High-Throughput Method for Automated Colony and Cell Counting by Digital Image Analysis Based on Edge Detection

PubMed Central

Choudhry, Priya

2016-01-01

Counting cells and colonies is an integral part of high-throughput screens and quantitative cellular assays. Due to its subjective and time-intensive nature, manual counting has hindered the adoption of cellular assays such as tumor spheroid formation in high-throughput screens. The objective of this study was to develop an automated method for quick and reliable counting of cells and colonies from digital images. For this purpose, I developed an ImageJ macro Cell Colony Edge and a CellProfiler Pipeline Cell Colony Counting, and compared them to other open-source digital methods and manual counts. The ImageJ macro Cell Colony Edge is valuable in counting cells and colonies, and measuring their area, volume, morphology, and intensity. In this study, I demonstrate that Cell Colony Edge is superior to other open-source methods, in speed, accuracy and applicability to diverse cellular assays. It can fulfill the need to automate colony/cell counting in high-throughput screens, colony forming assays, and cellular assays. PMID:26848849

Sense and sensibility: the use of cell death biomarker assays in high-throughput anticancer drug screening and monitoring treatment responses.

PubMed

Shoshan, Maria C; Havelka, Associate Professor Principal Investigator Aleksandra Mandic; Neumann, Frank; Linder, Stig

2006-11-01

Cell-based screening allows identification of biologically active compounds, for example, potential anticancer drugs. In this review, various screening assays are discussed in terms of what they measure and how this affects interpretation and relevance. High-throughput (HT) assays of viability based on the reduction of exogenous substrates do not always reflect viability or cell number levels. Membrane integrity assays can be used for HT quantification of cell death, but are non-specific as to the death mode. Several HT assays monitor end point apoptosis. Screening libraries at a single concentration (micromolar) can prevent detection of potent apoptosis inducers, as high concentrations may induce mainly necrosis. Using monolayer cultures limits the significance of cell-based screening as the properties of monolayer cells differ from tumours in vivo. Spheroid cultures are more physiological, but are impractical for screening by conventional methods. The authors have developed an assay quantifying accumulation of a caspase-cleaved protein specific for epithelial cells. It provides an integrated measure of apoptosis in two- and three-dimensional cultures and can be used as a blood biomarker assay for tumour apoptosis in vivo.

Constraint factor graph cut-based active contour method for automated cellular image segmentation in RNAi screening.

PubMed

Chen, C; Li, H; Zhou, X; Wong, S T C

2008-05-01

Image-based, high throughput genome-wide RNA interference (RNAi) experiments are increasingly carried out to facilitate the understanding of gene functions in intricate biological processes. Automated screening of such experiments generates a large number of images with great variations in image quality, which makes manual analysis unreasonably time-consuming. Therefore, effective techniques for automatic image analysis are urgently needed, in which segmentation is one of the most important steps. This paper proposes a fully automatic method for cells segmentation in genome-wide RNAi screening images. The method consists of two steps: nuclei and cytoplasm segmentation. Nuclei are extracted and labelled to initialize cytoplasm segmentation. Since the quality of RNAi image is rather poor, a novel scale-adaptive steerable filter is designed to enhance the image in order to extract long and thin protrusions on the spiky cells. Then, constraint factor GCBAC method and morphological algorithms are combined to be an integrated method to segment tight clustered cells. Compared with the results obtained by using seeded watershed and the ground truth, that is, manual labelling results by experts in RNAi screening data, our method achieves higher accuracy. Compared with active contour methods, our method consumes much less time. The positive results indicate that the proposed method can be applied in automatic image analysis of multi-channel image screening data.

Combining structure-based pharmacophore modeling, virtual screening, and in silico ADMET analysis to discover novel tetrahydro-quinoline based pyruvate kinase isozyme M2 activators with antitumor activity

PubMed Central

Chen, Can; Wang, Ting; Wu, Fengbo; Huang, Wei; He, Gu; Ouyang, Liang; Xiang, Mingli; Peng, Cheng; Jiang, Qinglin

2014-01-01

Compared with normal differentiated cells, cancer cells upregulate the expression of pyruvate kinase isozyme M2 (PKM2) to support glycolytic intermediates for anabolic processes, including the synthesis of nucleic acids, amino acids, and lipids. In this study, a combination of the structure-based pharmacophore modeling and a hybrid protocol of virtual screening methods comprised of pharmacophore model-based virtual screening, docking-based virtual screening, and in silico ADMET (absorption, distribution, metabolism, excretion and toxicity) analysis were used to retrieve novel PKM2 activators from commercially available chemical databases. Tetrahydroquinoline derivatives were identified as potential scaffolds of PKM2 activators. Thus, the hybrid virtual screening approach was applied to screen the focused tetrahydroquinoline derivatives embedded in the ZINC database. Six hit compounds were selected from the final hits and experimental studies were then performed. Compound 8 displayed a potent inhibitory effect on human lung cancer cells. Following treatment with Compound 8, cell viability, apoptosis, and reactive oxygen species (ROS) production were examined in A549 cells. Finally, we evaluated the effects of Compound 8 on mice xenograft tumor models in vivo. These results may provide important information for further research on novel PKM2 activators as antitumor agents. PMID:25214764

Clinical perspective of cell-free DNA testing for fetal aneuploidies.

PubMed

Gratacós, Eduard; Nicolaides, Kypros

2014-01-01

Cell-free DNA testing in maternal blood provides the most effective method of screening for trisomy 21, with a reported detection rate of 99% and a false positive rate of less than 0.1%. After many years of research, this method is now commercially available and is carried out in an increasing number of patients, and there is an expanding number of conditions that can be screened for. However, the application of these methods in clinical practice requires a careful analysis. Current first-trimester screening strategies are based on a complex combination of tests, aiming at detecting fetal defects and predicting the risk of main pregnancy complications. It is therefore necessary to define the optimal way of combining cell-free DNA testing with current first-trimester screening methods. In this concise review we describe the basis of cell-free DNA testing and discuss the potential approaches for its implementation in combination with current tests in the first trimester. © 2014 S. Karger AG, Basel.

An Integrated In Silico Method to Discover Novel Rock1 Inhibitors: Multi- Complex-Based Pharmacophore, Molecular Dynamics Simulation and Hybrid Protocol Virtual Screening.

PubMed

Chen, Haining; Li, Sijia; Hu, Yajiao; Chen, Guo; Jiang, Qinglin; Tong, Rongsheng; Zang, Zhihe; Cai, Lulu

2016-01-01

Rho-associated, coiled-coil containing protein kinase 1 (ROCK1) is an important regulator of focal adhesion, actomyosin contraction and cell motility. In this manuscript, a combination of the multi-complex-based pharmacophore (MCBP), molecular dynamics simulation and a hybrid protocol of a virtual screening method, comprised of multipharmacophore- based virtual screening (PBVS) and ensemble docking-based virtual screening (DBVS) methods were used for retrieving novel ROCK1 inhibitors from the natural products database embedded in the ZINC database. Ten hit compounds were selected from the hit compounds, and five compounds were tested experimentally. Thus, these results may provide valuable information for further discovery of more novel ROCK1 inhibitors.

Metabolomics Approach for Toxicity Screening of Volatile Substances

EPA Science Inventory

In 2007 the National Research Council envisioned the need for inexpensive, high throughput, cell based toxicity testing methods relevant to human health. High Throughput Screening (HTS) in vitro screening approaches have addressed these problems by using robotics. However, the ch...

A High-Throughput Assay for Screening of Natural Products that Enhanced Tumoricidal Activity of NK Cells.

PubMed

Gong, Chenyuan; Ni, Zhongya; Yao, Chao; Zhu, Xiaowen; Ni, Lulu; Wang, Lixin; Zhu, Shiguo

2015-01-01

Recently, immunotherapy has shown a lot of promise in cancer treatment and different immune cell types are involved in this endeavor. Among different immune cell populations, NK cells are also an important component in unleashing the therapeutic activity of immune cells. Therefore, in order to enhance the tumoricidal activity of NK cells, identification of new small-molecule natural products is important. Despite the availability of different screening methods for identification of natural products, a simple, economic and high-throughput method is lacking. Hence, in this study, we have developed a high-throughput assay for screening and indentifying natural products that can enhance NK cell-mediated killing of cancer cells. We expanded human NK cell population from human peripheral blood mononuclear cells (PBMCs) by culturing these PBMCs with membrane-bound IL-21 and CD137L engineered K562 cells. Next, expanded NK cells were co-cultured with non-small cell lung cancer (NSCLC) cells with or without natural products and after 24 h of co-culturing, harvested supernatants were analyzed for IFN-γ secretions by ELISA method. We screened 502 natural products and identified that 28 candidates has the potential to induce IFN-γ secretion by NK cells to varying degrees. Among the 28 natural product candidates, we further confirmed and analyzed the potential of one molecule, andrographolide. It actually increased IFN-γ secretion by NK cells and enhanced NK cell-mediated killing of NSCLC cells. Our results demonstrated that this IFN-γ based high-throughput assay for screening of natural products for NK cell tumoricidal activity is a simple, economic and reliable method.

Achieving accurate compound concentration in cell-based screening: validation of acoustic droplet ejection technology.

PubMed

Grant, Richard John; Roberts, Karen; Pointon, Carly; Hodgson, Clare; Womersley, Lynsey; Jones, Darren Craig; Tang, Eric

2009-06-01

Compound handling is a fundamental and critical step in compound screening throughout the drug discovery process. Although most compound-handling processes within compound management facilities use 100% DMSO solvent, conventional methods of manual or robotic liquid-handling systems in screening workflows often perform dilutions in aqueous solutions to maintain solvent tolerance of the biological assay. However, the use of aqueous media in these applications can lead to suboptimal data quality due to compound carryover or precipitation during the dilution steps. In cell-based assays, this effect is worsened by the unpredictable physical characteristics of compounds and the low DMSO tolerance within the assay. In some cases, the conventional approaches using manual or automated liquid handling resulted in variable IC(50) dose responses. This study examines the cause of this variability and evaluates the accuracy of screening data in these case studies. A number of liquid-handling options have been explored to address the issues and establish a generic compound-handling workflow to support cell-based screening across our screening functions. The authors discuss the validation of the Labcyte Echo reformatter as an effective noncontact solution for generic compound-handling applications against diverse compound classes using triple-quad liquid chromatography/mass spectrometry. The successful validation and implementation challenges of this technology for direct dosing onto cells in cell-based screening is discussed.

Directed differentiation of embryonic stem cells using a bead-based combinatorial screening method.

PubMed

Tarunina, Marina; Hernandez, Diana; Johnson, Christopher J; Rybtsov, Stanislav; Ramathas, Vidya; Jeyakumar, Mylvaganam; Watson, Thomas; Hook, Lilian; Medvinsky, Alexander; Mason, Chris; Choo, Yen

2014-01-01

We have developed a rapid, bead-based combinatorial screening method to determine optimal combinations of variables that direct stem cell differentiation to produce known or novel cell types having pre-determined characteristics. Here we describe three experiments comprising stepwise exposure of mouse or human embryonic cells to 10,000 combinations of serum-free differentiation media, through which we discovered multiple novel, efficient and robust protocols to generate a number of specific hematopoietic and neural lineages. We further demonstrate that the technology can be used to optimize existing protocols in order to substitute costly growth factors with bioactive small molecules and/or increase cell yield, and to identify in vitro conditions for the production of rare developmental intermediates such as an embryonic lymphoid progenitor cell that has not previously been reported.

Raman spectral signatures of cervical exfoliated cells from liquid-based cytology samples

NASA Astrophysics Data System (ADS)

Kearney, Padraig; Traynor, Damien; Bonnier, Franck; Lyng, Fiona M.; O'Leary, John J.; Martin, Cara M.

2017-10-01

It is widely accepted that cervical screening has significantly reduced the incidence of cervical cancer worldwide. The primary screening test for cervical cancer is the Papanicolaou (Pap) test, which has extremely variable specificity and sensitivity. There is an unmet clinical need for methods to aid clinicians in the early detection of cervical precancer. Raman spectroscopy is a label-free objective method that can provide a biochemical fingerprint of a given sample. Compared with studies on infrared spectroscopy, relatively few Raman spectroscopy studies have been carried out to date on cervical cytology. The aim of this study was to define the Raman spectral signatures of cervical exfoliated cells present in liquid-based cytology Pap test specimens and to compare the signature of high-grade dysplastic cells to each of the normal cell types. Raman spectra were recorded from single exfoliated cells and subjected to multivariate statistical analysis. The study demonstrated that Raman spectroscopy can identify biochemical signatures associated with the most common cell types seen in liquid-based cytology samples; superficial, intermediate, and parabasal cells. In addition, biochemical changes associated with high-grade dysplasia could be identified suggesting that Raman spectroscopy could be used to aid current cervical screening tests.

High-throughput screening of hybridoma supernatants using multiplexed fluorescent cell barcoding on live cells.

PubMed

Lu, Mei; Chan, Brian M; Schow, Peter W; Chang, Wesley S; King, Chadwick T

2017-12-01

With current available assay formats using either immobilized protein (ELISA, enzyme-linked immunosorbent assay) or immunostaining of fixed cells for primary monoclonal antibody (mAb) screening, researchers often fail to identify and characterize antibodies that recognize the native conformation of cell-surface antigens. Therefore, screening using live cells has become an integral and important step contributing to the successful identification of therapeutic antibody candidates. Thus the need for developing high-throughput screening (HTS) technologies using live cells has become a major priority for therapeutic mAb discovery and development. We have developed a novel technique called Multiplexed Fluorescent Cell Barcoding (MFCB), a flow cytometry-based method based upon the Fluorescent Cell Barcoding (FCB) technique and the Luminex fluorescent bead array system, but is applicable to high-through mAb screens on live cells. Using this technique in our system, we can simultaneously identify or characterize the antibody-antigen binding of up to nine unique fluorescent labeled cell populations in the time that it would normally take to process a single population. This has significantly reduced the amount of time needed for the identification of potential lead candidates. This new technology enables investigators to conduct large-scale primary hybridoma screens using flow cytometry. This in turn has allowed us to screen antibodies more efficiently than before and streamline identification and characterization of lead molecules. Copyright © 2017 Elsevier B.V. All rights reserved.

Microfluidic cell chips for high-throughput drug screening

PubMed Central

Chi, Chun-Wei; Ahmed, AH Rezwanuddin; Dereli-Korkut, Zeynep; Wang, Sihong

2016-01-01

The current state of screening methods for drug discovery is still riddled with several inefficiencies. Although some widely used high-throughput screening platforms may enhance the drug screening process, their cost and oversimplification of cell–drug interactions pose a translational difficulty. Microfluidic cell-chips resolve many issues found in conventional HTS technology, providing benefits such as reduced sample quantity and integration of 3D cell culture physically more representative of the physiological/pathological microenvironment. In this review, we introduce the advantages of microfluidic devices in drug screening, and outline the critical factors which influence device design, highlighting recent innovations and advances in the field including a summary of commercialization efforts on microfluidic cell chips. Future perspectives of microfluidic cell devices are also provided based on considerations of present technological limitations and translational barriers. PMID:27071838

In Vitro Toxicity Screening Technique for Volatile Substances Using Flow-Through System#

EPA Science Inventory

In 2007 the National Research Council envisioned the need for inexpensive, high throughput, cell based toxicity testing methods relevant to human health. High Throughput Screening (HTS) in vitro screening approaches have addressed these problems by using robotics. However the cha...

Smart Plasmonic Glucose Nanosensors as Generic Theranostic Agents for Targeting-Free Cancer Cell Screening and Killing.

PubMed

Chen, Limei; Li, Haijuan; He, Haili; Wu, Haoxi; Jin, Yongdong

2015-07-07

Fast and accurate identification of cancer cells from healthy normal cells in a simple, generic way is very crucial for early cancer detection and treatment. Although functional nanoparticles, like fluorescent quantum dots and plasmonic Au nanoparticles (NPs), have been successfully applied for cancer cell imaging and photothermal therapy, they suffer from the main drawback of needing time-consuming targeting preparation for specific cancer cell detection and selective ablation. The lack of a generic and effective method therefore limits their potential high-throughput cancer cell preliminary screening and theranostic applications. We report herein a generic in vitro method for fast, targeting-free (avoiding time-consuming preparations of targeting moiety for specific cancer cells) visual screening and selective killing of cancer cells from normal cells, by using glucose-responsive/-sensitive glucose oxidase-modified Ag/Au nanoshells (Ag/Au-GOx NSs) as a smart plasmonic theranostic agent. The method is generic to some extent since it is based on the distinct localized surface plasmon resonance (LSPR) responses (and colors) of the smart nanoprobe with cancer cells (typically have a higher glucose uptake level) and normal cells.

A multilayer microdevice for cell-based high-throughput drug screening

NASA Astrophysics Data System (ADS)

Liu, Chong; Wang, Lei; Xu, Zheng; Li, Jingmin; Ding, Xiping; Wang, Qi; Chunyu, Li

2012-06-01

A multilayer polydimethylsiloxane microdevice for cell-based high-throughput drug screening is described in this paper. This established microdevice was based on a modularization method and it integrated a drug/medium concentration gradient generator (CGG), pneumatic microvalves and a cell culture microchamber array. The CGG was able to generate five steps of linear concentrations with the same outlet flow rate. The medium/drug flowed through CGG and then into the pear-shaped cell culture microchambers vertically. This vertical perfusion mode was used to reduce the impact of the shear stress on the physiology of cells induced by the fluid flow in the microchambers. Pear-shaped microchambers with two arrays of miropillars at each outlet were adopted in this microdevice, which were beneficial to cell distribution. The chemotherapeutics Cisplatin (DDP)-induced Cisplatin-resistant cell line A549/DDP apoptotic experiments were performed well on this platform. The results showed that this novel microdevice could not only provide well-defined and stable conditions for cell culture, but was also useful for cell-based high-throughput drug screening with less reagents and time consumption.

High-efficient and high-content cytotoxic recording via dynamic and continuous cell-based impedance biosensor technology.

PubMed

Hu, Ning; Fang, Jiaru; Zou, Ling; Wan, Hao; Pan, Yuxiang; Su, Kaiqi; Zhang, Xi; Wang, Ping

2016-10-01

Cell-based bioassays were effective method to assess the compound toxicity by cell viability, and the traditional label-based methods missed much information of cell growth due to endpoint detection, while the higher throughputs were demanded to obtain dynamic information. Cell-based biosensor methods can dynamically and continuously monitor with cell viability, however, the dynamic information was often ignored or seldom utilized in the toxin and drug assessment. Here, we reported a high-efficient and high-content cytotoxic recording method via dynamic and continuous cell-based impedance biosensor technology. The dynamic cell viability, inhibition ratio and growth rate were derived from the dynamic response curves from the cell-based impedance biosensor. The results showed that the biosensors has the dose-dependent manners to diarrhetic shellfish toxin, okadiac acid based on the analysis of the dynamic cell viability and cell growth status. Moreover, the throughputs of dynamic cytotoxicity were compared between cell-based biosensor methods and label-based endpoint methods. This cell-based impedance biosensor can provide a flexible, cost and label-efficient platform of cell viability assessment in the shellfish toxin screening fields.

Review of the Potential of the Ni/Cu Plating Technique for Crystalline Silicon Solar Cells

PubMed Central

Rehman, Atteq ur; Lee, Soo Hong

2014-01-01

Developing a better method for the metallization of silicon solar cells is integral part of realizing superior efficiency. Currently, contact realization using screen printing is the leading technology in the silicon based photovoltaic industry, as it is simple and fast. However, the problem with metallization of this kind is that it has a lower aspect ratio and higher contact resistance, which limits solar cell efficiency. The mounting cost of silver pastes and decreasing silicon wafer thicknesses encourages silicon solar cell manufacturers to develop fresh metallization techniques involving a lower quantity of silver usage and not relying pressing process of screen printing. In recent times nickel/copper (Ni/Cu) based metal plating has emerged as a metallization method that may solve these issues. This paper offers a detailed review and understanding of a Ni/Cu based plating technique for silicon solar cells. The formation of a Ni seed layer by adopting various deposition techniques and a Cu conducting layer using a light induced plating (LIP) process are appraised. Unlike screen-printed metallization, a step involving patterning is crucial for opening the masking layer. Consequently, experimental procedures involving patterning methods are also explicated. Lastly, the issues of adhesion, back ground plating, process complexity and reliability for industrial applications are also addressed. PMID:28788516

Review of the Potential of the Ni/Cu Plating Technique for Crystalline Silicon Solar Cells.

PubMed

Rehman, Atteq Ur; Lee, Soo Hong

2014-02-18

Developing a better method for the metallization of silicon solar cells is integral part of realizing superior efficiency. Currently, contact realization using screen printing is the leading technology in the silicon based photovoltaic industry, as it is simple and fast. However, the problem with metallization of this kind is that it has a lower aspect ratio and higher contact resistance, which limits solar cell efficiency. The mounting cost of silver pastes and decreasing silicon wafer thicknesses encourages silicon solar cell manufacturers to develop fresh metallization techniques involving a lower quantity of silver usage and not relying pressing process of screen printing. In recent times nickel/copper (Ni/Cu) based metal plating has emerged as a metallization method that may solve these issues. This paper offers a detailed review and understanding of a Ni/Cu based plating technique for silicon solar cells. The formation of a Ni seed layer by adopting various deposition techniques and a Cu conducting layer using a light induced plating (LIP) process are appraised. Unlike screen-printed metallization, a step involving patterning is crucial for opening the masking layer. Consequently, experimental procedures involving patterning methods are also explicated. Lastly, the issues of adhesion, back ground plating, process complexity and reliability for industrial applications are also addressed.

Directed Differentiation of Embryonic Stem Cells Using a Bead-Based Combinatorial Screening Method

PubMed Central

Tarunina, Marina; Hernandez, Diana; Johnson, Christopher J.; Rybtsov, Stanislav; Ramathas, Vidya; Jeyakumar, Mylvaganam; Watson, Thomas; Hook, Lilian; Medvinsky, Alexander; Mason, Chris; Choo, Yen

2014-01-01

We have developed a rapid, bead-based combinatorial screening method to determine optimal combinations of variables that direct stem cell differentiation to produce known or novel cell types having pre-determined characteristics. Here we describe three experiments comprising stepwise exposure of mouse or human embryonic cells to 10,000 combinations of serum-free differentiation media, through which we discovered multiple novel, efficient and robust protocols to generate a number of specific hematopoietic and neural lineages. We further demonstrate that the technology can be used to optimize existing protocols in order to substitute costly growth factors with bioactive small molecules and/or increase cell yield, and to identify in vitro conditions for the production of rare developmental intermediates such as an embryonic lymphoid progenitor cell that has not previously been reported. PMID:25251366

Engineering 'cell robots' for parallel and highly sensitive screening of biomolecules under in vivo conditions.

PubMed

Song, Lifu; Zeng, An-Ping

2017-11-09

Cells are capable of rapid replication and performing tasks adaptively and ultra-sensitively and can be considered as cheap "biological-robots". Here we propose to engineer cells for screening biomolecules in parallel and with high sensitivity. Specifically, we place the biomolecule variants (library) on the bacterial phage M13. We then design cells to screen the library based on cell-phage interactions mediated by a specific intracellular signal change caused by the biomolecule of interest. For proof of concept, we used intracellular lysine concentration in E. coli as a signal to successfully screen variants of functional aspartate kinase III (AK-III) under in vivo conditions, a key enzyme in L-lysine biosynthesis which is strictly inhibited by L-lysine. Comparative studies with flow cytometry method failed to distinguish the wild-type from lysine resistance variants of AK-III, confirming a higher sensitivity of the method. It opens up a new and effective way of in vivo high-throughput screening for functional molecules and can be easily implemented at low costs.

Node-pore sensing enables label-free surface-marker profiling of single cells.

PubMed

Balakrishnan, Karthik R; Whang, Jeremy C; Hwang, Richard; Hack, James H; Godley, Lucy A; Sohn, Lydia L

2015-03-03

Flow cytometry is a ubiquitous, multiparametric method for characterizing cellular populations. However, this method can grow increasingly complex with the number of proteins that need to be screened simultaneously: spectral emission overlap of fluorophores and the subsequent need for compensation, lengthy sample preparation, and multiple control tests that need to be performed separately must all be considered. These factors lead to increased costs, and consequently, flow cytometry is performed in core facilities with a dedicated technician operating the instrument. Here, we describe a low-cost, label-free microfluidic method that can determine the phenotypic profiles of single cells. Our method employs Node-Pore Sensing to measure the transit times of cells as they interact with a series of different antibodies, each corresponding to a specific cell-surface antigen, that have been functionalized in a single microfluidic channel. We demonstrate the capabilities of our method not only by screening two acute promyelocytic leukemia human cells lines (NB4 and AP-1060) for myeloid antigens, CD13, CD14, CD15, and CD33, simultaneously, but also by distinguishing a mixture of cells of similar size—AP-1060 and NALM-1—based on surface markers CD13 and HLA-DR. Furthermore, we show that our method can screen complex subpopulations in clinical samples: we successfully identified the blast population in primary human bone marrow samples from patients with acute myeloid leukemia and screened these cells for CD13, CD34, and HLA-DR. We show that our label-free method is an affordable, highly sensitive, and user-friendly technology that has the potential to transform cellular screening at the benchside.

[Study of Cervical Exfoliated Cell's DNA Quantitative Analysis Based on Multi-Spectral Imaging Technology].

PubMed

Wu, Zheng; Zeng, Li-bo; Wu, Qiong-shui

2016-02-01

The conventional cervical cancer screening methods mainly include TBS (the bethesda system) classification method and cellular DNA quantitative analysis, however, by using multiple staining method in one cell slide, which is staining the cytoplasm with Papanicolaou reagent and the nucleus with Feulgen reagent, the study of achieving both two methods in the cervical cancer screening at the same time is still blank. Because the difficulty of this multiple staining method is that the absorbance of the non-DNA material may interfere with the absorbance of DNA, so that this paper has set up a multi-spectral imaging system, and established an absorbance unmixing model by using multiple linear regression method based on absorbance's linear superposition character, and successfully stripped out the absorbance of DNA to run the DNA quantitative analysis, and achieved the perfect combination of those two kinds of conventional screening method. Through a series of experiment we have proved that between the absorbance of DNA which is calculated by the absorbance unmixxing model and the absorbance of DNA which is measured there is no significant difference in statistics when the test level is 1%, also the result of actual application has shown that there is no intersection between the confidence interval of the DNA index of the tetraploid cells which are screened by using this paper's analysis method when the confidence level is 99% and the DNA index's judging interval of cancer cells, so that the accuracy and feasibility of the quantitative DNA analysis with multiple staining method expounded by this paper have been verified, therefore this analytical method has a broad application prospect and considerable market potential in early diagnosis of cervical cancer and other cancers.

Cardiotoxicity evaluation using human embryonic stem cells and induced pluripotent stem cell-derived cardiomyocytes.

PubMed

Zhao, Qi; Wang, Xijie; Wang, Shuyan; Song, Zheng; Wang, Jiaxian; Ma, Jing

2017-03-09

Cardiotoxicity remains an important concern in drug discovery. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have become an attractive platform to evaluate cardiotoxicity. However, the consistency between human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in prediction of cardiotoxicity has yet to be elucidated. Here we screened the toxicities of four representative drugs (E-4031, isoprenaline, quinidine, and haloperidol) using both hESC-CMs and hiPSC-CMs, combined with an impedance-based bioanalytical method. It showed that both hESC-CMs and hiPSC-CMs can recapitulate cardiotoxicity and identify the effects of well-characterized compounds. The combined platform of hPSC-CMs and an impedance-based bioanalytical method could improve preclinical cardiotoxicity screening, holding great potential for increasing drug development accuracy.

Ensemble pharmacophore meets ensemble docking: a novel screening strategy for the identification of RIPK1 inhibitors

NASA Astrophysics Data System (ADS)

Fayaz, S. M.; Rajanikant, G. K.

2014-07-01

Programmed cell death has been a fascinating area of research since it throws new challenges and questions in spite of the tremendous ongoing research in this field. Recently, necroptosis, a programmed form of necrotic cell death, has been implicated in many diseases including neurological disorders. Receptor interacting serine/threonine protein kinase 1 (RIPK1) is an important regulatory protein involved in the necroptosis and inhibition of this protein is essential to stop necroptotic process and eventually cell death. Current structure-based virtual screening methods involve a wide range of strategies and recently, considering the multiple protein structures for pharmacophore extraction has been emphasized as a way to improve the outcome. However, using the pharmacophoric information completely during docking is very important. Further, in such methods, using the appropriate protein structures for docking is desirable. If not, potential compound hits, obtained through pharmacophore-based screening, may not have correct ranks and scores after docking. Therefore, a comprehensive integration of different ensemble methods is essential, which may provide better virtual screening results. In this study, dual ensemble screening, a novel computational strategy was used to identify diverse and potent inhibitors against RIPK1. All the pharmacophore features present in the binding site were captured using both the apo and holo protein structures and an ensemble pharmacophore was built by combining these features. This ensemble pharmacophore was employed in pharmacophore-based screening of ZINC database. The compound hits, thus obtained, were subjected to ensemble docking. The leads acquired through docking were further validated through feature evaluation and molecular dynamics simulation.

web cellHTS2: a web-application for the analysis of high-throughput screening data.

PubMed

Pelz, Oliver; Gilsdorf, Moritz; Boutros, Michael

2010-04-12

The analysis of high-throughput screening data sets is an expanding field in bioinformatics. High-throughput screens by RNAi generate large primary data sets which need to be analyzed and annotated to identify relevant phenotypic hits. Large-scale RNAi screens are frequently used to identify novel factors that influence a broad range of cellular processes, including signaling pathway activity, cell proliferation, and host cell infection. Here, we present a web-based application utility for the end-to-end analysis of large cell-based screening experiments by cellHTS2. The software guides the user through the configuration steps that are required for the analysis of single or multi-channel experiments. The web-application provides options for various standardization and normalization methods, annotation of data sets and a comprehensive HTML report of the screening data analysis, including a ranked hit list. Sessions can be saved and restored for later re-analysis. The web frontend for the cellHTS2 R/Bioconductor package interacts with it through an R-server implementation that enables highly parallel analysis of screening data sets. web cellHTS2 further provides a file import and configuration module for common file formats. The implemented web-application facilitates the analysis of high-throughput data sets and provides a user-friendly interface. web cellHTS2 is accessible online at http://web-cellHTS2.dkfz.de. A standalone version as a virtual appliance and source code for platforms supporting Java 1.5.0 can be downloaded from the web cellHTS2 page. web cellHTS2 is freely distributed under GPL.

78 FR 16513 - Application of Advances in Nucleic Acid and Protein Based Detection Methods to Multiplex...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-03-15

... Transfusion-Transmissible Agents and Blood Cell Antigens in Blood Donations; Public Workshop AGENCY: Food and... Methods to Multiplex Detection of Transfusion- Transmissible Agents and Blood Cell Antigens in Blood... and the use of these tests in blood donor screening and blood cell antigen typing. The public workshop...

Multi-component identification and target cell-based screening of potential bioactive compounds in toad venom by UPLC coupled with high-resolution LTQ-Orbitrap MS and high-sensitivity Qtrap MS.

PubMed

Ren, Wei; Han, Lingyu; Luo, Mengyi; Bian, Baolin; Guan, Ming; Yang, Hui; Han, Chao; Li, Na; Li, Tuo; Li, Shilei; Zhang, Yangyang; Zhao, Zhenwen; Zhao, Haiyu

2018-04-28

Traditional Chinese medicines (TCMs) are undoubtedly treasured natural resources for discovering effective medicines in treating and preventing various diseases. However, it is still extremely difficult for screening the bioactive compounds due to the tremendous constituents in TCMs. In this work, the chemical composition of toad venom was comprehensively analyzed using ultra-high performance liquid chromatography (UPLC) coupled with high-resolution LTQ-Orbitrap mass spectrometry and 93 compounds were detected. Among them, 17 constituents were confirmed by standard substances and 8 constituents were detected in toad venom for the first time. Further, a compound database of toad venom containing the fullest compounds was further constructed using UPLC coupled with high-sensitivity Qtrap MS. Then a target cell-based approach for screening potential bioactive compounds from toad venom was developed by analyzing the target cell extracts. The reliability of this method was validated by negative controls and positive controls. In total, 17 components in toad venom were discovered to interact with the target cancer cells. Further, in vitro pharmacological trials were performed to confirm the anti-cancer activity of four of them. The results showed that the six bufogenins and seven bufotoxins detected in our research represented a promising resource to explore bufogenins/bufotoxins-based anticancer agents with low cardiotoxic effect. The target cell-based screening method coupled with the compound database of toad venom constructed by UPLC-Qtrap-MS with high sensitivity provide us a new strategy to rapidly screen and identify the potential bioactive constituents with low content in natural products, which was beneficial for drug discovery from other TCMs. ᅟ Graphical abstract.

Fluorescence-based assay as a new screening tool for toxic chemicals

PubMed Central

Moczko, Ewa; Mirkes, Evgeny M.; Cáceres, César; Gorban, Alexander N.; Piletsky, Sergey

2016-01-01

Our study involves development of fluorescent cell-based diagnostic assay as a new approach in high-throughput screening method. This highly sensitive optical assay operates similarly to e-noses and e-tongues which combine semi-specific sensors and multivariate data analysis for monitoring biochemical processes. The optical assay consists of a mixture of environmental-sensitive fluorescent dyes and human skin cells that generate fluorescence spectra patterns distinctive for particular physico-chemical and physiological conditions. Using chemometric techniques the optical signal is processed providing qualitative information about analytical characteristics of the samples. This integrated approach has been successfully applied (with sensitivity of 93% and specificity of 97%) in assessing whether particular chemical agents are irritating or not for human skin. It has several advantages compared with traditional biochemical or biological assays and can impact the new way of high-throughput screening and understanding cell activity. It also can provide reliable and reproducible method for assessing a risk of exposing people to different harmful substances, identification active compounds in toxicity screening and safety assessment of drugs, cosmetic or their specific ingredients. PMID:27653274

Fluorescence-based assay as a new screening tool for toxic chemicals.

PubMed

Moczko, Ewa; Mirkes, Evgeny M; Cáceres, César; Gorban, Alexander N; Piletsky, Sergey

2016-09-22

Our study involves development of fluorescent cell-based diagnostic assay as a new approach in high-throughput screening method. This highly sensitive optical assay operates similarly to e-noses and e-tongues which combine semi-specific sensors and multivariate data analysis for monitoring biochemical processes. The optical assay consists of a mixture of environmental-sensitive fluorescent dyes and human skin cells that generate fluorescence spectra patterns distinctive for particular physico-chemical and physiological conditions. Using chemometric techniques the optical signal is processed providing qualitative information about analytical characteristics of the samples. This integrated approach has been successfully applied (with sensitivity of 93% and specificity of 97%) in assessing whether particular chemical agents are irritating or not for human skin. It has several advantages compared with traditional biochemical or biological assays and can impact the new way of high-throughput screening and understanding cell activity. It also can provide reliable and reproducible method for assessing a risk of exposing people to different harmful substances, identification active compounds in toxicity screening and safety assessment of drugs, cosmetic or their specific ingredients.

Fluorescence-based assay as a new screening tool for toxic chemicals

NASA Astrophysics Data System (ADS)

Moczko, Ewa; Mirkes, Evgeny M.; Cáceres, César; Gorban, Alexander N.; Piletsky, Sergey

2016-09-01

Our study involves development of fluorescent cell-based diagnostic assay as a new approach in high-throughput screening method. This highly sensitive optical assay operates similarly to e-noses and e-tongues which combine semi-specific sensors and multivariate data analysis for monitoring biochemical processes. The optical assay consists of a mixture of environmental-sensitive fluorescent dyes and human skin cells that generate fluorescence spectra patterns distinctive for particular physico-chemical and physiological conditions. Using chemometric techniques the optical signal is processed providing qualitative information about analytical characteristics of the samples. This integrated approach has been successfully applied (with sensitivity of 93% and specificity of 97%) in assessing whether particular chemical agents are irritating or not for human skin. It has several advantages compared with traditional biochemical or biological assays and can impact the new way of high-throughput screening and understanding cell activity. It also can provide reliable and reproducible method for assessing a risk of exposing people to different harmful substances, identification active compounds in toxicity screening and safety assessment of drugs, cosmetic or their specific ingredients.

Prenatal Cell-Free DNA Screening

MedlinePlus

Prenatal cell-free DNA screening Overview Prenatal cell-free DNA (cfDNA) screening, also known as noninvasive prenatal screening, is a method to screen ... in a developing baby. During prenatal cell-free DNA screening, DNA from the mother and fetus is ...

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wenzel, Carsten; Riefke, Björn; Gründemann, Stephan

Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygenmore » and nutrient gradients as well as the development of dormant tumor regions. We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions. - Highlights: • Establishment of a novel method for 3D cell culture based high-content screening. • First reported high-content screen for the identification of compounds that target cells in dormant tumor spheroid regions. • Metabolite profiling by NMR spectroscopy on 3D tumor spheroids. • Identification of respiratory chain inhibitors to specifically induce cell death in inner tumor spheroid regions. • Respiratory chain inhibitors enhance cytostatic based therapy in vitro.« less

High-throughput screening of small molecules in miniaturized mammalian cell-based assays involving post-translational modifications.

PubMed

Stockwell, B R; Haggarty, S J; Schreiber, S L

1999-02-01

Fully adapting a forward genetic approach to mammalian systems requires efficient methods to alter systematically gene products without prior knowledge of gene sequences, while allowing for the subsequent characterization of these alterations. Ideally, these methods would also allow function to be altered in a temporally controlled manner. We report the development of a miniaturized cell-based assay format that enables a genetic-like approach to understanding cellular pathways in mammalian systems using small molecules, rather than mutations, as the source of gene-product alterations. This whole-cell immunodetection assay can sensitively detect changes in specific cellular macromolecules in high-density arrays of mammalian cells. Furthermore, it is compatible with screening large numbers of small molecules in nanoliter to microliter culture volumes. We refer to this assay format as a 'cytoblot', and demonstrate the use of cytoblotting to monitor biosynthetic processes such as DNA synthesis, and post-translational processes such as acetylation and phosphorylation. Finally, we demonstrate the applicability of these assays to natural-product screening through the identification of marine sponge extracts exhibiting genotype-specific inhibition of 5-bromodeoxyuridine incorporation and suppression of the anti-proliferative effect of rapamycin. We show that cytoblots can be used for high-throughput screening of small molecules in cell-based assays. Together with small-molecule libraries, the cytoblot assay can be used to perform chemical genetic screens analogous to those used in classical genetics and thus should be applicable to understanding a wide variety of cellular processes, especially those involving post-transitional modifications.

Polyglutamine Disease Modeling: Epitope Based Screen for Homologous Recombination using CRISPR/Cas9 System.

PubMed

An, Mahru C; O'Brien, Robert N; Zhang, Ningzhe; Patra, Biranchi N; De La Cruz, Michael; Ray, Animesh; Ellerby, Lisa M

2014-04-15

We have previously reported the genetic correction of Huntington's disease (HD) patient-derived induced pluripotent stem cells using traditional homologous recombination (HR) approaches. To extend this work, we have adopted a CRISPR-based genome editing approach to improve the efficiency of recombination in order to generate allelic isogenic HD models in human cells. Incorporation of a rapid antibody-based screening approach to measure recombination provides a powerful method to determine relative efficiency of genome editing for modeling polyglutamine diseases or understanding factors that modulate CRISPR/Cas9 HR.

Quantitative screening of yeast surface-displayed polypeptide libraries by magnetic bead capture.

PubMed

Yeung, Yik A; Wittrup, K Dane

2002-01-01

Magnetic bead capture is demonstrated here to be a feasible alternative for quantitative screening of favorable mutants from a cell-displayed polypeptide library. Flow cytometric sorting with fluorescent probes has been employed previously for high throughput screening for either novel binders or improved mutants. However, many laboratories do not have ready access to this technology as a result of the limited availability and high cost of cytometers, restricting the use of cell-displayed libraries. Using streptavidin-coated magnetic beads and biotinylated ligands, an alternative approach to cell-based library screening for improved mutants was developed. Magnetic bead capture probability of labeled cells is shown to be closely correlated with the surface ligand density. A single-pass enrichment ratio of 9400 +/- 1800-fold, at the expense of 85 +/- 6% binder losses, is achieved from screening a library that contains one antibody-displaying cell (binder) in 1.1 x 10(5) nondisplaying cells. Additionally, kinetic screening for an initial high affinity to low affinity (7.7-fold lower) mutant ratio of 1:95,000, the magnetic bead capture method attains a single-pass enrichment ratio of 600 +/- 200-fold with a 75 +/- 24% probability of loss for the higher affinity mutant. The observed high loss probabilities can be straightforwardly compensated for by library oversampling, given the inherently parallel nature of the screen. Overall, these results demonstrate that magnetic beads are capable of quantitatively screening for novel binders and improved mutants. The described methods are directly analogous to procedures in common use for phage display and should lower the barriers to entry for use of cell surface display libraries.

Large-Scale Chemical Similarity Networks for Target Profiling of Compounds Identified in Cell-Based Chemical Screens

PubMed Central

Lo, Yu-Chen; Senese, Silvia; Li, Chien-Ming; Hu, Qiyang; Huang, Yong; Damoiseaux, Robert; Torres, Jorge Z.

2015-01-01

Target identification is one of the most critical steps following cell-based phenotypic chemical screens aimed at identifying compounds with potential uses in cell biology and for developing novel disease therapies. Current in silico target identification methods, including chemical similarity database searches, are limited to single or sequential ligand analysis that have limited capabilities for accurate deconvolution of a large number of compounds with diverse chemical structures. Here, we present CSNAP (Chemical Similarity Network Analysis Pulldown), a new computational target identification method that utilizes chemical similarity networks for large-scale chemotype (consensus chemical pattern) recognition and drug target profiling. Our benchmark study showed that CSNAP can achieve an overall higher accuracy (>80%) of target prediction with respect to representative chemotypes in large (>200) compound sets, in comparison to the SEA approach (60–70%). Additionally, CSNAP is capable of integrating with biological knowledge-based databases (Uniprot, GO) and high-throughput biology platforms (proteomic, genetic, etc) for system-wise drug target validation. To demonstrate the utility of the CSNAP approach, we combined CSNAP's target prediction with experimental ligand evaluation to identify the major mitotic targets of hit compounds from a cell-based chemical screen and we highlight novel compounds targeting microtubules, an important cancer therapeutic target. The CSNAP method is freely available and can be accessed from the CSNAP web server (http://services.mbi.ucla.edu/CSNAP/). PMID:25826798

[Screening of anti-lung cancer bioactive compounds from Curcuma longa by target cell extraction and UHPLC/LTQ Orbitrap MS].

PubMed

Zhou, Jian-Liang; Wu, Ye-Qing; Tan, Chun-Mei; Zhu, Ming; Ma, Lin-Ke

2016-10-01

A target cell extraction-chemical profiling method based on human alveolar adenocarcinoma cell line (A549 cells) and UHPLC/LTQ Orbitrap MS for screening the anti-lung cancer bioactive compounds from Curcuma longa has been developed in this paper. According to the hypothesis that when cells are incubated together with the extract of Curcuma longa, the potential bioactive compounds in the extract should selectively combine with the cells, then the cell-binding compounds could be separated and analyzed by LC-MS. The bioactive compounds in C. longa are lipophilic components. They intend to be absorbed on the inner wall of cell culture flask when they were incubated with A549 cells, which will produce interference in the blank solution. In this paper, by using cells digestion and multi-step centrifugation and transfer strategy, the interference problem has been solved. Finally, using the developed method, three cell-binding compounds were screened out and were identified as bisdemethoxycurcumin, demethoxycurcumin, and curcumin. These compounds are the main bioactive compounds with anti-lung cancer bioactivity in C. longa. The improved method developed in this paper could avoid the false positive results due to the absorption of lipophilic compounds on the inner wall of cell culture flask, which will to be an effective complementary method for current target cell extraction-chemical profiling technology. Copyright© by the Chinese Pharmaceutical Association.

In Vitro Toxicity Screening Technique for Volatile Substances Using Flow-Through System@@

EPA Science Inventory

In 2007, the National Research Council envisioned the need for inexpensive, rapid, cell-based toxicity testing methods relevant to human health. in vitro screening approaches have largely addressed these problems by using robotics and automation. However, the challenge is that ma...

In silico approaches to identify novel myeloid cell leukemia-1 (Mcl-1) inhibitors for treatment of cancer.

PubMed

Ren, Ji-Xia; Li, Cheng-Ping; Zhou, Xiu-Ling; Cao, Xue-Song; Xie, Yong

2017-08-22

Myeloid cell leukemia-1 (Mcl-1) has been a validated and attractive target for cancer therapy. Over-expression of Mcl-1 in many cancers allows cancer cells to evade apoptosis and contributes to the resistance to current chemotherapeutics. Here, we identified new Mcl-1 inhibitors using a multi-step virtual screening approach. First, based on two different ligand-receptor complexes, 20 pharmacophore models were established by simultaneously using 'Receptor-Ligand Pharmacophore Generation' method and manual build feature method, and then carefully validated by a test database. Then, pharmacophore-based virtual screening (PB-VS) could be performed by using the 20 pharmacophore models. In addition, docking study was used to predict the possible binding poses of compounds, and the docking parameters were optimized before performing docking-based virtual screening (DB-VS). Moreover, a 3D QSAR model was established by applying the 55 aligned Mcl-1 inhibitors. The 55 inhibitors sharing the same scaffold were docked into the Mcl-1 active site before alignment, then the inhibitors with possible binding conformations were aligned. For the training set, the 3D QSAR model gave a correlation coefficient r 2 of 0.996; for the test set, the correlation coefficient r 2 was 0.812. Therefore, the developed 3D QSAR model was a good model, which could be applied for carrying out 3D QSAR-based virtual screening (QSARD-VS). After the above three virtual screening methods orderly filtering, 23 potential inhibitors with novel scaffolds were identified. Furthermore, we have discussed in detail the mapping results of two potent compounds onto pharmacophore models, 3D QSAR model, and the interactions between the compounds and active site residues.

Nonlinear mixed effects dose response modeling in high throughput drug screens: application to melanoma cell line analysis.

PubMed

Ding, Kuan-Fu; Petricoin, Emanuel F; Finlay, Darren; Yin, Hongwei; Hendricks, William P D; Sereduk, Chris; Kiefer, Jeffrey; Sekulic, Aleksandar; LoRusso, Patricia M; Vuori, Kristiina; Trent, Jeffrey M; Schork, Nicholas J

2018-01-12

Cancer cell lines are often used in high throughput drug screens (HTS) to explore the relationship between cell line characteristics and responsiveness to different therapies. Many current analysis methods infer relationships by focusing on one aspect of cell line drug-specific dose-response curves (DRCs), the concentration causing 50% inhibition of a phenotypic endpoint (IC 50 ). Such methods may overlook DRC features and do not simultaneously leverage information about drug response patterns across cell lines, potentially increasing false positive and negative rates in drug response associations. We consider the application of two methods, each rooted in nonlinear mixed effects (NLME) models, that test the relationship relationships between estimated cell line DRCs and factors that might mitigate response. Both methods leverage estimation and testing techniques that consider the simultaneous analysis of different cell lines to draw inferences about any one cell line. One of the methods is designed to provide an omnibus test of the differences between cell line DRCs that is not focused on any one aspect of the DRC (such as the IC 50 value). We simulated different settings and compared the different methods on the simulated data. We also compared the proposed methods against traditional IC 50 -based methods using 40 melanoma cell lines whose transcriptomes, proteomes, and, importantly, BRAF and related mutation profiles were available. Ultimately, we find that the NLME-based methods are more robust, powerful and, for the omnibus test, more flexible, than traditional methods. Their application to the melanoma cell lines reveals insights into factors that may be clinically useful.

Differentially pumped spray deposition as a rapid screening tool for organic and perovskite solar cells.

PubMed

Jung, Yen-Sook; Hwang, Kyeongil; Scholes, Fiona H; Watkins, Scott E; Kim, Dong-Yu; Vak, Doojin

2016-02-08

We report a spray deposition technique as a screening tool for solution processed solar cells. A dual-feed spray nozzle is introduced to deposit donor and acceptor materials separately and to form blended films on substrates in situ. Using a differential pump system with a motorised spray nozzle, the effect of film thickness, solution flow rates and the blend ratio of donor and acceptor materials on device performance can be found in a single experiment. Using this method, polymer solar cells based on poly(3-hexylthiophene) (P3HT):(6,6)-phenyl C61 butyric acid methyl ester (PC61BM) are fabricated with numerous combinations of thicknesses and blend ratios. Results obtained from this technique show that the optimum ratio of materials is consistent with previously reported values confirming this technique is a very useful and effective screening method. This high throughput screening method is also used in a single-feed configuration. In the single-feed mode, methylammonium iodide solution is deposited on lead iodide films to create a photoactive layer of perovskite solar cells. Devices featuring a perovskite layer fabricated by this spray process demonstrated a power conversion efficiencies of up to 7.9%.

Differentially pumped spray deposition as a rapid screening tool for organic and perovskite solar cells

PubMed Central

Jung, Yen-Sook; Hwang, Kyeongil; Scholes, Fiona H.; Watkins, Scott E.; Kim, Dong-Yu; Vak, Doojin

2016-01-01

We report a spray deposition technique as a screening tool for solution processed solar cells. A dual-feed spray nozzle is introduced to deposit donor and acceptor materials separately and to form blended films on substrates in situ. Using a differential pump system with a motorised spray nozzle, the effect of film thickness, solution flow rates and the blend ratio of donor and acceptor materials on device performance can be found in a single experiment. Using this method, polymer solar cells based on poly(3-hexylthiophene) (P3HT):(6,6)-phenyl C61 butyric acid methyl ester (PC61BM) are fabricated with numerous combinations of thicknesses and blend ratios. Results obtained from this technique show that the optimum ratio of materials is consistent with previously reported values confirming this technique is a very useful and effective screening method. This high throughput screening method is also used in a single-feed configuration. In the single-feed mode, methylammonium iodide solution is deposited on lead iodide films to create a photoactive layer of perovskite solar cells. Devices featuring a perovskite layer fabricated by this spray process demonstrated a power conversion efficiencies of up to 7.9%. PMID:26853266

Large-scale generation of human iPSC-derived neural stem cells/early neural progenitor cells and their neuronal differentiation.

PubMed

D'Aiuto, Leonardo; Zhi, Yun; Kumar Das, Dhanjit; Wilcox, Madeleine R; Johnson, Jon W; McClain, Lora; MacDonald, Matthew L; Di Maio, Roberto; Schurdak, Mark E; Piazza, Paolo; Viggiano, Luigi; Sweet, Robert; Kinchington, Paul R; Bhattacharjee, Ayantika G; Yolken, Robert; Nimgaonka, Vishwajit L; Nimgaonkar, Vishwajit L

2014-01-01

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented opportunity to perform high-throughput screening of novel drugs for neurological and neurodegenerative diseases. Such screenings require a robust and scalable method for generating large numbers of mature, differentiated neuronal cells. Currently available methods based on differentiation of embryoid bodies (EBs) or directed differentiation of adherent culture systems are either expensive or are not scalable. We developed a protocol for large-scale generation of neuronal stem cells (NSCs)/early neural progenitor cells (eNPCs) and their differentiation into neurons. Our scalable protocol allows robust and cost-effective generation of NSCs/eNPCs from iPSCs. Following culture in neurobasal medium supplemented with B27 and BDNF, NSCs/eNPCs differentiate predominantly into vesicular glutamate transporter 1 (VGLUT1) positive neurons. Targeted mass spectrometry analysis demonstrates that iPSC-derived neurons express ligand-gated channels and other synaptic proteins and whole-cell patch-clamp experiments indicate that these channels are functional. The robust and cost-effective differentiation protocol described here for large-scale generation of NSCs/eNPCs and their differentiation into neurons paves the way for automated high-throughput screening of drugs for neurological and neurodegenerative diseases.

DeepPap: Deep Convolutional Networks for Cervical Cell Classification.

PubMed

Zhang, Ling; Le Lu; Nogues, Isabella; Summers, Ronald M; Liu, Shaoxiong; Yao, Jianhua

2017-11-01

Automation-assisted cervical screening via Pap smear or liquid-based cytology (LBC) is a highly effective cell imaging based cancer detection tool, where cells are partitioned into "abnormal" and "normal" categories. However, the success of most traditional classification methods relies on the presence of accurate cell segmentations. Despite sixty years of research in this field, accurate segmentation remains a challenge in the presence of cell clusters and pathologies. Moreover, previous classification methods are only built upon the extraction of hand-crafted features, such as morphology and texture. This paper addresses these limitations by proposing a method to directly classify cervical cells-without prior segmentation-based on deep features, using convolutional neural networks (ConvNets). First, the ConvNet is pretrained on a natural image dataset. It is subsequently fine-tuned on a cervical cell dataset consisting of adaptively resampled image patches coarsely centered on the nuclei. In the testing phase, aggregation is used to average the prediction scores of a similar set of image patches. The proposed method is evaluated on both Pap smear and LBC datasets. Results show that our method outperforms previous algorithms in classification accuracy (98.3%), area under the curve (0.99) values, and especially specificity (98.3%), when applied to the Herlev benchmark Pap smear dataset and evaluated using five-fold cross validation. Similar superior performances are also achieved on the HEMLBC (H&E stained manual LBC) dataset. Our method is promising for the development of automation-assisted reading systems in primary cervical screening.

Photovoltaic and Impedance Spectroscopy Study of Screen-Printed TiO₂ Based CdS Quantum Dot Sensitized Solar Cells.

PubMed

Atif, M; Farooq, W A; Fatehmulla, Amanullah; Aslam, M; Ali, Syed Mansoor

2015-01-19

Cadmium sulphide (CdS) quantum dot sensitized solar cells (QDSSCs) based on screen-printed TiO₂ were assembled using a screen-printing technique. The CdS quantum dots (QDs) were grown by using the Successive Ionic Layer Adsorption and Reaction (SILAR) method. The optical properties were studied by UV-Vis absorbance spectroscopy. Photovoltaic characteristics and impedance spectroscopic measurements of CdS QDSSCs were carried out under air mass 1.5 illuminations. The experimental results of capacitance against voltage indicate a trend from positive to negative capacitance because of the injection of electrons from the Fluorine doped tin oxide (FTO) electrode into TiO₂.

QUANTITATIVE IN VITRO MEASUREMENT OF CELLULAR PROCESSES CRITICAL TO THE DEVELOPMENT OF NEURAL CONNECTIVITY USING HCA.

EPA Science Inventory

New methods are needed to screen thousands of environmental chemicals for toxicity, including developmental neurotoxicity. In vitro, cell-based assays that model key cellular events have been proposed for high throughput screening of chemicals for developmental neurotoxicity. Whi...

Direct analysis of prostaglandin-E2 and -D2 produced in an inflammatory cell reaction and its application for activity screening and potency evaluation using turbulent flow chromatography liquid chromatography-high resolution mass spectrometry.

PubMed

Shin, Jeong-Sook; Peng, Lei; Kang, Kyungsu; Choi, Yongsoo

2016-09-09

Direct analysis of prostaglandin-E2 (PGE2) and -D2 (PGD2) produced from a RAW264.7 cell-based reaction was performed by liquid chromatography high-resolution mass spectrometry (LC-HRMS), which was online coupled with turbulent flow chromatography (TFC). The capability of this method to accurately measure PG levels in cell reaction medium containing cytokines or proteins as a reaction byproduct was cross-validated by two conventional methods. Two methods, including an LC-HRMS method after liquid-liquid extraction (LLE) of the sample and a commercial PGE2 enzyme-linked immunosorbent assay (ELISA), showed PGE2 and/or PGD2 levels almost similar to those obtained by TFC LC-HRMS over the reaction time after LPS stimulation. After the cross-validation, significant analytical throughputs, allowing simultaneous screening and potency evaluation of 80 natural products including 60 phytochemicals and 20 natural product extracts for the inhibition of the PGD2 produced in the cell-based inflammatory reaction, were achieved using the TFC LC-HRMS method developed. Among the 60 phytochemicals screened, licochalcone A and formononetin inhibited PGD2 production the most with IC50 values of 126 and 151nM, respectively. For a reference activity, indomethacin and diclofenac were used, measuring IC50 values of 0.64 and 0.21nM, respectively. This method also found a butanol extract of Akebia quinata Decne (AQ) stem as a promising natural product for PGD2 inhibition. Direct and accurate analysis of PGs in the inflammatory cell reaction using the TFC LC-HRMS method developed enables the high-throughput screening and potency evaluation of as many as 320 samples in less than 48h without changing a TFC column. Copyright © 2016 Elsevier B.V. All rights reserved.

Real-time, label-free monitoring of cell viability based on cell adhesion measurements with an atomic force microscope.

PubMed

Yang, Fang; Riedel, René; Del Pino, Pablo; Pelaz, Beatriz; Said, Alaa Hassan; Soliman, Mahmoud; Pinnapireddy, Shashank R; Feliu, Neus; Parak, Wolfgang J; Bakowsky, Udo; Hampp, Norbert

2017-03-22

The adhesion of cells to an oscillating cantilever sensitively influences the oscillation amplitude at a given frequency. Even early stages of cytotoxicity cause a change in the viscosity of the cell membrane and morphology, both affecting their adhesion to the cantilever. We present a generally applicable method for real-time, label free monitoring and fast-screening technique to assess early stages of cytotoxicity recorded in terms of loss of cell adhesion. We present data taken from gold nanoparticles of different sizes and surface coatings as well as some reference substances like ethanol, cadmium chloride, and staurosporine. Measurements were recorded with two different cell lines, HeLa and MCF7 cells. The results obtained from gold nanoparticles confirm earlier findings and attest the easiness and effectiveness of the method. The reported method allows to easily adapt virtually every AFM to screen and assess toxicity of compounds in terms of cell adhesion with little modifications as long as a flow cell is available. The sensitivity of the method is good enough indicating that even single cell analysis seems possible.

A chemical genetic screen for mTOR pathway inhibitors based on 4E-BP-dependent nuclear accumulation of eIF4E.

PubMed

Livingstone, Mark; Larsson, Ola; Sukarieh, Rami; Pelletier, Jerry; Sonenberg, Nahum

2009-12-24

The signal transduction pathway wherein mTOR regulates cellular growth and proliferation is an active target for drug discovery. The search for new mTOR inhibitors has recently yielded a handful of promising compounds that hold therapeutic potential. This search has been limited by the lack of a high-throughput assay to monitor the phosphorylation of a direct rapamycin-sensitive mTOR substrate in cells. Here we describe a novel cell-based chemical genetic screen useful for efficiently monitoring mTOR signaling to 4E-BPs in response to stimuli. The screen is based on the nuclear accumulation of eIF4E, which occurs in a 4E-BP-dependent manner specifically upon inhibition of mTOR signaling. Using this assay in a small-scale screen, we have identified several compounds not previously known to inhibit mTOR signaling, demonstrating that this method can be adapted to larger screens. Copyright 2009 Elsevier Ltd. All rights reserved.

Whole Organism High-Content Screening by Label-Free, Image-Based Bayesian Classification for Parasitic Diseases

PubMed Central

Paveley, Ross A.; Mansour, Nuha R.; Hallyburton, Irene; Bleicher, Leo S.; Benn, Alex E.; Mikic, Ivana; Guidi, Alessandra; Gilbert, Ian H.; Hopkins, Andrew L.; Bickle, Quentin D.

2012-01-01

Sole reliance on one drug, Praziquantel, for treatment and control of schistosomiasis raises concerns about development of widespread resistance, prompting renewed interest in the discovery of new anthelmintics. To discover new leads we designed an automated label-free, high content-based, high throughput screen (HTS) to assess drug-induced effects on in vitro cultured larvae (schistosomula) using bright-field imaging. Automatic image analysis and Bayesian prediction models define morphological damage, hit/non-hit prediction and larval phenotype characterization. Motility was also assessed from time-lapse images. In screening a 10,041 compound library the HTS correctly detected 99.8% of the hits scored visually. A proportion of these larval hits were also active in an adult worm ex-vivo screen and are the subject of ongoing studies. The method allows, for the first time, screening of large compound collections against schistosomes and the methods are adaptable to other whole organism and cell-based screening by morphology and motility phenotyping. PMID:22860151

Fully automated screening of immunocytochemically stained specimens for early cancer detection

NASA Astrophysics Data System (ADS)

Bell, André A.; Schneider, Timna E.; Müller-Frank, Dirk A. C.; Meyer-Ebrecht, Dietrich; Böcking, Alfred; Aach, Til

2007-03-01

Cytopathological cancer diagnoses can be obtained less invasive than histopathological investigations. Cells containing specimens can be obtained without pain or discomfort, bloody biopsies are avoided, and the diagnosis can, in some cases, even be made earlier. Since no tissue biopsies are necessary these methods can also be used in screening applications, e.g., for cervical cancer. Among the cytopathological methods a diagnosis based on the analysis of the amount of DNA in individual cells achieves high sensitivity and specificity. Yet this analysis is time consuming, which is prohibitive for a screening application. Hence, it will be advantageous to retain, by a preceding selection step, only a subset of suspicious specimens. This can be achieved using highly sensitive immunocytochemical markers like p16 ink4a for preselection of suspicious cells and specimens. We present a method to fully automatically acquire images at distinct positions at cytological specimens using a conventional computer controlled microscope and an autofocus algorithm. Based on the thus obtained images we automatically detect p16 ink4a-positive objects. This detection in turn is based on an analysis of the color distribution of the p16 ink4a marker in the Lab-colorspace. A Gaussian-mixture-model is used to describe this distribution and the method described in this paper so far achieves a sensitivity of up to 90%.

The novel measuring method for screening and assessing chromium content in clothes and shoes materials

NASA Astrophysics Data System (ADS)

Salerno-Kochan, R.

2017-10-01

The aim of this paper is to propose the bioindicative measuring method for screening and assessing the safety of textile and leather materials in relation to chemical threats. This method is based on toxicological assay in which Tetrahymena pyriformis, unicellular organism belonging to protozoans, is used as a test organism. For the realization of the research goal the sensitivity threshold of test organisms to chromium(VI) solutions was identified. The changes in cell development of test organisms in chromium solutions were registered by colorimetric measurements in the presence of alamarBlue® cell viability reagent. Empirical data enabled to fit logistic curves on the base of which the level of chromium toxicity was estimated. In the second step, harmfulness of aqueous extracts obtained from textile and leather samples containing chromium in relation to test organisms was evaluated. The performed research confirmed the high efficiency of the proposed method in screening and assessing chromium content in clothes and shoes materials and showed possibilities of using it in safety assessment of products with regard to chemical risks.

Economic evaluation of DNA ploidy analysis vs liquid-based cytology for cervical screening.

PubMed

Nghiem, V T; Davies, K R; Beck, J R; Follen, M; MacAulay, C; Guillaud, M; Cantor, S B

2015-06-09

DNA ploidy analysis involves automated quantification of chromosomal aneuploidy, a potential marker of progression toward cervical carcinoma. We evaluated the cost-effectiveness of this method for cervical screening, comparing five ploidy strategies (using different numbers of aneuploid cells as cut points) with liquid-based Papanicolaou smear and no screening. A state-transition Markov model simulated the natural history of HPV infection and possible progression into cervical neoplasia in a cohort of 12-year-old females. The analysis evaluated cost in 2012 US$ and effectiveness in quality-adjusted life-years (QALYs) from a health-system perspective throughout a lifetime horizon in the US setting. We calculated incremental cost-effectiveness ratios (ICERs) to determine the best strategy. The robustness of optimal choices was examined in deterministic and probabilistic sensitivity analyses. In the base-case analysis, the ploidy 4 cell strategy was cost-effective, yielding an increase of 0.032 QALY and an ICER of $18 264/QALY compared to no screening. For most scenarios in the deterministic sensitivity analysis, the ploidy 4 cell strategy was the only cost-effective strategy. Cost-effectiveness acceptability curves showed that this strategy was more likely to be cost-effective than the Papanicolaou smear. Compared to the liquid-based Papanicolaou smear, screening with a DNA ploidy strategy appeared less costly and comparably effective.

Development of a 3D Tissue Culture-Based High-Content Screening Platform That Uses Phenotypic Profiling to Discriminate Selective Inhibitors of Receptor Tyrosine Kinases.

PubMed

Booij, Tijmen H; Klop, Maarten J D; Yan, Kuan; Szántai-Kis, Csaba; Szokol, Balint; Orfi, Laszlo; van de Water, Bob; Keri, Gyorgy; Price, Leo S

2016-10-01

3D tissue cultures provide a more physiologically relevant context for the screening of compounds, compared with 2D cell cultures. Cells cultured in 3D hydrogels also show complex phenotypes, increasing the scope for phenotypic profiling. Here we describe a high-content screening platform that uses invasive human prostate cancer cells cultured in 3D in standard 384-well assay plates to study the activity of potential therapeutic small molecules and antibody biologics. Image analysis tools were developed to process 3D image data to measure over 800 phenotypic parameters. Multiparametric analysis was used to evaluate the effect of compounds on tissue morphology. We applied this screening platform to measure the activity and selectivity of inhibitors of the c-Met and epidermal growth factor (EGF) receptor (EGFR) tyrosine kinases in 3D cultured prostate carcinoma cells. c-Met and EGFR activity was quantified based on the phenotypic profiles induced by their respective ligands, hepatocyte growth factor and EGF. The screening method was applied to a novel collection of 80 putative inhibitors of c-Met and EGFR. Compounds were identified that induced phenotypic profiles indicative of selective inhibition of c-Met, EGFR, or bispecific inhibition of both targets. In conclusion, we describe a fully scalable high-content screening platform that uses phenotypic profiling to discriminate selective and nonselective (off-target) inhibitors in a physiologically relevant 3D cell culture setting. © 2016 Society for Laboratory Automation and Screening.

Ultra-high-throughput screening method for the directed evolution of glucose oxidase.

PubMed

Ostafe, Raluca; Prodanovic, Radivoje; Nazor, Jovana; Fischer, Rainer

2014-03-20

Glucose oxidase (GOx) is used in many industrial processes that could benefit from improved versions of the enzyme. Some improvements like higher activity under physiological conditions and thermal stability could be useful for GOx applications in biosensors and biofuel cells. Directed evolution is one of the currently available methods to engineer improved GOx variants. Here, we describe an ultra-high-throughput screening system for sorting the best enzyme variants generated by directed evolution that incorporates several methodological refinements: flow cytometry, in vitro compartmentalization, yeast surface display, fluorescent labeling of the expressed enzyme, delivery of glucose substrate to the reaction mixture through the oil phase, and covalent labeling of the cells with fluorescein-tyramide. The method enables quantitative screening of gene libraries to identify clones with improved activity and it also allows cells to be selected based not only on the overall activity but also on the specific activity of the enzyme. Copyright © 2014 Elsevier Ltd. All rights reserved.

High-expression β(1) adrenergic receptor/cell membrane chromatography method based on a target receptor to screen active ingredients from traditional Chinese medicines.

PubMed

Yue, Yuan; Xue, Hui; Wang, Xin; Yang, Qian; Song, Yanhong; Li, Xiaoni

2014-02-01

β-Adrenergic receptors are important targets for drug discovery. We have developed a new β1 -adrenergic receptor cell membrane chromatography (β1 AR-CMC) with offline ultra-performance LC (UPLC) and MS method for screening active ingredients from traditional Chinese medicines. In this study, Chinese hamster ovary-S cells with high β1 AR expression levels were established and used to prepare a cell membrane stationary phase in a β1 AR-CMC model. The retention fractions were separated and identified by the UPLC-MS system. The screening results found that isoimperatorin from Rhizoma et Radix Notopterygii was the targeted component that could act on β1 AR in similar manner of metoprolol as a control drug. In addition, the biological effects of active component were also investigated in order to search for a new type of β1 AR antagonist. It will be a useful method for drug discovery as a leading compound resource. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identifying genes that extend life span using a high-throughput screening system.

PubMed

Chen, Cuiying; Contreras, Roland

2007-01-01

We developed a high-throughput functional genomic screening system that allows identification of genes prolonging lifespan in the baker's yeast Saccharomyces cerevisiae. The method is based on isolating yeast mother cells with a higher than average number of cell divisions as indicated by the number of bud scars on their surface. Fluorescently labeled wheat germ agglutinin (WGA) was used for specific staining of chitin, a major component of bud scars. The critical new steps in our bud-scar-sorting system are the use of small microbeads, which allows successive rounds of purification and regrowth of the mother cells (M-cell), and utilization of flow cytometry to sort and isolate cells with a longer lifespan based on the number of bud scars specifically labeled with WGA.

Validation of a Medium-Throughput Method for Evaluation of Intracellular Growth of Mycobacterium tuberculosis▿

PubMed Central

Eklund, Daniel; Welin, Amanda; Schön, Thomas; Stendahl, Olle; Huygen, Kris; Lerm, Maria

2010-01-01

Intracellular pathogens such as Mycobacterium tuberculosis have adapted to a life inside host cells, in which they utilize host nutrients to replicate and spread. Ineffective methods for the evaluation of growth of intracellular pathogens in their true environment pose an obstacle for basic research and drug screening. Here we present the validation of a luminometry-based method for the analysis of intramacrophage growth of M. tuberculosis. The method, which is performed in a medium-throughput format, can easily be adapted for studies of other intracellular pathogens and cell types. The use of host cells in drug-screening assays dedicated to find antimicrobials effective against intracellular pathogens permits the discovery of not only novel antibiotics but also compounds with immunomodulatory and virulence-impairing activities, which may be future alternatives or complements to antibiotics. PMID:20107000

A microscopy-based screen employing multiplex genome sequencing identifies cargo-specific requirements for dynein velocity

PubMed Central

Tan, Kaeling; Roberts, Anthony J.; Chonofsky, Mark; Egan, Martin J.; Reck-Peterson, Samara L.

2014-01-01

The timely delivery of membranous organelles and macromolecules to specific locations within the majority of eukaryotic cells depends on microtubule-based transport. Here we describe a screening method to identify mutations that have a critical effect on intracellular transport and its regulation using mutagenesis, multicolor-fluorescence microscopy, and multiplex genome sequencing. This screen exploits the filamentous fungus Aspergillus nidulans, which has many of the advantages of yeast molecular genetics but uses long-range microtubule-based transport in a manner more similar to metazoan cells. Using this method, we identified seven mutants that represent novel alleles of components of the intracellular transport machinery: specifically, kinesin-1, cytoplasmic dynein, and the dynein regulators Lis1 and dynactin. The two dynein mutations identified in our screen map to dynein's AAA+ catalytic core. Single-molecule studies reveal that both mutations reduce dynein's velocity in vitro. In vivo these mutants severely impair the distribution and velocity of endosomes, a known dynein cargo. In contrast, another dynein cargo, the nucleus, is positioned normally in these mutants. These results reveal that different dynein functions have distinct stringencies for motor performance. PMID:24403603

Development of a Cell-Based Functional Assay for the Detection of Clostridium botulinum Neurotoxin Types A and E

PubMed Central

Basavanna, Uma; Muruvanda, Tim; Brown, Eric W.; Sharma, Shashi K.

2013-01-01

The standard procedure for definitive detection of BoNT-producing Clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (MBA). The mouse bioassay is highly sensitive and specific, but it is expensive and time-consuming, and there are ethical concerns due to use of laboratory animals. Cell-based assays provide an alternative to the MBA in screening for BoNT-producing Clostridia. Here, we describe a cell-based assay utilizing a fluorescence reporter construct expressed in a neuronal cell model to study toxin activity in situ. Our data indicates that the assay can detect as little as 100 pM BoNT/A activity within living cells, and the assay is currently being evaluated for the analysis of BoNT in food matrices. Among available in vitro assays, we believe that cell-based assays are widely applicable in high-throughput screenings and have the potential to at least reduce and refine animal assays if not replace it. PMID:23533420

Automatic Segmentation of High-Throughput RNAi Fluorescent Cellular Images

PubMed Central

Yan, Pingkum; Zhou, Xiaobo; Shah, Mubarak; Wong, Stephen T. C.

2010-01-01

High-throughput genome-wide RNA interference (RNAi) screening is emerging as an essential tool to assist biologists in understanding complex cellular processes. The large number of images produced in each study make manual analysis intractable; hence, automatic cellular image analysis becomes an urgent need, where segmentation is the first and one of the most important steps. In this paper, a fully automatic method for segmentation of cells from genome-wide RNAi screening images is proposed. Nuclei are first extracted from the DNA channel by using a modified watershed algorithm. Cells are then extracted by modeling the interaction between them as well as combining both gradient and region information in the Actin and Rac channels. A new energy functional is formulated based on a novel interaction model for segmenting tightly clustered cells with significant intensity variance and specific phenotypes. The energy functional is minimized by using a multiphase level set method, which leads to a highly effective cell segmentation method. Promising experimental results demonstrate that automatic segmentation of high-throughput genome-wide multichannel screening can be achieved by using the proposed method, which may also be extended to other multichannel image segmentation problems. PMID:18270043

CellCognition: time-resolved phenotype annotation in high-throughput live cell imaging.

PubMed

Held, Michael; Schmitz, Michael H A; Fischer, Bernd; Walter, Thomas; Neumann, Beate; Olma, Michael H; Peter, Matthias; Ellenberg, Jan; Gerlich, Daniel W

2010-09-01

Fluorescence time-lapse imaging has become a powerful tool to investigate complex dynamic processes such as cell division or intracellular trafficking. Automated microscopes generate time-resolved imaging data at high throughput, yet tools for quantification of large-scale movie data are largely missing. Here we present CellCognition, a computational framework to annotate complex cellular dynamics. We developed a machine-learning method that combines state-of-the-art classification with hidden Markov modeling for annotation of the progression through morphologically distinct biological states. Incorporation of time information into the annotation scheme was essential to suppress classification noise at state transitions and confusion between different functional states with similar morphology. We demonstrate generic applicability in different assays and perturbation conditions, including a candidate-based RNA interference screen for regulators of mitotic exit in human cells. CellCognition is published as open source software, enabling live-cell imaging-based screening with assays that directly score cellular dynamics.

Stem cell-derived kidney cells and organoids: Recent breakthroughs and emerging applications.

PubMed

Chuah, Jacqueline Kai Chin; Zink, Daniele

The global rise in the numbers of kidney patients and the shortage in transplantable organs have led to an increasing interest in kidney-specific regenerative therapies, renal disease modelling and bioartificial kidneys. Sources for large quantities of high-quality renal cells and tissues would be required, also for applications in in vitro platforms for compound safety and efficacy screening. Stem cell-based approaches for the generation of renal-like cells and tissues would be most attractive, but such methods were not available until recently. This situation has drastically changed since 2013, and various protocols for the generation of renal-like cells and precursors from pluripotent stem cells (PSC) have been established. The most recent breakthroughs were related to the establishment of various protocols for the generation of PSC-derived kidney organoids. In combination with recent advances in genome editing, bioprinting and the establishment of predictive renal screening platforms this results in exciting new possibilities. This review will give a comprehensive overview over current PSC-based protocols for the generation of renal-like cells, precursors and organoids, and their current and potential applications in regenerative medicine, compound screening, disease modelling and bioartificial organs. Copyright © 2016 Elsevier Inc. All rights reserved.

Next-generation libraries for robust RNA interference-based genome-wide screens

PubMed Central

Kampmann, Martin; Horlbeck, Max A.; Chen, Yuwen; Tsai, Jordan C.; Bassik, Michael C.; Gilbert, Luke A.; Villalta, Jacqueline E.; Kwon, S. Chul; Chang, Hyeshik; Kim, V. Narry; Weissman, Jonathan S.

2015-01-01

Genetic screening based on loss-of-function phenotypes is a powerful discovery tool in biology. Although the recent development of clustered regularly interspaced short palindromic repeats (CRISPR)-based screening approaches in mammalian cell culture has enormous potential, RNA interference (RNAi)-based screening remains the method of choice in several biological contexts. We previously demonstrated that ultracomplex pooled short-hairpin RNA (shRNA) libraries can largely overcome the problem of RNAi off-target effects in genome-wide screens. Here, we systematically optimize several aspects of our shRNA library, including the promoter and microRNA context for shRNA expression, selection of guide strands, and features relevant for postscreen sample preparation for deep sequencing. We present next-generation high-complexity libraries targeting human and mouse protein-coding genes, which we grouped into 12 sublibraries based on biological function. A pilot screen suggests that our next-generation RNAi library performs comparably to current CRISPR interference (CRISPRi)-based approaches and can yield complementary results with high sensitivity and high specificity. PMID:26080438

Direct detection of a BRAF mutation in total RNA from melanoma cells using cantilever arrays

NASA Astrophysics Data System (ADS)

Huber, F.; Lang, H. P.; Backmann, N.; Rimoldi, D.; Gerber, Ch.

2013-02-01

Malignant melanoma, the deadliest form of skin cancer, is characterized by a predominant mutation in the BRAF gene. Drugs that target tumours carrying this mutation have recently entered the clinic. Accordingly, patients are routinely screened for mutations in this gene to determine whether they can benefit from this type of treatment. The current gold standard for mutation screening uses real-time polymerase chain reaction and sequencing methods. Here we show that an assay based on microcantilever arrays can detect the mutation nanomechanically without amplification in total RNA samples isolated from melanoma cells. The assay is based on a BRAF-specific oligonucleotide probe. We detected mutant BRAF at a concentration of 500 pM in a 50-fold excess of the wild-type sequence. The method was able to distinguish melanoma cells carrying the mutation from wild-type cells using as little as 20 ng µl-1 of RNA material, without prior PCR amplification and use of labels.

