Automation Highlights from the Literature

Abstract

Laboratory Automation and High-Throughput ChemistryLarge-Scale Cytological Profiling for Functional Analysis of Bioactive CompoundsIn this study, Woehrmann et al. from the Department of Biomolecular Engineering of the University of Santa Cruz, California, report that cytological profiling (CP) is an unbiased image-based screening technique that uses automated microscopy and image analysis to profile compounds based on numerous quantifiable phenotypic features. They use CP to evaluate a library of nearly 500 compounds with documented mechanisms of action (MOAs) spanning a wide range of biological pathways. They develop informatics techniques for generating dosage-independent phenotypic “fingerprints” for each compound and for quantifying the likelihood that a compound’s CP fingerprint corresponds to its annotated MOA.The authors identify groups of features that distinguish classes with closely related phenotypes, such as microtubule poisons versus HSP90 inhibitors and DNA synthesis versus proteasome inhibitors. Several cases are tested in which cytological profiles indicated novel mechanisms, including a tyrphostin kinase inhibitor involved in mitochondrial uncoupling, novel microtubule poisons, and a nominal PPAR-gamma ligand that acts as a proteasome inhibitor, using independent biochemical assays to confirm the MOAs predicted by the CP signatures. The authors also apply maximal-information statistics to identify correlations between cytological features and kinase inhibitory activities by combining the CP fingerprints of 24 kinase inhibitors with published data on their specificities against a diverse panel of kinases. The resulting analysis suggests a strategy for probing the biological functions of specific kinases by compiling cytological data from inhibitors of varying specificities. (Woehrmann et al., Mol. Biosyst. 2013, 9, 2604–2617)Microplates in Liquid Chromatography: New Solution in Clinical Research? A ReviewMicroplates are routinely used in radio- or immunoassays. Recently, microplates have found use not only in analytical but also in the preanalytical phase in bioanalyses (sample storage, sample preparation). A new connection of this technology to liquid chromatography could be an economical, fast, and simple solution for many routine laboratories handling large sequences of biological samples. This review summarizes the application of microplates in bioanalytical laboratories. Different types of sorbents, materials, and shapes of microplates are discussed, and the main advantages and disadvantages of microplates used in clinical research are presented. (Krcmova et al., Talanta 2013, 115, 973–979)New High-Throughput Screening Method for Drug Release MeasurementsIn the field of drug delivery systems, microparticles made of polymeric matrix appear as an attractive approach. The in vitro release kinetic profile is crucial information when developing new particulate formulations. These data are essential for batch-to-batch comparisons, quality control, and anticipation of in vivo behavior to select the best formulation to go further in preclinical investigations. The methods available present common drawbacks such as time and compound consumption that does not fit with formulation screening requirements in early development stages.In this study, a new microscale high-throughput screening (HTS) method is developed to investigate drug release kinetic from piroxicam-loaded polylactic acid and polylactic-co-glycolic acid microparticles. The method is sample and separation based, in which separation is performed by filtration using 96-well micro filter plates. Ninety-six experiments can therefore be performed on one plate at one time in a fully automated way and with a very low sample and particle consumption. The influence of different parameters controlling release profiles also is investigated using this technique. The HTS method gives the same release profile as the standard dialysis method. Shaking, particle concentration, and the nature of the release medium are found to be of influence. The HTS method appears to be a reliable method to evaluate drug release from particles with a smaller standard deviation and less consumption of material. (Pelczarska et al., Eur. J. Pharm. Biopharm. 