Impact of RNA-Guided Technologies for Target Identification and Deconvolution
For well over a decade, RNA interference (RNAi) has provided a powerful tool for investigators to query specific gene targets in an easily modulated loss-of-function setting, both in vitro and in vivo. Hundreds of publications have demonstrated the utility of RNAi in arrayed and pooled-based formats, in a wide variety of cell-based systems, including clonal, stem, transformed, and primary cells. Over the years, there have been significant improvements in the design of target-specific small-interfering RNA (siRNA) and short-hairpin RNA (shRNA), expression vectors, methods for mitigating off-target effects, and accurately in...
Source: Journal of Biomolecular Screening - November 20, 2014 Category: Molecular Biology Authors: Fennell, M., Xiang, Q., Hwang, A., Chen, C., Huang, C.-H., Chen, C.-C., Pelossof, R., Garippa, R. J. Tags: Review Article Source Type: research
Software, Database, and Information Services
(Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - September 19, 2014 Category: Molecular Biology Authors: Mattheakis, L. Tags: Product Focus Source Type: research
Rank Ordering Plate Data Facilitates Data Visualization and Normalization in High-Throughput Screening
High-throughput screening (HTS) of chemical and microbial strain collections is an indispensable tool for modern chemical and systems biology; however, HTS data sets have inherent systematic and random error, which may lead to false-positive or false-negative results. Several methods of normalization of data exist; nevertheless, due to the limitations of each, no single method has been universally adopted. Here, we present a method of data visualization and normalization that is effective, intuitive, and easy to implement in a spreadsheet program. For each plate, the data are ordered by ascending values and a plot thereof ...
Source: Journal of Biomolecular Screening - September 19, 2014 Category: Molecular Biology Authors: Mangat, C. S., Bharat, A., Gehrke, S. S., Brown, E. D. Tags: Application Note Source Type: research
Plasmid-Based shRNA Lentiviral Particle Production for RNAi Applications
In this report, we focus exclusively on this step and compare the overall impact of the commercial transfection reagent FuGENE 6 with FuGENE HD. We found that FuGENE HD resulted in at least 5-fold improvement in viral particle titer as assessed by the p24 standard enzyme-linked immunosorbent assay. We present the complete optimized workflow and demonstrate this utility in which a single modification of this transfection step improved the lentiviral particle production. (Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - September 19, 2014 Category: Molecular Biology Authors: Shum, D., Djaballah, H. Tags: Technical Note Source Type: research
Compound Precipitation in High-Concentration DMSO Solutions
This study also investigated whether particulates were present from the time of initial solubilization in DMSO or whether precipitation occurs during freeze-thaw cycling. (Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - September 19, 2014 Category: Molecular Biology Authors: Popa-Burke, I., Russell, J. Tags: Original Research Source Type: research
High-Throughput Screening and Stability Optimization of Anti-Streptavidin IgG1 and IgG2 Formulations
In this study, high-throughput stress and characterization techniques were applied to 96 formulations of anti-streptavidin antibodies (an IgG1 and an IgG2) to choose optimal formulations. Stress and analytical methods applied in this study were 96-well plate based using an automated liquid handling system to prepare the different formulations and sample plates. Aggregation and clipping propensity were evaluated by temperature and mechanical stresses. Multivariate regression analysis of high-throughput data was performed to find statistically significant formulation factors that alter measured parameters such as monomer per...
Source: Journal of Biomolecular Screening - September 19, 2014 Category: Molecular Biology Authors: Alekseychyk, L., Su, C., Becker, G. W., Treuheit, M. J., Razinkov, V. I. Tags: Original Research Source Type: research
Effects of a Ferulate-Derived Dihydrobenzofuran Neolignan on Angiogenesis, Steroidogenesis, and Redox Status in a Swine Cell Model
In the ongoing search for new therapeutic compounds, lignans and neolignans, which are widely distributed in plants, deserve special attention because of their interactions with several biological targets. Searching for potential antiangiogenic agents related to natural lignans/neolignans, we were attracted by a previously studied synthetic dihydrobenzofuran neolignan. We synthesized the compound by means of an eco-friendly, enzyme-mediated biomimetic coupling of the methyl ester of ferulic acid, and the present study was aimed to deeply investigate its effect in angiogenesis bioassays validated in our laboratory. In addit...
Source: Journal of Biomolecular Screening - September 19, 2014 Category: Molecular Biology Authors: Basini, G., Spatafora, C., Tringali, C., Bussolati, S., Grasselli, F. Tags: Original Research Source Type: research
Echinomycin, a Potential Binder of FKBP12, Shows Minor Effect on Calcineurin Activity
In this study, we investigated whether echinomycin interacts with the FKBP12 protein. Molecular docking was used, and the predicted binding energy was –10.61 kcal/mol. Moreover, surface plasmon resonance imaging and fluorescence quenching techniques were used to validate this interaction. Echinomycin binds to FKBP12 with a strong binding affinity comparable with rapamycin. Furthermore, the echinomycin-FKBP12 complex has been shown to affect calcineurin activity when tested in a calcineurin phosphatase inhibition assay. All of these studies have shown that echinomycin may have a double impact on HIF signaling by direc...
