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You, Kyusuk, Heping Zhu, and John Paul Abbott. "Assessment of Fluorescent Dye Brilliant Sulfaflavine Deposition on Stainless Steel Screens as Spray Droplet Collectors." Transactions of the ASABE 62, no. 2 (2019): 495–503. http://dx.doi.org/10.13031/trans.13136.
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Abstract. The fluorescent dye Brilliant Sulfaflavine (BSF, CAS 2391-30-2) was investigated to determine its photo-stability and recovery on five spray deposition collectors: white plastic plate, nylon screen, and stainless steel (SS) screens of three mesh sizes (40, 60, and 80). The photo-stability of the deposited dye was determined by measuring the variance in fluorescence intensity after daylight exposure. The recovery rates were investigated with statically dispensed droplets and dynamically discharged droplets. In addition, droplet penetration through the screen collectors and the amount of unrecovered dye on reprocessed collectors were assessed to better understand the differences in dye recovery rates among different collector types. Photo-degradation tests verified that all collector types were insignificant in fluorescence degradation (<3.1%) after 120 min of solar exposure. Nylon screens had the lowest dye recovery rate (87.0%) for statically dispensed droplets, whereas plastic plates and SS screens recovered more than 90% of deposited dye. For dynamically discharged droplets, the 60-mesh and 80-mesh SS screens recovered more than 70% of the deposited dye, whereas nylon screens showed less than 50% recovery rate. These results were substantiated visually with a high-speed imaging system that detected droplets penetrating more frequently through screens with larger mesh openings. Throughout ten continuous reprocessing cycles of the fluorimetry test, the fluorescence intensity on reprocessed nylon and 80-mesh SS screen collectors increased by 16.8% and 12.7%, respectively, while there was less than 1% change in fluorescence intensity on the 40-mesh and 60-mesh SS screens. These results were clarified through dye residue verification using digital image analysis. Keywords: Fluorescence intensity, Photo-degradation, Recovery rate, Spray deposition assessment, Stainless steel screen.
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Schaaf, Tory M., Evan Kleinboehl, Samantha L. Yuen, Lauren N. Roelike, Bengt Svensson, Andrew R. Thompson, Razvan L. Cornea, and David D. Thomas. "Live-Cell Cardiac-Specific High-Throughput Screening Platform for Drug-Like Molecules That Enhance Ca2+ Transport." Cells 9, no. 5 (May 8, 2020): 1170. http://dx.doi.org/10.3390/cells9051170.
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We engineered a concatenated fluorescent biosensor and dual-wavelength fluorescence lifetime (FLT) detection, to perform high-throughput screening (HTS) in living cells for discovery of potential heart-failure drugs. Heart failure is correlated with insufficient activity of the sarcoplasmic reticulum Ca-pump (SERCA2a), often due to excessive inhibition by phospholamban (PLB), a small transmembrane protein. We sought to discover small molecules that restore SERCA2a activity by disrupting this inhibitory interaction between PLB and SERCA2a. Our approach was to fluorescently tag the two proteins and measure fluorescence resonance energy transfer (FRET) to detect changes in binding or structure of the complex. To optimize sensitivity to these changes, we engineered a biosensor that concatenates the two fluorescently labeled proteins on a single polypeptide chain. This SERCA2a-PLB FRET biosensor construct is functionally active and effective for HTS. By implementing 2-wavelength FLT detection at extremely high speed during primary HTS, we culled fluorescent compounds as false-positive Hits. In pilot screens, we identified Hits that alter the SERCA2a-PLB interaction, and a newly developed secondary calcium uptake assay revealed both activators and inhibitors of Ca-transport. We are implementing this approach for large-scale screens to discover new drug-like modulators of SERCA2a-PLB interactions for heart failure therapeutic development.
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Ljosa, Vebjorn, and Anne E. Carpenter. "High-throughput screens for fluorescent dye discovery." Trends in Biotechnology 26, no. 10 (October 2008): 527–30. http://dx.doi.org/10.1016/j.tibtech.2008.06.008.
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Horvath, Peter, Thomas Wild, Ulrike Kutay, and Gabor Csucs. "Machine Learning Improves the Precision and Robustness of High-Content Screens." Journal of Biomolecular Screening 16, no. 9 (August 1, 2011): 1059–67. http://dx.doi.org/10.1177/1087057111414878.
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Imaging-based high-content screens often rely on single cell-based evaluation of phenotypes in large data sets of microscopic images. Traditionally, these screens are analyzed by extracting a few image-related parameters and use their ratios (linear single or multiparametric separation) to classify the cells into various phenotypic classes. In this study, the authors show how machine learning–based classification of individual cells outperforms those classical ratio-based techniques. Using fluorescent intensity and morphological and texture features, they evaluated how the performance of data analysis increases with increasing feature numbers. Their findings are based on a case study involving an siRNA screen monitoring nucleoplasmic and nucleolar accumulation of a fluorescently tagged reporter protein. For the analysis, they developed a complete analysis workflow incorporating image segmentation, feature extraction, cell classification, hit detection, and visualization of the results. For the classification task, the authors have established a new graphical framework, the Advanced Cell Classifier, which provides a very accurate high-content screen analysis with minimal user interaction, offering access to a variety of advanced machine learning methods.
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Kolb, Janet M., Gregory Yamanaka, and Susan P. Manly. "Use of a Novel Homogeneous Fluorescent Technology in High Throughput Screening." Journal of Biomolecular Screening 1, no. 4 (June 1996): 203–10. http://dx.doi.org/10.1177/108705719600100407.
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A new fluorescent technology called homogeneous time-resolved fluorescence (HTRF) is sensitive, homogeneous, and quite tolerant to extremes in reaction conditions. These characteristics make this technique an attractive candidate for use in high throughput screens. The assay system uses a pair of fluorescent compounds to label biomolecules. The long-lived nature of the fluorescence of one of them, europium cryptate, facilitates the homogeneous nature of the assay. Furthermore, the introduction of a time delay in reading the signal eliminates the principal difficulty in applying fluorescence to screening formats, that of endogenous fluorescence of samples tested (especially natural products). This technique is robust and sensitive, and we report here its utility in a high throughput screening format.
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George, Jeanette, Michelle L. Teear, Christopher G. Norey, and D. Dougal Burns. "Evaluation of an Imaging Platform during the Development of a FRET Protease Assay." Journal of Biomolecular Screening 8, no. 1 (January 2003): 72–80. http://dx.doi.org/10.1177/1087057102239778.
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Synthetic peptide substrates labeled with a fluorescent donor and quenching moiety flanking an enzyme cleavage site provide a reliable method for monitoring enzyme activity. The dye pair Mca/Dnp has been widely used for this purpose, but poor solubility characteristics, combined with fluorescence emission in the region of the spectrum associated with interference from bi-ologicals and library compounds, can limit the usefulness of Mca/Dnp substrates in a high-throughput screening (HTS) environment. Peptide Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH2 is a matrix-metalloproteinase 3 (MMP-3) enzyme substrate that the authors have labeled with a CyDye pair, Cy3/Cy5Q. The Mca/Dnp- and CyDye-labeled substrates were compared during the development of an MMP-3 inhibitor assay. The results obtained showed that although the peptide substrates behaved similarly throughout the development of the MMP-3 assay, during a test screen of 934 compounds randomly selected from a collection of more than 70,000 compounds, the CyDye substrate was considerably more reliable. Screen Z factor values of 0.84 and 0.15 were obtained using the CyDye and Mca/Dnp peptides respectively, and the authors found that although < 1% of the test compounds were auto-fluorescent at Cy3 wavelengths, > 10% could not be screened using the Mca/Dnp substrate because of compound auto-fluorescence and interference. During this study, the authors used a PMTbased fluorescence plate reader and at the same time evaluated a charged couple device (CCD)—based imaging platform specifically optimized for use with CyDye reagents. The imaging platform gave improved read accuracy and faster plate processing times compared with the PMT reader. Overall, the results presented here highlight the potential benefit of employing the red-shifted CyDye reagents and imaging technology during the development and execution of HTS protease screens. (Journal of Biomolecular Screening 2003:72-80)
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Scott, Benjamin M., Leanne E. Wybenga-Groot, C. Jane McGlade, Elise Heon, Sergio G. Peisajovich, and Belinda S. W. Chang. "Screening of Chemical Libraries Using a Yeast Model of Retinal Disease." SLAS DISCOVERY: Advancing the Science of Drug Discovery 24, no. 10 (September 26, 2019): 969–77. http://dx.doi.org/10.1177/2472555219875934.