An HTS-compatible 3D colony formation assay to identify tumor-specific chemotherapeutics.

PubMed

Horman, Shane R; To, Jeremy; Orth, Anthony P

2013-12-01

There has been increasing interest in the development of cellular behavior models that take advantage of three-dimensional (3D) cell culture. To enable assessment of differential perturbagen impacts on cell growth in 2D and 3D, we have miniaturized and adapted for high-throughput screening (HTS) the soft agar colony formation assay, employing a laser-scanning cytometer to image and quantify multiple cell types simultaneously. The assay is HTS compatible, providing high-quality, image-based, replicable data for multiple, co-cultured cell types. As proof of concept, we subjected colorectal carcinoma colonies in 3D soft agar to a mini screen of 1528 natural product compounds. Hit compounds from the primary screen were rescreened in an HTS 3D co-culture matrix containing colon stromal cells and cancer cells. By combining tumor cells and normal, nontransformed colon epithelial cells in one primary screening assay, we were able to obtain differential IC50 data, thereby distinguishing tumor-specific compounds from general cytotoxic compounds. Moreover, we were able to identify compounds that antagonized tumor colony formation in 3D only, highlighting the importance of this assay in identifying agents that interfere with 3D tumor structural growth. This screening platform provides a fast, simple, and robust method for identification of tumor-specific agents in a biologically relevant microenvironment.

Identifying Novel Molecular Structures for Advanced Melanoma by Ligand-Based Virtual Screening

PubMed Central

Wang, Zhao; Lu, Yan; Seibel, William; Miller, Duane D.; Li, Wei

2009-01-01

We recently discovered a new class of thiazole analogs that are highly potent against melanoma cells. To expand the structure-activity relationship study and to explore potential new molecular scaffolds, we performed extensive ligand-based virtual screening against a compound library containing 342,910 small molecules. Two different approaches of virtual screening were carried out using the structure of our lead molecule: 1) connectivity-based search using Scitegic Pipeline Pilot from Accelerys and 2) molecular shape similarity search using Schrodinger software. Using a testing compound library, both approaches can rank similar compounds very high and rank dissimilar compounds very low, thus validating our screening methods. Structures identified from these searches were analyzed, and selected compounds were tested in vitro to assess their activity against melanoma cancer cell lines. Several molecules showed good anticancer activity. While none of the identified compounds showed better activity than our lead compound, they provided important insight into structural modifications for our lead compound and also provided novel platforms on which we can optimize new classes of anticancer compounds. One of the newly synthesized analogs based on this virtual screening has improved potency and selectivity against melanoma. PMID:19445498

A permutation-based non-parametric analysis of CRISPR screen data.

PubMed

Jia, Gaoxiang; Wang, Xinlei; Xiao, Guanghua

2017-07-19

Clustered regularly-interspaced short palindromic repeats (CRISPR) screens are usually implemented in cultured cells to identify genes with critical functions. Although several methods have been developed or adapted to analyze CRISPR screening data, no single specific algorithm has gained popularity. Thus, rigorous procedures are needed to overcome the shortcomings of existing algorithms. We developed a Permutation-Based Non-Parametric Analysis (PBNPA) algorithm, which computes p-values at the gene level by permuting sgRNA labels, and thus it avoids restrictive distributional assumptions. Although PBNPA is designed to analyze CRISPR data, it can also be applied to analyze genetic screens implemented with siRNAs or shRNAs and drug screens. We compared the performance of PBNPA with competing methods on simulated data as well as on real data. PBNPA outperformed recent methods designed for CRISPR screen analysis, as well as methods used for analyzing other functional genomics screens, in terms of Receiver Operating Characteristics (ROC) curves and False Discovery Rate (FDR) control for simulated data under various settings. Remarkably, the PBNPA algorithm showed better consistency and FDR control on published real data as well. PBNPA yields more consistent and reliable results than its competitors, especially when the data quality is low. R package of PBNPA is available at: https://cran.r-project.org/web/packages/PBNPA/ .

Facile generation of cell microarrays using vacuum degassing and coverslip sweeping.

PubMed

Wang, Min S; Luo, Zhen; Cherukuri, Sundar; Nitin, Nitin

2014-07-15

A simple method to generate cell microarrays with high-percentage well occupancy and well-defined cell confinement is presented. This method uses a synergistic combination of vacuum degassing and coverslip sweeping. The vacuum degassing step dislodges air bubbles from the microwells, which in turn enables the cells to enter the microwells, while the physical sweeping step using a glass coverslip removes the excess cells outside the microwells. This low-cost preparation method provides a simple solution to generating cell microarrays that can be performed in basic research laboratories and point-of-care settings for routine cell-based screening assays. Copyright © 2014 Elsevier Inc. All rights reserved.

Novel KCNQ2 channel activators discovered using fluorescence-based and automated patch-clamp-based high-throughput screening techniques

PubMed Central

Yue, Jin-feng; Qiao, Guan-hua; Liu, Ni; Nan, Fa-jun; Gao, Zhao-bing

2016-01-01

Aim: To establish an improved, high-throughput screening techniques for identifying novel KCNQ2 channel activators. Methods: KCNQ2 channels were stably expressed in CHO cells (KCNQ2 cells). Thallium flux assay was used for primary screening, and 384-well automated patch-clamp IonWorks Barracuda was used for hit validation. Two validated activators were characterized using a conventional patch-clamp recording technique. Results: From a collection of 80 000 compounds, the primary screening revealed a total of 565 compounds that potentiated the fluorescence signals in thallium flux assay by more than 150%. When the 565 hits were examined in IonWorks Barracuda, 38 compounds significantly enhanced the outward currents recorded in KCNQ2 cells, and were confirmed as KCNQ2 activators. In the conventional patch-clamp recordings, two validated activators ZG1732 and ZG2083 enhanced KCNQ2 currents with EC50 values of 1.04±0.18 μmol/L and 1.37±0.06 μmol/L, respectively. Conclusion: The combination of thallium flux assay and IonWorks Barracuda assay is an efficient high-throughput screening (HTS) route for discovering KCNQ2 activators. PMID:26725738

Prevalence of sickle cell disease among Grenadian newborns.

PubMed

Antoine, Magdalene; Lee, Ketty; Donald, Tyhiesia; Belfon, Yonni; Drigo, Ali; Polson, Sharon; Martin, Francis; Mitchell, George; Etienne-Julan, Maryse; Hardy-Dessources, Marie-Dominique

2018-03-01

Objective To establish the birth prevalence of sickle cell disease in Grenada, with a view to assess the requirement for a population-based neonatal screening programme. Methods A two-year pilot neonatal screening programme, involving the Ministry of Health of Grenada, the Sickle Cell Association of Grenada, and the diagnostic laboratory of hemoglobinopathies of the University Hospital of Guadeloupe, was implemented in 2014-2015 under the auspices of the Caribbean Network of Researchers on Sickle Cell Disease and Thalassemia. Results Analysis of 1914 samples processed identified the following abnormal phenotypes: 10 FS, 2 FSC, 183 FAS, 63 FAC. These data indicate β s and β c allele frequencies of 0.054 and 0.018, respectively. Conclusion Neonatal screening conducted in the framework of this Caribbean cooperation can allow rapid detection and earlier management of affected children.

False negative rates in Drosophila cell-based RNAi screens: a case study

PubMed Central

2011-01-01

Background High-throughput screening using RNAi is a powerful gene discovery method but is often complicated by false positive and false negative results. Whereas false positive results associated with RNAi reagents has been a matter of extensive study, the issue of false negatives has received less attention. Results We performed a meta-analysis of several genome-wide, cell-based Drosophila RNAi screens, together with a more focused RNAi screen, and conclude that the rate of false negative results is at least 8%. Further, we demonstrate how knowledge of the cell transcriptome can be used to resolve ambiguous results and how the number of false negative results can be reduced by using multiple, independently-tested RNAi reagents per gene. Conclusions RNAi reagents that target the same gene do not always yield consistent results due to false positives and weak or ineffective reagents. False positive results can be partially minimized by filtering with transcriptome data. RNAi libraries with multiple reagents per gene also reduce false positive and false negative outcomes when inconsistent results are disambiguated carefully. PMID:21251254

Product-induced gene expression, a product-responsive reporter assay used to screen metagenomic libraries for enzyme-encoding genes.

PubMed

Uchiyama, Taku; Miyazaki, Kentaro

2010-11-01

A reporter assay-based screening method for enzymes, which we named product-induced gene expression (PIGEX), was developed and used to screen a metagenomic library for amidases. A benzoate-responsive transcriptional activator, BenR, was placed upstream of the gene encoding green fluorescent protein and used as a sensor. Escherichia coli sensor cells carrying the benR-gfp gene cassette fluoresced in response to benzoate concentrations as low as 10 μM but were completely unresponsive to the substrate benzamide. An E. coli metagenomic library consisting of 96,000 clones was grown in 96-well format in LB medium containing benzamide. The library cells were then cocultivated with sensor cells. Eleven amidase genes were recovered from 143 fluorescent wells; eight of these genes were homologous to known bacterial amidase genes while three were novel genes. In addition to their activity toward benzamide, the enzymes were active toward various substrates, including d- and l-amino acid amides, and displayed enantioselectivity. Thus, we demonstrated that PIGEX is an effective approach for screening novel enzymes based on product detection.

siRNA screen identifies QPCT as a druggable target for Huntington's disease.

PubMed

Jimenez-Sanchez, Maria; Lam, Wun; Hannus, Michael; Sönnichsen, Birte; Imarisio, Sara; Fleming, Angeleen; Tarditi, Alessia; Menzies, Fiona; Dami, Teresa Ed; Xu, Catherine; Gonzalez-Couto, Eduardo; Lazzeroni, Giulia; Heitz, Freddy; Diamanti, Daniela; Massai, Luisa; Satagopam, Venkata P; Marconi, Guido; Caramelli, Chiara; Nencini, Arianna; Andreini, Matteo; Sardone, Gian Luca; Caradonna, Nicola P; Porcari, Valentina; Scali, Carla; Schneider, Reinhard; Pollio, Giuseppe; O'Kane, Cahir J; Caricasole, Andrea; Rubinsztein, David C

2015-05-01

Huntington's disease (HD) is a currently incurable neurodegenerative condition caused by an abnormally expanded polyglutamine tract in huntingtin (HTT). We identified new modifiers of mutant HTT toxicity by performing a large-scale 'druggable genome' siRNA screen in human cultured cells, followed by hit validation in Drosophila. We focused on glutaminyl cyclase (QPCT), which had one of the strongest effects on mutant HTT-induced toxicity and aggregation in the cell-based siRNA screen and also rescued these phenotypes in Drosophila. We found that QPCT inhibition induced the levels of the molecular chaperone αB-crystallin and reduced the aggregation of diverse proteins. We generated new QPCT inhibitors using in silico methods followed by in vitro screening, which rescued the HD-related phenotypes in cell, Drosophila and zebrafish HD models. Our data reveal a new HD druggable target affecting mutant HTT aggregation and provide proof of principle for a discovery pipeline from druggable genome screen to drug development.

Bioinformatics: Cheap and robust method to explore biomaterial from Indonesia biodiversity

NASA Astrophysics Data System (ADS)

Widodo

2015-02-01

Indonesia has a huge amount of biodiversity, which may contain many biomaterials for pharmaceutical application. These resources potency should be explored to discover new drugs for human wealth. However, the bioactive screening using conventional methods is very expensive and time-consuming. Therefore, we developed a methodology for screening the potential of natural resources based on bioinformatics. The method is developed based on the fact that organisms in the same taxon will have similar genes, metabolism and secondary metabolites product. Then we employ bioinformatics to explore the potency of biomaterial from Indonesia biodiversity by comparing species with the well-known taxon containing the active compound through published paper or chemical database. Then we analyze drug-likeness, bioactivity and the target proteins of the active compound based on their molecular structure. The target protein was examined their interaction with other proteins in the cell to determine action mechanism of the active compounds in the cellular level, as well as to predict its side effects and toxicity. By using this method, we succeeded to screen anti-cancer, immunomodulators and anti-inflammation from Indonesia biodiversity. For example, we found anticancer from marine invertebrate by employing the method. The anti-cancer was explore based on the isolated compounds of marine invertebrate from published article and database, and then identified the protein target, followed by molecular pathway analysis. The data suggested that the active compound of the invertebrate able to kill cancer cell. Further, we collect and extract the active compound from the invertebrate, and then examined the activity on cancer cell (MCF7). The MTT result showed that the methanol extract of marine invertebrate was highly potent in killing MCF7 cells. Therefore, we concluded that bioinformatics is cheap and robust way to explore bioactive from Indonesia biodiversity for source of drug and another pharmaceutical material.

Fragment screening by SPR and advanced application to GPCRs.

PubMed

Shepherd, Claire A; Hopkins, Andrew L; Navratilova, Iva

2014-01-01

Surface plasmon resonance (SPR) is one of the primary biophysical methods for the screening of low molecular weight 'fragment' libraries, due to its low protein consumption and 'label-free' methodology. SPR biosensor interaction analysis is employed to both screen and confirm the binding of compounds in fragment screening experiments, as it provides accurate information on the affinity and kinetics of molecular interactions. The most advanced application of the use of SPR for fragment screening is against membrane protein drug targets, such G-protein coupled receptors (GPCRs). Biophysical GPCR assays using SPR have been validated with pharmacological measurements approximate to cell-based methods, yet provide the advantage of biophysical methods in their ability to measure the weak affinities of low molecular weight fragments. A number of SPR fragment screens against GPCRs have now been disclosed in the literature. SPR fragment screening is proving versatile to screen both thermostabilised GPCRs and solubilised wild type receptors. In this chapter, we discuss the state-of-the-art in GPCR fragment screening by SPR and the technical considerations in performing such experiments. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Transcranial Doppler Screening Among Children and Adolescents With Sickle Cell Anemia.

PubMed

Reeves, Sarah L; Madden, Brian; Freed, Gary L; Dombkowski, Kevin J

2016-06-01

With transcranial Doppler (TCD) screening, we can identify children and adolescents with sickle cell anemia who are at the highest risk of stroke. An accurate claims-based method for identifying children and adolescents with sickle cell anemia was recently developed and validated that establishes the necessary groundwork to enable large population-based assessments of health services utilization among children and adolescents with sickle cell anemia using administrative claims data. To assess the feasibility of using administrative claims data to identify and describe the receipt of TCD screening among children and adolescents with sickle cell anemia and to characterize opportunities for intervention. Retrospective cross-sectional study using Medicaid claims data from 2005 to 2010. Medicaid claims data were obtained from the following states: Florida, Illinois, Louisiana, Michigan, South Carolina, and Texas. Children and adolescents 2 to 16 years of age with sickle cell anemia were identified by the presence of 3 or more Medicaid claims with a diagnosis of sickle cell anemia within a calendar year (2005-2010). A total of 4775 children and adolescents contributed 10 787 person-years throughout the study period. Data were analyzed in 2015. A subset of children and adolescents enrolled for 2 or more consecutive years was identified to examine potential predictors of TCD screening, which included age, sex, previous receipt of TCD screening, state of residence, and health services utilization (well-child visits, outpatient visits, emergency department visits, and inpatient visits). Receipt of TCD screening was assessed by year and state. Using logistic regression with generalized estimating equations, we included associated predictors in a multivariable model to estimate odds of TCD screening. For a total of 4775 children and adolescents 2 to 16 years of age, TCD screening rates increased over the 6-year study period from 22% to 44% (P < .001); rates varied substantially across states. A subset of 2388 children and adolescents with sickle cell anemia (50%) was enrolled for 2 or more consecutive years. Each year of increasing age was associated with 3% lower odds of TCD screening (odds ratio, 0.97 [95% CI, 0.95-0.98]; P = .002). Previous receipt of TCD screening (odds ratio, 2.44 [95% CI, 2.11-2.81]; P < .001) and well-child visits (odds ratio, 1.10 [95% CI, 1.03-1.18]; P = .007) were associated with higher odds of receiving a TCD screening. Despite national recommendations, TCD screening rates remain low. Successful strategies to improve TCD screening rates may capitalize on the numerous health care interactions among children and adolescents with sickle cell anemia.

Segmentation of nuclear images in automated cervical cancer screening

NASA Astrophysics Data System (ADS)

Dadeshidze, Vladimir; Olsson, Lars J.; Domanik, Richard A.

1995-08-01

This paper describes an efficient method of segmenting cell nuclei from complex scenes based upon the use of adaptive region growing in conjuction with nucleus-specific filters. Results of segmenting potentially abnormal (cancer or neoplastic) cell nuclei in Papanicolaou smears from 0.8 square micrometers resolution images are also presented.

Detection of Antibiotics and Evaluation of Antibacterial Activity with Screen-Printed Electrodes

PubMed Central

Titoiu, Ana Maria; Marty, Jean-Louis

2018-01-01

This review provides a brief overview of the fabrication and properties of screen-printed electrodes and details the different opportunities to apply them for the detection of antibiotics, detection of bacteria and antibiotic susceptibility. Among the alternative approaches to costly chromatographic or ELISA methods for antibiotics detection and to lengthy culture methods for bacteria detection, electrochemical biosensors based on screen-printed electrodes present some distinctive advantages. Chemical and (bio)sensors for the detection of antibiotics and assays coupling detection with screen-printed electrodes with immunomagnetic separation are described. With regards to detection of bacteria, the emphasis is placed on applications targeting viable bacterial cells. While the electrochemical sensors and biosensors face many challenges before replacing standard analysis methods, the potential of screen-printed electrodes is increasingly exploited and more applications are anticipated to advance towards commercial analytical tools. PMID:29562637

Cytosine-based nucleoside analogs are selectively lethal to DNA mismatch repair-deficient tumour cells by enhancing levels of intracellular oxidative stress

PubMed Central

Hewish, M; Martin, S A; Elliott, R; Cunningham, D; Lord, C J; Ashworth, A

2013-01-01

Background: DNA mismatch repair deficiency is present in a significant proportion of a number of solid tumours and is associated with distinct clinical behaviour. Methods: To identify the therapeutic agents that might show selectivity for mismatch repair-deficient tumour cells, we screened a pair of isogenic MLH1-deficient and MLH1-proficient tumour cell lines with a library of clinically used drugs. To test the generality of hits in the screen, selective agents were retested in cells deficient in the MSH2 mismatch repair gene. Results: We identified cytarabine and other related cytosine-based nucleoside analogues as being selectively toxic to MLH1 and MSH2-deficient tumour cells. The selective cytotoxicity we observed was likely caused by increased levels of cellular oxidative stress, as it could be abrogated by antioxidants. Conclusion: We propose that cytarabine-based chemotherapy regimens may represent a tumour-selective treatment strategy for mismatch repair-deficient cancers. PMID:23361057

Fluorescence Reporter-Based Genome-Wide RNA Interference Screening to Identify Alternative Splicing Regulators.

PubMed

Misra, Ashish; Green, Michael R

2017-01-01

Alternative splicing is a regulated process that leads to inclusion or exclusion of particular exons in a pre-mRNA transcript, resulting in multiple protein isoforms being encoded by a single gene. With more than 90 % of human genes known to undergo alternative splicing, it represents a major source for biological diversity inside cells. Although in vitro splicing assays have revealed insights into the mechanisms regulating individual alternative splicing events, our global understanding of alternative splicing regulation is still evolving. In recent years, genome-wide RNA interference (RNAi) screening has transformed biological research by enabling genome-scale loss-of-function screens in cultured cells and model organisms. In addition to resulting in the identification of new cellular pathways and potential drug targets, these screens have also uncovered many previously unknown mechanisms regulating alternative splicing. Here, we describe a method for the identification of alternative splicing regulators using genome-wide RNAi screening, as well as assays for further validation of the identified candidates. With modifications, this method can also be adapted to study the splicing regulation of pre-mRNAs that contain two or more splice isoforms.

Use of screening tests to assess cancer risk and to estimate the risk of adult T-cell leukemia/lymphoma.

PubMed Central

Yanagawa, T; Tokudome, S

1990-01-01

We developed methods to assess the cancer risks by screening tests. These methods estimate the size of the high risk group adjusted for the characteristics of screening tests and estimate the incidence rates of cancer among the high risk group adjusted for the characteristics of the tests. A method was also developed for selecting the cut-off point of a screening test. Finally, the methods were applied to estimate the risk of the adult T-cell leukemia/lymphoma. PMID:2269244

Combinatorial electrochemical cell array for high throughput screening of micro-fuel-cells and metal/air batteries.

PubMed

Jiang, Rongzhong

2007-07-01

An electrochemical cell array was designed that contains a common air electrode and 16 microanodes for high throughput screening of both fuel cells (based on polymer electrolyte membrane) and metal/air batteries (based on liquid electrolyte). Electrode materials can easily be coated on the anodes of the electrochemical cell array and screened by switching a graphite probe from one cell to the others. The electrochemical cell array was used to study direct methanol fuel cells (DMFCs), including high throughput screening of electrode catalysts and determination of optimum operating conditions. For screening of DMFCs, there is about 6% relative standard deviation (percentage of standard deviation versus mean value) for discharge current from 10 to 20 mAcm(2). The electrochemical cell array was also used to study tin/air batteries. The effect of Cu content in the anode electrode on the discharge performance of the tin/air battery was investigated. The relative standard deviations for screening of metal/air battery (based on zinc/air) are 2.4%, 3.6%, and 5.1% for discharge current at 50, 100, and 150 mAcm(2), respectively.

Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format.

PubMed

Paulissen, Scott M; Huang, Linda S

2016-09-17

During times of nutritional stress, Saccharomyces cerevisiae undergoes gametogenesis, known as sporulation. Diploid yeast cells that are starved for nitrogen and carbon will initiate the sporulation process. The process of sporulation includes meiosis followed by spore formation, where the haploid nuclei are packaged into environmentally resistant spores. We have developed methods for the efficient sporulation of budding yeast in 96 multiwell plates, to increase the throughput of screening yeast cells for sporulation phenotypes. These methods are compatible with screening with yeast containing plasmids requiring nutritional selection, when appropriate minimal media is used, or with screening yeast with genomic alterations, when a rich presporulation regimen is used. We find that for this method, aeration during sporulation is critical for spore formation, and have devised techniques to ensure sufficient aeration that are compatible with the 96 multiwell plate format. Although these methods do not achieve the typical ~80% level of sporulation that can be achieved in large-volume flask based experiments, these methods will reliably achieve about 50-60% level of sporulation in small-volume multiwell plates.

A phenotypic screening approach to identify anticancer compounds derived from marine fungi.

PubMed

Ellinger, Bernhard; Silber, Johanna; Prashar, Anjali; Landskron, Johannes; Weber, Jonas; Rehermann, Sarah; Müller, Franz-Josef; Smith, Stephen; Wrigley, Stephen; Taskén, Kjetil; Gribbon, Philip; Labes, Antje; Imhoff, Johannes F

2014-04-01

This study covers the isolation, testing, and identification of natural products with anticancer properties. Secondary metabolites were isolated from fungal strains originating from a variety of marine habitats. Strain culture protocols were optimized with respect to growth media composition and fermentation conditions. From these producers, isolated compounds were screened for their effect on the viability and proliferation of a subset of the NCI60 panel of cancer cell lines. Active compounds of interest were identified and selected for detailed assessments and structural elucidation using nuclear magnetic resonance. This revealed the majority of fungal-derived compounds represented known anticancer chemotypes, confirming the integrity of the process and the ability to identify suitable compounds. Examination of effects of selected compounds on cancer-associated cell signaling pathways used phospho flow cytometry in combination with 3D fluorescent cell barcoding. In parallel, the study addressed the logistical aspects of maintaining multiple cancer cell lines in culture simultaneously. A potential solution involving microbead-based cell culture was investigated (BioLevitator, Hamilton). Selected cell lines were cultured in microbead and 2D methods and cell viability tests showed comparable compound inhibition in both methods (R2=0.95). In a further technology assessment, an image-based assay system was investigated for its utility as a possible complement to ATP-based detection for quantifying cell growth and viability in a label-free manner.

Automated live cell screening system based on a 24-well-microplate with integrated micro fluidics.

PubMed

Lob, V; Geisler, T; Brischwein, M; Uhl, R; Wolf, B

2007-11-01

In research, pharmacologic drug-screening and medical diagnostics, the trend towards the utilization of functional assays using living cells is persisting. Research groups working with living cells are confronted with the problem, that common endpoint measurement methods are not able to map dynamic changes. With consideration of time as a further dimension, the dynamic and networked molecular processes of cells in culture can be monitored. These processes can be investigated by measuring several extracellular parameters. This paper describes a high-content system that provides real-time monitoring data of cell parameters (metabolic and morphological alterations), e.g., upon treatment with drug compounds. Accessible are acidification rates, the oxygen consumption and changes in adhesion forces within 24 cell cultures in parallel. Addressing the rising interest in biomedical and pharmacological high-content screening assays, a concept has been developed, which integrates multi-parametric sensor readout, automated imaging and probe handling into a single embedded platform. A life-maintenance system keeps important environmental parameters (gas, humidity, sterility, temperature) constant.

Enhanced Imaging of Specific Cell-Surface Glycosylation Based on Multi-FRET.

PubMed

Yuan, Baoyin; Chen, Yuanyuan; Sun, Yuqiong; Guo, Qiuping; Huang, Jin; Liu, Jianbo; Meng, Xiangxian; Yang, Xiaohai; Wen, Xiaohong; Li, Zenghui; Li, Lie; Wang, Kemin

2018-05-15

Cell-surface glycosylation contains abundant biological information that reflects cell physiological state, and it is of great value to image cell-surface glycosylation to elucidate its functions. Here we present a hybridization chain reaction (HCR)-based multifluorescence resonance energy transfer (multi-FRET) method for specific imaging of cell-surface glycosylation. By installing donors through metabolic glycan labeling and acceptors through aptamer-tethered nanoassemblies on the same glycoconjugate, intramolecular multi-FRET occurs due to near donor-acceptor distance. Benefiting from amplified effect and spatial flexibility of the HCR nanoassemblies, enhanced multi-FRET imaging of specific cell-surface glycosylation can be obtained. With this HCR-based multi-FRET method, we achieved obvious contrast in imaging of protein-specific GalNAcylation on 7211 cell surfaces. In addition, we demonstrated the general applicability of this method by visualizing the protein-specific sialylation on CEM cell surfaces. Furthermore, the expression changes of CEM cell-surface protein-specific sialylation under drug treatment was accurately monitored. This developed imaging method may provide a powerful tool in researching glycosylation functions, discovering biomarkers, and screening drugs.

Label-free chemical imaging of live Euglena gracilis by high-speed SRS spectral microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Wakisaka, Yoshifumi; Suzuki, Yuta; Tokunaga, Kyoya; Hirose, Misa; Domon, Ryota; Akaho, Rina; Kuroshima, Mai; Tsumura, Norimichi; Shimobaba, Tomoyoshi; Iwata, Osamu; Suzuki, Kengo; Nakashima, Ayaka; Goda, Keisuke; Ozeki, Yasuyuki

2016-03-01

Microbes, especially microalgae, have recently been of great interest for developing novel biofuels, drugs, and biomaterials. Imaging-based screening of live cells can provide high selectivity and is attractive for efficient bio-production from microalgae. Although conventional cellular screening techniques use cell labeling, labeling of microbes is still under development and can interfere with their cellular functions. Furthermore, since live microbes move and change their shapes rapidly, a high-speed imaging technique is required to suppress motion artifacts. Stimulated Raman scattering (SRS) microscopy allows for label-free and high-speed spectral imaging, which helps us visualize chemical components inside biological cells and tissues. Here we demonstrate high-speed SRS imaging, with temporal resolution of 0.14 seconds, of intracellular distributions of lipid, polysaccharide, and chlorophyll concentrations in rapidly moving Euglena gracilis, a unicellular phytoflagellate. Furthermore, we show that our method allows us to analyze the amount of chemical components inside each living cell. Our results indicate that SRS imaging may be applied to label-free screening of living microbes based on chemical information.

Phenotype-Based Screening of Small Molecules to Modify Plant Cell Walls Using BY-2 Cells.

PubMed

Okubo-Kurihara, Emiko; Matsui, Minami

2018-01-01

The plant cell wall is an important and abundant biomass with great potential for use as a modern recyclable resource. For effective utilization of this cellulosic biomass, its ability to degrade efficiently is key point. With the aim of modifying the cell wall to allow easy decomposition, we used chemical biological technology to alter its structure. As a first step toward evaluating the chemicals in the cell wall we employed a phenotype-based approach using high-throughput screening. As the plant cell wall is essential in determining cell morphology, phenotype-based screening is particularly effective in identifying compounds that bring about alterations in the cell wall. For rapid and reproducible screening, tobacco BY-2 cell is an excellent system in which to observe cell morphology. In this chapter, we provide a detailed chemical biological methodology for studying cell morphology using tobacco BY-2 cells.

Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR library

PubMed Central

Zhu, Shiyou; Li, Wei; Liu, Jingze; Chen, Chen-Hao; Liao, Qi; Xu, Ping; Xu, Han; Xiao, Tengfei; Cao, Zhongzheng; Peng, Jingyu; Yuan, Pengfei; Brown, Myles; Liu, Xiaole Shirley; Wei, Wensheng

2017-01-01

CRISPR/Cas9 screens have been widely adopted to analyse coding gene functions, but high throughput screening of non-coding elements using this method is more challenging, because indels caused by a single cut in non-coding regions are unlikely to produce a functional knockout. A high-throughput method to produce deletions of non-coding DNA is needed. Herein, we report a high throughput genomic deletion strategy to screen for functional long non-coding RNAs (lncRNAs) that is based on a lentiviral paired-guide RNA (pgRNA) library. Applying our screening method, we identified 51 lncRNAs that can positively or negatively regulate human cancer cell growth. We individually validated 9 lncRNAs using CRISPR/Cas9-mediated genomic deletion and functional rescue, CRISPR activation or inhibition, and gene expression profiling. Our high-throughput pgRNA genome deletion method should enable rapid identification of functional mammalian non-coding elements. PMID:27798563

Micromachined nanocalorimetric sensor for ultra-low-volume cell-based assays.

PubMed

Johannessen, Erik A; Weaver, John M R; Bourova, Lenka; Svoboda, Petr; Cobbold, Peter H; Cooper, Jonathan M

2002-05-01

Current strategies for cell-based screening generally focus on the development of highly specific assays, which require an understanding of the nature of the signaling molecules and cellular pathways involved. In contrast, changes in temperature of cells provides a measure of altered cellular metabolism that is not stimulus specific and hence could have widespread applications in cell-based screening of receptor agonists and antagonists, as well as in the assessment of toxicity of new lead compounds. Consequently, we have developed a micromachined nanocalorimetric biological sensor using a small number of isolated living cells integrated within a subnanoliter format, which is capable of detecting 13 nW of generated power from the cells, upon exposure to a chemical or pharmaceutical stimulus. The sensor comprises a 10-junction gold and nickel thermopile, integrated on a silicon chip which was back-etched to span a 800-nm-thick membrane of silicon nitride. The thin-film membrane, which supported the sensing junctions of the thermoelectric transducer, gave the system a temperature resolution of 0.125 mK, a low heat capacity of 1.2 nJ mK(-1), and a rapid (unfiltered) response time of 12 ms. The application of the system in ultra-low-volume cell-based assays could provide a rapid endogenous screen. It offers important additional advantages over existing methods in that it is generic in nature, it does not require the use of recombinant cell lines or of detailed assay development, and finally, it can enable the use of primary cell lines or tissue biopsies.

Screening and identification of novel biologically active natural compounds.

PubMed

Newman, David

2017-01-01

With the advent of very rapid and cheap genome analyses and the linkage of these plus microbial metabolomics to potential compound structures came the realization that there was an immense sea of novel agents to be mined and tested. In addition, it is now recognized that there is significant microbial involvement in many natural products isolated from "nominally non-microbial sources". This short review covers the current screening methods that have evolved and one might even be tempted to say "devolved" in light of the realization that target-based screens had problems when the products entered clinical testing, with off-target effects being the major ones. Modern systems include, but are not limited to, screening in cell lines utilizing very modern techniques (a high content screen) that are designed to show interactions within cells when treated with an "agent". The underlying principle(s) used in such systems dated back to unpublished attempts in the very early 1980s by the pharmaceutical industry to show toxic interactions within animal cells by using automated light microscopy. Though somewhat successful, the technology was not adequate for any significant commercialization. Somewhat later, mammalian cell lines that were "genetically modified" to alter signal transduction cascades, either up or down, and frequently linked to luciferase readouts, were then employed in a 96-well format. In the case of microbes, specific resistance parameters were induced in isogenic cell lines from approximately the mid-1970s. In the latter two cases, comparisons against parent and sibling cell lines were used in order that a rapid determination of potential natural product "hits" could be made. Obviously, all of these assay systems could also be, and were, used for synthetic molecules. These methods and their results have led to a change in what the term "screening for bioactivity" means. In practice, versions of phenotypic screening are returning, but in a dramatically different scientific environment from the 1970s, as I hope to demonstrate in the short article that follows.

Alginate based 3D hydrogels as an in vitro co-culture model platform for the toxicity screening of new chemical entities.

PubMed

Lan, Shih-Feng; Starly, Binil

2011-10-01

Prediction of human response to potential therapeutic drugs is through conventional methods of in vitro cell culture assays and expensive in vivo animal testing. Alternatives to animal testing require sophisticated in vitro model systems that must replicate in vivo like function for reliable testing applications. Advancements in biomaterials have enabled the development of three-dimensional (3D) cell encapsulated hydrogels as in vitro drug screening tissue model systems. In this study, we have developed an in vitro platform to enable high density 3D culture of liver cells combined with a monolayer growth of target breast cancer cell line (MCF-7) in a static environment as a representative example of screening drug compounds for hepatotoxicity and drug efficacy. Alginate hydrogels encapsulated with serial cell densities of HepG2 cells (10(5)-10(8) cells/ml) are supported by a porous poly-carbonate disc platform and co-cultured with MCF-7 cells within standard cell culture plates during a 3 day study period. The clearance rates of drug transformation by HepG2 cells are measured using a coumarin based pro-drug. The platform was used to test for HepG2 cytotoxicity 50% (CT(50)) using commercially available drugs which further correlated well with published in vivo LD(50) values. The developed test platform allowed us to evaluate drug dose concentrations to predict hepatotoxicity and its effect on the target cells. The in vitro 3D co-culture platform provides a scalable and flexible approach to test multiple-cell types in a hybrid setting within standard cell culture plates which may open up novel 3D in vitro culture techniques to screen new chemical entity compounds. Copyright © 2011 Elsevier Inc. All rights reserved.

Concise review: modeling central nervous system diseases using induced pluripotent stem cells.

PubMed

Zeng, Xianmin; Hunsberger, Joshua G; Simeonov, Anton; Malik, Nasir; Pei, Ying; Rao, Mahendra

2014-12-01

Induced pluripotent stem cells (iPSCs) offer an opportunity to delve into the mechanisms underlying development while also affording the potential to take advantage of a number of naturally occurring mutations that contribute to either disease susceptibility or resistance. Just as with any new field, several models of screening are being explored, and innovators are working on the most efficient methods to overcome the inherent limitations of primary cell screens using iPSCs. In the present review, we provide a background regarding why iPSCs represent a paradigm shift for central nervous system (CNS) disease modeling. We describe the efforts in the field to develop more biologically relevant CNS disease models, which should provide screening assays useful for the pharmaceutical industry. We also provide some examples of successful uses for iPSC-based screens and suggest that additional development could revolutionize the field of drug discovery. The development and implementation of these advanced iPSC-based screens will create a more efficient disease-specific process underpinned by the biological mechanism in a patient- and disease-specific manner rather than by trial-and-error. Moreover, with careful and strategic planning, shared resources can be developed that will enable exponential advances in the field. This will undoubtedly lead to more sensitive and accurate screens for early diagnosis and allow the identification of patient-specific therapies, thus, paving the way to personalized medicine. ©AlphaMed Press.

Cell-based flavivirus infection (CFI) assay for the evaluation of dengue antiviral candidates using high-content imaging.

PubMed

Tan, Kah Hin; Ki, Kitti Chan Wing; Watanabe, Satoru; Vasudevan, Subhash G; Krishnan, Manoj

2014-01-01

Large-scale screening of antiviral compounds that target dengue virus life cycle requires a robust cell-based assay that is rapid, easy to conduct, and sensitive enough to be able to assess viral infectivity and cell viability so that antiviral efficacy can be measured. In this chapter we describe a method that uses high-content imaging to evaluate the in vitro antiviral efficacy in a modification to the cell-based flavivirus immunodetection (CFI) assay that was described previously in Wang et al. (Antimicrob Agents Chemother 53(5):1823-1831, 2009).

High-Density Droplet Microarray of Individually Addressable Electrochemical Cells.

PubMed

Zhang, Huijie; Oellers, Tobias; Feng, Wenqian; Abdulazim, Tarik; Saw, En Ning; Ludwig, Alfred; Levkin, Pavel A; Plumeré, Nicolas

2017-06-06

Microarray technology has shown great potential for various types of high-throughput screening applications. The main read-out methods of most microarray platforms, however, are based on optical techniques, limiting the scope of potential applications of such powerful screening technology. Electrochemical methods possess numerous complementary advantages over optical detection methods, including its label-free nature, capability of quantitative monitoring of various reporter molecules, and the ability to not only detect but also address compositions of individual compartments. However, application of electrochemical methods for the purpose of high-throughput screening remains very limited. In this work, we develop a high-density individually addressable electrochemical droplet microarray (eDMA). The eDMA allows for the detection of redox-active reporter molecules irrespective of their electrochemical reversibility in individual nanoliter-sized droplets. Orthogonal band microelectrodes are arranged to form at their intersections an array of three-electrode systems for precise control of the applied potential, which enables direct read-out of the current related to analyte detection. The band microelectrode array is covered with a layer of permeable porous polymethacrylate functionalized with a highly hydrophobic-hydrophilic pattern, forming spatially separated nanoliter-sized droplets on top of each electrochemical cell. Electrochemical characterization of single droplets demonstrates that the underlying electrode system is accessible to redox-active molecules through the hydrophilic polymeric pattern and that the nonwettable hydrophobic boundaries can spatially separate neighboring cells effectively. The eDMA technology opens the possibility to combine the high-throughput biochemical or living cell screenings using the droplet microarray platform with the sequential electrochemical read-out of individual droplets.

Integration of ARTP mutagenesis with biosensor-mediated high-throughput screening to improve L-serine yield in Corynebacterium glutamicum.

PubMed

Zhang, Xin; Zhang, Xiaomei; Xu, Guoqiang; Zhang, Xiaojuan; Shi, Jinsong; Xu, Zhenghong

2018-05-03

L-Serine is widely used in the pharmaceutical, food, and cosmetics industries. Although direct fermentative production of L-serine from sugar in Corynebacterium glutamicum has been achieved, the L-serine yield remains relatively low. In this study, atmospheric and room temperature plasma (ARTP) mutagenesis was used to improve the L-serine yield based on engineered C. glutamicum ΔSSAAI strain. Subsequently, we developed a novel high-throughput screening method using a biosensor constructed based on NCgl0581, a transcriptional factor specifically responsive to L-serine, so that L-serine concentration within single cell of C. glutamicum can be monitored via fluorescence-activated cell sorting (FACS). Novel L-serine-producing mutants were isolated from a large library of mutagenized cells. The mutant strain A36-pDser was screened from 1.2 × 10 5 cells, and the magnesium ion concentration in the medium was optimized specifically for this mutant. C. glutamicum A36-pDser accumulated 34.78 g/L L-serine with a yield of 0.35 g/g sucrose, which were 35.9 and 66.7% higher than those of the parent C. glutamicum ΔSSAAI-pDser strain, respectively. The L-serine yield achieved in this mutant was the highest of all reported L-serine-producing strains of C. glutamicum. Moreover, the whole-genome sequencing identified 11 non-synonymous mutations of genes associated with metabolic and transport pathways, which might be responsible for the higher L-serine production and better cell growth in C. glutamicum A36-pDser. This study explored an effective mutagenesis strategy and reported a novel high-throughput screening method for the development of L-serine-producing strains.

Visual detection technique for efficient screening and isolation of Salmonella based on a novel enrichment assay using chromatography membrane.

PubMed

Tang, F; Xiong, Y; Zhang, H; Wu, K; Xiang, Y; Shao, J-B; Ai, H-W; Xiang, Y-P; Zheng, X-L; Lv, J-R; Sun, H; Bao, L-S; Zhang, Z; Hu, H-B; Zhang, J-Y; Chen, L; Lu, J; Liu, W-Y; Mei, H; Ma, Y; Xu, C-F; Fang, A-Y; Gu, M; Xu, C-Y; Chen, Y; Chen, Z; Sun, Z-Y

2016-03-01

To detect Salmonella more efficiently and isolate strains more easily, a novel and simple detection method that uses an enrichment assay and two chromogenic reactions on a chromatography membrane was developed. Grade 3 chromatography paper is used as functionalized solid phase support (SPS), which contains specially optimized medium. One reaction for screening is based on the sulfate-reducing capacity of Salmonella. Hydrogen sulfide (H2S) generated by Salmonella reacts with ammonium ferric citrate to produce black colored ferrous sulfide. Another reaction is based on Salmonella C8 esterase that is unique for Enterobacteriaceae except Serratia and interacts with 4-methylumbelliferyl caprylate (MUCAP) to produce fluorescent umbelliferone, which is visible under ultraviolet light. A very low detection limit (10(1) CFU ml(-1)) for Salmonella was achieved on the background of 10(5) CFU ml(-1) Escherichia coli. More importantly, testing with more than 1,000 anal samples indicated that our method has a high positive detection rate and is relatively low cost, compared with the traditional culture-based method. It took only 1 day for the preliminary screening and 2 days to efficiently isolate the Salmonella cells, indicating that the new assay is specific, rapid, and simple for Salmonella detection. In contrast to the traditional culture-based method, this method can be easily used to screen and isolate targeted strains with the naked eye. The results of quantitative and comparative experiments showed that the visual detection technique is an efficient alternative method for the screening of Salmonella spp. in many applications of large-sized samples related to public health surveillance.

ASSESSMENT OF PC12 CELL DIFFERENTIATION AND NEURITE GROWTH: A COMPARISON OF MORPHOLOGICAL AND NEUROCHEMICAL MEASURES.

EPA Science Inventory

In order to screen large numbers of chemicals for their potential to produce developmental neurotoxicity new, in vitro methods are needed. One approach is to develop methods based on the biologic processes which underlie brain development including the growth and maturation of ce...

High-throughput bioluminescence screening of ubiquitin-proteasome pathway inhibitors from chemical and natural sources.

PubMed

Ausseil, Frederic; Samson, Arnaud; Aussagues, Yannick; Vandenberghe, Isabelle; Creancier, Laurent; Pouny, Isabelle; Kruczynski, Anna; Massiot, Georges; Bailly, Christian

2007-02-01

To discover original inhibitors of the ubiquitin-proteasome pathway, the authors have developed a cell-based bioluminescent assay and used it to screen collections of plant extracts and chemical compounds. They first established a DLD-1 human colon cancer cell line that stably expresses a 4Ubiquitin-Luciferase (4Ub-Luc) reporter protein, efficiently targeted to the ubiquitin-proteasome degradation pathway. The assay was then adapted to 96- and 384-well plate formats and calibrated with reference proteasome inhibitors. Assay robustness was carefully assessed, particularly cell toxicity, and the statistical Z factor value was calculated to 0.83, demonstrating a good performance level of the assay. A total of 18,239 molecules and 15,744 plant extracts and fractions thereof were screened for their capacity to increase the luciferase activity in DLD-1 4Ub-Luc cells, and 21 molecules and 66 extracts inhibiting the ubiquitin-proteasome pathway were identified. The fractionation of an active methanol extract of Physalis angulata L. aerial parts was performed to isolate 2 secosteroids known as physalin B and C. In a cell-based Western blot assay, the ubiquitinated protein accumulation was confirmed after a physalin treatment confirming the accuracy of the screening process. The method reported here thus provides a robust approach to identify novel ubiquitin-proteasome pathway inhibitors in large collections of chemical compounds and natural products.

A study of cellular counting to determine minimum thresholds for adequacy for liquid-based cervical cytology using a survey and counting protocol.

PubMed

Kitchener, Henry C; Gittins, Matthew; Desai, Mina; Smith, John H F; Cook, Gary; Roberts, Chris; Turnbull, Lesley

2015-03-01

Liquid-based cytology (LBC) for cervical screening would benefit from laboratory practice guidelines that define specimen adequacy for reporting of slides. The evidence base required to define cell adequacy should incorporate both ThinPrep™ (TP; Hologic, Inc., Bedford, MA, USA) and SurePath™ (SP; BD Diagnostics, Burlington, NC, USA), the two LBC systems used in the UK cervical screening programmes. The objectives of this study were to determine (1) current practice for reporting LBC in England, Wales and Scotland, (2) a reproducible method for cell counting, (3) the cellularity of slides classified as inadequate, negative or abnormal and (4) the impact of varying cellularity on the likelihood of detecting cytological abnormalities. The study involved four separate arms to pursue each of the four objectives. (1) A questionnaire survey of laboratories was conducted. (2) A standard counting protocol was developed and used by three experienced cytopathologists to determine a reliable and reproducible cell counting method. (3) Slide sets which included a range of cytological abnormalities were each sent to three laboratories for cell counting to study the correlation between cell counts and reported cytological outcomes. (4) Dilution of LBC samples by fluid only (unmixed) or by dilution with a sample containing normal cells (mixed) was performed to study the impact on reporting of reducing either the total cell count or the relative proportion of abnormal to normal cells. The study was conducted within the cervical screening programmes in England, Wales and Scotland, using routinely obtained cervical screening samples, and in 56 participating NHS cervical cytology laboratories. The study involved only routinely obtained cervical screening samples. There was no clinical intervention. The main outcome measures were (1) reliability of counting method, (2) correlation of reported cytology grades with cellularity and (3) levels of detection of abnormal cells in progressively diluted cervical samples. Laboratory practice varied in terms of threshold of cellular adequacy and of morphological markers of adequacy. While SP laboratories generally used a minimum acceptable cell count (MACC) of 15,000, the MACC employed by TP laboratories varied between 5000 and 15,000. The cell counting study showed that a standard protocol achieved moderate to strong inter-rater reproducibility. Analysis of slide reporting from laboratories revealed that a large proportion of the samples reported as inadequate had cell counts above a threshold of 15,000 for SP, and 5000 and 10,000 for TP. Inter-rater unanimity was greater among more cellular preparations. Dilution studies demonstrated greater detection of abnormalities in slides with counts above the MACC and among slides with more than 25 dyskaryotic cells. Variation in laboratory practice demonstrates a requirement for evidence-based standards for designating a MACC. This study has indicated that a MACC of 15,000 and 5000 for SP and TP, respectively, achieves a balance in terms of maintaining sensitivity and low inadequacy rates. The findings of this study should inform the development of laboratory practice guidelines. The National Institute for Health Research Health Technology Assessment programme.

Screening attenuation of coaxial cables determined in GTEM-cells

NASA Astrophysics Data System (ADS)

Knobloch, A.; Garbe, H.

2004-05-01

This paper describes the determination of the screening attenuation with a GTEM cell. An analytical part gives the link between the voltage at the cell port and the total radiated power. The next section investigates the optimal cable setup in the cell. With a measurement of the common mode current on the cable and a simulation of the radiation resistance the loop antenna characteristic of the cable setup could be verified. It is shown that the use of ferrit cores decrease the difference between the maximum and the minimum screening attenuation. The determination of great screening attenuation could be improved with the use of N-type measurement cables. A comparison between this GTEM cell method and the standard methods shows a good agreement.

In silico identification of novel ligands for G-quadruplex in the c- MYC promoter

NASA Astrophysics Data System (ADS)

Kang, Hyun-Jin; Park, Hyun-Ju

2015-04-01

G-quadruplex DNA formed in NHEIII1 region of oncogene promoter inhibits transcription of the genes. In this study, virtual screening combining pharmacophore-based search and structure-based docking screening was conducted to discover ligands binding to G-quadruplex in promoter region of c- MYC. Several hit ligands showed the selective PCR-arresting effects for oligonucleotide containing c- MYC G-quadruplex forming sequence. Among them, three hits selectively inhibited cell proliferation and decreased c- MYC mRNA level in Ramos cells, where NHEIII1 is included in translocated c- MYC gene for overexpression. Promoter assay using two kinds of constructs with wild-type and mutant sequences showed that interaction of these ligands with the G-quadruplex resulted in turning-off of the reporter gene. In conclusion, combined virtual screening methods were successfully used for discovery of selective c- MYC promoter G-quadruplex binders with anticancer activity.

Fluorescence dye-based detection of mAb aggregates in CHO culture supernatants.

PubMed

Paul, Albert Jesuran; Schwab, Karen; Prokoph, Nina; Haas, Elena; Handrick, René; Hesse, Friedemann

2015-06-01

Product yields, efficacy, and safety of monoclonal antibodies (mAbs) are reduced by the formation of higher molecular weight aggregates during upstream processing. In-process characterization of mAb aggregate formation is a challenge since there is a lack of a fast detection method to identify mAb aggregates in cell culture. In this work, we present a rapid method to characterize mAb aggregate-containing Chinese hamster ovary (CHO) cell culture supernatants. The fluorescence dyes thioflavin T (ThT) and 4-4-bis-1-phenylamino-8-naphthalene sulfonate (Bis-ANS) enabled the detection of soluble as well as large mAb aggregates. Partial least square (PLS) regression models were used to evaluate the linearity of the dye-based mAb aggregate detection in buffer down to a mAb aggregate concentration of 2.4 μg mL(-1). Furthermore, mAb aggregates were detected in bioprocess medium using Bis-ANS and ThT. Dye binding to aggregates was stable for 60 min, making the method robust and reliable. Finally, the developed method using 10 μmol L(-1) Bis-ANS enabled discrimination between CHO cell culture supernatants containing different levels of mAb aggregates. The method can be adapted for high-throughput screening, e.g., to screen for cell culture conditions influencing mAb product quality, and hence can contribute to the improvement of production processes of biopharmaceuticals in mammalian cell culture.

Automated Analysis of siRNA Screens of Virus Infected Cells Based on Immunofluorescence Microscopy

NASA Astrophysics Data System (ADS)

Matula, Petr; Kumar, Anil; Wörz, Ilka; Harder, Nathalie; Erfle, Holger; Bartenschlager, Ralf; Eils, Roland; Rohr, Karl

We present an image analysis approach as part of a high-throughput microscopy screening system based on cell arrays for the identification of genes involved in Hepatitis C and Dengue virus replication. Our approach comprises: cell nucleus segmentation, quantification of virus replication level in cells, localization of regions with transfected cells, cell classification by infection status, and quality assessment of an experiment. The approach is fully automatic and has been successfully applied to a large number of cell array images from screening experiments. The experimental results show a good agreement with the expected behavior of positive as well as negative controls and encourage the application to screens from further high-throughput experiments.

Localized transfection on arrays of magnetic beads coated with PCR products.

PubMed

Isalan, Mark; Santori, Maria Isabel; Gonzalez, Cayetano; Serrano, Luis

2005-02-01

High-throughput gene analysis would benefit from new approaches for delivering DNA or RNA into cells. Here we describe a simple system that allows any molecular biology laboratory to carry out multiple, parallel cell transfections on microscope coverslip arrays. By using magnetically defined positions and PCR product-coated paramagnetic beads, we achieved transfection in a variety of cell lines. Beads may be added to the cells at any time, allowing both spatial and temporal control of transfection. Because the beads may be coated with more than one gene construct, the method can be used to achieve cotransfection within single cells. Furthermore, PCR-generated mutants may be conveniently screened, bypassing cloning and plasmid purification steps. We illustrated the applicability of the method by screening combinatorial peptide libraries, fused to GFP, to identify previously unknown cellular localization motifs. In this way, we identified several localizing peptides, including structured localization signals based around the scaffold of a single C2H2 zinc finger.

Drug and bioactive molecule screening based on a bioelectrical impedance cell culture platform

PubMed Central

Ramasamy, Sakthivel; Bennet, Devasier; Kim, Sanghyo

2014-01-01

This review will present a brief discussion on the recent advancements of bioelectrical impedance cell-based biosensors, especially the electric cell-substrate impedance sensing (ECIS) system for screening of various bioactive molecules. The different technical integrations of various chip types, working principles, measurement systems, and applications for drug targeting of molecules in cells are highlighted in this paper. Screening of bioactive molecules based on electric cell-substrate impedance sensing is a trial-and-error process toward the development of therapeutically active agents for drug discovery and therapeutics. In general, bioactive molecule screening can be used to identify active molecular targets for various diseases and toxicity at the cellular level with nanoscale resolution. In the innovation and screening of new drugs or bioactive molecules, the activeness, the efficacy of the compound, and safety in biological systems are the main concerns on which determination of drug candidates is based. Further, drug discovery and screening of compounds are often performed in cell-based test systems in order to reduce costs and save time. Moreover, this system can provide more relevant results in in vivo studies, as well as high-throughput drug screening for various diseases during the early stages of drug discovery. Recently, MEMS technologies and integration with image detection techniques have been employed successfully. These new technologies and their possible ongoing transformations are addressed. Select reports are outlined, and not all the work that has been performed in the field of drug screening and development is covered. PMID:25525360

Fragment-based screening in tandem with phenotypic screening provides novel antiparasitic hits.

PubMed

Blaazer, Antoni R; Orrling, Kristina M; Shanmugham, Anitha; Jansen, Chimed; Maes, Louis; Edink, Ewald; Sterk, Geert Jan; Siderius, Marco; England, Paul; Bailey, David; de Esch, Iwan J P; Leurs, Rob

2015-01-01

Methods to discover biologically active small molecules include target-based and phenotypic screening approaches. One of the main difficulties in drug discovery is elucidating and exploiting the relationship between drug activity at the protein target and disease modification, a phenotypic endpoint. Fragment-based drug discovery is a target-based approach that typically involves the screening of a relatively small number of fragment-like (molecular weight <300) molecules that efficiently cover chemical space. Here, we report a fragment screening on TbrPDEB1, an essential cyclic nucleotide phosphodiesterase (PDE) from Trypanosoma brucei, and human PDE4D, an off-target, in a workflow in which fragment hits and a series of close analogs are subsequently screened for antiparasitic activity in a phenotypic panel. The phenotypic panel contained T. brucei, Trypanosoma cruzi, Leishmania infantum, and Plasmodium falciparum, the causative agents of human African trypanosomiasis (sleeping sickness), Chagas disease, leishmaniasis, and malaria, respectively, as well as MRC-5 human lung cells. This hybrid screening workflow has resulted in the discovery of various benzhydryl ethers with antiprotozoal activity and low toxicity, representing interesting starting points for further antiparasitic optimization. © 2014 Society for Laboratory Automation and Screening.

In Vitro Toxicity Screening Technique for Volatile Substances ...

EPA Pesticide Factsheets

In 2007 the National Research Council envisioned the need for inexpensive, high throughput, cell based toxicity testing methods relevant to human health. High Throughput Screening (HTS) in vitro screening approaches have addressed these problems by using robotics. However the challenge is that many of these chemicals are volatile and not amenable to HTS robotic liquid handling applications. We assembled an in vitro cell culture apparatus capable of screening volatile chemicals for toxicity with potential for miniaturization for high throughput. BEAS-2B lung cells were grown in an enclosed culture apparatus under air-liquid interface (ALI) conditions, and exposed to an array of xenobiotics in 5% CO2. Use of ALI conditions allows direct contact of cells with a gas xenobiotic, as well as release of endogenous gaseous molecules without interference by medium on the apical surface. To identify potential xenobiotic-induced perturbations in cell homeostasis, we monitored for alterations of endogenously-produced gaseous molecules in air directly above the cells, termed “headspace”. Alterations in specific endogenously-produced gaseous molecules (e.g., signaling molecules nitric oxide (NO) and carbon monoxide (CO) in headspace is indicative of xenobiotic-induced perturbations of specific cellular processes. Additionally, endogenously produced volatile organic compounds (VOCs) may be monitored in a nonspecific, discovery manner to determine whether cell processes are

Yeast as a potential vehicle for neglected tropical disease drug discovery.

PubMed

Denny, P W; Steel, P G

2015-01-01

High-throughput screening (HTS) efforts for neglected tropical disease (NTD) drug discovery have recently received increased attention because several initiatives have begun to attempt to reduce the deficit in new and clinically acceptable therapies for this spectrum of infectious diseases. HTS primarily uses two basic approaches, cell-based and in vitro target-directed screening. Both of these approaches have problems; for example, cell-based screening does not reveal the target or targets that are hit, whereas in vitro methodologies lack a cellular context. Furthermore, both can be technically challenging, expensive, and difficult to miniaturize for ultra-HTS [(u)HTS]. The application of yeast-based systems may overcome some of these problems and offer a cost-effective platform for target-directed screening within a eukaryotic cell context. Here, we review the advantages and limitations of the technologies that may be used in yeast cell-based, target-directed screening protocols, and we discuss how these are beginning to be used in NTD drug discovery. © 2014 Society for Laboratory Automation and Screening.

A method to identify differential expression profiles of time-course gene data with Fourier transformation.

PubMed

Kim, Jaehee; Ogden, Robert Todd; Kim, Haseong

2013-10-18

Time course gene expression experiments are an increasingly popular method for exploring biological processes. Temporal gene expression profiles provide an important characterization of gene function, as biological systems are both developmental and dynamic. With such data it is possible to study gene expression changes over time and thereby to detect differential genes. Much of the early work on analyzing time series expression data relied on methods developed originally for static data and thus there is a need for improved methodology. Since time series expression is a temporal process, its unique features such as autocorrelation between successive points should be incorporated into the analysis. This work aims to identify genes that show different gene expression profiles across time. We propose a statistical procedure to discover gene groups with similar profiles using a nonparametric representation that accounts for the autocorrelation in the data. In particular, we first represent each profile in terms of a Fourier basis, and then we screen out genes that are not differentially expressed based on the Fourier coefficients. Finally, we cluster the remaining gene profiles using a model-based approach in the Fourier domain. We evaluate the screening results in terms of sensitivity, specificity, FDR and FNR, compare with the Gaussian process regression screening in a simulation study and illustrate the results by application to yeast cell-cycle microarray expression data with alpha-factor synchronization.The key elements of the proposed methodology: (i) representation of gene profiles in the Fourier domain; (ii) automatic screening of genes based on the Fourier coefficients and taking into account autocorrelation in the data, while controlling the false discovery rate (FDR); (iii) model-based clustering of the remaining gene profiles. Using this method, we identified a set of cell-cycle-regulated time-course yeast genes. The proposed method is general and can be potentially used to identify genes which have the same patterns or biological processes, and help facing the present and forthcoming challenges of data analysis in functional genomics.

Anti-pulmonary fibrotic activity of salvianolic acid B was screened by a novel method based on the cyto-biophysical properties

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Miao; Zheng, Mingjing; Xu, Hanying

Various methods have been used to evaluate anti-fibrotic activity of drugs. However, most of them are complicated, labor-intensive and lack of efficiency. This study was intended to develop a rapid method for anti-fibrotic drugs screening based on biophysical properties. A549 cells in vitro were stimulated with transforming growth factor-β1 (TGF-β1), and fibrogenesis was confirmed by conventional immunological assays. Meanwhile, the alterations of cyto-biophysical properties including morphology, roughness and stiffness were measured utilizing atomic force microscopy (AFM). It was found that fibrogenesis was accompanied with changes of cellular biophysical properties. TGF-β1-stimulated A549 cells became remarkably longer, rougher and stiffer than the control.more » Then, the effect of N-acetyl-L-cysteine (NAC) as a positive drug on ameliorating fibrogenesis in TGF-β1-stimulated A549 cells was verified respectively by immunological and biophysical markers. The result of Principal Component Analysis showed that stiffness was a leading index among all biophysical markers during fibrogenesis. Salvianolic acid B (SalB), a natural anti-oxidant, was detected by AFM to protect TGF-β1-stimulated A549 cells against stiffening. Then, SalB treatment was provided in preventive mode on a rat model of bleomycin (BLM) -induced pulmonary fibrosis. The results showed that SalB treatment significantly ameliorated BLM-induced histological alterations, blocked collagen accumulations and reduced α-SMA expression in lung tissues. All these results revealed the anti-pulmonary fibrotic activity of SalB. Detection of cyto-biophysical properties were therefore recommended as a rapid method for anti-pulmonary fibrotic drugs screening. - Highlights: • Fibrogenesis was accompanied with the changes of cyto-biophysical properties. • Cyto-biophysical properties could be markers for anti-fibrotic drugs screening. • Stiffness is a leading index among all biophysical markers. • SalB was detected to protect TGF-β1-stimulated A549 cells against stiffening. • SalB treatment ameliorated pulmonary fibrosis induced by BLM in rats.« less

Chapter 17 Sterile Plate-Based Vitrification of Adherent Human Pluripotent Stem Cells and Their Derivatives Using the TWIST Method.

PubMed

Neubauer, Julia C; Stracke, Frank; Zimmermann, Heiko

2017-01-01

Due to their high biological complexity, e.g., their close cell-to-cell contacts, cryopreservation of human pluripotent stem cells with standard slow-rate protocols often is inefficient and can hardly be standardized. Vitrification that means ultrafast freezing already showed very good viability and recovery rates for this sensitive cell system, but is only applicable for low cell numbers, bears a high risk of contamination, and can hardly be implemented under GxP regulations. In this chapter, a sterile plate-based vitrification method for adherent pluripotent stem cells and their derivatives is presented based on a procedure and device for human embryonic stem cells developed by Beier et al. (Cryobiology 66:8-16, 2013). This protocol overcomes the limitations of conventional vitrification procedures resulting in the highly efficient preservation of ready-to-use adherent pluripotent stem cells with the possibility of vitrifying cells in multi-well formats for direct application in high-throughput screenings.

Somatostatin displayed on filamentous phage as a receptor-specific agonist

PubMed Central

Rousch, Mat; Lutgerink, Jan T; Coote, James; de Bruïne, Adriaan; Arends, Jan-Willem; Hoogenboom, Hennie R

1998-01-01

In search of methods to identify bio-active ligands specific for G protein-coupled receptors with seven transmembrane spanning regions, we have developed a filamentous phage-based selection and functional screening method. First, methods for panning peptide phage on cells were established, using the hormone somatostatin as a model. Somatostatin was displayed on the surface of filamentous phage by cloning into phage(mid) vectors and fusion to either pIII or pVIII viral coat proteins. Peptide displaying phage bound to a polyclonal anti-somatostatin serum, and, more importantly, to several somatostatin receptor subtypes (Sst) expressed on transfected CHO-K1 cells, in a pattern which was dependent on the used display method. Binding was competed with somatostatin, with an IC50 in the nanomolar range. The phage were specifically enriched by panning on cells, establishing conditions for cell selections of phage libraries. Binding of somatostatin displaying phage to sst2 on a reporter cell line, in which binding of natural ligand reduces secretion of alkaline phosphatase (via a cyclic AMP responsive element sensitive promoter), proved that the phage particles act as receptor-specific agonists. Less than 100 phage particles per cell were required for this activity, which is approximately 1000 fold less than soluble somatostatin, suggesting that phage binding interferes with normal receptor desensitization and/or recycling. The combination of biopanning of phage libraries on cells with functional screening of phage particles for receptor triggering activity, may be used to select novel, bio-active ligands from phage libraries of random peptides, antibody fragments, or libraries based on the natural receptor ligand. PMID:9776337

Detection of Methylated Circulating DNA as Noninvasive Biomarkers for Breast Cancer Diagnosis

PubMed Central

Cheuk, Isabella Wai Yin; Shin, Vivian Yvonne

2017-01-01

Internationally, breast cancer is the most common female cancer, and is induced by a combination of environmental, genetic, and epigenetic risk factors. Despite the advancement of imaging techniques, invasive sampling of breast epithelial cells is the only definitive diagnostic procedure for patients with breast cancer. To date, molecular biomarkers with high sensitivity and specificity for the screening and early detection of breast cancer are lacking. Recent evidence suggests that the detection of methylated circulating cell-free DNA in the peripheral blood of patients with cancer may be a promising quantitative and noninvasive method for cancer diagnosis. Methylation detection based on a multi-gene panel, rather than on the methylation status of a single gene, may be used to increase the sensitivity and specificity of breast cancer screening. In this review, the results of 14 relevant studies, investigating the efficacy of cell-free DNA methylation screening for breast cancer diagnosis, have been summarized. The genetic risk factors for breast cancer, the methods used for breast cancer detection, and the techniques and limitations related to the detection of cell-free DNA methylation status, have also been reviewed and discussed. From this review, we conclude that the analysis of peripheral blood or other samples to detect differentially methylated cell-free DNA is a promising technique for use in clinical settings, and may improve the sensitivity of screening for both, early detection and disease relapse, and thus improve the future prognosis of patients with breast cancer. PMID:28382090

Testing Of An Ultraviolet (UV)-Transparent Polymer-Based Passive Sampler for Rapid, Ultra-Low-Cost EDC Screening Applications

EPA Science Inventory

A new passive sampling method with rapid low-cost spectral detection has recently been developed. The method makes use of an ultraviolet (UV)-transparent polymer which serves as both a concentrator for dissolved compounds, and an optical cell for UV spectral detection. Because ...

Detection of dopamine in dopaminergic cell using nanoparticles-based barcode DNA analysis.

PubMed

An, Jeung Hee; Kim, Tae-Hyung; Oh, Byung-Keun; Choi, Jeong Woo

2012-01-01

Nanotechnology-based bio-barcode-amplification analysis may be an innovative approach to dopamine detection. In this study, we evaluated the efficacy of this bio-barcode DNA method in detecting dopamine from dopaminergic cells. Herein, a combination DNA barcode and bead-based immunoassay for neurotransmitter detection with PCR-like sensitivity is described. This method relies on magnetic nanoparticles with antibodies and nanoparticles that are encoded with DNA, and antibodies that can sandwich the target protein captured by the nanoparticle-bound antibodies. The aggregate sandwich structures are magnetically separated from solution, and treated in order to remove the conjugated barcode DNA. The DNA barcodes were then identified via PCR analysis. The dopamine concentration in dopaminergic cells can be readily and rapidly detected via the bio-barcode assay method. The bio-barcode assay method is, therefore, a rapid and high-throughput screening tool for the detection of neurotransmitters such as dopamine.

Droplet microfluidic technology for single-cell high-throughput screening.

PubMed

Brouzes, Eric; Medkova, Martina; Savenelli, Neal; Marran, Dave; Twardowski, Mariusz; Hutchison, J Brian; Rothberg, Jonathan M; Link, Darren R; Perrimon, Norbert; Samuels, Michael L

2009-08-25

We present a droplet-based microfluidic technology that enables high-throughput screening of single mammalian cells. This integrated platform allows for the encapsulation of single cells and reagents in independent aqueous microdroplets (1 pL to 10 nL volumes) dispersed in an immiscible carrier oil and enables the digital manipulation of these reactors at a very high-throughput. Here, we validate a full droplet screening workflow by conducting a droplet-based cytotoxicity screen. To perform this screen, we first developed a droplet viability assay that permits the quantitative scoring of cell viability and growth within intact droplets. Next, we demonstrated the high viability of encapsulated human monocytic U937 cells over a period of 4 days. Finally, we developed an optically-coded droplet library enabling the identification of the droplets composition during the assay read-out. Using the integrated droplet technology, we screened a drug library for its cytotoxic effect against U937 cells. Taken together our droplet microfluidic platform is modular, robust, uses no moving parts, and has a wide range of potential applications including high-throughput single-cell analyses, combinatorial screening, and facilitating small sample analyses.

Single tube genotyping of sickle cell anaemia using PCR-based SNP analysis.

PubMed

Waterfall, C M; Cobb, B D

2001-12-01

Allele-specific amplification (ASA) is a generally applicable technique for the detection of known single nucleotide polymorphisms (SNPs), deletions, insertions and other sequence variations. Conventionally, two reactions are required to determine the zygosity of DNA in a two-allele system, along with significant upstream optimisation to define the specific test conditions. Here, we combine single tube bi-directional ASA with a 'matrix-based' optimisation strategy, speeding up the whole process in a reduced reaction set. We use sickle cell anaemia as our model SNP system, a genetic disease that is currently screened using ASA methods. Discriminatory conditions were rapidly optimised enabling the unambiguous identification of DNA from homozygous sickle cell patients (HbS/S), heterozygous carriers (HbA/S) or normal DNA in a single tube. Simple downstream mathematical analyses based on product yield across the optimisation set allow an insight into the important aspects of priming competition and component interactions in this competitive PCR. This strategy can be applied to any polymorphism, defining specific conditions using a multifactorial approach. The inherent simplicity and low cost of this PCR-based method validates bi-directional ASA as an effective tool in future clinical screening and pharmacogenomic research where more expensive fluorescence-based approaches may not be desirable.

FRET and BRET-based biosensors in live cell compound screens.

PubMed

Robinson, Katie Herbst; Yang, Jessica R; Zhang, Jin

2014-01-01

Live cell compound screening with genetically encoded fluorescence or bioluminescence-based biosensors offers a potentially powerful approach to identify novel regulators of a signaling event of interest. In particular, compound screening in living cells has the added benefit that the entire signaling network remains intact, and thus the screen is not just against a single molecule of interest but against any molecule within the signaling network that may modulate the distinct signaling event reported by the biosensor in use. Furthermore, only molecules that are cell permeable or act at cell surface receptors will be identified as "hits," thus reducing further optimization of the compound in terms of cell penetration. Here we discuss a detailed protocol for using genetically encoded biosensors in living cells in a 96-well format for the execution of high throughput compound screens and the identification of small molecules which modulate a signaling event of interest.

Development of a novel non-radioactive cell-based method for the screening of SGLT1 and SGLT2 inhibitors using 1-NBDG.

PubMed

Chang, Hung-Chi; Yang, Su-Fu; Huang, Ching-Chun; Lin, Tzung-Sheng; Liang, Pi-Hui; Lin, Chun-Jung; Hsu, Lih-Ching

2013-08-01

Sodium-coupled glucose co-transporters SGLT1 and SGLT2 play important roles in intestinal absorption and renal reabsorption of glucose, respectively. Blocking SGLT2 is a novel mechanism for lowering the blood glucose level by inhibiting renal glucose reabsorption and selective SGLT2 inhibitors are under development for treatment of type 2 diabetes. Furthermore, it has been reported that perturbation of SGLT1 is associated with cardiomyopathy and cancer. Therefore, both SGLT1 and SGLT2 are potential therapeutic targets. Here we report the development of a non-radioactive cell-based method for the screening of SGLT inhibitors using COS-7 cells transiently expressing human SGLT1 (hSGLT1), CHO-K1 cells stably expressing human SGLT2 (hSGLT2), and a novel fluorescent d-glucose analogue 1-NBDG as a substrate. Our data indicate that 1-NBDG can be a good replacement for the currently used isotope-labeled SGLT substrate, (14)C-AMG. The Michaelis constant of 1-NBDG transport (0.55 mM) is similar to that of d-glucose (0.51 mM) and AMG (0.40 mM) transport through hSGLT1. The IC50 values of a SGLT inhibitor phlorizin for hSGLT1 obtained using 1-NBDG and (14)C-AMG were identical (0.11 μM) in our cell-based system. The IC50 values of dapagliflozin, a well-known selective SGLT2 inhibitor, for hSGLT2 and hSGLT1 determined using 1-NBDG were 1.86 nM and 880 nM, respectively, which are comparable to the published results obtained using (14)C-AMG. Compared to (14)C-AMG, the use of 1-NBDG is cost-effective, convenient and potentially more sensitive. Taken together, a non-radioactive system using 1-NBDG has been validated as a rapid and reliable method for the screening of SGLT1 and SGLT2 inhibitors.

In silico activity profiling reveals the mechanism of action of antimalarials discovered in a high-throughput screen

PubMed Central

Plouffe, David; Brinker, Achim; McNamara, Case; Henson, Kerstin; Kato, Nobutaka; Kuhen, Kelli; Nagle, Advait; Adrián, Francisco; Matzen, Jason T.; Anderson, Paul; Nam, Tae-gyu; Gray, Nathanael S.; Chatterjee, Arnab; Janes, Jeff; Yan, S. Frank; Trager, Richard; Caldwell, Jeremy S.; Schultz, Peter G.; Zhou, Yingyao; Winzeler, Elizabeth A.

2008-01-01

The growing resistance to current first-line antimalarial drugs represents a major health challenge. To facilitate the discovery of new antimalarials, we have implemented an efficient and robust high-throughput cell-based screen (1,536-well format) based on proliferation of Plasmodium falciparum (Pf) in erythrocytes. From a screen of ≈1.7 million compounds, we identified a diverse collection of ≈6,000 small molecules comprised of >530 distinct scaffolds, all of which show potent antimalarial activity (<1.25 μM). Most known antimalarials were identified in this screen, thus validating our approach. In addition, we identified many novel chemical scaffolds, which likely act through both known and novel pathways. We further show that in some cases the mechanism of action of these antimalarials can be determined by in silico compound activity profiling. This method uses large datasets from unrelated cellular and biochemical screens and the guilt-by-association principle to predict which cellular pathway and/or protein target is being inhibited by select compounds. In addition, the screening method has the potential to provide the malaria community with many new starting points for the development of biological probes and drugs with novel antiparasitic activities. PMID:18579783

Cyquant cell proliferation assay as a fluorescence-based method for in vitro screening of antimalarial activity.

PubMed

Sriwilaijaroen, Nongluk; Kelly, Jane Xu; Riscoe, Michael; Wilairat, Prapon

2004-12-01

The appearance of drug resistant parasites and the absence of an effective vaccine have resulted in the need for new effective antimalarial drugs. Consequently, a convenient method for in vitro screening of large numbers of antimalarial drug candidates has become apparent. The CyQUANT cell proliferation assay is a highly sensitive fluorescence-based method for quantitation of cell number by measuring the strong fluorescence produced when green GR dye binds to nucleic acids. We have applied the CyQUANT assay method to evaluate the growth of Plasmodium falciparum D6 strain in culture. The GR-nucleic acid fluorescence linearly correlated with percent parasitemia at both 0.75 or 1 percent hematocrit with the same correlation coefficient of r2 = 0.99. The sensitivity of P. falciparum D6 strain to chloroquine and to 3,6-bis-omega-diethylaminoamyloxyxanthone, a novel antimalarial, determined by the CyQUANT assay were comparable to those obtained by the traditional [3H]-ethanolamine assay: IC50 value of chloroquine was 54 nM and 51 nM by the CyQUANT and [3H]-ethanolamine assay, respectively; IC50 value for 3,6-bis-omega-diethylaminoamyloxyxanthone was 254 nM and 223 nM by the CyQUANT and [3H]-ethanolamine assay, respectively. This procedure requires no radioisotope, uses simple equipment, and is an easy and convenient procedure, with no washing and harvesting steps. Moreover, all procedures can be set up continuously and thus, the CyQUANT assay is suitable in automatic high through-put drug screening of antimalarial drugs.

Interrogation of ethnomedicinal plants for synthetic lethality effects in combination with deficiency in the DNA repair endonuclease RAD1 using a yeast cell-based assay.

PubMed

Aung, Hsu Mon; Huangteerakul, Chananya; Panvongsa, Wittaya; Jensen, Amornrat N; Chairoungdua, Arthit; Sukrong, Suchada; Jensen, Laran T

2018-09-15

Plant materials used in this study were selected based on the ethnobotanical literature. Plants have either been utilized by Thai practitioners as alternative treatments for cancer or identified to exhibit anti-cancer properties. To screen ethnomedicinal plants using a yeast cell-based assay for synthetic lethal interactions with cells deleted for RAD1, the yeast homologue of human ERCC4 (XPF) MATERIALS AND METHODS: Ethanolic extracts from thirty-two species of medicinal plants utilized in Thai traditional medicine were screened for synthetic lethal/sick interactions using a yeast cell-based assay. Cell growth was compared between the parental strain and rad1∆ yeast following exposure to select for specific toxicity of plant extracts. Candidate extracts were further examined for the mode of action using genetic and biochemical approaches. Screening a library of ethanolic extracts from medicinal plants identified Bacopa monnieri and Colubrina asiatica as having synthetic lethal effects in the rad1∆ cells but not the parental strain. Synthetic lethal effects for B. monneiri extracts were more apparent and this plant was examined further. Genetic analysis indicates that pro-oxidant activities and defective excision repair pathways do not significantly contribute to enhanced sensitivity to B. monneiri extracts. Exposure to B. monneiri extracts resulted in nuclear fragmentation and elevated levels of ethidium bromide staining in rad1∆ yeast suggesting promotion of an apoptosis-like event. Growth inhibition also observed in the human Caco-2 cell line suggesting the effects of B. monnieri extracts on both yeast and human cells may be similar. B. monneiri extracts may have utility in treatment of colorectal cancers that exhibit deficiency in ERCC4 (XPF). Copyright © 2018 Elsevier B.V. All rights reserved.

