High-throughput RNAi screening by time-lapse imaging of live human cells

RNA interference (RNAi) is a powerful tool to study gene function in cultured cells. Transfected cell microarrays in principle allow high-throughput phenotypic analysis after gene knockdown by microscopy. But bottlenecks in imaging and data analysis have limited such high-content screens to endpoint assays in fixed cells and determination of global parameters such as viability. Here we have overcome these limitations and developed an automated platform for high-content RNAi screening by time-lapse fluorescence microscopy of live HeLa cells expressing histone-GFP to report on chromosome segregation and structure. We automated all steps, including printing transfection-ready small interfering RNA (siRNA) microarrays, fluorescence imaging and computational phenotyping of digital images, in a high-throughput workflow. We validated this method in a pilot screen assaying cell division and delivered a sensitive, time-resolved phenoprint for each of the 49 endogenous genes we suppressed. This modular platform is scalable and makes the power of time-lapse microscopy available for genome-wide RNAi screens.     

Single-cell microliter-droplet screening system (MISS Cell): An integrated platform for automated high-throughput microbial monoclonal cultivation and picking

Solid plates have been used for microbial monoclonal isolation, cultivation, and colony picking since 1881. However, the process is labor- and resource-intensive for high-throughput requirements. Currently, several instruments have been integrated for automated and high-throughput picking, but complicated and expensive. To address these issues, we report a novel integrated platform, the single-cell microliter-droplet screening system (MISS Cell), for automated, high-throughput microbial monoclonal colony cultivation and picking. We verified the monoclonality of droplet cultures in the MISS Cell and characterized culture performance. Compared with solid plates, the MISS Cell generated a larger number of monoclonal colonies with higher initial growth rates using fewer resources. Finally, we established a workflow for automated high-throughput screening of Corynebacterium glutamicum using the MISS Cell and identified high glutamate-producing strains. The MISS Cell can serve as a universal platform to efficiently produce monoclonal colonies in high-throughput applications, overcoming the limitations of solid plates to promote rapid development in biotechnology.     

High-throughput screening by mass spectrometry: comparison with the scintillation proximity assay with a focused-file screen of AKT1/PKB alpha

Mass spectrometry is an emerging format for label-free high-throughput screening. The main limitation of mass spectrometry is throughput, due to the requirement to purify samples prior to ionization. Here the authors compare an automated high-throughput mass spectrometry (HTMS) system (RapidFire) with the scintillation proximity assay (SPA). The cancer therapy target AKT1/PKBalpha was screened against a focused library of kinase inhibitors and IC50 values determined for all compounds that exhibit > 50% inhibition. A selection of additional compounds that exhibited 相似文献   

The FAF-Drugs2 server: a multistep engine to prepare electronic chemical compound collections

SUMMARY: The FAF-Drugs2 server is a web application that prepares chemical compound libraries prior to virtual screening or that assists hit selection/lead optimization before chemical synthesis or ordering. The FAF-Drugs2 web server is an enhanced version of the FAF-Drugs2 package that now includes Pan Assay Interference Compounds detection. This online toolkit has been designed through a user-centered approach with emphasis on user-friendliness. This is a unique online tool allowing to prepare large compound libraries with in house or user-defined filtering parameters. AVAILABILITY: The FAF-Drugs2 server is freely available at http://bioserv.rpbs.univ-paris-diderot.fr/FAF-Drugs/.     

Development and optimization of a process for automated recovery of single cells identified by microengraving

Microfabricated devices are useful tools for manipulating and interrogating large numbers of single cells in a rapid and cost‐effective manner, but connecting these systems to the existing platforms used in routine high‐throughput screening of libraries of cells remains challenging. Methods to sort individual cells of interest from custom microscale devices to standardized culture dishes in an efficient and automated manner without affecting the viability of the cells are critical. Combining a commercially available instrument for colony picking (CellCelector, AVISO GmbH) and a customized software module, we have established an optimized process for the automated retrieval of individual antibody‐producing cells, secreting desirable antibodies, from dense arrays of subnanoliter containers. The selection of cells for retrieval is guided by data obtained from a high‐throughput, single‐cell screening method called microengraving. Using this system, 100 clones from a mixed population of two cell lines secreting different antibodies (12CA5 and HYB099‐01) were sorted with 100% accuracy (50 clones of each) in ～2 h, and the cells retained viability. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Probing the primary screening efficiency by multiple replicate testing: a quantitative analysis of hit confirmation and false screening results of a biochemical assay