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Retinitis pigmentosa (RP) is a degenerative retinal disease, often caused by mutations in the G-protein-coupled receptor rhodopsin. The majority of pathogenic rhodopsin mutations cause rhodopsin to misfold, including P23H, disrupting its crucial ability to respond to light. Previous screens to discover pharmacological chaperones of rhodopsin have primarily been based on rescuing rhodopsin trafficking and localization to the plasma membrane. Here, we present methods utilizing a yeast-based assay to screen for compounds that rescue the ability of rhodopsin to activate an associated downstream G-protein signaling cascade. We engineered a yeast strain in which human rhodopsin variants were genomically integrated, and were able to demonstrate functional coupling to the yeast mating pathway, leading to fluorescent protein expression. We confirmed that a known pharmacological chaperone, 9- cis retinal, could partially rescue light-dependent activation of a disease-associated rhodopsin mutation (P23H) expressed in yeast. These novel yeast strains were used to perform a phenotypic screen of 4280 compounds from the LOPAC1280 library and a peptidomimetic library, to discover novel pharmacological chaperones of rhodopsin. The fluorescence-based assay was robust in a 96-well format, with a Z′ factor of 0.65 and a signal-to-background ratio of above 14. One compound was selected for additional analysis, but it did not appear to rescue rhodopsin function in yeast. The methods presented here are amenable to future screens of small-molecule libraries, as they are robust and cost-effective. We also discuss how these methods could be further modified or adapted to perform screens of more compounds in the future.
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Giakoumakis, G. E., and D. M. Miliotis. "Light angular distribution of fluorescent screens excited by x-rays." Physics in Medicine and Biology 30, no. 1 (January 1, 1985): 21–29. http://dx.doi.org/10.1088/0031-9155/30/1/003.
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Talbot, Jan B. "From the Editor: Let There Be Light." Electrochemical Society Interface 7, no. 2 (June 1, 1998): 3. http://dx.doi.org/10.1149/2.001982if.
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Certain materials can absorb energy from such sources as X-rays, ultraviolet light, or electrons, then re-emit the energy as visible light. These luminescent materials, called “phosphors,” are ubiquitous. Phosphors light up fluorescent light bulbs, television screens, computer screens, and signs and displays everywhere we look. These are the materials of interest to the Luminescence and Display Materials Division, featured in this issue.
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FANELLI, ANTHONY J., JOAN V. BURLEW, and MINA K. GABRIEL. "Protection of Milk Packaged in High Density Polyethylene Against Photodegradation by Fluorescent Light." Journal of Food Protection 48, no. 2 (February 1, 1985): 112–17. http://dx.doi.org/10.4315/0362-028x-48.2.112.
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The effectiveness of visible and UV light screens, compounded in polyethylene dairy resin to protect vitamins in milk from photodegradation, was investigated. Three pigments and three UV absorbers were chosen for testing on the basis of their commercial availability, FDA approval for contact with food, and advertised compatibility with polyolefins. In this study, vitamin decomposition was accelerated over what would be experienced in a commercial milk container in order to expedite the testing program and exaggerate differences in effectiveness of the various light screens. Good protection of vitamin A and riboflavin was provided by 0.3 wt % FD&C yellow #5. Protection of ascorbic acid was marginal. Two of the UV absorbers, Cyasorb 531 and Tinuvin 326, afforded protection of vitamin A, but not riboflavin or ascorbic acid. Visible and UV spectra are presented for the vitamins and light screens used in this work.
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Efthymiou, Anastasia, Atossa Shaltouki, Joseph P. Steiner, Balendu Jha, Sabrina M. Heman-Ackah, Andrzej Swistowski, Xianmin Zeng, Mahendra S. Rao, and Nasir Malik. "Functional Screening Assays with Neurons Generated from Pluripotent Stem Cell–Derived Neural Stem Cells." Journal of Biomolecular Screening 19, no. 1 (September 9, 2013): 32–43. http://dx.doi.org/10.1177/1087057113501869.
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Rapid and effective drug discovery for neurodegenerative disease is currently impeded by an inability to source primary neural cells for high-throughput and phenotypic screens. This limitation can be addressed through the use of pluripotent stem cells (PSCs), which can be derived from patient-specific samples and differentiated to neural cells for use in identifying novel compounds for the treatment of neurodegenerative diseases. We have developed an efficient protocol to culture pure populations of neurons, as confirmed by gene expression analysis, in the 96-well format necessary for screens. These differentiated neurons were subjected to viability assays to illustrate their potential in future high-throughput screens. We have also shown that organelles such as nuclei and mitochondria could be live-labeled and visualized through fluorescence, suggesting that we should be able to monitor subcellular phenotypic changes. Neurons derived from a green fluorescent protein–expressing reporter line of PSCs were live-imaged to assess markers of neuronal maturation such as neurite length and co-cultured with astrocytes to demonstrate further maturation. These studies confirm that PSC-derived neurons can be used effectively in viability and functional assays and pave the way for high-throughput screens on neurons derived from patients with neurodegenerative disorders.
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Orengo, James P., Donnie Bundman, and Thomas A. Cooper. "A bichromatic fluorescent reporter for cell-based screens of alternative splicing." Nucleic Acids Research 34, no. 22 (November 16, 2006): e148-e148. http://dx.doi.org/10.1093/nar/gkl967.
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Jain, Shushant, David Sondervan, Patrizia Rizzu, Zoltan Bochdanovits, Daniel Caminada, and Peter Heutink. "The Complete Automation of Cell Culture." Journal of Biomolecular Screening 16, no. 8 (July 20, 2011): 932–39. http://dx.doi.org/10.1177/1087057111413920.
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Genomic approaches provide enormous amounts of raw data with regard to genetic variation, the diversity of RNA species, and protein complement. High-throughput (HT) and high-content (HC) cellular screens are ideally suited to contextualize the information gathered from other “omic” approaches into networks and can be used for the identification of therapeutic targets. Current methods used for HT–HC screens are laborious, time-consuming, and prone to human error. The authors thus developed an automated high-throughput system with an integrated fluorescent imager for HC screens called the AI.CELLHOST. The implementation of user-defined culturing and assay plate setup parameters allows parallel operation of multiple screens in diverse mammalian cell types. The authors demonstrate that such a system is able to successfully maintain different cell lines in culture for extended periods of time as well as significantly increasing throughput, accuracy, and reproducibility of HT and HC screens.
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Loll-Krippleber, Raphaël, Adeline Feri, Marie Nguyen, Corinne Maufrais, Jennifer Yansouni, Christophe d'Enfert, and Mélanie Legrand. "A FACS-Optimized Screen Identifies Regulators of Genome Stability in Candida albicans." Eukaryotic Cell 14, no. 3 (January 16, 2015): 311–22. http://dx.doi.org/10.1128/ec.00286-14.
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ABSTRACTLoss of heterozygosity (LOH) plays important roles in genome dynamics, notably, during tumorigenesis. In the fungal pathogenCandida albicans, LOH contributes to the acquisition of antifungal resistance. In order to investigate the mechanisms that regulate LOH inC. albicans, we have established a novel method combining an artificial heterozygous locus harboring the blue fluorescent protein and green fluorescent protein markers and flow cytometry to detect LOH events at the single-cell level. Using this fluorescence-based method, we have confirmed that elevated temperature, treatment with methyl methanesulfonate, and inactivation of the Mec1 DNA damage checkpoint kinase triggered an increase in the frequency of LOH. Taking advantage of this system, we have searched forC. albicansgenes whose overexpression triggered an increase in LOH and identified four candidates, some of which are known regulators of genome dynamics with human homologues contributing to cancer progression. Hence, the approach presented here will allow the implementation of new screens to identify genes that are important for genome stability inC. albicansand more generally in eukaryotic cells.
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Adam, Gregory C., Juncai Meng, Joseph M. Rizzo, Adam Amoss, Jeffrey W. Lusen, Amita Patel, Daniel Riley, et al. "Use of High-Throughput Mass Spectrometry to Reduce False Positives in Protease uHTS Screens." Journal of Biomolecular Screening 20, no. 2 (October 21, 2014): 212–22. http://dx.doi.org/10.1177/1087057114555832.
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As a label-free technology, mass spectrometry (MS) enables assays to be generated that monitor the conversion of substrates with native sequences to products without the requirement for substrate modifications or indirect detection methods. Although traditional liquid chromatography (LC)–MS methods are relatively slow for a high-throughput screening (HTS) paradigm, with cycle times typically ≥60 s per sample, the Agilent RapidFire High-Throughput Mass Spectrometry (HTMS) System, with a cycle time of 5–7 s per sample, enables rapid analysis of compound numbers compatible with HTS. By monitoring changes in mass directly, HTMS assays can be used as a triaging tool by eliminating large numbers of false positives resulting from fluorescent compound interference or from compounds interacting with hydrophobic fluorescent dyes appended to substrates. Herein, HTMS assays were developed for multiple protease programs, including cysteine, serine, and aspartyl proteases, and applied as a confirmatory assay. The confirmation rate for each protease assay averaged <30%, independent of the primary assay technology used (i.e., luminescent, fluorescent, and time-resolved fluorescent technologies). Importantly, >99% of compounds designed to inhibit the enzymes were confirmed by the corresponding HTMS assay. Hence, HTMS is an effective tool for removing detection-based false positives from ultrahigh-throughput screening, resulting in hit lists enriched in true actives for downstream dose response titrations and hit-to-lead efforts.