Methodologies for screening of bacteria-carbohydrate interactions: anti-adhesive milk oligosaccharides as a case study.

PubMed

Lane, Jonathan A; Mariño, Karina; Rudd, Pauline M; Carrington, Stephen D; Slattery, Helen; Hickey, Rita M

2012-07-01

Many studies have demonstrated the capacity of glycan-based compounds to disrupt microbial binding to mucosal epithelia. Therefore, oligosaccharides have potential application in the prevention of certain bacterial diseases. However, current screening methods for the identification of anti-adhesive oligosaccharides have limitations: they are time-consuming and require large amounts of oligosaccharides. There is a need to develop analytical techniques which can quickly screen for, and structurally define, anti-adhesive oligosaccharides prior to using human cell line models of infection. Considering this, we have developed a rapid method for screening complex oligosaccharide mixtures for potential anti-adhesive activity against bacteria. Our approach involves the use of whole bacterial cells to "deplete" free oligosaccharides from solution. As a case study, the free oligosaccharides from the colostrum of Holstein Friesian cows were screened for interactions with whole Escherichia coli cells. Reductions in oligosaccharide concentrations were determined by High pH Anion Exchange Chromatography and Hydrophilic Interaction Liquid Chromatography (HILIC-HPLC). Oligosaccharide structures were confirmed by a combination of HILIC-HPLC, exoglycosidase digestion and off-line negative ion mode MS/MS. The depletion assay confirmed selective bacterial interaction with certain bovine oligosaccharides which in previous studies, by other methodologies, had been shown to interact with E. coli. In particular, the bacterial cells depleted the following oligosaccharides in a population dependent manner: 3'-sialyllactose, disialyllactose, and 6'-sialyllactosamine. The assay methodology was further validated by studies in which we demonstrated the inhibitory activity of 3'-sialyllactose, and a mixture of bovine colostrum oligosaccharides, on E. coli adhesion to differentiated HT-29 cells. Copyright © 2012 Elsevier B.V. All rights reserved.

Human iPSC-derived cardiomyocytes and tissue engineering strategies for disease modeling and drug screening

PubMed Central

Smith, Alec S.T.; Macadangdang, Jesse; Leung, Winnie; Laflamme, Michael A.; Kim, Deok-Ho

2016-01-01

Improved methodologies for modeling cardiac disease phenotypes and accurately screening the efficacy and toxicity of potential therapeutic compounds are actively being sought to advance drug development and improve disease modeling capabilities. To that end, much recent effort has been devoted to the development of novel engineered biomimetic cardiac tissue platforms that accurately recapitulate the structure and function of the human myocardium. Within the field of cardiac engineering, induced pluripotent stem cells (iPSCs) are an exciting tool that offer the potential to advance the current state of the art, as they are derived from somatic cells, enabling the development of personalized medical strategies and patient specific disease models. Here we review different aspects of iPSC-based cardiac engineering technologies. We highlight methods for producing iPSC-derived cardiomyocytes (iPSC-CMs) and discuss their application to compound efficacy/toxicity screening and in vitro modeling of prevalent cardiac diseases. Special attention is paid to the application of micro- and nano-engineering techniques for the development of novel iPSC-CM based platforms and their potential to advance current preclinical screening modalities. PMID:28007615

Protein Transduction Based Therapies for Breast Cancer

DTIC Science & Technology

2006-07-01

we also have developed a method for screening for tissue-targeted transduction peptides using an M13 peptide phage display library. Using this...Instead the focus was on the ability to identify a tumor specific peptide. Task 4. An M13 peptide phage display library will be used for...cancespecific tumor lines by screening a peptide phage display library both in cell culture as well as in nude micebearing xenografts. Initial results in

A novel screen-printed mast cell-based electrochemical sensor for detecting spoilage bacterial quorum signaling molecules (N-acyl-homoserine-lactones) in freshwater fish.

PubMed

Jiang, Donglei; Liu, Yan; Jiang, Hui; Rao, Shengqi; Fang, Wu; Wu, Mangang; Yuan, Limin; Fang, Weiming

2018-04-15

A novel screen-printed cell-based electrochemical sensor was developed to assess bacterial quorum signaling molecules, N-acylhomoserine lactones (AHLs). Screen-printed carbon electrode (SPCE), which possesses excellent properties such as low-cost, disposable and energy-efficient, was modified with multi-walled carbon nanotubes (MWNTs) to improve electrochemical signals and enhance the sensitivity. Rat basophilic leukemia (RBL-2H3) mast cells encapsulated in alginate/graphene oxide (NaAgl/GO) hydrogel were immobilized on the MWNTs/SPCE to serve as recognition element. Electrochemical impedance spectroscopy (EIS) was employed to record the cell impedance signal as-influenced by Pseudomonas aeruginosa quorum-sensing molecule, N-3-oxododecanoyl homoserine lactone (3OC 12 -HSL). Experimental results show that 3OC 12 -HSL caused a significant decrease in cell viability in a dose dependent manner. The EIS value decreased with concentrations of 3OC 12 -HSL in the range of 0.1-1μM, and the detection limit for 3OC 12 -HSL was calculated to be 0.094μM. These results were confirmed via cell viability, SEM, TEM analysis. Next, the sensor was successfully applied to monitoring the production of AHLs by spoilage bacteria in three different freshwater fish juice samples which efficiently proved the practicability of this cell based method. Therefore, the proposed cell sensor may serve as an innovative and effective approach to the measurement of quorum signaling molecule and thus provides a new avenue for real-time monitoring the spoilage bacteria in freshwater fish production. Copyright © 2017 Elsevier B.V. All rights reserved.

Enhanced HTS hit selection via a local hit rate analysis.

PubMed

Posner, Bruce A; Xi, Hualin; Mills, James E J

2009-10-01

The postprocessing of high-throughput screening (HTS) results is complicated by the occurrence of false positives (inactive compounds misidentified as active by the primary screen) and false negatives (active compounds misidentified as inactive by the primary screen). An activity cutoff is frequently used to select "active" compounds from HTS data; however, this approach is insensitive to both false positives and false negatives. An alternative method that can minimize the occurrence of these artifacts will increase the efficiency of hit selection and therefore lead discovery. In this work, rather than merely using the activity of a given compound, we look at the presence and absence of activity among all compounds in its "chemical space neighborhood" to give a degree of confidence in its activity. We demonstrate that this local hit rate (LHR) analysis method outperforms hit selection based on ranking by primary screen activity values across ten diverse high throughput screens, spanning both cell-based and biochemical assay formats of varying biology and robustness. On average, the local hit rate analysis method was approximately 2.3-fold and approximately 1.3-fold more effective in identifying active compounds and active chemical series, respectively, than selection based on primary activity alone. Moreover, when applied to finding false negatives, this method was 2.3-fold better than ranking by primary activity alone. In most cases, novel hit series were identified that would have otherwise been missed. Additional uses of and observations regarding this HTS analysis approach are also discussed.

Systematic screening of isogenic cancer cells identifies DUSP6 as context-specific synthetic lethal target in melanoma

PubMed Central

Wittig-Blaich, Stephanie; Wittig, Rainer; Schmidt, Steffen; Lyer, Stefan; Bewerunge-Hudler, Melanie; Gronert-Sum, Sabine; Strobel-Freidekind, Olga; Müller, Carolin; List, Markus; Jaskot, Aleksandra; Christiansen, Helle; Hafner, Mathias; Schadendorf, Dirk; Block, Ines; Mollenhauer, Jan

2017-01-01

Next-generation sequencing has dramatically increased genome-wide profiling options and conceptually initiates the possibility for personalized cancer therapy. State-of-the-art sequencing studies yield large candidate gene sets comprising dozens or hundreds of mutated genes. However, few technologies are available for the systematic downstream evaluation of these results to identify novel starting points of future cancer therapies. We improved and extended a site-specific recombination-based system for systematic analysis of the individual functions of a large number of candidate genes. This was facilitated by a novel system for the construction of isogenic constitutive and inducible gain- and loss-of-function cell lines. Additionally, we demonstrate the construction of isogenic cell lines with combinations of the traits for advanced functional in vitro analyses. In a proof-of-concept experiment, a library of 108 isogenic melanoma cell lines was constructed and 8 genes were identified that significantly reduced viability in a discovery screen and in an independent validation screen. Here, we demonstrate the broad applicability of this recombination-based method and we proved its potential to identify new drug targets via the identification of the tumor suppressor DUSP6 as potential synthetic lethal target in melanoma cell lines with BRAF V600E mutations and high DUSP6 expression. PMID:28423600

High-throughput screening with nanoimprinting 3D culture for efficient drug development by mimicking the tumor environment.

PubMed

Yoshii, Yukie; Furukawa, Takako; Waki, Atsuo; Okuyama, Hiroaki; Inoue, Masahiro; Itoh, Manabu; Zhang, Ming-Rong; Wakizaka, Hidekatsu; Sogawa, Chizuru; Kiyono, Yasushi; Yoshii, Hiroshi; Fujibayashi, Yasuhisa; Saga, Tsuneo

2015-05-01

Anti-cancer drug development typically utilizes high-throughput screening with two-dimensional (2D) cell culture. However, 2D culture induces cellular characteristics different from tumors in vivo, resulting in inefficient drug development. Here, we report an innovative high-throughput screening system using nanoimprinting 3D culture to simulate in vivo conditions, thereby facilitating efficient drug development. We demonstrated that cell line-based nanoimprinting 3D screening can more efficiently select drugs that effectively inhibit cancer growth in vivo as compared to 2D culture. Metabolic responses after treatment were assessed using positron emission tomography (PET) probes, and revealed similar characteristics between the 3D spheroids and in vivo tumors. Further, we developed an advanced method to adopt cancer cells from patient tumor tissues for high-throughput drug screening with nanoimprinting 3D culture, which we termed Cancer tissue-Originated Uniformed Spheroid Assay (COUSA). This system identified drugs that were effective in xenografts of the original patient tumors. Nanoimprinting 3D spheroids showed low permeability and formation of hypoxic regions inside, similar to in vivo tumors. Collectively, the nanoimprinting 3D culture provides easy-handling high-throughput drug screening system, which allows for efficient drug development by mimicking the tumor environment. The COUSA system could be a useful platform for drug development with patient cancer cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

Phenotypic feature quantification of patient derived 3D cancer spheroids in fluorescence microscopy image

NASA Astrophysics Data System (ADS)

Kang, Mi-Sun; Rhee, Seon-Min; Seo, Ji-Hyun; Kim, Myoung-Hee

2017-03-01

Patients' responses to a drug differ at the cellular level. Here, we present an image-based cell phenotypic feature quantification method for predicting the responses of patient-derived glioblastoma cells to a particular drug. We used high-content imaging to understand the features of patient-derived cancer cells. A 3D spheroid culture formation resembles the in vivo environment more closely than 2D adherent cultures do, and it allows for the observation of cellular aggregate characteristics. However, cell analysis at the individual level is more challenging. In this paper, we demonstrate image-based phenotypic screening of the nuclei of patient-derived cancer cells. We first stitched the images of each well of the 384-well plate with the same state. We then used intensity information to detect the colonies. The nuclear intensity and morphological characteristics were used for the segmentation of individual nuclei. Next, we calculated the position of each nucleus that is appeal of the spatial pattern of cells in the well environment. Finally, we compared the results obtained using 3D spheroid culture cells with those obtained using 2D adherent culture cells from the same patient being treated with the same drugs. This technique could be applied for image-based phenotypic screening of cells to determine the patient's response to the drug.

Flow Cytometry Enables Multiplexed Measurements of Genetically Encoded Intramolecular FRET Sensors Suitable for Screening.

PubMed

Doucette, Jaimee; Zhao, Ziyan; Geyer, Rory J; Barra, Melanie M; Balunas, Marcy J; Zweifach, Adam

2016-07-01

Genetically encoded sensors based on intramolecular FRET between CFP and YFP are used extensively in cell biology research. Flow cytometry has been shown to offer a means to measure CFP-YFP FRET; we suspected it would provide a unique way to conduct multiplexed measurements from cells expressing different FRET sensors, which is difficult to do with microscopy, and that this could be used for screening. We confirmed that flow cytometry accurately measures FRET signals using cells transiently transfected with an ERK activity reporter, comparing responses measured with imaging and cytometry. We created polyclonal long-term transfectant lines, each expressing a different intramolecular FRET sensor, and devised a way to bar-code four distinct populations of cells. We demonstrated the feasibility of multiplexed measurements and determined that robust multiplexed measurements can be conducted in plate format. To validate the suitability of the method for screening, we measured responses from a plate of bacterial extracts that in unrelated experiments we had determined contained the protein kinase C (PKC)-activating compound teleocidin A-1. The multiplexed assay correctly identifying the teleocidin A-1-containing well. We propose that multiplexed cytometric FRET measurements will be useful for analyzing cellular function and for screening compound collections. © 2016 Society for Laboratory Automation and Screening.

A grid matrix-based Raman spectroscopic method to characterize different cell milieu in biopsied axillary sentinel lymph nodes of breast cancer patients.

PubMed

Som, Dipasree; Tak, Megha; Setia, Mohit; Patil, Asawari; Sengupta, Amit; Chilakapati, C Murali Krishna; Srivastava, Anurag; Parmar, Vani; Nair, Nita; Sarin, Rajiv; Badwe, R

2016-01-01

Raman spectroscopy which is based upon inelastic scattering of photons has a potential to emerge as a noninvasive bedside in vivo or ex vivo molecular diagnostic tool. There is a need to improve the sensitivity and predictability of Raman spectroscopy. We developed a grid matrix-based tissue mapping protocol to acquire cellular-specific spectra that also involved digital microscopy for localizing malignant and lymphocytic cells in sentinel lymph node biopsy sample. Biosignals acquired from specific cellular milieu were subjected to an advanced supervised analytical method, i.e., cross-correlation and peak-to-peak ratio in addition to PCA and PC-LDA. We observed decreased spectral intensity as well as shift in the spectral peaks of amides and lipid bands in the completely metastatic (cancer cells) lymph nodes with high cellular density. Spectral library of normal lymphocytes and metastatic cancer cells created using the cellular specific mapping technique can be utilized to create an automated smart diagnostic tool for bench side screening of sampled lymph nodes. Spectral library of normal lymphocytes and metastatic cancer cells created using the cellular specific mapping technique can be utilized to develop an automated smart diagnostic tool for bench side screening of sampled lymph nodes supported by ongoing global research in developing better technology and signal and big data processing algorithms.

Solar cell anomaly detection method and apparatus

NASA Technical Reports Server (NTRS)

Miller, Emmett L. (Inventor); Shumka, Alex (Inventor); Gauthier, Michael K. (Inventor)

1981-01-01

A method is provided for detecting cracks and other imperfections in a solar cell, which includes scanning a narrow light beam back and forth across the cell in a raster pattern, while monitoring the electrical output of the cell to find locations where the electrical output varies significantly. The electrical output can be monitored on a television type screen containing a raster pattern with each point on the screen corresponding to a point on the solar cell surface, and with the brightness of each point on the screen corresponding to the electrical output from the cell which was produced when the light beam was at the corresponding point on the cell. The technique can be utilized to scan a large array of interconnected solar cells, to determine which ones are defective.

[HTRF-based high-throughput PGE2 release prohibition model and application in discovering traditional Chinese medicine active ingredients].

PubMed

Bai, Zhi-Ru; Fei, Hong-Qiang; Li, Na; Cao, Liang; Zhang, Chen-Feng; Wang, Tuan-Jie; Ding, Gang; Wang, Zhen-Zhong; Xiao, Wei

2016-02-01

Prostaglandin (PG) E2 is an active substance in pathological and physiological mechanisms, such as inflammation and pain. The in vitro high-throughput assay for screening the inhibitors of reducing PEG2 production is a useful method for finding out antiphlogistic and analgesic candidates. The assay was based on LPS-induced PGE2 production model using a homogeneous time-resolved fluorescence(HTRF) PGE2 testing kit combined with liquid handling automation and detection instruments. The critical steps, including the cell density optimization and IC50 values determination of a positive compound, were taken to verify the stability and sensibility of the assay. Low intra-plate, inter-plate and day-to-day variability were observed in this 384-well, high-throughput format assay. Totally 5 121 samples were selected from the company's traditional Chinese medicine(TCM) material base library and used to screen PGE2 inhibitors. In this model, the cell plating density was 2 000 cells for each well; the average IC₅₀ value for positive compounds was (7.3±0.1) μmol; the Z' factor for test plates was more than 0.5 and averaged at 0.7. Among the 5 121 samples, 228 components exhibited a PGE2 production prohibition rate of more than 50%, and 23 components exhibited more than 80%. This model reached the expected standards in data stability and accuracy, indicating the reliability and authenticity of the screening results. The automated screening system was introduced to make the model fast and efficient, with a average daily screening amount exceeding 14 000 data points and provide a new model for discovering new anti-inflammatory and analgesic drug and quickly screening effective constituents of TCM in the early stage. Copyright© by the Chinese Pharmaceutical Association.

High content analysis of phagocytic activity and cell morphology with PuntoMorph.

PubMed

Al-Ali, Hassan; Gao, Han; Dalby-Hansen, Camilla; Peters, Vanessa Ann; Shi, Yan; Brambilla, Roberta

2017-11-01

Phagocytosis is essential for maintenance of normal homeostasis and healthy tissue. As such, it is a therapeutic target for a wide range of clinical applications. The development of phenotypic screens targeting phagocytosis has lagged behind, however, due to the difficulties associated with image-based quantification of phagocytic activity. We present a robust algorithm and cell-based assay system for high content analysis of phagocytic activity. The method utilizes fluorescently labeled beads as a phagocytic substrate with defined physical properties. The algorithm employs statistical modeling to determine the mean fluorescence of individual beads within each image, and uses the information to conduct an accurate count of phagocytosed beads. In addition, the algorithm conducts detailed and sophisticated analysis of cellular morphology, making it a standalone tool for high content screening. We tested our assay system using microglial cultures. Our results recapitulated previous findings on the effects of microglial stimulation on cell morphology and phagocytic activity. Moreover, our cell-level analysis revealed that the two phenotypes associated with microglial activation, specifically cell body hypertrophy and increased phagocytic activity, are not highly correlated. This novel finding suggests the two phenotypes may be under the control of distinct signaling pathways. We demonstrate that our assay system outperforms preexisting methods for quantifying phagocytic activity in multiple dimensions including speed, accuracy, and resolution. We provide a framework to facilitate the development of high content assays suitable for drug screening. For convenience, we implemented our algorithm in a standalone software package, PuntoMorph. Copyright © 2017 Elsevier B.V. All rights reserved.

Label-free resistive-pulse cytometry.

PubMed

Chapman, M R; Sohn, L L

2011-01-01

Numerous methods have recently been developed to characterize cells for size, shape, and specific cell-surface markers. Most of these methods rely upon exogenous labeling of the cells and are better suited for large cell populations (>10,000). Here, we review a label-free method of characterizing and screening cells based on the Coulter-counter technique of particle sizing: an individual cell transiting a microchannel (or "pore") causes a downward pulse in the measured DC current across that "pore". Pulse magnitude corresponds to the cell size, pulse width to the transit time needed for the cell to pass through the pore, and pulse shape to how the cell traverses across the pore (i.e., rolling or tumbling). When the pore is functionalized with an antibody that is specific to a surface-epitope of interest, label-free screening of a specific marker is possible, as transient binding between the two results in longer time duration than when the pore is unfunctionalized or functionalized with a nonspecific antibody. While this method cannot currently compete with traditional technology in terms of throughput, there are a number of applications for which this technology is better suited than current commercial cytometry systems. Applications include the rapid and nondestructive analysis of small cell populations (<100), which is not possible with current technology, and a platform for providing true point-of-care clinical diagnostics, due to the simplicity of the device, low manufacturing costs, and ease of use. Copyright © 2011 Elsevier Inc. All rights reserved.

A cell cycle-independent, conditional gene inactivation strategy for differentially tagging wild-type and mutant cells.

PubMed

Nagarkar-Jaiswal, Sonal; Manivannan, Sathiya N; Zuo, Zhongyuan; Bellen, Hugo J

2017-05-31

Here, we describe a novel method based on intronic MiMIC insertions described in Nagarkar-Jaiswal et al. (2015) to perform conditional gene inactivation in Drosophila . Mosaic analysis in Drosophila cannot be easily performed in post-mitotic cells. We therefore, therefore, developed Flip-Flop, a flippase -dependent in vivo cassette-inversion method that marks wild-type cells with the endogenous EGFP-tagged protein, whereas mutant cells are marked with mCherry upon inversion. We document the ease and usefulness of this strategy in differential tagging of wild-type and mutant cells in mosaics. We use this approach to phenotypically characterize the loss of SNF4Aγ , encoding the γ subunit of the AMP Kinase complex. The Flip-Flop method is efficient and reliable, and permits conditional gene inactivation based on both spatial and temporal cues, in a cell cycle-, and developmental stage-independent fashion, creating a platform for systematic screens of gene function in developing and adult flies with unprecedented detail.

CRISPR/Cas9-mediated gene knockout screens and target identification via whole-genome sequencing uncover host genes required for picornavirus infection.

PubMed

Kim, Heon Seok; Lee, Kyungjin; Bae, Sangsu; Park, Jeongbin; Lee, Chong-Kyo; Kim, Meehyein; Kim, Eunji; Kim, Minju; Kim, Seokjoong; Kim, Chonsaeng; Kim, Jin-Soo

2017-06-23

Several groups have used genome-wide libraries of lentiviruses encoding small guide RNAs (sgRNAs) for genetic screens. In most cases, sgRNA expression cassettes are integrated into cells by using lentiviruses, and target genes are statistically estimated by the readout of sgRNA sequences after targeted sequencing. We present a new virus-free method for human gene knockout screens using a genome-wide library of CRISPR/Cas9 sgRNAs based on plasmids and target gene identification via whole-genome sequencing (WGS) confirmation of authentic mutations rather than statistical estimation through targeted amplicon sequencing. We used 30,840 pairs of individually synthesized oligonucleotides to construct the genome-scale sgRNA library, collectively targeting 10,280 human genes ( i.e. three sgRNAs per gene). These plasmid libraries were co-transfected with a Cas9-expression plasmid into human cells, which were then treated with cytotoxic drugs or viruses. Only cells lacking key factors essential for cytotoxic drug metabolism or viral infection were able to survive. Genomic DNA isolated from cells that survived these challenges was subjected to WGS to directly identify CRISPR/Cas9-mediated causal mutations essential for cell survival. With this approach, we were able to identify known and novel genes essential for viral infection in human cells. We propose that genome-wide sgRNA screens based on plasmids coupled with WGS are powerful tools for forward genetics studies and drug target discovery. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Automated analysis of siRNA screens of cells infected by hepatitis C and dengue viruses based on immunofluorescence microscopy images

NASA Astrophysics Data System (ADS)

Matula, Petr; Kumar, Anil; Wörz, Ilka; Harder, Nathalie; Erfle, Holger; Bartenschlager, Ralf; Eils, Roland; Rohr, Karl

2008-03-01

We present an image analysis approach as part of a high-throughput microscopy siRNA-based screening system using cell arrays for the identification of cellular genes involved in hepatitis C and dengue virus replication. Our approach comprises: cell nucleus segmentation, quantification of virus replication level in the neighborhood of segmented cell nuclei, localization of regions with transfected cells, cell classification by infection status, and quality assessment of an experiment and single images. In particular, we propose a novel approach for the localization of regions of transfected cells within cell array images, which combines model-based circle fitting and grid fitting. By this scheme we integrate information from single cell array images and knowledge from the complete cell arrays. The approach is fully automatic and has been successfully applied to a large number of cell array images from screening experiments. The experimental results show a good agreement with the expected behaviour of positive as well as negative controls and encourage the application to screens from further high-throughput experiments.

First steps towards the successful surface-based cultivation of human embryonic stem cells in hanging drop systems.

PubMed

Schulz, Julia C; Stumpf, Patrick S; Katsen-Globa, Alisa; Sachinidis, Agapios; Hescheler, Jürgen; Zimmermann, Heiko

2012-11-01

Miniaturization and parallelization of cell culture procedures are in focus of research in order to develop test platforms with low material consumption and increased standardization for toxicity and drug screenings. The cultivation in hanging drops (HDs) is a convenient and versatile tool for biological applications and represents an interesting model system for the screening applications due to its uniform shape, the advantageous gas supply, and the small volume. However, its application has so far been limited to non-adherent and aggregate forming cells. Here, we describe for the first time the proof-of-principle regarding the adherent cultivation of human embryonic stem cells in HD. For this microcarriers were added to the droplet as dynamic cultivation surfaces resulting in a maintained pluripotency and proliferation capacity for 10 days. This enables the HD technique to be extended to the cultivation of adherence-dependent stem cells. Also, the possible automation of this method by implementation of liquid handling systems opens new possibilities for miniaturized screenings, the improvement of cultivation and differentiation conditions, and toxicity and drug development.

First steps towards the successful surface-based cultivation of human embryonic stem cells in hanging drop systems

PubMed Central

Schulz, Julia C; Stumpf, Patrick S; Katsen-Globa, Alisa; Sachinidis, Agapios; Hescheler, Jürgen; Zimmermann, Heiko

2012-01-01

Miniaturization and parallelization of cell culture procedures are in focus of research in order to develop test platforms with low material consumption and increased standardization for toxicity and drug screenings. The cultivation in hanging drops (HDs) is a convenient and versatile tool for biological applications and represents an interesting model system for the screening applications due to its uniform shape, the advantageous gas supply, and the small volume. However, its application has so far been limited to non‐adherent and aggregate forming cells. Here, we describe for the first time the proof-of-principle regarding the adherent cultivation of human embryonic stem cells in HD. For this microcarriers were added to the droplet as dynamic cultivation surfaces resulting in a maintained pluripotency and proliferation capacity for 10 days. This enables the HD technique to be extended to the cultivation of adherence-dependent stem cells. Also, the possible automation of this method by implementation of liquid handling systems opens new possibilities for miniaturized screenings, the improvement of cultivation and differentiation conditions, and toxicity and drug development. PMID:23486530

Fourier-transform infrared spectroscopy for rapid screening and live-cell monitoring: application to nanotoxicology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sundaram, S. K.; Sacksteder, Colette A.; Weber, T. J.

2013-01-01

A significant challenge to realize the full potential of nanotechnology for therapeutic and diagnostic applications is to understand and evaluate how live-cells interact with an external stimulus, e.g., a nanosized particle (NSP), and the toxicity and broad risk associated with these stimuli. NSPs are increasingly used in medicine with largely undetermined hazards in complex cell dynamics and environments. It is difficult to capture the complexity and dynamics of these interactions by following an omics-based approach exclusively, which are expensive and time-consuming. Additionally, this approach needs destructive sampling methods. Live-cell attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectrometry is well suited tomore » provide noninvasive approach to provide rapid screening of cellular responses to potentially toxic NSPs or any stimuli. Herein we review the technical basis of the approach, the instrument configuration and interface with the biological media, and various effects that impact the data, data analysis, and toxicity. Our preliminary results on live-cell monitoring show promise for rapid screening the NSPs.« less

Single tube genotyping of sickle cell anaemia using PCR-based SNP analysis

PubMed Central

Waterfall, Christy M.; Cobb, Benjamin D.

2001-01-01

Allele-specific amplification (ASA) is a generally applicable technique for the detection of known single nucleotide polymorphisms (SNPs), deletions, insertions and other sequence variations. Conventionally, two reactions are required to determine the zygosity of DNA in a two-allele system, along with significant upstream optimisation to define the specific test conditions. Here, we combine single tube bi-directional ASA with a ‘matrix-based’ optimisation strategy, speeding up the whole process in a reduced reaction set. We use sickle cell anaemia as our model SNP system, a genetic disease that is currently screened using ASA methods. Discriminatory conditions were rapidly optimised enabling the unambiguous identification of DNA from homozygous sickle cell patients (HbS/S), heterozygous carriers (HbA/S) or normal DNA in a single tube. Simple downstream mathematical analyses based on product yield across the optimisation set allow an insight into the important aspects of priming competition and component interactions in this competitive PCR. This strategy can be applied to any polymorphism, defining specific conditions using a multifactorial approach. The inherent simplicity and low cost of this PCR-based method validates bi-directional ASA as an effective tool in future clinical screening and pharmacogenomic research where more expensive fluorescence-based approaches may not be desirable. PMID:11726702

Comparison of cytology, HPV DNA testing and HPV 16/18 genotyping alone or combined targeting to the more balanced methodology for cervical cancer screening.

PubMed

Chatzistamatiou, Kimon; Moysiadis, Theodoros; Moschaki, Viktoria; Panteleris, Nikolaos; Agorastos, Theodoros

2016-07-01

The objective of the present study was to identify the most effective cervical cancer screening algorithm incorporating different combinations of cytology, HPV testing and genotyping. Women 25-55years old recruited for the "HERMES" (HEllenic Real life Multicentric cErvical Screening) study were screened in terms of cytology and high-risk (hr) HPV testing with HPV 16/18 genotyping. Women positive for cytology or/and hrHPV were referred for colposcopy, biopsy and treatment. Ten screening algorithms based on different combinations of cytology, HPV testing and HPV 16/18 genotyping were investigated in terms of diagnostic accuracy. Three clusters of algorithms were formed according to the balance between effectiveness and harm caused by screening. The cluster showing the best balance included two algorithms based on co-testing and two based on HPV primary screening with HPV 16/18 genotyping. Among these, hrHPV testing with HPV 16/18 genotyping and reflex cytology (atypical squamous cells of undetermined significance - ASCUS threshold) presented the optimal combination of sensitivity (82.9%) and specificity relative to cytology alone (0.99) with 1.26 false positive rate relative to cytology alone. HPV testing with HPV 16/18 genotyping, referring HPV 16/18 positive women directly to colposcopy, and hrHPV (non 16/18) positive women to reflex cytology (ASCUS threshold), as a triage method to colposcopy, reflects the best equilibrium between screening effectiveness and harm. Algorithms, based on cytology as initial screening method, on co-testing or HPV primary without genotyping, and on HPV primary with genotyping but without cytology triage, are not supported according to the present analysis. Copyright © 2016 Elsevier Inc. All rights reserved.

Combining Computational Methods for Hit to Lead Optimization in Mycobacterium tuberculosis Drug Discovery

PubMed Central

Ekins, Sean; Freundlich, Joel S.; Hobrath, Judith V.; White, E. Lucile; Reynolds, Robert C

2013-01-01

Purpose Tuberculosis treatments need to be shorter and overcome drug resistance. Our previous large scale phenotypic high-throughput screening against Mycobacterium tuberculosis (Mtb) has identified 737 active compounds and thousands that are inactive. We have used this data for building computational models as an approach to minimize the number of compounds tested. Methods A cheminformatics clustering approach followed by Bayesian machine learning models (based on publicly available Mtb screening data) was used to illustrate that application of these models for screening set selections can enrich the hit rate. Results In order to explore chemical diversity around active cluster scaffolds of the dose-response hits obtained from our previous Mtb screens a set of 1924 commercially available molecules have been selected and evaluated for antitubercular activity and cytotoxicity using Vero, THP-1 and HepG2 cell lines with 4.3%, 4.2% and 2.7% hit rates, respectively. We demonstrate that models incorporating antitubercular and cytotoxicity data in Vero cells can significantly enrich the selection of non-toxic actives compared to random selection. Across all cell lines, the Molecular Libraries Small Molecule Repository (MLSMR) and cytotoxicity model identified ~10% of the hits in the top 1% screened (>10 fold enrichment). We also showed that seven out of nine Mtb active compounds from different academic published studies and eight out of eleven Mtb active compounds from a pharmaceutical screen (GSK) would have been identified by these Bayesian models. Conclusion Combining clustering and Bayesian models represents a useful strategy for compound prioritization and hit-to lead optimization of antitubercular agents. PMID:24132686

Luciferase reporter assay in Drosophila and mammalian tissue culture cells

PubMed Central

Yun, Chi

2014-01-01

Luciferase reporter gene assays are one of the most common methods for monitoring gene activity. Because of their sensitivity, dynamic range, and lack of endogenous activity, luciferase assays have been particularly useful for functional genomics in cell-based assays, such as RNAi screening. This unit describes delivery of two luciferase reporters with other nucleic acids (siRNA /dsRNA), measurement of the dual luciferase activities, and analysis of data generated. The systematic query of gene function (RNAi) combined with the advances in luminescent technology have made it possible to design powerful whole genome screens to address diverse and significant biological questions. PMID:24652620

A novel in-cell ELISA method for screening of compounds inhibiting TRKA phosphorylation, using KM12 cell line harboring TRKA rearrangement.

PubMed

Pandre, Manoj Kumar; Shaik, Shama; Satya Pratap, Veera Venkata Valluri; Yadlapalli, Prasad; Yanamandra, Mahesh; Mitra, Sayan

2018-03-15

Tropomyosin-related kinase A (TRKA) fusion was originally detected in colorectal carcinoma that had resulted in expression of the oncogenic chimeric protein TPM3-TRKA. Lately, many more rearrangements in TRK family of kinases generating oncogenic fusion proteins have been identified. These genetic rearrangements usually result in fusion of cytoplasmic kinase domain of TRK to another gene of interest resulting in constitutive kinase activity. Estimation of TRK inhibitor potency in a cellular context is required for drug discovery programs and is measured by receptor phosphorylation levels upon compound administration. However, since a large chunk of the TRK protein is lost in this rearrangement, it's difficult to set up sandwich ELISA for detection of receptor phosphorylation in any cell assay harboring these fusion proteins. In order to address this issue, we developed a novel and robust in-cell ELISA method which quantifies the phosphorylation of TRK kinase (Tyr 674/675) within the KM12 cells. This cell based method is more versatile & economical than conventional ELISA using engineered overexpressing cell line and/or western blot methods. Performance reliability & robustness for the validated assay were determined by %CV and Z factor in assays with reference molecule larotrectinib. This in-cell ELISA method can be used with any TRKA rearranged oncogenic fusion cell type and can be extended to other TRK isoforms as well. We have used this assay to screen novel molecules in KM12 cells and to study pharmacodynamic properties of compounds in TRKA signaling. Copyright © 2018 Elsevier Inc. All rights reserved.

Droplet Microarray Based on Superhydrophobic-Superhydrophilic Patterns for Single Cell Analysis.

PubMed

Jogia, Gabriella E; Tronser, Tina; Popova, Anna A; Levkin, Pavel A

2016-12-09

Single-cell analysis provides fundamental information on individual cell response to different environmental cues and is a growing interest in cancer and stem cell research. However, current existing methods are still facing challenges in performing such analysis in a high-throughput manner whilst being cost-effective. Here we established the Droplet Microarray (DMA) as a miniaturized screening platform for high-throughput single-cell analysis. Using the method of limited dilution and varying cell density and seeding time, we optimized the distribution of single cells on the DMA. We established culturing conditions for single cells in individual droplets on DMA obtaining the survival of nearly 100% of single cells and doubling time of single cells comparable with that of cells cultured in bulk cell population using conventional methods. Our results demonstrate that the DMA is a suitable platform for single-cell analysis, which carries a number of advantages compared with existing technologies allowing for treatment, staining and spot-to-spot analysis of single cells over time using conventional analysis methods such as microscopy.

Automation-assisted cervical cancer screening in manual liquid-based cytology with hematoxylin and eosin staining.

PubMed

Zhang, Ling; Kong, Hui; Ting Chin, Chien; Liu, Shaoxiong; Fan, Xinmin; Wang, Tianfu; Chen, Siping

2014-03-01

Current automation-assisted technologies for screening cervical cancer mainly rely on automated liquid-based cytology slides with proprietary stain. This is not a cost-efficient approach to be utilized in developing countries. In this article, we propose the first automation-assisted system to screen cervical cancer in manual liquid-based cytology (MLBC) slides with hematoxylin and eosin (H&E) stain, which is inexpensive and more applicable in developing countries. This system consists of three main modules: image acquisition, cell segmentation, and cell classification. First, an autofocusing scheme is proposed to find the global maximum of the focus curve by iteratively comparing image qualities of specific locations. On the autofocused images, the multiway graph cut (GC) is performed globally on the a* channel enhanced image to obtain cytoplasm segmentation. The nuclei, especially abnormal nuclei, are robustly segmented by using GC adaptively and locally. Two concave-based approaches are integrated to split the touching nuclei. To classify the segmented cells, features are selected and preprocessed to improve the sensitivity, and contextual and cytoplasm information are introduced to improve the specificity. Experiments on 26 consecutive image stacks demonstrated that the dynamic autofocusing accuracy was 2.06 μm. On 21 cervical cell images with nonideal imaging condition and pathology, our segmentation method achieved a 93% accuracy for cytoplasm, and a 87.3% F-measure for nuclei, both outperformed state of the art works in terms of accuracy. Additional clinical trials showed that both the sensitivity (88.1%) and the specificity (100%) of our system are satisfyingly high. These results proved the feasibility of automation-assisted cervical cancer screening in MLBC slides with H&E stain, which is highly desirable in community health centers and small hospitals. © 2013 International Society for Advancement of Cytometry.

Aneuploidy screening of embryonic stem cell clones by metaphase karyotyping and droplet digital polymerase chain reaction.

PubMed

Codner, Gemma F; Lindner, Loic; Caulder, Adam; Wattenhofer-Donzé, Marie; Radage, Adam; Mertz, Annelyse; Eisenmann, Benjamin; Mianné, Joffrey; Evans, Edward P; Beechey, Colin V; Fray, Martin D; Birling, Marie-Christine; Hérault, Yann; Pavlovic, Guillaume; Teboul, Lydia

2016-08-05

Karyotypic integrity is essential for the successful germline transmission of alleles mutated in embryonic stem (ES) cells. Classical methods for the identification of aneuploidy involve cytological analyses that are both time consuming and require rare expertise to identify mouse chromosomes. As part of the International Mouse Phenotyping Consortium, we gathered data from over 1,500 ES cell clones and found that the germline transmission (GLT) efficiency of clones is compromised when over 50 % of cells harbour chromosome number abnormalities. In JM8 cells, chromosomes 1, 8, 11 or Y displayed copy number variation most frequently, whilst the remainder generally remain unchanged. We developed protocols employing droplet digital polymerase chain reaction (ddPCR) to accurately quantify the copy number of these four chromosomes, allowing efficient triage of ES clones prior to microinjection. We verified that assessments of aneuploidy, and thus decisions regarding the suitability of clones for microinjection, were concordant between classical cytological and ddPCR-based methods. Finally, we improved the method to include assay multiplexing so that two unstable chromosomes are counted simultaneously (and independently) in one reaction, to enhance throughput and further reduce the cost. We validated a PCR-based method as an alternative to classical karyotype analysis. This technique enables laboratories that are non-specialist, or work with large numbers of clones, to precisely screen ES cells for the most common aneuploidies prior to microinjection to ensure the highest level of germline transmission potential. The application of this method allows early exclusion of aneuploid ES cell clones in the ES cell to mouse conversion process, thus improving the chances of obtaining germline transmission and reducing the number of animals used in failed microinjection attempts. This method can be applied to any other experiments that require accurate analysis of the genome for copy number variation (CNV).

Inhibitors of Helicobacter pylori Protease HtrA Found by ‘Virtual Ligand’ Screening Combat Bacterial Invasion of Epithelia

PubMed Central

Schneider, Petra; Hoy, Benjamin; Wessler, Silja; Schneider, Gisbert

2011-01-01

Background The human pathogen Helicobacter pylori (H. pylori) is a main cause for gastric inflammation and cancer. Increasing bacterial resistance against antibiotics demands for innovative strategies for therapeutic intervention. Methodology/Principal Findings We present a method for structure-based virtual screening that is based on the comprehensive prediction of ligand binding sites on a protein model and automated construction of a ligand-receptor interaction map. Pharmacophoric features of the map are clustered and transformed in a correlation vector (‘virtual ligand’) for rapid virtual screening of compound databases. This computer-based technique was validated for 18 different targets of pharmaceutical interest in a retrospective screening experiment. Prospective screening for inhibitory agents was performed for the protease HtrA from the human pathogen H. pylori using a homology model of the target protein. Among 22 tested compounds six block E-cadherin cleavage by HtrA in vitro and result in reduced scattering and wound healing of gastric epithelial cells, thereby preventing bacterial infiltration of the epithelium. Conclusions/Significance This study demonstrates that receptor-based virtual screening with a permissive (‘fuzzy’) pharmacophore model can help identify small bioactive agents for combating bacterial infection. PMID:21483848

Screening anti-allergic components of Astragali Radix using LAD2 cell membrane chromatography coupled online with UHPLC-ESI-MS/MS method.

PubMed

Lv, Yanni; Sun, Yueming; Fu, Jia; Kong, Liyun; Han, Shengli

2017-02-01

Huangqi (Astragali Radix), a traditional Chinese herb, is widely used in clinical therapy in China. In addition, an anti-allergic effect of constituents in Huangqi has been reported in the scientific literature. In the present study, cell membrane chromatography coupled online with UHPLC-ESI-MS/MS method was developed to screen, analyze and identify the anti-allergic components of Huangqi. The Laboratory of Allergic Disease 2 (LAD2) cell was used to establish cell membrane chromatography, which was combined with UHPLC-ESI-MS/MS. The coupled system was then used to screen anti-allergic components from Huangqi. Effects of active components were verified by histamine release assay. A component retained on the LAD2 cell membrane chromatography was identified as formononetin. Bioactivity of formononetin was investigated by histamine release assay in LAD2 cells, and it was found that formononetin could inhibit histamine release in a dose-dependent manner from 1 to 100 μm. The LAD2 cell membrane chromatography online with UHPLC-ESI-MS/MS method is an effective technique for screening the anti-allergic components of Huangqi. Copyright © 2016 John Wiley & Sons, Ltd.

Cytotoxic and aryl hydrocarbon hydroxylase-inducing effects of laboratory rodent diets. A cell culture study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Toerroenen, R.; Pelkonen, K.; Kaerenlampi, S.

1991-01-01

Extracts of several rodent diets were studied for their cytotoxic and aryl hydrocarbon hydroxylase-inducing properties by an in vitro method. The cell culture system based on a mouse hepatoma cell line (Hepa-1) was shown to be convenient and sensitive method for screening of diets for these parameters implying the presence of compounds potentially harmful in vivo. Considerable differences among diets and batches were detected. Smallest effects were observed with a semipurified diet and with the unrefined diet which - contrary to other four unrefined diets - contained no fish.

In Vitro Toxicity Screening Technique for Volatile Substances Using Flow-Through System

EPA Science Inventory

In 2007 the National Research Council envisioned the need for inexpensive, rapid, cell based toxicity testing methods relevant to human health. Recent advances in robotics, automation, and miniaturization have been used to address these problems. However, one challenge is that ma...

In Vitro Toxicity Screening Technique for Volatile Substances Using Flow-Through System##

EPA Science Inventory

In 2007 the National Research Council envisioned the need for inexpensive, rapid, cell based toxicity testing methods relevant to human health. Recent advances in robotics, automation, and miniaturization have been used to address this challenge. However, one drawback to currentl...

A dinoflagellate mutant with higher frequency of multiple fission.

PubMed

Lam, C M; Chong, C; Wong, J T

2001-01-01

The dinoflagellate Crypthecodinium cohnii Biecheler propagates by both binary and multiple fission. By a newly developed mutagenesis protocol based on using ethyl methanesulfonate and a cell size screening method, a cell cycle mutant, mf2, was isolated with giant cells which predominantly divide by multiple fission. The average cell size of the mutant mf2 is larger than the control C. cohnii. Cell cycle synchronization experiments suggest that mutant mf2, when compared with the control strain, has a prolonged G1 phase with a corresponding delay of the G2 + M phase.

Personalized drug discovery: HCA approach optimized for rare diseases at Tel Aviv University.

PubMed

Solmesky, Leonardo J; Weil, Miguel

2014-03-01

The Cell screening facility for personalized medicine (CSFPM) at Tel Aviv University in Israel is devoted to screening small molecules libraries for finding new drugs for rare diseases using human cell based models. The main strategy of the facility is based on smartly reducing the size of the compounds collection in similarity clusters and at the same time keeping high diversity of pharmacophores. This strategy allows parallel screening of several patient derived - cells in a personalized screening approach. The tested compounds are repositioned drugs derived from collections of phase III and FDA approved small molecules. In addition, the facility carries screenings using other chemical libraries and toxicological characterizations of nanomaterials.

Tumor-Initiating Cells and Methods of Use

NASA Technical Reports Server (NTRS)

Hlatky, Lynn (Inventor)

2014-01-01

Provided herein are an isolated or enriched population of tumor initiating cells derived from normal cells, cells susceptible to neoplasia, or neoplastic cells. Methods of use of the cells for screening for anti-hyperproliferative agents, and use of the cells for animal models of hyperproliferative disorders including metastatic cancer, diagnostic methods, and therapeutic methods are provided.

Identification and Correction of Additive and Multiplicative Spatial Biases in Experimental High-Throughput Screening.

PubMed

Mazoure, Bogdan; Caraus, Iurie; Nadon, Robert; Makarenkov, Vladimir

2018-06-01

Data generated by high-throughput screening (HTS) technologies are prone to spatial bias. Traditionally, bias correction methods used in HTS assume either a simple additive or, more recently, a simple multiplicative spatial bias model. These models do not, however, always provide an accurate correction of measurements in wells located at the intersection of rows and columns affected by spatial bias. The measurements in these wells depend on the nature of interaction between the involved biases. Here, we propose two novel additive and two novel multiplicative spatial bias models accounting for different types of bias interactions. We describe a statistical procedure that allows for detecting and removing different types of additive and multiplicative spatial biases from multiwell plates. We show how this procedure can be applied by analyzing data generated by the four HTS technologies (homogeneous, microorganism, cell-based, and gene expression HTS), the three high-content screening (HCS) technologies (area, intensity, and cell-count HCS), and the only small-molecule microarray technology available in the ChemBank small-molecule screening database. The proposed methods are included in the AssayCorrector program, implemented in R, and available on CRAN.

Facile high-throughput forward chemical genetic screening by in situ monitoring of glucuronidase-based reporter gene expression in Arabidopsis thaliana

PubMed Central

Halder, Vivek; Kombrink, Erich

2015-01-01

The use of biologically active small molecules to perturb biological functions holds enormous potential for investigating complex signaling networks. However, in contrast to animal systems, the search for and application of chemical tools for basic discovery in the plant sciences, generally referred to as “chemical genetics,” has only recently gained momentum. In addition to cultured cells, the well-characterized, small-sized model plant Arabidopsis thaliana is suitable for cultivation in microplates, which allows employing diverse cell- or phenotype-based chemical screens. In such screens, a chemical's bioactivity is typically assessed either through scoring its impact on morphological traits or quantifying molecular attributes such as enzyme or reporter activities. Here, we describe a facile forward chemical screening methodology for intact Arabidopsis seedlings harboring the β-glucuronidase (GUS) reporter by directly quantifying GUS activity in situ with 4-methylumbelliferyl-β-D-glucuronide (4-MUG) as substrate. The quantitative nature of this screening assay has an obvious advantage over the also convenient histochemical GUS staining method, as it allows application of statistical procedures and unbiased hit selection based on threshold values as well as distinction between compounds with strong or weak bioactivity. At the same time, the in situ bioassay is very convenient requiring less effort and time for sample handling in comparison to the conventional quantitative in vitro GUS assay using 4-MUG, as validated with several Arabidopsis lines harboring different GUS reporter constructs. To demonstrate that the developed assays is particularly suitable for large-scale screening projects, we performed a pilot screen for chemical activators or inhibitors of salicylic acid-mediated defense signaling using the Arabidopsis PR1p::GUS line. Importantly, the screening methodology provided here can be adopted for any inducible GUS reporter line. PMID:25688251

Simple and fast screening of G-quadruplex ligands with electrochemical detection system.

PubMed

Fan, Qiongxuan; Li, Chao; Tao, Yaqin; Mao, Xiaoxia; Li, Genxi

2016-11-01

Small molecules that may facilitate and stabilize the formation of G-quadruplexes can be used for cancer treatments, because the G-quadruplex structure can inhibit the activity of telomerase, an enzyme over-expressed in many cancer cells. Therefore, there is considerable interest in developing a simple and high-performance method for screening small molecules binding to G-quadruplex. Here, we have designed a simple electrochemical approach to screen such ligands based on the fact that the formation and stabilization of G-quadruplex by ligand may inhibit electron transfer of redox species to electrode surface. As a proof-of-concept study, two types of classical G-quadruplex ligands, TMPyP4 and BRACO-19, are studied in this work, which demonstrates that this method is fast and robust and it may be applied to screen G-quadruplex ligands for anticancer drugs testing and design in the future. Copyright © 2016 Elsevier B.V. All rights reserved.

Jeffamine derivatized TentaGel beads and poly(dimethylsiloxane) microbead cassettes for ultrahigh-throughput in situ releasable solution-phase cell-based screening of one-bead-one-compound combinatorial small molecule libraries.

PubMed

Townsend, Jared B; Shaheen, Farzana; Liu, Ruiwu; Lam, Kit S

2010-09-13

A method to efficiently immobilize and partition large quantities of microbeads in an array format in microfabricated poly(dimethylsiloxane) (PDMS) cassette for ultrahigh-throughput in situ releasable solution-phase cell-based screening of one-bead-one-compound (OBOC) combinatorial libraries is described. Commercially available Jeffamine triamine T-403 (∼440 Da) was derivatized such that two of its amino groups were protected by Fmoc and the remaining amino group capped with succinic anhydride to generate a carboxyl group. This resulting trifunctional hydrophilic polymer was then sequentially coupled two times to the outer layer of topologically segregated bilayer TentaGel (TG) beads with solid phase peptide synthesis chemistry resulting in beads with increased loading capacity, hydrophilicity, and porosity at the outer layer. We have found that such bead configuration can facilitate ultrahigh-throughput in situ releasable solution-phase screening of OBOC libraries. An encoded releasable OBOC small molecule library was constructed on Jeffamine derivatized TG beads with library compounds tethered to the outer layer via a disulfide linker and coding tags in the interior of the beads. Compound-beads could be efficiently loaded (5-10 min) into a 5 cm diameter Petri dish containing a 10,000-well PDMS microbead cassette, such that over 90% of the microwells were each filled with only one compound-bead. Jurkat T-lymphoid cancer cells suspended in Matrigel were then layered over the microbead cassette to immobilize the compound-beads. After 24 h of incubation at 37 °C, dithiothreitol was added to trigger the release of library compounds. Forty-eight hours later, MTT reporter assay was used to identify regions of reduced cell viability surrounding each positive bead. From a total of about 20,000 beads screened, 3 positive beads were detected and physically isolated for decoding. A strong consensus motif was identified for these three positive compounds. These compounds were resynthesized and found to be cytotoxic (IC(50) 50-150 μM) against two T-lymphoma cell lines and less so against the MDA-MB 231 breast cancer cell line. This novel ultrahigh-throughput OBOC releasable method can potentially be adapted to many existing 96- or 384-well solution-phase cell-based or biochemical assays.

Jeffamine Derivatized TentaGel Beads and PDMS Microbead Cassettes for Ultra-high Throughput in situ Releasable Solution-Phase Cell-based Screening of OBOC Combinatorial Small Molecule Libraries

PubMed Central

Townsend, Jared B.; Shaheen, Farzana; Liu, Ruiwu; Lam, Kit S.

2011-01-01

A method to efficiently immobilize and partition large quantities of microbeads in an array format in microfabricated polydimethylsiloxane (PDMS) cassette for high-throughput in situ releasable solution-phase cell-based screening of one-bead-one-compound (OBOC) combinatorial libraries is described. Commercially available Jeffamine triamine T-403 (∼440 Da) was derivatized such that two of its amino groups were protected by Fmoc and the remaining amino group capped with succinic anhydride to generate a carboxyl group. This resulting tri-functional hydrophilic polymer was then sequentially coupled two times to the outer layer of topologically segregated bilayer TentaGel (TG) beads with solid phase peptide synthesis chemistry, resulting in beads with increased loading capacity, hydrophilicity and porosity at the outer layer. We have found that such bead configuration can facilitate ultra high-throughput in situ releasable solution-phase screening of OBOC libraries. An encoded releasable OBOC small molecule library was constructed on Jeffamine derivatized TG beads with library compounds tethered to the outer layer via a disulfide linker and coding tags in the interior of the beads. Compound-beads could be efficiently loaded (5-10 minutes) into a 5 cm diameter Petri dish containing a 10,000-well PDMS microbead cassette, such that over 90% of the microwells were each filled with only one compound-bead. Jurkat T-lymphoid cancer cells suspended in Matrigel® were then layered over the microbead cassette to immobilize the compound-beads. After 24 hours of incubation at 37°C, dithiothreitol was added to trigger the release of library compounds. Forty-eight hours later, MTT reporter assay was used to identify regions of reduced cell viability surrounding each positive bead. From a total of about 20,000 beads screened, 3 positive beads were detected and physically isolated for decoding. A strong consensus motif was identified for these three positive compounds. These compounds were re-synthesized and found to be cytotoxic (IC50 50-150 μM) against two T-lymphoma cell lines and less so against the MDA-MB 231 breast cancer cell line. This novel ultra high-throughput OBOC releasable method can potentially be adapted to many existing 96- or 384-well solution-phase cell-based or biochemical assays. PMID:20593859

A filter paper dry blood spot procedure for acute intermittent porphyria population screening by use of whole blood uroporphyrinogen-I-synthase assay.

PubMed

Johansson, L; Thunell, S; Wetterberg, L

1984-03-13

A filter paper dry blood spot procedure for the determination of whole blood uroporphyrinogen-I-synthase (UIS) activity is presented. The method is based on the concept of enzyme specific activity, the enzyme activity being related to the haemoglobin concentration of the assay sample. The diagnostic capacity with regard to the acute intermittent porphyria (AIP) gene carrier state is shown to be equivalent to that of a washed red cell reference method. On grounds of easy capillary blood sampling, uncomplicated and safe mail specimen transport and simple laboratory reception routines, the method is stated to be well adapted for use in AIP preadolescent population screening.

Simultaneous screening of four epidermal growth factor receptor antagonists from Curcuma longa via cell membrane chromatography online coupled with HPLC-MS.

PubMed

Sun, Meng; Ma, Wei-na; Guo, Ying; Hu, Zhi-gang; He, Lang-chong

2013-07-01

The epidermal growth factor receptors (EGFRs) are significant targets for screening active compounds. In this work, an analytical method was established for rapid screening, separation, and identification of EGFRs antagonists from Curcuma longa. Human embryonic kidney 293 cells with a steadily high expression of EGFRs were used to prepare the cell membrane stationary phase in a cell membrane chromatography model for screening active compounds. Separation and identification of the retention chromatographic peaks was achieved by HPLC-MS. The active sites, docking extents and inhibitory effects of the active compounds were also demonstrated. The screening result found that ar-turmerone, curcumin, demethoxycurcumin, and bisdemethoxycurcumin from Curcuma longa could be active components in a similar manner to gefitinib. Biological trials showed that all of four compounds can inhibit EGFRs protein secretion and cell growth in a dose-dependent manner, and downregulate the phosphorylation of EGFRs. This analytical method demonstrated fast and effective characteristics for screening, separation and identification of the active compounds from a complex system and should be useful for drug discovery with natural medicinal herbs. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Alternative Methods for the Detection of Emerging Marine Toxins: Biosensors, Biochemical Assays and Cell-Based Assays

PubMed Central

Reverté, Laia; Soliño, Lucía; Carnicer, Olga; Diogène, Jorge; Campàs, Mònica

2014-01-01

The emergence of marine toxins in water and seafood may have a considerable impact on public health. Although the tendency in Europe is to consolidate, when possible, official reference methods based on instrumental analysis, the development of alternative or complementary methods providing functional or toxicological information may provide advantages in terms of risk identification, but also low cost, simplicity, ease of use and high-throughput analysis. This article gives an overview of the immunoassays, cell-based assays, receptor-binding assays and biosensors that have been developed for the screening and quantification of emerging marine toxins: palytoxins, ciguatoxins, cyclic imines and tetrodotoxins. Their advantages and limitations are discussed, as well as their possible integration in research and monitoring programs. PMID:25431968

Comparative Analysis of 3D Bladder Tumor Spheroids Obtained by Forced Floating and Hanging Drop Methods for Drug Screening.

PubMed

Amaral, Robson L F; Miranda, Mariza; Marcato, Priscyla D; Swiech, Kamilla

2017-01-01

Introduction: Cell-based assays using three-dimensional (3D) cell cultures may reflect the antitumor activity of compounds more accurately, since these models reproduce the tumor microenvironment better. Methods: Here, we report a comparative analysis of cell behavior in the two most widely employed methods for 3D spheroid culture, forced floating (Ultra-low Attachment, ULA, plates), and hanging drop (HD) methods, using the RT4 human bladder cancer cell line as a model. The morphology parameters and growth/metabolism of the spheroids generated were first characterized, using four different cell-seeding concentrations (0.5, 1.25, 2.5, and 3.75 × 10 4 cells/mL), and then, subjected to drug resistance evaluation. Results: Both methods generated spheroids with a smooth surface and round shape in a spheroidization time of about 48 h, regardless of the cell-seeding concentration used. Reduced cell growth and metabolism was observed in 3D cultures compared to two-dimensional (2D) cultures. The optimal range of spheroid diameter (300-500 μm) was obtained using cultures initiated with 0.5 and 1.25 × 10 4 cells/mL for the ULA method and 2.5 and 3.75 × 10 4 cells/mL for the HD method. RT4 cells cultured under 3D conditions also exhibited a higher resistance to doxorubicin (IC 50 of 1.00 and 0.83 μg/mL for the ULA and HD methods, respectively) compared to 2D cultures (IC 50 ranging from 0.39 to 0.43). Conclusions: Comparing the results, we concluded that the forced floating method using ULA plates was considered more suitable and straightforward to generate RT4 spheroids for drug screening/cytotoxicity assays. The results presented here also contribute to the improvement in the standardization of the 3D cultures required for widespread application.

Comparative Analysis of 3D Bladder Tumor Spheroids Obtained by Forced Floating and Hanging Drop Methods for Drug Screening

PubMed Central

Amaral, Robson L. F.; Miranda, Mariza; Marcato, Priscyla D.; Swiech, Kamilla

2017-01-01

Introduction: Cell-based assays using three-dimensional (3D) cell cultures may reflect the antitumor activity of compounds more accurately, since these models reproduce the tumor microenvironment better. Methods: Here, we report a comparative analysis of cell behavior in the two most widely employed methods for 3D spheroid culture, forced floating (Ultra-low Attachment, ULA, plates), and hanging drop (HD) methods, using the RT4 human bladder cancer cell line as a model. The morphology parameters and growth/metabolism of the spheroids generated were first characterized, using four different cell-seeding concentrations (0.5, 1.25, 2.5, and 3.75 × 104 cells/mL), and then, subjected to drug resistance evaluation. Results: Both methods generated spheroids with a smooth surface and round shape in a spheroidization time of about 48 h, regardless of the cell-seeding concentration used. Reduced cell growth and metabolism was observed in 3D cultures compared to two-dimensional (2D) cultures. The optimal range of spheroid diameter (300–500 μm) was obtained using cultures initiated with 0.5 and 1.25 × 104 cells/mL for the ULA method and 2.5 and 3.75 × 104 cells/mL for the HD method. RT4 cells cultured under 3D conditions also exhibited a higher resistance to doxorubicin (IC50 of 1.00 and 0.83 μg/mL for the ULA and HD methods, respectively) compared to 2D cultures (IC50 ranging from 0.39 to 0.43). Conclusions: Comparing the results, we concluded that the forced floating method using ULA plates was considered more suitable and straightforward to generate RT4 spheroids for drug screening/cytotoxicity assays. The results presented here also contribute to the improvement in the standardization of the 3D cultures required for widespread application. PMID:28878686

Localization-based super-resolution imaging meets high-content screening.

PubMed

Beghin, Anne; Kechkar, Adel; Butler, Corey; Levet, Florian; Cabillic, Marine; Rossier, Olivier; Giannone, Gregory; Galland, Rémi; Choquet, Daniel; Sibarita, Jean-Baptiste

2017-12-01

Single-molecule localization microscopy techniques have proven to be essential tools for quantitatively monitoring biological processes at unprecedented spatial resolution. However, these techniques are very low throughput and are not yet compatible with fully automated, multiparametric cellular assays. This shortcoming is primarily due to the huge amount of data generated during imaging and the lack of software for automation and dedicated data mining. We describe an automated quantitative single-molecule-based super-resolution methodology that operates in standard multiwell plates and uses analysis based on high-content screening and data-mining software. The workflow is compatible with fixed- and live-cell imaging and allows extraction of quantitative data like fluorophore photophysics, protein clustering or dynamic behavior of biomolecules. We demonstrate that the method is compatible with high-content screening using 3D dSTORM and DNA-PAINT based super-resolution microscopy as well as single-particle tracking.

An in vivo multiplexed small molecule screening platform

PubMed Central

Yang, Dian; Ogasawara, Daisuke; Dix, Melissa M.; Rogers, Zoë N.; Chuang, Chen-Hua; McFarland, Christopher D.; Chiou, Shin-Heng; Brown, J. Mark; Cravatt, Benjamin F.; Bogyo, Matthew; Winslow, Monte M.

2016-01-01

Phenotype-based small molecule screening is a powerful method to identify regulators of cellular function. However, such screens are generally performed in vitro using conditions that do not necessarily model complex physiological conditions or disease states. Here, we use molecular cell barcoding to enable direct in vivo phenotypic screening of libraries of small molecules. The multiplexed nature of this approach allows rapid in vivo analysis of hundreds to thousands of compounds. Using this platform, we screened >700 covalent inhibitors directed towards hydrolases for their effect on pancreatic cancer metastatic seeding. We identified multiple hits and confirmed the relevant target of one compound as the lipase ABHD6. Pharmacological and genetic studies confirmed the role of this enzyme as a regulator of metastatic fitness. Our results highlight the applicability of this multiplexed screening platform for investigating complex processes in vivo. PMID:27617390

Sinking towards destiny: High throughput measurement of phytoplankton sinking rates through time-resolved fluorescence plate spectroscopy.

PubMed

Bannon, Catherine C; Campbell, Douglas A

2017-01-01

Diatoms are marine primary producers that sink in part due to the density of their silica frustules. Sinking of these phytoplankters is crucial for both the biological pump that sequesters carbon to the deep ocean and for the life strategy of the organism. Sinking rates have been previously measured through settling columns, or with fluorimeters or video microscopy arranged perpendicularly to the direction of sinking. These side-view techniques require large volumes of culture, specialized equipment and are difficult to scale up to multiple simultaneous measures for screening. We established a method for parallel, large scale analysis of multiple phytoplankton sinking rates through top-view monitoring of chlorophyll a fluorescence in microtitre well plates. We verified the method through experimental analysis of known factors that influence sinking rates, including exponential versus stationary growth phase in species of different cell sizes; Thalassiosira pseudonana CCMP1335, chain-forming Skeletonema marinoi RO5A and Coscinodiscus radiatus CCMP312. We fit decay curves to an algebraic transform of the decrease in fluorescence signal as cells sank away from the fluorometer detector, and then used minimal mechanistic assumptions to extract a sinking rate (m d-1) using an RStudio script, SinkWORX. We thereby detected significant differences in sinking rates as larger diatom cells sank faster than smaller cells, and cultures in stationary phase sank faster than those in exponential phase. Our sinking rate estimates accord well with literature values from previously established methods. This well plate-based method can operate as a high throughput integrative phenotypic screen for factors that influence sinking rates including macromolecular allocations, nutrient availability or uptake rates, chain-length or cell size, degree of silification and progression through growth stages. Alternately the approach can be used to phenomically screen libraries of mutants.

Identification of Potential Pharmacoperones Capable of Rescuing the Functionality of Misfolded Vasopressin 2 Receptor Involved in Nephrogenic Diabetes Insipidus.

PubMed

Smith, Emery; Janovick, Jo Ann; Bannister, Thomas D; Shumate, Justin; Scampavia, Louis; Conn, P Michael; Spicer, Timothy P

2016-09-01

Pharmacoperones correct the folding of otherwise misfolded protein mutants, restoring function (i.e., providing "rescue") by correcting their trafficking. Currently, most pharmacoperones possess intrinsic antagonist activity because they were identified using methods initially aimed at discovering such functions. Here, we describe an ultra-high-throughput homogeneous cell-based assay with a cAMP detection system, a method specifically designed to identify pharmacoperones of the vasopressin type 2 receptor (V2R), a GPCR that, when mutated, is associated with nephrogenic diabetes insipidus. Previously developed methods to identify compounds capable of altering cellular trafficking of V2R were modified and used to screen a 645,000 compound collection by measuring the ability of library compounds to rescue a mutant hV2R [L83Q], using a cell-based luminescent detection system. The campaign initially identified 3734 positive modulators of cAMP. The confirmation and counterscreen identified only 147 of the active compounds with an EC50 of ≤5 µM. Of these, 83 were reconfirmed as active through independently obtained pure samples and were also inactive in a relevant counterscreen. Active and tractable compounds within this set can be categorized into three predominant structural clusters, described here, in the first report detailing the results of a large-scale pharmacoperone high-throughput screening campaign. © 2016 Society for Laboratory Automation and Screening.

Inhibition of Retinoblastoma Protein Inactivation

DTIC Science & Technology

2017-11-01

SUBJECT TERMS cell cycle, Retinoblastoma protein, E2F transcription factor, high throughput screen, drug discovery, x-ray crystallography 16. SECURITY...screening by x-ray crystallography . 2.0 KEYWORDS Retinoblastoma (Rb) pathway, E2F transcription factor, cancer, cell-cycle inhibition, activation...modulation, inhibition, high throughput screening, fragment-based screening, x-ray crystallography . 3.0 ACCOMPLISHMENTS Summary: We

Gγ recruitment system incorporating a novel signal amplification circuit to screen transient protein-protein interactions.

PubMed

Fukuda, Nobuo; Ishii, Jun; Kondo, Akihiko

2011-09-01

Weak and transient protein-protein interactions are associated with biological processes, but many are still undefined because of the difficulties in their identification. Here, we describe a redesigned method to screen transient protein-protein interactions by using a novel signal amplification circuit, which is incorporated into yeast to artificially magnify the signal responding to the interactions. This refined method is based on the previously established Gγ recruitment system, which utilizes yeast G-protein signaling and mating growth selection to screen interacting protein pairs. In the current study, to test the capability of our method, we chose mutants of the Z-domain derived from Staphylococcus aureus protein A as candidate proteins, and the Fc region of human IgG as the counterpart. By introduction of an artificial signal amplifier into the previous Gγ recruitment system, the signal transduction responding to transient interactions between Z-domain mutants and the Fc region with significantly low affinity (8.0 × 10(3) M(-1)) was successfully amplified in recombinant haploid yeast cells. As a result of zygosis with the opposite mating type of wild-type haploid cells, diploid colonies were vigorously and selectively generated on the screening plates, whereas our previous system rarely produced positive colonies. This new approach will be useful for exploring the numerous transient interactions that remain undefined because of the lack of powerful screening tools for their identification. © 2011 The Authors Journal compilation © 2011 FEBS.

Research on major antitumor active components in Zi-Cao-Cheng-Qi decoction based on hollow fiber cell fishing with high performance liquid chromatography.

PubMed

Li, Miaomiao; Hu, Shuang; Chen, Xuan; Wang, Runqin; Bai, Xiaohong

2018-02-05

Hollow fiber cell fishing (HFCF) based on hepatoma HepG-2 cells, human renal tubular ACHN cells or human cervical carcinoma HeLa cells, coupled with high-performance liquid chromatography (HPLC), was developed and employed to research the major active components in Zi-Cao-Cheng-Qi decoction both in vitro and in vivo. The research showed that the active components, such as hesperidin, magnolol, honokiol, shikonin, emodin and β,β'-dimethylacrylshikonin were screened out by HFCF based on the cancer cells in vitro, furthermore they can be absorbed into blood and reach in the target organ, and some of the active components can be fished by the cells and maintain effective concentrations. Before application of HFCF with HPLC, cell growth state, cell survival rate, positive effect on screening results binding between active centers on the fiber and target components, repeatability of retention times and relative peak areas of the target analytes were analysed and investigated. In short, HFCF with HPLC is a simple, inexpensive, effective, and reliable method that can be used in researching active components from traditional Chinese medicine (TCM) and its formula both in vitro and in vivo, elucidating preliminarily the TCM characteristics of multiple components and multiple targets, laying a foundation for expounding the antitumor efficacy material basis in TCM. Copyright © 2017 Elsevier B.V. All rights reserved.

Non-invasive screening for Alzheimer's disease by sensing salivary sugar using Drosophila cells expressing gustatory receptor (Gr5a) immobilized on an extended gate ion-sensitive field-effect transistor (EG-ISFET) biosensor.

PubMed

Lau, Hui-Chong; Lee, In-Kyu; Ko, Pan-Woo; Lee, Ho-Won; Huh, Jeung-Soo; Cho, Won-Ju; Lim, Jeong-Ok

2015-01-01

Body fluids are often used as specimens for medical diagnosis. With the advent of advanced analytical techniques in biotechnology, the diagnostic potential of saliva has been the focus of many studies. We recently reported the presence of excess salivary sugars, in patients with Alzheimer's disease (AD). In the present study, we developed a highly sensitive, cell-based biosensor to detect trehalose levels in patient saliva. The developed biosensor relies on the overexpression of sugar sensitive gustatory receptors (Gr5a) in Drosophila cells to detect the salivary trehalose. The cell-based biosensor was built on the foundation of an improved extended gate ion-sensitive field-effect transistor (EG-ISFET). Using an EG-ISFET, instead of a traditional ion-sensitive field-effect transistor (ISFET), resulted in an increase in the sensitivity and reliability of detection. The biosensor was designed with the gate terminals segregated from the conventional ISFET device. This design allows the construction of an independent reference and sensing region for simultaneous and accurate measurements of samples from controls and patients respectively. To investigate the efficacy of the cell-based biosensor for AD screening, we collected 20 saliva samples from each of the following groups: participants diagnosed with AD, participants diagnosed with Parkinson's disease (PD), and a control group composed of healthy individuals. We then studied the response generated from the interaction of the salivary trehalose of the saliva samples and the Gr5a in the immobilized cells on an EG-ISFET sensor. The cell-based biosensor significantly distinguished salivary sugar, trehalose of the AD group from the PD and control groups. Based on these findings, we propose that salivary trehalose, might be a potential biomarker for AD and could be detected using our cell-based EG-ISFET biosensor. The cell-based EG-ISFET biosensor provides a sensitive and direct approach for salivary sugar detection and may be used in the future as a screening method for AD.

A method to identify differential expression profiles of time-course gene data with Fourier transformation

PubMed Central

2013-01-01

Background Time course gene expression experiments are an increasingly popular method for exploring biological processes. Temporal gene expression profiles provide an important characterization of gene function, as biological systems are both developmental and dynamic. With such data it is possible to study gene expression changes over time and thereby to detect differential genes. Much of the early work on analyzing time series expression data relied on methods developed originally for static data and thus there is a need for improved methodology. Since time series expression is a temporal process, its unique features such as autocorrelation between successive points should be incorporated into the analysis. Results This work aims to identify genes that show different gene expression profiles across time. We propose a statistical procedure to discover gene groups with similar profiles using a nonparametric representation that accounts for the autocorrelation in the data. In particular, we first represent each profile in terms of a Fourier basis, and then we screen out genes that are not differentially expressed based on the Fourier coefficients. Finally, we cluster the remaining gene profiles using a model-based approach in the Fourier domain. We evaluate the screening results in terms of sensitivity, specificity, FDR and FNR, compare with the Gaussian process regression screening in a simulation study and illustrate the results by application to yeast cell-cycle microarray expression data with alpha-factor synchronization. The key elements of the proposed methodology: (i) representation of gene profiles in the Fourier domain; (ii) automatic screening of genes based on the Fourier coefficients and taking into account autocorrelation in the data, while controlling the false discovery rate (FDR); (iii) model-based clustering of the remaining gene profiles. Conclusions Using this method, we identified a set of cell-cycle-regulated time-course yeast genes. The proposed method is general and can be potentially used to identify genes which have the same patterns or biological processes, and help facing the present and forthcoming challenges of data analysis in functional genomics. PMID:24134721

Microarray platform affords improved product analysis in mammalian cell growth studies

PubMed Central

Li, Lingyun; Migliore, Nicole; Schaefer, Eugene; Sharfstein, Susan T.; Dordick, Jonathan S.; Linhardt, Robert J.

2014-01-01

High throughput (HT) platforms serve as cost-efficient and rapid screening method for evaluating the effect of cell culture conditions and screening of chemicals. The aim of the current study was to develop a high-throughput cell-based microarray platform to assess the effect of culture conditions on Chinese hamster ovary (CHO) cells. Specifically, growth, transgene expression and metabolism of a GS/MSX CHO cell line, which produces a therapeutic monoclonal antibody, was examined using microarray system in conjunction with conventional shake flask platform in a non-proprietary medium. The microarray system consists of 60 nl spots of cells encapsulated in alginate and separated in groups via an 8-well chamber system attached to the chip. Results show the non-proprietary medium developed allows cell growth, production and normal glycosylation of recombinant antibody and metabolism of the recombinant CHO cells in both the microarray and shake flask platforms. In addition, 10.3 mM glutamate addition to the defined base media results in lactate metabolism shift in the recombinant GS/MSX CHO cells in the shake flask platform. Ultimately, the results demonstrate that the high-throughput microarray platform has the potential to be utilized for evaluating the impact of media additives on cellular processes, such as, cell growth, metabolism and productivity. PMID:24227746

Human iPSC-derived cardiomyocytes and tissue engineering strategies for disease modeling and drug screening.

PubMed

Smith, Alec S T; Macadangdang, Jesse; Leung, Winnie; Laflamme, Michael A; Kim, Deok-Ho

Improved methodologies for modeling cardiac disease phenotypes and accurately screening the efficacy and toxicity of potential therapeutic compounds are actively being sought to advance drug development and improve disease modeling capabilities. To that end, much recent effort has been devoted to the development of novel engineered biomimetic cardiac tissue platforms that accurately recapitulate the structure and function of the human myocardium. Within the field of cardiac engineering, induced pluripotent stem cells (iPSCs) are an exciting tool that offer the potential to advance the current state of the art, as they are derived from somatic cells, enabling the development of personalized medical strategies and patient specific disease models. Here we review different aspects of iPSC-based cardiac engineering technologies. We highlight methods for producing iPSC-derived cardiomyocytes (iPSC-CMs) and discuss their application to compound efficacy/toxicity screening and in vitro modeling of prevalent cardiac diseases. Special attention is paid to the application of micro- and nano-engineering techniques for the development of novel iPSC-CM based platforms and their potential to advance current preclinical screening modalities. Published by Elsevier Inc.

High-throughput spectral and lifetime-based FRET screening in living cells to identify small-molecule effectors of SERCA

PubMed Central

Schaaf, Tory M.; Peterson, Kurt C.; Grant, Benjamin D.; Bawaskar, Prachi; Yuen, Samantha; Li, Ji; Muretta, Joseph M.; Gillispie, Gregory D.; Thomas, David D.

2017-01-01

A robust high-throughput screening (HTS) strategy has been developed to discover small-molecule effectors targeting the sarco/endoplasmic reticulum calcium ATPase (SERCA), based on a fluorescence microplate reader that records both the nanosecond decay waveform (lifetime mode) and the complete emission spectrum (spectral mode), with high precision and speed. This spectral unmixing plate reader (SUPR) was used to screen libraries of small molecules with a fluorescence resonance energy transfer (FRET) biosensor expressed in living cells. Ligand binding was detected by FRET associated with structural rearrangements of green (GFP, donor) and red (RFP, acceptor) fluorescent proteins fused to the cardiac-specific SERCA2a isoform. The results demonstrate accurate quantitation of FRET along with high precision of hit identification. Fluorescence lifetime analysis resolved SERCA’s distinct structural states, providing a method to classify small-molecule chemotypes on the basis of their structural effect on the target. The spectral analysis was also applied to flag interference by fluorescent compounds. FRET hits were further evaluated for functional effects on SERCA’s ATPase activity via both a coupled-enzyme assay and a FRET-based calcium sensor. Concentration-response curves indicated excellent correlation between FRET and function. These complementary spectral and lifetime FRET detection methods offer an attractive combination of precision, speed, and resolution for HTS. PMID:27899691

Fast Estimation of Dietary Fiber Content in Apple.

PubMed

Le Gall, Sophie; Even, Sonia; Lahaye, Marc

2016-02-17

Dietary fibers (DF) are one of the nutritional benefits of fleshy fruit consumption that is becoming a quality criterion for genetic selection by breeders. However, the AOAC total DF content determination is not readily amenable for screening large fruit collections. A new screening method of DF content in an apple collection based on the automated preparation of cell wall material as an alcohol-insoluble residue (AIR) is proposed. The yield of AIR from 27 apple genotypes was compared with DF measured according to AOAC method 985.29. Although residual protein content in AIRs did not affect DF measurement, subtraction of starch content above 3% dry weight in AIRs was needed to agree with AOAC measured DF. A fast colorimetric screening of starch in AIR was developed to detect samples needing correction. The proposed method may prove useful for the rapid determination of DF in collections of other fleshy fruit besides apple.

