of DPC. Several genes might be cooperatively involved in the homologous aggregation, proliferation and cycle control of DPC. Among these genes, capping protein and palladin might be closely related to the aggregative behavior of dermal papilla cells, and VEGF and HSPC related clone would be responsible for the status of higher proliferation of dermal papilla cells.

  10. Bilateral Image Subtraction and Multivariate Models for the Automated Triaging of Screening Mammograms

    Directory of Open Access Journals (Sweden)

    José Celaya-Padilla

    2015-01-01

    Full Text Available Mammography is the most common and effective breast cancer screening test. However, the rate of positive findings is very low, making the radiologic interpretation monotonous and biased toward errors. This work presents a computer-aided diagnosis (CADx method aimed to automatically triage mammogram sets. The method coregisters the left and right mammograms, extracts image features, and classifies the subjects into risk of having malignant calcifications (CS, malignant masses (MS, and healthy subject (HS. In this study, 449 subjects (197 CS, 207 MS, and 45 HS from a public database were used to train and evaluate the CADx. Percentile-rank (p-rank and z-normalizations were used. For the p-rank, the CS versus HS model achieved a cross-validation accuracy of 0.797 with an area under the receiver operating characteristic curve (AUC of 0.882; the MS versus HS model obtained an accuracy of 0.772 and an AUC of 0.842. For the z-normalization, the CS versus HS model achieved an accuracy of 0.825 with an AUC of 0.882 and the MS versus HS model obtained an accuracy of 0.698 and an AUC of 0.807. The proposed method has the potential to rank cases with high probability of malignant findings aiding in the prioritization of radiologists work list.

  11. Early developability screen of therapeutic antibody candidates using Taylor dispersion analysis and UV area imaging detection.

    Science.gov (United States)

    Lavoisier, Alexandra; Schlaeppi, Jean-Marc

    2015-01-01

    Therapeutic antibodies represent one of the fastest growing segments in the pharmaceutical market. They are used in a broad range of disease fields, such as autoimmune diseases, cancer, inflammation and infectious diseases. The growth of the segment has necessitated development of new analytical platforms for faster and better antibody selection and characterization. Early quality control and risk assessment of biophysical parameters help prevent failure in later stages of antibody development, and thus can reduce costs and save time. Critical parameters such as aggregation, conformational stability, colloidal stability and hydrophilicity, are measured during the early phase of antibody generation and guide the selection process of the best lead candidates in terms of technical developability. We report on the use of a novel instrument (ActiPix/Viscosizer) for measuring both the hydrodynamic radius and the absolute viscosity of antibodies based on Taylor dispersion analysis and UV area imaging. The looped microcapillary-based method combines low sample consumption, fast throughput and high precision compared to other conventional methods. From a random panel of 130 antibodies in the early selection process, we identified some with large hydrodynamic radius outside the normal distribution and others with non-Gaussian Taylor dispersion profiles. The antibodies with such abnormal properties were confirmed later in the selection process to show poor developability profiles. Moreover, combining these results with those of the viscosity measurements at high antibody concentrations allows screening, with limited amounts of materials, candidates with potential issues in pre-formulation development.

  12. Tetrandrine identified in a small molecule screen to activate mesenchymal stem cells for enhanced immunomodulation.

    Science.gov (United States)

    Yang, Zijiang; Concannon, John; Ng, Kelvin S; Seyb, Kathleen; Mortensen, Luke J; Ranganath, Sudhir; Gu, Fangqi; Levy, Oren; Tong, Zhixiang; Martyn, Keir; Zhao, Weian; Lin, Charles P; Glicksman, Marcie A; Karp, Jeffrey M

    2016-07-26

    Pre-treatment or priming of mesenchymal stem cells (MSC) prior to transplantation can significantly augment the immunosuppressive effect of MSC-based therapies. In this study, we screened a library of 1402 FDA-approved bioactive compounds to prime MSC. We identified tetrandrine as a potential hit that activates the secretion of prostaglandin E2 (PGE2), a potent immunosuppressive agent, by MSC. Tetrandrine increased MSC PGE2 secretion through the NF-κB/COX-2 signaling pathway. When co-cultured with mouse macrophages (RAW264.7), tetrandrine-primed MSC attenuated the level of TNF-α secreted by RAW264.7. Furthermore, systemic transplantation of primed MSC into a mouse ear skin inflammation model significantly reduced the level of TNF-α in the inflamed ear, compared to unprimed cells. Screening of small molecules to pre-condition cells prior to transplantation represents a promising strategy to boost the therapeutic potential of cell therapy.

  13. The gender of cell lines matters when screening for novel anti-cancer drugs.

    Science.gov (United States)

    Nunes, Larissa M; Robles-Escajeda, Elisa; Santiago-Vazquez, Yahaira; Ortega, Nora M; Lema, Carolina; Muro, Almendra; Almodovar, Gladys; Das, Umashankar; Das, Swagatika; Dimmock, Johnatan R; Aguilera, Renato J; Varela-Ramirez, Armando

    2014-07-01

    Current reports indicated that the gender origin of cells is important in all facets of experimental biology. To explore this matter using an anticancer high throughput screening platform, seven male- and seven female-derived human cell lines, six from cancer patients in each group, were exposed to 81 novel cytotoxins. In this screen, the findings revealed that 79 out of 81 of the compounds consistently inflicted higher levels of toxicity towards male derived cells, emphasizing that there is indeed a gender-related difference in cell sensitivity to these anti-neoplastic agents. This gender-related drug sensitivity and toxicity explored at the molecular and cellular level emerged from a drug discovery enterprise.

  14. A genetic screen for components of the mammalian RNA interference pathway in Bloom-deficient mouse embryonic stem cells

    OpenAIRE

    2009-01-01

    Genetic screens performed in model organisms have helped identify key components of the RNA interference (RNAi) pathway. Recessive genetic screens have recently become feasible through the use of mouse embryonic stem (ES) cells that are Bloom's syndrome protein (Blm) deficient. Here, we developed and performed a recessive genetic screen to identify components of the mammalian RNAi pathway in Blm-deficient ES cells. Genome-wide mutagenesis using a retroviral gene trap strategy resulted in the ...

  15. Multiplex shRNA Screening of Germ Cell Development by in Vivo Transfection of Mouse Testis

    Directory of Open Access Journals (Sweden)

    Nicholas R. Y. Ho

    2017-01-01

    Full Text Available Spermatozoa are one of the few mammalian cell types that cannot be fully derived in vitro, severely limiting the application of modern genomic techniques to study germ cell biology. The current gold standard approach of characterizing single-gene knockout mice is slow as generation of each mutant line can take 6–9 months. Here, we describe an in vivo approach to rapid functional screening of germline genes based on a new nonsurgical, nonviral in vivo transfection method to deliver nucleic acids into testicular germ cells. By coupling multiplex transfection of short hairpin RNA (shRNA constructs with pooled amplicon sequencing as a readout, we were able to screen many genes for spermatogenesis function in a quick and inexpensive experiment. We transfected nine mouse testes with a pilot pool of RNA interference (RNAi against well-characterized genes to show that this system is highly reproducible and accurate. With a false negative rate of 18% and a false positive rate of 12%, this method has similar performance as other RNAi screens in the well-described Drosophila model system. In a separate experiment, we screened 26 uncharacterized genes computationally predicted to be essential for spermatogenesis and found numerous candidates for follow-up studies. Finally, as a control experiment, we performed a long-term selection screen in neuronal N2a cells, sampling shRNA frequencies at five sequential time points. By characterizing the effect of both libraries on N2a cells, we show that our screening results from testis are tissue-specific. Our calculations indicate that the current implementation of this approach could be used to screen thousands of protein-coding genes simultaneously in a single mouse testis. The experimental protocols and analysis scripts provided will enable other groups to use this procedure to study diverse aspects of germ cell biology ranging from epigenetics to cell physiology. This approach also has great promise as an

  16. Parameter estimation method for blurred cell images from fluorescence microscope

    Science.gov (United States)

    He, Fuyun; Zhang, Zhisheng; Luo, Xiaoshu; Zhao, Shulin

    2016-10-01

    Microscopic cell image analysis is indispensable to cell biology. Images of cells can easily degrade due to optical diffraction or focus shift, as this results in low signal-to-noise ratio (SNR) and poor image quality, hence affecting the accuracy of cell analysis and identification. For a quantitative analysis of cell images, restoring blurred images to improve the SNR is the first step. A parameter estimation method for defocused microscopic cell images based on the power law properties of the power spectrum of cell images is proposed. The circular radon transform (CRT) is used to identify the zero-mode of the power spectrum. The parameter of the CRT curve is initially estimated by an improved differential evolution algorithm. Following this, the parameters are optimized through the gradient descent method. Using synthetic experiments, it was confirmed that the proposed method effectively increased the peak SNR (PSNR) of the recovered images with high accuracy. Furthermore, experimental results involving actual microscopic cell images verified that the superiority of the proposed parameter estimation method for blurred microscopic cell images other method in terms of qualitative visual sense as well as quantitative gradient and PSNR.

  17. False Negative Cell-Free DNA Screening Result in a Newborn with Trisomy 13

    Directory of Open Access Journals (Sweden)

    Yang Cao

    2016-01-01

    Full Text Available Background. Noninvasive prenatal screening (NIPS is revolutionizing prenatal screening as a result of its increased sensitivity, specificity. NIPS analyzes cell-free fetal DNA (cffDNA circulating in maternal plasma to detect fetal chromosome abnormalities. However, cffDNA originates from apoptotic placental trophoblast; therefore cffDNA is not always representative of the fetus. Although the published data for NIPS testing states that the current technique ensures high sensitivity and specificity for aneuploidy detection, false positives are possible due to isolated placental mosaicism, vanishing twin or cotwin demise, and maternal chromosome abnormalities or malignancy. Results. We report a case of false negative cell-free DNA (cfDNA screening due to fetoplacental mosaicism. An infant male with negative cfDNA screening result was born with multiple congenital abnormalities. Postnatal chromosome and FISH studies on a blood specimen revealed trisomy 13 in 20/20 metaphases and 100% interphase nuclei, respectively. FISH analysis on tissues collected after delivery revealed extraembryonic mosaicism. Conclusions. Extraembryonic tissue mosaicism is likely responsible for the false negative cfDNA screening result. This case illustrates that a negative result does not rule out the possibility of a fetus affected with a trisomy, as cffDNA is derived from the placenta and therefore may not accurately represent the fetal genetic information.

  18. A three-dimensional collagen scaffold cell culture system for screening anti-glioma therapeutics

    Science.gov (United States)

    Lv, Donglai; Yu, Shi-cang; Ping, Yi-fang; Wu, Haibo; Zhao, Xilong; Zhang, Huarong; Cui, Youhong; Chen, Bing; Zhang, Xia; Dai, Jianwu

    2016-01-01

    Three-dimensional (3D) culture, which can simulate in vivo microenvironments, has been increasingly used to study tumor cell biology. Since most preclinical anti-glioma drug tests still rely on conventional 2D cell culture, we established a collagen scaffold for 3D glioma cell culture. Glioma cells cultured on these 3D scaffolds showed greater degree of dedifferentiation and quiescence than cells in 2D culture. 3D-cultured cells also exhibited enhanced resistance to chemotherapeutic alkylating agents, with a much higher proportion of glioma stem cells and upregulation of O6-methylguanine DNA methyltransferase (MGMT). Importantly, tumor cells in 3D culture showed chemotherapy resistance patterns similar to those observed in glioma patients. Our results suggest that 3D collagen scaffolds are promising in vitro research platforms for screening new anti-glioma therapeutics. PMID:27486877

  19. Characterization of mortality in children with sickle cell disease diagnosed through the Newborn Screening Program

    Directory of Open Access Journals (Sweden)

    Alessandra P. Sabarense

    2015-06-01

    Full Text Available OBJECTIVE: To characterize the deaths of 193 children with sickle cell disease screened by a neonatal program from 1998 to 2012 and contrast the initial years with the final years. METHODS: Deaths were identified by active surveillance of children absent to scheduled appointments in Blood Bank Clinical Centers (Hemominas. Clinical and epidemiological data came from death certificates, neonatal screening database, medical records, and family interviews. RESULTS: Between 1998 and 2012, 3,617,919 children were screened and 2,591 had sickle cell disease (1:1,400. There were 193 deaths (7.4%: 153 with SS/Sß0-talassemia, 34 SC and 6 Sß+thalassemia; 76.7% were younger than five years; 78% died in the hospital and 21% at home or in transit. The main causes of death were infection (45%, indeterminate (28%, and acute splenic sequestration (14%. In 46% of death certificates, the term "sickle cell" was not recorded. Seven-year death rate for children born between 1998 and 2005 was 5.43% versus 5.12% for those born between 2005 and 2012 (p = 0.72. Medical care was provided to 75% of children; 24% were unassisted. Medical care was provided within 6 hours of symptom onset in only half of the interviewed cases. In 40.5% of cases, death occurred within the first 24 hours. Low family income was recorded in 90% of cases, and illiteracy in 5%. CONCLUSIONS: Although comprehensive and effective, neonatal screening for sickle cell disease was not sufficient to significantly reduce mortality in a newborn screening program. Economic and social development and increase of the knowledge on sickle cell disease among health professionals and family are needed to overcome excessive mortality.

  20. Evaluation of the importance of astrocytes when screening for acute toxicity in neuronal cell systems.

    Science.gov (United States)

    Woehrling, E K; Hill, E J; Coleman, M D

    2010-02-01

    Reliable, high throughput, in vitro preliminary screening batteries have the potential to greatly accelerate the rate at which regulatory neurotoxicity data is generated. This study evaluated the importance of astrocytes when predicting acute toxic potential using a neuronal screening battery of pure neuronal (NT2.N) and astrocytic (NT2.A) and integrated neuronal/astrocytic (NT2.N/A) cell systems derived from the human NT2.D1 cell line, using biochemical endpoints (mitochondrial membrane potential (MMP) depolarisation and ATP and GSH depletion). Following exposure for 72 h, the known acute human neurotoxicants trimethyltin-chloride, chloroquine and 6-hydroxydopamine were frequently capable of disrupting biochemical processes in all of the cell systems at non-cytotoxic concentrations. Astrocytes provide key metabolic and protective support to neurons during toxic challenge in vivo and generally the astrocyte containing cell systems showed increased tolerance to toxicant insult compared with the NT2.N mono-culture in vitro. Whilst there was no consistent relationship between MMP, ATP and GSH log IC(50) values for the NT2.N/A and NT2.A cell systems, these data did provide preliminary evidence of modulation of the acute neuronal toxic response by astrocytes. In conclusion, the suitability of NT2 neurons and astrocytes as cell systems for acute toxicity screening deserves further investigation.

  1. Screening and target identification of bioactive compounds that modulate cell migration and autophagy.

    Science.gov (United States)

    Tashiro, Etsu; Imoto, Masaya

    2016-08-01

    Cell migration is a fundamental step for embryonic development, wound repair, immune responses, and tumor cell invasion and metastasis. It is well known that protrusive structures, namely filopodia and lamellipodia, can be observed at the leading edge of migrating cells. The formation of these structures is necessary for cell migration; however, the molecular mechanisms behind the formation of these structures remain largely unclear. Therefore, bioactive compounds that modulate protrusive structures are extremely powerful tools for studying the mechanisms behind the formation of these structures and subsequent cell migration. Therefore, we have screened for bioactive compounds that inhibit the formation of filopodia, lamellipodia, or cell migration from natural products, and attempted to identify the target molecules of our isolated compounds. Additionally, autophagy is a bulk, non-specific protein degradation system that is involved in the pathogenesis of cancer and neurodegenerative disorders. Recent extensive studies have revealed the molecular mechanisms of autophagy, however, they also remain largely unclear. Thus, we also have screened for bioactive compounds that modulate autophagy, and identified the target molecules. In the present article, we introduce the phenotypic screening system and target identification of four bioactive compounds.

  2. Adaptive Neuro-Fuzzy Inference System Approach for the Automatic Screening of Diabetic Retinopathy in Fundus Images

    Directory of Open Access Journals (Sweden)

    S. Kavitha

    2011-01-01

    Full Text Available Problem statement: Diabetic retinopathy is one of the most significant factors contributing to blindness and so early diagnosis and timely treatment is particularly important to prevent visual loss. Approach: An integrated approach for extraction of blood vessels and exudates detection was proposed to screen diabetic retinopathy. An automated classifier was developed based on Adaptive Neuro-Fuzzy Inference System (ANFIS to differentiate between normal and nonproliferative eyes from the quantitative assessment of monocular fundus images. Feature extraction was performed on the preprocessed fundus images. Structure of Blood vessels was extracted using Multiscale analysis. Hard Exudates were detected using CIE Color channel transformation, Entropy Thresholding and Improved Connected Component Analysis from the fundus images. Features like Wall to Lumen ratio in blood vessels, Texture, Homogeneity properties and area occupied by Hard Exudates, were given as input to ANFIS.ANFIS was trained with Back propagation in combination with the least squares method. Proposed method was evaluated on 200 real time images comprising 70 normal and 130 retinopathic eyes. Results and Conclusion: All of the results were validated with ground truths obtained from expert ophthalmologists. Quantitative performance of the method, detected exudates with an accuracy of 99.5%. Receiver operating characteristic curve evaluated for real time images produced better results compared to the other state of the art methods. ANFIS provides best classification and can be used as a screening tool in the analysis and diagnosis of retinal images.

  3. Screening of Small Molecule Microarrays for Ligands Targeted to the Extracellular Epitopes of Living Cells

    Directory of Open Access Journals (Sweden)

    Jeong Heon Lee

    2015-02-01

    Full Text Available The screening of living cells using high-throughput microarrays is technically challenging. Great care must be taken in the chemical presentation of potential ligands and the number of collisions that cells make with them. To overcome these issues, we have developed a glass slide-based microarray system to discover small molecule ligands that preferentially bind to one cell type over another, including when the cells differ by only a single receptor. Chemical spots of 300 ± 10 µm in diameter are conjugated covalently to glass slides using an arraying robot, and novel near-infrared fluorophores with peak emission at 700 nm and 800 nm are used to label two different cell types. By carefully optimizing incubation conditions, including cell density, motion, kinetics, detection, etc. we demonstrate that cell-ligand binding occurs, and that the number of cells bound per chemical spot correlates with ligand affinity and specificity. This screening system lays the foundation for high-throughput discovery of novel ligands to the cell surface.

  4. The Effect of Rapid Thermal Annealing Towards the Performance of Screen-Printed Si Solar Cell

    Directory of Open Access Journals (Sweden)

    Shahrul Anizan

    2011-01-01

    Full Text Available Problem statement: Solar cells are used to capture the photons which generate the energy. However the efficiency of the cells to turn the amount of photon to electricity needs to be high and so the cells enhancement is needed. This involved the whole process of the developing of the cells, thus annealing process is one of the important steps that needs to be optimised. Approach: Only Si solar cells will be discussed and the processes involved would be metal contact screen printing and metal paste co-firing. The contacts were first screen printed with Al paste for the rear side and Ag paste for the front side of the cell. Cells are then fired in the annealing furnace using selected temperature profile. Few sets of temperature profiles were used in every cycle. Results: After the IV characteristics were measured such as Voc, Isc, Pmax and fill factor, it shows that when higher annealing temperature of the profile was used, all the parameter will increase accordingly. However, profile with the highest annealing temperature will burn the paste as it will decrease the quality of the cell. This is considered as over heat to the paste. Conclusion: So by optimising the thermal treatment of the annealing process does improve the performance of the Si solar cell.

  5. Breast cancer screening with imaging: recommendations from the Society of Breast Imaging and the ACR on the use of mammography, breast MRI, breast ultrasound, and other technologies for the detection of clinically occult breast cancer.

    Science.gov (United States)

    Lee, Carol H; Dershaw, D David; Kopans, Daniel; Evans, Phil; Monsees, Barbara; Monticciolo, Debra; Brenner, R James; Bassett, Lawrence; Berg, Wendie; Feig, Stephen; Hendrick, Edward; Mendelson, Ellen; D'Orsi, Carl; Sickles, Edward; Burhenne, Linda Warren

    2010-01-01

    Screening for breast cancer with mammography has been shown to decrease mortality from breast cancer, and mammography is the mainstay of screening for clinically occult disease. Mammography, however, has well-recognized limitations, and recently, other imaging including ultrasound and magnetic resonance imaging have been used as adjunctive screening tools, mainly for women who may be at increased risk for the development of breast cancer. The Society of Breast Imaging and the Breast Imaging Commission of the ACR are issuing these recommendations to provide guidance to patients and clinicians on the use of imaging to screen for breast cancer. Wherever possible, the recommendations are based on available evidence. Where evidence is lacking, the recommendations are based on consensus opinions of the fellows and executive committee of the Society of Breast Imaging and the members of the Breast Imaging Commission of the ACR.

  6. Screening differentially expressed genes in mouse hepatocarcinoma ascites cell line with high potential of lymphatic metastasis

    Institute of Scientific and Technical Information of China (English)

    Xiao-Nan Cui; Jian-Wu Tang; Li Hou; Bo Song; Li Li; Ji-Wei Liu

    2005-01-01

    AIM: To screen genes differentially expressed in mouse hepatocarcinoma ascites cell line with high potential of lymphatic metastasis.METHODS: A subtracted cDNA library of mouse hepatocarcinoma cell line with high potential of lymphatic metastatic Hca-F and its synogenetic cell line Hca-P with a low metastatic potential was constructed by suppression subtracted hybridization(SSH) method. The screened clones of the subtracted library were sequenced and GeneBank homology search was performed.RESULTS: Fourteen differentially expressed cDNA fragments of Hca-F were obtained with two novel genes.CONCLUSION: SSH is a useful technique to detect differentially expressioned genes and an effective method to clone novel genes.

  7. X-ray diffraction imaging with the Multiple Inverse Fan Beam topology: principles, performance and potential for security screening.

    Science.gov (United States)

    Harding, G; Fleckenstein, H; Kosciesza, D; Olesinski, S; Strecker, H; Theedt, T; Zienert, G

    2012-07-01

    The steadily increasing number of explosive threat classes, including home-made explosives (HMEs), liquids, amorphous and gels (LAGs), is forcing up the false-alarm rates of security screening equipment. This development can best be countered by increasing the number of features available for classification. X-ray diffraction intrinsically offers multiple features for both solid and LAGs explosive detection, and is thus becoming increasingly important for false-alarm and cost reduction in both carry-on and checked baggage security screening. Following a brief introduction to X-ray diffraction imaging (XDI), which synthesizes in a single modality the image-forming and material-analysis capabilities of X-rays, the Multiple Inverse Fan Beam (MIFB) XDI topology is described. Physical relationships obtaining in such MIFB XDI components as the radiation source, collimators and room-temperature detectors are presented with experimental performances that have been achieved. Representative X-ray diffraction profiles of threat substances measured with a laboratory MIFB XDI system are displayed. The performance of Next-Generation (MIFB) XDI relative to that of the 2nd Generation XRD 3500TM screener (Morpho Detection Germany GmbH) is assessed. The potential of MIFB XDI, both for reducing the exorbitant cost of false alarms in hold baggage screening (HBS), as well as for combining "in situ" liquid and solid explosive detection in carry-on luggage screening is outlined.

  8. A Small-Molecule Screen for Enhanced Homing of Systemically Infused Cells

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    Oren Levy

    2015-03-01

    Full Text Available Poor homing of systemically infused cells to disease sites may limit the success of exogenous cell-based therapy. In this study, we screened 9,000 signal-transduction modulators to identify hits that increase mesenchymal stromal cell (MSC surface expression of homing ligands that bind to intercellular adhesion molecule 1 (ICAM-1, such as CD11a. Pretreatment of MSCs with Ro-31-8425, an identified hit from this screen, increased MSC firm adhesion to an ICAM-1-coated substrate in vitro and enabled targeted delivery of systemically administered MSCs to inflamed sites in vivo in a CD11a- (and other ICAM-1-binding domains-dependent manner. This resulted in a heightened anti-inflammatory response. This represents a new strategy for engineering cell homing to enhance therapeutic efficacy and validates CD11a and ICAM-1 as potential targets. Altogether, this multi-step screening process may significantly improve clinical outcomes of cell-based therapies.

  9. Electrochemical estrogen screen method based on the electrochemical behavior of MCF-7 cells.

    Science.gov (United States)

    Li, Jinlian; Song, Jia; Bi, Sheng; Zhou, Shi; Cui, Jiwen; Liu, Jiguang; Wu, Dongmei

    2016-08-05

    It was an urgent task to develop quick, cheap and accurate estrogen screen method for evaluating the estrogen effect of the booming chemicals. In this study, the voltammetric behavior between the estrogen-free and normal fragmented MCF-7 cell suspensions were compared, and the electrochemical signal (about 0.68V attributed by xanthine and guanine) of the estrogen-free fragmented MCF-7 cell suspension was obviously lower than that of the normal one. The electrochemistry detection of ex-secretion purines showed that the ability of ex-secretion purines of cells sharp decreased due to the removing of endogenous estrogen. The results indicated that the electrochemical signal of MCF-7 cells was related to the level of intracellular estrogen. When the level of intracellular estrogen was down-regulated, the concentrations of the xanthine and hypoxanthine decreased, which led to the electrochemical signal of MCF-7 cells fall. Based on the electrochemical signal, the electrochemical estrogen screen method was established. The estrogen effect of estradiol, nonylphenol and bisphenol A was evaluated with the electrochemical method, and the result was accordant with that of MTT assay. The electrochemical estrogen screen method was simple, quickly, cheap, objective, and it exploits a new way for the evaluation of estrogenic effects of chemicals.

  10. MiRNA mimic screen for improved expression of functional neurotensin receptor from HEK293 cells.

    Science.gov (United States)

    Xiao, Su; Chen, Yu-Chi; Betenbaugh, Michael J; Martin, Scott E; Shiloach, Joseph

    2015-08-01

    Obtaining adequate quantities of functional mammalian membrane proteins has been a bottleneck in their structural and functional studies because the expression of these proteins from mammalian cells is relatively low. To explore the possibility of enhancing expression of these proteins using miRNA, a stable T-REx-293 cell line expressing the neurotensin receptor type 1 (NTSR1), a hard-to-express G protein-coupled receptor (GPCR), was constructed. The cell line was then subjected to human miRNA mimic library screening. In parallel, an HEK293 cell line expressing luciferase was also screened with the same human miRNA mimic library. Five microRNA mimics: hsa-miR-22-5p, hsa-miR-18a-5p, hsa-miR-22-3p, hsa-miR-429, and hsa-miR-2110were identified from both screens. They led to 48% increase in the expression of functional NTSR1 and to 239% increase of luciferase expression. These miRNAs were also effective in enhancing the expression of secretedglypican-3 hFc-fusion protein from HEK293 cells.The results indicate that these molecules may have a wide role in enhancing the production of proteins with biomedical interest.

  11. Screening system of blocking agents of the receptor for advanced glycation endproducts in cells using fluorescence.

    Science.gov (United States)

    Jung, Dong Ho; Kim, Young Sook; Kim, Jin Sook

    2012-01-01

    Activation of the receptor for advanced glycation endproducts (RAGE) triggers cellular responses implicated in the pathogenesis of diabetic complications; blockade of RAGE has been shown to inhibit the development of diabetic complications. To develop a screening system to identify novel disruptors of advanced glycation endproducts (AGE)-RAGE binding, we used an AGE-RAGE binding system in RAGE-overexpressing cells; test compounds were screened using this system. To construct human RAGE-overexpressing cells, mouse mesangial cells (MMCs) were stably transfected with the pcDNA-human RAGE (hRAGE) vector and selected under 1 mg/mL gentamicin (G418). RAGE expression in hRAGE-overexpressing MMCs was analyzed by Western blotting with specific RAGE antibody. To identify novel disruptors of AGE-RAGE binding, 50 single compounds and AGE-bovine serum albumin (BSA)-Alexa 488 (AGE-BSA labeled with Alexa 488) were treated to the hRAGE-overexpressing MMCs. Nonbinding AGE-BSA-Alexa 488 was washed and fluorescence measured by microtiter plate reader (excitation wavelength, 485 nm; emission wavelength, 528 nm). In hRAGE-overexpressing cells, only treatment with AGE-BSA-Alexa 488 significantly increased fluorescence intensity in a dose-dependent manner. Of 50 compounds tested, genistein disrupted AGE-RAGE binding in a dose-dependent manner. This AGE-RAGE binding system using AGE-BSA-Alexa 488 in hRAGE-overexpressing cells was suitable for screening of agents that disrupt AGE-hRAGE binding.

  12. Live cell imaging to understand monocyte, macrophage, and dendritic cell function in atherosclerosis.

    Science.gov (United States)

    McArdle, Sara; Mikulski, Zbigniew; Ley, Klaus

    2016-06-27

    Intravital imaging is an invaluable tool for understanding the function of cells in healthy and diseased tissues. It provides a window into dynamic processes that cannot be studied by other techniques. This review will cover the benefits and limitations of various techniques for labeling and imaging myeloid cells, with a special focus on imaging cells in atherosclerotic arteries. Although intravital imaging is a powerful tool for understanding cell function, it alone does not provide a complete picture of the cell. Other techniques, such as flow cytometry and transcriptomics, must be combined with intravital imaging to fully understand a cell's phenotype, lineage, and function.

  13. Impact of Cell-Free Fetal DNA Screening on Patients’ Choice of Invasive Procedures after a Positive California Prenatal Screen Result

    Directory of Open Access Journals (Sweden)

    Forum T. Shah

    2014-07-01

    Full Text Available Until recently, maternal serum analyte levels paired with sonographic fetal nuchal translucency measurement was the most accurate prenatal screen available for Trisomies 18 and 21, (91% and 94% detection and false positive rates of 0.31% and 4.5% respectively. Women with positive California Prenatal Screening Program (CPSP results have the option of diagnostic testing to determine definitively if the fetus has a chromosomal abnormality. Cell-free fetal (cff- DNA screening for Trisomies 13, 18, and 21 was first offered in 2012, allowing women with positive screens to choose additional screening before diagnostic testing. Cff-DNA sensitivity rates are as high as 99.9% and 99.1%, with false positive rates of 0.4% and 0.1%, for Trisomies 18 and 21, respectively. A retrospective chart review was performed in 2012 on 500 CPSP referrals at the University of California, San Diego Thornton Hospital. Data were collected prior to and after the introduction of cff-DNA. There was a significant increase in the number of participants who chose to pursue additional testing and a decrease in the number of invasive procedures performed after cff-DNA screening was available. We conclude that as fetal aneuploidy screening improves, the number of invasive procedures will continue to decrease.

  14. Disease modeling and drug screening for neurological diseases using human induced pluripotent stem cells.

    Science.gov (United States)

    Xu, Xiao-hong; Zhong, Zhong

    2013-06-01

    With the general decline of pharmaceutical research productivity, there are concerns that many components of the drug discovery process need to be redesigned and optimized. For example, the human immortalized cell lines or animal primary cells commonly used in traditional drug screening may not faithfully recapitulate the pathological mechanisms of human diseases, leading to biases in assays, targets, or compounds that do not effectively address disease mechanisms. Recent advances in stem cell research, especially in the development of induced pluripotent stem cell (iPSC) technology, provide a new paradigm for drug screening by permitting the use of human cells with the same genetic makeup as the patients without the typical quantity constraints associated with patient primary cells. In this article, we will review the progress made to date on cellular disease models using human stem cells, with a focus on patient-specific iPSCs for neurological diseases. We will discuss the key challenges and the factors that associated with the success of using stem cell models for drug discovery through examples from monogenic diseases, diseases with various known genetic components, and complex diseases caused by a combination of genetic, environmental and other factors.

  15. A High-Throughput Screening Model of the Tumor Microenvironment for Ovarian Cancer Cell Growth.

    Science.gov (United States)

    Lal-Nag, Madhu; McGee, Lauren; Guha, Rajarshi; Lengyel, Ernst; Kenny, Hilary A; Ferrer, Marc

    2017-01-01

    The tumor microenvironment plays an important role in the processes of tumor growth, metastasis, and drug resistance. We have used a multilayered 3D primary cell culture model that reproduces the human ovarian cancer metastatic microenvironment to study the effect of the microenvironment on the pharmacological responses of different classes of drugs on cancer cell proliferation. A collection of oncology drugs was screened to identify compounds that inhibited the proliferation of ovarian cancer cells growing as monolayers or forming spheroids, on plastic and on a 3D microenvironment culture model of the omentum metastatic site, and also cells already in preformed spheroids. Target-based analysis of the pharmacological responses revealed that several classes of targets were more efficacious in cancer cells growing in the absence of the metastatic microenvironment, and other target classes were less efficacious in cancer cells in preformed spheres compared to forming spheroid cultures. These findings show that both the cellular context of the tumor microenvironment and cell adhesion mode have an essential role in cancer cell drug resistance. Therefore, it is important to perform screens for new drugs using model systems that more faithfully recapitulate the tissue composition at the site of tumor growth and metastasis.

  16. Establishment of a cell model for screening antibody drugs against rheumatoid arthritis with ADCC and CDC.

    Science.gov (United States)

    Yan, Li; Hu, Rui; Tu, Song; Cheng, Wen-Jun; Zheng, Qiong; Wang, Jun-Wen; Kan, Wu-Sheng; Ren, Yi-Jun

    2015-01-01

    TNFα played a dominant role in the development and progression of rheumatoid arthritis (RA). Clinical trials proved the efficacies of anti-TNFα agents for curing RA. However, most researchers were concentrating on their abilities of neutralizing TNFα, the potencies of different anti-TNFα agents varied a lot due to the antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). For better understanding and differentiating the potentiality of various candidate anti-TNF reagents at the stage of new drug research and development, present study established a cell model expressing the transmembrane TNFα for usage in in vitro ADCC or CDC assay, meanwhile, the assay protocol described here could provide guidelines for screening macromolecular antibody drugs. A stable cell subline bearing transmembrane TNFα was first established by conventional transfection method, the expression of transmembrane TNFα was approved by flow cytometer, and the performance of the stable subline in ADCC and CDC assay was evaluated, using human peripheral blood mononuclear cells as effector cells, and Adalimumab as the anti-TNFα reagent. The stable cell subline demonstrated high level of surface expression of transmembrane TNFα, and Adalimumab exerted both ADCC and CDC effects on this cell model. In conclusion, the stable cell line we established in present research could be used in ADCC or CDC assay for screening antibody drugs, which would provide in-depth understanding of the potencies of candidate antibody drugs in addition to the traditional TNFα neutralizing assay.

  17. Disease modeling and drug screening for neurological diseases using human induced pluripotent stem cells

    Institute of Scientific and Technical Information of China (English)

    Xiao-hong XU; Zhong ZHONG

    2013-01-01

    With the general decline of pharmaceutical research productivity,there are concerns that many components of the drug discovery process need to be redesigned and optimized.For example,the human immortalized cell lines or animal primary cells commonly used in traditional drug screening may not faithfully recapitulate the pathological mechanisms of human diseases,leading to biases in assays,targets,or compounds that do not effectively address disease mechanisms.Recent advances in stem cell research,especially in the development of induced pluripotent stem cell (iPSC) technology,provide a new paradigm for drug screening by permitting the use of human cells with the same genetic makeup as the patients without the typical quantity constraints associated with patient primary cells.In this article,we will review the progress made to date on cellular disease models using human stem cells,with a focus on patient-specific iPSCs for neurological diseases.We will discuss the key challenges and the factors that associated with the success of using stem cell models for drug discovery through examples from monogenic diseases,diseases with various known genetic components,and complex diseases caused by a combination of genetic,environmental and other factors.

  18. A Paper-Based Test for Screening Newborns for Sickle Cell Disease

    Science.gov (United States)

    Piety, Nathaniel Z.; George, Alex; Serrano, Sonia; Lanzi, Maria R.; Patel, Palka R.; Noli, Maria P.; Kahan, Silvina; Nirenberg, Damian; Camanda, João F.; Airewele, Gladstone; Shevkoplyas, Sergey S.

