scholarly journals Population Patch-Clamp Electrophysiology Analysis of Recombinant GABAA α1β3γ2 Channels Expressed in HEK-293 Cells

2009 ◽  
Vol 14 (7) ◽  
pp. 769-780 ◽  
Author(s):  
Emma C. Hollands ◽  
Tim J. Dale ◽  
Andrew W. Baxter ◽  
Helen J. Meadows ◽  
Andrew J. Powell ◽  
...  
Keyword(s):  
Patch Clamp ◽  
High Throughput Screening ◽  
Rational Design ◽  
Rank Order ◽  
Hek293 Cells ◽  
Small Scale ◽  
Single Shot ◽  
Hek 293 ◽  
Planar Array ◽  
Receptor Pharmacology

γ-Amino butyric acid (GABA)—activated Cl— channels are critical mediators of inhibitory postsynaptic potentials in the CNS. To date, rational design efforts to identify potent and selective GABAA subtype ligands have been hampered by the absence of suitable high-throughput screening approaches. The authors describe 384-well population patch-clamp (PPC) planar array electrophysiology methods for the study of GABAA receptor pharmacology. In HEK293 cells stably expressing human α1β3γ2 GABAA channels, GABA evoked outward currents at 0 mV of 1.05 ± 0.08 nA, measured 8 s post GABA addition. The IGABA was linear and reversed close to the theoretical ECl (—56 mV). Concentration-response curve analysis yielded a mean pEC50 value of 5.4 and Hill slope of 1.5, and for a series of agonists, the rank order of potency was muscimol > GABA > isoguvacine. A range of known positive modulators, including diazepam and pentobarbital, produced concentration-dependent augmentation of the GABA EC 20 response (1 µM). The competitive antagonists bicuculline and gabazine produced concentration-dependent, parallel, rightward displacement of GABA curves with pA2 and slope values of 5.7 and 1.0 and 6.7 and 1.0, respectively. In contrast, picrotoxin (0.2-150 µM) depressed the maximal GABA response, implying a non-competitive antagonism. Overall, the pharmacology of human α1β3γ2 GABAA determined by PPC was highly similar to that obtained by conventional patch-clamp methods. In small-scale single-shot screens, Z′ values of >0.5 were obtained in agonist, modulator, and antagonist formats with hit rates of 0% to 3%. The authors conclude that despite the inability of the method to resolve the peak agonist responses, PPC can rapidly and usefully quantify pharmacology for the α1β3γ2 GABAA isoform. These data suggest that PPC may be a valuable approach for a focused set and secondary screening of GABAA receptors and other slow ligand-gated ion channels. ( Journal of Biomolecular Screening 2009:769-780)

INCUBATION TYPE PLANAR PATCH CLAMP AS A NEW POTENTIAL TECHNOLOGY FOR DEVELOPING NEURONAL NETWORK HIGH THROUGHPUT SCREENING DEVICES

2016 ◽  
Vol 28 (03) ◽  
pp. 1630002
Author(s):  
Tsuneo Urisu
Keyword(s):  
Patch Clamp ◽  
Ion Channel ◽  
High Throughput ◽  
Neuronal Network ◽  
High Throughput Screening ◽  
Hek293 Cells ◽  
Base Line ◽  
Channel Current ◽  
Trpv1 Channels ◽  
Probability Number

Ion-channel current recordings based on an incubation type planar patch clamp were first reported in 2008, using HEK293 cells expressed with TRPV1 channels and capsaicin as ligand molecules. At first the success probability (number of devices which normally worked/total number of devices fabricated) was extremely low (several %). Several years later, we have succeeded in significantly decreasing the base line noise by using a salt-bridge-type Ag/AgCl electrode and successfully demonstrated the application of an incubation type planar patch clamp to ligand gated ion-channel biosensors and light gated ion-channel biosensors using HEK293 cells expressed with ChRWR. Furthermore, a spontaneous ion-channel current from a neuronal network was successfully observed by using a planar patch clamp chip, on which the neuronal network was occasionally formed with a soma of a neuron on a micro through-hole. Although the neuronal network was not controlled, this success shows the high potential of realizing a high throughput screening device on the basis of channel current measurements, which contain the most important information on network conditions.