Human pluripotent stem cells: Prospects and challenges as a source of cardiomyocytes for in vitro modeling and cell-based cardiac repair.

PubMed

Hartman, Matthew E; Dai, Dao-Fu; Laflamme, Michael A

2016-01-15

Human pluripotent stem cells (PSCs) represent an attractive source of cardiomyocytes with potential applications including disease modeling, drug discovery and safety screening, and novel cell-based cardiac therapies. Insights from embryology have contributed to the development of efficient, reliable methods capable of generating large quantities of human PSC-cardiomyocytes with cardiac purities ranging up to 90%. However, for human PSCs to meet their full potential, the field must identify methods to generate cardiomyocyte populations that are uniform in subtype (e.g. homogeneous ventricular cardiomyocytes) and have more mature structural and functional properties. For in vivo applications, cardiomyocyte production must be highly scalable and clinical grade, and we will need to overcome challenges including graft cell death, immune rejection, arrhythmogenesis, and tumorigenic potential. Here we discuss the types of human PSCs, commonly used methods to guide their differentiation into cardiomyocytes, the phenotype of the resultant cardiomyocytes, and the remaining obstacles to their successful translation. Copyright © 2015 Elsevier B.V. All rights reserved.

Differentially Coexpressed Disease Gene Identification Based on Gene Coexpression Network.

PubMed

Jiang, Xue; Zhang, Han; Quan, Xiongwen

2016-01-01

Screening disease-related genes by analyzing gene expression data has become a popular theme. Traditional disease-related gene selection methods always focus on identifying differentially expressed gene between case samples and a control group. These traditional methods may not fully consider the changes of interactions between genes at different cell states and the dynamic processes of gene expression levels during the disease progression. However, in order to understand the mechanism of disease, it is important to explore the dynamic changes of interactions between genes in biological networks at different cell states. In this study, we designed a novel framework to identify disease-related genes and developed a differentially coexpressed disease-related gene identification method based on gene coexpression network (DCGN) to screen differentially coexpressed genes. We firstly constructed phase-specific gene coexpression network using time-series gene expression data and defined the conception of differential coexpression of genes in coexpression network. Then, we designed two metrics to measure the value of gene differential coexpression according to the change of local topological structures between different phase-specific networks. Finally, we conducted meta-analysis of gene differential coexpression based on the rank-product method. Experimental results demonstrated the feasibility and effectiveness of DCGN and the superior performance of DCGN over other popular disease-related gene selection methods through real-world gene expression data sets.

Methods for efficient high-throughput screening of protein expression in recombinant Pichia pastoris strains.

PubMed

Camattari, Andrea; Weinhandl, Katrin; Gudiminchi, Rama K

2014-01-01

The methylotrophic yeast Pichia pastoris is becoming one of the favorite industrial workhorses for protein expression. Due to the widespread use of integration vectors, which generates significant clonal variability, screening methods allowing assaying hundreds of individual clones are of particular importance. Here we describe methods to detect and analyze protein expression, developed in a 96-well format for high-throughput screening of recombinant P. pastoris strains. The chapter covers essentially three common scenarios: (1) an enzymatic assay for proteins expressed in the cell cytoplasm, requiring cell lysis; (2) a whole-cell assay for a fungal cytochrome P450; and (3) a nonenzymatic assay for detection and quantification of tagged protein secreted into the supernatant.

Current Technologies and Recent Developments for Screening of HPV-Associated Cervical and Oropharyngeal Cancers

PubMed Central

Shah, Sunny S.; Senapati, Satyajyoti; Klacsmann, Flora; Miller, Daniel L.; Johnson, Jeff J.; Chang, Hsueh-Chia; Stack, M. Sharon

2016-01-01

Mucosal infection by the human papillomavirus (HPV) is responsible for a growing number of malignancies, predominantly represented by cervical cancer and oropharyngeal squamous cell carcinoma. Because of the prevalence of the virus, persistence of infection, and long latency period, novel and low-cost methods are needed for effective population level screening and monitoring. We review established methods for screening of cervical and oral cancer as well as commercially-available techniques for detection of HPV DNA. We then describe the ongoing development of microfluidic nucleic acid-based biosensors to evaluate circulating host microRNAs that are produced in response to an oncogenic HPV infection. The goal is to develop an ideal screening platform that is low-cost, portable, and easy to use, with appropriate signal stability, sensitivity and specificity. Advances in technologies for sample lysis, pre-treatment and concentration, and multiplexed nucleic acid detection are provided. Continued development of these devices provides opportunities for cancer screening in low resource settings, for point-of-care diagnostics and self-screening, and for monitoring response to vaccination or surgical treatment. PMID:27618102

Bacterial Microcolonies in Gel Beads for High-Throughput Screening of Libraries in Synthetic Biology.

PubMed

Duarte, José M; Barbier, Içvara; Schaerli, Yolanda

2017-11-17

Synthetic biologists increasingly rely on directed evolution to optimize engineered biological systems. Applying an appropriate screening or selection method for identifying the potentially rare library members with the desired properties is a crucial step for success in these experiments. Special challenges include substantial cell-to-cell variability and the requirement to check multiple states (e.g., being ON or OFF depending on the input). Here, we present a high-throughput screening method that addresses these challenges. First, we encapsulate single bacteria into microfluidic agarose gel beads. After incubation, they harbor monoclonal bacterial microcolonies (e.g., expressing a synthetic construct) and can be sorted according their fluorescence by fluorescence activated cell sorting (FACS). We determine enrichment rates and demonstrate that we can measure the average fluorescent signals of microcolonies containing phenotypically heterogeneous cells, obviating the problem of cell-to-cell variability. Finally, we apply this method to sort a pBAD promoter library at ON and OFF states.

Conditional screening for ultra-high dimensional covariates with survival outcomes

PubMed Central

Hong, Hyokyoung G.; Li, Yi

2017-01-01

Identifying important biomarkers that are predictive for cancer patients’ prognosis is key in gaining better insights into the biological influences on the disease and has become a critical component of precision medicine. The emergence of large-scale biomedical survival studies, which typically involve excessive number of biomarkers, has brought high demand in designing efficient screening tools for selecting predictive biomarkers. The vast amount of biomarkers defies any existing variable selection methods via regularization. The recently developed variable screening methods, though powerful in many practical setting, fail to incorporate prior information on the importance of each biomarker and are less powerful in detecting marginally weak while jointly important signals. We propose a new conditional screening method for survival outcome data by computing the marginal contribution of each biomarker given priorily known biological information. This is based on the premise that some biomarkers are known to be associated with disease outcomes a priori. Our method possesses sure screening properties and a vanishing false selection rate. The utility of the proposal is further confirmed with extensive simulation studies and analysis of a diffuse large B-cell lymphoma dataset. We are pleased to dedicate this work to Jack Kalbfleisch, who has made instrumental contributions to the development of modern methods of analyzing survival data. PMID:27933468

Exclusion-Based Capture and Enumeration of CD4+ T Cells from Whole Blood for Low-Resource Settings.

PubMed

Howard, Alexander L; Pezzi, Hannah M; Beebe, David J; Berry, Scott M

2014-06-01

In developing countries, demand exists for a cost-effective method to evaluate human immunodeficiency virus patients' CD4(+) T-helper cell count. The TH (CD4) cell count is the current marker used to identify when an HIV patient has progressed to acquired immunodeficiency syndrome, which results when the immune system can no longer prevent certain opportunistic infections. A system to perform TH count that obviates the use of costly flow cytometry will enable physicians to more closely follow patients' disease progression and response to therapy in areas where such advanced equipment is unavailable. Our system of two serially-operated immiscible phase exclusion-based cell isolations coupled with a rapid fluorescent readout enables exclusion-based isolation and accurate counting of T-helper cells at lower cost and from a smaller volume of blood than previous methods. TH cell isolation via immiscible filtration assisted by surface tension (IFAST) compares well against the established Dynal T4 Quant Kit and is sensitive at CD4 counts representative of immunocompromised patients (less than 200 TH cells per microliter of blood). Our technique retains use of open, simple-to-operate devices that enable IFAST as a high-throughput, automatable sample preparation method, improving throughput over previous low-resource methods. © 2013 Society for Laboratory Automation and Screening.

EVALUATION OF HUMAN NEURAL PROGENITOR CELLS FOR DEVELOPMENTAL NEUROTOXICITY SCREENING: TIME COURSE OF EFFECTS ON CELL PROLIFERATION AND VIABILITY.

EPA Science Inventory

Current testing methods for developmental neurotoxicity (DNT) make evaluation of the effects of large numbers of chemicals impractical and prohibitively expensive. As such, we are evaluating human neural progenitor cells (NPCs) as a screen for DNT. ReNcell CX (ReN CX) cells are a...

The Harvard Clean Energy Project: High-throughput screening of organic photovoltaic materials using cheminformatics, machine learning, and pattern recognition

NASA Astrophysics Data System (ADS)

Olivares-Amaya, Roberto; Hachmann, Johannes; Amador-Bedolla, Carlos; Daly, Aidan; Jinich, Adrian; Atahan-Evrenk, Sule; Boixo, Sergio; Aspuru-Guzik, Alán

2012-02-01

Organic photovoltaic devices have emerged as competitors to silicon-based solar cells, currently reaching efficiencies of over 9% and offering desirable properties for manufacturing and installation. We study conjugated donor polymers for high-efficiency bulk-heterojunction photovoltaic devices with a molecular library motivated by experimental feasibility. We use quantum mechanics and a distributed computing approach to explore this vast molecular space. We will detail the screening approach starting from the generation of the molecular library, which can be easily extended to other kinds of molecular systems. We will describe the screening method for these materials which ranges from descriptor models, ubiquitous in the drug discovery community, to eventually reaching first principles quantum chemistry methods. We will present results on the statistical analysis, based principally on machine learning, specifically partial least squares and Gaussian processes. Alongside, clustering methods and the use of the hypergeometric distribution reveal moieties important for the donor materials and allow us to quantify structure-property relationships. These efforts enable us to accelerate materials discovery in organic photovoltaics through our collaboration with experimental groups.

Toward a systems-level analysis of infection biology: a new method for conducting genetic screens in human cells.

PubMed

Stanley, Sarah A; Hung, Deborah T

2009-12-16

Loss-of-function genetic screens have facilitated great strides in our understanding of the biology of model organisms but have not been possible in diploid human cells. A recent report by Brummelkamp's group in Science describes the use of insertional mutagenesis to generate loss-of-function alleles in a largely haploid human cell line and demonstrates the versatility of this method in screens designed to investigate the host/pathogen interaction. This approach has strengths that are complementary to existing strategies and will facilitate progress toward a systems-level understanding of infectious disease and ultimately the development of new therapeutics.

A new effective assay to detect antimicrobial activity of filamentous fungi.

PubMed

Pereira, Eric; Santos, Ana; Reis, Francisca; Tavares, Rui M; Baptista, Paula; Lino-Neto, Teresa; Almeida-Aguiar, Cristina

2013-01-15

The search for new antimicrobial compounds and the optimization of production methods turn the use of antimicrobial susceptibility tests a routine. The most frequently used methods are based on agar diffusion assays or on dilution in agar or broth. For filamentous fungi, the most common antimicrobial activity detection methods comprise the co-culture of two filamentous fungal strains or the use of fungal extracts to test against single-cell microorganisms. Here we report a rapid, effective and reproducible assay to detect fungal antimicrobial activity against single-cell microorganisms. This method allows an easy way of performing a fast antimicrobial screening of actively growing fungi directly against yeast. Because it makes use of an actively growing mycelium, this bioassay also provides a way for studying the production dynamics of antimicrobial compounds by filamentous fungi. The proposed assay is less time consuming and introduces the innovation of allowing the direct detection of fungal antimicrobial properties against single cell microorganisms without the prior isolation of the active substance(s). This is particularly useful when performing large screenings for fungal antimicrobial activity. With this bioassay, antimicrobial activity of Hypholoma fasciculare against yeast species was observed for the first time. Copyright © 2012 Elsevier GmbH. All rights reserved.

Evaluation of Impermeant, DNA-Binding Dye Fluorescence as a Real-Time Readout of Eukaryotic Cell Toxicity in a High Throughput Screening Format

PubMed Central

Chiaraviglio, Lucius

2014-01-01

Abstract Interpretation of high throughput screening (HTS) data in cell-based assays may be confounded by cytotoxic properties of screening compounds. Therefore, assessing cell toxicity in real time during the HTS process itself would be highly advantageous. Here, we investigate the potential of putatively impermeant, fluorescent, DNA-binding dyes to give cell toxicity readout during HTS. Amongst 19 DNA-binding dyes examined, three classes were identified that were (1) permeant, (2) cytotoxic, or (3) neither permeant nor cytotoxic during 3-day incubation with a macrophage cell line. In the last class, four dyes (SYTOX Green, CellTox Green, GelGreen, and EvaGreen) gave highly robust cytotoxicity data in 384-well screening plates. As proof of principle, successful combination with a luminescence-based assay in HTS format was demonstrated. Here, both intracellular growth of Legionella pneumophila (luminescence) and host cell viability (SYTOX Green exclusion) were assayed in the same screening well. Incorporation of membrane-impermeant, DNA-binding, fluorescent dyes in HTS assays should prove useful by allowing evaluation of cytotoxicity in real time, eliminating reagent addition steps and effort associated with endpoint cell viability analysis, and reducing the need for follow-up cytotoxicity screening. PMID:24831788

Large-scale image-based profiling of single-cell phenotypes in arrayed CRISPR-Cas9 gene perturbation screens.

PubMed

de Groot, Reinoud; Lüthi, Joel; Lindsay, Helen; Holtackers, René; Pelkmans, Lucas

2018-01-23

High-content imaging using automated microscopy and computer vision allows multivariate profiling of single-cell phenotypes. Here, we present methods for the application of the CISPR-Cas9 system in large-scale, image-based, gene perturbation experiments. We show that CRISPR-Cas9-mediated gene perturbation can be achieved in human tissue culture cells in a timeframe that is compatible with image-based phenotyping. We developed a pipeline to construct a large-scale arrayed library of 2,281 sequence-verified CRISPR-Cas9 targeting plasmids and profiled this library for genes affecting cellular morphology and the subcellular localization of components of the nuclear pore complex (NPC). We conceived a machine-learning method that harnesses genetic heterogeneity to score gene perturbations and identify phenotypically perturbed cells for in-depth characterization of gene perturbation effects. This approach enables genome-scale image-based multivariate gene perturbation profiling using CRISPR-Cas9. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

High throughput assay for cytochrome P450 BM3 for screening libraries of substrates and combinatorial mutants.

PubMed

Tsotsou, Georgia Eleni; Cass, Anthony Edward George; Gilardi, Gianfranco

2002-01-01

A rapid method for identifying compounds that are potential substrates for the drug metabolising enzyme cytochrome P450 is described. The strategy is based on the detection of a degradation product of NAD(P)H oxidation during substrate turnover by the enzyme expressed in Escherichia coli cells spontaneously lysed under the experimental conditions. The performance of the method has been tested on two known substrates of the wild-type cytochrome P450 BM3, arachidonic (AA) and lauric (LA) acids, and two substrates with environmental significance, the anionic surfactant sodium dodecyl sulfate (SDS), and the solvent 1,1,2,2-tetrachloroethane (TCE). The minimal background signal given from cells expressing cytochrome P450 BM3 in the absence of added substrate is only 3% of the signal in the presence of saturating substrate. Control experiments have proven that this method is specifically detecting NADPH oxidation by catalytic turnover of P450 BM3. The assay has been adapted to a microtitre plate format and used to screen a series of furazan derivatives as potential substrates. Three derivatives were identified as substrates. The method gave a significant different signal for two isomeric furazan derivatives. All results found on the cell lysate were verified and confirmed with the purified enzyme. This strategy opens the way to automated high throughput screening of NAD(P)H-linked enzymatic activity of molecules of pharmacological and biotechnological interest and libraries of random mutants of NAD(P)H-dependent biocatalysts.

Structure-based virtual screening and characterization of a novel IL-6 antagonistic compound from synthetic compound database.

PubMed

Wang, Jing; Qiao, Chunxia; Xiao, He; Lin, Zhou; Li, Yan; Zhang, Jiyan; Shen, Beifen; Fu, Tinghuan; Feng, Jiannan

2016-01-01

According to the three-dimensional (3D) complex structure of (hIL-6⋅hIL-6R⋅gp 130) 2 and the binding orientation of hIL-6, three compounds with high affinity to hIL-6R and bioactivity to block hIL-6 in vitro were screened theoretically from the chemical databases, including 3D-Available Chemicals Directory (ACD) and MDL Drug Data Report (MDDR), by means of the computer-guided virtual screening method. Using distance geometry, molecular modeling and molecular dynamics trajectory analysis methods, the binding mode and binding energy of the three compounds were evaluated theoretically. Enzyme-linked immunosorbent assay analysis demonstrated that all the three compounds could block IL-6 binding to IL-6R specifically. However, only compound 1 could effectively antagonize the function of hIL-6 and inhibit the proliferation of XG-7 cells in a dose-dependent manner, whereas it showed no cytotoxicity to SP2/0 or L929 cells. These data demonstrated that the compound 1 could be a promising candidate of hIL-6 antagonist.

A Phenotype-Based RNAi Screening for Ras-ERK/MAPK Signaling-Associated Stem Cell Regulators in C. elegans.

PubMed

Lee, Myon-Hee; Yoon, Dong Suk

2017-01-01

Stem cells have the ability to self-renew and to generate differentiated cell types. A regulatory network that controls this balance is critical for stem cell homeostasis and normal animal development. Particularly, Ras-ERK/MAPK signaling pathway is critical for stem cell self-renewal and differentiation in mammals, including humans. Aberrant regulation of Ras-ERK/MAPK signaling pathway results in either stem cell or overproliferation. Therefore, the identification of Ras-ERK/MAPK signaling pathway-associated regulators is critical to understand the mechanism of stem cell (possibly cancer stem cell) control. In this report, using the nematode C. elegans mutants, we developed a methodology for a phenotype-based RNAi screening that identifies stem cell regulator genes associated with Ras-ERK/MAPK signaling within the context of a whole organism. Importantly, this phenotype-based RNAi screening can be applied for other stem cell-associated signaling pathways such as Wnt/β-catenin and Notch using the C. elegans.

Cognitive context detection in UAS operators using eye-gaze patterns on computer screens

NASA Astrophysics Data System (ADS)

Mannaru, Pujitha; Balasingam, Balakumar; Pattipati, Krishna; Sibley, Ciara; Coyne, Joseph

2016-05-01

In this paper, we demonstrate the use of eye-gaze metrics of unmanned aerial systems (UAS) operators as effective indices of their cognitive workload. Our analyses are based on an experiment where twenty participants performed pre-scripted UAS missions of three different difficulty levels by interacting with two custom designed graphical user interfaces (GUIs) that are displayed side by side. First, we compute several eye-gaze metrics, traditional eye movement metrics as well as newly proposed ones, and analyze their effectiveness as cognitive classifiers. Most of the eye-gaze metrics are computed by dividing the computer screen into "cells". Then, we perform several analyses in order to select metrics for effective cognitive context classification related to our specific application; the objective of these analyses are to (i) identify appropriate ways to divide the screen into cells; (ii) select appropriate metrics for training and classification of cognitive features; and (iii) identify a suitable classification method.

Expression screening using a Medaka cDNA library identifies evolutionarily conserved regulators of the p53/Mdm2 pathway.

PubMed

Zhang, Ping; Kratz, Anne Sophie; Salama, Mohammed; Elabd, Seham; Heinrich, Thorsten; Wittbrodt, Joachim; Blattner, Christine; Davidson, Gary

2015-10-08

The p53 tumor suppressor protein is mainly regulated by alterations in the half-life of the protein, resulting in significant differences in p53 protein levels in cells. The major regulator of this process is Mdm2, which ubiquitinates p53 and targets it for proteasomal degradation. This process can be enhanced or reduced by proteins that associate with p53 or Mdm2 and several proteins have been identified with such an activity. Furthermore, additional ubiquitin ligases for p53 have been identified in recent years. Nevertheless, our understanding of how p53 abundance and Mdm2 activity are regulated remains incomplete. Here we describe a cell culture based overexpression screen to identify evolutionarily conserved regulators of the p53/Mdm2 circuit. The results from this large-scale screening method will contribute to a better understanding of the regulation of these important proteins. Expression screening was based on co-transfection of H1299 cells with pools of cDNA's from a Medaka library together with p53, Mdm2 and, as internal control, Ror2. After cell lysis, SDS-PAGE/WB analysis was used to detect alterations in these proteins. More than one hundred hits that altered the abundance of either p53, Mdm2, or both were identified in the primary screen. Subscreening of the library pools that were identified in the primary screen identified several potential novel regulators of p53 and/or Mdm2. We also tested whether the human orthologues of the Medaka genes regulate p53 and/or Mdm2 abundance. All human orthologues regulated p53 and/or Mdm2 abundance in the same manner as the proteins from Medaka, which underscores the suitability of this screening methodology for the identification of new modifiers of p53 and Mdm2. Despite enormous efforts in the last two decades, many unknown regulators for p53 and Mdm2 abundance are predicted to exist. This cross-species approach to identify evolutionarily conserved regulators demonstrates that our Medaka unigene cDNA library represents a powerful tool to screen for these novel regulators of the p53/Mdm2 pathway.

Bidirectional reporter assay using HAL promoter and TOPFLASH improves specificity in high-throughput screening of Wnt inhibitors.

PubMed

Yamaguchi, Kiyoshi; Zhu, Chi; Ohsugi, Tomoyuki; Yamaguchi, Yuko; Ikenoue, Tsuneo; Furukawa, Yoichi

2017-12-01

Constitutive activation of Wnt signaling plays an important role in colorectal and liver tumorigenesis. Cell-based assays using synthetic TCF/LEF (T-cell factor/lymphoid enhancer factor) reporters, as readouts of β-catenin/TCF-dependent transcriptional activity, have contributed greatly to the discovery of small molecules that modulate Wnt signaling. In the present study, we report a novel screening method, called a bidirectional dual reporter assay. Integrated transcriptome analysis identified a histidine ammonia-lyase gene (HAL) that was negatively regulated by β-catenin/TCF-dependent transcriptional activity. We leveraged a promoter region of the HAL gene as another transcriptional readout of Wnt signaling. Cells stably expressing both an optimized HAL reporter and the TCF/LEF reporter enabled bidirectional reporter activities in response to Wnt signaling. Increased HAL reporter activity and decreased TCF/LEF reporter activity were observed simultaneously in the cells when β-catenin/TCF7L2 was inhibited. Notably, this method could decrease the number of false positives observed when screening an inhibitor library compared with the conventional TCF/LEF assay. We found that Brefeldin A, a disruptor of the Golgi apparatus, inhibited the Wnt/β-catenin signaling pathway. The utility of our system could be expanded to examine other disease-associated pathways beyond the Wnt/β-catenin signaling pathway. © 2017 Wiley Periodicals, Inc.

Novel method for screening of enteric film coatings properties with magnetic resonance imaging.

PubMed

Dorożyński, Przemysław; Jamróz, Witold; Niwiński, Krzysztof; Kurek, Mateusz; Węglarz, Władysław P; Jachowicz, Renata; Kulinowski, Piotr

2013-11-18

The aim of the study is to present the concept of novel method for fast screening of enteric coating compositions properties without the need of preparation of tablets batches for fluid bed coating. Proposed method involves evaluation of enteric coated model tablets in specially designed testing cell with application of MRI technique. The results obtained in the testing cell were compared with results of dissolution studies of mini-tablets coated in fluid bed apparatus. The method could be useful in early stage of formulation development for screening of film coating properties that will shorten and simplify the development works. Copyright © 2013 Elsevier B.V. All rights reserved.

Molecular Building Block-Based Electronic Charges for High-Throughput Screening of Metal-Organic Frameworks for Adsorption Applications.

PubMed

Argueta, Edwin; Shaji, Jeena; Gopalan, Arun; Liao, Peilin; Snurr, Randall Q; Gómez-Gualdrón, Diego A

2018-01-09

Metal-organic frameworks (MOFs) are porous crystalline materials with attractive properties for gas separation and storage. Their remarkable tunability makes it possible to create millions of MOF variations but creates the need for fast material screening to identify promising structures. Computational high-throughput screening (HTS) is a possible solution, but its usefulness is tied to accurate predictions of MOF adsorption properties. Accurate adsorption simulations often require an accurate description of electrostatic interactions, which depend on the electronic charges of the MOF atoms. HTS-compatible methods to assign charges to MOF atoms need to accurately reproduce electrostatic potentials (ESPs) and be computationally affordable, but current methods present an unsatisfactory trade-off between computational cost and accuracy. We illustrate a method to assign charges to MOF atoms based on ab initio calculations on MOF molecular building blocks. A library of building blocks with built-in charges is thus created and used by an automated MOF construction code to create hundreds of MOFs with charges "inherited" from the constituent building blocks. The molecular building block-based (MBBB) charges are similar to REPEAT charges-which are charges that reproduce ESPs obtained from ab initio calculations on crystallographic unit cells of nanoporous crystals-and thus similar predictions of adsorption loadings, heats of adsorption, and Henry's constants are obtained with either method. The presented results indicate that the MBBB method to assign charges to MOF atoms is suitable for use in computational high-throughput screening of MOFs for applications that involve adsorption of molecules such as carbon dioxide.

Dynamics of subcellular compartmentalization of steroid receptors in living cells as a strategic screening method to determine the biological impact of suspected endocrine disruptors.

PubMed

Tyagi, Rakesh Kumar

2003-04-01

Although a number of screening methods being used for identifying potential endocrine disruptors have generated a wealth of information, a search for alternative combination of methods is still needed to overcome experimental artefacts. There are no generally accepted or validated screening methods for monitoring and studying impact of environmental endocrine disruptors. Also, no single assay can accurately predict all the deleterious effects of endocrine disruptors. For this reason various environmental protection agencies, mainly European and US, have urged that a battery of tests in current use need to be designed to assess their adequacy in detecting the effects of endocrine disruptors. Some details about endocrine disruptors and screening programs can be found at http://www.epa.gov/scipoly/oscpendo/whatis.htm. Several studies in recent years have used fusion proteins between steroid receptors (estrogen, androgen, progesterone, etc.) and green fluorescent protein (GFP) that can serve as an alternative potent screening method to study intracellular dynamics of receptors in living cells. An approach employing nucleocytoplasmic trafficking of steroid receptors as a parameter in response to potential xenobiotic chemicals in living cells may prove to be promising in terms of being direct, fast, reliable, simple and inexpensive. Copyright 2003 Elsevier Science Ltd.

A droplet-based heterogeneous immunoassay for screening single cells secreting antigen-specific antibodies.

PubMed

Akbari, Samin; Pirbodaghi, Tohid

2014-09-07

High throughput heterogeneous immunoassays that screen antigen-specific antibody secreting cells are essential to accelerate monoclonal antibody discovery for therapeutic applications. Here, we introduce a heterogeneous single cell immunoassay based on alginate microparticles as permeable cell culture chambers. Using a microfluidic device, we encapsulated single antibody secreting cells in 35-40 μm diameter alginate microbeads. We functionalized the alginate to capture the secreted antibodies inside the microparticles, enabling single cell analysis and preventing the cross-talk between the neighboring encapsulated cells. We demonstrated non-covalent functionalization of alginate microparticles by adding three secondary antibodies to the alginate solution to form high molecular weight complexes that become trapped in the porous nanostructure of alginate and capture the secreted antibodies. We screened anti-TNF-alpha antibody-secreting cells from a mixture of antibody-secreting cells.

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy.

PubMed

Allan, Kristina J; Mahoney, Douglas J; Baird, Stephen D; Lefebvre, Charles A; Stojdl, David F

2018-04-03

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

Purification of human adipose-derived stem cells from fat tissues using PLGA/silk screen hybrid membranes.

PubMed

Chen, Da-Chung; Chen, Li-Yu; Ling, Qing-Dong; Wu, Meng-Hsueh; Wang, Ching-Tang; Suresh Kumar, S; Chang, Yung; Munusamy, Murugan A; Alarfajj, Abdullah A; Wang, Han-Chow; Hsu, Shih-Tien; Higuchi, Akon

2014-05-01

The purification of human adipose-derived stem cells (hADSCs) from human adipose tissue cells (stromal vascular fraction) was investigated using membrane filtration through poly(lactide-co-glycolic acid)/silk screen hybrid membranes. Membrane filtration methods are attractive in regenerative medicine because they reduce the time required to purify hADSCs (i.e., less than 30 min) compared with conventional culture methods, which require 5-12 days. hADSCs expressing the mesenchymal stem cell markers CD44, CD73, and CD90 were concentrated in the permeation solution from the hybrid membranes. Expression of the surface markers CD44, CD73, and CD99 on the cells in the permeation solution from the hybrid membranes, which were obtained using 18 mL of feed solution containing 50 × 10⁴ cells, was statistically significantly higher than that of the primary adipose tissue cells, indicating that the hADSCs can be purified in the permeation solution by the membrane filtration method. Cells expressing the stem cell-associated marker CD34 could be successfully isolated in the permeation solution, whereas CD34⁺ cells could not be purified by the conventional culture method. The hADSCs in the permeation solution demonstrated a superior capacity for osteogenic differentiation based on their alkali phosphatase activity, their osterix gene expression, and the results of mineralization analysis by Alizarin Red S and von Kossa staining compared with the cells from the suspension of human adipose tissue. These results suggest that the hADSCs capable of osteogenic differentiation preferentially permeate through the hybrid membranes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Nuclear morphology for the detection of alterations in bronchial cells from lung cancer: an attempt to improve sensitivity and specificity.

PubMed

Fafin-Lefevre, Mélanie; Morlais, Fabrice; Guittet, Lydia; Clin, Bénédicte; Launoy, Guy; Galateau-Sallé, Françoise; Plancoulaine, Benoît; Herlin, Paulette; Letourneux, Marc

2011-08-01

To identify which morphologic or densitometric parameters are modified in cell nuclei from bronchopulmonary cancer based on 18 parameters involving shape, intensity, chromatin, texture, and DNA content and develop a bronchopulmonary cancer screening method relying on analysis of sputum sample cell nuclei. A total of 25 sputum samples from controls and 22 bronchial aspiration samples from patients presenting with bronchopulmonary cancer who were professionally exposed to cancer were used. After Feulgen staining, 18 morphologic and DNA content parameters were measured on cell nuclei, via image cytom- etry. A method was developed for analyzing distribution quantiles, compared with simply interpreting mean values, to characterize morphologic modifications in cell nuclei. Distribution analysis of parameters enabled us to distinguish 13 of 18 parameters that demonstrated significant differences between controls and cancer cases. These parameters, used alone, enabled us to distinguish two population types, with both sensitivity and specificity > 70%. Three parameters offered 100% sensitivity and specificity. When mean values offered high sensitivity and specificity, comparable or higher sensitivity and specificity values were observed for at least one of the corresponding quantiles. Analysis of modification in morphologic parameters via distribution analysis proved promising for screening bronchopulmonary cancer from sputum.

Application of semiconductor diffusants to solar cells by screen printing

NASA Technical Reports Server (NTRS)

Evans, J. C., Jr.; Brandhorst, H. W., Jr.; Mazaris, G. A.; Scudder, L. R. (Inventor)

1978-01-01

Diffusants were applied onto semiconductor solar cell substrates, using screen printing techniques. The method was applicable to square and rectangular cells and can be used to apply dopants of opposite types to the front and back of the substrate. Then, simultaneous diffusion of both dopants can be performed with a single furnace pass.

Screening of Small Molecule Interactor Library by Using In-Cell NMR Spectroscopy (SMILI-NMR)

PubMed Central

Xie, Jingjing; Thapa, Rajiv; Reverdatto, Sergey; Burz, David S.; Shekhtman, Alexander

2011-01-01

We developed an in-cell NMR assay for screening small molecule interactor libraries (SMILI-NMR) for compounds capable of disrupting or enhancing specific interactions between two or more components of a biomolecular complex. The method relies on the formation of a well-defined biocomplex and utilizes in-cell NMR spectroscopy to identify the molecular surfaces involved in the interaction at atomic scale resolution. Changes in the interaction surface caused by a small molecule interfering with complex formation are used as a read-out of the assay. The in-cell nature of the experimental protocol insures that the small molecule is capable of penetrating the cell membrane and specifically engaging the target molecule(s). Utility of the method was demonstrated by screening a small dipeptide library against the FKBP–FRB protein complex involved in cell cycle arrest. The dipeptide identified by SMILI-NMR showed biological activity in a functional assay in yeast. PMID:19422228

Tandem screening of toxic compounds on GFP-labeled bacteria and cancer cells in microtiter plates.

PubMed

Montoya, Jessica; Varela-Ramirez, Armando; Shanmugasundram, Muthian; Martinez, Luis E; Primm, Todd P; Aguilera, Renato J

2005-09-23

A 96-well fluorescence-based assay has been developed for the rapid screening of potential cytotoxic and bacteriocidal compounds. The assay is based on detection of green fluorescent protein (GFP) in HeLa human carcinoma cells as well as gram negative (Escherichia coli) and gram positive bacteria (Mycobacterium avium). Addition of a toxic compound to the GFP marked cells resulted in the loss of the GFP fluorescence which was readily detected by fluorometry. Thirty-nine distinct naphthoquinone derivatives were screened and several of these compounds were found to be toxic to all cell types. Apart from differences in overall toxicity, two general types of toxic compounds were detected, those that exhibited toxicity to two or all three of the cell types and those that were primarily toxic to the HeLa cells. Our results demonstrate that the parallel screening of both eukaryotic and prokaryotic cells is not only feasible and reproducible but also cost effective.

A Reverse-phase Protein Microarray-based Screen Identifies Host Signaling Dynamics upon Burkholderia spp. Infection

DTIC Science & Technology

2015-07-27

University, Manassas, VA, USA, 4 PerkinElmer, Inc., Waltham, MA, USA Burkholderia is a diverse genus of gram-negative bacteria that causes high...Burkholderia spp. infections. Materials and Methods Bacterial Strains Bm ATCC 23344, NCTC 10247, NCTC 10229, NCTC 3708, NCTC 3709, 2002721278 (Burtnick...macrophage cell line RAW264.7 was obtained from ATCC (Manassas, VA). Cells were cultured in DMEM (Life Technologies) supplemented with 10% fetal

An in vitro screening method to evaluate chemicals as potential chemotherapeutants to control Aeromonas hydrophila infection in channel catfish

USDA-ARS?s Scientific Manuscript database

Using catfish gill cells G1B and four chemicals (hydrogen peroxide, sodium chloride, potassium permanganate, and D-mannose), the feasibility of using an in vitro screening method to identify potential effective chemotherapeutants was evaluated in this study. In vitro screening results revealed that,...

Isolation of tumor antigen-specific single-chain variable fragments using a chimeric antigen receptor bicistronic retroviral vector in a Mammalian screening protocol.

PubMed

Lipowska-Bhalla, Grazyna; Gilham, David E; Hawkins, Robert E; Rothwell, Dominic G

2013-12-01

The clinical potential of chimeric antigen receptors in adoptive cellular therapy is beginning to be realized with several recent clinical trials targeting CD19 showing promising results in advanced B cell malignancies. This increased efficacy corresponds with improved engineering of the chimeric receptors with the latest-generation receptors eliciting greater signaling and proliferation potential. However, the antigen-binding single-chain variable fragment (scFv) domain of the receptors is critical in determining the activity of the chimeric receptor-expressing T cells, as this determines specificity and affinity to the tumor antigen. In this study, we describe a mammalian T cell line screening protocol employing a 2A-based bicistronic retroviral vector to isolate functional scFvs. This approach involves expression of the scFv library in a chimeric antigen receptor, and is based on selection of clones capable of stimulating CD69 upregulation in a T cell line and has a number of advantages over previously described methods in that the use of a 2A cassette ensures the exclusion of nonexpressing scFvs and the screening using a chimeric receptor in a mammalian T cell line ensures selection in the optimum context for therapeutic use. Proof-of-principle experiments show that the protocol was capable of a 10(5)-fold enrichment of positive clones after three rounds of selection. Furthermore, an antigen-specific clone was successfully isolated from a partially enriched scFv library, confirming the strength of the protocol. This approach has the potential to identify novel scFvs of use in adoptive T cell therapy and, potentially, wider antibody-based applications.

A Multiplexed High-Content Screening Approach Using the Chromobody Technology to Identify Cell Cycle Modulators in Living Cells.

PubMed

Schorpp, Kenji; Rothenaigner, Ina; Maier, Julia; Traenkle, Bjoern; Rothbauer, Ulrich; Hadian, Kamyar

2016-10-01

Many screening hits show relatively poor quality regarding later efficacy and safety. Therefore, small-molecule screening efforts shift toward high-content analysis providing more detailed information. Here, we describe a novel screening approach to identify cell cycle modulators with low toxicity by combining the Cell Cycle Chromobody (CCC) technology with the CytoTox-Glo (CTG) cytotoxicity assay. The CCC technology employs intracellularly functional single-domain antibodies coupled to a fluorescent protein (chromobodies) to visualize the cell cycle-dependent redistribution of the proliferating cell nuclear antigen (PCNA) in living cells. This image-based cell cycle analysis was combined with determination of dead-cell protease activity in cell culture supernatants by the CTG assay. We adopted this multiplex approach to high-throughput format and screened 960 Food and Drug Administration (FDA)-approved drugs. By this, we identified nontoxic compounds, which modulate different cell cycle stages, and validated selected hits in diverse cell lines stably expressing CCC. Additionally, we independently validated these hits by flow cytometry as the current state-of-the-art format for cell cycle analysis. This study demonstrates that CCC imaging is a versatile high-content screening approach to identify cell cycle modulators, which can be multiplexed with cytotoxicity assays for early elimination of toxic compounds during screening. © 2016 Society for Laboratory Automation and Screening.

Live cell imaging compatible immobilization of Chlamydomonas reinhardtii in microfluidic platform for biodiesel research.

PubMed

Park, Jae Woo; Na, Sang Cheol; Nguyen, Thanh Qua; Paik, Sang-Min; Kang, Myeongwoo; Hong, Daewha; Choi, Insung S; Lee, Jae-Hyeok; Jeon, Noo Li

2015-03-01

This paper describes a novel surface immobilization method for live-cell imaging of Chlamydomonas reinhardtii for continuous monitoring of lipid droplet accumulation. Microfluidics allows high-throughput manipulation and analysis of single cells in precisely controlled microenvironment. Fluorescence imaging based quantitative measurement of lipid droplet accumulation in microalgae had been difficult due to their intrinsic motile behavior. We present a simple surface immobilization method using gelatin coating as the "biological glue." We take advantage of hydroxyproline (Hyp)-based non-covalent interaction between gelatin and the outer cell wall of microalgae to anchor the cells inside the microfluidic device. We have continuously monitored single microalgal cells for up to 6 days. The immobilized microalgae remain viable (viability was comparable to bulk suspension cultured controls). When exposed to wall shear stress, most of the cells remain attached up to 0.1 dyne/cm(2) . Surface immobilization allowed high-resolution, live-cell imaging of mitotic process in real time-which followed previously reported stages in mitosis of suspension cultured cells. Use of gelatin coated microfluidics devices can result in better methods for microalgae strain screening and culture condition optimization that will help microalgal biodiesel become more economically viable. © 2014 Wiley Periodicals, Inc.

Etiology and early pathogenesis of malignant testicular germ cell tumors: towards possibilities for preinvasive diagnosis

PubMed Central

Elzinga-Tinke, Jenny E; Dohle, Gert R; Looijenga, Leendert HJ

2015-01-01

Malignant testicular germ cell tumors (TGCT) are the most frequent cancers in Caucasian males (20–40 years) with an 70% increasing incidence the last 20 years, probably due to combined action of (epi)genetic and (micro)environmental factors. It is expected that TGCT have carcinoma in situ (CIS) as their common precursor, originating from an embryonic germ cell blocked in its maturation process. The overall cure rate of TGCT is more than 90%, however, men surviving TGCT can present long-term side effects of systemic cancer treatment. In contrast, men diagnosed and treated for CIS only continue to live without these long-term side effects. Therefore, early detection of CIS has great health benefits, which will require an informative screening method. This review described the etiology and early pathogenesis of TGCT, as well as the possibilities of early detection and future potential of screening men at risk for TGCT. For screening, a well-defined risk profile based on both genetic and environmental risk factors is needed. Since 2009, several genome wide association studies (GWAS) have been published, reporting on single-nucleotide polymorphisms (SNPs) with significant associations in or near the genes KITLG, SPRY4, BAK1, DMRT1, TERT, ATF7IP, HPGDS, MAD1L1, RFWD3, TEX14, and PPM1E, likely to be related to TGCT development. Prenatal, perinatal, and postnatal environmental factors also influence the onset of CIS. A noninvasive early detection method for CIS would be highly beneficial in a clinical setting, for which specific miRNA detection in semen seems to be very promising. Further research is needed to develop a well-defined TGCT risk profile, based on gene-environment interactions, combined with noninvasive detection method for CIS. PMID:25791729

A Policy Impact Analysis of the Mandatory NCAA Sickle Cell Trait Screening Program

PubMed Central

Tarini, Beth A; Brooks, Margaret Alison; Bundy, David G

2012-01-01

Objective To estimate the impact of the mandatory National Collegiate Athletic Association (NCAA) sickle cell trait (SCT) screening policy on the identification of sickle cell carriers and prevention of sudden death. Data Source We used NCAA reports, population-based SCT prevalence estimates, and published risks for exercise-related sudden death attributable to SCT. Study Design We estimated the number of sickle cell carriers identified and the number of potentially preventable sudden deaths with mandatory SCT screening of NCAA Division I athletes. We calculated the number of student-athletes with SCT using a conditional probability based upon SCT prevalence data and self-identified race/ethnicity status. We estimated sudden deaths over 10 years based on published attributable risk of exercise-related sudden death due to SCT. Principal Findings We estimate that over 2,000 NCAA Division I student-athletes with SCT will be identified under this screening policy and that, without intervention, about seven NCAA Division I student-athletes would die suddenly as a complication of SCT over a 10-year period. Conclusion Universal sickle cell screening of NCAA Division I student-athletes will identify a substantial number of sickle cell carriers. A successful intervention could prevent about seven deaths over a decade. PMID:22150647

Advanced Cell Classifier: User-Friendly Machine-Learning-Based Software for Discovering Phenotypes in High-Content Imaging Data.

PubMed

Piccinini, Filippo; Balassa, Tamas; Szkalisity, Abel; Molnar, Csaba; Paavolainen, Lassi; Kujala, Kaisa; Buzas, Krisztina; Sarazova, Marie; Pietiainen, Vilja; Kutay, Ulrike; Smith, Kevin; Horvath, Peter

2017-06-28

High-content, imaging-based screens now routinely generate data on a scale that precludes manual verification and interrogation. Software applying machine learning has become an essential tool to automate analysis, but these methods require annotated examples to learn from. Efficiently exploring large datasets to find relevant examples remains a challenging bottleneck. Here, we present Advanced Cell Classifier (ACC), a graphical software package for phenotypic analysis that addresses these difficulties. ACC applies machine-learning and image-analysis methods to high-content data generated by large-scale, cell-based experiments. It features methods to mine microscopic image data, discover new phenotypes, and improve recognition performance. We demonstrate that these features substantially expedite the training process, successfully uncover rare phenotypes, and improve the accuracy of the analysis. ACC is extensively documented, designed to be user-friendly for researchers without machine-learning expertise, and distributed as a free open-source tool at www.cellclassifier.org. Copyright © 2017 Elsevier Inc. All rights reserved.

HTS techniques for patch clamp-based ion channel screening - advances and economy.

PubMed

Farre, Cecilia; Fertig, Niels

2012-06-01

Ten years ago, the first publication appeared showing patch clamp recordings performed on a planar glass chip instead of using a conventional patch clamp pipette. "Going planar" proved to revolutionize ion channel drug screening as we know it, by allowing high quality measurements of ion channels and their effectors at a higher throughput and at the same time de-skilling the highly laborious technique. Over the years, platforms evolved in response to user requirements regarding experimental features, data handling plus storage, and suitable target diversity. This article gives a snapshot image of patch clamp-based ion channel screening with focus on platforms developed to meet requirements of high-throughput screening environments. The commercially available platforms are described, along with their benefits and drawbacks in ion channel drug screening. Automated patch clamp (APC) platforms allow faster investigation of a larger number of ion channel active compounds or cell clones than previously possible. Since patch clamp is the only method allowing direct, real-time measurements of ion channel activity, APC holds the promise of picking up high quality leads, where they otherwise would have been overseen using indirect methods. In addition, drug candidate safety profiling can be performed earlier in the drug discovery process, avoiding late-phase compound withdrawal due to safety liability issues, which is highly costly and inefficient.

Screening soy hydrolysates for the production of a recombinant therapeutic protein in commercial cell line by combined approach of near-infrared spectroscopy and chemometrics.

PubMed

Li, Guiyang; Wen, Zai-Qing

2013-03-01

Soy hydrolysates are widely used as the major nutrient sources for cell culture processes for industrial manufacturing of therapeutic recombinant proteins. The primary goal of this study was to develop a spectroscopy based chemometric method, a partial least squares (PLS), to screen soy hydrolysates for better yield of protein production (titers) in cell culture medium. Harvest titer values of 29 soy hydrolysate lots with production yield between 490 and 1,350 mg/L were obtained from shake flask models or from manufacture engineering runs. The soy hydrolysate samples were measured by near-infrared (NIR) in reflectance mode using an infrared fiber optic probe. The fiber optic probe could easily enable in situ measurement of the soy hydrolysates for convenient raw material screening. The best PLS calibration has a determination coefficient of R (2) = 0.887 utilizing no spectral preprocessing, the two spectral ranges of 10,000-5,376 cm(-1) and 4,980-4,484 cm(-1), and a rank of 6 factors. The cross-validation of the model resulted in a determination coefficient of R (2) = 0.741 between the predicted and actual titer values with an average standard deviation of 72 mg/L. Compared with the resource demanding shake flask model, the combination of NIR and chemometric modeling provides a convenient method for soy hydrolysate screening with the advantage of fast speed, low cost and non-destructive.

3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions.

PubMed

Wenzel, Carsten; Riefke, Björn; Gründemann, Stephan; Krebs, Alice; Christian, Sven; Prinz, Florian; Osterland, Marc; Golfier, Sven; Räse, Sebastian; Ansari, Nariman; Esner, Milan; Bickle, Marc; Pampaloni, Francesco; Mattheyer, Christian; Stelzer, Ernst H; Parczyk, Karsten; Prechtl, Stefan; Steigemann, Patrick

2014-04-15

Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients as well as the development of dormant tumor regions. We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Cytotoxicity Testing: Cell Experiments

NASA Astrophysics Data System (ADS)

Grünert, Renate; Westendorf, Aron; Buczkowska, Magdalena; Hänsch, Mareike; Grüunert, Sybil; Bednarski, Patrick J.

Screening for new anticancer agents has traditionally been done with in vitro cell culture methods. Even in the genomic era of target-driven drug design, screening for cytotoxic activity is still a standard tool in the search for new anticancer agents, especially if the mode of action of a substance is not yet known. A wide variety of cell culture methods with unique end-points are available for testing the anticancer potential of a substance. Each has its advantages and disadvantages, which must be weighed in the decision to use a particular method. Often several complementary methods are used to gain information on the mode of action of a substance.

Strategy for Identifying Repurposed Drugs for the Treatment of Cerebral Cavernous Malformation

PubMed Central

Gibson, Christopher C.; Zhu, Weiquan; Davis, Chadwick T.; Bowman-Kirigin, Jay A.; Chan, Aubrey C.; Ling, Jing; Walker, Ashley E.; Goitre, Luca; Monache, Simona Delle; Retta, Saverio Francesco; Shiu, Yan-Ting E.; Grossmann, Allie H.; Thomas, Kirk R.; Donato, Anthony J.; Lesniewski, Lisa A.; Whitehead, Kevin J.; Li, Dean Y.

2014-01-01

Background Cerebral cavernous malformation (CCM) is a hemorrhagic stroke disease affecting up to 0.5% of North Americans with no approved non-surgical treatment. A subset of patients have a hereditary form of the disease due primarily to loss-of-function mutations in KRIT1, CCM2, or PDCD10. We sought to identify known drugs that could be repurposed to treat CCM. Methods and Results We developed an unbiased screening platform based on both cellular and animal models of loss-of-function of CCM2. Our discovery strategy consisted of four steps: an automated immunofluorescence and machine-learning-based primary screen of structural phenotypes in human endothelial cells deficient in CCM2; a secondary screen of functional changes in endothelial stability in these same cells; a rapid in vivo tertiary screen of dermal microvascular leak in mice lacking endothelial Ccm2; and finally a quaternary screen of CCM lesion burden in these same mice. We screened 2,100 known drugs and bioactive compounds, and identified two candidates for further study, cholecalciferol (Vitamin D3) and tempol (a scavenger of superoxide). Each drug decreased lesion burden in a mouse model of CCM vascular disease by approximately 50%. Conclusions By identifying known drugs as potential therapeutics for CCM, we have decreased the time, cost, and risk of bringing treatments to patients. Each drug also prompts additional exploration of biomarkers of CCM disease. We further suggest that the structure-function screening platform presented here may be adapted and scaled to facilitate drug discovery for diverse loss-of-function genetic vascular disease. PMID:25486933

Using silver and bighead carp cell lines for the identification of a unique metabolite fingerprint from thiram-specific chemical exposure

USGS Publications Warehouse

Putnam, Joel G.; Nelson, Justine; Leis, Eric M; Erickson, Richard A.; Hubert, Terrance D.; Amberg, Jon J.

2017-01-01

Conservation biology often requires the control of invasive species. One method is the development and use of biocides. Identifying new chemicals as part of the biocide registration approval process can require screening millions of compounds. Traditionally, screening new chemicals has been done in vivo using test organisms. Using in vitro (e.g., cell lines) and in silico (e.g., computer models) methods decrease test organism requirements and increase screening speed and efficiency. These methods, however, would be greatly improved by better understanding how individual fish species metabolize selected compounds.We combined cell assays and metabolomics to create a powerful tool to facilitate the identification of new control chemicals. Specifically, we exposed cell lines established from bighead carp and silver carp larvae to thiram (7 concentrations) then completed metabolite profiling to assess the dose-response of the bighead carp and silver carp metabolome to thiram. Forty one of the 700 metabolomic markers identified in bighead carp exhibited a dose-response to thiram exposure compared to silver carp in which 205 of 1590 metabolomic markers exhibited a dose-response. Additionally, we identified 11 statistically significant metabolomic markers based upon volcano plot analysis common between both species. This smaller subset of metabolites formed a thiram-specific metabolomic fingerprint which allowed for the creation of a toxicant specific, rather than a species-specific, metabolomic fingerprint. Metabolomic fingerprints may be used in biocide development and improve our understanding of ecologically significant events, such as mass fish kills.

An approach for cell viability online detection based on the characteristics of lensfree cell diffraction fingerprint.

PubMed

Li, Guoxiao; Zhang, Rongbiao; Yang, Ning; Yin, Changsheng; Wei, Mingji; Zhang, Yecheng; Sun, Jian

2018-06-01

To overcome the drawbacks such as low automation and high cost, an approach for cell viability online detection is proposed, based on the extracted lensfree cell diffraction fingerprint characteristics. The cell fingerprints are acquired by a constructed large field-of-view (FOV) diffraction imaging platform without any lenses. The approach realizes distinguishing live and dead cells online and calculating cell viability index based on the number of live cells. With theoretical analysis and simulation, diffraction fingerprints of cells with different morphology are simulated and two characteristics are discovered to be able to reflect cell viability status effectively. Two parameters, fringe intensity contrast (FIC) and fringe dispersion (FD), are defined to quantify these two characteristics. They are verified to be reliable to identify live cells. In a cytotoxicity assay of different methyl mercury concentration on BRL cells, the proposed approach is used to detect cell viability. MTT method is also employed and the results of correlational analysis and Bland-Altman analysis prove the validity of the proposed approach. By comparison, it can be revealed that the proposed approach has some advantages over other present techniques. Therefore it may be widely used as a cell viability measurement method in drug screening, nutritional investigation and cell toxicology studies. Copyright © 2018 Elsevier B.V. All rights reserved.

A Digital PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells

PubMed Central

Berman, Jennifer R.; Postovit, Lynne-Marie

2016-01-01

The rapid adoption of gene editing tools such as CRISPRs and TALENs for research and eventually therapeutics necessitates assays that can rapidly detect and quantitate the desired alterations. Currently, the most commonly used assay employs “mismatch nucleases” T7E1 or “Surveyor” that recognize and cleave heteroduplexed DNA amplicons containing mismatched base-pairs. However, this assay is prone to false positives due to cancer-associated mutations and/or SNPs and requires large amounts of starting material. Here we describe a powerful alternative wherein droplet digital PCR (ddPCR) can be used to decipher homozygous from heterozygous mutations with superior levels of both precision and sensitivity. We use this assay to detect knockout inducing alterations to stem cell associated proteins, NODAL and SFRP1, generated using either TALENs or an “all-in-one” CRISPR/Cas plasmid that we have modified for one-step cloning and blue/white screening of transformants. Moreover, we highlight how ddPCR can be used to assess the efficiency of varying TALEN-based strategies. Collectively, this work highlights how ddPCR-based screening can be paired with CRISPR and TALEN technologies to enable sensitive, specific, and streamlined approaches to gene editing and validation. PMID:27089539

Droplet based microfluidics for highthroughput screening of antibody secreting cells

NASA Astrophysics Data System (ADS)

Cai, Liheng; Heyman, John; Mazutis, Linas; Ung, Lloyd; Guerra, Rodrigo; Aubrecht, Donald; Weitz, David

2014-03-01

We present a droplet based microfluidic platform that allows highthroughput screening of antibody secreting cells. We coencapsulate single cells, fluorescent probes, and detection beads into emulsion droplets with diameter of 40 micron. The beads capture antibodies secreted by cells, resulting in a pronounced fluorescent signal that activates dielectrophoresis sorting at rate about 500 droplets per second. Moreover, we demonstrate that Reverse Transcription Polymerase Chain Reaction (RT-PCR) can be successfully applied to the cell encapsulated in a single sorted droplet. Our work highlights the potential of droplet based microfluidics as a platform to generate recombinant antibodies.

High-throughput, label-free, single-cell, microalgal lipid screening by machine-learning-equipped optofluidic time-stretch quantitative phase microscopy.

PubMed

Guo, Baoshan; Lei, Cheng; Kobayashi, Hirofumi; Ito, Takuro; Yalikun, Yaxiaer; Jiang, Yiyue; Tanaka, Yo; Ozeki, Yasuyuki; Goda, Keisuke

2017-05-01

The development of reliable, sustainable, and economical sources of alternative fuels to petroleum is required to tackle the global energy crisis. One such alternative is microalgal biofuel, which is expected to play a key role in reducing the detrimental effects of global warming as microalgae absorb atmospheric CO 2 via photosynthesis. Unfortunately, conventional analytical methods only provide population-averaged lipid amounts and fail to characterize a diverse population of microalgal cells with single-cell resolution in a non-invasive and interference-free manner. Here high-throughput label-free single-cell screening of lipid-producing microalgal cells with optofluidic time-stretch quantitative phase microscopy was demonstrated. In particular, Euglena gracilis, an attractive microalgal species that produces wax esters (suitable for biodiesel and aviation fuel after refinement), within lipid droplets was investigated. The optofluidic time-stretch quantitative phase microscope is based on an integration of a hydrodynamic-focusing microfluidic chip, an optical time-stretch quantitative phase microscope, and a digital image processor equipped with machine learning. As a result, it provides both the opacity and phase maps of every single cell at a high throughput of 10,000 cells/s, enabling accurate cell classification without the need for fluorescent staining. Specifically, the dataset was used to characterize heterogeneous populations of E. gracilis cells under two different culture conditions (nitrogen-sufficient and nitrogen-deficient) and achieve the cell classification with an error rate of only 2.15%. The method holds promise as an effective analytical tool for microalgae-based biofuel production. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Comparison of traditional trigger tool to data warehouse based screening for identifying hospital adverse events.

PubMed

O'Leary, Kevin J; Devisetty, Vikram K; Patel, Amitkumar R; Malkenson, David; Sama, Pradeep; Thompson, William K; Landler, Matthew P; Barnard, Cynthia; Williams, Mark V

2013-02-01

Research supports medical record review using screening triggers as the optimal method to detect hospital adverse events (AE), yet the method is labour-intensive. This study compared a traditional trigger tool with an enterprise data warehouse (EDW) based screening method to detect AEs. We created 51 automated queries based on 33 traditional triggers from prior research, and then applied them to 250 randomly selected medical patients hospitalised between 1 September 2009 and 31 August 2010. Two physicians each abstracted records from half the patients using a traditional trigger tool and then performed targeted abstractions for patients with positive EDW queries in the complementary half of the sample. A third physician confirmed presence of AEs and assessed preventability and severity. Traditional trigger tool and EDW based screening identified 54 (22%) and 53 (21%) patients with one or more AE. Overall, 140 (56%) patients had one or more positive EDW screens (total 366 positive screens). Of the 137 AEs detected by at least one method, 86 (63%) were detected by a traditional trigger tool, 97 (71%) by EDW based screening and 46 (34%) by both methods. Of the 11 total preventable AEs, 6 (55%) were detected by traditional trigger tool, 7 (64%) by EDW based screening and 2 (18%) by both methods. Of the 43 total serious AEs, 28 (65%) were detected by traditional trigger tool, 29 (67%) by EDW based screening and 14 (33%) by both. We found relatively poor agreement between traditional trigger tool and EDW based screening with only approximately a third of all AEs detected by both methods. A combination of complementary methods is the optimal approach to detecting AEs among hospitalised patients.

Comparative analysis of machine learning methods in ligand-based virtual screening of large compound libraries.

PubMed

Ma, Xiao H; Jia, Jia; Zhu, Feng; Xue, Ying; Li, Ze R; Chen, Yu Z

2009-05-01

Machine learning methods have been explored as ligand-based virtual screening tools for facilitating drug lead discovery. These methods predict compounds of specific pharmacodynamic, pharmacokinetic or toxicological properties based on their structure-derived structural and physicochemical properties. Increasing attention has been directed at these methods because of their capability in predicting compounds of diverse structures and complex structure-activity relationships without requiring the knowledge of target 3D structure. This article reviews current progresses in using machine learning methods for virtual screening of pharmacodynamically active compounds from large compound libraries, and analyzes and compares the reported performances of machine learning tools with those of structure-based and other ligand-based (such as pharmacophore and clustering) virtual screening methods. The feasibility to improve the performance of machine learning methods in screening large libraries is discussed.

A Systematic Evaluation of Field-Based Screening Methods for the Assessment of Anterior Cruciate Ligament (ACL) Injury Risk.

PubMed

Fox, Aaron S; Bonacci, Jason; McLean, Scott G; Spittle, Michael; Saunders, Natalie

2016-05-01

Laboratory-based measures provide an accurate method to identify risk factors for anterior cruciate ligament (ACL) injury; however, these methods are generally prohibitive to the wider community. Screening methods that can be completed in a field or clinical setting may be more applicable for wider community use. Examination of field-based screening methods for ACL injury risk can aid in identifying the most applicable method(s) for use in these settings. The objective of this systematic review was to evaluate and compare field-based screening methods for ACL injury risk to determine their efficacy of use in wider community settings. An electronic database search was conducted on the SPORTDiscus™, MEDLINE, AMED and CINAHL databases (January 1990-July 2015) using a combination of relevant keywords. A secondary search of the same databases, using relevant keywords from identified screening methods, was also undertaken. Studies identified as potentially relevant were independently examined by two reviewers for inclusion. Where consensus could not be reached, a third reviewer was consulted. Original research articles that examined screening methods for ACL injury risk that could be undertaken outside of a laboratory setting were included for review. Two reviewers independently assessed the quality of included studies. Included studies were categorized according to the screening method they examined. A description of each screening method, and data pertaining to the ability to prospectively identify ACL injuries, validity and reliability, recommendations for identifying 'at-risk' athletes, equipment and training required to complete screening, time taken to screen athletes, and applicability of the screening method across sports and athletes were extracted from relevant studies. Of 1077 citations from the initial search, a total of 25 articles were identified as potentially relevant, with 12 meeting all inclusion/exclusion criteria. From the secondary search, eight further studies met all criteria, resulting in 20 studies being included for review. Five ACL-screening methods-the Landing Error Scoring System (LESS), Clinic-Based Algorithm, Observational Screening of Dynamic Knee Valgus (OSDKV), 2D-Cam Method, and Tuck Jump Assessment-were identified. There was limited evidence supporting the use of field-based screening methods in predicting ACL injuries across a range of populations. Differences relating to the equipment and time required to complete screening methods were identified. Only screening methods for ACL injury risk were included for review. Field-based screening methods developed for lower-limb injury risk in general may also incorporate, and be useful in, screening for ACL injury risk. Limited studies were available relating to the OSDKV and 2D-Cam Method. The LESS showed predictive validity in identifying ACL injuries, however only in a youth athlete population. The LESS also appears practical for community-wide use due to the minimal equipment and set-up/analysis time required. The Clinic-Based Algorithm may have predictive value for ACL injury risk as it identifies athletes who exhibit high frontal plane knee loads during a landing task, but requires extensive additional equipment and time, which may limit its application to wider community settings.

High impact technologies for natural products screening.

PubMed

Koehn, Frank E

2008-01-01

Natural products have historically been a rich source of lead molecules in drug discovery. However, natural products have been de-emphasized as high throughput screening resources in the recent past, in part because of difficulties in obtaining high quality natural products screening libraries, or in applying modern screening assays to these libraries. In addition, natural products programs based on screening of extract libraries, bioassay-guided isolation, structure elucidation and subsequent production scale-up are challenged to meet the rapid cycle times that are characteristic of the modern HTS approach. Fortunately, new technologies in mass spectrometry, NMR and other spectroscopic techniques can greatly facilitate the first components of the process - namely the efficient creation of high-quality natural products libraries, bimolecular target or cell-based screening, and early hit characterization. The success of any high throughput screening campaign is dependent on the quality of the chemical library. The construction and maintenance of a high quality natural products library, whether based on microbial, plant, marine or other sources is a costly endeavor. The library itself may be composed of samples that are themselves mixtures - such as crude extracts, semi-pure mixtures or single purified natural products. Each of these library designs carries with it distinctive advantages and disadvantages. Crude extract libraries have lower resource requirements for sample preparation, but high requirements for identification of the bioactive constituents. Pre-fractionated libraries can be an effective strategy to alleviate interferences encountered with crude libraries, and may shorten the time needed to identify the active principle. Purified natural product libraries require substantial resources for preparation, but offer the advantage that the hit detection process is reduced to that of synthetic single component libraries. Whether the natural products library consists of crude or partially fractionated mixtures, the library contents should be profiled to identify the known components present - a process known as dereplication. The use of mass spectrometry and HPLC-mass spectrometry together with spectral databases is a powerful tool in the chemometric profiling of bio-sources for natural product production. High throughput, high sensitivity flow NMR is an emerging tool in this area as well. Whether by cell based or biomolecular target based assays, screening of natural product extract libraries continues to furnish novel lead molecules for further drug development, despite challenges in the analysis and prioritization of natural products hits. Spectroscopic techniques are now being used to directly screen natural product and synthetic libraries. Mass spectrometry in the form of methods such as ESI-ICRFTMS, and FACS-MS as well as NMR methods such as SAR by NMR and STD-NMR have been utilized to effectively screen molecular libraries. Overall, emerging advances in mass spectrometry, NMR and other technologies are making it possible to overcome the challenges encountered in screening natural products libraries in today's drug discovery environment. As we apply these technologies and develop them even further, we can look forward to increased impact of natural products in the HTS based drug discovery.

Biomimetic three-dimensional tissue models for advanced high-throughput drug screening

PubMed Central

Nam, Ki-Hwan; Smith, Alec S.T.; Lone, Saifullah; Kwon, Sunghoon; Kim, Deok-Ho

2015-01-01

Most current drug screening assays used to identify new drug candidates are 2D cell-based systems, even though such in vitro assays do not adequately recreate the in vivo complexity of 3D tissues. Inadequate representation of the human tissue environment during a preclinical test can result in inaccurate predictions of compound effects on overall tissue functionality. Screening for compound efficacy by focusing on a single pathway or protein target, coupled with difficulties in maintaining long-term 2D monolayers, can serve to exacerbate these issues when utilizing such simplistic model systems for physiological drug screening applications. Numerous studies have shown that cell responses to drugs in 3D culture are improved from those in 2D, with respect to modeling in vivo tissue functionality, which highlights the advantages of using 3D-based models for preclinical drug screens. In this review, we discuss the development of microengineered 3D tissue models which accurately mimic the physiological properties of native tissue samples, and highlight the advantages of using such 3D micro-tissue models over conventional cell-based assays for future drug screening applications. We also discuss biomimetic 3D environments, based-on engineered tissues as potential preclinical models for the development of more predictive drug screening assays for specific disease models. PMID:25385716

Methods to identify and analyze gene products involved in neuronal intracellular transport using Drosophila

PubMed Central

Neisch, Amanda L.; Avery, Adam W.; Machame, James B.; Li, Min-gang; Hays, Thomas S.

2017-01-01

Proper neuronal function critically depends on efficient intracellular transport and disruption of transport leads to neurodegeneration. Molecular pathways that support or regulate neuronal transport are not fully understood. A greater understanding of these pathways will help reveal the pathological mechanisms underlying disease. Drosophila melanogaster is the premier model system for performing large-scale genetic functional screens. Here we describe methods to carry out primary and secondary genetic screens in Drosophila aimed at identifying novel gene products and pathways that impact neuronal intracellular transport. These screens are performed using whole animal or live cell imaging of intact neural tissue to ensure integrity of neurons and their cellular environment. The primary screen is used to identify gross defects in neuronal function indicative of a disruption in microtubule-based transport. The secondary screens, conducted in both motoneurons and dendritic arborization neurons, will confirm the function of candidate gene products in intracellular transport. Together, the methodologies described here will support labs interested in identifying and characterizing gene products that alter intracellular transport in Drosophila. PMID:26794520

Characterization of Human Neural Progenitor Cell Models for Developmental Neurotoxicity Screening

EPA Science Inventory

Current testing methods for developmental neurotoxicity (DNT) make evaluation of the effects of large numbers of chemicals impractical and prohibitively expensive. As such, we are evaluating two different human neural progenitor cell (hNPC) models for their utility in screens for...

Bioprinting for cancer research.

PubMed

Knowlton, Stephanie; Onal, Sevgi; Yu, Chu Hsiang; Zhao, Jean J; Tasoglu, Savas

2015-09-01

Bioprinting offers the ability to create highly complex 3D architectures with living cells. This cutting-edge technique has significantly gained popularity and applicability in several fields. Bioprinting methods have been developed to effectively and rapidly pattern living cells, biological macromolecules, and biomaterials. These technologies hold great potential for applications in cancer research. Bioprinted cancer models represent a significant improvement over previous 2D models by mimicking 3D complexity and facilitating physiologically relevant cell-cell and cell-matrix interactions. Here we review bioprinting methods based on inkjet, microextrusion, and laser technologies and compare 3D cancer models with 2D cancer models. We discuss bioprinted models that mimic the tumor microenvironment, providing a platform for deeper understanding of cancer pathology, anticancer drug screening, and cancer treatment development. Copyright © 2015 Elsevier Ltd. All rights reserved.

Arrayed antibody library technology for therapeutic biologic discovery.

PubMed

Bentley, Cornelia A; Bazirgan, Omar A; Graziano, James J; Holmes, Evan M; Smider, Vaughn V

2013-03-15

Traditional immunization and display antibody discovery methods rely on competitive selection amongst a pool of antibodies to identify a lead. While this approach has led to many successful therapeutic antibodies, targets have been limited to proteins which are easily purified. In addition, selection driven discovery has produced a narrow range of antibody functionalities focused on high affinity antagonism. We review the current progress in developing arrayed protein libraries for screening-based, rather than selection-based, discovery. These single molecule per microtiter well libraries have been screened in multiplex formats against both purified antigens and directly against targets expressed on the cell surface. This facilitates the discovery of antibodies against therapeutically interesting targets (GPCRs, ion channels, and other multispanning membrane proteins) and epitopes that have been considered poorly accessible to conventional discovery methods. Copyright © 2013. Published by Elsevier Inc.

Towards early detection of cervical cancer: Fractal dimension of AFM images of human cervical epithelial cells at different stages of progression to cancer.

PubMed

Guz, Nataliia V; Dokukin, Maxim E; Woodworth, Craig D; Cardin, Andrew; Sokolov, Igor

2015-10-01

We used AFM HarmoniX modality to analyse the surface of individual human cervical epithelial cells at three stages of progression to cancer, normal, immortal (pre-malignant) and carcinoma cells. Primary cells from 6 normal strains, 6 cancer, and 6 immortalized lines (derived by plasmid DNA-HPV-16 transfection of cells from 6 healthy individuals) were tested. This cell model allowed for good control of the cell phenotype down to the single cell level, which is impractical to attain in clinical screening tests (ex-vivo). AFM maps of physical (nonspecific) adhesion are collected on fixed dried cells. We show that a surface parameter called fractal dimension can be used to segregate normal from both immortal pre-malignant and malignant cells with sensitivity and specificity of more than 99%. The reported method of analysis can be directly applied to cells collected in liquid cytology screening tests and identified as abnormal with regular optical methods to increase sensitivity. Despite cervical smear screening, sometimes it is very difficult to differentiate cancers cells from pre-malignant cells. By using AFM to analyze the surface properties of human cervical epithelial cells, the authors were able to accurately identify normal from abnormal cells. This method could augment existing protocols to increase diagnostic accuracy. Copyright © 2015. Published by Elsevier Inc.

Introduction to thematic minireview series: Development of human therapeutics based on induced pluripotent stem cell (iPSC) technology.

PubMed

Rao, Mahendra; Gottesfeld, Joel M

2014-02-21

With the advent of human induced pluripotent stem cell (hiPSC) technology, it is now possible to derive patient-specific cell lines that are of great potential in both basic research and the development of new therapeutics for human diseases. Not only do hiPSCs offer unprecedented opportunities to study cellular differentiation and model human diseases, but the differentiated cell types obtained from iPSCs may become therapeutics themselves. These cells can also be used in the screening of therapeutics and in toxicology assays for potential liabilities of therapeutic agents. The remarkable achievement of transcription factor reprogramming to generate iPSCs was recognized by the award of the Nobel Prize in Medicine to Shinya Yamanaka in 2012, just 6 years after the first publication of reprogramming methods to generate hiPSCs (Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. (2007) Cell 131, 861-872). This minireview series highlights both the promises and challenges of using iPSC technology for disease modeling, drug screening, and the development of stem cell therapeutics.

Short tandem repeat DNA typing provides an international reference standard for authentication of human cell lines.

PubMed

Dirks, Wilhelm Gerhard; Faehnrich, Silke; Estella, Isabelle Annick Janine; Drexler, Hans Guenter

2005-01-01

Cell lines have wide applications as model systems in the medical and pharmaceutical industry. Much drug and chemical testing is now first carried out exhaustively on in vitro systems, reducing the need for complicated and invasive animal experiments. The basis for any research, development or production program involving cell lines is the choice of an authentic cell line. Microsatellites in the human genome that harbour short tandem repeat (STR) DNA markers allow individualisation of established cell lines at the DNA level. Fluorescence polymerase chain reaction amplification of eight highly polymorphic microsatellite STR loci plus gender determination was found to be the best tool to screen the uniqueness of DNA profiles in a fingerprint database. Our results demonstrate that cross-contamination and misidentification remain chronic problems in the use of human continuous cell lines. The combination of rapidly generated DNA types based on single-locus STR and their authentication or individualisation by screening the fingerprint database constitutes a highly reliable and robust method for the identification and verification of cell lines.

Functional single-cell hybridoma screening using droplet-based microfluidics.

PubMed

El Debs, Bachir; Utharala, Ramesh; Balyasnikova, Irina V; Griffiths, Andrew D; Merten, Christoph A

2012-07-17

Monoclonal antibodies can specifically bind or even inhibit drug targets and have hence become the fastest growing class of human therapeutics. Although they can be screened for binding affinities at very high throughput using systems such as phage display, screening for functional properties (e.g., the inhibition of a drug target) is much more challenging. Typically these screens require the generation of immortalized hybridoma cells, as well as clonal expansion in microtiter plates over several weeks, and the number of clones that can be assayed is typically no more than a few thousand. We present here a microfluidic platform allowing the functional screening of up to 300,000 individual hybridoma cell clones within less than a day. This approach should also be applicable to nonimmortalized primary B-cells, as no cell proliferation is required: Individual cells are encapsulated into aqueous microdroplets and assayed directly for the release of antibodies inhibiting a drug target based on fluorescence. We used this system to perform a model screen for antibodies that inhibit angiotensin converting enzyme 1, a target for hypertension and congestive heart failure drugs. When cells expressing these antibodies were spiked into an unrelated hybridoma cell population in a ratio of 1:10,000 we observed a 9,400-fold enrichment after fluorescence activated droplet sorting. A wide variance in antibody expression levels at the single-cell level within a single hybridoma line was observed and high expressors could be successfully sorted and recultivated.

Structure Based Virtual Screening Studies to Identify Novel Potential Compounds for GPR142 and Their Relative Dynamic Analysis for Study of Type 2 Diabetes

NASA Astrophysics Data System (ADS)

Kaushik, Aman C.; Kumar, Sanjay; Wei, Dong Q.; Sahi, Shakti

2018-02-01

GPR142 (G protein receptor 142) is a novel orphan GPCR (G protein coupled receptor) belonging to ‘Class A’ of GPCR family and expressed in beta cells of pancreas. In this study, we reported the structure based virtual screening to identify the hit compounds which can be developed as leads for potential agonists. The results were validated through induced fit docking, pharmacophore modeling and system biology approaches. Since, there is no solved crystal structure of GPR142, we attempted to predict the 3D structure followed by validation and then identification of active site using threading and ab initio methods. Also, structure based virtual screening was performed against a total of 1171519 compounds from different libraries and only top 20 best hit compounds were screened and analyzed. Moreover, the biochemical pathway of GPR142 complex with screened compound2 was also designed and compared with experimental data. Interestingly, compound2 showed an increase in insulin production via Gq mediated signaling pathway suggesting the possible role of novel GPR142 agonists in therapy against type 2 diabetes.

Identification of a ligand for tumor necrosis factor receptor from Chinese herbs by combination of surface plasmon resonance biosensor and UPLC-MS.

PubMed

Cao, Yan; Li, Ying-Hua; Lv, Di-Ya; Chen, Xiao-Fei; Chen, Lang-Dong; Zhu, Zhen-Yu; Chai, Yi-Feng; Zhang, Jun-Ping

2016-07-01

Identification of bioactive compounds directly from complex herbal extracts is a key issue in the study of Chinese herbs. The present study describes the establishment and application of a sensitive, efficient, and convenient method based on surface plasmon resonance (SPR) biosensors for screening active ingredients targeting tumor necrosis factor receptor type 1 (TNF-R1) from Chinese herbs. Concentration-adjusted herbal extracts were subjected to SPR binding assay, and a remarkable response signal was observed in Rheum officinale extract. Then, the TNF-R1-bound ingredients were recovered, enriched, and analyzed by UPLC-QTOF/MS. As a result, physcion-8-O-β-D-monoglucoside (PMG) was identified as a bioactive compound, and the affinity constant of PMG to TNF-R1 was determined by SPR affinity analysis (K D  = 376 nM). Pharmacological assays revealed that PMG inhibited TNF-α-induced cytotoxicity and apoptosis in L929 cells via TNF-R1. Although PMG was a trace component in the chemical constituents of the R. officinale extract, it had considerable anti-inflammatory activities. It was found for the first time that PMG was a ligand for TNF receptor from herbal medicines. The proposed SPR-based screening method may prove to be an effective solution to analyzing bioactive components of Chinese herbs and other complex drug systems. Graphical abstract Scheme of the method based on SPR biosensor for screening and recovering active ingredients from complex herbal extracts and UPLC-MS for identifying them. Scheme of the method based on SPR biosensor for screening and recovering active ingredients from complex herbal extracts and UPLC-MS for identifying them.

Detecting Cancer Quickly and Accurately

NASA Astrophysics Data System (ADS)

Gourley, Paul; McDonald, Anthony; Hendricks, Judy; Copeland, Guild; Hunter, John; Akhil, Ohmar; Capps, Heather; Curry, Marc; Skirboll, Steve

2000-03-01

We present a new technique for high throughput screening of tumor cells in a sensitive nanodevice that has the potential to quickly identify a cell population that has begun the rapid protein synthesis and mitosis characteristic of cancer cell proliferation. Currently, pathologists rely on microscopic examination of cell morphology using century-old staining methods that are labor-intensive, time-consuming and frequently in error. New micro-analytical methods for automated, real time screening without chemical modification are critically needed to advance pathology and improve diagnoses. We have teamed scientists with physicians to create a microlaser biochip (based upon our R&D award winning bio-laser concept)1 which evaluates tumor cells by quantifying their growth kinetics. The key new discovery was demonstrating that the lasing spectra are sensitive to the biomolecular mass in the cell, which changes the speed of light in the laser microcavity. Initial results with normal and cancerous human brain cells show that only a few hundred cells -- the equivalent of a billionth of a liter -- are required to detect abnormal growth. The ability to detect cancer in such a minute tissue sample is crucial for resecting a tumor margin or grading highly localized tumor malignancy. 1. P. L. Gourley, NanoLasers, Scientific American, March 1998, pp. 56-61. This work supported under DOE contract DE-AC04-94AL85000 and the Office of Basic Energy Sciences.

