High-Content Functional Screening of AEG-1 and AKR1C2 for the Promotion of Metastasis in Liver Cancer
Liver cancer is one of the most lethal cancer types in humans, but our understanding of the molecular mechanisms underlying this process remains insufficient. Here, we conducted high-content screening of the potential genes involved in liver cancer metastasis, which we selected from the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, based on the SAMcell method and RNA interference technology. We identified two powerful genes in the liver cancer metastasis process, AEG-1 and AKR1C2, both of which proved to be positive regulators in promoting metastasis in liver cancer. Further clinical results verified the...
Source: Journal of Biomolecular Screening - December 17, 2015 Category: Molecular Biology Authors: Li, C., Wu, X., Zhang, W., Li, J., Liu, H., Hao, M., Wang, J., Zhang, H., Yang, G., Hao, M., Sheng, S., Sun, Y., Long, J., Li, J., Zhuang, F., Hu, C., Li, L., Zheng, J. Tags: Technical Notes Source Type: research
A Surface Plasmon Resonance Spectroscopy Method for Characterizing Small-Molecule Binding to Nerve Growth Factor
This study details for the first time a methodology that can be used to directly characterize the binding event between small-molecule inhibitors and NGF. (Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - December 17, 2015 Category: Molecular Biology Authors: Kennedy, A. E., Sheffield, K. S., Eibl, J. K., Murphy, M. B., Vohra, R., Scott, J. A., Ross, G. M. Tags: Technical Notes Source Type: research
Understanding ForteBios Sensors for High-Throughput Kinetic and Epitope Screening for Purified Antibodies and Yeast Culture Supernatant
Real-time and label-free antibody screening systems are becoming more popular because of the increasing output of purified antibodies and antibody supernatant from many antibody discovery platforms. However, the properties of the biosensor can greatly affect the kinetic and epitope binning results generated by these label-free screening systems. ForteBio human-specific ProA, anti-human IgG quantitation (AHQ), anti-human Fc capture (AHC) sensors, and custom biotinylated-anti-human Fc capture (b-AHFc) sensors were evaluated in terms of loading ability, regeneration, kinetic characterization, and epitope binning with both pur...
Source: Journal of Biomolecular Screening - December 17, 2015 Category: Molecular Biology Authors: Yu, Y., Mitchell, S., Lynaugh, H., Brown, M., Nobrega, R. P., Zhi, X., Sun, T., Caffry, I., Cao, Y., Yang, R., Burnina, I., Xu, Y., Estep, P. Tags: Technical Notes Source Type: research
Discovery of Small-Molecule Nonfluorescent Inhibitors of Fluorogen-Fluorogen Activating Protein Binding Pair
A new class of biosensors, fluorogen activating proteins (FAPs), has been successfully used to track receptor trafficking in live cells. Unlike the traditional fluorescent proteins (FPs), FAPs do not fluoresce unless bound to their specific small-molecule fluorogens, and thus FAP-based assays are highly sensitive. Application of the FAP-based assay for protein trafficking in high-throughput flow cytometry resulted in the discovery of a new class of compounds that interferes with the binding between fluorogens and FAP, thus blocking the fluorescence signal. These compounds are high-affinity, nonfluorescent analogs of fluoro...
Source: Journal of Biomolecular Screening - December 17, 2015 Category: Molecular Biology Authors: Wu, Y., Stauffer, S. R., Stanfield, R. L., Tapia, P. H., Ursu, O., Fisher, G. W., Szent-Gyorgyi, C., Evangelisti, A., Waller, A., Strouse, J. J., Carter, M. B., Bologa, C., Gouveia, K., Poslusney, M., Waggoner, A. S., Lindsley, C. W., Jarvik, J. W., Sklar Tags: Original Research Source Type: research
Development of a Highly Sensitive Cell-Based Assay for Detecting Botulinum Neurotoxin Type A through Neural Culture Media Optimization
Botulinum neurotoxin (BoNT) is the most lethal naturally produced neurotoxin. Due to the extreme toxicity, BoNTs are implicated in bioterrorism, while the specific mechanism of action and long-lasting effect was found to be medically applicable in treating various neurological disorders. Therefore, for both public and patient safety, a highly sensitive, physiologic, and specific assay is needed. In this paper, we show a method for achieving a highly sensitive cell-based assay for BoNT/A detection using the motor neuron–like continuous cell line NG108-15. To achieve high sensitivity, we performed a media optimization ...