Despite a large body of references on assay development, assay optimization, strategies, and methodologies for high-throughput screening (HTS), there have been few reports on investigations of the efficiency of primary screening in a systematic and quantitative manner for a typical HTS process. Recently, the authors investigated the primary hit comparison and the effect of measurement variability by screening a library of approximately 25,000 random compounds in multiple replicate tests in a nuclear receptor recruitment assay with 2 different assay detection technologies. In this report, we utilized these sets of multiple replicate screening data from a different perspective and conducted a systematic data analysis in order to gain some insights into the hit-finding efficiency of a typical primary screening process. Specifically, hit confirmation, false-positive (declaration) rates, and false-negative rates at different hit cutoff limits were explored and calculated from the 2 different assay formats. Results and analyses provided some quantitative estimation regarding the reliability and efficiency of the primary screening process. For the 2 assay formats tested in this report, the confirmation rate (activity repeated at or above a certain hit limit) was found to be 65% or above. It was also suggested that, at least in this case, applying some hit-selection strategies, it is possible to decrease the number of false-negative or false-positive hits without significantly increasing the efforts in primary screening.     

A heuristic based batching and assigning method for online customer orders

In an online order picking system, customer orders arrive in real time and the picking information is updated dynamically. One challenging problem is how to process customer orders in a timely manner. In this paper, a nonparametric heuristic method, Green Area, is presented to address the real-time online order batching problems. By nonparametric, we mean that our method is independent of the parameters of a warehouse layout and the characteristics of customer orders; these parameters facilitates the implementation in real life. The advantages of this method are verified under different scenarios by simulations. Specifically, the influences of the arrival rate, the number of order pickers and the number of orders in the order service time are discussed. The results demonstrate that the Green Area method leads to shorter order service times than traditional methods for optimal batch sizes. Finally, we demonstrate that the Green Area method can be applied to online order picking systems with variable arrival rates.     

A novel multiplex cell viability assay for high-throughput RNAi screening

Cell-based high-throughput RNAi screening has become a powerful research tool in addressing a variety of biological questions. In RNAi screening, one of the most commonly applied assay system is measuring the fitness of cells that is usually quantified using fluorescence, luminescence and absorption-based readouts. These methods, typically implemented and scaled to large-scale screening format, however often only yield limited information on the cell fitness phenotype due to evaluation of a single and indirect physiological indicator. To address this problem, we have established a cell fitness multiplexing assay which combines a biochemical approach and two fluorescence-based assaying methods. We applied this assay in a large-scale RNAi screening experiment with siRNA pools targeting the human kinome in different modified HEK293 cell lines. Subsequent analysis of ranked fitness phenotypes assessed by the different assaying methods revealed average phenotype intersections of 50.7±2.3%-58.7±14.4% when two indicators were combined and 40-48% when a third indicator was taken into account. From these observations we conclude that combination of multiple fitness measures may decrease false-positive rates and increases confidence for hit selection. Our robust experimental and analytical method improves the classical approach in terms of time, data comprehensiveness and cost.     

A sensitive colorimetric high-throughput screening method for lipase synthetic activity assay

A sensitive and practical high-throughput screening method for assaying lipase synthetic activity is described. Lipase-catalyzed transesterification between vinyl acetate and n-butanol in n-hexane was chosen as a model reaction. The released acetaldehyde was determined by the colorimetric method using 3-methyl-2-benzothialinone (MBTH) derivatization. In comparison with other methods, the major advantages of this process include high sensitivity, simple detection, inexpensive reagents, and low requirements for instruments.     

Direct screening of lyophilised biological fluids for bile acids using an evaporative light scattering detector

The usefulness of lyophilisation for the direct screening of biological fluids for bile acids was investigated. Human serum and urine were lyophilised without losses of the target compounds and further extracted with n-hexane in acidic medium under magnetic stirring. An integrated flow injection-liquid chromatographic system coupled to an evaporative light scattering detector (ELSD) was used for automated screening/confirmation. The continuous module allows sequential filtration of the organic phase, solvent changeover and solid-phase extraction for clean-up and preconcentration purposes. Retained bile acids were eluted with an acetonitrile-methanol (65:35, v/v) stream. For screening purposes, the effluent was directly introduced in the ELSD detector and the total bile acid content of the sample determined. For confirmatory analysis, another aliquot of the sample was processed in the screening module but the effluent was directed to the chromatographic columns, which provided the free bile acid profile of the sample. Fasting serum and urine samples obtained from healthy individuals were lyophilised and processed. Good agreement was obtained in the analysis of the two matrices assayed following the screening and confirmatory methods.     

