Journal articles on the topic 'Miniaturized receptors'
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1
Hodder, Peter, Rebecca Mull, Jason Cassaday, Kurtis Berry, and Berta Strulovici. "Miniaturization of Intracellular Calcium Functional Assays to 1536-Well Plate Format Using a Fluorometric Imaging Plate Reader." Journal of Biomolecular Screening 9, no. 5 (August 2004): 417–26. http://dx.doi.org/10.1177/1087057104264038.
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The measurement of intracellular calcium response transients in living mammalian cells is a popular functional assay for identification of agonists and antagonists to receptors or channels of pharmacological interest. In recent years, advances in fluorescence-based detection techniques and automation technologies have facilitated the adaptation of this assay to 384-well microplate format high-throughput screening (HTS) assays. However, the cost and time required performing the intracellular calcium HTS assays in the 384-well format can be prohibitive for HTS campaigns of greater than 1 × 106 wells. For these reasons, it is attractive to miniaturize intracellular calcium functional assays to the 1536-well microplate format, where assay volumes and plate throughput can be decreased by several fold. The focus of the research described in this article is the miniaturization of an intracellular calcium assay to 1536-well plate format. This was accomplished by modifying the hardware and software of a fluorometric imaging plate reader (FLIPR) to enable transfer of nanoliters of test compound directly to a 1536-well assay plate, and measure the resulting calcium response from all 1536 wells simultaneously. An intracellular calcium functional assay against the rat muscarinic acetylcholine receptor subtype 1 (rmAchR1) G-protein coupled receptor (GPCR) was miniaturized and executed on this modified instrument. In experiments measuring the activity of known muscarinic receptor agonists and antagonists, the miniaturized FLIPR assay gave EC50 and IC50 values and rank order potency comparable to the 384-well format assays. Calculated Z′ factors for the miniaturized agonist and antagonist assays were, respectively, 0.56 ± 0.21 and 0.53 ± 0.22, which were slightly higher (Z′agonist = 0.55 ± 0.33) and lower (Z′antagonist = 0.70 ± 0.18) than the corresponding values in the 384-well assays. A mock agonist HTS campaign against the muscarinic receptor in miniaturized format was able to identify all wells spiked with the rmAchR1 agonist carbachol.
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Klumpp, Martin, Andreas Scheel, Eloisa Lopez-Calle, Michael Busch, Kenneth J. Murray, and Andrew J. Pope. "Ligand Binding to Transmembrane Receptors on Intact Cells or Membrane Vesicles Measured in a Homogeneous 1-Microliter Assay Format." Journal of Biomolecular Screening 6, no. 3 (June 2001): 159–70. http://dx.doi.org/10.1177/108705710100600306.
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We have developed homogeneous miniaturized assays to measure ligand binding to either intact cells or receptor-containing membrane fragments by analysis of particle brightness. As an example, the affinities and inhibition constants of fluorescently labeled interleukin-8 (IL-8) and a low-molecular-weight antagonist toward the receptors CXCR1 and CXCR2, which belong to the superfamily of G protein-coupled receptors (GPCRs), were determined. Although the results were generally comparable between the two approaches, the cell-based measurements revealed a more complex pattern of both ligand and inhibitor titration curves, pointing to the influence of intracellular regulatory events. Both the vesicle- and cell-based membrane receptor assays were successfully miniaturized to a total volume of 1,l without compromising their sensitivity, indicating that screening of transmembrane receptors in these formats is feasible. This is the first report of a cellular ligand-binding assay performed in such low volumes. The resulting savings in reagent could potentially enable the use of primary cells for future HTS/ultra-HTS efforts.
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Scheel, Andreas A., Bettina Funsch, Michael Busch, Gabriele Gradl, Johannes Pschorr, and Martin J. Lohse. "Receptor-Ligand Interactions Studied with Homogeneous Fluorescence-Based Assays Suitable for Miniaturized Screening." Journal of Biomolecular Screening 6, no. 1 (February 2001): 11–18. http://dx.doi.org/10.1177/108705710100600103.
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Cell membrane receptors play a central role in controlling cellular functions, making them the target of drugs for a wide variety of diseases. This report describes how a recently developed method, fluorescence intensity distribution analysis (FIDA), can be used to develop homogeneous, nonradioactive high throughput screening assays for membrane receptors. With FIDA, free ligand and ligand accumulated on receptor-bearing membrane vesicles can be distinguished on the basis of their particle brightness. This allows the concentration of both bound and free ligand to be determined reliably from a single measurement, without any separation. We demonstrate that ligand affinity, receptor expression level, and potency of inhibitors can be determined using the epidermal growth factor and β2-adrenergic receptors as model systems. Highly focused confocal optics enable single-molecule sensitivity, and sample volumes can thus be reduced to 1,IL without affecting the quality of the fluorescence signal. Our results demonstrate that FIDA is an ideal method for membrane receptor assays offering substantial benefits for assay development and high throughput pharmaceutical screening.
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Xu, Chen-Yan, Kang-Ping Ning, Zheng Wang, Yao Yao, Qin Xu, and Xiao-Ya Hu. "Flexible Electrochemical Platform Coupled with In Situ Prepared Synthetic Receptors for Sensitive Detection of Bisphenol A." Biosensors 12, no. 12 (November 25, 2022): 1076. http://dx.doi.org/10.3390/bios12121076.
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A flexible electrochemical sensor based on the carbon felt (CF) functionalized with Bisphenol A (BPA) synthetic receptors was developed. The artificial Bisphenol A receptors were grafted on the CF by a simple thermal polymerization molecular imprinting process. Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and electrochemical characterizations were used to analyze the receptors. Characterization results demonstrated that the Bisphenol A synthetic receptors successfully formed on the CFs surface. Because the synthetic receptor and the porous CFs were successfully combined, the sensor displayed a better current response once Bisphenol A was identified. The sensor’s linear range was determined to be from 0.5 to 8.0 nM and 10.0 to 300.0 nM, with a detection limit of 0.36 nM. Even after being bent and stretched repeatedly, the electrode’s performance was unaffected, demonstrating the robustness, adaptability and viability of installing the sensor on flat or curved surfaces for on-site detection. The designed electrochemical sensor has been used successfully to identify Bisphenol A in milk samples with satisfactory results. This work provided a promising platform for the design of implantable, portable and miniaturized sensors.
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Kornienko, Oleg, Raul Lacson, Priya Kunapuli, Jonathan Schneeweis, Ira Hoffman, Todd Smith, Melissa Alberts, James Inglese, and Berta Strulovici. "Miniaturization of Whole Live Cell-Based GPCR Assays Using Microdispensing and Detection Systems." Journal of Biomolecular Screening 9, no. 3 (April 2004): 186–95. http://dx.doi.org/10.1177/1087057103260070.
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Cell-based β-lactamase reporter gene assays designed to measure the functional responses of G-protein-coupled receptors (GPCRs) were miniaturized to less than 2 μL total assay volume in a 3456-well microplate. Studies were done to evaluate both receptor agonists and antagonists. The pharmacology of agonists and antagonists for target GPCRs originally developed in a 96-well format was recapitulated in a 3456-well microplate format without compromising data quality or EC50/IC50 precision. These assays were employed in high-throughput screening campaigns, allowing the testing of more than 150,000 compounds in 8 h. The instrumentation used and practical aspects of the assay development are discussed.
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Polreichova, Miroslava, Usman Latif, and Franz L. Dickert. "Functionalized Polymers as Receptors for Detection of Cells." Australian Journal of Chemistry 64, no. 9 (2011): 1256. http://dx.doi.org/10.1071/ch11181.
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Mass sensitive sensors were applied for fast and label-free detection of bio-analytes. Robust and miniaturized sensor devices were fabricated by combining bio-mimetic imprinted surfaces with quartz crystal microbalances for the analysis of yeast and bacteria cells. These sensors allow us to differentiate between different growing stages of yeast cells. Moreover, the viability of cells was detected by structuring quartz crystal microbalance electrodes like a grid. Artificial yeast cells were produced to pattern the recognition layer, giving reversible enrichment of the respective bio-analytes. This approach was followed to ensure the reproducibility of the identical sensitive material in each case, because the properties of each cell depend on its growth stage, which varies over time. The strategy was further applied to develop a sensitive system for Escherichia coli. Structuring of these materials by soft lithography allows differentiation between cell strains, e.g. E. coli (strain W & B) with a five-fold selectivity.
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Iglesias, Alba, Sonia Lage, Maria Isabel Cadavid, Maria Isabel Loza, and José Brea. "Development of a Multiplex Assay for Studying Functional Selectivity of Human Serotonin 5-HT2A Receptors and Identification of Active Compounds by High-Throughput Screening." Journal of Biomolecular Screening 21, no. 8 (July 10, 2016): 816–23. http://dx.doi.org/10.1177/1087057116644162.
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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 phospholipase C (PLC) signaling pathways. We aimed to develop a multiplex functional assay in 96-well plates for the simultaneous measurement of the PLA2 and PLC pathways coupled to 5-HT2A receptors; this approach enables the detection of either functional selectivity or cooperativity phenomena in early drug screening stages. The suitability of the method for running screening campaigns was tested using the Prestwick Chemical Library, and 22 confirmed hits with activities of more than 90% were identified; 11 of these hits produced statistically significant differences between the two effector pathways. Thus, we have developed a miniaturized multiplex assay in 96-well plates to measure functional selectivity for 5-HT2A receptors in the early stages of the drug discovery process.
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Lecas, Lucile, Lucie Hartmann, Lydia Caro, Sarah Mohamed-Bouteben, Claire Raingeval, Isabelle Krimm, Renaud Wagner, Vincent Dugas, and Claire Demesmay. "Miniaturized weak affinity chromatography for ligand identification of nanodiscs-embedded G-protein coupled receptors." Analytica Chimica Acta 1113 (May 2020): 26–35. http://dx.doi.org/10.1016/j.aca.2020.03.062.