2013, 85, 151–157).High-Throughput Method for the Analysis of Ethylenethiourea with Direct Injection of Hydrolyzed Urine Using Online On-Column Extraction Liquid Chromatography and Triple Quadrupole Mass SpectrometryEthylenethiourea (ETU) is of major toxicological concern, because in experimental animal studies, ETU has shown a large spectrum of adverse effects. High occupational exposure can be found among agricultural workers or during manufacturing of ethylenbisdithiocarbamates. For the general public, sources of environmental exposure may be residues of ETU in commercial products, food, and beverages.For the determination of ETU in human urine, Ekman et al. present a high-throughput online on-column extraction liquid chromatography triple quadrupole mass spectrometry method using direct injection of hydrolyzed urine samples. This method is simple and user and environmentally friendly, and all sample preparation is performed in 96-well plates. A labeled ETU internal standard is used for quantification. The method shows a good sensitivity with a limit of quantification of 0.5 ng ETU/mL urine, and the calibration curve is linear in the range of 0.25 to 200 ng ETU/mL urine. The within-run, between-run, and between-batch precision is between 6% and 13%. Alkaline hydrolysis considerably increases the levels of ETU, indicating a potential conjugate.This method is applied in an experimental dermal exposure study in humans, with sample concentrations ranging from 0.4 to 5.0 ng ETU/mL urine. The excretion in urine is 10% of the applied dose. The elimination profile seems to differ between the two individuals. The results show an estimated half-life of ETU between 34 and 72 h. Although the experiment is limited to two individuals, the data provide valuable and new information regarding the toxicokinetics of ETU after dermal exposure. (Ekman et al., J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2013, 934, 53–59)Microfluidic Chip Technology and Microreactor TechnologyAccessing New Chemical Entities through Microfluidic SystemsFlow systems are successfully used for a wide variety of applications in chemical research and development, including the miniaturization of (bio)analytical methods and synthetic (bio)organic chemistry. Currently, the authors are witnessing the growing use of microfluidic technologies for the discovery of new chemical entities. As a consequence, chemical biology and molecular medicine research are being reshaped by this technique.In this mini review, Rodrigues et al. portray the state of the art, including the most recent advances in the application of microchip reactors as well as the micro- and mesoscale coil reactor-assisted synthesis of bioactive small molecules, and they forecast the potential future use of this promising technology. (Rodrigues et al., Angew. Chem. Int. Ed. Engl. 2014, 53, 5750–5758)An Automated Microfluidic Device for Assessment of Mammalian Cell Genetic StabilitySingle-cell transcriptome contains reliable gene regulatory relationships because gene-gene interactions happen only within a mammalian cell. Although the study of gene-gene interactions enables us to understand the molecular mechanism of cellular events and to evaluate molecular characteristics of a mammalian cell population, its complexity requires an analysis of a large number of single cells at various stages. However, many existing microfluidic platforms cannot process single cells effectively for routine molecular analysis. To address these challenges, Chen et al. demonstrate an integrated system with an individual controller for effective single-cell transcriptome analysis.In this article, the authors report an integrated microfluidic approach to rapidly measure gene expression in individual cells for genetic stability assessment of a cell population. Inside this integrated microfluidic device, the cells are individually manipulated and isolated in an array using microsieve structures, then transferred into different nanoliter reaction chambers for parallel processing of single-cell transcriptome analysis. This device enables the manipulation of individual single-cells in a nanoliter reactor with a high recovery rate. The authors perform gene expression analysis for a large number of HeLa cells and 293T cells expanded from a single cell. The data show that even the housekeeping genes are expressed at heterogeneous levels within a clone of cells. The heterogeneity of actin expression reflects the genetic stability, and the expression distribution is different between cancer cells (HeLa) and immortalized 293T cells. The result demonstrates that this platform has the potential for assessment of genetic stability in cancer diagnosis. (Chen et al., Lab Chip 2012, 12, 3930–3935)Individually Addressable Multi-Chamber Electroporation Platform with Dielectrophoresis and Alternating-Current-Electro-Osmosis Assisted Cell PositioningPark et al., from the Micro and Nanofluidics Laboratory of the Technion-Israel Institute of Technology, report a multifunctional microfluidic platform fabricated to demonstrate the feasibility of on-chip electroporation integrated with dielectrophoresis (DEP) and alternating-current-electro-osmosis (ACEO)–assisted cell/particle manipulation. A spatial gradient of electroporation parameters is generated within a microchamber array and validated using normal human dermal fibroblast cells and red fluorescent protein-expressing human umbilical vein endothelial cells with various fluorescent indicators.The edge of the bottom electrode, coinciding with the microchamber entrance, may act as an on-demand gate, functioning under either positive or negative DEP. In addition, at