    2017-01-01

    The high cost, complexity and reliance on electricity, specialized equipment and supplies associated with conventional diagnostic methods limit the scope and sustainability of newborn screening for sickle cell disease (SCD) in sub-Saharan Africa and other resource-limited areas worldwide. Here we describe the development of a simple, low-cost, rapid, equipment- and electricity-free paper-based test capable of detecting sickle hemoglobin (HbS) in newborn blood samples with a limit of detection of 2% HbS. We validated this newborn paper-based test in a cohort of 159 newborns at an obstetric hospital in Cabinda, Angola. Newborn screening results using the paper-based test were compared to conventional isoelectric focusing (IEF). The test detected the presence of HbS with 81.8% sensitivity and 83.3% specificity, and identified SCD newborns with 100.0% sensitivity and 70.7% specificity. The use of the paper-based test in a two-stage newborn screening process could have excluded about 70% of all newborns from expensive confirmatory testing by IEF, without missing any of the SCD newborns in the studied cohort. This study demonstrates the potential utility of the newborn paper-based test for reducing the overall cost of screening newborns for SCD and thus increasing the practicality of universal newborn SCD screening programs in resource-limited settings. PMID:28367971

  19. High-throughput screens in mammalian cells using the CRISPR-Cas9 system.

    Science.gov (United States)

    Peng, Jingyu; Zhou, Yuexin; Zhu, Shiyou; Wei, Wensheng

    2015-06-01

    As a powerful genome-editing tool, the clustered regularly interspaced short palindromic repeats (CRISPR)-clustered regularly interspaced short palindromic repeats-associated protein 9 (Cas9) system has been quickly developed into a large-scale function-based screening strategy in mammalian cells. This new type of genetic library is constructed through the lentiviral delivery of single-guide RNA collections that direct Cas9 or inactive dead Cas9 fused with effectors to interrogate gene function or regulate gene transcription in targeted cells. Compared with RNA interference screening, the CRISPR-Cas9 system demonstrates much higher levels of effectiveness and reliability with respect to both loss-of-function and gain-of-function screening. Unlike the RNA interference strategy, a CRISPR-Cas9 library can target both protein-coding sequences and regulatory elements, including promoters, enhancers and elements transcribing microRNAs and long noncoding RNAs. This powerful genetic tool will undoubtedly accelerate the mechanistic discovery of various biological processes. In this mini review, we summarize the general procedure of CRISPR-Cas9 library mediated functional screening, system optimization strategies and applications of this new genetic toolkit.

  20. A genetic screen to isolate Toxoplasma gondii host-cell egress mutants.

    Science.gov (United States)

    Coleman, Bradley I; Gubbels, Marc-Jan

    2012-02-08

    The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understood at the molecular level. The signaling pathways underlying egress have been characterized through the use of pharmacological agents acting on different aspects of the pathways. As such, several independent triggers of egress have been identified which all converge on the release of intracellular Ca(2+), a signal that is also critical for host cell invasion. This insight informed a candidate gene approach which led to the identification of plant like calcium dependent protein kinase (CDPK) involved in egress. In addition, several recent breakthroughs in understanding egress have been made using (chemical) genetic approaches. To combine the wealth of pharmacological information with the increasing genetic accessibility of Toxoplasma we recently established a screen permitting the enrichment for parasite mutants with a defect in host cell egress. Although chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) or ethyl methanesulfonate (EMS) has been used for decades in the study of Toxoplasma biology, only recently has genetic mapping of mutations underlying the phenotypes become routine. Furthermore, by generating temperature-sensitive mutants, essential processes can be dissected and the underlying genes directly identified. These mutants behave as wild-type under the permissive temperature (35 °C), but fail to proliferate at the restrictive temperature (40 °C) as a result of the mutation in question. Here we illustrate a new phenotypic screening method to isolate mutants

  1. Adapting human pluripotent stem cells to high-throughput and high-content screening.

    Science.gov (United States)

    Desbordes, Sabrina C; Studer, Lorenz

    2013-01-01

    The increasing use of human pluripotent stem cells (hPSCs) as a source of cells for drug discovery, cytotoxicity assessment and disease modeling requires their adaptation to large-scale culture conditions and screening formats. Here, we describe a simple and robust protocol for the adaptation of human embryonic stem cells (hESCs) to high-throughput screening (HTS). This protocol can also be adapted to human induced pluripotent stem cells (hiPSCs) and high-content screening (HCS). We also describe a 7-d assay to identify compounds with an effect on hESC self-renewal and differentiation. This assay can be adapted to a variety of applications. The procedure involves the culture expansion of hESCs, their adaptation to 384-well plates, the addition of small molecules or other factors, and finally data acquisition and processing. In this protocol, the optimal number of hESCs plated in 384-well plates has been adapted to HTS/HCS assays of 7 d.

  2. Occlusion of the middle cerebral artery Guidance by screen imaging using an EDA-H portable medium-soft electronic endoscope

    Institute of Scientific and Technical Information of China (English)

    Xingbao Zhu; Junli Luo; Song Liu; Dongping Li; Min Li; Quanshui Fan

    2011-01-01

    The present study aimed to verify the practicability of performing screen-based surgical operations under the guidance of a real-time viewing and recording system (RTVRS) using a portable medium-soft electronic endoscope (pmsEE). The middle cerebral artery in rats was occluded under screen-imaging guidance using a pmsEE RTVRS to reproduce an animal model of human cerebral infarction. The screen imaging of the pmsEE RTVRS was clear, life-like, stereoscopic and synchronous with the actual operation. Screen-imaging guidance led to an accurate, smooth, minimally invasive and comparatively easy surgical procedure. The surgical success rate, time of model establishment, neurological function scores, and infarct volume were similar to those using an operating microscope. These results indicate that the self-designed pmsEE RTVRS could be utilized for portable endoscopic screen-based surgical operations.

  3. Live-cell imaging of mitosis in Caenorhabditis elegans embryos.

    Science.gov (United States)

    Powers, James A

    2010-06-01

    Caenorhabditis elegans is a wonderful model system for live imaging studies of mitosis. A huge collection of research tools is readily available to facilitate experimentation. For imaging, C. elegans embryos provide large clear cells, an invariant pattern of cell division, only six chromosomes, a very short cell cycle, and remain healthy and happy at room temperature. Mitosis is a complicated process and the types of research questions being asked about the mechanisms involved are continuously expanding. For each experiment, the details of imaging methods need to be tailored to the question. Specific imaging methods will depend on the microscopy hardware and software available to each researcher. This article presents points to consider when choosing a microscope, designing an imaging experiment, or selecting appropriate worm strains for imaging. A method for mounting C. elegans embryos and guidelines for fluorescence and differential interference contrast imaging of mitosis in live embryos are presented.

  4. Label-free classification of cultured cells through diffraction imaging.

    Science.gov (United States)

    Dong, Ke; Feng, Yuanming; Jacobs, Kenneth M; Lu, Jun Q; Brock, R Scott; Yang, Li V; Bertrand, Fred E; Farwell, Mary A; Hu, Xin-Hua

    2011-06-01

    Automated classification of biological cells according to their 3D morphology is highly desired in a flow cytometer setting. We have investigated this possibility experimentally and numerically using a diffraction imaging approach. A fast image analysis software based on the gray level co-occurrence matrix (GLCM) algorithm has been developed to extract feature parameters from measured diffraction images. The results of GLCM analysis and subsequent classification demonstrate the potential for rapid classification among six types of cultured cells. Combined with numerical results we show that the method of diffraction imaging flow cytometry has the capacity as a platform for high-throughput and label-free classification of biological cells.

  5. INFECTED HALLER CELL. RADIOLOGY IMAGE OF THE ISSUE

    Directory of Open Access Journals (Sweden)

    Balasubramanian Thiagarajan

    2012-08-01

    Full Text Available Haller cells are also known as infraorbital ethmoidal cells / maxilla ethmoidal cells. These cellsextend into the inferomedial portion of orbital floor. They are seen in 40% of patients. 1 This article discusses the imaging features of haller cell as seen in coronal CT scan.

  6. miRNAs modified by dietary lipids in Caco-2 cells. A microarray screening

    Directory of Open Access Journals (Sweden)

    Lidia Daimiel

    2015-09-01

    Full Text Available We performed a screening of miRNAs regulated by dietary lipids in a cellular model of enterocytes, Caco-2 cells. Our aim was to describe new lipid-modified miRNAs with an implication in lipid homeostasis and cardiovascular disease [1,2]. For that purpose, we treated differentiated Caco-2 cells with micelles containing the assayed lipids (cholesterol, conjugated linoleic acid and docosahexaenoic acid and the screening of miRNAs was carried out by microarray using the μParaflo®Microfluidic Biochip Technology of LC Sciences (Huston, TX, USA. Experimental design, microarray description and raw data have been made available in the GEO database with the reference number of GSE59153. Here we described in detail the experimental design and methods used to obtain the relative expression data.

  7. Chemical biology drug sensitivity screen identifies sunitinib as synergistic agent with disulfiram in prostate cancer cells.

    Directory of Open Access Journals (Sweden)

    Kirsi Ketola

    Full Text Available BACKGROUND: Current treatment options for castration- and treatment-resistant prostate cancer are limited and novel approaches are desperately needed. Our recent results from a systematic chemical biology sensitivity screen covering most known drugs and drug-like molecules indicated that aldehyde dehydrogenase inhibitor disulfiram is one of the most potent cancer-specific inhibitors of prostate cancer cell growth, including TMPRSS2-ERG fusion positive cancers. However, the results revealed that disulfiram alone does not block tumor growth in vivo nor induce apoptosis in vitro, indicating that combinatorial approaches may be required to enhance the anti-neoplastic effects. METHODS AND FINDINGS: In this study, we utilized a chemical biology drug sensitivity screen to explore disulfiram mechanistic details and to identify compounds potentiating the effect of disulfiram in TMPRSS2-ERG fusion positive prostate cancer cells. In total, 3357 compounds including current chemotherapeutic agents as well as drug-like small molecular compounds were screened alone and in combination with disulfiram. Interestingly, the results indicated that androgenic and antioxidative compounds antagonized disulfiram effect whereas inhibitors of receptor tyrosine kinase, proteasome, topoisomerase II, glucosylceramide synthase or cell cycle were among compounds sensitizing prostate cancer cells to disulfiram. The combination of disulfiram and an antiangiogenic agent sunitinib was studied in more detail, since both are already in clinical use in humans. Disulfiram-sunitinib combination induced apoptosis and reduced androgen receptor protein expression more than either of the compounds alone. Moreover, combinatorial exposure reduced metastatic characteristics such as cell migration and 3D cell invasion as well as induced epithelial differentiation shown as elevated E-cadherin expression. CONCLUSIONS: Taken together, our results propose novel combinatorial approaches to inhibit

  8. Chemical Biology Drug Sensitivity Screen Identifies Sunitinib as Synergistic Agent with Disulfiram in Prostate Cancer Cells

    Science.gov (United States)

    Ketola, Kirsi; Kallioniemi, Olli; Iljin, Kristiina

    2012-01-01

    Background Current treatment options for castration- and treatment-resistant prostate cancer are limited and novel approaches are desperately needed. Our recent results from a systematic chemical biology sensitivity screen covering most known drugs and drug-like molecules indicated that aldehyde dehydrogenase inhibitor disulfiram is one of the most potent cancer-specific inhibitors of prostate cancer cell growth, including TMPRSS2-ERG fusion positive cancers. However, the results revealed that disulfiram alone does not block tumor growth in vivo nor induce apoptosis in vitro, indicating that combinatorial approaches may be required to enhance the anti-neoplastic effects. Methods and Findings In this study, we utilized a chemical biology drug sensitivity screen to explore disulfiram mechanistic details and to identify compounds potentiating the effect of disulfiram in TMPRSS2-ERG fusion positive prostate cancer cells. In total, 3357 compounds including current chemotherapeutic agents as well as drug-like small molecular compounds were screened alone and in combination with disulfiram. Interestingly, the results indicated that androgenic and antioxidative compounds antagonized disulfiram effect whereas inhibitors of receptor tyrosine kinase, proteasome, topoisomerase II, glucosylceramide synthase or cell cycle were among compounds sensitizing prostate cancer cells to disulfiram. The combination of disulfiram and an antiangiogenic agent sunitinib was studied in more detail, since both are already in clinical use in humans. Disulfiram-sunitinib combination induced apoptosis and reduced androgen receptor protein expression more than either of the compounds alone. Moreover, combinatorial exposure reduced metastatic characteristics such as cell migration and 3D cell invasion as well as induced epithelial differentiation shown as elevated E-cadherin expression. Conclusions Taken together, our results propose novel combinatorial approaches to inhibit prostate cancer cell

  9. Molecular Imaging in Stem Cell Therapy for Spinal Cord Injury

    Directory of Open Access Journals (Sweden)

    Fahuan Song

    2014-01-01

    Full Text Available Spinal cord injury (SCI is a serious disease of the center nervous system (CNS. It is a devastating injury with sudden loss of motor, sensory, and autonomic function distal to the level of trauma and produces great personal and societal costs. Currently, there are no remarkable effective therapies for the treatment of SCI. Compared to traditional treatment methods, stem cell transplantation therapy holds potential for repair and functional plasticity after SCI. However, the mechanism of stem cell therapy for SCI remains largely unknown and obscure partly due to the lack of efficient stem cell trafficking methods. Molecular imaging technology including positron emission tomography (PET, magnetic resonance imaging (MRI, optical imaging (i.e., bioluminescence imaging (BLI gives the hope to complete the knowledge concerning basic stem cell biology survival, migration, differentiation, and integration in real time when transplanted into damaged spinal cord. In this paper, we mainly review the molecular imaging technology in stem cell therapy for SCI.

  10. XRIndex: A brief screening tool for individual differences in security threat detection in x-ray images

    Directory of Open Access Journals (Sweden)

    Elena eRusconi

    2015-08-01

    Full Text Available X-ray imaging is a cost-effective technique at security checkpoints that typically require the presence of human operators. We have previously shown that self-reported Attention to Detail can predict threat detection performance with small-vehicle x-ray images (Rusconi et al., 2012. Here we provide evidence for the generality of such a link by having a large sample of naïve participants screen more typical dual-energy x-ray images of hand luggage. The results show that the Attention to Detail score is a linear predictor of threat detection accuracy. We then develop and fine-tune a novel self-report scale for security screening: the XRIndex, which improves on the Attention to Detail scale for predictive power and opacity to interpretation. The XRIndex is not redundant with any of the Big Five personality traits. We validate the XRIndex against security x-ray images with an independent sample of untrained participants and suggest that the XRIndex may be a useful aid for the identification of suitable candidates for professional security training with a focus on x-ray threat detection. Further studies are needed to determine whether this can also apply to trained professionals.

  11. Non-invasive Imaging of Human Embryonic Stem Cells

    OpenAIRE

    Hong, Hao; Yang, Yunan; Zhang, Yin; Cai, Weibo

    2010-01-01

    Human embryonic stem cells (hESCs) hold tremendous therapeutic potential in a variety of diseases. Over the last decade, non-invasive imaging techniques have proven to be of great value in tracking transplanted hESCs. This review article will briefly summarize the various techniques used for non-invasive imaging of hESCs, which include magnetic resonance imaging (MRI), bioluminescence imaging (BLI), fluorescence, single-photon emission computed tomography (SPECT), positron emission tomography...

  12. Functional screening of intracardiac cell transplants using two-photon fluorescence microscopy.

    Science.gov (United States)

    Tao, Wen; Soonpaa, Mark H; Field, Loren J; Chen, Peng-Sheng; Firulli, Anthony B; Shou, Weinian; Rubart, Michael

    2012-08-01

    Although the adult mammalian myocardium exhibits a limited ability to undergo regenerative growth, its intrinsic renewal rate is insufficient to compensate for myocyte loss during cardiac disease. Transplantation of donor cardiomyocytes or cardiomyogenic stem cells is considered a promising strategy for reconstitution of cardiac mass, provided the engrafted cells functionally integrate with host myocardium and actively contribute to its contractile force. The authors previously developed a two-photon fluorescence microscopy-based assay that allows in situ screening of donor cell function after intracardiac delivery of the cells. This report reviews the techniques of two-photon fluorescence microscopy and summarizes its application for quantifying the extent to which a variety of donor cell types stably and functionally couple with the recipient myocardium.

  13. Induction of iodide uptake in transformed thyrocytes: a compound screening in cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Froehlich, Eleonore [University of Tuebingen, Department of Endocrinology, Metabolism, Nephrology and Clinical Chemistry, Internal Medicine, Tuebingen (Germany); Brossart, Peter [University of Tuebingen, Department of Haematology, Oncology, Immunology and Rheumatology, Internal Medicine, Tuebingen (Germany); Wahl, Richard [University of Tuebingen, Department of Endocrinology, Metabolism, Nephrology and Clinical Chemistry, Internal Medicine, Tuebingen (Germany); Department IV, Internal Medicine, Tuebingen (Germany)

    2009-05-15

    Retinoic acid presently is the most advanced agent able to improve the efficacy of radioiodine therapy in differentiated thyroid carcinoma. In order to identify compounds with higher efficacy a panel of pharmacologically well-characterized compounds with antitumour action in solid cancer cell lines was screened. The effects of the compounds on iodide uptake, cell number, proliferation and apoptosis were evaluated. In general, compounds were more effective in cell lines derived from more aggressive tumours. The effectiveness in terms of number of responsive cell lines and maximal increase in iodide uptake achieved decreased in the order: APHA > valproic acid {approx} sirolimus {approx} arsenic trioxide > retinoic acid {approx} lovastatin > apicidine {approx} azacytidine {approx} retinol {approx} rosiglitazone {approx} bortezomib. We hypothesize that testing of cells from primary tumours or metastases in patients may be a way to identify compounds with optimum therapeutic efficacy for individualized treatment. (orig.)

  14. Proposed low-cost premarital screening program for prevention of sickle cell and thalassemia in Yemen.

    Science.gov (United States)

    Al-Nood, Hafiz; Al-Hadi, Abdulrahman

    2013-01-01

    In Yemen, the prevalence of sickle cell trait and β-thalassemia trait are high. The aim of this premarital program is to identify sickle cell and thalassemia carrier couples in Yemen before completing marriages proposal, in order to prevent affected birth. This can be achieved by applying a low-cost premarital screening program using simple blood tests compatible with the limited health resources of the country. If microcytosis or positive sickle cell is found in both or one partner has microcytosis and the other has positive sickle cell, so their children at high risk of having sickle cell or/and thalassemia diseases. Carrier couples will be referred to genetic counseling. The outcomes of this preventive program are predicted to decrease the incidence of affected birth and reduce the health burden of these disorders. The success of this program also requires governmental, educational and religious supports.

  15. Metabolite profiling of microfluidic cell culture conditions for droplet based screening

    DEFF Research Database (Denmark)

    2015-01-01

    We investigate the impact of droplet culture conditions on cell metabolic state by determining key metabolite concentrations in S. cerevisiae cultures in different microfluidic droplet culture formats. Control of culture conditions is critical for single cell/clone screening in droplets....... Metabolite profiling provides a more nuanced estimate of cell state compared to proliferation studies alone. We show that the choice of droplet incubation format impacts cell proliferation and metabolite production. The standard syringe incubation of droplets exhibited metabolite profiles similar to oxygen...... limited cultures, whereas the metabolite profiles of cells cultured in the alternative wide tube droplet incubation format resemble those from aerobic culture. Furthermore, we demonstrate retained droplet stability and size in the new better oxygenated droplet incubation format....

  16. Proposed low-cost premarital screening program for prevention of sickle cell and thalassemia in Yemen

    Science.gov (United States)

    Al-Nood, Hafiz; Al-Hadi, Abdulrahman

    2013-01-01

    In Yemen, the prevalence of sickle cell trait and β-thalassemia trait are high. The aim of this premarital program is to identify sickle cell and thalassemia carrier couples in Yemen before completing marriages proposal, in order to prevent affected birth. This can be achieved by applying a low-cost premarital screening program using simple blood tests compatible with the limited health resources of the country. If microcytosis or positive sickle cell is found in both or one partner has microcytosis and the other has positive sickle cell, so their children at high risk of having sickle cell or/and thalassemia diseases. Carrier couples will be referred to genetic counseling. The outcomes of this preventive program are predicted to decrease the incidence of affected birth and reduce the health burden of these disorders. The success of this program also requires governmental, educational and religious supports. PMID:25003062

  17. Droplet-based microfluidic platforms for the encapsulation and screening of Mammalian cells and multicellular organisms.

    Science.gov (United States)

    Clausell-Tormos, Jenifer; Lieber, Diana; Baret, Jean-Christophe; El-Harrak, Abdeslam; Miller, Oliver J; Frenz, Lucas; Blouwolff, Joshua; Humphry, Katherine J; Köster, Sarah; Duan, Honey; Holtze, Christian; Weitz, David A; Griffiths, Andrew D; Merten, Christoph A

    2008-05-01

    High-throughput, cell-based assays require small sample volumes to reduce assay costs and to allow for rapid sample manipulation. However, further miniaturization of conventional microtiter plate technology is problematic due to evaporation and capillary action. To overcome these limitations, we describe droplet-based microfluidic platforms in which cells are grown in aqueous microcompartments separated by an inert perfluorocarbon carrier oil. Synthesis of biocompatible surfactants and identification of gas-permeable storage systems allowed human cells, and even a multicellular organism (C. elegans), to survive and proliferate within the microcompartments for several days. Microcompartments containing single cells could be reinjected into a microfluidic device after incubation to measure expression of a reporter gene. This should open the way for high-throughput, cell-based screening that can use >1000-fold smaller assay volumes and has approximately 500x higher throughput than conventional microtiter plate assays.

  18. Genome-wide screen for differential DNA methylation associated with neural cell differentiation in mouse.

    Directory of Open Access Journals (Sweden)

    Rene Cortese

    Full Text Available Cellular differentiation involves widespread epigenetic reprogramming, including modulation of DNA methylation patterns. Using Differential Methylation Hybridization (DMH in combination with a custom DMH array containing 51,243 features covering more than 16,000 murine genes, we carried out a genome-wide screen for cell- and tissue-specific differentially methylated regions (tDMRs in undifferentiated embryonic stem cells (ESCs, in in-vitro induced neural stem cells (NSCs and 8 differentiated embryonic and adult tissues. Unsupervised clustering of the generated data showed distinct cell- and tissue-specific DNA methylation profiles, revealing 202 significant tDMRs (p1.96 enrichment for genes involved in neural differentiation, including, for example, Jag1 and Tcf4. Our results provide robust evidence for the relevance of DNA methylation in early neural development and identify novel marker candidates for neural cell differentiation.

  19. A novel yeast cell-based screen identifies flavone as a tankyrase inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Yashiroda, Yoko, E-mail: ytyy@riken.jp [Chemical Genomics Research Group/Chemical Genetics Laboratory, RIKEN Advanced Science Institute, Wako, Saitama 351-0198 (Japan); Okamoto, Reika [Chemical Genomics Research Group/Chemical Genetics Laboratory, RIKEN Advanced Science Institute, Wako, Saitama 351-0198 (Japan); Japan Biological Informatics Consortium (JBIC), Koto-ku, Tokyo 135-8073 (Japan); Hatsugai, Kaori [Division of Molecular Biotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Koto-ku, Tokyo 135-8550 (Japan); Division of Chemotherapy, Graduate School of Pharmaceutical Sciences, Keio University, Minato-ku, Tokyo 105-8512 (Japan); Takemoto, Yasushi [Chemical Genomics Research Group/Chemical Genetics Laboratory, RIKEN Advanced Science Institute, Wako, Saitama 351-0198 (Japan); Goshima, Naoki [National Institute of Advanced Industrial Science and Technology, Koto-ku, Tokyo 135-0064 (Japan); Saito, Tamio [Chemical Biology Core Facility/Antibiotics Laboratory, RIKEN Advanced Science Institute, Wako, Saitama 351-0198 (Japan); Hamamoto, Makiko [Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Kanagawa 214-8571 (Japan); Sugimoto, Yoshikazu [Division of Chemotherapy, Graduate School of Pharmaceutical Sciences, Keio University, Minato-ku, Tokyo 105-8512 (Japan); Osada, Hiroyuki [Chemical Biology Core Facility/Antibiotics Laboratory, RIKEN Advanced Science Institute, Wako, Saitama 351-0198 (Japan); Seimiya, Hiroyuki [Division of Molecular Biotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Koto-ku, Tokyo 135-8550 (Japan); Yoshida, Minoru [Chemical Genomics Research Group/Chemical Genetics Laboratory, RIKEN Advanced Science Institute, Wako, Saitama 351-0198 (Japan); CREST Research Project, Japan Science and Technology Corporation, Saitama 332-0012 (Japan)

    2010-04-09

    The telomere-associated protein tankyrase 1 is a poly(ADP-ribose) polymerase and is considered to be a promising target for cancer therapy, especially for BRCA-associated cancers. However, an efficient assay system for inhibitor screening has not been established, mainly due to the difficulty of efficient preparation of the enzyme and its substrate. Here, we report a cell-based assay system for detecting inhibitory activity against tankyrase 1. We found that overexpression of the human tankyrase 1 gene causes a growth defect in the fission yeast Schizosaccharomyces pombe. Chemicals that restore the growth defect phenotype can be identified as potential tankyrase 1 inhibitors. We performed a high-throughput screen using this system, and identified flavone as a compound that restores the growth of yeast cells overexpressing tankyrase 1. Indeed, flavone inhibited poly(ADP-ribosyl)ation of proteins caused by overexpression of tankyrase 1 in yeast cells. This system allows rapid identification of inhibitory activity against tankyrase 1 and is amenable to high-throughput screening using robotics.

  20. Screen printed silver top electrode for efficient inverted organic solar cells

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Junwoo [Department of Printed Electronics, Korea Institute of Machinery & Materials (KIMM), Daejeon (Korea, Republic of); Duraisamy, Navaneethan [Department of Mechatronics Engineering, Jeju National University, Jeju (Korea, Republic of); Lee, Taik-Min [Department of Printed Electronics, Korea Institute of Machinery & Materials (KIMM), Daejeon (Korea, Republic of); Kim, Inyoung, E-mail: ikim@kimm.re.kr [Department of Printed Electronics, Korea Institute of Machinery & Materials (KIMM), Daejeon (Korea, Republic of); Choi, Kyung-Hyun, E-mail: amm@jejunu.ac.kr [Department of Mechatronics Engineering, Jeju National University, Jeju (Korea, Republic of)

    2015-10-15

    Highlights: • Screen printing of silver pattern. • X-ray diffraction pattern confirmed the face centered cubic structure of silver. • Uniform surface morphology of silver pattern with sheet resistance of 0.06 Ω/sq. • The power conversion efficiency of fabricated solar cell is found to be 2.58%. - Abstract: The present work is mainly focused on replacement of the vacuum process for top electrode fabrication in organic solar cells. Silver top electrode deposited through solution based screen printing on pre-deposited polymeric thin film. The solution based printing technology provides uniform top electrode without damaging the underlying organic layers. The surface crystallinity and surface morphology of silver top electrode are examined through X-ray diffraction, field-emission scanning electron microscope and atomic force microscope. The purity of silver is examined through X-ray energy dispersive spectroscopy. The top electrode exhibits face centered cubic structure with homogeneous morphology. The sheet resistance of top electrode is found to be 0.06 Ω/sq and an average pattern thickness of ∼15 μm. The power conversion efficiency is 2.58%. Our work demonstrates that the solution based screen printing is a significant role in the replacement of vacuum process for the fabrication of top electrode in organic solar cells.

  1. Mechanisms of cell cycle control revealed by a systematic and quantitative overexpression screen in S. cerevisiae.

    Directory of Open Access Journals (Sweden)

    Wei Niu

    2008-07-01

    Full Text Available Regulation of cell cycle progression is fundamental to cell health and reproduction, and failures in this process are associated with many human diseases. Much of our knowledge of cell cycle regulators derives from loss-of-function studies. To reveal new cell cycle regulatory genes that are difficult to identify in loss-of-function studies, we performed a near-genome-wide flow cytometry assay of yeast gene overexpression-induced cell cycle delay phenotypes. We identified 108 genes whose overexpression significantly delayed the progression of the yeast cell cycle at a specific stage. Many of the genes are newly implicated in cell cycle progression, for example SKO1, RFA1, and YPR015C. The overexpression of RFA1 or YPR015C delayed the cell cycle at G2/M phases by disrupting spindle attachment to chromosomes and activating the DNA damage checkpoint, respectively. In contrast, overexpression of the transcription factor SKO1 arrests cells at G1 phase by activating the pheromone response pathway, revealing new cross-talk between osmotic sensing and mating. More generally, 92%-94% of the genes exhibit distinct phenotypes when overexpressed as compared to their corresponding deletion mutants, supporting the notion that many genes may gain functions upon overexpression. This work thus implicates new genes in cell cycle progression, complements previous screens, and lays the foundation for future experiments to define more precisely roles for these genes in cell cycle progression.

  2. A novel approach for immunization, screening and characterization of selected scFv libraries using membrane fractions of tumor cells.

    Science.gov (United States)

    Tur, Mehmet Kemal; Rothe, Achim; Huhn, Michael; Goerres, Ute; Klimka, Alexander; Stöcker, Michael; Engert, Andreas; Fischer, Rainerr; Finner, Ricarda; Barth, Stefan

    2003-04-01

    Isolation of cell-surface specific antibodies prerequisites the functional expressing of antigens on intact cells, which are maintained routinely by cell culturing. However, long-term culturing of tumor cells could alter their antigen expression patterns and stable fixation of whole cells is not guaranteed on plastic surfaces during stringent screening procedures. We prepared functional breast cancer cell-membrane fractions that express surface molecules in their native conformation. Specific binding phages were isolated from phage antibody libraries constructed from the spleen messenger RNA of mice immunized with breast cancer cell-membrane fractions. After negative selection on non-mammary carcinoma cells and four rounds of positive selection on breast carcinoma cell lines, phage antibodies were enriched that bound specifically to breast cancer cell lines as confirmed by phage enzyme linked immunosorbent assay (ELISA) using 96-well plates coated with breast cancer cell membranes. The isolated phage antibodies were highly specific for the breast cancer cell line 8701-BC but not on other carcinoma such as the Hodgkin-derived cell line L540Cy as demonstrated by ELISA and flow cytometry. This report describes a rapid and more versatile method for isolating antibody fragments compared to whole cell screening procedures. One single membrane preparation can be stored for at least 15 months at -80 degrees C and used to immunize mice or for screening of antibody libraries. The selection and screening strategy used should be generally applicable to identify novel cell-surface antigens and their corresponding antibodies.

  3. Cytotoxicity screening of Bangladeshi medicinal plant extracts on pancreatic cancer cells

    Directory of Open Access Journals (Sweden)

    Abbasi Atiya

    2010-09-01

    Full Text Available Abstract Background There has been a long standing interest in the identification of medicinal plants and derived natural products for developing cancer therapeutics. Our study focuses upon pancreatic cancer, due to its high mortality rate, that is attributed in part to the lack of an effective chemotherapeutic agent. Previous reports on the use of medicinal plant extracts either alone or alongside conventional anticancer agents in the treatment of this cancer have shown promising results. This work aims to investigate the therapeutic properties of a library of medicinal plants from Bangladesh. Methods 56 extracts of 44 unique medicinal plants were studied. The extracts were screened for cytotoxicity against the pancreatic adenocarcinoma cell line Panc-1, using a label-free biosensor assay. The top cytotoxic extracts identified in this screen were tested on two additional pancreatic cancer cell lines (Mia-Paca2 and Capan-1 and a fibroblast cell line (Hs68 using an MTT proliferation assay. Finally, one of the most promising extracts was studied using a caspase-3 colorimetric assay to identify induction of apoptosis. Results Crude extracts of Petunia punctata, Alternanthera sessilis, and Amoora chittagonga showed cytotoxicity to three cancer cell lines with IC50 values ranging between 20.3 - 31.4 μg/mL, 13.08 - 34.9 μg/mL, and 42.8 - 49.8 μg/mL, respectively. Furthermore, treatment of Panc-1 cells with Petunia punctata was shown to increase caspase-3 activity, indicating that the observed cytotoxicity was mediated via apoptosis. Only Amoora chittagonga showed low cytotoxicity to fibroblast cells with an IC50 value > 100 μg/mL. Conclusion Based upon the initial screening work reported here, further studies aimed at the identification of active components of these three extracts and the elucidation of their mechanisms as cancer therapeutics are warranted.

  4. Quantification of cell viability and rapid screening anti-cancer drug utilizing nanomechanical fluctuation.

    Science.gov (United States)

    Wu, Shangquan; Liu, Xiaoli; Zhou, Xiarong; Liang, Xin M; Gao, Dayong; Liu, Hong; Zhao, Gang; Zhang, Qingchuan; Wu, Xiaoping

    2016-03-15

    Cancer is a serious threat to human health. Although numerous anti-cancer drugs are available clinically, many have shown toxic side effects due to poor tumor-selectivity, and reduced effectiveness due to cancers rapid development of resistance to treatment. The development of new highly efficient and practical methods to quantify cell viability and its change under drug treatment is thus of significant importance in both understanding of anti-cancer mechanism and anti-cancer drug screening. Here, we present an approach of utilizing a nanomechanical fluctuation based highly sensitive microcantilever sensor, which is capable of characterizing the viability of cells and quantitatively screening (within tens of minutes) their responses to a drug with the obvious advantages of a rapid, label-free, quantitative, noninvasive, real-time and in-situ assay. The microcantilever sensor operated in fluctuation mode was used in evaluating the paclitaxel effectiveness on breast cancer cell line MCF-7. This study demonstrated that the nanomechanical fluctuations of the microcantilever sensor are sensitive enough to detect the dynamic variation in cellular force which is provided by the cytoskeleton, using cell metabolism as its energy source, and the dynamic instability of microtubules plays an important role in the generation of the force. We propose that cell viability consists of two parts: biological viability and mechanical viability. Our experimental results suggest that paclitaxel has little effect on biological viability, but has a significant effect on mechanical viability. This new method provides a new concept and strategy for the evaluation of cell viability and the screening of anti-cancer drugs.

  5. Optimizing fuel cell parts by using 3D screen printed metals

    Energy Technology Data Exchange (ETDEWEB)

    Studnitzky, Thomas [Fraunhofer-Institue for Manufacturing and Advanced Materials, Dresden (Germany). Dept. of Powder Metallurgy and Composite Materials; Strauss, Alexander [Centre for Fuel Cell Technology, Duisburg (Germany). Dept. of Microsystems

    2010-07-01

    Miniature fuel cells have the potential to extend the runtime of various portable applications. In this context, sufficient energy densities have to be achieved within a stack in order to build fuel cell systems competitive to established battery technologies. Metallic bipolar plates composed of stainless steels permit the construction of thin and mechanically robust cells. Moreover the utilisation of the presented manufacturing method enables a new freedom in design of Bipolar Plates and their integrated flow field structures for future improvements of cell efficiency. In this study different miniature PEM fuel cells have been designed and tested. Bases for their construction are micro structured plates composed of 316L stainless steel. This design can include very fine walls down to 60 {mu}m as well as undercuts, which is impossible with other manufacturing methods. These designs were adapted by Fraunhofer IFAM for the screen printing process. As a first result a proof of concept has been established for 3D screen printing as a method for the manufacturing of fuel cell bipolar elements. To this end, a modified screen printing process is used to manufacture 3-dimensional parts layer-on-layer by depositing a suitable metallic powder which is mixed with a binder. The resulting green parts might include closed channels and channel wall thicknesses may reach 80 {mu}m or less. The green parts are debindered and sintered in order to obtain purely metallic structures. The manufactured bipolar plates show promising electric behaviour. In the current state of the project, bipolar plates with undercuts and new materials combinations are in the centre of the investigations. (orig.)

  6. Cell-based therapies and imaging in cardiology

    Energy Technology Data Exchange (ETDEWEB)

    Bengel, Frank M. [Technische Universitaet Muenchen, Nuklearmedizinische Klinik und Poliklinik, Munich (Germany); Schachinger, Volker; Dimmeler, Stefanie [University of Frankfurt, Department of Molecular Cardiology, Frankfurt (Germany)

    2005-12-01

    Cell therapy for cardiac repair has emerged as one of the most exciting and promising developments in cardiovascular medicine. Evidence from experimental and clinical studies is increasing that this innovative treatment will influence clinical practice in the future. But open questions and controversies with regard to the basic mechanisms of this therapy continue to exist and emphasise the need for specific techniques to visualise the mechanisms and success of therapy in vivo. Several non-invasive imaging approaches which aim at tracking of transplanted cells in the heart have been introduced. Among these are direct labelling of cells with radionuclides or paramagnetic agents, and the use of reporter genes for imaging of cell transplantation and differentiation. Initial studies have suggested that these molecular imaging techniques have great potential. Integration of cell imaging into studies of cardiac cell therapy holds promise to facilitate further growth of the field towards a broadly clinically useful application. (orig.)

  7. Tracking immune cells in vivo using magnetic resonance imaging.

    Science.gov (United States)

    Ahrens, Eric T; Bulte, Jeff W M

    2013-10-01

    The increasing complexity of in vivo imaging technologies, coupled with the development of cell therapies, has fuelled a revolution in immune cell tracking in vivo. Powerful magnetic resonance imaging (MRI) methods are now being developed that use iron oxide- and ¹⁹F-based probes. These MRI technologies can be used for image-guided immune cell delivery and for the visualization of immune cell homing and engraftment, inflammation, cell physiology and gene expression. MRI-based cell tracking is now also being applied to evaluate therapeutics that modulate endogenous immune cell recruitment and to monitor emerging cellular immunotherapies. These recent uses show that MRI has the potential to be developed in many applications to follow the fate of immune cells in vivo.