On the prediction of statistical parameters in high-throughput screening using resampling techniques

A severe drawback in the high-throughput screening (HTS) process is the unintentional (random) presence of false positives and negatives. Their rates depend, among others, on the screening process being applied and the target class. Although false positives can be sorted out in subsequent process steps, their occurrence can lead to increased project cost. More fundamentally, it is not possible to rescue false nonhits. In this article, we investigate the prediction of the primary hit rate, hit confirmation rate, and false-positive and false-negative rates. Results for approximately 2800 compounds are considered that are tested as a pilot screen ahead of the primary screening work. This pilot screen is done at several concentrations and in replicates. The rates are predicted as a function of the proposed hit threshold by having the replicates serve as each other's confirmers, and confidence limits to the prediction are attached by means of a resampling scheme. A comparison of the rates resulting from the resampling with the primary hit rate and the confirmation rates obtained during the screening campaign shows how accurate this method is. Hence, the "optimal" compound concentration for the screen as well as the optimal hit threshold corresponding to low false rates can be determined prior to starting the subsequent screening campaign.     

Automated screening for biological weapons in homeland defense

Biological threat detection programs that collect air samples and monitor for large-scale release of biowarfare agents generate large numbers of samples that must be quickly and accurately screened for the presence of biological agents. An impediment to the rapid analysis of large numbers of environmental biological samples is that manual laboratory processes are time-consuming and require resources to maintain infrastructure, trained personnel, and adequate supplies of testing reagents. An ideal screening system would be capable of processing multiple samples rapidly, cost-effectively, and with minimal personnel. In the present study, we evaluated the Automated Biological Agent Testing System (ABATS) to explore the capability of automation to increase sample throughput, maximize system accuracy, and reduce the analysis costs associated with biological threat agent screening in environmental samples. This study demonstrates the utility of this concept and the potential of an automated system to address the growing environmental monitoring needs of the United States.     

Advances in membrane receptor screening and analysis

During the last decade there has been significant progress in the development of analytical techniques for the screening of ligand binding to membranes and membrane receptors. This review focuses on developments using label-free assays that facilitate ligand-membrane-receptor screening without the need for chemical-, biological- or radiological-labelled reagents. These assays include acoustic, optical surface plasmon resonance biosensing, sedimentation (analytical ultracentrifugation), chromatographic assays, isothermal titration calorimetry and differential scanning calorimetry. The merits and applications of cell-based screening systems and of different model membrane systems, including planar supported lipid layers, bead-supported membranes and lipid micro-arrays, are discussed. Recent advances involving more established techniques including intrinsic fluorescence, FRET spectroscopy, scintillation proximity assays and automated patch clamping are presented along with applications to peripheral membrane proteins, ion channels and G protein-coupled receptors. Novel high-throughput assays for determination of drug- and protein-partitioning in membranes are also highlighted. To aid the experimenter, a brief synopsis of the techniques commonly employed to purify and reconstitute membranes and membrane receptors is included.     

To 5D and beyond: quantitative fluorescence microscopy in the postgenomic era

Digital fluorescence microscopy is now a standard technology for assaying molecular localisation in cells and tissues. The choice of laser scanning (LSM) and wide-field microscopes (WFM) largely depends on the type of sample, with LSMs performing best on thick samples and WFMs performing best on thin ones. These systems are increasingly used to collect large multidimensional datasets. We propose a unified image structure that considers space, time, and fluorescence wavelength as integral parts of the image. Moreover, the application of fluorescence imaging to large-scale screening means that large datasets are now routinely acquired. We propose that analysis of these data requires querying tools based on relational databases and describe one such system.     

Nmrglue: an open source Python package for the analysis of multidimensional NMR data

Nmrglue, an open source Python package for working with multidimensional NMR data, is described. When used in combination with other Python scientific libraries, nmrglue provides a highly flexible and robust environment for spectral processing, analysis and visualization and includes a number of common utilities such as linear prediction, peak picking and lineshape fitting. The package also enables existing NMR software programs to be readily tied together, currently facilitating the reading, writing and conversion of data stored in Bruker, Agilent/Varian, NMRPipe, Sparky, SIMPSON, and Rowland NMR Toolkit file formats. In addition to standard applications, the versatility offered by nmrglue makes the package particularly suitable for tasks that include manipulating raw spectrometer data files, automated quantitative analysis of multidimensional NMR spectra with irregular lineshapes such as those frequently encountered in the context of biomacromolecular solid-state NMR, and rapid implementation and development of unconventional data processing methods such as covariance NMR and other non-Fourier approaches. Detailed documentation, install files and source code for nmrglue are freely available at http://nmrglue.com. The source code can be redistributed and modified under the New BSD license.     