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Fares, Hanna, and Iva Greenwald. "Genetic Analysis of Endocytosis in Caenorhabditis elegans: Coelomocyte Uptake Defective Mutants." Genetics 159, no. 1 (September 1, 2001): 133–45. http://dx.doi.org/10.1093/genetics/159.1.133.
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Abstract The coelomocytes of Caenorhabditis elegans are scavenger cells that continuously and nonspecifically endocytose fluid from the pseudocoelom (body cavity). Green fluorescent protein (GFP) secreted into the pseudocoelom from body wall muscle cells is endocytosed and degraded by coelomocytes. We show that toxin-mediated ablation of coelomocytes results in viable animals that fail to endocytose pseudocoelomic GFP, indicating that endocytosis by coelomocytes is not essential for growth or survival of C. elegans under normal laboratory conditions. We examined known viable endocytosis mutants, and performed RNAi for other known endocytosis genes, for coelomocyte uptake defective (Cup) phenotypes. We also screened for new genes involved in endocytosis by isolating viable mutants with Cup defects; this screen identified 14 different genes, many with multiple alleles. A variety of Cup terminal phenotypes were observed, consistent with defects at various steps in the endocytic pathway. Available molecular information indicates that the Cup mutant screen has identified novel components of the endocytosis machinery that are conserved in mammals but not in Saccharomyces cerevisiae, the only other organism for which large-scale genetic screens for endocytosis mutants have been performed.
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Rose, Alan B., Jiayang Li, and Robert L. Last. "An Allelic Series of Blue Fluorescent trp1 Mutants of Arabidopsis thaliana." Genetics 145, no. 1 (January 1, 1997): 197–205. http://dx.doi.org/10.1093/genetics/145.1.197.
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Nine blue fluorescent mutants of the flowering plant Arabidopsis thaliana were isolated by genetic selections and fluorescence screens. Each was shown to contain a recessive allele of trp1, a previously described locus that encodes the tryptophan biosynthetic enzyme phosphoribosylanthranilate transferase (PAT, called trpD in bacteria). The trp1 mutants consist of two groups, tryptophan auxotrophs and prototrophs, that differ significantly in growth rate, morphology, and fertility. The trp1 alleles cause plants to accumulate varying amounts of blue fluorescent anthranilate compounds, and only the two least severely affected of the prototrophs have any detectable PAT enzyme activity. All four of the trp1 mutations that were sequenced are G to A or C to T transitions that cause an amino acid change, but in only three of these is the affected residue phylogenetically conserved. There is an unusually high degree of sequence divergence in the single-copy gene encoding PAT from the wild-type Columbia and Landsberg erecta ecotypes of Arabidopsis.
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Barker, Sarah L., Linda Lee, B. Daniel Pierce, Lymarie Maldonado-Báez, David G. Drubin, and Beverly Wendland. "Interaction of the Endocytic Scaffold Protein Pan1 with the Type I Myosins Contributes to the Late Stages of Endocytosis." Molecular Biology of the Cell 18, no. 8 (August 2007): 2893–903. http://dx.doi.org/10.1091/mbc.e07-05-0436.
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The yeast endocytic scaffold Pan1 contains an uncharacterized proline-rich domain (PRD) at its carboxy (C)-terminus. We report that the pan1-20 temperature-sensitive allele has a disrupted PRD due to a frame-shift mutation in the open reading frame of the domain. To reveal redundantly masked functions of the PRD, synthetic genetic array screens with a pan1ΔPRD strain found genetic interactions with alleles of ACT1, LAS17 and a deletion of SLA1. Through a yeast two-hybrid screen, the Src homology 3 domains of the type I myosins, Myo3 and Myo5, were identified as binding partners for the C-terminus of Pan1. In vitro and in vivo assays validated this interaction. The relative timing of recruitment of Pan1-green fluorescent protein (GFP) and Myo3/5-red fluorescent protein (RFP) at nascent endocytic sites was revealed by two-color real-time fluorescence microscopy; the type I myosins join Pan1 at cortical patches at a late stage of internalization, preceding the inward movement of Pan1 and its disassembly. In cells lacking the Pan1 PRD, we observed an increased lifetime of Myo5-GFP at the cortex. Finally, Pan1 PRD enhanced the actin polymerization activity of Myo5–Vrp1 complexes in vitro. We propose that Pan1 and the type I myosins interactions promote an actin activity important at a late stage in endocytic internalization.
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Smylla, Thomas, Krystina Wagner, and Armin Huber. "Application of Fluorescent Proteins for Functional Dissection of the Drosophila Visual System." International Journal of Molecular Sciences 22, no. 16 (August 19, 2021): 8930. http://dx.doi.org/10.3390/ijms22168930.
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The Drosophila eye has been used extensively to study numerous aspects of biological systems, for example, spatio-temporal regulation of differentiation, visual signal transduction, protein trafficking and neurodegeneration. Right from the advent of fluorescent proteins (FPs) near the end of the millennium, heterologously expressed fusion proteins comprising FPs have been applied in Drosophila vision research not only for subcellular localization of proteins but also for genetic screens and analysis of photoreceptor function. Here, we summarize applications for FPs used in the Drosophila eye as part of genetic screens, to study rhodopsin expression patterns, subcellular protein localization, membrane protein transport or as genetically encoded biosensors for Ca2+ and phospholipids in vivo. We also discuss recently developed FPs that are suitable for super-resolution or correlative light and electron microscopy (CLEM) approaches. Illustrating the possibilities provided by using FPs in Drosophila photoreceptors may aid research in other sensory or neuronal systems that have not yet been studied as well as the Drosophila eye.
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Bilsland, Elizabeth, Andrew Sparkes, Kevin Williams, Harry J. Moss, Michaela de Clare, Pınar Pir, Jem Rowland, et al. "Yeast-based automated high-throughput screens to identify anti-parasitic lead compounds." Open Biology 3, no. 2 (February 2013): 120158. http://dx.doi.org/10.1098/rsob.120158.
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We have developed a robust, fully automated anti-parasitic drug-screening method that selects compounds specifically targeting parasite enzymes and not their host counterparts, thus allowing the early elimination of compounds with potential side effects. Our yeast system permits multiple parasite targets to be assayed in parallel owing to the strains’ expression of different fluorescent proteins. A strain expressing the human target is included in the multiplexed screen to exclude compounds that do not discriminate between host and parasite enzymes. This form of assay has the advantages of using known targets and not requiring the in vitro culture of parasites. We performed automated screens for inhibitors of parasite dihydrofolate reductases, N -myristoyltransferases and phosphoglycerate kinases, finding specific inhibitors of parasite targets. We found that our ‘hits’ have significant structural similarities to compounds with in vitro anti-parasitic activity, validating our screens and suggesting targets for hits identified in parasite-based assays. Finally, we demonstrate a 60 per cent success rate for our hit compounds in killing or severely inhibiting the growth of Trypanosoma brucei , the causative agent of African sleeping sickness.
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Cintra, Giovana A. S., Brenno A. D. Neto, Pedro H. P. R. Carvalho, Carolina B. Moraes, and Lucio H. Freitas-Junior. "Expanding the Biological Application of Fluorescent Benzothiadiazole Derivatives: A Phenotypic Screening Strategy for Anthelmintic Drug Discovery Using Caenorhabditis elegans." SLAS DISCOVERY: Advancing the Science of Drug Discovery 24, no. 7 (June 10, 2019): 755–65. http://dx.doi.org/10.1177/2472555219851130.
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The current methodologies used to identify promising new anthelmintic compounds rely on subjective microscopic examination of worm motility or involve genetic modified organisms. We describe a new methodology to detect worm viability that takes advantage of the differential incorporation of the fluorescent molecular marker propidium iodide and the 2,1,3-benzothiadiazole core, which has been widely applied in light technology. The new assay developed could be validated using the “Pathogen Box” library. By use of this bioassay, it was possible to identify three molecules with activity against Caenorhabditis elegans that were previously described as effective in in vitro assays against other pathogens, such as Schistosoma mansoni, Mycobacterium tuberculosis, and Plasmodium falciparum, accelerating the identification of molecules with anthelmintic potential. The current fluorescence-based bioassay may be used for assessing C. elegans viability. The described methodology replaces the subjectivity of previous assays and provides an enabling technology that is useful for rapid in vitro screens of both natural and synthetic compound libraries. It is expected that the results obtained from these robust in vitro screens would select the most effective compounds for follow-up in vivo experimentation with pathogenic helminths.
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Burnett, Paul, Janet K. Robertson, Jeffrey M. Palmer, Richard R. Ryan, Adrienne E. Dubin, and Robert A. Zivin. "Fluorescence Imaging of Electrically Stimulated Cells." Journal of Biomolecular Screening 8, no. 6 (December 2003): 660–67. http://dx.doi.org/10.1177/1087057103258546.