  8. Nonlinear Microwave Imaging for Breast-Cancer Screening Using Gauss–Newton's Method and the CGLS Inversion Algorithm

    DEFF Research Database (Denmark)

    Rubæk, Tonny; Meaney, P. M.; Meincke, Peter;

    2007-01-01

    Breast-cancer screening using microwave imaging is emerging as a new promising technique as a supplement to X-ray mammography. To create tomographic images from microwave measurements, it is necessary to solve a nonlinear inversion problem, for which an algorithm based on the iterative Gauss-Newton...... method has been developed at Dartmouth College. This algorithm determines the update values at each iteration by solving the set of normal equations of the problem using the Tikhonov algorithm. In this paper, a new algorithm for determining the iteration update values in the Gauss-Newton algorithm...... algorithm is compared to the Gauss-Newton algorithm with Tikhonov regularization and is shown to reconstruct images of similar quality using fewer iterations....

  9. Fabrication of Large-Scale Microlens Arrays Based on Screen Printing for Integral Imaging 3D Display.

    Science.gov (United States)

    Zhou, Xiongtu; Peng, Yuyan; Peng, Rong; Zeng, Xiangyao; Zhang, Yong-Ai; Guo, Tailiang

    2016-09-14

    The low-cost large-scale fabrication of microlens arrays (MLAs) with precise alignment, great uniformity of focusing, and good converging performance are of great importance for integral imaging 3D display. In this work, a simple and effective method for large-scale polymer microlens arrays using screen printing has been successfully presented. The results show that the MLAs possess high-quality surface morphology and excellent optical performances. Furthermore, the microlens' shape and size, i.e., the diameter, the height, and the distance between two adjacent microlenses of the MLAs can be easily controlled by modifying the reflowing time and the size of open apertures of the screen. MLAs with the neighboring microlenses almost tangent can be achieved under suitable size of open apertures of the screen and reflowing time, which can remarkably reduce the color moiré patterns caused by the stray light between the blank areas of the MLAs in the integral imaging 3D display system, exhibiting much better reconstruction performance.

  10. Whole-cell microtiter plate screening assay for terminal hydroxylation of fatty acids by P450s.

    Science.gov (United States)

    Weissenborn, Martin J; Notonier, Sandra; Lang, Sarah-Luise; Otte, Konrad B; Herter, Susanne; Turner, Nicholas J; Flitsch, Sabine L; Hauer, Bernhard

    2016-05-04

    A readily available galactose oxidase (GOase) variant was used to develop a whole cell screening assay. This endpoint detection system was applied in a proof-of-concept approach by screening a focussed mutant library. This led to the discovery of the thus far most active P450 Marinobacter aquaeolei mutant catalysing the terminal hydroxylation of fatty acids.

  11. High-Throughput Accurate Single-Cell Screening of Euglena gracilis with Fluorescence-Assisted Optofluidic Time-Stretch Microscopy

    Science.gov (United States)

    Guo, Baoshan; Lei, Cheng; Ito, Takuro; Jiang, Yiyue; Ozeki, Yasuyuki; Goda, Keisuke

    2016-01-01

    The development of reliable, sustainable, and economical sources of alternative fuels is an important, but challenging goal for the world. As an alternative to liquid fossil fuels, algal biofuel is expected to play a key role in alleviating global warming since algae absorb atmospheric CO2 via photosynthesis. Among various algae for fuel production, Euglena gracilis is an attractive microalgal species as it is known to produce wax ester (good for biodiesel and aviation fuel) within lipid droplets. To date, while there exist many techniques for inducing microalgal cells to produce and accumulate lipid with high efficiency, few analytical methods are available for characterizing a population of such lipid-accumulated microalgae including E. gracilis with high throughout, high accuracy, and single-cell resolution simultaneously. Here we demonstrate high-throughput, high-accuracy, single-cell screening of E. gracilis with fluorescence-assisted optofluidic time-stretch microscopy–a method that combines the strengths of microfluidic cell focusing, optical time-stretch microscopy, and fluorescence detection used in conventional flow cytometry. Specifically, our fluorescence-assisted optofluidic time-stretch microscope consists of an optical time-stretch microscope and a fluorescence analyzer on top of a hydrodynamically focusing microfluidic device and can detect fluorescence from every E. gracilis cell in a population and simultaneously obtain its image with a high throughput of 10,000 cells/s. With the multi-dimensional information acquired by the system, we classify nitrogen-sufficient (ordinary) and nitrogen-deficient (lipid-accumulated) E. gracilis cells with a low false positive rate of 1.0%. This method holds promise for evaluating cultivation techniques and selective breeding for microalgae-based biofuel production. PMID:27846239

  12. Circulating cell-free microRNA as biomarkers for screening, diagnosis and monitoring of neurodegenerative diseases and other neurologic pathologies

    Directory of Open Access Journals (Sweden)

    Kira S Sheinerman

    2013-09-01

    Full Text Available Many neurodegenerative diseases, such as Alzheimer’s disease, Parkinson disease, vascular and frontotemporal dementias, as well as other chronic neurological pathologies, are characterized by slow development with a long asymptomatic period followed by a stage with mild clinical symptoms. As a consequence, these serious pathologies are diagnosed late in the course of a disease, when massive death of neurons has already occurred and effective therapeutic intervention is problematic. Thus, the development of screening tests capable of detecting neurodegenerative diseases during early, preferably asymptomatic, stages is a high unmet need. Since such tests are to be used for screening of large populations, they should be non-invasive and relatively inexpensive. Further, while subjects identified by screening tests can be further tested with more invasive and expensive methods, e.g. analysis of cerebrospinal fluid or imaging techniques, to be of practical utility screening tests should have high sensitivity and specificity. In this review, we discuss advantages and disadvantages of various approaches to developing screening tests based on analysis of circulating cell-free miRNA. Applications of circulating miRNA-based tests for diagnosis of acute and chronic brain pathologies, for research of normal brain aging, and for disease and treatment monitoring are also discussed.

  13. Highly sensitive quantitative imaging for monitoring single cancer cell growth kinetics and drug response.

    Directory of Open Access Journals (Sweden)

    Mustafa Mir

    Full Text Available The detection and treatment of cancer has advanced significantly in the past several decades, with important improvements in our understanding of the fundamental molecular and genetic basis of the disease. Despite these advancements, drug-screening methodologies have remained essentially unchanged since the introduction of the in vitro human cell line screen in 1990. Although the existing methods provide information on the overall effects of compounds on cell viability, they are restricted by bulk measurements, large sample sizes, and lack capability to measure proliferation kinetics at the individual cell level. To truly understand the nature of cancer cell proliferation and to develop personalized adjuvant therapies, there is a need for new methodologies that provide quantitative information to monitor the effect of drugs on cell growth as well as morphological and phenotypic changes at the single cell level. Here we show that a quantitative phase imaging modality known as spatial light interference microscopy (SLIM addresses these needs and provides additional advantages over existing proliferation assays. We demonstrate these capabilities through measurements on the effects of the hormone estradiol and the antiestrogen ICI182,780 (Faslodex on the growth of MCF-7 breast cancer cells. Along with providing information on changes in the overall growth, SLIM provides additional biologically relevant information. For example, we find that exposure to estradiol results in rapidly growing cells with lower dry mass than the control population. Subsequently blocking the estrogen receptor with ICI results in slower growing cells, with lower dry masses than the control. This ability to measure changes in growth kinetics in response to environmental conditions provides new insight on growth regulation mechanisms. Our results establish the capabilities of SLIM as an advanced drug screening technology that provides information on changes in proliferation

  14. Features of undiagnosed breast cancers at screening breast MR imaging and potential utility of computer-aided evaluation

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Mirinae; Cho, Nariya; Bea, Min Sun; Koo, Hye Ryoung; Kim, Won Hwa; Lee, Su Hyun; Chu, A Jung [Dept. of Radiology, Seoul National University Hospital, Seoul (Korea, Republic of)

    2016-02-15

    To retrospectively evaluate the features of undiagnosed breast cancers on prior screening breast magnetic resonance (MR) images in patients who were subsequently diagnosed with breast cancer, as well as the potential utility of MR-computer-aided evaluation (CAE). Between March 2004 and May 2013, of the 72 consecutive pairs of prior negative MR images and subsequent MR images with diagnosed cancers (median interval, 32.8 months; range, 5.4-104.6 months), 36 (50%) had visible findings (mean size, 1.0 cm; range, 0.3-5.2 cm). The visible findings were divided into either actionable or under threshold groups by the blinded review by 5 radiologists. MR imaging features, reasons for missed cancer, and MR-CAE features according to actionability were evaluated. Of the 36 visible findings on prior MR images, 33.3% (12 of 36) of the lesions were determined to be actionable and 66.7% (24 of 36) were underthreshold; 85.7% (6 of 7) of masses and 31.6% (6 of 19) of non-mass enhancements were classified as actionable lesions. Mimicking physiologic enhancements (27.8%, 10 of 36) and small lesion size (27.8%, 10 of 36) were the most common reasons for missed cancer. Actionable findings tended to show more washout or plateau kinetic patterns on MR-CAE than underthreshold findings, as the 100% of actionable findings and 46.7% of underthreshold findings showed washout or plateau (p = 0.008). MR-CAE has the potential for reducing the number of undiagnosed breast cancers on screening breast MR images, the majority of which are caused by mimicking physiologic enhancements or small lesion size.

  15. Rapid screening of potential autophagic inductor agents using mammalian cell lines.

    Science.gov (United States)

    Martins, Waleska K; Severino, Divinomar; Souza, Cleidiane; Stolf, Beatriz S; Baptista, Maurício S

    2013-06-01

    Recent progress in understanding the molecular basis of autophagy has demonstrated its importance in several areas of human health. Affordable screening techniques with higher sensitivity and specificity to identify autophagy are, however, needed to move the field forward. In fact, only laborious and/or expensive methodologies such as electron microscopy, dye-staining of autophagic vesicles, and LC3-II immunoblotting or immunoassaying are available for autophagy identification. Aiming to fulfill this technical gap, we describe here the association of three widely used assays to determine cell viability - Crystal Violet staining (CVS), 3-[4, 5-dimethylthiaolyl]-2, 5-diphenyl-tetrazolium bromide (MTT) reduction, and neutral red uptake (NRU) - to predict autophagic cell death in vitro. The conceptual framework of the method is the superior uptake of NR in cells engaging in autophagy. NRU was then weighted by the average of MTT reduction and CVS allowing the calculation of autophagic arbitrary units (AAU), a numeric variable that correlated specifically with the autophagic cell death. The proposed strategy is very useful for drug discovery, allowing the investigation of potential autophagic inductor agents through a rapid screening using mammalian cell lines B16-F10, HaCaT, HeLa, MES-SA, and MES-SA/Dx5 in a unique single microplate.

  16. 19.4%-efficient large-area fully screen-printed silicon solar cells

    Energy Technology Data Exchange (ETDEWEB)

    Gatz, Sebastian; Hannebauer, Helge; Hesse, Rene; Werner, Florian; Schmidt, Arne; Dullweber, Thorsten; Bothe, Karsten [Institute for Solar Energy Hamelin (ISFH), Am Ohrberg 1, 31860 Emmerthal (Germany); Schmidt, Jan; Brendel, Rolf [Institute for Solar Energy Hamelin (ISFH), Am Ohrberg 1, 31860 Emmerthal (Germany); Institute of Solid-State Physics, University of Hannover, Appelstrasse 2, 30167 Hannover (Germany)

    2011-04-15

    We demonstrate industrially feasible large-area solar cells with passivated homogeneous emitter and rear achieving energy conversion efficiencies of up to 19.4% on 125 x 125 mm{sup 2} p-type 2-3 {omega} cm boron-doped Czochralski silicon wafers. Front and rear metal contacts are fabricated by screen-printing of silver and aluminum paste and firing in a conventional belt furnace. We implement two different dielectric rear surface passivation stacks: (i) a thermally grown silicon dioxide/silicon nitride stack and (ii) an atomic-layer-deposited aluminum oxide/silicon nitride stack. The dielectrics at the rear result in a decreased surface recombination velocity of S{sub rear} = 70 cm/s and 80 cm/s, and an increased internal IR reflectance of up to 91% corresponding to an improved J{sub sc} of up to 38.9 mA/cm{sup 2} and V{sub oc} of up to 664 mV. We observe an increase in cell efficiency of 0.8% absolute for the cells compared to 18.6% efficient reference solar cells featuring a full-area aluminum back surface field. To our knowledge, the energy conversion efficiency of 19.4% is the best value reported so far for large area screen-printed solar cells. (copyright 2011 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  17. Optimizing human embryonic stem cells differentiation efficiency by screening size-tunable homogenous embryoid bodies.

    Science.gov (United States)

    Moon, Sung-Hwan; Ju, Jongil; Park, Soon-Jung; Bae, Daekyeong; Chung, Hyung-Min; Lee, Sang-Hoon

    2014-07-01

    Human embryonic stem cells (hESCs) are generally induced to differentiate by forming spherical structures termed embryoid bodies (EBs) in the presence of soluble growth factors. hEBs are generated by suspending small clumps of hESC colonies; however, the resulting hEBs are heterogeneous because this method lacks the ability to control the number of cells in individual EBs. This heterogeneity affects factors that influence differentiation such as cell-cell contact and the diffusion of soluble factors, and consequently, the differentiation capacity of each EB varies. Here, we fabricated size-tunable concave microwells to control the physical environment, thereby regulating the size of EBs formed from single hESCs. Defined numbers of single hESCs were forced to aggregate and generate uniformly sized EBs with high fidelity, and the size of the EBs was controlled using concave microwells of different diameters. Differentiation patterns in H9- and CHA15-hESCs were affected by EB size in both the absence and presence of growth factors. By screening EB size in the presence of various BMP4 concentrations, a two-fold increase in endothelial cell differentiation was achieved. Because each hESC line has unique characteristics, the findings of this study demonstrate that concave microwells could be used to screen different EB sizes and growth factor concentrations to optimize differentiation for each hESC line.

  18. Modulators of prostate cancer cell proliferation and viability identified by short-hairpin RNA library screening.

    Directory of Open Access Journals (Sweden)

    Kimberly Brown Dahlman

    Full Text Available There is significant need to identify novel prostate cancer drug targets because current hormone therapies eventually fail, leading to a drug-resistant and fatal disease termed castration-resistant prostate cancer. To functionally identify genes that, when silenced, decrease prostate cancer cell proliferation or induce cell death in combination with antiandrogens, we employed an RNA interference-based short hairpin RNA barcode screen in LNCaP human prostate cancer cells. We identified and validated four candidate genes (AKT1, PSMC1, STRADA, and TTK that impaired growth when silenced in androgen receptor positive prostate cancer cells and enhanced the antiproliferative effects of antiandrogens. Inhibition of AKT with a pharmacologic inhibitor also induced apoptosis when combined with antiandrogens, consistent with recent evidence for PI3K and AR pathway crosstalk in prostate cancer cells. Recovery of hairpins targeting a known prostate cancer pathway validates the utility of shRNA library screening in prostate cancer as a broad strategy to identify new candidate drug targets.

  19. Modulators of prostate cancer cell proliferation and viability identified by short-hairpin RNA library screening.

    Science.gov (United States)

    Dahlman, Kimberly Brown; Parker, Joel S; Shamu, Tambudzai; Hieronymus, Haley; Chapinski, Caren; Carver, Brett; Chang, Kenneth; Hannon, Gregory J; Sawyers, Charles L

    2012-01-01

    There is significant need to identify novel prostate cancer drug targets because current hormone therapies eventually fail, leading to a drug-resistant and fatal disease termed castration-resistant prostate cancer. To functionally identify genes that, when silenced, decrease prostate cancer cell proliferation or induce cell death in combination with antiandrogens, we employed an RNA interference-based short hairpin RNA barcode screen in LNCaP human prostate cancer cells. We identified and validated four candidate genes (AKT1, PSMC1, STRADA, and TTK) that impaired growth when silenced in androgen receptor positive prostate cancer cells and enhanced the antiproliferative effects of antiandrogens. Inhibition of AKT with a pharmacologic inhibitor also induced apoptosis when combined with antiandrogens, consistent with recent evidence for PI3K and AR pathway crosstalk in prostate cancer cells. Recovery of hairpins targeting a known prostate cancer pathway validates the utility of shRNA library screening in prostate cancer as a broad strategy to identify new candidate drug targets.

  20. Bioprinting 3D cell-laden hydrogel microarray for screening human periodontal ligament stem cell response to extracellular matrix.

    Science.gov (United States)

    Ma, Yufei; Ji, Yuan; Huang, Guoyou; Ling, Kai; Zhang, Xiaohui; Xu, Feng

    2015-12-22

    Periodontitis is an inflammatory disease negatively affecting up to 15% of adults worldwide. Periodontal ligament stem cells (PDLSCs) hold great promises for periodontal tissue regeneration, where it is necessary to find proper extracellular matrix (ECM) materials (e.g., composition, concentration). In this study, we proposed a bioprinting-based approach to generate nano-liter sized three-dimensional (3D) cell-laden hydrogel array with gradient of ECM components, through controlling the volume ratio of two hydrogels, such as gelatin methacrylate (GelMA) and poly(ethylene glycol) (PEG) dimethacrylate. The resulting cell-laden array with a gradient of GelMA/PEG composition was used to screen human PDLSC response to ECM. The behavior (e.g., cell viability, spreading) of human PDLSCs in GelMA/PEG array were found to be depended on the volume ratios of GelMA/PEG, with cell viability and spreading area decreased along with increasing the ratio of PEG. The developed approach would be useful for screening cell-biomaterial interaction in 3D and promoting regeneration of functional tissue.

  1. Microfluidic geometric metering-based multi-reagent mixture generator for robust live cell screening array.

    Science.gov (United States)

    Wang, Han; Kim, Jeongyun; Jayaraman, Arul; Han, Arum

    2014-12-01

    Microfluidic live cell arrays with integrated concentration gradient or mixture generators have been utilized in screening cellular responses to various biomolecular cues. Microfluidic network-based gradient generators that can create concentration gradients by repeatedly splitting and mixing different solutions using networks of serpentine channels are commonly used. However, in this method the generation of concentration gradients relies on the continuous flow of sample solutions at optimized flow rates, which poses challenges in maintaining the pressure and flow stability throughout the entire assay period. Here we present a microfluidic live cell screening array with an on-demand multi-reagent mixture generator where the mixing ratios, thus generated concentrations, are hard-wired into the chip itself through a geometric metering method. This platform showed significantly improved robustness and repeatability in generating concentration gradients of fluorescent dyes (average coefficient of variance C.V. = 9 %) compared to the conventional network-based gradient generators (average C.V. = 21 %). In studying the concentration dependent effects of the environmental toxicant 3-methylcholanthrene (3MC) on the activation of cytochrome P450 1A1 (Cyp 1A1) enzyme in H4IIE rat hepatoma cells, statistical variation of the Cyp 1A1 response was significantly lower (C.V. = 5 %) when using the developed mixture generator compared to that using the conventional gradient generator (C.V. = 12 %). Reduction in reagent consumption by 12-times was also achieved. This robust, accurate, and scalable multi-reagent mixture generator integrated with a cell culture array as a live cell assay platform can be readily implemented into various screening applications where repeatability, robustness, and low reagent consumptions over long periods of assay time are of importance.

  2. Hollow fiber cell fishing with high performance liquid chromatography for screening bioactive compounds from traditional Chinese medicines.

    Science.gov (United States)

    Xue, Xue; Li, Lihua; Chen, Xuan; Hu, Shuang; Bai, Xiaohong

    2013-03-08

    A novel hollow fiber cell fishing method with high performance liquid chromatography was proposed and used to screen, isolate, and analyze bioactive compounds from Traditional Chinese Medicines (TCMs). The active compounds that interact with the living cells acceptor inside the hollow fiber lumen were screened and isolated from the TCM extracts in phosphate buffer solution (pH 7.4). Subsequently, the active compounds bound to the cells were desorbed with methanol, and were analyzed using HPLC. HFCF with HPLC was introduced for the screening and analysis of lignans in Schisandra chinensis (Turcz) Baill and coumarins in Fructus Cnidii and Fructus Psoraleae. The surface properties of the hollow fibers filled with living cells were characterized. The nonspecific binding between the active centers of the hollow fibers and the bioactive compounds were investigated. The cell survival rates were determined before and after the screening. The repeatability of the method was tested. Some structures of the lignans and coumarins screened from TCMs were identified by the comparison to the retention times of the reference substances. HFCF-HPLC is a simple, fast, effective, and reliable method for the screening and analysis of bioactive compounds, and it can be extended to screen other bioactive compounds from TCMs.

  3. Measurement of cell traction forces with ImageJ.

    Science.gov (United States)

    Martiel, Jean-Louis; Leal, Aldo; Kurzawa, Laetitia; Balland, Martial; Wang, Irene; Vignaud, Timothée; Tseng, Qingzong; Théry, Manuel

    2015-01-01

    The quantification of cell traction forces requires three key steps: cell plating on a deformable substrate, measurement of substrate deformation, and the numerical estimation of the corresponding cell traction forces. The computing steps to measure gel deformation and estimate the force field have somehow limited the adoption of this method in cell biology labs. Here we propose a set of ImageJ plug-ins so that every lab equipped with a fluorescent microscope can measure cell traction forces.

  4. Immunomagnetic separation of tumor initiating cells by screening two surface markers

    Science.gov (United States)

    Sun, Chen; Hsieh, Yuan-Pang; Ma, Sai; Geng, Shuo; Cao, Zhenning; Li, Liwu; Lu, Chang

    2017-01-01

    Isolating tumor initiating cells (TICs) often requires screening of multiple surface markers, sometimes with opposite preferences. This creates a challenge for using bead-based immunomagnetic separation (IMS) that typically enriches cells based on one abundant marker. Here, we propose a new strategy that allows isolation of CD44+/CD24− TICs by IMS involving both magnetic beads coated by anti-CD44 antibody and nonmagnetic beads coated by anti-CD24 antibody (referred to as two-bead IMS). Cells enriched with our approach showed significant enhancement in TIC marker expression (examined by flow cytometry) and improved tumorsphere formation efficiency. Our method will extend the application of IMS to cell subsets characterized by multiple markers. PMID:28074882

  5. An efficient detection agent for the high throughput screening of recombinant manufacturing cell lines.

    Science.gov (United States)

    Duverger, Valérie; Sauvage, Christophe; Kobr, Michel; Imhof, Markus O

    2013-12-31

    To ensure the selection of high producing recombinant cell lines, a number of screening processes were developed in the presence of detection agents. Here, CHO cell lines secreting recombinant antibodies were detected in semi-solid medium containing detection agents. The aim was to compare two protein A-derived detection agents to two commercial fluorescent antibodies directed against the Fc part of the antibody of interest: the protein A derived Z domain fused to the red fluorescent protein and protein A labelled with a fluorescent Dylight™ 488 dye. All of these agents were compatible with cell recovery and colony formation, and specifically detected colonies secreting recombinant antibodies. Optimisation of the concentration of the fluorescent protein A allowed the identification of a higher number of good producers. Thus these data demonstrate that fluorescently labelled protein A-derivatives can be used for the selection of high producer cells.

  6. Automatic segmentation of HeLa cell images

    CERN Document Server

    Urban, Jan

    2011-01-01

    In this work, the possibilities for segmentation of cells from their background and each other in digital image were tested, combined and improoved. Lot of images with young, adult and mixture cells were able to prove the quality of described algorithms. Proper segmentation is one of the main task of image analysis and steps order differ from work to work, depending on input images. Reply for biologicaly given question was looking for in this work, including filtration, details emphasizing, segmentation and sphericity computing. Order of algorithms and way to searching for them was also described. Some questions and ideas for further work were mentioned in the conclusion part.

  7. In vivo SPECT reporter gene imaging of regulatory T cells.

    Directory of Open Access Journals (Sweden)

    Ehsan Sharif-Paghaleh

    Full Text Available Regulatory T cells (Tregs were identified several years ago and are key in controlling autoimmune diseases and limiting immune responses to foreign antigens, including alloantigens. In vivo imaging techniques including intravital microscopy as well as whole body imaging using bioluminescence probes have contributed to the understanding of in vivo Treg function, their mechanisms of action and target cells. Imaging of the human sodium/iodide symporter via Single Photon Emission Computed Tomography (SPECT has been used to image various cell types in vivo. It has several advantages over the aforementioned imaging techniques including high sensitivity, it allows non-invasive whole body studies of viable cell migration and localisation of cells over time and lastly it may offer the possibility to be translated to the clinic. This study addresses whether SPECT/CT imaging can be used to visualise the migratory pattern of Tregs in vivo. Treg lines derived from CD4(+CD25(+FoxP3(+ cells were retrovirally transduced with a construct encoding for the human Sodium Iodide Symporter (NIS and the fluorescent protein mCherry and stimulated with autologous DCs. NIS expressing self-specific Tregs were specifically radiolabelled in vitro with Technetium-99m pertechnetate ((99mTcO(4(- and exposure of these cells to radioactivity did not affect cell viability, phenotype or function. In addition adoptively transferred Treg-NIS cells were imaged in vivo in C57BL/6 (BL/6 mice by SPECT/CT using (99mTcO(4(-. After 24 hours NIS expressing Tregs were observed in the spleen and their localisation was further confirmed by organ biodistribution studies and flow cytometry analysis. The data presented here suggests that SPECT/CT imaging can be utilised in preclinical imaging studies of adoptively transferred Tregs without affecting Treg function and viability thereby allowing longitudinal studies within disease models.

  8. Genome-Wide Screen for Haploinsufficient Cell Size Genes in the Opportunistic Yeast Candida albicans

    Directory of Open Access Journals (Sweden)

    Julien Chaillot

    2017-02-01

    Full Text Available One of the most critical but still poorly understood aspects of eukaryotic cell proliferation is the basis for commitment to cell division in late G1 phase, called Start in yeast and the Restriction Point in metazoans. In all species, a critical cell size threshold coordinates cell growth with cell division and thereby establishes a homeostatic cell size. While a comprehensive survey of cell size genetic determinism has been performed in the saprophytic yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, very little is known in pathogenic fungi. As a number of critical Start regulators are haploinsufficient for cell size, we applied a quantitative analysis of the size phenome, using elutriation-barcode sequencing methodology, to 5639 barcoded heterozygous deletion strains of the opportunistic yeast Candida albicans. Our screen identified conserved known regulators and biological processes required to maintain size homeostasis in the opportunistic yeast C. albicans. We also identified novel C. albicans-specific size genes and provided a conceptual framework for future mechanistic studies. Interestingly, some of the size genes identified were required for fungal pathogenicity suggesting that cell size homeostasis may be elemental to C. albicans fitness or virulence inside the host.

  9. Genome-Wide Screen for Haploinsufficient Cell Size Genes in the Opportunistic Yeast Candida albicans

    Science.gov (United States)

    Chaillot, Julien; Cook, Michael A.; Corbeil, Jacques; Sellam, Adnane

    2016-01-01

    One of the most critical but still poorly understood aspects of eukaryotic cell proliferation is the basis for commitment to cell division in late G1 phase, called Start in yeast and the Restriction Point in metazoans. In all species, a critical cell size threshold coordinates cell growth with cell division and thereby establishes a homeostatic cell size. While a comprehensive survey of cell size genetic determinism has been performed in the saprophytic yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, very little is known in pathogenic fungi. As a number of critical Start regulators are haploinsufficient for cell size, we applied a quantitative analysis of the size phenome, using elutriation-barcode sequencing methodology, to 5639 barcoded heterozygous deletion strains of the opportunistic yeast Candida albicans. Our screen identified conserved known regulators and biological processes required to maintain size homeostasis in the opportunistic yeast C. albicans. We also identified novel C. albicans-specific size genes and provided a conceptual framework for future mechanistic studies. Interestingly, some of the size genes identified were required for fungal pathogenicity suggesting that cell size homeostasis may be elemental to C. albicans fitness or virulence inside the host. PMID:28040776

  10. Preparation of Single Cells for Imaging Mass Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Berman, E S; Fortson, S L; Kulp, K S; Checchi, K D; Wu, L; Felton, J S; Wu, K J

    2007-10-24

    Characterizing chemical changes within single cells is important for determining fundamental mechanisms of biological processes that will lead to new biological insights and improved disease understanding. Imaging biological systems with mass spectrometry (MS) has gained popularity in recent years as a method for creating precise chemical maps of biological samples. In order to obtain high-quality mass spectral images that provide relevant molecular information about individual cells, samples must be prepared so that salts and other cell-culture components are removed from the cell surface and the cell contents are rendered accessible to the desorption beam. We have designed a cellular preparation protocol for imaging MS that preserves the cellular contents for investigation and removes the majority of the interfering species from the extracellular matrix. Using this method, we obtain excellent imaging results and reproducibility in three diverse cell types: MCF7 human breast cancer cells, Madin-Darby canine kidney (MDCK) cells, and NIH/3T3 mouse fibroblasts. This preparation technique allows routine imaging MS analysis of cultured cells, allowing for any number of experiments aimed at furthering scientific understanding of molecular processes within individual cells.

  11. Bioorthogonal SERS Nanoprobes for Mulitplex Spectroscopic Detection, Tumor Cell Targeting, and Tissue Imaging.

    Science.gov (United States)

    Wu, Junzhou; Liang, Duanwei; Jin, Qingqing; Liu, Jie; Zheng, Meiling; Duan, Xuanming; Tang, Xinjing

    2015-09-07

    A surface-enhanced Raman scattering (SERS) technique shows extraordinary features for a range of biological and biomedical applications. Herein, a series of novel bioorthogonal SERS nanoprobes were constructed with Gold nanoflower (AuNF) and Raman reporters, the signals of which were located in a Raman-silent region of biological samples. AS1411 aptamer was also co-conjugated with AuNF through a self-assembled monolayer coverage strategy. Multiplex SERS imaging using these nanoprobes with three different bioorthogonal small-molecule Raman reporters is successfully achieved with high multiplexing capacity in a biologically Raman-silent region. These Raman nanoprobes co-conjugated with AS1411 showed high affinity for tumor cells with overexpressed nucleolin and can be used for selective tumor cell screening and tissue imaging.

  12. Coupling of a scanning flow cell with online electrochemical mass spectrometry for screening of reaction selectivity

    Science.gov (United States)

    Grote, Jan-Philipp; Zeradjanin, Aleksandar R.; Cherevko, Serhiy; Mayrhofer, Karl J. J.

    2014-10-01

    In this work the online coupling of a miniaturized electrochemical scanning flow cell (SFC) to a mass spectrometer is introduced. The system is designed for the determination of reaction products in dependence of the applied potential and/or current regime as well as fast and automated change of the sample. The reaction products evaporate through a hydrophobic PTFE membrane into a small vacuum probe, which is positioned only 50-100 μm away from the electrode surface. The probe is implemented into the SFC and directly connected to the mass spectrometer. This unique configuration enables fast parameter screening for complex electrochemical reactions, including investigation of operation conditions, composition of electrolyte, and material composition. The technical developments of the system are validated by initial measurements of hydrogen evolution during water electrolysis and electrochemical reduction of CO2 to various products, showcasing the high potential for systematic combinatorial screening by this approach.

  13. Genetic Screening for Bacterial Mutants in Liquid Growth Media By Fluorescence-Activated Cell Sorting

    Science.gov (United States)

    Abuaita, Basel H.; Withey, Jeffrey H.

    2010-01-01

    Many bacterial pathogens have defined in vitro virulence inducing conditions in liquid media which lead to production of virulence factors important during an infection. Identifying mutants that no longer respond to virulence inducing conditions will increase our understanding of bacterial pathogenesis. However, traditional genetic screens require growth on solid media. Bacteria in a single colony are in every phase of the growth curve, which complicates the analysis and make screens for growth phase-specific mutants problematic. Here, we utilize fluorescence-activated cell sorting in conjunction with random transposon mutagenesis to isolate bacteria grown in liquid media that are defective in virulence activation. This method permits analysis of an entire bacterial population in real time and selection of individual bacterial mutants with the desired gene expression profile at any time point after induction. We have used this method to identify Vibrio cholerae mutants defective in virulence induction. PMID:21094189

  14. Screening of Compounds Toxicity against Human Monocytic cell line-THP-1 by Flow Cytometry

    Directory of Open Access Journals (Sweden)

    Pick Neora

    2004-01-01

    Full Text Available The worldwide rapid increase in bacterial resistance to numerous antibiotics requires on-going development of new drugs to enter the market. As the development of new antibiotics is lengthy and costly, early monitoring of compound's toxicity is essential in the development of novel agents. Our interest is in a rapid, simple, high throughput screening method to assess cytotoxicity induced by potential agents. Some intracellular pathogens, such as Mycobacterium tuberculosis primary site of infection is human alveolar macrophages. Thus, evaluation of candidate drugs for macrophage toxicity is crucial. Protocols for high throughput drug toxicity screening of macrophages using flow cytometry are lacking in the literature. For this application we modified a preexisting technique, propidium iodide (PI exclusion staining and utilized it for rapid toxicity tests. Samples were prepared in 96 well plates and analyzed by flow cytometry, which allowed for rapid, inexpensive and precise assessment of compound's toxicity associated with cell death.

  15. Silicon solar cells textured by reactive ion etching and processed with screen printing

    Energy Technology Data Exchange (ETDEWEB)

    Burgers, A.R.; Tool, C.J.J.; Hylton, J.D.; Weeber, A.W. [ECN Solar and Wind, Netherlands Energy Research Foundation ECN, Petten (Netherlands); Verholen, A.G.B.J.; Gardeniers, J.G.E.; De Boer, M.J.; Elwenspoek, M.C. [MESA Research Institute for Micro Electronics, Materials- and Molecular Engineering, and Sensors and Actuators, University of Twente, Enschede (Netherlands)

    1998-07-01

    Reactive Ion Etching (RIE) can texture multicrystalline silicon wafers very well. RIE has the advantage over alkaline etches that it is not sensitive to the crystallographic orientation. RIE has the advantage over acid etches that it can be controlled much better. This work describes multicrystalline silicon solar cells textured by RIE and processed with an industrial process sequence consisting of mainly screen-printing steps. We also made mini-modules of the RIE textured cells. The RIE texturing parameters can be varied to give different textures. Care must be taken that the texture is sufficiently rough to give a low encapsulated reflectance but not so rough that carriers generated by light absorbed in the texture recombine at the surface and cannot be collected at the p-n junction. The screen printing on RIE textured cells was successful. We were able to make cells with both low reflectance and good quantum efficiency. Due to encapsulation problems we were not able to demonstrate an efficiency gain yet. 10 refs.

  16. Screening of Differently Expressed Genes in Human Prostate Cancer Cell Lines with Different Metastasis Potentials

    Institute of Scientific and Technical Information of China (English)

    SONG Anping; LIAO Guoning; WU Mingfu; LU Yunping; MA Ding

    2007-01-01

    In order to screen the genes differentially expressed in two human prostate cancer cells with different metastasis potentials, suppression subtractive hybridization (SSH) was done twice on human prostate cancer cell line with high potential of metastasis PC3M-1E8 and its synogenetic cell line PC3M-2B4 with low metastasis potential. In the first subtraction PC3M-2B4 was used as tester and PC3M-1E8 as driver and the forward subtractive library was constructed. In the second one the tester and driver were interchanged and the reverse subtractive library was constructed. The screened clones of both libraries were sequenced and Gene Bank homology search was performed. Some clones were confirmed by quantitative real-time PCR. The results showed that two subtrac-tive libraries containing 238 positive clones were constructed. Analysis of 16 sequenced clones ran-domly picked from two libraries showed that 4 differentially expressed gene fragments were identi-fied as new EST with unknown functions. It was concluded that two subtractive libraries of human prostate cancer cell lines with different metastasis potentials were constructed successfully.

  17. Development of a cell-based, high-throughput screening assay for ATM kinase inhibitors.

    Science.gov (United States)

    Guo, Kexiao; Shelat, Anang A; Guy, R Kiplin; Kastan, Michael B

    2014-04-01

    The ATM (ataxia-telangiectasia, mutated) protein kinase is a major regulator of cellular responses to DNA double-strand breaks (DSBs), DNA lesions that can be caused by ionizing irradiation (IR), oxidative damage, or exposure to certain chemical agents. In response to DSBs, the ATM kinase is activated and subsequently phosphorylates numerous downstream substrates, including p53, Chk2, BRCA1, and KAP1, which affect processes such as cell cycle progression and DNA repair. Numerous studies have demonstrated that loss of ATM function results in enhanced sensitivity to ionizing irradiation in clinically relevant dose ranges, suggesting that ATM kinase is an attractive therapeutic target for enhancing tumor cell kill with radiotherapy. Previously identified small-molecule ATM kinase inhibitors, such as CP466722 and Ku55933, were identified using in vitro kinase assays carried out with recombinant ATM kinase isolated from mammalian cells. Since it has not been feasible to express full-length recombinant ATM in bacterial or baculovirus systems, a robust in vitro screening tool has been lacking. We have developed a cell-based assay that is robust, straightforward, and sensitive. Using this high-throughput assay, we screened more than 7000 compounds and discovered additional small molecules that inhibit the ATM kinase and further validated these hits by secondary assays.