Early phase drug discovery: Cheminformatics and computational techniques in identifying lead series

Early drug discovery processes rely on hit finding procedures followed by extensive experimental confirmation in order to select high priority hit series which then undergo further scrutiny in hit-to-lead studies. The experimental cost and the risk associated with poor selection of lead series can be greatly reduced by the use of many different computational and cheminformatic techniques to sort and prioritize compounds. We describe the steps in typical hit identification and hit-to-lead programs and then describe how cheminformatic analysis assists this process. In particular, scaffold analysis, clustering and property calculations assist in the design of high-throughput screening libraries, the early analysis of hits and then organizing compounds into series for their progression from hits to leads. Additionally, these computational tools can be used in virtual screening to design hit-finding libraries and as procedures to help with early SAR exploration.     

Quantitative assessment of hit detection and confirmation in single and duplicate high-throughput screenings

The process of identifying active targets (hits) in high-throughput screening (HTS) usually involves 2 steps: first, removing or adjusting for systematic variation in the measurement process so that extreme values represent strong biological activity instead of systematic biases such as plate effect or edge effect and, second, choosing a meaningful cutoff on the calculated statistic to declare positive compounds. Both false-positive and false-negative errors are inevitable in this process. Common control or estimation of error rates is often based on an assumption of normal distribution of the noise. The error rates in hit detection, especially false-negative rates, are hard to verify because in most assays, only compounds selected in primary screening are followed up in confirmation experiments. In this article, the authors take advantage of a quantitative HTS experiment in which all compounds are tested 42 times over a wide range of 14 concentrations so true positives can be found through a dose-response curve. Using the activity status defined by dose curve, the authors analyzed the effect of various data-processing procedures on the sensitivity and specificity of hit detection, the control of error rate, and hit confirmation. A new summary score is proposed and demonstrated to perform well in hit detection and useful in confirmation rate estimation. In general, adjusting for positional effects is beneficial, but a robust test can prevent overadjustment. Error rates estimated based on normal assumption do not agree with actual error rates, for the tails of noise distribution deviate from normal distribution. However, false discovery rate based on empirically estimated null distribution is very close to observed false discovery proportion.     

Novel Microarrays for Simultaneous Serodiagnosis of Multiple Antiviral Antibodies

We developed an automated diagnostic system for the detection of virus-specific immunoglobulin Gs (IgGs) that was based on a microarray platform. We compared efficacies of our automated system with conventional enzyme immunoassays (EIAs). Viruses were immobilized to microarrays using a radical cross-linking reaction that was induced by photo-irradiation. A new photoreactive polymer containing perfluorophenyl azide (PFPA) and poly(ethylene glycol) methacrylate was prepared and coated on plates. Inactivated measles, rubella, mumps, Varicella-Zoster and recombinant Epstein-Barr viruse antigen were added to coated plates, and irradiated with ultraviolet light to facilitate immobilization. Virus-specific IgGs in healthy human sera were assayed using these prepared microarrays and the results obtained compared with those from conventional EIAs. We observed high correlation (0.79–0.96) in the results between the automated microarray technique and EIAs. The microarray-based assay was more rapid, involved less reagents and sample, and was easier to conduct compared with conventional EIA techniques. The automated microarray system was further improved by introducing reagent storage reservoirs inside the chamber, thereby conserving the use of expensive reagents and antibodies. We considered the microarray format to be suitable for rapid and multiple serological diagnoses of viral diseases that could be developed further for clinical applications.     

Long-term treatment with a beta-blocker timolol attenuates renal-damage in diabetic rats via enhancing kidney antioxidant-defense system

Mutations in Ras isoforms such as K-Ras, N-Ras, and H-Ras contribute to roughly 85, 15, and 1 % of human cancers, respectively. Proper membrane targeting of these Ras isoforms, a prerequisite for Ras activity, requires farnesylation or geranylgeranylation at the C-terminal CAAX box. We devised an in vivo screening strategy based on monitoring Ras activation and phenotypic physiological outputs for assaying synthetic Ras function inhibitors (RFI). Ras activity was visualized by the translocation of RBD _Raf1 -GFP to activated Ras at the plasma membrane. By using this strategy, we screened one synthetic farnesyl substrate analog (AGOH) along with nine putative inhibitors and found that only m-CN-AGOH inhibited Ras activation. Phenotypic analysis of starving cells could be used to monitor polarization, motility, and the inability of these treated cells to aggregate properly during fruiting body formation. Incorporation of AGOH and m-CN-AGOH to cellular proteins was detected by western blot. These screening assays can be incorporated into a high throughput screening format using Dictyostelium discoideum and automated microscopy to determine effective RFIs. These RFI candidates can then be further tested in mammalian systems.