sufficiently low activation frequencies, ACEO vortices can complement the DEP to contribute to a rapid trapping/alignment of particles. As such, results clearly indicate that the microfluidic platform has the potential to achieve high-throughput screening for electroporation with spatial control and uniformity, assisted by DEP and ACEO manipulation/trapping of particles/cells into individual microchambers. (Park, Biomicrofluidics 2014, 8, 024117)Microfluidic Single-Cell Whole-Transcriptome SequencingSingle-cell whole-transcriptome analysis is a powerful tool for quantifying gene expression heterogeneity in populations of cells. Many techniques have been recently developed to perform transcriptome sequencing (RNA-Seq) on individual cells. To probe subtle biological variation between samples with limiting amounts of RNA, more precise and sensitive methods are still required.Streets et al. adapt a previously developed strategy for single-cell RNA-Seq that shows promise for superior sensitivity and implements the chemistry in a microfluidic platform for single-cell whole-transcriptome analysis. In this approach, single cells are captured and lysed in a microfluidic device, in which mRNAs with poly(A) tails are reverse transcribed into cDNA. Double-stranded cDNA is then collected and sequenced using a next-generation sequencing platform.Streets et al. prepare 94 libraries consisting of single mouse embryonic cells and technical replicates of extracted RNA and thoroughly characterize the performance of this technology. Microfluidic implementation increases mRNA detection sensitivity as well as improves measurement precision compared with tube-based protocols. With 0.2 M reads per cell, the authors reconstruct a majority of the bulk transcriptome with 10 single cells. The authors also quantify variation between and within different types of mouse embryonic cells and find that enhanced measurement precision, detection sensitivity, and experimental throughput aid the distinction between biological variability and technical noise. With this work, the authors validate the advantages of an early approach to single-cell RNA-Seq and show that the benefits of combining microfluidic technology with high-throughput sequencing will be valuable for large-scale efforts in single-cell transcriptome analysis. (Streets et al., Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 7048–7053)Next-Generation MUT-MAP, a High-Sensitivity High-Throughput Microfluidics Chip-Based Mutation Analysis PanelMolecular profiling of tumor tissue to detect alterations, such as oncogenic mutations, plays a vital role in determining treatment options in oncology. Hence, there is an increasing need for a robust and high-throughput technology to detect oncogenic hotspot mutations. Although commercial assays are available to detect genetic alterations in single genes, only a limited amount of tissue is often available from patients, requiring multiplexing to allow for simultaneous detection of mutations in many genes using low DNA input. Even though next-generation sequencing (NGS) platforms provide powerful tools for this purpose, they face challenges such as high cost, large DNA input requirement, complex data analysis, and long turnaround times, limiting their use in clinical settings.Schleifman et al. report the development of the next-generation mutation multianalyte panel (MUT-MAP), a high-throughput, microfluidic panel for detecting 120 somatic mutations across 11 genes of therapeutic interest (AKT1, BRAF, EGFR, FGFR3, FLT3, HRAS, KIT, KRAS, MET, NRAS, and PIK3CA) using allele-specific PCR and Taqman technology. This mutation panel requires as little as 2 ng of high-quality DNA from fresh frozen or 100 ng of DNA from formalin-fixed paraffin-embedded (FFPE) tissues. Mutation calls, including an automated data analysis process, are implemented to run 88 samples per day. Validation of this platform using plasmids shows robust signal and low cross-reactivity in all of the newly added assays, and mutation calls in cell line samples are found to be consistent with the Catalogue of Somatic Mutations in Cancer (COSMIC) database, allowing for direct comparison of the authors’ platform to Sanger sequencing. High correlation with NGS when compared with the SuraSeq500 panel run on the Ion Torrent platform in an FFPE dilution experiment shows assay sensitivity down to 0.45%. This multiplexed mutation panel is a valuable tool for high-throughput biomarker discovery in personalized medicine and cancer drug development. (Schleifman et al., PLoS One 2014, 21, e90761)High-Throughput AnalyticsLarge-Scale Automated Image Analysis for Computational Profiling of Brain Tissue Surrounding Implanted Neuroprosthetic Devices Using PythonIn this article, Ray-Villamizar et al. describe the use of Python for large-scale automated server-based bio-image analysis in FARSIGHT, a free and open-source toolkit of image analysis methods for quantitative studies of complex and dynamic