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Cauteruccio, Silvia, Valentina Pelliccioli, Sara Grecchi, Roberto Cirilli, Emanuela Licandro, and Serena Arnaboldi. "Bipolar Electrochemical Analysis of Chirality in Complex Media through Miniaturized Stereoselective Light-Emitting Systems." Chemosensors 11, no. 2 (February 13, 2023): 131. http://dx.doi.org/10.3390/chemosensors11020131.
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Environmentally relevant contaminants endowed with chirality may include pharmaceutical compounds, flame retardants, perfluoroalkyl chemicals, pesticides, and polychlorinated biphenyls. Despite having similar physicochemical properties, enantiomers may differ in their biochemical interactions with enzymes, receptors, and other chiral molecules leading to different biological responses. In this work, we have designed a wireless miniaturized stereoselective light-emitting system able to qualitatively detect a chiral contaminant (3,4-dihydroxyphenylalanine, DOPA) dissolved in reduced volumes (in the microliters range), through bipolar electrochemistry. The diastereomeric environment was created by mixing the enantiomers of an inherently chiral inductor endowed with helical shape (7,8-dipropyltetrathia[7]helicene) and the chiral probe (DOPA) in micro-solutions of a commercial ionic liquid. The synergy between the inductor, the applied electric field, and the chiral pollutant was transduced by the light emission produced from a miniaturized light-emitting diode (LED) exploited in such an approach as a bipolar electrode.
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Rudiger, Martin, Ulrich Haupts, Keith J. Moore, and Andrew J. Pope. "Single-Molecule Detection Technologies in Miniaturized High Throughput Screening: Binding Assays for G Protein-Coupled Receptors Using Fluorescence Intensity Distribution Analysis and Fluorescence Anisotropy." Journal of Biomolecular Screening 6, no. 1 (February 2001): 29–37. http://dx.doi.org/10.1177/108705710100600105.
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G Protein-coupled receptors (GPCRs) represent one of the most important target classes for drug discovery. Various assay formats are currently applied to screen large compound libraries for agonists or antagonists. However, the development of nonradioactive, miniaturizable assays that are compatible with the requirements of ultra-high throughput screening (uHTS) has so far been slow. In this report we describe homogeneous fluorescence-based binding assays that are highly amenable to miniaturization. Fluorescence intensity distribution analysis (FIDA) is a single-molecule detection method that is sensitive to brightness changes of individual particles, such as those induced by binding of fluorescent ligands to membrane particles with multiple receptor sites. As a confocal detection technology, FIDA inherently allows reduction of the assay volume to the microliter range and below without any loss of signal. Binding and displacement experiments are demonstrated for various types of GPCRs, such as chemokine, peptide hormone, or small-molecule ligand receptors, demonstrating the broad applicability of this method. The results correlate quantitatively with radioligand binding data. We compare FIDA with fluorescence anisotropy (FA), which is based on changes of molecular rotation rates upon binding of fluorescent ligands to membranes. While FA requires a higher degree of binding, FIDA is sensitive down to lower levels of receptor expression. Both methods are, within these boundary conditions, applicable to uHTS.
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Gilchrist, Mark A., Angela Cacace, and David G. Harden. "Characterization of the 5-HT2b Receptor in Evaluation of Aequorin Detection of Calcium Mobilization for Miniaturized GPCR High-Throughput Screening." Journal of Biomolecular Screening 13, no. 6 (July 2008): 486–93. http://dx.doi.org/10.1177/1087057108319212.
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Fluorescent detection of calcium mobilization has been used successfully to identify modulators of G-protein—coupled receptors (GPCRs); however, inherent issues with fluorescence may limit its potential for high-throughput screening miniaturization. The data presented here demonstrate that the calcium-sensitive photoprotein aequorin (AequoScreen™), when compared with FLUO-4 in the same cellular background, allows for miniaturization of functional kinetic calcium flux assays, in which the rank order of potency and efficacy was maintained for a series of diverse small-molecule modulators. Small-volume (<10 µL) 384- and 1536-well aequorin assays were implemented by integration of acoustic dispensing (Echo 550™) and kinetic flash luminometry (CyBi Lumax™). The enhanced high signal-to-background ratios observed relative to fluorescence were readily manipulated by altering per-well cell densities and yielded acceptable screening statistics in miniaturized format for both agonist and antagonist screening scenarios. In addition, the authors demonstrate the feasibility of using agonist concentrations less than EC50 in a miniaturized antagonist assay. These features, coupled with improved sample handling, should enhance sensitivity and provide the benefits of miniaturization including cost reduction and throughput gains. ( Journal of Biomolecular Screening 2008:486-493)
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Hilal, Tarek, Vera Puetter, Christiane Otto, Karsten Parczyk, and Benjamin Bader. "A Dual Estrogen Receptor TR-FRET Assay for Simultaneous Measurement of Steroid Site Binding and Coactivator Recruitment." Journal of Biomolecular Screening 15, no. 3 (February 11, 2010): 268–78. http://dx.doi.org/10.1177/1087057109359196.
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The human estrogen receptors (hER) are members of the nuclear hormone receptor (NHR) superfamily and represent important drug targets for the pharmaceutical industry. Initially, ligand binding assays were used to identify novel ligands using receptors purified from native tissues. With the advent of molecular cloning techniques, cell-based transactivation assays have been the gold standard for many years of drug discovery. With the elucidation of the structural mechanisms underlying the activation of NHRs, cell-free assays with purified receptors have become important tools to directly assess different binding sites (e.g., the hormone binding site or the cofactor binding site). The available cell-free assays have so far facilitated the study of one binding site at a time. With the introduction of Terbium (Tb3+)–based time-resolved fluorescence energy transfer (TR-FRET), it has become possible to measure 2 different interactions within 1 test tube in parallel. The authors have applied this technology to develop a dual readout system for the simultaneous monitoring of steroid hormone site binding and cofactor peptide recruitment. They took advantage of a commercially available fluorescent tracer as an indicator for classical steroid site binding and designed a novel peptide derived from the peroxisome proliferator-activated receptor gamma coactivator-1a (PGC1a) as an indicator for functional agonistic behavior of a test compound. The established assay is able to differentiate between agonists, antagonists, partial agonists, and compounds binding to the cofactor recruitment site. The IC50 values obtained for a number of reference compounds in the multiplexed assay are in concordance with published data. The simple 1-step mix-and-measure protocol gives excellent quality and robustness and can be miniaturized to 5-µL volume.
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Glickman, J. Fraser, Xiang Wu, Robert Mercuri, Chantal Illy, Benjamin R. Bowen, Yang He, and Matthew Sills. "A Comparison of ALPHAScreen, TR-FRET, and TRF as Assay Methods for FXR Nuclear Receptors." Journal of Biomolecular Screening 7, no. 1 (February 2002): 3–10. http://dx.doi.org/10.1177/108705710200700102.
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New developments in detection technologies are providing a variety of biomolecular screening strategies from which to choose. Consequently, we performed a detailed analysis of both separation-based and non-separation-based formats for screening nuclear receptor ligands. In this study, time-resolved fluorescence resonance energy transfer (TR-FRET), ALPHAScreen, and time-resolved fluorescence (TRF) assays were optimized and compared with respect to sensitivity, reproducibility, and miniaturization capability. The results showed that the ALPHAScreen system had the best sensitivity and dynamic range. The TRF assay was more time consuming because of the number of wash steps necessary. The TR-FRET assay had less interwell variation, most likely because of ratiometric measurement. Both the ALPHAScreen and the TR-FRET assays were miniaturized to 8-μl volumes. Of the photomultiplier tube-based readers, the ALPHAScreen reader (ALPHAQuest) presented the advantage of faster reading times through simultaneous reading with four photomultiplier tubes.
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Gustafsdottir, Sigrun M., Stefan Wennström, Simon Fredriksson, Edith Schallmeiner, Andrew D. Hamilton, Said M. Sebti, and Ulf Landegren. "Use of Proximity Ligation to Screen for Inhibitors of Interactions between Vascular Endothelial Growth Factor A and Its Receptors." Clinical Chemistry 54, no. 7 (July 1, 2008): 1218–25. http://dx.doi.org/10.1373/clinchem.2007.099424.
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Abstract Background: Improved methods are required to screen drug candidates for their influences on protein interactions. There is also a compelling need for miniaturization of screening assays, with attendant reductions in reagent consumption and assay costs. Methods: We used sensitive, miniaturized proximity ligation assays (PLAs) to monitor binding of vascular endothelial growth factor A (VEGF-A) to 2 of its receptors, VEGFR-1 and VEGFR-2. We measured the effects of proteins and low molecular weight compounds capable of disrupting these interactions and compared the results with those obtained by immunoblot analysis. We analyzed 6 different inhibitors: a DNA aptamer, a mixed DNA/RNA aptamer, a monoclonal VEGF-A neutralizing antibody, a monoclonal antibody directed against VEGFR-2, a recombinant competitive protein, and a low molecular weight synthetic molecule. Results: The PLAs were successful for monitoring the formation and inhibition of VEGF-A–receptor complexes, and the results correlated well with those obtained by measuring receptor phosphorylation. The total PLA time is just 3 hours, with minimal manual work and reagent additions. The method allows evaluation of the apparent affinity [half-maximal inhibitory concentration (IC50)] from a dose–response curve. Conclusions: The PLA may offer significant advantages over conventional methods for screening the interactions of ligands with their receptors. The assay may prove useful for parallel analyses of large numbers of samples in the screening of inhibitor libraries for promising agents. The technique provides dose–response curves, allowing IC50 values to be calculated.