Optofluidic time-stretch microscopy: recent advances

NASA Astrophysics Data System (ADS)

Lei, Cheng; Nitta, Nao; Ozeki, Yasuyuki; Goda, Keisuke

2018-06-01

Flow cytometry is an indispensable method for valuable applications in numerous fields such as immunology, pathology, pharmacology, molecular biology, and marine biology. Optofluidic time-stretch microscopy is superior to conventional flow cytometry methods for its capability to acquire high-quality images of single cells at a high-throughput exceeding 10,000 cells per second. This makes it possible to extract copious information from cellular images for accurate cell detection and analysis with the assistance of machine learning. Optofluidic time-stretch microscopy has proven its effectivity in various applications, including microalga-based biofuel production, evaluation of thrombotic disorders, as well as drug screening and discovery. In this review, we discuss the principles and recent advances of optofluidic time-stretch microscopy.

Optofluidic time-stretch microscopy: recent advances

NASA Astrophysics Data System (ADS)

Lei, Cheng; Nitta, Nao; Ozeki, Yasuyuki; Goda, Keisuke

2018-04-01

Flow cytometry is an indispensable method for valuable applications in numerous fields such as immunology, pathology, pharmacology, molecular biology, and marine biology. Optofluidic time-stretch microscopy is superior to conventional flow cytometry methods for its capability to acquire high-quality images of single cells at a high-throughput exceeding 10,000 cells per second. This makes it possible to extract copious information from cellular images for accurate cell detection and analysis with the assistance of machine learning. Optofluidic time-stretch microscopy has proven its effectivity in various applications, including microalga-based biofuel production, evaluation of thrombotic disorders, as well as drug screening and discovery. In this review, we discuss the principles and recent advances of optofluidic time-stretch microscopy.

Rapid screening of serum-free media for the growth of adherent Vero cells by using a small-scale and non-invasive tool.

PubMed

Petiot, Emma; Fournier, Frantz; Gény, Cécile; Pinton, Hervé; Marc, Annie

2010-03-01

The paper proposes a rapid screening method for a first step improvement of an animal component-free medium dedicated to the growth of the anchorage-dependent Vero cell line. A new, rapid, and non-invasive technique is presented to specifically monitor cultures of adherent cells in 96-well plates. The operating conditions of an image analyzer are adapted to take into account the decrease of cell size when the attached cell density increases. An experimental design is carried out to assess the influence of ten component groups in the original medium. Two groups including protein extracts, growth factor, insulin, glucose, and pyruvate show significant positive effects. The groups with vitamins and molecules related to nitrogenous bases display a less pronounced influence. The mixture of amino acids, B(1) vitamin, magnesium sulfate, and sodium phosphate as well as the couple sodium citrate and ferric chloride lead to a downward trend. The screening results are proved to be scalable in stirred cultures with cells on microcarriers. An improved serum-free medium, with some component groups being removed or added, can be rapidly formulated to reach respectively similar or 1.6 times higher cell density than in the original medium. The results from this global approach could be helpful to further focus experiments on identified medium components.

Implementation and Challenges of Direct Acoustic Dosing into Cell-Based Assays.

PubMed

Roberts, Karen; Callis, Rowena; Ikeda, Tim; Paunovic, Amalia; Simpson, Carly; Tang, Eric; Turton, Nick; Walker, Graeme

2016-02-01

Since the adoption of Labcyte Echo Acoustic Droplet Ejection (ADE) technology by AstraZeneca in 2005, ADE has become the preferred method for compound dosing into both biochemical and cell-based assays across AstraZeneca research and development globally. The initial implementation of Echos and the direct dosing workflow provided AstraZeneca with a unique set of challenges. In this article, we outline how direct Echo dosing has evolved over the past decade in AstraZeneca. We describe the practical challenges of applying ADE technology to 96-well, 384-well, and 1536-well assays and how AstraZeneca developed and applied software and robotic solutions to generate fully automated and effective cell-based assay workflows. © 2015 Society for Laboratory Automation and Screening.

Automated cell analysis tool for a genome-wide RNAi screen with support vector machine based supervised learning

NASA Astrophysics Data System (ADS)

Remmele, Steffen; Ritzerfeld, Julia; Nickel, Walter; Hesser, Jürgen

2011-03-01

RNAi-based high-throughput microscopy screens have become an important tool in biological sciences in order to decrypt mostly unknown biological functions of human genes. However, manual analysis is impossible for such screens since the amount of image data sets can often be in the hundred thousands. Reliable automated tools are thus required to analyse the fluorescence microscopy image data sets usually containing two or more reaction channels. The herein presented image analysis tool is designed to analyse an RNAi screen investigating the intracellular trafficking and targeting of acylated Src kinases. In this specific screen, a data set consists of three reaction channels and the investigated cells can appear in different phenotypes. The main issue of the image processing task is an automatic cell segmentation which has to be robust and accurate for all different phenotypes and a successive phenotype classification. The cell segmentation is done in two steps by segmenting the cell nuclei first and then using a classifier-enhanced region growing on basis of the cell nuclei to segment the cells. The classification of the cells is realized by a support vector machine which has to be trained manually using supervised learning. Furthermore, the tool is brightness invariant allowing different staining quality and it provides a quality control that copes with typical defects during preparation and acquisition. A first version of the tool has already been successfully applied for an RNAi-screen containing three hundred thousand image data sets and the SVM extended version is designed for additional screens.

Single-Cell Whole-Genome Amplification and Sequencing: Methodology and Applications.

PubMed

Huang, Lei; Ma, Fei; Chapman, Alec; Lu, Sijia; Xie, Xiaoliang Sunney

2015-01-01

We present a survey of single-cell whole-genome amplification (WGA) methods, including degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), multiple displacement amplification (MDA), and multiple annealing and looping-based amplification cycles (MALBAC). The key parameters to characterize the performance of these methods are defined, including genome coverage, uniformity, reproducibility, unmappable rates, chimera rates, allele dropout rates, false positive rates for calling single-nucleotide variations, and ability to call copy-number variations. Using these parameters, we compare five commercial WGA kits by performing deep sequencing of multiple single cells. We also discuss several major applications of single-cell genomics, including studies of whole-genome de novo mutation rates, the early evolution of cancer genomes, circulating tumor cells (CTCs), meiotic recombination of germ cells, preimplantation genetic diagnosis (PGD), and preimplantation genomic screening (PGS) for in vitro-fertilized embryos.

Is alkaline phosphatase the smoking gun for highly refractory primitive leukemic cells?

PubMed Central

Rico, Laura G.; Juncà, Jordi; Ward, Mike D.; Bradford, Jolene; Petriz, Jordi

2016-01-01

With the aim to detect candidate malignant primitive progenitor populations, we modified an original alkaline phosphatase (ALP) stem cell detection method based on the identification of alkaline phosphatase fluorescent cells in combination with flow cytometry immunophenotyping. Over a period of one year, we have been using this technique to study its activity in patients with leukemia and lymphoma, showing that changes in the alkaline phosphatase levels can be used to detect rare populations of highly refractory malignant cells. By screening different blood cancers, we have observed that this activity is not always restricted to CD34+ leukemic cells, and can be overexpressed in CD34 negative leukemia. We have verified that this method gives accurate and reproducible measurements and our preliminary results suggest that CD34+/ALPhigh cells appear to sustain leukemogenesis over time. PMID:27732563

A deep learning and novelty detection framework for rapid phenotyping in high-content screening

PubMed Central

Sommer, Christoph; Hoefler, Rudolf; Samwer, Matthias; Gerlich, Daniel W.

2017-01-01

Supervised machine learning is a powerful and widely used method for analyzing high-content screening data. Despite its accuracy, efficiency, and versatility, supervised machine learning has drawbacks, most notably its dependence on a priori knowledge of expected phenotypes and time-consuming classifier training. We provide a solution to these limitations with CellCognition Explorer, a generic novelty detection and deep learning framework. Application to several large-scale screening data sets on nuclear and mitotic cell morphologies demonstrates that CellCognition Explorer enables discovery of rare phenotypes without user training, which has broad implications for improved assay development in high-content screening. PMID:28954863

Inference of RhoGAP/GTPase regulation using single-cell morphological data from a combinatorial RNAi screen.

PubMed

Nir, Oaz; Bakal, Chris; Perrimon, Norbert; Berger, Bonnie

2010-03-01

Biological networks are highly complex systems, consisting largely of enzymes that act as molecular switches to activate/inhibit downstream targets via post-translational modification. Computational techniques have been developed to perform signaling network inference using some high-throughput data sources, such as those generated from transcriptional and proteomic studies, but comparable methods have not been developed to use high-content morphological data, which are emerging principally from large-scale RNAi screens, to these ends. Here, we describe a systematic computational framework based on a classification model for identifying genetic interactions using high-dimensional single-cell morphological data from genetic screens, apply it to RhoGAP/GTPase regulation in Drosophila, and evaluate its efficacy. Augmented by knowledge of the basic structure of RhoGAP/GTPase signaling, namely, that GAPs act directly upstream of GTPases, we apply our framework for identifying genetic interactions to predict signaling relationships between these proteins. We find that our method makes mediocre predictions using only RhoGAP single-knockdown morphological data, yet achieves vastly improved accuracy by including original data from a double-knockdown RhoGAP genetic screen, which likely reflects the redundant network structure of RhoGAP/GTPase signaling. We consider other possible methods for inference and show that our primary model outperforms the alternatives. This work demonstrates the fundamental fact that high-throughput morphological data can be used in a systematic, successful fashion to identify genetic interactions and, using additional elementary knowledge of network structure, to infer signaling relations.

Screening of Osteogenic-Enhancing Short Peptides from BMPs for Biomimetic Material Applications

PubMed Central

Kanie, Kei; Kurimoto, Rio; Tian, Jing; Ebisawa, Katsumi; Narita, Yuji; Honda, Hiroyuki; Kato, Ryuji

2016-01-01

Bone regeneration is an important issue in many situations, such as bone fracture and surgery. Umbilical cord mesenchymal stem cells (UC-MSCs) are promising cell sources for bone regeneration. Bone morphogenetic proteins and their bioactive peptides are biomolecules known to enhance the osteogenic differentiation of MSCs. However, fibrosis can arise during the development of implantable biomaterials. Therefore, it is important to control cell organization by enhancing osteogenic proliferation and differentiation and inhibiting fibroblast proliferation. Thus, we focused on the screening of such osteogenic-enhancing peptides. In the present study, we developed new peptide array screening platforms to evaluate cell proliferation and alkaline phosphatase activity in osteoblasts, UC-MSCs and fibroblasts. The conditions for the screening platform were first defined using UC-MSCs and an osteogenic differentiation peptide known as W9. Next, in silico screening to define the candidate peptides was carried out to evaluate the homology of 19 bone morphogenetic proteins. Twenty-five candidate 9-mer peptides were selected for screening. Finally, the screening of osteogenic-enhancing (osteogenic cell-selective proliferation and osteogenic differentiation) short peptide was carried out using the peptide array method, and three osteogenic-enhancing peptides were identified, confirming the validity of this screening. PMID:28773850

CRISPR-UMI: single-cell lineage tracing of pooled CRISPR-Cas9 screens.

PubMed

Michlits, Georg; Hubmann, Maria; Wu, Szu-Hsien; Vainorius, Gintautas; Budusan, Elena; Zhuk, Sergei; Burkard, Thomas R; Novatchkova, Maria; Aichinger, Martin; Lu, Yiqing; Reece-Hoyes, John; Nitsch, Roberto; Schramek, Daniel; Hoepfner, Dominic; Elling, Ulrich

2017-12-01

Pooled CRISPR screens are a powerful tool for assessments of gene function. However, conventional analysis is based exclusively on the relative abundance of integrated single guide RNAs (sgRNAs) between populations, which does not discern distinct phenotypes and editing outcomes generated by identical sgRNAs. Here we present CRISPR-UMI, a single-cell lineage-tracing methodology for pooled screening to account for cell heterogeneity. We generated complex sgRNA libraries with unique molecular identifiers (UMIs) that allowed for screening of clonally expanded, individually tagged cells. A proof-of-principle CRISPR-UMI negative-selection screen provided increased sensitivity and robustness compared with conventional analysis by accounting for underlying cellular and editing-outcome heterogeneity and detection of outlier clones. Furthermore, a CRISPR-UMI positive-selection screen uncovered new roadblocks in reprogramming mouse embryonic fibroblasts as pluripotent stem cells, distinguishing reprogramming frequency and speed (i.e., effect size and probability). CRISPR-UMI boosts the predictive power, sensitivity, and information content of pooled CRISPR screens.

High-content screening of small compounds on human embryonic stem cells.

PubMed

Barbaric, Ivana; Gokhale, Paul J; Andrews, Peter W

2010-08-01

Human ES (embryonic stem) cells and iPS (induced pluripotent stem) cells have been heralded as a source of differentiated cells that could be used in the treatment of degenerative diseases, such as Parkinson's disease or diabetes. Despite the great potential for their use in regenerative therapy, the challenge remains to understand the basic biology of these remarkable cells, in order to differentiate them into any functional cell type. Given the scale of the task, high-throughput screening of agents and culture conditions offers one way to accelerate these studies. The screening of small-compound libraries is particularly amenable to such high-throughput methods. Coupled with high-content screening technology that enables simultaneous assessment of multiple cellular features in an automated and quantitative way, this approach is proving powerful in identifying both small molecules as tools for manipulating stem cell fates and novel mechanisms of differentiation not previously associated with stem cell biology. Such screens performed on human ES cells also demonstrate the usefulness of human ES/iPS cells as cellular models for pharmacological testing of drug efficacy and toxicity, possibly a more imminent use of these cells than in regenerative medicine.

Rapid formation of size-controllable multicellular spheroids via 3D acoustic tweezers.

PubMed

Chen, Kejie; Wu, Mengxi; Guo, Feng; Li, Peng; Chan, Chung Yu; Mao, Zhangming; Li, Sixing; Ren, Liqiang; Zhang, Rui; Huang, Tony Jun

2016-07-05

The multicellular spheroid is an important 3D cell culture model for drug screening, tissue engineering, and fundamental biological research. Although several spheroid formation methods have been reported, the field still lacks high-throughput and simple fabrication methods to accelerate its adoption in drug development industry. Surface acoustic wave (SAW) based cell manipulation methods, which are known to be non-invasive, flexible, and high-throughput, have not been successfully developed for fabricating 3D cell assemblies or spheroids, due to the limited understanding on SAW-based vertical levitation. In this work, we demonstrated the capability of fabricating multicellular spheroids in the 3D acoustic tweezers platform. Our method used drag force from microstreaming to levitate cells in the vertical direction, and used radiation force from Gor'kov potential to aggregate cells in the horizontal plane. After optimizing the device geometry and input power, we demonstrated the rapid and high-throughput nature of our method by continuously fabricating more than 150 size-controllable spheroids and transferring them to Petri dishes every 30 minutes. The spheroids fabricated by our 3D acoustic tweezers can be cultured for a week with good cell viability. We further demonstrated that spheroids fabricated by this method could be used for drug testing. Unlike the 2D monolayer model, HepG2 spheroids fabricated by the 3D acoustic tweezers manifested distinct drug resistance, which matched existing reports. The 3D acoustic tweezers based method can serve as a novel bio-manufacturing tool to fabricate complex 3D cell assembles for biological research, tissue engineering, and drug development.

Drug screens based on the newly found role of dystroglycan proteolysis and restoration of dystroglycan function thereof

DOEpatents

Bissell, Mina J.; Muschler, John L.

2010-02-23

The present invention provides methods and compositions for the diagnosis and treatment of cells lacking normal growth arresting characteristic. The present invention demonstrates that many tumor cells lack normal cell surface .alpha.-dystroglycan and thereby lack dystroglycan function. Dystroglycan can be lost from the cell surface by proteolytic shedding of a fragment of .alpha.-dystroglycan into the surrounding medium. Upon restoration of dystroglycan function and over-expression of the dystroglycan gene, the once tumorigenic cells revert to non-tumorigenic cells which polarize and arrest cell growth in the presence of basement membrane proteins, demonstrating that dystroglycan functions as a tumor marker and suppressor.

A robust screening method for dietary agents that activate tumour-suppressor microRNAs

PubMed Central

Hagiwara, Keitaro; Gailhouste, Luc; Yasukawa, Ken; Kosaka, Nobuyoshi; Ochiya, Takahiro

2015-01-01

Certain dietary agents, such as natural products, have been reported to show anti-cancer effects. However, the underlying mechanisms of these substances in human cancer remain unclear. We recently found that resveratrol exerts an anti-cancer effect by upregulating tumour-suppressor microRNAs (miRNAs). In the current study, we aimed to identify new dietary products that have the ability to activate tumour-suppressor miRNAs and that therefore may serve as novel tools for the prevention and treatment of human cancers. We describe the generation and use of an original screening system based on a luciferase-based reporter vector for monitoring miR-200c tumour-suppressor activity. By screening a library containing 139 natural substances, three natural compounds — enoxolone, magnolol and palmatine chloride — were identified as being capable of inducing miR-200c expression in breast cancer cells at 10 μM. Moreover, these molecules suppressed the invasiveness of breast cancer cells in vitro. Next, we identified a molecular pathway by which the increased expression of miR-200c induced by natural substances led to ZEB1 inhibition and E-cadherin induction. These results indicate that our method is a valuable tool for a fast identification of natural molecules that exhibit tumour-suppressor activity in human cancer through miRNA activation. PMID:26423775

Rapid and Facile Microwave-Assisted Surface Chemistry for Functionalized Microarray Slides

PubMed Central

Lee, Jeong Heon; Hyun, Hoon; Cross, Conor J.; Henary, Maged; Nasr, Khaled A.; Oketokoun, Rafiou; Choi, Hak Soo; Frangioni, John V.

2011-01-01

We describe a rapid and facile method for surface functionalization and ligand patterning of glass slides based on microwave-assisted synthesis and a microarraying robot. Our optimized reaction enables surface modification 42-times faster than conventional techniques and includes a carboxylated self-assembled monolayer, polyethylene glycol linkers of varying length, and stable amide bonds to small molecule, peptide, or protein ligands to be screened for binding to living cells. We also describe customized slide racks that permit functionalization of 100 slides at a time to produce a cost-efficient, highly reproducible batch process. Ligand spots can be positioned on the glass slides precisely using a microarraying robot, and spot size adjusted for any desired application. Using this system, we demonstrate live cell binding to a variety of ligands and optimize PEG linker length. Taken together, the technology we describe should enable high-throughput screening of disease-specific ligands that bind to living cells. PMID:23467787

20180312 - Application of a Multiplexed High Content Imaging (HCI) Based Cell Viability and Apoptosis Chemical Screening Assay with Results in MCF-7 Cells (SOT)

EPA Science Inventory

The NCCT high throughput transcriptomics (HTTr) screening program uses whole transcriptome profiling assay in human-derived cells to collect concentration-response data for large numbers (100s-1000s) of environmental chemicals. To contextualize HTTr data, chemical effects on cell...

Rapid screening of bioactive compounds from natural products by integrating 5-channel parallel chromatography coupled with on-line mass spectrometry and microplate based assays.

PubMed

Zhang, Yufeng; Xiao, Shun; Sun, Lijuan; Ge, Zhiwei; Fang, Fengkai; Zhang, Wen; Wang, Yi; Cheng, Yiyu

2013-05-13

A high throughput method was developed for rapid screening and identification of bioactive compounds from traditional Chinese medicine, marine products and other natural products. The system, integrated with five-channel chromatographic separation and dual UV-MS detection, is compatible with in vitro 96-well microplate based bioassays. The stability and applicability of the proposed method was validated by testing radical scavenging capability of a mixture of seven known compounds (rutin, dihydroquercetin, salvianolic acid A, salvianolic acid B, glycyrrhizic acid, rubescensin A and tangeretin). Moreover, the proposed method was successfully applied to the crude extracts of traditional Chinese medicine and a marine sponge from which 12 bioactive compounds were screened and characterized based on their anti-oxidative or anti-tumor activities. In particular, two diterpenoid derivatives, agelasine B and (-)-agelasine D, were identified for the first time as anti-tumor compounds from the sponge Agelas mauritiana, showing a considerable activity toward MCF-7 cells (IC50 values of 7.84±0.65 and 10.48±0.84 μM, respectively). Our findings suggested that the integrated system of 5-channel parallel chromatography coupled with on-line mass spectrometry and microplate based assays can be a versatile and high efficient approach for the discovery of active compounds from natural products. Copyright © 2013 Elsevier B.V. All rights reserved.

Is high pressure liquid chromatography an effective screening tool for characterization of molecular defects in hemoglobinopathies?

PubMed

Moorchung, Nikhil; Phillip, Joseph; Sarkar, Ravi Shankar; Prasad, Rupesh; Dutta, Vibha

2013-01-01

Hemoglobinopathies constitute entities that are generated by either abnormal hemoglobin or thalassemias. high pressure liquid chromatography (HPLC) is one of the best methods for screening and detection of various hemoglobinopathies but it has intrinsic interpretive problems. The study was designed to evaluate the different mutations seen in cases of hemoglobinopathies and compare the same with screening tests. 68 patients of hemoglobinopathies were screened by HPLC. Mutation studies in the beta globin gene was performed using the polymerase chain reaction (PCR)-based allele-specific Amplification Refractory Mutation System (ARMS). Molecular analysis for the sickle cell mutation was done by standard methods. The IVS 1/5 mutation was the commonest mutation seen and it was seen in 26 (38.23%) of the cases. This was followed by the IVS 1/1, codon 41/42, codon 8/9, del 22 mutation, codon 15 mutation and the -619 bp deletion. No mutation was seen in eight cases. There was a 100% concordance between the sickle cell trait as diagnosed by HPLC and genetic testing. Our study underlies the importance of molecular testing in all cases of hemoglobinopathies. Although HPLC is a useful screening tool, molecular testing is very useful in accurately diagnosing the mutations. Molecular testing is especially applicable in cases with an abnormal hemoglobin (HbD, HbE and HbS) because there may be a concomitant inheritance of a beta thalassemia mutation. Molecular testing is the gold standard when it comes to the diagnosis of hemoglobinopathies.

In vitro culture and characterization of human lung cancer circulating tumor cells isolated by size exclusion from an orthotopic nude-mouse model expressing fluorescent protein.

PubMed

Kolostova, Katarina; Zhang, Yong; Hoffman, Robert M; Bobek, Vladimir

2014-09-01

In the present study, we demonstrate an animal model and recently introduced size-based exclusion method for circulating tumor cells (CTCs) isolation. The methodology enables subsequent in vitro CTC-culture and characterization. Human lung cancer cell line H460, expressing red fluorescent protein (H460-RFP), was orthotopically implanted in nude mice. CTCs were isolated by a size-based filtration method and successfully cultured in vitro on the separating membrane (MetaCell®), analyzed by means of time-lapse imaging. The cultured CTCs were heterogeneous in size and morphology even though they originated from a single tumor. The outer CTC-membranes were blebbing in general. Abnormal mitosis resulting in three daughter cells was frequently observed. The expression of RFP ensured that the CTCs originated from lung tumor. These readily isolatable, identifiable and cultivable CTCs can be used to characterize individual patient cancers and for screening of more effective treatment.

Optimization of a polydopamine (PD)-based coating method and polydimethylsiloxane (PDMS) substrates for improved mouse embryonic stem cell (ESC) pluripotency maintenance and cardiac differentiation.

PubMed

Fu, Jiayin; Chuah, Yon Jin; Ang, Wee Tong; Zheng, Nan; Wang, Dong-An

2017-05-30

Myocardiocyte derived from pluripotent stem cells, such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), is a promising cell source for cardiac tissue engineering. Combined with microfluidic technologies, a heart-on-a-chip is very likely to be developed and function as a platform for high throughput drug screening. Polydimethylsiloxane (PDMS) silicone elastomer is a widely-used biomaterial for the investigation of cell-substrate interactions and biochip fabrication. However, the intrinsic PDMS surface hydrophobicity inhibits cell adhesion on the PDMS surface, and PDMS surface modification is required for effective cell adhesion. Meanwhile, the formulation of PDMS also affects the behaviors of the cells. To fabricate PDMS-based biochips for ESC pluripotency maintenance and cardiac differentiation, PDMS surface modification and formulation were optimized in this study. We found that a polydopamine (PD) with gelatin coating greatly improved the ESC adhesion, proliferation and cardiac differentiation on its surface. In addition, different PDMS substrates varied in their surface properties, which had different impacts on ESCs, with the 40 : 1 PDMS substrate being more favorable for ESC adhesion and proliferation as well as embryoid body (EB) attachment than the other PDMS substrates. Moreover, the ESC pluripotency was best maintained on the 5 : 1 PDMS substrate, while the cardiac differentiation of the ESCs was optimal on the 40 : 1 PDMS substrate. Based on the optimized coating method and PDMS formulation, biochips with two different designs were fabricated and evaluated. Compared to the single channels, the multiple channels on the biochips could provide larger areas and accommodate more nutrients to support improved ESC pluripotency maintenance and cardiac differentiation. These results may contribute to the development of a real heart-on-a-chip for high-throughput drug screening in the future.

Renilla Luciferase-LC3 Based Reporter Assay for Measuring Autophagic Flux.

PubMed

Farkas, T; Jäättelä, M

2017-01-01

Macroautophagy (autophagy) is a dynamic intracellular degradation pathway. Monitoring the flux through the autophagy pathway is experimentally challenging but obviously a prerequisite for the proper investigation of the process. Here, we present an indirect autophagy flux assay based on monitoring the degradation of an autophagosome-associated fusion protein Rluc-LC3 by luminescence detection. The method is suitable for screening purposes with a high number of parallel samples and can be used for measurements in cell lysates as well as in living cells. The Rluc-LC3 assay has proven useful for the identification of genes, miRNAs, and small molecules that regulate autophagy flux in mammalian cells. © 2017 Elsevier Inc. All rights reserved.

High-throughput label-free screening of euglena gracilis with optofluidic time-stretch quantitative phase microscopy

NASA Astrophysics Data System (ADS)

Guo, Baoshan; Lei, Cheng; Ito, Takuro; Yaxiaer, Yalikun; Kobayashi, Hirofumi; Jiang, Yiyue; Tanaka, Yo; Ozeki, Yasuyuki; Goda, Keisuke

2017-02-01

The development of reliable, sustainable, and economical sources of alternative fuels is an important, but challenging goal for the world. As an alternative to liquid fossil fuels, microalgal biofuel is expected to play a key role in reducing the detrimental effects of global warming since microalgae absorb atmospheric CO2 via photosynthesis. Unfortunately, conventional analytical methods only provide population-averaged lipid contents and fail to characterize a diverse population of microalgal cells with single-cell resolution in a noninvasive and interference-free manner. Here we demonstrate high-throughput label-free single-cell screening of lipid-producing microalgal cells with optofluidic time-stretch quantitative phase microscopy. In particular, we use Euglena gracilis - an attractive microalgal species that produces wax esters (suitable for biodiesel and aviation fuel after refinement) within lipid droplets. Our optofluidic time-stretch quantitative phase microscope is based on an integration of a hydrodynamic-focusing microfluidic chip, an optical time-stretch phase-contrast microscope, and a digital image processor equipped with machine learning. As a result, it provides both the opacity and phase contents of every single cell at a high throughput of 10,000 cells/s. We characterize heterogeneous populations of E. gracilis cells under two different culture conditions to evaluate their lipid production efficiency. Our method holds promise as an effective analytical tool for microalgaebased biofuel production.

Screening of Peptide Libraries against Protective Antigen of Bacillus anthracis in a Disposable Microfluidic Cartridge

PubMed Central

Kogot, Joshua M.; Zhang, Yanting; Moore, Stephen J.; Pagano, Paul; Stratis-Cullum, Dimitra N.; Chang-Yen, David; Turewicz, Marek; Pellegrino, Paul M.; de Fusco, Andre; Soh, H. Tom; Stagliano, Nancy E.

2011-01-01

Bacterial surface peptide display has gained popularity as a method of affinity reagent generation for a wide variety of applications ranging from drug discovery to pathogen detection. In order to isolate the bacterial clones that express peptides with high affinities to the target molecule, multiple rounds of manual magnetic activated cell sorting (MACS) followed by multiple rounds of fluorescence activated cell sorting (FACS) are conventionally used. Although such manual methods are effective, alternative means of library screening which improve the reproducibility, reduce the cost, reduce cross contamination, and minimize exposure to hazardous target materials are highly desired for practical application. Toward this end, we report the first semi-automated system demonstrating the potential for screening bacterially displayed peptides using disposable microfluidic cartridges. The Micro-Magnetic Separation platform (MMS) is capable of screening a bacterial library containing 3×1010 members in 15 minutes and requires minimal operator training. Using this system, we report the isolation of twenty-four distinct peptide ligands that bind to the protective antigen (PA) of Bacilus anthracis in three rounds of selection. A consensus motif WXCFTC was found using the MMS and was also found in one of the PA binders isolated by the conventional MACS/FACS approach. We compared MMS and MACS rare cell recovery over cell populations ranging from 0.1% to 0.0000001% and found that both magnetic sorting methods could recover cells down to 0.0000001% initial cell population, with the MMS having overall lower standard deviation of cell recovery. We believe the MMS system offers a compelling approach towards highly efficient, semi-automated screening of molecular libraries that is at least equal to manual magnetic sorting methods and produced, for the first time, 15-mer peptide binders to PA protein that exhibit better affinity and specificity than peptides isolated using conventional MACS/FACS. PMID:22140433

An on-chip imaging droplet-sorting system: a real-time shape recognition method to screen target cells in droplets with single cell resolution

NASA Astrophysics Data System (ADS)

Girault, Mathias; Kim, Hyonchol; Arakawa, Hisayuki; Matsuura, Kenji; Odaka, Masao; Hattori, Akihiro; Terazono, Hideyuki; Yasuda, Kenji

2017-01-01

A microfluidic on-chip imaging cell sorter has several advantages over conventional cell sorting methods, especially to identify cells with complex morphologies such as clusters. One of the remaining problems is how to efficiently discriminate targets at the species level without labelling. Hence, we developed a label-free microfluidic droplet-sorting system based on image recognition of cells in droplets. To test the applicability of this method, a mixture of two plankton species with different morphologies (Dunaliella tertiolecta and Phaeodactylum tricornutum) were successfully identified and discriminated at a rate of 10 Hz. We also examined the ability to detect the number of objects encapsulated in a droplet. Single cell droplets sorted into collection channels showed 91 ± 4.5% and 90 ± 3.8% accuracy for D. tertiolecta and P. tricornutum, respectively. Because we used image recognition to confirm single cell droplets, we achieved highly accurate single cell sorting. The results indicate that the integrated method of droplet imaging cell sorting can provide a complementary sorting approach capable of isolating single target cells from a mixture of cells with high accuracy without any staining.

An on-chip imaging droplet-sorting system: a real-time shape recognition method to screen target cells in droplets with single cell resolution.

PubMed

Girault, Mathias; Kim, Hyonchol; Arakawa, Hisayuki; Matsuura, Kenji; Odaka, Masao; Hattori, Akihiro; Terazono, Hideyuki; Yasuda, Kenji

2017-01-06

A microfluidic on-chip imaging cell sorter has several advantages over conventional cell sorting methods, especially to identify cells with complex morphologies such as clusters. One of the remaining problems is how to efficiently discriminate targets at the species level without labelling. Hence, we developed a label-free microfluidic droplet-sorting system based on image recognition of cells in droplets. To test the applicability of this method, a mixture of two plankton species with different morphologies (Dunaliella tertiolecta and Phaeodactylum tricornutum) were successfully identified and discriminated at a rate of 10 Hz. We also examined the ability to detect the number of objects encapsulated in a droplet. Single cell droplets sorted into collection channels showed 91 ± 4.5% and 90 ± 3.8% accuracy for D. tertiolecta and P. tricornutum, respectively. Because we used image recognition to confirm single cell droplets, we achieved highly accurate single cell sorting. The results indicate that the integrated method of droplet imaging cell sorting can provide a complementary sorting approach capable of isolating single target cells from a mixture of cells with high accuracy without any staining.

An on-chip imaging droplet-sorting system: a real-time shape recognition method to screen target cells in droplets with single cell resolution

PubMed Central

Girault, Mathias; Kim, Hyonchol; Arakawa, Hisayuki; Matsuura, Kenji; Odaka, Masao; Hattori, Akihiro; Terazono, Hideyuki; Yasuda, Kenji

2017-01-01

A microfluidic on-chip imaging cell sorter has several advantages over conventional cell sorting methods, especially to identify cells with complex morphologies such as clusters. One of the remaining problems is how to efficiently discriminate targets at the species level without labelling. Hence, we developed a label-free microfluidic droplet-sorting system based on image recognition of cells in droplets. To test the applicability of this method, a mixture of two plankton species with different morphologies (Dunaliella tertiolecta and Phaeodactylum tricornutum) were successfully identified and discriminated at a rate of 10 Hz. We also examined the ability to detect the number of objects encapsulated in a droplet. Single cell droplets sorted into collection channels showed 91 ± 4.5% and 90 ± 3.8% accuracy for D. tertiolecta and P. tricornutum, respectively. Because we used image recognition to confirm single cell droplets, we achieved highly accurate single cell sorting. The results indicate that the integrated method of droplet imaging cell sorting can provide a complementary sorting approach capable of isolating single target cells from a mixture of cells with high accuracy without any staining. PMID:28059147

Stem cells: a model for screening, discovery and development of drugs.

PubMed

Kitambi, Satish Srinivas; Chandrasekar, Gayathri

2011-01-01

The identification of normal and cancerous stem cells and the recent advances made in isolation and culture of stem cells have rapidly gained attention in the field of drug discovery and regenerative medicine. The prospect of performing screens aimed at proliferation, directed differentiation, and toxicity and efficacy studies using stem cells offers a reliable platform for the drug discovery process. Advances made in the generation of induced pluripotent stem cells from normal or diseased tissue serves as a platform to perform drug screens aimed at developing cell-based therapies against conditions like Parkinson's disease and diabetes. This review discusses the application of stem cells and cancer stem cells in drug screening and their role in complementing, reducing, and replacing animal testing. In addition to this, target identification and major advances in the field of personalized medicine using induced pluripotent cells are also discussed.

Combinatorial high-throughput optical screening of high performance Pd alloy cathode for hybrid Li-air battery.

PubMed

Jun, Young Jin; Park, Sung Hyeon; Woo, Seong Ihl

2014-12-08

Combinatorial high-throughput optical screening method was developed to find the optimum composition of highly active Pd-based catalysts at the cathode of the hybrid Li-air battery. Pd alone, which is one-third the cost of Pt, has difficulty in replacing Pt; therefore, the integration of other metals was investigated to improve its performance toward oxygen reduction reaction (ORR). Among the binary Pd-based catalysts, the composition of Pd-Ir derived catalysts had higher performance toward ORR compared to other Pd-based binary combinations. The composition at 88:12 at. % (Pd: Ir) showed the highest activity toward ORR at the cathode of the hybrid Li-air battery. The prepared Pd(88)Ir(12)/C catalyst showed a current density of -2.58 mA cm(-2) at 0.8 V (vs RHE), which was around 30% higher compared to that of Pd/C (-1.97 mA cm(-2)). When the prepared Pd(88)Ir(12)/C catalyst was applied to the hybrid Li-air battery, the polarization of the cell was reduced and the energy efficiency of the cell was about 30% higher than that of the cell with Pd/C.

Laser-emission imaging of nuclear biomarkers for high-contrast cancer screening and immunodiagnosis

PubMed Central

Chen, Yu-Cheng; Tan, Xiaotian; Sun, Qihan; Chen, Qiushu; Wang, Wenjie; Fan, Xudong

2017-01-01

Detection of nuclear biomarkers such as nucleic acids and nuclear proteins is critical for early-stage cancer diagnosis and prognosis. Conventional methods relying on morphological assessment of cell nuclei in histopathology slides may be subjective, whereas colorimetric immunohistochemical and fluorescence-based imaging are limited by strong light absorption, broad-emission bands and low contrast. Here, we describe the development and use of a scanning laser-emission-based microscope that maps lasing emissions from nuclear biomarkers in human tissues. 41 tissue samples from 35 patients labelled with site-specific and biomarker-specific antibody-conjugated dyes were sandwiched in a Fabry-Pérot microcavity while an excitation laser beam built a laser-emission image. We observed multiple sub-cellular lasing emissions from cancer cell nuclei, with a threshold of tens of μJ/mm2, sub-micron resolution (<700 nm), and a lasing band in the few-nanometre range. Different lasing thresholds of nuclei in cancer and normal tissues enabled the identification and multiplexed detection of nuclear proteomic biomarkers, with a high sensitivity for early-stage cancer diagnosis. Laser-emission-based cancer screening and immunodiagnosis might find use in precision medicine and facilitate research in cell biology. PMID:29204310

Multiparametric Phenotypic Screening System for Profiling Bioactive Compounds Using Human Fetal Hippocampal Neural Stem/Progenitor Cells.

PubMed

Tabata, Yoshikuni; Murai, Norio; Sasaki, Takeo; Taniguchi, Sachie; Suzuki, Shuichi; Yamazaki, Kazuto; Ito, Masashi

2015-10-01

Stem cell research has been progressing rapidly, contributing to regenerative biology and regenerative medicine. In this field, small-molecule compounds affecting stem cell proliferation/differentiation have been explored to understand stem cell biology and support regenerative medicine. In this study, we established a multiparametric screening system to detect bioactive compounds affecting the cell fate of human neural stem/progenitor cells (NSCs/NPCs), using human fetal hippocampal NSCs/NPCs, HIP-009 cells. We examined effects of 410 compounds, which were collected based on mechanisms of action (MOAs) and chemotypes, on HIP-009's cell fate (self-renewal, neuronal and astrocytic differentiation) and morphology by automated multiparametric assays and profiled induced cellular phenotypes. We found that this screening classified compounds with the same MOAs into subgroups according to additional pharmacological effects (e.g., mammalian target of rapamycin complex 1 [mTORC1] inhibitors and mTORC1/mTORC2 dual inhibitors among mTOR inhibitors). Moreover, it identified compounds that have off-target effects under matrix analyses of MOAs and structure similarities (e.g., neurotropic effects of amitriptyline among tri- and tetracyclic compounds). Therefore, this automated, medium-throughput and multiparametric screening system is useful for finding compounds that affect the cell fate of human NSCs/NPCs for supporting regenerative medicine and to fingerprint compounds based on human stem cells' multipotency, leading to understanding of stem cell biology. © 2015 Society for Laboratory Automation and Screening.

Active Learning Strategies for Phenotypic Profiling of High-Content Screens.

PubMed

Smith, Kevin; Horvath, Peter

2014-06-01

High-content screening is a powerful method to discover new drugs and carry out basic biological research. Increasingly, high-content screens have come to rely on supervised machine learning (SML) to perform automatic phenotypic classification as an essential step of the analysis. However, this comes at a cost, namely, the labeled examples required to train the predictive model. Classification performance increases with the number of labeled examples, and because labeling examples demands time from an expert, the training process represents a significant time investment. Active learning strategies attempt to overcome this bottleneck by presenting the most relevant examples to the annotator, thereby achieving high accuracy while minimizing the cost of obtaining labeled data. In this article, we investigate the impact of active learning on single-cell-based phenotype recognition, using data from three large-scale RNA interference high-content screens representing diverse phenotypic profiling problems. We consider several combinations of active learning strategies and popular SML methods. Our results show that active learning significantly reduces the time cost and can be used to reveal the same phenotypic targets identified using SML. We also identify combinations of active learning strategies and SML methods which perform better than others on the phenotypic profiling problems we studied. © 2014 Society for Laboratory Automation and Screening.

Microfluidic devices for the controlled manipulation of small volumes

DOEpatents

Ramsey, J Michael [Knoxville, TN; Jacobson, Stephen C [Knoxville, TN

2003-02-25

A method for conducting a broad range of biochemical analyses or manipulations on a series of nano- to subnanoliter reaction volumes and an apparatus for carrying out the same are disclosed. The method and apparatus are implemented on a fluidic microchip to provide high serial throughput. The method and device of the invention also lend themselves to multiple parallel analyses and manipulation to provide greater throughput for the generation of biochemical information. In particular, the disclosed device is a microfabricated channel device that can manipulate nanoliter or subnanoliter biochemical reaction volumes in a controlled manner to produce results at rates of 1 to 10 Hz per channel. The individual reaction volumes are manipulated in serial fashion analogous to a digital shift register. The method and apparatus according to this invention have application to such problems as screening molecular or cellular targets using single beads from split-synthesis combinatorial libraries, screening single cells for RNA or protein expression, genetic diagnostic screening at the single cell level, or performing single cell signal transduction studies.

Characterization and Separation of Cancer Cells with a Wicking Fiber Device.

PubMed

Tabbaa, Suzanne M; Sharp, Julia L; Burg, Karen J L

2017-12-01

Current cancer diagnostic methods lack the ability to quickly, simply, efficiently, and inexpensively screen cancer cells from a mixed population of cancer and normal cells. Methods based on biomarkers are unreliable due to complexity of cancer cells, plasticity of markers, and lack of common tumorigenic markers. Diagnostics are time intensive, require multiple tests, and provide limited information. In this study, we developed a novel wicking fiber device that separates cancer and normal cell types. To the best of our knowledge, no previous work has used vertical wicking of cells through fibers to identify and isolate cancer cells. The device separated mouse mammary tumor cells from a cellular mixture containing normal mouse mammary cells. Further investigation showed the device separated and isolated human cancer cells from a heterogeneous mixture of normal and cancerous human cells. We report a simple, inexpensive, and rapid technique that has potential to identify and isolate cancer cells from large volumes of liquid samples that can be translated to on-site clinic diagnosis.

Risks of CIN 2+, CIN 3+, and Cancer by Cytology and Human Papillomavirus Status: The Foundation of Risk-Based Cervical Screening Guidelines

PubMed Central

Demarco, Maria; Lorey, Thomas S.; Fetterman, Barbara; Cheung, Li C.; Guido, Richard S.; Wentzensen, Nicolas; Kinney, Walter K.; Poitras, Nancy E.; Befano, Brian; Castle, Philip E.; Schiffman, Mark

2017-01-01

Objectives The next round of the American Society for Colposcopy and Cervical Pathology (ASCCP)-sponsored cervical cancer screening and management guidelines will recommend clinical actions based on risk, rather than test-based algorithms. This article gives preliminary risk estimates for the screening setting, showing combinations of the 2 most important predictors, human papillomavirus (HPV) status and cytology result. Materials and Methods Among 1,262,713 women aged 25 to 77 years co-tested with HC2 (Qiagen) and cytology at Kaiser Permanente Northern California, we estimated 0–5-year cumulative risk of cervical intraepithelial neoplasia (CIN) 2+, CIN 3+, and cancer for combinations of cytology (negative for intraepithelial lesion or malignancy [NILM], atypical squamous cells of undetermined significance [ASC-US], low-grade squamous intraepithelial lesion [LSIL], atypical squamous cells cannot exclude HSIL [ASC-H], high-grade squamous intraepithelial lesion [HSIL], atypical glandular cells [AGC]) and HPV status. Results Ninety percent of screened women had HPV-negative NILM and an extremely low risk of subsequent cancer. Five-year risks of CIN 3+ were lower after HPV negativity (0.12%) than after NILM (0.25%). Among HPV-negative women, 5-year risks for CIN 3+ were 0.10% for NILM, 0.44% for ASC-US, 1.8% for LSIL, 3.0% for ASC-H, 1.2% for AGC, and 29% for HSIL+ cytology (which was very rare). Among HPV-positive women, 5-year risks were 4.0% for NILM, 6.8% for ASC-US, 6.1% for LSIL, 28% for ASC-H, 30% for AGC, and 50% for HSIL+ cytology. Conclusions As a foundation for the next guidelines revision, we confirmed with additional precision the risk estimates previously reported for combinations of HPV and cytology. Future analyses will estimate risks for women being followed in colposcopy clinic and posttreatment and will consider the role of risk modifiers such as age, HPV vaccine status, HPV type, and screening and treatment history. PMID:28953116

A microfluidic perfusion platform for cultivation and screening study of motile microalgal cells

PubMed Central

Eu, Young-Jae; Park, Hye-Sun; Kim, Dong-Pyo; Wook Hong, Jong

2014-01-01

Systematic screening of algal cells is getting huge interest due to their capability of producing lipid-based biodiesel. Here, we introduce a new microfluidic platform composed of an array of perfusion chambers designed for long-term cultivation and preliminary screening of motile microalgal cells through loading and releasing of cells to and from the chambers. The chemical environment in each perfusion chamber was independently controlled for 5 days. The effect of nitrogen-depletion on the lipid production, phototaxis behavior in the absence of Ca2+, and cytotoxic effect of herbicide on microalgal cells was successfully monitored and compared with simultaneous control experiments on the platform. The present methodology could be extended to effective screening of algal cells and various cell lines for the production of biodiesel and other useful chemicals. PMID:24803962

Microfabricated Electrochemical Cell-Based Biosensors for Analysis of Living Cells In Vitro

PubMed Central

Wang, Jun; Wu, Chengxiong; Hu, Ning; Zhou, Jie; Du, Liping; Wang, Ping

2012-01-01

Cellular biochemical parameters can be used to reveal the physiological and functional information of various cells. Due to demonstrated high accuracy and non-invasiveness, electrochemical detection methods have been used for cell-based investigation. When combined with improved biosensor design and advanced measurement systems, the on-line biochemical analysis of living cells in vitro has been applied for biological mechanism study, drug screening and even environmental monitoring. In recent decades, new types of miniaturized electrochemical biosensor are emerging with the development of microfabrication technology. This review aims to give an overview of the microfabricated electrochemical cell-based biosensors, such as microelectrode arrays (MEA), the electric cell-substrate impedance sensing (ECIS) technique, and the light addressable potentiometric sensor (LAPS). The details in their working principles, measurement systems, and applications in cell monitoring are covered. Driven by the need for high throughput and multi-parameter detection proposed by biomedicine, the development trends of electrochemical cell-based biosensors are also introduced, including newly developed integrated biosensors, and the application of nanotechnology and microfluidic technology. PMID:25585708

Fragment-Based Phenotypic Lead Discovery: Cell-Based Assay to Target Leishmaniasis.

PubMed

Ayotte, Yann; Bilodeau, François; Descoteaux, Albert; LaPlante, Steven R

2018-05-02

A rapid and practical approach for the discovery of new chemical matter for targeting pathogens and diseases is described. Fragment-based phenotypic lead discovery (FPLD) combines aspects of traditional fragment-based lead discovery (FBLD), which involves the screening of small-molecule fragment libraries to target specific proteins, with phenotypic lead discovery (PLD), which typically involves the screening of drug-like compounds in cell-based assays. To enable FPLD, a diverse library of fragments was first designed, assembled, and curated. This library of soluble, low-molecular-weight compounds was then pooled to expedite screening. Axenic cultures of Leishmania promastigotes were screened, and single hits were then tested for leishmanicidal activity against intracellular amastigote forms in infected murine bone-marrow-derived macrophages without evidence of toxicity toward mammalian cells. These studies demonstrate that FPLD can be a rapid and effective means to discover hits that can serve as leads for further medicinal chemistry purposes or as tool compounds for identifying known or novel targets. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The interdependence between screening methods and screening libraries.

PubMed

Shelat, Anang A; Guy, R Kiplin

2007-06-01

The most common methods for discovery of chemical compounds capable of manipulating biological function involves some form of screening. The success of such screens is highly dependent on the chemical materials - commonly referred to as libraries - that are assayed. Classic methods for the design of screening libraries have depended on knowledge of target structure and relevant pharmacophores for target focus, and on simple count-based measures to assess other properties. The recent proliferation of two novel screening paradigms, structure-based screening and high-content screening, prompts a profound rethink about the ideal composition of small-molecule screening libraries. We suggest that currently utilized libraries are not optimal for addressing new targets by high-throughput screening, or complex phenotypes by high-content screening.

Evaluation of a QuECHERS-like extraction approach for the determination of PBDEs in mussels by immuno-assay-based screening methods

USDA-ARS?s Scientific Manuscript database

A sample preparation method was evaluated for the determination of polybrominated diphenyl ethers (PBDEs) in mussel samples, by using colorimetric and electrochemical immunoassay-based screening methods. A simple sample preparation in conjunction with a rapid screening method possesses the desired c...

A flow-cytometry-based method to simplify the analysis and quantification of protein association to chromatin in mammalian cells

PubMed Central

Forment, Josep V.; Jackson, Stephen P.

2016-01-01

Protein accumulation on chromatin has traditionally been studied using immunofluorescence microscopy or biochemical cellular fractionation followed by western immunoblot analysis. As a way to improve the reproducibility of this kind of analysis, make it easier to quantify and allow a stream-lined application in high-throughput screens, we recently combined a classical immunofluorescence microscopy detection technique with flow cytometry1. In addition to the features described above, and by combining it with detection of both DNA content and DNA replication, this method allows unequivocal and direct assignment of cell-cycle distribution of protein association to chromatin without the need for cell culture synchronization. Furthermore, it is relatively quick (no more than a working day from sample collection to quantification), requires less starting material compared to standard biochemical fractionation methods and overcomes the need for flat, adherent cell types that are required for immunofluorescence microscopy. PMID:26226461

The preservation of living cells with biocompatible microparticles

NASA Astrophysics Data System (ADS)

Yang, Jing; Zhu, Yingnan; Xu, Tong; Pan, Chao; Cai, Nana; Huang, He; Zhang, Lei

2016-07-01

Biomedical applications of living cells have rapidly expanded in many fields such as toxic detection, drug screening, and regenerative medicine, etc. Efficient methods to support cell survival and maintain activity in vitro have become increasingly important. However, traditional cryopreservation for living cell-based applications is limited by several problems. Here, we report that magnetic hydrogel microparticles can physically assemble into a 3D environment for efficient cell preservation in physiological conditions, avoiding any chemical reactions that would damage the cells. Two representative cell lines (loosely and firmly adherent) were tested to evaluate the versatility of this method. The results showed that cell longevity was significantly extended to at least 15 days, while the control cell samples without microparticles quickly died within 3 days. Moreover, after preservation, cells can be easily retrieved by applying a magnet to separate the magnetic particles. This strategy can also inhibit cell over-proliferation while avoiding the use of temperature extremes or toxic cryoprotectants that are essential in cryopreservation.

A novel automatic quantification method for high-content screening analysis of DNA double strand-break response.

PubMed

Feng, Jingwen; Lin, Jie; Zhang, Pengquan; Yang, Songnan; Sa, Yu; Feng, Yuanming

2017-08-29

High-content screening is commonly used in studies of the DNA damage response. The double-strand break (DSB) is one of the most harmful types of DNA damage lesions. The conventional method used to quantify DSBs is γH2AX foci counting, which requires manual adjustment and preset parameters and is usually regarded as imprecise, time-consuming, poorly reproducible, and inaccurate. Therefore, a robust automatic alternative method is highly desired. In this manuscript, we present a new method for quantifying DSBs which involves automatic image cropping, automatic foci-segmentation and fluorescent intensity measurement. Furthermore, an additional function was added for standardizing the measurement of DSB response inhibition based on co-localization analysis. We tested the method with a well-known inhibitor of DSB response. The new method requires only one preset parameter, which effectively minimizes operator-dependent variations. Compared with conventional methods, the new method detected a higher percentage difference of foci formation between different cells, which can improve measurement accuracy. The effects of the inhibitor on DSB response were successfully quantified with the new method (p = 0.000). The advantages of this method in terms of reliability, automation and simplicity show its potential in quantitative fluorescence imaging studies and high-content screening for compounds and factors involved in DSB response.

In vitro flow cytometry-based screening platform for cellulase engineering

PubMed Central

Körfer, Georgette; Pitzler, Christian; Vojcic, Ljubica; Martinez, Ronny; Schwaneberg, Ulrich

2016-01-01

Ultrahigh throughput screening (uHTS) plays an essential role in directed evolution for tailoring biocatalysts for industrial applications. Flow cytometry-based uHTS provides an efficient coverage of the generated protein sequence space by analysis of up to 107 events per hour. Cell-free enzyme production overcomes the challenge of diversity loss during the transformation of mutant libraries into expression hosts, enables directed evolution of toxic enzymes, and holds the promise to efficiently design enzymes of human or animal origin. The developed uHTS cell-free compartmentalization platform (InVitroFlow) is the first report in which a flow cytometry-based screened system has been combined with compartmentalized cell-free expression for directed cellulase enzyme evolution. InVitroFlow was validated by screening of a random cellulase mutant library employing a novel screening system (based on the substrate fluorescein-di-β-D-cellobioside), and yielded significantly improved cellulase variants (e.g. CelA2-H288F-M1 (N273D/H288F/N468S) with 13.3-fold increased specific activity (220.60 U/mg) compared to CelA2 wildtype: 16.57 U/mg). PMID:27184298

Search for biomarkers of asbestos exposure and asbestos-induced cancers in investigations of the immunological effects of asbestos.

PubMed

Matsuzaki, Hidenori; Kumagai-Takei, Naoko; Lee, Suni; Maeda, Megumi; Sada, Nagisa; Hatayama, Tamayo; Yamamoto, Shoko; Ikeda, Miho; Yoshitome, Kei; Min, Yu; Nishimura, Yasumitsu; Otsuki, Takemi

2017-06-09

The immunological effects of asbestos exposure on various lymphocytes such as the regulatory T cell (Treg), responder CD4+ T helper cell (Tresp), CD8+ cytotoxic T lymphocytes (CTL), and natural killer (NK) cells were investigated. Results show that asbestos exposure impairs antitumor immunity through enhancement of regulatory T cell function and volume, reduction of CXCR3 chemokine receptor in responder CD4+ T helper cells, and impairment of the killing activities of CD8+ cytotoxic T lymphocytes (CTL) and NK cells. These findings were used to explore biological markers associated with asbestos exposure and asbestos-induced cancers and suggested the usefulness of serum/plasma IL-10 and TGF-β, surface CXCR3 expression in Tresp, the secreting potential of IFN-γ in Tresp, intracellular perforin level in CTL, and surface expression NKp46 in NK cells. Although other unexplored cytokines in serum/plasma and molecules in these immunological cells, including Th17, should be investigated by experimental procedures in addition to a comprehensive analysis of screening methods, biomarkers based on immunological alterations may be helpful in clinical situations to screen the high-risk population exposed to asbestos and susceptible to asbestos-related cancers such as mesothelioma.

The Isolation of Novel Phage Display-Derived Human Recombinant Antibodies Against CCR5, the Major Co-Receptor of HIV

PubMed Central

Shimoni, Moria; Herschhorn, Alon; Britan-Rosich, Yelena; Kotler, Moshe; Benhar, Itai

2013-01-01

Abstract Selecting for antibodies against specific cell-surface proteins is a difficult task due to many unrelated proteins that are expressed on the cell surface. Here, we describe a method to screen antibody-presenting phage libraries against native cell-surface proteins. We applied this method to isolate antibodies that selectively recognize CCR5, which is the major co-receptor for HIV entry (consequently, playing a pivotal role in HIV transmission and pathogenesis). We employed a phage screening strategy by using cells that co-express GFP and CCR5, along with an excess of control cells that do not express these proteins (and are otherwise identical to the CCR5-expressing cells). These control cells are intended to remove most of the phages that bind the cells nonspecifically; thus leading to an enrichment of the phages presenting anti-CCR5-specific antibodies. Subsequently, the CCR5-presenting cells were quantitatively sorted by flow cytometry, and the bound phages were eluted, amplified, and used for further successive selection rounds. Several different clones of human single-chain Fv antibodies that interact with CCR5-expressing cells were identified. The most specific monoclonal antibody was converted to a full-length IgG and bound the second extracellular loop of CCR5. The experimental approach presented herein for screening for CCR5-specific antibodies can be applicable to screen antibody-presenting phage libraries against any cell-surface expressed protein of interest. PMID:23941674

Development of New Front Side Metallization Method of Aluminum Electroplating for Silicon Solar Cell

NASA Astrophysics Data System (ADS)

Willis, Megan D.

In this thesis, the methods of aluminum electroplating in an ionic liquid for silicon solar cell front side metallization were studied. It focused on replacing the current silver screen printing with an alternative metallization technology using a low-cost Earth-abundant metal for mass production, due to the high cost and limited availability of silver. A conventional aluminum electroplating method was employed for silicon solar cells fabrication on both p-type and n-type substrates. The highest efficiency of 17.9% was achieved in the n-type solar cell with a rear junction, which is comparable to that of the same structure cell with screen printed silver electrodes from industrial production lines. It also showed better spiking resistant performance than the common structure p-type solar cell. Further efforts were put on the development of a novel light-induced plating of aluminum technique. The aluminum was deposited directly on a silicon substrate without the assistance of a conductive seed layer, thus simplified and reduced the process cost. The plated aluminum has good adhesion to the silicon surface with the resistivity as low as 4x10-6 Ω-cm. A new demo tool was designed and set up for the light-induced plating experiment, aiming to utilize this technique in large-size solar cells fabrication and mass production. Besides the metallization methods, a comprehensive sensitivity analysis for the efficiency dispersion in the production of crystalline-Si solar cells was presented based on numerical simulations. Temperature variation in the diffusion furnace was the most significant cause of the efficiency dispersion. It was concluded that a narrow efficiency range of +/-0.5% absolute is achievable if the emitter diffusion temperature is confined to a 13°C window, while other cell parameters vary within their normal windows. Possible methods to minimize temperature variation in emitter diffusion were proposed.

DHAD variants and methods of screening

DOEpatents

Kelly, Kristen J.; Ye, Rick W.

2017-02-28

Methods of screening for dihydroxy-acid dehydratase (DHAD) variants that display increased DHAD activity are disclosed, along with DHAD variants identified by these methods. Such enzymes can result in increased production of compounds from DHAD requiring biosynthetic pathways. Also disclosed are isolated nucleic acids encoding the DHAD variants, recombinant host cells comprising the isolated nucleic acid molecules, and methods of producing butanol.

Identification of Novel Pro-Migratory, Cancer-Associated Genes Using Quantitative, Microscopy-Based Screening

PubMed Central

Naffar-Abu-Amara, Suha; Shay, Tal; Galun, Meirav; Cohen, Naomi; Isakoff, Steven J.; Kam, Zvi; Geiger, Benjamin

2008-01-01

Background Cell migration is a highly complex process, regulated by multiple genes, signaling pathways and external stimuli. To discover genes or pharmacological agents that can modulate the migratory activity of cells, screening strategies that enable the monitoring of diverse migratory parameters in a large number of samples are necessary. Methodology In the present study, we describe the development of a quantitative, high-throughput cell migration assay, based on a modified phagokinetic tracks (PKT) procedure, and apply it for identifying novel pro-migratory genes in a cancer-related gene library. In brief, cells are seeded on fibronectin-coated 96-well plates, covered with a monolayer of carboxylated latex beads. Motile cells clear the beads, located along their migratory paths, forming tracks that are visualized using an automated, transmitted-light screening microscope. The tracks are then segmented and characterized by multi-parametric, morphometric analysis, resolving a variety of morphological and kinetic features. Conclusions In this screen we identified 4 novel genes derived from breast carcinoma related cDNA library, whose over-expression induces major alteration in the migration of the stationary MCF7 cells. This approach can serve for high throughput screening for novel ways to modulate cellular migration in pathological states such as tumor metastasis and invasion. PMID:18213366

Screening for Developmental Neurotoxicity Using PC12 Cells: Comparisons of Organophosphates with a Carbamate, an Organochlorine, and Divalent Nickel

PubMed Central

Slotkin, Theodore A.; MacKillop, Emiko A.; Ryde, Ian T.; Tate, Charlotte A.; Seidler, Frederic J.

2007-01-01

Background In light of the large number of chemicals that are potential developmental neurotoxicants, there is a need to develop rapid screening techniques. Objectives We exposed undifferentiated and differentiating neuronotypic PC12 cells to different organophosphates (chlorpyrifos, diazinon, parathion), a carbamate (physostigmine), an organochlorine (dieldrin), and a metal (divalent nickel; Ni2+) and examined indices of cell replication and differentiation for both short- and long-term exposures. Results In undifferentiated cells, all the agents inhibited DNA synthesis, with the greatest effect for diazinon, but physostigmine eventually produced the largest deficits in the total number of cells after prolonged exposure. The onset of differentiation intensified the adverse effects on DNA synthesis and changed the rank order in keeping with a shift away from noncholinergic mechanisms and toward cholinergic mechanisms. Differentiation also worsened the effects of each agent on cell number after prolonged exposure, whereas cell growth was not suppressed, nor were there any effects on viability as assessed with trypan blue. Nevertheless, differentiating cells displayed signs of oxidative stress from all of the test compounds except Ni2+, as evidenced by measurements of lipid peroxidation. Finally, all of the toxicants shifted the transmitter fate of the cells away from the cholinergic phenotype and toward the catecholaminergic phenotype. Conclusions These studies point out the feasibility of developing cell-based screening methods that enable the detection of multiple end points that may relate to mechanisms associated with developmental neurotoxicity, revealing some common targets for disparate agents. PMID:17366826

A gravimetric simplified method for nucleated marrow cell counting using an injection needle.

PubMed

Saitoh, Toshiki; Fang, Liu; Matsumoto, Kiyoshi

2005-08-01

A simplified gravimetric marrow cell counting method for rats is proposed for a regular screening method. After fresh bone marrow was aspirated by an injection needle, the marrow cells were suspended in carbonate buffered saline. The nucleated marrow cell count (NMC) was measured by an automated multi-blood cell analyzer. When this gravimetric method was applied to rats, the NMC of the left and right femurs had essentially identical values due to careful handling. The NMC at 4 to 10 weeks of age in male and female Crj:CD(SD)IGS rats was 2.72 to 1.96 and 2.75 to 1.98 (x10(6) counts/mg), respectively. More useful information for evaluation could be obtained by using this gravimetric method in addition to myelogram examination. However, some difficulties with this method include low NMC due to blood contamination and variation of NMC due to handling. Therefore, the utility of this gravimetric method for screening will be clarified by the accumulation of the data on myelotoxicity studies with this method.

Performance of prenatal screening using maternal serum and ultrasound markers for Down syndrome in Chinese women: a systematic review and meta-analysis.

PubMed

Tu, S; Rosenthal, M; Wang, D; Huang, J; Chen, Y

2016-09-01

Controversies about the performance of conventional prenatal screening using maternal serum and ultrasound markers (PSMSUM) in detecting Down syndrome (DS) have been raised as a result of a recently available noninvasive prenatal test based on cell-free fetal DNA sequencing. To evaluate the screening performance of PSMSUM in detecting DS in Chinese women. An exhaustive literature search of MEDLINE, Embase, the Cochrane Library, ISI Web of Science and China BioMedical Disc. Primary studies, published from January 2004 to November 2014, which examined the screening accuracy of PSMSUM in pregnant Chinese women, compared with a reference standard, either chromosomal verification or inspection of the newborn. Data were extracted as screening positive/negative results for Down and non-Down syndrome pregnancies, allowing estimation of sensitivities and specificities. Risks of bias within and across studies were assessed. Screening accuracy measures were pooled using a bivariate random effects regression model. Seventy-eight studies, involving six categories of PSMSUM, were included. Second-trimester double serum [pooled sensitivity (SEN) = 0.80, pooled specificity (SPE) = 0.95] and triple-serum (pooled SEN = 0.79, pooled SPE = 0.96) screening were the predominant PSMSUM methods. The screening performances of these methods achieved the national standard but varied enormously across studies. First-trimester combined screening (pooled SEN = 0.92, pooled SPE = 0.93) and second-trimester quadruple serum screening (median SEN = 0.86, median SPE = 0.96) performed better, but were rarely used. Second-trimester maternal serum screening has the potential to achieve satisfactory screening performance in middle- and low-income countries. The reported enormous range in screening performance of second-trimester PSMSUM calls for urgent implementation of methods for performance optimization. Meta-analysis results show good accuracy of maternal serum and ultrasound screening for trisomy 21 in Chinese women. © 2016 Royal College of Obstetricians and Gynaecologists.

A Network-Based Method to Assess the Statistical Significance of Mild Co-Regulation Effects

PubMed Central

Horvát, Emőke-Ágnes; Zhang, Jitao David; Uhlmann, Stefan; Sahin, Özgür; Zweig, Katharina Anna

2013-01-01

Recent development of high-throughput, multiplexing technology has initiated projects that systematically investigate interactions between two types of components in biological networks, for instance transcription factors and promoter sequences, or microRNAs (miRNAs) and mRNAs. In terms of network biology, such screening approaches primarily attempt to elucidate relations between biological components of two distinct types, which can be represented as edges between nodes in a bipartite graph. However, it is often desirable not only to determine regulatory relationships between nodes of different types, but also to understand the connection patterns of nodes of the same type. Especially interesting is the co-occurrence of two nodes of the same type, i.e., the number of their common neighbours, which current high-throughput screening analysis fails to address. The co-occurrence gives the number of circumstances under which both of the biological components are influenced in the same way. Here we present SICORE, a novel network-based method to detect pairs of nodes with a statistically significant co-occurrence. We first show the stability of the proposed method on artificial data sets: when randomly adding and deleting observations we obtain reliable results even with noise exceeding the expected level in large-scale experiments. Subsequently, we illustrate the viability of the method based on the analysis of a proteomic screening data set to reveal regulatory patterns of human microRNAs targeting proteins in the EGFR-driven cell cycle signalling system. Since statistically significant co-occurrence may indicate functional synergy and the mechanisms underlying canalization, and thus hold promise in drug target identification and therapeutic development, we provide a platform-independent implementation of SICORE with a graphical user interface as a novel tool in the arsenal of high-throughput screening analysis. PMID:24039936

Diagnostic Peptide Discovery: Prioritization of Pathogen Diagnostic Markers Using Multiple Features

PubMed Central

Carmona, Santiago J.; Sartor, Paula A.; Leguizamón, María S.; Campetella, Oscar E.; Agüero, Fernán

2012-01-01

The availability of complete pathogen genomes has renewed interest in the development of diagnostics for infectious diseases. Synthetic peptide microarrays provide a rapid, high-throughput platform for immunological testing of potential B-cell epitopes. However, their current capacity prevent the experimental screening of complete “peptidomes”. Therefore, computational approaches for prediction and/or prioritization of diagnostically relevant peptides are required. In this work we describe a computational method to assess a defined set of molecular properties for each potential diagnostic target in a reference genome. Properties such as sub-cellular localization or expression level were evaluated for the whole protein. At a higher resolution (short peptides), we assessed a set of local properties, such as repetitive motifs, disorder (structured vs natively unstructured regions), trans-membrane spans, genetic polymorphisms (conserved vs. divergent regions), predicted B-cell epitopes, and sequence similarity against human proteins and other potential cross-reacting species (e.g. other pathogens endemic in overlapping geographical locations). A scoring function based on these different features was developed, and used to rank all peptides from a large eukaryotic pathogen proteome. We applied this method to the identification of candidate diagnostic peptides in the protozoan Trypanosoma cruzi, the causative agent of Chagas disease. We measured the performance of the method by analyzing the enrichment of validated antigens in the high-scoring top of the ranking. Based on this measure, our integrative method outperformed alternative prioritizations based on individual properties (such as B-cell epitope predictors alone). Using this method we ranked 10 million 12-mer overlapping peptides derived from the complete T. cruzi proteome. Experimental screening of 190 high-scoring peptides allowed the identification of 37 novel epitopes with diagnostic potential, while none of the low scoring peptides showed significant reactivity. Many of the metrics employed are dependent on standard bioinformatic tools and data, so the method can be easily extended to other pathogen genomes. PMID:23272069

Manipulation-free cultures of human iPSC-derived cardiomyocytes offer a novel screening method for cardiotoxicity.

PubMed

Rajasingh, Sheeja; Isai, Dona Greta; Samanta, Saheli; Zhou, Zhi-Gang; Dawn, Buddhadeb; Kinsey, William H; Czirok, Andras; Rajasingh, Johnson

2018-04-05

Induced pluripotent stem cell (iPSC)-based cardiac regenerative medicine requires the efficient generation, structural soundness and proper functioning of mature cardiomyocytes, derived from the patient's somatic cells. The most important functional property of cardiomyocytes is the ability to contract. Currently available methods routinely used to test and quantify cardiomyocyte function involve techniques that are labor-intensive, invasive, require sophisticated instruments or can adversely affect cell vitality. We recently developed optical flow imaging method analyses and quantified cardiomyocyte contractile kinetics from video microscopic recordings without compromising cell quality. Specifically, our automated particle image velocimetry (PIV) analysis of phase-contrast video images captured at a high frame rate yields statistical measures characterizing the beating frequency, amplitude, average waveform and beat-to-beat variations. Thus, it can be a powerful assessment tool to monitor cardiomyocyte quality and maturity. Here we demonstrate the ability of our analysis to characterize the chronotropic responses of human iPSC-derived cardiomyocytes to a panel of ion channel modulators and also to doxorubicin, a chemotherapy agent with known cardiotoxic side effects. We conclude that the PIV-derived beat patterns can identify the elongation or shortening of specific phases in the contractility cycle, and the obtained chronotropic responses are in accord with known clinical outcomes. Hence, this system can serve as a powerful tool to screen the new and currently available pharmacological compounds for cardiotoxic effects.

First quantitative high-throughput screen in zebrafish identifies novel pathways for increasing pancreatic β-cell mass

PubMed Central

Wang, Guangliang; Rajpurohit, Surendra K; Delaspre, Fabien; Walker, Steven L; White, David T; Ceasrine, Alexis; Kuruvilla, Rejji; Li, Ruo-jing; Shim, Joong S; Liu, Jun O; Parsons, Michael J; Mumm, Jeff S

2015-01-01

Whole-organism chemical screening can circumvent bottlenecks that impede drug discovery. However, in vivo screens have not attained throughput capacities possible with in vitro assays. We therefore developed a method enabling in vivo high-throughput screening (HTS) in zebrafish, termed automated reporter quantification in vivo (ARQiv). In this study, ARQiv was combined with robotics to fully actualize whole-organism HTS (ARQiv-HTS). In a primary screen, this platform quantified cell-specific fluorescent reporters in >500,000 transgenic zebrafish larvae to identify FDA-approved (Federal Drug Administration) drugs that increased the number of insulin-producing β cells in the pancreas. 24 drugs were confirmed as inducers of endocrine differentiation and/or stimulators of β-cell proliferation. Further, we discovered novel roles for NF-κB signaling in regulating endocrine differentiation and for serotonergic signaling in selectively stimulating β-cell proliferation. These studies demonstrate the power of ARQiv-HTS for drug discovery and provide unique insights into signaling pathways controlling β-cell mass, potential therapeutic targets for treating diabetes. DOI: http://dx.doi.org/10.7554/eLife.08261.001 PMID:26218223

Discrimination of Avian Influenza Virus using Host-cell Infection Fingerprinting by Sulfinate-based Fluorescence Superoxide Probe.

PubMed

Hong, Seong Cheol; Murale, Dhiraj P; Jang, Se-Young; Haque, Md Mamunul; Seo, Minah; Lee, Seok; Woo, Deok Ha; Kwon, Junghoon; Song, Chang-Seon; Kim, Yun Kyung; Lee, Jun-Seok

2018-06-22

Avian Influenza (AI) caused an annual epidemic outbreak that led to destroying tens of millions of poultry worldwide. Current gold standard AI diagnosis method is an embryonic egg-based hemagglutination assay followed by immunoblotting or PCR sequencing to confirm subtypes. It requires, however, specialized facilities to handle egg inoculation and incubation, and the subtyping methods relied on costly reagents. Here, we demonstrated the first differential sensing approach to distinguish AI subtypes using series of cell lines and fluorescent sensor. Susceptibility of AI virus differs depending on genetic backgrounds of host cells. Thus, we examined cells from different organ origin, and the infection patterns against a panel of cells were utilized for AI virus subtyping. To quantify AI infection, we designed a highly cell-permeable fluorescent superoxide sensor to visualize infection. Though many AI monitoring strategies relied on sophisticated antibody have been extensively studied, our differential sensing strategy successfully proved discriminations of AI subtypes and demonstrated as a useful primary screening platform to monitor a large number of samples. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Discovery of Potent Myeloid Cell Leukemia 1 (Mcl-1) Inhibitors Using Fragment-Based Methods and Structure-Based Design

DOE Office of Scientific and Technical Information (OSTI.GOV)

Friberg, Anders; Vigil, Dominico; Zhao, Bin

2012-12-17

Myeloid cell leukemia 1 (Mcl-1), a member of the Bcl-2 family of proteins, is overexpressed and amplified in various cancers and promotes the aberrant survival of tumor cells that otherwise would undergo apoptosis. Here we describe the discovery of potent and selective Mcl-1 inhibitors using fragment-based methods and structure-based design. NMR-based screening of a large fragment library identified two chemically distinct hit series that bind to different sites on Mcl-1. Members of the two fragment classes were merged together to produce lead compounds that bind to Mcl-1 with a dissociation constant of <100 nM with selectivity for Mcl-1 over Bcl-xLmore » and Bcl-2. Structures of merged compounds when complexed to Mcl-1 were obtained by X-ray crystallography and provide detailed information about the molecular recognition of small-molecule ligands binding Mcl-1. The compounds represent starting points for the discovery of clinically useful Mcl-1 inhibitors for the treatment of a wide variety of cancers.« less

High-throughput identification of genotype-specific cancer vulnerabilities in mixtures of barcoded tumor cell lines

PubMed Central

Yu, Channing; Mannan, Aristotle M.; Yvone, Griselda Metta; Ross, Kenneth N.; Zhang, Yan-Ling; Marton, Melissa A.; Taylor, Bradley R.; Crenshaw, Andrew; Gould, Joshua Z.; Tamayo, Pablo; Weir, Barbara A.; Tsherniak, Aviad; Wong, Bang; Garraway, Levi A.; Shamji, Alykhan F.; Palmer, Michelle A.; Foley, Michael A.; Winckler, Wendy; Schreiber, Stuart L.; Kung, Andrew L.; Golub, Todd R.

2016-01-01

Hundreds of genetically characterized cell lines are available for the discovery of genotype-specific cancer vulnerabilities. However, screening large numbers of compounds against large numbers of cell lines is currently impractical, and such experiments are often difficult to control1-4. Here, we report a method called PRISM that allows pooled screening of mixtures of cancer cell lines by labeling each cell line with 24-nucleotide barcodes. PRISM displayed the expected patterns of cell killing seen in conventional (unpooled) assays. In a screen of 102 cell lines across 8,400 compounds, PRISM led to the identification of BRD-7880 as a potent and highly specific inhibitor of aurora kinases B and C. Cell line pools also efficiently formed tumors as xenografts, and PRISM recapitulated the expected pattern of erlotinib sensitivity in vivo. PMID:26928769

In silico cloning and B/T cell epitope prediction of triosephosphate isomerase from Echinococcus granulosus.

PubMed

Wang, Fen; Ye, Bin

2016-10-01

Cystic echinococcosis is a worldwide zoonosis caused by Echinococcus granulosus. Because the methods of diagnosis and treatment for cystic echinococcosis were limited, it is still necessary to screen target proteins for the development of new anti-hydatidosis vaccine. In this study, the triosephosphate isomerase gene of E. granulosus was in silico cloned. The B cell and T cell epitopes were predicted by bioinformatics methods. The cDNA sequence of EgTIM was composition of 1094 base pairs, with an open reading frame of 753 base pairs. The deduced amino acid sequences were composed of 250 amino acids. Five cross-reactive epitopes, locating on 21aa-35aa, 43aa-57aa, 94aa-107aa, 115-129aa, and 164aa-183aa, could be expected to serve as candidate epitopes in the development of vaccine against E. granulosus. These results could provide bases for gene cloning, recombinant expression, and the designation of anti-hydatidosis vaccine.

Hydrogel tissue construct-based high-content compound screening.

PubMed

Lam, Vy; Wakatsuki, Tetsuro

2011-01-01

Current pharmaceutical compound screening systems rely on cell-based assays to identify therapeutic candidates and potential toxicities. However, cells grown on 2D substrata or in suspension do not exhibit the mechanical or physiological properties of cells in vivo. To address this limitation, the authors developed an in vitro, high-throughput, 3D hydrogel tissue construct (HTC)-based assay system to quantify cell and tissue mechanical properties and multiple parameters of physiology. HTC mechanics was quantified using an automated device, and physiological status was assessed using spectroscopy-based indicators that were read on microplate readers. To demonstrate the application of this system, the authors screened 4 test compounds--rotenone (ROT), cytochalasin D (CD), 2,4-dinitrophenol (DNP), and Rho kinase inhibitor (H-1152)--for their ability to modulate HTC contractility without affecting actin integrity, mitochondrial membrane potential (MMP), or viability. All 4 compounds dose-dependently reduced HTC contractility. However, ROT was toxic, DNP dissipated MMP, and CD reduced both intracellular F-actin and viability. H-1152 was found to be the best candidate compound since it reduced HTC contractility with minimal side effects. The authors propose that their HTC-based assay system can be used to screen for compounds that modulate HTC contractility and assess the underlying physiological mechanism(s) of compound activity and toxicity.