scholarly journals Novel 384-Well Population Patch Clamp Electrophysiology Assays for Ca2+-Activated K+ Channels

2006 ◽  
Vol 12 (1) ◽  
pp. 50-60 ◽  
Author(s):  
Victoria H. John ◽  
Tim J. Dale ◽  
Emma C. Hollands ◽  
Mao Xiang Chen ◽  
Leanne Partington ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Rank Order ◽  
Large Cell ◽  
Activator Concentration ◽  
Response Curves ◽  
Channel Expression ◽  
Planar Array ◽  
Patch Clamp Electrophysiology ◽  
Assay Precision ◽  
Voltage Gated Ion Channels

Planar array electrophysiology techniques were applied to assays for modulators of recombinant hIK and hSK3 Ca2+-activated K+ channels. In CHO-hIK—expressing cells, under asymmetric K+ gradients, small-molecule channel activators evoked time- and voltage-independent currents characteristic of those previously described by classical patch clamp electrophysiology methods. In single-hole (cell) experiments, the large cell-to-cell heterogeneity in channel expression rendered it difficult to generate activator concentration-response curves. However, in population patch clamp mode, in which signals are averaged from up to 64 cells, well-to-well variation was substantially reduced such that concentration-response curves could be easily constructed. The absolute EC50 values and rank order of potency for a range of activators, including 1-EBIO and DC-EBIO, corresponded well with conventional patch clamp data. Activator responses of hIK and hSK3 channels could be fully and specifically blocked by the selective inhibitors TRAM-34 and apamin, with IC50 values of 0.31 μM and 3 nM, respectively. To demonstrate assay precision and robustness, a test set of 704 compounds was screened in a 384-well format of the hIK assay. All plates had Z′ values greater than 0.6, and the statistical cutoff for activity was 8%. Eleven hits (1.6%) were identified from this set, in addition to the randomly spiked wells with known activators. Overall, our findings demonstrate that population patch clamp is a powerful and enabling method for screening Ca2+-activated K+ channels and provides significant advantages over single-cell electrophysiology (IonWorksHT) and other previously published approaches. Moreover, this work demonstrates for the 1st time the utility of population patch clamp for ion channel activator assays and for non—voltage-gated ion channels.

Novel 1,2,3-Triazole-functionalized pyrido[3',2':4,5]furo[3,2-d]pyrimidin- 4(3H)-one Derivatives: Synthesis, Anticancer Activity, CoMFA and CoMSIA Studies

2020 ◽  
Vol 17 (12) ◽  
pp. 969-978
Author(s):  
Balakishan Vadla ◽  
Sailu Betala
Keyword(s):  
Anticancer Activity ◽  
Cell Line ◽  
Normal Cell ◽  
Rational Design ◽  
Human Cancer ◽  
Strong Basis ◽  
Hek 293 ◽  
Normal Cell Line ◽  
Human Cancer Cell Lines ◽  
Mcf 7

A series of novel triazole functionalized pyrido [3',2':4,5] furo[3,2-d] pyrimidin-4 (3H)-one derivatives 7a-p were prepared from ethyl furo[2,3-b]pyridine-2-carboxylate 3 on reaction with ammonia to afford furo[2,3-b]pyridine-2-carboxamide 4. This compound, on reaction with triethyl orthoformate TEOF, gave compound 5. Compound 5 on propargylation, followed by a reaction with substituted aryl azides under Sharpless reaction conditions, furnished triazole tagged pyrido [3',2':4,5]furo[3,2-d] pyrimidin-4(3H)-one derivatives. All the products 7a-p were screened against four human cancer cell lines, such as HeLa - Cervical cancer (CCL-2), COLO 205- Colon cancer (CCL-222), HepG2- Liver cancer (HB-8065), and MCF7 - Breast cancer (HTB-22) and one normal cell line (HEK 293). Compounds 7b, 7n, 7o and 7p, which showed promising anticancer activity, were identified and found to be non-toxic to normal cell line. Studies for HeLa, COLO205, HepG2, and MCF-7 using CoMFA and CoMSIA were carried out . Models from 3D-QSAR provided a strong basis for future rational design of more active and selective HeLa, COLO205, HepG2, and MCF-7 cell line inhibitors.