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Titus, Steve, Susanne Neumann, Wei Zheng, Noel Southall, Sam Michael, Carleen Klumpp, Adam Yasgar, et al. "Quantitative High-Throughput Screening Using a Live-Cell cAMP Assay Identifies Small-Molecule Agonists of the TSH Receptor." Journal of Biomolecular Screening 13, no. 2 (January 23, 2008): 120–27. http://dx.doi.org/10.1177/1087057107313786.
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The thyroid-stimulating hormone (TSH; thyrotropin) receptor belongs to the glycoprotein hormone receptor subfamily of 7-transmembrane spanning receptors. TSH receptor (TSHR) is expressed mainly in thyroid follicular cells and is activated by TSH, which regulates the growth and function of thyroid follicular cells. Recombinant TSH is used in diagnostic screens for thyroid cancer, especially in patients after thyroid cancer surgery. Currently, no selective small-molecule agonists of the TSHR are available. To screen for novel TSHR agonists, the authors miniaturized a commercially available cell-based cyclic adenosine 3′,5′ monophosphate (cAMP) assay into a 1536-well plate format. This assay uses an HEK293 cell line stably transfected with the TSHR coupled to a cyclic nucleotide gated ion channel as a biosensor. From a quantitative high-throughput screen of 73,180 compounds in parallel with a parental cell line (without the TSHR), 276 primary active compounds were identified. The activities of the selected active compounds were further confirmed in an orthogonal homogeneous time-resolved fluorescence cAMP-based assay. Forty-nine compounds in several structural classes have been confirmed as the small-molecule TSHR agonists that will serve as a starting point for chemical optimization and studies of thyroid physiology in health and disease. ( Journal of Biomolecular Screening 2008:120-127)
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Lindenau, Kristen L., Jeffrey L. Barr, Christopher R. Higgins, Kevin T. Sporici, Eugen Brailoiu, and Gabriela C. Brailoiu. "Blood–Brain Barrier Disruption Mediated by FFA1 Receptor—Evidence Using Miniscope." International Journal of Molecular Sciences 23, no. 4 (February 18, 2022): 2258. http://dx.doi.org/10.3390/ijms23042258.
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Omega-3 polyunsaturated fatty acids (n-3 PUFAs), obtained from diet and dietary supplements, have been tested in clinical trials for the prevention or treatment of several diseases. n-3 PUFAs exert their effects by activation of free fatty acid (FFA) receptors. FFA1 receptor, expressed in the pancreas and brain, is activated by medium- to long-chain fatty acids. Despite some beneficial effects on cognition, the effects of n-3 PUFAs on the blood–brain barrier (BBB) are not clearly understood. We examined the effects of FFA1 activation on BBB permeability in vitro, using rat brain microvascular endothelial cells (RBMVEC), and in vivo, by assessing Evans Blue extravasation and by performing live imaging of brain microcirculation in adult rats. AMG837, a synthetic FFA1 agonist, produced a dose-dependent decrease in RBMVEC monolayer resistance assessed with Electric Cell–Substrate Impedance Sensing (ECIS); the effect was attenuated by the FFA1 antagonist, GW1100. Immunofluorescence studies revealed that AMG837 produced a disruption in tight and adherens junction proteins. AMG837 increased Evans Blue content in the rat brain in a dose-dependent manner. Live imaging studies of rat brain microcirculation with miniaturized fluorescence microscopy (miniscope) showed that AMG837 increased extravasation of sodium fluorescein. Taken together, our results demonstrate that FFA1 receptor activation reduced RBMVEC barrier function and produced a transient increase in BBB permeability.
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Fottner, C., E. Mettler, M. Goetz, E. Schirrmacher, M. Anlauf, D. Strand, R. Schirrmacher, et al. "In Vivo Molecular Imaging of Somatostatin Receptors in Pancreatic Islet Cells and Neuroendocrine Tumors by Miniaturized Confocal Laser-Scanning Fluorescence Microscopy." Endocrine Reviews 31, no. 2 (April 1, 2010): 260. http://dx.doi.org/10.1210/edrv.31.2.9991.
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Fottner, C., E. Mettler, M. Goetz, E. Schirrmacher, M. Anlauf, D. Strand, R. Schirrmacher, et al. "In Vivo Molecular Imaging of Somatostatin Receptors in Pancreatic Islet Cells and Neuroendocrine Tumors by Miniaturized Confocal Laser-Scanning Fluorescence Microscopy." Journal of Clinical Endocrinology & Metabolism 95, no. 4 (April 1, 2010): 2005. http://dx.doi.org/10.1210/jcem.95.4.9988.
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Fottner, C., E. Mettler, M. Goetz, E. Schirrmacher, M. Anlauf, D. Strand, R. Schirrmacher, et al. "In Vivo Molecular Imaging of Somatostatin Receptors in Pancreatic Islet Cells and Neuroendocrine Tumors by Miniaturized Confocal Laser-Scanning Fluorescence Microscopy." Endocrinology 151, no. 5 (March 16, 2010): 2179–88. http://dx.doi.org/10.1210/en.2009-1313.
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The aim of the study was to evaluate real time in vivo molecular imaging of somatostatin receptors (sstrs) using a handheld miniaturized confocal laser scan microscope (CLM) in conjunction with fluorescein-labeled octreotate (OcF) in healthy mice and murine models of neuroendocrine tumors. For CLM a small rigid probe (diameter 7 mm) with an integrated single line laser (488 nm) was used (optical slice thickness 7 μm; lateral resolution 0.7 μm). OcF was synthesized via Fmoc solid-phase peptide synthesis and purified by HPLC showing high-affinity binding to the sstr2 (IC50 6.2 nmol). For in vitro evaluation, rat and human pancreatic cancer cells were used and characterized with respect to its sstr subtype expression and functional properties. For in vivo confocal imaging, healthy mouse pancreatic islet and renal tubular cells as well as immunoincompetent nude mice harboring sstr-expressing tumors were evaluated. Incubation of sstr-positive cells with OcF showed a specific time- and dose-dependent staining of sstr-positive cells. CLM showed rapid internalization and homogenous cytoplasmatic distribution. After systemic application to mice (n = 8), specific time-dependent internalization and cytoplasmatic distribution into pancreatic islet cells and tubular cells of the renal cortex was recorded. After injection in tumor-harboring nude mice (n = 8), sstr-positive cells selectively displayed a cell surface and cytoplasmatic staining. CLM-targeted biopsies detected sstr-positive tumor cells with a sensitivity of 87.5% and a specificity of 100% as correlated with ex vivo immunohistochemistry. CLM with OcF permits real-time molecular, functional, and morphological imaging of sstr-expressing cell structures, allowing the specific visualization of pancreatic islet cells and neuroendocrine tumors in vivo.
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Brandish, Philip E., Chi-Sung Chiu, Jonathan Schneeweis, Nicholas J. Brandon, Clare L. Leech, Oleg Kornienko, Edward M. Scolnick, Berta Strulovici, and Wei Zheng. "A Cell-Based Ultra-High-Throughput Screening Assay for Identifying Inhibitors of D-Amino Acid Oxidase." Journal of Biomolecular Screening 11, no. 5 (April 28, 2006): 481–87. http://dx.doi.org/10.1177/1087057106288181.
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Enzymes are often considered less “druggable” targets than ligand-regulated proteins such as G-protein-coupled receptors, ion channels, or other hormone receptors. Reasons for this include cellular location (intracellular vs. cell surface), typically lower affinities for the binding of small molecules compared to ligand-specific receptors, and binding (catalytic) sites that are often charged or highly polar. A practical drawback to the discovery of compounds targeting enzymes is that screening of compound libraries is typically carried out in cell-free activity assays using purified protein in an inherently artificial environment. Cell-based assays, although often arduous to design for enzyme targets, are the preferred discovery tool for the screening of large compound libraries. The authors have recently described a novel cell-based approach to screening for inhibitors of a phosphatase enzyme and now report on the development and implementation of a homogeneous 3456-well plate assay for D-amino acid oxidase (DAO). Human DAO was stably expressed in Chinese hamster ovary (CHO) cells, and its activity was measured as the amount of hydrogen peroxide detected in the growth medium following feeding the cells with D-serine. In less than 12 weeks, the authors proved the concept in 96-and then 384-well formats, miniaturized the assay to the 3456-well (nanoplate) scale, and screened a library containing more than 1 million compounds. They have identified several cell-permeable inhibitors of DAO from this cell-based high-throughput screening, which provided the discovery program with a few novel and attractive lead structures.
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Miglione, Antonella, Maria Napoletano, and Stefano Cinti. "Electrochemical Biosensors for Tracing Cyanotoxins in Food and Environmental Matrices." Biosensors 11, no. 9 (September 4, 2021): 315. http://dx.doi.org/10.3390/bios11090315.
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The adoption of electrochemical principles to realize on-field analytical tools for detecting pollutants represents a great possibility for food safety and environmental applications. With respect to the existing transduction mechanisms, i.e., colorimetric, fluorescence, piezoelectric etc., electrochemical mechanisms offer the tremendous advantage of being easily miniaturized, connected with low cost (commercially available) readers and unaffected by the color/turbidity of real matrices. In particular, their versatility represents a powerful approach for detecting traces of emerging pollutants such as cyanotoxins. The combination of electrochemical platforms with nanomaterials, synthetic receptors and microfabrication makes electroanalysis a strong starting point towards decentralized monitoring of toxins in diverse matrices. This review gives an overview of the electrochemical biosensors that have been developed to detect four common cyanotoxins, namely microcystin-LR, anatoxin-a, saxitoxin and cylindrospermopsin. The manuscript provides the readers a quick guide to understand the main electrochemical platforms that have been realized so far, and the presence of a comprehensive table provides a perspective at a glance.