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Designing high-throughput screens for voltage-gated ion channels has been a tremendous challenge for the pharmaceutical industry because channel activity is dependent on the transmembrane voltage gradient, a stimulus unlike ligand binding to G-protein-coupled receptors or ligand-gated ion channels. To achieve an acceptable throughput, assays to screen for voltage-gated ion channel modulators that are employed today rely on pharmacological intervention to activate these channels. These interventions can introduce artifacts. Ideally, a high-throughput screen should not compromise physiological relevance. Hence, a more appropriate method would activate voltage-gated ion channels by altering plasma membrane potential directly, via electrical stimulation, while simultaneously recordingthe operation of the channel in populations of cells. The authors present preliminary results obtained from a device that is designed to supply precise and reproducible electrical stimuli to populations of cells. Changes in voltage-gated ion channel activity were monitored using a digital fluorescent microscope. The prototype electric field stimulation (EFS) device provided real-time analysis of cellular responsiveness to physiological and pharmacological stimuli. Voltage stimuli applied to SK-N-SH neuroblastoma cells cultured on the EFS device evoked membrane potential changes that were dependent on activation of voltage-gated sodium channels. Data obtained using digital fluorescence microscopy suggests suitability of this system for HTS.
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Ge, Y., D. Zhang, X. Zhou, and Z. Zhang. "High-content Analysis in Monastrol Suppressor Screens." Methods of Information in Medicine 50, no. 03 (2011): 265–72. http://dx.doi.org/10.3414/me09-01-0030.
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SummaryObjectives: High-content screening (HCS) via automated fluorescent microscopy is a powerful technology for the effective expression of cellular processes. However, HCS will generally produce tremendous image datasets, which leads to difficulties of handling and analyzing. We proposed an automatic classification approach for simultaneous feature extraction and cell phenotype recognition of monoaster and bipolar cells in HCS system.Methods: The proposed approach was composed of image segmentation, feature extraction, and classification. The image segmentation was based on the Laplacian of Gaussian (LoG) edge detection method. For the reduction of noise effect on cellular images, we employed an adaptive threshold in microtubule channel. The principal component analysis was used in the feature selection process. The classification was performed with a back-propagation neural network (BPNN). Using the current approach, the cell phases were distinguished from three-channel acquisitions of cellular images and the numbers of bipolar and monoaster cells were automatically counted.Results: The validity of this approach was examined by the application of screening the response of drug compounds in suppressing Monastrol. Our results indicate that the proposed algorithm could improve the recognition rates of monoaster and bipolar cells to 97.98% and 93.12%, respectively, compared with 97.02% and 86.96% obtained from the same samples by multi-phenotypic mitotic analysis (MMA).Conclusions: We have shown that BPNN is a valuable tool to classify cell phenotype. To further improve the classification performance, more test data, more optimized feature selection approaches, and advanced classifier may be required and will be investigated in future works.
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Chandra, V. K., B. P. Chandra, and Piyush Jha. "Organic Light - Emitting Diodes and their Applications." Defect and Diffusion Forum 357 (July 2014): 29–93. http://dx.doi.org/10.4028/www.scientific.net/ddf.357.29.
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Organic light emitting diodes (OLEDs) have been the focus of intense study since the late 1980s, when the low voltage organic electroluminescence in small organic molecules such as Alq3, and large organic molecules such as polymers (PPV), was reported. Since that time, research has continued to demonstrate the potential of OLEDs as viable systems for displays and eco-friendly lighting applications. OLEDs offer full colour display, reduced manufacturing cost, larger viewing angle, more flexible, lower power consumption, better contrast, slimmer, etc. which help in replacing the other technologies such as LCD. The operation of OLEDs involves injection of charge carriers into organic semiconducting layers, recombination of charge carriers, formation of singlet and triplet excitons, and emission of light during decay of excitons. The maximum internal quantum efficiency of fluorescent OLEDs consisting of the emissive layer of fluorescent organic material is 25% because in this case only the 25% singlet excitons can emit light. The maximum internal quantum efficiency of phosphorescent OLEDs consisting of the emissive layer of fluorescent organic material mixed with phosphorescent material of heavy metal complexes such as platinum complexes, iridium complexes, etc. is nearly 100% because in this case both the 25% singlet excitons and 75% triplet excitons emit light. Recently, a new class of OLEDs based on thermally activated delayed fluorescence (TADF) has been reported, in which the energy gap between the singlet and triplet excited states is minimized by design, thereby promoting highly efficient spin up-conversion from non-radiative triplet states to radiative singlet states while maintaining high radiative decay rates of more than 106decays per second. These molecules harness both singlet and triplet excitons for light emission through fluorescence decay channels and provides an intrinsic fluorescence efficiency in excess of 90 per cent and a very high external electroluminescence efficiency of more than 19 per cent, which is comparable to that achieved in high-efficiency phosphorescence-based OLEDs.The OLED technology can be used to make screens large enough for laptop, cell phones, desktop computers, televisions, etc. OLED materials could someday be applied to plastic and other materials to create wall-size video panels, roll-up screens for laptops, automotive displays, and even head wearable displays. Presently, the OLEDs are opening up completely new design possibilities for lighting in the world of tomorrow whereby the offices and living rooms could be illuminated by lighting panels on the ceiling. The present paper describes the salient features of OLEDs and discusses the applications of OLEDs in displays and solid state lighting devices. Finally, the challenges in the field of OLEDs are explored. Contents of Paper
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Sedeeq, Mohammed, Ahmed Maklad, Nuri Gueven, and Iman Azimi. "Development of a High-throughput Agar Colony Formation Assay to Identify Drug Candidates against Medulloblastoma." Pharmaceuticals 13, no. 11 (November 5, 2020): 368. http://dx.doi.org/10.3390/ph13110368.
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Medulloblastoma (MB) is the most common malignant childhood brain cancer. High-risk MB tumours have a high incidence of metastasis and result in poor patient survival. Drug screens, commonly used to identify potential novel therapeutic agents against MB, focus on 2D cell proliferation and viability assays given that these assays are easily adaptable to high-throughput regimes. However, 2D models fail to address invasive characteristics that are crucial to MB metastasis and are thus not representative of tumour growth in vivo. In this study, we developed a 3D 384-well agar colony formation assay using MB cells of molecular subgroup 3 that is associated with the highest level of metastasis. Two fluorescence substrates, resazurin and glycyl-phenylalanyl-aminofluorocoumarin (GF-AFC) that measure cell viability via distinct mechanisms were used to assess the growth of MB cells in the agar matrix. The assay was optimised for seeding density, growth period, substrate incubation time and homogeneity of the fluorescent signals within individual wells. Our data demonstrate the feasibility to multiplex the two fluorescent substrates without detectable signal interference. This assay was validated by assessing the concentration-dependent effect of two commonly used chemotherapeutic agents clinically used for MB treatment, vincristine and lomustine. Subsequently, a panel of plasma membrane calcium channel modulators was screened for their effect on the 3D growth of D341 MB cells, which identified modulators of T-type voltage gated and ORAI calcium channels as selective growth modulators. Overall, this 3D assay provides a reproducible, time and cost-effective assay for high-throughput screening to identify potential drugs against MB.
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Lindner, Benjamin, Tobias Burkard, and Michael Schuler. "Phagocytosis assays with different pH-sensitive fluorescent particles and various readouts." BioTechniques 68, no. 5 (May 2020): 245–50. http://dx.doi.org/10.2144/btn-2020-0003.
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Phagocytosis is a fundamental mechanism of innate immunity and its impairment is associated with severe chronic diseases, for example, chronic obstructive pulmonary disease. Investigating phagocytosis requires flexible tools and assay conditions, such as different fluorescent particle types, detection colors and readouts. We comprehensively evaluated and optimized phagocytosis assays using particles labeled with fluorescent pH-sensitive pHrodo® dyes, facilitating the specific detection of phagocytosed particles. Beads, bacterial and yeast particles labeled with pHrodo red and green were tested for their uptake by THP-1 cells and primary human macrophages by flow cytometry and high-content imaging. Whereas the latter allowed kinetic phagocytosis measurement, the former demonstrated the feasibility of using cell sorting for periods of up to 6 h, enabling downstream applications such as pooled genetic screens.
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Gubbels, Marc-Jan, and Boris Striepen. "Studying the Cell Biology of Apicomplexan Parasites Using Fluorescent Proteins." Microscopy and Microanalysis 10, no. 5 (October 2004): 568–79. http://dx.doi.org/10.1017/s1431927604040899.
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The ability to transfect Apicomplexan parasites has revolutionized the study of this important group of pathogens. The function of specific genes can be explored by disruption of the locus or more subtly by introduction of altered or tagged versions. Using the transgenic reporter gene green fluorescent protein (GFP), cell biological processes can now be studied in living parasites and in real time. We review recent advances made using GFP-based experiments in the understanding of protein trafficking, organelle biogenesis, and cell division inToxoplasma gondiiandPlasmodium falciparum. A technical section provides a collection of basic experimental protocols for fluorescent protein expression inT. gondii. The combination of thein vivomarker GFP with an increasingly diverse genetic toolbox forT. gondiiopens many exciting experimental opportunities, and emerging applications of GFP in genetic and pharmacological screens are discussed.
