In Vivo Chemical Screen in Zebrafish Embryos Identifies Regulators of Hematopoiesis Using a Semiautomated Imaging Assay
Hematopoietic stem and progenitor cells (HSPCs) generate all cell types of the blood and are crucial for homeostasis of all blood lineages in vertebrates. Hematopoietic stem cell transplantation (HSCT) is a rapidly evolving technique that offers potential cure for hematologic cancers, such as leukemia or lymphoma. HSCT may be autologous or allogenic. Successful HSCT depends critically on the abundance of engraftment-competent HSPCs, which are currently difficult to obtain in large numbers. Therefore, finding compounds that enhance either the number or the activity of HSPCs could improve prognosis for patients undergoing HS...
Source: Journal of Biomolecular Screening - September 19, 2016 Category: Molecular Biology Authors: Arulmozhivarman, G., Stöter, M., Bickle, M., Kräter, M., Wobus, M., Ehninger, G., Stölzel, F., Brand, M., Bornhäuser, M., Shayegi, N. Tags: Original Research Source Type: research
Ranking Differential Drug Activities from Dose-Response Synthetic Lethality Screens
Synthetic lethal screens are used to discover new combination treatments for cancer. In traditional high-throughput synthetic lethal screens, compounds are tested at a single dose, and hit selection is based on threshold activity values from the variance of the efficacy of the compounds tested. The limitation of the single-dose screening for synthetic lethal screens is that it does not allow for the robust detection of differential activities from compound collections with a broad range of potencies and efficacies. There is therefore a need to develop screening approaches that enable the identification of compounds with sy...
Source: Journal of Biomolecular Screening - September 19, 2016 Category: Molecular Biology Authors: Guha, R., Mathews Griner, L. A., Keller, J. M., Zhang, X., Fitzgerald, D., Antignani, A., Pastan, I., Thomas, C. J., Ferrer, M. Tags: Original Research Source Type: research
Assay Establishment and Validation of a High-Throughput Screening Platform for Three-Dimensional Patient-Derived Colon Cancer Organoid Cultures
The application of patient-derived three-dimensional culture systems as disease-specific drug sensitivity models has enormous potential to connect compound screening and clinical trials. However, the implementation of complex cell-based assay systems in drug discovery requires reliable and robust screening platforms. Here we describe the establishment of an automated platform in 384-well format for three-dimensional organoid cultures derived from colon cancer patients. Single cells were embedded in an extracellular matrix by an automated workflow and subsequently self-organized into organoid structures within 4 days of cul...
Source: Journal of Biomolecular Screening - September 19, 2016 Category: Molecular Biology Authors: Boehnke, K., Iversen, P. W., Schumacher, D., Lallena, M. J., Haro, R., Amat, J., Haybaeck, J., Liebs, S., Lange, M., Schäfer, R., Regenbrecht, C. R. A., Reinhard, C., Velasco, J. A. Tags: Original Research Source Type: research
Reproducibility of Uniform Spheroid Formation in 384-Well Plates: The Effect of Medium Evaporation
In this study, we examined the effect of evaporation on the reproducibility of spheroids of tumor and nontumor cell lines in 384-well plates, and show that culture conditions that prevent evaporation-induced medium loss result in the formation of uniform spheroids across the plate. Additionally, we also present a few technical improvements to increase the scalability of the liquid-overlay spheroid culturing technique in MPs, together with a simple software routine for the quantification of spheroid size. We believe that these cost-effective improvements will aid in further improvement of spheroid cultures for HTS drug disc...