  18. Suitability of Cell-Based Label-Free Detection for Cytotoxicity Screening of Carbon Nanotubes

    Directory of Open Access Journals (Sweden)

    Claudia Meindl

    2013-01-01

    Full Text Available Cytotoxicity testing of nanoparticles (NPs by conventional screening assays is often complicated by interference. Carbon nanotubes (CNTs are particularly difficult to assess. To test the suitability of cell-based label-free techniques for this application, a panel of CNTs with different diameters and surface functionalizations was assessed by impedance-based technique (xCELLigence RTCA and automated microscopy (Cell-IQ compared to formazan bioreduction (MTS assay. For validation of the label-free systems different concentrations of ethanol and of amine (AMI polystyrene NPs were used. CNTs were evaluated in various cell lines, but only endothelial EAhy926 cells and L929 and V79 fibroblasts could be evaluated in all systems. Polystyrene particles obtained similar results in all assays. All systems identified thin (20 nm CNTs, but detection by xCELLigence system was less sensitive to CNT-induced cytotoxicity. Despite advantages, such as continuous monitoring and more detailed analysis of cytotoxic effects, label-free techniques cannot be generally recommended for cytotoxicity screening of NPs.

  19. Identification of microprocessor-dependent cancer cells allows screening for growth-sustaining micro-RNAs.

    Science.gov (United States)

    Peric, D; Chvalova, K; Rousselet, G

    2012-04-19

    Micro-RNAs are deregulated in cancer cells, and some are either tumor suppressive or oncogenic. In addition, a link has been established between decreased expression of micro-RNAs and transformation, and several proteins of the RNA interference pathway have been shown to be haploinsufficient tumor suppressors. Oncogenic micro-RNAs (oncomiRs) could represent new therapeutic targets, and their identification is therefore crucial. However, structural and functional redundancy between micro-RNAs hampers approaches relying on individual micro-RNA inhibition. We reasoned that in cancer cells that depend on oncomiRs, impairing the micro-RNA pathway could lead to growth perturbation rather than increased tumorigenesis. Identifying such cells could allow functional analyses of individual micro-RNAs by complementation of the phenotypes observed upon global micro-RNA inhibition. Therefore, we developed episomal vectors coding for small hairpin RNAs targeting either Drosha or DGCR8, the two components of the microprocessor, the nuclear micro-RNA maturation complex. We identified cancer cell lines in which both vectors induced colony growth arrest. We then screened for individual micro-RNAs complementing this growth arrest, and identified miR-19a, miR-19b, miR-20a and miR-27b as major growth-sustaining micro-RNAs. However, the effect of miR-19a and miR-19b was only transient. In addition, embryonic stem cell-derived micro-RNAs with miR-20a seeds were much less efficient than miR-20a in sustaining cancer cell growth, a finding that contrasted with results obtained in stem cells. Finally, we showed that the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10, a shared target of miR-19 and miR-20, was functionally involved in the growth arrest induced by microprocessor inhibition. We conclude that our approach allowed to identify microprocessor-dependent cancer cells, which could be used to screen for growth-sustaining micro-RNAs. This complementation screen

  20. A whole-genome RNA interference screen for human cell factors affecting myxoma virus replication.

    Science.gov (United States)

    Teferi, Wondimagegnehu M; Dodd, Kristopher; Maranchuk, Rob; Favis, Nicole; Evans, David H

    2013-04-01

    Myxoma virus (MYXV) provides an important model for investigating host-pathogen interactions. Recent studies have also highlighted how mutations in transformed human cells can expand the host range of this rabbit virus. Although virus growth depends upon interactions between virus and host proteins, the nature of these interactions is poorly understood. To address this matter, we performed small interfering RNA (siRNA) screens for genes affecting MYXV growth in human MDA-MB-231 cells. By using siRNAs targeting the whole human genome (21,585 genes), a subset of human phosphatases and kinases (986 genes), and also a custom siRNA library targeting selected statistically significant genes ("hits") and nonsignificant genes ("nonhits") of the whole human genome screens (88 genes), we identified 711 siRNA pools that promoted MYXV growth and 333 that were inhibitory. Another 32 siRNA pools (mostly targeting the proteasome) were toxic. The overall overlap in the results was about 25% for the hits and 75% for the nonhits. These pro- and antiviral genes can be clustered into pathways and related groups, including well-established inflammatory and mitogen-activated protein kinase pathways, as well as clusters relating to β-catenin and the Wnt signaling cascade, the cell cycle, and cellular metabolism. The validity of a subset of these hits was independently confirmed. For example, treating cells with siRNAs that might stabilize cells in G(1), or inhibit passage into S phase, stimulated MYXV growth, and these effects were reproduced by trapping cells at the G(1)/S boundary with an inhibitor of cyclin-dependent kinases 4/6. By using 2-deoxy-D-glucose and plasmids carrying the gene for phosphofructokinase, we also confirmed that infection is favored by aerobic glycolytic metabolism. These studies provide insights into how the growth state and structure of cells affect MYXV growth and how these factors might be manipulated to advantage in oncolytic virus therapy.

  1. Establishment of a cell-based assay to screen regulators for Klotho gene promoter

    Institute of Scientific and Technical Information of China (English)

    Zhi-liang XU; Hong GAO; Ke-qing OU-YANG; Shao-xi CAI; Ying-he HU

    2004-01-01

    AIM: To discover compounds which can regulate Klotho promoter activity. Klotho is an aging suppressor gene. A defect in Klotho gene expression in the mouse results in the phenotype similar to human aging. Recombinant Klotho protein improves age-associated diseases in animal models. It has been proposed that up-regulation of Klotho gene expression may have anti-aging effects. METHODS: Klotho promoter was cloned into a vector containing luciferase gene, and the reporter gene vector was transfected into HEK293 cells to make a stable cell line (HEK293/KL). A model for cellular aging was established by treating HEK293/KL cells with H2O2. These cells were treated with extracts from Traditional Chinese Medicines (TCMs). The luciferase activity was detected to identify compounds that can regulate Klotho promoter. RESULTS:The expression of luciferase in these cells was under control of Klotho promoter and down-regulated after H2O2 treatment The down-regulation of luciferase expression was H2O2 concentration-dependent with an IC50 at approximately 0.006 %. This result demonstrated that the Klotho gene promoter was regulated by oxidative stress. Using the cell-based reporter gene assay, we screened natural product extracts for regulation of Klotho gene promoter. Several extracts were identified that could rescue the H2O2effects and up-regulated Klotho promoter activity. CONCLUSION: A cell -based assay for high-throughput drug screening was established to identify compounds that regulate Klotho promoter activity, and several hits were discovered from natural products. Further characterization of these active extracts could help to investigate Klotho function and aging mechanisms.

  2. Imaging Cells in Flow Cytometer Using Spatial-Temporal Transformation.

    Science.gov (United States)

    Han, Yuanyuan; Lo, Yu-Hwa

    2015-08-18

    Flow cytometers measure fluorescence and light scattering and analyze multiple physical characteristics of a large population of single cells as cells flow in a fluid stream through an excitation light beam. Although flow cytometers have massive statistical power due to their single cell resolution and high throughput, they produce no information about cell morphology or spatial resolution offered by microscopy, which is a much wanted feature missing in almost all flow cytometers. In this paper, we invent a method of spatial-temporal transformation to provide flow cytometers with cell imaging capabilities. The method uses mathematical algorithms and a spatial filter as the only hardware needed to give flow cytometers imaging capabilities. Instead of CCDs or any megapixel cameras found in any imaging systems, we obtain high quality image of fast moving cells in a flow cytometer using PMT detectors, thus obtaining high throughput in manners fully compatible with existing cytometers. To prove the concept, we demonstrate cell imaging for cells travelling at a velocity of 0.2 m/s in a microfluidic channel, corresponding to a throughput of approximately 1,000 cells per second.

  3. Novel hepatitis C virus reporter replicon cell lines enable efficient antiviral screening against genotype 1a.

    Science.gov (United States)

    Robinson, Margaret; Yang, Huiling; Sun, Siu-Chi; Peng, Betty; Tian, Yang; Pagratis, Nikos; Greenstein, Andrew E; Delaney, William E

    2010-08-01

    The hepatitis C virus (HCV) subgenomic replicon is the primary tool for evaluating the activity of anti-HCV compounds in drug discovery research. Despite the prevalence of HCV genotype 1a (approximately 70% of U.S. HCV patients), few genotype 1a reporter replicon cell lines have been described; this is presumably due to the low replication capacity of such constructs in available Huh-7 cells. In this report, we describe the selection of highly permissive Huh-7 cell lines that support robust replication of genotype 1a subgenomic replicons harboring luciferase reporter genes. These novel cell lines support the replication of multiple genotype 1a replicons (including the H77 and SF9 strains), are significantly more permissive to genotype 1a HCV replication than parental Huh7-Lunet cells, and maintain stable genotype 1a replication levels suitable for antiviral screening. We found that the sensitivity of genotype 1a luciferase replicons to known antivirals was highly consistent between individual genotype 1a clonal cell lines but could vary significantly between genotypes 1a and 1b. Sequencing of the nonstructural region of 12 stable replicon cell clones suggested that the enhanced permissivity is likely due to cellular component(s) in these new cell lines rather than the evolution of novel adaptive mutations in the replicons. These new reagents will enhance drug discovery efforts targeting genotype 1a and facilitate the profiling of compound activity among different HCV genotypes and subtypes.

  4. Stereoscopic uncooled thermal imaging with autostereoscopic 3D flat-screen display in military driving enhancement systems

    Science.gov (United States)

    Haan, H.; Münzberg, M.; Schwarzkopf, U.; de la Barré, R.; Jurk, S.; Duckstein, B.

    2012-06-01

    Thermal cameras are widely used in driver vision enhancement systems. However, in pathless terrain, driving becomes challenging without having a stereoscopic perception. Stereoscopic imaging is a well-known technique already for a long time with understood physical and physiological parameters. Recently, a commercial hype has been observed, especially in display techniques. The commercial market is already flooded with systems based on goggle-aided 3D-viewing techniques. However, their use is limited for military applications since goggles are not accepted by military users for several reasons. The proposed uncooled thermal imaging stereoscopic camera with a geometrical resolution of 640x480 pixel perfectly fits to the autostereoscopic display with a 1280x768 pixels. An eye tracker detects the position of the observer's eyes and computes the pixel positions for the left and the right eye. The pixels of the flat panel are located directly behind a slanted lenticular screen and the computed thermal images are projected into the left and the right eye of the observer. This allows a stereoscopic perception of the thermal image without any viewing aids. The complete system including camera and display is ruggedized. The paper discusses the interface and performance requirements for the thermal imager as well as for the display.

  5. Human skin-derived stem cells as a novel cell source for in vitro hepatotoxicity screening of pharmaceuticals.

    Science.gov (United States)

    Rodrigues, Robim M; De Kock, Joery; Branson, Steven; Vinken, Mathieu; Meganathan, Kesavan; Chaudhari, Umesh; Sachinidis, Agapios; Govaere, Olivier; Roskams, Tania; De Boe, Veerle; Vanhaecke, Tamara; Rogiers, Vera

    2014-01-01

    Human skin-derived precursors (hSKP) are postnatal stem cells with neural crest properties that reside in the dermis of human skin. These cells can be easily isolated from small (fore) skin segments and have the capacity to differentiate into multiple cell types. In this study, we show that upon exposure to hepatogenic growth factors and cytokines, hSKP acquire sufficient hepatic features that could make these cells suitable in vitro tools for hepatotoxicity screening of new chemical entities and already existing pharmaceutical compounds. Indeed, hepatic differentiated hSKP [hSKP-derived hepatic progenitor cells (hSKP-HPC)] express hepatic progenitor cell markers (EPCAM, NCAM2, PROM1) and adult hepatocyte markers (ALB), as well as key biotransformation enzymes (CYP1B1, FMO1, GSTA4, GSTM3) and influx and efflux drug transporters (ABCC4, ABCA1, SLC2A5). Using a toxicogenomics approach, we could demonstrate that hSKP-HPC respond to acetaminophen exposure in a comparable way to primary human hepatocytes in culture. The toxicological responses "liver damage", "liver proliferation", "liver necrosis" and "liver steatosis" were found to be significantly enriched in both in vitro models. Also genes associated with either cytotoxic responses or induction of apoptosis (BCL2L11, FOS, HMOX1, TIMP3, and AHR) were commonly upregulated and might represent future molecular biomarkers for hepatotoxicity. In conclusion, our data gives a first indication that hSKP-HPC might represent a suitable preclinical model for in vitro screening of hepatotoxicity. To the best of our knowledge, this is the first report in which human postnatal stem cells derived from skin are described as a potentially relevant cell source for in vitro hepatotoxicity testing of pharmaceutical compounds.

  6. Dynamic imaging of cell-free and cell-associated viral capture in mature dendritic cells.

    Science.gov (United States)

    Izquierdo-Useros, Nuria; Esteban, Olga; Rodriguez-Plata, Maria T; Erkizia, Itziar; Prado, Julia G; Blanco, Julià; García-Parajo, Maria F; Martinez-Picado, Javier

    2011-12-01

    Dendritic cells (DCs) capture human immunodeficiency virus (HIV) through a non-fusogenic mechanism that enables viral transmission to CD4(+) T cells, contributing to in vivo viral dissemination. Although previous studies have provided important clues to cell-free viral capture by mature DCs (mDCs), dynamic and kinetic insight on this process is still missing. Here, we used three-dimensional video microscopy and single-particle tracking approaches to dynamically dissect both cell-free and cell-associated viral capture by living mDCs. We show that cell-free virus capture by mDCs operates through three sequential phases: virus binding through specific determinants expressed in the viral particle, polarized or directional movements toward concrete regions of the cell membrane and virus accumulation in a sac-like structure where trapped viral particles display a hindered diffusive behavior. Moreover, real-time imaging of cell-associated viral transfer to mDCs showed a similar dynamics to that exhibited by cell-free virus endocytosis leading to viral accumulation in compartments. However, cell-associated HIV type 1 transfer to mDCs was the most effective pathway, boosted throughout enhanced cellular contacts with infected CD4(+) T cells. Our results suggest that in lymphoid tissues, mDC viral uptake could occur either by encountering cell-free or cell-associated virus produced by infected cells generating the perfect scenario to promote HIV pathogenesis and impact disease progression.

  7. Magnetic Resonance Imaging as a Biomarker for Renal Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Yan Wu

    2015-01-01

    Full Text Available As the most common neoplasm arising from the kidney, renal cell carcinoma (RCC continues to have a significant impact on global health. Conventional cross-sectional imaging has always served an important role in the staging of RCC. However, with recent advances in imaging techniques and postprocessing analysis, magnetic resonance imaging (MRI now has the capability to function as a diagnostic, therapeutic, and prognostic biomarker for RCC. For this narrative literature review, a PubMed search was conducted to collect the most relevant and impactful studies from our perspectives as urologic oncologists, radiologists, and computational imaging specialists. We seek to cover advanced MR imaging and image analysis techniques that may improve the management of patients with small renal mass or metastatic renal cell carcinoma.

  8. Single Molecule Imaging in Living Cell with Optical Method

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Significance, difficult, international developing actuality and our completed works for single molecules imaging in living cell with optical method are described respectively. Additionally we give out some suggestions for the technology development further.

  9. Cellular transfer and AFM imaging of cancer cells using Bioimprint

    Directory of Open Access Journals (Sweden)

    Melville DOS

    2006-01-01

    Full Text Available Abstract A technique for permanently capturing a replica impression of biological cells has been developed to facilitate analysis using nanometer resolution imaging tools, namely the atomic force microscope (AFM. The method, termed Bioimprint™, creates a permanent cell 'footprint' in a non-biohazardous Poly (dimethylsiloxane (PDMS polymer composite. The transfer of nanometer scale biological information is presented as an alternative imaging technique at a resolution beyond that of optical microscopy. By transferring cell topology into a rigid medium more suited for AFM imaging, many of the limitations associated with scanning of biological specimens can be overcome. Potential for this technique is demonstrated by analyzing Bioimprint™ replicas created from human endometrial cancer cells. The high resolution transfer of this process is further detailed by imaging membrane morphological structures consistent with exocytosis. The integration of soft lithography to replicate biological materials presents an enhanced method for the study of biological systems at the nanoscale.

  10. Cloud Screening and Quality Control Algorithm for Star Photometer Data: Assessment with Lidar Measurements and with All-sky Images

    Science.gov (United States)

    Ramirez, Daniel Perez; Lyamani, H.; Olmo, F. J.; Whiteman, D. N.; Navas-Guzman, F.; Alados-Arboledas, L.

    2012-01-01

    This paper presents the development and set up of a cloud screening and data quality control algorithm for a star photometer based on CCD camera as detector. These algorithms are necessary for passive remote sensing techniques to retrieve the columnar aerosol optical depth, delta Ae(lambda), and precipitable water vapor content, W, at nighttime. This cloud screening procedure consists of calculating moving averages of delta Ae() and W under different time-windows combined with a procedure for detecting outliers. Additionally, to avoid undesirable Ae(lambda) and W fluctuations caused by the atmospheric turbulence, the data are averaged on 30 min. The algorithm is applied to the star photometer deployed in the city of Granada (37.16 N, 3.60 W, 680 ma.s.l.; South-East of Spain) for the measurements acquired between March 2007 and September 2009. The algorithm is evaluated with correlative measurements registered by a lidar system and also with all-sky images obtained at the sunset and sunrise of the previous and following days. Promising results are obtained detecting cloud-affected data. Additionally, the cloud screening algorithm has been evaluated under different aerosol conditions including Saharan dust intrusion, biomass burning and pollution events.

  11. Cloud screening and quality control algorithm for star photometer data: assessment with lidar measurements and with all-sky-images

    Directory of Open Access Journals (Sweden)

    D. Pérez-Ramírez

    2012-02-01

    Full Text Available This paper present the development and set up of a cloud screening and data quality control algorithm for a star photometer based on CCD camera as detector. This kind of algorithms is necessary for passive remote sensing techniques to retrieve the columnar aerosol optical depth, δAe(λ, and precipitable water vapor content, W, at night-time. This cloud screening procedure consists of calculating moving averages of δAe(λ and W under different time-windows combined with a procedure for detecting outliers. Additionally, to avoid undesirable δAe(λ and W fluctuations caused by the atmospheric turbulence, the data are averaged on 30 min. The algorithm is applied to the star photometer deployed in the city of Granada (37.16° N, 3.60° W, 680 m a.s.l.; South-East of Spain for the measurements acquired between March 2007 and September 2009. The algorithm is evaluated with correlative measurements registered by a lidar system and also with all-sky images obtained at the sunset and sunrise of the previous and following days. Promising results are obtained detecting cloud-affected data. Additionally, the cloud screening algorithm has been evaluated under different aerosol conditions including Saharan dust intrusion, biomass burning and pollution events.

  12. Cloud screening and quality control algorithm for star photometer data: assessment with lidar measurements and with all-sky images

    Directory of Open Access Journals (Sweden)

    D. Pérez-Ramírez

    2012-07-01

    Full Text Available This paper presents the development and set up of a cloud screening and data quality control algorithm for a star photometer based on CCD camera as detector. These algorithms are necessary for passive remote sensing techniques to retrieve the columnar aerosol optical depth, δAe(λ, and precipitable water vapor content, W, at nighttime. This cloud screening procedure consists of calculating moving averages of δAe(λ and W under different time-windows combined with a procedure for detecting outliers. Additionally, to avoid undesirable δAe(λ and W fluctuations caused by the atmospheric turbulence, the data are averaged on 30 min. The algorithm is applied to the star photometer deployed in the city of Granada (37.16° N, 3.60° W, 680 m a.s.l.; South-East of Spain for the measurements acquired between March 2007 and September 2009. The algorithm is evaluated with correlative measurements registered by a lidar system and also with all-sky images obtained at the sunset and sunrise of the previous and following days. Promising results are obtained detecting cloud-affected data. Additionally, the cloud screening algorithm has been evaluated under different aerosol conditions including Saharan dust intrusion, biomass burning and pollution events.

  13. Molecular Imaging and Therapy of Merkel Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Volkan Beylergil

    2014-04-01

    Full Text Available Several molecular imaging modalities have been evaluated in the management of Merkel cell carcinoma (MCC, a rare and aggressive tumor with a high tendency to metastasize. Continuous progress in the field of molecular imaging might improve management in these patients. The authors review the current modalities and their impact on MCC in this brief review article.

  14. Fluorescence lifetime imaging of oxygen in living cells

    NARCIS (Netherlands)

    Gerritsen, H.C.; Sanders, R.; Draaijer, A.; Ince, C.; Levine, Y.K.

    1997-01-01

    The usefulness of the fluorescent probe ruthenium tris(2,2′-dipyridyl) dichloride hydrate (RTDP) for the quantitative imaging of oxygen in single cells was investigated utilizing fluorescence life-time imaging. The results indicate that the fluorescence behavior of RTDP in the presence of oxygen can

  15. On generating cell exemplars for detection of mitotic cells in breast cancer histopathology images.

    Science.gov (United States)

    Aloraidi, Nada A; Sirinukunwattana, Korsuk; Khan, Adnan M; Rajpoot, Nasir M

    2014-01-01

    Mitotic activity is one of the main criteria that pathologists use to decide the grade of the cancer. Computerised mitotic cell detection promises to bring efficiency and accuracy into the grading process. However, detection and classification of mitotic cells in breast cancer histopathology images is a challenging task because of the large intra-class variation in the visual appearance of mitotic cells in various stages of cell division life cycle. In this paper, we test the hypothesis that cells in histopathology images can be effectively represented using cell exemplars derived from sub-images of various kinds of cells in an image for the purposes of mitotic cell classification. We compare three methods for generating exemplar cells. The methods have been evaluated in terms of classification performance on the MITOS dataset. The experimental results demonstrate that eigencells combined with support vector machines produce reasonably high detection accuracy among all the methods.

  16. Photoacoustic imaging of single circulating melanoma cells in vivo

    Science.gov (United States)

    Wang, Lidai; Yao, Junjie; Zhang, Ruiying; Xu, Song; Li, Guo; Zou, Jun; Wang, Lihong V.

    2015-03-01

    Melanoma, one of the most common types of skin cancer, has a high mortality rate, mainly due to a high propensity for tumor metastasis. The presence of circulating tumor cells (CTCs) is a potential predictor for metastasis. Label-free imaging of single circulating melanoma cells in vivo provides rich information on tumor progress. Here we present photoacoustic microscopy of single melanoma cells in living animals. We used a fast-scanning optical-resolution photoacoustic microscope to image the microvasculature in mouse ears. The imaging system has sub-cellular spatial resolution and works in reflection mode. A fast-scanning mirror allows the system to acquire fast volumetric images over a large field of view. A 500-kHz pulsed laser was used to image blood and CTCs. Single circulating melanoma cells were imaged in both capillaries and trunk vessels in living animals. These high-resolution images may be used in early detection of CTCs with potentially high sensitivity. In addition, this technique enables in vivo study of tumor cell extravasation from a primary tumor, which addresses an urgent pre-clinical need.

  17. A Cell-based High-throughput Screening Assay for Farnesoid X Recepter Agonist

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To develop a high-throughput screening assay for Farnesoid X receptor (FXR) agonists based on mammalian one-hybrid system (a chimera receptor gene system) for the purpose of identifying new lead compounds for dyslipidaemia drug from the chemical library. Methods cDNA encoding the human FXR ligand binding domain (LBD) was amplified by RT-PCR from a human liver total mRNA and fused to the DNA binding domain (DBD) of yeast GAL4 of pBIND to construct a GAL4-FXR (LBD) chimera expression plasmid. Five copies of the GAL4 DNA binding site were synthesized and inserted into upstream of the SV40 promoter of pGL3-promoter vector to construct a reporter plasmid pG5-SV40 Luc. The assay was developed by transient co-transfection with pG5-SV40 Luc reporter plasmid and pBIND-FXR-LBD (189-472) chimera expression plasmid. Results After optimization, CDCA, a FXR natural agonist, could induce expression of the luciferase gene in a dose-dependent manner, and had a signal/noise ratio of 10 and Z'factor value of 0.65. Conclusion A stable and sensitive cell-based high-throughput screening model can be used in high-throughput screening for FXR agonists from the synthetic and natural compound library.

  18. A cell-free microtiter plate screen for improved [FeFe] hydrogenases.

    Directory of Open Access Journals (Sweden)

    James A Stapleton

    Full Text Available BACKGROUND: [FeFe] hydrogenase enzymes catalyze the production and dissociation of H(2, a potential renewable fuel. Attempts to exploit these catalysts in engineered systems have been hindered by the biotechnologically inconvenient properties of the natural enzymes, including their extreme oxygen sensitivity. Directed evolution has been used to improve the characteristics of a range of natural catalysts, but has been largely unsuccessful for [FeFe] hydrogenases because of a lack of convenient screening platforms. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe an in vitro screening technology for oxygen-tolerant and highly active [FeFe] hydrogenases. Despite the complexity of the protocol, we demonstrate a level of reproducibility that allows moderately improved mutants to be isolated. We have used the platform to identify a mutant of the Chlamydomonas reinhardtii [FeFe] hydrogenase HydA1 with a specific activity approximately 4 times that of the wild-type enzyme. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate the feasibility of using the screen presented here for large-scale efforts to identify improved biocatalysts for energy applications. The system is based on our ability to activate these complex enzymes in E. coli cell extracts, which allows unhindered access to the protein maturation and assay environment.

  19. Screening the stage-specific expression gene from different germ cells of rat

    Institute of Scientific and Technical Information of China (English)

    贾孟春; 娄利霞; 裴开颜; 赵龙梅; 石心泉

    2002-01-01

    Objective: To screen the stage-specific expression genes from rat spermatogonia, pachytene spermatocytes and round spermatids. Methods: Highly purified spermatogonia were isolated from 9-day-old rats, pachytene spermatocytes and round spermatids from adult rats by sedimentation velocity at unit gravity, using 2%-4% BSA gradient in DMEM/F12 medium. A mRNA differential display method was used for screening the stage-specific expression gene. Results: Nineteen differentially expressed cDNA fragments were obtained. After excluding the false positive cDNA fragments by dot blot, 13 cDNAs were selected to clone and sequence. To obtain longer cDNAs, six ESTs were used to screen the rat testis λ-zap II cDNA library. Two longer cDNA fragments, designated as LY21 and LM66, were obtained. The analysis with DNAMAN software indicated that LY21 had a long open reading frame coding 372 amino acids while LM66 had no long open reading frame. LY21 were highly homologous with hnRNP H1. To observe the expression patterns of LY21 gene in the testicular cells, we performed in situ hybridization on testis sections from adult rats. The LY21 gene expression was found in the spermatogonia and primary spermatocytes.Conclusion: This study indicated that LY21 gene was associated with spermatogenesis. Further studies will be needed to explore the function of LY21.

  20. Somatic and germ cell cytogenetic studies and AZF microdeletion screening in infertile men

    Directory of Open Access Journals (Sweden)

    Rita C. V. Carrara

    2004-01-01

    Full Text Available Clinical and cytogenetic studies were performed in 65 infertile individuals, and 56 of them were also screened for microdeletions in Yq11 (AZF region. Relevant environmental etiological factors were identified in 10 cases (15.4%. Sertoli-cell-only syndrome was diagnosed in six patients (9,2%. Karyotype abnormalities were detected in six individuals, and five other patients presented desynapsis of bivalents in meiosis. Three out of the 56 patients studied were carriers of microdeletions in the AZF region, one of them also presenting a chromosomal mosaicism for an extra i(22p.

  1. Phantom experiments with a microwave imaging system for breast-cancer screening

    DEFF Research Database (Denmark)

    Rubæk, Tonny; Zhurbenko, Vitaliy

    2009-01-01

    Microwave imaging is emerging as a promising technique for breast-cancer detection. In this paper, the microwave imaging system currently being developed at the Technical University of Denmark is introduced. This system consists of 32 antennas positioned in a cylindrical setup, each equipped...

  2. In vivo imaging of immune cell trafficking in cancer.

    Science.gov (United States)

    Ottobrini, Luisa; Martelli, Cristina; Trabattoni, Daria Lucia; Clerici, Mario; Lucignani, Giovanni

    2011-05-01

    Tumour establishment, progression and regression can be studied in vivo using an array of imaging techniques ranging from MRI to nuclear-based and optical techniques that highlight the intrinsic behaviour of different cell populations in the physiological context. Clinical in vivo imaging techniques and preclinical specific approaches have been used to study, both at the macroscopic and microscopic level, tumour cells, their proliferation, metastasisation, death and interaction with the environment and with the immune system. Fluorescent, radioactive or paramagnetic markers were used in direct protocols to label the specific cell population and reporter genes were used for genetic, indirect labelling protocols to track the fate of a given cell subpopulation in vivo. Different protocols have been proposed to in vivo study the interaction between immune cells and tumours by different imaging techniques (intravital and whole-body imaging). In particular in this review we report several examples dealing with dendritic cells, T lymphocytes and macrophages specifically labelled for different imaging procedures both for the study of their physiological function and in the context of anti-neoplastic immunotherapies in the attempt to exploit imaging-derived information to improve and optimise anti-neoplastic immune-based treatments.

  3. Labeling and imaging cells in the zebrafish hindbrain.

    Science.gov (United States)

    Jayachandran, Pradeepa; Hong, Elim; Brewster, Rachel

    2010-07-25

    Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells (1) . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos (2). The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

  4. Computational Validation of a 3-D Microwave Imaging System for Breast-Cancer Screening

    DEFF Research Database (Denmark)

    Rubæk, Tonny; Kim, Oleksiy S.; Meincke, Peter

    2009-01-01

    -of-moments solution of the associated forward scattering problem. A cylindrical multistatic antenna setup with 32 horizontally oriented antennas is used for collecting the data. It has been found that formulating the imaging algorithm in terms of the logarithm of the amplitude and the unwrapped phase of the measured......The microwave imaging system currently being developed at the Technical University of Denmark is described and its performance tested on simulated data. The system uses an iterative Newton-based imaging algorithm for reconstructing the images in conjunction with an efficient method...... in the measurement system is shown by imaging the same breast model using a measurement setup in which the antennas are vertically oriented....

  5. Serum-Free Generation of Multipotent Mesoderm (Kdr-positive) Progenitor Cells in Mouse Embryonic Stem Cells For Functional Genomics Screening

    OpenAIRE

    2012-01-01

    This unit describes a robust protocol for producing multipotent Kdr-expressing mesoderm progenitor cells in serum-free conditions and functional genomics screening using these cells. Kdr-positive cells are known to be able to differentiate into a wide array of mesoderm derivatives including, vascular endothelial cells, cardiomyocytes, hematopietic progenitors and smooth muscle cells. The efficient generation of such progenitor cells is of particular interest because it permits subsequent step...

  6. Preliminary screening and identification of stem cell-like sphere clones in a gallbladder cancer cell line GBC-SD*

    Institute of Scientific and Technical Information of China (English)

    Bao-bing YIN; Shuang-jie WU; Hua-jie ZONG; Bao-jin MA; Duan CAI

    2011-01-01

    This paper aims to screen and identify sphere clone cells with characteristics similar to cancer stem cellsin human gallbladder cancer cell line GBC-SD. GBC-SD cells were cultured in a serum-free culture medium with different concentrations of the chemotherapeutic drug cisplatin for generating sphere clones. The mRNA expressions of stem cell-related genes CD133, OCT-4, Nanog, and drug resistance genes ABCG2 and MDR-1 in sphere clones were detected by quantitative real-time polymerase chain reaction (PCR). Stem cell markers were also analyzed by flow cytometry and immunofluorescent staining. Different amounts of sphere clones were injected into nude mice to test their abilities to form tumors. Sphere clones were formed in serum-free culture medium containing cisplatin (30 pmol/L).Flow cytometry results demonstrated that the sphere clones expressed high levels of stem cell markers CD133+ (97.6%) and CD44+ (77.9%) and low levels of CD24+ (2.3%). These clones also overexpressed the drug resistance genes ABCG2 and MDR-1. Quantitative real-time PCR showed that sphere clones expressed stem cell genes Nanog and OCT-4 284 and 266 times, respectively, more than those in the original GBC-SD cells. Immunofluorescent staining showed that sphere clones overexpressed OCT-4, Nanog, and SOX-2, and Iow expressed MUG1 and vimentin. Tumor formation experiments showed that 1 x 103 sphere clone cells could induce much larger tumors in nude mice than 1 x 105 GBC-SD cells. In conclusion, sphere clones of gallbladder cancer with stem cell-like characteristics can be obtained using suspension cultures of GBC-SD cells in serum-free culture medium containing cisplatin.

  7. A prospective newborn screening and treatment program for sickle cell anemia in Luanda, Angola.

    Science.gov (United States)

    McGann, Patrick T; Ferris, Margaret G; Ramamurthy, Uma; Santos, Brigida; de Oliveira, Vysolela; Bernardino, Luis; Ware, Russell E

    2013-12-01

    Over 300,000 infants are born annually with sickle cell anemia (SCA) in sub-Saharan Africa, and >50% die young from infection or anemia, usually without diagnosis of SCA. Early identification by newborn screening (NBS), followed by simple interventions dramatically reduced the mortality of SCA in the United States, but this strategy is not yet established in Africa. We designed and implemented a proof-of-principle NBS and treatment program for SCA in Angola, with focus on capacity building and local ownership. Dried bloodspots from newborns were collected from five birthing centers. Hemoglobin identification was performed using isoelectric focusing; samples with abnormal hemoglobin patterns were analyzed by capillary electrophoresis. Infants with abnormal FS or FSC patterns were enrolled in a newborn clinic to initiate penicillin prophylaxis and receive education, pneumococcal immunization, and insecticide-treated bed nets. A total of 36,453 infants were screened with 77.31% FA, 21.03% FAS, 1.51% FS, and 0.019% FSC. A majority (54.3%) of affected infants were successfully contacted and brought to clinical care. Compliance in the newborn clinic was excellent (96.6%). Calculated first-year mortality rate for babies with SCA compares favorably to the national infant mortality rate (6.8 vs. 9.8%). The SCA burden is extremely high in Angola, but NBS is feasible. Capacity building and training provide local healthcare workers with skills needed for a functional screening program and clinic. Contact and retrieval of all affected SCA infants remains a challenge, but families are compliant with clinic appointments and treatment. Early mortality data suggest screening and early preventive care saves lives.

  8. Neonatal Screening and the Clinical Outcome in Children with Sickle Cell Disease in Central India.

    Directory of Open Access Journals (Sweden)

    Dipti S Upadhye

    Full Text Available Sickle cell disease (SCD is a major health burden in India. The objective of the study was to establish a neonatal screening program and to understand the clinical course of children with SCD in central India.Pregnant mothers were screened for sickle hemoglobin using the solubility test. Babies were screened by high performance liquid chromatography if the mother was positive for sickle hemoglobin. The diagnosis was confirmed by molecular analysis. They received early prophylactic treatment and vaccination. Of 2134 newborns screened, 104 were sickle homozygous (SS, seven had sickle β-thalassemia (S-β thal and 978 were sickle heterozygous (AS. The other hemoglobin abnormalities detected included HbS-δβ thalassemia-1, HbSD disease-2, HbE traits-5, β-thalassemia traits-4, alpha chain variants-3 and HbH disease-1.These babies were followed up regularly for hematological and clinical evaluation. Pain, severe anemia requiring blood transfusions and acute febrile illness were the major complications with 59.7, 45.1 and 42.6 cases per 100 person years. Fetal hemoglobin (HbF levels were inversely associated with vaso-oclussive crisis (VOC and severe anemia while presence of alpha thalassemia increased the rate of painful events and sepsis. Six early deaths occurred among the SS babies.A systematic follow up of this first newborn SCD cohort in central India showed that 47% of babies presented within 1 year of age. In spite of the presence of the Arab-Indian haplotype many babies had severe manifestations.

  9. PET molecular imaging in stem cell therapy for neurological diseases

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jiachuan; Zhang, Hong [Second Affiliated Hospital of Zhejiang University School of Medicine, Department of Nuclear Medicine, Hangzhou, Zhejiang (China); Zhejiang University, Medical PET Center, Hangzhou (China); Institute of Nuclear Medicine and Molecular Imaging of Zhejiang University, Hangzhou (China); Key Laboratory of Medical Molecular Imaging of Zhejiang Province, Hangzhou (China); Tian, Mei [University of Texas, M.D. Anderson Cancer Center, Department of Experimental Diagnostic Imaging, Houston, TX (United States)

    2011-10-15

    Human neurological diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, spinal cord injury and multiple sclerosis are caused by loss of different types of neurons and glial cells in the brain and spinal cord. At present, there are no effective therapies against these disorders. Discovery of the therapeutic potential of stem cells offers new strategies for the treatment of neurological diseases. Direct assessment of stem cells' survival, interaction with the host and impact on neuronal functions after transplantation requires advanced in vivo imaging techniques. Positron emission tomography (PET) is a potential molecular imaging modality to evaluate the viability and function of transplanted tissue or stem cells in the nervous system. This review focuses on PET molecular imaging in stem cell therapy for neurological diseases. (orig.)