scholarly journals High-throughput screening and rational design of biofunctionalized surfaces with optimized biocompatibility and antimicrobial activity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhou Fang ◽  
Junjian Chen ◽  
Ye Zhu ◽  
Guansong Hu ◽  
Haoqian Xin ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Rational Design ◽  
Click Reaction ◽  
Reaction Times ◽  
Great Promise ◽  
Biomaterial Surfaces

AbstractPeptides are widely used for surface modification to develop improved implants, such as cell adhesion RGD peptide and antimicrobial peptide (AMP). However, it is a daunting challenge to identify an optimized condition with the two peptides showing their intended activities and the parameters for reaching such a condition. Herein, we develop a high-throughput strategy, preparing titanium (Ti) surfaces with a gradient in peptide density by click reaction as a platform, to screen the positions with desired functions. Such positions are corresponding to optimized molecular parameters (peptide densities/ratios) and associated preparation parameters (reaction times/reactant concentrations). These parameters are then extracted to prepare nongradient mono- and dual-peptide functionalized Ti surfaces with desired biocompatibility or/and antimicrobial activity in vitro and in vivo. We also demonstrate this strategy could be extended to other materials. Here, we show that the high-throughput versatile strategy holds great promise for rational design and preparation of functional biomaterial surfaces.

Improved SSD-assisted algorithm for surface defect detection of electromagnetic luminescence

2021 ◽  
pp. 1748006X2199538
Author(s):  
Zhenying Xu ◽  
Ziqian Wu ◽  
Wei Fan
Keyword(s):  
Defect Detection ◽  
Feature Fusion ◽  
Recognition Rate ◽  
Detection Methods ◽  
Small Scale ◽  
Detection Accuracy ◽  
Single Shot ◽  
Surface Defect Detection ◽  
Feature Pyramid ◽  
Small Feature

Defect detection of electromagnetic luminescence (EL) cells is the core step in the production and preparation of solar cell modules to ensure conversion efficiency and long service life of batteries. However, due to the lack of feature extraction capability for small feature defects, the traditional single shot multibox detector (SSD) algorithm performs not well in EL defect detection with high accuracy. Consequently, an improved SSD algorithm with modification in feature fusion in the framework of deep learning is proposed to improve the recognition rate of EL multi-class defects. A dataset containing images with four different types of defects through rotation, denoising, and binarization is established for the EL. The proposed algorithm can greatly improve the detection accuracy of the small-scale defect with the idea of feature pyramid networks. An experimental study on the detection of the EL defects shows the effectiveness of the proposed algorithm. Moreover, a comparison study shows the proposed method outperforms other traditional detection methods, such as the SIFT, Faster R-CNN, and YOLOv3, in detecting the EL defect.

scholarly journals Cytotoxic Profiling of Annotated and Diverse Chemical Libraries Using Quantitative High-Throughput Screening

2019 ◽  
Vol 25 (1) ◽  
pp. 9-20 ◽  
Author(s):  
Olivia W. Lee ◽  
Shelley Austin ◽  
Madison Gamma ◽  
Dorian M. Cheff ◽  
Tobie D. Lee ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
High Throughput ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Large Scale ◽  
Early Stage ◽  
Cancer Cell Line ◽  
Hek 293 ◽  
Cytotoxic Compounds