tissue microenvironments imaged by modern optical microscopes, including confocal, multispectral, multiphoton, and time-lapse systems. The core FARSIGHT modules for image segmentation, feature extraction, tracking, and machine learning, are written in C++, leveraging widely used libraries including ITK, VTK, Boost, and Qt. For solving complex image analysis tasks, these modules must be combined into scripts using Python.As a concrete example, the authors consider the problem of analyzing three-dimensional multispectral images of brain tissue surrounding implanted neuroprosthetic devices, acquired using high-throughput multispectral spinning disk step-and-repeat confocal microscopy. The resulting images typically contain five fluorescent channels. Each channel consists of 6000 × 10,000 × 500 voxels with 16 bits/voxel, implying image sizes exceeding 250 GB. These images must be mosaicked, preprocessed to overcome imaging artifacts, and segmented to enable cellular-scale feature extraction. The features are used to identify cell types and to perform large-scale analysis for identifying spatial distributions of specific cell types relative to the device. Python is used to build a server-based script (Dell 910 PowerEdge servers with four sockets/server with 10 cores each, two threads per core, and 1 TB of RAM running on Red Hat Enterprise Linux linked to a RAID 5 SAN) capable of routinely handling image data sets at this scale and performing all of these processing steps in a collaborative multiuser, multiplatform environment. The Python script enables efficient data storage and movement between computers and storage servers, logs all the processing steps, and performs full multithreaded execution of all codes, including open- and closed-source third-party libraries. (Rey-Villamizar et al., Front Neuroinform. 2014, 8, 39)From Human Monocytes to Genome-Wide Binding Sites—A Protocol for Small Amounts of Blood: Monocyte Isolation/ChIP-Protocol/Library Amplification/Genome Wide Computational Data AnalysisChromatin immunoprecipitation in combination with a genome-wide analysis via high-throughput sequencing is the state-of-the-art method to gain genome-wide representation of histone modification or transcription factor binding profiles. However, chromatin immunoprecipitation analysis in the context of human experimental samples is limited, especially in the case of blood cells. The typically extremely low yields of precipitated DNA are usually not compatible with library amplification for next-generation sequencing.Weiterer et al. develop a highly reproducible protocol to present a guideline from the first step of isolating monocytes from a blood sample to analyze the distribution of histone modifications in a genome-wide manner. They conclude that the protocol describes the whole workflow from isolating monocytes from human blood samples followed by a high-sensitivity and small-scale chromatin immunoprecipitation assay with guidance for generating libraries compatible with next-generation sequencing from small amounts of immunoprecipitated DNA. (Weiterer et al., PLoS One 2014, 9, e94164)Advances in Automated ELISAsComparison of Two Automated Immunoassays for the Determination of Puumalavirus IgM and IgGThe puumala virus (PUUV), a member of the genus Hantavirus, can cause nephropathia epidemica, a mild form of hemorrhagic fever with renal syndrome. The method of choice for the serodiagnosis of hantavirus infections are enzyme-linked immunosorbent assays (ELISAs). Muyldermans et al. compare two commercially available PUUV ELISA kits: hantavirus (puumala) IgM/IgG ELISA (Progen, Heidelberg, Germany) and PUUMALA IgM and IgG EIA AutoM (Reagena, Toivala, Finland). The sensitivity of the ELISA kits is evaluated with a panel of 55 serum samples from patients with an acute (n = 27) or past (n = 28) infection based on Progen or Reagena. A panel of 56 serum samples is composed to evaluate the specificity: samples with potentially false-positive Progen puumala IgM results (n = 12), seronegative samples for puumala IgG/IgM with Progen (n = 20), and potentially cross-reacting samples (n = 24). Discrepancies between the two assays are resolved with strip immunoblot. As measure of agreement between Progen and Reagena results, the Cohen kappa coefficient is calculated.As a result, Reagena shows a higher specificity (IgM 100%, IgG 100%) than Progen puumala (IgM 73.21%, IgG 100%). However, Reagena shows a slightly lower sensitivity (IgM 96.15%, IgG 97.78%) compared with Progen (IgM 100%, IgG 100%). Substantial agreement with a Cohen kappa of 0.67 and 0.76 is found between the two assays for puumala IgM and IgG, respectively.This study shows a higher specificity of Reagena in comparison to Progen with a lower sensitivity, which is probably caused by selection bias. Despite Reagena’s lower sensitivity, no acute infection is missed with this assay. (Muyldermans et al., J. Clin. Virol. 