Generation of human pluripotent stem cell-derived hepatocyte-like cells for drug toxicity screening.

PubMed

Takayama, Kazuo; Mizuguchi, Hiroyuki

2017-02-01

Because drug-induced liver injury is one of the main reasons for drug development failures, it is important to perform drug toxicity screening in the early phase of pharmaceutical development. Currently, primary human hepatocytes are most widely used for the prediction of drug-induced liver injury. However, the sources of primary human hepatocytes are limited, making it difficult to supply the abundant quantities required for large-scale drug toxicity screening. Therefore, there is an urgent need for a novel unlimited, efficient, inexpensive, and predictive model which can be applied for large-scale drug toxicity screening. Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are able to replicate indefinitely and differentiate into most of the body's cell types, including hepatocytes. It is expected that hepatocyte-like cells generated from human ES/iPS cells (human ES/iPS-HLCs) will be a useful tool for drug toxicity screening. To apply human ES/iPS-HLCs to various applications including drug toxicity screening, homogenous and functional HLCs must be differentiated from human ES/iPS cells. In this review, we will introduce the current status of hepatocyte differentiation technology from human ES/iPS cells and a novel method to predict drug-induced liver injury using human ES/iPS-HLCs. Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Obligate multivalent recognition of cell surface tomoregulin following selection from a multivalent phage antibody library.

PubMed

Heitner, Tara; Satozawa, Noboru; McLean, Kirk; Vogel, David; Cobb, Ronald R; Liu, Bing; Mahmoudi, Mithra; Finster, Silke; Larsen, Brent; Zhu, Ying; Zhou, Hongxing; Müller-Tiemann, Beate; Monteclaro, Felipe; Zhao, Xiao-Yan; Light, David R

2006-12-01

A therapeutic antibody candidate (AT-19) isolated using multivalent phage display binds native tomoregulin (TR) as a mul-timer not as a monomer. This report raises the importance of screening and selecting phage antibodies on native antigen and reemphasizes the possibility that potentially valuable antibodies are discarded when a monomeric phage display system is used for screening. A detailed live cell panning selection and screening method to isolate multivalently active antibodies is described. AT-19 is a fully human antibody recognizing the cell surface protein TR, a proposed prostate cancer target for therapeutic antibody internalization. AT-19 was isolated from a multivalent single-chain variable fragment (scFv) antibody library rescued with hyperphage. The required multivalency for isolation of AT-19 is supported by fluorescence activated cell sorting data demonstrating binding of the multivalent AT-19 phage particles at high phage concentrations and failure of monovalent particles to bind. Pure monomeric scFv AT-19 does not bind native receptor on cells, whereas dimeric scFv or immunoglobulin G binds with nanomolar affinity. The isolation of AT-19 antibody with obligate bivalent binding activity to native TR is attributed to the use of a multivalent display of scFv on phage and the method for selecting and screening by alternate use of 2 recombinant cell lines.

A multiplexed method for kinetic measurements of apoptosis and proliferation using live-content imaging.

PubMed

Artymovich, Katherine; Appledorn, Daniel M

2015-01-01

In vitro cell proliferation and apoptosis assays are widely used to study cancer cell biology. Commonly used methodologies are however performed at a single, user-defined endpoint. We describe a kinetic multiplex assay incorporating the CellPlayer(TM) NucLight Red reagent to measure proliferation and the CellPlayer(TM) Caspase-3/7 reagent to measure apoptosis using the two-color, live-content imaging platform, IncuCyte(TM) ZOOM. High-definition phase-contrast images provide an additional qualitative validation of cell death based on morphological characteristics. The kinetic data generated using this strategy can be used to derive informed pharmacology measurements to screen potential cancer therapeutics.

Automated Protocol for Large-Scale Modeling of Gene Expression Data.

PubMed

Hall, Michelle Lynn; Calkins, David; Sherman, Woody

2016-11-28

With the continued rise of phenotypic- and genotypic-based screening projects, computational methods to analyze, process, and ultimately make predictions in this field take on growing importance. Here we show how automated machine learning workflows can produce models that are predictive of differential gene expression as a function of a compound structure using data from A673 cells as a proof of principle. In particular, we present predictive models with an average accuracy of greater than 70% across a highly diverse ∼1000 gene expression profile. In contrast to the usual in silico design paradigm, where one interrogates a particular target-based response, this work opens the opportunity for virtual screening and lead optimization for desired multitarget gene expression profiles.

[Identification of human monoclonal HIV-1-neutralizing antibodies from phage antibody library by cell-based screening].

PubMed

Zhang, Na; Man, Lai; Sun, Jian-ping; Meng, Jia-zi; He, Yu-xian

2013-09-01

To identify human monoclonal HIV-l-neutralizing antibodies from an HIV-1 CRF07BC specific phage display antibody library by cell-based screening. 293T cells were transfected by pCH064. 2-Env plas mid and then used to biopan the phage antibody library. The positive phage clones were screened by cell based ELISA and sequenced for the variable region of heavy (VH) and light (VL) chains. The expressed Fabs were purified by Ni(+2) -NTA column and analyzed by SDS-PAGE. The cell- and gp120 protein-based ELISA as well as flow cytometry were used to measure Fab's binding activity. The neutralizing activity of Fabs was assessed by HIV-1 pseudoviruses. After 4-round biopanning, the binding phages to transfected cells were enriched about 650-folds. A total of 28 positive clones were screened out by cell ELISA and sequence analysis identified 5 different Fabs possessing unique VH and VL (2801, 2837, 2863, 2870 and 2920). Interestingly, these Fabs reacted with the Env-transfected 293T cells but not soluble gp120 proteins, suggesting that they might target conformation-dependent epitopes presenting on viral Env complex. We found that three Fabs (2801, 2863, 2870) exhibited potent neutralizing activity against CRF07_BC isolate CH120. 6 with IC50 of 2.24, 0.89 and 3.09 microg/mL respectively, and that 2801 and 2863 cross-neutral ized the subtype B isolate SF162 at IC50 of 0.69 and 3.52 microg/mL respectively. In conclusion, the HIV-1 Env-transfected 293T cells can be used to efficiently enrich and screen the phage antibody library and isolate human monoclonal HIV-1-neutralizing Fabs that target the Env complex-dependent conformational epitopes. Therefore, our studies provide a powerful platform for exploring the mechanism of HIV-1 neu tralizing response and for designing AIDS vaccines.

Constellation Pharmacology: A new paradigm for drug discovery

PubMed Central

Schmidt, Eric W.; Olivera, Baldomero M.

2015-01-01

Constellation Pharmacology is a cell-based high-content phenotypic-screening platform that utilizes subtype-selective pharmacological agents to elucidate the cell-specific combinations (“constellations”) of key signaling proteins that define specific cell types. Heterogeneous populations of native cells, in which the different individual cell types have been identified and characterized, are the foundation for this screening platform. Constellation Pharmacology is useful for screening small molecules or for deconvoluting complex mixtures of biologically-active natural products. This platform has been used to purify natural products and discover their molecular mechanisms. In the on-going development of Constellation Pharmacology, there is a positive-feedback loop between the pharmacological characterization of cell types and screening for new drug candidates. As Constellation Pharmacology is used to discover compounds with novel targeting-selectivity profiles, those new compounds then further help to elucidate the constellations of specific cell types, thereby increasing the content of this high-content platform. PMID:25562646

Optical diagnostics of osteoblast cells and osteogenic drug screening

NASA Astrophysics Data System (ADS)

Kolanti, Elayaraja; Veerla, Sarath C.; Khajuria, Deepak K.; Roy Mahapatra, D.

2016-02-01

Microfluidic device based diagnostics involving optical fibre path, in situ imaging and spectroscopy are gaining importance due to recent advances in diagnostics instrumentation and methods, besides other factors such as low amount of reagent required for analysis, short investigation times, and potential possibilities to replace animal model based study in near future. It is possible to grow and monitor tissues in vitro in microfluidic lab-on-chip. It may become a transformative way of studying how cells interact with drugs, pathogens and biomaterials in physiologically relevant microenvironments. To a large extent, progress in developing clinically viable solutions has been constrained because of (i) contradiction between in vitro and in vivo results and (ii) animal model based and clinical studies which is very expensive. Our study here aims to evaluate the usefulness of microfluidic device based 3D tissue growth and monitoring approach to better emulate physiologically and clinically relevant microenvironments in comparison to conventional in vitro 2D culture. Moreover, the microfluidic methodology permits precise high-throughput investigations through real-time imaging while using very small amounts of reagents and cells. In the present study, we report on the details of an osteoblast cell based 3D microfluidic platform which we employ for osteogenic drug screening. The drug formulation is functionalized with fluorescence and other biomarkers for imaging and spectroscopy, respectively. Optical fibre coupled paths are used to obtain insight regarding the role of stress/flow pressure fluctuation and nanoparticle-drug concentration on the osteoblast growth and osteogenic properties of bone.

Detection of tyrosine hydroxylase in dopaminergic neuron cell using gold nanoparticles-based barcode DNA.

PubMed

An, Jeung Hee; Oh, Byung-Keun; Choi, Jeong Woo

2013-04-01

Tyrosine hydroxylase, the rate-limiting enzyme of catecholamine biosysthesis, is predominantly expressed in several cell groups within the brain, including the dopaminergic neurons of the substantia nigra and ventral tegmental area. We evaluated the efficacy of this protein-detection method in detecting tyrosine hydroxylase in normal and oxidative stress damaged dopaminergic cells. In this study, a coupling of DNA barcode and bead-based immnunoassay for detecting tyrosine hydroxylaser with PCR-like sensitivity is reported. The method relies on magnetic nanoparticles with antibodies and nanoparticles that are encoded with DNA and antibodies that can sandwich the target protein captured by the nanoparticle-bound antibodies. The aggregate sandwich structures are magnetically separated from solution, and treated to remove the conjugated barcode DNA. The DNA barcodes were identified by PCR analysis. The concentration of tyrosine hydroxylase in dopaminergic cell can be easily and rapidly detected using bio-barcode assay. The bio-barcode assay is a rapid and high-throughput screening tool to detect of neurotransmitter such as dopamine.

Alu–based cell-free DNA: a novel biomarker for screening of gastric cancer

PubMed Central

Zhao, Jianmei; Shen, Xianjuan; Jing, Rongrong; Yu, Juan; Li, Li; Shi, Yingjuan; Zhang, Lurong; Wang, Zhiwei; Cong, Hui

2017-01-01

Gastric cancer (GC) is the fourth most common cancer and the second major cause of cancer-related deaths worldwide. In our previous study, a novel and sensitive method for quantifying cell-free DNA (CFD) in human blood was established and tested for its ability to predict patients with tumor. We want to investigate CFD expression in the sera of GC patients in an attempt to explore the clinical significance of CFD in improving the early screening of GC and monitoring GC progression by the branched DNA (bDNA)-based Alu assay. The concentration of CFD was quantitated by bDNA-based Alu assay. CEA, CA19-9, C72-4 and CA50 concentrations were determined by ABBOTT ARCHITECT I2000 SR. We found the CFD concentrations have significant differences between GC patients, benign gastric disease (BGD) patients and healthy controls (P < 0.05). CFD were weakly correlated with CEA (r = −0.197, P < 0.05) or CA50 (r = 0.206, P < 0.05), and no correlation with CA19-9 (r = −0.061, P > 0.05) or CA72-4 (r = 0.011, P > 0.05). In addition, CFD concentrations were significantly higher in stage I GC patients than BGD patients and healthy controls (P < 0.05), but there was no significant difference in CEA, CA19-9 and CA50 among the three traditional tumor markers (P > 0.05). Our analysis showed that CFD was more sensitive than CEA, CA19-9, CA72-4 or CA50 in early screening of GC. Compared with CEA, CA19-9, CA72-4 and CA50, CFD may prove to be a better biomarker for the screening of GC, thus providing a sensitive biomarker for screening and monitoring progression of GC. PMID:28903321

Incorporating interaction networks into the determination of functionally related hit genes in genomic experiments with Markov random fields

PubMed Central

Robinson, Sean; Nevalainen, Jaakko; Pinna, Guillaume; Campalans, Anna; Radicella, J. Pablo; Guyon, Laurent

2017-01-01

Abstract Motivation: Incorporating gene interaction data into the identification of ‘hit’ genes in genomic experiments is a well-established approach leveraging the ‘guilt by association’ assumption to obtain a network based hit list of functionally related genes. We aim to develop a method to allow for multivariate gene scores and multiple hit labels in order to extend the analysis of genomic screening data within such an approach. Results: We propose a Markov random field-based method to achieve our aim and show that the particular advantages of our method compared with those currently used lead to new insights in previously analysed data as well as for our own motivating data. Our method additionally achieves the best performance in an independent simulation experiment. The real data applications we consider comprise of a survival analysis and differential expression experiment and a cell-based RNA interference functional screen. Availability and implementation: We provide all of the data and code related to the results in the paper. Contact: sean.j.robinson@utu.fi or laurent.guyon@cea.fr Supplementary information: Supplementary data are available at Bioinformatics online. PMID:28881978

Generation of an arrayed CRISPR-Cas9 library targeting epigenetic regulators: from high-content screens to in vivo assays

PubMed Central

2017-01-01

ABSTRACT The CRISPR-Cas9 system has revolutionized genome engineering, allowing precise modification of DNA in various organisms. The most popular method for conducting CRISPR-based functional screens involves the use of pooled lentiviral libraries in selection screens coupled with next-generation sequencing. Screens employing genome-scale pooled small guide RNA (sgRNA) libraries are demanding, particularly when complex assays are used. Furthermore, pooled libraries are not suitable for microscopy-based high-content screens or for systematic interrogation of protein function. To overcome these limitations and exploit CRISPR-based technologies to comprehensively investigate epigenetic mechanisms, we have generated a focused sgRNA library targeting 450 epigenetic regulators with multiple sgRNAs in human cells. The lentiviral library is available both in an arrayed and pooled format and allows temporally-controlled induction of gene knock-out. Characterization of the library showed high editing activity of most sgRNAs and efficient knock-out at the protein level in polyclonal populations. The sgRNA library can be used for both selection and high-content screens, as well as for targeted investigation of selected proteins without requiring isolation of knock-out clones. Using a variety of functional assays we show that the library is suitable for both in vitro and in vivo applications, representing a unique resource to study epigenetic mechanisms in physiological and pathological conditions. PMID:29327641

Pilot Screening to Determine Antimicrobial Synergies in a Multidrug-Resistant Bacterial Strain Library

PubMed Central

Kim, Si-Hyun; Park, Chulmin; Chun, Hye-Sun; Choi, Jae-Ki; Lee, Hyo-Jin; Cho, Sung-Yeon; Park, Sun Hee; Choi, Su-Mi; Choi, Jung-Hyun; Yoo, Jin-Hong

2016-01-01

With the rise in multidrug-resistant (MDR) bacterial infections, there has been increasing interest in combinations of ≥2 antimicrobial agents with synergistic effects. We established an MDR bacterial strain library to screen for in vitro antimicrobial synergy by using a broth microdilution checkerboard method and high-throughput luciferase-based bacterial cell viability assay. In total, 39 MDR bacterial strains, including 23 carbapenem-resistant gram-negative bacteria, 9 vancomycin-intermediate Staphylococcus aureus, and 7 vancomycin-resistant Enterococcus faecalis, were used to screen for potential antimicrobial synergies. Synergies were more frequently identified with combinations of imipenem plus trimethoprim–sulfamethoxazole for carbapenem-resistant Acinetobacter baumannii in the library. To verify this finding, we tested 34 A. baumannii clinical isolates resistant to both imipenem and trimethoprim–sulfamethoxazole by the checkerboard method. The imipenem plus trimethoprim–sulfamethoxazole combination showed synergy in the treatment of 21 (62%) of the clinical isolates. The results indicate that pilot screening for antimicrobial synergy in the MDR bacterial strain library could be valuable in the selection of combination therapeutic regimens to treat MDR bacterial infections. Further studies are warranted to determine whether this screening system can be useful to screen for the combined effects of conventional antimicrobials and new-generation antimicrobials or nonantimicrobials. PMID:26974861

Use of Dried Capillary Blood Sampling for Islet Autoantibody Screening in Relatives: A Feasibility Study

PubMed Central

Rafkin, Lisa E.; Matheson, Della; Steck, Andrea K.; Yu, Liping; Henderson, Courtney; Beam, Craig A.; Boulware, David C.

2015-01-01

Abstract Background: Islet autoantibody testing provides the basis for assessment of risk of progression to type 1 diabetes. We set out to determine the feasibility and acceptability of dried capillary blood spot–based screening to identify islet autoantibody–positive relatives potentially eligible for inclusion in prevention trials. Materials and Methods: Dried blood spot (DBS) and venous samples were collected from 229 relatives participating in the TrialNet Pathway to Prevention Study. Both samples were tested for glutamic acid decarboxylase, islet antigen 2, and zinc transporter 8 autoantibodies, and venous samples were additionally tested for insulin autoantibodies and islet cell antibodies. We defined multiple autoantibody positive as two or more autoantibodies in venous serum and DBS screen positive if one or more autoantibodies were detected. Participant questionnaires compared the sample collection methods. Results: Of 44 relatives who were multiple autoantibody positive in venous samples, 42 (95.5%) were DBS screen positive, and DBS accurately detected 145 of 147 autoantibody-negative relatives (98.6%). Capillary blood sampling was perceived as more painful than venous blood draw, but 60% of participants would prefer initial screening using home fingerstick with clinic visits only required if autoantibodies were found. Conclusions: Capillary blood sampling could facilitate screening for type 1 diabetes prevention studies. PMID:26375197

Combining Phage and Yeast Cell Surface Antibody Display to Identify Novel Cell Type-Selective Internalizing Human Monoclonal Antibodies.

PubMed

Bidlingmaier, Scott; Su, Yang; Liu, Bin

2015-01-01

Using phage antibody display, large libraries can be generated and screened to identify monoclonal antibodies with affinity for target antigens. However, while library size and diversity is an advantage of the phage display method, there is limited ability to quantitatively enrich for specific binding properties such as affinity. One way of overcoming this limitation is to combine the scale of phage display selections with the flexibility and quantitativeness of FACS-based yeast surface display selections. In this chapter we describe protocols for generating yeast surface antibody display libraries using phage antibody display selection outputs as starting material and FACS-based enrichment of target antigen-binding clones from these libraries. These methods should be widely applicable for the identification of monoclonal antibodies with specific binding properties.

High content analysis platform for optimization of lipid mediated CRISPR-Cas9 delivery strategies in human cells.

PubMed

Steyer, Benjamin; Carlson-Stevermer, Jared; Angenent-Mari, Nicolas; Khalil, Andrew; Harkness, Ty; Saha, Krishanu

2016-04-01

Non-viral gene-editing of human cells using the CRISPR-Cas9 system requires optimized delivery of multiple components. Both the Cas9 endonuclease and a single guide RNA, that defines the genomic target, need to be present and co-localized within the nucleus for efficient gene-editing to occur. This work describes a new high-throughput screening platform for the optimization of CRISPR-Cas9 delivery strategies. By exploiting high content image analysis and microcontact printed plates, multi-parametric gene-editing outcome data from hundreds to thousands of isolated cell populations can be screened simultaneously. Employing this platform, we systematically screened four commercially available cationic lipid transfection materials with a range of RNAs encoding the CRISPR-Cas9 system. Analysis of Cas9 expression and editing of a fluorescent mCherry reporter transgene within human embryonic kidney cells was monitored over several days after transfection. Design of experiments analysis enabled rigorous evaluation of delivery materials and RNA concentration conditions. The results of this analysis indicated that the concentration and identity of transfection material have significantly greater effect on gene-editing than ratio or total amount of RNA. Cell subpopulation analysis on microcontact printed plates, further revealed that low cell number and high Cas9 expression, 24h after CRISPR-Cas9 delivery, were strong predictors of gene-editing outcomes. These results suggest design principles for the development of materials and transfection strategies with lipid-based materials. This platform could be applied to rapidly optimize materials for gene-editing in a variety of cell/tissue types in order to advance genomic medicine, regenerative biology and drug discovery. CRISPR-Cas9 is a new gene-editing technology for "genome surgery" that is anticipated to treat genetic diseases. This technology uses multiple components of the Cas9 system to cut out disease-causing mutations in the human genome and precisely suture in therapeutic sequences. Biomaterials based delivery strategies could help transition these technologies to the clinic. The design space for materials based delivery strategies is vast and optimization is essential to ensuring the safety and efficacy of these treatments. Therefore, new methods are required to rapidly and systematically screen gene-editing efficacy in human cells. This work utilizes an innovative platform to generate and screen many formulations of synthetic biomaterials and components of the CRISPR-Cas9 system in parallel. On this platform, we watch genome surgery in action using high content image analysis. These capabilities enabled us to identify formulation parameters for Cas9-material complexes that can optimize gene-editing in a specific human cell type. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Microfluidic cell culture systems for drug research.

PubMed

Wu, Min-Hsien; Huang, Song-Bin; Lee, Gwo-Bin

2010-04-21

In pharmaceutical research, an adequate cell-based assay scheme to efficiently screen and to validate potential drug candidates in the initial stage of drug discovery is crucial. In order to better predict the clinical response to drug compounds, a cell culture model that is faithful to in vivo behavior is required. With the recent advances in microfluidic technology, the utilization of a microfluidic-based cell culture has several advantages, making it a promising alternative to the conventional cell culture methods. This review starts with a comprehensive discussion on the general process for drug discovery and development, the role of cell culture in drug research, and the characteristics of the cell culture formats commonly used in current microfluidic-based, cell-culture practices. Due to the significant differences in several physical phenomena between microscale and macroscale devices, microfluidic technology provides unique functionality, which is not previously possible by using traditional techniques. In a subsequent section, the niches for using microfluidic-based cell culture systems for drug research are discussed. Moreover, some critical issues such as cell immobilization, medium pumping or gradient generation in microfluidic-based, cell-culture systems are also reviewed. Finally, some practical applications of microfluidic-based, cell-culture systems in drug research particularly those pertaining to drug toxicity testing and those with a high-throughput capability are highlighted.

Fragment-based discovery of potent inhibitors of the anti-apoptotic MCL-1 protein.

PubMed

Petros, Andrew M; Swann, Steven L; Song, Danying; Swinger, Kerren; Park, Chang; Zhang, Haichao; Wendt, Michael D; Kunzer, Aaron R; Souers, Andrew J; Sun, Chaohong

2014-03-15

Apoptosis is regulated by the BCL-2 family of proteins, which is comprised of both pro-death and pro-survival members. Evasion of apoptosis is a hallmark of malignant cells. One way in which cancer cells achieve this evasion is thru overexpression of the pro-survival members of the BCL-2 family. Overexpression of MCL-1, a pro-survival protein, has been shown to be a resistance factor for Navitoclax, a potent inhibitor of BCL-2 and BCL-XL. Here we describe the use of fragment screening methods and structural biology to drive the discovery of novel MCL-1 inhibitors from two distinct structural classes. Specifically, cores derived from a biphenyl sulfonamide and salicylic acid were uncovered in an NMR-based fragment screen and elaborated using high throughput analog synthesis. This culminated in the discovery of selective and potent inhibitors of MCL-1 that may serve as promising leads for medicinal chemistry optimization efforts. Copyright © 2014 Elsevier Ltd. All rights reserved.

A Rapid Method for Quantifying Viable Mycobacterium avium subsp. paratuberculosis in Cellular Infection Assays

PubMed Central

Pooley, Hannah B.; de Silva, Kumudika; Purdie, Auriol C.; Begg, Douglas J.; Whittington, Richard J.

2016-01-01

ABSTRACT Determining the viability of bacteria is a key outcome of in vitro cellular infection assays. Currently, this is done by culture, which is problematic for fastidious slow-growing bacteria such as Mycobacterium avium subsp. paratuberculosis, where it can take up to 4 months to confirm growth. This study aimed to identify an assay that can rapidly quantify the number of viable M. avium subsp. paratuberculosis cells in a cellular sample. Three commercially available bacterial viability assays along with a modified liquid culture method coupled with high-throughput quantitative PCR growth detection were assessed. Criteria for assessment included the ability of each assay to differentiate live and dead M. avium subsp. paratuberculosis organisms and their accuracy at low bacterial concentrations. Using the culture-based method, M. avium subsp. paratuberculosis growth was reliably detected and quantified within 2 weeks. There was a strong linear association between the 2-week growth rate and the initial inoculum concentration. The number of viable M. avium subsp. paratuberculosis cells in an unknown sample was quantified based on the growth rate, by using growth standards. In contrast, none of the commercially available viability assays were suitable for use with samples from in vitro cellular infection assays. IMPORTANCE Rapid quantification of the viability of Mycobacterium avium subsp. paratuberculosis in samples from in vitro cellular infection assays is important, as it allows these assays to be carried out on a large scale. In vitro cellular infection assays can function as a preliminary screening tool, for vaccine development or antimicrobial screening, and also to extend findings derived from experimental animal trials. Currently, by using culture, it takes up to 4 months to obtain quantifiable results regarding M. avium subsp. paratuberculosis viability after an in vitro infection assay; however, with the quantitative PCR and liquid culture method developed, reliable results can be obtained at 2 weeks. This method will be important for vaccine and antimicrobial screening work, as it will allow a greater number of candidates to be screened in the same amount of time, which will increase the likelihood that a favorable candidate will be found to be subjected to further testing. PMID:27371585

Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library.

PubMed

Koike-Yusa, Hiroko; Li, Yilong; Tan, E-Pien; Velasco-Herrera, Martin Del Castillo; Yusa, Kosuke

2014-03-01

Identification of genes influencing a phenotype of interest is frequently achieved through genetic screening by RNA interference (RNAi) or knockouts. However, RNAi may only achieve partial depletion of gene activity, and knockout-based screens are difficult in diploid mammalian cells. Here we took advantage of the efficiency and high throughput of genome editing based on type II, clustered, regularly interspaced, short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems to introduce genome-wide targeted mutations in mouse embryonic stem cells (ESCs). We designed 87,897 guide RNAs (gRNAs) targeting 19,150 mouse protein-coding genes and used a lentiviral vector to express these gRNAs in ESCs that constitutively express Cas9. Screening the resulting ESC mutant libraries for resistance to either Clostridium septicum alpha-toxin or 6-thioguanine identified 27 known and 4 previously unknown genes implicated in these phenotypes. Our results demonstrate the potential for efficient loss-of-function screening using the CRISPR-Cas9 system.

Machine Learning Approach to Automated Quality Identification of Human Induced Pluripotent Stem Cell Colony Images.

PubMed

Joutsijoki, Henry; Haponen, Markus; Rasku, Jyrki; Aalto-Setälä, Katriina; Juhola, Martti

2016-01-01

The focus of this research is on automated identification of the quality of human induced pluripotent stem cell (iPSC) colony images. iPS cell technology is a contemporary method by which the patient's cells are reprogrammed back to stem cells and are differentiated to any cell type wanted. iPS cell technology will be used in future to patient specific drug screening, disease modeling, and tissue repairing, for instance. However, there are technical challenges before iPS cell technology can be used in practice and one of them is quality control of growing iPSC colonies which is currently done manually but is unfeasible solution in large-scale cultures. The monitoring problem returns to image analysis and classification problem. In this paper, we tackle this problem using machine learning methods such as multiclass Support Vector Machines and several baseline methods together with Scaled Invariant Feature Transformation based features. We perform over 80 test arrangements and do a thorough parameter value search. The best accuracy (62.4%) for classification was obtained by using a k-NN classifier showing improved accuracy compared to earlier studies.

Evaluation of a Screening System for Obesogenic Compounds: Screening of Endocrine Disrupting Compounds and Evaluation of the PPAR Dependency of the Effect

PubMed Central

Pereira-Fernandes, Anna; Demaegdt, Heidi; Vandermeiren, Karine; Hectors, Tine L. M.; Jorens, Philippe G.; Blust, Ronny; Vanparys, Caroline

2013-01-01

Recently the environmental obesogen hypothesis has been formulated, proposing a role for endocrine disrupting compounds (EDCs) in the development of obesity. To evaluate this hypothesis, a screening system for obesogenic compounds is urgently needed. In this study, we suggest a standardised protocol for obesogen screening based on the 3T3-L1 cell line, a well-characterised adipogenesis model, and direct fluorescent measurement using Nile red lipid staining technique. In a first phase, we characterised the assay using the acknowledged obesogens rosiglitazone and tributyltin. Based on the obtained dose-response curves for these model compounds, a lipid accumulation threshold value was calculated to ensure the biological relevance and reliability of statistically significant effects. This threshold based method was combined with the well described strictly standardized mean difference (SSMD) method for classification of non-, weak- or strong obesogenic compounds. In the next step, a range of EDCs, used in personal and household care products (parabens, musks, phthalates and alkylphenol compounds), were tested to further evaluate the obesogenicity screening assay for its discriminative power and sensitivity. Additionally, the peroxisome proliferator activated receptor γ (PPARγ) dependency of the positive compounds was evaluated using PPARγ activation and antagonist experiments. Our results showed the adipogenic potential of all tested parabens, several musks and phthalate compounds and bisphenol A (BPA). PPARγ activation was associated with adipogenesis for parabens, phthalates and BPA, however not required for obesogenic effects induced by Tonalide, indicating the role of other obesogenic mechanisms for this compound. PMID:24155963

High content screening of ToxCast compounds using Vala Sciences’ complex cell culturing systems (SOT)

EPA Science Inventory

US EPA’s ToxCast research program evaluates bioactivity for thousands of chemicals utilizing high-throughput screening assays to inform chemical testing decisions. Vala Sciences provides high content, multiplexed assays that utilize quantitative cell-based digital image analysis....

Microfluidic microscopy-assisted label-free approach for cancer screening: automated microfluidic cytology for cancer screening.

PubMed

Jagannadh, Veerendra Kalyan; Gopakumar, G; Subrahmanyam, Gorthi R K Sai; Gorthi, Sai Siva

2017-05-01

Each year, about 7-8 million deaths occur due to cancer around the world. More than half of the cancer-related deaths occur in the less-developed parts of the world. Cancer mortality rate can be reduced with early detection and subsequent treatment of the disease. In this paper, we introduce a microfluidic microscopy-based cost-effective and label-free approach for identification of cancerous cells. We outline a diagnostic framework for the same and detail an instrumentation layout. We have employed classical computer vision techniques such as 2D principal component analysis-based cell type representation followed by support vector machine-based classification. Analogous to criminal face recognition systems implemented with help of surveillance cameras, a signature-based approach for cancerous cell identification using microfluidic microscopy surveillance is demonstrated. Such a platform would facilitate affordable mass screening camps in the developing countries and therefore help decrease cancer mortality rate.

Human endogenous retrovirus K(HML-2) Gag and Env specific T-cell responses are not detected in HTLV-I-infected subjects using standard peptide screening methods.

PubMed

Jones, R Brad; Leal, Fabio E; Hasenkrug, Aaron M; Segurado, Aluisio C; Nixon, Douglas F; Ostrowski, Mario A; Kallas, Esper G

2013-01-10

An estimated 10-20 million individuals are infected with the retrovirus human T-cell leukemia virus type 1 (HTLV-1). While the majority of these individuals remain asymptomatic, 0.3-4% develop a neurodegenerative inflammatory disease, termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HAM/TSP results in the progressive demyelination of the central nervous system and is a differential diagnosis of multiple sclerosis (MS). The etiology of HAM/TSP is unclear, but evidence points to a role for CNS-inflitrating T-cells in pathogenesis. Recently, the HTLV-1-Tax protein has been shown to induce transcription of the human endogenous retrovirus (HERV) families W, H and K. Intriguingly, numerous studies have implicated these same HERV families in MS, though this association remains controversial. Here, we explore the hypothesis that HTLV-1-infection results in the induction of HERV antigen expression and the elicitation of HERV-specific T-cells responses which, in turn, may be reactive against neurons and other tissues. PBMC from 15 HTLV-1-infected subjects, 5 of whom presented with HAM/TSP, were comprehensively screened for T-cell responses to overlapping peptides spanning HERV-K(HML-2) Gag and Env. In addition, we screened for responses to peptides derived from diverse HERV families, selected based on predicted binding to predicted optimal epitopes. We observed a lack of responses to each of these peptide sets. Thus, although the limited scope of our screening prevents us from conclusively disproving our hypothesis, the current study does not provide data supporting a role for HERV-specific T-cell responses in HTLV-1 associated immunopathology.

Drug screening of cancer cell lines and human primary tumors using droplet microfluidics.

PubMed

Wong, Ada Hang-Heng; Li, Haoran; Jia, Yanwei; Mak, Pui-In; Martins, Rui Paulo da Silva; Liu, Yan; Vong, Chi Man; Wong, Hang Cheong; Wong, Pak Kin; Wang, Haitao; Sun, Heng; Deng, Chu-Xia

2017-08-22

Precision Medicine in Oncology requires tailoring of therapeutic strategies to individual cancer patients. Due to the limited quantity of tumor samples, this proves to be difficult, especially for early stage cancer patients whose tumors are small. In this study, we exploited a 2.4 × 2.4 centimeters polydimethylsiloxane (PDMS) based microfluidic chip which employed droplet microfluidics to conduct drug screens against suspended and adherent cancer cell lines, as well as cells dissociated from primary tumor of human patients. Single cells were dispersed in aqueous droplets and imaged within 24 hours of drug treatment to assess cell viability by ethidium homodimer 1 staining. Our results showed that 5 conditions could be screened for every 80,000 cells in one channel on our chip under current circumstances. Additionally, screening conditions have been adapted to both suspended and adherent cancer cells, giving versatility to potentially all types of cancers. Hence, this study provides a powerful tool for rapid, low-input drug screening of primary cancers within 24 hours after tumor resection from cancer patients. This paves the way for further technological advancement to cutting down sample size and increasing drug screening throughput in advent to personalized cancer therapy.

Overview of online two-dimensional liquid chromatography based on cell membrane chromatography for screening target components from traditional Chinese medicines.

PubMed

Muhammad, Saqib; Han, Shengli; Xie, Xiaoyu; Wang, Sicen; Aziz, Muhammad Majid

2017-01-01

Cell membrane chromatography is a simple, specific, and time-saving technique for studying drug-receptor interactions, screening of active components from complex mixtures, and quality control of traditional Chinese medicines. However, the short column life, low sensitivity, low column efficiency (so cannot resolve satisfactorily mixture of compounds), low peak capacity, and inefficient in structure identification were bottleneck in its application. Combinations of cell membrane chromatography with multidimensional chromatography such as two-dimensional liquid chromatography and high sensitivity detectors like mass have significantly reduced many of the above-mentioned shortcomings. This paper provides an overview of the current advances in online two-dimensional-based cell membrane chromatography for screening target components from traditional Chinese medicines with particular emphasis on the instrumentation, preparation of cell membrane stationary phase, advantages, and disadvantages compared to alternative approaches. The last section of the review summarizes the applications of the online two-dimensional high-performance liquid chromatography based cell membrane chromatography reported since its emergence to date (2010-June 2016). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Seed-effect modeling improves the consistency of genome-wide loss-of-function screens and identifies synthetic lethal vulnerabilities in cancer cells.

PubMed

Jaiswal, Alok; Peddinti, Gopal; Akimov, Yevhen; Wennerberg, Krister; Kuznetsov, Sergey; Tang, Jing; Aittokallio, Tero

2017-06-01

Genome-wide loss-of-function profiling is widely used for systematic identification of genetic dependencies in cancer cells; however, the poor reproducibility of RNA interference (RNAi) screens has been a major concern due to frequent off-target effects. Currently, a detailed understanding of the key factors contributing to the sub-optimal consistency is still a lacking, especially on how to improve the reliability of future RNAi screens by controlling for factors that determine their off-target propensity. We performed a systematic, quantitative analysis of the consistency between two genome-wide shRNA screens conducted on a compendium of cancer cell lines, and also compared several gene summarization methods for inferring gene essentiality from shRNA level data. We then devised novel concepts of seed essentiality and shRNA family, based on seed region sequences of shRNAs, to study in-depth the contribution of seed-mediated off-target effects to the consistency of the two screens. We further investigated two seed-sequence properties, seed pairing stability, and target abundance in terms of their capability to minimize the off-target effects in post-screening data analysis. Finally, we applied this novel methodology to identify genetic interactions and synthetic lethal partners of cancer drivers, and confirmed differential essentiality phenotypes by detailed CRISPR/Cas9 experiments. Using the novel concepts of seed essentiality and shRNA family, we demonstrate how genome-wide loss-of-function profiling of a common set of cancer cell lines can be actually made fairly reproducible when considering seed-mediated off-target effects. Importantly, by excluding shRNAs having higher propensity for off-target effects, based on their seed-sequence properties, one can remove noise from the genome-wide shRNA datasets. As a translational application case, we demonstrate enhanced reproducibility of genetic interaction partners of common cancer drivers, as well as identify novel synthetic lethal partners of a major oncogenic driver, PIK3CA, supported by a complementary CRISPR/Cas9 experiment. We provide practical guidelines for improved design and analysis of genome-wide loss-of-function profiling and demonstrate how this novel strategy can be applied towards improved mapping of genetic dependencies of cancer cells to aid development of targeted anticancer treatments.

High-Throughput Accurate Single-Cell Screening of Euglena gracilis with Fluorescence-Assisted Optofluidic Time-Stretch Microscopy.

PubMed

Guo, Baoshan; Lei, Cheng; Ito, Takuro; Jiang, Yiyue; Ozeki, Yasuyuki; Goda, Keisuke

2016-01-01

The development of reliable, sustainable, and economical sources of alternative fuels is an important, but challenging goal for the world. As an alternative to liquid fossil fuels, algal biofuel is expected to play a key role in alleviating global warming since algae absorb atmospheric CO2 via photosynthesis. Among various algae for fuel production, Euglena gracilis is an attractive microalgal species as it is known to produce wax ester (good for biodiesel and aviation fuel) within lipid droplets. To date, while there exist many techniques for inducing microalgal cells to produce and accumulate lipid with high efficiency, few analytical methods are available for characterizing a population of such lipid-accumulated microalgae including E. gracilis with high throughout, high accuracy, and single-cell resolution simultaneously. Here we demonstrate high-throughput, high-accuracy, single-cell screening of E. gracilis with fluorescence-assisted optofluidic time-stretch microscopy-a method that combines the strengths of microfluidic cell focusing, optical time-stretch microscopy, and fluorescence detection used in conventional flow cytometry. Specifically, our fluorescence-assisted optofluidic time-stretch microscope consists of an optical time-stretch microscope and a fluorescence analyzer on top of a hydrodynamically focusing microfluidic device and can detect fluorescence from every E. gracilis cell in a population and simultaneously obtain its image with a high throughput of 10,000 cells/s. With the multi-dimensional information acquired by the system, we classify nitrogen-sufficient (ordinary) and nitrogen-deficient (lipid-accumulated) E. gracilis cells with a low false positive rate of 1.0%. This method holds promise for evaluating cultivation techniques and selective breeding for microalgae-based biofuel production.

RNAi Screening with Self-Delivering, Synthetic siRNAs for Identification of Genes That Regulate Primary Human T Cell Migration.

PubMed

Freeley, Michael; Derrick, Emily; Dempsey, Eugene; Hoff, Antje; Davies, Anthony; Leake, Devin; Vermeulen, Annaleen; Kelleher, Dermot; Long, Aideen

2015-09-01

Screening of RNA interference (RNAi) libraries in primary T cells is labor-intensive and technically challenging because these cells are hard to transfect. Chemically modified, self-delivering small interfering RNAs (siRNAs) offer a solution to this problem, because they enter hard-to-transfect cell types without needing a delivery reagent and are available in library format for RNAi screening. In this study, we have screened a library of chemically modified, self-delivering siRNAs targeting the expression of 72 distinct genes in conjunction with an image-based high-content-analysis platform as a proof-of-principle strategy to identify genes involved in lymphocyte function-associated antigen-1 (LFA-1)-mediated migration in primary human T cells. Our library-screening strategy identified the small GTPase RhoA as being crucial for T cell polarization and migration in response to LFA-1 stimulation and other migratory ligands. We also demonstrate that multiple downstream assays can be performed within an individual RNAi screen and have used the remainder of the cells for additional assays, including cell viability and adhesion to ICAM-1 (the physiological ligand for LFA-1) in the absence or presence of the chemokine SDF-1α. This study therefore demonstrates the ease and benefits of conducting siRNA library screens in primary human T cells using self-delivering, chemically modified siRNAs, and it emphasizes the feasibility and potential of this approach for elucidating the signaling pathways that regulate T cell function. © 2015 Society for Laboratory Automation and Screening.

High throughput ion-channel pharmacology: planar-array-based voltage clamp.

PubMed

Kiss, Laszlo; Bennett, Paul B; Uebele, Victor N; Koblan, Kenneth S; Kane, Stefanie A; Neagle, Brad; Schroeder, Kirk

2003-02-01

Technological advances often drive major breakthroughs in biology. Examples include PCR, automated DNA sequencing, confocal/single photon microscopy, AFM, and voltage/patch-clamp methods. The patch-clamp method, first described nearly 30 years ago, was a major technical achievement that permitted voltage-clamp analysis (membrane potential control) of ion channels in most cells and revealed a role for channels in unimagined areas. Because of the high information content, voltage clamp is the best way to study ion-channel function; however, throughput is too low for drug screening. Here we describe a novel breakthrough planar-array-based HT patch-clamp technology developed by Essen Instruments capable of voltage-clamping thousands of cells per day. This technology provides greater than two orders of magnitude increase in throughput compared with the traditional voltage-clamp techniques. We have applied this method to study the hERG K(+) channel and to determine the pharmacological profile of QT prolonging drugs.

A methodological overview on molecular preimplantation genetic diagnosis and screening: a genomic future?

PubMed

Vendrell, Xavier; Bautista-Llácer, Rosa

2012-12-01

The genetic diagnosis and screening of preimplantation embryos generated by assisted reproduction technology has been consolidated in the prenatal care framework. The rapid evolution of DNA technologies is tending to molecular approaches. Our intention is to present a detailed methodological view, showing different diagnostic strategies based on molecular techniques that are currently applied in preimplantation genetic diagnosis. The amount of DNA from one single, or a few cells, obtained by embryo biopsy is a limiting factor for the molecular analysis. In this sense, genetic laboratories have developed molecular protocols considering this restrictive condition. Nevertheless, the development of whole-genome amplification methods has allowed preimplantation genetic diagnosis for two or more indications simultaneously, like the selection of histocompatible embryos plus detection of monogenic diseases or aneuploidies. Moreover, molecular techniques have permitted preimplantation genetic screening to progress, by implementing microarray-based comparative genome hybridization. Finally, a future view of the embryo-genetics field based on molecular advances is proposed. The normalization, cost-effectiveness analysis, and new technological tools are the next topics for preimplantation genetic diagnosis and screening. Concomitantly, these additions to assisted reproduction technologies could have a positive effect on the schedules of preimplantation studies.

Antibody-based detection of protein phosphorylation status to track the efficacy of novel therapies using nanogram protein quantities from stem cells and cell lines.

PubMed

Aspinall-O'Dea, Mark; Pierce, Andrew; Pellicano, Francesca; Williamson, Andrew J; Scott, Mary T; Walker, Michael J; Holyoake, Tessa L; Whetton, Anthony D

2015-01-01

This protocol describes a highly reproducible antibody-based method that provides protein level and phosphorylation status information from nanogram quantities of protein cell lysate. Nanocapillary isoelectric focusing (cIEF) combines with UV-activated linking chemistry to detect changes in phosphorylation status. As an example application, we describe how to detect changes in response to tyrosine kinase inhibitors (TKIs) in the phosphorylation status of the adaptor protein CrkL, a major substrate of the oncogenic tyrosine kinase BCR-ABL in chronic myeloid leukemia (CML), using highly enriched CML stem cells and mature cell populations in vitro. This protocol provides a 2.5 pg/nl limit of protein detection (<0.2% of a stem cell sample containing <10(4) cells). Additional assays are described for phosphorylated tyrosine 207 (pTyr207)-CrkL and the protein tyrosine phosphatase PTPRC/CD45; these assays were developed using this protocol and applied to CML patient samples. This method is of high throughput, and it can act as a screen for in vitro cancer stem cell response to drugs and novel agents.

Advance in phage display technology for bioanalysis.

PubMed

Tan, Yuyu; Tian, Tian; Liu, Wenli; Zhu, Zhi; J Yang, Chaoyong

2016-06-01

Phage display technology has emerged as a powerful tool for target gene expression and target-specific ligand selection. It is widely used to screen peptides, proteins and antibodies with the advantages of simplicity, high efficiency and low cost. A variety of targets, including ions, small molecules, inorganic materials, natural and biological polymers, nanostructures, cells, bacteria, and even tissues, have been demonstrated to generate specific binding ligands by phage display. Phages and target-specific ligands screened by phage display have been widely used as affinity reagents in therapeutics, diagnostics and biosensors. In this review, comparisons of different types of phage display systems are first presented. Particularly, microfluidic-based phage display, which enables screening with high throughput, high efficiency and integration, is highlighted. More importantly, we emphasize the advances in biosensors based on phages or phage-derived probes, including nonlytic phages, lytic phages, peptides or proteins screened by phage display, phage assemblies and phage-nanomaterial complexes. However, more efficient and higher throughput phage display methods are still needed to meet an explosion in demand for bioanalysis. Furthermore, screening of cyclic peptides and functional peptides will be the hotspot in bioanalysis. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of Small Molecule Inhibitors of Clostridium perfringens ε-Toxin Cytotoxicity Using a Cell-Based High-Throughput Screen.

PubMed

Lewis, Michelle; Weaver, Charles David; McClain, Mark S

2010-07-01

The Clostridium perfringens epsilon toxin, a select agent, is responsible for a severe, often fatal enterotoxemia characterized by edema in the heart, lungs, kidney, and brain. The toxin is believed to be an oligomeric pore-forming toxin. Currently, there is no effective therapy for countering the cytotoxic activity of the toxin in exposed individuals. Using a robust cell-based high-throughput screening (HTS) assay, we screened a 151,616-compound library for the ability to inhibit ε-toxin-induced cytotoxicity. Survival of MDCK cells exposed to the toxin was assessed by addition of resazurin to detect metabolic activity in surviving cells. The hit rate for this screen was 0.6%. Following a secondary screen of each hit in triplicate and assays to eliminate false positives, we focused on three structurally-distinct compounds: an N-cycloalkylbenzamide, a furo[2,3-b]quinoline, and a 6H-anthra[1,9-cd]isoxazol. None of the three compounds appeared to inhibit toxin binding to cells or the ability of the toxin to form oligomeric complexes. Additional assays demonstrated that two of the inhibitory compounds inhibited ε-toxin-induced permeabilization of MDCK cells to propidium iodide. Furthermore, the two compounds exhibited inhibitory effects on cells pre-treated with toxin. Structural analogs of one of the inhibitors identified through the high-throughput screen were analyzed and provided initial structure-activity data. These compounds should serve as the basis for further structure-activity refinement that may lead to the development of effective anti-ε-toxin therapeutics.

Identification of Small Molecule Inhibitors of Clostridium perfringens ε-Toxin Cytotoxicity Using a Cell-Based High-Throughput Screen

PubMed Central

Lewis, Michelle; Weaver, Charles David; McClain, Mark S.

2010-01-01

The Clostridium perfringens epsilon toxin, a select agent, is responsible for a severe, often fatal enterotoxemia characterized by edema in the heart, lungs, kidney, and brain. The toxin is believed to be an oligomeric pore-forming toxin. Currently, there is no effective therapy for countering the cytotoxic activity of the toxin in exposed individuals. Using a robust cell-based high-throughput screening (HTS) assay, we screened a 151,616-compound library for the ability to inhibit ε-toxin-induced cytotoxicity. Survival of MDCK cells exposed to the toxin was assessed by addition of resazurin to detect metabolic activity in surviving cells. The hit rate for this screen was 0.6%. Following a secondary screen of each hit in triplicate and assays to eliminate false positives, we focused on three structurally-distinct compounds: an N-cycloalkylbenzamide, a furo[2,3-b]quinoline, and a 6H-anthra[1,9-cd]isoxazol. None of the three compounds appeared to inhibit toxin binding to cells or the ability of the toxin to form oligomeric complexes. Additional assays demonstrated that two of the inhibitory compounds inhibited ε-toxin-induced permeabilization of MDCK cells to propidium iodide. Furthermore, the two compounds exhibited inhibitory effects on cells pre-treated with toxin. Structural analogs of one of the inhibitors identified through the high-throughput screen were analyzed and provided initial structure-activity data. These compounds should serve as the basis for further structure-activity refinement that may lead to the development of effective anti-ε-toxin therapeutics. PMID:20721308

Cell and small animal models for phenotypic drug discovery.

PubMed

Szabo, Mihaly; Svensson Akusjärvi, Sara; Saxena, Ankur; Liu, Jianping; Chandrasekar, Gayathri; Kitambi, Satish S

2017-01-01

The phenotype-based drug discovery (PDD) approach is re-emerging as an alternative platform for drug discovery. This review provides an overview of the various model systems and technical advances in imaging and image analyses that strengthen the PDD platform. In PDD screens, compounds of therapeutic value are identified based on the phenotypic perturbations produced irrespective of target(s) or mechanism of action. In this article, examples of phenotypic changes that can be detected and quantified with relative ease in a cell-based setup are discussed. In addition, a higher order of PDD screening setup using small animal models is also explored. As PDD screens integrate physiology and multiple signaling mechanisms during the screening process, the identified hits have higher biomedical applicability. Taken together, this review highlights the advantages gained by adopting a PDD approach in drug discovery. Such a PDD platform can complement target-based systems that are currently in practice to accelerate drug discovery.

Integrated Magneto-Electrochemical Sensor for Exosome Analysis.

PubMed

Jeong, Sangmoo; Park, Jongmin; Pathania, Divya; Castro, Cesar M; Weissleder, Ralph; Lee, Hakho

2016-02-23

Extracellular vesicles, including exosomes, are nanoscale membrane particles that carry molecular information on parental cells. They are being pursued as biomarkers of cancers that are difficult to detect or serially follow. Here we present a compact sensor technology for rapid, on-site exosome screening. The sensor is based on an integrated magneto-electrochemical assay: exosomes are immunomagnetically captured from patient samples and profiled through electrochemical reaction. By combining magnetic enrichment and enzymatic amplification, the approach enables (i) highly sensitive, cell-specific exosome detection and (ii) sensor miniaturization and scale-up for high-throughput measurements. As a proof-of-concept, we implemented a portable, eight-channel device and applied it to screen extracellular vesicles in plasma samples from ovarian cancer patients. The sensor allowed for the simultaneous profiling of multiple protein markers within an hour, outperforming conventional methods in assay sensitivity and speed.

Integrated Magneto-Electrochemical Sensor for Exosome Analysis

PubMed Central

Jeong, Sangmoo; Park, Jongmin; Pathania, Divya; Castro, Cesar M.; Weissleder, Ralph; Lee, Hakho

2016-01-01

Extracellular vesicles, including exosomes, are nanoscale vesicles that carry molecular information of parental cells. They are being pursued as biomarkers of cancers that are difficult to detect or serially follow. Here we present a compact sensor technology for rapid, on-site exosome screening. The sensor is based on an integrated magnetic-electrochemical assay: exosomes are immunomagnetically captured from patient samples, and profiled through electrochemical reaction. By combining magnetic enrichment and enzymatic amplification, the approach enables i) highly sensitive, cell-specific exosome detection, and ii) sensor miniaturization and scale-up for high throughput measurements. As a proof-of-concept, we implemented a portable, eight-channel device, and applied it to screen extracellular vesicles in plasma samples from ovarian cancer patients. The sensor allowed for the profiling of multiple protein markers simultaneously within an hour, outperforming conventional methods in assay sensitivity and speed. PMID:26808216

Adapting CRISPR/Cas9 for functional genomics screens.

PubMed

Malina, Abba; Katigbak, Alexandra; Cencic, Regina; Maïga, Rayelle Itoua; Robert, Francis; Miura, Hisashi; Pelletier, Jerry

2014-01-01

The use of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) for targeted genome editing has been widely adopted and is considered a "game changing" technology. The ease and rapidity by which this approach can be used to modify endogenous loci in a wide spectrum of cell types and organisms makes it a powerful tool for customizable genetic modifications as well as for large-scale functional genomics. The development of retrovirus-based expression platforms to simultaneously deliver the Cas9 nuclease and single guide (sg) RNAs provides unique opportunities by which to ensure stable and reproducible expression of the editing tools and a broad cell targeting spectrum, while remaining compatible with in vivo genetic screens. Here, we describe methods and highlight considerations for designing and generating sgRNA libraries in all-in-one retroviral vectors for such applications.

Prevalence of Sickle Cell Trait in the Southern Suburb of Beirut, Lebanon.

PubMed

El Ariss, Abdel Badih; Younes, Mohamad; Matar, Jad; Berjaoui, Zeina

2016-01-01

The objective of this study was to assess the prevalence, gender differences, and time trends of Sickle Cell Trait in the Southern Suburb of Beirut, Lebanon, as well as to highlight the importance of screening for Sickle Cell Trait carriers in this population. Another objective was to describe a new screening technique for Sickle Cell Trait carriers. This was a retrospective cohort study carried out at a private laboratory in the Southern Suburb of Beirut, Lebanon between 2002 and 2014. The sickling test was carried out for each patient using two methods: the classical "sodium metabisulfite sickling test", and the new "sickling test method" used in the private lab. As a confirmatory test, hemoglobin electrophoresis was run on a random sample of 223 cases which were found to be positive using the two sickling tests. A total of 899 cases were found to be positive for the sickle cell trait out of 184,105 subjects screened during the 12-year period, prevalence = 0.49% (95% CI: 0.46 - 0.52). Among the total sample, females were found to have higher prevalence, where no time trend over the studied period was noted. The haemoglobin electrophoresis method confirmed the results of this new sickling test technique among the random sample of the 223 cases. We found that the prevalence of sickle cell trait is lower as compared to other Arab countries, higher in females, with no significant time trend. The sickle cell test was found to be an accurate, simple and cheap test that could be easily added as a requirement for the pre-marital testing to screen for Sickle Cell Trait carriers.

Prevalence of Sickle Cell Trait in the Southern Suburb of Beirut, Lebanon

PubMed Central

El Ariss, Abdel Badih; Younes, Mohamad; Matar, Jad; Berjaoui, Zeina

2016-01-01

Objective The objective of this study was to assess the prevalence, gender differences, and time trends of Sickle Cell Trait in the Southern Suburb of Beirut, Lebanon, as well as to highlight the importance of screening for Sickle Cell Trait carriers in this population. Another objective was to describe a new screening technique for Sickle Cell Trait carriers. Methods This was a retrospective cohort study carried out at a private laboratory in the Southern Suburb of Beirut, Lebanon between 2002 and 2014. The sickling test was carried out for each patient using two methods: the classical “sodium metabisulfite sickling test”, and the new “sickling test method” used in the private lab. As a confirmatory test, hemoglobin electrophoresis was run on a random sample of 223 cases which were found to be positive using the two sickling tests. Results A total of 899 cases were found to be positive for the sickle cell trait out of 184,105 subjects screened during the 12-year period, prevalence = 0.49% (95% CI: 0.46 – 0.52). Among the total sample, females were found to have higher prevalence, where no time trend over the studied period was noted. The haemoglobin electrophoresis method confirmed the results of this new sickling test technique among the random sample of the 223 cases. Conclusion We found that the prevalence of sickle cell trait is lower as compared to other Arab countries, higher in females, with no significant time trend. The sickle cell test was found to be an accurate, simple and cheap test that could be easily added as a requirement for the pre-marital testing to screen for Sickle Cell Trait carriers. PMID:26977274

Rapid contrast evaluation method based on affinity beads and backscattered electron imaging for the screening of electron stains.

PubMed

Kaku, Hiroki; Inoue, Kanako; Muranaka, Yoshinori; Park, Pyoyun; Ikeda, Kenichi

2015-10-01

Uranyl salts are toxic and radioactive; therefore, several studies have been conducted to screen for substitutes of electron stains. In this regard, the contrast evaluation process is time consuming and the results obtained are inconsistent. In this study, we developed a novel contrast evaluation method using affinity beads and a backscattered electron image (BSEI), obtained using scanning electron microscopy. The contrast ratios of BSEI in each electron stain treatment were correlated with those of transmission electron microscopic images. The affinity beads bound to cell components independently. Protein and DNA samples were enhanced by image contrast treated with electron stains; however, this was not observed for sugars. Protein-conjugated beads showed an additive effect of image contrast when double-stained with lead. However, additive effect of double staining was not observed in DNA-conjugated beads. The varying chemical properties of oligopeptides showed differences in image contrast when treated with each electron stain. This BSEI-based evaluation method not only enables screening for alternate electron stains, but also helps analyze the underlying mechanisms of electron staining of cellular structures. © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry

PubMed Central

Gaber, Rok; Majerle, Andreja; Jerala, Roman; Benčina, Mojca

2013-01-01

To effectively fight against the human immunodeficiency virus infection/acquired immunodeficiency syndrome (HIV/AIDS) epidemic, ongoing development of novel HIV protease inhibitors is required. Inexpensive high-throughput screening assays are needed to quickly scan large sets of chemicals for potential inhibitors. We have developed a Förster resonance energy transfer (FRET)-based, HIV protease-sensitive sensor using a combination of a fluorescent protein pair, namely mCerulean and mCitrine. Through extensive in vitro characterization, we show that the FRET-HIV sensor can be used in HIV protease screening assays. Furthermore, we have used the FRET-HIV sensor for intracellular quantitative detection of HIV protease activity in living cells, which more closely resembles an actual viral infection than an in vitro assay. We have developed a high-throughput method that employs a ratiometric flow cytometry for analyzing large populations of cells that express the FRET-HIV sensor. The method enables FRET measurement of single cells with high sensitivity and speed and should be used when subpopulation-specific intracellular activity of HIV protease needs to be estimated. In addition, we have used a confocal microscopy sensitized emission FRET technique to evaluate the usefulness of the FRET-HIV sensor for spatiotemporal detection of intracellular HIV protease activity. PMID:24287545

Micropillar arrays as a high-throughput screening platform for therapeutics in multiple sclerosis.

PubMed

Mei, Feng; Fancy, Stephen P J; Shen, Yun-An A; Niu, Jianqin; Zhao, Chao; Presley, Bryan; Miao, Edna; Lee, Seonok; Mayoral, Sonia R; Redmond, Stephanie A; Etxeberria, Ainhoa; Xiao, Lan; Franklin, Robin J M; Green, Ari; Hauser, Stephen L; Chan, Jonah R

2014-08-01

Functional screening for compounds that promote remyelination represents a major hurdle in the development of rational therapeutics for multiple sclerosis. Screening for remyelination is problematic, as myelination requires the presence of axons. Standard methods do not resolve cell-autonomous effects and are not suited for high-throughput formats. Here we describe a binary indicant for myelination using micropillar arrays (BIMA). Engineered with conical dimensions, micropillars permit resolution of the extent and length of membrane wrapping from a single two-dimensional image. Confocal imaging acquired from the base to the tip of the pillars allows for detection of concentric wrapping observed as 'rings' of myelin. The platform is formatted in 96-well plates, amenable to semiautomated random acquisition and automated detection and quantification. Upon screening 1,000 bioactive molecules, we identified a cluster of antimuscarinic compounds that enhance oligodendrocyte differentiation and remyelination. Our findings demonstrate a new high-throughput screening platform for potential regenerative therapeutics in multiple sclerosis.

Flow Cytometry: Impact on Early Drug Discovery.

PubMed

Edwards, Bruce S; Sklar, Larry A

2015-07-01

Modern flow cytometers can make optical measurements of 10 or more parameters per cell at tens of thousands of cells per second and more than five orders of magnitude dynamic range. Although flow cytometry is used in most drug discovery stages, "sip-and-spit" sampling technology has restricted it to low-sample-throughput applications. The advent of HyperCyt sampling technology has recently made possible primary screening applications in which tens of thousands of compounds are analyzed per day. Target-multiplexing methodologies in combination with extended multiparameter analyses enable profiling of lead candidates early in the discovery process, when the greatest numbers of candidates are available for evaluation. The ability to sample small volumes with negligible waste reduces reagent costs, compound usage, and consumption of cells. Improved compound library formatting strategies can further extend primary screening opportunities when samples are scarce. Dozens of targets have been screened in 384- and 1536-well assay formats, predominantly in academic screening lab settings. In concert with commercial platform evolution and trending drug discovery strategies, HyperCyt-based systems are now finding their way into mainstream screening labs. Recent advances in flow-based imaging, mass spectrometry, and parallel sample processing promise dramatically expanded single-cell profiling capabilities to bolster systems-level approaches to drug discovery. © 2015 Society for Laboratory Automation and Screening.

ASSESSMENT OF SYNAPSE FORMATION IN RAT PRIMARY NEURAL CELL CULTURE USING HIGH CONTENT MICROSCOPY.

EPA Science Inventory

Cell-based assays can model neurodevelopmental processes including neurite growth and synaptogenesis, and may be useful for screening and evaluation of large numbers of chemicals for developmental neurotoxicity. This work describes the use of high content screening (HCS) to dete...

Niche-based screening identifies small-molecule inhibitors of leukemia stem cells.

PubMed

Hartwell, Kimberly A; Miller, Peter G; Mukherjee, Siddhartha; Kahn, Alissa R; Stewart, Alison L; Logan, David J; Negri, Joseph M; Duvet, Mildred; Järås, Marcus; Puram, Rishi; Dancik, Vlado; Al-Shahrour, Fatima; Kindler, Thomas; Tothova, Zuzana; Chattopadhyay, Shrikanta; Hasaka, Thomas; Narayan, Rajiv; Dai, Mingji; Huang, Christina; Shterental, Sebastian; Chu, Lisa P; Haydu, J Erika; Shieh, Jae Hung; Steensma, David P; Munoz, Benito; Bittker, Joshua A; Shamji, Alykhan F; Clemons, Paul A; Tolliday, Nicola J; Carpenter, Anne E; Gilliland, D Gary; Stern, Andrew M; Moore, Malcolm A S; Scadden, David T; Schreiber, Stuart L; Ebert, Benjamin L; Golub, Todd R

2013-12-01

Efforts to develop more effective therapies for acute leukemia may benefit from high-throughput screening systems that reflect the complex physiology of the disease, including leukemia stem cells (LSCs) and supportive interactions with the bone marrow microenvironment. The therapeutic targeting of LSCs is challenging because LSCs are highly similar to normal hematopoietic stem and progenitor cells (HSPCs) and are protected by stromal cells in vivo. We screened 14,718 compounds in a leukemia-stroma co-culture system for inhibition of cobblestone formation, a cellular behavior associated with stem-cell function. Among those compounds that inhibited malignant cells but spared HSPCs was the cholesterol-lowering drug lovastatin. Lovastatin showed anti-LSC activity in vitro and in an in vivo bone marrow transplantation model. Mechanistic studies demonstrated that the effect was on target, via inhibition of HMG-CoA reductase. These results illustrate the power of merging physiologically relevant models with high-throughput screening.

Niche-based screening identifies small-molecule inhibitors of leukemia stem cells

PubMed Central

Mukherjee, Siddhartha; Kahn, Alissa R; Stewart, Alison L; Logan, David J; Negri, Joseph M; Duvet, Mildred; Järås, Marcus; Puram, Rishi; Dancik, Vlado; Al-Shahrour, Fatima; Kindler, Thomas; Tothova, Zuzana; Chattopadhyay, Shrikanta; Hasaka, Thomas; Narayan, Rajiv; Dai, Mingji; Huang, Christina; Shterental, Sebastian; Chu, Lisa P; Haydu, J Erika; Shieh, Jae Hung; Steensma, David P; Munoz, Benito; Bittker, Joshua A; Shamji, Alykhan F; Clemons, Paul A; Tolliday, Nicola J; Carpenter, Anne E; Gilliland, D Gary; Stern, Andrew M; Moore, Malcolm A S; Scadden, David T; Schreiber, Stuart L; Ebert, Benjamin L; Golub, Todd R

2014-01-01

Efforts to develop more effective therapies for acute leukemia may benefit from high-throughput screening systems that reflect the complex physiology of the disease, including leukemia stem cells (LSCs) and supportive interactions with the bone-marrow microenvironment. The therapeutic targeting of LSCs is challenging because LSCs are highly similar to normal hematopoietic stem and progenitor cells (HSPCs) and are protected by stromal cells in vivo. We screened 14,718 compounds in a leukemia-stroma co-culture system for inhibition of cobblestone formation, a cellular behavior associated with stem-cell function. Among those that inhibited malignant cells but spared HSPCs was the cholesterol-lowering drug lovastatin. Lovastatin showed anti-LSC activity in vitro and in an in vivo bone marrow transplantation model. Mechanistic studies demonstrated that the effect was on-target, via inhibition of HMGCoA reductase. These results illustrate the power of merging physiologically-relevant models with high-throughput screening. PMID:24161946

Novel flowcytometry-based approach of malignant cell detection in body fluids using an automated hematology analyzer

PubMed Central

Tabe, Yoko; Takemura, Hiroyuki; Kimura, Konobu; Takahashi, Toshihiro; Yang, Haeun; Tsuchiya, Koji; Konishi, Aya; Uchihashi, Kinya; Horii, Takashi; Ohsaka, Akimichi

2018-01-01

Morphological microscopic examinations of nucleated cells in body fluid (BF) samples are performed to screen malignancy. However, the morphological differentiation is time-consuming and labor-intensive. This study aimed to develop a new flowcytometry-based gating analysis mode “XN-BF gating algorithm” to detect malignant cells using an automated hematology analyzer, Sysmex XN-1000. XN-BF mode was equipped with WDF white blood cell (WBC) differential channel. We added two algorithms to the WDF channel: Rule 1 detects larger and clumped cell signals compared to the leukocytes, targeting the clustered malignant cells; Rule 2 detects middle sized mononuclear cells containing less granules than neutrophils with similar fluorescence signal to monocytes, targeting hematological malignant cells and solid tumor cells. BF samples that meet, at least, one rule were detected as malignant. To evaluate this novel gating algorithm, 92 various BF samples were collected. Manual microscopic differentiation with the May-Grunwald Giemsa stain and WBC count with hemocytometer were also performed. The performance of these three methods were evaluated by comparing with the cytological diagnosis. The XN-BF gating algorithm achieved sensitivity of 63.0% and specificity of 87.8% with 68.0% for positive predictive value and 85.1% for negative predictive value in detecting malignant-cell positive samples. Manual microscopic WBC differentiation and WBC count demonstrated 70.4% and 66.7% of sensitivities, and 96.9% and 92.3% of specificities, respectively. The XN-BF gating algorithm can be a feasible tool in hematology laboratories for prompt screening of malignant cells in various BF samples. PMID:29425230

Novel flowcytometry-based approach of malignant cell detection in body fluids using an automated hematology analyzer.

PubMed

Ai, Tomohiko; Tabe, Yoko; Takemura, Hiroyuki; Kimura, Konobu; Takahashi, Toshihiro; Yang, Haeun; Tsuchiya, Koji; Konishi, Aya; Uchihashi, Kinya; Horii, Takashi; Ohsaka, Akimichi

2018-01-01

Morphological microscopic examinations of nucleated cells in body fluid (BF) samples are performed to screen malignancy. However, the morphological differentiation is time-consuming and labor-intensive. This study aimed to develop a new flowcytometry-based gating analysis mode "XN-BF gating algorithm" to detect malignant cells using an automated hematology analyzer, Sysmex XN-1000. XN-BF mode was equipped with WDF white blood cell (WBC) differential channel. We added two algorithms to the WDF channel: Rule 1 detects larger and clumped cell signals compared to the leukocytes, targeting the clustered malignant cells; Rule 2 detects middle sized mononuclear cells containing less granules than neutrophils with similar fluorescence signal to monocytes, targeting hematological malignant cells and solid tumor cells. BF samples that meet, at least, one rule were detected as malignant. To evaluate this novel gating algorithm, 92 various BF samples were collected. Manual microscopic differentiation with the May-Grunwald Giemsa stain and WBC count with hemocytometer were also performed. The performance of these three methods were evaluated by comparing with the cytological diagnosis. The XN-BF gating algorithm achieved sensitivity of 63.0% and specificity of 87.8% with 68.0% for positive predictive value and 85.1% for negative predictive value in detecting malignant-cell positive samples. Manual microscopic WBC differentiation and WBC count demonstrated 70.4% and 66.7% of sensitivities, and 96.9% and 92.3% of specificities, respectively. The XN-BF gating algorithm can be a feasible tool in hematology laboratories for prompt screening of malignant cells in various BF samples.

Gold Nanoparticles-Based Barcode Analysis for Detection of Norepinephrine.

PubMed

An, Jeung Hee; Lee, Kwon-Jai; Choi, Jeong-Woo

2016-02-01

Nanotechnology-based bio-barcode amplification analysis offers an innovative approach for detecting neurotransmitters. We evaluated the efficacy of this method for detecting norepinephrine in normal and oxidative-stress damaged dopaminergic cells. Our approach use a combination of DNA barcodes and bead-based immunoassays for detecting neurotransmitters with surface-enhanced Raman spectroscopy (SERS), and provides polymerase chain reaction (PCR)-like sensitivity. This method relies on magnetic Dynabeads containing antibodies and nanoparticles that are loaded both with DNA barcords and with antibodies that can sandwich the target protein captured by the Dynabead-bound antibodies. The aggregate sandwich structures are magnetically separated from the solution and treated to remove the conjugated barcode DNA. The DNA barcodes are then identified by SERS and PCR analysis. The concentration of norepinephrine in dopaminergic cells can be readily detected using the bio-barcode assay, which is a rapid, high-throughput screening tool for detecting neurotransmitters.

Triazole-based Zn²⁺-specific molecular marker for fluorescence bioimaging.

PubMed

Sinha, Sougata; Mukherjee, Trinetra; Mathew, Jomon; Mukhopadhyay, Subhra K; Ghosh, Subrata

2014-04-25

Fluorescence bioimaging potential, both in vitro and in vivo, of a yellow emissive triazole-based molecular marker has been investigated and demonstrated. Three different kinds of cells, viz Bacillus thuringiensis, Candida albicans, and Techoma stans pollen grains were used to investigate the intracellular zinc imaging potential of 1 (in vitro studies). Fluorescence imaging of translocation of zinc through the stem of small herb, Peperomia pellucida, having transparent stem proved in vivo bioimaging capability of 1. This approach will enable in screening cell permeability and biostability of a newly developed probe. Similarly, the current method for detection and localization of zinc in Gram seed sprouts could be an easy and potential alternative of the existing analytical methods to investigate the efficiency of various strategies applied for increasing zinc-content in cereal crops. The probe-zinc ensemble has efficiently been applied for detecting phosphate-based biomolecules. Copyright © 2014 Elsevier B.V. All rights reserved.

Cost-Effectiveness of HIV Screening in STD Clinics, Emergency Departments, and Inpatient Units: A Model-Based Analysis

PubMed Central

Prabhu, Vimalanand S.; Farnham, Paul G.; Hutchinson, Angela B.; Soorapanth, Sada; Heffelfinger, James D.; Golden, Matthew R.; Brooks, John T.; Rimland, David; Sansom, Stephanie L.

2011-01-01

Background Identifying and treating persons with human immunodeficiency virus (HIV) infection early in their disease stage is considered an effective means of reducing the impact of the disease. We compared the cost-effectiveness of HIV screening in three settings, sexually transmitted disease (STD) clinics serving men who have sex with men, hospital emergency departments (EDs), settings where patients are likely to be diagnosed early, and inpatient diagnosis based on clinical manifestations. Methods and Findings We developed the Progression and Transmission of HIV/AIDS model, a health state transition model that tracks index patients and their infected partners from HIV infection to death. We used program characteristics for each setting to compare the incremental cost per quality-adjusted life year gained from early versus late diagnosis and treatment. We ran the model for 10,000 index patients for each setting, examining alternative scenarios, excluding and including transmission to partners, and assuming HAART was initiated at a CD4 count of either 350 or 500 cells/µL. Screening in STD clinics and EDs was cost-effective compared with diagnosing inpatients, even when including only the benefits to the index patients. Screening patients in STD clinics, who have less-advanced disease, was cost-effective compared with ED screening when treatment with HAART was initiated at a CD4 count of 500 cells/µL. When the benefits of reduced transmission to partners from early diagnosis were included, screening in settings with less-advanced disease stages was cost-saving compared with screening later in the course of infection. The study was limited by a small number of observations on CD4 count at diagnosis and by including transmission only to first generation partners of the index patients. Conclusions HIV prevention efforts can be advanced by screening in settings where patients present with less-advanced stages of HIV infection and by initiating treatment with HAART earlier in the course of infection. PMID:21625489

A low density microarray method for the identification of human papillomavirus type 18 variants.

PubMed

Meza-Menchaca, Thuluz; Williams, John; Rodríguez-Estrada, Rocío B; García-Bravo, Aracely; Ramos-Ligonio, Ángel; López-Monteon, Aracely; Zepeda, Rossana C

2013-09-26

We describe a novel microarray based-method for the screening of oncogenic human papillomavirus 18 (HPV-18) molecular variants. Due to the fact that sequencing methodology may underestimate samples containing more than one variant we designed a specific and sensitive stacking DNA hybridization assay. This technology can be used to discriminate between three possible phylogenetic branches of HPV-18. Probes were attached covalently on glass slides and hybridized with single-stranded DNA targets. Prior to hybridization with the probes, the target strands were pre-annealed with the three auxiliary contiguous oligonucleotides flanking the target sequences. Screening HPV-18 positive cell lines and cervical samples were used to evaluate the performance of this HPV DNA microarray. Our results demonstrate that the HPV-18's variants hybridized specifically to probes, with no detection of unspecific signals. Specific probes successfully reveal detectable point mutations in these variants. The present DNA oligoarray system can be used as a reliable, sensitive and specific method for HPV-18 variant screening. Furthermore, this simple assay allows the use of inexpensive equipment, making it accessible in resource-poor settings.

A Low Density Microarray Method for the Identification of Human Papillomavirus Type 18 Variants

PubMed Central

Meza-Menchaca, Thuluz; Williams, John; Rodríguez-Estrada, Rocío B.; García-Bravo, Aracely; Ramos-Ligonio, Ángel; López-Monteon, Aracely; Zepeda, Rossana C.

2013-01-01

We describe a novel microarray based-method for the screening of oncogenic human papillomavirus 18 (HPV-18) molecular variants. Due to the fact that sequencing methodology may underestimate samples containing more than one variant we designed a specific and sensitive stacking DNA hybridization assay. This technology can be used to discriminate between three possible phylogenetic branches of HPV-18. Probes were attached covalently on glass slides and hybridized with single-stranded DNA targets. Prior to hybridization with the probes, the target strands were pre-annealed with the three auxiliary contiguous oligonucleotides flanking the target sequences. Screening HPV-18 positive cell lines and cervical samples were used to evaluate the performance of this HPV DNA microarray. Our results demonstrate that the HPV-18's variants hybridized specifically to probes, with no detection of unspecific signals. Specific probes successfully reveal detectable point mutations in these variants. The present DNA oligoarray system can be used as a reliable, sensitive and specific method for HPV-18 variant screening. Furthermore, this simple assay allows the use of inexpensive equipment, making it accessible in resource-poor settings. PMID:24077317

Hydrogel-Based Fluorescent Dual pH and Oxygen Sensors Loaded in 96-Well Plates for High-Throughput Cell Metabolism Studies.

PubMed

Wu, Shanshan; Wu, Siying; Yi, Zheyuan; Zeng, Fei; Wu, Weizhen; Qiao, Yuan; Zhao, Xingzhong; Cheng, Xing; Tian, Yanqing

2018-02-13

In this study, we developed fluorescent dual pH and oxygen sensors loaded in multi-well plates for in-situ and high-throughput monitoring of oxygen respiration and extracellular acidification during microbial cell growth for understanding metabolism. Biocompatible PHEMA-co-PAM materials were used as the hydrogel matrix. A polymerizable oxygen probe (OS2) derived from PtTFPP and a polymerizable pH probe (S2) derived from fluorescein were chemically conjugated into the matrix to solve the problem of the probe leaching from the matrix. Gels were allowed to cure directly on the bottom of 96-well plates at room-temperature via redox polymerization. The influence of matrix's composition on the sensing behaviors was investigated to optimize hydrogels with enough robustness for repeatable use with good sensitivity. Responses of the dual sensing hydrogels to dissolved oxygen (DO) and pH were studied. These dual oxygen-pH sensing plates were successfully used for microbial cell-based screening assays, which are based on the measurement of fluorescence intensity changes induced by cellular oxygen consumption and pH changes during microbial growth. This method may provide a real-time monitoring of cellular respiration, acidification, and a rapid kinetic assessment of multiple samples for cell viability as well as high-throughput drug screening. All of these assays can be carried out by a conventional plate reader.