  10. Information management for high content live cell imaging

    Directory of Open Access Journals (Sweden)

    White Michael RH

    2009-07-01

    Full Text Available Abstract Background High content live cell imaging experiments are able to track the cellular localisation of labelled proteins in multiple live cells over a time course. Experiments using high content live cell imaging will generate multiple large datasets that are often stored in an ad-hoc manner. This hinders identification of previously gathered data that may be relevant to current analyses. Whilst solutions exist for managing image data, they are primarily concerned with storage and retrieval of the images themselves and not the data derived from the images. There is therefore a requirement for an information management solution that facilitates the indexing of experimental metadata and results of high content live cell imaging experiments. Results We have designed and implemented a data model and information management solution for the data gathered through high content live cell imaging experiments. Many of the experiments to be stored measure the translocation of fluorescently labelled proteins from cytoplasm to nucleus in individual cells. The functionality of this database has been enhanced by the addition of an algorithm that automatically annotates results of these experiments with the timings of translocations and periods of any oscillatory translocations as they are uploaded to the repository. Testing has shown the algorithm to perform well with a variety of previously unseen data. Conclusion Our repository is a fully functional example of how high throughput imaging data may be effectively indexed and managed to address the requirements of end users. By implementing the automated analysis of experimental results, we have provided a clear impetus for individuals to ensure that their data forms part of that which is stored in the repository. Although focused on imaging, the solution provided is sufficiently generic to be applied to other functional proteomics and genomics experiments. The software is available from: fhttp://code.google.com/p/livecellim/

  11. Bioluminescent, Nonlytic, Real-Time Cell Viability Assay and Use in Inhibitor Screening.

    Science.gov (United States)

    Duellman, Sarah J; Zhou, Wenhui; Meisenheimer, Poncho; Vidugiris, Gediminas; Cali, James J; Gautam, Prson; Wennerberg, Krister; Vidugiriene, Jolanta

    2015-10-01

    Real-time continuous monitoring of cellular processes offers distinct advantages over traditional endpoint assays. A comprehensive representation of the changes occurring in live cells over the entire length of an experiment provides information about the biological status of the cell and informs decisions about the timing of treatments or the use of other functional endpoint assays. We describe a homogeneous, nonlytic, bioluminescent assay that measures cell viability in real time. This time-dependent measurement allowed us to monitor cell health for 72 h from the same test samples, distinguish differential cell growth, and investigate drug mechanism of action by analyzing time- and dose-dependent drug effects. The real-time measurements also allowed us to detect cell death immediately (>75% signal decrease within 15 min of digitonin addition), analyze drug potency versus efficacy, and identify cytostatic versus toxic drug effects. We screened an oncology compound library (Z' = 0.7) and identified compounds with varying activity at different time points (1.6% of the library showed activity within 3 h, whereas 35.4% showed a response by 47 h). The assay compared well with orthogonal endpoint cell viability assays and additionally provided data at multiple time points and the opportunity to multiplex assays on the same cells. To test the advantage of time-dependent measurements to direct optimal timing of downstream applications, we used the real-time cell viability assay to determine the ideal time to measure caspase activity by monitoring the onset of cell death and multiplexing a luminescent caspase activation assay on the same test samples.

  12. Optical imaging for stem cell differentiation to neuronal lineage.

    Science.gov (United States)

    Hwang, Do Won; Lee, Dong Soo

    2012-03-01

    In regenerative medicine, the prospect of stem cell therapy holds great promise for the recovery of injured tissues and effective treatment of intractable diseases. Tracking stem cell fate provides critical information to understand and evaluate the success of stem cell therapy. The recent emergence of in vivo noninvasive molecular imaging has enabled assessment of the behavior of grafted stem cells in living subjects. In this review, we provide an overview of current optical imaging strategies based on cell- or tissue-specific reporter gene expression and of in vivo methods to monitor stem cell differentiation into neuronal lineages. These methods use optical reporters either regulated by neuron-specific promoters or containing neuron-specific microRNA binding sites. Both systems revealed dramatic changes in optical reporter imaging signals in cells differentiating into a neuronal lineage. The detection limit of weak promoters or reporter genes can be greatly enhanced by adopting a yeast GAL4 amplification system or an engineering-enhanced luciferase reporter gene. Furthermore, we propose an advanced imaging system to monitor neuronal differentiation during neurogenesis that uses in vivo multiplexed imaging techniques capable of detecting several targets simultaneously.

  13. Optical Imaging for Stem Cell Differentiation to Neuronal Lineage

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, Do Won; Lee, Dong Soo [Seoul National Univ., Seoul (Korea, Republic of)

    2012-03-15

    In regenerative medicine, the prospect of stem cell therapy hold great promise for the recovery of injured tissues and effective treatment of intractable diseases. Tracking stem cell fate provides critical information to understand and evaluate the success of stem cell therapy. The recent emergence of in vivo noninvasive molecular imaging has enabled assessment of the behavior of grafted stem cells in living subjects. In this review, we provide an overview of current optical imaging strategies based on cell or tissue specific reporter gene expression and of in vivo methods to monitor stem cell differentiation into neuronal lineages. These methods use optical reporters either regulated by neuron-specific promoters or containing neuron-specific microRNA binding sites. Both systems revealed dramatic changes in optical reporter imaging signals in cells differentiating a yeast GAL4 amplification system or an engineering-enhanced luciferase reported gene. Furthermore, we propose an advanced imaging system to monitor neuronal differentiation during neurogenesis that uses in vivo multiplexed imaging techniques capable of detecting several targets simultaneously.

  14. Small molecule probes for plant cell wall polysaccharide imaging

    Directory of Open Access Journals (Sweden)

    Ian eWallace

    2012-05-01

    Full Text Available Plant cell walls are composed of interlinked polymer networks consisting of cellulose, hemicelluloses, pectins, proteins, and lignin. The ordered deposition of these components is a dynamic process that critically affects the development and differentiation of plant cells. However, our understanding of cell wall synthesis and remodeling, as well as the diverse cell wall architectures that result from these processes, has been limited by a lack of suitable chemical probes that are compatible with live-cell imaging. In this review, we summarize the currently available molecular toolbox of probes for cell wall polysaccharide imaging in plants, with particular emphasis on recent advances in small molecule-based fluorescent probes. We also discuss the potential for further development of small molecule probes for the analysis of cell wall architecture and dynamics.

  15. Dissecting Immune Circuits by Linking CRISPR-Pooled Screens with Single-Cell RNA-Seq.

    Science.gov (United States)

    Jaitin, Diego Adhemar; Weiner, Assaf; Yofe, Ido; Lara-Astiaso, David; Keren-Shaul, Hadas; David, Eyal; Salame, Tomer Meir; Tanay, Amos; van Oudenaarden, Alexander; Amit, Ido

    2016-12-15

    In multicellular organisms, dedicated regulatory circuits control cell type diversity and responses. The crosstalk and redundancies within these circuits and substantial cellular heterogeneity pose a major research challenge. Here, we present CRISP-seq, an integrated method for massively parallel single-cell RNA sequencing (RNA-seq) and clustered regularly interspaced short palindromic repeats (CRISPR)-pooled screens. We show that profiling the genomic perturbation and transcriptome in the same cell enables us to simultaneously elucidate the function of multiple factors and their interactions. We applied CRISP-seq to probe regulatory circuits of innate immunity. By sampling tens of thousands of perturbed cells in vitro and in mice, we identified interactions and redundancies between developmental and signaling-dependent factors. These include opposing effects of Cebpb and Irf8 in regulating the monocyte/macrophage versus dendritic cell lineages and differential functions for Rela and Stat1/2 in monocyte versus dendritic cell responses to pathogens. This study establishes CRISP-seq as a broadly applicable, comprehensive, and unbiased approach for elucidating mammalian regulatory circuits.

  16. A loss-of-function genetic screening identifies novel mediators of thyroid cancer cell viability

    Science.gov (United States)

    Cantisani, Maria Carmela; Parascandolo, Alessia; Perälä, Merja; Allocca, Chiara; Fey, Vidal; Sahlberg, Niko; Merolla, Francesco; Basolo, Fulvio; Laukkanen, Mikko O.; Kallioniemi, Olli Pekka; Santoro, Massimo; Castellone, Maria Domenica

    2016-01-01

    RET, BRAF and other protein kinases have been identified as major molecular players in thyroid cancer. To identify novel kinases required for the viability of thyroid carcinoma cells, we performed a RNA interference screening in the RET/PTC1(CCDC6-RET)-positive papillary thyroid cancer cell line TPC1 using a library of synthetic small interfering RNAs (siRNAs) targeting the human kinome and related proteins. We identified 14 hits whose silencing was able to significantly reduce the viability and the proliferation of TPC1 cells; most of them were active also in BRAF-mutant BCPAP (papillary thyroid cancer) and 8505C (anaplastic thyroid cancer) and in RAS-mutant CAL62 (anaplastic thyroid cancer) cells. These included members of EPH receptor tyrosine kinase family as well as SRC and MAPK (mitogen activated protein kinases) families. Importantly, silencing of the identified hits did not affect significantly the viability of Nthy-ori 3-1 (hereafter referred to as NTHY) cells derived from normal thyroid tissue, suggesting cancer cell specificity. The identified proteins are worth exploring as potential novel druggable thyroid cancer targets. PMID:27058903

  17. Combinatorial Screening of Mesenchymal Stem Cell Adhesion and Differentiation Using Polymer Pen Lithography

    Science.gov (United States)

    Cabezas, Maria D.; Eichelsdoerfer, Daniel J.; Brown, Keith A.; Mrksich, Milan; Mirkin, Chad A.

    2014-01-01

    The extracellular matrix (ECM) is a complex, spatially inhomogeneous environment that is host to myriad cell–receptor interactions that promote changes in cell behavior. These biological systems can be probed and simulated with engineered surfaces,but doing so demands careful control over the arrangement of ligands. Here, we describe how such surfaces can be fabricated by utilizing polymer pen lithography (PPL), which is a cantilever-free scanning probe lithographic method that utilizes polymeric pen arrays to generate patterns over large areas. With the advent of PPL, fundamental questions in cell biology can be answered by recapitulating cell–ECM interactions to explore how these interactions lead to changes in cell behavior. Here, we describe an approach for the combinatorial screening of cell adhesion behavior to gain understanding of how ECM protein feature size dictates osteogenic differentiation of mesenchymal stem cells. The technique outlined here is generalizable to other biological systems and can be paired with quantitative analytical methods to probe important processes such as cell polarization, proliferation, signaling, and differentiation. PMID:24439289

  18. Collection of sequential imaging events for research in breast cancer screening

    Science.gov (United States)

    Patel, M. N.; Young, K.; Halling-Brown, M. D.

    2016-03-01

    Due to the huge amount of research involving medical images, there is a widely accepted need for comprehensive collections of medical images to be made available for research. This demand led to the design and implementation of a flexible image repository, which retrospectively collects images and data from multiple sites throughout the UK. The OPTIMAM Medical Image Database (OMI-DB) was created to provide a centralized, fully annotated dataset for research. The database contains both processed and unprocessed images, associated data, annotations and expert-determined ground truths. Collection has been ongoing for over three years, providing the opportunity to collect sequential imaging events. Extensive alterations to the identification, collection, processing and storage arms of the system have been undertaken to support the introduction of sequential events, including interval cancers. These updates to the collection systems allow the acquisition of many more images, but more importantly, allow one to build on the existing high-dimensional data stored in the OMI-DB. A research dataset of this scale, which includes original normal and subsequent malignant cases along with expert derived and clinical annotations, is currently unique. These data provide a powerful resource for future research and has initiated new research projects, amongst which, is the quantification of normal cases by applying a large number of quantitative imaging features, with a priori knowledge that eventually these cases develop a malignancy. This paper describes, extensions to the OMI-DB collection systems and tools and discusses the prospective applications of having such a rich dataset for future research applications.

  19. Automatic recognition of abnormal cells in cytological tests using multispectral imaging

    Science.gov (United States)

    Gertych, A.; Galliano, G.; Bose, S.; Farkas, D. L.

    2010-03-01

    Cervical cancer is the leading cause of gynecologic disease-related death worldwide, but is almost completely preventable with regular screening, for which cytological testing is a method of choice. Although such testing has radically lowered the death rate from cervical cancer, it is plagued by low sensitivity and inter-observer variability. Moreover, its effectiveness is still restricted because the recognition of shape and morphology of nuclei is compromised by overlapping and clumped cells. Multispectral imaging can aid enhanced morphological characterization of cytological specimens. Features including spectral intensity and texture, reflecting relevant morphological differences between normal and abnormal cells, can be derived from cytopathology images and utilized in a detection/classification scheme. Our automated processing of multispectral image cubes yields nuclear objects which are subjected to classification facilitated by a library of spectral signatures obtained from normal and abnormal cells, as marked by experts. Clumps are processed separately with reduced set of signatures. Implementation of this method yields high rate of successful detection and classification of nuclei into predefined malignant and premalignant types and correlates well with those obtained by an expert. Our multispectral approach may have an impact on the diagnostic workflow of cytological tests. Abnormal cells can be automatically highlighted and quantified, thus objectivity and performance of the reading can be improved in a way which is currently unavailable in clinical setting.

  20. On-chip electrical impedance tomography for imaging biological cells.

    Science.gov (United States)

    Sun, Tao; Tsuda, Soichiro; Zauner, Klaus-Peter; Morgan, Hywel

    2010-01-15

    Electrical impedance tomography is an imaging technology that spatially characterizes the electrical properties of an object. We present a miniaturized electrical impedance tomography system that can image the electrical conductivity distribution within a two-dimensional cell culture. A chip containing a circular 16-electrode array was fabricated using printed circuit board developing technology and used to inject current and to measure spatial voltage across the object. The signal stimulation and voltage data acquisition were performed using an impedance analyzer, operating in four-electrode mode. An open source software, EIDORS was used for image reconstruction. Finite element modelling was used to simulate the image reconstruction process by imaging two ellipsoidal phantoms in the circular 16-electrode array. The effect of the regularization parameter in the reconstruction algorithm and the influence from noise on the fidelity of the images has been numerically analyzed. Experimentally, we show reconstructed images of a multi-nuclear single cellular organism, Physarum Polycephalum, demonstrating the first step towards impedance imaging of single cells in culture. Our system provides a non-invasive lab-on-a-chip technology for spatially mapping the electrical properties of single cells, which would be significant and useful for diagnostic and clinical applications.

  1. Monitoring of adherent live cells morphology using the undecimated wavelet transform multivariate image analysis (UWT-MIA).

    Science.gov (United States)

    Juneau, Pierre-Marc; Garnier, Alain; Duchesne, Carl

    2017-01-01

    Cell morphology is an important macroscopic indicator of cellular physiology and is increasingly used as a mean of probing culture state in vitro. Phase contrast microscopy (PCM) is a valuable tool for observing live cells morphology over long periods of time with minimal culture artifact. Two general approaches are commonly used to analyze images: individual object segmentation and characterization by pattern recognition. Single-cell segmentation is difficult to achieve in PCM images of adherent cells since their contour is often irregular and blurry, and the cells bundle together when the culture reaches confluence. Alternatively, pattern recognition approaches such as the undecimated wavelet transform multivariate image analysis (UWT-MIA), allow extracting textural features from PCM images that are correlated with cellular morphology. A partial least squares (PLS) regression model built using textural features from a set of 200 ground truth images was shown to predict the distribution of cellular morphological features (major and minor axes length, orientation, and roundness) with good accuracy for most images. The PLS models were then applied on a large dataset of 631,136 images collected from live myoblast cell cultures acquired under different conditions and grown in two different culture media. The method was found sensitive to morphological changes due to cell growth (culture time) and those introduced by the use of different culture media, and was able to distinguish both sources of variations. The proposed approach is promising for application on large datasets of PCM live-cell images to assess cellular morphology and growth kinetics in real-time which could be beneficial for high-throughput screening as well as automated cell culture kinetics assessment and control applications. Biotechnol. Bioeng. 2017;114: 141-153. © 2016 Wiley Periodicals, Inc.

  2. Combinatorial synthesis and screening of cancer cell-specific nanomedicines targeted via phage fusion proteins

    Directory of Open Access Journals (Sweden)

    James W. Gillespie

    2015-06-01

    Full Text Available Active tumor targeting of nanomedicines has recently shown significant improvements in the therapeutic activity of currently existing drug delivery systems, such as liposomal doxorubicin (Doxil/Caelyx/Lipodox. Previously, we have shown that isolated pVIII major coat proteins of the fd tet filamentous phage vector, containing cancer cell-specific peptide fusions at their N terminus, can be used as active targeting ligands in a liposomal doxorubicin delivery system in vitro and in vivo. Here, we show a novel major coat protein isolation procedure in 2-propanol that allows spontaneous incorporation of the hydrophobic protein core into preformed liposomal doxorubicin with minimal damage or drug loss while still retaining the targeting ligand exposed for cell-specific targeting. Using a panel of 12 structurally unique ligands with specificity towards breast, lung, and/or pancreatic cancer, we showed the feasibility of pVIII major coat proteins to significantly increase the throughput of targeting ligand screening in a common nanomedicine core. Phage protein-modified Lipodox samples showed an average doxorubicin recovery of 82.8% across all samples with 100% of protein incorporation in the correct orientation (N-terminus exposed. Following cytotoxicity screening in a doxorubicin-sensitive breast cancer line (MCF-7, three major groups of ligands were identified. Ligands showing the most improved cytotoxicity included: DMPGTVLP, ANGRPSMT, VNGRAEAP, and ANDVYLD showing a 25-fold improvement (p < 0.05 in toxicity. Similarly DGQYLGSQ, ETYNQPYL, and GSSEQLYL ligands with specificity towards a doxorubicin-insensitive pancreatic cancer line (PANC-1 showed significant increases in toxicity (2-fold; p < 0.05. Thus, we demonstrated proof-of-concept that pVIII major coat proteins can be screened in significantly higher throughput to identify novel ligands displaying improved therapeutic activity in a desired cancer phenotype.

  3. High resolution imaging of surface patterns of single bacterial cells

    Energy Technology Data Exchange (ETDEWEB)

    Greif, Dominik; Wesner, Daniel [Experimental Biophysics and Applied Nanoscience, Bielefeld University, Universitaetsstrasse 25, 33615 Bielefeld (Germany); Regtmeier, Jan, E-mail: jan.regtmeier@physik.uni-bielefeld.de [Experimental Biophysics and Applied Nanoscience, Bielefeld University, Universitaetsstrasse 25, 33615 Bielefeld (Germany); Anselmetti, Dario [Experimental Biophysics and Applied Nanoscience, Bielefeld University, Universitaetsstrasse 25, 33615 Bielefeld (Germany)

    2010-09-15

    We systematically studied the origin of surface patterns observed on single Sinorhizobium meliloti bacterial cells by comparing the complementary techniques atomic force microscopy (AFM) and scanning electron microscopy (SEM). Conditions ranged from living bacteria in liquid to fixed bacteria in high vacuum. Stepwise, we applied different sample modifications (fixation, drying, metal coating, etc.) and characterized the observed surface patterns. A detailed analysis revealed that the surface structure with wrinkled protrusions in SEM images were not generated de novo but most likely evolved from similar and naturally present structures on the surface of living bacteria. The influence of osmotic stress to the surface structure of living cells was evaluated and also the contribution of exopolysaccharide and lipopolysaccharide (LPS) by imaging two mutant strains of the bacterium under native conditions. AFM images of living bacteria in culture medium exhibited surface structures of the size of single proteins emphasizing the usefulness of AFM for high resolution cell imaging.

  4. High resolution ultrasound and photoacoustic imaging of single cells

    Directory of Open Access Journals (Sweden)

    Eric M. Strohm

    2016-03-01

    Full Text Available High resolution ultrasound and photoacoustic images of stained neutrophils, lymphocytes and monocytes from a blood smear were acquired using a combined acoustic/photoacoustic microscope. Photoacoustic images were created using a pulsed 532 nm laser that was coupled to a single mode fiber to produce output wavelengths from 532 nm to 620 nm via stimulated Raman scattering. The excitation wavelength was selected using optical filters and focused onto the sample using a 20× objective. A 1000 MHz transducer was co-aligned with the laser spot and used for ultrasound and photoacoustic images, enabling micrometer resolution with both modalities. The different cell types could be easily identified due to variations in contrast within the acoustic and photoacoustic images. This technique provides a new way of probing leukocyte structure with potential applications towards detecting cellular abnormalities and diseased cells at the single cell level.

  5. Molecular imaging of cell-mediated cancer immunotherapy.

    Science.gov (United States)

    Lucignani, Giovanni; Ottobrini, Luisa; Martelli, Cristina; Rescigno, Maria; Clerici, Mario

    2006-09-01

    New strategies based on the activation of a patient's immune response are being sought to complement present conventional exogenous cancer therapies. Elucidating the trafficking pathways of immune cells in vivo, together with their migratory properties in relation to their differentiation and activation status, is useful for understanding how the immune system interacts with cancer. Methods based on tissue sampling to monitor immune responses are inadequate for repeatedly characterizing the responses of the immune system in different organs. A solution to this problem might come from molecular and cellular imaging - a branch of biomedical sciences that combines biotechnology and imaging methods to characterize, in vivo, the molecular and cellular processes involved in normal and pathologic states. The general concepts of noninvasive imaging of targeted cells as well as the technology and probes applied to cell-mediated cancer immunotherapy imaging are outlined in this review.

  6. High resolution ultrasound and photoacoustic imaging of single cells.

    Science.gov (United States)

    Strohm, Eric M; Moore, Michael J; Kolios, Michael C

    2016-03-01

    High resolution ultrasound and photoacoustic images of stained neutrophils, lymphocytes and monocytes from a blood smear were acquired using a combined acoustic/photoacoustic microscope. Photoacoustic images were created using a pulsed 532 nm laser that was coupled to a single mode fiber to produce output wavelengths from 532 nm to 620 nm via stimulated Raman scattering. The excitation wavelength was selected using optical filters and focused onto the sample using a 20× objective. A 1000 MHz transducer was co-aligned with the laser spot and used for ultrasound and photoacoustic images, enabling micrometer resolution with both modalities. The different cell types could be easily identified due to variations in contrast within the acoustic and photoacoustic images. This technique provides a new way of probing leukocyte structure with potential applications towards detecting cellular abnormalities and diseased cells at the single cell level.

  7. MISSION LentiPlex pooled shRNA library screening in mammalian cells.

    Science.gov (United States)

    Coussens, Matthew J; Corman, Courtney; Fischer, Ashley L; Sago, Jack; Swarthout, John

    2011-12-21

    RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to knockdown gene expression levels in loss of gene function studies. There are two main methods for performing RNAi. The first is the use of small interfering RNAs (siRNAs) that are chemically synthesized, and the second utilizes short-hairpin RNAs (shRNAs) encoded within plasmids. The latter can be transfected into cells directly or packaged into replication incompetent lentiviral particles. The main advantages of using lentiviral shRNAs is the ease of introduction into a wide variety of cell types, their ability to stably integrate into the genome for long term gene knockdown and selection, and their efficacy in conducting high-throughput loss of function screens. To facilitate this we have created the LentiPlex pooled shRNA library. The MISSION LentiPlex Human shRNA Pooled Library is a genome-wide lentiviral pool produced using a proprietary process. The library consists of over 75,000 shRNA constructs from the TRC collection targeting 15,000+ human genes. Each library is tested for shRNA representation before product release to ensure robust library coverage. The library is provided in a ready-to-use lentiviral format at titers of at least 5 x 10(8) TU/ml via p24 assay and is pre-divided into ten subpools of approximately 8,000 shRNA constructs each. Amplification and sequencing primers are also provided for downstream target identification. Previous studies established a synergistic antitumor activity of TRAIL when combined with Paclitaxel in A549 cells, a human lung carcinoma cell line. In this study we demonstrate the application of a pooled LentiPlex shRNA library to rapidly conduct a positive selection screen for genes involved in the cytotoxicity of A549 cells when exposed to TRAIL and Paclitaxel. One barrier often encountered with high-throughput screens is the cost and difficulty in deconvolution; we

  8. A cell-based screen reveals that the albendazole metabolite, albendazole sulfone, targets Wolbachia.

    Science.gov (United States)

    Serbus, Laura R; Landmann, Frederic; Bray, Walter M; White, Pamela M; Ruybal, Jordan; Lokey, R Scott; Debec, Alain; Sullivan, William

    2012-09-01

    Wolbachia endosymbionts carried by filarial nematodes give rise to the neglected diseases African river blindness and lymphatic filariasis afflicting millions worldwide. Here we identify new Wolbachia-disrupting compounds by conducting high-throughput cell-based chemical screens using a Wolbachia-infected, fluorescently labeled Drosophila cell line. This screen yielded several Wolbachia-disrupting compounds including three that resembled Albendazole, a widely used anthelmintic drug that targets nematode microtubules. Follow-up studies demonstrate that a common Albendazole metabolite, Albendazole sulfone, reduces intracellular Wolbachia titer both in Drosophila melanogaster and Brugia malayi, the nematode responsible for lymphatic filariasis. Significantly, Albendazole sulfone does not disrupt Drosophila microtubule organization, suggesting that this compound reduces titer through direct targeting of Wolbachia. Accordingly, both DNA staining and FtsZ immunofluorescence demonstrates that Albendazole sulfone treatment induces Wolbachia elongation, a phenotype indicative of binary fission defects. This suggests that the efficacy of Albendazole in treating filarial nematode-based diseases is attributable to dual targeting of nematode microtubules and their Wolbachia endosymbionts.

  9. High-Throughput Chemical Screens Identify Disulfiram as an Inhibitor of Human Glioblastoma Stem Cells

    Science.gov (United States)

    Hothi, Parvinder; Martins, Timothy J.; Chen, LiPing; Deleyrolle, Loic; Yoon, Jae-Geun; Reynolds, Brent; Foltz, Greg

    2012-01-01

    Glioblastoma Multiforme (GBM) continues to have a poor patient prognosis despite optimal standard of care. Glioma stem cells (GSCs) have been implicated as the presumed cause of tumor recurrence and resistance to therapy. With this in mind, we screened a diverse chemical library of 2,000 compounds to identify therapeutic agents that inhibit GSC proliferation and therefore have the potential to extend patient survival. High-throughput screens (HTS) identified 78 compounds that repeatedly inhibited cellular proliferation, of which 47 are clinically approved for other indications and 31 are experimental drugs. Several compounds (such as digitoxin, deguelin, patulin and phenethyl caffeate) exhibited high cytotoxicity, with half maximal inhibitory concentrations (IC50) in the low nanomolar range. In particular, the FDA approved drug for the treatment of alcoholism, disulfiram (DSF), was significantly potent across multiple patient samples (IC50 of 31.1 nM). The activity of DSF was potentiated by copper (Cu), which markedly increased GSC death. DSF–Cu inhibited the chymotrypsin-like proteasomal activity in cultured GSCs, consistent with inactivation of the ubiquitin-proteasome pathway and the subsequent induction of tumor cell death. Given that DSF is a relatively non-toxic drug that can penetrate the blood-brain barrier, we suggest that DSF should be tested (as either a monotherapy or as an adjuvant) in pre-clinical models of human GBM. Data also support targeting of the ubiquitin-proteasome pathway as a therapeutic approach in the treatment of GBM. PMID:23165409

  10. Enzyme- and affinity biomolecule-mediated polymerization systems for biological signal amplification and cell screening.

    Science.gov (United States)

    Malinowska, Klara H; Nash, Michael A

    2016-06-01

    Enzyme-mediated polymerization and polymerization-based signal amplification have emerged as two closely related techniques that are broadly applicable in the nanobio sciences. We review recent progress on polymerization systems mediated by biological molecules (e.g., affinity molecules and enzymes), and highlight newly developed formats and configurations of these systems to perform such tasks as non-instrumented biodetection, synthesis of core-shell nanomaterials, isolation of rare cells, and high-throughput screening. We discuss useful features of biologically mediated polymerization systems, such as multiple mechanisms of amplification (e.g., enzymatic, radical chain propagation), and the ability to localize structures at interfaces and at cell surfaces with microscopic spatial confinement. We close with a perspective on desirable improvements that need to be addressed to adapt these molecular systems to future applications.

  11. Screening of adulterants in powdered foods and ingredients using line-scan Raman chemical imaging

    Science.gov (United States)

    Qin, Jianwei; Chao, Kuanglin; Kim, Moon S.

    2015-05-01

    A newly developed line-scan Raman imaging system using a 785 nm line laser was used to authenticate powdered foods and ingredients. The system was used to collect hyperspectral Raman images in a wavenumber range of 102-2865 cm-1 from three representative food powders mixed with selected adulterants with a concentration of 0.5%, including milk and melamine, flour and benzoyl peroxide, and starch and maleic anhydride. An acoustic mixer was used to create food adulterant mixtures. All the mixed samples were placed in sample holders with a surface area of 50 mm×50 mm. Spectral and image processing algorithms were developed based on single-band images at unique Raman peaks of the individual adulterants. Chemical images were created to show identification, spatial distribution, and morphological features of the adulterant particles mixed in the food powders. The potential of estimating mass concentrations of the adulterants using the percentages of the adulterant pixels in the chemical images was also demonstrated.

  12. Screening cDNA Libraries Using Partial Probes to Isolate Full-Length cDNAs from Vascular Cells.

    Science.gov (United States)

    Csortos, C; Lazar, V; Garcia, J G

    1999-01-01

    The purpose of screening cDNA libraries is to isolate a particular cDNA clone encoding a mRNA and by implication, a protein, of interest. The screening is based on identification of the desired clone among a large number of recombinant clones within the library selected (1,2). As an example of both the utility and power of library screening, we will relate our own library screening efforts utilized to isolate the nonmuscle high molecular weight myosin light chain kinase isoform from a human umbilical vein endothelial cell cDNA library (3). This unique nonmuscle myosin light chain kinase isoform phosphorylates myosin light chains, thereby playing an essential role in agonist-mediated endothelial cell contraction, paracellular gap formation and increased vascular permeability. We are hopeful that this step-by-step approach will help the reader to understand the discussed methods.

  13. Quantitative neuroanatomy of all Purkinje cells with light sheet microscopy and high-throughput image analysis

    Directory of Open Access Journals (Sweden)

    Ludovico eSilvestri

    2015-05-01

    Full Text Available Characterizing the cytoarchitecture of mammalian central nervous system on a brain-wide scale is becoming a compelling need in neuroscience. For example, realistic modeling of brain activity requires the definition of quantitative features of large neuronal populations in the whole brain. Quantitative anatomical maps will also be crucial to classify the cytoarchtitectonic abnormalities associated with neuronal pathologies in a high reproducible and reliable manner. In this paper, we apply recent advances in optical microscopy and image analysis to characterize the spatial distribution of Purkinje cells across the whole cerebellum. Light sheet microscopy was used to image with micron-scale resolution a fixed and cleared cerebellum of an L7-GFP transgenic mouse, in which all Purkinje cells are fluorescently labeled. A fast and scalable algorithm for fully automated cell identification was applied on the image to extract the position of all the fluorescent Purkinje cells. This vectorized representation of the cell population allows a thorough characterization of the complex three-dimensional distribution of the neurons, highlighting the presence of gaps inside the lamellar organization of Purkinje cells, whose density is believed to play a significant role in autism spectrum disorders. Furthermore, clustering analysis of the localized somata permits dividing the whole cerebellum in groups of Purkinje cells with high spatial correlation, suggesting new possibilities of anatomical partition. The quantitative approach presented here can be extended to study the distribution of different types of cell in many brain regions and across the whole encephalon, providing a robust base for building realistic computational models of the brain, and for unbiased morphological tissue screening in presence of pathologies and/or drug treatments.

  14. Expression profiling of colon cancer cell lines and colon biopsies: Towards a screening system for potential cancer-preventive compounds

    NARCIS (Netherlands)

    Erk, van M.J.; Krul, C.A.M.; Caldenhoven, E.; Stierum, R.H.; Peters, W.H.; Woutersen, R.A.; Ommen, van B.

    2005-01-01

    Interest in mechanisms of colon cancer prevention by food compounds is strong and research in this area is often performed with cultured colon cancer cells. In order to assess utility for screening of potential cancer-preventive (food) compounds, expression profiles of 14 human cell lines derived fr

  15. Production of large-area polymer solar cells by industrial silk screen printing, lifetime considerations and lamination with polyethyleneterephthalate

    DEFF Research Database (Denmark)

    Krebs, Frederik C; Alstrup, J.; Spanggaard, H.

    2004-01-01

    ). The half-life based on I-sc in air for the devices were 63 h. The cells were laminated in a 125 mum PET encasement. Lamination had a negative effect on the lifetime. We demonstrate the feasibility of industrial production of large area solar cells (1 m(2)) by silk screen printing and envisage...

  16. Expression profiling of colon cancer cell lines and colon biopsies: towards a screening system for potential cancer-preventive compounds.

    NARCIS (Netherlands)

    Erk, M.J. van; Krul, C.A.; Caldenhoven, E.; Stierum, R.H.; Peters, W.H.M.; Woutersen, R.A.; Ommen, B.

    2005-01-01

    Interest in mechanisms of colon cancer prevention by food compounds is strong and research in this area is often performed with cultured colon cancer cells. In order to assess utility for screening of potential cancer-preventive (food) compounds, expression profiles of 14 human cell lines derived fr

  17. Aqueous biphasic cancer cell migration assay enables robust, high-throughput screening of anti-cancer compounds.

    Science.gov (United States)

    Lemmo, Stephanie; Nasrollahi, Samila; Tavana, Hossein

    2014-03-01

    Migration of tumor cells is a fundamental event implicated in metastatic progression of cancer. Therapeutic compounds with the ability to inhibit the motility of cancer cells are critical for preventing cancer metastasis. Achieving this goal requires new technologies that enable high-throughput drug screening against migration of cancer cells and expedite drug discovery. We report an easy-to-implement, robotically operated, cell migration microtechnology with the capability of simultaneous screening of multiple compounds. The technology utilizes a fully biocompatible polymeric aqueous two-phase system to pattern a monolayer of cells containing a cell-excluded gap that serves as the migration niche. We adapted this technology to a standard 96-well plate format and parametrically optimized it to generate highly consistent migration niches. The analysis of migration is done automatically using computerized schemes. We use statistical metrics and show the robustness of this assay for drug screening and its sensitivity to identify effects of different drug compounds on migration of cancer cells. This technology can be employed in core centers, research laboratories, and pharmaceutical industries to evaluate the efficacy of compounds against migration of various types of metastatic cancer cells prior to expensive animal tests and thus, streamline anti-migratory drug screening.

  18. Induced pluripotent stem cell-derived cardiomyocytes for cardiovascular disease modeling and drug screening.

    Science.gov (United States)

    Sharma, Arun; Wu, Joseph C; Wu, Sean M

    2013-12-24

    Human induced pluripotent stem cells (hiPSCs) have emerged as a novel tool for drug discovery and therapy in cardiovascular medicine. hiPSCs are functionally similar to human embryonic stem cells (hESCs) and can be derived autologously without the ethical challenges associated with hESCs. Given the limited regenerative capacity of the human heart following myocardial injury, cardiomyocytes derived from hiPSCs (hiPSC-CMs) have garnered significant attention from basic and translational scientists as a promising cell source for replacement therapy. However, ongoing issues such as cell immaturity, scale of production, inter-line variability, and cell purity will need to be resolved before human clinical trials can begin. Meanwhile, the use of hiPSCs to explore cellular mechanisms of cardiovascular diseases in vitro has proven to be extremely valuable. For example, hiPSC-CMs have been shown to recapitulate disease phenotypes from patients with monogenic cardiovascular disorders. Furthermore, patient-derived hiPSC-CMs are now providing new insights regarding drug efficacy and toxicity. This review will highlight recent advances in utilizing hiPSC-CMs for cardiac disease modeling in vitro and as a platform for drug validation. The advantages and disadvantages of using hiPSC-CMs for drug screening purposes will be explored as well.

  19. Cytotoxicity screening of Melastoma malabathricum extracts on human breast cancer cell lines in vitro

    Institute of Scientific and Technical Information of China (English)

    Nurfariza; Ahmad; Roslen; Nur; Aizura; Mat; Alewi; Hadji; Ahamada; Mohammad; Syaiful; Bahari; Abdull; Rasad

    2014-01-01

    Objective:To screen the cytotoxic activity of Melasloma malabathricum(M,malubathricum)against human breast carreer cell line(MCF-7)in vitro.Methods:A three steps extraction protocol using n-hexane,chloroform and methanol as the solvents systems was carried out on leaves,stems and flowers of M.nalabathricum.Dimethyl sulfoxide was used in extracts dilution and serial dilutions were conducted to obtain five different extract concentrations(100μg/mL,50μg/mL,25μg/mL,123μg/rnL and 6.25μg/mL).The evaluation of cell growth was determined using methylene blue assay.Results:Methanol extract from the leaves showed significant anticancer activity against MCF-7cell lines with the TC50value of 7.14μg/ml while methanol and chloroform extract from the flowers exhibited a moderate activity towards MCF-7 cell line,with the IC50value of 33.63μg/mL and 45.76μg/mL respectively after 72 h of treatment.Conclusions:The extracts from leaves and flowers of M.nulabatkricum showed promising anticancer activity toward human breast cancer cell lines with the lowest IC50at 7.14μg/mL while the extracts from stems showed less growth inhibition activity.