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Rüdiger, Martin, Ulrich Haupts, Keith J. Moore, and Andrew J. Pope. "Single-Molecule Detection Technologies in Miniaturized High Throughput Screening: Binding Assays for G Protein–Coupled Receptors Using Fluorescence Intensity Distribution Analysis and Fluorescence Anisotropy." Journal of Biomolecular Screening 6, no. 1 (February 1, 2001): 29–37. http://dx.doi.org/10.1089/108705701750067006.
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Larauche, Muriel, Guillaume Gourcerol, Lixin Wang, Karina Pambukchian, Stefan Brunnhuber, David W. Adelson, Jean Rivier, Mulugeta Million, and Yvette Taché. "Cortagine, a CRF1 agonist, induces stresslike alterations of colonic function and visceral hypersensitivity in rodents primarily through peripheral pathways." American Journal of Physiology-Gastrointestinal and Liver Physiology 297, no. 1 (July 2009): G215—G227. http://dx.doi.org/10.1152/ajpgi.00072.2009.
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Corticotropin-releasing factor (CRF) 1 receptor (CRF1) activation in the brain is a core pathway orchestrating the stress response. Anatomical data also support the existence of CRF signaling components within the colon. We investigated the colonic response to intraperitoneal (ip) injection of cortagine, a newly developed selective CRF1 peptide agonist. Colonic motor function and visceral motor response (VMR) were monitored by using a modified miniaturized pressure transducer catheter in adult conscious male Sprague-Dawley rats and C57Bl/6 mice. Colonic permeability was monitored by the Evans blue method and myenteric neurons activation by Fos immunohistochemistry. Compared with vehicle, cortagine (10 μg/kg ip) significantly decreased the distal colonic transit time by 45% without affecting gastric transit, increased distal and transverse colonic contractility by 35.6 and 66.2%, respectively, and induced a 7.1-fold increase in defecation and watery diarrhea in 50% of rats during the first hour postinjection whereas intracerebroventricular (icv) cortagine (3 μg/rat) had lesser effects. Intraperitoneal (ip) cortagine also increased colonic permeability, activated proximal and distal colonic myenteric neurons, and induced visceral hypersensitivity to a second set of phasic colorectal distention (CRD). The CRF antagonist astressin (10 μg/kg ip) abolished ip cortagine-induced hyperalgesia whereas injected icv it had no effect. In mice, cortagine (30 μg/kg ip) stimulated defecation by 7.8-fold, induced 60% incidence of diarrhea, and increased VMR to CRD. Stresslike colonic alterations induced by ip cortagine in rats and mice through restricted activation of peripheral CRF1 receptors support a role for peripheral CRF1 signaling as the local arm of the colonic response to stress.
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Fan, Yi, Joseph G. Naglich, Jennifer D. Koenitzer, Humberto Ribeiro, Jonathan Lippy, Jordan Blum, Xin Li, et al. "Miniaturized High-Throughput Multiparameter Flow Cytometry Assays Measuring In Vitro Human Dendritic Cell Maturation and T-Cell Activation in Mixed Lymphocyte Reactions." SLAS DISCOVERY: Advancing the Science of Drug Discovery 23, no. 7 (June 6, 2018): 742–50. http://dx.doi.org/10.1177/2472555218775409.
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Enhancing antitumor activities of the human immune system is a clinically proven approach with the advent of monoclonal antibodies recognizing programmed cell death protein-1 (PD1) receptors on immune cell surfaces. Historically, using flow cytometry as a means to assess next-generation agent activities was underused, largely due to limits on cell number and assay sensitivity. Here, we leveraged an IntelliCyt high-throughput flow cytometry platform to monitor human dendritic cell maturation and lymphocyte proliferation in mixed lymphocyte reactions. Specifically, we established flow cytometry–based immunophenotyping and screening methodologies capable of measuring T-cell activation as a result of cell-associated antigens presented on dendritic cell surfaces, as indicated by cell proliferation, cytokine secretion, and surface marker expression. Together, the overall novelty of this 384-well platform is its capability to measure multiple functional readouts in one well and consistently evaluate large numbers of compounds in a single study, as well as its ability to show increased assay sensitivity requiring considerably fewer primary cells and less reagents compared to more traditional 96-well flow cytometry methods.
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Krantis, Anthony, Kamal Mattar, and Ian Glasgow. "Rat gastroduodenal motility in vivo: interaction of GABA and VIP in control of spontaneous relaxations." American Journal of Physiology-Gastrointestinal and Liver Physiology 275, no. 5 (November 1, 1998): G897—G903. http://dx.doi.org/10.1152/ajpgi.1998.275.5.g897.
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Spontaneous relaxations occurring within motor activity in the rat gastroduodenum in vivo can be distinguished by their dependence on either nitric oxide (NO) or ATP. We examined the interaction of γ-aminobutyric acid (GABA) and vasoactive intestinal peptide (VIP) within pathways controlling this activity in the antrum (S) and duodenum (D) of anesthetized Sprague-Dawley rats, using miniaturized extraluminal foil strain gauges oriented perpendicular to (S1, D1) or in the axis of (S2) the circular smooth muscle. The NO synthase inhibitor N G-nitro-l-arginine methyl ester (l-NAME; 10 mg/kg iv) attenuated ( P < 0.05) antral relaxations and, in the duodenum, nonpropagating “intergroup” relaxations. The GABAA receptor antagonist bicuculline (350 μg/kg sc) had similar effects. The GABAA agonist 3-amino-1-propanesulfonic acid stimulatedl-NAME-sensitive relaxations at S1 and D1. Propagating “grouped” responses were unchanged. VIP (6 μg/kg iv) always induced a relaxation of the duodenum, which was attenuated by bicuculline andl-NAME. VIP caused simultaneous responses at S1 and S2; however, the antrum displayed either contraction or relaxation in response to VIP. All antral relaxations in response to VIP were attenuated ( P < 0.05) byl-NAME; however, only VIP-induced relaxations at S1 were sensitive to bicuculline. VIP-induced contractions were also unaffected. GABAA receptors mediate the pathway(s) controlling NO-related spontaneous relaxations of the antrum and duodenal circular muscle. All VIP-induced relaxations are mediated by NO. Spontaneous relaxations of the rat gastroduodenum include responses that involve a GABAAergic NO-related pathway, which is targeted by VIP. In addition, VIP can target NO relaxations of the antrum via other pathways.
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Pandit, Santosh, Mengyue Li, Yanyan Chen, Shadi Rahimi, Vrss Mokkapati, Alessandra Merlo, August Yurgens, and Ivan Mijakovic. "Graphene-Based Sensor for Detection of Bacterial Pathogens." Sensors 21, no. 23 (December 3, 2021): 8085. http://dx.doi.org/10.3390/s21238085.
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Microbial colonization to biomedical surfaces and biofilm formation is one of the key challenges in the medical field. Recalcitrant biofilms on such surfaces cause serious infections which are difficult to treat using antimicrobial agents, due to their complex structure. Early detection of microbial colonization and monitoring of biofilm growth could turn the tide by providing timely guidance for treatment or replacement of biomedical devices. Hence, there is a need for sensors, which could generate rapid signals upon bacterial colonization. In this study, we developed a simple prototype sensor based on pristine, non-functionalized graphene. The detection principle is a change in electrical resistance of graphene upon exposure to bacterial cells. Without functionalization with specific receptors, such sensors cannot be expected to be selective to certain bacteria. However, we demonstrated that two different bacterial species can be detected and differentiated by our sensor due to their different growth dynamics, adherence pattern, density of adhered bacteria and microcolonies formation. These distinct behaviors of tested bacteria depicted distinguishable pattern of resistance change, resistance versus gate voltage plot and hysteresis effect. This sensor is simple to fabricate, can easily be miniaturized, and can be effective in cases when precise identification of species is not needed.
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Masoumi, Saeid, Hassan Hajghassem, Alireza Erfanian, and Ahmad Molaei Rad. "Design and manufacture of TNT explosives detector sensors based on CNTFET." Sensor Review 36, no. 4 (September 19, 2016): 414–20. http://dx.doi.org/10.1108/sr-01-2016-0014.
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Purpose Miniaturized smart sensors that can perform sensitive and selective real-time monitoring of target analytes are tremendously valuable for various sensing applications. So, the purpose of this paper is to provide details of sensors based on selective nanocoatings by combining trinitrotoluene (TNT) receptors bound to conjugated polydiacetylene (PDA) polymers with single-walled carbon nanotube field-effect transistors (CNTFETs) for detecting explosives TNT. Design/methodology/approach Following an introduction, this paper describes the way of creating an FET with CNTs, which are functionalized by the peptide based on TNT molecule recognition elements and PDA, to offer a system which has the capability of answering the presence of related target molecules (TNT). Finally, brief conclusions are drawn. Findings Single-wall nanotubes and reduced graphene oxide are interesting materials for creating biosensors of FETs at nanoscale because of unique electrical, mechanical, geometrical and biocompatible properties. Therefore, this sensor is designed and manufactured, and the results of applying TNT to sensor show good sensitivity and selectivity response. Originality/value In this timeframe of history, sensors based on CNTFET are required for different uses, including clinical diagnosis technologies, environmental tests and bioterrorism recognition technologies, that correspond to the military conflicts and terrorism. So, CNTFET sensor design provides real-time detection of TNT explosives.
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Meinen, Sarina, Patrizia Barzaghi, Shuo Lin, Hanns Lochmüller, and Markus A. Ruegg. "Linker molecules between laminins and dystroglycan ameliorate laminin-α2–deficient muscular dystrophy at all disease stages." Journal of Cell Biology 176, no. 7 (March 26, 2007): 979–93. http://dx.doi.org/10.1083/jcb.200611152.