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Bukesov, S. A. "Electrophysical characteristics and low-energy cathodoluminescence of vacuum fluorescent display and field emission display screens." Journal of Vacuum Science & Technology B: Microelectronics and Nanometer Structures 16, no. 4 (July 1998): 2082. http://dx.doi.org/10.1116/1.590131.
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Lindström, Jan, and Gudrun Alm Carlsson. "A simple model for estimating the particle size dependence of absolute efficiency of fluorescent screens." Physics in Medicine and Biology 44, no. 5 (January 1, 1999): 1353–67. http://dx.doi.org/10.1088/0031-9155/44/5/319.
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Jaeger, Philipp A., Cameron McElfresh, Lily R. Wong, and Trey Ideker. "Beyond Agar: Gel Substrates with Improved Optical Clarity and Drug Efficiency and Reduced Autofluorescence for Microbial Growth Experiments." Applied and Environmental Microbiology 81, no. 16 (June 12, 2015): 5639–49. http://dx.doi.org/10.1128/aem.01327-15.
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ABSTRACTAgar, a seaweed extract, has been the standard support matrix for microbial experiments for over a century. Recent developments in high-throughput genetic screens have created a need to reevaluate the suitability of agar for use as colony support, as modern robotic printing systems now routinely spot thousands of colonies within the area of a single microtiter plate. Identifying optimal biophysical, biochemical, and biological properties of the gel support matrix in these extreme experimental conditions is instrumental to achieving the best possible reproducibility and sensitivity. Here we systematically evaluate a range of gelling agents by using the yeastSaccharomyces cerevisiaeas a model microbe. We find that carrageenan and Phytagel have superior optical clarity and reduced autofluorescence, crucial for high-resolution imaging and fluorescent reporter screens. Nutrient choice and use of refined Noble agar or pure agarose reduce the effective dose of numerous selective drugs by >50%, potentially enabling large cost savings in genetic screens. Using thousands of mutant yeast strains to compare colony growth between substrates, we found no evidence of significant growth or nutrient biases between gel substrates, indicating that researchers could freely pick and choose the optimal gel for their respective application and experimental condition.
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Wei, Xian Fu, Shao Hong Gao, Bei Qing Huang, and Wan Zhang. "Research on the Imaging Mechanism of Additive Color of Fluorescent Ink-Jet Ink." Applied Mechanics and Materials 262 (December 2012): 22–26. http://dx.doi.org/10.4028/www.scientific.net/amm.262.22.
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The formation of color images are generally divided into two methods: additive color imaging based on color light mixture and subtractive color imaging based on colorant mixture. Additive color imaging refers to the color image mixed by a certain percentage of the three primary color lights: red (R), green (G) and blue (B), such as LED display, cell phone screens, digital cameras and others. Subtractive color imaging refers to the color image generated by light mixture reflected by each colorant, due to some light of different wavelength in the white light separately absorbed by certain colorant after mixing a certain percentage of the three primary colorant: cyan (C), magenta (M) and yellow (Y). Inkjet, color laser printing, inkjet printing, etc. belong to the subtractive color imaging. Most commonly, printing also belongs to the subtractive color imaging. This thesis is based on the characteristics that fluorescent materials can produce colors under UV excitation, designing and preparing three fluorescent inkjet inks which can respectively produce red (R), green (G), blue (B) under UV light excitation, using inkjet printers to print color images, investigating the imaging mechanism of the additive color based on fluorescence inkjet printing, and observing the imaging results. The results show that color effect of monochrome image is good, and the color deviation is large after overlapping different colors.
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Johnson, J. E. "Image quality vs data obtainment in biological electron microscopy." Proceedings, annual meeting, Electron Microscopy Society of America 44 (August 1986): 42–43. http://dx.doi.org/10.1017/s0424820100141950.
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In the early years of biological electron microscopy, scientists had their hands full attempting to describe the cellular microcosm that was suddenly before them on the fluorescent screen. Mitochondria, Golgi, endoplasmic reticulum, and other myriad organelles were being examined, micrographed, and documented in the literature. A major problem of that early period was the development of methods to cut sections thin enough to study under the electron beam. A microtome designed in 1943 moved the specimen toward a rotary “Cyclone” knife revolving at 12,500 RPM, or 1000 times as fast as an ordinary microtome. It was claimed that no embedding medium was necessary or that soft embedding media could be used. Collecting the sections thus cut sounded a little precarious: “The 0.1 micron sections cut with the high speed knife fly out at a tangent and are dispersed in the air. They may be collected... on... screens held near the knife“.
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Marra, Andrea, Jyoti Asundi, Magdalena Bartilson, Stacey Lawson, Flora Fang, Jillian Christine, Cedric Wiesner, Daniel Brigham, William P. Schneider, and Alexander E. Hromockyj. "Differential Fluorescence Induction Analysis of Streptococcus pneumoniae Identifies Genes Involved in Pathogenesis." Infection and Immunity 70, no. 3 (March 2002): 1422–33. http://dx.doi.org/10.1128/iai.70.3.1422-1433.2002.
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ABSTRACT Differential fluorescence induction (DFI) technology was used to identify promoters of Streptococcus pneumoniae induced under various in vitro and in vivo conditions. A promoter-trap library using green fluorescent protein as the reporter was constructed in S. pneumoniae, and the entire library was screened for clones exhibiting increased gfp expression under the chosen conditions. The in vitro conditions used were chosen to mimic aspects of the in vivo environment encountered by the pathogen once it enters a host: changes in temperature, osmolarity, oxygen, and iron concentration, as well as blood. In addition, the library was used to infect animals in three different models, and clones induced in these environments were identified. Several promoters were identified in multiple screens, and genes whose promoters were induced twofold or greater under the inducing condition were mutated to assess their roles in virulence. A total of 25 genes were mutated, and the effects of the mutations were assessed in at least two different infection models. Over 50% of these mutants were attenuated in at least one infection model. We show that DFI is a useful tool for identifying bacterial virulence factors as well as a means of elucidating the microenvironment encountered by pathogens upon infection.
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Zon, Leonard I. "Modeling Diamond Blackfan Anemia and p53 Activation in the Zebrafish." Blood 128, no. 22 (December 2, 2016): SCI—43—SCI—43. http://dx.doi.org/10.1182/blood.v128.22.sci-43.sci-43.
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Marrow dysplasias are pre-leukemic conditions that include ribosomopathies, a group of rare genetic diseases, or myelodysplasia preceded by clonal expansion in older adults with somatic mutations. To discover novel therapies for ribosomopathies, we performed two chemical suppressor screens using a ribosomal protein mutant zebrafish and human induced pluripotent stem cells (iPSCs) derived from ribosomopathy patients. In the zebrafish screen, we found calmodulin inhibitors rescued the anemia in zebrafish and also rescued the erythroid defect of the mouse (in vivo) and human (in vitro) models of marrow failure. The screen in RPS19 mutant human iPSCs identified a compound, SMER28, which enhanced erythropoiesis in anemia models through an autophagy-dependent mechanism. The compounds identified in our chemicals screens have provided new insight into the etiology of marrow failure, and calmodulin inhibitors will soon be studied in a clinical trial with adult Diamond-Blackfan anemia patients. To study the abnormal clonal hematopoiesis associated with myelodysplasia, we have generated a new system using the zebrafish in which single stem cells and their progeny express unique combinations of fluorescent proteins, allowing them to be tracked over time by their specific color. Mosaic mutagenesis induced by clustered regularly interspaced short palindromic repeats /cas9 and/or gene overexpression in this system led to expansion of specifically colored blood, modeling the clonal hematopoiesis of human myelodysplasia. Our studies should provide new insight into the ribosomopathies and myelodysplasia. Disclosures Zon: Marauder Therapeutics: Equity Ownership, Other: Founder; Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Fate, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.
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Bibic, Lucka, Volker Herzig, Glenn F. King, and Leanne Stokes. "Development of High-Throughput Fluorescent-Based Screens to Accelerate Discovery of P2X Inhibitors from Animal Venoms." Journal of Natural Products 82, no. 9 (September 18, 2019): 2559–67. http://dx.doi.org/10.1021/acs.jnatprod.9b00410.
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Shen, Quan, Ping Guo, and Baofeng Chai. "pDsRed-EGFPmtag-, an effective dual fluorescent reporter system for cell-based screens of premature termination codon." Cytotechnology 67, no. 6 (June 17, 2014): 931–37. http://dx.doi.org/10.1007/s10616-014-9728-x.
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De Conti, Giulia, Alicja M. Gruszka, Debora Valli, Andrea Umberto Cammarata, Matteo Righi, Massimiliano Mazza, and Pier Giuseppe Pelicci. "A Novel Platform to Test In Vivo Single Gene Dependencies in t(8,21) and t(15,17) AML Confirms Zeb2 as Leukemia Target." Cancers 12, no. 12 (December 14, 2020): 3768. http://dx.doi.org/10.3390/cancers12123768.
