scholarly journals Dose-Response Modeling of High-Throughput Screening Data

2009 ◽  
Vol 14 (10) ◽  
pp. 1216-1227 ◽  
Author(s):  
Fred Parham ◽  
Chris Austin ◽  
Noel Southall ◽  
Ruili Huang ◽  
Raymond Tice ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Statistical Approach ◽  
Statistical Tests ◽  
Cell Types ◽  
Large Numbers ◽  
Rodent Cell ◽  
Response Modeling ◽  
Significant Concentration

The National Toxicology Program is developing a high-throughput screening (HTS) program to set testing priorities for compounds of interest, to identify mechanisms of action, and potentially to develop predictive models for human toxicity. This program will generate extensive data on the activity of large numbers of chemicals in a wide variety of biochemical- and cell-based assays. The first step in relating patterns of response among batteries of HTS assays to in vivo toxicity is to distinguish between positive and negative compounds in individual assays. Here, the authors report on a statistical approach developed to identify compounds positive or negative in an HTS cytotoxicity assay based on data collected from screening 1353 compounds for concentration-response effects in 9 human and 4 rodent cell types. In this approach, the authors develop methods to normalize the data (removing bias due to the location of the compound on the 1536-well plates used in the assay) and to analyze for concentration-response relationships. Various statistical tests for identifying significant concentration-response relationships and for addressing reproducibility are developed and presented.

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.

scholarly journals A Microfluidic Spheroid Culture Device with a Concentration Gradient Generator for High-Throughput Screening of Drug Efficacy

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3355 ◽  
Author(s):  
Wanyoung Lim ◽  
Sungsu Park
Keyword(s):  
High Throughput ◽  
Concentration Gradient ◽  
High Throughput Screening ◽  
3D Cell Culture ◽  
Drug Efficacy ◽  
Cancer Drug ◽  
Spheroid Culture ◽  
Gradient Generator ◽  
Micro Channels

Three-dimensional (3D) cell culture is considered more clinically relevant in mimicking the structural and physiological conditions of tumors in vivo compared to two-dimensional cell cultures. In recent years, high-throughput screening (HTS) in 3D cell arrays has been extensively used for drug discovery because of its usability and applicability. Herein, we developed a microfluidic spheroid culture device (μFSCD) with a concentration gradient generator (CGG) that enabled cells to form spheroids and grow in the presence of cancer drug gradients. The device is composed of concave microwells with several serpentine micro-channels which generate a concentration gradient. Once the colon cancer cells (HCT116) formed a single spheroid (approximately 120 μm in diameter) in each microwell, spheroids were perfused in the presence of the cancer drug gradient irinotecan for three days. The number of spheroids, roundness, and cell viability, were inversely proportional to the drug concentration. These results suggest that the μFSCD with a CGG has the potential to become an HTS platform for screening the efficacy of cancer drugs.

scholarly journals Systematic comparison of high-throughput single-cell RNA-seq methods for immune cell profiling

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Tracy M. Yamawaki ◽  
Daniel R. Lu ◽  
Daniel C. Ellwanger ◽  
Dev Bhatt ◽  
Paolo Manzanillo ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Immune Cell ◽  
Cell Types ◽  
Data Interpretation ◽  
Detection Sensitivity ◽  
Rna Seq ◽  
Cell Recovery

Abstract Background Elucidation of immune populations with single-cell RNA-seq has greatly benefited the field of immunology by deepening the characterization of immune heterogeneity and leading to the discovery of new subtypes. However, single-cell methods inherently suffer from limitations in the recovery of complete transcriptomes due to the prevalence of cellular and transcriptional dropout events. This issue is often compounded by limited sample availability and limited prior knowledge of heterogeneity, which can confound data interpretation. Results Here, we systematically benchmarked seven high-throughput single-cell RNA-seq methods. We prepared 21 libraries under identical conditions of a defined mixture of two human and two murine lymphocyte cell lines, simulating heterogeneity across immune-cell types and cell sizes. We evaluated methods by their cell recovery rate, library efficiency, sensitivity, and ability to recover expression signatures for each cell type. We observed higher mRNA detection sensitivity with the 10x Genomics 5′ v1 and 3′ v3 methods. We demonstrate that these methods have fewer dropout events, which facilitates the identification of differentially-expressed genes and improves the concordance of single-cell profiles to immune bulk RNA-seq signatures. Conclusion Overall, our characterization of immune cell mixtures provides useful metrics, which can guide selection of a high-throughput single-cell RNA-seq method for profiling more complex immune-cell heterogeneity usually found in vivo.

scholarly journals Complex Tumor Spheroids, a Tissue-Mimicking Tumor Model, for Drug Discovery and Precision Medicine

2021 ◽  
pp. 247255522110383
Author(s):  
Gurmeet Kaur ◽  
David M. Evans ◽  
Beverly A. Teicher ◽  
Nathan P. Coussens
Keyword(s):  
Drug Discovery ◽  
Precision Medicine ◽  
Cell Lines ◽  
High Throughput ◽  
High Throughput Screening ◽  
Cell Types ◽  
Response To Therapy ◽  
Cancer Drug ◽  
Malignant Cells ◽  
Pancreatic Cell