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Mutations in laminin-α2 cause a severe congenital muscular dystrophy, called MDC1A. The two main receptors that interact with laminin-α2 are dystroglycan and α7β1 integrin. We have previously shown in mouse models for MDC1A that muscle-specific overexpression of a miniaturized form of agrin (mini-agrin), which binds to dystroglycan but not to α7β1 integrin, substantially ameliorates the disease (Moll, J., P. Barzaghi, S. Lin, G. Bezakova, H. Lochmuller, E. Engvall, U. Muller, and M.A. Ruegg. 2001. Nature. 413:302–307; Bentzinger, C.F., P. Barzaghi, S. Lin, and M.A. Ruegg. 2005. Matrix Biol. 24:326–332.). Now we show that late-onset expression of mini-agrin still prolongs life span and improves overall health, although not to the same extent as early expression. Furthermore, a chimeric protein containing the dystroglycan-binding domain of perlecan has the same activities as mini-agrin in ameliorating the disease. Finally, expression of full-length agrin also slows down the disease. These experiments are conceptual proof that linking the basement membrane to dystroglycan by specifically designed molecules or by endogenous ligands, could be a means to counteract MDC1A at a progressed stage of the disease, and thus opens new possibilities for the development of treatment options for this muscular dystrophy.
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Romero, Celeste, Lester J. Lambert, Douglas J. Sheffler, Laurent J. S. De Backer, Dhanya Raveendra-Panickar, Maria Celeridad, Stefan Grotegut, et al. "A cellular target engagement assay for the characterization of SHP2 (PTPN11) phosphatase inhibitors." Journal of Biological Chemistry 295, no. 9 (January 17, 2020): 2601–13. http://dx.doi.org/10.1074/jbc.ra119.010838.
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The nonreceptor protein-tyrosine phosphatase (PTP) SHP2 is encoded by the proto-oncogene PTPN11 and is a ubiquitously expressed key regulator of cell signaling, acting on a number of cellular processes and components, including the Ras/Raf/Erk, PI3K/Akt, and JAK/STAT pathways and immune checkpoint receptors. Aberrant SHP2 activity has been implicated in all phases of tumor initiation, progression, and metastasis. Gain-of-function PTPN11 mutations drive oncogenesis in several leukemias and cause developmental disorders with increased risk of malignancy such as Noonan syndrome. Until recently, small molecule-based targeting of SHP2 was hampered by the failure of orthosteric active-site inhibitors to achieve selectivity and potency within a useful therapeutic window. However, new SHP2 allosteric inhibitors with excellent potency and selectivity have sparked renewed interest in the selective targeting of SHP2 and other PTP family members. Crucially, drug discovery campaigns focusing on SHP2 would greatly benefit from the ability to validate the cellular target engagement of candidate inhibitors. Here, we report a cellular thermal shift assay that reliably detects target engagement of SHP2 inhibitors. Using this assay, based on the DiscoverX InCell Pulse enzyme complementation technology, we characterized the binding of several SHP2 allosteric inhibitors in intact cells. Moreover, we demonstrate the robustness and reliability of a 384-well miniaturized version of the assay for the screening of SHP2 inhibitors targeting either WT SHP2 or its oncogenic E76K variant. Finally, we provide an example of the assay's ability to identify and characterize novel compounds with specific cellular potency for either WT or mutant SHP2.
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Carter, Mark, Sarita Chauhan, John O’Rourke, and Nina Senutovitch. "Development of a cells and bead-based, advanced flow cytometry assay for exhausted T cell quantitation." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 86.29. http://dx.doi.org/10.4049/jimmunol.204.supp.86.29.
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Abstract T cells can become exhausted due to persistent antigen exposure during chronic infections and cancer. Exhausted T cells exhibit decreased effector functions and express inhibitory receptors (IRs). The exhausted phenotype may be protective in the context of chronic infection, however, it allows tumors to avoid immune surveillance and immune cell mediated killing. Recent therapies block the IRs to reinvigorate exhausted T cells. A miniaturized, multiplexed flow cytometry assay was developed to measure expression of IRs on T cells while simultaneously measuring viability, proliferation, and cytokine secretion in microtiter plates. Data was acquired on the Intellicyt® iQue platform and analyzed using Forecyt®. Templates were prepared for ease of data analysis, to auto-generate gating, standard curves and sample quantitation and rapid, actionable results. For proof of concept studies, human PBMCs, purified CD3+, or in vitro exhausted purified CD3+ cells were treated with anti-CD3/CD28 and cells were analyzed at 1, 2, and 3 days post-stimulation. Non-exhausted cells showed a concentration and time dependent increase in IR expression. Persistent, high levels of the IR markers PD-1, LAG-3 and TIM-3 were measured in exhausted cells. Stimulated exhausted T cells secreted stable and moderate levels of IFN-γ (<1650 pg/ml) and TNF (<750 pg/ml) over time whereas non-exhausted cells showed time and concentration dependent increases in IFN-γ (13,319 pg/ml) and TNF (3,711 pg/ml) upon stimulation. The ability of this assay to examine T cell response allows screening for molecules that reverse T cell exhaustion, screening of modified cells such as CAR-Ts or selection of clones that exhibit low levels of IRs and show robust response to stimulation.
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Schwenk, Jochen M., Oliver Poetz, Robert Zeillinger, and Thomas O. Joos. "A Miniaturized Ligand Binding Assay for EGFR." International Journal of Proteomics 2012 (April 8, 2012): 1–5. http://dx.doi.org/10.1155/2012/247059.
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In order to study receptor abundance and its function in solutions or in homogenates from clinical specimen, methods such as sandwich or radioimmunoassays are most commonly employed. For the determination of epidermal growth factor receptor (EGFR), we describe the development of a miniaturized bead-based ligand binding assay using its ligand EGF as immobilized capture reagent. This assay was used to analyze lysates from cell lines, and the ligand-bound EGFR was detected using an EGFR-specific antibody combined with a fluorescence-based reporter system. In a proof-of concept study with lysates from breast biopsies, the assay allowed to classify breast cancer samples in accordance to clinically the relevant EGFR cut-off level. The study suggests that such a ligand binding receptor assay could become an integral part of protein profiling procedures to provide additional information about receptor functionality in addition to its abundance.
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Weiss-Wichert, Ch, M. Smetazko, M. Valina-Saba, and Th Schalkhammer. "A New Analytical Device Based on Gated Ion Channels: A Peptide-Channel Biosensor." Journal of Biomolecular Screening 2, no. 1 (February 1997): 11–18. http://dx.doi.org/10.1177/108705719700200104.
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Binding of a protein at a ligand-modified ion channel can lead to closure or distortion of the channel, resulting in a digital on/off response of the ion flux. This principle could be applied as a strategy for sensor design using artificial membrane ligands or receptors. Important details of modeling and a technique of realization of the new molecular electronic sensor device are presented. The response of the sensor induced by analyte binding depends on the analyte's size and ability to close or distort the ion channel. Testing different ion channels in a typical single molecule-binding event modifies the membrane current by about 0.2 to 20 pA. The gated channel-based sensor is set up by using elements from different fields. It consists of a stable transmembrane channel (for example, a chemically engineered 6.3 helix peptide antibiotic) with a ligand covalently bound at the peptide channel entrance, an electrochemical sensor chip with a photostructurized hydrophobic polymer frame, a hydrophilic ion-conducting membrane support, a lipid membrane incorporating the engineered ion channel, and a sensitive membrane current amplifier or a sensitive fluorescence monitor. Detection of channel opening or closure can either be obtained directly by monitoring membrane conductivity or by monitoring a transient change in pH or ion concentration within the membrane compartment. This transient change can be induced by various means, and its decay is directly correlated to the ion permeability of the membrane. Nonspecific binding at a lipid membrane does not interfere with the ion flux through ion channels. This is a basic advantage of this principle compared to that of enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or hybridization of labeled DNA/RNA probes. Furthermore, because nonspecific binding events are mainly transient, specific binding events directly at the ion channel can be separated from them by evaluating the current peak time scale. Theoretically, the sensitivity of such a device can approach one molecule. Under practical conditions, the sensitivity is limited by the diffusion process of the analyte to the receptor ligand but not by the molecular electronic device. The new type of biosensor combines sensor design and technology from immunoassays and biorecognition assays with automated analysis systems and knowledge from the field of nanotechnology. Based on the detection principle, miniaturized portable sensor chips with either electronic or optical detection are under construction. These may have capabilities exceeding those of existing analytical instruments (for example, ELISA tests, medical analyzer, and high-pressure liquid chromatography [HPLC].
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Hernandez Ambato, Valeria, and Hugo Moreno Avilés. "Miniaturización de una Antena Microstrip Aplicando Diseño Fractal T-cuadrado." Revista Perspectivas 3, no. 2 (July 12, 2021): 36–44. http://dx.doi.org/10.47187/perspectivas.vol3iss2.pp36-44.2021.
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Este documento describe el diseño e implementación de una antena fractal microstrip miniaturizada con frecuencia de operación a 2.4 GHz para la aplicación en dispositivos portables. La antena se desarrolló considerando la frecuencia de operación y las características del material. El material utilizado es RO4003C con permitividad dieléctrica εr = 3.38 y espesor h = 0.254 mm. Se aplicó dos técnicas de miniaturización: por la forma y ranura en el parche. La forma del parche miniaturizado se definió en la segunda iteración del diseño fractal del T-cuadrado. El proceso de optimización del diseño de la antena se realizó mediante simulaciones y se utilizó la herramienta Ansoft Designer. Se implementó el diseño de la antena final que alcanzó el valor del parámetro S de -21 dB. Las mediciones se realizaron con la antena transmisora y receptora separadas a 15 cm y con potencia de entrada de 15 dBm. El diseño del parche final presentó el 46,65 % de miniaturización del tamaño con respecto al parche original. La optimización del diseño final de la antena presentó el 62,2 % de reducción en el tamaño con respecto a la antena microstrip inicial.