  20. Cytotoxicity screening of Melastoma malabathricum extracts on human breast cancer cell lines in vitro

    Institute of Scientific and Technical Information of China (English)

    Nurfariza Ahmad Roslen; Nur Aizura Mat Alewi; Hadji Ahamada

    2014-01-01

    Objective: To screen the cytotoxic activity of Melastoma malabathricum (M. malabathricum) against human breast cancer cell line (MCF-7) in vitro. Methods: A three steps extraction protocol using n-hexane, chloroform and methanol as the solvents systems was carried out on leaves, stems and flowers of M. malabathricum. Dimethyl sulfoxide was used in extracts dilution and serial dilutions were conducted to obtain five different extract concentrations (100 µg/mL, 50 µg/mL, 25 µg/mL, 12.5 µg/mL and 6.25 µg/mL). The evaluation of cell growth was determined using methylene blue assay.Results:Methanol extract from the leaves showed significant anticancer activity against MCF-7 cell lines with the IC50 value of 7.14 µg/ml while methanol and chloroform extract from the flowers exhibited a moderate activity towards MCF-7 cell line with the IC50 value of 33.63 µg/mL and 45.76 µg/mL respectively after 72 h of treatment.Conclusions:The extracts from leaves and flowers of M. malabathricum showed promising anticancer activity toward human breast cancer cell lines with the lowest IC50 at 7.14 µg/mL while the extracts from stems showed less growth inhibition activity.

  1. Hepatic differentiation of human pluripotent stem cells in miniaturized format suitable for high-throughput screen

    Directory of Open Access Journals (Sweden)

    Arnaud Carpentier

    2016-05-01

    Full Text Available The establishment of protocols to differentiate human pluripotent stem cells (hPSCs including embryonic (ESC and induced pluripotent (iPSC stem cells into functional hepatocyte-like cells (HLCs creates new opportunities to study liver metabolism, genetic diseases and infection of hepatotropic viruses (hepatitis B and C viruses in the context of specific genetic background. While supporting efficient differentiation to HLCs, the published protocols are limited in terms of differentiation into fully mature hepatocytes and in a smaller-well format. This limitation handicaps the application of these cells to high-throughput assays. Here we describe a protocol allowing efficient and consistent hepatic differentiation of hPSCs in 384-well plates into functional hepatocyte-like cells, which remain differentiated for more than 3 weeks. This protocol affords the unique opportunity to miniaturize the hPSC-based differentiation technology and facilitates screening for molecules in modulating liver differentiation, metabolism, genetic network, and response to infection or other external stimuli.

  2. Live-cell imaging of mammalian RNAs with Spinach2

    Science.gov (United States)

    Strack, Rita L.; Jaffrey, Samie R.

    2015-01-01

    The ability to monitor RNAs of interest in living cells is crucial to understanding the function, dynamics, and regulation of this important class of molecules. In recent years, numerous strategies have been developed with the goal of imaging individual RNAs of interest in living cells, each with their own advantages and limitations. This chapter provides an overview of current methods of live-cell RNA imaging, including a detailed discussion of genetically encoded strategies for labeling RNAs in mammalian cells. This chapter then focuses on the development and use of “RNA mimics of GFP” or Spinach technology for tagging mammalian RNAs, and includes a detailed protocol for imaging 5S and CGG60 RNA with the recently described Spinach2 tag. PMID:25605384

  3. Imaging the Beta-cell mass: why and how

    DEFF Research Database (Denmark)

    Saudek, Frantisek; Brogren, Carl-Henrik; Manohar, Srirang

    2008-01-01

    of the native beta-cell mass is still limited to autopsy studies. Endeavors to find a biological structure specific for beta-cells led to the discovery of potential candidates that have been tested for noninvasive imaging. Among them are the ligand to the vesicular monoamine transporter type 2 (VMAT-2), which......Diabetes is a disorder characterized by beta-cell loss or exhaustion and insulin deficiency. At present, knowledge is lacking on the underlying causes and for the therapeutic recovery of the beta-cell mass. A better understanding of diabetes pathogenesis could be obtained through exact monitoring...... of the fate of beta-cells under disease and therapy conditions. This could pave the way for a new era of intervention by islet replacement and regeneration regimens. Monitoring the beta-cell mass requires a reliable method for noninvasive in vivo imaging. Such a method is not available at present due...

  4. High-content live cell imaging with RNA probes: advancements in high-throughput antimalarial drug discovery

    Directory of Open Access Journals (Sweden)

    Cervantes Serena

    2009-06-01

    Full Text Available Abstract Background Malaria, a major public health issue in developing nations, is responsible for more than one million deaths a year. The most lethal species, Plasmodium falciparum, causes up to 90% of fatalities. Drug resistant strains to common therapies have emerged worldwide and recent artemisinin-based combination therapy failures hasten the need for new antimalarial drugs. Discovering novel compounds to be used as antimalarials is expedited by the use of a high-throughput screen (HTS to detect parasite growth and proliferation. Fluorescent dyes that bind to DNA have replaced expensive traditional radioisotope incorporation for HTS growth assays, but do not give additional information regarding the parasite stage affected by the drug and a better indication of the drug's mode of action. Live cell imaging with RNA dyes, which correlates with cell growth and proliferation, has been limited by the availability of successful commercial dyes. Results After screening a library of newly synthesized stryrl dyes, we discovered three RNA binding dyes that provide morphological details of live parasites. Utilizing an inverted confocal imaging platform, live cell imaging of parasites increases parasite detection, improves the spatial and temporal resolution of the parasite under drug treatments, and can resolve morphological changes in individual cells. Conclusion This simple one-step technique is suitable for automation in a microplate format for novel antimalarial compound HTS. We have developed a new P. falciparum RNA high-content imaging growth inhibition assay that is robust with time and energy efficiency.

  5. Real-time imaging of microparticles and living cells with CMOS nanocapacitor arrays

    Science.gov (United States)

    Laborde, C.; Pittino, F.; Verhoeven, H. A.; Lemay, S. G.; Selmi, L.; Jongsma, M. A.; Widdershoven, F. P.

    2015-09-01

    Platforms that offer massively parallel, label-free biosensing can, in principle, be created by combining all-electrical detection with low-cost integrated circuits. Examples include field-effect transistor arrays, which are used for mapping neuronal signals and sequencing DNA. Despite these successes, however, bioelectronics has so far failed to deliver a broadly applicable biosensing platform. This is due, in part, to the fact that d.c. or low-frequency signals cannot be used to probe beyond the electrical double layer formed by screening salt ions, which means that under physiological conditions the sensing of a target analyte located even a short distance from the sensor (∼1 nm) is severely hampered. Here, we show that high-frequency impedance spectroscopy can be used to detect and image microparticles and living cells under physiological salt conditions. Our assay employs a large-scale, high-density array of nanoelectrodes integrated with CMOS electronics on a single chip and the sensor response depends on the electrical properties of the analyte, allowing impedance-based fingerprinting. With our platform, we image the dynamic attachment and micromotion of BEAS, THP1 and MCF7 cancer cell lines in real time at submicrometre resolution in growth medium, demonstrating the potential of the platform for label/tracer-free high-throughput screening of anti-tumour drug candidates.

  6. In vivo imaging of cancer cells with electroporation of quantum dots and multispectral imaging

    Science.gov (United States)

    Yoo, Jung Sun; Won, Nayoun; Kim, Hong Bae; Bang, Jiwon; Kim, Sungjee; Ahn, Saeyoung; Soh, Kwang-Sup

    2010-06-01

    Our understanding of dissemination and growth of cancer cells is limited by our inability for long-term followup of this process in vivo. Fluorescence molecular imaging has the potential to track cancer cells with high contrast and sensitivity in living animals. For this purpose, intracellular delivery of near-infrared fluorescence quantum dots (QDs) by electroporation offers considerable advantages over organic fluorophores and other cell tagging methods. In this research we developed a multispectral imaging system that could eliminate two major parameters compromising in vivo fluorescence imaging performance, i.e., variations in the tissue optical properties and tissue autofluorescence. We demonstrated that electroporation of QDs and multispectral imaging allowed in vivo assessment of cancer development and progression in the xenograft mouse tumor model for more than 1 month, providing a powerful means to learn more about the biology of cancer and metastasis.

  7. Imaging of blood cells based on snapshot Hyper-Spectral Imaging systems

    Science.gov (United States)

    Robison, Christopher J.; Kolanko, Christopher; Bourlai, Thirimachos; Dawson, Jeremy M.

    2015-05-01

    Snapshot Hyper-Spectral imaging systems are capable of capturing several spectral bands simultaneously, offering coregistered images of a target. With appropriate optics, these systems are potentially able to image blood cells in vivo as they flow through a vessel, eliminating the need for a blood draw and sample staining. Our group has evaluated the capability of a commercial Snapshot Hyper-Spectral imaging system, the Arrow system from Rebellion Photonics, in differentiating between white and red blood cells on unstained blood smear slides. We evaluated the imaging capabilities of this hyperspectral camera; attached to a microscope at varying objective powers and illumination intensity. Hyperspectral data consisting of 25, 443x313 hyperspectral bands with ~3nm spacing were captured over the range of 419 to 494nm. Open-source hyper-spectral data cube analysis tools, used primarily in Geographic Information Systems (GIS) applications, indicate that white blood cells features are most prominent in the 428-442nm band for blood samples viewed under 20x and 50x magnification over a varying range of illumination intensities. These images could potentially be used in subsequent automated white blood cell segmentation and counting algorithms for performing in vivo white blood cell counting.

  8. Development of droplets‐based microfluidic systems for single­‐cell high‐throughput screening

    DEFF Research Database (Denmark)

    Chen, Jun; Jensen, Thomas Glasdam; Godina, Alexei

    2014-01-01

    High-throughput screening (HTS) plays an important role in the development of microbial cell factories. One of the most popular approaches is to use microplates combined with the application of robotics, liquid handling and sophisticated detection methods. However, these workstations require larg...... picoliter aqueous droplets surround by an immiscible fluorinated oil phase. Our aim is to use this system to facilitate the screening process for both the biotechnology and food industry....

  9. Cell culture monitoring for drug screening and cancer research: a transparent, microfluidic, multi-sensor microsystem.

    Science.gov (United States)

    Weltin, Andreas; Slotwinski, Kinga; Kieninger, Jochen; Moser, Isabella; Jobst, Gerhard; Wego, Marcus; Ehret, Ralf; Urban, Gerald A

    2014-01-07

    We present a novel, multiparametric microphysiometry system for the dynamic online monitoring of human cancer cell metabolism. The optically transparent, modular, hybrid microsystem is based on a glass chip and combines a cell cultivation chamber, microfluidics and metabolic monitoring with fully integrated chemo- and biosensors. pH and oxygen are measured in the cell culture area, and biosensors for lactate and glucose are connected downstream by microfluidics. The wafer-level fabrication features thin-film platinum and iridium oxide microelectrodes on a glass chip, microfluidics in an epoxy resist, a hybrid assembly and an on-chip reference electrode. The reliable analytical performance of the sensors in cell culture medium was demonstrated. The pH sensors exhibit a long-term stable, linear response. The oxygen sensors show a linear behaviour, which is also observed for low oxygen concentrations. Glucose and lactate measurements show a linear, long-term stable, selective and reversible behaviour in the desired range. T98G human brain cancer cells were cultivated and cell culture metabolism was measured on-chip. Stop/flow cycles were applied and extracellular acidification, respiration, glucose consumption and lactate production were quantified. Long-term metabolic rates were determined and all parameters could be measured in the outlet channel. A placement downstream of the cell cultivation area for biosensors was realised. A highly effective medium exchange and undiluted sampling from the cell culture chamber with low flow rates (2 μl min(-1)) and low volumes (15 μl per cycle) were achieved. The drug screening application was demonstrated by detecting alteration and recovery effects of cellular metabolism induced by the addition of substances to the medium.

  10. AFM imaging of fenestrated liver sinusoidal endothelial cells.

    Science.gov (United States)

    Braet, F; Wisse, E

    2012-12-01

    Each microscope with its dedicated sample preparation technique provides the investigator with a specific set of data giving an instrument-determined (or restricted) insight into the structure and function of a tissue, a cell or parts thereof. Stepwise improvements in existing techniques, both instrumental and preparative, can sometimes cross barriers in resolution and image quality. Of course, investigators get really excited when completely new principles of microscopy and imaging are offered in promising new instruments, such as the AFM. The present paper summarizes a first phase of studies on the thin endothelial cells of the liver. It describes the preparation-dependent differences in AFM imaging of these cells after isolation. Special point of interest concerned the dynamics of the fenestrae, thought to filter lipid-carrying particles during their transport from the blood to the liver cells. It also describes the attempts to image the details of these cells when alive in cell cultures. It explains what physical conditions, mainly contributed to the scanning stylus, are thought to play a part in the limitations in imaging these cells. The AFM also offers promising specifications to those interested in cell surface details, such as membrane-associated structures, receptors, coated pits, cellular junctions and molecular aggregations or domains. The AFM also offers nano-manipulation possibilities, strengths and elasticity measurements, force interactions, affinity measurements, stiffness and other physical aspects of membranes and cytoskeleton. The potential for molecular approaches is there. New developments in cantilever construction and computer software promise to bring real time video imaging to the AFM. Home made accessories for the first generation of AFM are now commodities in commercial instruments and make the life of the AFM microscopist easier. Also, the combination of different microscopies, such as AFM and TEM, or AFM and SEM find their way to the

  11. Glucagon-Secreting Alpha Cell Selective Two-Photon Fluorescent Probe TP-α: For Live Pancreatic Islet Imaging.

    Science.gov (United States)

    Agrawalla, Bikram Keshari; Chandran, Yogeswari; Phue, Wut-Hmone; Lee, Sung-Chan; Jeong, Yun-Mi; Wan, Si Yan Diana; Kang, Nam-Young; Chang, Young-Tae

    2015-04-29

    Two-photon (TP) microscopy has an advantage for live tissue imaging which allows a deeper tissue penetration up to 1 mm comparing to one-photon (OP) microscopy. While there are several OP fluorescence probes in use for pancreatic islet imaging, TP imaging of selective cells in live islet still remains a challenge. Herein, we report the discovery of first TP live pancreatic islet imaging probe; TP-α (Two Photon-alpha) which can selectively stain glucagon secreting alpha cells. Through fluorescent image based screening using three pancreatic cell lines, we discovered TP-α from a TP fluorescent dye library TPG (TP-Green). In vitro fluorescence test showed that TP-α have direct interaction and appear glucagon with a significant fluorescence increase, but not with insulin or other hormones/analytes. Finally, TP-α was successfully applied for 3D imaging of live islets by staining alpha cell directly. The newly developed TP-α can be a practical tool to evaluate and identify live alpha cells in terms of localization, distribution and availability in the intact islets.

  12. A genetic screen for components of the mammalian RNA interference pathway in Bloom-deficient mouse embryonic stem cells.

    Science.gov (United States)

    Trombly, Melanie I; Su, Hong; Wang, Xiaozhong

    2009-03-01

    Genetic screens performed in model organisms have helped identify key components of the RNA interference (RNAi) pathway. Recessive genetic screens have recently become feasible through the use of mouse embryonic stem (ES) cells that are Bloom's syndrome protein (Blm) deficient. Here, we developed and performed a recessive genetic screen to identify components of the mammalian RNAi pathway in Blm-deficient ES cells. Genome-wide mutagenesis using a retroviral gene trap strategy resulted in the isolation of putative homozygous RNAi mutant cells. Candidate clones were confirmed by an independent RNAi-based reporter assay and the causative gene trap integration site was identified using molecular techniques. Our screen identified multiple mutant cell lines of Argonaute 2 (Ago2), a known essential component of the RNAi pathway. This result demonstrates that true RNAi components can be isolated by this screening strategy. Furthermore, Ago2 homozygous mutant ES cells provide a null genetic background to perform mutational analyses of the Ago2 protein. Using genetic rescue, we resolve an important controversy regarding the role of two phenylalanine residues in Ago2 activity.

  13. Multimodality Molecular Imaging of Cardiac Cell Transplantation: Part II. In Vivo Imaging of Bone Marrow Stromal Cells in Swine with PET/CT and MR Imaging.

    Science.gov (United States)

    Parashurama, Natesh; Ahn, Byeong-Cheol; Ziv, Keren; Ito, Ken; Paulmurugan, Ramasamy; Willmann, Jürgen K; Chung, Jaehoon; Ikeno, Fumiaki; Swanson, Julia C; Merk, Denis R; Lyons, Jennifer K; Yerushalmi, David; Teramoto, Tomohiko; Kosuge, Hisanori; Dao, Catherine N; Ray, Pritha; Patel, Manishkumar; Chang, Ya-Fang; Mahmoudi, Morteza; Cohen, Jeff Eric; Goldstone, Andrew Brooks; Habte, Frezghi; Bhaumik, Srabani; Yaghoubi, Shahriar; Robbins, Robert C; Dash, Rajesh; Yang, Phillip C; Brinton, Todd J; Yock, Paul G; McConnell, Michael V; Gambhir, Sanjiv S

    2016-09-01

    Purpose To quantitatively determine the limit of detection of marrow stromal cells (MSC) after cardiac cell therapy (CCT) in swine by using clinical positron emission tomography (PET) reporter gene imaging and magnetic resonance (MR) imaging with cell prelabeling. Materials and Methods Animal studies were approved by the institutional administrative panel on laboratory animal care. Seven swine received 23 intracardiac cell injections that contained control MSC and cell mixtures of MSC expressing a multimodality triple fusion (TF) reporter gene (MSC-TF) and bearing superparamagnetic iron oxide nanoparticles (NP) (MSC-TF-NP) or NP alone. Clinical MR imaging and PET reporter gene molecular imaging were performed after intravenous injection of the radiotracer fluorine 18-radiolabeled 9-[4-fluoro-3-(hydroxyl methyl) butyl] guanine ((18)F-FHBG). Linear regression analysis of both MR imaging and PET data and nonlinear regression analysis of PET data were performed, accounting for multiple injections per animal. Results MR imaging showed a positive correlation between MSC-TF-NP cell number and dephasing (dark) signal (R(2) = 0.72, P = .0001) and a lower detection limit of at least approximately 1.5 × 10(7) cells. PET reporter gene imaging demonstrated a significant positive correlation between MSC-TF and target-to-background ratio with the linear model (R(2) = 0.88, P = .0001, root mean square error = 0.523) and the nonlinear model (R(2) = 0.99, P = .0001, root mean square error = 0.273) and a lower detection limit of 2.5 × 10(8) cells. Conclusion The authors quantitatively determined the limit of detection of MSC after CCT in swine by using clinical PET reporter gene imaging and clinical MR imaging with cell prelabeling. (©) RSNA, 2016 Online supplemental material is available for this article.

  14. 3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions

    Energy Technology Data Exchange (ETDEWEB)

    Wenzel, Carsten; Riefke, Björn; Gründemann, Stephan; Krebs, Alice; Christian, Sven; Prinz, Florian; Osterland, Marc; Golfier, Sven; Räse, Sebastian [Bayer Pharma AG, Global Drug Discovery, Muellerstrasse 178, 13353 Berlin (Germany); Ansari, Nariman [Physical Biology Group, Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt (Germany); Esner, Milan; Bickle, Marc [Max Planck Institute of Molecular Cell Biology and Genetics, High-Throughput Technology Development Studio (TDS), Dresden (Germany); Pampaloni, Francesco; Mattheyer, Christian; Stelzer, Ernst H. [Physical Biology Group, Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt (Germany); Parczyk, Karsten; Prechtl, Stefan [Bayer Pharma AG, Global Drug Discovery, Muellerstrasse 178, 13353 Berlin (Germany); Steigemann, Patrick, E-mail: Patrick.Steigemann@bayer.com [Bayer Pharma AG, Global Drug Discovery, Muellerstrasse 178, 13353 Berlin (Germany)

    2014-04-15

    Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients as well as the development of dormant tumor regions. We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions. - Highlights: • Establishment of a novel method for 3D cell culture based high-content screening. • First reported high

  15. Feasibility Study on Prenatal Cardiac Screening Using Four-Dimensional Ultrasound with Spatiotemporal Image Correlation: A Multicenter Study.

    Directory of Open Access Journals (Sweden)

    Liqing Zhao

    Full Text Available This study aimed at investigating the feasibility of using the spatiotemporal image correlation (STIC technology for prenatal cardiac screening, finding factors that influence the offline evaluation of reconstructed fetal heart, and establishing an optimal acquisition scheme.The study included 452 gravidae presenting for routine screening at 3 maternity centers at 20-38 gestational weeks. The factors influencing the quality of STIC volume data were evaluated using t test, chi-square test, and logistic regression analysis. The predictive power was evaluated using the receiver operating characteristic (ROC curve.Among the 452 fetuses enrolled, 353 (78.1% were identified as successful and 99 (21.9% as failure of evaluation of the reconstructed fetal heart. The total success rate of qualified STIC images was 78.1%. The display rates of reconstructed cardiac views were 86.5% (four-chamber view, 92.5% (left ventricular outflow tract view, 92.7% (right ventricular outflow tract view, 89.9% (three-vessel trachea view, 63.9% (aortic arch view, 81.4% (ductal arch view, 81% (short-axis view of great vessels, 80.1% (long-cava view, and 86.9% (abdominal view. A logistic regression analysis showed that more than 28 gestational weeks [OR = 0.39 (CI 95% 0.16, 0.19, P = 0.035], frequent fetal movements [OR = 0.37 (CI 95% 0.16, 0.87, P = 0.022], shadowing [OR = 0.36 (CI 95% 0.19, 0.72, P = 0.004], spine location at 10-2 o'clock [OR = 0.08 (CI 95% 0.02, 0.27, P = 0.0], and original cardiac view [OR = 0.51 (0.25, 0.89, P = 0.019] had a significant impact on the quality of STIC. The area under the ROC curve was 0.775.Fetal cardiac-STIC seems a feasible tool for prenatal screening of congenital heart diseases. The influence factors on the quality of STIC images included the intensity of training, gestational age, fetal conditions and parameter settings. The optimal acquisition scheme may improve the application and widespread use of cardiac STIC.

  16. Non-invasive imaging of human embryonic stem cells.

    Science.gov (United States)

    Hong, Hao; Yang, Yunan; Zhang, Yin; Cai, Weibo

    2010-09-01

    Human embryonic stem cells (hESCs) hold tremendous therapeutic potential in a variety of diseases. Over the last decade, non-invasive imaging techniques have proven to be of great value in tracking transplanted hESCs. This review article will briefly summarize the various techniques used for non-invasive imaging of hESCs, which include magnetic resonance imaging (MRI), bioluminescence imaging (BLI), fluorescence, single-photon emission computed tomography (SPECT), positron emission tomography (PET), and multimodality approaches. Although the focus of this review article is primarily on hESCs, the labeling/tracking strategies described here can be readily applied to other (stem) cell types as well. Non-invasive imaging can provide convenient means to monitor hESC survival, proliferation, function, as well as overgrowth (such as teratoma formation), which could not be readily investigated previously. The requirement for hESC tracking techniques depends on the clinical scenario and each imaging technique will have its own niche in preclinical/clinical research. Continued evolvement of non-invasive imaging techniques will undoubtedly contribute to significant advances in understanding stem cell biology and mechanisms of action.

  17. Tumor-stem cells interactions by fluorescence imaging

    Science.gov (United States)

    Meleshina, Aleksandra V.; Cherkasova, Elena I.; Sergeeva, Ekaterina; Turchin, Ilya V.; Kiseleva, Ekaterina V.; Dashinimaev, Erdem B.; Shirmanova, Marina V.; Zagaynova, Elena V.

    2013-02-01

    Recently, great deal of interest is investigation the function of the stem cells (SC) in tumors. In this study, we studied «recipient-tumor- fluorescent stem cells » system using the methods of in vivo imaging and laser scanning microscopy (LSM). We used adipose-derived adult stem (ADAS) cells of human lentiviral transfected with the gene of fluorescent protein Turbo FP635. ADAS cells were administrated into nude mice with transplanted tumor HeLa Kyoto (human cervical carcinoma) at different stages of tumor growth (0-8 days) intravenously or into tumor. In vivo imaging was performed on the experimental setup for epi - luminescence bioimaging (IAP RAS, Nizhny Novgorod). The results of the imaging showed localization of fluorophore tagged stem cells in the spleen on day 5-9 after injection. The sensitivity of the technique may be improved by spectral separation autofluorescence and fluorescence of stem cells. We compared the results of in vivo imaging and confocal laser scanning microscopy (LSM 510 META, Carl Zeiss, Germany). Internal organs of the animals and tumor tissue were investigated. It was shown that with i.v. injection of ADAS, bright fluorescent structures with spectral characteristics corresponding to TurboFP635 protein are locally accumulated in the marrow, lungs and tumors of animals. These findings indicate that ADAS cells integrate in the animal body with transplanted tumor and can be identified by fluorescence bioimaging techniques in vivo and ex vivo.

  18. 3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions.

    Science.gov (United States)

    Wenzel, Carsten; Riefke, Björn; Gründemann, Stephan; Krebs, Alice; Christian, Sven; Prinz, Florian; Osterland, Marc; Golfier, Sven; Räse, Sebastian; Ansari, Nariman; Esner, Milan; Bickle, Marc; Pampaloni, Francesco; Mattheyer, Christian; Stelzer, Ernst H; Parczyk, Karsten; Prechtl, Stefan; Steigemann, Patrick

    2014-04-15

    Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients as well as the development of dormant tumor regions. We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions.

  19. Magnetic resonance imaging of water content across the Nafion membrane in an operational PEM fuel cell

    Science.gov (United States)

    Zhang, Ziheng; Martin, Jonathan; Wu, Jinfeng; Wang, Haijiang; Promislow, Keith; Balcom, Bruce J.

    2008-08-01

    Water management is critical to optimize the operation of polymer electrolyte membrane fuel cells. At present, numerical models are employed to guide water management in such fuel cells. Accurate measurements of water content variation in polymer electrolyte membrane fuel cells are required to validate these models and to optimize fuel cell behavior. We report a direct water content measurement across the Nafion membrane in an operational polymer electrolyte membrane fuel cell, employing double half k-space spin echo single point imaging techniques. The MRI measurements with T2 mapping were undertaken with a parallel plate resonator to avoid the effects of RF screening. The parallel plate resonator employs the electrodes inherent to the fuel cell to create a resonant circuit at RF frequencies for MR excitation and detection, while still operating as a conventional fuel cell at DC. Three stages of fuel cell operation were investigated: activation, operation and dehydration. Each profile was acquired in 6 min, with 6 μm nominal resolution and a SNR of better than 15.

  20. Red blood cell cluster separation from digital images for use in sickle cell disease.

    Science.gov (United States)

    González-Hidalgo, Manuel; Guerrero-Peña, F A; Herold-García, S; Jaume-I-Capó, Antoni; Marrero-Fernández, P D

    2015-07-01

    The study of cell morphology is an important aspect of the diagnosis of some diseases, such as sickle cell disease, because red blood cell deformation is caused by these diseases. Due to the elongated shape of the erythrocyte, ellipse adjustment and concave point detection are applied widely to images of peripheral blood samples, including during the detection of cells that are partially occluded in the clusters generated by the sample preparation process. In the present study, we propose a method for the analysis of the shape of erythrocytes in peripheral blood smear samples of sickle cell disease, which uses ellipse adjustments and a new algorithm for detecting notable points. Furthermore, we apply a set of constraints that allow the elimination of significant image preprocessing steps proposed in previous studies. We used three types of images to validate our method: artificial images, which were automatically generated in a random manner using a computer code; real images from peripheral blood smear sample images that contained normal and elongated erythrocytes; and synthetic images generated from real isolated cells. Using the proposed method, the efficiency of detecting the two types of objects in the three image types exceeded 99.00%, 98.00%, and 99.35%, respectively. These efficiency levels were superior to the results obtained with previously proposed methods using the same database, which is available at http://erythrocytesidb.uib.es/. This method can be extended to clusters of several cells and it requires no user inputs.

  1. Quantitative high-throughput screening identifies 8-hydroxyquinolines as cell-active histone demethylase inhibitors.

    Directory of Open Access Journals (Sweden)

    Oliver N F King

    Full Text Available BACKGROUND: Small molecule modulators of epigenetic processes are currently sought as basic probes for biochemical mechanisms, and as starting points for development of therapeutic agents. N(ε-Methylation of lysine residues on histone tails is one of a number of post-translational modifications that together enable transcriptional regulation. Histone lysine demethylases antagonize the action of histone methyltransferases in a site- and methylation state-specific manner. N(ε-Methyllysine demethylases that use 2-oxoglutarate as co-factor are associated with diverse human diseases, including cancer, inflammation and X-linked mental retardation; they are proposed as targets for the therapeutic modulation of transcription. There are few reports on the identification of templates that are amenable to development as potent inhibitors in vivo and large diverse collections have yet to be exploited for the discovery of demethylase inhibitors. PRINCIPAL FINDINGS: High-throughput screening of a ∼236,000-member collection of diverse molecules arrayed as dilution series was used to identify inhibitors of the JMJD2 (KDM4 family of 2-oxoglutarate-dependent histone demethylases. Initial screening hits were prioritized by a combination of cheminformatics, counterscreening using a coupled assay enzyme, and orthogonal confirmatory detection of inhibition by mass spectrometric assays. Follow-up studies were carried out on one of the series identified, 8-hydroxyquinolines, which were shown by crystallographic analyses to inhibit by binding to the active site Fe(II and to modulate demethylation at the H3K9 locus in a cell-based assay. CONCLUSIONS: These studies demonstrate that diverse compound screening can yield novel inhibitors of 2OG dependent histone demethylases and provide starting points for the development of potent and selective agents to interrogate epigenetic regulation.

  2. Role of serum angiopoietin-2 level in screening for esophageal squamous cell cancer and its precursors

    Institute of Scientific and Technical Information of China (English)

    ZHOU Ying-zhi; FANG Xue-qiang; LI Hao; DIAO Yu-tao; YANG Yan-fang; ZHAO De-li; WU Kan; LI Hui-qing

    2007-01-01

    Background Angiopoietin-2 (Ang-2) is one of the critical regulators of tumor angiogenesis. Studies have shown a significant correlation of Ang-2 expression to tumor invasion and metastasis in various human cancers, but little is known about the serum Ang-2 (sAng-2) levels in esophageal squamous cell cancer (ESCC) and its precursors. In this study, we aimed to investigate its role in screening for ESCC and its precursors.Methods We carried out a free endoscopic screening in Feicheng City, a high ESCC incidence area in Shandong Province of China. Serum samples were collected as follows: 91 from normal subjects, 44 from patients with esophagitis,85 from patients with hyperplasia, and 13 from patients with early ESCC. In addition, 28 serum samples were obtained from patients with invasive ESCC undergoing surgery in People's Hospital of Feicheng City. All the subjects of the five groups were diagnosed by histopathology. The sAng-2 levels were tested and compared, and the diagnostic power in early or/and invasive ESCC was calculated in terms of sensitivity and other parameters.Results The sAng-2 levels were (22.0±5.5), (21.3±3.2), (20.5±3.3), (24.0±5.0), and (29.8±5.0) U/ml in normal,esophagitis, hyperplasia, early ESCC, and invasive ESCC groups respectively. It was significantly higher in early ESCC than inhyperplasia group (P=0.009). The invasive ESCC group showed the highest Ang-2 level among all groups (all P=0.000). The sensitivities of sAng-2 to early and invasive ESCC were 23.1% and 78.6% respectively.Conclusion sAng-2 level is related to carcinogenesis and progression of ESCC, but it can not be used to screen for early ESCC.

  3. Autofluorescence-based diagnostic UV imaging of tissues and cells

    Science.gov (United States)

    Renkoski, Timothy E.

    Cancer is the second leading cause of death in the United States, and its early diagnosis is critical to improving treatment options and patient outcomes. In autofluorescence (AF) imaging, light of controlled wavelengths is projected onto tissue, absorbed by specific molecules, and re-emitted at longer wavelengths. Images of re-emitted light are used together with spectral information to infer tissue functional information and diagnosis. This dissertation describes AF imaging studies of three different organs using data collected from fresh human surgical specimens. In the ovary study, illumination was at 365 nm, and images were captured at 8 emission wavelengths. Measurements from a multispectral imaging system and fiber optic probe were used to map tissue diagnosis at every image pixel. For the colon and pancreas studies, instrumentation was developed extending AF imaging capability to sub-300 nm excitation. Images excited in the deep UV revealed tryptophan and protein content which are believed to change with disease state. Several excitation wavelength bands from 280 nm to 440 nm were investigated. Microscopic AF images collected in the pancreas study included both cultured and primary cells. Several findings are reported. A method of transforming fiber optic probe spectra for direct comparison with imager spectra was devised. Normalization of AF data by green reflectance data was found useful in correcting hemoglobin absorption. Ratio images, both AF and reflectance, were formulated to highlight growths in the colon. Novel tryptophan AF images were found less useful for colon diagnostics than the new ratio techniques. Microscopic tryptophan AF images produce useful visualization of cellular protein content, but their diagnostic value requires further study.

  4. Screening of plants acting against Heterometrus laoticus scorpion venom activity on fibroblast cell lysis.

    Science.gov (United States)

    Uawonggul, Nunthawun; Chaveerach, Arunrat; Thammasirirak, Sompong; Arkaravichien, Tarinee; Chuachan, Chattong; Daduang, Sakda

    2006-01-16

    The aqueous extracts of 64 plant species, listed as animal- or insect-bite antidotes in old Thai drug recipes were screened for their activity against fibroblast cell lysis after Heterometrus laoticus scorpion venom treatment. The venom was preincubated with plant extract for 30 min and furthered treated to confluent fibroblast cells for 30 min. More than 40% efficiency (test/control) was obtained from cell treatment with venom preincubated with extracts of Andrographis paniculata Nees (Acanthaceae), Barringtonia acutangula (L.) Gaertn. (Lecythidaceae), Calamus sp. (Palmae), Clinacanthus nutans Lindau (Acanthaceae), Euphorbia neriifolia L. (Euphorbiaceae), Ipomoea aquatica Forssk (Convolvulaceae), Mesua ferrea L. (Guttiferae), Passiflora laurifolia L. (Passifloraceae), Plectranthus amboinicus (Lour.) Spreng. (Labiatae), Ricinus communis L. (Euphorbiaceae), Rumex sp. (Polygonaceae) and Sapindus rarak DC. (Sapindaceae), indicating that they had a tendency to be scorpion venom antidotes. However, only Andrographis paniculata and Barringtonia acutangula extracts provided around 50% viable cells from extract treatments without venom preincubation. These two plant extracts are expected to be scorpion venom antidotes with low cytotoxicity.

  5. RNAi screen reveals an Abl kinase-dependent host cell pathway involved in Pseudomonas aeruginosa internalization.

    Directory of Open Access Journals (Sweden)

    Julia F Pielage

    2008-03-01

    Full Text Available Internalization of the pathogenic bacterium Pseudomonas aeruginosa by non-phagocytic cells is promoted by rearrangements of the actin cytoskeleton, but the host pathways usurped by this bacterium are not clearly understood. We used RNAi-mediated gene inactivation of approximately 80 genes known to regulate the actin cytoskeleton in Drosophila S2 cells to identify host molecules essential for entry of P. aeruginosa. This work revealed Abl tyrosine kinase, the adaptor protein Crk, the small GTPases Rac1 and Cdc42, and p21-activated kinase as components of a host signaling pathway that leads to internalization of P. aeruginosa. Using a variety of complementary approaches, we validated the role of this pathway in mammalian cells. Remarkably, ExoS and ExoT, type III secreted toxins of P. aeruginosa, target this pathway by interfering with GTPase function and, in the case of ExoT, by abrogating P. aeruginosa-induced Abl-dependent Crk phosphorylation. Altogether, this work reveals that P. aeruginosa utilizes the Abl pathway for entering host cells and reveals unexpected complexity by which the P. aeruginosa type III secretion system modulates this internalization pathway. Our results furthermore demonstrate the applicability of using RNAi screens to identify host signaling cascades usurped by microbial pathogens that may be potential targets for novel therapies directed against treatment of antibiotic-resistant infections.