2014, 60, 165–167)A Simple and Fast Non-Radioactive Bridging Immunoassay for Insulin AutoantibodiesType 1 diabetes (T1D) is an autoimmune disease that results from the destruction of pancreatic beta cells. Autoantibodies directed against islet antigens are valuable diagnostic tools. Insulin autoantibodies (IAAs) are usually the first to appear and also the most difficult to detect among the four major islet autoantibodies.A nonradioactive IAA bridging ELISA is developed to this end. In this assay, one site of the IAAs from serum samples is bound to a hapten-labeled insulin (GC300-insulin), which is subsequently captured on anti-GC300 antibody-coated 96-well plates. The other site of the IAAs is bound to biotinylated insulin, allowing the complex to be detected by an enzyme-streptavidin conjugate.In the study, 50 serum samples from patients with newly diagnosed T1D and 100 control sera from nondiabetic individuals are analyzed with the new assay, and the results are correlated with an IAA radioimmunoassay (RIA). Using IAA bridging ELISA, IAAs are detected in 32 of 50 T1D children, whereas with IAA RIA, 41 of 50 children with newly diagnosed T1D are scored as positive. In conclusion, the IAA bridging ELISA could serve as an attractive approach for rapid and automated detection of IAAs in T1D patients for diagnostic purposes. (Kikkas et al., PLoS One 2013, 8, e69021) Laboratory Automation and High-Throughput ChemistryLarge-Scale Cytological Profiling for Functional Analysis of Bioactive CompoundsIn this study, Woehrmann et al. from the Department of Biomolecular Engineering of the University of Santa Cruz, California, report that cytological profiling (CP) is an unbiased image-based screening technique that uses automated microscopy and image analysis to profile compounds based on numerous quantifiable phenotypic features. They use CP to evaluate a library of nearly 500 compounds with documented mechanisms of action (MOAs) spanning a wide range of biological pathways. They develop informatics techniques for generating dosage-independent phenotypic “fingerprints” for each compound and for quantifying the likelihood that a compound’s CP fingerprint corresponds to its annotated MOA.The authors identify groups of features that distinguish classes with closely related phenotypes, such as microtubule poisons versus HSP90 inhibitors and DNA synthesis versus proteasome inhibitors. Several cases are tested in which cytological profiles indicated novel mechanisms, including a tyrphostin kinase inhibitor involved in mitochondrial uncoupling, novel microtubule poisons, and a nominal PPAR-gamma ligand that acts as a proteasome inhibitor, using independent biochemical assays to confirm the MOAs predicted by the CP signatures. The authors also apply maximal-information statistics to identify correlations between cytological features and kinase inhibitory activities by combining the CP fingerprints of 24 kinase inhibitors with published data on their specificities against a diverse panel of kinases. The resulting analysis suggests a strategy for probing the biological functions of specific kinases by compiling cytological data from inhibitors of varying specificities. (Woehrmann et al., Mol. Biosyst. 2013, 9, 2604–2617)Microplates in Liquid Chromatography: New Solution in Clinical Research? A ReviewMicroplates are routinely used in radio- or immunoassays. Recently, microplates have found use not only in analytical but also in the preanalytical phase in bioanalyses (sample storage, sample preparation). A new connection of this technology to liquid chromatography could be an economical, fast, and simple solution for many routine laboratories handling large sequences of biological samples. This review summarizes the application of microplates in bioanalytical laboratories. Different types of sorbents, materials, and shapes of microplates are discussed, and the main advantages and disadvantages of microplates used in clinical research are presented. (Krcmova et al., Talanta 2013, 115, 973–979)New High-Throughput Screening Method for Drug Release MeasurementsIn the field of drug delivery systems, microparticles made of polymeric matrix appear as an attractive approach. The in vitro release kinetic profile is crucial information when developing new particulate formulations. These data are essential for batch-to-batch comparisons, quality control, and anticipation of in vivo behavior to select the best formulation to go further in preclinical investigations. The methods available present common drawbacks such as time and compound consumption that does not fit with formulation screening requirements in early development stages.In this study, a new microscale high-throughput screening (HTS) method is developed to investigate drug release kinetic from piroxicam-loaded polylactic acid and polylactic-co-glycolic acid microparticles. The method is sample and separation based, in which separation is performed by filtration using 96-well micro filter plates. Ninety-six experiments can therefore be performed on one plate at one time in a fully automated way and with a very low sample and particle consumption. The influence of different parameters controlling release profiles also is investigated using this technique. The HTS method gives the same release profile as the standard dialysis method. Shaking, particle concentration, and the nature of the release medium are found to be of influence. The HTS method appears to be a reliable method to evaluate drug release from particles with a smaller standard deviation and less consumption of material. (Pelczarska et al., Eur. J. Pharm. Biopharm. 