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Benjamin, Elfrida R., Sarah L. Haftl, Dimitris N. Xanthos, Gregg Crumley, Mohamed Hachicha, and Kenneth J. Valenzano. "A Miniaturized Column Chromatography Method for Measuring Receptor-Mediated Inositol Phosphate Accumulation." Journal of Biomolecular Screening 9, no. 4 (June 2004): 343–53. http://dx.doi.org/10.1177/1087057103262841.
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Inositol phosphates (IPs), such as 1,4,5-inositol-trisphosphate (IP3), comprise a ubiquitous intracellular signaling cascade initiated in response to G protein-coupled receptor-mediated activation of phospholipase C. Classical methods for measuring intracellular accumulation of these molecules include time-consuming high-performance liquid chromatography (HPLC) separation or large-volume, gravity-fed anion-exchange column chromatography. More recent approaches, such as radio-receptor and AlphaScreen™ assays, offer higher throughput. However, these techniques rely on measurement of IP3 itself, rather than its accumulation with other downstream IPs, and often suffer from poor signal-to-noise ratios due to the transient nature of IP3. The authors have developed a miniaturized, anion-exchange chromatography method for measuring inositol phosphate accumulation in cells that takes advantage of signal amplification achieved through measuring IP3 and downstream IPs. This assay uses centrifugation of 96-well-formatted anion-exchange mini-columns for the isolation of radiolabeled inositol phosphates from cell extracts, followed by low-background dry-scintillation counting. This improved assay method measures receptor-mediated IP accumulation with signal-to-noise and pharmacological values comparable to the classical large-volume, column-based methods. Assay validation data for recombinant muscarinic receptor 1, galanin receptor 2, and rat astrocyte metabotropic glutamate receptor 5 are presented. This miniaturized protocol reduces reagent usage and assay time as compared to large-column methods and is compatible with standard 96-well scintillation counters.
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Schubert, RL, and D. Puett. "Single-chain human chorionic gonadotropin analogs containing the determinant loop of the beta-subunit linked to the alpha-subunit." Journal of Molecular Endocrinology 31, no. 1 (August 1, 2003): 157–68. http://dx.doi.org/10.1677/jme.0.0310157.
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Human chorionic gonadotropin (hCG) is a member of the family of glycoprotein hormones containing a common alpha-subunit and distinct beta-subunits that confer hormonal specificity. hCG binds to the relatively large ectodomain of the human luteinizing hormone receptor (hLHR), a member of the G protein-coupled receptor superfamily, leading to increased intracellular production of cAMP. Using protein engineering, two miniaturized versions of hCGbeta have been separately fused to the N-terminus of the alpha-subunit to give N-des[1-91]hCGbeta-alpha-C and N-des[1-91,110-114]hCGbeta-alpha-C, i.e. fusion proteins of the hCGbeta determinant loop (extended to include the complete seat belt and carboxy-terminal peptide) coupled to the alpha-subunit. Bioactivity of these single-chain gonadotropin analogs was assessed in two systems following transient transfections into HEK 293 cells and subsequent cAMP measurements. In one, each mini-beta-alpha cDNA was fused to that of hLHR and transfected into cells to create yoked miniaturized hCG-hLHR complexes; in the other, the cDNA of each single chain mini-beta-alpha was co-transfected with that of hLHR in an effort to produce non-covalent miniaturized hCG-hLHR complexes. Using yoked hCG-hLHR and hLHR as positive and negative controls respectively, expression of each mini-hCG-hLHR complex was confirmed using antibody and ligand binding assays. The two mini-hCGs led to minimal activation of hLHR, suggesting weak intrinsic activity of the mini-beta-alpha fusion proteins. These results suggest that potent agonists and antagonists will require the presence of other portions of hCGbeta in addition to the determinant loop/seat belt.
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Harris, Alison, Sarah Cox, Dougal Burns, and Christopher Norey. "Miniaturization of Fluorescence Polarization Receptor-Binding Assays Using CyDye-Labeled Ligands." Journal of Biomolecular Screening 8, no. 4 (August 2003): 410–20. http://dx.doi.org/10.1177/1087057103256319.
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Fluorescence polarization (FP) is an established technique for the study of biological interactions and is frequently used in the high-throughput screening (HTS) of potential new drug targets. This work describes the miniaturization of FP receptor assays to 1536-well formats for use in HTS. The FP assays were initially developed in 384-well microplates using CyDye-labeled nonpeptide and peptide ligands. Receptor expression levels varied from ∼1 to 10 pmols receptor per mg protein, and ligand concentrations were in the 0.5- to 1.0-nM range. The FP assays were successfully miniaturized to 1536-well formats using Cy3B-labeled ligands, significantly reducing reagent consumption, particularly the receptor source, without compromising assay reliability. Z' factor values determined for the FP receptor assays in both 384- and 1536-well formats were found to be > 0.5, indicating the assays to be robust, reliable, and suitable for HTS purposes.
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Heise, Denise, Alicia Derrac Soria, Selina Hansen, Christine Dambietz, Mohammad Akbarzadeh, Anna F. Berg, Georg H. Waetzig, et al. "Selective inhibition of IL-6 trans-signaling by a miniaturized, optimized chimeric soluble gp130 inhibits TH17 cell expansion." Science Signaling 14, no. 696 (August 17, 2021): eabc3480. http://dx.doi.org/10.1126/scisignal.abc3480.
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The cytokine interleukin-6 (IL-6) signals through three mechanisms called classic signaling, trans-signaling, and trans-presentation. IL-6 trans-signaling is distinctly mediated through a soluble form of its transmembrane receptor IL-6R (sIL-6R) and the coreceptor gp130 and is implicated in multiple autoimmune diseases. Although a soluble form of gp130 (sgp130) inhibits only IL-6 trans-signaling, it also blocks an analogous trans-signaling mechanism of IL-11 and its soluble receptor sIL-11R. Here, we report miniaturized chimeric soluble gp130 variants that efficiently trap IL-6:sIL-6R but not IL-11:sIL-11R complexes. We designed a novel IL-6 trans-signaling trap by fusing a miniaturized sgp130 variant to an IL-6:sIL-6R complex–binding nanobody and the Fc portion of immunoglobulin G (IgG). This trap, called cs-130Fc, exhibited improved inhibition of as well as increased selectivity for IL-6 trans-signaling compared to the conventional fusion protein sgp130Fc. We introduced affinity-enhancing mutations in cs-130Fc and sgp130Fc that further improved selectivity toward IL-6 trans-signaling. Moreover, cs-130Fc efficiently inhibited the expansion of T helper 17 (TH17) cells in cultures of mouse CD4+ T cells treated with IL-6:sIL-6R. Thus, these variants may provide or lead to the development of more precisely targeted therapeutics for inflammatory disorders associated with IL-6 trans-signaling.
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Yang, Hao, Weipin Qian, Lily Yang, Huikai Xie, and Huabei Jiang. "In Vivo Evaluation of a Miniaturized Fluorescence Molecular Tomography (FMT) Endoscope for Breast Cancer Detection Using Targeted Nanoprobes." International Journal of Molecular Sciences 21, no. 24 (December 9, 2020): 9389. http://dx.doi.org/10.3390/ijms21249389.
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In this study, in vivo animal experiments with 12 nude mice bearing breast-cancer-patient-tissue-derived xenograft (PDX) tumors were performed aiming to verify the imaging capability of a novel miniaturized fluorescence molecular tomography (FMT) endoscope, in combination with targeted nanoparticle–near-infrared (NIR) dye conjugates. Tumor-bearing mice were divided into two groups by systematic injection with urokinase plasminogen activator receptor-targeted (n = 7) and nontargeted (n = 5) imaging nanoprobes as a contrast agent, respectively. Each mouse was imaged at 6, 24, and 48 h following the injection of nanoprobes using the FMT endoscope. The results show that systemic delivery of targeted nanoprobes produced a 4-fold enhancement in fluorescence signals from tumors, compared with tumors that received nontargeted nanoprobes. This study indicates that our miniaturized FMT endoscope, coupled with the targeted nanoparticle–NIR dye conjugates as a contrast agent, has high sensitivity and specificity, and thus great potential to be used for image-guided detection and removal of a primary tumor and local metastatic tumors during surgery.
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Carter, Clare M. Scaramellini, Juliet R. Leighton-Davies, and Steven J. Charlton. "Miniaturized Receptor Binding Assays: Complications Arising from Ligand Depletion." Journal of Biomolecular Screening 12, no. 2 (January 11, 2007): 255–66. http://dx.doi.org/10.1177/1087057106297788.
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The advent of miniaturized assay formats has made possible the screening of large numbers of compounds against a single target, known as high-throughput screening. Despite this clear advantage, assay miniaturization also increases the risk of ligand depletion, where the actual concentration of free ligand is significantly lower than that added. This, in turn, complicates the interpretation of data from such assays, potentially introducing significant error if not recognized. In this study, the effects of reducing assay volume on radioligand Kd and competitor Ki values have been investigated, using the muscarinic M3 receptor as a model system. It was found that assay miniaturization caused dramatic effects, with up to a 30-fold underestimation of ligand affinity. A theoretical model was developed and shown to accurately predict both the degree of ligand depletion in any given assay volume and the effect of this depletion on affinity estimates for competing ligands. Importantly, it was found that in most cases, errors introduced by ligand depletion could be largely corrected for by the use of appropriate analysis methods. In addition to those previously described by others, the authors propose a simple method capable of correcting errors in competition binding experiments performed in conditions of ligand depletion.