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The increased usage of high-throughput technologies in cancer research, including genetic and drug screens, generates large sets of candidate targets that need to be functionally validated for their roles in tumor development. Thus, reliable and robust in vivo model systems are needed to perform reverse genetic experiments. Ideally, these models should allow for a conditional silencing of the target and an unambiguous identification of engineered cancer cells. Here, we present a platform consisting of: (i) t(8;21) and t(15;17) driven acute myeloid leukemia (AML) transgenic mice with constitutive expression of green fluorescent protein (GFP) and inducible expression of Cre recombinase, and (ii) REX, a modified pSico lentiviral vector for inducible shRNA expression and red fluorescent protein (RFP) as a selection marker. In this system, leukemic cells from transgenic mice are transduced with REX, flow sorted, and transplanted into syngeneic hosts. Gene interference is induced in established tumors by tamoxifen treatment. Dual-color cell fluorescence guides the in vivo identification of shRNA interfered AML cells, monitoring engraftment and disease progression. We tested the platform by inducing knockdown of Zeb2, a gene upregulated by AML1-ETO and PML-RARα oncogenes in pre-leukemic hematopoietic stem cell compartment, and observed a significant delay in leukemia onset. This proves the power and utility of the platform and confirms Zeb2 contribution to the pathogenesis of AML.
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Caì, Yíngyún, Masaharu Iwasaki, Brett Beitzel, Shuīqìng Yú, Elena Postnikova, Beatrice Cubitt, Lisa DeWald, et al. "Recombinant Lassa Virus Expressing Green Fluorescent Protein as a Tool for High-Throughput Drug Screens and Neutralizing Antibody Assays." Viruses 10, no. 11 (November 20, 2018): 655. http://dx.doi.org/10.3390/v10110655.
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Lassa virus (LASV), a mammarenavirus, infects an estimated 100,000–300,000 individuals yearly in western Africa and frequently causes lethal disease. Currently, no LASV-specific antivirals or vaccines are commercially available for prevention or treatment of Lassa fever, the disease caused by LASV. The development of medical countermeasure screening platforms is a crucial step to yield licensable products. Using reverse genetics, we generated a recombinant wild-type LASV (rLASV-WT) and a modified version thereof encoding a cleavable green fluorescent protein (GFP) as a reporter for rapid and quantitative detection of infection (rLASV-GFP). Both rLASV-WT and wild-type LASV exhibited similar growth kinetics in cultured cells, whereas growth of rLASV-GFP was slightly impaired. GFP reporter expression by rLASV-GFP remained stable over several serial passages in Vero cells. Using two well-characterized broad-spectrum antivirals known to inhibit LASV infection, favipiravir and ribavirin, we demonstrate that rLASV-GFP is a suitable screening tool for the identification of LASV infection inhibitors. Building on these findings, we established a rLASV-GFP-based high-throughput drug discovery screen and an rLASV-GFP-based antibody neutralization assay. Both platforms, now available as a standard tool at the IRF-Frederick (an international resource), will accelerate anti-LASV medical countermeasure discovery and reduce costs of antiviral screens in maximum containment laboratories.
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Schaaf, Tory, Ang Li, Benjamin Grant, Kurt Peterson, Samantha Yuen, Prachi Bawaskar, Evan Kleinboehl, Ji Li, David Thomas, and Gregory Gillispie. "Red-Shifted FRET Biosensors for High-Throughput Fluorescence Lifetime Screening." Biosensors 8, no. 4 (October 24, 2018): 99. http://dx.doi.org/10.3390/bios8040099.
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We have developed fluorescence resonance energy transfer (FRET) biosensors with red-shifted fluorescent proteins (FP), yielding improved characteristics for time-resolved (lifetime) fluorescence measurements. In comparison to biosensors with green and red FRET pairs (GFP/RFP), FPs that emit at longer wavelengths (orange and maroon, OFP/MFP) increased the FRET efficiency, dynamic range, and signal-to-background of high-throughput screening (HTS). OFP and MFP were fused to specific sites on the human cardiac calcium pump (SERCA2a) for detection of structural changes due to small-molecule effectors. When coupled with a recently improved HTS fluorescence lifetime microplate reader, this red-shifted FRET biosensor enabled high-precision nanosecond-resolved fluorescence decay measurements from microliter sample volumes at three minute read times per 1536-well-plate. Pilot screens with a library of small-molecules demonstrate that the OFP/MFP FRET sensor substantially improves HTS assay quality. These high-content FRET methods detect minute FRET changes with high precision, as needed to elucidate novel structural mechanisms from small-molecule or peptide regulators discovered through our ongoing HTS efforts. FRET sensors that emit at longer wavelengths are highly attractive to the FRET biosensor community for drug discovery and structural interrogation of new therapeutic targets.
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Bykov, Yury S., Nir Cohen, Natalia Gabrielli, Hetty Manenschijn, Sonja Welsch, Petr Chlanda, Wanda Kukulski, Kiran R. Patil, Maya Schuldiner, and John A. G. Briggs. "High-throughput ultrastructure screening using electron microscopy and fluorescent barcoding." Journal of Cell Biology 218, no. 8 (July 9, 2019): 2797–811. http://dx.doi.org/10.1083/jcb.201812081.
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Genetic screens using high-throughput fluorescent microscopes have generated large datasets, contributing many cell biological insights. Such approaches cannot tackle questions requiring knowledge of ultrastructure below the resolution limit of fluorescent microscopy. Electron microscopy (EM) reveals detailed cellular ultrastructure but requires time-consuming sample preparation, limiting throughput. Here we describe a robust method for screening by high-throughput EM. Our approach uses combinations of fluorophores as barcodes to uniquely mark each cell type in mixed populations and correlative light and EM (CLEM) to read the barcode of each cell before it is imaged by EM. Coupled with an easy-to-use software workflow for correlation, segmentation, and computer image analysis, our method, called “MultiCLEM,” allows us to extract and analyze multiple cell populations from each EM sample preparation. We demonstrate several uses for MultiCLEM with 15 different yeast variants. The methodology is not restricted to yeast, can be scaled to higher throughput, and can be used in multiple ways to enable EM to become a powerful screening technique.
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Wang, Ai. "The Design and Progress of Organic Light-Emitting Diode." Highlights in Science, Engineering and Technology 27 (December 27, 2022): 343–48. http://dx.doi.org/10.54097/hset.v27i.3776.
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Organic light emitting diode (OLED) is one of the main lighting devices and can be used as mobile phone screens, so its performance enhancement is worthy of an in-depth study. This paper discusses three types of devices with different light emitting principles in the history of OLED development in chronological order and their performances, as well as ways to enhance their external quantum efficiency (EQE). For fluorescent, phosphorescent and thermally activated delayed fluorescent (TADF) OLEDs, adding additional layers and doping are effective means of improving the performance. For fluorescent OLED, the EQE can be increased up to 11.5% by adding an efficiency enhancement layer and doping the emitting layer with a new blue dopant with a higher orientation coefficient. For phosphorescent OLED, a hole transport layer is utilized to block excitons within the FIrpic-doped emissive layer leading to an EQE of 16.7%. For TADF OLED, the soluble doped TADF OLEDs is helpful at improving the quantum efficiency up to 18.3%. This paper looks forward to the maturation of these strategies and their practical application, and the identification of more technologies that can enhance the performance of OLED devices to help make it more usable.
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McKinstry-Wu, Andrew R., Weiming Bu, Ganesha Rai, Wendy A. Lea, Brian P. Weiser, David F. Liang, Anton Simeonov, Ajit Jadhav, David J. Maloney, and Roderic G. Eckenhoff. "Discovery of a Novel General Anesthetic Chemotype Using High-throughput Screening." Anesthesiology 122, no. 2 (February 1, 2015): 325–33. http://dx.doi.org/10.1097/aln.0000000000000505.
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Abstract Background: The development of novel anesthetics has historically been a process of combined serendipity and empiricism, with most recent new anesthetics developed via modification of existing anesthetic structures. Methods: Using a novel high-throughput screen employing the fluorescent anesthetic 1-aminoanthracene and apoferritin as a surrogate for on-pathway anesthetic protein target(s), we screened a 350,000 compound library for competition with 1-aminoanthracene–apoferritin binding. Hit compounds meeting structural criteria had their binding affinities for apoferritin quantified with isothermal titration calorimetry and were tested for γ-aminobutyric acid type A receptor binding using a flunitrazepam binding assay. Chemotypes with a strong presence in the top 700 and exhibiting activity via isothermal titration calorimetry were selected for medicinal chemistry optimization including testing for anesthetic potency and toxicity in an in vivo Xenopus laevis tadpole assay. Compounds with low toxicity and high potency were tested for anesthetic potency in mice. Results: From an initial chemical library of more than 350,000 compounds, we identified 2,600 compounds that potently inhibited 1-aminoanthracene binding to apoferritin. A subset of compounds chosen by structural criteria (700) was successfully reconfirmed using the initial assay. Based on a strong presence in both the initial and secondary screens the 6-phenylpyridazin-3(2H)-one chemotype was assessed for anesthetic activity in tadpoles. Medicinal chemistry efforts identified four compounds with high potency and low toxicity in tadpoles, two were found to be effective novel anesthetics in mice. Conclusion: The authors demonstrate the first use of a high-throughput screen to successfully identify a novel anesthetic chemotype and show mammalian anesthetic activity for members of that chemotype.