Source: Journal of Biomolecular Screening - December 17, 2015 Category: Molecular Biology Authors: Hong, W. S., Pezzi, H. M., Schuster, A. R., Berry, S. M., Sung, K. E., Beebe, D. J. Tags: Original Research Source Type: research
Establishment of a Human Neuronal Network Assessment System by Using a Human Neuron/Astrocyte Co-Culture Derived from Fetal Neural Stem/Progenitor Cells
In this study, we applied HIP-009 cells to a multielectrode array (MEA) system to detect neuronal signals as neuronal network functions. We observed spontaneous firings of HIP-009 neurons, and validated functional formation of neuronal networks pharmacologically. By using this assay system, we investigated effects of several reference compounds, including agonists and antagonists of glutamate and -aminobutyric acid receptors, and sodium, potassium, and calcium channels, on neuronal network functions using firing and burst numbers, and synchrony as readouts. These results indicate that the HIP-009/MEA assay system is applic...
Source: Journal of Biomolecular Screening - December 17, 2015 Category: Molecular Biology Authors: Fukushima, K., Miura, Y., Sawada, K., Yamazaki, K., Ito, M. Tags: Original Research Source Type: research
Screening and Identification of a Phage Display Derived Peptide That Specifically Binds to the CD44 Protein Region Encoded by Variable Exons
CD44, especially the isoforms with variable exons (CD44v), is a promising biomarker for the detection of cancer. To develop a CD44v-specific probe, we screened a 7-mer phage peptide library against the CD44v3-v10 protein using an improved subtractive method. The consensus sequences with the highest frequency (designated CV-1) emerged after four rounds of panning. The binding affinity and specificity of the CV-1 phage and the synthesized peptide for the region of CD44 encoded by the variable exons were confirmed using enzyme-linked immunosorbent assay and competitive inhibition assays. Furthermore, the binding of the CV-1 p...
Source: Journal of Biomolecular Screening - December 17, 2015 Category: Molecular Biology Authors: Zhang, D., Jia, H., Li, W., Hou, Y., Lu, S., He, S. Tags: Original Research Source Type: research
Construction of a Semisynthetic Human VH Single-Domain Antibody Library and Selection of Domain Antibodies against {alpha}-Crystalline of Mycobacterium tuberculosis
The use of human variable heavy (VH) domain antibodies has been on the rise due to their small scaffold size and simple folding mechanism. A highly diverse library is largely dependent on the diversity introduced within the complementarity-determining region (CDR) cassettes. Here we introduced diversity with the use of a single framework diversifying all three CDRs using tailored codons consisting of degenerate trinucleotides (NNK). The length of the degeneracy in the CDRs was also taken into consideration based on the most frequently occurring length of CDRs and the canonical confirmation for each antibody subfamily. The ...
Source: Journal of Biomolecular Screening - December 17, 2015 Category: Molecular Biology Authors: Hairul Bahara, N. H., Chin, S. T., Choong, Y. S., Lim, T. S. Tags: Original Research Source Type: research
Isolation of Potent CGRP Neutralizing Antibodies Using Four Simple Assays
Calcitonin gene-related peptide (CGRP) is a small neuropeptide and a potent vasodilator that is widely associated with chronic pain and migraine. An antibody that inhibits CGRP function would be a potential therapeutic for treatment of these disorders. Here we describe the isolation of highly potent antibodies to CGRP from phage and ribosome display libraries and characterization of their epitope, species cross-reactivity, kinetics, and functional activity. Homogenous time-resolved fluorescence (HTRF) binding assays identified antibodies with the desired species cross-reactivity from naïve libraries, and HTRF epito...
Source: Journal of Biomolecular Screening - December 17, 2015 Category: Molecular Biology Authors: Neal, F., Arnold, J., Rossant, C. J., Podichetty, S., Lowne, D., Dobson, C., Wilkinson, T., Colley, C., Howes, R., Vaughan, T. J. Tags: Original Research Source Type: research
High-Throughput Screening for Internalizing Antibodies by Homogeneous Fluorescence Imaging of a pH-Activated Probe
Antibody-drug conjugates (ADCs) represent a rapidly growing class of biotherapeutics that deliver drugs specifically to target cells by binding of the antibody component to surface receptors. The majority of ADCs require receptor internalization depending on intrinsic features of the specific ADC-antigen interaction. The development of potent ADCs would greatly benefit from the identification of efficiently internalizing antibodies at early stages of discovery. We developed a highly sensitive and rapid antibody internalization assay using an indirect Cypher5E label. The pH-activated CypHer5E label becomes fluorescent upon ...