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Nijmeijer, Saskia, Henry F. Vischer, Anders F. Rudebeck, Frank Fleurbaaij, David Falck, Rob Leurs, Wilfried M. A. Niessen, and Jeroen Kool. "Development of a Profiling Strategy for Metabolic Mixtures by Combining Chromatography and Mass Spectrometry with Cell-Based GPCR Signaling." Journal of Biomolecular Screening 17, no. 10 (June 26, 2012): 1329–38. http://dx.doi.org/10.1177/1087057112451922.
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In this study, we developed an in-line methodology that combines analytical with pharmacological techniques to characterize metabolites of human histamine H4 receptor (hH4R) ligands. Liquid chromatographic separation of metabolic mixtures is coupled to high-resolution fractionation into 96- or 384-well plates and directly followed by a cell-based reporter gene assay to measure receptor signaling. The complete methodology was designed, optimized, validated, and ultimately miniaturized into a high-density well plate format. Finally, the methodology was demonstrated in a metabolic profiling setting for three hH4R lead compounds and the drug clozapine. This new methodology comprises integrated analytical separations, mass spectrometry, and a cell-based signal transduction–driven reporter gene assay that enables the implementation of comprehensive metabolic profiling earlier in the drug discovery process.
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Li Pira, Giuseppina, Federico Ivaldi, Nadia Starc, Fabiola Landi, Franco Locatelli, Sergio Rutella, Gino Tripodi, and Fabrizio Manca. "Miniaturized and High-Throughput Assays for Analysis of T-Cell Immunity Specific for Opportunistic Pathogens and HIV." Clinical and Vaccine Immunology 21, no. 4 (January 29, 2014): 488–95. http://dx.doi.org/10.1128/cvi.00660-13.
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ABSTRACTMonitoring of antigen-specific T-cell responses is valuable in numerous conditions that include infectious diseases, vaccinations, and opportunistic infections associated with acquired or congenital immune defects. A variety of assays that make use of peripheral lymphocytes to test activation markers, T-cell receptor expression, or functional responses are currently available. The last group of assays calls for large numbers of functional lymphocytes. The number of cells increases with the number of antigens to be tested. Consequently, cells may be the limiting factor, particularly in lymphopenic subjects and in children, the groups that more often require immune monitoring. We have developed immunochemical assays that measure secreted cytokines in the same wells in which peripheral blood mononuclear cells (PBMC) are cultured. This procedure lent itself to miniaturization and automation. Lymphoproliferation and the enzyme-linked immunosorbent spot (ELISPOT) assay have been adapted to a miniaturized format. Here we provide examples of immune profiles and describe a comparison between miniaturized assays based on cytokine secretion or proliferation. We also demonstrate that these assays are convenient for use in testing antigen specificity in established T-cell lines, in addition to analysis of PBMC. In summary, the applicabilities of miniaturization to save cells and reagents and of automation to save time and increase accuracy were demonstrated in this study using different methodological approaches valuable in the clinical immunology laboratory.
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Hong, Yulong, Brian L. Webb, Sadashiva Pai, Ann Ferrie, Jinlin Peng, Fang Lai, Joydeep Lahiri, et al. "G-Protein-Coupled Receptor Microarrays for Multiplexed Compound Screening." Journal of Biomolecular Screening 11, no. 4 (April 28, 2006): 435–38. http://dx.doi.org/10.1177/1087057106287139.
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Conventional assay methods for discovering and profiling drug-target interactions are typically developed on a target-by-target basis and hence can be cumbersome to enable and orchestrate. Herein the authors report a solid-state ligand-binding assay that operates in a multiplexed mode to report compound activity against a micorarray-configured panel of G-protein-coupled receptor (GPCR) targets. The pharmacological fidelity of the system is high, and its miniaturized “plug-and-play” format provides improved efficiency both in terms of execution time and reagent consumption. Taken together, these features make the system ideally suited to explore the structure-activity relationship of compounds across a broad region of target class space.
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Graham, Debbie L., Nicola Bevan, Peter N. Lowe, Michelle Palmer, and Stephen Rees. "Application of β-Galactosidase Enzyme Complementation Technology as a High Throughput Screening Format for Antagonists of the Epidermal Growth Factor Receptor." Journal of Biomolecular Screening 6, no. 6 (December 2001): 401–11. http://dx.doi.org/10.1177/108705710100600606.
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We have applied enzyme complementation technology to develop a screen for antagonists of the epidermal growth factor (EGF) receptor. Chimeric proteins containing two weakly complementing deletion mutants of Escherichia coli β-galactosidase (β-gal), each fused to the EGF receptor extracellular and transmembrane domains, have been stably expressed in C2C12 cells. In this cell line, formation of active β-gal is dependent on agonist-stimulated dimerization of the EGF receptor. We have developed a homogenous 384-well assay protocol and have applied this to characterize the pharmacology of the receptor and to develop a high throughput screen (HTS) for EGF receptor antagonists. The assay is tolerant to DMSO concentrations of up to 2% and, across 21 passages in culture, exhibits an EC50 for EGF of 5.4 ± 3.6 ng/ml (n = 11) and a Z' of 0.55 ± 0.13 (n = 11). A random set of 1,280 compounds was screened in duplicate at 11 μM to examine the robustness of enzyme complementation technology and to characterize the false-positive hit rate in the assay. Using a cutoff of 40% inhibition of EGF-promoted β-gal activity, the hit rate on day 1 was 2.5% and on day 2 was 1.9%. After retesting the active compounds, the hit rate was reduced to 0.4%, of which one of the compounds was identified as a β-gal inhibitor and the remainder appeared to be nonspecific inhibitors in the assay. This technology is amenable to automated screen workstations, there are highly sensitive chemiluminescent and fluorescent β-gal assay reagents amenable to detection in miniaturized plate formats, and the assay benefits from a low false-positive hit rate. Enzyme complementation technology may have wide application within the HTS environment for the detection of modulators of receptor activation or inhibitors of protein-protein interactions in mammalian cells.
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Grisoni, Francesca, Berend J. H. Huisman, Alexander L. Button, Michael Moret, Kenneth Atz, Daniel Merk, and Gisbert Schneider. "Combining generative artificial intelligence and on-chip synthesis for de novo drug design." Science Advances 7, no. 24 (June 2021): eabg3338. http://dx.doi.org/10.1126/sciadv.abg3338.
Full textAbstract:
Automating the molecular design-make-test-analyze cycle accelerates hit and lead finding for drug discovery. Using deep learning for molecular design and a microfluidics platform for on-chip chemical synthesis, liver X receptor (LXR) agonists were generated from scratch. The computational pipeline was tuned to explore the chemical space of known LXRα agonists and generate novel molecular candidates. To ensure compatibility with automated on-chip synthesis, the chemical space was confined to the virtual products obtainable from 17 one-step reactions. Twenty-five de novo designs were successfully synthesized in flow. In vitro screening of the crude reaction products revealed 17 (68%) hits, with up to 60-fold LXR activation. The batch resynthesis, purification, and retesting of 14 of these compounds confirmed that 12 of them were potent LXR agonists. These results support the suitability of the proposed design-make-test-analyze framework as a blueprint for automated drug design with artificial intelligence and miniaturized bench-top synthesis.
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Solemani Zadeh, Arghavan, Alissa Grässer, Heiko Dinter, Maximilian Hermes, and Katharina Schindowski. "Efficient Construction and Effective Screening of Synthetic Domain Antibody Libraries." Methods and Protocols 2, no. 1 (February 14, 2019): 17. http://dx.doi.org/10.3390/mps2010017.
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Phage display is a powerful technique for drug discovery in biomedical research in particular for antibody libraries. But, several technical challenges are associated with the selection process. For instance, during the panning step, the successful elution of the phages bound to the antigen is critical in order to avoid losing the most promising binders. Here, we present an efficient protocol to establish, screen and select synthetic libraries of domain antibodies using phage display. We do not only present suitable solutions to the above-mentioned challenges to improve elution by 50-fold, but we also present a step by step in-depth protocol with miniaturized volumes and optimized procedures to save material, costs and time for a successful phage display with domain antibodies. Hence, this protocol improves the selection process for an efficient handling process. The here presented library is based on the variable domain (vNAR) of the naturally occurring novel antibody receptor (IgNAR) from cartilage fishes. Diversity was introduced in the Complementarity-Determining Region 3 (CDR3) of the antigen-binding site with different composition and length.
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Maheshwari, Hiralal G., Suzan S. Pezzoli, Asad Rahim, Stephen M. Shalet, Michael O. Thorner, and Gerhard Baumann. "Pulsatile growth hormone secretion persists in genetic growth hormone-releasing hormone resistance." American Journal of Physiology-Endocrinology and Metabolism 282, no. 4 (April 1, 2002): E943—E951. http://dx.doi.org/10.1152/ajpendo.00537.2001.
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Growth hormone (GH) secretion is regulated by GH-releasing hormone (GHRH), somatostatin, and possibly ghrelin, but uncertainty remains about the relative contributions of these hypophysiotropic factors to GH pulsatility. Patients with genetic GHRH receptor (GHRH-R) deficiency present an opportunity to examine GH secretory dynamics in the selective absence of GHRH input. We studied circadian GH profiles in four young men homozygous for a null mutation in the GHRH-R gene by use of an ultrasensitive GH assay. Residual GH secretion was pulsatile, with normal pulse frequency, but severely reduced amplitude (<1% normal) and greater than normal process disorder (as assessed by approximate entropy). Nocturnal GH secretion, both basal and pulsatile, was enhanced compared with daytime. We conclude that rhythmic GH secretion persists in an amplitude-miniaturized version in the absence of a GHRH-R signal. The nocturnal enhancement of GH secretion is likely mediated by decreased somatostatin tone. Pulsatility of residual GH secretion may be caused by oscillations in somatostatin and/or ghrelin; it may also reflect intrinsic oscillations in somatotropes.