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Wei, Min, Peiyuan Wang, and Guojun Zhang. "Abstract P3-02-08: Diagnosis, navigation, treatment: A versatile biocompatible and biodegradable mesoporous silica platform of NIR II fluorescence imaging for early diagnosis, breast cancer precisely surgery, and radiosensitization." Cancer Research 82, no. 4_Supplement (February 15, 2022): P3–02–08—P3–02–08. http://dx.doi.org/10.1158/1538-7445.sabcs21-p3-02-08.
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Abstract Background: Triple-negative breast cancer (TNBC) has the highest recurrence and distant metastasis rates among breast cancer molecular types. In breast-conserving surgery, the insensitivity discrimination of negative margins correlating with locoregional recurrence and poor patient outcomes. As for immunotherapy, only 20 %-40 % of the patients respond to the atezolizumab treatment due to the change of PDL1 receptor. Meanwhile, TNBC has radiotherapy resistance. Novel radiosensitization targets need to be found to enhance both the local and systematic benefits of radiotherapy. Objectives and rationale: We aim to construct an “all in one” multi-functional nanoprobe (MSN-Gd@Aptamer-ICG, NPs) using mesoporous silica as the framework. The mesoporous silica nanospheres are co-doped with gadolinium (Gd3+), loads indocyanine green (ICG) in its mesoporous channel, and conjugates with a specific PDL1 targeting aptamer on the surface. The biocompatible and biodegradable NPs are capable of release ICG by the reaction of intracellular high GSH and tetrasulfide bonds in silica frame. In the diagnosis, NIR II fluorescent imaging of ICG with the high resolution and deep tissue penetration depth screens out PDL1 highly expressed tumors. In surgical resection, it can show a real-time cancerous tissues margin in intraoperative scenarios and the tumors are successfully remove under the navigation of NIR II fluorescent imaging. During metastasis therapy of post-operation, the released Gd3+ effectively deposits X-rays and produces abundant hydroxyl radicals (•OH) and oxidative stress to induce radiosensitizing effects. More importantly, the therapy efficiency is monitored by NIR II fluorescent imaging. Therefore, our multifunctional NPs have roles in the accurate diagnosis of PDL1 expression of tumor in early stage, intraoperative navigation for radical excision, and metastasis eradication with radiosensitization efficiency enhanced, which lays a solid foundation for clinical TNBC therapy. Methods: The probe was 50 nm and it was successfully synthesized and certified. MTT assay suggested that it was nontoxic to normal breast cells and TNBC cells. Transmission Electron Microscope images showed that the probe was stable at the extracellular fluid and was degradable at tumor intracellular conditions. The leaching of ICG and Gd3+ was negligible at the extracellular fluid. The tumor targeting capability of the probe in vitro was evaluated on 4T1 (TNBC, PDL1 low expressed) and MDA-MB-231 (TNBC, PDL1 high expressed) cells by flow cytometry and laser confocal microscope. In vivo, the NIR II fluorescence imaging with a high resolution and tissue penetration depth successfully distinguished 4T1 and MDA-MB-231 bearing mice indicated the superior diagnostic capability of PDL1 via aptamer. In the surgical resection, the NIR II fluorescence imaging of NPs guided to remove the tumor with precisely discriminate the boundary of the tumor. When combined with RT, IHC staining of Ki67 showed that the NPs exhibited superior radiosensitizing effects under NIR II fluorescent imaging guidance in comparison with undoped Gd mesoporous silica nanoprobes. TUNEL fluorescent staining and H&E staining further confirmed that NPs radiation therapy induced more •OH and extensive DNA double-strand breaks. Results and Conclusion: Our biocompatible and biodegradable platform is nontoxic, can screen out PDL1 high expression tumors, and navigate the precise resection in the operation. Besides, it enhances the radioimmunotherapy efficacy of breast metastasis. Our platform is potential to provide new hope for breast cancer diagnostic, guiding resection, and radiosensitization under NIR II fluorescent imaging. Citation Format: Min Wei, Peiyuan Wang, Guojun Zhang. Diagnosis, navigation, treatment: A versatile biocompatible and biodegradable mesoporous silica platform of NIR II fluorescence imaging for early diagnosis, breast cancer precisely surgery, and radiosensitization [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-02-08.
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Gutierrez, Alejandro, Hui Feng, Prochownik Edward, John Kanki, and A. Thomas Look. "A Tamoxifen-Dependent Conditional Model of MYC-Induced T Cell Acute Lymphoblastic Leukemia in the Zebrafish." Blood 110, no. 11 (November 16, 2007): 2808. http://dx.doi.org/10.1182/blood.v110.11.2808.2808.
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Abstract The MYC oncogene plays a central role in the pathogenesis of human T cell acute lymphoblastic leukemia (T-ALL), and our laboratory has previously developed a zebrafish model of Myc-induced T-ALL. The primary strength of the zebrafish as a model system for human disease lies in is its suitability for unbiased forward genetic and small molecule screens. Our central hypothesis is that forward screens performed using our zebrafish model of MYC-induced T-ALL will lead to the identification of entirely novel genes and pathways that play critical roles in MYC-induced leukemogenesis. However, zebrafish from our original line develop rapidly progressive T-ALL prior to achieving reproductive maturity, making this line poorly suited for the performance of large-scale screens. Therefore, a conditional model was required. We have now generated a transgenic zebrafish line that expresses a human MYC-estrogen receptor fusion construct under the control of the zebrafish recombination activating gene 2 (Rag2) promoter, which is lymphocyte-specific. When mated against fish transgenic for a Rag2-GFP transgene, the development and progression of T-ALL can be readily tracked in live fish by fluorescent microscopy. Upon treatment with 4-hydroxytamoxifen (4HT), zebrafish from this line develop fully penetrant T-ALL, with a mean time to tumor onset of 8 weeks. Additionally, removal from 4HT invariably led to complete morphologic remission in leukemic zebrafish from this line, and all of these fish remained alive and were able to mate successfully for greater than 6 months after removal from 4HT. This conditional zebrafish model of MYC-induced T-ALL will now allow the successful performance of forward genetic and small molecule screens to identify known and novel genes and pathways that play critical roles in T-ALL leukemogenesis and MYC-induced transformation. Figure Figure
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Madan, Babita, Vikas Madan, Odile Weber, Philippe Tropel, Carmen Blum, Emmanuelle Kieffer, Stéphane Viville, and Hans Jörg Fehling. "The Pluripotency-Associated Gene Dppa4 Is Dispensable for Embryonic Stem Cell Identity and Germ Cell Development but Essential for Embryogenesis." Molecular and Cellular Biology 29, no. 11 (March 30, 2009): 3186–203. http://dx.doi.org/10.1128/mcb.01970-08.
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ABSTRACT Dppa4 (developmental pluripotency-associated 4) has been identified in several high-profile screens as a gene that is expressed exclusively in pluripotent cells. It encodes a nuclear protein with an SAP-like domain and appears to be associated preferentially with transcriptionally active chromatin. Its exquisite expression pattern and results of RNA interference experiments have led to speculation that Dppa4, as well as its nearby homolog Dppa2, might play essential roles in embryonic stem (ES) cell function and/or germ cell development. To rigorously assess suggested roles, we have generated Dppa4-deficient and Dppa4/Dppa2 doubly deficient ES cells, as well as mice lacking Dppa4. Contrary to predictions, we find that Dppa4 is completely dispensable for ES cell identity and germ cell development. Instead, loss of Dppa4 in mice results in late embryonic/perinatal death and striking skeletal defects with partial penetrance. Thus, surprisingly, Dppa4-deficiency affects tissues that apparently never transcribed the gene, and at least some loss-of-function defects manifest phenotypically at an embryonic stage long after physiologic Dppa4 expression has ceased. Concomitant with targeted gene inactivation, we have introduced into the Dppa4 locus a red fluorescent marker (tandem-dimer red fluorescent protein) that is compatible with green fluorescent proteins and allows noninvasive visualization of pluripotent cells and reprogramming events.
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Asghar, Z., G. H. Zahid, E. Ahmad, Rafi ud Din, Muhammad Noaman ul Haq, T. Subhani, Z. Hussain, and S. Badshah. "Effect of particle morphology and coating thickness on fluorescent behavior of Ce doped yttrium aluminium garnet phosphor screens." Journal of Materials Science: Materials in Electronics 26, no. 9 (June 13, 2015): 6744–49. http://dx.doi.org/10.1007/s10854-015-3279-6.