Source: Journal of Biomolecular Screening - September 19, 2014 Category: Molecular Biology Authors: Singh, V., Nand, A., Chen, C., Li, Z., Li, S.-J., Wang, S., Yang, M., Merino, A., Zhang, L., Zhu, J. Tags: Original Research Source Type: research
High-Throughput Fluorescence-Based Screening Assays for Tryptophan-Catabolizing Enzymes
Indoleamine 2,3-dioxygenase (IDO1) and tryptophan 2,3-dioxygenase (TDO) are two structurally different enzymes that have a different tissue distribution and physiological roles, but both catalyze the conversion of tryptophan to N-formylkynurenine (NFK). IDO1 has been clinically validated as a small-molecule drug target for cancer, while preclinical studies indicate that TDO may be a target for cancer immunotherapy and neurodegenerative disease. We have developed a high-throughput screening assay for IDO1 and TDO based on a novel chemical probe, NFK Green, that reacts specifically with NFK to form a green fluorescent molecu...
Source: Journal of Biomolecular Screening - September 19, 2014 Category: Molecular Biology Authors: Seegers, N., van Doornmalen, A. M., Uitdehaag, J. C. M., de Man, J., Buijsman, R. C., Zaman, G. J. R. Tags: Original Research Source Type: research
Identification of Selective Agonists and Positive Allosteric Modulators for {micro}- and {delta}-Opioid Receptors from a Single High-Throughput Screen
Hetero-oligomeric complexes of G protein–coupled receptors (GPCRs) may represent novel therapeutic targets exhibiting different pharmacology and tissue- or cell-specific site of action compared with receptor monomers or homo-oligomers. An ideal tool for validating this concept pharmacologically would be a hetero-oligomer selective ligand. We set out to develop and execute a 1536-well high-throughput screen of over 1 million compounds to detect potential hetero-oligomer selective ligands using a β-arrestin recruitment assay in U2OS cells coexpressing recombinant µ- and -opioid receptors. Hetero-oligomer sel...
Source: Journal of Biomolecular Screening - September 19, 2014 Category: Molecular Biology Authors: Burford, N. T., Wehrman, T., Bassoni, D., O'Connell, J., Banks, M., Zhang, L., Alt, A. Tags: Original Research Source Type: research
Usefulness of Monitoring {gamma}-H2AX and Cell Cycle Arrest in HepG2 Cells for Estimating Genotoxicity Using a High-Content Analysis System
In this study, we examined -H2AX formation and cell cycle arrest using high-content screening (HCS) as a method for determining genotoxicity. HepG2 cells were treated with a panel of compounds and then stained with Hoechst 33342 and anti--H2AX, anti-phospho-histone H3, and anti-tubulin antibodies. In total, 19 genotoxic and 7 nongenotoxic compounds were tested in this study. -H2AX production was observed within 1 h posttreatment for the majority of Ames-positive compounds, topoisomerase inhibitors, and DNA polymerase inhibitors. Cell cycle arrest in either the S or G2 phase was detected for all DNA-damaging compounds 24 h ...
Source: Journal of Biomolecular Screening - September 19, 2014 Category: Molecular Biology Authors: Ando, M., Yoshikawa, K., Iwase, Y., Ishiura, S. Tags: Original Research Source Type: research
Detection of PI3K Inhibition in Human Neuroblastoma Using Multiplex Luminex Bead Immunoassay: A Targeted Approach for Pathway Analysis
Neuroblastoma (NB) is a common solid tumor in children. Outcomes for advanced stage NB have not improved, at least in part because of multimodality therapy resistance. Better comprehension of novel molecular targets will likely lead to improved therapies with specific cytotoxic agents. For instance, the role of deregulated IGF-1R/AKT/PI3K/mTOR (PI3K) pathway activity has attracted much attention across several tumors, including NB. Thus, modulating this pathway via anti-PI3K drugs has taken center stage in many cancer clinical trials. However, varied clinical effects have hampered the precise application of these agents. T...
Source: Journal of Biomolecular Screening - September 19, 2014 Category: Molecular Biology Authors: Lang, W. H., Sandoval, J. A. Tags: Original Research Source Type: research
Corrigendum
Ouimet, T;, Duquesnoy, S.; Poras, H.; Fournié-Zaluski, M.-C.; et al. Comparison of Fluorigenic Peptide Substrates PL50, SNAPtide, and BoTest A/E for BoNT/A Detection and Quantification: Exosite Binding Confers High-Assay Sensitivity. J. Biomol. Screen. 2013, 18(6), 726-735. (Original doi: 10.1177/1087057113476089) (Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - August 5, 2014 Category: Molecular Biology Tags: Regular Columns Source Type: research
Microplates, Assay Reagents, Screening Consumables, and Kits
(Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - August 5, 2014 Category: Molecular Biology Authors: Hart, C. Tags: Product Focus Source Type: research
Self-Checking Cell-Based Assays for GPCR Desensitization and Resensitization
G protein–coupled receptors (GPCRs) play stimulatory or modulatory roles in numerous physiological states and processes, including growth and development, vision, taste and olfaction, behavior and learning, emotion and mood, inflammation, and autonomic functions such as blood pressure, heart rate, and digestion. GPCRs constitute the largest protein superfamily in the human and are the largest target class for prescription drugs, yet most are poorly characterized, and of the more than 350 nonolfactory human GPCRs, over 100 are orphans for which no endogenous ligand has yet been convincingly identified. We here describ...
Source: Journal of Biomolecular Screening - August 5, 2014 Category: Molecular Biology Authors: Fisher, G. W., Fuhrman, M. H., Adler, S. A., Szent-Gyorgyi, C., Waggoner, A. S., Jarvik, J. W. Tags: Technical Notes Source Type: research