Source: Journal of Biomolecular Screening - December 17, 2015 Category: Molecular Biology Authors: Riedl, T., van Boxtel, E., Bosch, M., Parren, P. W. H. I., Gerritsen, A. F. Tags: Original Research Source Type: research
A New Perspective in the Field of Cardiac Safety Testing through the Comprehensive In Vitro Proarrhythmia Assay Paradigm
For the past decade, cardiac safety screening to evaluate the propensity of drugs to produce QT interval prolongation and Torsades de Pointes (TdP) arrhythmia has been conducted according to ICH S7B and ICH E14 guidelines. Central to the existing approach are hERG channel assays and in vivo QT measurements. Although effective, the present paradigm carries a risk of unnecessary compound attrition and high cost, especially when considering costly thorough QT (TQT) studies conducted later in drug development. The Comprehensive In Vitro Proarrhythmia Assay (CiPA) initiative is a public-private collaboration with the aim of upd...
Source: Journal of Biomolecular Screening - December 17, 2015 Category: Molecular Biology Authors: Fermini, B., Hancox, J. C., Abi-Gerges, N., Bridgland-Taylor, M., Chaudhary, K. W., Colatsky, T., Correll, K., Crumb, W., Damiano, B., Erdemli, G., Gintant, G., Imredy, J., Koerner, J., Kramer, J., Levesque, P., Li, Z., Lindqvist, A., Obejero-Paz, C. A., Tags: Review Article Source Type: research
A High-Throughput Assay for Rho Guanine Nucleotide Exchange Factors Based on the Transcreener GDP Assay
Ras homologous (Rho) family GTPases act as molecular switches controlling cell growth, movement, and gene expression by cycling between inactive guanosine diphosphate (GDP)- and active guanosine triphosphate (GTP)-bound conformations. Guanine nucleotide exchange factors (GEFs) positively regulate Rho GTPases by accelerating GDP dissociation to allow formation of the active, GTP-bound complex. Rho proteins are directly involved in cancer pathways, especially cell migration and invasion, and inhibiting GEFs holds potential as a therapeutic strategy to diminish Rho-dependent oncogenesis. Methods for measuring GEF activity sui...
Source: Journal of Biomolecular Screening - November 20, 2015 Category: Molecular Biology Authors: Reichman, M., Schabdach, A., Kumar, M., Zielinski, T., Donover, P. S., Laury-Kleintop, L. D., Lowery, R. G. Tags: Technical Notes Source Type: research
A Comparison of Real-Time and Endpoint Cell Viability Assays for Improved Synthetic Lethal Drug Validation
In this study, we compared the performance of three endpoint assays (resazurin reduction, CellTiter-Glo, and nuclei enumeration) and two real-time systems (IncuCyte and xCELLigence). Of the endpoint approaches, both the resazurin reduction and CellTiter-Glo assays showed higher cell viabilities when compared directly to stained nuclei counts. The IncuCyte and xCELLigence real-time systems were comparable, and both were particularly effective at tracking the effects of drug treatment on cell proliferation at sub-confluent growth. However, the real-time systems failed to evaluate contrasting cell densities between drug-treat...
Source: Journal of Biomolecular Screening - November 20, 2015 Category: Molecular Biology Authors: Single, A., Beetham, H., Telford, B. J., Guilford, P., Chen, A. Tags: Technical Notes Source Type: research
Development of an ELISA-Based HDAC Activity Assay for Characterization of Isoform-Selective Inhibitors
We report here a robust HDAC activity assay for screening selective HDAC inhibitors, which is inspired by the traditional enzyme-linked immunosorbent assay (ELISA). The key feature of the ELISA-based HDAC activity assay is use of mammalian cell–derived HDAC isoforms instead of recombinant proteins. Importantly, the assay was validated with several known HDAC inhibitors. The ELISA-based HDAC activity assay will facilitate the characterization of isoform-selective HDAC inhibitors against mammalian cell–derived HDAC proteins, which will enhance HDAC-centered cancer research and provide a foundation for anticancer ...
Source: Journal of Biomolecular Screening - November 20, 2015 Category: Molecular Biology Authors: Padige, G., Negmeldin, A. T., Pflum, M. K. H. Tags: Original Research Source Type: research
Fit-for-Purpose Radio Receptor Assay for the Determination of Growth Hormone Secretagogues in Urine
The everlasting pharmacological development is continuously producing new substances with potential doping abuse. Among these, secretagogues are very prone to misuse by athletes for their properties to release growth hormone (GH) and some limitations in the actual analytical methods to detect them. In this paper, an in-depth study on the key variables of the radio receptor method previously developed by our group is performed and a fit-for-purpose protocol is established. Thus, this sensitive and robust screening method is proposed as an intelligent and preventive antidoping method to detect new growth hormone secretagogue...
Source: Journal of Biomolecular Screening - November 20, 2015 Category: Molecular Biology Authors: Ferro, P., Gutierrez-Gallego, R., Bosch, J., Farre, M., Segura, J. Tags: Original Research Source Type: research