Source: Journal of Biomolecular Screening - September 19, 2016 Category: Molecular Biology Authors: Das, V., Fürst, T., Gurska, S., Dzubak, P., Hajduch, M. Tags: Special Collection Source Type: research
Development of a 3D Tissue Culture-Based High-Content Screening Platform That Uses Phenotypic Profiling to Discriminate Selective Inhibitors of Receptor Tyrosine Kinases
In conclusion, we describe a fully scalable high-content screening platform that uses phenotypic profiling to discriminate selective and nonselective (off-target) inhibitors in a physiologically relevant 3D cell culture setting. (Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - September 19, 2016 Category: Molecular Biology Authors: Booij, T. H., Klop, M. J. D., Yan, K., Szantai-Kis, C., Szokol, B., Orfi, L., van de Water, B., Keri, G., Price, L. S. Tags: Special Collection Source Type: research
High-Throughput Platform for Identifying Molecular Factors Involved in Phenotypic Stabilization of Primary Human Hepatocytes In Vitro
Liver disease is a leading cause of morbidity worldwide and treatment options are limited, with organ transplantation being the only form of definitive management. Cell-based therapies have long held promise as alternatives to whole-organ transplantation but have been hindered by the rapid loss of liver-specific functions over a period of days in cultured hepatocytes. Hypothesis-driven studies have identified a handful of factors that modulate hepatocyte functions in vitro, but our understanding of the mechanisms involved remains incomplete. We thus report here the development of a high-throughput platform to enable system...
Source: Journal of Biomolecular Screening - September 19, 2016 Category: Molecular Biology Authors: Shan, J., Logan, D. J., Root, D. E., Carpenter, A. E., Bhatia, S. N. Tags: Special Collection Source Type: research
A Novel Automated High-Content Analysis Workflow Capturing Cell Population Dynamics from Induced Pluripotent Stem Cell Live Imaging Data
We present an efficient tool set for automated high-content analysis suitable for cells with challenging morphology. This approach has potentially widespread applications for human pluripotent stem cells and other cell types. (Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - September 19, 2016 Category: Molecular Biology Authors: Kerz, M., Folarin, A., Meleckyte, R., Watt, F. M., Dobson, R. J., Danovi, D. Tags: Special Collection Source Type: research
SLAS Europe High-Content Screening Conference in Dresden: A Glimpse of the Future?
(Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - September 19, 2016 Category: Molecular Biology Authors: Montoya, M., Dorval, T., Bickle, M. Tags: Special Collection Source Type: research
Development of a Human Whole Blood Screening Platform to Monitor JAK/STAT Signaling Using High-Throughput Flow Cytometry
Oral agents targeting Janus-associated kinases (JAKs) are promising new agents in clinical development. To better understand the relationship between JAK inhibition and biological outcome, compounds targeting JAKs were evaluated in peripheral human whole blood. To date, these analyses are low throughput and costly. Here, we developed a robust 384-well, high-throughput flow-based assay approach to screen small molecules for JAK/STAT signaling inhibition in human whole blood. This assay platform provides a highly sensitive analysis of signaling events in blood and facilitates measurement of target engagement. Further, the au...
Source: Journal of Biomolecular Screening - August 7, 2016 Category: Molecular Biology Authors: Fereshteh, M. P., Li, X., Li, S., Fan, Y., Zhang, R., Farr, G. A., Kolodin, G., Lippy, J., Naglich, J. G., Schieven, G., Schweizer, L., Zhang, L. Tags: Technical Note Source Type: research
Flow Cytometric Method for the Detection of Flavonoids in Cell Lines
Here, we describe an easy-to-use flow cytometric method using diphenylboric acid 2-amino ethyl ester (DPBA) stain for the detection of flavonoids in cells from human/animal origin. Flavonoid bioavailability and bioactivity depend on structure, conjugation and the cell type to which they are presented. We have studied cellular uptake of five flavonoids with different structures and conjugation forms. First, parameters including fixation method, technical and batch variability, and concentration were optimized. Second, uptake of two aglycones—quercetin and hesperetin—and their corresponding glycosides—rutin...
Source: Journal of Biomolecular Screening - August 7, 2016 Category: Molecular Biology Authors: Grootaert, C., Gonzales, G. B., Vissenaekens, H., Van de Wiele, T., Raes, K., Smagghe, G., Van Camp, J. Tags: Original Research Source Type: research
A Graphene Oxide-Based Sensing Platform for the Determination of Methicillin-Resistant Staphylococcus aureus Based on Strand-Displacement Polymerization Recycling and Synchronous Fluorescent Signal Amplification
To develop new technology for detecting methicillin-resistant Staphylococcus aureus (MRSA), a novel fluorescent biosensor based on Klenow fragment (KF)–assisted target recycling amplification and synchronous fluorescence analysis was created. Carboxy-fluorescein (FAM)–labeled single-stranded DNA (ssDNA) containing a capture probe and a signal probe was adsorbed onto the surface of graphene oxide (GO) via -stacking interactions, resulting in the fluorescence quenching of the dye. When target and primer were introduced, the fluorescence was restored due to P0 being completely released from the surface of the GO. ...