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Mullokandov, Gavriel, Gayathri Vijayakumar, Paul Leon, Carole Henry, Patrick C. Wilson, Florian Krammer, Peter Palese, and Brian D. Brown. "High-complexity extracellular barcoding using a viral hemagglutinin." Proceedings of the National Academy of Sciences 117, no. 6 (January 27, 2020): 2767–69. http://dx.doi.org/10.1073/pnas.1919182117.
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While single-cell sequencing technologies have revealed tissue heterogeneity, resolving mixed cellular libraries into cellular clones is essential for many pooled screens and clonal lineage tracing. Fluorescent proteins are limited in number, while DNA barcodes can only be read after cell lysis. To overcome these limitations, we used influenza virus hemagglutinins to engineer a genetically encoded cell-surface protein barcoding system. Using antibodies paired to hemagglutinins carrying combinations of escape mutations, we developed an exponential protein barcoding system which can label 128 clones using seven antibodies. This study provides a proof of principle for a strategy to create protein-level cell barcodes that can be used in vivo in mice to track clonal populations.
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Huang, Shu-Gui. "Development of a High Throughput Screening Assay for Mitochondrial Membrane Potential in Living Cells." Journal of Biomolecular Screening 7, no. 4 (August 2002): 383–89. http://dx.doi.org/10.1177/108705710200700411.
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The mitochondrion plays a pivotal role in energy metabolism in eukaryotic cells. The electrochemical potential across the mitochondrial inner membrane is regulated to cope with cellular energy needs and thus reflects the bioenergetic state of the cell. Traditional assays for mitochondrial membrane potential are not amenable to high-throughput drug screening. In this paper, I describe a high-throughput assay that measures the mitochondrial membrane potential of living cells in 96- or 384-well plates. Cells were first treated with test compounds and then with a fluorescent potentiometric probe, the cationic-lipophilic dye tetramethylrhodamine methyl ester (TMRM). The cells were then washed to remove free compounds and probe. The amount of TMRM retained in the mitochondria, which is proportional to the mitochondrial membrane potential, was measured on an LJL Analyst fluorescence reader. Under optimal conditions, the assay measured only the mitochondrial membrane potential. The chemical uncouplers carbonylcyanide m-chlorophenyl hydrazone and dinitrophenol decreased fluorescence intensity, with IC50 values (concentration at 50% inhibition) similar to those reported in the literature. A Z' factor of greater than 0.5 suggests that this cell-based assay can be adapted for high-throughput screening of chemical libraries. This assay may be used in screens for drugs to treat metabolic disorders such as obesity and diabetes, as well as cancer and neurodegenerative diseases.
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Makowski, Michal, Paweł Piątek, and Mateusz Grynkiewicz. "Projection of holographic images in volumetric fluorescent fluids for near-eye displays." Photonics Letters of Poland 11, no. 4 (December 31, 2019): 99. http://dx.doi.org/10.4302/plp.v11i4.936.
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The optical setup for holographic projection on the scatterings in fluorescent liquids is presented. Such media can be used as volumetric screens for near-eye holographic displays, solving the problem of speckle noise and very small exit pupils in existing setups. Three different oils (canola, olive and engine oil) with 532 nm laser and tonic water with 405 nm laser are used for projecting holographic fields, the quality of such images is investigated. The laser wavelength is cut out from acquisition on a camera and only filtered fluorescent light is observed. The best and brightest results are obtained with engine oil. Full Text: PDF ReferencesX. Li, C. P. Chen, H. Gao, et al. "Video-Rate Holographic Display Using Azo-Dye-Doped Liquid Crystal", Journal of display technology 10(6), 438-443 (2014). CrossRef X. Li, Z. Song, F. Li, X. Dong, W. Liu, "79‐3: Video‐rate Holographic Display in ZnSe layer‐assisted Quantum Dot Doped Liquid Crystal with High‐photorefractive Sensitivity", SID Symposium Digest of Technical Papers. Vol. 48. No. 1. 2017, CrossRef Sasaki, Takeo, et al. "Real-time dynamic hologram in photorefractive ferroelectric liquid crystal with two-beam coupling gain coefficient of over 800 cm–1 and response time of 8 ms", Applied Physics Letters 6(2) (2013) CrossRef N. Tsutsumi, K. Kinashi, A. Nomura, W. Sasaki, "Quickly Updatable Hologram Images Using Poly(N-vinyl Carbazole) (PVCz) Photorefractive Polymer Composite", Materials 5.8: 1477-1486 (2012) CrossRef M. Makowski, "Simple holographic projection in color", et al. Optics express 20.22: 25130-25136 (2012) CrossRef A. Yagi, M. Imura, Y, Kuroda, O. Oshiro, "360-degree fog projection interactive display", SIGGRAPH Asia 2011 Emerging Technologies. ACM, (2011) CrossRef C.H. Hsu, K. L. Hua, W. H. Cheng. "Omni-Tube: a low-cost portable omnidirectional interactive 3D display", SIGGRAPH Asia 2012 Posters. ACM, (2012) CrossRef Z. Zeng, H. Zheng, X. Lu, H. Gao, Y. Yu, "Dynamic holographic three-dimensional projection based on liquid crystal spatial light modulator and cylindrical fog screen", Opt Rev (2015) 22: 853 CrossRef I. Rakkolainen, "Feasible mid-air virtual reality with the immaterial projection screen technology", 3DTV-Conference, Tampere (2010) CrossRef S. Yanfeng, et al. "A multi-plane optical see-through holographic three-dimensional display for augmented reality applications", Optik 157: 190-196 (2018) CrossRef G. Li, D. Lee, Y. Jeong, J. Cho, B. Lee, "Holographic display for see-through augmented reality using mirror-lens holographic optical element", Opt. Lett. 41(11), 2486-2489 (2016) CrossRef C. L. Lin, Y. Z. Su, M. W. Hung, K. C. Huang "Augmented reality system", Proc. SPIE 7798, Applications of Digital Image Processing XXXIII, 779826 (2010) CrossRef A. Maimone, A. Georgiou, J. S. Kollin, "Holographic near-eye displays for virtual and augmented reality", ACM Trans. Graph. 36, 4, 1-16 (2017) CrossRef M. Quinten, Optical properties of nanoparticle systems: Mie and beyond (John Wiley & Sons 2010). CrossRef J.-W. Liaw, S.-W. Tsai, H.-H. Lin, T.-C. Yen, B.-R. Chen, "Wavelength-dependent Faraday–Tyndall effect on laser-induced microbubble in gold colloid", Journal of Quantitative Spectroscopy and Radiative Transfer 113(17), 2234-2242 (2012), CrossRef T. Mu et al. "Classification of edible oils using 532 nm laser-induced fluorescence combined with support vector machine", Anal. Methods 5, 6960 (2013) CrossRef T. Mu et al. "Classification of Motor Oil Using Laser-Induced Fluorescence and Phosphorescence", Analytical Letters 49:8, 1233-1239 (2015) CrossRef V. Rostampour, M. J. Lynch, "Quantitative Techniques To Discriminate Petroleum Oils Using LED-induced Fluorescence", WIT Transactions on Ecology and the Environment 95, 265 262 (2006) CrossRef F. Wyrowski and O. Bryngdahl, "Iterative Fourier-transform algorithm applied to computer holography", Opt. Soc. Am. A 5(7), 1058-1065 (1988) CrossRef
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Mifsud, Antoinette, Daphne Peelen, Patricia Brincat, Sylvana Abela, Neville Debattista, Stefan Laspina, Daniel Zammit, David J. Camilleri, and Alex Gatt. "A feasibility study on the effects of Triton X-100 for the in vitro inactivation of Ebola virus on haematological assays." Journal of Clinical Pathology 69, no. 7 (December 15, 2015): 637–42. http://dx.doi.org/10.1136/jclinpath-2015-203331.
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AimsThe aim of this study was to check the effect of Triton X-100 on various, commonly used haematology test parameters.MethodsAnonymised blood samples were treated with 10 µL of 10% Triton X-100 per 1 mL of blood. Treated and untreated samples were tested in parallel for blood film morphology, complete blood counts (CBCs), flow cytometry, blood grouping and antibody screens. Samples were also taken in 3.2% citrate tubes for coagulation test analyses.ResultsStatistical differences were noted in all CBC parameters apart from the mean cell volume, eosinophil and basophil counts. Platelet counts were significantly different with an apparent rise after the addition of Triton X-100. Samples were noted to have a high red cell fragmentation index. Immunological platelet counting methods using flow cytometry and fluorescent methods showed no significant differences and gave reliable results. Neither flow cytometry for T-cell subsets nor blood grouping/antibody screens were affected by Triton X-100. However, coagulation samples were severely haemolysed prohibiting analysis.ConclusionsWe have demonstrated that the addition of Triton X-100 to haematology blood samples impacts mainly on platelet counts and coagulation studies due to haemolysis. The platelet count is spuriously raised probably due to the presence of red cell fragments. The latter can be circumvented by the use of immunological platelet counting technology.