  6. RNAi screen in Drosophila cells reveals the involvement of the Tom complex in Chlamydia infection.

    Directory of Open Access Journals (Sweden)

    Isabelle Derré

    2007-10-01

    Full Text Available Chlamydia spp. are intracellular obligate bacterial pathogens that infect a wide range of host cells. Here, we show that C. caviae enters, replicates, and performs a complete developmental cycle in Drosophila SL2 cells. Using this model system, we have performed a genome-wide RNA interference screen and identified 54 factors that, when depleted, inhibit C. caviae infection. By testing the effect of each candidate's knock down on L. monocytogenes infection, we have identified 31 candidates presumably specific of C. caviae infection. We found factors expected to have an effect on Chlamydia infection, such as heparansulfate glycosaminoglycans and actin and microtubule remodeling factors. We also identified factors that were not previously described as involved in Chlamydia infection. For instance, we identified members of the Tim-Tom complex, a multiprotein complex involved in the recognition and import of nuclear-encoded proteins to the mitochondria, as required for C. caviae infection of Drosophila cells. Finally, we confirmed that depletion of either Tom40 or Tom22 also reduced C. caviae infection in mammalian cells. However, C. trachomatis infection was not affected, suggesting that the mechanism involved is C. caviae specific.

  7. Quality assessment of images displayed on LCD screen with local backlight dimming

    DEFF Research Database (Denmark)

    Mantel, Claire; Burini, Nino; Korhonen, Jari;

    2013-01-01

    This paper presents a subjective experiment collecting quality assessment of images displayed on a LCD with local backlight dimming using two methodologies: absolute category ratings and paired-comparison. Some well-known objective quality metrics are then applied to the stimuli and their respect...

  8. Development of a biosensor microarray towards food screening, using imaging surface plasmon resonance

    NARCIS (Netherlands)

    Raz, Sabina Rebe; Bremer, Maria G. E. G.; Giesbers, Marcel; Norde, Willem

    2008-01-01

    In this study we examined the possibilities of implementing direct and competitive immunoassay formats for small and large molecule detection on a microarray, using IBIS imaging surface plasmon resonance (iSPR) system. First, IBIS iSPR optics performance was evaluated. Using a glycerol calibration c

  9. Development of a biosensor microarray towards food screening using imaging surface plasmon resonance

    NARCIS (Netherlands)

    Rebe, S.; Bremer, M.G.E.G.; Giesbers, M.; Norde, W.

    2008-01-01

    In this study we examined the possibilities of implementing direct and competitive immunoassay formats for small and large molecule detection on a microarray, using IBIS imaging surface plasmon resonance (iSPR) system. First, IBIS iSPR optics performance was evaluated. Using a glycerol calibration c

  10. Multiparametric Phenotypic Screening System for Profiling Bioactive Compounds Using Human Fetal Hippocampal Neural Stem/Progenitor Cells.

    Science.gov (United States)

    Tabata, Yoshikuni; Murai, Norio; Sasaki, Takeo; Taniguchi, Sachie; Suzuki, Shuichi; Yamazaki, Kazuto; Ito, Masashi

    2015-10-01

    Stem cell research has been progressing rapidly, contributing to regenerative biology and regenerative medicine. In this field, small-molecule compounds affecting stem cell proliferation/differentiation have been explored to understand stem cell biology and support regenerative medicine. In this study, we established a multiparametric screening system to detect bioactive compounds affecting the cell fate of human neural stem/progenitor cells (NSCs/NPCs), using human fetal hippocampal NSCs/NPCs, HIP-009 cells. We examined effects of 410 compounds, which were collected based on mechanisms of action (MOAs) and chemotypes, on HIP-009's cell fate (self-renewal, neuronal and astrocytic differentiation) and morphology by automated multiparametric assays and profiled induced cellular phenotypes. We found that this screening classified compounds with the same MOAs into subgroups according to additional pharmacological effects (e.g., mammalian target of rapamycin complex 1 [mTORC1] inhibitors and mTORC1/mTORC2 dual inhibitors among mTOR inhibitors). Moreover, it identified compounds that have off-target effects under matrix analyses of MOAs and structure similarities (e.g., neurotropic effects of amitriptyline among tri- and tetracyclic compounds). Therefore, this automated, medium-throughput and multiparametric screening system is useful for finding compounds that affect the cell fate of human NSCs/NPCs for supporting regenerative medicine and to fingerprint compounds based on human stem cells' multipotency, leading to understanding of stem cell biology.

  11. Cardiac magnetic resonance imaging after ventricular tachyarrhythmias increases diagnostic precision and reduces the need for family screening for inherited cardiac disease

    DEFF Research Database (Denmark)

    Marstrand, Peter; Axelsson, Anna; Thune, Jens Jakob

    2017-01-01

    -CAG) (81%), exercise stress test (47%), late potentials (54%), electrophysiological study (44%), pharmacological provocation (44%), and/or myocardial biopsy (16%). Family screening was indicated for 53 probands (67%) prior to CMR. After full workup, only 43 cases (54%) warranted evaluation of relatives (19...... magnetic resonance imaging re-defines the cardiac diagnoses in a significant proportion of cases and reduces the number of patients in whom family screening is warranted. Cardiac magnetic resonance imaging is highly relevant for optimal care and resource allocation when an inherited heart disease...

  12. Usefulness of Infrared Thermal Imaging Camera for Screening of Postoperative Surgical Site Infection after the Nuss Procedure

    Directory of Open Access Journals (Sweden)

    Kenya Fujita

    2013-01-01

    Full Text Available Introduction and Objective. The Nuss procedure is widely used in the treatment of pectus excavatum worldwide. Postoperative pectus bar infection is one of the most serious complications associated with this procedure. Therefore, early detection of signs of implant infection is very important. However, this is difficult, and effective methods have yet to be established. Methods. We use a handheld infrared thermal imaging camera to screen patients for postoperative infection following the Nuss procedure. Here, we report a 28-year-old man with recurrent postoperative (Ravitch procedure pectus excavatum. Results. Infrared thermography camera clearly indicated slight cellulitis in the right chest. Conclusion. Our technique may assist in preventing postoperative bar infection and removal caused by severe bar infection. Furthermore, this camera is potentially suitable for many situations in infection monitoring following subcutaneous implant surgery.

  13. Microfluidic Cell Deformability Assay for Rapid and Efficient Kinase Screening with the CRISPR-Cas9 System.

    Science.gov (United States)

    Han, Xin; Liu, Zongbin; Zhao, Li; Wang, Feng; Yu, Yang; Yang, Jianhua; Chen, Rui; Qin, Lidong

    2016-07-18

    Herein we report a CRISPR-Cas9-mediated loss-of-function kinase screen for cancer cell deformability and invasive potential in a high-throughput microfluidic chip. In this microfluidic cell separation platform, flexible cells with high deformability and metastatic propensity flowed out, while stiff cells remained trapped. Through deep sequencing, we found that loss of certain kinases resulted in cells becoming more deformable and invasive. High-ranking candidates identified included well-reported tumor suppressor kinases, such as chk2, IKK-α, p38 MAPKs, and DAPK2. A high-ranking candidate STK4 was chosen for functional validation and identified to play an important role in the regulation of cell deformability and tumor suppression. Collectively, we have demonstrated that CRISPR-based on-chip mechanical screening is a potentially powerful strategy to facilitate systematic genetic analyses.

  14. Challenges in imaging cell surface receptor clusters

    Science.gov (United States)

    Medda, Rebecca; Giske, Arnold; Cavalcanti-Adam, Elisabetta Ada

    2016-01-01

    Super-resolution microscopy offers unique tools for visualizing and resolving cellular structures at the molecular level. STED microscopy is a purely optical method where neither complex sample preparation nor mathematical post-processing is required. Here we present the use of STED microscopy for imaging receptor cluster composition. We use two-color STED to further determine the distribution of two different receptor subunits of the family of receptor serine/threonine kinases in the presence or absence of their ligands. The implications of receptor clustering on the downstream signaling are discussed, and future challenges are also presented.

  15. The use of magnetic nanoparticles in thermal therapy monitoring and screening: Localization and imaging (invited).

    Science.gov (United States)

    Weaver, John B

    2012-04-01

    Magnetic nanoparticles have many diagnostic and therapeutic applications. A method termed magnetic spectroscopy of nanoparticle Brownian motion (MSB) was developed to interrogate in vivo the microscopic environment surrounding magnetic nanoparticles. We can monitor several effects that are important in thermal therapy and screening including temperature measurement and the bound state distribution. Here we report on simulations of nanoparticle localization. Measuring the spatial distribution of nanoparticles would allow us to identify ovarian cancer much earlier when it is still curable or monitor thermal therapies more accurately. We demonstrate that with well-designed equipment superior signal to noise ratio (SNR) can be achieved using only two harmonics rather than using all the harmonics containing signal. Alternatively, smaller magnetic field amplitudes can be used to achieve the same SNR. The SNR is improved using fewer harmonics because the noise is limited.

  16. Quantitative phase imaging for cell culture quality control.

    Science.gov (United States)

    Kastl, Lena; Isbach, Michael; Dirksen, Dieter; Schnekenburger, Jürgen; Kemper, Björn

    2017-03-06

    The potential of quantitative phase imaging (QPI) with digital holographic microscopy (DHM) for quantification of cell culture quality was explored. Label-free QPI of detached single cells in suspension was performed by Michelson interferometer-based self-interference DHM. Two pancreatic tumor cell lines were chosen as cellular model and analyzed for refractive index, volume, and dry mass under varying culture conditions. Firstly, adequate cell numbers for reliable statistics were identified. Then, to characterize the performance and reproducibility of the method, we compared results from independently repeated measurements and quantified the cellular response to osmolality changes of the cell culture medium. Finally, it was demonstrated that the evaluation of QPI images allows the extraction of absolute cell parameters which are related to cell layer confluence states. In summary, the results show that QPI enables label-free imaging cytometry, which provides novel complementary integral biophysical data sets for sophisticated quantification of cell culture quality with minimized sample preparation. © 2017 International Society for Advancement of Cytometry.

  17. Electrical impedance tomographic imaging of a single cell electroporation.

    Science.gov (United States)

    Meir, Arie; Rubinsky, Boris

    2014-06-01

    A living cell placed in a high strength electric field, can undergo a process known as electroporation. It is believed that during electroporation nano-scale defects (pores) occur in the membrane of the cell, causing dramatic changes to the permeability of its membrane. Electroporation is an important technique in biotechnology and medicine and numerous methods are being developed to improve the understanding and use of the technology. We propose to extend the toolbox available for studying electroporation by generating impedance distribution images of the cell as it undergoes electroporation using Electrical Impedance Tomography (EIT). To investigate the feasibility of this concept, we develop a mathematical model of the process of electroporation in a single cell and of EIT of the process and show simulation results of a computer-based finite element model (FEM). Our work is an attempt to develop a new imaging tool for visualizing electroporation in a single cell, offering a different temporal and spatial resolution compared to the state of the art, which includes bulk measurements of electrical properties during single cell electroporation, patch clamp and voltage clamp measurement in single cells and optical imaging with colorimetric dyes during single cell electroporation. This paper is a preliminary theoretic feasibility study.

  18. A Cell-Based Approach for the Biosynthesis/Screening of Cyclic Peptide Libraries against Bacterial Toxins

    Energy Technology Data Exchange (ETDEWEB)

    Camarero, J A; Kimura, R; Woo, Y; Cantor, J; Steenblock, E

    2007-10-24

    Available methods for developing and screening small drug-like molecules able to knockout toxins or pathogenic microorganisms have some limitations. In order to be useful, these new methods must provide high-throughput analysis and identify specific binders in a short period of time. To meet this need, we are developing an approach that uses living cells to generate libraries of small biomolecules, which are then screened inside the cell for activity. Our group is using this new, combined approach to find highly specific ligands capable of disabling anthrax Lethal Factor (LF) as proof of principle. Key to our approach is the development of a method for the biosynthesis of libraries of cyclic peptides, and an efficient screening process that can be carried out inside the cell.

  19. A Digital PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells.

    Directory of Open Access Journals (Sweden)

    Scott D Findlay

    Full Text Available The rapid adoption of gene editing tools such as CRISPRs and TALENs for research and eventually therapeutics necessitates assays that can rapidly detect and quantitate the desired alterations. Currently, the most commonly used assay employs "mismatch nucleases" T7E1 or "Surveyor" that recognize and cleave heteroduplexed DNA amplicons containing mismatched base-pairs. However, this assay is prone to false positives due to cancer-associated mutations and/or SNPs and requires large amounts of starting material. Here we describe a powerful alternative wherein droplet digital PCR (ddPCR can be used to decipher homozygous from heterozygous mutations with superior levels of both precision and sensitivity. We use this assay to detect knockout inducing alterations to stem cell associated proteins, NODAL and SFRP1, generated using either TALENs or an "all-in-one" CRISPR/Cas plasmid that we have modified for one-step cloning and blue/white screening of transformants. Moreover, we highlight how ddPCR can be used to assess the efficiency of varying TALEN-based strategies. Collectively, this work highlights how ddPCR-based screening can be paired with CRISPR and TALEN technologies to enable sensitive, specific, and streamlined approaches to gene editing and validation.

  20. Impact of prematurity and immigration on neonatal screening for sickle cell disease

    Science.gov (United States)

    Cortés-Castell, Ernesto; Pla, Carolina; Goicoechea, Mercedes; Rizo-Baeza, María Mercedes; Juste, Mercedes; Gil-Guillén, Vicente Francisco

    2017-01-01

    Background Others have described a relationship between hemoglobin A levels and gestational age, gender and ethnicity. However, studies are needed to determine normal cut-off points considering these factors. To address this issue we designed a study to determine the percentiles of normality of neonatal hemoglobin A levels taking these factors into account. Methods This cross-sectional study involved 16,025 samples for sickle cell disease screening in the province of Alicante, Spain, which has a high immigration rate. The primary variable was hemoglobin A, and the secondary variables were gender, gestational age (preterm and full term) and maternal origin (Spain, the rest of Europe, North Africa, Sub-Saharan Africa, Latin America and Asia). Percentiles of normality (1 and 99) were obtained by origin, gender and gestational age using quantile regression models and bootstrap samples. The association between these percentiles of normality and altered levels (≥1%) of hemoglobin E was analyzed. We obtained the percentiles of normality (1 and 99) for each maternal origin, gender and gestational age. Results Of a total of 88 possible E carriers, 65 had above-normal hemoglobin A levels (74%). The levels of normality for hemoglobin A varied greatly according to the maternal origin and gestational age. Conclusion With the levels of normality that we established it is possible to discard samples with unrecorded blood transfusions. Our methodology could be applied to other diseases in the neonatal screening. PMID:28170418

  1. A Digital PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells

    Science.gov (United States)

    Berman, Jennifer R.; Postovit, Lynne-Marie

    2016-01-01

    The rapid adoption of gene editing tools such as CRISPRs and TALENs for research and eventually therapeutics necessitates assays that can rapidly detect and quantitate the desired alterations. Currently, the most commonly used assay employs “mismatch nucleases” T7E1 or “Surveyor” that recognize and cleave heteroduplexed DNA amplicons containing mismatched base-pairs. However, this assay is prone to false positives due to cancer-associated mutations and/or SNPs and requires large amounts of starting material. Here we describe a powerful alternative wherein droplet digital PCR (ddPCR) can be used to decipher homozygous from heterozygous mutations with superior levels of both precision and sensitivity. We use this assay to detect knockout inducing alterations to stem cell associated proteins, NODAL and SFRP1, generated using either TALENs or an “all-in-one” CRISPR/Cas plasmid that we have modified for one-step cloning and blue/white screening of transformants. Moreover, we highlight how ddPCR can be used to assess the efficiency of varying TALEN-based strategies. Collectively, this work highlights how ddPCR-based screening can be paired with CRISPR and TALEN technologies to enable sensitive, specific, and streamlined approaches to gene editing and validation. PMID:27089539

  2. Redefining circulating tumor cells by image processing

    NARCIS (Netherlands)

    Ligthart, S.T.

    2012-01-01

    Circulating tumor cells (CTC) in the blood of patients with metastatic carcinomas are associated with poor survival and can be used to guide therapy. However, CTC are very heterogeneous in size and shape, and are present at very low frequencies. Missing or misjudging a few events may have great cons

  3. High-throughput library screening identifies two novel NQO1 inducers in human lung cells.

    Science.gov (United States)

    Tan, Xiang-Lin; Marquardt, Gaby; Massimi, Aldo B; Shi, Miao; Han, Weiguo; Spivack, Simon D

    2012-03-01

    Many phytochemicals possess antioxidant and cancer-preventive properties, some putatively through antioxidant response element-mediated phase II metabolism, entailing mutagen/oxidant quenching. In our recent studies, however, most candidate phytochemical agents were not potent in inducing phase II genes in normal human lung cells. In this study, we applied a messenger RNA (mRNA)-specific gene expression-based high throughput in vitro screening approach to discover new, potent plant-derived phase II inducing chemopreventive agents. Primary normal human bronchial epithelial (NHBE) cells and immortalized human bronchial epithelial cells (HBECs) were exposed to 800 individual compounds in the MicroSource Natural Products Library. At a level achievable in humans by diet (1.0 μM), 2,3-dihydroxy-4-methoxy-4'-ethoxybenzophenone (DMEBP), triacetylresveratrol (TRES), ivermectin, sanguinarine sulfate, and daunorubicin induced reduced nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase 1 (NQO1) mRNA and protein expression in NHBE cells. DMEBP and TRES were the most attractive agents as coupling potency and low toxicity for induction of NQO1 (mRNA level, ≥3- to 10.8-fold that of control; protein level, ≥ two- to fourfold that of control). Induction of glutathione S-transferase pi mRNA expression was modest, and none was apparent for glutathione S-transferase pi protein expression. Measurements of reactive oxygen species and glutathione/oxidized glutathione ratio showed an antioxidant effect for DMEBP, but no definite effect was found for TRES in NHBE cells. Exposure of NHBE cells to H(2)O(2) induced nuclear translocation of nuclear factor erythroid 2-related factor 2, but this translocation was not significantly inhibited by TRES and DMEBP. These studies show that potency and low toxicity may align for two potential NQO1-inducing agents, DMEBP and TRES.

  4. Quantitative photoacoustic imaging of nanoparticles in cells and tissues.

    Science.gov (United States)

    Cook, Jason R; Frey, Wolfgang; Emelianov, Stanislav

    2013-02-26

    Quantitative visualization of nanoparticles in cells and tissues, while preserving the spatial information, is very challenging. A photoacoustic imaging technique to depict the presence and quantity of nanoparticles is presented. This technique is based on the dependence of the photoacoustic signal on both the nanoparticle quantity and the laser fluence. Quantitative photoacoustic imaging is a robust technique that does not require knowledge of the local fluence, but a relative change in the fluence. This eliminates the need for sophisticated methods or models to determine the energy distribution of light in turbid media. Quantitative photoacoustic imaging was first applied to nanoparticle-loaded cells, and quantitation was validated by inductively coupled plasma mass spectrometry. Quantitative photoacoustic imaging was then extended to xenograft tumor tissue sections, and excellent agreement with traditional histopathological analysis was demonstrated. Our results suggest that quantitative photoacoustic imaging may be used in many applications including the determination of the efficiency and effectiveness of molecular targeting strategies for cell studies and animal models, the quantitative assessment of photoacoustic contrast agent biodistribution, and the validation of in vivo photoacoustic imaging.

  5. How I report breast magnetic resonance imaging studies for breast cancer staging and screening.

    Science.gov (United States)

    Vinnicombe, Sarah

    2016-07-25

    Magnetic resonance imaging (MRI) of the breast is the most sensitive imaging technique for the diagnosis and local staging of primary breast cancer and yet, despite the fact that it has been in use for 20 years, there is little evidence that its widespread uncritical adoption has had a positive impact on patient-related outcomes.This has been attributed previously to the low specificity that might be expected with such a sensitive modality, but with modern techniques and protocols, the specificity and positive predictive value for malignancy can exceed that of breast ultrasound and mammography. A more likely explanation is that historically, clinicians have acted on MRI findings and altered surgical plans without prior histological confirmation. Furthermore, modern adjuvant therapy for breast cancer has improved so much that it has become a very tall order to show a an improvement in outcomes such as local recurrence rates.In order to obtain clinically useful information, it is necessary to understand the strengths and weaknesses of the technique and the physiological processes reflected in breast MRI. An appropriate indication for the scan, proper patient preparation and good scan technique, with rigorous quality assurance, are all essential prerequisites for a diagnostically relevant study.The use of recognised descriptors from a standardised lexicon is helpful, since assessment can then dictate subsequent recommendations for management, as in the American College of Radiology BI-RADS (Breast Imaging Reporting and Data System) lexicon (Morris et al., ACR BI-RADS® Atlas, Breast Imaging Reporting and Data System, 2013). It also enables audit of the service. However, perhaps the most critical factor in the generation of a meaningful report is for the reporting radiologist to have a thorough understanding of the clinical question and of the findings that will influence management. This has never been more important than at present, when we are in the throes of a

  6. Development of an Automated Modality-Independent Elastographic Image Analysis System for Tumor Screening

    Science.gov (United States)

    2007-02-01

    quantity of polymer solution with an imaging contrast agent. Before the phantom fully polymerizes through freezing, a hypodermic needle is used to create...approximation in favor of one compatible with large deformations. The difference in solutions between small and large deformation theory can be...difference in the linear model among Fig. 2a and 2b, 2b is the reverse of 2a (this is a characteristic of linear theory ). However, the lack of this

  7. Teratogen screening using transcriptome profiling of differentiating human embryonic stem cells.

    Science.gov (United States)

    Mayshar, Yoav; Yanuka, Ofra; Benvenisty, Nissim

    2011-06-01

    Teratogens are substances that may cause defects in normal embryonic development while not necessarily being toxic in adults. Identification of possible teratogenic compounds has been historically beset by the species-specific nature of the teratogen response. To examine teratogenic effects on early human development we performed non-biased expression profiling of differentiating human embryonic and induced pluripotent stem cells treated with several drugs--ethanol, lithium, retinoic acid (RA), caffeine and thalidomide, which is known to be highly species specific. Our results point to the potency of specific teratogens and their affected tissues and pathways. Specifically, we could show that ethanol caused dramatic increase in endodermal differentiation, RA caused misregulation of neural development and thalidomide affected both these processes. We thus propose this method as a valuable addition to currently available animal screening approaches.

  8. Development of a screening approach for exploring cell factory potential through metabolic flux analysis and physiology

    DEFF Research Database (Denmark)

    Knudsen, Peter Boldsen; Nielsen, Kristian Fog; Thykær, Jette

    2012-01-01

    The recent developments within the field of metabolic engineering have significantly increased the speed by which fungal recombinant strains are being constructed, pushing focus towards physiological characterisation and analysis. This raises demand for a tool for diligent analysis of the recombi...... and work-load connected with screening and selection of potential cell factories with attractive properties, compared with more “traditional” methodologies where metabolic flux analysis is applied at a much later state in the characterisation process.......The recent developments within the field of metabolic engineering have significantly increased the speed by which fungal recombinant strains are being constructed, pushing focus towards physiological characterisation and analysis. This raises demand for a tool for diligent analysis...... on a Hamilton robotic system. This method aimed at characterising physiology at two levels: (1) An approach focusing on the traditional growth related parameters, i.e. growth rate, yield coefficients and extracellular metabolites. (2) 13C-labelling experiments, where metabolic fluxes are quantified...

  9. Screening and Extraction of Plant Pigments and Fabrication of Dye Sensitized Solar Cells

    Directory of Open Access Journals (Sweden)

    Gaanappriya Mohan

    2016-12-01

    Full Text Available Photovoltaic devices are constructed on the concept of charge separation at an interface of two different materials with different conduction mechanism. Till date, this area of research has been dominated by solid-state junction devices that are usually made of silicon, and profiting from the experience and material availability resulting from the semiconductor industry. In the present study, research is undertaken to unravel the typical coloring pigments from a variety of plant species that can be used for photochemical conversion of the solar energy. Eleven plants’ samples were taken for the screening procedure. Spectrophotometrial analyses were carried out for all the natural dyes extracted. Based on the absorption spectra two dyes were found to be possessing absorption spectra of the permissible range. These two samples were used for the assemblages of Dye Sensitized Solar Cells(DSSC. The photovoltaic characters were analyzed. The Conversion efficiency is reported and discussed.

  10. Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity

    Science.gov (United States)

    Terekhov, Stanislav S.; Smirnov, Ivan V.; Stepanova, Anastasiya V.; Bobik, Tatyana V.; Mokrushina, Yuliana A.; Ponomarenko, Natalia A.; Belogurov, Alexey A.; Rubtsova, Maria P.; Kartseva, Olga V.; Gomzikova, Marina O.; Moskovtsev, Alexey A.; Bukatin, Anton S.; Dubina, Michael V.; Kostryukova, Elena S.; Babenko, Vladislav V.; Vakhitova, Maria T.; Manolov, Alexander I.; Malakhova, Maja V.; Kornienko, Maria A.; Tyakht, Alexander V.; Vanyushkina, Anna A.; Ilina, Elena N.; Masson, Patrick; Gabibov, Alexander G.; Altman, Sidney

    2017-01-01

    Ultrahigh-throughput screening (uHTS) techniques can identify unique functionality from millions of variants. To mimic the natural selection mechanisms that occur by compartmentalization in vivo, we developed a technique based on single-cell encapsulation in droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (MDE). Biocompatible MDE enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions. This platform was probed with uHTS for biocatalysts anchored to yeast with enrichment close to the theoretically calculated limit and cell-to-cell interactions. MDE–FACS allowed the identification of human butyrylcholinesterase mutants that undergo self-reactivation after inhibition by the organophosphorus agent paraoxon. The versatility of the platform allowed the identification of bacteria, including slow-growing oral microbiota species that suppress the growth of a common pathogen, Staphylococcus aureus, and predicted which genera were associated with inhibitory activity. PMID:28202731

  11. Disease Modeling and Phenotypic Drug Screening for Diabetic Cardiomyopathy using Human Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Faye M. Drawnel

    2014-11-01

    Full Text Available Diabetic cardiomyopathy is a complication of type 2 diabetes, with known contributions of lifestyle and genetics. We develop environmentally and genetically driven in vitro models of the condition using human-induced-pluripotent-stem-cell-derived cardiomyocytes. First, we mimic diabetic clinical chemistry to induce a phenotypic surrogate of diabetic cardiomyopathy, observing structural and functional disarray. Next, we consider genetic effects by deriving cardiomyocytes from two diabetic patients with variable disease progression. The cardiomyopathic phenotype is recapitulated in the patient-specific cells basally, with a severity dependent on their original clinical status. These models are incorporated into successive levels of a screening platform, identifying drugs that preserve cardiomyocyte phenotype in vitro during diabetic stress. In this work, we present a patient-specific induced pluripotent stem cell (iPSC model of a complex metabolic condition, showing the power of this technique for discovery and testing of therapeutic strategies for a disease with ever-increasing clinical significance.

  12. Raman hyperspectral imaging of iron transport across membranes in cells

    Science.gov (United States)

    Das, Anupam; Costa, Xavier Felipe; Khmaladze, Alexander; Barroso, Margarida; Sharikova, Anna

    2016-09-01

    Raman scattering microscopy is a powerful imaging technique used to identify chemical composition, structural and conformational state of molecules of complex samples in biology, biophysics, medicine and materials science. In this work, we have shown that Raman techniques allow the measurement of the iron content in protein mixtures and cells. Since the mechanisms of iron acquisition, storage, and excretion by cells are not completely understood, improved knowledge of iron metabolism can offer insight into many diseases in which iron plays a role in the pathogenic process, such as diabetes, neurodegenerative diseases, cancer, and metabolic syndrome. Understanding of the processes involved in cellular iron metabolism will improve our knowledge of cell functioning. It will also have a big impact on treatment of diseases caused by iron deficiency (anemias) and iron overload (hereditary hemochromatosis). Previously, Raman studies have shown substantial differences in spectra of transferrin with and without bound iron, thus proving that it is an appropriate technique to determine the levels of bound iron in the protein mixture. We have extended these studies to obtain hyperspectral images of transferrin in cells. By employing a Raman scanning microscope together with spectral detection by a highly sensitive back-illuminated cooled CCD camera, we were able to rapidly acquire and process images of fixed cells with chemical selectivity. We discuss and compare various methods of hyperspectral Raman image analysis and demonstrate the use of these methods to characterize cellular iron content without the need for dye labeling.

  13. Nonlinear chemical imaging microscopy: near-field third harmonic generation imaging of human red blood cells.

    Science.gov (United States)

    Schaller, R D; Johnson, J C; Saykally, R J

    2000-11-01

    Third harmonic generation (THG) imaging using a near-field scanning optical microscope (NSOM) is demonstrated for the first time. A femtosecond, tunable near-infrared laser was used to generate both nonresonant and resonantly enhanced third harmonic radiation in human red blood cells. We show that resonantly enhanced THG is a chemically specific bulk probe in NSOM imaging by tuning the excitation source onto and off of resonance with the Soret transition of oxyhemoglobin. Additionally, we provide evidence that tightly focused, nonresonant, far-field THG imaging experiments do not produce contrast that is truly surface specific.

  14. A high-throughput screen for teratogens using human pluripotent stem cells.

    Science.gov (United States)

    Kameoka, Sei; Babiarz, Joshua; Kolaja, Kyle; Chiao, Eric

    2014-01-01

    There is need in the pharmaceutical and chemical industries for high-throughput human cell-based assays for identifying hazardous chemicals, thereby reducing the overall reliance on animal studies for predicting the risk of toxic responses in humans. Despite instances of human-specific teratogens such as thalidomide, the use of human cell-teratogenicity assays has just started to be explored. Herein, a human pluripotent stem cell test (hPST) for identifying teratogens is described, benchmarking the in vitro findings to traditional preclinical toxicology teratogenicity studies and when available to teratogenic outcomes in humans. The hPST method employs a 3-day monolayer directed differentiation of human embryonic stem cells. The teratogenic risk of a compound is gauged by measuring the reduction in nuclear translocation of the transcription factor SOX17 in mesendodermal cells. Decreased nuclear SOX17 in the hPST model was strongly correlated with in vivo teratogenicity. Specifically, 71 drug-like compounds with known in vivo effects, including thalidomide, were examined in the hPST. A threshold of 5 μM demonstrated 94% accuracy (97% sensitivity and 92% specificity). Furthermore, 15 environmental toxicants with physicochemical properties distinct from small molecule pharmaceutical agents were examined and a similarly strong concordance with teratogenicity outcomes from in vivo studies was observed. Finally, to assess the suitability of the hPST for high-throughput screens, a small library of 300 kinase inhibitors was tested, demonstrating the hPST platform's utility for interrogating teratogenic mechanisms and drug safety prediction. Thus, the hPST assay is a robust predictor of teratogenicity and appears to be an improvement over existing in vitro models.

  15. Screening for the Most Suitable Reference Genes for Gene Expression Studies in Equine Milk Somatic Cells.

    Directory of Open Access Journals (Sweden)

    Jakub Cieslak

    Full Text Available Apart from the well-known role of somatic cell count as a parameter reflecting the inflammatory status of the mammary gland, the composition of cells isolated from milk is considered as a valuable material for gene expression studies in mammals. Due to its unique composition, in recent years an increasing interest in mare's milk consumption has been observed. Thus, investigating the genetic background of horse's milk variability presents and interesting study model. Relying on 39 milk samples collected from mares representing three breeds (Polish Primitive Horse, Polish Cold-blooded Horse, Polish Warmblood Horse we aimed to investigate the utility of equine milk somatic cells as a source of mRNA and to screen the best reference genes for RT-qPCR using geNorm and NormFinder algorithms. The results showed that despite relatively low somatic cell counts in mare's milk, the amount and the quality of the extracted RNA are sufficient for gene expression studies. The analysis of the utility of 7 potential reference genes for RT-qPCR experiments for the normalization of equine milk somatic cells revealed some differences between the outcomes of the applied algorithms, although in both cases the KRT8 and TOP2B genes were pointed as the most stable. Analysis by geNorm showed that the combination of 4 reference genes (ACTB, GAPDH, TOP2B and KRT8 is required for apropriate RT-qPCR experiments normalization, whereas NormFinder algorithm pointed the combination of KRT8 and RPS9 genes as the most suitable. The trial study of the relative transcript abundance of the beta-casein gene with the use of various types and numbers of internal control genes confirmed once again that the selection of proper reference gene combinations is crucial for the final results of each real-time PCR experiment.

  16. NSC23925, identified in a high-throughput cell-based screen, reverses multidrug resistance.

    Directory of Open Access Journals (Sweden)

    Zhenfeng Duan

    Full Text Available BACKGROUND: Multidrug resistance (MDR is a major factor which contributes to the failure of cancer chemotherapy, and numerous efforts have been attempted to overcome MDR. To date, none of these attempts have yielded a tolerable and effective therapy to reverse MDR; thus, identification of new agents would be useful both clinically and scientifically. METHODOLOGY/PRINCIPAL FINDINGS: To identify small molecule compounds that can reverse chemoresistance, we developed a 96-well plate high-throughput cell-based screening assay in a paclitaxel resistant ovarian cancer cell line. Coincubating cells with a sublethal concentration of paclitaxel in combination with each of 2,000 small molecule compounds from the National Cancer Institute Diversity Set Library, we identified a previously uncharacterized molecule, NSC23925, that inhibits Pgp1 and reverses MDR1 (Pgp1 but does not inhibit MRP or BCRP-mediated MDR. The cytotoxic activity of NSC23925 was further evaluated using a panel of cancer cell lines expressing Pgp1, MRP, and BCRP. We found that at a concentration of >10 microM NSC23925 moderately inhibits the proliferation of both sensitive and resistant cell lines with almost equal activity, but its inhibitory effect was not altered by co-incubation with the Pgp1 inhibitor, verapamil, suggesting that NSC23925 itself is not a substrate of Pgp1. Additionally, NSC23925 increases the intracellular accumulation of Pgp1 substrates: calcein AM, Rhodamine-123, paclitaxel, mitoxantrone, and doxorubicin. Interestingly, we further observed that, although NSC23925 directly inhibits the function of Pgp1 in a dose-dependent manner without altering the total expression level of Pgp1, NSC23925 actually stimulates ATPase activity of Pgp, a phenomenon seen in other Pgp inhibitors. CONCLUSIONS/SIGNIFICANCE: The ability of NSC23925 to restore sensitivity to the cytotoxic effects of chemotherapy or to prevent resistance could significantly benefit cancer patients.

  17. Nylon-3 copolymers that generate cell-adhesive surfaces identified by library screening.

    Science.gov (United States)

    Lee, Myung-Ryul; Stahl, Shannon S; Gellman, Samuel H; Masters, Kristyn S

    2009-11-25

    Polymers in the nylon-3 family contain subunits derived from beta-amino acids, which are linked to one another via amide bonds. Thus, the nylon-3 backbone is homologous to the alpha-amino acid-based backbone of proteins. This molecular-level homology suggests that nylon-3 materials might be intrinsically protein-mimetic. The experiments described here explore this prospect in the context of cell adhesion, with tissue engineering as a long-range goal. We have evaluated a small library of sequence-random nylon-3 copolymers for the ability to render surfaces attractive to NIH 3T3 fibroblast adhesion and spreading. Library screening was accomplished in a high-throughput, parallel mode via attachment of the copolymers in a two-dimensional array to a modified glass surface. Significant variations in fibroblast adhesion and spreading were observed as a function of nylon-3 subunit identity and proportion. Several of the nylon-3 copolymers supported cell adhesion and morphology that was comparable, or even superior, to that achieved on positive control substrates such as tissue culture polystyrene and collagen-coated glass. Moreover, studies conducted under serum-free conditions demonstrated that specific nylon-3 derivatives supported cell adhesion independently of serum protein adsorption. Although cell adhesion was diminished in the absence of serum, particular copolymers demonstrated an ability to support substantially greater cell adhesion than any of the other conditions, including the positive controls. The nylon-3 copolymers that were most effective at promoting adhesion to a modified glass surface proved also to be effective at promoting adhesion when attached to a PEG-based hydrogel, demonstrating the potential for these copolymers to be used in tissue engineering applications.

  18. Automated image analysis with the potential for process quality control applications in stem cell maintenance and differentiation.

    Science.gov (United States)

    Smith, David; Glen, Katie; Thomas, Robert

    2016-01-01

    The translation of laboratory processes into scaled production systems suitable for manufacture is a significant challenge for cell based therapies; in particular there is a lack of analytical methods that are informative and efficient for process control. Here the potential of image analysis as one part of the solution to this issue is explored, using pluripotent stem cell colonies as a valuable and challenging exemplar. The Cell-IQ live cell imaging platform was used to build image libraries of morphological culture attributes such as colony "edge," "core periphery" or "core" cells. Conventional biomarkers, such as Oct3/4, Nanog, and Sox-2, were shown to correspond to specific morphologies using immunostaining and flow cytometry techniques. Quantitative monitoring of these morphological attributes in-process using the reference image libraries showed rapid sensitivity to changes induced by different media exchange regimes or the addition of mesoderm lineage inducing cytokine BMP4. The imaging sample size to precision relationship was defined for each morphological attribute to show that this sensitivity could be achieved with a relatively low imaging sample. Further, the morphological state of single colonies could be correlated to individual colony outcomes; smaller colonies were identified as optimum for homogenous early mesoderm differentiation, while larger colonies maintained a morphologically pluripotent core. Finally, we show the potential of the same image libraries to assess cell number in culture with accuracy comparable to sacrificial digestion and counting. The data supports a potentially powerful role for quantitative image analysis in the setting of in-process specifications, and also for screening the effects of process actions during development, which is highly complementary to current analysis in optimization and manufacture.