Cell-based phenotypic screening is a commonly used approach to discover biological pathways, novel drug targets, chemical probes, and high-quality hit-to-lead molecules. Many hits identified from high-throughput screening campaigns are ruled out through a series of follow-up potency, selectivity/specificity, and cytotoxicity assays. Prioritization of molecules with little or no cytotoxicity for downstream evaluation can influence the future direction of projects, so cytotoxicity profiling of screening libraries at an early stage is essential for increasing the likelihood of candidate success. In this study, we assessed the cell-based cytotoxicity of nearly 10,000 compounds in the National Institutes of Health, National Center for Advancing Translational Sciences annotated libraries and more than 100,000 compounds in a diversity library against four normal cell lines (HEK 293, NIH 3T3, CRL-7250, and HaCat) and one cancer cell line (KB 3-1, a HeLa subline). This large-scale library profiling was analyzed for overall screening outcomes, hit rates, pan-activity, and selectivity. For the annotated library, we also examined the primary targets and mechanistic pathways regularly associated with cell death. To our knowledge, this is the first study to use high-throughput screening to profile a large screening collection (>100,000 compounds) for cytotoxicity in both normal and cancer cell lines. The results generated here constitute a valuable resource for the scientific community and provide insight into the extent of cytotoxic compounds in screening libraries, allowing for the identification and avoidance of compounds with cytotoxicity during high-throughput screening campaigns.

scholarly journals Evaluation of Division-Arrested Cells for Cell-Based High-Throughput Screening and Profiling

2005 ◽  
Vol 10 (6) ◽  
pp. 615-623 ◽  
Author(s):  
Mary Ellen Digan ◽  
Chantevy Pou ◽  
Honglin Niu ◽  
Ji-Hu Zhang
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Rank Order ◽  
Kinase Assay ◽  
Just In Time ◽  
Reporter Assay ◽  
A431 Cells ◽  
Dividing Cells ◽  
Epidermal Growth ◽  
Assay Conditions

Just-in-time cell supply for cell-based high-throughput screening (HTS) is frequently problematic. In addition to scheduling and logistical issues, quality issues and variability due to passage effect, cell cycle, or confluency contribute to day-to-day signal variability in the course of cell-based HTS campaigns. Cell division-arrest and cryopreservation technologies permit the use of cells as assay-ready reagents for HTS and other cell-based profiling and structure-activity studies. In this report, the authors compare division-arrested and dividing cells in 2 assay types that are dependent on movement of proteins within or through cell membranes: a receptor tyrosine kinase assay involving A431 cells responsive to epidermal growth factor, and a secretion reporter assay, which measures secretion of a reporter gene, secreted alkaline phosphatase. In both assays, dividing and division-arrested cells yielded similar basal and maximal signals at a given cell density. Similar IC50s were obtained for reference inhibitors in each assay, type in both dividing and division-arrested cells. In addition, for the secretion reporter assay, when comparing IC50s obtained from 44 compounds randomly chosen from a primary screening hit list, the rank order of potency obtained from dividing cells and division-arrested cells was essentially identical. Furthermore, the results show that, under certain assay conditions, data generated using division-arrested cells are less variable than those generated using dividing cells. In summary, the results suggest that, in many cases, division-arrested cells can substitute for dividing cells and offer certain advantages for cell-based assays.

scholarly journals Research fronts of Chemical Biology

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Shanshan Lv
Keyword(s):  
High Throughput Screening ◽  
Drug Targets ◽  
Chemical Biology ◽  
Rational Design ◽  
Future Research ◽  
Design Strategies ◽  
Binding Assays ◽  
Cellular Mechanisms ◽  
Diagnostic Technologies

Abstract Over the past decades, researchers have witnessed substantially increasing and ever-growing interests and efforts in Chemical Biology studies, thanks to the development of genome and epi-genome sequencing (revealing potential drug targets), synthetic chemistry (producing new medicines), bioorthogonal chemistry (chemistry in living systems) and high-throughput screening technologies (in vitro cell systems, protein binding assays and phenotypic assays). This report presents literature search results for current research in Chemical Biology, to explore basic principles, summarize recent advances, identify key challenges, and provide suggestions for future research (with a focus on Chemical Biology in the context of human health and diseases). Chemical Biology research can positively contribute to delivering a better understanding of the molecular and cellular mechanisms that accompany pathology underlying diseases, as well as developing improved methods for diagnosis, drug discovery, and therapeutic delivery. While much progress has been made, as shown in this report, there are still further needs and opportunities. For instance, pressing challenges still exist in selecting appropriate targets in biological systems and adopting more rational design strategies for the development of innovative and sustainable diagnostic technologies and medical treatments. Therefore, more than ever, researchers from different disciplines need to collaborate to address the challenges in Chemical Biology.

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