Immobilized OBOC combinatorial bead array to facilitate multiplicative screening.

PubMed

Xiao, Wenwu; Bononi, Fernanda C; Townsend, Jared; Li, Yuanpei; Liu, Ruiwu; Lam, Kit S

2013-07-01

One-bead-one-compound (OBOC) combinatorial library screening has been broadly utilized for the last two decades to identify small molecules, peptides or peptidomimetics targeting variable screening probes such as cell surface receptors, bacteria, protein kinases, phosphatases, proteases etc. In previous screening methods, library beads were suspended in solution and screened against one single probe. Only the positive beads were tracked and isolated for additional screens and finally selected for chemical decoding. During this process, the remaining negative beads were not tracked and discarded. Here we report a novel bead immobilization method such that a bead library array can be conveniently prepared and screened in its entirety, sequentially many times with a series of distinct probes. This method not only allows us to increase the screening efficiency but also permits us to determine the binding profile of each and every library bead against a large number of target receptors. As proof of concept, we serially screened a random OBOC disulfide containing cyclic heptapeptide library with three water soluble dyes as model probes: malachite green, bromocresol purple and indigo carmine. This multiplicative screening approach resulted in a rapid determination of the binding profile of each and every bead respective to each of the three dyes. Beads that interacted with malachite green only, bromocresol purple only, or both indigo carmine and bromocresol purple were isolated, and their peptide sequences were determined with microsequencer. Ultimately, the novel OBOC multiplicative screening approach could play a key role in the enhancement of existing on-bead assays such as whole cell binding, bacteria binding, protein binding, posttranslational modifications etc. with increased efficiency, capacity, and specificity.

Whole organism high content screening identifies stimulators of pancreatic beta-cell proliferation.

PubMed

Tsuji, Naoki; Ninov, Nikolay; Delawary, Mina; Osman, Sahar; Roh, Alex S; Gut, Philipp; Stainier, Didier Y R

2014-01-01

Inducing beta-cell mass expansion in diabetic patients with the aim to restore glucose homeostasis is a promising therapeutic strategy. Although several in vitro studies have been carried out to identify modulators of beta-cell mass expansion, restoring endogenous beta-cell mass in vivo has yet to be achieved. To identify potential stimulators of beta-cell replication in vivo, we established transgenic zebrafish lines that monitor and allow the quantification of cell proliferation by using the fluorescent ubiquitylation-based cell cycle indicator (FUCCI) technology. Using these new reagents, we performed an unbiased chemical screen, and identified 20 small molecules that markedly increased beta-cell proliferation in vivo. Importantly, these structurally distinct molecules, which include clinically-approved drugs, modulate three specific signaling pathways: serotonin, retinoic acid and glucocorticoids, showing the high sensitivity and robustness of our screen. Notably, two drug classes, retinoic acid and glucocorticoids, also promoted beta-cell regeneration after beta-cell ablation. Thus, this study establishes a proof of principle for a high-throughput small molecule-screen for beta-cell proliferation in vivo, and identified compounds that stimulate beta-cell proliferation and regeneration.

Discovery of potent KIFC1 inhibitors using a method of integrated high-throughput synthesis and screening.

PubMed

Yang, Bin; Lamb, Michelle L; Zhang, Tao; Hennessy, Edward J; Grewal, Gurmit; Sha, Li; Zambrowski, Mark; Block, Michael H; Dowling, James E; Su, Nancy; Wu, Jiaquan; Deegan, Tracy; Mikule, Keith; Wang, Wenxian; Kaspera, Rüdiger; Chuaqui, Claudio; Chen, Huawei

2014-12-11

KIFC1 (HSET), a member of the kinesin-14 family of motor proteins, plays an essential role in centrosomal bundling in cancer cells, but its function is not required for normal diploid cell division. To explore the potential of KIFC1 as a therapeutic target for human cancers, a series of potent KIFC1 inhibitors featuring a phenylalanine scaffold was developed from hits identified through high-throughput screening (HTS). Optimization of the initial hits combined both design-synthesis-test cycles and an integrated high-throughput synthesis and biochemical screening method. An important aspect of this integrated method was the utilization of DMSO stock solutions of compounds registered in the corporate compound collection as synthetic reactants. Using this method, over 1500 compounds selected for structural diversity were quickly assembled in assay-ready 384-well plates and were directly tested after the necessary dilutions. Our efforts led to the discovery of a potent KIFC1 inhibitor, AZ82, which demonstrated the desired centrosome declustering mode of action in cell studies.

[Adult T-cell leukemia/lymphoma associated with unusual positivity of anti-ATLA (adult T-cell leukemia-cell-associated antigen) antibodies].

PubMed

Eto, T; Okamura, H; Okamura, T; Gondo, H; Kudo, J; Shibuya, T; Harada, M; Niho, Y

1990-03-01

A 56-year-old female was admitted because of generalized lymphadenopathy. Based upon histological findings of biopsied lymph node, malignant lymphoma, diffuse large cell type was diagnosed. The surface marker analysis showed that malignant cells were positive for CD4 and CD2 but negative for CD8. Although anti-ATLA (adult T-cell leukemia associated antigen) antibody was negative with the use of a gelatin particle agglutination method (P.A.), other methods such as an indirect immunofluorescence assay (I.F.), an enzyme-linked immunosorbent assay (E.I.A.) and a Western blotting assay revealed the positivity for anti-ATLA antibody. Adult T-cell leukemia/lymphoma (ATL/L) was confirmed by the presence of monoclonal integration of HTLV-I proviral DNA in biopsied specimen. This case, showing a pattern of P.A. (-) and I.F. (+), is extremely unusual, because I.F. and P.A. show highly close correlation. Thus, it is important to employ different methods for screening of anti-ATLA antibodies in the diagnosis of ATL/L.

Chip Based Magnetic Imager for Molecular Profiling of Ovarian Cancer Cells

DTIC Science & Technology

2016-12-01

2015) Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis. Cell 160:1246-1260. PMC4380877, PMID:25748654. Acknowledgement of...Weissleder R, Lee H, Zhang F, Sharp PA (2015) Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis. Cell 160:1246-1260. 5. Im H, Shao H...Lett 32(10):1229–1231. 6 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1501815112 Im et al. Resource Genome-wide CRISPR Screen in a Mouse Model of Tumor

PGD and aneuploidy screening for 24 chromosomes: advantages and disadvantages of competing platforms.

PubMed

Bisignano, A; Wells, D; Harton, G; Munné, S

2011-12-01

Diagnosis of embryos for chromosome abnormalities, i.e. aneuploidy screening, has been invigorated by the introduction of microarray-based testing methods allowing analysis of 24 chromosomes in one test. Recent data have been suggestive of increased implantation and pregnancy rates following microarray testing. Preimplantation genetic diagnosis for infertility aims to test for gross chromosome changes with the hope that identification and transfer of normal embryos will improve IVF outcomes. Testing by some methods, specifically single-nucleotide polymorphism (SNP) microarrays, allow for more information and potential insight into parental origin of aneuploidy and uniparental disomy. The usefulness and validity of reporting this information is flawed. Numerous papers have shown that the majority of meiotic errors occur in the egg, while mitotic errors in the embryo affect parental chromosomes at random. Potential mistakes made in assigning an error as meiotic or mitotic may lead to erroneous reporting of results with medical consequences. This study's data suggest that the bioinformatic cleaning used to 'fix' the miscalls that plague single-cell whole-genome amplification provides little improvement in the quality of useful data. Based on the information available, SNP-based aneuploidy screening suffers from a number of serious issues that must be resolved. Copyright © 2011 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Utilizing high throughput screening data for predictive toxicology models: protocols and application to MLSCN assays

NASA Astrophysics Data System (ADS)

Guha, Rajarshi; Schürer, Stephan C.

2008-06-01

Computational toxicology is emerging as an encouraging alternative to experimental testing. The Molecular Libraries Screening Center Network (MLSCN) as part of the NIH Molecular Libraries Roadmap has recently started generating large and diverse screening datasets, which are publicly available in PubChem. In this report, we investigate various aspects of developing computational models to predict cell toxicity based on cell proliferation screening data generated in the MLSCN. By capturing feature-based information in those datasets, such predictive models would be useful in evaluating cell-based screening results in general (for example from reporter assays) and could be used as an aid to identify and eliminate potentially undesired compounds. Specifically we present the results of random forest ensemble models developed using different cell proliferation datasets and highlight protocols to take into account their extremely imbalanced nature. Depending on the nature of the datasets and the descriptors employed we were able to achieve percentage correct classification rates between 70% and 85% on the prediction set, though the accuracy rate dropped significantly when the models were applied to in vivo data. In this context we also compare the MLSCN cell proliferation results with animal acute toxicity data to investigate to what extent animal toxicity can be correlated and potentially predicted by proliferation results. Finally, we present a visualization technique that allows one to compare a new dataset to the training set of the models to decide whether the new dataset may be reliably predicted.

Screening and identification of genetic loci involved in producing more/denser inclusion bodies in Escherichia coli

PubMed Central

2013-01-01

Background Many proteins and peptides have been used in therapeutic or industrial applications. They are often produced in microbial production hosts by fermentation. Robust protein production in the hosts and efficient downstream purification are two critical factors that could significantly reduce cost for microbial protein production by fermentation. Producing proteins/peptides as inclusion bodies in the hosts has the potential to achieve both high titers in fermentation and cost-effective downstream purification. Manipulation of the host cells such as overexpression/deletion of certain genes could lead to producing more and/or denser inclusion bodies. However, there are limited screening methods to help to identify beneficial genetic changes rendering more protein production and/or denser inclusion bodies. Results We report development and optimization of a simple density gradient method that can be used for distinguishing and sorting E. coli cells with different buoyant densities. We demonstrate utilization of the method to screen genetic libraries to identify a) expression of glyQS loci on plasmid that increased expression of a peptide of interest as well as the buoyant density of inclusion body producing E. coli cells; and b) deletion of a host gltA gene that increased the buoyant density of the inclusion body produced in the E. coli cells. Conclusion A novel density gradient sorting method was developed to screen genetic libraries. Beneficial host genetic changes could be exploited to improve recombinant protein expression as well as downstream protein purification. PMID:23638724

Screening and identification of genetic loci involved in producing more/denser inclusion bodies in Escherichia coli.

PubMed

Pandey, Neeraj; Sachan, Annapurna; Chen, Qi; Ruebling-Jass, Kristin; Bhalla, Ritu; Panguluri, Kiran Kumar; Rouviere, Pierre E; Cheng, Qiong

2013-05-02

Many proteins and peptides have been used in therapeutic or industrial applications. They are often produced in microbial production hosts by fermentation. Robust protein production in the hosts and efficient downstream purification are two critical factors that could significantly reduce cost for microbial protein production by fermentation. Producing proteins/peptides as inclusion bodies in the hosts has the potential to achieve both high titers in fermentation and cost-effective downstream purification. Manipulation of the host cells such as overexpression/deletion of certain genes could lead to producing more and/or denser inclusion bodies. However, there are limited screening methods to help to identify beneficial genetic changes rendering more protein production and/or denser inclusion bodies. We report development and optimization of a simple density gradient method that can be used for distinguishing and sorting E. coli cells with different buoyant densities. We demonstrate utilization of the method to screen genetic libraries to identify a) expression of glyQS loci on plasmid that increased expression of a peptide of interest as well as the buoyant density of inclusion body producing E. coli cells; and b) deletion of a host gltA gene that increased the buoyant density of the inclusion body produced in the E. coli cells. A novel density gradient sorting method was developed to screen genetic libraries. Beneficial host genetic changes could be exploited to improve recombinant protein expression as well as downstream protein purification.

A High Content Screening (HCS) Assay for the Identification of Chemical Inducers of PML Oncogenic Domains (PODs)

PubMed Central

Yip, Kenneth W.; Cuddy, Michael; Pinilla, Clemencia; Giulanotti, Marc; Heynen-Genel, Susanne; Matsuzawa, Shu-ichi; Reed, John C.

2014-01-01

PML is a tumor suppressor that promotes apoptosis through both p53-dependent and - independent mechanisms, participates in Rb-mediated cell cycle arrest, inhibits neoangiogenesis, and contributes to maintenance of genomic stability. PML also plays a role in host defense against viruses, conferring antiviral activity. When active, PML localizes to subnuclear structures named PML oncogenic domains (PODs) or PML nuclear bodies (PML-NBs), whereas inactive PML is located diffusely throughout the nucleus of cells, thus providing a morphological indicator. Known activators of PML include arsenicals and interferons, however, these agents induce a plethora of toxic effects, limiting their effectiveness. The objective of the current study was to develop a high content screening (HCS) assay for the identification of chemical activators of PML. We describe methods for automated analysis of POD formation using high throughput microscopy (HTM) to localize PML immunofluorescence in conjunction with image analysis software for POD quantification. Using this HCS assay in 384 well format, we performed pilot screens of a small synthetic chemical library and mixture-based combinatorial libraries, demonstrating the robust performance of the assay. HCS counter-screening assays were also developed for hit characterization, based on immunofluorescence analyses of the subcellular location of phosphorylated H2AX or phosphorylated CHK1, which increase in a punctate nuclear pattern in response to DNA damage. Thus, the HCS assay devised here represents a high throughput screen that can be utilized to discover POD-inducing compounds that may restore the tumor suppressor activity of PML in cancers or possibly promote anti-viral states. PMID:21233309

Single gene-based distinction of individual microbial genomes from a mixed population of microbial cells.

PubMed

Tamminen, Manu V; Virta, Marko P J

2015-01-01

Recent progress in environmental microbiology has revealed vast populations of microbes in any given habitat that cannot be detected by conventional culturing strategies. The use of sensitive genetic detection methods such as CARD-FISH and in situ PCR have been limited by the cell wall permeabilization requirement that cannot be performed similarly on all cell types without lysing some and leaving some nonpermeabilized. Furthermore, the detection of low copy targets such as genes present in single copies in the microbial genomes, has remained problematic. We describe an emulsion-based procedure to trap individual microbial cells into picoliter-volume polyacrylamide droplets that provide a rigid support for genetic material and therefore allow complete degradation of cellular material to expose the individual genomes. The polyacrylamide droplets are subsequently converted into picoliter-scale reactors for genome amplification. The amplified genomes are labeled based on the presence of a target gene and differentiated from those that do not contain the gene by flow cytometry. Using the Escherichia coli strains XL1 and MC1061, which differ with respect to the presence (XL1), or absence (MC1061) of a single copy of a tetracycline resistance gene per genome, we demonstrate that XL1 genomes present at 0.1% of MC1061 genomes can be differentiated using this method. Using a spiked sediment microbial sample, we demonstrate that the method is applicable to highly complex environmental microbial communities as a target gene-based screen for individual microbes. The method provides a novel tool for enumerating functional cell populations in complex microbial communities. We envision that the method could be optimized for fluorescence-activated cell sorting to enrich genetic material of interest from complex environmental samples.

siMacro: A Fast and Easy Data Processing Tool for Cell-Based Genomewide siRNA Screens.

PubMed

Singh, Nitin Kumar; Seo, Bo Yeun; Vidyasagar, Mathukumalli; White, Michael A; Kim, Hyun Seok

2013-03-01

Growing numbers of studies employ cell line-based systematic short interfering RNA (siRNA) screens to study gene functions and to identify drug targets. As multiple sources of variations that are unique to siRNA screens exist, there is a growing demand for a computational tool that generates normalized values and standardized scores. However, only a few tools have been available so far with limited usability. Here, we present siMacro, a fast and easy-to-use Microsoft Office Excel-based tool with a graphic user interface, designed to process single-condition or two-condition synthetic screen datasets. siMacro normalizes position and batch effects, censors outlier samples, and calculates Z-scores and robust Z-scores, with a spreadsheet output of >120,000 samples in under 1 minute.

siMacro: A Fast and Easy Data Processing Tool for Cell-Based Genomewide siRNA Screens

PubMed Central

Singh, Nitin Kumar; Seo, Bo Yeun; Vidyasagar, Mathukumalli; White, Michael A.

2013-01-01

Growing numbers of studies employ cell line-based systematic short interfering RNA (siRNA) screens to study gene functions and to identify drug targets. As multiple sources of variations that are unique to siRNA screens exist, there is a growing demand for a computational tool that generates normalized values and standardized scores. However, only a few tools have been available so far with limited usability. Here, we present siMacro, a fast and easy-to-use Microsoft Office Excel-based tool with a graphic user interface, designed to process single-condition or two-condition synthetic screen datasets. siMacro normalizes position and batch effects, censors outlier samples, and calculates Z-scores and robust Z-scores, with a spreadsheet output of >120,000 samples in under 1 minute. PMID:23613684

Target Abundance-Based Fitness Screening (TAFiS) Facilitates Rapid Identification of Target-Specific and Physiologically Active Chemical Probes

PubMed Central

Butts, Arielle; DeJarnette, Christian; Peters, Tracy L.; Parker, Josie E.; Kerns, Morgan E.; Eberle, Karen E.; Kelly, Steve L.

2017-01-01

ABSTRACT Traditional approaches to drug discovery are frustratingly inefficient and have several key limitations that severely constrain our capacity to rapidly identify and develop novel experimental therapeutics. To address this, we have devised a second-generation target-based whole-cell screening assay based on the principles of competitive fitness, which can rapidly identify target-specific and physiologically active compounds. Briefly, strains expressing high, intermediate, and low levels of a preselected target protein are constructed, tagged with spectrally distinct fluorescent proteins (FPs), and pooled. The pooled strains are then grown in the presence of various small molecules, and the relative growth of each strain within the mixed culture is compared by measuring the intensity of the corresponding FP tags. Chemical-induced population shifts indicate that the bioactivity of a small molecule is dependent upon the target protein’s abundance and thus establish a specific functional interaction. Here, we describe the molecular tools required to apply this technique in the prevalent human fungal pathogen Candida albicans and validate the approach using two well-characterized drug targets—lanosterol demethylase and dihydrofolate reductase. However, our approach, which we have termed target abundance-based fitness screening (TAFiS), should be applicable to a wide array of molecular targets and in essentially any genetically tractable microbe. IMPORTANCE Conventional drug screening typically employs either target-based or cell-based approaches. The first group relies on biochemical assays to detect modulators of a purified target. However, hits frequently lack drug-like characteristics such as membrane permeability and target specificity. Cell-based screens identify compounds that induce a desired phenotype, but the target is unknown, which severely restricts further development and optimization. To address these issues, we have developed a second-generation target-based whole-cell screening approach that incorporates the principles of both chemical genetics and competitive fitness, which enables the identification of target-specific and physiologically active compounds from a single screen. We have chosen to validate this approach using the important human fungal pathogen Candida albicans with the intention of pursuing novel antifungal targets. However, this approach is broadly applicable and is expected to dramatically reduce the time and resources required to progress from screening hit to lead compound. PMID:28989971

Discovery of potent DOT1L inhibitors by AlphaLISA based High Throughput Screening assay.

PubMed

Song, Yakai; Li, Linjuan; Chen, Yantao; Liu, Jingqiu; Xiao, Senhao; Lian, Fulin; Zhang, Naixia; Ding, Hong; Zhang, Yuanyuan; Chen, Kaixian; Jiang, Hualiang; Zhang, Chenhua; Liu, Yu-Chih; Chen, Shijie; Luo, Cheng

2018-05-01

DOT1L (the disruptor of telomeric silencing 1-like), through its methyltransferase activity of H3K79, plays essential roles in transcriptional regulation, cell cycle regulation, and DNA damage response. In addition, DOT1L is believed to be involved in the development of MLL-rearranged leukemia driven by the MLL (mixed-lineage leukemia) fusion proteins, which thus to be a crucial target for leukemia therapy. Hence, discovering of novel DOT1L inhibitors has been in a great demand. In this study, we initiated the discovering process from setting up the AlphaLISA based High Throughput Screening (HTS) assay of DOT1L. Combining with radioactive inhibition assay and Surface Plasmon Resonance (SPR) binding assay, we identified compound 3 and its active analogues as novel DOT1L inhibitors with IC 50 values range from 7 μM to 20 μM in vitro. Together with the analysis of structure activity relationships (SAR) and binding modes of these compounds, we provided clues to assist in the future development of more potent DOT1L inhibitors. Moreover, compounds 3 and 9 effectively inhibited the proliferation of MLL-rearranged leukemia cells MV4-11, which could induce cell cycle arrest and apoptosis. In conclusion, we developed a HTS platform based on AlphaLISA method for screening and discovery of DOT1L novel inhibitor, through which we discovered compound 3 and its analogues as potent DOT1L inhibitors with promising MLL-rearranged leukemia therapeutic application. Copyright © 2018 Elsevier Ltd. All rights reserved.

Kinetic assay for high-throughput screening of in vitro transthyretin amyloid fibrillogenesis inhibitors.

PubMed

Dolado, Ignacio; Nieto, Joan; Saraiva, Maria João M; Arsequell, Gemma; Valencia, Gregori; Planas, Antoni

2005-01-01

Stabilization of tetrameric transthyretin (TTR) by binding of small ligands is a current strategy aimed at inhibiting amyloid fibrillogenesis in transthyretin-associated pathologies, such as senile systemic amyloidosis (SSA) and familial amyloidotic polyneuropathy (FAP). A kinetic assay is developed for rapid evaluation of compounds as potential in vitro inhibitors in a high-throughput screening format. It is based on monitoring the time-dependent increase of absorbance due to turbidity occurring by acid-induced protein aggregation. The method uses the highly amyloidogenic Y78F mutant of human transthyretin (heterogously expressed in Escherichia coli cells). Initial rates of protein aggregation at different inhibitor concentrations follow a monoexponential dose-response curve from which inhibition parameters are calculated. For the assay development, thyroid hormones and nonsteroidal antiinflamatory drugs were chosen among other reference compounds. Some of them are already known to be in vitro inhibitors of TTR amyloidogenesis. Analysis time is optimized to last 1.5 h, and the method is implemented in microtiter plates for screening of libraries of potential fibrillogenesis inhibitors.

Screening and large-scale expression of membrane proteins in mammalian cells for structural studies.

PubMed

Goehring, April; Lee, Chia-Hsueh; Wang, Kevin H; Michel, Jennifer Carlisle; Claxton, Derek P; Baconguis, Isabelle; Althoff, Thorsten; Fischer, Suzanne; Garcia, K Christopher; Gouaux, Eric

2014-11-01

Structural, biochemical and biophysical studies of eukaryotic membrane proteins are often hampered by difficulties in overexpression of the candidate molecule. Baculovirus transduction of mammalian cells (BacMam), although a powerful method to heterologously express membrane proteins, can be cumbersome for screening and expression of multiple constructs. We therefore developed plasmid Eric Gouaux (pEG) BacMam, a vector optimized for use in screening assays, as well as for efficient production of baculovirus and robust expression of the target protein. In this protocol, we show how to use small-scale transient transfection and fluorescence-detection size-exclusion chromatography (FSEC) experiments using a GFP-His8-tagged candidate protein to screen for monodispersity and expression level. Once promising candidates are identified, we describe how to generate baculovirus, transduce HEK293S GnTI(-) (N-acetylglucosaminyltransferase I-negative) cells in suspension culture and overexpress the candidate protein. We have used these methods to prepare pure samples of chicken acid-sensing ion channel 1a (cASIC1) and Caenorhabditis elegans glutamate-gated chloride channel (GluCl) for X-ray crystallography, demonstrating how to rapidly and efficiently screen hundreds of constructs and accomplish large-scale expression in 4-6 weeks.

Screening and large-scale expression of membrane proteins in mammalian cells for structural studies

PubMed Central

Goehring, April; Lee, Chia-Hsueh; Wang, Kevin H.; Michel, Jennifer Carlisle; Claxton, Derek P.; Baconguis, Isabelle; Althoff, Thorsten; Fischer, Suzanne; Garcia, K. Christopher; Gouaux, Eric

2014-01-01

Structural, biochemical and biophysical studies of eukaryotic membrane proteins are often hampered by difficulties in over-expression of the candidate molecule. Baculovirus transduction of mammalian cells (BacMam), although a powerful method to heterologously express membrane proteins, can be cumbersome for screening and expression of multiple constructs. We therefore developed plasmid Eric Gouaux (pEG) BacMam, a vector optimized for use in screening assays, as well as for efficient production of baculovirus and robust expression of the target protein. In this protocol we show how to use small-scale transient transfection and fluorescence-detection, size-exclusion chromatography (FSEC) experiments using a GFP-His8 tagged candidate protein to screen for monodispersity and expression level. Once promising candidates are identified, we describe how to generate baculovirus, transduce HEK293S GnTI− (N-acetylglucosaminyltransferase I-negative) cells in suspension culture, and over-express the candidate protein. We have used these methods to prepare pure samples of chicken acid-sensing ion channel 1a (cASIC1) and Caenorhabditis elegans glutamate-gated chloride channel (GluCl), for X-ray crystallography, demonstrating how to rapidly and efficiently screen hundreds of constructs and accomplish large-scale expression in 4-6 weeks. PMID:25299155

Microplate-based active/inactive 1 screen for biomass degrading enzyme library purification and gene discovery

USDA-ARS?s Scientific Manuscript database

We present here a whole-cell and permeabilized E. coli cell 1' active/inactive microplate screen for ß-D-xylosidase, xylanase, endocellulase, and ferulic acid esterase enzyme activities which are critical for the enzymatic deconstruction of biomass for fuels and chemicals. Transformants from genomic...

Comparison of PC12 and Cerebellar Granule Cell Cultures for Evaluating Neurite Outgrowth Using High Content Screening

EPA Science Inventory

Development of high-throughput assays for chemical screening and hazard identification is a pressing priority worldwide. One approach uses in vitro, cell-based assays which recapitulate biological events observed in vivo. Neurite outgrowth is one such critical cellular process un...

COMPARISON OF NEUROSCREEN-1 AND CEREBELLAR GRANULE CELL CULTURES FOR EVALUATING NEURITE OUTGROWTH USING THE ARRAYSCAN HIGH CONTENT ANALYSIS SYSTEM

EPA Science Inventory

A major challenge facing the Environmental Protection Agency is the development of high-throughput screening assays amendable to resource-efficient developmental neurotoxicity for chemical screening and toxicity prioritization. One approach uses in vitro, cell-based assays which...

A gene expression biomarker accurately predicts estrogen ...

EPA Pesticide Factsheets

The EPA’s vision for the Endocrine Disruptor Screening Program (EDSP) in the 21st Century (EDSP21) includes utilization of high-throughput screening (HTS) assays coupled with computational modeling to prioritize chemicals with the goal of eventually replacing current Tier 1 screening tests. The ToxCast program currently includes 18 HTS in vitro assays that evaluate the ability of chemicals to modulate estrogen receptor α (ERα), an important endocrine target. We propose microarray-based gene expression profiling as a complementary approach to predict ERα modulation and have developed computational methods to identify ERα modulators in an existing database of whole-genome microarray data. The ERα biomarker consisted of 46 ERα-regulated genes with consistent expression patterns across 7 known ER agonists and 3 known ER antagonists. The biomarker was evaluated as a predictive tool using the fold-change rank-based Running Fisher algorithm by comparison to annotated gene expression data sets from experiments in MCF-7 cells. Using 141 comparisons from chemical- and hormone-treated cells, the biomarker gave a balanced accuracy for prediction of ERα activation or suppression of 94% or 93%, respectively. The biomarker was able to correctly classify 18 out of 21 (86%) OECD ER reference chemicals including “very weak” agonists and replicated predictions based on 18 in vitro ER-associated HTS assays. For 114 chemicals present in both the HTS data and the MCF-7 c

Small molecules enhance CRISPR genome editing in pluripotent stem cells.

PubMed

Yu, Chen; Liu, Yanxia; Ma, Tianhua; Liu, Kai; Xu, Shaohua; Zhang, Yu; Liu, Honglei; La Russa, Marie; Xie, Min; Ding, Sheng; Qi, Lei S

2015-02-05

The bacterial CRISPR-Cas9 system has emerged as an effective tool for sequence-specific gene knockout through non-homologous end joining (NHEJ), but it remains inefficient for precise editing of genome sequences. Here we develop a reporter-based screening approach for high-throughput identification of chemical compounds that can modulate precise genome editing through homology-directed repair (HDR). Using our screening method, we have identified small molecules that can enhance CRISPR-mediated HDR efficiency, 3-fold for large fragment insertions and 9-fold for point mutations. Interestingly, we have also observed that a small molecule that inhibits HDR can enhance frame shift insertion and deletion (indel) mutations mediated by NHEJ. The identified small molecules function robustly in diverse cell types with minimal toxicity. The use of small molecules provides a simple and effective strategy to enhance precise genome engineering applications and facilitates the study of DNA repair mechanisms in mammalian cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Screening of pollution control and clean-up materials for river chemical spills using the multiple case-based reasoning method with a difference-driven revision strategy.

PubMed

Liu, Rentao; Jiang, Jiping; Guo, Liang; Shi, Bin; Liu, Jie; Du, Zhaolin; Wang, Peng

2016-06-01

In-depth filtering of emergency disposal technology (EDT) and materials has been required in the process of environmental pollution emergency disposal. However, an urgent problem that must be solved is how to quickly and accurately select the most appropriate materials for treating a pollution event from the existing spill control and clean-up materials (SCCM). To meet this need, the following objectives were addressed in this study. First, the material base and a case base for environment pollution emergency disposal were established to build a foundation and provide material for SCCM screening. Second, the multiple case-based reasoning model method with a difference-driven revision strategy (DDRS-MCBR) was applied to improve the original dual case-based reasoning model method system, and screening and decision-making was performed for SCCM using this model. Third, an actual environmental pollution accident from 2012 was used as a case study to verify the material base, case base, and screening model. The results demonstrated that the DDRS-MCBR method was fast, efficient, and practical. The DDRS-MCBR method changes the passive situation in which the choice of SCCM screening depends only on the subjective experience of the decision maker and offers a new approach to screening SCCM.

Characterization of inhibitory constituents of NO production from Catalpa ovata using LC-MS coupled with a cell-based assay.

PubMed

Park, Sangmin; Shin, Hyeji; Park, Yeeun; Choi, Ilgyu; Park, Byoungduck; Lee, Ki Yong

2018-05-25

An effective screening method for inhibitors of NO production in natural products using LC-QTOF MS/MS coupled with a cell-based assay was proposed. The ethyl acetate fraction of Catalpa ovata exhibited a strong inhibitory effect on NO production in lipopolysaccharide-induced BV2 microglia cells. We attempted to identify the active constituents of C. ovata by using LC-QTOF MS/MS coupled with a cell-based assay. Peaks at approximately 14-15 min on the MS chromatogram were estimated to be the bioactive constituents. A new iridoid compound, 6-O-trans-feruloyl-3β-hydroxy-7-deoxyrehamaglutin A (4), and nine known compounds (1-3, 5-10) were isolated from the ethyl acetate fraction of C. ovata by repeated column chromatography. Compounds 3, 4, 5, 7, and 8 significantly attenuated lipopolysaccharide-stimulated NO production in BV2 cells. Our results indicate that LC-QTOF MS/MS coupled with a cell-based NO production inhibitory assay successfully predicted active compounds without a time-consuming isolation process. Copyright © 2018 Elsevier Inc. All rights reserved.

Screening Test for Shed Skin Cells by Measuring the Ratio of Human DNA to Staphylococcus epidermidis DNA.

PubMed

Nakanishi, Hiroaki; Ohmori, Takeshi; Hara, Masaaki; Takahashi, Shirushi; Kurosu, Akira; Takada, Aya; Saito, Kazuyuki

2016-05-01

A novel screening method for shed skin cells by detecting Staphylococcus epidermidis (S. epidermidis), which is a resident bacterium on skin, was developed. Staphylococcus epidermidis was detected using real-time PCR. Staphylococcus epidermidis was detected in all 20 human skin surface samples. Although not present in blood and urine samples, S. epidermidis was detected in 6 of 20 saliva samples, and 5 of 18 semen samples. The ratio of human DNA to S. epidermidisDNA was significantly smaller in human skin surface samples than in saliva and semen samples in which S. epidermidis was detected. Therefore, although skin cells could not be identified by detecting only S. epidermidis, they could be distinguished by measuring the S. epidermidis to human DNA ratio. This method could be applied to casework touch samples, which suggests that it is useful for screening whether skin cells and human DNA are present on potential evidentiary touch samples. © 2016 American Academy of Forensic Sciences.

A fully automated primary screening system for the discovery of therapeutic antibodies directly from B cells.

PubMed

Tickle, Simon; Howells, Louise; O'Dowd, Victoria; Starkie, Dale; Whale, Kevin; Saunders, Mark; Lee, David; Lightwood, Daniel

2015-04-01

For a therapeutic antibody to succeed, it must meet a range of potency, stability, and specificity criteria. Many of these characteristics are conferred by the amino acid sequence of the heavy and light chain variable regions and, for this reason, can be screened for during antibody selection. However, it is important to consider that antibodies satisfying all these criteria may be of low frequency in an immunized animal; for this reason, it is essential to have a mechanism that allows for efficient sampling of the immune repertoire. UCB's core antibody discovery platform combines high-throughput B cell culture screening and the identification and isolation of single, antigen-specific IgG-secreting B cells through a proprietary technique called the "fluorescent foci" method. Using state-of-the-art automation to facilitate primary screening, extremely efficient interrogation of the natural antibody repertoire is made possible; more than 1 billion immune B cells can now be screened to provide a useful starting point from which to identify the rare therapeutic antibody. This article will describe the design, construction, and commissioning of a bespoke automated screening platform and two examples of how it was used to screen for antibodies against two targets. © 2014 Society for Laboratory Automation and Screening.

Quantitative electrophysiological monitoring of anti-histamine drug effects on live cells via reusable sensor platforms.

PubMed

Pham Ba, Viet Anh; Cho, Dong-Guk; Kim, Daesan; Yoo, Haneul; Ta, Van-Thao; Hong, Seunghun

2017-08-15

We demonstrated the quantitative electrophysiological monitoring of histamine and anti-histamine drug effects on live cells via reusable sensor platforms based on carbon nanotube transistors. This method enabled us to monitor the real-time electrophysiological responses of a single HeLa cell to histamine with different concentrations. The measured electrophysiological responses were attributed to the activity of histamine type 1 receptors on a HeLa cell membrane by histamine. Furthermore, the effects of anti-histamine drugs such as cetirizine or chlorphenamine on the electrophysiological activities of HeLa cells were also evaluated quantitatively. Significantly, we utilized only a single device to monitor the responses of multiple HeLa cells to each drug, which allowed us to quantitatively analyze the antihistamine drug effects on live cells without errors from the device-to-device variation in device characteristics. Such quantitative evaluation capability of our method would promise versatile applications such as drug screening and nanoscale bio sensor researches. Copyright © 2017 Elsevier B.V. All rights reserved.

Chip-based generation of carbon nanodots via electrochemical oxidation of screen printed carbon electrodes and the applications for efficient cell imaging and electrochemiluminescence enhancement

NASA Astrophysics Data System (ADS)

Xu, Yuanhong; Liu, Jingquan; Zhang, Jizhen; Zong, Xidan; Jia, Xiaofang; Li, Dan; Wang, Erkang

2015-05-01

A portable lab-on-a-chip methodology to generate ionic liquid-functionalized carbon nanodots (CNDs) was developed via electrochemical oxidation of screen printed carbon electrodes. The CNDs can be successfully applied for efficient cell imaging and solid-state electrochemiluminescence sensor fabrication on the paper-based chips.A portable lab-on-a-chip methodology to generate ionic liquid-functionalized carbon nanodots (CNDs) was developed via electrochemical oxidation of screen printed carbon electrodes. The CNDs can be successfully applied for efficient cell imaging and solid-state electrochemiluminescence sensor fabrication on the paper-based chips. Electronic supplementary information (ESI) available: Experimental section; Fig. S1. XPS spectra of the as-prepared CNDs after being dialyzed for 72 hours; Fig. S2. LSCM images showing time-dependent fluorescence signals of HeLa cells treated by the as-prepared CNDs; Tripropylamine analysis using the Nafion/CNDs modified ECL sensor. See DOI: 10.1039/c5nr01765c

A fast boosting-based screening method for large-scale association study in complex traits with genetic heterogeneity.

PubMed

Wang, Lu-Yong; Fasulo, D

2006-01-01

Genome-wide association study for complex diseases will generate massive amount of single nucleotide polymorphisms (SNPs) data. Univariate statistical test (i.e. Fisher exact test) was used to single out non-associated SNPs. However, the disease-susceptible SNPs may have little marginal effects in population and are unlikely to retain after the univariate tests. Also, model-based methods are impractical for large-scale dataset. Moreover, genetic heterogeneity makes the traditional methods harder to identify the genetic causes of diseases. A more recent random forest method provides a more robust method for screening the SNPs in thousands scale. However, for more large-scale data, i.e., Affymetrix Human Mapping 100K GeneChip data, a faster screening method is required to screening SNPs in whole-genome large scale association analysis with genetic heterogeneity. We propose a boosting-based method for rapid screening in large-scale analysis of complex traits in the presence of genetic heterogeneity. It provides a relatively fast and fairly good tool for screening and limiting the candidate SNPs for further more complex computational modeling task.

EZ spheres: a stable and expandable culture system for the generation of pre-rosette multipotent stem cells from human ESCs and iPSCs.

PubMed

Ebert, Allison D; Shelley, Brandon C; Hurley, Amanda M; Onorati, Marco; Castiglioni, Valentina; Patitucci, Teresa N; Svendsen, Soshana P; Mattis, Virginia B; McGivern, Jered V; Schwab, Andrew J; Sareen, Dhruv; Kim, Ho Won; Cattaneo, Elena; Svendsen, Clive N

2013-05-01

We have developed a simple method to generate and expand multipotent, self-renewing pre-rosette neural stem cells from both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) without utilizing embryoid body formation, manual selection techniques, or complex combinations of small molecules. Human ESC and iPSC colonies were lifted and placed in a neural stem cell medium containing high concentrations of EGF and FGF-2. Cell aggregates (termed EZ spheres) could be expanded for long periods using a chopping method that maintained cell-cell contact. Early passage EZ spheres rapidly down-regulated OCT4 and up-regulated SOX2 and nestin expression. They retained the potential to form neural rosettes and consistently differentiated into a range of central and peripheral neural lineages. Thus, they represent a very early neural stem cell with greater differentiation flexibility than other previously described methods. As such, they will be useful for the rapidly expanding field of neurological development and disease modeling, high-content screening, and regenerative therapies based on pluripotent stem cell technology. Copyright © 2013 Elsevier B.V. All rights reserved.

3 dimensional cell cultures: a comparison between manually and automatically produced alginate beads.

PubMed

Lehmann, R; Gallert, C; Roddelkopf, T; Junginger, S; Wree, A; Thurow, K

2016-08-01

Cancer diseases are a common problem of the population caused by age and increased harmful environmental influences. Herein, new therapeutic strategies and compound screenings are necessary. The regular 2D cultivation has to be replaced by three dimensional cell culturing (3D) for better simulation of in vivo conditions. The 3D cultivation with alginate matrix is an appropriate method for encapsulate cells to form cancer constructs. The automated manufacturing of alginate beads might be an ultimate method for large-scaled manufacturing constructs similar to cancer tissue. The aim of this study was the integration of full automated systems for the production, cultivation and screening of 3D cell cultures. We compared the automated methods with the regular manual processes. Furthermore, we investigated the influence of antibiotics on these 3D cell culture systems. The alginate beads were formed by automated and manual procedures. The automated steps were processes by the Biomek(®) Cell Workstation (celisca, Rostock, Germany). The proliferation and toxicity were manually and automatically evaluated at day 14 and 35 of cultivation. The results visualized an accumulation and expansion of cell aggregates over the period of incubation. However, the proliferation and toxicity were faintly and partly significantly decreased on day 35 compared to day 14. The comparison of the manual and automated methods displayed similar results. We conclude that the manual production process could be replaced by the automation. Using automation, 3D cell cultures can be produced in industrial scale and improve the drug development and screening to treat serious illnesses like cancer.

High-throughput Screening of ToxCast" Phase I Chemicals in an Embryonic Stem Cell Assay Reveals Potential Disruption of a Critical Developmental Signaling Pathway

EPA Science Inventory

Little is known about the developmental toxicity of the expansive chemical landscape in existence today. Significant efforts are being made to apply novel methods to predict developmental activity of chemicals utilizing high-throughput screening (HTS) and high-content screening (...

Automatic detection of spermatozoa for laser capture microdissection.

PubMed

Vandewoestyne, Mado; Van Hoofstat, David; Van Nieuwerburgh, Filip; Deforce, Dieter

2009-03-01

In sexual assault crimes, differential extraction of spermatozoa from vaginal swab smears is often ineffective, especially when only a few spermatozoa are present in an overwhelming amount of epithelial cells. Laser capture microdissection (LCM) enables the precise separation of spermatozoa and epithelial cells. However, standard sperm-staining techniques are non-specific and rely on sperm morphology for identification. Moreover, manual screening of the microscope slides is time-consuming and labor-intensive. Here, we describe an automated screening method to detect spermatozoa stained with Sperm HY-LITER. Different ratios of spermatozoa and epithelial cells were used to assess the automatic detection method. In addition, real postcoital samples were also screened. Detected spermatozoa were isolated using LCM and DNA analysis was performed. Robust DNA profiles without allelic dropout could be obtained from as little as 30 spermatozoa recovered from postcoital samples, showing that the staining had no significant influence on DNA recovery.

Mass spectrometric-based stable isotopic 2-aminobenzoic acid glycan mapping for rapid glycan screening of biotherapeutics.

PubMed

Prien, Justin M; Prater, Bradley D; Qin, Qiang; Cockrill, Steven L

2010-02-15

Fast, sensitive, robust methods for "high-level" glycan screening are necessary during various stages of a biotherapeutic product's lifecycle, including clone selection, process changes, and quality control for lot release testing. Traditional glycan screening involves chromatographic or electrophoretic separation-based methods, and, although reproducible, these methods can be time-consuming. Even ultrahigh-performance chromatographic and microfluidic integrated LC/MS systems, which work on the tens of minute time scale, become lengthy when hundreds of samples are to be analyzed. Comparatively, a direct infusion mass spectrometry (MS)-based glycan screening method acquires data on a millisecond time scale, exhibits exquisite sensitivity and reproducibility, and is amenable to automated peak annotation. In addition, characterization of glycan species via sequential mass spectrometry can be performed simultaneously. Here, we demonstrate a quantitative high-throughput MS-based mapping approach using stable isotope 2-aminobenzoic acid (2-AA) for rapid "high-level" glycan screening.

Parallel RNAi screens across different cell lines identify generic and cell type-specific regulators of actin organization and cell morphology.

PubMed

Liu, Tao; Sims, David; Baum, Buzz

2009-01-01

In recent years RNAi screening has proven a powerful tool for dissecting gene functions in animal cells in culture. However, to date, most RNAi screens have been performed in a single cell line, and results then extrapolated across cell types and systems. Here, to dissect generic and cell type-specific mechanisms underlying cell morphology, we have performed identical kinome RNAi screens in six different Drosophila cell lines, derived from two distinct tissues of origin. This analysis identified a core set of kinases required for normal cell morphology in all lines tested, together with a number of kinases with cell type-specific functions. Most significantly, the screen identified a role for minibrain (mnb/DYRK1A), a kinase associated with Down's syndrome, in the regulation of actin-based protrusions in CNS-derived cell lines. This cell type-specific requirement was not due to the peculiarities in the morphology of CNS-derived cells and could not be attributed to differences in mnb expression. Instead, it likely reflects differences in gene expression that constitute the cell type-specific functional context in which mnb/DYRK1A acts. Using parallel RNAi screens and gene expression analyses across cell types we have identified generic and cell type-specific regulators of cell morphology, which include mnb/DYRK1A in the regulation of protrusion morphology in CNS-derived cell lines. This analysis reveals the importance of using different cell types to gain a thorough understanding of gene function across the genome and, in the case of kinases, the difficulties of using the differential gene expression to predict function.

A holistic high-throughput screening framework for biofuel feedstock assessment that characterises variations in soluble sugars and cell wall composition in Sorghum bicolor

PubMed Central

2013-01-01

Background A major hindrance to the development of high yielding biofuel feedstocks is the ability to rapidly assess large populations for fermentable sugar yields. Whilst recent advances have outlined methods for the rapid assessment of biomass saccharification efficiency, none take into account the total biomass, or the soluble sugar fraction of the plant. Here we present a holistic high-throughput methodology for assessing sweet Sorghum bicolor feedstocks at 10 days post-anthesis for total fermentable sugar yields including stalk biomass, soluble sugar concentrations, and cell wall saccharification efficiency. Results A mathematical method for assessing whole S. bicolor stalks using the fourth internode from the base of the plant proved to be an effective high-throughput strategy for assessing stalk biomass, soluble sugar concentrations, and cell wall composition and allowed calculation of total stalk fermentable sugars. A high-throughput method for measuring soluble sucrose, glucose, and fructose using partial least squares (PLS) modelling of juice Fourier transform infrared (FTIR) spectra was developed. The PLS prediction was shown to be highly accurate with each sugar attaining a coefficient of determination (R 2 ) of 0.99 with a root mean squared error of prediction (RMSEP) of 11.93, 5.52, and 3.23 mM for sucrose, glucose, and fructose, respectively, which constitutes an error of <4% in each case. The sugar PLS model correlated well with gas chromatography–mass spectrometry (GC-MS) and brix measures. Similarly, a high-throughput method for predicting enzymatic cell wall digestibility using PLS modelling of FTIR spectra obtained from S. bicolor bagasse was developed. The PLS prediction was shown to be accurate with an R 2 of 0.94 and RMSEP of 0.64 μg.mgDW-1.h-1. Conclusions This methodology has been demonstrated as an efficient and effective way to screen large biofuel feedstock populations for biomass, soluble sugar concentrations, and cell wall digestibility simultaneously allowing a total fermentable yield calculation. It unifies and simplifies previous screening methodologies to produce a holistic assessment of biofuel feedstock potential. PMID:24365407

Isolation of New Gravitropic Mutants under Hypergravity Conditions.

PubMed

Mori, Akiko; Toyota, Masatsugu; Shimada, Masayoshi; Mekata, Mika; Kurata, Tetsuya; Tasaka, Masao; Morita, Miyo T

2016-01-01

Forward genetics is a powerful approach used to link genotypes and phenotypes, and mutant screening/analysis has provided deep insights into many aspects of plant physiology. Gravitropism is a tropistic response in plants, in which hypocotyls and stems sense the direction of gravity and grow upward. Previous studies of gravitropic mutants have suggested that shoot endodermal cells in Arabidopsis stems and hypocotyls are capable of sensing gravity (i.e., statocytes). In the present study, we report a new screening system using hypergravity conditions to isolate enhancers of gravitropism mutants, and we also describe a rapid and efficient genome mapping method, using next-generation sequencing (NGS) and single nucleotide polymorphism (SNP)-based markers. Using the endodermal-amyloplast less 1 ( eal1 ) mutant, which exhibits defective development of endodermal cells and gravitropism, we found that hypergravity (10 g) restored the reduced gravity responsiveness in eal1 hypocotyls and could, therefore, be used to obtain mutants with further reduction in gravitropism in the eal1 background. Using the new screening system, we successfully isolated six ene ( enhancer of eal1 ) mutants that exhibited little or no gravitropism under hypergravity conditions, and using NGS and map-based cloning with SNP markers, we narrowed down the potential causative genes, which revealed a new genetic network for shoot gravitropism in Arabidopsis .

Isolation of New Gravitropic Mutants under Hypergravity Conditions

PubMed Central

Mori, Akiko; Toyota, Masatsugu; Shimada, Masayoshi; Mekata, Mika; Kurata, Tetsuya; Tasaka, Masao; Morita, Miyo T.

2016-01-01

Forward genetics is a powerful approach used to link genotypes and phenotypes, and mutant screening/analysis has provided deep insights into many aspects of plant physiology. Gravitropism is a tropistic response in plants, in which hypocotyls and stems sense the direction of gravity and grow upward. Previous studies of gravitropic mutants have suggested that shoot endodermal cells in Arabidopsis stems and hypocotyls are capable of sensing gravity (i.e., statocytes). In the present study, we report a new screening system using hypergravity conditions to isolate enhancers of gravitropism mutants, and we also describe a rapid and efficient genome mapping method, using next-generation sequencing (NGS) and single nucleotide polymorphism (SNP)-based markers. Using the endodermal-amyloplast less 1 (eal1) mutant, which exhibits defective development of endodermal cells and gravitropism, we found that hypergravity (10 g) restored the reduced gravity responsiveness in eal1 hypocotyls and could, therefore, be used to obtain mutants with further reduction in gravitropism in the eal1 background. Using the new screening system, we successfully isolated six ene (enhancer of eal1) mutants that exhibited little or no gravitropism under hypergravity conditions, and using NGS and map-based cloning with SNP markers, we narrowed down the potential causative genes, which revealed a new genetic network for shoot gravitropism in Arabidopsis. PMID:27746791

Development of an XTT tetrazolium salt-based assay for detection of specific hyperthermia sensitizers in a high-flux screening programme.

PubMed

Lechpammer, S; Asea, A; Mallick, R; Zhong, R; Sherman, M Y; Calderwood, S K

2002-01-01

It is now possible to search for new drugs using high-throughput screening of chemical libraries accumulated over the past few years. To detect potential new hyperthermia sensitizers, we are screening for chemical inhibitors of thermotolerance. For the screening of a large chemical library, a rapid and simple assay based on the XTT-tetrazolium salt with the addition of intermediate electron acceptor, phenazine methosulphate (PMS) as a promoter, was developed. It was found that the sensitivity of the XTT/PMS assay is sufficient for assessing thermal cell killing and thermotolerance, although it was highly dependent on cell number and type. When the formazan assay system was challenged with the bioflavonoid drug quercetin (up to 25mm) and validated against the clonogenic cell survival assay, significant decreases in thermotolerant cell viability were observed, directly reflecting inhibition of thermotolerance. Although short-term assays can, in some instances, underestimate overall cell killing, the dose dependency of inhibition of thermotolerance by quercetin recorded in this study by clonogenic and XTT/PMS assays was similar. Application of the XTT/PMS assay in chemical library screening was highly effective in differentiating potential thermotolerance inhibitors from both chemicals with lack of efficacy and from toxic compounds. Taken together, these results show that the XTT/PMS assay, when carried out under careful conditions, is well suited for primary high-flux screen of many thousands of compounds, thus opening up new areas for discovery of hyperthermia sensitizers.

Computational methods for evaluation of cell-based data assessment--Bioconductor.

PubMed

Le Meur, Nolwenn

2013-02-01

Recent advances in miniaturization and automation of technologies have enabled cell-based assay high-throughput screening, bringing along new challenges in data analysis. Automation, standardization, reproducibility have become requirements for qualitative research. The Bioconductor community has worked in that direction proposing several R packages to handle high-throughput data including flow cytometry (FCM) experiment. Altogether, these packages cover the main steps of a FCM analysis workflow, that is, data management, quality assessment, normalization, outlier detection, automated gating, cluster labeling, and feature extraction. Additionally, the open-source philosophy of R and Bioconductor, which offers room for new development, continuously drives research and improvement of theses analysis methods, especially in the field of clustering and data mining. This review presents the principal FCM packages currently available in R and Bioconductor, their advantages and their limits. Copyright © 2012 Elsevier Ltd. All rights reserved.

Digital microfluidic three-dimensional cell culture and chemical screening platform using alginate hydrogels

PubMed Central

2015-01-01

Electro wetting-on-dielectric (EWOD) digital microfluidics (DMF) can be used to develop improved chemical screening platforms using 3-dimensional (3D) cell culture. Alginate hydrogels are one common method by which a 3D cell culture environment is created. This paper presents a study of alginate gelation on EWOD DMF and investigates designs to obtain uniform alginate hydrogels that can be repeatedly addressed by any desired liquids. A design which allows for gels to be retained in place during liquid delivery and removal without using any physical barriers or hydrophilic patterning of substrates is presented. A proof of concept screening platform is demonstrated by examining the effects of different concentrations of a test chemical on 3D cells in alginate hydrogels. In addition, the temporal effects of the various chemical concentrations on different hydrogel posts are demonstrated, thereby establishing the benefits of an EWOD DMF 3D cell culture and chemical screening platform using alginate hydrogels. PMID:25945142

Functional video-based analysis of 3D cardiac structures generated from human embryonic stem cells.

PubMed

Nitsch, Scarlett; Braun, Florian; Ritter, Sylvia; Scholz, Michael; Schroeder, Insa S

2018-05-01

Human embryonic stem cells (hESCs) differentiated into cardiomyocytes (CM) often develop into complex 3D structures that are composed of various cardiac cell types. Conventional methods to study the electrophysiology of cardiac cells are patch clamp and microelectrode array (MEAs) analyses. However, these methods are not suitable to investigate the contractile features of 3D cardiac clusters that detach from the surface of the culture dishes during differentiation. To overcome this problem, we developed a video-based motion detection software relying on the optical flow by Farnebäck that we call cBRA (cardiac beat rate analyzer). The beating characteristics of the differentiated cardiac clusters were calculated based on the local displacement between two subsequent images. Two differentiation protocols, which profoundly differ in the morphology of cardiac clusters generated and in the expression of cardiac markers, were used and the resulting CM were characterized. Despite these differences, beat rates and beating variabilities could be reliably determined using cBRA. Likewise, stimulation of β-adrenoreceptors by isoproterenol could easily be identified in the hESC-derived CM. Since even subtle changes in the beating features are detectable, this method is suitable for high throughput cardiotoxicity screenings. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Discovery of novel hedgehog antagonists from cell-based screening: Isosteric modification of p38 bisamides as potent inhibitors of SMO.

PubMed

Yang, Bin; Hird, Alexander W; Russell, Daniel John; Fauber, Benjamin P; Dakin, Les A; Zheng, Xiaolan; Su, Qibin; Godin, Robert; Brassil, Patrick; Devereaux, Erik; Janetka, James W

2012-07-15

Cell-based subset screening of compounds using a Gli transcription factor reporter cell assay and shh stimulated cell differentiation assay identified a series of bisamide compounds as hedgehog pathway inhibitors with good potency. Using a ligand-based optimization strategy, heteroaryl groups were utilized as conformationally restricted amide isosteres replacing one of the amides which significantly increased their potency against SMO and the hedgehog pathway while decreasing activity against p38α kinase. We report herein the identification of advanced lead compounds such as imidazole 11c and 11f encompassing good p38α selectivity, low nanomolar potency in both cell assays, excellent physiochemical properties and in vivo pharmacokinetics. Copyright © 2012 Elsevier Ltd. All rights reserved.

Newborn Screening for Severe Primary Immunodeficiency Diseases in Sweden-a 2-Year Pilot TREC and KREC Screening Study.

PubMed

Barbaro, Michela; Ohlsson, Annika; Borte, Stephan; Jonsson, Susanne; Zetterström, Rolf H; King, Jovanka; Winiarski, Jacek; von Döbeln, Ulrika; Hammarström, Lennart

2017-01-01

Newborn screening for severe primary immunodeficiencies (PID), characterized by T and/or B cell lymphopenia, was carried out in a pilot program in the Stockholm County, Sweden, over a 2-year period, encompassing 58,834 children. T cell receptor excision circles (TREC) and kappa-deleting recombination excision circles (KREC) were measured simultaneously using a quantitative PCR-based method on DNA extracted from dried blood spots (DBS), with beta-actin serving as a quality control for DNA quantity. Diagnostic cutoff levels enabling identification of newborns with milder and reversible T and/or B cell lymphopenia were also evaluated. Sixty-four children were recalled for follow-up due to low TREC and/or KREC levels, and three patients with immunodeficiency (Artemis-SCID, ATM, and an as yet unclassified T cell lymphopenia/hypogammaglobulinemia) were identified. Of the positive samples, 24 were associated with prematurity. Thirteen children born to mothers treated with immunosuppressive agents during pregnancy (azathioprine (n = 9), mercaptopurine (n = 1), azathioprine and tacrolimus (n = 3)) showed low KREC levels at birth, which spontaneously normalized. Twenty-nine newborns had no apparent cause identified for their abnormal results, but normalized with time. Children with trisomy 21 (n = 43) showed a lower median number of both TREC (104 vs. 174 copies/μL blood) and KREC (45 vs. 100 copies/3.2 mm blood spot), but only one, born prematurely, fell below the cutoff level. Two children diagnosed with DiGeorge syndrome were found to have low TREC levels, but these were still above the cutoff level. This is the first large-scale screening study with a simultaneous detection of both TREC and KREC, allowing identification of newborns with both T and B cell defects.

Parallel production and verification of protein products using a novel high-throughput screening method.

PubMed

Tegel, Hanna; Yderland, Louise; Boström, Tove; Eriksson, Cecilia; Ukkonen, Kaisa; Vasala, Antti; Neubauer, Peter; Ottosson, Jenny; Hober, Sophia

2011-08-01

Protein production and analysis in a parallel fashion is today applied in laboratories worldwide and there is a great need to improve the techniques and systems used for this purpose. In order to save time and money, a fast and reliable screening method for analysis of protein production and also verification of the protein product is desired. Here, a micro-scale protocol for the parallel production and screening of 96 proteins in plate format is described. Protein capture was achieved using immobilized metal affinity chromatography and the product was verified using matrix-assisted laser desorption ionization time-of-flight MS. In order to obtain sufficiently high cell densities and product yield in the small-volume cultivations, the EnBase® cultivation technology was applied, which enables cultivation in as small volumes as 150 μL. Here, the efficiency of the method is demonstrated by producing 96 human, recombinant proteins, both in micro-scale and using a standard full-scale protocol and comparing the results in regard to both protein identity and sample purity. The results obtained are highly comparable to those acquired through employing standard full-scale purification protocols, thus validating this method as a successful initial screening step before protein production at a larger scale. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Shape-Based Virtual Screening with Volumetric Aligned Molecular Shapes

PubMed Central

Koes, David Ryan; Camacho, Carlos J.

2014-01-01

Shape-based virtual screening is an established and effective method for identifying small molecules that are similar in shape and function to a reference ligand. We describe a new method of shape-based virtual screening, volumetric aligned molecular shapes (VAMS). VAMS uses efficient data structures to encode and search molecular shapes. We demonstrate that VAMS is an effective method for shape-based virtual screening and that it can be successfully used as a pre-filter to accelerate more computationally demanding search algorithms. Unique to VAMS is a novel minimum/maximum shape constraint query for precisely specifying the desired molecular shape. Shape constraint searches in VAMS are particularly efficient and millions of shapes can be searched in a fraction of a second. We compare the performance of VAMS with two other shape-based virtual screening algorithms a benchmark of 102 protein targets consisting of more than 32 million molecular shapes and find that VAMS provides a competitive trade-off between run-time performance and virtual screening performance. PMID:25049193

A sensitive method to quantify human cell-free circulating DNA in blood: relevance to myocardial infarction screening.

PubMed

Jing, Rong-Rong; Wang, Hui-Min; Cui, Ming; Fang, Meng-Kang; Qiu, Xiao-Jun; Wu, Xin-Hua; Qi, Jin; Wang, Yue-Guo; Zhang, Lu-Rong; Zhu, Jian-Hua; Ju, Shao-Qing

2011-09-01

Human cell-free circulating DNA (cf-DNA) derived mainly from cell apoptosis and necrosis can be measured by a variety of laboratory techniques, but almost all of these methods require sample preparation. We have developed a branched DNA (bDNA)-based Alu assay for quantifying cf-DNA in myocardial infarction (MI) patients. A total of 82 individuals were included in the study; 22 MI and 60 normal controls. cf-DNA was quantified using a bDNA-based Alu assay. cf-DNA was higher in serum compared to plasma and there was a difference between genders. cf-DNA was significantly higher in MI patients compared to the controls. There was no correlation between cf-DNA and creatine kinase-MB (CK-MB), troponin I (cTnI) or myoglobin (MYO). In serial specimens, cf-DNA was sensitive and peaked earlier than cTnI. The bDNA-based Alu assay is a novel method for quantifying human cf-DNA. Increased cf-DNA in MI patients might complement cTnI, CK-MB and MYO in a multiple marker format. Copyright © 2011 The Canadian Society of Clinical Chemists. All rights reserved.

Evaluation of anemia diagnosis based on elastic light scattering (Conference Presentation)

NASA Astrophysics Data System (ADS)

Tong, Lieshu; Wang, Xinrui; Xie, Dengling; Chen, Xiaoya; Chu, Kaiqin; Dou, Hu; Smith, Zachary J.

2017-03-01

Currently, one-third of humanity is still suffering from anemia. In China the most common forms of anemia are iron deficiency and Thalassemia minor. Differentiating these two is the key to effective treatment. Iron deficiency is caused by malnutrition and can be cured by iron supplementation. Thalassemia is a hereditary disease in which the hemoglobin β chain is lowered or absent. Iron therapy is not effective, and there is evidence that iron therapy may be harmful to patients with Thalassemia. Both anemias can be diagnosed using red blood cell morphology: Iron deficiency presents a smaller mean cell volume compared to normal cells, but with a wide distribution; Thalassemia, meanwhile, presents a very small cell size and tight particle size distribution. Several researchers have proposed diagnostic indices based on red cell morphology to differentiate these two diseases. However, these indices lack sensitivity and specificity and are constructed without statistical rigor. Using multivariate methods we demonstrate a new classification method based on red cell morphology that diagnoses anemia in a Chinese population with enough accuracy for its use as a screening method. We further demonstrate a low cost instrument that precisely measures red cell morphology using elastic light scattering. This instrument is combined with an automated analysis program that processes scattering data to report red cell morphology without the need for user intervention. Despite using consumer-grade components, when comparing our experimental results with gold-standard measurements, the device can still achieve the high precision required for sensing clinically significant changes in red cell morphology.

Determination of Tolerance to Internal Shorts and Its Screening in Lithium-ion Cells NASA - JSC Method

NASA Technical Reports Server (NTRS)

Jeevarajan, Judith

2009-01-01

This slide presentation reviews the method developed by the NASA Johnson Space Center (JSC) to determine tolerances to internal shorts and screening for problems in commercial off the shelf (COTS) Lithium-ion batteries. The test apparatus is shown and several examples of the usage and results of the test are discussed.

Paper-based microreactor array for rapid screening of cell signaling cascades.

PubMed

Huang, Chia-Hao; Lei, Kin Fong; Tsang, Ngan-Ming

2016-08-07

Investigation of cell signaling pathways is important for the study of pathogenesis of cancer. However, the related operations used in these studies are time consuming and labor intensive. Thus, the development of effective therapeutic strategies may be hampered. In this work, gel-free cell culture and subsequent immunoassay has been successfully integrated and conducted in a paper-based microreactor array. Study of the activation level of different kinases of cells stimulated by different conditions, i.e., IL-6 stimulation, starvation, and hypoxia, was demonstrated. Moreover, rapid screening of cell signaling cascades after the stimulations of HGF, doxorubicin, and UVB irradiation was respectively conducted to simultaneously screen 40 kinases and transcription factors. Activation of multi-signaling pathways could be identified and the correlation between signaling pathways was discussed to provide further information to investigate the entire signaling network. The present technique integrates most of the tedious operations using a single paper substrate, reduces sample and reagent consumption, and shortens the time required by the entire process. Therefore, it provides a first-tier rapid screening tool for the study of complicated signaling cascades. It is expected that the technique can be developed for routine protocol in conventional biological research laboratories.

Screening of bioactive peptides using an embryonic stem cell-based neurodifferentiation assay.

PubMed

Xu, Ruodan; Feyeux, Maxime; Julien, Stéphanie; Nemes, Csilla; Albrechtsen, Morten; Dinnyés, Andras; Krause, Karl-Heinz

2014-05-01

Differentiation of pluripotent stem cells, PSCs, towards neural lineages has attracted significant attention, given the potential use of such cells for in vitro studies and for regenerative medicine. The present experiments were designed to identify bioactive peptides which direct PSC differentiation towards neural cells. Fifteen peptides were designed based on NCAM, FGFR, and growth factors sequences. The effect of peptides was screened using a mouse embryonic stem cell line expressing luciferase dual reporter construct driven by promoters for neural tubulin and for elongation factor 1. Cell number was estimated by measuring total cellular DNA. We identified five peptides which enhanced activities of both promoters without relevant changes in cell number. We selected the two most potent peptides for further analysis: the NCAM-derived mimetic FGLL and the synthetic NCAM ligand, Plannexin. Both compounds induced phenotypic neuronal differentiation, as evidenced by increased neurite outgrowth. In summary, we used a simple, but sensitive screening approach to identify the neurogenic peptides. These peptides will not only provide new clues concerning pathways of neurogenesis, but they may also be interesting biotechnology tools for in vitro generation of neurons.

A novel tandem mass spectrometry method for first-line screening of mainly beta-thalassemia from dried blood spots.

PubMed

Yu, Chaowen; Huang, Shuodan; Wang, Ming; Zhang, Juan; Liu, Hao; Yuan, Zhaojian; Wang, Xingbin; He, Xiaoyan; Wang, Jie; Zou, Lin

2017-02-10

Traditional methods for thalassemia screening are time-consuming and easily affected by cell hemolysis or hemoglobin degradation in stored blood samples. Tandem mass spectrometry (MS/MS) proved to be an effective technology for sickle cell disorders (SCD) screening. Here, we developed a novel MS/MS method for β-thalassemia screening from dried blood spots (DBS). Stable isotopic-labeled peptides were used as internal standards for quantification and calculation of the α:β-globin ratios. We used the α:β-globin ratio cutoffs to differentiate between normal individuals and patients with thalassemia. About 781 patients and 300 normal individuals were analyzed. The α:β-globin ratios showed significant difference between normal and β-thalassemia patients (P<0.01), particularly when the disease was homozygous or double heterozygous with another α- or β-thalassemia mutation. In the parallel study, all cases screened for suspected thalassemia from six hundred DBS samples by using this MS/MS method were successfully confirmed by genotyping. The intra-assay and inter-assay CVs of the ratios ranged from 2.4% to 3.9% and 4.7% to 7.1%, and there was no significant sample carryover or matrix effect for this MS/MS method. Combined with SCD screening, this MS/MS method could be used as a first-line screening assay for both structural and expression abnormalities of human hemoglobin. Traditional methods for thalassemia screening were depending on the structural integrity of tetramers and could be affected by hemolysis and degradation of whole blood samples, especially when stored. We used proteospecific peptides produced by the tryptic digestion of each globin to evaluate the production ratio between α- and β-globin chains, which turned out to be quite stable even when stored for more than two months. Though most of the peptides were specific to α-globin or β-globin, we only chose four most informative peptides and its stable isotopic-labeled peptides as internal standards for analysis, which could obtain a high accuracy. Currently, we are the first to address the application of MS/MS for thalassemia screening, when combined with SCD screening, this MS/MS method could be used as a first-line screening assay for both structural and expression abnormalities of human hemoglobin. Copyright © 2016. Published by Elsevier B.V.

Identification of Inhibitory Compounds Against Singapore Grouper Iridovirus Infection by Cell Viability-Based Screening Assay and Droplet Digital PCR.

PubMed

Jia, Kuntong; Yuan, Yongming; Liu, Wei; Liu, Lan; Qin, Qiwei; Yi, Meisheng

2018-02-01

Singapore grouper iridovirus (SGIV) is one of the major causative agents of fish diseases and has caused significant economic losses in the aquaculture industry. There is currently no commercial vaccine or effective antiviral treatment against SGIV infection. Annually, an increasing number of small molecule compounds from various sources have been produced, and many are proved to be potential inhibitors against viruses. Here, a high-throughput in vitro cell viability-based screening assay was developed to identify antiviral compounds against SGIV using the luminescent-based CellTiter-Glo reagent in cultured grouper spleen cells by quantificational measurement of the cytopathic effects induced by SGIV infection. This assay was utilized to screen for potential SGIV inhibitors from five customized compounds which had been reported to be capable of inhibiting other viruses and 30 compounds isolated from various marine organisms, and three of them [ribavirin, harringtonine, and 2-hydroxytetradecanoic acid (2-HOM)] were identified to be effective on inhibiting SGIV infection, which was further confirmed with droplet digital PCR (ddPCR). In addition, the ddPCR results revealed that ribavirin and 2-HOM inhibited SGIV replication and entry in a dose-dependent manner, and harringtonine could reduce SGIV replication rather than entry at the working concentration without significant toxicity. These findings provided an easy and reliable cell viability-based screening assay to identify compounds with anti-SGIV effect and a way of studying the anti-SGIV mechanism of compounds.

CrossCheck: an open-source web tool for high-throughput screen data analysis.

PubMed

Najafov, Jamil; Najafov, Ayaz

2017-07-19

Modern high-throughput screening methods allow researchers to generate large datasets that potentially contain important biological information. However, oftentimes, picking relevant hits from such screens and generating testable hypotheses requires training in bioinformatics and the skills to efficiently perform database mining. There are currently no tools available to general public that allow users to cross-reference their screen datasets with published screen datasets. To this end, we developed CrossCheck, an online platform for high-throughput screen data analysis. CrossCheck is a centralized database that allows effortless comparison of the user-entered list of gene symbols with 16,231 published datasets. These datasets include published data from genome-wide RNAi and CRISPR screens, interactome proteomics and phosphoproteomics screens, cancer mutation databases, low-throughput studies of major cell signaling mediators, such as kinases, E3 ubiquitin ligases and phosphatases, and gene ontological information. Moreover, CrossCheck includes a novel database of predicted protein kinase substrates, which was developed using proteome-wide consensus motif searches. CrossCheck dramatically simplifies high-throughput screen data analysis and enables researchers to dig deep into the published literature and streamline data-driven hypothesis generation. CrossCheck is freely accessible as a web-based application at http://proteinguru.com/crosscheck.

Treatment of Infants Identified by Newborn Screening for Severe Combined Immunodeficiency

PubMed Central

Dorsey, Morna J.; Dvorak, Christopher C.; Cowan, Morton J.; Puck, Jennifer M.

2017-01-01

Background Severe combined immunodeficiency (SCID) is characterized by severely impaired T cell development and is fatal without treatment. Newborn screening (NBS) for SCID permits identification of affected infants before development of opportunistic infections and other complications. Substantial variation exists between treatment centers with regard to pre-transplant care and transplant protocols for NBS identified SCID infants, as well as for infants with other T lymphopenic disorders detected by NBS. Methods We developed approaches to management based on the study of infants identified by SCID NBS who received care at UCSF. Results From August 2010 through October 2016, 32 NBS SCID and leaky SCID cases from California and other states were treated and 42 NBS identified non-SCID T cell lymphopenia (TCL) cases were followed. Conclusions Our center’s approach supports successful outcomes; systematic review of our practice provides a framework for diagnosis and management, recognizing that more data will continue to shape best practices. PMID:28270365

Fluorophore Absorption Size Exclusion Chromatography (FA-SEC): An Alternative Method for High-Throughput Detergent Screening of Membrane Proteins.

PubMed

Lin, Sung-Yao; Sun, Xing-Han; Hsiao, Yu-Hsuan; Chang, Shao-En; Li, Guan-Syun; Hu, Nien-Jen

2016-01-01

Membrane proteins play key roles in many fundamental functions in cells including ATP synthesis, ion and molecule transporter, cell signalling and enzymatic reactions, accounting for ~30% genes of whole genomes. However, the hydrophobic nature of membrane proteins frequently hampers the progress of structure determination. Detergent screening is the critical step in obtaining stable detergent-solubilized membrane proteins and well-diffracting protein crystals. Fluorescence Detection Size Exclusion Chromatography (FSEC) has been developed to monitor the extraction efficiency and monodispersity of membrane proteins in detergent micelles. By tracing the FSEC profiles of GFP-fused membrane proteins, this method significantly enhances the throughput of detergent screening. However, current methods to acquire FSEC profiles require either an in-line fluorescence detector with the SEC equipment or an off-line spectrofluorometer microplate reader. Here, we introduce an alternative method detecting the absorption of GFP (FA-SEC) at 485 nm, thus making this methodology possible on conventional SEC equipment through the in-line absorbance spectrometer. The results demonstrate that absorption is in great correlation with fluorescence of GFP. The comparably weaker absorption signal can be improved by using a longer path-length flow cell. The FA-SEC profiles were congruent with the ones plotted by FSEC, suggesting FA-SEC could be a comparable and economical setup for detergent screening of membrane proteins.

Development of an analytical method coupling cell membrane chromatography with gas chromatography-mass spectrometry via microextraction by packed sorbent and its application in the screening of volatile active compounds in natural products.

PubMed

Li, Miao; Wang, Sicen; He, Langchong

2015-01-01

Natural products (NPs) are important sources of lead compounds in modern drug discovery. To facilitate the screening of volatile active compounds in NPs, we have developed a new biochromatography method that uses rat vascular smooth muscle cells (VSMC), which are rich in L-type calcium channels (LCC), to prepare the stationary phase. This integrated method, which couples cell membrane chromatography (CMC) with gas chromatography-mass spectrometry (GC-MS) via microextraction by packed sorbent (MEPS) technology, has been termed VSMC/CMC-MEPS-GC-MS. Methodological validation confirmed its specificity, reliability and convenience. Screening results for Radix Angelicae Dahuricae and Fructus Cnidii obtained using VSMC/CMC-MEPS-GC-MS were consistent with those obtained using VSMC/CMC-offline-GC-MS. MEPS connection plays as simplified solid-phase extraction and replaces the uncontrollable evaporation operation in reported offline connections, so our new method is supposed to be more efficient and reliable than the offline ones, especially for compounds that are volatile, thermally unstable or difficult to purify. In application, senkyunolide A and ligustilide were preliminary identified as the volatile active components in Rhizoma Chuanxiong. We have thus confirmed the suitability of VSMC/CMC-MEPS-GC-MS for volatile active compounds screening in NP. Copyright © 2014 Elsevier B.V. All rights reserved.

Using Functional Signature Ontology (FUSION) to Identify Mechanisms of Action for Natural Products

PubMed Central

Potts, Malia B.; Kim, Hyun Seok; Fisher, Kurt W.; Hu, Youcai; Carrasco, Yazmin P.; Bulut, Gamze Betul; Ou, Yi-Hung; Herrera-Herrera, Mireya L.; Cubillos, Federico; Mendiratta, Saurabh; Xiao, Guanghua; Hofree, Matan; Ideker, Trey; Xie, Yang; Huang, Lily Jun-shen; Lewis, Robert E.; MacMillan, John B.; White, Michael A.

2014-01-01

A challenge for biomedical research is the development of pharmaceuticals that appropriately target disease mechanisms. Natural products can be a rich source of bioactive chemicals for medicinal applications but can act through unknown mechanisms and can be difficult to produce or obtain. To address these challenges, we developed a new marine-derived, renewable natural products resource and a method for linking bioactive derivatives of this library to the proteins and biological processes that they target in cells. We used cell-based screening and computational analysis to match gene expression signatures produced by natural products to those produced by siRNA and synthetic microRNA libraries. With this strategy, we matched proteins and microRNAs with diverse biological processes and also identified putative protein targets and mechanisms of action for several previously undescribed marine-derived natural products. We confirmed mechanistic relationships for selected short-interfering RNAs, microRNAs, and compounds with functional roles in autophagy, chemotaxis mediated by discoidin domain receptor 2, or activation of the kinase AKT. Thus, this approach may be an effective method for screening new drugs while simultaneously identifying their targets. PMID:24129700

A high-throughput platform for population reformatting and mammalian expression of phage display libraries to enable functional screening as full-length IgG.

PubMed

Xiao, Xiaodong; Douthwaite, Julie A; Chen, Yan; Kemp, Ben; Kidd, Sara; Percival-Alwyn, Jennifer; Smith, Alison; Goode, Kate; Swerdlow, Bonnie; Lowe, David; Wu, Herren; Dall'Acqua, William F; Chowdhury, Partha S

Phage display antibody libraries are a rich resource for discovery of potential therapeutic antibodies. Single-chain variable fragment (scFv) libraries are the most common format due to the efficient display of scFv by phage particles and the ease by which soluble scFv antibodies can be expressed for high-throughput screening. Typically, a cascade of screening and triaging activities are performed, beginning with the assessment of large numbers of E. coli-expressed scFv, and progressing through additional assays with individual reformatting of the most promising scFv to full-length IgG. However, use of high-throughput screening of scFv for the discovery of full-length IgG is not ideal because of the differences between these molecules. Furthermore, the reformatting step represents a bottle neck in the process because each antibody has to be handled individually to preserve the unique VH and VL pairing. These problems could be resolved if populations of scFv could be reformatted to full-length IgG before screening without disrupting the variable region pairing. Here, we describe a novel strategy that allows the reformatting of diverse populations of scFv from phage selections to full-length IgG in a batch format. The reformatting process maintains the diversity and variable region pairing with high fidelity, and the resulted IgG pool enables high-throughput expression of IgG in mammalian cells and cell-based functional screening. The improved process led to the discovery of potent candidates that are comparable or better than those obtained by traditional methods. This strategy should also be readily applicable to Fab-based phage libraries. Our approach, Screening in Product Format (SiPF), represents a substantial improvement in the field of antibody discovery using phage display.

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells.