Source: Journal of Biomolecular Screening - August 7, 2016 Category: Molecular Biology Authors: Ning, Y., Gao, Q., Zhang, X., Wei, K., Chen, L. Tags: Original Research Source Type: research
Simultaneous High-Throughput Conformational and Colloidal Stability Screening Using a Fluorescent Molecular Rotor Dye, 4-(4-(Dimethylamino)styryl)-N-Methylpyridinium Iodide (DASPMI)
Technologies to improve the throughput for screening protein formulations are continuously evolving. The purpose of this article is to highlight novel applications of a molecular rotor dye, 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (DASPMI) in screening for the conformational stability, colloidal stability, and subtle pretransition dynamics of protein structures during early formulation development. The measurement of the apparent unfolding temperature (Tm) for a monoclonal antibody in the presence of Tween 80 was conducted and data were compared to the results of differential scanning calorimetry (DSC) measure...
Source: Journal of Biomolecular Screening - August 7, 2016 Category: Molecular Biology Authors: Wong, J. J. H., Wright, S. K., Ghozalli, I., Mehra, R., Furuya, K., Katayama, D. S. Tags: Original Research Source Type: research
An Automatic Quality Control Pipeline for High-Throughput Screening Hit Identification
The correction or removal of signal errors in high-throughput screening (HTS) data is critical to the identification of high-quality lead candidates. Although a number of strategies have been previously developed to correct systematic errors and to remove screening artifacts, they are not universally effective and still require fair amount of human intervention. We introduce a fully automated quality control (QC) pipeline that can correct generic interplate systematic errors and remove intraplate random artifacts. The new pipeline was first applied to ~100 large-scale historical HTS assays; in silico analysis showed auto-Q...
Source: Journal of Biomolecular Screening - August 7, 2016 Category: Molecular Biology Authors: Zhai, Y., Chen, K., Zhong, Y., Zhou, B., Ainscow, E., Wu, Y.-T., Zhou, Y. Tags: Original Research Source Type: research
Identification of Potential Pharmacoperones Capable of Rescuing the Functionality of Misfolded Vasopressin 2 Receptor Involved in Nephrogenic Diabetes Insipidus
Pharmacoperones correct the folding of otherwise misfolded protein mutants, restoring function (i.e., providing "rescue") by correcting their trafficking. Currently, most pharmacoperones possess intrinsic antagonist activity because they were identified using methods initially aimed at discovering such functions. Here, we describe an ultra-high-throughput homogeneous cell-based assay with a cAMP detection system, a method specifically designed to identify pharmacoperones of the vasopressin type 2 receptor (V2R), a GPCR that, when mutated, is associated with nephrogenic diabetes insipidus. Previously developed methods to id...
Source: Journal of Biomolecular Screening - August 7, 2016 Category: Molecular Biology Authors: Smith, E., Janovick, J. A., Bannister, T. D., Shumate, J., Scampavia, L., Conn, P. M., Spicer, T. P. Tags: Original Research Source Type: research
Development of a Multiplex Assay for Studying Functional Selectivity of Human Serotonin 5-HT2A Receptors and Identification of Active Compounds by High-Throughput Screening
G protein–coupled receptors (GPCRs) exist as collections of conformations in equilibrium, and the efficacy of drugs has been proposed to be associated with their absolute and relative affinities for these different conformations. The serotonin 2A (5-HT2A) receptor regulates multiple physiological functions, is involved in the pathophysiology of schizophrenia, and serves as an important target of atypical antipsychotic drugs. This receptor was one of the first GPCRs for which the functional selectivity phenomenon was observed, with its various ligands exerting differential effects on the phospholipase A2 (PLA2) and ph...
Source: Journal of Biomolecular Screening - August 7, 2016 Category: Molecular Biology Authors: Iglesias, A., Lage, S., Cadavid, M. I., Loza, M. I., Brea, J. Tags: Original Research Source Type: research