2013, 85, 151–157).High-Throughput Method for the Analysis of Ethylenethiourea with Direct Injection of Hydrolyzed Urine Using Online On-Column Extraction Liquid Chromatography and Triple Quadrupole Mass SpectrometryEthylenethiourea (ETU) is of major toxicological concern, because in experimental animal studies, ETU has shown a large spectrum of adverse effects. High occupational exposure can be found among agricultural workers or during manufacturing of ethylenbisdithiocarbamates. For the general public, sources of environmental exposure may be residues of ETU in commercial products, food, and beverages.For the determination of ETU in human urine, Ekman et al. present a high-throughput online on-column extraction liquid chromatography triple quadrupole mass spectrometry method using direct injection of hydrolyzed urine samples. This method is simple and user and environmentally friendly, and all sample preparation is performed in 96-well plates. A labeled ETU internal standard is used for quantification. The method shows a good sensitivity with a limit of quantification of 0.5 ng ETU/mL urine, and the calibration curve is linear in the range of 0.25 to 200 ng ETU/mL urine. The within-run, between-run, and between-batch precision is between 6% and 13%. Alkaline hydrolysis considerably increases the levels of ETU, indicating a potential conjugate.This method is applied in an experimental dermal exposure study in humans, with sample concentrations ranging from 0.4 to 5.0 ng ETU/mL urine. The excretion in urine is 10% of the applied dose. The elimination profile seems to differ between the two individuals. The results show an estimated half-life of ETU between 34 and 72 h. Although the experiment is limited to two individuals, the data provide valuable and new information regarding the toxicokinetics of ETU after dermal exposure. (Ekman et al., J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2013, 934, 53–59) Large-Scale Cytological Profiling for Functional Analysis of Bioactive CompoundsIn this study, Woehrmann et al. from the Department of Biomolecular Engineering of the University of Santa Cruz, California, report that cytological profiling (CP) is an unbiased image-based screening technique that uses automated microscopy and image analysis to profile compounds based on numerous quantifiable phenotypic features. They use CP to evaluate a library of nearly 500 compounds with documented mechanisms of action (MOAs) spanning a wide range of biological pathways. They develop informatics techniques for generating dosage-independent phenotypic “fingerprints” for each compound and for quantifying the likelihood that a compound’s CP fingerprint corresponds to its annotated MOA.The authors identify groups of features that distinguish classes with closely related phenotypes, such as microtubule poisons versus HSP90 inhibitors and DNA synthesis versus proteasome inhibitors. Several cases are tested in which cytological profiles indicated novel mechanisms, including a tyrphostin kinase inhibitor involved in mitochondrial uncoupling, novel microtubule poisons, and a nominal PPAR-gamma ligand that acts as a proteasome inhibitor, using independent biochemical assays to confirm the MOAs predicted by the CP signatures. The authors also apply maximal-information statistics to identify correlations between cytological features and kinase inhibitory activities by combining the CP fingerprints of 24 kinase inhibitors with published data on their specificities against a diverse panel of kinases. The resulting analysis suggests a strategy for probing the biological functions of specific kinases by compiling cytological data from inhibitors of varying specificities. (Woehrmann et al., Mol. Biosyst. 2013, 9, 2604–2617) In this study, Woehrmann et al. from the Department of Biomolecular Engineering of the University of Santa Cruz, California, report that cytological profiling (CP) is an unbiased image-based screening technique that uses automated microscopy and image analysis to profile compounds based on numerous quantifiable phenotypic features. They use CP to evaluate a library of nearly 500 compounds with documented mechanisms of action (MOAs) spanning a wide range of biological pathways. They develop informatics techniques for generating dosage-independent phenotypic “fingerprints” for each compound and for quantifying the likelihood that a compound’s CP fingerp