  19. First quantitative high-throughput screen in zebrafish identifies novel pathways for increasing pancreatic β-cell mass.

    Science.gov (United States)

    Wang, Guangliang; Rajpurohit, Surendra K; Delaspre, Fabien; Walker, Steven L; White, David T; Ceasrine, Alexis; Kuruvilla, Rejji; Li, Ruo-Jing; Shim, Joong S; Liu, Jun O; Parsons, Michael J; Mumm, Jeff S

    2015-07-28

    Whole-organism chemical screening can circumvent bottlenecks that impede drug discovery. However, in vivo screens have not attained throughput capacities possible with in vitro assays. We therefore developed a method enabling in vivo high-throughput screening (HTS) in zebrafish, termed automated reporter quantification in vivo (ARQiv). In this study, ARQiv was combined with robotics to fully actualize whole-organism HTS (ARQiv-HTS). In a primary screen, this platform quantified cell-specific fluorescent reporters in >500,000 transgenic zebrafish larvae to identify FDA-approved (Federal Drug Administration) drugs that increased the number of insulin-producing β cells in the pancreas. 24 drugs were confirmed as inducers of endocrine differentiation and/or stimulators of β-cell proliferation. Further, we discovered novel roles for NF-κB signaling in regulating endocrine differentiation and for serotonergic signaling in selectively stimulating β-cell proliferation. These studies demonstrate the power of ARQiv-HTS for drug discovery and provide unique insights into signaling pathways controlling β-cell mass, potential therapeutic targets for treating diabetes.

  20. Methods, compositions and kits for imaging cells and tissues using nanoparticles and spatial frequency heterodyne imaging

    Energy Technology Data Exchange (ETDEWEB)

    Rose-Petruck, Christoph; Wands, Jack R.; Rand, Danielle; Derdak, Zoltan; Ortiz, Vivian

    2016-04-19

    Methods, compositions, systems, devices and kits are provided herein for preparing and using a nanoparticle composition and spatial frequency heterodyne imaging for visualizing cells or tissues. In various embodiments, the nanoparticle composition includes at least one of: a nanoparticle, a polymer layer, and a binding agent, such that the polymer layer coats the nanoparticle and is for example a polyethylene glycol, a polyelectrolyte, an anionic polymer, or a cationic polymer, and such that the binding agent that specifically binds the cells or the tissue. Methods, compositions, systems, devices and kits are provided for identifying potential therapeutic agents in a model using the nanoparticle composition and spatial frequency heterodyne imaging.

  1. Screening and Monitoring Response to Treatment Using Subsecond Molecular Imaging and Hyperpolarized Contrast Agents

    Science.gov (United States)

    2013-05-01

    OP) cell, 2” OD. The design and production using 3D printer is shown on the left and the exploded view is shown on the right. 4. Electromagnet...NMR and MRI. Proc. Natl. Acad. Sci. U. S. A., conditionally accepted (2013). Funding applied for based on work supported by this award 1. 3D Printer for

  2. One-step seeding of neural stem cells with vitronectin-supplemented medium for high throughput screening assays

    Science.gov (United States)

    Dai, Sheng; Li, Rong; Long, Yan; Titus, Steve; Zhao, Jinghua; Huang, Ruili; Xia, Menghang; Zheng, Wei

    2017-01-01

    Human neuronal cells differentiated from induced pluripotent cells have emerged as a new model system for the study of disease pathophysiology and evaluation of drug efficacy. Differentiated neuronal cells are more similar in genetics and biological content to the human brain cells than other animal disease models. However, culture of neuronal cells in assay plates requires a labor-intensive procedure of plate pre-coating, hampering its applications in high throughput screening (HTS). We developed a simplified method with one-step seeding of neural stem cells in assay plates by supplementing the medium with a recombinant human vitronectin (VTN), thus avoiding plate pre-coating. Robust results were obtained from cell viability, calcium response, and neurite outgrowth assays using this new method. Our data demonstrate that this approach greatly simplifies high throughput assays using neuronal cells differentiated from human stem cells for translational research. PMID:27647668

  3. In Vivo Imaging of Natural Killer Cell Trafficking in Tumors

    NARCIS (Netherlands)

    Galli, Filippo; Rapisarda, Anna Serafina; Stabile, Helena; Malviya, Gaurav; Manni, Isabella; Bonanno, Elena; Piaggio, Giulia; Gismondi, Angela; Santoni, Angela; Signore, Alberto

    2015-01-01

    Natural killer cells (NKs) are important effectors of the innate immune system, with marked antitumor activity. Imaging NK trafficking in vivo may be relevant to following up the efficacy of new therapeutic approaches aiming at increasing tumor-infiltrating NKs (TINKs). The specific aims of present

  4. Unraveling cell processes: interference imaging interwoven with data analysis

    DEFF Research Database (Denmark)

    Brazhe, Nadezda; Brazhe, Alexey; Pavlov, A N;

    2006-01-01

    The paper presents results on the application of interference microscopy and wavelet-analysis for cell visualization and studies of cell dynamics. We demonstrate that interference imaging of erythrocytes can reveal reorganization of the cytoskeleton and inhomogenity in the distribution of hemoglo......The paper presents results on the application of interference microscopy and wavelet-analysis for cell visualization and studies of cell dynamics. We demonstrate that interference imaging of erythrocytes can reveal reorganization of the cytoskeleton and inhomogenity in the distribution...... properties differ from cell type to cell type and depend on the cellular compartment. Our results suggest that low frequency variations (0.1-0.6 Hz) result from plasma membrane processes and that higher frequency variations (20-26 Hz) are related to the movement of vesicles. Using double-wavelet analysis, we...... study the modulation of the 1 Hz rhythm in neurons and reveal its changes under depolarization and hyperpolarization of the plasma membrane. We conclude that interference microscopy combined with wavelet analysis is a useful technique for non-invasive cell studies, cell visualization, and investigation...

  5. Risk of invasive cervical cancer after atypical glandular cells in cervical screening: nationwide cohort study

    Science.gov (United States)

    Andrae, Bengt; Sundström, Karin; Ström, Peter; Ploner, Alexander; Elfström, K Miriam; Arnheim-Dahlström, Lisen; Dillner, Joakim; Sparén, Pär

    2016-01-01

    Objectives To investigate the risks of invasive cervical cancer after detection of atypical glandular cells (AGC) during cervical screening. Design Nationwide population based cohort study. Setting Cancer and population registries in Sweden. Participants 3 054 328 women living in Sweden at any time between 1 January 1980 and 1 July 2011 who had any record of cervical cytological testing at ages 23-59. Of these, 2 899 968 women had normal cytology results at the first screening record. The first recorded abnormal result was atypical glandular cells (AGC) in 14 625, high grade squamous intraepithelial lesion (HSIL) in 65 633, and low grade squamous intraepithelial lesions (LSIL) in 244 168. Main outcome measures Cumulative incidence of invasive cervical cancer over 15.5 years; proportion of invasive cervical cancer within six months of abnormality (prevalence); crude incidence rates for invasive cervical cancer over 0.5-15.5 years of follow-up; incidence rate ratios compared with women with normal cytology, estimated with Poisson regression adjusted for age and stratified by histopathology of cancer; distribution of clinical assessment within six months after the abnormality. Results The prevalence of cervical cancer was 1.4% for women with AGC, which was lower than for women with HSIL (2.5%) but higher than for women with LSIL (0.2%); adenocarcinoma accounted for 73.2% of the prevalent cases associated with AGC. The incidence rate of invasive cervical cancer after AGC was significantly higher than for women with normal results on cytology for up to 15.5 years and higher than HSIL and LSIL for up to 6.5 years. The incidence rate of adenocarcinoma was 61 times higher than for women with normal results on cytology in the first screening round after AGC, and remained nine times higher for up to 15.5 years. Incidence and prevalence of invasive cervical cancer was highest when AGC was found at ages 30-39. Only 54% of women with AGC underwent histology assessment

  6. Immune cell-based screening assay for response to anticancer agents: applications in pharmacogenomics

    Directory of Open Access Journals (Sweden)

    Frick A

    2015-02-01

    Full Text Available Amber Frick,1 Yuri Fedoriw,2 Kristy Richards,3,4 Blossom Damania,3,5 Bethany Parks,6 Oscar Suzuki,1 Cristina S Benton,1 Emmanuel Chan,1 Russell S Thomas,7 Tim Wiltshire1,3 1Division of Pharmacotherapy and Experimental Therapeutics, Eshelman School of Pharmacy, 2Department of Pathology and Laboratory Medicine, School of Medicine, 3Lineberger Comprehensive Cancer Center, School of Medicine, 4Department of Genetics, School of Medicine, 5Department of Microbiology and Immunology, School of Medicine, University of North Carolina, Chapel Hill, NC, USA; 6The Hamner Institutes for Health Sciences, 7Environmental Protection Agency, Research Triangle Park, NC, USA Background: Interpatient variability in immune and chemotherapeutic cytotoxic responses is likely due to complex genetic differences and is difficult to ascertain in humans. Through the use of a panel of genetically diverse mouse inbred strains, we developed a drug screening platform aimed at examining interstrain differences in viability on normal, noncancerous immune cells following chemotherapeutic cytotoxic insult. Drug effects were investigated by comparing selective chemotherapeutic agents, such as BEZ-235 and selumetinib, against conventional cytotoxic agents targeting multiple pathways, including doxorubicin and idarubicin. Methods: Splenocytes were isolated from 36 isogenic strains of mice using standard procedures. Of note, the splenocytes were not stimulated to avoid attributing responses to pathways involved with cellular stimulation rather than toxicity. Cells were incubated with compounds on a nine-point logarithmic dosing scale ranging from 15 nM to 100 µM (37°C, 5% CO2. At 4 hours posttreatment, cells were labeled with antibodies and physiological indicator dyes and fixed with 4% paraformaldehyde. Cellular phenotypes (eg, viability were collected and analyzed using flow cytometry. Dose-response curves with response normalized to the zero dose as a function of log concentration

  7. High-throughput screening identifies idarubicin as a preferential inhibitor of smooth muscle versus endothelial cell proliferation.

    Directory of Open Access Journals (Sweden)

    Shakti A Goel

    Full Text Available Intimal hyperplasia is the cause of the recurrent occlusive vascular disease (restenosis. Drugs currently used to treat restenosis effectively inhibit smooth muscle cell (SMC proliferation, but also inhibit the growth of the protective luminal endothelial cell (EC lining, leading to thrombosis. To identify compounds that selectively inhibit SMC versus EC proliferation, we have developed a high-throughput screening (HTS format using human cells and have employed this to screen a multiple compound collection (NIH Clinical Collection. We developed an automated, accurate proliferation assay in 96-well plates using human aortic SMCs and ECs. Using this HTS format we screened a 447-drug NIH Clinical Library. We identified 11 compounds that inhibited SMC proliferation greater than 50%, among which idarubicin exhibited a unique feature of preferentially inhibiting SMC versus EC proliferation. Concentration-response analysis revealed this differential effect most evident over an ∼10 nM-5 µM window. In vivo testing of idarubicin in a rat carotid injury model at 14 days revealed an 80% reduction of intimal hyperplasia and a 45% increase of lumen size with no significant effect on re-endothelialization. Taken together, we have established a HTS assay of human vascular cell proliferation, and identified idarubicin as a selective inhibitor of SMC versus EC proliferation both in vitro and in vivo. Screening of larger and more diverse compound libraries may lead to the discovery of next-generation therapeutics that can inhibit intima hyperplasia without impairing re-endothelialization.

  8. Imaging Cutaneous T-Cell Lymphoma with Optical Coherence Tomography

    Directory of Open Access Journals (Sweden)

    Hans Christian Ring

    2012-07-01

    Full Text Available Aim: To investigate the presentation of a patch-stage cutaneous T-cell lymphoma (CTCL using optical coherence tomography (OCT. Methods: A patient with a patch caused by CTCL was photographed digitally, OCT-scanned and biopsied. A normal skin area adjacent to the patch was OCT-scanned for comparison, but not biopsied. The OCT image and the histological image were compared. Results: The OCT images illustrated a thickened and hyperreflective stratum corneum. OCT also demonstrated several elongated hyporeflective structures in the dermis. The largest structure was measured to have a width of 0.13 mm. A good immediate correlation was found between histology and OCT imaging of the sample. Conclusion: The aetiology of the elongated structures is thought to be lymphomatous infiltrates. Similar findings have been described in ocular lymphoma and may therefore be an important characteristic of cutaneous lymphoma. It may further be speculated that the differences in OCT images may reflect the biological behaviour of the infiltrate. This observation therefore suggests that OCT imaging may be a relevant tool for the in vivo investigation of mycosis fungoides and other CTCLs, but in order to verify these observed patterns in OCT imaging, further investigations will be required.

  9. Comparison of narrow-band imaging and conventional nasopharyngoscopy for the screening of unaffected members of families with nasopharyngeal carcinoma.

    Science.gov (United States)

    Ho, Ching-Yin; Chan, Kee-Tak; Chu, Pen-Yuan

    2013-09-01

    Familial aggregation of nasopharyngeal carcinoma (NPC) has been widely reported. The excess risk is about 4-8-fold among first-degree relatives of NPC patients compared with those without a family history of the disease. We used nasopharyngoscopy and a narrow-band image system (NBI) to screen NPC high-risk patients and identify a good tool for the early detection of NPC in these high-risk groups. We recruited all available, affected blood relations of the patients. When NPC patients were more distant relatives, such as cousins, we recruited their shared second-degree relatives, such as unaffected aunts and uncles, to genetically connect the NPC cases. We performed transnasal endoscopy, first in white-light mode, then under the NBI system. There were two NBI patterns in NPC: microvascular proliferation and engorged blood vessels. The NBI pattern in normal nasopharyngeal mucosa was a regular cobblestone pattern. A prospective study included 211 asymptomatic members from 154 NPC families. We found four cases of NPC, all with a tumor stage of T1. In one patient (1/4), MRI revealed a 2-cm-diameter neck lymphadenopathy (N1). The correlation between conventional nasopharyngoscopy and NBI was very high (κ = 0.798, P = 0.000). In conclusions, NBI is not superior to conventional nasopharyngoscopy for the early detection of NPC in unaffected members of families with NPC history. The long-term follow-up is necessary in high-risk NPC patients. Further studies will be needed to determine which screening tool-conventional nasopharyngoscopy, NBI, or EB virus titer-is most effective.

  10. Performance characteristics of two automated solid-phase red cell adherence systems for pretransfusion antibody screening: a cautionary tale.

    Science.gov (United States)

    Quillen, K; Caron, J; Murphy, K

    2012-01-01

    Out institution has implemented two instruments, the Galileo and the Echo, that use different solid-phase red cell adherence assays for antibody screening in pretransfusion compatibility testing.During the initial implementation of these two instruments, we noticed very different problems: falsely positive results on the Galileo, and falsely negative results and lack of reproducibility on the Echo. Comparison of falsely positive antibody screen results from approximately equivalent numbers of samples run on the Galileo and samples tested by standard manual tube technique using low-ionic-strength saline enhancement showed a false-positive rate of 1.4 percent on the Galileo (defined as a positive screen with a negative panel). Testing using the Echo identified four cases of falsely negative antibody screens, (defined as a negative screen on a patient sample subsequently shown to be positive by the same method). In addition, we note a lack of reproducibility on the Echo, which emphasizes the importance of replicate testing during validation of automated antibody screening platforms.

  11. Follicular Dendritic Cell Sarcoma of the Abdomen: the Imaging Findings

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Tae Wook; Lee, Soon Jin; Song, Hye Jong [Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of)

    2010-04-15

    Follicular dendritic cell sarcoma is a rare neoplasm that originates from follicular dendritic cells in lymphoid follicles. This disease usually involves the lymph nodes, and especially the head and neck area. Rarely, extranodal sites may be affected, including tonsil, the oral cavity, liver, spleen and the gastrointestinal tract. We report here on the imaging findings of follicular dendritic cell sarcoma of the abdomen that involved the retroperitoneal lymph nodes and colon. It shows as a well-defined, enhancing homogenous mass with internal necrosis and regional lymphadenopathy.

  12. Serum-Free Generation of Multipotent Mesoderm (Kdr-positive) Progenitor Cells in Mouse Embryonic Stem Cells For Functional Genomics Screening

    Science.gov (United States)

    McKeithan, Wesley; Colas, Alexandre; Bushway, Paul J.; Ray, Saugata; Mercola, Mark

    2013-01-01

    This unit describes a robust protocol for producing multipotent Kdr-expressing mesoderm progenitor cells in serum-free conditions and functional genomics screening using these cells. Kdr-positive cells are known to be able to differentiate into a wide array of mesoderm derivatives including, vascular endothelial cells, cardiomyocytes, hematopietic progenitors and smooth muscle cells. The efficient generation of such progenitor cells is of particular interest because it permits subsequent steps in cardiovascular development to be analyzed in detail, including deciphering the mechanisms that direct differentiation. The oligonucleotide transfection protocol used to functionally screen siRNA and microRNA libraries is a powerful tool to reveal networks of genes, signaling proteins and microRNAs that control the diversification of cardiovascular lineages from multipotent progenitors. The discussion addresses technical limitations, troubleshooting and potential applications. PMID:23154934

  13. In vitro screening assay for teratogens using growth inhibition of human embryonic cells.

    Science.gov (United States)

    Pratt, R M; Willis, W D

    1985-01-01

    We have tested 35 teratogenic and 20 nonteratogenic chemicals or drugs in a short-term, in vitro assay that identifies teratogens by their ability to inhibit growth of an established line of human embryonic palatal mesenchymal cells. Only those chemicals that exhibited a dose-dependent inhibition of growth at concentrations less than 1 mM were classified as inhibitory. An Aroclor-induced rat liver S-9 system was effective in metabolizing cyclophosphamide to its teratogenic form in culture. We suggest that this assay, along with the complementary tumor cell-attachment assay of Braun et al. [Braun, A. G., Emerson, D. J. & Nichinson, B. B. (1979) Nature (London) 282, 507-509] may be useful as a short-term in vitro battery for assessment of the teratogenic potential in environmental agents and to prioritize those chemicals which merit further testing in vivo. Images PMID:3862095

  14. Super-resolution Microscopy in Plant Cell Imaging.

    Science.gov (United States)

    Komis, George; Šamajová, Olga; Ovečka, Miroslav; Šamaj, Jozef

    2015-12-01

    Although the development of super-resolution microscopy methods dates back to 1994, relevant applications in plant cell imaging only started to emerge in 2010. Since then, the principal super-resolution methods, including structured-illumination microscopy (SIM), photoactivation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and stimulated emission depletion microscopy (STED), have been implemented in plant cell research. However, progress has been limited due to the challenging properties of plant material. Here we summarize the basic principles of existing super-resolution methods and provide examples of applications in plant science. The limitations imposed by the nature of plant material are reviewed and the potential for future applications in plant cell imaging is highlighted.

  15. Gold nanocages for imaging and therapy of prostate cancer cells

    Science.gov (United States)

    Sironi, Laura; Avvakumova, Svetlana; Galbiati, Elisabetta; Locarno, Silvia A.; Macchi, Chiara; D'Alfonso, Laura; Ruscica, Massimiliano; Magni, Paolo; Collini, Maddalena; Romeo, Sergio; Chirico, Giuseppe; Prosperi, Davide

    2016-04-01

    Gold nanocages (AuNCs) have been shown to be a useful tool both for imaging and hyperthermia therapy of cancer, thanks to their outstanding optical properties, low toxicity and facile functionalization with targeting molecules, including peptides and antibodies. In particular, hyperthermia is a minimally invasive therapy which takes advantage of the peculiar properties of gold nanoparticles to efficiently convert the absorbed light into heat. Here, we use AuNCs for the selective targeting and imaging of prostate cancer cells. Moreover, we report the hyperthermic effect characterization of the AuNCs both in solution and internalized in cells. Prostate cancer cells were irradiated at different exposure times, with a pulsed near infrared laser, and the cellular viability was evaluated by confocal microscopy.

  16. Segmentation of fluorescence microscopy cell images using unsupervised mining.

    Science.gov (United States)

    Du, Xian; Dua, Sumeet

    2010-05-28

    The accurate measurement of cell and nuclei contours are critical for the sensitive and specific detection of changes in normal cells in several medical informatics disciplines. Within microscopy, this task is facilitated using fluorescence cell stains, and segmentation is often the first step in such approaches. Due to the complex nature of cell issues and problems inherent to microscopy, unsupervised mining approaches of clustering can be incorporated in the segmentation of cells. In this study, we have developed and evaluated the performance of multiple unsupervised data mining techniques in cell image segmentation. We adapt four distinctive, yet complementary, methods for unsupervised learning, including those based on k-means clustering, EM, Otsu's threshold, and GMAC. Validation measures are defined, and the performance of the techniques is evaluated both quantitatively and qualitatively using synthetic and recently published real data. Experimental results demonstrate that k-means, Otsu's threshold, and GMAC perform similarly, and have more precise segmentation results than EM. We report that EM has higher recall values and lower precision results from under-segmentation due to its Gaussian model assumption. We also demonstrate that these methods need spatial information to segment complex real cell images with a high degree of efficacy, as expected in many medical informatics applications.

  17. An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics.

    Science.gov (United States)

    Martinelli, Roberta; Carman, Christopher V

    2015-12-24

    Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells ('APCs') referred to as 'immunological synapses'. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of 'semi-professional APCs'. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic 'planar cellular APC model' for improved imaging and interrogation of fundamental antigenic signaling processes will be described.

  18. New concepts for building vocabulary for cell image ontologies

    Directory of Open Access Journals (Sweden)

    Plant Anne L

    2011-12-01

    Full Text Available Abstract Background There are significant challenges associated with the building of ontologies for cell biology experiments including the large numbers of terms and their synonyms. These challenges make it difficult to simultaneously query data from multiple experiments or ontologies. If vocabulary terms were consistently used and reused across and within ontologies, queries would be possible through shared terms. One approach to achieving this is to strictly control the terms used in ontologies in the form of a pre-defined schema, but this approach limits the individual researcher's ability to create new terms when needed to describe new experiments. Results Here, we propose the use of a limited number of highly reusable common root terms, and rules for an experimentalist to locally expand terms by adding more specific terms under more general root terms to form specific new vocabulary hierarchies that can be used to build ontologies. We illustrate the application of the method to build vocabularies and a prototype database for cell images that uses a visual data-tree of terms to facilitate sophisticated queries based on a experimental parameters. We demonstrate how the terminology might be extended by adding new vocabulary terms into the hierarchy of terms in an evolving process. In this approach, image data and metadata are handled separately, so we also describe a robust file-naming scheme to unambiguously identify image and other files associated with each metadata value. The prototype database http://sbd.nist.gov/ consists of more than 2000 images of cells and benchmark materials, and 163 metadata terms that describe experimental details, including many details about cell culture and handling. Image files of interest can be retrieved, and their data can be compared, by choosing one or more relevant metadata values as search terms. Metadata values for any dataset can be compared with corresponding values of another dataset through logical

  19. Human IL-12 p40 as a reporter gene for high-throughput screening of engineered mouse embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Shaffer Benjamin

    2008-06-01

    Full Text Available Abstract Background Establishing a suitable level of exogenous gene expression in mammalian cells in general, and embryonic stem (ES cells in particular, is an important aspect of understanding pathways of cell differentiation, signal transduction and cell physiology. Despite its importance, this process remains challenging because of the poor correlation between the presence of introduced exogenous DNA and its transcription. Consequently, many transfected cells must be screened to identify those with an appropriate level of expression. To improve the screening process, we investigated the utility of the human interleukin 12 (IL-12 p40 cDNA as a reporter gene for studies of mammalian gene expression and for high-throughput screening of engineered mouse embryonic stem cells. Results A series of expression plasmids were used to study the utility of IL-12 p40 as an accurate reporter of gene activity. These studies included a characterization of the IL-12 p40 expression system in terms of: (i a time course of IL-12 p40 accumulation in the medium of transfected cells; (ii the dose-response relationship between the input DNA and IL-12 p40 mRNA levels and IL-12 p40 protein secretion; (iii the utility of IL-12 p40 as a reporter gene for analyzing the activity of cis-acting genetic elements; (iv expression of the IL-12 p40 reporter protein driven by an IRES element in a bicistronic mRNA; (v utility of IL-12 p40 as a reporter gene in a high-throughput screening strategy to identify successful transformed mouse embryonic stem cells; (vi demonstration of pluripotency of IL-12 p40 expressing ES cells in vitro and in vivo; and (vii germline transmission of the IL-12 p40 reporter gene. Conclusion IL-12 p40 showed several advantages as a reporter gene in terms of sensitivity and ease of the detection procedure. The IL-12 p40 assay was rapid and simple, in as much as the reporter protein secreted from the transfected cells was accurately measured by ELISA using

  20. Dual-Modal Nanoprobes for Imaging of Mesenchymal Stem Cell Transplant by MRI and Fluorescence Imaging

    Energy Technology Data Exchange (ETDEWEB)

    Sung, Chang Kyu; Hong, Kyung Ah; Lin, Shun Mei [Seoul Metropolitan Boramae Medical Center, Seoul (Korea, Republic of)] (and others)

    2009-12-15

    To determine the feasibility of labeling human mesenchymal stem cells (hMSCs) with bifunctional nanoparticles and assessing their potential as imaging probes in the monitoring of hMSC transplantation. The T1 and T2 relaxivities of the nanoparticles (MNP SiO{sub 2}[RITC]-PEG) were measured at 1.5T and 3T magnetic resonance scanner. Using hMSCs and the nanoparticles, labeling efficiency, toxicity, and proliferation were assessed. Confocal laser scanning microscopy and transmission electron microscopy were used to specify the intracellular localization of the endocytosed iron nanoparticles. We also observed in vitro and in vivo visualization of the labeled hMSCs with a 3T MR scanner and optical imaging. MNP SiO{sub 2}(RITC)-PEG showed both superparamagnetic and fluorescent properties. The r{sub 1} and r{sub 2} relaxivity values of the MNP SiO{sub 2}(RITC)-PEG were 0.33 and 398 mM{sup -1} s{sup -1} at 1.5T, respectively, and 0.29 and 453 mM{sup -1} s{sup -1} at 3T, respectively. The effective internalization of MNP SiO{sub 2}(RITC)-PEG into hMSCs was observed by confocal laser scanning fluorescence microscopy. The transmission electron microscopy images showed that MNP SiO{sub 2}(RITC)-PEG was internalized into the cells and mainly resided in the cytoplasm. The viability and proliferation of MNP SiO{sub 2}(RITC)-PEG-labeled hMSCs were not significantly different from the control cells. MNP SiO{sub 2}(RITC)-PEG-labeled hMSCs were observed in vitro and in vivo with optical and MR imaging. MNP SiO{sub 2}(RITC)-PEG can be a useful contrast agent for stem cell imaging, which is suitable for a bimodal detection by MRI and optical imaging.

  1. A commentary on iPS cells: potential applications in autologous transplantation, study of illnesses and drug screening.

    Science.gov (United States)

    Romano, Gaetano; Morales, Fátima; Marino, Ignazio R; Giordano, Antonio

    2014-02-01

    Undoubtedly, the focus of the field of stem cell research is predominantly aimed at the artificial reprogramming of human somatic cells for the production of induced pluripotent stem (iPS) cells. This relatively new technology may circumvent the ethical issues of using human embryonic stem (hES) cells for the potential applications in cell replacement therapy. Besides such ethical issues, iPS cell technology offers the advantage of obtaining patient-derived tissues and/or cells, which may be utilized for autologous transplantation and tissue regeneration, investigation of a variety of human illnesses and for the screening of new drugs. The field of stem cell research has placed a major emphasis in understanding the genetic and epigenetic codes for pluripotency, in order to control and optimize autologous transplantation techniques and avoid teratoma formation.

  2. Dialkoxyquinazolines: Screening Epidermal Growth Factor ReceptorTyrosine Kinase Inhibitors for Potential Tumor Imaging Probes

    Energy Technology Data Exchange (ETDEWEB)

    VanBrocklin, Henry F.; Lim, John K.; Coffing, Stephanie L.; Hom,Darren L.; Negash, Kitaw; Ono, Michele Y.; Hanrahan, Stephen M.; Taylor,Scott E.; Vanderpoel, Jennifer L.; Slavik, Sarah M.; Morris, Andrew B.; Riese II, David J.

    2005-09-01

    The epidermal growth factor receptor (EGFR), a long-standingdrug development target, is also a desirable target for imaging. Sixteendialkoxyquinazoline analogs, suitable for labeling with positron-emittingisotopes, have been synthesized and evaluated in a battery of in vitroassays to ascertain their chemical and biological properties. Thesecharacteristics provided the basis for the adoption of a selection schemato identify lead molecules for labeling and in vivo evaluation. A newEGFR tyrosine kinase radiometric binding assay revealed that all of thecompounds possessed suitable affinity (IC50 = 0.4 - 51 nM) for the EGFRtyrosine kinase. All of the analogs inhibited ligand-induced EGFRtyrosine phosphorylation (IC50 = 0.8 - 20 nM). The HPLC-estimatedoctanol/water partition coefficients ranged from 2.0-5.5. Four compounds,4-(2'-fluoroanilino)- and 4-(3'-fluoroanilino)-6,7-diethoxyquinazoline aswell as 4-(3'-chloroanilino)- and4-(3'-bromoanilino)-6,7-dimethoxyquinazoline, possess the bestcombination of characteristics that warrant radioisotope labeling andfurther evaluation in tumor-bearing mice.

  3. The screening and morbidity pattern of sickle cell anemia in chhattisgarh.

    Science.gov (United States)

    Panigrahi, Sumanta; Patra, P K; Khodiar, P K

    2015-03-01

    Our objective was to find out prevalence of sickle cell anemia among the population of three districts (Kanker, Dantewada and Raigarh) of Chhattisgarh with clinical and hematological profile of sickle cell disease patients. A cross sectional study was done. A total of 15,701 persons collectively from three districts voluntarily attended the mobile camp and were screened for sickle cell anemia. First solubility test were done and were confirmed by Hb electrophoresis. The prevalence of sickle cell trait (HbAS) was 1,672 (10.6 %), sickle cell disease (HbSS) and inconclusive band was 97 (0.66 %). The HbSS and inconclusive band were subjected to HPLC. Among them 12 (0.076 %) cases were double heterozygous for Hb-S and beta thalassemia minor (SB+), 2 (0.012 %) cases were double heterozygous for Hb-S and Hb-E (S/HBE), 1 (0.006 %) case was double heterozygous for Hb-S and Hb-D Punjab (S/HBD) and 22 (0.14 %) cases had Hb-S with Hb-F level more than 20 % (SSF). Maximum number of HbSS cases were 13 (2.29 %) out of 567 children in the age group 0-5 years and HbAS cases were 124 (15.6 %) out of 794 persons in the age group 21-25 years. On comparison between vaso-occlusive and steady state, homozygous patients showed decrease in Hb, HCT, MCH, RBC in vaso-occlusive crises (p < 0.001) than steady state. Also there was one moderate negative correlation in number of blood transfusion (r = 0.46) with fetal hemoglobin (HbF) level. Patients with high HbF can have severe disease. This happens due to uneven distribution of fetal hemoglobin in F-cells with mean HbF remaining constant but in our study, those who had HbF level above 15-20 % were having fewer crises.

  4. Chest radiographic image quality: comparison of asymmetric screen-film, digital storage phosphor, and digital selenium drum systems--preliminary study.

    Science.gov (United States)

    Beute, G H; Flynn, M J; Eyler, W R; Samei, E; Spizarny, D L; Zylak, C J

    1998-01-01

    Conventional screen-film radiography does not display all regions of the thorax satisfactorily. Three chest radiographic techniques display both the lung and the mediastinum with good contrast. These techniques are asymmetric screen-film (ASF), digital storage phosphor (DSP), and digital selenium drum (DSD) imaging. ASF systems use two asymmetric screen-film combinations to produce a wide-latitude image of the thorax with good contrast in the lungs. In DSP systems, image data are acquired digitally with a wide dynamic range by using the optical output of a photostimulable phosphor plate; in DSD systems, the wide-range digital image data are acquired by using the electronic charge generated on a drum coated with a thin layer of amorphous selenium. The appearance of a DSP or DSD radiograph is then determined by user-selected image processing operations: tone scaling, spatial frequency processing, and dynamic range compensation. Digital chest radiographs processed with strong regional equalization provide both excellent contrast in the lungs and effective display of the mediastinum and chest wall. At visual comparison, the high lung contrast and good mediastinal, retrocardiac, and subdiaphragmatic detail provided by the DSD method distinguish it from the other two methods.

  5. A high throughput screen identifies Nefopam as targeting cell proliferation in β-catenin driven neoplastic and reactive fibroproliferative disorders.

    Directory of Open Access Journals (Sweden)

    Raymond Poon

    Full Text Available Fibroproliferative disorders include neoplastic and reactive processes (e.g. desmoid tumor and hypertrophic scars. They are characterized by activation of β-catenin signaling, and effective pharmacologic approaches are lacking. Here we undertook a high throughput screen using human desmoid tumor cell cultures to identify agents that would inhibit cell viability in tumor cells but not normal fibroblasts. Agents were then tested in additional cell cultures for an effect on cell proliferation, apoptosis, and β-catenin protein level. Ultimately they were tested in Apc1638N mice, which develop desmoid tumors, as well as in wild type mice subjected to full thickness skin wounds. The screen identified Neofopam, as an agent that inhibited cell numbers to 42% of baseline in cell cultures from β-catenin driven fibroproliferative disorders. Nefopam decreased cell proliferation and β-catenin protein level to 50% of baseline in these same cell cultures. The half maximal effective concentration in-vitro was 0.5 uM and there was a plateau in the effect after 48 hours of treatment. Nefopam caused a 45% decline in tumor number, 33% decline in tumor volume, and a 40% decline in scar size when tested in mice. There was also a 50% decline in β-catenin level in-vivo. Nefopam targets β-catenin protein level in mesenchymal cells in-vitro and in-vivo, and may be an effective therapy for neoplastic and reactive processes driven by β-catenin mediated signaling.

  6. A water soluble vitamin B12-ReI fluorescent conjugate for cell uptake screens: use in the confirmation of cubilin in the lung cancer line A549.

    Science.gov (United States)

    Vortherms, Anthony R; Kahkoska, Anna R; Rabideau, Amy E; Zubieta, Jon; Andersen, Louise Lund; Madsen, Mette; Doyle, Robert P

    2011-09-21

    A water soluble vitamin B(12)-rhenium conjugate was synthesized and used in concert with intrinsic factor to screen for cubilin receptor-mediated uptake in lung cancer cells. Internalization of the conjugate demonstrated that it could be used to rapidly screen for the cubilin receptor in living cells, subsequently confirmed with Western blotting and RT-PCR.

  7. Islet-selectivity of G-protein coupled receptor ligands evaluated for PET imaging of pancreatic {beta}-cell mass

    Energy Technology Data Exchange (ETDEWEB)

    Cline, Gary W., E-mail: gary.cline@yale.edu [Yale University School of Medicine (United States); Zhao, Xiaojian [Yale University School of Medicine (United States); Jakowski, Amy B.; Soeller, Walter C.; Treadway, Judith L. [Pfizer Global Research and Development, Pfizer Inc., Groton CT (United States)

    2011-09-02

    Highlights: {yields} We screened G-protein coupled receptors for imaging pancreatic. {yields} Database mining and immunohistochemistry identified GPCRs enriched in {beta}-cells. {yields} In vitro and in vivo assays were used to determine exocrine vs endocrine specificity. {yields} GPCR candidates for imaging of {beta}-cell mass are Prokineticin-1R, mGluR5, and GLP-1R. -- Abstract: A critical unmet need exists for methods to quantitatively measure endogenous pancreatic {beta}-cell mass (BCM) for the clinical evaluation of therapies to prevent or reverse loss of BCM and diabetes progression. Our objective was to identify G-protein coupled receptors (GPCRs) that are expressed with a high degree of specificity to islet {beta}-cells for receptor-targeted imaging of BCM. GPCRs enriched in pancreatic islets relative to pancreas acinar and hepatic tissue were identified using a database screen. Islet-specific expression was confirmed by human pancreas immunohistochemistry (IHC). In vitro selectivity assessment was determined from the binding and uptake of radiolabeled ligands to the rat insulinoma INS-1 832/13 cell line and isolated rat islets relative to the exocrine pancreas cell-type, PANC-1. Tail-vein injections of radioligands into rats were used to determine favorable image criteria of in vivo biodistribution to the pancreas relative to other internal organs (i.e., liver, spleen, stomach, and lungs). Database and IHC screening identified four candidate receptors for further in vitro and in vivo evaluation for PET imaging of BCM: prokineticin-1 receptor (PK-1R), metabotropic glutamate receptor type-5 (mGluR5