Malignant tumors are complex tissues composed of malignant cells, vascular cells, structural mesenchymal cells including pericytes and carcinoma-associated fibroblasts, infiltrating immune cells, and others, collectively called the tumor stroma. The number of stromal cells in a tumor is often much greater than the number of malignant cells. The physical associations among all these cell types are critical to tumor growth, survival, and response to therapy. Most cell-based screens for cancer drug discovery and precision medicine validation use malignant cells in isolation as monolayers, embedded in a matrix, or as spheroids in suspension. Medium- and high-throughput screening with multiple cell lines requires a scalable, reproducible, robust cell-based assay. Complex spheroids include malignant cells and two normal cell types, human umbilical vein endothelial cells and highly plastic mesenchymal stem cells, which rapidly adapt to the malignant cell microenvironment. The patient-derived pancreatic adenocarcinoma cell line, K24384-001-R, was used to explore complex spheroid structure and response to anticancer agents in a 96-well format. We describe the development of the complex spheroid assay as well as the growth and structure of complex spheroids over time. Subsequently, we demonstrate successful assay miniaturization to a 384-well format and robust performance in a high-throughput screen. Implementation of the complex spheroid assay was further demonstrated with 10 well-established pancreatic cell lines. By incorporating both human stromal and tumor components, complex spheroids might provide an improved model for tumor response in vivo.

scholarly journals A High-Throughput Cellular Screening Assay for Small-Molecule Inhibitors and Activators of Cytoplasmic Dynein-1-Based Cargo Transport

2020 ◽  
Vol 25 (9) ◽  
pp. 985-999
Author(s):  
John Vincent ◽  
Marian Preston ◽  
Elizabeth Mouchet ◽  
Nicolas Laugier ◽  
Adam Corrigan ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Cytoplasmic Dynein ◽  
Lead Discovery ◽  
Screening Assay ◽  
Age Related ◽  
The Uk

Cytoplasmic dynein-1 (hereafter dynein) is a six-subunit motor complex that transports a variety of cellular components and pathogens along microtubules. Dynein’s cellular functions are only partially understood, and potent and specific small-molecule inhibitors and activators of this motor would be valuable for addressing this issue. It has also been hypothesized that an inhibitor of dynein-based transport could be used in antiviral or antimitotic therapy, whereas an activator could alleviate age-related neurodegenerative diseases by enhancing microtubule-based transport in axons. Here, we present the first high-throughput screening (HTS) assay capable of identifying both activators and inhibitors of dynein-based transport. This project is also the first collaborative screening report from the Medical Research Council and AstraZeneca agreement to form the UK Centre for Lead Discovery. A cellular imaging assay was used, involving chemically controlled recruitment of activated dynein complexes to peroxisomes. Such a system has the potential to identify molecules that affect multiple aspects of dynein biology in vivo. Following optimization of key parameters, the assay was developed in a 384-well format with semiautomated liquid handling and image acquisition. Testing of more than 500,000 compounds identified both inhibitors and activators of dynein-based transport in multiple chemical series. Additional analysis indicated that many of the identified compounds do not affect the integrity of the microtubule cytoskeleton and are therefore candidates to directly target the transport machinery.

Patterned superhydrophobic surfaces to process and characterize biomaterials and 3D cell culture

2018 ◽  
Vol 5 (3) ◽  
pp. 379-393 ◽  
Author(s):  
A. I. Neto ◽  
P. A. Levkin ◽  
J. F. Mano
Keyword(s):  
Cell Culture ◽  
High Throughput ◽  
High Throughput Screening ◽  
3D Cell Culture ◽  
Superhydrophobic Surfaces ◽  
Technological Breakthrough ◽  
Large Numbers

Microarrays are a technological breakthrough for high-throughput screening of large numbers of assays.

scholarly journals In Vivo, High-Throughput Selection of Thermostable Cyclohexanone Monooxygenase (CHMO)

Catalysts ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 935
Author(s):  
Sarah Maxel ◽  
Linyue Zhang ◽  
Edward King ◽  
Ana Paula Acosta ◽  
Ray Luo ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Rational Design ◽  
Random Mutagenesis ◽  
Residual Activity ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Coli Strain ◽  
Screening Tools ◽  
Cyclohexanone Monooxygenase

Cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871 is characterized as having wide substrate versatility for the biooxidation of (cyclic) ketones into esters and lactones with high stereospecificity. Despite industrial potential, CHMO usage is restricted by poor thermostability. Limited high-throughput screening tools and challenges in rationally engineering thermostability have impeded CHMO engineering efforts. We demonstrate the application of an aerobic, high-throughput growth selection platform in Escherichia coli (strain MX203) for the discovery of thermostability enhancing mutations for CHMO. The selection employs growth for the easy readout of CHMO activity in vivo, by requiring nicotinamide adenine dinucleotide phosphate (NADPH)-consuming enzymes to restore cellular redox balance. In the presence of the native substrate cyclohexanone, variant CHMO GV (A245G-A288V) was discovered from a random mutagenesis library screened at 42 °C. This variant retained native activity, exhibited ~4.4-fold improvement in residual activity after 30 °C incubation, and demonstrated ~5-fold higher cyclohexanone conversion at 37 °C compared to the wild type. Molecular modeling indicates that CHMO GV experiences more favorable residue packing and supports additional backbone hydrogen bonding. Further rational design resulted in CHMO A245G-A288V-T415C with improved thermostability at 45 °C. Our platform for oxygenase evolution enabled the rapid engineering of protein stability critical for industrial scalability.

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