PubMed

Casanova, Alain; Low, Shyan H; Emmenlauer, Mario; Conde-Alvarez, Raquel; Salcedo, Suzana P; Gorvel, Jean-Pierre; Dehio, Christoph

2016-08-05

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections. Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring. In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells. In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular bacteria based on the GFP signal, with only intracellular bacteria being able to express GFP. This allows the robust detection of single intracellular bacteria before intracellular proliferation is initiated.

Identification of Inhibitors of the Association of ZAP-70 with the T Cell Receptor by High-Throughput Screen

DOE PAGES

Visperas, Patrick R.; Wilson, Christopher G.; Winger, Jonathan A.; ...

2016-12-13

ZAP-70 is a critical molecule in the transduction of T cell antigen receptor signaling and the activation of T cells. Upon activation of the T cell antigen receptor, ZAP-70 is recruited to the intracellular ζ-chains of the T cell receptor, where ZAP-70 is activated and colocalized with its substrates. Inhibitors of ZAP-70 could potentially function as treatments for autoimmune diseases or organ transplantation. In this work, we present the design, optimization, and implementation of a screen for inhibitors that would disrupt the interaction between ZAP-70 and the T cell antigen receptor. Finally, the screen is based on a fluorescence polarizationmore » assay for peptide binding to ZAP-70.« less

A dual reporter cell assay for identifying serotype and drug susceptibility of herpes simplex virus.

PubMed

Lu, Wen-Wen; Sun, Jun-Ren; Wu, Szu-Sian; Lin, Wan-Hsuan; Kung, Szu-Hao

2011-08-15

A dual reporter cell assay (DRCA) that allows real-time detection of herpes simplex virus (HSV) infection was developed. This was achieved by stable transfection of cells with an expression cassette that contains the dual reporter genes, secreted alkaline phosphatase (SEAP) and enhanced green fluorescent protein (EGFP), under the control of an HSV early gene promoter. Baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cell lines were used as parental cell lines because the former is permissive for both HSV serotypes, HSV-1 and HSV-2, whereas the latter is susceptible to infection only by HSV-2. The DRCA permitted differential detection of HSV-1 and HSV-2 by observation of EGFP-positive cells, as substantiated by screening a total of 35 samples. The BHK-based cell line is sensitive to a viral titer as low as a single plaque-forming unit with a robust assay window as measured by a chemiluminescent assay. Evaluations of the DRCA with representative acyclovir-sensitive and acyclovir-resistant HSV strains demonstrated that their drug susceptibilities were accurately determined by a 48-h format. In summary, this novel DRCA is a useful means for serotyping of HSV in real time as well as a rapid screening method for determining anti-HSV susceptibilities. Copyright © 2011 Elsevier Inc. All rights reserved.

Immune cell screening of a nanoparticle library improves atherosclerosis therapy

PubMed Central

Baxter, Samantha; Menon, Arjun; Alaarg, Amr; Sanchez-Gaytan, Brenda L.; Fay, Francois; Zhao, Yiming; Ouimet, Mireille; Braza, Mounia S.; Longo, Valerie A.; Abdel-Atti, Dalya; Duivenvoorden, Raphael; Calcagno, Claudia; Storm, Gert; Tsimikas, Sotirios; Moore, Kathryn J.; Swirski, Filip K.; Nahrendorf, Matthias; Fisher, Edward A.; Pérez-Medina, Carlos; Fayad, Zahi A.; Reiner, Thomas; Mulder, Willem J. M.

2016-01-01

Immunological complexity in atherosclerosis warrants targeted treatment of specific inflammatory cells that aggravate the disease. With the initiation of large phase III trials investigating immunomodulatory drugs for atherosclerosis, cardiovascular disease treatment enters a new era. We here propose a radically different approach: implementing and evaluating in vivo a combinatorial library of nanoparticles with distinct physiochemical properties and differential immune cell specificities. The library’s nanoparticles are based on endogenous high-density lipoprotein, which can preferentially deliver therapeutic compounds to pathological macrophages in atherosclerosis. Using the apolipoprotein E-deficient (Apoe−/−) mouse model of atherosclerosis, we quantitatively evaluated the library’s immune cell specificity by combining immunological techniques and in vivo positron emission tomography imaging. Based on this screen, we formulated a liver X receptor agonist (GW3965) and abolished its liver toxicity while still preserving its therapeutic function. Screening the immune cell specificity of nanoparticles can be used to develop tailored therapies for atherosclerosis and other inflammatory diseases. PMID:27791119

High-throughput identification of small molecules that affect human embryonic vascular development

PubMed Central

Vazão, Helena; Rosa, Susana; Barata, Tânia; Costa, Ricardo; Pitrez, Patrícia R.; Honório, Inês; de Vries, Margreet R.; Papatsenko, Dimitri; Benedito, Rui; Saris, Daniel; Khademhosseini, Ali; Quax, Paul H. A.; Pereira, Carlos F.; Mercader, Nadia; Ferreira, Lino

2017-01-01

Birth defects, which are in part caused by exposure to environmental chemicals and pharmaceutical drugs, affect 1 in every 33 babies born in the United States each year. The current standard to screen drugs that affect embryonic development is based on prenatal animal testing; however, this approach yields low-throughput and limited mechanistic information regarding the biological pathways and potential adverse consequences in humans. To develop a screening platform for molecules that affect human embryonic development based on endothelial cells (ECs) derived from human pluripotent stem cells, we differentiated human pluripotent stem cells into embryonic ECs and induced their maturation under arterial flow conditions. These cells were then used to screen compounds that specifically affect embryonic vasculature. Using this platform, we have identified two compounds that have higher inhibitory effect in embryonic than postnatal ECs. One of them was fluphenazine (an antipsychotic), which inhibits calmodulin kinase II. The other compound was pyrrolopyrimidine (an antiinflammatory agent), which inhibits vascular endothelial growth factor receptor 2 (VEGFR2), decreases EC viability, induces an inflammatory response, and disrupts preformed vascular networks. The vascular effect of the pyrrolopyrimidine was further validated in prenatal vs. adult mouse ECs and in embryonic and adult zebrafish. We developed a platform based on human pluripotent stem cell-derived ECs for drug screening, which may open new avenues of research for the study and modulation of embryonic vasculature. PMID:28348206

High-throughput identification of small molecules that affect human embryonic vascular development.

PubMed

Vazão, Helena; Rosa, Susana; Barata, Tânia; Costa, Ricardo; Pitrez, Patrícia R; Honório, Inês; de Vries, Margreet R; Papatsenko, Dimitri; Benedito, Rui; Saris, Daniel; Khademhosseini, Ali; Quax, Paul H A; Pereira, Carlos F; Mercader, Nadia; Fernandes, Hugo; Ferreira, Lino

2017-04-11

Birth defects, which are in part caused by exposure to environmental chemicals and pharmaceutical drugs, affect 1 in every 33 babies born in the United States each year. The current standard to screen drugs that affect embryonic development is based on prenatal animal testing; however, this approach yields low-throughput and limited mechanistic information regarding the biological pathways and potential adverse consequences in humans. To develop a screening platform for molecules that affect human embryonic development based on endothelial cells (ECs) derived from human pluripotent stem cells, we differentiated human pluripotent stem cells into embryonic ECs and induced their maturation under arterial flow conditions. These cells were then used to screen compounds that specifically affect embryonic vasculature. Using this platform, we have identified two compounds that have higher inhibitory effect in embryonic than postnatal ECs. One of them was fluphenazine (an antipsychotic), which inhibits calmodulin kinase II. The other compound was pyrrolopyrimidine (an antiinflammatory agent), which inhibits vascular endothelial growth factor receptor 2 (VEGFR2), decreases EC viability, induces an inflammatory response, and disrupts preformed vascular networks. The vascular effect of the pyrrolopyrimidine was further validated in prenatal vs. adult mouse ECs and in embryonic and adult zebrafish. We developed a platform based on human pluripotent stem cell-derived ECs for drug screening, which may open new avenues of research for the study and modulation of embryonic vasculature.

Genetic and epigenetic markers in colorectal cancer screening: recent advances.

PubMed

Singh, Manish Pratap; Rai, Sandhya; Suyal, Shradha; Singh, Sunil Kumar; Singh, Nand Kumar; Agarwal, Akash; Srivastava, Sameer

2017-07-01

Colorectal cancer (CRC) is a heterogenous disease which develops from benign intraepithelial lesions known as adenomas to malignant carcinomas. Acquired alterations in Wnt signaling, TGFβ, MAPK pathway genes and clonal propagation of altered cells are responsible for this transformation. Detection of adenomas or early stage cancer in asymptomatic patients and better prognostic and predictive markers is important for improving the clinical management of CRC. Area covered: In this review, the authors have evaluated the potential of genetic and epigenetic alterations as markers for early detection, prognosis and therapeutic predictive potential in the context of CRC. We have discussed molecular heterogeneity present in CRC and its correlation to prognosis and response to therapy. Expert commentary: Molecular marker based CRC screening methods still fail to gain trust of clinicians. Invasive screening methods, molecular heterogeneity, chemoresistance and low quality test samples are some key challenges which need to be addressed in the present context. New sequencing technologies and integrated omics data analysis of individual or population cohort results in GWAS. MPE studies following a GWAS could be future line of research to establish accurate correlations between CRC and its risk factors. This strategy would identify most reliable biomarkers for CRC screening and management.

A Novel Dual Expression Platform for High Throughput Functional Screening of Phage Libraries in Product like Format.

PubMed

Xiao, Xiaodong; Chen, Yan; Mugabe, Sheila; Gao, Changshou; Tkaczyk, Christine; Mazor, Yariv; Pavlik, Peter; Wu, Herren; Dall'Acqua, William; Chowdhury, Partha Sarathi

2015-01-01

High throughput screenings of single chain Fv (scFv) antibody phage display libraries are currently done as soluble scFvs produced in E.coli. Due to endotoxin contaminations from bacterial cells these preparations cannot be reliably used in mammalian cell based assays. The monovalent nature and lack of Fc in soluble scFvs prevent functional assays that are dependent on target cross linking and/or Fc functions. A convenient approach is to convert scFvs into scFv.Fc fusion proteins and express them in mammalian cell lines for screening. This approach is low throughput and is only taken after primary screening of monovalent scFvs that are expressed in bacteria. There is no platform at present that combines the benefits of both bacterial and mammalian expression system for screening phage library output. We have, therefore, developed a novel dual expression vector, called pSplice, which can be used to express scFv.Fc fusion proteins both in E.coli and mammalian cell lines. The hallmark of the vector is an engineered intron which houses the bacterial promoter and signal peptide for expression and secretion of scFv.Fc in E.coli. When the vector is transfected into a mammalian cell line, the intron is efficiently spliced out resulting in a functional operon for expression and secretion of the scFv.Fc fusion protein into the culture medium. By applying basic knowledge of mammalian introns and splisosome, we designed this vector to enable screening of phage libraries in a product like format. Like IgG, the scFv.Fc fusion protein is bi-valent for the antigen and possesses Fc effector functions. Expression in E.coli maintains the speed of the bacterial expression platform and is used to triage clones based on binding and other assays that are not sensitive to endotoxin. Triaged clones are then expressed in a mammalian cell line without the need for any additional cloning steps. Conditioned media from the mammalian cell line containing the fusion proteins are then used for different types of cell based assays. Thus this system retains the speed of the current screening system for phage libraries and adds additional functionality to it.

Biomek Cell Workstation: A Variable System for Automated Cell Cultivation.

PubMed

Lehmann, R; Severitt, J C; Roddelkopf, T; Junginger, S; Thurow, K

2016-06-01

Automated cell cultivation is an important tool for simplifying routine laboratory work. Automated methods are independent of skill levels and daily constitution of laboratory staff in combination with a constant quality and performance of the methods. The Biomek Cell Workstation was configured as a flexible and compatible system. The modified Biomek Cell Workstation enables the cultivation of adherent and suspension cells. Until now, no commercially available systems enabled the automated handling of both types of cells in one system. In particular, the automated cultivation of suspension cells in this form has not been published. The cell counts and viabilities were nonsignificantly decreased for cells cultivated in AutoFlasks in automated handling. The proliferation of manual and automated bioscreening by the WST-1 assay showed a nonsignificant lower proliferation of automatically disseminated cells associated with a mostly lower standard error. The disseminated suspension cell lines showed different pronounced proliferations in descending order, starting with Jurkat cells followed by SEM, Molt4, and RS4 cells having the lowest proliferation. In this respect, we successfully disseminated and screened suspension cells in an automated way. The automated cultivation and dissemination of a variety of suspension cells can replace the manual method. © 2015 Society for Laboratory Automation and Screening.

Rapid screening and identification of dominant B cell epitopes of HBV surface antigen by quantum dot-based fluorescence polarization assay

NASA Astrophysics Data System (ADS)

Meng, Zhongji; Song, Ruihua; Chen, Yue; Zhu, Yang; Tian, Yanhui; Li, Ding; Cui, Daxiang

2013-03-01

A method for quickly screening and identifying dominant B cell epitopes was developed using hepatitis B virus (HBV) surface antigen as a target. Eleven amino acid fragments from HBV surface antigen were synthesized by 9-fluorenylmethoxy carbonyl solid-phase peptide synthesis strategy, and then CdTe quantum dots were used to label the N-terminals of all peptides. After optimizing the factors for fluorescence polarization (FP) immunoassay, the antigenicities of synthetic peptides were determined by analyzing the recognition and combination of peptides and standard antibody samples. The results of FP assays confirmed that 10 of 11 synthetic peptides have distinct antigenicities. In order to screen dominant antigenic peptides, the FP assays were carried out to investigate the antibodies against the 10 synthetic peptides of HBV surface antigen respectively in 159 samples of anti-HBV surface antigen-positive antiserum. The results showed that 3 of the 10 antigenic peptides may be immunodominant because the antibodies against them existed more widely among the samples and their antibody titers were higher than those of other peptides. Using three dominant antigenic peptides, 293 serum samples were detected for HBV infection by FP assays; the results showed that the antibody-positive ratio was 51.9% and the sensitivity and specificity were 84.3% and 98.2%, respectively. In conclusion, a quantum dot-based FP assay is a very simple, rapid, and convenient method for determining immunodominant antigenic peptides and has great potential in applications such as epitope mapping, vaccine designing, or clinical disease diagnosis in the future.

Developing a novel fiber optic fluorescence device for multiplexed high-throughput cytotoxic screening.

PubMed

Lee, Dennis; Barnes, Stephen

2010-01-01

The need for new pharmacological agents is unending. Yet the drug discovery process has changed substantially over the past decade and continues to evolve in response to new technologies. There is presently a high demand to reduce discovery time by improving specific lab disciplines and developing new technology platforms in the area of cell-based assay screening. Here we present the developmental concept and early stage testing of the Ab-Sniffer, a novel fiber optic fluorescence device for high-throughput cytotoxicity screening using an immobilized whole cell approach. The fused silica fibers are chemically functionalized with biotin to provide interaction with fluorescently labeled, streptavidin functionalized alginate-chitosan microspheres. The microspheres are also functionalized with Concanavalin A to facilitate binding to living cells. By using lymphoma cells and rituximab in an adaptation of a well-known cytotoxicity protocol we demonstrate the utility of the Ab-Sniffer for functional screening of potential drug compounds rather than indirect, non-functional screening via binding assay. The platform can be extended to any assay capable of being tied to a fluorescence response including multiple target cells in each well of a multi-well plate for high-throughput screening.

Curcumin Based Drug Screening for Inhibitors of NF kappa B in a Cell Model of Prostate Cancer Progression

DTIC Science & Technology

2008-02-01

West Society of Toxicology in Breckenridge, CO in September 2007: “Identification of Curcumin Analogs Toxic against Prostate Cancer Cells Through...quantitative structure-activity relationship ( QSAR ) and ligand-based virtual screening (LBVS) to explore the possibility of improving their efficacy...Student in my laboratory has presented part of this data at the 25th Annual Meeting of the Mountain West Society of Toxicology in Breckenridge, CO in

Visual and efficient immunosensor technique for advancing biomedical applications of quantum dots on Salmonella detection and isolation

NASA Astrophysics Data System (ADS)

Tang, Feng; Pang, Dai-Wen; Chen, Zhi; Shao, Jian-Bo; Xiong, Ling-Hong; Xiang, Yan-Ping; Xiong, Yan; Wu, Kai; Ai, Hong-Wu; Zhang, Hui; Zheng, Xiao-Li; Lv, Jing-Rui; Liu, Wei-Yong; Hu, Hong-Bing; Mei, Hong; Zhang, Zhen; Sun, Hong; Xiang, Yun; Sun, Zi-Yong

2016-02-01

It is a great challenge in nanotechnology for fluorescent nanobioprobes to be applied to visually detect and directly isolate pathogens in situ. A novel and visual immunosensor technique for efficient detection and isolation of Salmonella was established here by applying fluorescent nanobioprobes on a specially-designed cellulose-based swab (a solid-phase enrichment system). The selective and chromogenic medium used on this swab can achieve the ultrasensitive amplification of target bacteria and form chromogenic colonies in situ based on a simple biochemical reaction. More importantly, because this swab can serve as an attachment site for the targeted pathogens to immobilize and immunologically capture nanobioprobes, our mAb-conjugated QD bioprobes were successfully applied on the solid-phase enrichment system to capture the fluorescence of targeted colonies under a designed excitation light instrument based on blue light-emitting diodes combined with stereomicroscopy or laser scanning confocal microscopy. Compared with the traditional methods using 4-7 days to isolate Salmonella from the bacterial mixture, this method took only 2 days to do this, and the process of initial screening and preliminary diagnosis can be completed in only one and a half days. Furthermore, the limit of detection can reach as low as 101 cells per mL Salmonella on the background of 105 cells per mL non-Salmonella (Escherichia coli, Proteus mirabilis or Citrobacter freundii, respectively) in experimental samples, and even in human anal ones. The visual and efficient immunosensor technique may be proved to be a favorable alternative for screening and isolating Salmonella in a large number of samples related to public health surveillance.It is a great challenge in nanotechnology for fluorescent nanobioprobes to be applied to visually detect and directly isolate pathogens in situ. A novel and visual immunosensor technique for efficient detection and isolation of Salmonella was established here by applying fluorescent nanobioprobes on a specially-designed cellulose-based swab (a solid-phase enrichment system). The selective and chromogenic medium used on this swab can achieve the ultrasensitive amplification of target bacteria and form chromogenic colonies in situ based on a simple biochemical reaction. More importantly, because this swab can serve as an attachment site for the targeted pathogens to immobilize and immunologically capture nanobioprobes, our mAb-conjugated QD bioprobes were successfully applied on the solid-phase enrichment system to capture the fluorescence of targeted colonies under a designed excitation light instrument based on blue light-emitting diodes combined with stereomicroscopy or laser scanning confocal microscopy. Compared with the traditional methods using 4-7 days to isolate Salmonella from the bacterial mixture, this method took only 2 days to do this, and the process of initial screening and preliminary diagnosis can be completed in only one and a half days. Furthermore, the limit of detection can reach as low as 101 cells per mL Salmonella on the background of 105 cells per mL non-Salmonella (Escherichia coli, Proteus mirabilis or Citrobacter freundii, respectively) in experimental samples, and even in human anal ones. The visual and efficient immunosensor technique may be proved to be a favorable alternative for screening and isolating Salmonella in a large number of samples related to public health surveillance. Electronic supplementary information (ESI) available: One additional figure (Fig. S1), two additional tables (Tables S1 and S2) and additional information. See DOI: 10.1039/c5nr07424j

High-Content, High-Throughput Screening for the Identification of Cytotoxic Compounds Based on Cell Morphology and Cell Proliferation Markers

PubMed Central

Martin, Heather L.; Adams, Matthew; Higgins, Julie; Bond, Jacquelyn; Morrison, Ewan E.; Bell, Sandra M.; Warriner, Stuart; Nelson, Adam; Tomlinson, Darren C.

2014-01-01

Toxicity is a major cause of failure in drug discovery and development, and whilst robust toxicological testing occurs, efficiency could be improved if compounds with cytotoxic characteristics were identified during primary compound screening. The use of high-content imaging in primary screening is becoming more widespread, and by utilising phenotypic approaches it should be possible to incorporate cytotoxicity counter-screens into primary screens. Here we present a novel phenotypic assay that can be used as a counter-screen to identify compounds with adverse cellular effects. This assay has been developed using U2OS cells, the PerkinElmer Operetta high-content/high-throughput imaging system and Columbus image analysis software. In Columbus, algorithms were devised to identify changes in nuclear morphology, cell shape and proliferation using DAPI, TOTO-3 and phosphohistone H3 staining, respectively. The algorithms were developed and tested on cells treated with doxorubicin, taxol and nocodazole. The assay was then used to screen a novel, chemical library, rich in natural product-like molecules of over 300 compounds, 13.6% of which were identified as having adverse cellular effects. This assay provides a relatively cheap and rapid approach for identifying compounds with adverse cellular effects during screening assays, potentially reducing compound rejection due to toxicity in subsequent in vitro and in vivo assays. PMID:24505478

Rapid identification and quantification of tumor cells using an electrocatalytic method based on gold nanoparticles.

PubMed

de la Escosura-Muñiz, Alfredo; Sánchez-Espinel, Christian; Díaz-Freitas, Belén; González-Fernández, Africa; Maltez-da Costa, Marisa; Merkoçi, Arben

2009-12-15

There is a high demand for simple, rapid, efficient, and user-friendly alternative methods for the detection of cells in general and, in particular, for the detection of cancer cells. A biosensor able to detect cells would be an all-in-one dream device for such applications. The successful integration of nanoparticles into cell detection assays could allow for the development of this novel class of cell sensors. Indeed, their application could well have a great future in diagnostics, as well as other fields. As an example of a novel biosensor, we report here an electrocatalytic device for the specific identification of tumor cells that quantifies gold nanoparticles (AuNPs) coupled with an electrotransducing platform/sensor. Proliferation and adherence of tumor cells are achieved on the electrotransducer/detector, which consists of a mass-produced screen-printed carbon electrode (SPCE). In situ identification/quantification of tumor cells is achieved with a detection limit of 4000 cells per 700 microL of suspension. This novel and selective cell-sensing device is based on the reaction of cell surface proteins with specific antibodies conjugated with AuNPs. Final detection requires only a couple of minutes, taking advantage of the catalytic properties of AuNPs on hydrogen evolution. The proposed detection method does not require the chemical agents used in most existing assays for the detection of AuNPs. It allows for the miniaturization of the system and is much cheaper than other expensive and sophisticated methods used for tumor cell detection. We envisage that this device could operate in a simple way as an immunosensor or DNA sensor. Moreover, it could be used, even by inexperienced staff, for the detection of protein molecules or DNA strands.

Non-invasive imaging of oxygen extraction fraction in adults with sickle cell anaemia.

PubMed

Jordan, Lori C; Gindville, Melissa C; Scott, Allison O; Juttukonda, Meher R; Strother, Megan K; Kassim, Adetola A; Chen, Sheau-Chiann; Lu, Hanzhang; Pruthi, Sumit; Shyr, Yu; Donahue, Manus J

2016-03-01

Sickle cell anaemia is a monogenetic disorder with a high incidence of stroke. While stroke screening procedures exist for children with sickle cell anaemia, no accepted screening procedures exist for assessing stroke risk in adults. The purpose of this study is to use novel magnetic resonance imaging methods to evaluate physiological relationships between oxygen extraction fraction, cerebral blood flow, and clinical markers of cerebrovascular impairment in adults with sickle cell anaemia. The specific goal is to determine to what extent elevated oxygen extraction fraction may be uniquely present in patients with higher levels of clinical impairment and therefore may represent a candidate biomarker of stroke risk. Neurological evaluation, structural imaging, and the non-invasive T2-relaxation-under-spin-tagging magnetic resonance imaging method were applied in sickle cell anaemia (n = 34) and healthy race-matched control (n = 11) volunteers without sickle cell trait to assess whole-brain oxygen extraction fraction, cerebral blood flow, degree of vasculopathy, severity of anaemia, and presence of prior infarct; findings were interpreted in the context of physiological models. Cerebral blood flow and oxygen extraction fraction were elevated (P < 0.05) in participants with sickle cell anaemia (n = 27) not receiving monthly blood transfusions (interquartile range cerebral blood flow = 46.2-56.8 ml/100 g/min; oxygen extraction fraction = 0.39-0.50) relative to controls (interquartile range cerebral blood flow = 40.8-46.3 ml/100 g/min; oxygen extraction fraction = 0.33-0.38). Oxygen extraction fraction (P < 0.0001) but not cerebral blood flow was increased in participants with higher levels of clinical impairment. These data provide support for T2-relaxation-under-spin-tagging being able to quickly and non-invasively detect elevated oxygen extraction fraction in individuals with sickle cell anaemia with higher levels of clinical impairment. Our results support the premise that magnetic resonance imaging-based assessment of elevated oxygen extraction fraction might be a viable screening tool for evaluating stroke risk in adults with sickle cell anaemia. © The Author (2016). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Non-invasive imaging of oxygen extraction fraction in adults with sickle cell anaemia

PubMed Central

Gindville, Melissa C.; Scott, Allison O.; Juttukonda, Meher R.; Strother, Megan K.; Kassim, Adetola A.; Chen, Sheau-Chiann; Lu, Hanzhang; Pruthi, Sumit; Shyr, Yu; Donahue, Manus J.

2016-01-01

Sickle cell anaemia is a monogenetic disorder with a high incidence of stroke. While stroke screening procedures exist for children with sickle cell anaemia, no accepted screening procedures exist for assessing stroke risk in adults. The purpose of this study is to use novel magnetic resonance imaging methods to evaluate physiological relationships between oxygen extraction fraction, cerebral blood flow, and clinical markers of cerebrovascular impairment in adults with sickle cell anaemia. The specific goal is to determine to what extent elevated oxygen extraction fraction may be uniquely present in patients with higher levels of clinical impairment and therefore may represent a candidate biomarker of stroke risk. Neurological evaluation, structural imaging, and the non-invasive T2-relaxation-under-spin-tagging magnetic resonance imaging method were applied in sickle cell anaemia (n = 34) and healthy race-matched control (n = 11) volunteers without sickle cell trait to assess whole-brain oxygen extraction fraction, cerebral blood flow, degree of vasculopathy, severity of anaemia, and presence of prior infarct; findings were interpreted in the context of physiological models. Cerebral blood flow and oxygen extraction fraction were elevated (P < 0.05) in participants with sickle cell anaemia (n = 27) not receiving monthly blood transfusions (interquartile range cerebral blood flow = 46.2–56.8 ml/100 g/min; oxygen extraction fraction = 0.39–0.50) relative to controls (interquartile range cerebral blood flow = 40.8–46.3 ml/100 g/min; oxygen extraction fraction = 0.33–0.38). Oxygen extraction fraction (P < 0.0001) but not cerebral blood flow was increased in participants with higher levels of clinical impairment. These data provide support for T2-relaxation-under-spin-tagging being able to quickly and non-invasively detect elevated oxygen extraction fraction in individuals with sickle cell anaemia with higher levels of clinical impairment. Our results support the premise that magnetic resonance imaging-based assessment of elevated oxygen extraction fraction might be a viable screening tool for evaluating stroke risk in adults with sickle cell anaemia. PMID:26823369

Evaluation of the antioxidants activities of four Slovene medicinal plant species by traditional and novel biosensory assays.

PubMed

Kintzios, Spiridon; Papageorgiou, Katerina; Yiakoumettis, Iakovos; Baricevic, Dea; Kusar, Anita

2010-11-02

We investigated the antioxidant activity of methanolic and water extracts of Slovene accessions of four medicinal plant species (Salvia officinalis, Achillea millefolium, Origanum vulgare subsp. vulgare and Gentiana lutea). Their free radical-scavenging activity against the DPPH. free radical was studied with a spectrophotometric assay, while their biological activity with the help of a laboratory-made biosensor based on immobilized fibroblast cells (assay duration: 3 min). The observed antioxidant activity of the extracts from the four investigated medicinal plant species was dependent on both the solvent used for extraction and the assay method (conventional or biosensor-based). Independently from the assay method and the solvent used for extraction, the lowest scavenging activity was observed in root extracts of G. lutea. Treatment of the immobilized cells with the plant extracts resulted in an increase of the cell membrane potential (membrane hyperpolarization), possibly due to the reduction of membrane damage due to oxidation. The novel cell biosensor could be utilized as a rapid, high throughput tool for screening the antioxidant properties of plant-derived compounds. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Short tandem repeat profiling: part of an overall strategy for reducing the frequency of cell misidentification.

PubMed

Nims, Raymond W; Sykes, Greg; Cottrill, Karin; Ikonomi, Pranvera; Elmore, Eugene

2010-12-01

The role of cell authentication in biomedical science has received considerable attention, especially within the past decade. This quality control attribute is now beginning to be given the emphasis it deserves by granting agencies and by scientific journals. Short tandem repeat (STR) profiling, one of a few DNA profiling technologies now available, is being proposed for routine identification (authentication) of human cell lines, stem cells, and tissues. The advantage of this technique over methods such as isoenzyme analysis, karyotyping, human leukocyte antigen typing, etc., is that STR profiling can establish identity to the individual level, provided that the appropriate number and types of loci are evaluated. To best employ this technology, a standardized protocol and a data-driven, quality-controlled, and publically searchable database will be necessary. This public STR database (currently under development) will enable investigators to rapidly authenticate human-based cultures to the individual from whom the cells were sourced. Use of similar approaches for non-human animal cells will require developing other suitable loci sets. While implementing STR analysis on a more routine basis should significantly reduce the frequency of cell misidentification, additional technologies may be needed as part of an overall authentication paradigm. For instance, isoenzyme analysis, PCR-based DNA amplification, and sequence-based barcoding methods enable rapid confirmation of a cell line's species of origin while screening against cross-contaminations, especially when the cells present are not recognized by the species-specific STR method. Karyotyping may also be needed as a supporting tool during establishment of an STR database. Finally, good cell culture practices must always remain a major component of any effort to reduce the frequency of cell misidentification.

Agent-Based Computational Modeling to Examine How Individual Cell Morphology Affects Dosimetry

EPA Science Inventory

Cell-based models utilizing high-content screening (HCS) data have applications for predictive toxicology. Evaluating concentration-dependent effects on cell fate and state response is a fundamental utilization of HCS data.Although HCS assays may capture quantitative readouts at ...

Pathway-selective sensitization of Mycobacterium tuberculosis for target-based whole-cell screening

PubMed Central

Abrahams, Garth L.; Kumar, Anuradha; Savvi, Suzana; Hung, Alvin W.; Wen, Shijun; Abell, Chris; Barry, Clifton E.; Sherman, David R.; Boshoff, Helena I.M.; Mizrahi, Valerie

2012-01-01

SUMMARY Whole-cell screening of Mycobacterium tuberculosis (Mtb) remains a mainstay of drug discovery but subsequent target elucidation often proves difficult. Conditional mutants that under-express essential genes have been used to identify compounds with known mechanism of action by target-based whole-cell screening (TB-WCS). Here, the feasibility of TB-WCS in Mtb was assessed by generating mutants that conditionally express pantothenate synthetase (panC), diaminopimelate decarboxylase (lysA) and isocitrate lyase (icl1). The essentiality of panC and lysA, and conditional essentiality of icl1 for growth on fatty acids, was confirmed. Depletion of PanC and Icl1 rendered the mutants hypersensitive to target-specific inhibitors. Stable reporter strains were generated for use in high-throughput screening, and their utility demonstrated by identifying compounds that display greater potency against a PanC-depleted strain. These findings illustrate the power of TB-WCS as a tool for tuberculosis drug discovery. PMID:22840772

Defined, serum/feeder-free conditions for expansion and drug screening of primary B-acute lymphoblastic leukemia.

PubMed

Jiang, Zhiwu; Wu, Di; Ye, Wei; Weng, Jianyu; Lai, Peilong; Shi, Pengcheng; Guo, Xutao; Huang, Guohua; Deng, Qiuhua; Tang, Yanlai; Zhao, Hongyu; Cui, Shuzhong; Lin, Simiao; Wang, Suna; Li, Baiheng; Wu, Qiting; Li, Yangqiu; Liu, Pentao; Pei, Duanqing; Du, Xin; Yao, Yao; Li, Peng

2017-12-05

Functional screening for compounds represents a major hurdle in the development of rational therapeutics for B-acute lymphoblastic leukemia (B-ALL). In addition, using cell lines as valid models for evaluating responses to novel drug therapies raises serious concerns, as cell lines are prone to genotypic/phenotypic drift and loss of heterogeneity in vitro . Here, we reported that OP9 cells, not OP9-derived adipocytes (OP9TA), support the growth of primary B-ALL cells in vitro . To identify the factors from OP9 cells that support the growth of primary B-ALL cells, we performed RNA-Seq to analyze the gene expression profiles of OP9 and OP9TA cells. We thus developed a defined, serum/feeder-free condition (FI76V) that can support the expansion of a range of clinically distinct primary B-ALL cells that still maintain their leukemia-initiating ability. We demonstrated the suitability of high-throughput drug screening based on our B-ALL cultured conditions. Upon screening 378 kinase inhibitors, we identified a cluster of 17 kinase inhibitors that can efficiently kill B-ALL cells in vitro . Importantly, we demonstrated the synergistic cytotoxicity of dinaciclib/BTG226 to B-ALL cells. Taken together, we developed a defined condition for the ex vivo expansion of primary B-ALL cells that is suitable for high-throughput screening of novel compounds.

High-throughput combinatorial cell co-culture using microfluidics.

PubMed

Tumarkin, Ethan; Tzadu, Lsan; Csaszar, Elizabeth; Seo, Minseok; Zhang, Hong; Lee, Anna; Peerani, Raheem; Purpura, Kelly; Zandstra, Peter W; Kumacheva, Eugenia

2011-06-01

Co-culture strategies are foundational in cell biology. These systems, which serve as mimics of in vivo tissue niches, are typically poorly defined in terms of cell ratios, local cues and supportive cell-cell interactions. In the stem cell niche, the ability to screen cell-cell interactions and identify local supportive microenvironments has a broad range of applications in transplantation, tissue engineering and wound healing. We present a microfluidic platform for the high-throughput generation of hydrogel microbeads for cell co-culture. Encapsulation of different cell populations in microgels was achieved by introducing in a microfluidic device two streams of distinct cell suspensions, emulsifying the mixed suspension, and gelling the precursor droplets. The cellular composition in the microgels was controlled by varying the volumetric flow rates of the corresponding streams. We demonstrate one of the applications of the microfluidic method by co-encapsulating factor-dependent and responsive blood progenitor cell lines (MBA2 and M07e cells, respectively) at varying ratios, and show that in-bead paracrine secretion can modulate the viability of the factor dependent cells. Furthermore, we show the application of the method as a tool to screen the impact of specific growth factors on a primary human heterogeneous cell population. Co-encapsulation of IL-3 secreting MBA2 cells with umbilical cord blood cells revealed differential sub-population responsiveness to paracrine signals (CD14+ cells were particularly responsive to locally delivered IL-3). This microfluidic co-culture platform should enable high throughput screening of cell co-culture conditions, leading to new strategies to manipulate cell fate. This journal is © The Royal Society of Chemistry 2011

Flow Cytometry: Impact On Early Drug Discovery

PubMed Central

Edwards, Bruce S.; Sklar, Larry A.

2015-01-01

Summary Modern flow cytometers can make optical measurements of 10 or more parameters per cell at tens-of-thousands of cells per second and over five orders of magnitude dynamic range. Although flow cytometry is used in most drug discovery stages, “sip-and-spit” sampling technology has restricted it to low sample throughput applications. The advent of HyperCyt sampling technology has recently made possible primary screening applications in which tens-of-thousands of compounds are analyzed per day. Target-multiplexing methodologies in combination with extended multi-parameter analyses enable profiling of lead candidates early in the discovery process, when the greatest numbers of candidates are available for evaluation. The ability to sample small volumes with negligible waste reduces reagent costs, compound usage and consumption of cells. Improved compound library formatting strategies can further extend primary screening opportunities when samples are scarce. Dozens of targets have been screened in 384- and 1536-well assay formats, predominantly in academic screening lab settings. In concert with commercial platform evolution and trending drug discovery strategies, HyperCyt-based systems are now finding their way into mainstream screening labs. Recent advances in flow-based imaging, mass spectrometry and parallel sample processing promise dramatically expanded single cell profiling capabilities to bolster systems level approaches to drug discovery. PMID:25805180

Screen printed silver top electrode for efficient inverted organic solar cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Junwoo; Duraisamy, Navaneethan; Lee, Taik-Min

2015-10-15

Highlights: • Screen printing of silver pattern. • X-ray diffraction pattern confirmed the face centered cubic structure of silver. • Uniform surface morphology of silver pattern with sheet resistance of 0.06 Ω/sq. • The power conversion efficiency of fabricated solar cell is found to be 2.58%. - Abstract: The present work is mainly focused on replacement of the vacuum process for top electrode fabrication in organic solar cells. Silver top electrode deposited through solution based screen printing on pre-deposited polymeric thin film. The solution based printing technology provides uniform top electrode without damaging the underlying organic layers. The surface crystallinitymore » and surface morphology of silver top electrode are examined through X-ray diffraction, field-emission scanning electron microscope and atomic force microscope. The purity of silver is examined through X-ray energy dispersive spectroscopy. The top electrode exhibits face centered cubic structure with homogeneous morphology. The sheet resistance of top electrode is found to be 0.06 Ω/sq and an average pattern thickness of ∼15 μm. The power conversion efficiency is 2.58%. Our work demonstrates that the solution based screen printing is a significant role in the replacement of vacuum process for the fabrication of top electrode in organic solar cells.« less

Impact of screening colonoscopy on outcomes in colorectal cancer.

PubMed

Matsuda, Takahisa; Ono, Akiko; Kakugawa, Yasuo; Matsumoto, Minori; Saito, Yutaka

2015-10-01

Colorectal cancer is one of the most common cancers in both men and women worldwide and a good candidate for screening programs. There are two modalities of colorectal cancer screening: (i) population-based screening and (ii) opportunistic screening. The first one is based on organized, well-coordinated, monitored and established programs with a systematic invitation covering the entire target population. In contrast, opportunistic screening tests are offered to people who are being examined for other reasons. Recently, a variety of colorectal cancer screening tests have become available; each country should make a choice, based on national demographics and resources, on the screening method to be used. Fecal occult blood test, especially the fecal immunochemical test, would be the best modality for decreasing colorectal cancer mortality through population-based screening. In contrast, if the aim includes the early detection of colorectal cancer and adenomas, endoscopic methods are more appropriate. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Thermal precipitation fluorescence assay for protein stability screening.

PubMed

Fan, Junping; Huang, Bo; Wang, Xianping; Zhang, Xuejun C

2011-09-01

A simple and reliable method of protein stability assessment is desirable for high throughput expression screening of recombinant proteins. Here we described an assay termed thermal precipitation fluorescence (TPF) which can be used to compare thermal stabilities of recombinant protein samples directly from cell lysate supernatants. In this assay, target membrane proteins are expressed as recombinant fusions with a green fluorescence protein tag and solubilized with detergent, and the fluorescence signals are used to report the quantity of the fusion proteins in the soluble fraction of the cell lysate. After applying a heat shock, insoluble protein aggregates are removed by centrifugation. Subsequently, the amount of remaining protein in the supernatant is quantified by in-gel fluorescence analysis and compared to samples without a heat shock treatment. Over 60 recombinant membrane proteins from Escherichia coli were subject to this screening in the presence and absence of a few commonly used detergents, and the results were analyzed. Because no sophisticated protein purification is required, this TPF technique is suitable to high throughput expression screening of recombinant membrane proteins as well as soluble ones and can be used to prioritize target proteins based on their thermal stabilities for subsequent large scale expression and structural studies. Copyright © 2011 Elsevier Inc. All rights reserved.

Generation of diverse neural cell types through direct conversion

PubMed Central

Petersen, Gayle F; Strappe, Padraig M

2016-01-01

A characteristic of neurological disorders is the loss of critical populations of cells that the body is unable to replace, thus there has been much interest in identifying methods of generating clinically relevant numbers of cells to replace those that have been damaged or lost. The process of neural direct conversion, in which cells of one lineage are converted into cells of a neural lineage without first inducing pluripotency, shows great potential, with evidence of the generation of a range of functional neural cell types both in vitro and in vivo, through viral and non-viral delivery of exogenous factors, as well as chemical induction methods. Induced neural cells have been proposed as an attractive alternative to neural cells derived from embryonic or induced pluripotent stem cells, with prospective roles in the investigation of neurological disorders, including neurodegenerative disease modelling, drug screening, and cellular replacement for regenerative medicine applications, however further investigations into improving the efficacy and safety of these methods need to be performed before neural direct conversion becomes a clinically viable option. In this review, we describe the generation of diverse neural cell types via direct conversion of somatic cells, with comparison against stem cell-based approaches, as well as discussion of their potential research and clinical applications. PMID:26981169

Cervical cancer patterns with automation-assisted and conventional cytological screening: a randomized study.

PubMed

Anttila, Ahti; Pokhrel, Arun; Kotaniemi-Talonen, Laura; Hakama, Matti; Malila, Nea; Nieminen, Pekka

2011-03-01

The purpose was to evaluate alternative cytological screening methods in population-based screening for cervical cancer up to cancer incidence and mortality outcome. Automation-assisted screening was compared to conventional cytological screening in a randomized design. The study was based on follow-up of 503,391 women invited in the Finnish cervical cancer screening program during 1999-2003. The endpoints were incident cervical cancer, severe intraepithelial neoplasia and deaths from cervical cancer. One third of the women had been randomly allocated to automation-assisted screening and two thirds to conventional cytology. Information on cervical cancer and severe neoplasia were obtained through 1999-2007 from a linkage between screening and cancer registry files. There were altogether 3.2 million woman-years at risk, and the average follow-up time was 6.3 years. There was no difference in the risk of cervical cancer between the automation-assisted and conventional screening methods; the relative risk (RR) of cervical cancer between the study and control arm was 1.00 (95% confidence interval [CI] = 0.76-1.29) among all invited and 1.08 (95% CI = 0.76-1.51) among women who were test negative at entry. Comparing women who were test negative with nonscreened, RR of cervical cancer incidence was 0.26, 95% CI = 0.19-0.36 and of mortality 0.24 (0.13-0.43). Both methods were valid for screening. Because cervical cancer is rare in our country, we cannot rule out small differences between methods. Evidence on alternative methods for cervical cancer screening is increasing and it is thus feasible to evaluate new methods in large-scale population-based screening programs up to cancer outcome. Copyright © 2010 UICC.

A screen-printed circular-type paper-based glucose/O2 biofuel cell

NASA Astrophysics Data System (ADS)

Shitanda, Isao; Nohara, Saki; Hoshi, Yoshinao; Itagaki, Masayuki; Tsujimura, Seiya

2017-08-01

The printable paper-based enzymatic biofuel cell (PBFC) to directly power small devices is an important objective for realizing cost-effective and disposable energy harvesting devices. In the present study, a screen-printed circular-type PBFC, composed of a series of 5 individual cells, was constructed. The PBFC exhibited the open circuit potential of 2.65 V and maximum power of 350 μW at 1.55 V, which were sufficient to illuminate an LED without requiring a booster circuit. The output voltage of this PBFC can also be easily adjusted as required.

A system and methodology for high-content visual screening of individual intact living cells in suspension

NASA Astrophysics Data System (ADS)

Renaud, Olivier; Heintzmann, Rainer; Sáez-Cirión, Asier; Schnelle, Thomas; Mueller, Torsten; Shorte, Spencer

2007-02-01

Three dimensional imaging provides high-content information from living intact biology, and can serve as a visual screening cue. In the case of single cell imaging the current state of the art uses so-called "axial through-stacking". However, three-dimensional axial through-stacking requires that the object (i.e. a living cell) be adherently stabilized on an optically transparent surface, usually glass; evidently precluding use of cells in suspension. Aiming to overcome this limitation we present here the utility of dielectric field trapping of single cells in three-dimensional electrode cages. Our approach allows gentle and precise spatial orientation and vectored rotation of living, non-adherent cells in fluid suspension. Using various modes of widefield, and confocal microscope imaging we show how so-called "microrotation" can provide a unique and powerful method for multiple point-of-view (three-dimensional) interrogation of intact living biological micro-objects (e.g. single-cells, cell aggregates, and embryos). Further, we show how visual screening by micro-rotation imaging can be combined with micro-fluidic sorting, allowing selection of rare phenotype targets from small populations of cells in suspension, and subsequent one-step single cell cloning (with high-viability). Our methodology combining high-content 3D visual screening with one-step single cell cloning, will impact diverse paradigms, for example cytological and cytogenetic analysis on haematopoietic stem cells, blood cells including lymphocytes, and cancer cells.

Advances in genetic modification of pluripotent stem cells.

PubMed

Fontes, Andrew; Lakshmipathy, Uma

2013-11-15

Genetically engineered stem cells aid in dissecting basic cell function and are valuable tools for drug discovery, in vivo cell tracking, and gene therapy. Gene transfer into pluripotent stem cells has been a challenge due to their intrinsic feature of growing in clusters and hence not amenable to common gene delivery methods. Several advances have been made in the rapid assembly of DNA elements, optimization of culture conditions, and DNA delivery methods. This has lead to the development of viral and non-viral methods for transient or stable modification of cells, albeit with varying efficiencies. Most methods require selection and clonal expansion that demand prolonged culture and are not suited for cells with limited proliferative potential. Choosing the right platform based on preferred length, strength, and context of transgene expression is a critical step. Random integration of the transgene into the genome can be complicated due to silencing or altered regulation of expression due to genomic effects. An alternative to this are site-specific methods that target transgenes followed by screening to identify the genomic loci that support long-term expression with stem cell proliferation and differentiation. A highly precise and accurate editing of the genome driven by homology can be achieved using traditional methods as well as the newer technologies such as zinc finger nuclease, TAL effector nucleases and CRISPR. In this review, we summarize the different genetic engineering methods that have been successfully used to create modified embryonic and induced pluripotent stem cells. © 2013. Published by Elsevier Inc. All rights reserved.

Discovery of a diaminoquinoxaline benzenesulfonamide antagonist of HIV-1 Nef function using a yeast-based phenotypic screen

PubMed Central

2013-01-01

Background HIV-1 Nef is a viral accessory protein critical for AIDS progression. Nef lacks intrinsic catalytic activity and binds multiple host cell signaling proteins, including Hck and other Src-family tyrosine kinases. Nef binding induces constitutive Hck activation that may contribute to HIV pathogenesis by promoting viral infectivity, replication and downregulation of cell-surface MHC-I molecules. In this study, we developed a yeast-based phenotypic screen to identify small molecules that inhibit the Nef-Hck complex. Results Nef-Hck interaction was faithfully reconstituted in yeast cells, resulting in kinase activation and growth arrest. Yeast cells expressing the Nef-Hck complex were used to screen a library of small heterocyclic compounds for their ability to rescue growth inhibition. The screen identified a dihydrobenzo-1,4-dioxin-substituted analog of 2-quinoxalinyl-3-aminobenzene-sulfonamide (DQBS) as a potent inhibitor of Nef-dependent HIV-1 replication and MHC-I downregulation in T-cells. Docking studies predicted direct binding of DQBS to Nef which was confirmed in differential scanning fluorimetry assays with recombinant purified Nef protein. DQBS also potently inhibited the replication of HIV-1 NL4-3 chimeras expressing Nef alleles representative of all M-group HIV-1 clades. Conclusions Our findings demonstrate the utility of a yeast-based growth reversion assay for the identification of small molecule Nef antagonists. Inhibitors of Nef function discovered with this assay, such as DQBS, may complement the activity of current antiretroviral therapies by enabling immune recognition of HIV-infected cells through the rescue of cell surface MHC-I. PMID:24229420

High content screening of defined chemical libraries using normal and glioma-derived neural stem cell lines.

PubMed

Danovi, Davide; Folarin, Amos A; Baranowski, Bart; Pollard, Steven M

2012-01-01

Small molecules with potent biological effects on the fate of normal and cancer-derived stem cells represent both useful research tools and new drug leads for regenerative medicine and oncology. Long-term expansion of mouse and human neural stem cells is possible using adherent monolayer culture. These cultures represent a useful cellular resource to carry out image-based high content screening of small chemical libraries. Improvements in automated microscopy, desktop computational power, and freely available image processing tools, now means that such chemical screens are realistic to undertake in individual academic laboratories. Here we outline a cost effective and versatile time lapse imaging strategy suitable for chemical screening. Protocols are described for the handling and screening of human fetal Neural Stem (NS) cell lines and their malignant counterparts, Glioblastoma-derived neural stem cells (GNS). We focus on identification of cytostatic and cytotoxic "hits" and discuss future possibilities and challenges for extending this approach to assay lineage commitment and differentiation. Copyright © 2012 Elsevier Inc. All rights reserved.

Organic electrochemical transistors for cell-based impedance sensing

NASA Astrophysics Data System (ADS)

Rivnay, Jonathan; Ramuz, Marc; Leleux, Pierre; Hama, Adel; Huerta, Miriam; Owens, Roisin M.

2015-01-01

Electrical impedance sensing of biological systems, especially cultured epithelial cell layers, is now a common technique to monitor cell motion, morphology, and cell layer/tissue integrity for high throughput toxicology screening. Existing methods to measure electrical impedance most often rely on a two electrode configuration, where low frequency signals are challenging to obtain for small devices and for tissues with high resistance, due to low current. Organic electrochemical transistors (OECTs) are conducting polymer-based devices, which have been shown to efficiently transduce and amplify low-level ionic fluxes in biological systems into electronic output signals. In this work, we combine OECT-based drain current measurements with simultaneous measurement of more traditional impedance sensing using the gate current to produce complex impedance traces, which show low error at both low and high frequencies. We apply this technique in vitro to a model epithelial tissue layer and show that the data can be fit to an equivalent circuit model yielding trans-epithelial resistance and cell layer capacitance values in agreement with literature. Importantly, the combined measurement allows for low biases across the cell layer, while still maintaining good broadband signal.

Defining the taxonomic domain of applicability for mammalian-based high-throughput screening assays

EPA Science Inventory

Cell-based high throughput screening (HTS) technologies are becoming mainstream in chemical safety evaluations. The US Environmental Protection Agency (EPA) Toxicity Forecaster (ToxCastTM) and the multi-agency Tox21 Programs have been at the forefront in advancing this science, m...

Screening antiallergic components from Carthamus tinctorius using rat basophilic leukemia 2H3 cell membrane chromatography combined with high-performance liquid chromatography and tandem mass spectrometry.

PubMed

Han, Shengli; Huang, Jing; Cui, Ronghua; Zhang, Tao

2015-02-01

Carthamus tinctorius, used in traditional Chinese medicine, has many pharmacological effects, such as anticoagulant effects, antioxidant effects, antiaging effects, regulation of gene expression, and antitumor effects. However, there is no report on the antiallergic effects of the components in C. tinctorius. In the present study, we investigated the antiallergic components of C. tinctorius and its mechanism of action. A rat basophilic leukemia 2H3/cell membrane chromatography coupled online with high-performance liquid chromatography and tandem mass spectrometry method was developed to screen antiallergic components from C. tinctorius. The screening results showed that Hydroxysafflor yellow A, from C. tinctorius, was the targeted component that retained on the rat basophilic leukemia 2H3/cell membrane chromatography column. We measured the amount of β-hexosaminidase and histamine released in mast cells and the key markers of degranulation. The release assays showed that Hydroxysafflor yellow A could attenuate the immunoglobulin E induced release of allergic cytokines without affecting cell viability from 1.0 to 50.0 μM. In conclusion, the established rat basophilic leukemia 2H3 cell membrane chromatography coupled with online high-performance liquid chromatography and tandem mass spectrometry method successfully screened and identified Hydroxysafflor yellow A from C. tinctorius as a potential antiallergic component. Pharmacological analysis elucidated that Hydroxysafflor yellow A is an effective natural component for inhibiting immunoglobulin E-antigen-mediated degranulation. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[Targeted newborn screening for sickle-cell anemia: Sickling test (Emmel test) boundaries in the prenatal assessment in West African area].

PubMed

Diallo, D A; Guindo, A; Touré, B A; Sarro, Y S; Sima, M; Tessougué, O; Baraika, M A; Guindo, P; Traoré, M; Diallo, M; Dorie, A

2018-05-01

Newborn screening for sickle cell anemia is necessary in Africa where the disease is more frequent. Hemoglobin electrophoresis is used for screening, but is limited by a high cost and difficult access. Sickling test (Emmel test), which is more affordable and technically more accessible, is often requested for prenatal assessment of pregnant women in West African areas to reserve screening for newborns from mothers in whom the positive sickling test attests the presence of hemoglobin S. This study aims to evaluate the number of undetected sickle cell anemia newborns by a screening policy targeting only newborns from mothers in whom a sickling test would have been positive. From 2010 to 2012, in Bamako, Mali, West Africa, 2489 newborns were routinely screened for sickle cell anemia at the umbilical cord or heel by isoelectrofocusing and, if necessary, by high-performance liquid chromatography. These newborns were born from 2420 mothers whose hemoglobin was studied by isoelectrofocusing. The data was recorded and processed using Excel software version 14.0.0. We calculated the frequency of the sickle cell gene in mothers and newborns as well as the number of SCA newborns from heterozygous or C homozygous mothers. Of the 2489 newborns, 16 had sickle cell anemia (6 SS and 10 SC); 198 had the sickle cell trait; 139 were AC and 1 was CC. Of the 10 newborns with SC profile, 3 were born from mothers not carrying the S gene but the C gene of hemoglobin and in which an Emmel test would have been negative. Targeted newborn screening, based on the results of sickling test in pregnant women, would misdiagnose more than one of six sickle cell anemia newborns who would not benefit from early care. Cost-effectiveness studies of routine newborn screening for sickle cell anemia should lead to a better screening strategy in contexts where hemoglobin S and other hemoglobin defect genes coexist. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Establishment of A431 cell membrane chromatography-RPLC method for screening target components from Radix Caulophylli.

PubMed

Hou, Xiaofang; Wang, Sicen; Hou, Jingjing; He, Langchong

2011-03-01

We describe here an analytical method of A431 cell membrane chromatography (A431/CMC) (CMC, cell membrane chromatography) combined with RPLC for recognition, separation, and identification of target components from traditional Chinese medicines (TCMs) Radix Caulophylli. The A431 cells with high expressed epidermal growth factor receptor (EGFR) were used to prepare the stationary phase in the CMC model. Retention fractions on the A431-CMC model were collected using an automated fraction collection and injection module (FC/I). Each fraction was analyzed by RPLC under the optimized conditions. Gefitinib and erlotinib were used as standard compounds to investigate the suitability and reliability of the A431 cell membrane chromatography-RPLC method prior to screening target component from Radix Caulophylli total alkaloids. The results indicated that caulophine and taspine were the target component acting on the epidermal growth factor receptor. This method could be an efficient way in drug discovery using natural medicinal herbs as a source of novel compounds. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The third dimension bridges the gap between cell culture and live tissue.

PubMed

Pampaloni, Francesco; Reynaud, Emmanuel G; Stelzer, Ernst H K

2007-10-01

Moving from cell monolayers to three-dimensional (3D) cultures is motivated by the need to work with cellular models that mimic the functions of living tissues. Essential cellular functions that are present in tissues are missed by 'petri dish'-based cell cultures. This limits their potential to predict the cellular responses of real organisms. However, establishing 3D cultures as a mainstream approach requires the development of standard protocols, new cell lines and quantitative analysis methods, which include well-suited three-dimensional imaging techniques. We believe that 3D cultures will have a strong impact on drug screening and will also decrease the use of laboratory animals, for example, in the context of toxicity assays.

Genome stability of programmed stem cell products.

PubMed

Martin, Ulrich

2017-10-01

Inherited and acquired genomic abnormalities are known to cause genetic diseases and contribute to cancer formation. Recent studies demonstrated a substantial mutational load in mouse and human embryonic and induced pluripotent stem cells (ESCs and iPSCs). Single nucleotide variants, copy number variations, and larger chromosomal abnormalities may influence the differentiation capacity of pluripotent stem cells and the functionality of their derivatives in disease modeling and drug screening, and are considered a serious risk for cellular therapies based on ESC or iPSC derivatives. This review discusses the types and origins of different genetic abnormalities in pluripotent stem cells, methods for their detection, and the mechanisms of development and enrichment during reprogramming and culture expansion. Copyright © 2017 Elsevier B.V. All rights reserved.

c-Src activity is differentially required by cancer cell motility modes.

PubMed

Logue, Jeremy S; Cartagena-Rivera, Alexander X; Chadwick, Richard S

2018-04-01

Cancer cell migration requires that cells respond and adapt to their surroundings. In the absence of extracellular matrix cues, cancer cells will undergo a mesenchymal to ameboid transition, whereas a highly confining space will trigger a switch to "leader bleb-based" migration. To identify oncogenic signaling pathways mediating these transitions, we undertook a targeted screen using clinically useful inhibitors. Elevated Src activity was found to change actin and focal adhesion dynamics, whereas inhibiting Src triggered focal adhesion disassembly and blebbing. On non-adherent substrates and in collagen matrices, amoeboid-like, blebbing cells having high Src activity formed protrusions of the plasma membrane. To evaluate the role of Src in confined cells, we use a novel approach that places cells under a slab of polydimethylsiloxane (PDMS), which is held at a defined height. Using this method, we find that leader bleb-based migration is resistant to Src inhibition. High Src activity was found to markedly change the architecture of cortical actomyosin, reduce cell mechanical properties, and the percentage of cells that undergo leader bleb-based migration. Thus, Src is a signal transducer that can potently influence transitions between migration modes with implications for the rational development of metastasis inhibitors.

Potential Novel Antibiotics from HTS Targeting the Virulence-regulating Transcription Factor, VirF, from Shigella flexneri

PubMed Central

Emanuele, Anthony A.; Adams, Nancy E.; Chen, Yi-Chen; Maurelli, Anthony T.; Garcia, George A.

2014-01-01

VirF is an AraC-type transcriptional regulator responsible for activating the transcription of virulence genes required for the intracellular invasion and cell-to-cell spread of Shigella flexneri. Gene disruption studies have validated VirF as a potential target for an anti-virulence therapy to treat shigellosis by determining that VirF is necessary for virulence, but not required for bacterial viability. Using a bacteria-based, β-galactosidase reporter assay we completed a high-throughput screening (HTS) campaign monitoring VirF activity in the presence of over 140,000 small molecules. From our screening campaign we identified five lead compounds to pursue in tissue-culture-based invasion and cell-to-cell spread assays and toxicity screens. Our observations of activity in these models for infection have validated our approach of targeting virulence regulation and have allowed us to identify a promising chemical scaffold from our HTS for hit-to-lead development. Interestingly, differential effects on invasion versus cell-to-cell spread suggest that the compounds’ efficacies may depend, in part, on the specific promoter that VirF is recognizing. PMID:24549153

Models of Community-Based Hepatitis B Surface Antigen Screening Programs in the U.S. and Their Estimated Outcomes and Costs

PubMed Central

Rein, David B.; Lesesne, Sarah B.; Smith, Bryce D.; Weinbaum, Cindy M.

2011-01-01

Objectives Information on the process and method of service delivery is sparse for hepatitis B surface antigen (HBsAg) testing, and no systematic study has evaluated the relative effectiveness or cost-effectiveness of different HBsAg screening models. To address this need, we compared five specific community-based screening programs. Methods We funded five HBsAg screening programs to collect information on their design, costs, and outcomes of participants during a six-month observation period. We categorized programs into four types of models. For each model, we calculated the number screened, the number screened as per Centers for Disease Control and Prevention (CDC) recommendations, and the cost per screening. Results The models varied by cost per person screened and total number of people screened, but they did not differ meaningfully in the proportion of people screened following CDC recommendations, the proportion of those screened who tested positive, or the proportion of those who newly tested positive. Conclusions Integrating screening into outpatient service settings is the most cost-effective method but may not reach all people needing to be screened. Future research should examine cost-effective methods that expand the reach of screening into communities in outpatient settings. PMID:21800750

High-Throughput Screening Using a Whole-Cell Virus Replication Reporter Gene Assay to Identify Inhibitory Compounds against Rift Valley Fever Virus Infection.

PubMed

Islam, Md Koushikul; Baudin, Maria; Eriksson, Jonas; Öberg, Christopher; Habjan, Matthias; Weber, Friedemann; Överby, Anna K; Ahlm, Clas; Evander, Magnus

2016-04-01

Rift Valley fever virus (RVFV) is an emerging virus that causes serious illness in humans and livestock. There are no approved vaccines or treatments for humans. The purpose of the study was to identify inhibitory compounds of RVFV infection without any preconceived idea of the mechanism of action. A whole-cell-based high-throughput drug screening assay was developed to screen 28,437 small chemical compounds targeting RVFV infection. To accomplish both speed and robustness, a replication-competent NSs-deleted RVFV expressing a fluorescent reporter gene was developed. Inhibition of fluorescence intensity was quantified by spectrophotometry and related to virus infection in human lung epithelial cells (A549). Cell toxicity was assessed by the Resazurin cell viability assay. After primary screening, 641 compounds were identified that inhibited RVFV infection by ≥80%, with ≥50% cell viability at 50 µM concentration. These compounds were subjected to a second screening regarding dose-response profiles, and 63 compounds with ≥60% inhibition of RVFV infection at 3.12 µM compound concentration and ≥50% cell viability at 25 µM were considered hits. Of these, six compounds with high inhibitory activity were identified. In conclusion, the high-throughput assay could efficiently and safely identify several promising compounds that inhibited RVFV infection. © 2016 Society for Laboratory Automation and Screening.

Improving the apo-state detergent stability of NTS1 with CHESS for pharmacological and structural studies.

PubMed

Scott, Daniel J; Kummer, Lutz; Egloff, Pascal; Bathgate, Ross A D; Plückthun, Andreas

2014-11-01

The largest single class of drug targets is the G protein-coupled receptor (GPCR) family. Modern high-throughput methods for drug discovery require working with pure protein, but this has been a challenge for GPCRs, and thus the success of screening campaigns targeting soluble, catalytic protein domains has not yet been realized for GPCRs. Therefore, most GPCR drug screening has been cell-based, whereas the strategy of choice for drug discovery against soluble proteins is HTS using purified proteins coupled to structure-based drug design. While recent developments are increasing the chances of obtaining GPCR crystal structures, the feasibility of screening directly against purified GPCRs in the unbound state (apo-state) remains low. GPCRs exhibit low stability in detergent micelles, especially in the apo-state, over the time periods required for performing large screens. Recent methods for generating detergent-stable GPCRs, however, offer the potential for researchers to manipulate GPCRs almost like soluble enzymes, opening up new avenues for drug discovery. Here we apply cellular high-throughput encapsulation, solubilization and screening (CHESS) to the neurotensin receptor 1 (NTS1) to generate a variant that is stable in the apo-state when solubilized in detergents. This high stability facilitated the crystal structure determination of this receptor and also allowed us to probe the pharmacology of detergent-solubilized, apo-state NTS1 using robotic ligand binding assays. NTS1 is a target for the development of novel antipsychotics, and thus CHESS-stabilized receptors represent exciting tools for drug discovery. Copyright © 2014 Elsevier B.V. All rights reserved.

Advances in Reprogramming-Based Study of Neurologic Disorders

PubMed Central

Baldwin, Kristin K.

2015-01-01

The technology to convert adult human non-neural cells into neural lineages, through induced pluripotent stem cells (iPSCs), somatic cell nuclear transfer, and direct lineage reprogramming or transdifferentiation has progressed tremendously in recent years. Reprogramming-based approaches aimed at manipulating cellular identity have enormous potential for disease modeling, high-throughput drug screening, cell therapy, and personalized medicine. Human iPSC (hiPSC)-based cellular disease models have provided proof of principle evidence of the validity of this system. However, several challenges remain before patient-specific neurons produced by reprogramming can provide reliable insights into disease mechanisms or be efficiently applied to drug discovery and transplantation therapy. This review will first discuss limitations of currently available reprogramming-based methods in faithfully and reproducibly recapitulating disease pathology. Specifically, we will address issues such as culture heterogeneity, interline and inter-individual variability, and limitations of two-dimensional differentiation paradigms. Second, we will assess recent progress and the future prospects of reprogramming-based neurologic disease modeling. This includes three-dimensional disease modeling, advances in reprogramming technology, prescreening of hiPSCs and creating isogenic disease models using gene editing. PMID:25749371

Promoter library-based module combination (PLMC) technology for optimization of threonine biosynthesis in Corynebacterium glutamicum.

PubMed

Wei, Liang; Xu, Ning; Wang, Yiran; Zhou, Wei; Han, Guoqiang; Ma, Yanhe; Liu, Jun

2018-05-01

Due to the lack of efficient control elements and tools, the fine-tuning of gene expression in the multi-gene metabolic pathways is still a great challenge for engineering microbial cell factories, especially for the important industrial microorganism Corynebacterium glutamicum. In this study, the promoter library-based module combination (PLMC) technology was developed to efficiently optimize the expression of genes in C. glutamicum. A random promoter library was designed to contain the putative - 10 (NNTANANT) and - 35 (NNGNCN) consensus motifs, and refined through a three-step screening procedure to achieve numerous genetic control elements with different strength levels, including fluorescence-activated cell sorting (FACS) screening, agar plate screening, and 96-well plate screening. Multiple conventional strategies were employed for further precise characterizations of the promoter library, such as real-time quantitative PCR, sodium dodecyl sulfate polyacrylamide gel electrophoresis, FACS analysis, and the lacZ reporter system. These results suggested that the established promoter elements effectively regulated gene expression and showed varying strengths over a wide range. Subsequently, a multi-module combination technology was created based on the efficient promoter elements for combination and optimization of modules in the multi-gene pathways. Using this technology, the threonine biosynthesis pathway was reconstructed and optimized by predictable tuning expression of five modules in C. glutamicum. The threonine titer of the optimized strain was significantly improved to 12.8 g/L, an approximate 6.1-fold higher than that of the control strain. Overall, the PLMC technology presented in this study provides a rapid and effective method for combination and optimization of multi-gene pathways in C. glutamicum.

Development of a recombinant human ovarian (BG1) cell line containing estrogen receptor α and β for improved detection of estrogenic/antiestrogenic chemicals.

PubMed

Brennan, Jennifer C; Bassal, Arzoo; He, Guochun; Denison, Michael S

2016-01-01

Estrogenic endocrine-disrupting chemicals are found in environmental and biological samples, commercial and consumer products, food, and numerous other sources. Given their ubiquitous nature and potential for adverse effects, a critical need exists for rapidly detecting these chemicals. The authors developed an estrogen-responsive recombinant human ovarian (BG1Luc4E2) cell line recently accepted by the US Environmental Protection Agency (USEPA) and Organisation for Economic Co-operation and Development (OECD) as a bioanalytical method to detect estrogen receptor (ER) agonists/antagonists. Unfortunately, these cells appear to contain only 1 of the 2 known ER isoforms, ERα but not ERβ, and the differential ligand selectivity of these ERs indicates that the currently accepted screening method only detects a subset of total estrogenic chemicals. To improve the estrogen screening bioassay, BG1Luc4E2 cells were stably transfected with an ERβ expression plasmid and positive clones identified using ERβ-selective ligands (genistein and Br-ERβ-041). A highly responsive clone (BG1LucERβc9) was identified that exhibited greater sensitivity and responsiveness to ERβ-selective ligands than BG1Luc4E2 cells, and quantitative reverse-transcription polymerase chain reaction confirmed the presence of ERβ expression in these cells. Screening of pesticides and industrial chemicals identified chemicals that preferentially stimulated ERβ-dependent reporter gene expression. Together, these results not only demonstrate the utility of this dual-ER recombinant cell line for detecting a broader range of estrogenic chemicals than the current BG1Luc4E2 cell line, but screening with both cell lines allows identification of ERα- and ERβ-selective chemicals. © 2015 SETAC.

Development of a recombinant human ovarian (BG1) cell line containing estrogen receptor α and β for improved detection of estrogenic/antiestrogenic chemicals

PubMed Central

Brennan, Jennifer C.; Bassal, Arzoo; He, Guochun; Denison, Michael S.

2016-01-01

Estrogenic endocrine disrupting chemicals are found in environmental and biological samples, commercial and consumer products, food, and numerous other sources. Given their ubiquitous nature and potential for adverse effects, there is a critical need for rapidly detecting these chemicals. We developed an estrogen-responsive recombinant human ovarian (BG1Luc4E2) cell line recently accepted by the USEPA and OECD as a bioanalytical method to detect estrogen receptor (ER) agonists/antagonists. Unfortunately, these cells appear to contain only one of the two known ER isoforms, ERα but not ERβ, and the differential ligand selectivity of these ERs indicates that the currently accepted screening method only detects a subset of total estrogenic chemicals. To improve the estrogen screening bioassay, BG1Luc4E2 cells were stably transfected with an ERβ expression plasmid and positive clones identified using ERβ-selective ligands (genistein and Br-ERβ-041). A highly responsive clone (BG1LucERβc9) was identified that exhibited greater sensitivity and responsiveness to ERβ-selective ligands than BG1Luc4E2 cells and qRT-PCR confirmed the presence of ERβ expression in these cells. Screening of pesticides and industrial chemicals identified chemicals that preferentially stimulated ERβ-dependent reporter gene expression. Together, these results not only demonstrate the utility of this dual ER recombinant cell line for detecting a broader range of estrogenic chemicals than the current BG1Luc4E2 cell line, but screening with both cell lines allows identification of ERα and ERβ-selective chemicals. PMID:26139245

3-Dimensional culture systems for anti-cancer compound profiling and high-throughput screening reveal increases in EGFR inhibitor-mediated cytotoxicity compared to monolayer culture systems.

PubMed

Howes, Amy L; Richardson, Robyn D; Finlay, Darren; Vuori, Kristiina

2014-01-01

3-dimensional (3D) culture models have the potential to bridge the gap between monolayer cell culture and in vivo studies. To benefit anti-cancer drug discovery from 3D models, new techniques are needed that enable their use in high-throughput (HT) screening amenable formats. We have established miniaturized 3D culture methods robust enough for automated HT screens. We have applied these methods to evaluate the sensitivity of normal and tumorigenic breast epithelial cell lines against a panel of oncology drugs when cultured as monolayers (2D) and spheroids (3D). We have identified two classes of compounds that exhibit preferential cytotoxicity against cancer cells over normal cells when cultured as 3D spheroids: microtubule-targeting agents and epidermal growth factor receptor (EGFR) inhibitors. Further improving upon our 3D model, superior differentiation of EC50 values in the proof-of-concept screens was obtained by co-culturing the breast cancer cells with normal human fibroblasts and endothelial cells. Further, the selective sensitivity of the cancer cells towards chemotherapeutics was observed in 3D co-culture conditions, rather than as 2D co-culture monolayers, highlighting the importance of 3D cultures. Finally, we examined the putative mechanisms that drive the differing potency displayed by EGFR inhibitors. In summary, our studies establish robust 3D culture models of human cells for HT assessment of tumor cell-selective agents. This methodology is anticipated to provide a useful tool for the study of biological differences within 2D and 3D culture conditions in HT format, and an important platform for novel anti-cancer drug discovery.

3-Dimensional Culture Systems for Anti-Cancer Compound Profiling and High-Throughput Screening Reveal Increases in EGFR Inhibitor-Mediated Cytotoxicity Compared to Monolayer Culture Systems

PubMed Central

Howes, Amy L.; Richardson, Robyn D.; Finlay, Darren; Vuori, Kristiina

2014-01-01

3-dimensional (3D) culture models have the potential to bridge the gap between monolayer cell culture and in vivo studies. To benefit anti-cancer drug discovery from 3D models, new techniques are needed that enable their use in high-throughput (HT) screening amenable formats. We have established miniaturized 3D culture methods robust enough for automated HT screens. We have applied these methods to evaluate the sensitivity of normal and tumorigenic breast epithelial cell lines against a panel of oncology drugs when cultured as monolayers (2D) and spheroids (3D). We have identified two classes of compounds that exhibit preferential cytotoxicity against cancer cells over normal cells when cultured as 3D spheroids: microtubule-targeting agents and epidermal growth factor receptor (EGFR) inhibitors. Further improving upon our 3D model, superior differentiation of EC50 values in the proof-of-concept screens was obtained by co-culturing the breast cancer cells with normal human fibroblasts and endothelial cells. Further, the selective sensitivity of the cancer cells towards chemotherapeutics was observed in 3D co-culture conditions, rather than as 2D co-culture monolayers, highlighting the importance of 3D cultures. Finally, we examined the putative mechanisms that drive the differing potency displayed by EGFR inhibitors. In summary, our studies establish robust 3D culture models of human cells for HT assessment of tumor cell-selective agents. This methodology is anticipated to provide a useful tool for the study of biological differences within 2D and 3D culture conditions in HT format, and an important platform for novel anti-cancer drug discovery. PMID:25247711

Accelerating glioblastoma drug discovery: Convergence of patient-derived models, genome editing and phenotypic screening.

PubMed

O'Duibhir, Eoghan; Carragher, Neil O; Pollard, Steven M

2017-04-01

Patients diagnosed with glioblastoma (GBM) continue to face a bleak prognosis. It is critical that new effective therapeutic strategies are developed. GBM stem cells have molecular hallmarks of neural stem and progenitor cells and it is possible to propagate both non-transformed normal neural stem cells and GBM stem cells, in defined, feeder-free, adherent culture. These primary stem cell lines provide an experimental model that is ideally suited to cell-based drug discovery or genetic screens in order to identify tumour-specific vulnerabilities. For many solid tumours, including GBM, the genetic disruptions that drive tumour initiation and growth have now been catalogued. CRISPR/Cas-based genome editing technologies have recently emerged, transforming our ability to functionally annotate the human genome. Genome editing opens prospects for engineering precise genetic changes in normal and GBM-derived neural stem cells, which will provide more defined and reliable genetic models, with critical matched pairs of isogenic cell lines. Generation of more complex alleles such as knock in tags or fluorescent reporters is also now possible. These new cellular models can be deployed in cell-based phenotypic drug discovery (PDD). Here we discuss the convergence of these advanced technologies (iPS cells, neural stem cell culture, genome editing and high content phenotypic screening) and how they herald a new era in human cellular genetics that should have a major impact in accelerating glioblastoma drug discovery. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Discovery of a drug targeting microenvironmental support for lymphoma cells by screening using patient-derived xenograft cells

PubMed Central

Sugimoto, Keiki; Hayakawa, Fumihiko; Shimada, Satoko; Morishita, Takanobu; Shimada, Kazuyuki; Katakai, Tomoya; Tomita, Akihiro; Kiyoi, Hitoshi; Naoe, Tomoki

2015-01-01

Cell lines have been used for drug discovery as useful models of cancers; however, they do not recapitulate cancers faithfully, especially in the points of rapid growth rate and microenvironment independency. Consequently, the majority of conventional anti-cancer drugs are less sensitive to slow growing cells and do not target microenvironmental support, although most primary cancer cells grow slower than cell lines and depend on microenvironmental support. Here, we developed a novel high throughput drug screening system using patient-derived xenograft (PDX) cells of lymphoma that maintained primary cancer cell phenotype more than cell lines. The library containing 2613 known pharmacologically active substance and off-patent drugs were screened by this system. We could find many compounds showing higher cytotoxicity than conventional anti-tumor drugs. Especially, pyruvinium pamoate showed the highest activity and its strong anti-tumor effect was confirmed also in vivo. We extensively investigated its mechanism of action and found that it inhibited glutathione supply from stromal cells to lymphoma cells, implying the importance of the stromal protection from oxidative stress for lymphoma cell survival and a new therapeutic strategy for lymphoma. Our system introduces a primary cancer cell phenotype into cell-based phenotype screening and sheds new light on anti-cancer drug development. PMID:26278963

Apparatus for combinatorial screening of electrochemical materials

DOEpatents

Kepler, Keith Douglas [Belmont, CA; Wang, Yu [Foster City, CA

2009-12-15

A high throughput combinatorial screening method and apparatus for the evaluation of electrochemical materials using a single voltage source (2) is disclosed wherein temperature changes arising from the application of an electrical load to a cell array (1) are used to evaluate the relative electrochemical efficiency of the materials comprising the array. The apparatus may include an array of electrochemical cells (1) that are connected to each other in parallel or in series, an electronic load (2) for applying a voltage or current to the electrochemical cells (1), and a device (3), external to the cells, for monitoring the relative temperature of each cell when the load is applied.

Back to the Future: Lessons Learned in Modern Target-based and Whole-Cell Lead Optimization of Antimalarials

PubMed Central

Chatterjee, Arnab K; Yeung, Bryan KS

2012-01-01

Antimalarial drug discovery has historically benefited from the whole-cell (phenotypic) screening approach to identify lead molecules in the search for new drugs. However over the past two decades there has been a shift in the pharmaceutical industry to move away from whole-cell screening to target-based approaches. As part of a Wellcome Trust and Medicines for Malaria Venture (MMV) funded consortium to discover new blood-stage antimalarials, we used both approaches to identify new antimalarial chemotypes, two of which have progressed beyond the lead optimization phase and display excellent in vivo efficacy in mice. These two advanced series were identified through a cell-based optimization devoid of target information and in this review we summarize the advantages of this approach versus a target-based optimization. Although the each lead optimization required slightly different medicinal chemistry strategies, we observed some common issues across the different the scaffolds which could be applied to other cell based lead optimization programs. PMID:22242845