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Decker, Ann M., Kelly M. Mathews, Bruce E. Blough, and Brian P. Gilmour. "Validation of a High-Throughput Calcium Mobilization Assay for the Human Trace Amine-Associated Receptor 1." SLAS DISCOVERY: Advancing the Science of Drug Discovery 26, no. 1 (July 31, 2020): 140–50. http://dx.doi.org/10.1177/2472555220945279.
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The human trace amine-associated receptor 1 (hTAAR1) is a G protein-coupled receptor (GPCR) that is widely expressed in monoaminergic nuclei in the central nervous system and has therapeutic potential for multiple diseases, including drug addiction and schizophrenia. Thus, identification of novel hTAAR1 ligands is critical to advancing our knowledge of hTAAR1 function and to the development of therapeutics for a wide range of diseases. Herein we describe the development of a robust, 3-addition high-throughput screening (HTS) calcium mobilization assay using stable CHO-Gαq16-hTAAR1 cells, which functionally couple hTAAR1 to the promiscuous Gαq16 protein and thus allow signal transduction to occur through mobilization of internal calcium. Our previously established 96-well hTAAR1 assay was first miniaturized to the 384-well format and optimized to provide an assay with a Z′ factor of 0.84, which is indicative of a robust HTS assay. Using the 3-addition protocol, 22,000 compounds were screened and yielded a ~1% agonist hit rate and a ~0.2% antagonist hit rate. Of the antagonist hits, two confirmed hits are the most potent hTAAR1 antagonists identified to date (IC50 = 206 and 281 nM). While scientists have been studying hTAAR1 for years, the lack of suitable hTAAR1 antagonists has been a major roadblock for studying the basic pharmacology of hTAAR1. Thus, these new ligands will serve as valuable tools to study hTAAR1-mediated signaling mechanisms, therapeutic potential, and in vivo functions.
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Wang, Lili, Alex K. Shalek, Jellert Gaublomme, Michael Lawrence, Kristen E. Stevenson, Donna S. Neuberg, Nir Hacohen, et al. "Somatic Mutation As a Mechanism of Wnt/β-Catenin Pathway Activation in CLL." Blood 120, no. 21 (November 16, 2012): 559. http://dx.doi.org/10.1182/blood.v120.21.559.559.
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Abstract Abstract 559 One goal of cancer genome sequencing is to identify key genes and pathways that drive tumor pathogenesis. While many studies identify driver genes based on recurrence of mutations in individual genes, subsets of genes with non-recurrent mutations may also be defined as drivers if they affect components of a single biological pathway. By large-scale DNA sequencing, we recently identified the Wnt pathway as a significantly mutated pathway in chronic lymphocytic leukemia (CLL) (Wang et al NEJM 2011). This pathway is known to be critical for hematopoietic stem cell development and differentiation, and is highly dysregulated in CLL. However, the functional alterations generated by specific mutations in members of this pathway and their contributions to CLL pathogenesis have not been previously characterized. In our previously reported DNA sequencing of 91 CLL samples, we identified a broad spectrum of mutated Wnt pathway members. Fifteen non-synonymous coding mutations were identified in 13 CLL samples (1–2 mutations per sample) within core Wnt pathway members. These nonsilent coding mutations involved components of the entire Wnt pathway–ranging from extracellular factors (DKK2, WNT1, WNT10A, RSPO4, CSNK1E), to cell surface receptors (FZD5, RYK), cytoplasmic factors (CSNK1E, PRICKLE1) and nuclear transcriptional factors (CHD8, BRD7, CREBBP, BCL9). All of the identified Wnt pathway mutations localized to evolutionarily conserved sites, supporting a role for these mutations in perturbing Wnt pathway function. To assess the contribution of the Wnt pathway to CLL-B cell survival, we optimized a novel miniaturized transfection platform, silicon nanowires (SiNWs), to deliver siRNAs targeting core Wnt pathway members into normal and CLL-B cells. By exploring various nanowire sizes, lengths and densities, we achieved conditions to consistently deliver siRNAs into B cells (90%) without impacting cellular viability (>95%). Using SiNW-mediated delivery of gene-specific siRNAs, we established that silencing of the Wnt pathway members LEF1, CTTNB1, and DVL1 in CLL samples (n=3) resulted in greater loss of cell viability (by∼20–35%) compared to delivery of non-targeting controls (P=0.06, 0.01, and 0.005, respectively). These results demonstrate that perturbation of key nodes in the Wnt pathway reduce CLL-B cell survival, and support the importance of this pathway in CLL. We then explored whether mutated Wnt pathway members could functionally alter Wnt pathway signaling. We focused our studies on two mutated Wnt pathway members, DKK2-R197H and BCL9-G548S. We generated plasmids expressing either wildtype or mutant alleles and measured Wnt pathway activation in 293T cells using a TCF/LEF-dependent luciferase reporter system following coexpression of WNT1 with the wildtype or mutant gene or both. For both DKK2 and BCL9, mutated forms abolished their normal repressive effects on Wnt activation. Elimination of the repressive effects of the wildtype protein was also observed when the wildtype and mutant alleles were expressed together, suggesting a dominant activating effect of the mutated allele. We hypothesized that chronic pathway activation by these mutations could lead to dependence of patient CLL cells on Wnt signaling for their survival. We directly addressed this question by examining the survival of normal B cells (n=4) and patient CLL-B cells with or without (n=7) mutated DKK2 or BCL9 following SiNW-mediated delivery of gene-specific siRNAs. This revealed that a BCL9-mutated CLL sample was more dependent on BCL9 expression for survival than normal B cells (p=0.02) or CLL samples lacking Wnt pathway mutations (p=0.07). Similarly, silencing of DKK2 in a DKK2- mutated CLL sample led to greater cell death than gene silencing in normal B cells, or in CLL samples without Wnt pathway mutations (p=0.003). These results demonstrate that CLL samples harboring mutations in DKK2 or BCL9 exhibit greater dependency on Wnt pathway signaling than samples without Wnt pathway mutations, and support a driving role for these mutations in CLL. Taken together, our results demonstrate that somatic mutations in Wnt pathway components in CLL-B cells alter the activity of this pathway and increase its sensitivity to pathway inhibition. Our findings further support the notion that non-recurrent mutations at different nodes of the Wnt pathway contribute to CLL pathogenesis. Disclosures: Brown: Calistoga, Celgene, Genentech, Pharmacyclics, Novartis, Avila: Consultancy; Genzyme, Celgene: Research Funding.
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Maggi, Maristella, Greta Pessino, Isabella Guardamagna, Leonardo Lonati, Cristina Pulimeno, and Claudia Scotti. "A Targeted Catalytic Nanobody (T-CAN) with Asparaginolytic Activity." Cancers 13, no. 22 (November 11, 2021): 5637. http://dx.doi.org/10.3390/cancers13225637.
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E. coli L-asparaginase is an amidohydrolase (EC 3.5.1.1) which has been successfully used for the treatment of Acute Lymphoblastic Leukemia for over 50 years. Despite its efficacy, its side effects, and especially its intrinsic immunogenicity, hamper its usage in a significant subset of cases, thus limiting therapeutic options. Innovative solutions to improve on these drawbacks have been attempted, but none of them have been truly successful so far. In this work, we fully replaced the enzyme scaffold, generating an active, miniaturized form of L-asparaginase by protein engineering of a camel single domain antibody, a class of antibodies known to have a limited immunogenicity in humans. We then targeted it onto tumor cells by an antibody scFv fragment directed onto the CD19 B-cell surface receptor expressed on ALL cells. We named this new type of nanobody-based antibody-drug conjugate “Targeted Catalytic Nanobody” (T-CAN). The new molecule retains the catalytic activity and the binding capability of the original modules and successfully targets CD19 expressing cells in vitro. Thanks to its theoretically reduced immunogenic potential compared to the original molecule, the T-CAN can represent a novel approach to tackle current limitations in L-asparaginase usage.
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Hamman, Brian D., Brian A. Pollok, Todd Bennett, Janet Allen, and Roger Heim. "Binding of a Pleckstrin Homology Domain Protein to Phosphoinositide in Membranes: A Miniaturized FRET-Based Assay for Drug Screening." Journal of Biomolecular Screening 7, no. 1 (February 2002): 45–55. http://dx.doi.org/10.1177/108705710200700107.
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Pleckstrin homology (PH) domains are present in key proteins involved in many vital cell processes. For example, the PH domain of Bruton’s tyrosine kinase (Btk) binds to phosphatidylinositol triphosphate (PIP3) in the plasma membrane after stimulation of the B-cell receptor in B cells. Mutations in the Btk PH domain result in changes in its affinity for PIP3, with higher binding leading to cell transformation in vitro and lower binding leading to antibody deficiencies in both humans and mice. We describe here a fluorescence resonance energy transfer (FRET)-based biochemical assay that directly monitors the interaction of a PH domain with PIP3 at a membrane surface. We overexpressed a fusion protein consisting of an enhanced green fluorescent protein (GFP) and the N-terminal 170 amino acids of a Tec family kinase that contains its PH domain (PH170). Homogeneous unilamellar vesicles were made that contained PIP3 and octadecylrhodamine (OR), a lipophilic FRET acceptor for GFP. After optimization of both protein and vesicle components, we found that binding of the GFP-PH170 protein to PIP3 in vesicles that contain OR results in about a 90% reduction of GFP fluorescence. Using this assay to screen 1440 compounds, we identified three that efficiently inhibited binding of GFP-PH170 to PIP3 in vesicles. This biochemical assay readily miniaturized to 1.8-μl reaction volumes and was validated in a 3456-well screening format.